PUBLISHED ARTICLE

Citation*

Negussie A., Aerts R., Gebrehiwot K., Prinsen E., Muys B. 2009. Euphorbia

abyssinica latex promotes rooting of Boswellia cuttings. New Forests 37, 35-

42.

DOI:10.1007/s11056-008-9106-7

Authors: Aklilu Negussie1, Raf Aerts2*, Kindeya Gebrehiwot1, Els Prinsen3 and

Bart Muys2 1Land Resources Management and Environmental Protection

Department, Mekelle University, P.O. Box 231, Mekelle, Ethiopia 2Division Forest, Nature and Landscape, Katholieke Universiteit Leuven,

Celestijnenlaan 200E-2411, BE-3001 Leuven, Belgium 3Department of Biology, University of Antwerp, Groenenborgerlaan 171,

BE-2020 Antwerpen, Belgium

* Corresponding author: dr. ir. Raf Aerts, Celestijnenlaan 200E-2411,

BE-3001 Leuven (Belgium).

Tel. +32-16-329721; Fax: +32-16-329760

E-mail: raf.aerts@ees.kuleuven.be

Running title: Euphorbia latex and tree propagation

*Springer-Verlag authorized the authors to self-archive this article on their personal website,

with full bibliographic reference to the publication and a link to the published article on

Springer’s website www.springerlink.com (see DOI). The Authors ensure that the publication

by Springer-Verlag is properly credited and that the relevant copyright notice is repeated

verbatim.

Creative Commons Attribution-Noncommercial-Share Alike 2.0 Belgium License

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Euphorbia abyssinica latex promotes rooting 1

of Boswellia cuttings 2

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Aklilu Negussiea, Raf Aertsb*, Kindeya Gebrehiwota, Els Prinsenc and Bart Muysb 4 5 aLand Resources Management and Environmental Protection Department, Mekelle University, 6 P.O. Box 231, Mekelle, Ethiopia 7 bDivision Forest, Nature and Landscape, Katholieke Universiteit Leuven, Celestijnenlaan 200E-8 2411, BE-3001 Leuven, Belgium 9 cDepartment of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerpen, 10 Belgium 11 12

* Corresponding author and address for proofs: 13

dr. ir. Raf Aerts, Celestijnenlaan 200E-2411, BE-3001 Leuven (Belgium) 14

Tel. +32-16-329721 15

Fax: +32-16-329760 16

E-mail: raf.aerts@ees.kuleuven.be 17 18

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Euphorbia abyssinica latex promotes rooting 1

of Boswellia cuttings 2 3

Keywords: Auxins; Ethiopia; IAA; Indole acetic acid; Rooting response; 4

Traditional knowledge 5

6

Abstract 7

Traditional knowledge in northern Ethiopia points towards the root-stimulating properties of the 8 latex found in Euphorbia abyssinica (EAG). Stem cuttings of EAG as well as cuttings of other 9 species treated with EAG latex reportedly root easily in dry soil. This could be attributable to 10 endogenous auxins contained in the latex. We screened EAG latex for auxins and precursor 11 molecules using analytical LC-MS/MS, and tested the effect of EAG latex on the rooting of woody 12 cuttings of Boswellia papyrifera, a threatened multipurpose tree. 13

The EAG latex contained indole acetic acid (IAA) (0.06 µg/g latex), an auxin controlling apical 14 dominance and lateral rooting, as well as IAA metabolites and conjugates. Boswellia cuttings 15 treated with EAG latex rooted significantly sooner and showed higher survival ratios than 16 untreated controls. 17

EAG is widely available and its latex easy to obtain. Therefore it is a promising source of a 18 natural plant growth regulator which may be used for improving low technology vegetative 19 propagation of woody species in East and Northeast Africa, including the threatened Boswellia 20 tree. 21

