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Effect of antioxidants and associate changes in antioxidantenzymes in controlling browning and necrosis ofproliferating shoots of elite Jatropha curcas L

Pratibha Misra*, Dibya D. Toppo, Neha Gupta, Debasis Chakrabarty, Rakesh Tuli**

National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR), Rana Pratap Marg, Lucknow 226001, India

a r t i c l e i n f o

Article history:

Received 30 November 2009

Received in revised form

15 July 2010

Accepted 22 July 2010

Available online 21 August 2010

Keywords:

Antioxidant enzyme

Isoenzymes

Jatropha curcas

Long-term culture

Shoot necrosis

Abbreviations: AdS, adenine sulphate; APXCys, cysteine; EDTA, ethylene diaminetetraaform; GSSG, glutathione, oxidized form; H2ONADPH, b-nicotinamide adenine dinucleotidvinylpyrrolidone; PMSF, phenazine methosumutase; Toco, a-tocopherol; TCA, trichloroa* Corresponding author. Tel.: þ91 522 229792** Corresponding author. Present address:Punjab, India.

E-mail addresses: pratibhamisra@nbri.re0961-9534/$ e see front matter ª 2010 Elsevdoi:10.1016/j.biombioe.2010.07.027

a b s t r a c t

A high yielding elite plant of Jatropha curcas was established under aseptic condition from

field-grown nodal explants. Shoots were proliferated in MS medium supplemented with

0.5 mg dm�3 benzyladenine and 0.1 mg dm�3 indolebutyric acid along with 10 mg dm�3

adenine sulphate and a combination of 15 mg dm�3 each of L-glutamine and L-arginine.

However, within 15e20 d of culture incubation, tissue browning/necrosis leading to poor

plant regeneration in vitro was observed. A set of different antioxidants, namely, reduced

glutathione, ascorbic acid, tocopherol and cysteine were used in the medium individually

and in combination to solve the problem of tissue browning and necrosis. The addition of

antioxidants proved beneficial for the growth of the shoots. The optimum medium

comprised of 25 mg dm�3 reduced glutathione and 10 mg dm�3 ascorbic acid, where

proliferating shoots having highest leaf canopy area, remained fresh, green and regener-

ative up to 40 d of culture incubation without any subculture. The activities of antioxidant

enzymes, such as superoxide dismutase was higher in control shoots, indicating that

tissue browning/necrosis was associated with oxidative stress which was further sup-

ported by higher contents of H2O2 and phenolics in control shoots compared to the other

treatments. Similarly glutathione reductase, ascorbate peroxidase and guiacol peroxidase

was higher in treated shoots than control indicating that these shoots have developed

antioxidant enzymatic protective system which determine the ability to survive in oxida-

tive stress and up regulation of these enzymes would help to reduce the built up of reactive

oxygen species.

ª 2010 Elsevier Ltd. All rights reserved.

, ascorbate peroxidase; AA, ascorbic acid; BA, 6-benzyladenine; BSA, bovine serum albumin;cetic acid; GPX, guiacol peroxidase; GR, glutathione reductase; GSH, glutathione, reduced

2, hydrogen peroxide; IBA, indole-3-butyric acid; MS, Murashige and Skoog’s (1962) medium;e phosphate, reduced form; NBT, nitroblue tetrazolium; OH, hydroxyl radicals; PVP, poly-lfate; ROS, reactive oxygen species; SDS, sodium dodecyl sulphate; SOD, superoxide dis-cetic acid.3; fax: þ91 522 2205836/2205839.National Agrifood Biotechnology Institute (Department of Biotechnology), Mohali-160071,

s.in, pratibhaflora@yahoo.com (P. Misra).ier Ltd. All rights reserved.

b i om a s s an d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 91862

1. Introduction

Biofuel is a locally-available source of energy that not only can

provide energy to meet the increased energy demand derived

from the economic development of developing countries, but

also contributes to climate change mitigation and rural deve-

lopment. Jatropha curcas (Fam. Euphorbiaceae) plant has been

acknowledged as the preferred crop for the purpose which is

drought resistant, perennial and fast growing on poor soil [1]. It

produces oil from the seeds, which can be combusted as fuel

without being refined [2]. It burns with clear smoke-free flame,

tested successfully as fuel for simple diesel engine [3]. Although

there are other sources available as biofuel crops, but being

a non food crop Jatropha cultivation as biodiesel crop ranked

first among all, considering social, economic and humanitarian

reasons [4]. Jatrophahas a lot of interspecific genetic diversity [5]

