The contents of the neuro-excitatory amino acid β-ODAP (β- N-oxalyl- l-α,β-diaminopropionic...

Im proving the nutritional quality of grass pea (L athyrus sativus L.)

II

Improving the nutritional quality of Lathyrus sativus L. (grass pea)

for safer consumption

Asnake Fikre Woldemedhin

EIAR


III

A Promoters:

Prof. Dr. Godelieve Gheysen Ghent University Department of Molecular Biotechnology Coupure links 653, B-9000, Ghent, Belgium Prof. Dr. Ir. Fernand Lambein Ghent University Institute of Plant Biotechnology for Developing Countries (IPBO), K. L. Ledeganckstraat 35, B-9000, Ghent, Belgium Dean: Prof. Dr. Ir. Herman Van Langenhove

Rector: Prof. Dr. Paul Van Cauwenberge


IV

Asnake Fikre Woldemedhin

Improving the nutritional quality of Lathyrus sativus L. (grass pea) for safer consumption

Thesis submitted in partial fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences


V

Verbetering van de nutritionele kwaliteit van Lathyrus sativus voor een veiliger consumptie Cover illustration : Field grown grass pea in Ethiopia, the green unripe pods that is often consumed by children in fields and diverse seeds from different origin. Citation : Asnake Fikre. 2008. Improving the nutritional quality of Lathyrus sativus L. (grass pea) for safer consumption. PhD thesis. Ghent University, Belgium. ISBN- number: 978-90-5989-255-2 The author and promoter give the authorization to consult and to copy parts of this work for personal use only. No part is to be reproduced without the permission of the author.


VI

Dedicated to my very first teacher the late Sium Woldemichael who wished if among his students could attain such level, and equally to lathyrism victim children whose life remains in ruins.


VII

ACKNOWLEDGMENTS I thank God for making my efforts fruitful. I would like to extend my deepest thanks to professor Dr. Ir. Fernand Lambein whose concern for my work was extremely important. He taught me not only the academics of knowledgeable merit but as well the value of life experience. My deepest thanks is extended to my promoter professor Dr. Godelieve Gheysen whose scientific support was extremely important. I extend my heartfelt thanks to Dr. Yu-Haey Kuo who basically trained me in laboratory work and familiarization with lab equipments. She as well kept me in good caring that made my stay comfortable and in a family feeling. I am grateful to Dr. Nancy Terryn, Dr. Kibebew Asefa, Dr. Alemu Yami, Dr. Reta Leta, Mrs Marijke Van Moorhem and Mr. Hailemariam Teklewold for their comments and/or scientific support of my thesis work. My thanks go to Prof. Dr. Marc Van Montagu, founder of the Institute of Plant Biotechnology for Developing Countries (IPBO), institute established to assist the developing world. Dr. Seid Ahmed, Dr. Million Eshete, Mr. Lijalem Korbu, Mr. Melese Dadi, Mr. Asfaw Taddese, Mrs. Konjit Ayalew, Mr. Nega Alemu, Mr. Tekalign Taddesse, Mr. Wendimagegnehu Weldesemayat, Dr. Sylvia Burssens, Mrs. Veerle van Ongeval, Mrs. Bossena and the late Mr. Kirubel Kora are highly acknowledged for their technical and/or administrative support and facilitation during the course of my work. I thank the Department of Applied Analytical and Physical Chemistry, and Department of Food safety and Food Quality for analysing my samples for micronutrient and allowing me to analyse my samples for proximate composition respectively. I like to extend my greatest thanks to the members of the examination committee: Professor Dr. Els Van Damme, Professor Dr. Ir. Patrick Van Damme, Professor Dr. Patrick Van der Stuyft, Professor Dr. Marcelle Holsters and Professor Dr. Ir. Geert Angenon for their unreserved constructive suggestions and comments. I also extend my thanks to my beloved wife Sr. Meseret Bekele who kept our children, Nahom and Yesufekad, and tolerantly stayed alone in my absence. My thank also goes to my beloved parents Mr. Fikre Woldemedhin Dawitee and Mrs. Mulunesh Habteyohanis Haile, my sisters, my brothers and relatives who were on my side that gave me courage in my work. Last but not least, I appreciably give my thanks to Ethio-Belgian (EIAR-VLIR) Lathyrus Nutritional Improvement Project (2004-2008) which was the financial resource for my study to this end.


VIII

Abbreviations and acronyms AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ANF Anti Nutritional Factors ANOVA Analysis of Variances AOAC Association of Official Analytical Chemists ASL Above Sea Level BAPN ß-aminopropionitrile BD Bulk Density BIA β-(isoxazolin-5-on-2-yl)-alanine DABA L-2, 4-diaminobutyric acid DAPRO α,ß-diaminopropionic acid DDW Double Distilled Water DF Days to Flowering DM Days to Maturity DG Deep Green DTPA Diethylene Triamine Pentaacetic Acid DVM Doctor of Veterinary Medicine DZARC Debre Zeit Agricultural Research Centre EIAR Ethiopian Institute of Agricultural Research FAO Food and Agricultural Organization FCE Food Conversion Efficiency GFAAS Graphite Furnace Atomic Absorption Spectrophotometry GLM General Linear Model GO Glycolate Oxidase HPLC High-Performance Liquid Chromatography ICARDA International Centre for Agricultural Research in Dry Areas ICP Inductively Coupled Plasma IPGRI International Plant Genetic Resource Institute MCF Moisture Correction Factor MJ Mega joule NSRC National Soil Research Centre OM Organic Matter β-ODAP β-N-oxalyl-L-α,β-diaminopropionic acid pH Potential of Hydrogen PITC Phenyl-iso-thiocyanate SSH Sunshine Hours TN Total Nitrogen VLIR Flemish Interuniversity Council WHO World Health Organization


IX

Table of contents

OUTLINE............................................................................................................... XI CHAPTER I .............................................................................................................. 1 Literature review ....................................................................................................... 1

I.1. Introduction..................................................................................................... 3 I.1.1. Characteristic features of grass pea and its cultivation............................... 3 I.1.2. Grass pea consumption habits and neurolathyrism .................................... 7

I.1.2.1. Consumption...................................................................................... 7 I.1.2.2. Neurolathyrism .................................................................................. 7

I.1.3. Factors affecting the level of neuro-excitatory amino acid in grass pea ... 10 I.1.3.1. Climatic factors and ß-ODAP biosynthesis ...................................... 11 I.1.3.2. Growth media and associated changes in ß-ODAP........................... 13 I.1.3.3. Effect of grass pea processing on ß-ODAP reduction and nutritional quality ......................................................................................................... 14 I.1.3.4. Breeding or genetic engineering to reduce β-ODAP in grass pea...... 18

I.1.4. Effect of methionine on toxicity of grass pea .......................................... 21 I.1.5. Rationale of the study and motivation of the investigation ...................... 22 I.1.6. Overall and specific objectives of the study ............................................ 24

General objective......................................................................................... 24 Specific objectives....................................................................................... 24

CHAPTER II........................................................................................................... 25 Level of β-ODAP, methionine and other free and protein amino acids in the seeds of different grass pea (Lathyrus sativus L.) genotypes.................................................. 25

II.1. Introduction ................................................................................................. 27 II.2. Materials and methods ................................................................................. 29 II.3. Results ......................................................................................................... 32 II.4. Discussion.................................................................................................... 36

CHAPTER III ......................................................................................................... 39 Study of environmental factors (climatic, edaphic) affecting yield and toxicity of grass pea grown in Ethiopia in a multilocation experiment ............................................... 39

III.1. Introduction ................................................................................................ 42 III.2. Materials and methods ................................................................................ 44 III.3. Results........................................................................................................ 48 III.4. Discussion .................................................................................................. 55

CHAPTER IV ......................................................................................................... 60 Effect of methionine supplement on physical responses and neurological symptoms of broiler chicks fed grass pea based starter ration ....................................................... 61

IV.1. Introduction................................................................................................ 64 IV.2. Materials and Methods ...............................................................................65 IV.3. Results........................................................................................................ 67 IV.4. Discussion .................................................................................................. 73


X

CHAPTER V .......................................................................................................... 79 Effect of methionine supplement on serum amino acids status and trace elements level of broiler chicks fed with grass pea based starter ration ........................................... 79

V.1. Introduction ................................................................................................. 82 V.2. Materials and Methods................................................................................. 83 V.3. Results......................................................................................................... 85 V.4. Discussion ................................................................................................... 90

CHAPTER VI ......................................................................................................... 93 Identification of gamma irradiation derived mutant lines of Lathyrus sativus for higher methionine level ........................................................................................... 93

VI.1. Introduction................................................................................................ 95 VI.2. Materials and methods................................................................................ 97 VI. 3. Results....................................................................................................... 99 VI.4. Discussion .................................................................................................103

CHAPTER VII.......................................................................................................107 General discussion and conclusions........................................................................107

VII.1. Environmental influence on levels of amino acids and crop performances in L. sativus ............................................................................................................109 VII.2. Effect of methionine on toxicity of L. sativus ...........................................114 VII.3. Mutational methionine enrichment in L. sativus........................................117 VII.4. Recommendations and future perspectives ...............................................119

References..............................................................................................................123 Summary - Samenvatting .......................................................................................149

Summary............................................................................................................151 Samenvatting......................................................................................................155

Curriculum Vitae……………………………………………………………………156


XI

OUTLINE


XII

OUTLINE

Lathyrus sativus L. (grass pea), a drought tolerant legume originating from the Near

East, has emerged with the historical beginning of agricultural practices. However,

this crop, which has been used as a pulse for at least 8000 years, has made little

progress as a food crop during this time. Prolonged and monotonous over-

consumption carries the risk of neurolathyrism, a disease causing irreversible

crippling of the legs. Producers prefer grass pea to many other legumes as production

is insured even under environmental stresses such as drought and flooding. This pulse

crop needs little or no inputs, is cheaply available, has wide adaptability and it is used

as a multipurpose crop for food, feed, forage and green manure.

In CHAPTER I, an overview of the literature is presented. Concerted breeding

research efforts have been exhaustively directed towards reducing or removing the

neuro-excitatory free amino acid β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid)

from the plant. This nonprotein amino acid was discovered in 1964 and proposed as

the causal agent of neurolathyrism. The level of this metabolite is affected by

interdependent environmental factors, processing types and genetic factors. This

focused research approach in nutritional improvement of the crop neither resulted in

toxin free lines nor stopped neurolathyrism affliction. Also, other potential uses of the

crop have been under-exploited. In this work, experiments on comparative chemical

characterization of genotypes from different origin, on identification of important

environmental factors affecting grass pea toxicity and yield in Ethiopian context,

effect of methionine on grass pea toxicity, and in vivo methionine enrichment have

been undertaken with the following objectives: (i) Analyse factors affecting the level

of ß-ODAP, methionine and other amino acids in grass pea; (ii) assess the effect of

methionine on toxicity of grass pea in young broiler chicks; and (iii) mutagenize grass

pea seeds and select putative mutant lines with improved methionine content marker.

In CHAPTERS II & III, an extensive study into the influence of altitude, climatic

factors, geographic origin, growth media, genotypes and processing on the level of β-

ODAP, methionine and other amino acids was made. The problem statement was set

against the repeatedly published general belief that genetic variability and drought are

the only factors affecting toxicity, and therefore, hypothesized that there could be

other factors affecting toxicity and crop yield in Ethiopia. Both the free and protein

amino acids of the samples were quantitatively determined using HPLC analysis. Soil


XIII

nutrients were determined by flame ionisation analysis. Variability in amino acid

content of genotypes from different origin has been demonstrated. Important

environmental factors affecting the concentration of β-ODAP and yield were

investigated and possible interrelations were discussed. From the results, novel

conclusive remarks were made that several environmental factors interplay in

determining the level of β-ODAP and grass pea yield.

In CHAPTERS IV & V, the role of methionine on grass pea toxicity was studied by

examining chicks in a starter feeding experiment. The hypothesis was that methionine

addition could counteract grass pea toxicity and its aftermath. Performances of 450

chicks subdivided into two grass pea feed levels, each with four different levels of

DL-methionine supplement, were evaluated. Statistical analysis (ANOVA) was

carried out to compare treatment effects on feed intake, weight gain, and feed

conversion efficiency. Development of symptoms such as neck twisting was

investigated and phenotypically described. Serum amino acids and micronutrient

levels in the serum of the young chicks was determined against treatment levels and

time.

In CHAPTER VI, methionine enrichment of grass pea to improve its sulphur amino

acid deficient protein was attempted by gamma-irradiation mutagenesis. According to

Getahun et al. (2005) increased sulphur amino acid content of the diet contributed to

the protection of human consumers from neurolathyrism. The hypothesis is that in

vivo methionine enrichment in grass pea through mutation could be attained and that

it would contribute to food safety. Gamma ray mutagenized grass pea plants

exhibiting different phenotypical appearances were selected by observation in the

growing field. Methionine enhanced putative mutant lines were identified,

characterized and typical features were described. Deep green leaf was demonstrated

to have higher methionine level, therefore showing the typical morphological marker

of methionine enrichment as previously used by others in soybean.

Finally, in CHAPTER VII of the thesis, the main findings of the reported research

were discussed and conclusions drawn. Recommendations were derived and future

perspectives proposed.


1

CHAPTER I

Literature review


2

Lathyrus sativus L. (a) part of a flowering branch, (b) flower in front and side view, (c) dorsal petal (bottom),

wings and keel (from right), and (d) pod with seeds

Adopted from Campbell (1997).


3

CHAPTER I

Literature review

I.1. Introduction I.1.1. Characteristic features of grass pea and its cultivation

Lathyrus sativus L., commonly known as grass pea, chickling vetch or chickling pea,

is commercially an important legume in the tribe Viciae of the Fabaceae family. The

genus Lathyrus is characterized by the presence of leaf tendrils in the majority of its

members, an unusual arrangement of vascular tissue in the stem and characteristic

floral parts (Goyder, 1985). It comprises more than 150 species with 15 (Smartt et al.,

1994) or 13 (Kupicha, 1983) intragenic sections where many members, viz L. sativus,

L. hirsutus, L. odoratus, L. latifolius, L. cicera, L. clymenum, etc., have commercial

importance to mankind (Kupicha, 1983; Yamamoto et al., 1986; Yan et al., 2006).

Relationship studies between species in Lathyrus indicated that only two, i.e L.

amphicarpos and L. cicera, gave viable hybrids when crossed with L. sativus while

six others produced pods when crossed with the cultivated species (L. sativus and L.

cicera) which, however, contained either shrivelled seeds or were totally aborted

(Smartt, 1990; Smartt et al., 1994).

The epigrammatic taxonomic features of Lathyrus sativus have been reviewed by

several authors (Duke, 1981; Plitmann et al., 1985 and Campbell, 1997). Owing to the

large variation in several of its characters, only general common features are

discussed.

Grass pea is a herbaceous annual with straggling or climbing growth habit, branched

and with a sub-erect standing of 0.6-0.9 meters long. Stems are slender and

quadrangular with winged margins. Stipules are prominent, narrowly triangular to

ovate with a basal appendage.

The pinnately compounded opposite leaves of 30-150 mm long by 3-11 mm wide are

linear. Leaflets are lanceolate with simple or much-branched tendrils that are modified

from the terminal leaflets.


4

Flowers having a short (10-15 mm) and slender pedicel are solitary, axilliary, on a

peduncle of 30-60 mm; petals are erect and spreading, ovate 15 x 18 mm, finely

pubescent at upper margin, clawed, flat, and reddish-purple, pink, blue or white in

colour; wings are ovate, 14 x 8 mm, clawed and obtuse at top; keel is slightly twisted,

boat-shaped, 10 x 7 mm, entirely split dorsally, ventrally split near the base; calyx

teeth glabrous and longer than the calyx tube. Stamens are diadelphous (9+1) with

vexillary stamens free, 9 mm long, winged at base, apical part filiform, slightly

winged; anthers are elliptoid, 0.5 mm long and yellow; pollen 49-57 µm long; flowers

are predominantly self-pollinated. Ovary is sessile, thin, 6 mm long, pubescent with 5-

8 ovules; style is abruptly upturned, 6-7 mm long, widening at tip, and bearded below

the stigma; stigma is terminal, glandular-papillate and spatulate.

Pods are oblong, 2.5-4 cm long, flat, slightly curved, dorsally two-winged, with 2-5

seeds per pod. The seed is white, greyish-brown or yellowish, usually spotted or

mottled and sometimes smooth, 3-15 mm long by 7-9 mm diameter, round or angular

wedge-shaped; hilum is elliptic, with 1/15th to 1/16th of the circumference of a seed.

Germination is hypogeal, the epicotyls are purplish-green; the first leaf is small, scale-

like, often fused with two lateral stipulae. The second leaf is sublate, connected at the

base with the stipulae. Chromosomal number is 2x = 2n = 14 (Narayan, 1998). L.

sativus has a well-developed taproot with rootlets often covered by cylindrical

nodules.

The combination of archaeo-botanical and phyto-geographical evidence indicates that

the origin of L. sativus was in the Balkan Peninsula. It was already cultivated in the

early Neolithic period, at the beginning of the 6th millennium BC (Kislev, 1989). This

author also suggested that the practice of cropping annuals, including cereals and

legumes such as pea and lentil, introduced from the Near East around 6000 BC,

enabled domestication of L. sativus in this region. This means that L. sativus is

perhaps among the first crop domesticated in Europe as a consequence of expansion

of agriculture from the Near East. It is probably derived from the genetically nearest

wild species L. cicera (Hopf, 1986).


5

Man’s cultivation of grass pea dates back to the prehistoric era, and evidently

originated around the Mediterranean Sea. South East Asia and Ethiopia are traceable

early cultivation sites along with or right after its domestication period (Renfrew,

1979; Hopf, 1986; Kislev, 1989; Kockhar, 1992). Smartt (1984) found it rather

puzzling that a crop, which has been used as a pulse for at least 8000 years and is still

so used, should have made so little evolutionary progress as a grain crop during this

time. He considered that the lack of progress as a pulse crop might have been due to

its possibly more important use as a forage crop. Thus, in many ways, different

selection pressures imposed on the crop may have cancelled each other and must have

resulted in limited improvement of agronomic traits over this long period.

In some countries like Italy, grass pea is receiving renewed attention as a local and

traditional product; it is becoming an exclusive and fashionable food for which

consumers are prepared to pay a higher price than for other pulse products (Polignano

et al., 2005).

For the specific case of Ethiopia, grass pea has shown increasing trend both in

consumption and production. In the last decade alone its production area has

increased from about 80 thousand hectares to more than 120 thousand hectares

(Figure I.1). It has gained more than 100 % production increase over the last three to

four years, which likely resulted from both improving productivity and an increase in

production area. This could be attributed from the overall increasing trend, for

example, of total agricultural areas by 6 % and grass pea area by 4 % (Dadi et al.,

2003), and also due to increased preference for its insured production under low input

in marginal areas and with such unpredictable climatic situations. Since four years,

grass pea has got momentum of an average 8 % annual increase in production (Figure

I.1). Based on FAO grass pea production statistics (1996-2007), it can be calculated

that Ethiopia accounts for 75.3 % and 9 % in area cover and 85.4 % and 8 % in

production of Africa and the world, respectively.

The most interesting agronomical features of the species are its drought tolerance (as

it can grow with residual soil moisture alone), resistance to pests and diseases,

adaptability to different types of soil as well as to adverse climatic conditions (Noto et

al., 2001). The crop can be produced without any modern inputs whereas seeds


6

harvested from local cultivars are used for sowing the next season. In Ethiopia, L.

sativus stands between third and fifth in land cover among major pulses produced in

the country and is mainly grown on clayey vertisols and cambisols. However, a

considerable difference in yield between the national average (~1 t/ha) and the

productivity under improved management (4 t/ha) was demonstrated in Ethiopia

(Fikre et al., 2006). However, in spite of the many advantages this adaptable,

multipurpose crop can offer, it is inadequately exploited and studied.

It is important to underline that the consumption of L. sativus seeds by humans and

animals has been limited due to the presence of a neurotoxin known as β-N-oxalyl-L-

α,β-diaminopropionic acid (β-ODAP) in the plant. When consumed as a staple during

an uninterrupted period of several months, grass pea seeds can lead to

“neurolathyrism”, a disease causing permanent paralysis of the limbs (Eyzaguirre,

1999). For the last four decades, breeding programmes in many countries have been

focusing on toxin reduction and removal, however, without success in releasing toxin-

free or stable low toxin varieties and thus the problem persists to date.

0

20

40

60

80

100

120

140

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Years

Are

a

0 20 40 60 80 100 120 140 160 180 200

Pro

du

ctio

n

Area in 1000 ha Production in 1000 mt mmtmtMt

Figure I.1: Production trend of grass pea since 1996 in Ethiopia (Source: CSA 1996-

2007). Ha-hectare, mt-metric tone


7

I.1.2. Grass pea consumption habits and neurolathyrism

I.1.2.1. Consumption

Unlike other food legumes, grass pea is used as a staple food. It is an important source

of dietary proteins, calories and minerals for humans (Urga et al., 2005). Grass pea is

a nutritious and stress-surviving crop. Seed of Lathyrus sativus is prepared into

traditional dishes after dehusking and parching or milling. As a drought tolerant food

crop, it is a very promising source of protein, carbohydrate and minerals for drought

prone marginal lands with seeds containing an average of 27 % protein, 0.6 % fat,

58.2 % carbohydrate (about 35 % starch) (Duke, 1981; Williams et al., 1994; Urga et

al., 2005; Vaz Patto et al., 2006). The seeds contain 1.5 % sucrose, 6.8 % pentosans,

3.6 % phytin, 1.5 % lignin, 6.69 % albumin, 1.5 % prolamine, 13.3 % globulin, and

3.8 % glutelin. The concentration of essential amino acids in g per 16 g of N

(equivalent to 100 g of protein) reported were: arginine 7.85, histidine 2.51, leucine

6.57, isoleucine 6.59, lysine 6.94, methionine 0.38, phenylalanine 4.14, threonine

2.34, tryptophane 0.40, and valine 4.68 (Duke, 1981; Williams et al., 1994). Like

other cool season food legumes, grass pea is highly deficient in methionine, cysteine

and tryptophane.

Grass pea has been reported to have toxic metabolites causing irreversible crippling in

humans. Several substances that are toxic to laboratory animals have been identified

from seeds of Lathyrus species including ß-ODAP in L. sativus, the γ-glutamyl

derivative of ß-aminopropionitrile (BAPN) in L. odoratus, L. hirsutus, L. pussilus and

L. roseus, and L-2,4-diaminobutyric acid (DABA) in L. latifolius (Barrow et al.,

1974; Roy and Spencer, 1989; Foster, 1990). In addition to L. sativus, ß-ODAP is

produced in 17 species of Acacia and 13 species of Crotalaria (Quereshi et al., 1977),

and in only one non-legume: Panax ginseng (Long et al., 1996; Kuo et al., 2003).

I.1.2.2. Neurolathyrism

Neurolathyrism is an irreversible disease caused by prolonged dietary dependence on

grass pea (Getahun et al., 2005; Haimanot et al., 2005). A non-protein amino acid,

glutamate analogue, that was identified four decades ago (Rao et al., 1964), ß-N-


8

oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), was proposed as the causative

molecule.

COOH

CO

NH – CH2 – CH – COOH

NH2

Figure I.2. Chemical structure of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP)

Over thirty different physiological and biochemical activities have been ascribed to

this non-protein amino acid (Lambein et al., 2007). The best studied activity of ß-

ODAP is the excitation of a subclass of glutamate receptors on the neuronal cells

(Spencer et al., 1986). This activity triggers the release of the endogenous neuro-

excitant glutamate, whereas ß-ODAP also inhibits the re-uptake of glutamate by the

astrocytes in the central nervous system (CNS) that would ‘detoxify’ glutamate into

glutamine. This not only increases and prolongs the excitation of the receptors, but

also increases the production of nitric oxide (NO), a short-lived molecule that is

responsible for the production of reactive oxygen species (ROS) and oxidative stress.

An interesting activity of ß-ODAP in the plant is reported to be the protection of

glycolate oxidase, an enzyme in plant photosynthesis, against high light intensity. The

level of ß-ODAP in the plant increases with higher light intensity (Zhang et al., 2003).

Faced with drought-triggered crop devastation, people are confronted with a dilemma

of either starvation or survival on the only foods available; i.e. grass pea with its toxic

reputation, knowing that especially the young men, as the breadwinners in the family,

are most apt to succumb to neurolathyrism. The tragic consequences of its unbalanced

consumption may have been recognized shortly after its domestication.

Neurolathyrism mostly affects the poor subsistence farmers families who often are

illiterates and are least informed about the disease. There is a strong perception of the

public about this stigmatising disease that is not normally reported to medical or


9

administrative authorities (Getahun et al., 2002c). This underreporting of

neurolathyrism may in part be responsible for its status of ‘neglected disease’.

The typical feature of neurolathyrism is spastic paraparesis of the legs, whereby the

heels can hardly touch the ground, accompanied by the easily recognised ‘scissor

gate’ when the knees touch each other while walking (Ludolph et al., 1988). The term

’’motor neurone disease’’ encompasses independent or combined upper and lower

motor neurone disorders (Donaghy, 1999). There is increasing evidence that the

neurotoxic effects of excitatory amino acids and their analogues are part of the

pathogenesis of this neuronal degeneration primarily affecting corticospinal tracts

regulating the lower limbs (Ludolph et al., 1993). The presence of such a mechanism

in combination with oxidative stress could play a pathogenic role (Getahun, 2004;

Nunn et al., 2005).

Daily overconsumption of grass pea (up to 1 kg during 2-3 months) without

interruption and apparently with no admixture of herbs or condiments is considered to

lead to symmetric crippling of the legs (Getahun et al., 2005). The estimated threshold

intake of ß-ODAP per day per adult person is about 500 mg (Lambein et al., 2001).

The generally suggested cause of neurolathyrism is the agonistic activity of ß-ODAP

at glutamate receptors of neurons, but this cannot explain the extreme inter-species

and intra-species differences in susceptibility to ß-ODAP (Rao, 2001). The later

author indicated that differences in individual physiological activity ascribe for

differences in susceptibility or resistance to the disease in humans.

Neurolathyrism is an important public health problem in some developing countries:

e.g. India, Bangladesh, Nepal, Ethiopia and NW China, which are known for having

high populations and food insufficiency (Haque, 1997). Especially the subsistence

farmers in remote areas that are susceptible to droughts and where grass pea is

considered a survival food are at risk. Hundreds of thousands in the world and tens of

thousands in Ethiopia are living with neurolathyrism. Poverty, illiteracy, malnutrition,

recurrent natural calamities and traditional agriculture are common features of regions

with this problem. The predisposing factors and the sudden onset of neurolathyrism

symptoms are poorly understood, however. Particularily in Ethiopia, sex, age,

illiteracy, poverty and cooking of grass pea foods exclusively in traditional clay pots


10

are also reported to be risk factors of neurolathyrism (Getahun et al., 2002a).

Accordingly, they reported, male, young age, illiterates, poor and those using clay

cooking utensils were found to be more susceptible.

Patients of neurolathyrism complain of walking difficulties, leg stiffness and

heaviness of the lower limbs and of frequency of micturition (sense of urination). Gait

abnormalities include spasticity of the calf muscles, scissor gait, toe walking and the

need of walking sticks (Misra et al., 1993; Haque, 1997; Getahun et al., 2005).

Neurolathyrism is said to be a non- or little progressive disease. Neurologists

recognise four stages in the disease based on severity of the physical disabilities and

on typical symptoms and with Babinski sign (a specific reflex of the foot clonus)

(Haimanot et al., 1990).

Table I.1: The four stages of neurolathyrism (Getahun, 2004)

No-stick /mild = stage I One-stick = stage II Two-stick = stage III Crawling = stage IV

-Spastic gait with no stick

and no Babinski sign

-Only ankle and joint

movement restricted by

muscle spasm

-Rigid walk on ball of the

feet, tilting pelvis leading

to jerky movement

-More visible when running

-It remains or passes to the

next stage

-Severely affected

patients

-With adductor

spastic walk,

scissors gait

-Ankle clonus and

Babinski sign

present

-One crutch/stick

needed for

maintaining body

balance

-Aggravated condition of

stage II

-Muscular stiffness and

bending at knee joints

-Spastic cross adductor gait

-Ankle clonus and Babinski

sign present

-Severe muscular rigidity,

patients need two sticks for

support

-Most severe cases

-Patients develop chronic

paraplegia in flexion of

knee joints

-Extreme stiffness of lower

limb and considerable

bending of the knees

(contracture)

-Arms are strong and

pyramidal signs present

-Can not walk upright

hence crawling or

bedridden

I.1.3. Factors affecting the level of neuro-excitatory amino acid in grass pea

Environmental factors have been evidenced to affect the toxicity of grass pea seed.

The metabolite blamed for this toxicity is ß-N-oxalyl-L-α,ß-diaminopropionic acid

(ß-ODAP). The biosynthesis of this metabolite seems to be very responsive to the

environment (Haque, 1997). The risk for neurolathyrism is thus also dependent on the

environment and is much higher during drought, when crop failures result in less


11

balanced diets, food insufficiency and also increasing toxicity of the then possiblly

available grass pea. Based on the current annual production of 100,000 tons grass pea

seeds, the production of ß-ODAP could be in the order of 400 tons annually from

Ethiopian soil alone.

I.1.3.1. Climatic factors and ß-ODAP biosynthesis

Climatic factors play a crucial role in plant adaptation to environments that can be

expressed by growth, production and level of plant biosynthates. There appear clear

differences in concentrations not only for ß-ODAP but also for other antinutritional

factors (ANF) of grass peas such as tannins, phytic acid and trypsin inhibitor when

grown in different agro-ecologies (Urga et al., 2005). Also, some protein and non-

protein amino acids with similar but less potent activities are present in grass pea

(Ikegami et al., 1995). Among these are glutamate, aspartate and BIA or ß-

(isoxazolin-5-on-2-yl)-alanine that are synthesized in the grass pea plant. BIA can

reach high concentrations in the seedling stage (cotyledon removed): up to 2 % of the

dry weight (Lambein et al., 1976). Other metabolites such as total phenolics and

condensed tannins have been shown to be more correlated with cultivars than with

environment (Wang et al., 1998a).

Several authors confirmed the wide variation in ß-ODAP concentration both among

genotypes and environments independent of the method of analysis (Dahiya and

Jeswani, 1975; Leakey, 1979; Ramanujam et al., 1980; Campbell, 1997; DZARC,

2003). Based on an evaluation on three locations with different growing environments

of two common Lathyrus species (407 L. sativus and 96 L. cicera lines) collected

from three geographic origins (Ethiopian, Mediterranean and European) Hanbury and

co-workers (2000a) found that for both species, the genotype was the most important

determinant of ß-ODAP concentration and that environment had less influence.