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Introduction 1

Vegetative propagation of trees is an invaluable tool for reliable and cost-2

effective production of planting material for reforestation. The most 3

common method of vegetative propagation for nursery stock production is 4

the rooted cutting (Romero 2004). The method often involves treating the 5

cuttings with synthetic plant growth regulators (PGRs), typically the auxins 6

indole butyric acid (IBA) and naphthalene acetic acid (NAA) (e.g. Swamy 7

et al. 2002; Tchoundjeu et al. 2002; Husen and Pal 2007). Alternative 8

techniques to stimulate adventitious root formation in cuttings include the 9

inoculation of the rooting media with ectomycorrhizal fungi or rhizobacteria 10

that are able to produce PGRs (Niemi et al. 2002; Teixeira et al. 2007), 11

and the use of commercial concentrates of marine algae which are rich in 12

auxins (Crouch et al. 1992; Crouch and Vanstaden 1993). 13

On the other hand also wild or cultivated plants may be used as 14

PGR sources. In particular root tips, apical buds and young leaves but 15

also cotyledons and expanding leaves are rich in the natural auxin indole 16

acetic acid (IAA) (Ljung et al. 2001; 2005). Extracts from these plant 17

tissues could therefore be used as substitutes for synthetic PGRs and 18

improve the establishment of plants. In Mexico, for example, crude leaf-19

extracts of the Creosote bush (Larrea tridentata Coville) have been shown 20

to stimulate germination and growth of barley seedlings (De la Rosa-Ibarra 21

and Villareal 2000). For rural communities in developing countries, such 22

low technology propagation techniques would be much cheaper, more 23

practical and thus far more effective than the application of synthetic PGRs 24

or micro-organisms. 25

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In northern Ethiopia, traditional knowledge points towards Desert 1

candle (Euphorbia abyssinica J.F. Gmel.) (further referred to as EAG) as a 2

potential source of natural PGRs. This succulent tree of dry deciduous 3

and evergreen montane forest, woodland and shrub savanna is easily 4

propagated from untreated mature branch cuttings. It is therefore widely 5

used as a live fence and in hedge rows to control soil erosion (Fichtl and 6

Admasu 1994). Farmers attribute the ability of large EAG branches to 7

regenerate with ease in dry soil to the milky latex produced in all parts of 8

the plants. As a consequence, it is local (but far from widespread) practice 9

to treat branch cuttings from other tree species with EAG latex to stimulate 10

root formation. The technique is said to be most successful at the end of 11

the dry season. If EAG latex indeed promotes root formation in woody 12

cuttings, it may be related to natural plant growth regulator hormones such 13

as IAA contained in the latex, or to other compounds preventing infection 14

or decay of the cutting. Until now only rubber, certain waxes and resins 15

were identified as its major constituents (Uzabakiliho et al. 1987). 16

Therefore, the aim of this study was to screen EAG latex for 17

endogenous auxins, and to test the effect of the latex on the rooting ability 18

of woody cuttings of the Frankincense tree (Boswellia papyrifera Hochst.), 19

a multipurpose tree that co-occurs with EAG in northeast Africa and that is 20

threatened due to human pressure and environmental degradation 21

(Gebrehiwot et al. 2003; Negussie et al. 2008). 22

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Materials and methods 1

Analysis of endogenous IAA, IAA intermediates and IAA conjugates 2

EAG latex was collected from three locations in Tigray, northern Ethiopia 3

(Jijike, Hagere Selam and Mekelle), and transported in waterproof, dark 4

containers (designed to carry photographic film) to the University of 5

Antwerp (Antwerp, Belgium) for biochemical analysis. Indole acetic acid 6

(IAA), indole-3-ethanol (IEt), indole-3-lactate (ILA) and IAA conjugates 7

were purified from 1 g latex by a combined solid phase extraction 8

procedure following Prinsen et al. (1997). 100 ng 13C6-IAA (Cambridge 9

Isotope Laboratories Inc., Andover, Massachusetts, USA) was used for 10

isotope dilution purposes. The organic acids IAA and ILA in the extract 11

were methylated. 12

Samples containing indole compounds were analysed using a micro 13

HPLC system linked to a mass spectrometer equipped with an electro-14

spray interface (LC/(+)ES-MS/MS) (Quattro II, Waters, Milford, MA, USA). 15

Samples of 25 µl were injected onto a RP-C18 reversed phase column (5µ 16

ODS(3) 100A; 100 x 1.00 mm; Phenomenex, Torrance, CA, USA) and 17

eluted with a methanol – 0.01 M ammonium acetate gradient (1 minute at 18

20% methanol; linear gradient from 20% to 90% methanol in 2 minutes; 19

3.5 minutes at 90% methanol, 5 minutes at 20% methanol, flow 0.06 20

ml·min-1) (HPLC pump, BIO-TEK Instruments, System 522, Waters, 21

Milford, MA, USA) (based on Prinsen et al. 1998). 22

Quantification was performed by multiple reactant monitoring 23

(MRM) of the [MH+] ion and the appropriate product ion (Prinsen et al. 24

1997). The 13C6-IAA tracer was used as internal tracer for the 25

quantifications of ILA and IEt. IAA-conjugates (the total pool of IAA amino 26

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esters and IAA sugar esters) (analysed each from a one-third fraction of 1