However, Wuletaw (2003) concluded for L. sativus that genotype and its interaction

with the environment is the most significant determinant for ß-ODAP level. The

repeated multilocation trial of Debre Zeit Agricultural Research Centre (DZARC) on

fields ranging from lower altitude (altitude 1600 m asl, light soil, short rainy period

and a mean annual temperature of 23.3 oC) to higher altitude (altitude 2200 m asl,

vertisol soil, longer rainy period, and a mean annual temperature of 16.7 oC) areas


12

(DZARC, 2003) together with specific observations by Campbell (1997) indicated

that ß-ODAP was reduced by more than half for the same cultivars by varying the

environment from high to low drought stress.

A 25 % increment in ß-ODAP in the seeds was observed when planting date was

delayed by two to three weeks (Asfaw et al., 2003). This might have coincided with a

drop in residual moisture in the soil. Cocks and co-workers (2000) found that a

greater influence (up to +100 %) on seed neurotoxin levels resulted from drought

stress applied at early post-anthesis compared to the period of pre-anthesis.

Considering the seed as a physiological sink for ß-ODAP, the amount of grass pea

seed per plant affects the ß-ODAP level. A negative relationship between ß-ODAP

concentration in the seed and total amount of ß-ODAP in the plant was then plausible.

Under optimal growth conditions, the plant produces more pods and seeds than it does

under marginal conditions, in which the reduced ß-ODAP concentrations obtained

may be caused by a dilution of the available toxin over a greater number of seeds.

Even under identical unstressed conditions, plants with only 10 pods produced seeds

with 59 % more ß-ODAP than plants with 100 pods (Cocks et al., 2000).

Haque (1997) studied stress phenomena in grass pea and found a strong correlation

between arginine and homoarginine accumulation in the seed that could be linked to

almost any type of nutritional or environmental stress. He suggested that the non-

protein amino acid homoarginine might act as a central defence for L. sativus to

various stresses. He further proposed that the higher accumulation of ß-ODAP in

drought-stressed L. sativus plants and seeds during the ‘survival phase’ and ‘cell

disorganization phase’ might have resulted from disturbed nitrogen assimilation and

loss of specificity of particular enzymes that might lead to higher synthesis of

asparagine. Asparagine is considered the primary precursor of the isoxazolinone ring

of BIA which is the precursor for ß-ODAP (Kuo et al., 1994 and 1998). During plant

growth, the highest β-ODAP concentration in grass pea shifts from active vegetative

leaf tips to the sinks of the reproductive stage (embryos, cotyledons).

Many plants react to drought stress by increasing their concentration of amino acids

and other osmolytes. Lambein and co-workers (1990) postulated that the free amino


13

acids including ß-ODAP might have a role in conveying tolerance to drought that has

made grass pea such a useful species in drought-prone areas over the long period of

its domestication.

I.1.3.2. Growth media and associated changes in ß-ODAP

Grass pea grows on diverse soil types, ranging from nutritionally rich to depleted

marginal lands. In Ethiopia, it grows predominantly on heavy vertisols (that accounts

for about 12 million hectares in the country) under both excessive moisture with poor

rhizosphere aeration and drought stress with typically cracking soils. The

physiological mechanism for survival under such diverse moisture regimes and the

ability of the crop to supply the root with oxygen in poorly-aerated soil are not well-

understood. Eavis (1971) postulated that benefits from additional residual soil water

for plant growth, following a period of water logging or water saturation of the soil,

compensate for the effects of poor aeration. We found no scientific explanation why

grass pea roots that grow under submerged conditions can survive with little oxygen

from the surrounding soil and water or, as in the case of flooded rice and some bog

plants where specialised tissues can facilitate obtaining sufficient supplies of oxygen.

Greenwood (1968) postulated O2 transport through air passages from the aerial parts

to the roots. However, we found no reference to such specialised aerenchyma tissues

for grass pea.

Most soil micro- and macronutrients affect ß-ODAP synthesis and other related amino

acids in grass pea. Haque (1997) found that high Fe2+, Mn2+, B3+, Co2+, and Al3+

increased synthesis and accumulation, while low Zn2+, Mo6+, Cu2+ and Al3+ decreased

the value. High ratios of P2+/Zn2+ and Fe2+/Zn2+ increase the ß-ODAP production. ß-

ODAP has been proposed as carrier molecule for the zinc ion and, therefore, higher

Zn2+ could reduce its synthesis. The same author (Haque, 1997) indicated that

depletion of zinc from the soil results in higher ß-ODAP levels in the plant and in the

seeds.

Once germination is established, grass pea tolerates a certain level of soil salinity,

principally the presence of Na+, K+, Ca2+, and Mg2+. These cations are commonly

linked to the anions Cl- and SO42- (Jackson, 1973). Interestingly, moderate salinity has


14

a decreasing effect (up to 0.6 % NaCl of artificial salinity in hydroponic cultures and

in the field of EC = 13 m mohs/cm) while high salinity has an increasing effect on ß-

ODAP accumulation (Haque, 1997).

I.1.3.3. Effect of grass pea processing on ß-ODAP reduction and nutritional quality

A diversity of recipes that are made from grass peas alone or mixed with other pulses

exists in Ethiopia. Grass pea can be consumed in different ways: roasted, cooked,

boiled, fermented or raw. In various other parts of the world, recipes using grass pea

seeds or green shoot tips are prepared and consumed with condiments (herbs, spices,

salt). Some recipes are used for daily home consumption while some others are only

used on specific festive occasions.

Any grass pea seed processing seems to affect ß-ODAP level present in it (Srivastava

and Khokhar, 1996). The most popular processing of soaking and decanting the water

reduces ß-ODAP content, but this processing also results in the simultaneous

depletion of some other important amino acids and water-soluble vitamins and

mineral nutrients. As a rule, cooking, roasting and fermentation decrease protein

solubility but improve protein digestibility in legumes (Baik et al., 2004).

Fermentation of cereals and their blend with food legumes is a potentially important

processing method that can be expected to improve the balance of essential amino

acids while reducing antinutritional factors (Kuo et al., 1995; Yigzaw et al., 2001),

hence improving their nutritional value. It can decrease certain antinutritional factors

(ANF) like phytates, protease inhibitors or flatulence factors (Kuo et al., 1995).

Significant levels of ANF in soybean, such as trypsin inhibitors and phytic acid, leach

out or are destroyed during soaking and cooking, as well as during fermentation

(Tawali et al., 1998). Yigzaw and co-workers (2001) reported that fungal

fermentation of teff (Eragrostis teff)/grass pea mixtures (teff, a fine seeded crop, is the

traditional staple cereal in Ethiopia) in a ratio of 8/2 was found to be quite comparable

in essential amino acid profile to an ideal reference protein recommended for children

of 2-5 years of age (FAO/WHO, 1985). Solid state fungal fermentation of grass pea

using Rhizopus oligosporus and Aspergillus oryzae strains in succession has shown


15

that the neurotoxin β-ODAP was removed by 97 % on average from high-toxin

varieties and by up to 80 % from low-toxin varieties. It was also reported that

fermentation of grass peas the same way as teff is fermented during the preparation of

the Ethiopian traditional injera, is a protective factor in the epidemiology of

neurolathyrism (Getahun et al., 2003). However, the directly baked unleavened bread

or kita is less safe than injera prepared from grass pea as the latter involves

fermentation (Getahun et al., 2003).

Table I.2. Some traditional foods prepared from L. sativus

Cou

ntr

y

Traditional grass pea food

Brief description of the preparation for consumption

Ref

ere

nce

Shiro wot (sauce)

Sauce prepared from roasted L. sativus powder and eaten with pancake (injera) or unleavened bread (kita)

Kik wot Roasted, de-husked and split L. sativus seed made into sauce Kollo (roasted snack)

Roasted seed directly consumed

Nifro (boiled snack)

Boiled seed eaten directly

Kita (unleavened bread)

Unleavened bread usually made from unfermented dough of L. sativus consumed in time of acute food problem

Eshet (green peas)

The seed in the green pod before maturity, usually consumed by children while tending the farm field

Elbet (sauce) Powder of roasted grass pea well spiced and fermented, occationally consumed like in fasting periods of coptic Christians

Seteto Baked kitta after fermentation of grass pea dough usually on clay pans

Eth

iopi

a

Injera (pancake bread)

Fermented and baked, from powder usually adulterated with cereals

Hai

ma

not e

t al.,

19

93;

1995

; G

eta

hun et

al,

2005

; U

rga

et

al.,

200

5; F

ikre

, 200

7

Dahl Aqueous slurry cooked with spices Curry Cooked with vegetables and/or fish Bora Paste ball with onion and spices, deep fried in oil Chapatti /roti Unleavened bread of khesari powder

Ba

ngla

desh

Khichuri Cooked with rice in 3:1 ratio H

ussa

in,

199

4

Chapatti L. sativus flour is routinely used either alone or in combination with wheat, barley or Bengal gram as chapatti

Ghotu L. sativus flour and rice in ratio of 3:1 cooked in water to form a stiff porridge

Dal (soup- like dish)

Cooked whole split or de-husked grains into a soft semi-solid form and eaten as side dish with rice or chapatti

Indi

a

Snacks As substitute of pigeon pea and chick peas Ma

lek,

199

8

W.A

sia

Green vegetable

Young vegetative parts plucked (4-6 cm) and cooked as green vegetable also rolled and dried for off-season use Green pods/seeds eaten directly or salted, cooked and consumed as snacks (India, Bangladesh, Pakistan) C

am

pbe

ll, 1

997


16

The neurotoxic amino acid (β-ODAP) is water soluble. As a preventive measure by

consumers, aqueous removal of the soluble neurotoxin from grass pea seed seems to

have co-evolved with its consumption. Grass pea seeds are commonly boiled and

consumed as a pulse. Aqueous detoxification can be done by boiling or by soaking the

seeds overnight and subsequently draining out the supernatant (Bell, 1964; Mohan,

1966). A total replacement of Ethiopian Noug cake (the solids after extracting oil

from niger seed Guizotia abyssinica) by boiled grass pea in a chick diet was found not

to reduce the chicks’ performance significantly (Tadelle et al., 2003). The latter

authors also suggested cooking at 90 °C as a means to improve the nutritional quality

since it showed similar results to the commercial poultry ration (p<0.05) in terms of

feed efficiency. Padmajaprasad and associates (1997) demonstrated a significant

reduction of ß-ODAP by up to 90 % after boiling. Wang (2000) indicated that the

extent of isomerization of the toxin from the naturally occurring toxic ß-form to the

less toxic α-form during cooking might affect the toxicity of the preparation. This

isomerisation is time- and temperature-dependent and results in an equilibrium of 40

% α- and 60 % β-isomer. Toxicity of L. sativus seed due to ß-ODAP is only partially

removed during cooking and a significant proportion of the toxin (about 60%)

remains as the toxic β-form. From comparable experiments, Akalu and co-workers

(1998) reported a statistically significant reduction (57 %) after 60 minute pressure-

cooking at 150oC of grass pea flour and a 39 % reduction after a 30-minute

autoclaving of the dry seeds. Cho and co-workers (2008) reported a remarkable

reduction of the toxin by steaming, which may suggest the need to investigate the

presence or absence of ß-ODAP in the steam from boiling or steamed grass pea.

Roasting also reduces the neurotoxin concentration of grass peas. Akalu and co-

workers (1998) recorded a significant ß-ODAP reduction with up to 30 % after

roasting milled samples and up to 67 % after cooking presoaked seeds compared to

that of whole raw seeds. Girma (1999) indicated that ß-ODAP content in whole seeds

could be reduced by up to 87 % by using various combinations of roasting, cooking,

autoclaving and soaking.

Soaking grass pea seeds prior to cooking can leach out ß-ODAP and decrease the risk

of neurolathyrism by half (Getahun et al., 2005). Soaking (12 h) of the grits (1.5-2

mm) from the cotyledons and roasting at a temperature of 200 °C for a period of 37


17

minutes could be recommended for a significant reduction of ß-ODAP, while in vitro

digestibility of starch also improved (Girma, 1999). Likewise, Malek (1998) reported

that soaking seed in water for 7-8 hours and then decanting off the water removes

most ß-ODAP. He further claimed that soaking split seeds overnight and decanting

water made the dal (preparation of pulses that have been stripped of their outer hulls

and split; also referring to the thick, spicy stew prepared therefrom, a mainstay of

Indian, Pakistani, and Bangladeshi cuisine) toxin-free and safe for consumption.

However, when grass pea powder (besan) is used for making pakoras, chapatis or

dalpuri, the danger of neurolathyrism still remains. By soaking mature grass pea seeds

for 72 hours in different soaking media, Kelbessa and Mengistu (1993) found an 11

and 13 % increase of total protein after soaking in plain water and 1 mM HCl,

respectively, and 12 and 16 % decrease in 1 mM NaOH and 0.1 % wood ash

solutions, respectively. They noticed that this processing resulted in the loss of total

solids, non-protein nitrogen, total soluble sugars and reducing sugars. Previous

research indicates that sprouting legumes improved their nutritive value (ability of

food to provide a usable form of nutrients: protein, carbohydrates, vitamins and

minerals) due to an increase of enzyme activity, which breaks down storage protein

and starch in the seed into amino acids, peptides, and simpler carbohydrates needed

for the embryo to grow. Simultaneously, antinutritional factors such as enzyme

inhibitors and other anti-nutrients are brought down to insignificant levels (Vidal-

Valverde et al., 2002).

Extrusion of grass pea seed reduced the ANFs, such as tannins by 77 %, the trypsin

inhibitor to below detection limit, and ß-ODAP by 46.09 % (Masoero et al., 2004). In

peas (Pisum sativum) extrusion causes enzymatic degradability of starch from 11.80

% in meal to 39.70 % in the extruded product while in faba beans (Vicia faba)

extrusion increased starch digestibility from 11.39 % to 85.05 %, whereas in lupines

(Lupinus spp.) it caused a complete starch hydrolysis (Masoero et al., 2004). Some

ANFs like phytates and nutrient components were not affected by extrusion.

Obviously, the amino acid value of a mixed diet containing cereals rich in methionine

and legumes rich in lysine is more complete than either of them consumed

individually. An interesting illustration of this is the detailed report by Kessler (1947)

on a WWII prisoners of war camp in the Ukrainian town of Vapniarca. Inmates were


18

given the "horse fodder" left by the Russian cavalry consisting of grass pea. They

received daily rations of 200 g boiled grass pea seed and 200 g barley bread

containing 20 % chaff. The 150 inmates did not develop any sign of lathyrism during

3 to 6 months after which the diet was changed to 400 g boiled grass pea and the same

200 g bread. Within two months after this diet with a higher portion of grass pea was

initiated, cases of crippling started and after 4 months 60 % of the inmates had

developed various degrees of neurolathyrism. Lambein and associates (2001)

estimated a threshold intake level of 500 mg of ß-ODAP per day to develop this high

incidence of neurolathyrism under severe conditions of malnourishment and physical

exhaustion. In an Encyclopaedia of Plants published in 1855 (Loudon, 1855), it was

already reported that bread made from a 50/50 mixture of grass pea and wheat seems

to have no deleterious effect, while bread made only from grass pea caused paralysis

of the legs “when used in continuance”. Thus, if only 0.5 kg of grass pea is consumed

daily in combination with cereals, the tolerance level might be as high as about 2 g of

β-ODAP intake per day (Lambein et al., 2001). Mixing the food with gravy that

contains condiments with antioxidant activity reduced the neurolathyrism cases

manyfold (Getahun et al., 2005). The latter authors confirmed that consumption of

grass pea mixed with cereals rich in sulphur amino acids was also highly

neurolathyrism-protective. Getahun and co-workers (2003) investigated the impact of

cereal aid during drought-triggered famine and reported that consumption of boiled

grass pea and green immature grass pea without cereals was linked to higher

incidence of neurolathyrism.

I.1.3.4. Breeding or genetic engineering to reduce β-ODAP in grass pea

It is rather puzzling that a commonly used crop such as grass pea has made so little

progress as a grain crop in its long cultivation period (Smartt, 1984). The level of β-

ODAP in low toxin varieties is undoubtedly strongly influenced by the environment.

The 40 years of efforts to reduce the crop’s toxicity has produced varieties with a

remarkable reduction in β-ODAP but neither stable nor toxin-free varieties could be

established yet. An Ethiopian-released variety ”Wasie”, claimed to have β-ODAP

concentrations below toxic threshold level (<0.04 %) turned out to be higher in toxin

(>0.2 %) after growing in lowland areas with relatively higher drought stress.


19

Chromosome numbers of more than 60 species in the genus Lathyrus have been

reported, with only three species having more than 14 chromosomes (Narayan, 1998).

Successful inter-specific hybridisation in the genus has been shown to be extremely

rare. Following a report on the successful crossing of L. hirsutus x L. odoratus

(Barker, 1916), many researchers have attempted inter-specific hybridisation among

species in the genus Lathyrus. Inter-specific hybridisation involving L. sativus has

only been reported as successful with L. cicera (Lwin, 1956) and with L. amphicarpus

(only when the latter was used as female) (Khawaja, 1988). Cytological studies of F1

hybrids between L. amphicarpus x L. sativus, L. amphicarpus x L. cicera and L.

odoratus x L. chloranthus showed 50-70 % chromosome homology and pollen

fertility in conformity with meiotic pairing (Khawaja, 1988). To our knowledge, up

to now there has not been any line developed by crossing that is considered as having

a safe level of toxicity. Successful fertilization of certain cross-combinations

commonly showed embryo abortion during development. Therefore, embryo rescue

techniques need to be employed. This may imply that breeding strategies involving

genetic transfer for the improvement of grass pea should be developed. However,

sofar a high frequency transformation could not be achieved with L. sativus or with

the readily crossable Lathyrus species.

The floral biology of L. sativus is such that it favours self-pollination even if up to

27% outcrossing has been the concern of several breeders (Rahman et al, 1995;

Campbell, 1997). In most breeding programmes, crosses are done under controlled

conditions in the greenhouse or under insect-proof netting. Flowers are emasculated

by removing the anthers in the late bud stage, before flower opening. Male sterility is

uncommon in L. sativus. The morning after, styles are fertilized with pollen from a

pre-selected parental plant as soon as possible following dehiscence of the anthers.

The pollen at this time of the day is orange and rapidly becomes colourless as it loses

its viability. Although this manual pollination is a time-consuming process, it can

rapidly result in a large number of successful pollinations as 3-4 seeds develop from

each successful crossing.

No seed colour or flower colour has been confirmed to associate with the level of β-

ODAP. Also no other phenotypic character can be used as marker for low toxin. From

the variability of toxin levels in F2 populations of many studies, it is confirmed that β-


20

ODAP content is inherited quantitatively whereas the presence of cytoplasmic factors

suggested the parental effects on the β-ODAP score (Campbell, 1997). During the last

three decades, a number of low toxin varieties have been released in the world as a

result of conventional breeding. However, the toxin level in seeds of these lines could

vary due to the changes in the growing environment.

Grass pea has a limited secondary gene pool in related species. So far, grass pea only

crosses with L. cicera and L. amphicarpus and then produces a F1 generation to form

the secondary gene pool (Yunus and Jackson, 1991). Tertiary gene pool can be

exploited by crossing species using embryo rescue techniques or protoplast fusion

and/or biotechnological techniques to move genes between species (Smartt et al.,

1994; McCutchan et al., 1999; Durieu and Ochatt, 2000; Ochatt et al., 2001). After

the recent successful regeneration of complete plants from different parts of L.

sativus, somaclones were generated showing tremendous variability with respect to

leaf size, internodal length, flower and seed colour, seed weight, pod morphology and

β-ODAP score (Roy et al., 1991; Mehta and Santha, 1996; Santha et al., 1998; Abd El

Moneim et al., 1999; Zambre et al., 2002). So far, thousands of somaclonal lines

having a remarkable reduction of β-ODAP were developed in many laboratories,

although none were ever found to be toxin-free (Vaz Patto et al., 2006).

The advent of molecular marker techniques may facilitate breeding for improving

important agronomic traits including removal of unwanted metabolites or toxins.

Genetic engineering of the biosynthetic pathway for β–ODAP could be the most

direct method for producing a safe crop. Genetic transformation of L. sativus using

Agrobacterium tumefaciens, or A. rhizogenes, or biolistic techniques have been tried

repeatedly, but with very limited success (Vaz Patto et al., 2006) until the recent

report of an Agrobacterium-based transformation protocol (Barik et al., 2005).

Strategies to engineer the biosynthetic pathway (Kuo et al., 1998) and metabolic

breakdown (Datta, 1995; Metha and Santha, 1997) of β–ODAP have been proposed,

but the tools to achieve this have not yet been developed. Introgression of novel genes

for β-ODAP reduction and increased methionine or the use of antisense RNA

technology to silence or to block the biosynthetic pathway can create opportunities to

develop stable and safe varieties for consumers (Vaz Patto et al., 2006).


21

I.1.4. Effect of methionine on toxicity of grass pea

Even before the toxic compound was identified in grass pea, the low level of

methionine in urine of lathyrism patients having consumed grass pea had been

reported (Rudra and Chaudhury, 1952). More recently, Nunn and co-workers (2005)

rediscovered this phenomenon with human volunteers after consumption of grass pea

seed and suggested that a reduced flux of methionine to the central nervous system

(CNS) may sensitise motor neurons to the excito-toxic action of β-ODAP.

Maintaining the proportion of cereals (richer in sulphur amino acids) in the meal to at

least one third of a mixture with grass pea was identified as a protective factor for

neurolathyrism (Getahun et al., 2005).

Oxidative stress is thought to be important in the aetiology of neurolathyrism

development. Excitation of glutamate receptors in the CNS by β-ODAP produces

unstable nitric oxide (NO) that gives rise to free oxygen radicals (ROS) generation

that disrupt cell functions (Schinder et al., 1996). Several studies have clearly

indicated the protective effect of methionine against oxidative stresses caused by the

neurotoxicity of grass pea (Lambein et al., 2001; Nunn et al., 2005; Getahun et al.,

2005). However, the effective concentration level of methionine to sufficiently protect

against neurolathyrism has never been worked out or modelled, and this is one of the

study areas of the present thesis. It could be estimated that a total intake of about 200

g of β-ODAP present in grass pea consumed daily per person over a period of two

months can cripple over 50 % of males under the extreme hardships and malnutrition

of a Second World War camp prisoners in Vapniarca (Lambein et al., 2001), where

only males were present and males are known to be more susceptible to

neurolathyrism. Had it not been for the role of methionine and antioxidants present in

other food components such as teff (the traditional Ethiopian staple cereal) or

condiments used in sauce preparations, the incidence of neurolathyrism would have

been much higher than the dozen or so annual cases occurring during a normal year in

Ethiopia. This annual incidence can, however, get as high as up to hundreds or even

thousands during droughts and famine periods. When considering that the annual

production of β-ODAP in Ethiopia is about 400 tons (in 100,000 tons of grass pea

seed), this could in theory cripple more than a million people annually under extreme

conditions such as those experienced in the Vapniarca camp.


22

The consumption of grass pea mixed with cereals richer in sulphur amino acids than

legumes was demonstrated to be highly protective from neurolathyrism (Getahun et

al., 2005). The antioxidant activity and enhancing of liver functioning by addition of

Zn and methionine in the molybdenotic rat (receiving sub-lethal doses of

molybdenum) has been documented (Suresh and Archana, 2000). Inhibition of the

production of reactive oxygen species (ROS) was proposed as mode of action in this

experimental system. It was also indicated that antagonists to excitatory amino acid

receptors may offer a therapeutic opportunity for some neurological diseases

(Ludolph et al., 1993).

Nunn and co-workers (2005) noticed that consumption of grass pea seed caused

persistent plasma methionine deficiency in human volunteers. The normal

physiological response to consumption of a methionine-deficient meal would be a

reduced plasma methionine concentration as found after consuming methionine-

limited grass pea and lentils, where around 50 % reduction of plasma methionine was

found after 24 hours. A reduction in the plasma methionine concentration would

cause a reduced methionine flux into the central nervous system as a consequence of

consuming grass pea. This may sensitise these cells to the neurotoxin β-ODAP

present in the grass pea seed. The latter authors concluded that supplementing such a

diet with methionine might protect those at risk from neurolathyrism.

I.1.5. Rationale of the study and motivation of the investigation

Compared to many field legumes, grass pea is a nutritionally rich source of food and

feed, containing high levels of protein, micronutrients and energy. However, its

protein is not well-balanced for it lacks sufficient sulphur-containing amino acids. The

remarkable tolerance to biotic and abiotic stresses, and high nitrogen fixation activity

should make grass pea a promising crop for economic use and for amelioration of

marginal soils. Especially considering global warming, the unique hardiness to grow

under adverse environmental conditions suggests a potential future for this crop in the

already drought-stressed and deteriorating highlands of Ethiopia. At present, in the

developing world over one hundred million poor and often illiterate farmers make


23

their living on grass pea cultivation in the face of the hitherto poorly perceived risk

crippling.

Global research on grass pea improvement, unlike with other crops, has been biased

towards taking out the inherent toxic element. Exhaustively undertaken conventional

breeding has been successful in downscaling ß-ODAP manyfolds from the original

0.5 to 1.2 % in seeds to as low as 0.01 %. Due probably to the complexity of the

challenge, and considering the misconceptions prevailing among farmers and the

poorly understood aetiology of the disease, these low-toxin lines had neither the

impact nor the extent of utilization expected. On the other hand, the bias on toxicity

research has masked the other important agronomic and qualitative parameters of the

crop that were not extensively researched or exploited.

Genetic research including contemporary techniques is very little used on grass pea

compared to other field crops. Already in 2004, FAO raised concerns about the need

for gene revolution for so-called ‘orphan crops’ including grass pea if we have to feed

an additional 2 billion people in the next three decades from increasingly fragile

landmasses.

Having potential as an important food source, the holistic approach of improving the

diet needs more attention. Other than toxicity reduction in the plant, the study of the

role of sulphur amino acids and attempts to increase the level of methionine in the

plant could be a novel approach to fundamentally address the problems of the crop. In

addition, assessing environmental influences on toxin concentration could show the

way for improving field management options.

Thus it becomes imperative to look into the importance of methionine as a

counteracting principle against the risk for neurolathyrism from grass pea

consumption. In vivo enrichment of sulphur-containing amino acids and studying

environmental factors affecting biosynthesis and biosynthate levels in grass pea is

anticipated to improve the nutritional quality and the economic value of the crop.


24

I.1.6. Overall and specific objectives of the study

General objective

Improving the nutritional quality of grass pea for safer consumption and increased

economic benefit for the farmers.

Specific objectives

∎ Identification of gaps in our knowledge of grass pea and neurolathyrism by

reviewing related and relevant literature on the crop.

∎ Identification of climatic and edaphic factors affecting the level of ß-ODAP,

methionine and other amino acids in grass pea relevant to the risk for

neurolathyrism.

∎ To assess the protective effect of methionine on grass pea toxicity.

∎ To attempt increasing methionine in grass pea seed by gamma-irradiation

mutation and screening for methionine-rich mutant lines.


25

CHAPTER II

Level of ββββ-ODAP, methionine and other free and protein

amino acids in the seeds of different grass pea (Lathyrus

sativus L.) genotypes


26

L. sativus seeds from different origin


27

CHAPTER II

Level of ββββ-ODAP, methionine and other free and protein amino acids

in the seeds of different grass pea (Lathyrus sativus L.) genotypes

[Fikre A., Korbu L., Kuo Y.-H. and Lambein F. 2008. Food Chemistry 110: 422-427]

Abstract

Free and protein amino acids of nine different genotypes of grass pea (Lathyrus

sativus L.) seeds were analysed by HPLC with precolumn PITC (phenyl

isothiocyanate) derivatisation. Among the free amino acids, homoarginine is

quantitatively the most important (up to 0.8 % of dry seed weight) and stable, while

the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid),

second most abundant, showed highest variation (0.02 % - 0.54 %) in the nine

genotypes examined. For the protein amino acids, glutamate was quantitatively most

significant followed by aspartate, arginine, leucine, lysine and proline. The sulphur

amino acid methionine showed the lowest concentration (~0.1 %) in all L. sativus

genotypes, and also in lentil (Lens culinaris) and in soybean (Glycine max) seeds

analysed at the same time.

II.1. Introduction

Grass pea (Lathyrus sativus L.) is perhaps one of the most environmental stress

resistant legume crop with high tolerance to drought, flooding and insect attacks

(Campbell, 1997). During drought and failure of other crops in Ethiopia, grass pea is

survival food number one for the poor. However, overconsumption of grass pea as a

staple during 2-3 months might cause increased incidence of the crippling disease

neurolathyrism reaching up to 6 % of the rural population. A non-protein amino acid

β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) found in grass pea seeds was

suggested to be the causal agent of this upper motorneurone disease. A recent report

showed that the mechanism of neuro-toxicity of β-ODAP leading to neurolathyrism is


28

very complex and oxidative stress might be involved in the pathogenesis (Lambein et

al., 2007).

The ß-ODAP content of grass pea varies widely, both among genotypes and

environments (Dahiya and Jeswani, 1975; Leakey, 1979; Ramanujam et al., 1980;

Campbell, 1997; DZARC, 2003). Based on the evaluation from three Australian

locations growing 407 lines of Lathyrus sativus and 96 lines of Lathyrus cicera

collected from three geographic origins namely Ethiopian, Mediterranean and

European, Hanbury and co-workers (2000a) summarised that for both species,

genotype was the most important determinant of ß-ODAP concentration while

environment had less influence. However, Wuletaw (2003) studied the stability of ß-

ODAP content in Lathyrus sativus and reported the importance of genotype with

environment interaction. The multi-annual multi-location trial by Debre Zeit

Agricultural Research Centre in Ethiopia with growing conditions going from high

altitude 2200 m above sea level (asl), vertisol, low temperature of mean 16 ± 2 oC to

low altitude of 1600 m asl, light soil and higher temperature of mean 24 ± 2 oC

(DZARC, 2003), and also the specific observation from Campbell (1997), indicated

that ß-ODAP level doubles or more for the same grass pea cultivars as the varying

growing environment slides from lower to higher stress conditions. A large variability

of ß-ODAP was seen due to environmental changes (Fikre et al., 2006). Both

environment and genotypes could play an important role in the biosynthesis level of

the toxin.

There appear to be clear differences not only for ß-ODAP content but also for other

antinutritional factors (ANFs) of grass pea. Urga et al. (2005) reported variability in

level of tannins, phytic acid and trypsin inhibitor activities of grass pea grown in

different agro-ecological locations of Ethiopia. Levels of total phenolics and

condensed tannins were reported to be positively correlated and they are determined

by genotypes rather than by environment in grass pea (Wang et al., 1998a). The same

authors also reported that trypsin inhibitor activities in grass pea did not differ among

cultivars or environments (Wang et al., 1998b).