the initial sample extract) were analysed as IAA after alkaline hydrolysis in 2

respectively 7 N NaOH at 100°C for 3h (IAA-amino esters) or 1N NaOH at 3

room temperature for 1 hour (IAA-sugar esters) under a water saturated 4

nitrogen stream (Bialek and Cohen 1989; Prinsen et al. 2000). All data 5

were processed by Masslynx software (Waters, Milford, MA, USA). 6

7

Collection of woody plant material and preparation of cuttings 8

Healthy end shoots were obtained in May 2007 from a population of 9

mature Boswellia papyrifera trees in the woodlands of Jijike (13°14’–10

13°42’N; 38°38’–39°02’E; 1 400 to 1 650 m a.s.l), in the lower Geba river 11

catchment in northern Ethiopia. The plant material was transported to the 12

Endayesus tree nursery of Mekelle University (Mekelle, Ethiopia) and 112 13

top branch cuttings were prepared. Cuttings were clipped to a height of 25 14

cm, leaving the top intact. The cuttings had a mean basal diameter of 23 15

mm. 16

17

Treatment of cuttings and follow-up 18

EAG latex for the rooting experiment was obtained from plants growing in 19

the same site as the sampled Boswellia trees. Aqueous EAG 20

concentrations of 50 and 10% (v/v) were obtained by mixing pure latex 21

with distilled water. 22

A single treatment was applied to each cutting using the basal dip 23

method (dipping and immediate removal from the solution; cfr. Negash 24

2003a). There were three latex concentrations (100% EAG, 50% EAG, 25

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10% EAG) and a control (a mixture of methanol, ethanol and distilled 1

water), and each of these was applied to 28 cuttings. 2

After treatment, the cuttings were planted 4 cm deep into the 3

substrate in a non-mist propagator built following the design of Leakey et 4

al. (1990) on the Endayesus tree nursery of Mekelle University (Mekelle, 5

Ethiopia). The basis of the propagator was covered with layers of fine 6

sand (1 cm thick), stones (7 cm thick; stones 3–12 cm diameter) and 7

gravel (7 cm thick). The gravel layer was covered with 10 cm rinsed fine 8

sand (pH 8.7) to serve as a rooting medium that allowed for regular non-9

destructive inspection of rooting in cuttings. The water table in each 10

compartment was maintained at a height of 20 cm from the bottom. The 11

propagators were shaded by a grass roof. Humidity within the propagators 12

was maintained by intermittent water spraying. Mean minimum and 13

maximum temperatures in the propagators were 17 ± 2 °C and 23 ± 5 °C 14

and the relative humidity ranged between 70 and 100 %. 15

Every month, all cuttings were carefully dug up, and the number of 16

rooted cuttings, the number of roots per cuttings and the length of roots 17

were assessed. After measurement, the cuttings were placed back into 18

the substrate without damaging the roots (when present). 19

20

Statistical analysis 21

Rooting and cutting survival over time were analyzed using Kaplan-Meier 22

(KM) survival analysis and the log rank test (Bland and Altman 1998). 23

Non-parametric binomial tests (NPB) were applied to compare the final 24

probability of rooting for all treatments to the results of the control 25

treatment. The effect of treatments on final root number and average root 26

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length was analyzed using Kruskal-Wallis one-way analysis of variance 1

(KW). Statistical tests were performed using SPSS 15.0 (SPSS Inc., 2

Chicago, IL). 3

4

Results 5

Biochemical analysis 6

The EAG latex samples (n = 3) that were chemically analyzed contained 7

indole acetic acid (IAA). The mean concentration (and standard error of 8

the mean) was 350 ± 117 pmole/g latex, which corresponded to 0.06 µg/g 9

or 0.06 mg/l latex. Apart from IAA, the IAA metabolites indole lactate (ILA: 10

477 ± 12 pmole/g latex) and indole ethanol (IEt: 316 ± 73 pmole/g latex) 11

were detected. One sample also contained IAA conjugated to amino-acids 12

(108 pmole/g latex) and glucose (371 pmole/g latex). There were no 13

traces of endogenous IBA. 14

15

Cutting experiment 16

Overall survival of cuttings was 54% and 40% had rooted at the end of the 17

experiment. EAG latex had no significant effect on survival over time (KM 18

χ2 = 3.20; P =0.362) (Fig 1a), but rooting (KM χ2 = 8.39; P = 0.039) 19

differed significantly between treatments (Fig. 1b). Rooting was 20

significantly greater in cuttings treated with 10% EAG latex (NPB P = 21

0.002) and 50-100% EAG latex (NPB P < 0.001) than in the control. 22

23

(Insert Fig. 1) 24 25

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Different levels of EAG concentration did not have an effect on the 1