The ß-ODAP content of grass pea seeds analysed by different labs is not always

consistent. The latter variation might be caused by the different analytical methods


29

used which include OPT (O-phthalaldehyde) colourimetric assay, automatic amino

acid analyser, HPLC (High Performance Liquid Chromatography), CZE (Capillary

Zone Electrophoresis), NIR (Near Infrared Reflectance Spectroscopy) and GC-MS

(Gas Chromatography-Mass Spectrometry). Some of these methods cannot

differentiate between α- and β-ODAP. However, α-ODAP is present naturally in

minor ratios (< 5%) of the total and was reported to be without neuroexcitatory effect.

For this study, we have collected grass pea seeds from different origins and analysed

their content of β-ODAP and other free amino acids by HPLC with precolumn PITC

(phenyl isothiocyanate) derivatisation as reported previously (Kuo et al., 2003).

The average protein content in legume seeds is 21-25 %. These proteins are rich in

most of the essential amino acids particularly lysine, but usually poor in the sulphur-

containing amino acids methionine and cysteine (Kuo et al., 1995). Monsoor and

Yusuf (2002) reported that the crude protein content in grass pea seeds (29.93 ± 1.26

%) is higher than that of lentil (23.23 ± 1.07 %) and chickpea (20.40 ± 2.27 %). The

objective of this study was to compare the contents of free amino acids, as well as the

protein amino acids among different grass pea genotypes, with special attention for

the neuro-excitatory β-ODAP and for the essential sulphur amino acid methionine.

II.2. Materials and methods

Plant materials: Seeds from nine genotypes of Lathyrus sativus originating from

different parts of the world including Ethiopia (local cultivars purchased from markets

at Debre Zeit; Gonder; Wollo and Bahr Dar), and collections of Institute of Plant

Biotechnology for Developing Countries (IPBO) from India (received from Dr R.L.

Pandey, Indira Gandhi Agricultural University, Raipur), China (black eye cultivar,

received from prof. Yu Jin-Zhong, Soil and Fertility Institute, Yangling, Shaanxii

province), Poland (Derek variety, obtained from prof. Marian Milczak, Lublin) and

Canada (LS 82046 and LS 87124, two low toxin lines bred by Dr. Campbell,

Agriculture Canada, Morden). Two other grain legume seeds, lentil (Lens culinaris)

and soybean (Glycine max) obtained from Turkey and Canada respectively through

commercial sources, were also analysed at the same time for comparison.


30

Dry seeds were powdered in an electric coffee grinder and sieved. The seed powders

were used for further analysis.

Figure II.1. Variability in size, shape, texture and colour of L. sativus seeds from different origin

Preparation of seed extracts for free amino acids analysis: 200 mg of seed powder

was weighed and mixed with 10 ml of 70% ethanol. In each sample, 50 µl of DL-

allylglycine (100 µmol/ml, Sigma) was added as internal standard. The mixture was

allowed to stand at 4 °C overnight before centrifugation at 34,800 g for 20 min. The

supernatant was collected and the pellet was washed with 2 ml 70 % ethanol and

centrifuged as above. The pooled supernatant was concentrated to 0.5 ml extract with

a rotavapor under vacuum at 45 °C. An aliquot of 50 µl was used for derivatisation

with PITC for HPLC analysis.

Preparation of seed extract for protein amino acid analysis: 100 mg of seed

powder was weighed in a 2 ml glass vacule designed for hydrolysis (Wheaton, USA).

In each vacule, 2 ml of 6 N HCl, 0.01 % mercaptoethanol and 100 µl internal standard

norleucine (100 µmol/ml, Sigma) were added. The mixture was allowed to freeze by

placing the vacule in dry ice (carboglass) for 5-8 min. The vacule was then connected

to a vacuum system with gentle shake for about 10 minutes during the application of

the vacuum. The evacuated vacule was then sealed in a gas flame and placed in an

oven for hydrolysis at 110 °C for 18 hours.


31

The hydrolysate was transferred to an Eppendorf and centrifuged in an Eppendorf

centrifuge (Hawksley MBC, England). The supernatant was transferred to a conical

evaporatory flask and dried with rotary evaporator under vacuum at 45 °C. This

procedure was repeated twice by adding distilled water after drying to remove the

HCl. Two ml distilled water was added to the flask to dissolve the dried samples and

the mixture was centrifuged. The supernatant was collected and 50 µl aliquot was

used for derivatisation with PITC for HPLC analysis.

PITC (Phenyl isothiocyanate) derivatisation: each 50 µl sample was first dried

under vacuum in an Eppendorf Vacufuge concentrator (5301) at 45 °C. To each dried

sample, 20 µl of coupling buffer (methanol/water/triethylamine: 2/2/1 by volume) was

added, and the whole was mixed and dried in Eppendorf Vacufuge concentrator.

Finally, 30 µl of the PITC reagent

(methanol/water/triethylamine/phenylisothiocyanate: 7/1/1/1 by volume) was added

and left to react at room temperature for 20 min before concentrating to dryness.

To each PITC-derivatised sample, 500 µl of buffer A (0.1 M ammonium acetate, pH

6.5) were added, mixed well and centrifuged. The supernatant was filtered through a

Millipore Millex filter (0.45 µm) and 20 µl aliquot was injected into the HPLC for

analysis. A standard amino acid mixture (AA-S-18, Sigma), L-(+)-homoarginine (99+

%, Janssen Chimica) and synthetic ß-ODAP (obtained from Dr. Rao S.L.N, India)

were also derivatised and prepared as above and injected into the HPLC as standard.

High performance liquid chromatography (HPLC) for amino acid analysis

A Waters 625 LC system with Waters 991 photodiode array detectors was used, as

reported previously (Kuo et al., 2003). A gradient system with buffer A (0.1 M

NH4OAc, pH 6.5) and buffer B (0.1 M NH4OAc, containing acetonitrile and MeOH;

44/46/10 by volume, pH 6.5) with flow rate of 1 ml/min was used for the separation

of amino acids during 50 min. An Alltima C18 column (250 x 4.6 mm I.D., 5 µm

particle size, Alltech, USA) was used with column temperature at 43 °C during

analysis. Absorbance at 254 nm was used for calculations. Results were analysed by

Millennium software (Waters, version 1.10).


32

Statistics

All experiments were repeated twice and each sample was injected twice into the

HPLC. Results were expressed as mean ± SD. Data were analysed with ANOVA

using SPSS 12 software. Statistical significance of differences in mean values was

calculated with a confidence level of 95 %.

II.3. Results

Free amino acids profile

The grass pea seeds from different origins showed high variability in morphology,

especially size, shape and colour (Figure II. 1). In this study, we found that the non-

protein amino acid homoarginine (Har) was the most abundant amino acid in all the

genotypes of L. sativus seeds analysed (Table II.1), confirming previous results

obtained from one genotype (Lambein et al., 1992). In addition, this high

concentration (0.68-0.86 %) is rather stable among all genotypes analysed with no

statistical difference. This high level is a unique feature specific for grass pea seeds as

compared to other food legumes (Rozan et al., 2001; Kuo et al., 2004).

Table II.1: Free amino acids (% dry seed weight; n = 4; mean ± SD) in Lathyrus sativus seeds of different origin

Values in a row followed by the same letters are not significantly different (p< 0.05); Har-homoarginine

β-ODAP is quantitatively the second most important free amino acid in L. sativus

seeds but with greatest variation from 0.2 mg/g dry seed in LS-82046 (Canada) to 5.4

mg/g dry seed in Raipur (India). Earlier studies on the influence of nutrient supply on

β-ODAP content in grass pea seeds growing in hydroponic media indicated that

reduction of macro-nutrients such as NO3-, Mg2+, or K+ in Hoagland solution resulted

in a sharp increase of β-ODAP content of the ripe seeds (Kebede et al., 1994).

D.Zeit

(Shoa), Eth

Gonder,

Eth Wollo, Eth B.Dar, Eth India China Poland Ls82046 Ls 87124

Har 0.68±0.11a 0.78±0.16a 0.69±0.09a 0.86±0.18a 0.79±0.05a 0.68±0.20a 0.80±0.32a 0.74±0.10a 0.69±0.15a

ß-ODAP 0.35±0.11ab 0.28±0.09bc 0.35±0.07ab 0.48±0.18ab 0.54±0.06a 0.38±0.13ab 0.18±0.09c 0.02±0.01d 0.02±0.01d

Aspargine 0.04±0.01b 0.08±0.04b 0.06±0.10b 0.05±0.04b 0.03±0.02b 0.07±0.03b 0.15±0.06a 0.06±0.05b 0.03±0.01b

Arginine 0.05±0.07ab 0.03±0.02bc 0.05±0.01ab 0.03±0.03bc 0.06±0.04a 0.04±0.01bc 0.03±0.01bc 0.03±0.01bc 0.01±0.00c

Aspartate 0.02±0.02b 0.03±0.03ab 0.04±0.03a 0.03±0.03ab 0.04±0.04a 0.04±0.02a 0.04±0.01a 0.02±0.01b 0.01±0.00c

Glutamate 0.03±0.03b 0.08±0.06a 0.07±0.04a 0.04±0.03b 0.03±0.03b 0.04±0.02b 0.08±0.02a 0.04±0.01b 0.04±0.02b


33

00.10.20.30.40.50.60.70.80.9

Homoa

rgini

ne

Beta-

ODAP

Aspar

agine

Argini

ne

Aspar

tate

Glutam

ate

amino acids

% s

eed

wt

Figure II.2: Free amino acid profile in Lathyrus sativus (mean of 9 genotypes)

Besides Har and β-ODAP, the other free amino acids asparagine, glutamate, arginine

and aspartate were found in much lower concentrations. The free amino acids profile

of L. sativus as shown in Figure II.2 indicates a big variation among free amino acids,

especially β-ODAP.

Protein amino acids profile

In the seed hydrolysate of all grass pea genotypes studied, glutamate was found to

have the highest concentration followed by aspartate, arginine and lysine (Table II.2).

In the lentil and soybean seeds examined, a similar pattern was found except that

leucine showed a higher level than lysine. The sulphur amino acid methionine ranked

lowest among all the amino acids in all genotypes of L. sativus as well as in lentil and

soybean. The average concentration of methionine in L. sativus (around 0.1 % of the

dry seed weight) was similar to that in lentils (Table II.2), but only half of that in

soybean (ca 0.2 %), which is known as the richest protein source among legume crops

(FAO, 1985). The protein amino acids profile of grass pea, lentil and soybean are

summarised in Figure II.3.

The protein-bound methionine content seems rather stable among the nine grass pea

genotypes studied in spite of the high variation in β-ODAP. There is no significant

difference in protein methionine level between grass pea from India with high β-

ODAP content and Canadian LS 87124 with low β-ODAP content. Thus, the protein

methionine content seems not to be correlated with the level of β-ODAP in Lathyrus

sativus seeds (Figure II.4).


34

0

1

2

3

4

5

6

Aspar

tate

Glutam

ate

Serine

Glycine

Histidi

ne

Argini

ne

Threo

nine

Alanine

Prolin

e

Tyros

ine

Valine

Met

hionin

e

Isoleu

cine

Leuc

ine

Pheny

lalan

ine

Lysin

e

amino acids

% s

eed

wt

Glycine max Lens culinaris Lathyrus sativus

Figure II.3: Average protein amino acid profile in Lathyrus sativus (mean of nine genotypes), compared with lentil (Lens culinaris) and soybean (Glycine max)

0

0.1

0.2

0.3

0.4

0.5

0.6

India B.Dar,Eth

China D.Zeit,Eth

Wollo, Eth Gonder,Eth

Derek,Poland

Ls 87124 Ls82046

% s

eed

wt

Methionine Beta-ODAP

Figure II.4: Level of the free amino acid β-ODAP versus protein amino acid methionine in Lathyrus sativus seed

Impr

ovin

g th

e nu

trit

iona

l qu

alit

y of

gra

ss p

ea (

Lat

hyru

s sa

tivu

s L

.)

35

Tab

le II

.2:

Pro

tein

am

ino

aci

ds (

% d

ry s

eed

wei

ght;

mea

n ±

SD

; n

= 4

) in

th

e se

eds

of La

thyr

us s

ativ

us o

f diff

eren

t

ge

noty

pes

com

par

ed t

o t

wo

oth

er fo

od

legu

mes

i.e.

lent

il an

d so

ybe

an

Val

ues

in a

row

follo

wed

by

the

sam

e le

tter

are

not

sig

nifi

cant

ly d

iffe

rent

(p<

0.05

)

D

.Ze

it (S

hoa

), E

th

Go

nder

, E

th

Wo

llo,

Eth

B

.Dar

, Eth

In

dia

C

hin

a P

ola

nd

Ls8

20

46

Ls 8

712

4

Len

s cu

lina

ris

Gly

cine

max

Asp

art

ate

1

.40

±0.5

3e 1

.98

±0.5

7b 1

.50

±0.4

5de

1.7

5±0

.63cd

1

.92

±0.5

7bc

1.6

3±0

.55d

1.9

5±0

.65bc

1

.54

±0.3

8de

1.3

9±0

.46e

1.1

8±0

.45ef

3

.36

±1.3

0a

Glu

tam

ate

2

.3±0

.91ef

3.0

7±1

.10bc

2

.43

±0.8

3de

2.9

0±1

.19d

3.0

2±1

.13cd

2

.67

±0.9

3ef

2.9

8±1

.04cd

2

.64

±0.9

2e 2

.48

±0.9

5e 1

.94

±0.7

6f 5

.26

±2.2

9a

Ser

ine

0

.85

±0.3

4cd

0.9

1±0

.04cd

0

.75

±0.0

3e 0

.95

±0.2

4b 0

.94

±0.1

1bc

0.8

8±0

.05cd

0

.93

±0.0

4bc

0.8

6±0

.07cd

0

.63

±0.2

1f 0

.76

±0.0

9de

1.5

6±0

.51a

Gly

cine

0

.69

±0.3

1cd

0.7

1±0

.08bc

0

.62

±0.1

0cd

0.7

6±0

.17b

0.7

2±0

.03bc

0

.74

±0.0

8b 0

.79

±0.0

4b 0

.72

±0.0

3bc

0.5

2±0

.18e

0.6

5±0

.02cd

1

.21

±0.3

8a

His

tidin

e

0.4

5±0

.22cd

0

.50

±0.1

7bc

0.4

1±0

.18e

0.5

1±0

.18bc

0

.51

±0.1

2bc

0.4

9±0

.21cd

0

.52

±0.1

7b 0

.50

±0.1

3c 0

.43

±0.0

8cd

0.4

3±0

.09cd

0

.89

±0.2

7a

Arg

inin

e 1

.32

±0.4

5cd

1.4

7±0

.33bc

1

.21

±0.2

7cd

1.5

1±0

.58bc

1

.56

±0.4

3b 1

.43

±0.2

8c 1

.55

±0.3

6b 1

.41

±0.3

3bc

1.0

4±0

.30d

1.1

9±0

.29d

2.2

6±1

.00a

Thr

eon

ine

0.5

2±0

.23cd

0.6

2±0

.14bc

0

.52

±0.1

3cd

0.6

8±0

.20b

0.6

1±0

.11bc

0

.61

±0.2

1bc

0.6

6±0

.14b

0.5

9±0

.08c

0.4

8±0

.10d

0.5

6±0

.03c

1.0

6±0

.49a

Ala

nin

e

1.0

3±0

.40b

0.8

1±0

.04d

c 0

.83

±0.1

9d 0

.99

±0.4

3cd

0.8

1±0

.14d

e 1

.02

±0.2

8bc

0.9

7±0

.16cd

1

.01

±0.1

4c 0

.76

±0.1

8f 1

.04

±0.2

7b 1

.31

±0.6

2a

Pro

line

0

.90

±0.3

2cde

0.9

3±0

.16cd

0

.80

±0.0

7e 1

.01

±0.0

7bc

1.0

4±0

.27b

0.9

4±0

.21bc

d 1

.06

±0.2

4b 0

.95

±0.2

4c 0

.83

±0.2

6d 0

.67

±0.0

4e 1

.53

±0.7

8a

Tyr

osi

ne

0.4

4±0

.22cd

0.4

9±0

.17c

0.4

3±0

.16d

0.5

3±0

.16b

0.5

1±0

.10bc

0

.49

±0.2

0c 0

.51

±0.1

5bc

0.4

8±0

.12c

0.4

2±0

.08d

0.4

1±0

.08d

0.9

6±0

.55a

Va

line

0.7

3±0

.30cd

0

.79

±0.0

8c 0

.70

±0.1

2e 0

.84

±0.3

7b 0

.78

±0.0c

0.8

1±0

.06bc

0

.81

±0.0

4bc

0.8

1±0

.06bc

0

.66

±0.0

8e 0

.72

±0.0

9d 1

.30

±0.7

7a

Met

hio

nine

0

.11

±0.0

9c 0

.10

±0.0

3d 0

.10

±0.0

3d 0

.12

±0.0

4bc

0.0

9±0

.02e

0.1

2±0

.07bc

0

.10

±0.0

5d 0

.13

±0.0

3b 0

.09

±0.0

2e 0

.10

±0.0

2d 0

.24

±0.0

9a

Iso

leuc

ine

0.4

8±0

.16cd

0.5

8±0

.38b

0.4

9±0

.33cd

0

.57

±0.4

2bc

0.5

5±0

.35bc

0

.53

±0.3

8c 0

.56

±0.3

6bc

0.5

2±0

.34cd

0

.44

±0.2

8d 0

.44

±0.2

9d 0

.99

±0.6

7a

Leuc

ine

0

.96

±0.5

5d 1

.17

±0.3

6b 0

.97

±0.2

9d 1

.15

±0.3

1bc

1.1

5±0

.38bc

1

.07

±0.1

9cd

1.1

3±0

.32c

1.0

5±0

.32cd

0

.92

±0.3

0e 0

.91

±0.3

3e 2

.08

±0.6

1a

Phe

nyl

ala

nine

0

.82

±0.3

2cd

0

.91

±0.2

8b 0

.78

±0.2

6e 0

.91

±0.2

6b 0

.91

±0.3

1b 0

.82

±0.1

9cd

0.8

8±0

.23c

0.8

8±0

.17c

0.7

8±0

.17e

0.8

3±0

.21d

1.6

6±0

.45a

Lysi

ne

0.9

1±0

.59d

1.1

3±0

.22b

0.9

4±0

.23cd

1

.13

±0.2

3b 1

.10

±0.2

5bc

1.1

0±0

.19bc

1

.21

±0.0

2b 1

.01

±0.1

1cd

0.9

6±0

.12cd

0

.88

±0.1

6d 1

.81

±0.1

4ab

To

tal

14.8

8

16.1

7

12.9

8

16.2

9

16.2

2

15.3

5

16.6

1

15.

1

11.8

3

12.7

1

25.9

7


36

II.4. Discussion

Our analysis of free amino acids in the different genotypes demonstrated that

homoarginine (Har) was both the most abundant and stable free amino acid in grass pea.

Har has also been found in dry seeds and 4-day-old seedlings of several Lens species but

the amount was very low and negligible (Rozan et al., 2001). The study of the free amino

acids profile of some other common food legumes including pea, bean and lentil (Kuo et

al., 2004) also showed that Har was undetectable in the dry seeds while arginine was

quantitatively the most important free amino acid in pea and bean, and glutamic acid was

the most important amino acid in lentil. Haque (1997) reported that for the low toxin line

of L. sativus LS-82046, when grown in hydroponic solutions with a doubled supply of

trace elements Zn2+, Fe2+, B(OH)4- or Co2+, or with Al3+ at 2 x 10–6 M in the media, or

with increasing salinity up to 0.8 % NaCl (v/w), the level of Har in the seeds increased

dramatically.

Both Har and β-ODAP were detected in the blood plasma and in the urine after human

volunteers ingested a single meal of 200 g cooked grass pea seeds (Nunn et al., 1994).

Har has been reported to antagonise the neurotoxic action of β-ODAP fed to one-day-old

chicks (Yusuf et al., 1995). This might be explained by the modulating effect of L-Har on

nitric oxide synthase (Jyothi and Rao, 1999).

Jiao and co-workers (2006) reported that β-ODAP content in grass pea might be related

to the level of total free nitrogenous compounds whereas nitrogen and phosphate may be

the crucial nutrient factors influencing β-ODAP content under field conditions.

Environmental factors such as drought, zinc deficiency, iron oversupply and the presence

of heavy metals in the soil can considerably increase the level of β-ODAP in the seeds

(Haque, 1997).

The presence of β-ODAP in grass pea seeds was blamed for causing the crippling disease

neurolathyrism and the toxicity is possibly mediated by collective effects of L-β-ODAP

on the AMPA-type receptor, metabotropic glutamate receptors, and NO (nitric oxide)


37

production (Kusama-Eguchi et al., 2006). β-ODAP was found to chelate copper and

zinc, which might interfere with the redox homeostasis in the body after heavy

consumption (Davies et al., 1990). However, this multifunctional compound, named as

dencichine in traditional Chinese herbal medicine, was also detected in the longevity

promoting ginseng root (Kuo et al., 2003), and praised for its haemostatic and platelet

increasing properties (Xie et al., 2007).

Efforts in plant breeding research to eliminate β-ODAP from the seeds have produced a

large number of “low toxin” varieties but did not yet result in “toxin-free” varieties.

Post-harvest processing of grass pea seeds by solid state fermentation with Aspergillus

oryzae and Rhizopus microsporus dramatically reduced the level of β-ODAP to a

minimum amount of around 0.1 g kg–1 but could not remove the neurotoxin completely

even with the low toxin lines (Kuo et al., 1995; Kuo et al., 2000).

Grass pea seeds are the major protein source for the poor and become a survival food

during drought or when land productivity becomes marginal in rural areas. Gatta et al.

(2002) reported that the protein content of 161 accessions in the Bari (Bangladesh

Agricultural Research Institute) Lathyrus germplasm collection ranged from 23 to 29.9 %

(mean 26.3 %) with low coefficient of variation (4 %). On the other hand, the protein-

bound methionine content seems to be the least abundant amino acid as reported earlier

(Kuo et al., 1995) but stable among nine grass pea genotypes in spite of high variability

in β-ODAP. Moreover, Nunn and co-workers (2005) reported that consumption of these

seeds limiting in methionine (Kuo et al., 1995) as staple food for a long period leads to

the deprivation of methionine in the bloodstream. The long-term deficiency of

methionine in the bloodstream and thus reduced methionine flux into motor neuron cells

might lead to higher susceptibility of these cells to the excitatory effects of β-ODAP

(Nunn et al., 2005). The concentration of methionine (0.1 %) in the naturally growing

grass pea accounts for only about a fourth of the normal dietary requirement for an

average person and less than a fourth for children (FAO/WHO, 1985). Neurolathyrism is

believed to be caused by the neuro-excitotoxic action of β-ODAP and oxidative stress.

The deficiency of methionine contributes to this oxidative stress. Therefore, for the


38

nutritional improvement of Lathyrus sativus and the prevention of neurolathyrism there is

a crucial need for additional sulphur amino acid supplement in grass pea food or for in

vivo enrichment in the plant itself.


39

CHAPTER III

Study of environmental factors (climatic, edaphic) affecting

yield and toxicity of grass pea grown in Ethiopia in a

multilocation experiment


40

L. sativus growing on Ethiopian soil


41

CHAPTER III

Study of environmental factors (climatic, edaphic) affecting yield and

toxicity of grass pea grown in Ethiopia in a multilocation experiment

Abstract

Statistical analysis (path coefficient, ANOVA, correlation) was done to identify the

interrelations and the effects of environmental factors (climatic, edaphic, altitudinal) on

yield and β-ODAP level of grass pea grown in Ethiopia. Ten grass pea genotypes grown

for two years (2005- 2006) in five different agro-ecological locations of Ethiopia were

used. Environmental factors including rainfall, sunshine hours, temperature, soil nutrients

and physical soil factors linked to altitudes of the respective locations were studied in

relation to their interrelations or correlations with grass pea yield and β-ODAP level.

Analysis of varience indicated that crop yield and yield related parameters including β-

ODAP showed considerable variability among locations (altitudes), years and genotypes.

Path coefficient analysis indicated that days to maturity was the most influencing factor

having the largest and most positive direct effect on yield. Total rainfall during the

growth period had a large positive direct effect on yield. High levels of soil nutrients

Mn2+ and S2- seem to affect yield negatively at Denbi site. Path analysis for β-ODAP

revealed that Zn2+/P, days to maturity, yield and K+ were variables having a larger direct

effect on the response variable β-ODAP. The dominant variables with high total effect (r

> 0.75) on β-ODAP under field conditions were K+, sunshine hours (ssh) and days to

maturity. We also found that supply of Zn2+ can be optimised to minimise β-ODAP

content, but this is also affected by interactions with other essential soil nutrients. From

linear correlation analysis, we found that K+ and ssh positively, while pH, days to

maturity and yield negatively have the highest negative correlations (>0.70) with β-

ODAP levels among 35 factors considered in this study for Ethiopia. The analysis also

indicated the strongest correlation of sunshine hours with β-ODAP level during the phase

of crop maturity (ssh III). This may suggest that β-ODAP synthesis and translocation to

the final sink (seed) is mainly taking place during crop maturation or late reproductive


42

phase. From this we can propose that β-ODAP biosynthesis and its response to

environmental stress could be highest during postanthesis.

III.1. Introduction

Climatic and edaphic factors were found to affect the level of a number of biosynthates

and crop parameters in Lathyrus sativus. Significant differences were reported for crop

performances (plant size, days to maturity, days to flowering, total biomass and grain

production), β-ODAP and other antinutritional constituents of grass pea when grown in

different agro-ecologies. Many studies confirmed that both genotypic and environmental

factors, and their interaction affect the concentration of β-ODAP in grass pea (Campbell,

1997; Hanbury et al., 2000a; Cocks et al., 2000; Wuletaw, 2003; DZARC, 2003; Asfaw

et al., 2003).

Haque (1997) proposed that higher accumulation of β-ODAP in drought-stressed L.

sativus plants and seeds may result from disrupted nitrogen assimilation and loss of

specificity of particular nitrogen assimilating enzymes, leading to higher synthesis of

asparagine. Asparagine is considered the primary precursor of the isoxazolinone ring in

BIA, which was confirmed to be the in vivo and in vitro precursor of β-ODAP in L.

sativus. It was suggested that β-ODAP might be important for the plant by acting as a

defence to various stresses (Lambein et al., 1990; Haque, 1997). This β-ODAP may then

contribute to the hardiness of the crop. Hence, the plant apparently produces less toxin in

the seed under optimal growing conditions than when it grows under marginal conditions.

Under favourable growing conditions, the plant produces more pods and seeds with

reduced β-ODAP concentrations, possibly as a result of a toxin dilution effect. Under

identical unstressed conditions, plants with only 10 pods contained 59 % more β-ODAP

in the seed than plants with 100 pods (Cocks et al., 2000).

From hydroponic pot experiments, Haque (1997) reported that soil nutrients Fe2+, Mn2+,

B3+, Co2+ and high Al3+ were found to enhance accumulation of β-ODAP while Zn2+,


43

Mo6+, Cu2+ and low Al3+ decreased the level. The antagonistic effect of Fe2+ and P against

Zn2+availability seem to increase the level of β-ODAP in a manner that higher P/Zn2+ and

Fe2+/Zn2+ ratios would boost toxin production. According to Jiao et al. (2006) β-ODAP

accumulation in grass pea might be related to the level of total free nitrogenous

compounds, whereas nitrogen and phosphate may be the crucial nutrient factors

influencing β-ODAP synthesis and accumulation under field conditions. Grass pea is

reported to be tolerant to a certain level of soil salinity. Occurrence of water soluble salts

refers principally to the precence of four cations Na+, K+, Ca2+, and Mg2+ linked with the

anions Cl- and SO42- (Jackson, 1973). In artificial salinity in sandy soil, grass pea biomass

production increases up to 0.3 % NaCl (EC = 7.8 m.mohs/cm). However, this remains

controversial as salinity has a decreasing effect on β-ODAP accumulation at lower levels

of artificial salinity (up to 0.6% NaCl) and an increasing effect at higher levels of

artificial salinity (Haque, 1997).

Light also influences β-ODAP biosynthesis. Zhang and co-workers (2003) discovered

that β-ODAP can act as a scavenger for hydroxyl radicals in the plant and protects

glycolate oxidase (GO, an enzyme of photorespiration) under high light intensity. In the

Ethiopian highlands high light intensity can obuviously play a role. β-ODAP content

increases while GO activity decreases. The same authors (Zhang et al., 2003) also found

that oxalate, a precursor molecule for β-ODAP, increases β-ODAP content in the seed

when applied in the seedling stage.

Previous studies on grass pea mostly emphasized on genotype improvement with focus

on reducing neurotoxin content, while studies on environmental factors influencing this

metabolite synthesis and subsquent accumulation were hardly undertaken. As a rationale,

understanding the relative importance of environmental factors and their interrelation in

affecting β-ODAP content and crop yield would help developing possible crop

mangement measures aimed at crop improvement and maximizing farmer’s benefit.

Although experiments have previously been carried out in diverse environments in

relation to grass pea toxicity (particularly β-ODAP), those experiments failed to

adequately and inclusively analyse associated factors (climatic, edaphic) influencing seed


44

β-ODAP content or crop yield. Hence, the present study was designed to investigate the

effects of various growth factors on grass pea production and its toxicity from different

experimental plots operated by the Ethiopian Institute for Agricultural Research (EIAR).

Environmental and geographic parameters (climatic, edaphic, altitudinal, seasonal) were

measured and monitored. A statistical method called ‘Path Coefficient Analysis’ that was

developed for calculating such cause/effect or predictor/responsive variable relationships

(Gravois and McNew, 1993) was employed. Hence, the study was initiated to identify

correlative effects of environmental (climatic, and edaphic) and genotypic factors and to

determine their level of importance on yield and β-ODAP content of grass pea in

Ethiopia.

III.2. Materials and methods

Field experiments were conducted at five experimental stations (Fig III.1.) in Ethiopia

during the 2005-2006 seasons to measure seed yield and β-ODAP level in 10 genotypes

of grass pea, meteorological and edaphic data. Agronomic parameters and meteorological

data are from the two years (2005 and 2006), however, due to resource limitations, soil

nutrient and amino acid analysis data are presented from the 2005 season only and

analysis was done accordingly. Nine grass pea genotypes that were selected for their wide

environmental adaptation and low toxicity content by International Centre for

Agricultural Research in the Dry Areas (ICARDA), Allepo, Syria were used. These

include ILAT-Ls-590, ILAT-Ls-610, ILAT-Ls-690, ILAT-Ls-Ls-B1, ILAT-Ls-Ls-B2

(newly released variety in Ethiopia named Wasie), ILAT-Ls-k290, ILAT-Ls-k387, ILAT-

Ls-k190, ILAT-Ls-390. Furthermore, cultivar Debre Zeit was included as local control.

ICARDA is an international center that has the mandate for grass pea improvement

research. The experiment was arranged in a randomized complete block design with three

replications. Plot size was 1.6 m x 4.0 m or 6.4 m2. Spacing was 10 cm between plants

and 40 cm between rows. No fertilizer was applied. Plots were weeded as needed. Field

data: sowing date, plant emergence, pest infestation monitoring, days to flowering date,

days to maturity and seed yield were monitored and recorded. Harvesting was done from

the whole plot as plants grow intermingled and separation is very difficult.


45

Figure. III.1. Growing locations used for analysis in East Shoa Zone in the central high lands of Ethiopia (1*-Alem Tena, 2*-Debre Zeit, 3*-Denbi, 4*-Akaki, 5*-Chefe Donsa. Major roads (yellow) with water bodies (blue patches ) indicated. (See Table III.1 for coordinates). Source: google earth mapping Agro-ecological analysis

Locations altitude were determined using a pocket altimeter (Bariso, Finland) adjusted in

reference points and are given in meters above sea level (m asl). During the growth

period of the crop, the daily measurements of maximum and minimum temperature (oC)

by thermometer (CASSESLA, England), rainfall (mm) by rain gauge (Lamp Cht,

Germany) and daily sunshine hours (the time during which the direct solar radiation

5* 4* 3* 2* 1*


46

exceeds the level of 120 W/m2) by radiometer (SIAP, Italy) were taken from the

respective locations’ weather stations.

At different stages of development, the plant may have different sensitivity to stress

(Cocks et al., 2000), with different responses to β-ODAP synthesis levels. Hence, three

growth stages were differentiated and the climatic factor values were analyzed separately

based on these three growth stages of the plant. The growth stages considered were i) pre-

branching or seedling stage: the stage between sowing and branching (first 3-5 weeks); ii)

pre-anthesis (next 3-7 weeks): between branching and anthesis (maximum biomass

accumulation); and iii) post-anthesis (last 5-9 weeks), the period after flowers are fully

open (functional) until crop maturity. The climatic parameters (temperature, rainfall and

sunshine hours) recorded during the respective stages were labeled as tempI, tempII, and

tempIII, and RFI, RFII, RFIII, and sshI, sshII, sshIII.

Soil nutrient and amino acid analysis (2005 season)

Before planting, soil samples were taken using an Auger manual drill in the respective

growth locations (six samples/location) from the surface down to 60 cm depth. Analysis

of soil parameters was done in the national soil laboratory, Ethiopia. Soil micronutrients

(Zn2+, Mn2+, Fe2+, Cu2+) were extracted using the chelating agent Diethylene Triamine

Pentaacetic Acid (DTPA) after which their amount was determined using an atomic

absorption spectrophotometer (Milton Roy Spectronic model No 401, USA) together with

reference standards for each microelement. Published analytical procedures were

followed (Houba et al., 1989) and the content of microelements (Fe2+/Mn2+/Zn2+/Cu2+)

was calculated as:

(mg/kg soil) = (a-b) x 40/S x mcf;

where:

a = conc. of element in sample extract (mg/l)

b = conc. of element in the blank (mg/l)

40 = volume of extractant used per sample (ml)

S = weight of air dry soil (g)

mcf = moisture correction factor.


47

Sulphur concentration was determined following the procedure described by Cottenie

(1980). Available soil phosphorus content was determined by the 0.5 M sodium

bicarbonate extraction solution (pH 8.5) method of Olsen (1954). C:N ratio was

calculated from the separate determinations of organic carbon (OC) by weight loss on

ignition in a combustion furnace and total nitrogen (TN) using the Kjeldahl method. Soil

pH meter reading was made after suspension in water using calibrations at pH 4.00, 7.00

and 9.00.

Concentration of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), the neuro-

excitatory free amino acid in the plant known to cause irreversible crippling in humans,

was determined only for the 2005 harvested seed samples using O-phthalaldehyde (OPT)

color reaction as standardized by Rao (1978) at the reference laboratory in ICARDA by

Dr. Abd El Moniem. Protein hydrolysis for amino acids determination using HPLC was

performed as explained in Chapter II. All samples were hydrolysed in duplicate and each

derivatised hydrolysate was analysed twice by HPLC, giving four numbers per data point.

Agronomic parameter analysis

Crop response parameters considered were: days to maturity, 100 seeds weight (g) and

dry seed yield in kg/ha. Days to maturity were determined as the number of days from

seed germination to maturity (>95 pods changed yellow or pale) of the plants in the plot.

Hundred seed weight is the weight in g of 100 seeds taken randomly from the respective

harvested seed lots. Yield in kg/ha was estimated by extrapolation of the yield of all 160

plants of the plot.

Statistics

Analysis of variance for the means was done using statistical analysis software (SAS

Inst., Cary NC) (Kang, 1994). Determination of path coefficients for the path analysis

was done in a multiple step calculation using SPSS. Predictor variables for yield [days to

maturity, copper (Cu2+ in ppm), zinc (Zn2+ in ppm), total rain fall during stage I (RFI)]

and for β-ODAP [soil pH, available K+ (ppm), sshIII, days to maturity, yield (kg/ha), Fe2+


48

(ppm), Zn2+/Fe2+, Zn2+/P, soil bulk density (BD/cm3), tempII and RFII] were selected

using stepwise regression after they were found to have significant correlation with

respective dependent variable (yield or β-ODAP). Path coefficient analysis was selected

as the method of choice because it not only shows relationships between predictor and

response variables (direct effect of specific component), but also shows interrelationships

between predictor variables in their effects on the response variable (indirect effect of

specific component on the response variable via other components) (Gravoice and

McNew, 1993). By measuring the extent of effect of one variable on another using

standardized data or a correlation matrix as input, the model, path coefficient, enables

identification of environmental components affecting the response variable (in this case:

grass pea seed yield and β-ODAP level) and to estimate the strength of this effect.

III.3. Results As can be seen in Table III.1 and Figure III. 2, the locations have different altitudinal,

climatic and edaphic features. These differences were correspondingly reflected on the

growth period of grass pea (Table III.2 and Figure III.3) resulting in differences in days

to maturity and ultimate yield among the locations (Tables III.2 and III.4). According to

Bull’s (1996) agro-ecological classification based on length of growth period (L) and

thermal zone (T) as related to altitude, we identified two thermal and two growth length

groups [LI=<90 days, LII = 91-150 days, T3 = 1300-2000 m asl, T4 = 2000-3000 m asl]

in the five locations. This classification was adopted since it is found having important

relevance to the present study. Hence the locations can be grouped into ‘short growth

period LI’ as in Alem Tena and Denbi, and ‘long growth period LII’ as in Debre Zeit,

Akaki and Chefe Donsa (see Table III.1). This effect of the location on length of growth

period (days to maturity) is also found to vary associated with altitude, moisture regime,

temperature range and soil properties (Tables III.3 and III.4 and Figure III.2). In addition

to the light soil texture that may allow fast evapotranspiration, the levels of S2- and Mn2+

were found remarkably high at the poor yielding location of Denbi (Tables III.2 and III.

3).


49

Table III.1: Some characteristic features of the experimental fields in the study areas Descriptive geo-physical features of locations

Location names

Coordinates

Altitude in (m asl)

Mean annual temperature (°C)

Total rainfall during crop growth period (mm)

Soil texture & Electric conductivity range values in ds/m

Agro- ecological group (Bull, 1996) (see text III.3)

1 Alem Tena

Lat. 8˚ 18’ Long. 38˚ 56’

1650 23.3 368.0 Clay loam

LI T3

2 Debre Zeit Lat. 8˚ 45’ Long. 38˚ 57’

1900 18.7 366.9 Clay

LII T3

3 Denbi

Lat. 8˚ 44’ Long. 38˚ 57’

1960 18.6 362.7 Silt clay

LI T3

4 Akaki

Lat. 9˚ 02’ Long. 38˚ 45’

2200 16.7 500.4 Clay

LII T4

5 Chefe Donsa

Lat. 8˚ 58’ Long 39˚ 09’

2400 15.1 542.0 Clay

LII T4

050

100150200250300

July

augu

st sep oc

tno

vde

cjan

12345

Rainfall (mm)

Figure III.2 : Pattern in rainfall (monthly total in mm), sunshine hour (mean monthly) and temperature (monthly minimum and maximum °C) during the growth period (2005-2006) of grass pea at 1-Alem Tena, 2-Debre Zeit, 3-Denbi, 4-Akaki, 5-Chefe Donsa locations

0

5

10

15

20

25

30

July August Sep Oct Nov Dec Jan

Alem Tena0

5

10

15

20

25

30


Debre Zeit

0

5

10

15

20

25

30

Denbi

0

5

10

15

20

25

30


Akaki0

5

10

15

20

25

30


Chefe Donsa

02468

1012

July

augu

stse

p oct

nov

dec

jan

12345sunshine hour


50

Growth period Locations July August Sep Oct Nov Dec Jan 1 2 3 4 5 Figure III.3. Plant growth period at the five experimental locations (horizontal bar); plant growth stages are bounded by vertical slash Table III.2. Performances of selected grass pea genotypes and a local cultivar from Debre Zeit as affected by locations (1-Alem Tena, 2-Debre Zeit, 3-Denbi, 4-Akaki, 5-Chefe Donsa) for days to maturity, yield and β-ODAP content.

Days to maturity (mean for 2005-2006) Yield (kg/ha) (mean for 2005-2006) β-ODAP % seed weight (2005)

Genotypes 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

ILAT-IS-590 82c 120b 91c 139a 147a 1237d 2271c 783e 3898a 3219b 0.208a 0.198a 0.183ab 0.158b 0.132c

ILAT-IS-610 80c 128b 87c 143a 147a 1394d 2520c 810e 3738a 2821bc 0.202a 0.190a 0.198a 0.167b 0.122c

ILAT-IS-690 76c 121b 88c 140a 140a 1800d 2585c 929e 3847a 2843bc 0.186a 0.148c 0.182a 0.168b 0.150c

ILAT-Ls-Ls-B1 82c 119b 93c 143a 147a 1174d 2183ab 609e 2590a 1795bc 0.108a 0.086b 0.083b 0.080b 0.078b

ILAT-Ls-Ls-B2 81c 126b 91c 146a 153a 1182c 2232b 869cd 3047a 1967b 0.101a 0.085b 0.080b 0.080b 0.079b

ILAT-LS-K-290 80c 120b 84c 138a 141a 1444d 2496bc 933e 4320a 2845b 0.135a 0.121b 0.112b 0.121b 0.112b

ILAT-LS-K-387 82c 120b 88c 138a 147a 1137d 2383c 938d 2965b 3458a 0.151a 0.123b 0.122b 0.126b 0.122b

ILAT-LS-K-190 86c 123b 89c 142a 149a 1280c 2065b 899d 3270a 3026a 0.110a 0.087bc 0.093b 0.076c 0.083bc

ILAT-LS-K-390 78c 118b 89c 136a 139a 1358c 2427b 903d 3742a 2263b 0.206a 0.108b 0.101b 0.104b 0.101b

DZ local cultivar 86c 127b 95c 141a 149a 1247d 2710c 961d 3903a 3286b 0.338a 0.316a 0.251b 0.178c 0.122d Values in a row followed by the same letters are not significantly different at p< 0.05

Table III.3. Physico-chemical features of the soils from the trial locations as analyzed pre-planting (n=6 ± stdev, nutrients in ppm) in 2005 (BD = bulk density)

Locations Cu Fe Mn Zn S Available P

AvailableK C/N pH BD

Alem Tena 1.28±0.15 46.41±25 73.51±38.5 1.06±0.34 1.34±0.57 1.4±0.70 300±33 10.2±0.50 6.75±0.15 1.80±0.20

Debre Zeit 5.77±0.27 73.30±7 57.55±13.6 1.19±0.12 0.92±0.78 23.6±20.19 258±30 9.2±0.38 6.05±0.15 1.30±0.17

Denbi 2.99±0.36 70.15±15 133.00±8.7 0.78±0.38 1.96±0.23 10.0±3.63 223±31 11.2±0.32 6.40±0.20 1.29±0.12

Akaki 2.03±0.26 35.23±27 20.14±5.6 0.38±0.16 0.34±0.27 2.6±0.52 95±9 11.4±0.41 7.30±0.20 1.33±0.10

Chefe Donsa 2.07±0.68 34.63±26 19.75±18.1 0.48±0.47 0.35±0.40 3.4±0.85 96±7 11.1±0.63 7.60±0.45 1.27±0.10


51

In agronomic performance analysis, yield was shown to be significantly (p<0.05)

different in response to cropping year, genotype, location and interaction effects. We

found a significant (p<0.05) difference for genotypes (G), locations (L) and their

interaction (G x L) for yield (Table III.4).

Better yield performance was associated with locations at higher altitude (Akaki or Chefe

Donsa) where crop growth period (days to maturity) was relatively longer. Higher

temperature and lower total rain (drought stress) that might shorten the growth period at

Alem Tena, gave rise to lower yields. Despite having a similar agro-climatic

environment, Denbi has poorer yield performances compared to Debre Zeit (Table III.1),

which mainly could be attributed by edaphic factors (soil texture, nutrient factors) (Table

III.3) than any climatic features of the location.

There was a significant difference for days to maturity due to years, genotypes and

location effect. Both genotypes and locations and their interaction were significant for β-

ODAP content (Table III.4).

Table III.4. ANOVA of selected parameters for performances of grass pea grown at five locations

Source Degrees of freedom

Days to maturity

100 seed weight

Yield (kg/ha)

β-ODAP % weight

Year (Y) 1 * Ns * - Genotype (G) 9 * * ** ** Location (L) 4 ** Ns ** * Year X genotype 9 Ns Ns * - Genotype X location 36 Ns Ns ** ** Year X location 4 ** Ns ** -

**, * Significant at 0.01 and 0.05 probability level respectively; Ns-non significant

A mean contrast for crop performance attained was about quadripled for yield, about

doubled for days to maturity and about doubled for β-ODAP levels between best and

poorest performing locations (Table III.2). Variation in seed weight was small ranging

between 7.6 g to 8.5 g per hundred seeds and variation of the mean seed weight was

found to be significant (p ≤ 0.05) only for genotypes (Table III.4).


52

The correlation structure elucidated by the path analysis (Table III.5) with the path

coefficient in the diagonal indicates the level of influence each predictor variable causes

directly on the response variable (seed yield). The direct effects in the correlation reveal

that number of days to maturity has the highest direct effect value and also accounts for

most of the variation (0.94). The second highest value of direct effect was due to rainfall

(RFI) during early growth stage (0.31). Cu2+ and Zn2+ have modest negative direct effects,

whereas the Zn2+/S2- ratio has a very small direct effect. Since no path coefficint values

exceed unity, we conclude that multicolinearity has not produced any inflated path

coefficient values as described in Williams et al. (1990).

Considering the indirect effects on yield in Table III.5, the variable days to maturity have

the greatest indirect effect (-0.34, -0.63, -0.66, 0.43) on yield through other factors.

Rainfall has got modest indirect effects on yield (-0.22, 0.12, -0.18) through all other

factors except Cu2+ (-0.03) which has got the least indirect effect. Smaller indirect effects

(<0.1) were seen for Cu2+, Zn2+ and Zn2+/S2-. Correlation of predictor variables with the

response variable ranged from 0.38 to 0.91 and coefficient of determination was 0.97.

Table III.5: Path analysis of the direct effects (bold) and indirect effects of the most relevant parameters: days to maturity, copper (Cu2+ in ppm), zinc (Zn2+ in ppm), total rain fall during stage I (RFI) on seed yield in grass pea grown at five locations in Ethiopia

Maturity

Cu2+

Zn2+

RFI

Zn2+: S2- r R2

Maturity 0.94 0.07 0.09 -0.22 0.04 0.91** 0.97

Cu2+

-0.34 -0.19 -0.06 -0.03 -0.01 -0.63**

Zn2+

-0.63 -0.08 -0.13 0.12 -0.01 -0.73**

RFI -0.66 0.02 -0.05 0.31 -0.04 -0.43**

Zn2+

/ S2- 0.43 0.03 0.02 -0.18 0.08 0.38* *, **- Significant at respectively 0.05 and 0.01 probability

The higher concentration of micronutrients S2- and Mn2+, at Denbi could be additional

factors to be considered having a negative effect on the yield of the crop, as they appear

to have a negative association (Figure III.4). These nutrients also produced a higher ratio

with Fe2+ and Cu2+ at the same location.


53

From the graphically illustration of relative distribution of plant and soil parameters

(Figure III.4), it is rather complex to derive a simple linear relation, but some general

trends will be deducted. The concentration of β-ODAP increases from Chefe Donsa

(highland) to Alem Tena (lowland) whereas days to maturity and crop yield (except at

Denbi), decreased in the same direction. Zn2+/ S2- and Zn2+/Mn2+ are higher at higher

altitude whereas the ratios Zn2+/Cu2+ and Zn2+/Fe2+ are higher at locations where we

found higher β-ODAP in the seeds. Association of high Zn2+/Fe2+with high β-ODAP

might be in contradiction of the pot experimentl results by Haque (1997).

0%

20%

40%

60%

80%

100%

alt

B-oda

p %

met

hio m

g/g

pro

mat

urity

days

100s

w

yd kg

/ha

pH: H

2O

Cu ppm

Fe

ppm

Mn p

pm

Zn p

pm

S p

pmZn:C

uZn:F

e

Zn:Mn

Zn:S S:Cu

S:Fe

S:M

n

Mn:

Fe

Mn:

CuFe

:Cu

parameters

rela

tive

valu

es

Alem Tena

Debre Zeit

Denbi

Akaki

Chefe Donsa

Figure III.4. Graphical presentation for relative quantitative distribution of plant parameters and soil nutrients analysed at the five growing locations. Data for each parameter for the five locations were added and considered as 100% and relative values spatially plotted.

Path analysis with 11 predictors for β-ODAP, selected on the basis of stepwise regression

was run the results are shown in Table III.6. Among the selected parmeters, the largest

direct effects were found for Zn2+/P (-0.55), days to maturity (-0.44), yield (-0.39) and K+

(0.39). Smaller direct effects were found for sshIII (0.35), pH (-0.19), Fe2+(0.31), Zn2+/

Fe2+(0.25) soil bulk density (BD) (-0.21), TempII (-0.23) and RFII (0.22). Among larger

indirect effects, yield showed the highest indirect effect through days to maturity (-0.40),

days to maturity showed the highest indirect effect through K+ (-0.34) and K+ showed the


54

largest indirect effects through sshIII (0.36) on β-ODAP. Small indirect effects were

found for K+ through pH, maturity, Bulk Density (BD), TempII and RFII; sshIII through

pH, K+, maturity, yield, temp II and RFII; maturity through pH, sshII, BD, TempII and

RFII; yield through pH, K+, sshII, TempII and RFII; Zn2+/ P through sshII; TempII

through K+ and sshII; TempII through K+, sshII and maturity.

For some parameters such as Fe2+, Zn2+/Fe2+ and BD the direct effects were much higher

than their indirect effects through other predictor variables. From the path analysis

Zn2+/P, maturity, yield and K+ were found to have higher direct effects, indicating that

they probably are the dominant variables influencing the response variable β-ODAP in

the field. It is suggested that those parameters with lower values of indirect effect have

more independent (direct) effects on the response variable β-ODAP. In this case Fe2+ and

Zn2+/Fe2+ seem to have predominantly a direct effect compared to other variables.

Table III.6: Path analysis of direct effects (bold) and indirect effects for selected parameters: soil pH, available K+ (ppm), sshIII, days to maturity, yield (kg/ha), Fe2+ (ppm), Zn2+/Fe2+, Zn2+/P, soil bulk density (BD/cm3), tempII and RFII on β-ODAP level of grass pea grown during two years at five locations in Ethiopia. Data used were two years (2005 and 2006) for climatic and biological factors, and one year (2005) for edaphic factors.

pH K+

sshIII

Maturity

Yd (kg/ha) Fe

2+

Zn2+

/Fe2+

Zn2+

/P BD Temp II RFII r R2

pH -0.19 -0.26 -0.23 -0.30 0.30 -0.16 -0.01 0.15 0.00 0.13 -0.03 -0.73**

K+

0.14 0.39 0.32 0.37 -0.30 0.08 0.08 -0.12 -0.12 -0.19 0.14 0.91**

sshIII 0.13 0.36 0.35 0.31 -0.25 0.08 0.06 -0.22 -0.10 -0.19 0.12 0.90**

maturity -0.13 -0.34 -0.25 -0.44 0.36 -0.07 -0.10 -0.07 0.10 0.18 -0.13 -0.78**

Yield -0.15 -0.30 -0.22 -0.40 -0.39 -0.10 -0.07 -0.05 0.04 0.13 -0.08 -0.70**

Fe2+

0.10 0.10 0.10 0.09 -0.13 0.31 -0.14 -0.16 0.03 -0.04 -0.04 0.32*

Zn2+

/Fe2+

0.01 0.13 0.09 0.16 -0.11 -0.17 0.25 0.14 -0.10 -0.06 0.10 0.28*

Zn2+

/P 0.05 0.08 0.14 -0.06 0.03 0.09 -0.07 -0.55 0.02 -0.03 -0.04 0.32*

BD 0.00 0.22 0.17 0.20 -0.08 -0.04 0.12 0.04 -0.21 -0.12 0.16 0.41**

Temp II 0.11 0.32 0.28 0.34 -0.22 0.05 0.06 -0.08 -0.11 -0.23 0.15 0.75**

RFII 0.02 0.24 0.19 0.26 -0.14 -0.05 0.11 0.10 -0.15 -0.15 0.22 0.54** 0.92

Linear Pearson’s correlation coefficient values of β-ODAP with all possible growth-

affecting factors explored including climatic, biological factors, soil physico-chemical

and geographic factors is presented as a diagram in Figure III.6. Many of the factors have


55

a significant correlation with the level of β-ODAP in the seed. Altitude (-0.85), days to

maturity (-0.78), soil pH (-0.72) and crop yield (-0.71) have highly significant negative

correlations with β-ODAP content, while K+ (0.86) and ssh III (0.80) have a significant

positive correlation to β-ODAP. The lowest level of β-ODAP was found in neutral to

weakly basic soil pH (6.7-8) conditions. Haque (1997) reported that the lowest value of

β-ODAP was produced at a pH level around neutral. We found the factor sunshine hours

to have a strong positive correlation with β-ODAP, the greatest (r = 0.80** ) being

recorded during post-anthesis phases of the plant (ssh III).

R F II 0 .5 4 **

R F III -0 .5 3 **

S W -0 .5 2 **

te m p II 0 .5 2 **

S :F e -0 .5 2 **

S :C u 0 .5 1 **

Z n ;S -0 .4 9 ** S 0 .4 7 **

te m p I0 .4 4 ** C :N -0 .4 2 **

Z n ;C u 0 .4 1 ** B D 0 .3 9 **

P 0 .3 4 **

F e 0 .3 2 *

Z n :M n -0 .3 2 *

Z n :P 0 .3 1 *

Z n :F e 0 .2 9 *

C u 0 .2 4

Z n :K 0 .0 6 S M n 0 .1 5 F e :C u 0 .1 5

te m p III0 .5 4 ** R F I0 .5 5 *

M n :C u 0 .5 5 * M n :F e 0 .5 5 **

Z n 0 .6 0 ** M n 0 .6 2 ** y ie ld -0 .7 1 **

S S H II0 .7 1 ** P H -0 .7 2 **

S S H I0 .7 7 ** m a tu r ity -0 .7 8 **

S S H III0 .8 0 ** a lt-0 .8 5 ** K 0 .8 6 **

Figure III.6. Pearson’s correlation coefficients of the different environmental and plant variables with β-ODAP in L. sativus (with the strongest correlation for K+ and the weakest for Zn2+/K+)

III.4. Discussion

The experimental plots are located in agro-ecology areas of grass pea production and

hence, the results reflects aspects of grass pea growth conditions in Ethiopia. A number

of important environmental factors were found to affect crop yield and β-ODAP content

in Lathyrus sativus seeds. In general, the soil in the locations was neutral to slightly

acidic or slightly basic, which may favour grass pea growth. Previous work indicated

ββββ----ODAP


56

that the soils of the study locations have low levels of organic matter, total nitrogen, P,

Cu2+ and Zn2+ (Abayneh and Demeke, 2003).

The crop growing period gets longer with higher altitude. Higher altitude is also

associated with more cloudiness, an increment in total amount of rain and length of the

rainy period. The better moisture holding capacity of the soil, lower temperature and

smaller sunshine hours (see Table III.1 and Figure III.2) at those higher locations might

have allowed an extention of the reproductive period and thereby better yield. The

number of sunshine hours was less in the highland compared to lowland areas due mainly

to the longer periods of cloud cover in the highlands. Akaki followed by Chefe Donsa

location were found to be the best yielding with longer crop growing period among the

five locations. In addition, this could be associated with the narrower range of max-min

temperatures and the higher C/N that might create better conditions for plant performance

at Akaki.

Days to maturity and amount of rainfall seem to be the dominant effectors for crop yield

as they showed a higher level of direct effect than the other factors considered. The

longer days to maturity period might have allowed effective fertilization of a higher

number of flowers and a longer period of translocation of biosynthates to an increased

number of seed sinks. Thus higher yield would be a resultant effect both from a larger

number of pods and better seed filling. By contrast, a shorter growth period at Alem

Tena and Denbi could be related more to the light soil texture that allow more

evapotranspiration forcing the plant into early maturity and poorer yield. Significant

correlations have been reported between seed yield and length of seed filling period (r =

0.57; Hanson, 1985) whereas a long maturation period is generally associated with

increased seed size (Swank et al., 1987). This was also clear from our own path

coefficient analysis: the greatest effect on yield was found for days to maturity (0.94)

followed by rainfall during crop establishment (0.31). Likewise, in other experiments,

significant (P<0.05) yield variation was reported ranging between 1.2 to 3.9 t/ha due to

locations in Ethiopia (Fikre et al., 2006), between 1.2 to 1.8 in winter and between 1.1 to


57

2.2 in spring seasons in Turkey (Düsünceli, 1993) and between 0.1 to 2.5 due to genotype

effect in Bangladesh (Campbell, 1997).

The significance of G x L (genotype by location) interaction for yield suggests that the

different varieties respond differently to the diverse environments as previously reported

by Wuletaw (2003). We found that better yield performance was associated with

locations at higher altitude (Akaki) where the growing season was longer. The longer

growth period must have allowed sufficient translocation of biosynthates to the sink.

There is ample evidence that yield improvement in crops has come from a longer

reproductive phase duration, which leads to a higher seed harvest index (Nelson, 1986;

Smith and Nelson, 1986a and 1986b). In contrast, lower yield recorded in drought stress

environments could have contributed partially to forced earlier maturation, leading to

development of a reduced size of reproductive sink as a physiological phenomenon (Hall,

2004). This condition induces higher concentration of β-ODAP in the relatively smaller

sink size (Cocks et al., 2000).

Nutrient optimization is one crucial element in the soil complex for enhanced

productivity of the crop. If one nutrient is lacking, it may negatively affect the availability

of others. The response of crops to supplementation, in cases where deficiency exists, can

be very pronounced. Micronutrient levels may have negatively affected yield as revealed

by the observation that there is a strong negative correlation between higher

concentration of S2- and Mn2+ and poor yield. The higher abundance of S2- and Mn2+ at

Denbi might explain its toxic levels and possible interferance with the normal

physiological performance of the plants to have lowest yield.

Potassium is absorbed by plants in larger amounts than any other mineral element except

nitrogen and, in some cases, calcium. Yield, Zn2+/P, maturity and K+ have the largest

direct effect on β-ODAP level in the path analysis. The total effect of K+, sshII, days to

maturity, pH and TempII on β-ODAP appear to be considerably high, presupposing their

corresponding influence. However, since these factors have got a substantial level of

indirect effect through other variables, β-ODAP could not be predicted from a single or


58

very few factors only. In Pearson’s correlation coefficient analysis, K+ showed the

strongest positive correlation with β-ODAP among all factors considered. This may

suggest that K+, which is reported to be high in Ethiopian soils (Murphy, 1968), could be

an important edaphic factor affecting β-ODAP levels in Ethiopian grass pea. Haque

(1997) reported that excess salinity cations including K+ could enhance the β-ODAP level

while lower salinity levels had a decreasing effect.

Many reports proposed that high concentrations of Zn2+ in the growing medium decrease

the level of β-ODAP in the seed (Lambein et al., 1994; Haque, 1997), possibly by its

chelating action. We found a positive correlation between β-ODAP and Zn2+ per se,

however, this became negative when combined with other nutrients. This conflicting

output might emerge from overall deficiency of Zn2+ in the soil (Abayneh and Demeke,

2003) and/or plants growing in an open field of complex environment could not simply

be reproduced in performance from controlled pot experiments reported earlier (Haque,

1997). Hence, the nutrient balance in the complex field condition could have a more

important role than a single nutrient (Haque, 1997).

In agreement with the results obtained from pot experiments by Haque (1997) the weakly

alkaline soil pH (6.7 – 8.0) appeared to effectively decrease β-ODAP. This could be the

pH where nutrient availability and plant performances are optimized, and whereby

accumulation of the stress metabolite β-ODAP is reduced.

Zhang and co-workers (2003) reported that higher light intensity could promote β-ODAP

levels. In contrast, we found higher β-ODAP values in lowland areas (1600 m asl) with

less light intensity than in highland areas (2200 m asl). In our analysis, the length of shh

period was found to have a strong positive correlation with β-ODAP, the greatest

correlation (r = 0.80** ) being recorded in the maturation phase (ssh III) of the plant. We

propose that the higher light intensity in the highlands combined with shorter light hour

periods (mean = 5 hrs/day with solar irradiance > 120 W/m2) most likely result in lower

light stress than the lower intensity combined with longer light periods (mean = 9.5


59

hrs/day) of the lowlands during the crop growth period. In other words, the cloud cover in

the highlands was longer than in the lowlands like Alem Tena.

In general, the effect of stress due to climatic factors (temperature, rain fall, sunshine

hours) on β-ODAP accumulation was apparently stronger during post-anthesis or during

the reproductive phase than at early growth stages. Similarly, Cocks and co-workers

(2000) reported that β-ODAP doubled due to drought stress at post-anthesis compared to

drought stress at pre-anthesis. Likewise, soil factors seem to have substantial influence on

the crop yield and β-ODAP content. Hence, in addition to the effect of genotype, β-

ODAP accumulation in the seed of grass pea is also considerably influenced by

environmental (edaphic and climatic) factors in Ethiopia. These results can be part of the

information needed for better agricultural management to produce a grass pea crop in

more suitable agro-ecologies that may yield seed with better nutritional quality.