number of roots (KW χ2 = 1.535; P = 0.674). Average root lengths were 2

slightly lower in cuttings treated with diluted EAG (50 and 10%), but 3

overall, differences in mean root length were not significant between 4

treatments (KW χ2 = 2.402; P = 0.493) (Fig. 2). 5

6

(Insert Fig. 2) 7 8

Discussion 9

Euphorbia latex as natural auxin source 10

The IAA metabolites in EAG latex, ILA and IEt, can be converted to indole-11

3-pyruvic acid (IPA) and indole acetaldehyde (IAAld) respectively. These 12

molecules are produced during tryptophan dependent biosynthesis of 13

auxins in plants. For instance, in Arabidopsis thaliana Schur, de novo IAA 14

biosynthesis initiates from tryptophan (Trp) or Trp precursors (Woodward 15

and Bartel 2005). This does not mean that auxins are actually produced in 16

the EAG latex. Conjugates and intermediates can also be storage or 17

transport forms of hormones in higher plants (Woodward and Bartel 2005). 18

Phytohormones are after all mainly used by plants for long-range signaling 19

between distant organs (Mouchel and Leyser 2007). The presence of IAA 20

in the EAG latex along with its conjugates and intermediates suggests that 21

the latex is used by the plant for remote translocation of IAA from 22

producing sites (e.g. apical meristems) towards tissues where auxin action 23

is needed (e.g. for cell regeneration after wounding). 24

Auxin transport and storage is not a widely recognized function of 25

latex but it may help to explain why adventitious rooting of stem cuttings is 26

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10

fairly easy in some other latex-producing species such as epiphytic figs 1

(Ficus spp.) (e.g. Danthu et al. 2002). On the other hand, this is not a 2

general trend in latex-bearing plants, because endogenous latex can also 3

inhibit rooting of stem cuttings, as observed in for instance Japanese 4

umbrella pine (Sciadopitys verticillata Sieb. & Zucc.) (Yates et al. 2006). 5

Nevertheless, it may be worthwhile to investigate the latex of other 6

members of Euphorbia, a genus with worldwide distribution, or other latex-7

bearing plants such as figs for this interesting property. 8

9

Practical use of Euphorbia latex 10

Berhe and Negash (1998) measured rooting in East-African pencil cedar 11

(Juniperus procera Hochst. ex Endl.) cuttings treated with IAA, IBA, NAA 12

or 2,4-D in concentrations ranging from 10-3 to 10-9 M. Optimal rooting 13

(60% compared to 29% in the control) was obtained using IAA at 10-7 M, 14

which is about the concentration of IAA in pure EAG latex measured in our 15

samples (0.06 mg·l-1 or 3.4·10-7 M). Thus, pure EAG latex and aqueous 16

solutions down to 33% (v/v) are probably the most appropriate for treating 17

cuttings, because these have an IAA concentration in the order of 10-7 M. 18

In our Boswellia cutting experiment, the highest rooting and survival were 19

indeed obtained in the 50% EAG treatment (Fig. 1b). 20

EAG latex is easy to tap but should be handled with care – it is toxic 21

and may cause skin irritations and blindness when applied into the eyes, 22

features shared with the latex of many other members of the 23

Euphorbiaceae such as E. tirucalli L. (Bekele-Tessema et al. 1993). There 24

are other practical limitations to the use of the latex. IAA is unstable when 25

exposed to light or changing temperatures. These problems can be easily 26

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overcome by using fresh latex, transporting it in sealed and dark 1