60


61

CHAPTER IV

Effect of methionine supplement on physical responses and

neurological symptoms of broiler chicks fed grass pea based

starter ration


62

Young chicks feeding on grass pea diet, one of them with torticolis (neck bending) as

neurological symptoms


63

CHAPTER IV

Effect of methionine supplement on physical responses and neurological

symptoms of broiler chicks fed grass pea based starter ration

Abstract

Starter feeding experiments of broiler chicks with raw grass pea (Lathyrus sativus L.,

cultivar Debre Zeit local, containing 0.35 % neuro-excitatory amino acid ß-ODAP)

supplemented with different levels of methionine were undertaken during 2005 at EIAR

(Debre Zeit Center, Ethiopia) for 30 days to assess toxicity of grass pea based feed and

to correlate it with animal performances. Four hundred fifty one-day-old broiler chicks

were randomly divided into two groups, each group was given formulations containing

35 % (ration I) or 98.5 % (ration II) grass pea respectively. Each ration included controls

and treatments with four concentrations of methionine (0.025, 0.05, 0.075 and 0.1% of

the feed in ration I; 0.05, 0.1, 0.15 and 0.2 % of the feed in ration II). Each treatment was

conducted with 45 chicks, which were further randomly separated into three replicates of

15 chicks each. Performances of the chicks (feed intake, weight gain, feed conversion

efficiency) were much better in ration I than in ration II. Those parameters were

significantly improved by addition of methionine (0.075 % of the feed in ration I and 0.15

% in ration II respectively). Significant reduction (from 84 % to 2 %) of acute

neurological signs with addition of methionine was observed in grass pea ration II. The

feed intake tolerance for unprocessed grass pea was enhanced with increasing methionine

in the diet and with age. Despite a similarity in the initial intake, a significant (p≤0.05)

difference in the final feed intake level by the chicks with methionine addition compared

to the controls was found in both rations. The total weight gain per head improved by

methionine addition for up to 26.3 % and 65.5 % in ration I and II respectively.

Reversible convulsions developed in some chicks within five days after feeding, the

highest incidence was found in ration II (94 % of the total number of cases) with most


64

cases (89 %) in the controls (without addition of methionine) and in chicks on feed with

0.05 % methionine addition. These results suggest that methionine can improve a grass

pea-based diet for the growth of broiler chicks and also can protect young chicks from

neurological symptoms. This drought tolerant, cheap and protein-rich grass pea when

supplemented with optimal methionine can at least partially replace soybean in animal

feed during food crises.

IV.1. Introduction

Dependent consumption of grass pea (L. sativus L.) for more than two months can give

rise to the crippling neurolathyrism in humans, giving the stigma of toxicity to this crop.

Toxicity is blamed on the presence of a non-protein amino acid, β-N-oxalyl-L-α,β-

diaminopropionic acid (ß-ODAP), that acts as a potent agonist of the AMPA subclass of

neuronal glutamate receptors (Spencer et al., 1986). Like all legumes, grass pea is

deficient in the essential sulphur amino acids methionine and cysteine, a deficiency that

might play an important role in its toxicity (Nunn et al., 2005).

Neurological signs can be experimentally induced in animals. In young chicks these signs

are acute and reversible and do not necessarily reflect the aetiology of human

neurolathyrism. Young chicks show specific but reversible signs within one to five days

after grass pea consumption or injection of ß-ODAP; while for primates on a balanced

diet, much higher doses of ß-ODAP are needed for a longer period to develop central

motor neuron deficiency and hind limb spasticity (Spencer et al., 1986). Rao (2001)

demonstrated that there exist extreme inter-species and intra-species differences in

susceptibility to ß-ODAP that may be ascribed to individual metabolic differences.

Currently, both cellular (Kusama-Eguchi et al., 2003) and animal models (Kusama-

Eguchi et al., 2005) for human neurolathyrism are being developed.

Several authors suggested a role for oxidative stress in the aetiology of neurolathyrism

(Lambein et al., 2001; Nunn et al., 2005; Getahun et al., 2005). Depletion of methionine

and cysteine in the bloodstream and in the central nervous system (CNS) can jeopardize


65

the defence against oxidative stress and increase the risk of neurolathyrism. A low level

of methionine in urine of lathyrism patients consuming grass pea had been reported

(Rudra and Chaudhury, 1952) even before the toxic compound was identified in grass

pea. Consumption of grass pea by mixing with at least one third of cereals (richer in

sulphur amino acid than legumes) in the diet was identified as a protective factor for

neurolathyrism (Getahun et al., 2005).

Based on a study with human volunteers, Nunn et al. (2005) found that consumption of

grass pea seed resulted in a significant and persistent decrease in plasma methionine. The

normal physiological response to consuming a methionine-deficient meal is suggested to

be reduced plasma methionine concentration. Methionine-deficient grass peas and lentils

were seen to reduce plasma methionine by about 50 % persistently for 24 hours. High

methionine concentrations are found in rat motor neurons, which suggests a high

requirement for methionine by these cells, and perhaps an increased sensitivity to

methionine deficiency. A reduction in plasma methionine concentration could thus cause

a reduced methionine flux into the CNS. As a consequence of prolonged consumption of

grass pea seed as a staple, neuronal cells may be sensitised to the additional oxidative

stress produced by over-excitation of the motor neurons by ß-ODAP and the ensuing

production of nitric oxide (NO) and reactive oxygen species (ROS). Nunn and co-

workers (2005) proposed that supplementing such a diet with methionine might protect

those at risk from neurolathyrism.

The present study was initiated with the hypothesis that methionine plays aginst lathyrism

and lathyrism symptom development in chicks with possible implications in humans. The

objective of the present study was to assess the effect of methionine on grass pea toxicity

by studying neurolathyritic symptoms and general performance of broiler chicks fed with

rations containing grass pea.

IV.2. Materials and Methods The study area The experiments were conducted in the Poultry Research Department at

Debre Zeit Agricultural Research Centre (DZARC), Ethiopia.


66

Experimental animals and their management Four hundred fifty mixed sex one-day-

old broiler chicks of similar body weight were randomely divided into sets of 225 chicks.

These were subdivided into five groups of 45 receiving the control diet or the four

different levels of methionine addition to the diet. Each group was further randomly sub-

divided into three replicates of 15 chicks and placed in experimental pens at a density of

10 chicks/m2. Chicks on various rations were randomly assigned to pens. Birds were

provided daily with a known amount of feed early in the morning, refusals were weighed

by the end of the day, and the difference calculated as the daily intake value per sub-

group of 15 chicks. Water was provided ad libitum. Routine vaccination and health care

was given as necessary. Chicks’ body weight measurements were taken + 0.1 g for each

replicate group as a whole starting at the beginning of the trial and every week thereafter.

Symptom-developing individual chicks in a replication were marked with different

colours for further monitoring and characterization. No mortality occurred during the

experiment.

Feeding rations Broiler starter rations with two levels of raw grass pea (Debre Zeit local

cultivar containing 0.35 % β-ODAP) as diet were formulated. These served as control

rations. Treatment rations were formulated by adding different levels of DL-methionine

(≥99 %, feed grade) produced by Sumitomo Chemical Company, Tokyo, Japan. Raw

grass pea seeds were coarsely ground with an electric mill and homogeneously mixed

with the other ingredients yielding rations I and II, respectively. The composition of the

two rations is detailed below.

Ration I = 48 % maize, 35 % grass pea, 15.5 % noug cake (protein supplement widely

available in Ethiopia. It is the residue after extraction of oil from niger seed or Guizotia

abyssinica, Asteraceae), 1 % vitamin-mineral premix (commercial premix containing per

kg: 625000 IU vitamin A, 125000 IU vitamin D3, 1250 IU vitamin E, 1500 mg Fe, 1250

mg Cu, 3000 mg Zn, 3750 mg Mn, 15 mg Co and 10 mg Se), 0.5 % NaCl. Four levels of

methionine (as % of feed) were used: 0.025, 0.05, 0.075 and 0.10.

Ration II = 98.5 % grass pea, 1 % vitamin-mineral premix (as in ration I), and 0.5 %


67

NaCl. Four levels of methionine (as % of feed) were used: 0.05, 0.1, 0.15, and 0.2.

Chemical analysis Samples of the two feed rations were analysed for proximate

principles based on the standard procedures of AOAC (Williams, 1984) at the

Department of Analytical and Physical Chemistry, Faculty of Bioscience Engineering,

Ghent University. Dry matter (DM) and ash determination were done using the

procedures described in Williams (1984) and De Vleeshauwer et al. (1948). Protein and

fat content were determined using the Kjeldahl method as described in Egan et al. (1981)

and the Weibull method (Pearson’s, 1981) respectively. Carbohydrate values were

derived by differences whereas the energy values (mega joule/ kg) were calculated using

the physiological fuel values of carbohydrate, protein and fat (Table IV.1).

Concentrations (% seed weight) of β-ODAP in the feeding mixtures (ration I and II) were

analysed by HPLC with pre-column PITC derivatisation as described in the previous

chapter.

Table IV.1. Feed component analysis of a starter treatment ration for broiler chicks

% DM % Ash % Protein % Fat Energy (MJ/Kg)

% ß-ODAP

Ration I 89.63 ± 0.35 4.18± 0.02 16.71 ± 0.27 3.16 ± 0.78 16.6 ± 0.016 0.134 + 0.10 Ration II 89.32 ± 0.22 3.91± 0.38 22.81 ± 0.27 1.08 ± 0.46 16.3 ± 0.003 0.293+ 0.19

Statistical analysis

Experiment of the two rations was laid down in a Completely Randomised Design (CRD)

with three replications of 15 chicks each under the four treatment levels and control.

Variance between treatments was analysed using the General Linear Model (GLM)

procedure of the Statistical Analysis System (SAS, 2001). Duncan multiple range test

(Duncan, 1955) was applied to indicate when significant differences between treatments

means exist.

IV.3. Results With increasing methionine in the feed, the chicks’ performance, expressed as weight

gain, feed intake, feed conversion efficiency, showed improvement in both rations. The


68

overall performance of chicks in ration I were better than chicks in ration II. For ration I

(35 % grass pea) feed intake was significantly (p<0.05) higher when methionine was

added compared to control (Table IV. 2). However, there were no significant differences

between the intakes of various levels (0.025 to 0.10) of methionine addition. Significant

differences (p<0.05) were also found for weekly weight gain, total weight gain, final

body weight and feed conversion efficiency (FCE) at a methionine level of 0.075 %

compared to the control.

Table IV.2. Effect of methionine addition on different parameters of chicks in broiler starter ration I (35 % grass pea) with standard error (SE).

Methionine concentration added (% of feed) Parameter Control 0.025 0.05 0.075 0.10

SE

Mean feed intake (g/bird/day) 25.38a 27.09b 27.04b 27.80b 28.25b 0.488

Initial body wt. (g/bird) 41.88a 39.05a 39.29a 40.12a 39.34a 1.23

Final body wt. (g/bird) 245.13a 268.13b 256.77b 296.83c 263.97b 10.57

Mean weight gain (g/bird/week) 50.81a 57.27a 54.37a 64.18b 56.16a 0.521 Total weight gain/bird (g) 203.25a 229.08b 217.48ab 256.71c 224.63b 3.224 FCE (Feed: gain) 3.496a 3.311a 3.481a 3.032b 3.521a 0.012 *Mean values followed by different letters are significantly different (P<0.05) from each other; SE-standard error

Table IV.3. Effect of methionine addition on different parameters of chicks in broiler starter ration II (98.5 % grass pea) with standard error (SE).

Methionine concentration added (% of feed) Parameter Control 0.05 0.1 0.15 0.2

SE

Mean feed intake (g/bird/day) 14.33a 16.52b 16.60b 16.35b 16.50b 2.925 Initial body wt. (g/bird) 39.83a 39.44a 40.56a 39.81a 40.18a 0.251 Final body wt. (g/bird) 95.62a 128.04b 126.58b 132.15b 127.32b 5.288 Mean weight gain (g/bird/week) 13.95a 22.15b 21.51b 23.09b 21.78b 0.212 Total weight gain/bird (g) 55.79a 88.60b 86.02b 92.34b 87.14b 7.23 FCE (Feed: Gain) 7.191a 5.221ab 5.403 ab 4.958b 5.302ab 2.252 *Mean values followed by different letters are significantly different (P<0.05) from each other; SE-standard error

In the second ration with chicks receiving 98.5 % grass pea, overall under-performance of

birds compared to those on ration I was obvious. However, positive responses due to

methionine addition were found. There was a significant increase in feed intake by birds

on methionine-added feed compared to those fed on the control diet, but we found no

significant difference among various methionine levels. A significant difference with

controls (p<0.05) was found for final body weights, weight gains and feed conversion

efficiency (Table IV.3). In this experiment with ration II, most parameters showed


69

maximum values at methionine levels of 0.15 % of the feed with a declining trend with

further increasing methionine addition. Total weight gain per bird from 55.79 g to 92.34 g

or an increase of 65 % in this exclusive grass pea ration II proves the importance of

methionine addition. In general, feed conversion efficiency, as expressed by g weight

gain per g consumed feed, of this group was low compared to the chicks on ration I.

However, it was improved with increasing methionine addition. Overall under-

performance of chicks on ration II as compared to that obtained in the birds on ration I

could be the consequence of higher level of other antinutritional factors (ANFs) that are

present in grass pea (Urga et al., 2005) as compared to the higher cereals ratio (48 %

maize) in ration I. Improved feed conversion efficiency resulted in a considerably higher

weight gain per chick yielding a higher total body weight at the end of the four weeks

feeding experiment.

Daily feed intake (difference between offered and refusal) by the chicks showed a drop

during the first three days for the high grass pea feed (ration II) followed by immediate

recovery (Figure IV.1). In both rations, feed intake gradually increased during the

experimental period to reach a maximum by the end of the experiment. An overall low

feed intake in ration II could be the effect of the high amount of antinutritional factors,

which also resulted in poor feed conversion efficiency. Weekly weight gain was

substantially higher in the first ration compared to ration II (that showed poorer FCE)

irrespective of similarity in the daily intake trend of both rations. This weight gain

showed a significant increase with time advance (P<0.05), except for ration II of the last

week that showed a decrease. We can conclude that feed intake increased with age of the

chicks, and was also positively affected by increasing methionine addition in both rations.


70

Figure IV.1. Trend in evolution of daily food intake (g/head) I = mean intake in control and in methionine-added feed (mean value of 0.025-0.1 % treatments) of ration I; II = mean intake in control and in methionine-added feed (mean value of 0.05-0.2 % treatments) of ration II, [■ control, ▲ methionine-added]

Bars of a group with the same letters are not significantly different (P<0.05)

Figure IV.2. Weight gain (g/head/week) during the feeding period: I = mean weight gain in control and in methionine-added feed (mean value of 0.025-0.1 % treatments) of ration I; II = mean weight gain in control and in methionine-added feed (mean value of 0.05-0.2 % treatments) of ration II; [W-week]

Methionine addition also maximized total weight gain/head over the control by 26.3 %

and 65.5 % (Tables IV. 2 & IV. 3) in rations I and II, respectively. From the feed intake

level, we calculated that intake of ß-ODAP per head increased starting from the

methionine addition of 0.025 % and 0.05 %, respectively in the two rations. Difference

I

0

5

10

15

20

25

30

1 4 7 10 13 16 19 22 25 28days

daily

me

an

inta

ke

(g/h

d)

control methionine added

0

5

10

15

20

25

30

1 4 7 10 13 16 19 22 25 28days

daily

me

an

inta

ke

(g/h

d)

control methionine added II

0

10

20

30

40

50

60

70

80

W1 W2 W3 W4weeks

we

ight

ga

in (

g/hd

/we

ek)

Control Methionine addedII

aabb

c

a ab

c

0

10

20

30

40

50

60

70

80

W1 W2 W3 W4weeks

we

ight

ga

in (

g/hd

/we

ek)

Control Methionine addedIa a

b

c

a

b

c

d


71

between control and treatments with added methionine in ration I was not as big as in

ration II (Figure IV.3) for calculated ß-ODAP intake of the final phase. This may be due

to the better methionine content in ration I with the presence of cereals (48% maize)

compared to ration II. Clear intake difference in ration II was exhibited with advancing

age of the chicks.

*, ** Significant at 0.05 and 0.01 probability levels, respectively

Figure IV.3. Initial (mean of the first three days) and final (mean of the 4th week) level of ß-ODAP intake by chicks during the 30 days feeding period in the two rations with methionine addition (Top: ration I; bottom: ration II).

Despite a similarity in initial intake of ß-ODAP, a significant (p<0.05) difference in final

intake level of the chicks on feed with methionine addition compared to control, 12.8 %

in ration I and 37.9% in ration II, was found in both rations (Figure IV.3). The highest

methionine levels in ration I and all the levels in ration II showed significant differences

from control (no methionine addition). However, in ration II, the intake of feed with 0.2

% methionine addition was also significantly lower than that of lower methionine

addition levels and intake differences became more pronounced with time at the end of

the four weeks feeding period (Figure IV. 3). In ration II where differences were

0

2

4

6

8

1 0

1 2

1 4

1 6

0 0 .05 0 .1 0 .1 5 0 .2

m et h io n ine addit io n (% feed)

OD

AP

inta

ke (

mg

/da

y/h

d)

II

*

******

0

2

4

6

8

1 0

1 2

1 4

0 0 .0 25 0 .05 0 .07 5 0 .1

methionine addit ion (% feed)

OD

AP

inta

ke (

mg

/da

y/h

d)

in it ial ODAP in t ak e

f inal ODAP in t ake

I

*


72

apparent, the intake of ß-ODAP (concentration) expressed per g body weight increased

approximately five fold i.e. from 1 mg/40 g in the first week to 12 mg/90 g (i.e. 0.025 to

0.13) in the last week (Figure IV. 3). This result is in line with several previous reports on

chick experiments that indicated that there appeared to be some adaptation to the

antinutritional factors in grass pea and also increased toxicity tolerance with increased

age (Low et al., 1990; Tadelle et al., 2003; Yan et al., 2006). Addition of methionine

significantly improved ß-ODAP intake or tolerance of the chicks on ration II (Figure

IV.2).

Figure IV.4. Torticolis or neck twist (right or left), stargazing and ‘head downing’ were the most common symptoms developed by broiler chicks in the first two weeks of grass pea (98.5 %) feeding. Higher incidence of reversible convulsions was observed in chickens on ration II

accounting for 94 % of the total number of cases and only 6 % from chickens on ration I.

However, the symptoms decreased from 84 % to 2% with the increased addition of

methionine of ration II (Table IV.4). The severity of the convulsion varied (mild or

strong) among chickens within and between treatment levels. Most chicks started

showing symptoms within five days after feeding on grass pea, and these symptoms were

reversible within ten days. Throughout the entire experimental period 2 % of the chicks

on ration I and 31 % of the chicks on ration II developed symptoms. Most birds were

affected in the first two weeks and all symptoms disappeared after three weeks (Table IV.

4). The majority of cases (88 %) were found in chicks with control and with the lowest

methionine level (0.0-0.05 %) treatments during the first two weeks. Of the symptoms,

62.7 % showed right torticolis (head tilted to right side), 19.7 % had left torticolis or twist

(neck tilted to left side), 14 % had stargazing (head tilted upward) (Figure IV. 4) whereas


73

in less than 4 % of cases the head was tilted down with leg crippling.

Table IV.4. Number of visibly affected chicks that developed lathyritic symptoms (with percentage values in parenthesis) receiving diets with the indicated content of % methionine in the feed, during the four weeks feeding period (W: week)

IV.4. Discussion

The physiological disorder due to grass pea intake may be the result of the combined

effects of ß-ODAP and other antinutritional factors (ANFs) found in grass pea (Urga et

al., 2005). According to Urga et al. (2005), ANFs of Ethiopian grass pea seed collected

from 15 growing locations: ß-ODAP ranged from 0.518 % to 1.0 % of seed weight,

tannins from 558 to 766 mg/100g, trypsin inhibitor activity (TIA) from 15.53 to 18.99

TIU/mg and phytic acid from 547.27 to 1008.57 mg/100 g. Hence, overall under

performance in ration II as compared to the birds on ration I could be the consequence of

the higher level of additional antinutritional factors (ANFs) that are present in extremely

high grass pea proportion of ration II as compared to the higher cereals ratio (48 %

maize) in ration I. The effect of ANFs in L. cicera and L. sativus on animal performance

is not well understood and sometimes confounded with β-ODAP effects (Hanbury et al.,

2000b). Heating of grass pea seed will reduce levels of the proteinaceous ANFs and in

some cases of β-ODAP as well. Latif et al. (1976) reported reduced growth of broiler

chicks (14 day-old) by feeding diets containing 36.7% (367 g/kg) of grass pea; but when

autoclaved or heated grass pea was given at the same level in the diet this did not inhibit

Methionine 0 0.025 0.05 0.075 0.10W1 1 (2.2) 2 (4.4) 1 (2.2) 0 (0) 0 (0) W2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) W3 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Rat

ion

I

W4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) total 1 (2.2) 2 (4.4) 1 (2.2) 0 (0) 0 (0)

Methionine 0 0.05 0.10 0.15 0.20W1 22 (48.9) 16 (35.6) 2 (4.4) 1 (2.2) 1 (2.2) W2 15 (33.3) 8 (17.8) 3 (6.7) 0 (0) 1 (2.2) W3 1 (2.2) 1 (2.2) 0 (0) 0 (0) 0 (0)

Rat

ion

II

W4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) total 38 (84.4) 25 (55.6) 5 (11.1) 1 (2.2) 2 (4.4)


74

growth whereas showed the same result of a maize-soya diet. Tadelle et al. (2003) fed

broiler starters (0-29 days) with grass pea (at 35 % of the diet) heated in water at 60 oC,

75 oC, 90 oC and boiling temperature respectively, and found better growth performance

of the young chicks after treating the grass pea at increasing temperatures. Boiled grass

pea diet showed no significant difference (p< 0.05) with the diet that composed of

maize/wheat/noug cake. Low et al. (1990) reported that with 800 g/kg grass pea (80 % of

the feed) in the diet for growing chicks over a four week period, chicks’ performance

(feed intake, weight gain) was significantly poorer than that of wheat/soybean meal-based

diet (p<0.05) while it was markedly improved by dietary methionine supplementation at

0.25 % of the feed (p <0.05). Smulikowska et al. (2008) included grass pea var. Krab as

10 % of the feed for broiler chicken (age 8-40 days) and were able to show that growth

performance of the chicks did not differ from the controls that did not receive grass pea in

the diet. In this Polish experiment, birds fed with var. Krab seed had enlarged pancreases

(p< 0.05) and livers in comparison to controls. Rotter et al. (1991) evaluated low (ß-

ODAP 0.13 % of seed weight) and medium (0.22 % ß-ODAP) toxin lines of grass pea in

the diet to feed chicks and concluded that with 40 % grass pea (low or medium ODAP

lines) in the diet, the weight gain showed no significant difference (p<0.05) with the

chicks fed on a wheat-based diet. Hanbury et al. (2000b) observed a 40 % grass pea (0.27

% ß-ODAP) feed tolerance of chicks with insignificant weight reduction. Chowdhury et

al. (2005) studied the nutritional value of grass pea (0.4 % ß-ODAP) for growing and

laying pullets and concluded that inclusion of 10, 15 and 20% of grass pea in the feed,

supported growth performance in pullets at the same level or better than the control feed

(without grass pea). However, for egg-layers, grass pea in the feed was well tolerated

only up to 15 % grass pea without deleterious effects on egg production. A summary of

published feeding trials is presented in Table IV.5. Our study in ration II is the first report

for replacing grass pea as the only energy source (98.5 %) in the chick diet.


75

Table IV.5. A brief summary of earlier reports on chicks feeding experiments based on grass pea (Lathyrus sativus) diet Reference Test method Result and recommendation remarks

1 Latif et al. (1976)

Feeding broiler chicks with raw grass pea seeds at 100, 222, 367 g/kg meal for 14 days

-Birds grow slowly -When seed was autoclaved/heated, chicks fed 367 g/kg diet showed equal performance to a maize-soybean diet group

2 Low et al. (1990)

Feeding growing chicks with 800 g/kg grass pea during 4 weeks

-Poor performance compared to control -Methionine addition improved performance -Some adaptation to antinutritional factors observed

3 Rotter et al. (1991)

Comparative feeding low ODAP (1.3 g/kg seed) and medium ODAP (2.2 g/kg seed) grass pea lines

-No different effects between the two lines -Up to at least 400 g/kg diet can be used -Pre adaptation or dehulling had no effect on chicks

4 Chen et al. (1992)

Intraperitonial injection of 700 mg β-ODAP/kg bodyweight

-Lethal for 3 days old chicks

5 Hanbury et al. (2000b)

L. sativus/L. cicera inclusion in normal diet of poultry

-Inclusion of 40 % L. sativus or L.cicera in poultry diet was found safe and without growth reduction

6 Tadelle et al. (2003)

Heat treatment of high toxin grass pea seed for protein replacement in broilers diet

-Cooking grass pea at 90 oC and replacing it for protein source showed similar feed efficiency as commercial feed product and found economical -Treatment below this temperature found detrimental

7 Chowdhury et al. (2005)

Feeding raw grass pea seeds at rates 0, 100, 150 & 200 g/kg feed for 12-22 weeks age brown pullets

-Up to 150 g/kg dietary level can be used with even better performance of animals than the control

8 Yan et al. (2006)

Poultry grass pea feeding (review)

-Poultry tolerates up to 40 % of their diet with L. sativus of moderate toxicity

9 Smulikowska et al. (2008)

Comparative feeding of grass pea mutants and parental line for chicks to see effect

-Bone deformation (lathyrism indicator) shown on birds fed parental line than the mutant -Birds have lower pancreas and liver weight than those fed on the parental line

From the present grass pea feeding trial we can make some interesting suggestions and

conclusions with economic and socio-medical consequences. Apparently, in diets

containing mainly grass pea, the effect of increasing methionine appears to be significant

on a number of variables such as weight gain, feed conversion efficiency and feed intake.

In spite of the general under-performance for some parameters, the chicks essentially

showed positive improvement compared to the control with addition of methionine.

However, a diet of exclusively unprocessed grass pea seed gave substantially reduced

weight gain compared to the ration containing 35 % grass pea. This reduced feed

conversion efficiency suggests a difference in efficiency of protein utilization by birds

consuming different rations. We found higher effects of 0.075 % methionine addition in

the feed containing 35 % grass pea and around 0.15 % methionine addition in feed


76

containing grass pea as an exclusive ( 98.5 % of the feed) protein and energy source. In a

similar way, Molvig and co-workers (1997) mentioned that when a diet with lupine, that

contains ANFs, as major protein source was supplemented with methionine, significant

increases were found in growth of pigs and in efficiency of protein utilization in rats.

Because non-ruminant animals can convert methionine to cysteine, added methionine can

satisfy their total requirement for dietary sulphur containing amino acids. Al-Mayah

(2006) concluded that better immune response could be obtained with adequate

supplementation of methionine at 1 g/L in drinking water or as 0.1 % in the feed for

broiler chicks. This confirms that the compensatory action of methionine supplements for

sulphur amino acid-limited diets such as grass pea can maximize the safe utilization of

this under-utilised but protein rich legume crop (Lambein et al., 2001; Getahun et al.,

2005; Nunn et al., 2005).

The results from the two experiments with different rations containing respectively 35 %

and 98.5 % of grass pea suggest the need for optimising methionine content of feed

formulations for optimal feed conversion efficiency and economical weight gain. Result

also suggests that health feed formulations could be considered using the cheap grass pea

as ingredient.

An important observation was the increase in intake tolerance for unprocessed grass pea

containing ß-ODAP and other ANFs, with increasing methionine in the diet. Our

experiment shows that performance of animals on the exclusive Ethiopian grass pea diet

was improved substantially in food intake and in weight gain by addition of the essential

amino acid methionine. Higher intake of the high toxin grass pea with some

antinutritional factors present in raw seed could be attributed to increase in age (Low et

al., 1990; Chen et al., 1992; Tadelle et al., 2003; Yan et al., 2006) and the addition of

methionine. The added methionine might have suppressed the toxic effects of grass pea

feed. In ration II, methionine at level 0.1 % and above kept the animals safe from chronic

illness and convulsions (Table IV.4). Our results suggests that (1) methionine is an

effective antidote against grass pea toxicity; and (2) its optimisation would improve

chicks performances and thus increase economic gain of farmers.


77

We can estimate that chicks in ration II, with highest incidence of neurological

symptoms, were using an average of about 600 mg ß-ODAP per kilogram body weight

per day. The ß-ODAP concentration in the blood could be hypothetically considered to be

the amount that would be absorbed by the body to bring about the neurological problem.

Chen and co-workers (1992) reported that intraperitoneal injection of ~700 mg/ kg of

body weights can be lethal for 3-day old chicks.

Our result is in line with earlier suggestions that methionine-rich diet reduces the toxic

action of ß-ODAP ( Getahun et al., 2003, 2005). In agreement with previous work (Mehta

et al., 1979; Chen et al., 1992; Tadelle et al., 2003) also in this study acute neurotoxicity

was more frequent in younger chicks and decreased as age increased. The older the chick,

the less susceptible it is per unit body weight to the toxic amino acids and ANFs in L.

sativus seeds. The common acute convulsive manifestation in chicks was the deformation

or bending of the neck muscle unlike the symptoms seen in human neurolathyrism.

Similar symptoms of head moving downwards or bending, and stiffening of the neck has

been reported in one-day-old chicks by either giving oral doses of β-ODAP (0.1 mmol)

(Shamim et al., 2002) or by intraperitoneal injections of 0.2 to 0.6 ml of L. sativus extract

(5 gm dry seed/ml) per chick (Roy et al., 1963). A direct comparison of toxicity and

protection between the acute reversible signs in chicks and human neurolathyrism

produced by chronic intoxication by grass pea over-consumption (or chronic depletion of

methionine) may not be possible. But the general trend of methionine as protectant

against the toxin is likely to be valid also in humans. Consumption of methionine-rich

foods as protection against the neuro-excitatory action of ß-ODAP and against the

oxidative stress produced by its biochemical and physiological activities has been

proposed before (Lambein et al., 2001; Kusama-Eguchi et al., 2003; Getahun et al., 2005;

Nunn et al., 2005) and is supported by our findings.


78


79

CHAPTER V

Effect of methionine supplement on serum amino acids status

and trace elements level of broiler chicks fed with grass pea

based starter ration


80

Healthy chick after feeding on grass pea diet


81

CHAPTER V

Effect of methionine supplement on serum amino acids status and trace

elements level of broiler chicks fed with grass pea based starter diet

Abstract

Starter feeding experiments with broiler chicks using grass pea (Lathyrus sativus L.)

supplemented with methionine were undertaken for 4 weeks during 2005 at DZARC,

Ethiopia to (1) assess toxicity by analysing their serum and to (2) assess the evolution of

selected trace elements in serum. The objective was to study the effect of increasing

methionine supplements on grass pea toxicity. Four hundred fifty broiler chicks were

divided into two experimental sets or rations formulations containing (a) 35 % (ration I)

or (b) 98.5 % (ration II) raw grass pea. Each ration include control and four treatments

with methionine (0.025, 0.05, 0.075 and 0.1 % of the feed in ration I; and 0.05, 0.1, 0.15

and 0.2 % in ration II). Blood serum of the chicks of different treatments was collected

each week and analysed for its free amino acids and trace elements content. With addition

of methionine in the feed, some amino acids in the serum showed an increase while

others showed a decrease compared to control (without methionine supplement). ß-

ODAP level in chick serum decreased progressively with increasing methionine

supplement in the feed of ration I. The levels of β-ODAP, homoarginine (Har), arginine

and lysine were much higher in chick serum of ration II than that of ration I due to the

high proportion of grass pea in feed ration II. Trace elements including iron, copper, zinc

and manganese showed different evolution with increasing methionine supplement and

with increasing age. The content of Cu2+ and Fe2+ increased more than double while Zn2+

and Mn2+ showed no difference with control at the end of three weeks. These preliminary

results suggest that methionine supplement in the feed influenced chick’s amino acids

metabolism and trace elements level and this can be important as protective element in a

grass pea based diet.


82

V.1. Introduction

In Ethiopia, one of the important purposes of grass pea (Lathyrus sativus L) cultivation is

its use for animal feed (Fikre, 2007; Dadi et al., 2003). Grass pea is a cheap source of

protein and smallholder farmers usually use feed rations containing grass pea for

fattening animals.

Neurological symptoms can develop particularly in monogastric animals (Yan et al.,

2006) after prolonged consumption of grass pea. This toxicity is blamed on the presence

of a non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid or ß-ODAP

(Spencer et al., 1986). However, the acute and reversible neurological signs in young

chicks is totally different from the aetiology of human neurolathyrism commonly

manifested by irreversible symptoms of spastic paraparesis in legs. After consumption of

grass pea or injection of ß-ODAP, young chicks show specific but reversible neurological

signs such as torticolis (bending of the neck sidewards) or star gazing (bending of the

neck backward).

For primates on a balanced diet, massive doses of ß-ODAP are needed during an

extended period to develop central motor neuron and hind limb deficiency (Spencer et al.,

1986). Rao (2001) indicated that there exist extreme inter-species and intra-species

differences in susceptibility to ß-ODAP that may be ascribed to metabolic differences,

and he reported that urine samples collected from humans following a Lathyrus meal

showed very little urinary excretion of ß-ODAP (less than 2-5% of what is injested). Kuo

and co-workers (2007) demonstrated that the free amino acids aspartic acid, glycine, β-

aminoisobutyric acid, arginine, α-aminoadipic acid and phenylalanine were significantly

higher (p<0.05) in urine from patients with neurolathyrism than in urine from healthy

controls while β-ODAP was not detected because the consumption of grass pea had been

discontinued at the time of sampling. Nunn and co-workers (2005) found that

consumption of grass pea seed resulted in a significant and persistent decrease in plasma

methionine by about 50 %. A reduction in plasma methionine concentration would cause


83

a reduced methionine flux into the central nervous system. As a consequence, cells may

be sensitised to additional oxidative stresses. This oxidative stress and probably the

neuro-toxicity is aggravated by the inherent deficiency of essential sulphur amino acids

methionine and cysteine in grass pea (Nunn et al., 2005). Al-Mayah (2006) reported that

in soybean based feed supplementation of DL-methionine at 1 g/L of drinking water or at

1 g/kg of chick’s feed results in increment in a number of blood parameters: red blood

cell counts, mean cell volume, total serum and liver protein, albumin and globulin. He

suggested that better immune response of chicks could be obtained with adequate

supplementation of methionine in the poultry feed.

The analyses in this study have been conducted with the view that the physical responses

of animals on grass pea rations, low in sulphur amino acids and containing the neuro-

excitatory ß-ODAP, might reflect changes in the serum amino acids and trace elements

comparable to humans on a grass pea diet. Addition of methionine to grass pea rations

may have a modulating effect on these concentration changes and on other physiological

parameters related to redox homeostasis. Hence, the objective of this analytical part of the

feeding study with grass pea was to investigate and to compare the amino acids, β-ODAP

and relevant trace elements levels in the serum of chicks during the first three weeks

period.

V.2. Materials and Methods The study area, the starter broiler chicks, their management and two feeding rations used

(with 35 % and 98.5 % grass pea respectivelly) were as mentioned in Chapter IV.

Serum sampling: a specialized veterinarian collected serum from the wing vein using a 5

cc disposable syringe. This was done for each replication of the treatments on a weekly

basis. However, in the first week, at most four chicks per replication were sacrificed to

get enough volume of blood serum for the analysis. Samples were immediately

centrifuged (5000 rpm) for five minutes and the plasma supernatants was decanted in

standard tubes for freezing until the lab analyses for amino acids and trace elements.


84

Trace elements analysis: Trace elements were sequentially extracted by nitric acid

(HNO3) and hydrogen peroxide (H2O2) (Quevauviller et al.,1996). Cu2+ and Mn2+ were

determined with Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS)

while Fe2+ and Zn2+ were determined with Inductively Coupled Plasma mass

spectrometry (ICP). These analyses were performed by the Department of Analytical and

Physical Chemistry, Faculty of Bioscience Engineering, Ghent University.

Free amino acid analysis:

Serum samples: 100 µl of serum was mixed with 300 µl of 95 % ethanol. In each

sample, 5 µl of DL-allylglycine (100 µmol/ml, Sigma) was added as internal standard.

The mixture was allowed to stand at 4 °C overnight before centrifugation at 12,000 rpm

for 10 min in an Eppendorf centrifuge (Hawksley MBC). The supernatant was

concentrated in an Eppendorf Vacufuge concentrator 5301 (Germany) at 45 °C under

vacuum. 100 µl ion-free water was added to the concentrate, stirred, centrifuged and the

supernatant collected and dried for PITC derivatisation.

PITC (Phenyl isothiocyanate) derivatisation: in each dried sample, 20 µl of coupling

buffer (methanol/water/triethylamine: 2/2/1 by volume) was added, mixed and dried in

the concentrator. Finally, 30 µl of the PITC reagent (methanol/water/triethylamine/phenyl

isothiocyanate: 7/1/1/1 by volume) was added and reacted at room temperature for 20

min before concentrating to dryness.

To each PITC-derivatised sample, 500 µl of buffer A (0.1 M ammonium acetate, pH 6.5)

was added, mixed well and centrifuged. The supernatant was filtered through a Millipore

Millex filter (0.45 µm) and 20 µl aliquot was injected in the HPLC for analysis. A

standard amino acid mixture (AA-S-18, Sigma), L-homoarginine (99+ %, Janssen

Chimica) and synthetic ß-ODAP (a gift from Dr. Rao S.L.N, India) were also derivatised

and prepared as above and injected in HPLC as standard.

High-performance liquid chromatography (HPLC) for amino acid analysis: a Waters

625 LC system with Waters 991 photodiode array detector was used as previously


85

reported (Kuo et al., 2003). A gradient system with buffer A (0.1 M NH4OAc, pH 6.5)

and buffer B (0.1 M NH4OAc, containing acetonitrile and MeOH; 44/46/10 by volume,

pH 6.5) with flow rate of 1 ml/min was used for the separation of amino acids during 50

min. An Alltima C18 column (250x4.6 mm I.D., 5 µm particle size, Alltech, USA) was

used with column temperature at 43 °C during analysis. The absorbance at 254 nm was

used for calculations. Results were analysed by Millennium software (Waters, version

1.10).

Statistical analysis

Amino acids detected in chick serum of the first week for ration I and the three

consecutive weeks for ration II were analysed with ANOVA using SPSS 12 software.

Statistical significance of differences among means was determined using Tukey

Honestly Significant Difference set at 95 % confidence level. Amino acid concentrations

(µmol/100 ml) in serum were presented with their respective standard deviation.

Pearson’s correlation coefficients were calculated for micronutrients, β-ODAP, body

weight and FCE.

V.3. Results Free amino acids in the serum

The free amino acid profiles of chicken serum fed with grass pea ration I (1st week,

samples of second and third weeks could not be analysed) and II (1st, 2nd and 3rd week)

are given in Tables V.1 and V.2 respectively. A number of amino acids showed increase

or decrease along with increasing methionine levels. In ration I, β-ODAP, glutamic acid

(Glu) and homoarginine (Har) showed a decreasing trend with methionine supplements

while arginine (Arg), tyrosine (Tyr) and leucine (Leu) increased compared to the control

without methionine addition (Table V.1). Interestingly, with the increase in methionine

concentration in serum, the corresponding ß-ODAP level in the serum decreased

progressively in the ration I experiment.


86

Table V.1: Concentrations of free amino acids (µmol/100 ml) in chick serum (mean ± SD, average of 1st week, N = 4) fed with 35 % grass pea (ration I) with different levels of DL-methionine addition (0.025, 0.050, 0.075 and 0.100 % of feed)

0.0 0.025 0.05 0.075 0.1

Asp 10.4±5.8b 16.5±5.0a 10.8±2.8b 13.9±6.6ab 16.8±4.7a

ß-ODAP 16.5±7.3a 8.4±7.7b 7.2±6.2b 7.9±6.8b 4.9±3.0b

Glu 42.8±21.4a 32.3±7.2b 27.5±9.1c 30.0±9.8bc 31.4±5.9bc

α-AAA 8.8±5.4c 7.7±3.1c 14.0±7.1b 13.6±11.2b 22.9±12.8a

Ser 76.2±43b 76.4±29.8b 57.9±18.1c 56.1±25.0c 97.2±44.4a

Gly 75.7±15.9a 62.0±16.9bc 72.0±32.1a 61.6±23.2c 66.5±32.9b

Gln 65.3±46.6b 103.8±29.0a 54.3±25.1c 70.8±35.0b 59.8±29.3c

His 9.5±3.7a 10.1±2.8a 9.5±4.6a 9.2±1.4a 12.2±5.6a

Tau 33.2±17b 25.6±5.6c 27.9±15.6c 33.7±13.6b 70.0±31.8a

Arg 15.4±10.4c 38.7±25.5a 25.6±8.5b 23.7±12.5b 40.7±23.6a

Thr 46.2±35.6a 33.8±18.9b 41.4±11.7a 23.1±19.1c 25.7±16.6c

Ala 55.2±32.6c 55.2±29.8c 54.7±33.5c 73.1±26.0a 66.8±19.3b

Har 139.3±97.8a 66.4±45d 82.3±51.6b 74.7±42.6c 81.2±28.7b

Pro 38.5±24.1a 39.7±17.2a 36.8±21.2a 34.8±18.7b 38.4±20.1a

Tyr 6.9±4.2d 22.1±15.8b 19.7±15.7b 31.1±20.1a 13.5±6.7c

Val 16.7±7.3c 25.7±8.0ab 24.9±8.5b 16.5±9.2c 30.1±14.8a

Met 4.6±2.6b 5.9±1.7ab 3.8±1.8b 7.5±4.4a 6.3±1.3a

Ile 7.1±5.2b 10.5±3.5b 8.8±3.3b 7.7±3.5b 15.1±6.0a

Leu 11.0±5.2b 16.2±2.25a 16.1±3.9a 17.2±4.0a 17.2±10.7a

Phe 14.6±13.5a 14.7±9.1a 10.8±6.8ab 9.0±3.8b 8.9±6.6b

Trp 39.5±25.3a 18.3±10.8bc 16.2±4.8c 21.4±14.1b 37.2±22.1a

Lys 41.3±33.5a 41.5±31.5a 27.3±21.5c 35.5±23.0b 29.3±8.2c

α-AAA: α-amino adipic acid; Har: homoarginine

Values in a row followed by the same letters are not significantly different (P<0.05).

Impr

ovin

g th

e nu

trit

iona

l qu

alit

y of

gra

ss p

ea (

Lat

hyru

s sa

tivu

s L

.)

87

Tab

le V

.2:

Con

cent

ratio

ns o

f fr

ee a

min

o ac

ids

(µm

ol/1

00 m

l) in

chi

ck s

erum

(m

ean

± S

D,

aver

age

of 1

st,

2nd,

3rd w

eeks

, N

=4,

2 a

nd 2

for

w

eeks

1,

2 an

d 3

resp

ectiv

ely)

fed

with

98.

5% g

rass

pea

(ra

tio

n II)

with

diff

eren

t le

vel

of D

L-m

ethi

onin

e ad

ditio

n (0

.05,

0.1

0, 0

.15,

and

0.

20 %

of f

eed)

V

alue

s in

a r

ow fo

llow

ed b

y th

e sa

me

lett

ers

are

not s

igni

fica

ntly

dif

fere

nt (

P<

0.05

).

W

eek

1 W

eek

2 W

eek

3

0.

0 0.

05

0.1

0.15

0.

2 0.

0 0.

05

0.1

0.15

0.

2 0.

0 0.

05

0.1

0.15

0.

2

Asp

17

.7±0

.8b 14

.6±7

.3c 22

.4±1

2.7a

22.3

±8.0a

21.2

±8.3a

11.4

±1.8c

19.8

±2.6b

25.1

±3.4a

23.3

±1.2a

b 15

.4±1

.9bc

14.1

±2.8b

16.1

±11.

2b 18

.6±9

.2b 18

.6±4

.5b 25

.2±1

.7a

β-O

DA

P 22

.2±6

.2c 27

.8±2

1.4b

28.0

±7.3b

35.0

±27.

9a 29

.2±8

.2b 12

.3±1

.3c 12

.4±0

.4c 41

.3±4

.9a 18

.2±0

.3b 12

.4±3

.4c 14

.7±4

.4b 12

.1±0

.5b 15

.8±6

.0b 27

.8±1

5.9a

14.6

±10.

4b

Glu

36

.9±9

.6b 30

.9±6

.0c 44

.1±7

.6ab

23.6

±2.4d

47.6

±7.4a

75.2

±7.3a

42.9

±5.5b

36.8

±4.4c

35.3

±3.9cd

30

.2±2

.3d 35

.0±9

.5ab

32.0

±8.3b

42.3

±0.8a

38.5

±12.

5a 31

.5±6

.6b

α-A

AA

12

.5±8

.8a

8.7±

0.1a

b 8.

0±4.

9b 7.

7±0.

4b 11

.6±2

.1a 11

.1±1

.5b 9.

3±0.

5b 9.

0±1.

3b 10

.4±1

.4b 19

.3±0

.9a 11

.2±1

.3ab

10.4

±2.5a

b 10

.8±0

.8ab

8.3±

1.1b

14.3

±2.1a

Ser

59

.3±4

.5b 56

.3±1

8.1b

71.2

±5.3a

44.2

±4.6c

74.2

±25.

5a 32

.2±3

.6c 62

.2±7

.0b 60

.3±7

.4b 38

.7±2

.9c 75

.2±6

.2a 52

.3±7

.4b 50

.6±1

4.4b

59.4

±2.6b

50.1

±19.

2b 74

.3±1

0.3a

Gly

67

.2±3

.5b 57

.9±8

.1c 67

.5±8

.1b 67

.1±3

.3b 92

.9±2

.9a 73

.1±1

1.1a

b 78

.7±6

.9a 70

.1±6

.7b 55

.9±3

.5c 64

.7±3

.9bc

59.3

±1.1bc

51

.9±4

.5c 64

.6±6

.5b 62

.6±0

.4b 76

.4±8

.6a

Gln

50

.1±1

6.8d

80.3

±18.

8a 58

.0±0

.1c 66

.4±1

0.3b

58.9

±21.

2c 62

.6±2

5.2b

35.7

±4.5d

55.3

±6.8c

88.6

±6.4a

52.6

±4.1c

33.5

±2.0d

73.1

±17.

4a 43

.8±1

2.7c

67.2

±18.

4ab

65.0

±10.

6b

His

21

.6±1

0.3a

11.3

±2.7bc

8.

1±3.

1c 9.

7±2.

3c 13

.9±2

.9b 12

.8±1

.6a 10

.2±2

.8a 11

.2±2

.3a 8.

2±0.

3a 9.

9±0.

1a 5.

9±0.

7b 9.

8±1.

9a 7.

7±3.

1ab

10.3

±3.5a

12.3

±0.2a

Ta

u 49

.5±4

5.9a

45.2

±39.

2b 51

.1±3

9.1a

14.4

±8.6c

43.5

±8.4b

65.7

±6.1a

36.4

±8.8b

18.1

±2.8c

22.4

±1.1c

39.1

±1.2b

31.3

±23.

0ab

28.3

±12.

6b 35

.0±1

7.8a

40.1

±27.

2a 20

.0±1

3.7c

Arg

75

.1±4

5.2a

43.8

±8.2d

57.6

±14.

9c 41

.6±1

4.5d

63.7

±28.

0b 59

.0±6

.6a 41

.1±3

.7b 52

.0±5

.9ab

50.3

±3.7a

b 46

.5±4

.1b 40

.5±3

.5ab

38.4

±3.7b

50.3

±17.

4a 47

.9±3

.6a 47

.3±1

2.4a

Thr

70

.6±5

0.0a

52.1

±26.

2c 50

.6±2

1.2c

61.6

±4.7b

61.3

±26.

8b 10

0.9±

14.4a

88.0

±10.

3b 89

.3±1

5.6b

80.2

±7.2b

82.7

±10.

8bc

31.8

±22.

9e 86

.9±2

8.2b

44.8

±4.7d

67.6

±24.

4c 11

8.0±

23.1a

Ala

70

.6±5

1.5c

82.2

±16.

9b 96

.4±9

.8c 85

.7±2

.2b 93

.2±2

.8c 10

6.0±

15.9a

90.1

±14.

2b 10

5.1±

18.2a

76.8

±7.6c

78.8

±6.7c

56.6

±8.7c

83.7

±32.

3a 67

.9±2

4.6b

85.8

±19.

7a 84

.3±1

0.5a

Har

14

6.4±

5.2a

118.

1±12

.1b 13

8.9±

50.7a

143.

6±28

.6a 14

7.1±

21.4a

157.

5±18

.4b 11

3.3±

22.5c

178.

1±22

.1a 15

6.8±

13.4b

179.

8±16

.7a 59

.1±1

6.1c

81.1

±39.

6c 13

2.4±

43.4b

144.

0±16

.2ab

149.

8±52

.5a

Pro

48

.5±3

.3b 38

.5±7

.9c 50

.4±1

7.7b

43.7

±4.4c

56.3

±7.0a

48.9

±6.1b

52.5

±4.7b

63.1

±7.5a

47.8

±4.0b

52.1

±1.6b

42.4

±5.8b

65.4

±6.1a

46.6

±11.

7b 45

.4±4

.0b 59

.2±5

.4a

Tyr

14

.1±6

.4b 10

.0±7

.2c 21

.0±1

2.9a

12.9

±0.1bc

9.

9±2.

2c 13

.8±2

.2b 19

.8±1

5.3a

b 19

.9±1

2.9a

b 18

.9±1

.3b 24

.5±5

.7a 12

.9±0

.8c 11

.0±8

.5c 12

.8±0

.6c 18

.1±7

.2b 25

.2±5

.7a

Val

55

.8±6

.5a 32

.1±2

4.3b

49.8

±28.

6ab

50.0

±10.

6ab

52.7

±22.

0a 20

.9±3

.3d 44

.4±6

.6b 55

.8±1

0.2a

46.9

±4.8b

34.5

±2.1c

38.2

±17.

6b 44

.7±4

.5ab

44.4

±26.

4ab

48.4

±0.9a

53.0

±0.3a

Met

3.

8±1.

4b 4.

8±1.

2ab

6.4±

4.8a

4.5±

2.3a

b 4.

1±2.

3ab

2.0±

1.5b

3.2±

0.8b

6.7±

1.1a

4.6±

0.2b

8.3±

0.6a

3.2±

0.9b

7.1±

0.9a

5.8±

4.5a

5.5±

1.1a

6.3±

1.3a

Ile

18.7

±3.9b

18.2

±1.2b

23.8

±4.5a

17.9

±5.5bc

14

.3±1

0.1c

8.2±

5.7c

13.7

±1.3b

20.4

±2.5a

19.1

±0.8a

15.5

±4.6b

14.9

±6.8b

17.1

±1.1a

b 17

.5±1

0.4a

b 20

.1±0

.8a 20

.1±1

.3a

Leu

22.8

±2.1a

b 23

.9±0

.2a 25

.2±1

1.4a

22.1

±8.1a

b 20

.3±1

1.9b

13.5

±3.5c

18.9

±1.9

bc 26

.6±2

.5a 22

.6±1

.3ab

19.7

±13.

6b 18

.2±7

.7b 22

.1±1

.3ab

21.7

±12.

7ab

25.5

±0.7a

27.7

±1.5a

Phe

39

.1±2

7.6b

16.0

±12.

0c 44

.0±3

1.3a

37.0

±26.

9b 48

.0±2

4.1a

25.2

±22.

7c 49

.0±3

5.2a

24.0

±19.

9c 37

.7±2

.7b 18

.0±8

.9d 28

.1±1

7.3bc

20

.6±8

.1c 21

.6±1

2.2c

29.8

±4.5b

40.0

±28.

5a

Trp

50

.3±2

5.8b

39.0

±18.

5c 40

.0±3

5.4c

46.2

±1.7b

57.8

±67.

0a 17

.5±1

2.4c

41.7

±16.

6a 27

.9±1

9.4b

38.2

±16.

0a 42

.1±2

3.0a

42.3

±33.

5b 58

.5±1

8.0a

18.0

±13.

2c 36

.0±1

2.4bc

50

.0±1

3.0a

b

Lys

94.3

±6.8b

73.1

±6.6c

107.

7±37

.5a 93

.4±2

.4b 10

1.1±

34.2a

90.3

±63.

9b 68

.7±7

.2c 84

.9±1

1.5b

87.9

±10.

0b 10

8.1±

14.1a

67.2

±32.

8c 85

.0±3

5.7b

80.8

±52.

0b 87

.5±1

3.5b

112.

3±21

.7a


88

In ration II which contains 98.5 % grass pea, ß-ODAP level in the chick’s serum

increased with methionine supplements at the end of the first week while it decreased

with time or with age of the chicks later on (Table V.2). After the first week, a general

change trend from lower to higher levels along the methionine addition was found for β-

ODAP, aspartic acid (Asp) and glutamine (Gln) while histidine (His), arginine (Arg) and

threonine (Thr) showed a decreasing trend compared to control. The β-ODAP level was

high in the first week with methionine addition and gradually decreased after the second

week, irrespective of methionine addition. Obviously, chickens with 98.5 % grass pea in

the feed required a period of several weeks to adapt to such diet. Homoarginine (Har), the

main amino acid in grass pea seeds (see chapter II), was the most abundant amino acid

particularly in ration II serums.

Trace elements in the serum

Among the trace elements we analysed in the chick serum, Fe2+ showed the highest

concentration (2.70-19.0 mg/kg) followed by Zn2+ (1.57-4.04 mg/kg), Cu2+ (0.16-0.62

mg/kg) and Mn2+ (0.027-0.186 mg/kg). The concentrations of trace elements in chick

serum from ration II experiment seem to be affected by time (age) and methionine

concentration in the feed (Figure V.1 and Table V.3) although there is no clear overall

trend. Most changes were found at higher methionine supplement from 0.1 % to 0.2 %

between week 1 and week 3. At the end of the 3rd weeks, Cu2+ and Fe2+ increased more

than double in the chick serum while Zn2+ and Mn2+ had less change compared to the first

week.


89

Figure V.1. Contents of trace elements Cu2+, Zn2+, Mn2+ and Fe2+ (mg/kg) detected in chick serum with supplement of methionine in the feed (0, 0.05, 0.10, 0.15 and 0.20 %) after week 1 (W1), week 2 (W2) and week 3 (W3) in grass pea ration II fed broilers

Table V.3. Level of trace elements (mg/kg) detected (one sample) in chick serum in ration II with increasing methionine supplements at the end of 1st, 2nd and 3rd weeks

Met supplement 0 0.05 % 0.10 % 0.15 % 0.20 % Fe2+ Week 1 3.000 6.000 5.400 9.500 3.890 Week 2 7.620 7.790 8.200 19.000 14.300 Week 3 10.700 7.970 10.400 9.500 15.700 Mn2+ Week 1 0.027 0.052 0.042 0.170 0.047 Week 2 0.035 0.029 0.025 0.047 0.072 Week 3 0.033 0.031 0.043 0.060 0.046 Zn2+ Week 1 1.740 2.300 1.990 1.900 2.220 Week 2 2.120 3.070 4.040 2.850 2.830 Week 3 2.070 1.860 2.380 2.620 2.360 Cu2+ Week 1 0.300 0.220 0.170 0.200 0.160 Week 2 0.230 0.220 0.130 0.210 0.200 Week 3 0.370 0.290 0.380 0.490 0.400

00.020.040.060.08

0.10.120.140.160.18

0 0.05 0.1 0.15 0.2

Mn W 1 Mn W 2 Mn W 3

00.05

0.10.15

0.20.25

0.30.35

0.40.45

0.50.55

0 0.05 0.1 0.15 0.2

Cu W 1 Cu W 2 Cu W 3

02468

1 01 21 41 61 82 0

0 0 .0 5 0 .1 0 .1 5 0 .2

Fe W 1 Fe W 2 Fe W 3

00.5

11.5

22.5

33.5

44.5

0 0.05 0.1 0.15 0.2

Z n W 1 Z n W 2 Z n W 3


90

Table V.4: Correlations among serum methionine, trace elements, ß-ODAP levels and physical parameters of broilers fed with ration II. From mean values over three weeks and over treatments Methionine Fe2+ Mn2+ Zn2+ Cu2+ ß-ODAP Body wt FCE Methionine 1

Fe2+ 0.95* 1

Mn2+ 0.65 0.78 1

Zn2+ 0.46 0.17 -0.05 1

Cu2+ -0.59 -0.36 -0.27 -0.95* 1

ß-ODAP -0.31 0.18 0.40 0.71 -0.85 1

Body wt 0.72 0.62 0.55 0.71 -0.86 0.84 1

FCE 0.69 0.49 0.14 0.89* -0.88 0.63 0.86 1

* Significantly correlated

The correlation analysis of some physical and chemical parameters (Table V.4) indicates

that the content of methionine was positively and significantly correlated with Fe2+ while

Zn2+ seems to correlate well with FCE (food conversion efficiency) (r = 0.89*). The

serum content of ß-ODAP was negatively correlated with Cu2+ and methionine. Though

only significant for Zn2+, Cu2+ appeared to correlate negatively with all other parameters

studied. Body weight seems a function of FCE physiology, which by itself is positively

correlated with the content of methionine, Zn2+ and ß-ODAP (Table V.4).

V.4. Discussion To our knowledge, there is no previous report on serum amino acids including the toxic

metabolite β-ODAP and trace elements in chicks after feeding with grass pea. Also, our

study is the first experiment on chicks with feeding of such exceedingly high proportion

of grass pea (98.5 %). However, with the limited number of samples analysed for amino

acids and trace elements our results are preliminary but can at least give some indications

for further study.

An increasing level of ß-ODAP in serum with methionine addition in ration II apparently

comes from the higher intake. These improved tolerance value could be the result of the

methionine effect, which as well could have produced a synergetic effect with adaptation

(Low et al., 1990), age (Chen et al., 1992) and the genetic factors (Rao, 2001). The group


91

of chicks on ration II, having the highest incidence of neurological symptoms, were

taking on average about 600 mg ß-ODAP per kilogram body weight per day of the

feeding period. The ß-ODAP concentration level in the blood could reflect the amount

absorbed by the body. In comparison, Chen and co-workers (1992) reported that

intraperitoneal injection of ~700 mg/kg body weight can be lethal for 3-day old chicks.

A number of protein and non-protein amino acids assayed in serum of chicks consuming

grass pea feed showed differences between the control and methionine-added feeds.

However, the changes were neither unidirectional nor consistent for all of them. As

compared to controls, several amino acids showed an increase that may be indicating the

improvement in the animal’s physiological performances in response to methionine

addition. The much higher concentration of lysine and arginine found in serum of chicks

on ration II than that in chicks on ration I might be related to the higher intake of

homoarginine in ration II. Homoarginine can be metabolised into lysine and can also

replace arginine in some metabolic reactions. Homoarginine has been reported to

significantly depress feed intake and also to influence basic tissue amino acids

concentration and arginase activity in chickens (Angkanaporn et al., 1997). Har was also

reported to compete with lysine for transport across the blood brain barrier in rats (Tews

and Harper, 1986). The later authors found that feeding Har to the rats lowered the

concentration of lysine, ornithine and arginine in the brains, while glycine concentration

increased.

Consumption of methionine-rich foods as protection against the neuro-excitatory action

of ß-ODAP and the oxidative stress produced by its biochemical and physiological

activities has been proposed before (Lambein et al., 2001; Kusama-Eguchi et al., 2003;

Getahun et al., 2005; Nunn et al., 2005) and is supported by our findings. However,

additional experiments are needed to obtain statistically significant results.

Of all micronutrients determined in the serum samples, Fe2+ was found at the highest and

Mn2+ at the lowest concentrations. High Fe2+ intake might have an aggravating effect in

neurodegeneration as reported by Jellinger (1999). Cu2+ was reported to be significantly


92

higher (p < 0.05) in the urine of neurolathyrism patients (0.266 ± 0.117 mg/kg) compared

to healthy controls (0.042 ± 0.009 mg/kg) (Kuo et al., 2007). High copper content in

grass pea seeds compared to that in cereal grains like wheat and barley (Kuo et al., 2007)

might also lead to the higher concentrations of Cu2+ in chick serum after being fed

exclusively with grass pea in ration II. In general, however, the micronutrients level

detected in the serum did not show a clear trend with time and with the concentration of

methionine in the feed. Since only one serum sample of each treatment was analysed this

result should be considered preliminary. More data are needed for more clearer

conclusions.


93

CHAPTER VI

Identification of gamma irradiation derived mutant lines of

Lathyrus sativus for higher methionine level


94

Putative mutant of L. sativus with higher methionine in the seed


95

CHAPTER VI

Identification of gamma irradiation derived mutant lines of Lathyrus

sativus selected for higher methionine level

Abstract

Methionine is a limiting essential amino acid in legumes. Methionine supplement was

suggested to protect against neurolathyrism caused by Lathyrus sativus (grass pea) based

diets. Seeds of Lathyrus sativus from different origins were irradiated using 40 KRs

gamma cell irradiation at DZARC, Ethiopia during 2005. Procedures to identify

methionine-overproducing mutants by phenotypic performance analysis were adapted

from similar work on soybean (Glycine max). Common phenotypic features of mutants in

the field include plant size reduction, reduction of branching, colour intensity, leaf

deformation (curling), infertility and absence of flower setting. Mutagenic events were

estimated at a rate of 1-3 per thousand irradiated seeds. Three distinct phenotype mutants

meeting the typical changes of phenotypic features described for soybean mutants with

increased methionine were identified, propagated and further characterized. They all have

glittering deep green leaves, stem and pods, a relatively smaller plant size when

compared to the parent. It gave samaller but a sensible yield and seed size comparable to

the parental line (cv. Debre Zeit). Analysis of seeds revealed that the putative mutants

have about 60 % higher methionine levels compared to parental controls.