containers (which also improves its safety) and by diluting the latex only, if 2

necessary, immediately before use. 3

When stored for longer periods, the latex hardens. The total 4

amount of dry matter is between 30 and 40 % of the fresh weight as in 5

related species E. tirucalli and E. candelabrum Trem. ex Kotschy 6

(Uzabakiliho et al. 1987). The hardened latex is not soluble in water and 7

disintegrates in ethyl acetate, but auxins can still be extracted by 8

maceration in a mixture of methanol and ethanol. While this may be 9

interesting for laboratory use, the use of fresh EAG latex is recommended 10

for propagation in field conditions. 11

Several long-living Afromontane species, including J. procera 12

(Berhe and Negash 1998), Olea europaea ssp. cuspidata (Wal. ex G. Don) 13

Cif. (Negash 2003a), Afrocarpus falcatus (Thunb.) C.N. Page (Negash 14

2003b) and Prunus africana (Hook. f.) Kalkman (Tchoundjeu et al. 2002), 15

respond well to auxin treatment in vegetative propagation. Nevertheless, 16

cuttings of O. europaea, Cordia africana Lam. and the Ethiopian endemic 17

Millettia ferruginea Hochst. treated with pure EAG or an EAG maceration 18

showed higher mortality rates and lower rooting success than controls 19

(Aerts R., pers. obs.). As with synthetic auxins, optimal EAG latex 20

concentrations may be species-specific and therefore require further 21

investigation. 22

23

Boswellia woodland restoration 24

Boswellia papyrifera is the source of frankincense, an internationally 25

valued resin and important resource for rural communities in the semiarid 26

12

12

lower highlands of eastern Africa (Gebrehiwot et al. 2003). Throughout its 1

range Boswellia populations are declining due to human-induced fire, 2

improper forest use and agricultural land expansion (Ogbazghi et al. 2006; 3

Lemenih et al. 2007). Even in formally protected woodlands, regeneration 4

remains problematic due to reduced seed viability and seedling mortality 5

(Rijkers et al. 2006; Negussie et al. 2008). In both cases, rooted cuttings 6

may help to overcome these recruitment problems, by avoiding seed and 7

establishment limitation. EAG latex may therefore assist in Boswellia 8

woodland restoration by increasing rooting and survival of cuttings. 9

EAG latex may also be used to adapt the long cutting technique – 10

planting large vegetative stakes ~2-4 m tall (Zahawi 2008) – to dry 11

woodlands. In abandoned Costa Rican pastures, large planted cuttings 12

developed a more extensive rooting system and were more resistant to 13

drought than seedlings (Zahawi and Holl 2008). A priority for this 14

technique to be successful in semiarid ecosystems, is fast rooting of 15

stakes. The use of EAG latex may be a way to achieve this, and 16

therefore, substantially accelerate the return of a healthy population 17

structure in the threatened Boswellia woodlands (Negussie et al. 2008). 18

19

Conclusion 20

Many semiarid areas in northern Ethiopia require increased reforestation 21

efforts. In such areas, Euphorbia abyssinica is usually still widely available 22

because it has persisted environmental degradation and because it has 23

often been planted as a live fence. Its latex contains the endogenous root-24

stimulating auxin IAA, is easy to tap and can be applied to cuttings directly. 25

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Therefore, it may be used for improving vegetative propagation of woody 1

species, including the threatened Boswellia tree. Further research on the 2

auxin metabolism of EAG and other Euphorbia spp. (e.g. to identify plants 3

and plant parts with optimal IAA concentration in the latex) and on the 4

effect of Euphorbia latex on the rooting of stem cuttings of other tree 5

species is recommended to improve and encourage the wider application 6

of Euphorbia latex in traditional and rural forest management. 7

8

Acknowledgements 9

This research was funded by the K.U.Leuven Research Fund and the Flemish Interuniversity 10 Council (VLIR). The authors wish to thank Sevgi Öden for her skilful technical assistance and the 11 Botanical Garden of Leuven (Hortus Botanicus Lovaniensis) for providing Olea europaea cuttings 12 for additional EAG experiments. 13

Informal interviews held by AN with the Bureau of Agriculture and Natural Resources of 14 Maechew and the farmers of Jijike form the basis of this research. The authors explicitly 15 acknowledge the intellectual property rights regarding traditional knowledge of local communities 16 relevant for the conservation and sustainable use of biodiversity, as recommended in Article 8(j) of 17 the UN Convention on Biological Diversity (UNCBD), which aims at protecting these rights and 18 encourages the conservation and wider application of such knowledge. 19 20

References 21

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Figure captions 1

Fig. 1 Survival (A) and rooting (B) of leafy branch cuttings of Boswellia papyrifera (n = 112) 2 treated with different concentrations of Euphorbia abyssinica latex (EAG). 3 4

Fig. 2 Number of roots and average root length of leafy branch cuttings of Boswellia papyrifera 5 treated with different concentrations of Euphorbia abyssinica latex (EAG) after nine months. 6 Only rooted cuttings were used to calculate means and standard errors of means. 7

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Figures 1

Fig. 1 2

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Fig. 2 1

2