VI.1. Introduction

Lathyrus sativus L. (grass pea) is an environmentally hardy multipurpose legume

growing in many parts of the world since almost as long as the existence of agriculture.

Grass pea is endowed with environmental hardiness that ensures production even when

other crops fail. It is responsible for the survival of millions of subsistence farmers during

drought and famine. There is a strong positive association between drought stress and the


96

biosynthesis of a neurotoxic metabolite, β-N-oxalyl-L-α,β-diaminopropionic acid (β-

ODAP). Grass pea is the only legume used as a staple food when other crops fail.

Ethiopia is the largest producer (85 %) and consumer of grass pea in Africa and is also

the most affected by the neurotoxicity it induces (Fikre, 2007). Grass pea is a good source

of protein (27 - 32 %) (Urga et al., 2005) but this protein is limited in the essential amino

acids methionine and cysteine (Kuo et al., 1995), which counts only for a quarter of the

amount in standard egg proteins (Coates et al., 1985). It was mentioned that developing

high sulphur-containing protein soybean lines could save huge financial resources

commonly used to purchase synthetic methionine added to feed in the livestock industry

(Imsande, 2001).

Neurolathyrism is an irreversible crippling disease resulting from prolonged grass pea

overconsumption. The suggested causative molecule β-ODAP is a free amino acid and its

biosynthesis is affected both by genetic and environmental factors. The neurotoxicity

mechanism of β-ODAP is counteracted by adding methionine-rich cereals or condiments

(Getahun et al., 2005) that make a better balanced diet (Molvig et al., 1997). Methionine

(like cysteine) functions as an antioxidant and is a key component for the regulation of

cellular redox homeostasis. Methionine is readily oxidised to methionine sulfoxide by

reactive oxygen species (ROS). The oxidation of methionine residues exposed at the

surface of proteins serves to protect other functionally essential residues from oxidative

damage. Methionine sulfoxide reductase enzymes have the potential to reduce those

residues back to methionine, increasing the scavenging efficiency of the system.

Interconversion of methionine and methionine sulfoxide can regulate the biological

activity of proteins through alteration of the catalytic efficiency and modulation of the

hydrophobicity of the protein (Levine et al., 2000).

Cysteine is involved in methionine biosynthesis in plants and its internal concentration

may contribute to the regulation of methionine biosynthesis (Kim et al., 1999). It was

reported that in soybean the internal concentration of O-acetyl serine (OAS), a precursor

of cysteine, regulates the relative abundance of the β-subunit of β-conglycinin, a soybean

storage protein that is low in sulphur amino acid content (Kim et al., 1999). Rosa and co-


97

workers (2000) investigated the storage proteins from L. sativus. These represent 20 %

of the seed dry weight and are composed of globulins and albumins. A single, 24 kDa

polypeptide comprises more than half of the protein present in the albumin fraction. The

globulins may be fractionated into -lathyrin, β-lathyrin, and -lathyrin components. Two

major polypeptides are glycosylated and exhibit structural similarity with β-conglutin

from Lupinus albus. One of these is a lectin. The amino acid composition of these storage

proteins is not known. A strategically located mutation that would favour storage proteins

with higher methionine or in the genes encoding either cystathionine γ-synthase or serine

acetyl-transferase may promote higher methionine production and thus improve the

sulphur amino acid deficient grass pea. The aim of this study was to isolate methionine-

enriched grass pea mutant phenotypes by γ-irradiation of seeds and screening the plants

grown from the irradiated seeds for higher methionine content.

VI.2. Materials and methods

Gamma irradiation

A total of 17,800 seeds of Lathyrus sativus from different origins were gamma irradiated

at 40 kilorads at DZARC, Ethiopia in 2005. The effectiveness of the irradiation dose was

established from previous similar work experience in the Debre Zeit Agricultural

Research Center. The seeds used were from the collections of IPBO and EIAR: 3,360

seeds from India (Raipur), 4,370 seeds from Bangladesh (Mymensingh), 1870 seeds from

Poland, 660 seeds from China (black eye) and 7,540 seeds from Ethiopia (3000 Debre

Zeit local, 2,700 Bahir Dar, 970 Wello, 870 Gondar).

Irradiated seeds (M1) were planted in rows in the field with 10 cm between seeds and 40

cm between rows on a plot of size 4 m by 10 m. Parental line was planted in a separate

plot comparatively to monitor general performances. The climatic and soil conditions of

DZARC were as described in Chapter III. Field managements (weeding, cultivation)

were performed as needed. Progeny seeds (M2) from plots were bulk-harvested for the

next generation planting, from which selection would be made.


98

Selection for morpho-phenotypic appearance in the field

About half of the 140,000 obtained M2 seeds were planted along parental check in field

plots at DZARC. Field plot preparation and planting procedures were as described in the

above section. In comparison with parental check, plants grown from M2 seeds were

screened on the basis of morpho-phenotypic markers inspired from similar work on

soybean (Imsande, 2001). This established procedure in identifying soybean methionine-

rich lines was conceived to have a similar applicability in grass pea. Selections from the

field screening were planted in separate plots of smaller size in an insect proof screen

house for a further two subsequent generations (M3 and M4) to confirm whether the

altered phenotypes were stable mutants compared to the parents. However, the Chinese

and Polish cultivars did not give viable seeds and were excluded from further screening.

Plant parameters on mean basis were determined from randomly selected plants in the

plot. After germination, days to flowering was determined at 50 % of plants flowering in

the plot, whereas days to maturity is detrmined when 95 % of the pods changed to yellow

(pale). Plant height (cm) is measured from five ramdomely selected plants in the

respective plot at maturity. Seed number per plant is mean seed count from five plants.

Hundred seed weight (g) was measured twice from random 100 seeds of the respective

line and mean value taken. Yield per plant (g) was determined from total plant yield in

the plot divided by the number of plants. Leaflet number is the count of five plant leaflets

and expressed in mean. Mean leaf area, leaf width, leaf shape, leaf length were

determined using CI-202 Portable Leaf Area Meter (model 4901, CID,USA) by measuring

100 leaves. Leaf area index and leaf length to leaf width ratios were derived from

measured values. Descriptive statistics for the parameters were performed on the basis of

means for characterization of mutants against the parent.

Selection by ethionine resistance

Ethionine is the higher homologue of methionine and is toxic for plants and all living

systems. Hence, plants producing more methionine are assumed to be more tolerant to

ethionine (Imsande, 2001). The second half of the harvested progeny seeds (M2) were

taken for an in vitro screening, based on root growth in ethionine media following


99

procedure published in Imsande (2001) for soybean. DL-ethionine (99 %) was purchased

from Acros (Belgium) and dissolved in monodistilled water at a concentration range of

12.5, 50, and 100 ppm. However, this method could not be applied successfully in grass

pea mainly due to lack of fertility of the screened materials. So a workable screening

procedure for grass pea was failed although its application with soybean was reported

(Imsande, 2001). Therefore, there would not be further reports in the result section on

this.

Evaluation of the selections

After selections were identified and isolated, free and protein-bound amino acid contents

of randomly selected 10-14 M4 seeds and parental seeds were analysed using High

Performance Liquid Chromatography as described in Chapter II.

VI. 3. Results

Morpho-phenotypic variations were observed in plants grown from irradiated grass peas

in the field as indicated in Figure VI.1 and Table VI.1. Irradiation increased the range of

variation of traits in mutants obtained as compared to control. Mutagenic events observed

included decreased plant height, reduced branching, leaf colour intensity, leaf

deformation (leaf curling), infertility and lack of flower setting. The frequency of events

observed at M2 ranged between 1 and 3 per thousand irradiated seeds. More variations

were observed among the Ethiopian cultivars, which produced more biomass compared

to the Indian and Bangladesh cultivars. The later two cultivars behaved as dwarf types

under Ethiopian growth conditions.

The phenotypic feature of selections were the glittering deep green colour of leaf, stem

and pods (Figure VI.1) that was clearly observed in the field and was similar to what was

reported in soybean for methionine-enriched mutants (Imsande, 2001). From the progeny

of irradiated Debre Zeit parental seeds, three single plants were visually identified and

selected in the field exhibiting clearly distinctive phenotypes. They are sub-erect, have a

smaller biomass, smaller leaf size, comparable seed size, and slightly smaller grain


100

productivity compared to the parent line (Debre Zeit). They were selected for generation

advancement, characterization and were designated as deep greens (DGA, DGB and

DGC). These putative mutants are discussed in this chapter.

Figure VI.1: Phenotypic appearance of control (right) and one of the mutants (left) - DGA (M4). Differences in colour, glitterness and plant morphology were among phenotypic markers used as selection criteria in the field.


101

Figure VI.2: Chromatograms of protein amino acids; the methionine [↓] peak at elution time 37.3 min in the parent Debre Zeit seed, deep green mutant line and in soybean seeds, respectively Several agronomic and qualitative parameters analysed in the field showed variations

between parental and putative mutant lines. The parental line has got greater plant height

(97 + 5.6 cm) surpassing the mutants by about 61 %, 38 % and 15 % for DGA, DGB and

DGC, respectively (Table VI.1). Yield parameters showed a wide variation in the

selections with DGC scoring best for early flowering, albeit later than the parent, and

having the longest reproductive period (from flowering to maturity) that possibly allowed

more translocation and accumulation of photosynthates into the sink. With efficient

fertilization this line may have higher harvest index as can be seen from the higher

number of seeds per plant. Leaf area index, a quantitative indicator of photosynthesis, is


102

highest in variant DGC following the parental control. Seed size for DGA was greater by

about 10 % than for the other mutants and similar to the control value. Both DGA and

DGC recorded better yield performance per plant, as DGC could apparently compensate

lower seed weight with its higher seed number. They showed a 18 % reduction compared

to the parent, however, about double compared to DGB. Comparison of the mutants

showed poor yield for DGB, which had a shorter period between flowering and seed

maturity.

Table VI.1: Descriptive statistics for some characters of selected mutants (M4) and parental line (Debre Zeit local cultivar) Variables Variables mean values with standard deviation Parent DGA DGB DGC Days to flowering 51 76 82 60 Days to maturity 106 115 115 116 Plant height (cm) 97 ± 5.6 60 ± 12.0 70 ± 8.5 84 ± 8.6 Seeds/plant 265 ± 29.2 230 ± 38.2 147 ± 29.5 253 ± 39.5 Hundred seed weight (g) 7.6 ± 0.4 7.4 ± 0.5 6.7 ± 0.8 6.7 ± 0.18 Yield per plant (g) 20.2 ± 5.6 17.0 ± 2.8 9.8 ± 4.2 17.0 ± 3.9 Mean leaf area (cm2) 3.43 ± 0.5 1.21 ± 0.5 1.69 ± 0.4 1.60 ± 0.2 Number leaflet / plant 2258 ± 284.6 1427 ± 165.2 841 ± 276.2 1430 ± 305.1 Leaf area index (leaf area/ 100cm2 , at full flowering) 77.4 ± 3.5 17.2 ± 2.8 14.23 ± 1.9 22.8 ± 9.0

Leaf width (cm) 1.12 ± 0.2 0.61 ± 0.1 0.87 ± 0.1 0.74 ± 0.3 Leaf length (cm) 4.7 ± 0.5 3.4 ± 0.2 3.7 ± 0.6 3.8 ± 0.5 Leaf perimeter 11.25 ± 0.5 6.58±0.9 8.05 ± 0.8 7.96 ± 0.4 LL: LW 4.08 ± 0.4 6.00 ± 0.2 4.22 ± 0.5 4.93 ± 0.2 Leaf Shape (LA/LP), 1=circle 0.39 ± 0.05 0.33 ± 0.04 0.46 ± 0.9 0.32 ± 0.08

The preliminary protein amino acid analysis of hydrolysed seed meal showed that the

selected putative mutant lines have higher levels of the sulphur amino acid methionine

compared to the parental plants (Table VI.2). There are higher levels of essential amino

acids and total amino acids in the mutants compared to the parent line. The methionine

peak showed quantitative differences among the three presented in Figure VI.2: soybean

highest, the mutant variant of grass pea intermediate and the parental line remaining

lowest. The relative value of methionine, expressed as percentage of total amino acids

detected, was highest for DGC due to its lowest total amino acid value. The concentration

of the non-protein amino acid β-ODAP in the seeds of parent, DGA, DGB and DGC was


103

also determined by HPLC as 0.35 %, 0.38 %, 0.22 % and 0.36 % of seed weight

respectively.

Table VI.2: A preliminary result (single analyses) of protein amino acids (% seed dry weight) levels of mutants (DGA, DGB, DGC) and parental line (Debre Zeit local cultivar). Values in parenthesis are percentage of total amino acids detected

Parent DGA DGB DGC Aspartate 2.02 (11.50) 3.37 (13.00) 3.36 (11.92) 2.37 (11.54) Glutamate 3.15 (18.00) 4.24 (16.85) 5.06 (17.94) 3.29 (16.02) Serine 1.03 (5.88) 1.42 (5.64) 1.60 (5.67) 1.05 (5.11) Glycine 0.77 (4.40) 1.09 (4.33) 1.21 (4.29) 0.80 (3.90) Histidine 0.52 (2.97) 0.73 (2.90) 0.79 (2.80) 0.52 (2.53) Arginine 1.57 (8.97) 2.46 (9.78) 2.60 (9.21) 1.56 (7.60) Threonine 0.64 (3.66) 1.02 (4.05) 1.11 (3.93) 0.74 (3.60) Alanine 0.87 (4.97) 1.57 (6.40) 1.56 (5.53) 1.05 (5.11) Proline 1.23 (7.03) 1.59 (6.31) 1.87 (6.63) 1.15 (5.60) Tyrosine 0.54 (3.09) 0.75 (2.98) 0.78 (2.77) 0.61 (2.97) Valine 0.83 (4.74) 1.15 (4.57) 1.28 (4.53) 0.84 (4.09) Methionine 0.11 (0.61) 0.18 (0.71) 0.19 (0.67) 0.16 (0.77) Isoleucine 0.75 (4.29) 1.05 (4.17) 1.16 (4.11) 0.79 (3.85) Leucine 1.32 (7.54) 1.60 (6.36) 2.20 (7.80) 2.43 (11.83) Phenylalanine 0.86 (4.91) 1.17 (4.65) 1.32 (4.68) 0.88 (4.28) Lysine 1.31 (7.49) 1.77 (7.04) 2.05 (7.27) 1.30 (6.38) Total 17.50 (100) 25.16 (100) 28.21 (100) 20.54 (100)

VI.4. Discussion

In general, the traditional crop plant populations arose from natural variation and sexual

recombination. Contemporary plant breeding is based on creating variation, selection,

evaluation and multiplication of selected genotypes. One of the elements of variation

creation is spontaneous mutation, which naturally occurs at very low frequency and is

difficult to recognize. To create additional variation, the use of chemical and physical

mutagens is an interesting method, which has become an established technology

(Waghmare and Mehra, 2000).

Our results confirm the effectiveness of mutagens in inducing a wide variability of grass

pea traits, which was also observed by other investigators (Waghmare and Mehra, 2000;

Rybinski, 2001). The selected mutants exhibited variability in important yield

parameters. From an agricultural viewpoint, reduction in plant size with reasonable yield


104

as seen in mutants is highly needed as grass pea has important yield losses from the

inherent high lodging problem. The per plant basis yield of the mutant lines could be

acceptable, provided the methionine level is improved. Waghmare and Mehra (2000)

using gamma rays and EMS (ethyl methanesulfonate), induced polygenic variability in

grass pea and found plant heights of 26-120 cm in M2 and 37-118 cm in M3.

A visual phenotypic screening established for methionine overproduction in soybean

experiments (Imsande, 2001) appears to be applicable in grass pea. This method has

become instrumental in the identification and characterization of methionine-enriched

mutants. Selected mutants maintained the phenotypical markers over the subsequent

generations. The isolation of methionine overproducing lines both by phenotypic

screening procedures (Imsande, 2001) and chemical analyses resulted in three lines with

a promising increase of methionine. Considering that the sulfur amino acid limitation of

grass pea is believed to be an important factor in the aetiology of a neurodegenerative

disease (Getahun et al., 2005), this result may well have important medical consequences.

A promising increase in the level of methionine by mutagenesis is now reported for the

first time in grass pea, although more analyses should be done to confirm this result. This

would improve the methionine content of grass pea seed from one-fourth of the dietary

requirement of the standard diet (FAO/WHO/UNU-1985) to a more healthy level. This

could probably imply a considerable reduction of the potential toxicity effect in seeds of

grass pea. As methionine biosynthesis depends on sulphur fixation into the cysteine

pathway (Molvig et al., 1997), the mutagenic event might have altered gene expression

and/or enzyme activity in favor of cysteine/methionine biosynthesis.

Enhancing methionine levels via genetic engineering has shown to increase the nutritive

value of legume seeds (Molvig et al., 1997). The increased level of essential amino acids

including methionine in the mutants may considerably improve the nutritional quality of

grass pea towards a better-balanced food and feed. Mutants with lower total amino acid

values appear to have higher relative methionine levels. This relative value of essential

amino acids determines the quality of protein. Supplementary feeding experiments need


105

to verify the biological value of the protein from mutants with the improved methionine

level.

The higher methionine selections (DGA, DGB, DGC) still have elevated β-ODAP levels

that are similar to the parent line (around 0.35 % of seed weight). Simultaneous reduction

in the level of the neurotoxin needs to be worked out by crossing of our mutant selections

with the presently available low toxin lines.

Sufficient dietary methionine and even addition of synthetic methionine improves the

nutritional quality of a diet with pulses in general (Molvig et al., 1997). Even more

importantly, higher methionine can be protective against neurolathyrism for consumers of

grass pea (Getahun et al., 2005). Compared to the parental line, the putative mutants

obtained in this study have around 60 % higher methionine level, which would improve

their nutritional quality. This also implies that the antioxidant activity of food or feed

made from these seeds would be higher, with potentially lower risk for neurolathyrism.

This considerable improvement is, however, not yet sufficient to reach the optimal

balance of essential amino acids. Hence, selection of adaptive, methionine

overproducing, low β-ODAP lines is becoming a more promising field of research to

develop nutritionally improved grass pea lines that would be safe for consumers.

Evaluation of these mutants in different environmental conditions together with

nutritional or feeding tests need to be undertaken to determine comparative benefits. The

new lines could be further used in breeding programmes and for genetic analysis. Such

approaches need to be further strengthened to achieve the goal of preventing

neurolathyrism in Ethiopia and in other countries.


106


107

CHAPTER VII

General discussion and conclusions


108


109

CHAPTER VII

General discussion and conclusions

From the viewpoint of improving the nutritional quality of grass pea, it is felt that a

holistic approach integrating the social, environmental and biological components could

be chosen for solving the nutritional and toxicity problems of the crop. Aside from topics

presented in individual chapters of this thesis, the author has made important

observations while touring lathyrism-prone areas during field work in Ethiopia. It is

worth mentioning that grass pea consumption habit (with or without cereals or fish),

processing, level of education, immature green pea consumption frequency are all

variables existing in the society that seem to affect the incidence of neurolathyrism. The

present work describes observations and experiments that contribute to our understanding

of this crop, the aetiology of neurolathyrism and to its prevention. It also highlights some

properties of grass pea that are less frequently in focus of research on grass pea and

neurolathyrism.

VII.1. Environmental influence on levels of amino acids and crop performances in L. sativus

Efforts to improve this important legume crop have been underway since the discovery of

the neuro-excitatory amino acid β-ODAP in the seeds (Rao et al., 1964). Since this

discovery the focus was on the reduction of this metabolite. No holistic approach on the

more general improvement of the nutritional quality was performed and other merits of

the crop were neglected. As a consequence of the stigma of neurolathyrism and the lack

of incentive to improve the agronomic properties, not much progress has been achieved

to make grass pea a more productive food crop. Probably because the crop was normally

used for forage and only becoming important for human consumption during droughts

and famines, the crop has undergone very little evolutionary progress in its adaptive

environment.


110

Analysis of the free amino acids of grass pea seeds (chapter II) revealed that the non-

protein amino acid homoarginine (Har), which is absent from most other legumes, was

the most abundant amino acid among all grass pea genotypes from diverse origin,

supporting previous results (Lambein et al., 1992). The concentration of this metabolite

was rather similar among all the genotypes, which marks a unique feature specific to

grass pea seeds. The study of the profile of free amino acids in grass pea and in some

other common food legumes including pea, bean and lentil (Kuo et al., 2004) showed that

Har was undetectable in the dry seeds of these species where quantitatively the most

important free amino acid was arginine (in pea and bean) or glutamic acid (in lentil).

Haque (1997) reported that the level of Har in the seeds increased dramatically in the low

toxin line of L. sativus LS-82046, when growing in Hoagland solution under stress

conditions with supply of the double amount of the trace elements Zn2+, Fe2+, B(OH)-4

and Co2+ in hydroponic media or with Al3+ at 2 x 10 –6 M in the media, or with increasing

salinity up to 0.8 % NaCl (w/v).

Har has been reported to antagonise the neurotoxic action of β-ODAP fed to one-day-old

chicks (Yusuf et al., 1995; Shamim et al., 2002). This might be explained by the

modulating effect of L-Har on nitric oxide synthase (Jyothi & Rao, 1999).

β-ODAP is quantitatively the second most abundant free amino acid in L. sativus seeds

but with greatest variation from 0.2 mg/g dry seed in LS-82046 (Canada) to 5.4 mg/g dry

seed in Raipur (India) (chapter II and Fikre et al., 2008). The presence of β-ODAP in

grass pea seeds was blamed for causing the crippling disease neurolathyrism and the

toxicity is possibly mediated by collective effects of L-β-ODAP on the AMPA-type

receptor, metabotropic glutamate receptors, and nitric oxide production (Kusama-Eguchi

et al., 2006). This free amino acid is reported to be highly variable among genotypes and

environments (Campbell, 1997; Wuletaw, 2003). We have been able to demonstrate the

high variability of this molecule as its level is affected by changes in temperature,

moisture regime, light duration, plant phenology, soil nutrients and altitudinal variations

(chapter III). Increases up to 100 % in levels were exhibited by the same cultivars grown


111

in environments with a marked alteration of altitudinal and associated climatic and

edaphic factors in Ethiopia (chapter III).

Earlier studies on the influence of nutrient supply on the β-ODAP content in grass pea

seeds growing in hydroponic media indicated that reduction of the macro-nutrients such

as Mg2+, or K+ in the Hoagland solution resulted in sharp changes of β-ODAP content in

the ripe seeds (Kebede et al., 1994). Conversely, actual environmental factor analysis

(chapter III) showed that K+, known to be high in Ethiopian soil (Murphy, 1968), has a

strong positive correlation with β-ODAP. This strong dependency on environmental

factors indicates the potential for improved crop quality under better-controlled

environmental conditions. We have demonstrated that K+, Zn2+, and P are crucial nutrient

factors influencing β-ODAP content under field conditions while Jiao et al. (2006)

reported the same importance for N and P in Gansu province of China.

Drought stress was reported to have maximal influence on β-ODAP concentration in

grass pea (Lambein et al., 1990; Campbell, 1997; Haque, 1997; Cocks et al., 2000;

Asfaw et al., 2003; DZARC, 2003). From studies on interrelations between

environmental factors we propose that drought stress influences the β-ODAP level in a

multifaceted interplay of links to which the plant responds by over-synthesizing β-ODAP

as a protective molecule or a stress metabolite. The mechanism could be summarized as:

Directly Indirectly Plant responses/processes

Drought

stress

Rain fall distribution

Temperature and wind

Soil moisture availability

Altitude

Evapotranspiration

Nutrient uptake

pH

Root exploration restricted

Physiological retardation

Forced maturity

Yield and metabolites synthesis

Low productivity

High conc. of β-ODAP → seed

sink

Efforts in plant breeding research to eliminate β-ODAP from the seeds have produced

large numbers of “low toxin” varieties but did not yet result in “toxin-free” ones. A

number of post-harvest processing methods of grass pea seeds dramatically reduced the


112

level of β-ODAP but could not remove the neurotoxin completely (Srivastava and

Khokhar, 1996; Kuo et al., 2000).

In seed hydrolysates of all grass pea genotypes glutamate was found to have the highest

concentration followed by aspartate, arginine and lysine (chapter II). The sulphur amino

acid methionine ranked the lowest among all the amino acids in all genotypes of L.

sativus as well as in lentil and soybean. The average level of protein methionine in L.

sativus was 0.1 % of the seed weight, and seems rather stable among genotypes of

diverse origin in spite of the high variation of β-ODAP. There is no significant difference

in protein methionine level between grass pea genotypes from India with high β-ODAP

content and Canadian line LS 87124 with low β-ODAP content. Thus protein methionine

content seems not to be correlated with the level of β-ODAP in Lathyrus sativus seeds,

although a biochemical link seems to exist because O-acetylserine (OAS) is involved in

the biosynthesis of both cysteine and BIA (β-(isoxazolin-5-on-2-yl)-alanine) through

cysteine synthase (see Figure VII.1). BIA, present in seedlings of species of the genera

Pisum, Lens, most Lathyrus species and some Vicia species, was confirmed to be the

precursor of β-ODAP in grass pea (Ikegami et al., 1993; Kuo et al., 1998).

Figure VII.1: Biosynthetic pathways of the sulphur containing amino acids, Cystine and Methionine. The enzymes involved in the pathways are: CS (Cys synthase); Cgs (Cysthathionine g-synthase); Cbl (Cystathionine beta-lyase); MS (Met synthase); SAMS (S-adenosylmethionine synthase). Scheme adapted from Noji and Saito (2003).

Acetyl-CoA Ser

OAS CS BIA ↓ ODAP

SO42- ↓ ↓ S2- ↓ CS Cys ↓ Protein Protein

↓ Cgs Cysthathionine ↓ Cbl Homocystein ↓ MS ← Met ↓ SAMS SAM


113

Grass pea seeds are the major protein source for the poor and occasionally become the

survival food during crop failure. Gatta et al. (2002) reported that the protein content of

161 Bangladesh accessions ranged from 23 to 29.9 % with a mean value of 26.3 %. We

found 24.1 % protein content in Debre Zeit local cultivar and Urga et al. (2005) reported

a protein range of 27.3-32 % in fifteen cultivars collected in Ethiopia.

Agronomically, the dominant factors affecting the yield of grass pea in studied locations

of Ethiopia were the days to maturity and moisture (chapter III). Yield and various yield

components have shown variability in response to cropping seasons (years), genotype,

locations and their interactions. Better yield performance was associated to locations at

higher altitude where the crop growth period was relatively longer. The exceptional low

yield harvest at Denbi might be attributed to the shorter maturity period, and high

concentration of sulphur and manganese in the soil that showed a significant negative

correlation to yield. Despite their individual negative correlation the interaction Zn2+/S

produced a positive correlation to yield, suggesting that an optimal ratio can be found for

better yield realization.

In summarizing this part, it is very well established that grass pea is an important legume

grain food and a life saving crop (Fikre et al., 2006) but it is also controversial as the

cause of crippling lathyrism. It is well known that genotypic variability exists for the

level of the neuro-excitatory amino acid β-ODAP in the seed (Wuletaw, 2003). Those

earlier studies were done using a colourimetric method that cannot distinguish between

the non-toxic α-ODAP and the toxic β-ODAP. In our comparative study with two other

food legume species (lentil and soybean), the levels of β-ODAP, methionine and other

amino acids were determined using the more accurate HPLC-method (Fikre et al., 2008).

This is also the first comparative study on free and protein amino acids in a set of

genotypes collected from different parts of the world. However, the concentration of β-

ODAP (with a wide range) and methionine (narrow range) did not show any specific

correlation between these two amino acids, although one enzyme, cysteine synthase, is

involved in the biosynthesis of both.


114

In Ethiopia, recurrent climatic disasters (drought) are a common phenomenon, and this is

known to be associated with increasing β-ODAP in L. sativus (Asfaw et al., 2003) with a

potential increase of neurolathyrism incidence to epidemic proportions (Haimanot et al.,

2005). However, the role of environmental factors on the toxicity (β-ODAP level) of the

crop has never been comprehensively studied. From the present path analysis using two

growing seasons at five locations, a number of additional factors including both climatic

and edaphic ones were shown to have a strong relation with β-ODAP level and crop yield

in Ethiopia. Previously, other authors did not consider these factors. The results suggest

novel crop management options to better control the particular agro-ecology for safer

utilization and better exploitation of this underutilized crop. However, such short period

of data collection in the unstable and changing climatic condition of Ethiopia may not

allow long-term predictions. Also, the results may not be applicable to countries such as

Bangladesh with different edaphic (lowland sedimental soil as opposed to volcanic soil in

Ethiopia) and climatic (higher precipitation and even flooding) conditions and also with

different geographic locations (different light quality).

VII.2. Effect of methionine on toxicity of L. sativus

This study was initiated with the hypothesis that methionine could neutralize the

antinutritional nature of grass pea and would contribute better to its overall food quality

improvement than focusing only on toxicity reduction/elimination alone. When

comparing neurolathyrism with konzo (an irreversible crippling disease undistiguishable

from neurolathyrism and caused by prolonged overconsumption of cassava root

preparations) it was suggested that the common feature of the diets leading to those

similar diseases, namely methionine deficiency could play a role in the aetiology of

neurolathyrism (Defoort, 2004; Lambein et al., 2004). The impetus was then to confirm

the hypothesis that methionine would affect grass pea toxicity and also to estimate the

level of its effect. A feeding experiment with methionine supplements in the diet was

done with broiler chicks and the results are presented in section IV.3.


115

Nunn and co-workers (2005) reported that consumption of grass pea seeds deficient in

methionine as staple food for a long period leads to the deprivation of methionine in the

plasma. They suggested that long-term deficiency of methionine in plasma and thus

reduced methionine flux into motor neurone cells might lead to the higher susceptibility

of these cells to the excitatory effects of β-ODAP.

Among the essential amino acids in grass pea the level of methionine and cysteine in the

seed reaches only about 1/4th of the requirement for a normal balanced diet. Diets with

grass pea as the staple are therefore a poor source of methionine and cysteine amino acids

(Kuo et al., 1995). The antioxidant effect of methionine and its protection against the

oxidative stress induced by the neuro-excitatory activity of ß-ODAP leading to

neurolathyrism have been reported (Kumar and Rao, 1990; Getahun et al., 2005; Nunn et

al., 2005).

From the grass pea feeding trial (chapter IV) the effect of increasing methionine addition

was evident in bringing significant change to the chick performances and phenotypic

responses. In spite of the general under-performances for some parameters when the

chicks received exclusively grass pea feed (98.5 %), the broiler chicks showed

improvement along with the increasing addition of methionine. Although this study did

not address the economics of grass pea in animal husbandry, it has shown that a positive

outcome can be expected from a focussed economic study, which can be undertaken

using different formulations as treatment inputs.

The highest effects were found at methionine addition of 0.075 % when grass pea

accounts for 35 % of the feed and 0.15 % when grass pea (98.5 %) became almost the

exclusive feed source. If this would be valid also for humans, then this may suggest that

methionine optimisation in the diet could play a central role for the safe utilization of this

under-utilised but protein rich grass pea (Lambein et al., 2001; Getahun et al., 2005;

Nunn et al., 2005).


116

It was also demonstrated that the β-ODAP intake tolerance of chicks increased with the

addition of methionine in the diet (chapter IV). On average, the performance (feed intake,

weight gain) of the chicks fed on the high β-ODAP Ethiopian grass peas was

significantly improved. The improved performance in feed intake and body weight could

likely be the indirect consequence of enhanced FCE and/or from the fact that methionine

protects the animals from oxidative stress induced by ß-ODAP. In an earlier study

Hanbury and co-workers (2000b) had observed a tolerance in chicks of up to 40 % grass

pea (containing 0.27 % ß-ODAP) with minor weight reduction.

With the increase of methionine in the feed, ß-ODAP was found to decrease in serum of

chicks on ration I (35% grass pea), which may result from improved physiological

condition and metabolism of the chicks (Chapter V). Individual variability in tolerance to

acute neurological symptoms was seen in chicks having the same level of exposure. Rao

(2001) and Rudra and co-workers (2004) suggested that there exist extreme inter and

intra species differences in susceptibility to ß-ODAP that may be ascribed to individual

metabolic differences. To our knowledge a genetic basis for such differences has only

been proposed by Getahun and co-workers (2002b) who indicated that blood type could

be a factor of susceptibility to neurolathyrism in humans.

From the analysis of trace elements in chick’s serum (chapter V) we found a gradual

increase of Fe2+, Zn2+, and Cu2+ with time but not for Mn2+. In agreement with previous

studies, in this study acute neurological symptoms were observed more in younger chicks

and becoming less as age increased. From the data of feed intake over the feeding period

we calculated that the convulsive dose for young chicks was about 600 mg of ß-ODAP

per kilo of body weight in ration II (98.5% grass pea) while Chen and co-workers (1992)

reported that intraperitoneal injection of ~700 mg ß-ODAP/kg body weight is lethal for

3-day old chicks. Unlike the manifestations of neurolathyrism in humans, the common

convulsive symptoms in chicks were mainly spasticity of the neck muscles. Differences

also include the reversibility of symptoms in chicks and the irreversible symptoms of leg

spasticity in man with sudden onset after prolonged consumption of mainly grass pea

seed during two months or more.


117

Our study attempted to estimate the particular effect of methionine on grass pea toxicity

as feed. Deficiency of methionine in L. sativus (Kuo et al., 1995) and its potential

involvement in neurolathyrism were reported (Kumar and Rao, 1990; Nunn et al., 2005;

Getahun et al., 2005). The absence of a suitable animal model is claimed to be a barrier

for better understanding of neurolathyrism (Getahun et al., 2004). A limited study was

undertaken on the effect of methionine on chicks fed a L. sativus based ration (Table

IV.5). Unlike previous experiments, in this thesis the effect of methionine per se on grass

pea toxicity was studied not only from the viewpoint of animal performance, but also on

neurological symptoms. Chicks were symptomatically characterized, their performance

recorded and amino acids in the serum analyzed. The study contributes to the

understanding of the effect of methionine on L. sativus toxicity and confirms

unequivocally its protective effect against grass pea toxicity in young chicks. It also

indicates the possibility that methionine could be implicated as protective factor in other

animals and/or human neurolathyrism as well. However, the experimental setup did not

allow distinguishing between the toxic effect of β-ODAP and of other ANFs, as only the

raw unprocessed seeds were used.

VII.3. Mutational methionine enrichment in L. sativus

The sulphur containing amino acids methionine and cysteine are in general deficient in

legumes and consumption of legumes as the sole protein source in the human diet is not

advised. The deficiency of these amino acids (Kuo et al., 1995) is of particular

importance in neurolathyrism disease development that is caused by monotonous

consumption of L. sativus.

By adopting phenotypic markers for methionine enriched lines as has been established for

soybean (Imsande, 2001) we have identified and selected L. sativus variants in the field

from γ-irradiated seeds (chapter VI). The phenotypic variants exhibited phenological and

morphological appearances different from the parental line. Unlike the parent line they

have a smaller size, later maturity and glittering deep green phenotypic appearance.


118

Amino acid analysis of seeds from the mutants has shown a 60 % higher methionine

content compared to the parent. Unlike other legumes, the sulfur amino acid limitation of

grass pea apparently has medical implications by increasing the susceptibility of subjects

to a neurodegenerative disease caused by dependent consumption of the crop (Getahun et

al., 2005). Hence, it is assumed that in vivo methionine improvement potentially

enhances the nutritional quality as well as the safety of grass pea food preparations or

feed. However, the ß-ODAP level in the putative mutants we obtained is not lower than

that in the parent. Further work should aim at the simultaneous reduction of the β-ODAP

level and increase of methionine as a promissing goal. While genetic transformation

could be utilized to reach this goal, the present selections can at least be used in breeding

programs to achieve lines with increased nutritional quality and adapted to marginal or

drought prone lands.

In conclusion, in vivo methionine enrichment in L. sativus could be a plausible approach

in the process of making it a more healthy food. This has never been tried effectively thus

far. Among methods that could produce genetic alteration, mutagenesis was attempted by

irradiating several thousand seeds where mutants with a distinct phenotypic appearance

were advanced over four subsequent generations and were chemically and phenotypically

characterized. From the preliminary analysis, some selected putative mutants have

revealed higher methionine content and desirable agronomic performances. Though the

work is at its early phase, this novel approach might give promising prospects for

improvement of the crop with a better balance of essential amino acids. Moreover, the

potential introduction of such mutants would not be subjected to the stringent regulations

that virtually stop the use of even the most promising genetically modified food plants.

Limited experience in this area has put a challenge in the course of the work. The work is

not yet complete/conclusive as analysis and characterization has to go over several

generations to confirm this preliminary result. Supportive genetic and molecular studies

(mode of inheritance, molecular characterization) are needed to further characterize and

utilize the selections.


119

VII.4. Recommendations and future perspectives This thesis attempts to identify and study gaps in our knowledge of the traditional legume

crop plant Lathyrus sativus and the human crippling disease neurolathyrism. The ultimate

goal is to prevent neurolathyrism by establishing varieties with improved nutritional

quality without the risk for the irreversible crippling neurolathyrism. A secondary goal is

to suggest ways to optimise the use of this underutilized crop. The environmental and

biological aspects have been studied after a thorough literature review of already

established knowledge. Recommendations and suggestions of particular importance or

usefulness are forwarded.

In the interaction of various factors, the process of neurolathyrism development could be

summarized in the following simplified scheme (Figure VII.2).

Figure VII.2. Hypothetical scheme of neurolathyrism (NL) development by interaction of various factors (human, environment, socio-economics and grass pea plant). Some factors are directly linked to NL; others are via affecting β–ODAP.

The level of factor’s effect = * lowest, ***** highest A-age, B- sex, C- physical labor, D- health status, E- individual metabolism/genetics, F- moisture stress, G- soil nutrient balance, H- salinity, I-light hours, J- genetic interventions, K- crop management/ productivity, L- stressed growth, M- processing, N-green pea O- traditional perception and precaution measures Vs scientific information, P- poverty (± option), Q- consumption style and intensity, R- diet status (± methionine and antioxidant rich condiments). Our study demonstrated that crop parameters such as yield, ß-ODAP and other amino

acids of grass pea are affected by various environmental factors. Particularly, the unstable

ß-ODAP was reported to be affected by moisture stresses, nutrient levels, genetic factors

and processing. However, these parameter effects should also be studied in other areas

prone to neurolathyrism, as there could be certain specificity from country to country. In


120

this part of the study, interrelations between amino acid levels and environmental

conditions of grass pea have been demonstrated. Especially the great variability of ß-

ODAP level in response to varying environment, media and processing was verified

under Ethiopian conditions. As the sulphur containing amino acid methionine has been

proposed to protect cells against β-ODAP toxicity, we also focused on the level of

methonine in seeds (± 0.1 % of dry weight). It was shown that methionine levels are

much less variable compared to ß-ODAP in grass pea and it is present in concentrations

that may lead to dietary deficiency if grass pea is consumed as staple. Especially, this

study revealed that, among several factors considered, the soil factors (K+, pH) and the

photosynthetic factor (sunshine hours) have a strong positive relation with β-ODAP level

while days to maturity, yield and altitude have negative correlations. Further studies on

the fertilization and other factors affecting crop yield, Rhizobium nodulation and

adaptation across environments and resistance to biotic and abiotic stresses are

anticipated to provide better information to optimize the crop use. This drought tolerant

traditional crop is well suited for adaptation to the increasing water deficits Ethiopia may

face due to the global warming and to become an important asset to overcome food

insufficiency if the stigma of neurolathyrism can be removed.

The antioxidant activity of the essential sulphur containing amino acids cysteine and

methionine is confirmed in a variety of research reports. Especially the motor neurones

seem to require high methionine for their integrity. Some experimental studies indicated

that sufficient cereals (1/3rd of the diet) rich in methionine could protect against

neurolathyrism. There are also some results indicating that methionine antagonizes the

antinutritional effects of β-ODAP. In a broiler chick feeding experiment we have

demonstrated that methionine addition in grass pea feed improved feed intake, weight

gains and general animal performances. This is in support of the hypothesis that the

antioxidant activity of methionine counteracts the oxidative stresses induced by various

physiological and biochemical activities of β-ODAP. Developing a reproducible and

reliable animal model would allow more detailed studies to advance our knowledge on

the aetiology of human neurolathyrism with potential prevention implication. Such an

animal model would be important also for the study of other nutrition related


121

neurological diseases such as konzo (a neurological disease with symptoms

undistinguishable from neurolathyrism but caused by unbalanced overconsumption of

methionine-deficient cassava roots).

Apparently, as a potential protective factor against β-ODAP toxicity, methionine enriched

food such as cereals or addition of synthetic methionine or genetically enriched grass pea

varieties are believed to confer such protection. Methionine deficiency in legumes

including grass pea is long established. Grass pea can only supply about a fifth to a fourth

of the methionine required for an optimal balanced diet. There is hardly any ongoing

research on genetic enrichment of methionine in grass pea. We have demonstrated for the

first time a methionine-enriched selection after γ-irradiation of the seeds and we

described the mutant isolates. Phenotypically distinct putative mutants showed a medium

biomass, have comparably smaller and deeper-glittering green leaf, medium seed size and

reasonable grain productivity. This study is now in the M5 stage and mutant lines are

designated as deep greens (DGA, DGB, DGC) referring to their typical phenotype. They

have more than 50 % methionine increase against the parent line. A subsequent study into

its environmental adaptation, the mode of methionine inheritance, crossing ability and

molecular characterization of the genetic alteration would enhance the incentive to

change the crop into a promising healthy food and feed. Further studies on mutation and

molecular techniques are essential in grass pea research. Evaporates of grass pea have to

be studied for potential toxic constituent, as there is no clear evidence of this in spite of

the strongly entrenched traditional conception that the vapour from flowering plants or

from cooking the seed are toxic. Safe varieties of the drought tolerant grass pea with a

better-balanced complement of essential sulfur amino acids can become a wonder crop

for subsistence farmers in marginal areas of Ethiopia and South-East Asia. This would lift

the socio-economic status of many poor farmers and it would also be of enormous benefit

to the ecology of areas with marginal agricultural productivity or with adverse climates.


122


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Summary - Samenvatting


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Summary

Grass pea (Lathyrus sativus L.) is economically the most important species in the genus

Lathyrus. Hundreds of millions of people in Asia, Africa and the Near East use grass pea

in their daily diet. Typically and unlike other legumes, it is used as a staple food. Its

adaptation to environmental stresses, productivity under low inputs, and capacity to

restore soil fertility capacity underline its agricultural potential. Despite its hardiness it is

a dependable and promising source of protein, carbohydrate and minerals. Grass pea is

used as a pulse since the early history of agriculture and is still so used. However, it has

made little progress as a grain crop during this long history.

Unbalanced overconsumption of grass pea during months can lead to neurolathyrism, an

irreversible crippling of the legs in humans. The disease can become epidemic,

particularly if accompanied by malnutrition, drought, insufficient processing or raw

consumption. The causative metabolite is a neuro-excitatory amino acid β-N-oxalyl-L-α-

β-diaminopropionic acid (β-ODAP). In grass pea this metabolite is highly affected by

environmental factors, processing and genetic factors. Hence, the level of β-ODAP in the

seed seems to be related to a physiological reaction of the plant to environmental stresses

and may be considered as a stress metabolite. Methionine has a protective effect against

the toxicity of grass pea, but is very limited in this plant.

The study tries to integrate the environmental and biological aspects towards the same

goal of improving the nutritional quality of grass pea. In the first part of the thesis

interrelations between amino acids in grass pea and environmental conditions were

studied. Unique among legume species, grass pea is an important source of the non-

protein amino acids homoarginine, followed in concentration by the presumed cause of

neurolathyrism ß-ODAP. These metabolites are suggested to confer the plant with some

degree of tolerance against different biotic and abiotic stresses. There was big variation of

ß-ODAP level in response to genotypes and environments. Grass pea has a sufficient

level of most essential amino acids but methionine is extremely low, covering less than

25% of the recommended level for a healthy diet. Methionine levels are less variable than


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ß-ODAP in grass pea. Path-analysis with selected predictors for ß-ODAP showed a larger

direct effect due to K+, high light intensity during maturity phase (sshIII), maturity, yield

and Zn2+:P. Ethiopian soil is often very high in potassium, and this has been

demonstrated to have a strong enhancing activity for β-ODAP in Ethiopia. Pearson’s

correlation coefficient showed that altitude, maturity period, soil pH and crop yield have

a strong negative correlation while K+ and shh have strong positive correlation with β-

ODAP. High light intensity, supposedly found at higher elevation, is found to increase β-

ODAP biosynthesis. However, in this study the effect of high light intensity in high land

was modulated by the cloudiness and thus shorter period of high light intensity resulting

in higher ssh in lowlands producing higher β-ODAP than high lands with reduced total

ssh in the plant growth periods. Apart from these we found the greatest direct effect of

maturity period (0.94) followed by rainfall during crop establishment (0.31) for crop

yield. There was also a significance of Genotype x Environment combination for grass

pea yield, indicating the different responsiveness of varieties to the varying environment.

The effect of methionine on grass pea toxicity was investigated using broiler chicks. The

responses of chicks to dietary DL-methionine supplements to grass pea feed were

evaluated. This supported the hypothesis that the antioxidant activity of methionine

counteracted the oxidative stresses generated by various physiological activities of β-

ODAP. In agreement with other reports, some chicks developed reversible convulsions of

the neck region at early age and showed resistance afterwards. Symptoms occurred only

in maximum 1/3rd of chicks receiving the same exposure to β-ODAP, consistent with

earlier suggestions that individual differences exist. There was a much-reduced level of

β-ODAP in the chick’s serum compared to the corresponding intake level. This might

suggest the reduced uptake or increased metabolic breakdown in the presence of higher

methionine, which might also have individual variation. The improved performances of

the chicks in response to methionine supplementation was partially due to the indirect

effect through FCE improvement. It was learned that the tolerance level of the animals

remarkably improved with age, suggesting that adaptation and stronger detoxification

mechanism might have developed. There was a trend of ß-ODAP reduction with


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increasing methionine supplement, suggesting the counteracting nature of the two amino

acids.

Methionine enrichment in grass pea is perceived as a key element to reduce

neurolathyrism development. Mutation and selection for increased methionine has been

adapted from similar work with other legumes. Three phenotypically distinct putative

mutant lines have been selected from gamma-irradiated grass pea seeds. The M4

regenerants of these putative mutant lines have 63% higher methionine in the seed

compared to the control parental line. Characterization of the three selections showed that

they are sub-erect with medium biomass, have comparably smaller and deeper-glittering

green leaves, medium seed size, and reasonable grain productivity. They were designated

as deep greens (DGA, DGB, DGC) referring to their typical phenotype. However, the ß-

ODAP level in the new mutants is not different from the wild type. The methionine level

in the mutant lines may improve the nutritional quality and safety of the seed in food and

feed. Hence, the market price of grass pea and the financial situation of the farmers may

improve. These lines would serve as attractive genetic resource for future breeding.


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Samenvatting

Zaailatyrus (Lathyrus sativus L.) is economisch een van de meest interessante

peulvruchten binnen het genus Lathyrus. Het onderscheidt zich van andere peulvruchten

door het feit dat het door honderden miljoenen mensen in Azië, Afrika en het

Middenoosten dagelijks als voedsel gebruikt wordt. Het aanpassingsvermogen van deze

peulvrucht aan stress uit de omgeving, de hoge opbrengst die relatief weinig moeite kost,

en de capaciteit om de bodem te verbeteren, geeft zaailatyrus een toekomstperspectief in

het milieu van de toekomst met meer droogte en verzilting. Naast de mogelijkheid om in

extreme omstandigheden te overleven is het ook een rijke bron van proteïnen,

koolhydraten en mineralen. Toch heeft zaailatyrus, dat sinds millennia als peulvrucht

wordt gebruikt en tot nu toe weinig uitgebuit werd, tot op heden als landbouwgewas

weinig vooruitgang geboekt.

Wanneer mensen maandenlang afhankelijk zijn van de consumptie van zaailatyrus kan

dit leiden tot neurolathyrisme, een irreversibele verlamming van de onderste ledematen.

Deze aandoening kan voorkomen onder de vorm van een epidemie, voornamelijk in

combinatie met ondervoeding, droogte, inefficiënte verwerking of consumptie van de

rauwe zaden. Het vrij aminozuur β-N-oxalyl-L-α-β-diaminopropionzuur (β-ODAP) in

zaailatyrus wordt verantwoordelijk geacht voor die aandoening. De concentratie van dit

aminozuur in de zaden wordt sterk beïnvloed door omgevingsfactoren, de

bereidingswijze van de zaden of genetische factoren. Zo blijkt er een verband te bestaan

tussen de concentratie van die ongewone metaboliet en diverse omgevingsfactoren.

Methionine, dat deficiënt is in peulvruchten en vooral in latyrus kan bescherming bieden

tegen de neurotoxische werking van langdurige latyrus overconsumptie; waarschijnlijk

als beschermer tegen oxidatieve stress.

Deze studie probeert de socio-economische aspecten te integreren met de

omgevingsgerelateerde en biologische aspecten met het oog op de preventie van

neurolathyrisme en de verbetering van de voedingswaarde van zaailatyrus. In het eerste


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deel beschrijven we een grondig literatuuroverzicht waarbij de aandacht gaat naar

ontbrekende schakels in onze kennis van het lathyrisme en de consumptie van latyrus.

In het tweede deel van de studie werden onderlinge verbanden aangetoond tussen de

niveau’s van aminozuren in zaailatyrus en de omgeving. In vergelijking met andere

peulvruchten is zaailatyrus een unieke bron van het vrije aminozuren homoarginine,

kwantitatief gevolgd door het vermoedelijk toxische ß-ODAP. Deze moleculen worden

verondersteld het gewas een brede tolerantie te verlenen tegen verschillende vormen van

omgevingsstress. Het ß-ODAP gehalte varieerde sterk naargelang het genotype, ecotype

en de soort voedselbereiding. Zaailatyrus bevat voldoende essentiële aminozuren,

behalve methionine en cysteine. Het methioninegehalte is heel laag, namelijk minder dan

25% van de normale behoefte, en het gehalte is minder variabel dan dat van ß-ODAP. In

optimale groeimedia vertonen beide aminozuren een duidelijk tegengestelde gunstige

wijziging (methionine stijgt en ß-ODAP daalt), wijzend op de mogelijkheid tot simultane

verbetering van deze twee negatieve factoren in de voedingskwaliteit van de plant. De

stress-vrije conditie zou de plant minder stimuleren om ß-ODAP te synthetiseren en meer

aanzetten tot de biosynthese van essentiële aminozuren. Er werd ook aangetoond dat de

manier van bereiding van voedsel de twee aminozuren gunstig beïnvloedt, mogelijk door

het uitwassen van het toxine. Pad-analyse met geselecteerde predictoren toonde tevens

een groter direct effect van K+, uren zonlicht, maturiteit, opbrengst en de verhouding

Zn2+:P op de synthese van ß-ODAP. Er werd aangetoond dat K+, dat vaak in overmaat

aanwezig is in de Ethiopische bodem, de hoeveelheid ß-ODAP sterk kan verhogen. De

Pearson’s correlatiecoefficiënt toonde een sterk negatieve correlatie voor de hoogte, de

maturiteitsperiode, de pH van de bodem en de opbrengst van het gewas, terwijl K+ en shh

een sterk positieve correlatie met ß-ODAP vertonen. De hogere lichtintensiteit op grotere

hoogte, zou de biosynthese van ß-ODAP verhogen. Toch bleek in deze studie de invloed

van het licht redelijk compenserend te zijn, aangezien de lager gelegen percelen in totaal

meer uren zon hadden en de planten er meer ß-ODAP produceerden dan in het gebergte

met een verminderde totale duur zonneschijn in de groeiperiode van de plant. Naast deze

effecten waren de maturiteitsperiode (0.94), gevolgd door regenval tijdens de

ontwikkeling van het jonge het gewas (0.31), de voornaamste directe effecten op de


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opbrengst van het gewas. Ook de combinatie Genotype x Omgeving vertoonde een

significant effect op de opbrengst van zaailatyrus, wijzend op de verschillende reactie van

de variëteiten op een veranderende omgeving.

Het effect van methionine op β-ODAP toxiciteit werd onderzocht aan de hand van

kuikens die gevoed werden met zaailatyrus. De fysiologische en gedrags-responsen van

de kuikens op toevoeging van DL-methionine aan hun dieet van zaailatyrus werden

onderzocht. Dit bevestigde dat de antioxiderende werking van methionine de oxidatieve

stress van β-ODAP tegenwerkt. Naar analogie met andere rapporten verkregen de

kuikens het reversibel convulsief torticolis van de nek bij jonge leeftijd en vertoonden

resistentie bij oudere leeftijd. Symptomen traden enkel op bij een derde van dezelfde

groep kuikens die werd blootgesteld, consistent met eerdere suggesties dat er individuele

verschillen bestaan. Er was een sterk verminderde hoeveelheid β-ODAP in serum ten

opzichte van de ingenomen hoeveelheid, wat fysiologische mechanismen kan suggereren

die kunnen bijdragen tot individuele efficiëntie om zich te wapenen tegen de ziekte. De

verbeterde resistentie van de kuikens bij toevoeging van methionine was deels te wijten

aan de indirecte verbetering van de voedselconversie. Er werd aangetoond dat de graad

van tolerantie van de dieren opmerkelijk verbeterde met de leeftijd, wat kan wijzen op

aanpassing en ontwikkeling van sterkere detoxificatiemechanismen. Bij kippen met

neurologische symptomen werd veel meer β-ODAP aangetroffen in het serum naast een

veel lagere concentratie methionine, wat de antagonistische aard van deze twee

aminozuren suggereert.

Het verrijken van zaailatyrus met methionine wordt beschouwd als een cruciale factor om

het risico van neurolathyrisme te verlagen. Het draagt bij tot de verbetering van de

kwaliteit van het proteïne. Uit gamma-bestraalde zaden werden drie mutantlijnen

geselecteerd met een verschillend fenotype. De R4 regeneraties van de vermeende

mutantlijnen vertonen een verhoging tot 63% van de methionineconcentratie in de zaden

in vergelijking met de controle parentale lijn. Karakterisatie van de selecties toonde aan

dat ze minder rechtop staan met een medium biomassa, dat ze relatief kleinere en

glanzende groene bladeren hadden, medium zaadgrootte en een redelijke graanproductie.


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Ze werden gedefinieerd als diepgroen A-C (DGA, DGB, DGC) overeenkomstig met hun

eigen fenotype. Toch was het β-ODAP gehalte in de nieuwe mutanten niet verschillend

van het wildtype. Het methioninegehalte in de mutante lijnen zou de capaciteit van

zaailatyrus als voeding substantieel kunnen verbeteren en zijn nutritionele kwaliteit en

veiligheid bevorderen. Deze lijnen zouden in de toekomst kunnen gebruikt worden als

genetische bron voor verder onderzoek op de nutritionele kwaliteit van zaailathyrus.


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CURRICULUM VITAE

Full name: Asnake Fikre Woldemedhin Date of birth: 7/12/1972 Birth: Ethiopia, Arsi Citizenship: Ethiopian

Institution: Ethiopian Institute of Agricultural Research /Debre Zeit Center/ Training: Degree

University Duration (date/mon./year) Major field

B.Sc Alemaya Sep. 1, 1989- Sep. 5, 1992 Plant Science M.Sc Alemaya Sep. 1, 1997- July 10, 1999 Agronomy/Breeding PhD Ghent Oct. 2004- Sep. 2008 Applied biological Sciences Other trainings

Place Duration (date/mon./year)

Field of activity

Israel, Mashave

24Nov-15Dec 2004 Ecological Considerations for Sustainable Agricultural Development Projects

ICARDA, Syria

5-9, May, 2002

Specialized training course in pulse breeding and selection

EARO, Melkasa

24-29, Sep. 2001 Training workshop on research planning, monitoring and evaluation

DZARC Feb.-April (two months), 2001 Computer application program Work experience

Position Duration (month & year)

Field of work

Enumerator household survey Dec.-Feb., 1992 Survey on different livelihood aspects of the households in the rural areas data analysis

Junior expert Mar.1993-Oct.1995 Coffee agronomy Expert Dec. 1995- Jan.1997 Crop agronomy and protection Team leader in Mult. And Dissemination of horticultural crops

Feb.-Augst, 1998 Coordinating multiplication of horticultural crops & coffee seedlings

Head Zonal Planning and Economic Development department

March 1999-July 2000 Coordinating and leading integrated developmental activities among different sectors as per the TOR

Pulses breeder Aug 2000- Nov2002 Assistant researcher position design, plan and conducting experiments

National Lentil Project Coordinator and breeder (Asso. researcher)

Dec, 2001-Oct. 2004 Coordinating and leading the program at national level as per the TOR, and conducting breeding and agronomy experiments


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Publications

Fikre A ., Korbu L., Kuo Y.-H., Lambein F. (2008) The contents of the neuro-excitatory amino acid ß-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid), and other free and protein amino acids in the seeds of different genotypes of grass pea (Lathyrus sativus L.). Food Chemistry 110: 422-427. Fikre, A , F. Lambein, F, and Gheysen, G. 2006. A life saving food plant producing more neurotoxin under environmental stresses. Comm. Appl. Biol. Sci. Ghent University, Vol 71(1) 79-82. Fikre, A . 2007. Grass pea. A research review handbook. Ethiopian Institute of Agricultural Research, Addis, Ababa. Fikre, A. and Bejiga, G. Breeding lentil for wider adaptation. In Ali K, Keneni G., Ahmed S., Malhautra R., Bainwal S., Makkouk K and Halila M.H. (eds). 2006. Food and forage legumes of Ethiopia; progress and prospects. Proceeding of the workshop on food and forage legumes22-23 sep. 2003. Addis Ababa, Ethiopia. Fikre, A.,Sarker, A., Ahmed, S.,Ali, K., Halila, H. and Erskine, W. 2006. Science for the poor: an example of lentil research for the Ethiopian (in press), Syria, Aleppo. Getachew A., Asnake F. and Ayalew B. Soil fertility and crop management in highland pulses in central highland of Ethiopia. In Ali K, Keneni G., Ahmed S., Malhautra R., Bainwal S., Makkouk K and Halila M.H. (eds). 2006. Food and forage legumes of Ethiopia; progress and prospects. Proceeding of the workshop on food and forage legumes22-23 sep. 2003. Addis Ababa, Ethiopia. Legesse D., Senait R., Asnake F and Demisie M., 2005. Adoption of chickpea varieties in the central high lands of Ethiopia. Research reports no 62. EARO, Addis Ababa. Senait R., Legesse D., Demisie M., Asnake F and Aden A.H. 2005. Impact of research and technologies in selected lentil-growing areas of Ethiopia. Research reports no 67. EARO, Addis Ababa. Malhotra, R.S., Bejiga, G., Anbessa, Y., Eshete, M., Taddesse, N., Daba, K., Fikre, A ., Ahmed, S. and Khalaf, G. 2007. Registration of 'Teji' a kabuli Chick pea. Journal of plant registration. Crop Science Society of America, USA 1:111. Malhotra, R.S., Bejiga, G., Anbessa, Y., Eshete, M., Taddesse, N., Daba, K., Fikre, A., Ahmed, S. and Khalaf, G. 2007. Registration of 'Ejere' a kabuli Chick pea. Journal of plant registration. Crop Science Society of America, USA 1:112.


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Poster presentations and abstracts Lambein, F., Diasolua, D.N, Fikre, A , Banea Mayambu, J.-P and Kuo Y-H. 2008. Nutritional improvement of grass pea and cassava to prevent lathyrism and konzo. Science with Africa, March 3-7, 2008 Addis Ababa, Ethiopia. Abstract. Lambein, F., Fikre , A. Van Moorhem, M. and Kou, Y.-H. 2007. Lathyrus sativus (grass pea), from toxic plant to wonder crop. Integrating legume biology for sustainable agriculture. Lisbon, Portugal. Abstract. Fikre, A., Lambein, F., and Gheysen, G. 2006. A life saving food plant producing more neurotoxin under environmental stresses. Comm. Appl. Biol. Sci. Ghent University, Vol 71(1) 79-82. Poster presentation. Fikre, A . 2007. Intervention, gap analysis and applicability with regard to grass pea utilization in Ethiopian context. Seminar presentation. DZARC, Ethiopia, Aug 18-22, 2007. Fikre, A., Ahmad, S., Lambein, F. and Gheysen, G. 2007. The incidence of neurolathyrism is strongly linked to age, gender and grass pea food type in Ethiopia. First GAP Symposium: Heritage and/as reproduction in Africa: outcomes and limits, Ghent University. Poster presentation and abstract Fikre, A., Yami, A., Kuo, Y.-H. and Lambein, F. 2007. Protective effect of methionine on acute neurolathyrism in chicks. First GAP Symposium: Heritage and/as reproduction in Africa: outcomes and limits, Ghent University. Poster presentation and abstract. Van Moorhem, M., Fikre, A ., Lambein, F. and Leybaert, L. 2008. Lathyrus sativus (grass pea), from toxic plant to wonder crop. Science with Africa, March 3-7, 2008 Addis Ababa, Ethiopia. Abstract.

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