as well as diversity among different accessions of Indian vari-

eties [6]. The inter-variety spread of oil yield between Jatropha

varieties ranges between 30 and 37%. Therefore, use of elite

variety for planting material can assure a 20% more biodiesel

yield. The present study overcomes hitherto problem of

browning of in vitro cultures of Jatropha [7] and achieves a robust

protocol for large scale propagation of tissue raised plants of its

elite variety. There are few reports on in vitro shoot regenera-

tion from different seedling explants [8,9], nodal explants [10]

and from leaf explants [11]. In most of the reports in vitro

clonal propagation was achieved through organogenesis

[1,10,12], but few are through somatic embryogenesis [13,14].

Although shoot multiplication and their growth is not

a problem in J. curcas, but the regenerating shoots turned

brown/necrosed within 15e20 d of culture incubation. Due to

browning of tissue in Jatropha, it becomes difficult to continue

long-term cultures. Tissue browning generally inhibits the

growth and adventitious shoot formation [15]. The problem of

browning in callus cultures of Jatropha has recently been pub-

lished [7]. Reactive oxygen species such as superoxide radical,

H2O2 and OH radicals have a role in lipid peroxidation,

membrane damage and consequently in leaf senescence. It has

been shown that during leaf senescence, proteins, phospho-

lipids and pigments may be degraded by free radicals as free

Table 1 e Effect of antioxidants on browning of proliferating s

Antioxidantconcentrationmg dm�3

Period after which shootsstarted necrosis (d)

*Mean of numof shoots� S

Control 15 1.7 � 0.09

AA 10 20 2.3 � 0.12

20 17 2.0 � 0.10

GSH 25 25 2.5 � 0.08

40 25 3.3 � 0.09

Toco 40 25 2.3 � 0.08

60 20 2.7 � 0.03

Cyst 10 20 3.0 � 0.06

20 15 2.0 � 0.03

Culture incubation: 30 d.

*Mean of 5 replicate shoots.

AA e ascorbic acid; GSH e glutathione reduced; Toco e tocopherol; Cyst

‘þ’ sign denotes increasing amount, while ‘e’ sign denotes no callus pres

radical scavenging declines [16]. Free radicals have been

involved in programmed cell death, both in animal and in plant

cells [17]. Antioxidant protection involves compounds such as

carotenoids, ascorbic acid, a-tocopherol, glutathione, cysteine,

phenolics and flavonoids [18] and a number of enzymes

includingsuperoxidedismutase (SOD), guiacolperoxidase (GPX)

and ascorbate peroxidase (APX), catalase (CAT) and glutathione

reductase (GR) [19]. SOD is believed to play a crucial role in the

antioxidant systems as it catalyses the dismutation of O2e into

H2O2andO2 [20]. TheconversionofH2O2 intowater iscarriedout

in cytosol and chloroplasts by ascorbateeglutathione cycle,

which involves APX and GR [21].

In the present communication control of browning leading

to necrosis of shoots has been solved with the addition of

antioxidants in the medium. The objective of the present

study was to study changes in antioxidant enzyme activities,

pigment (chlorophylls and carotenoids) concentrations and

total phenolic compounds in relation to the growth and

development of shoots.

2. Material and methods

2.1. In vitro-proliferation of shoots

A high yielding, C2 accession of J. curcas (2 years of age)

procured from Central Salt and Marine Chemicals Research

Institute, Bhavnagar (Institute of CSIR), India, growing in NBRI

campus had been selected as elite tree for taking field-grown

nodal explants for establishment under aseptic condition.

Shoots were proliferated in MS [22] medium containing

15 mg dm�3 each of L-glutamine and L-arginine in the basal

media. A combination of 0.5 mg dm�3 benzyladenine (BA) and

0.1mg dm�3 indole-3-butyric acid (IBA) alongwith 10mg dm�3

adenine sulphate (AdS) was supplemented in the medium for

proliferation and growth of regenerated shoots.

2.2. Effects of antioxidants on growth of cultures

Initially, different antioxidants, like reduced glutathione,

tocopherol, ascorbic acid, and cysteine (two concentrations

hoots of J. curcas.

berE

*Mean of height ofshoots� SE (cm)

*Mean of number ofleaves shoot�1� SE

Inducedcallus

2.2 � 0.1 7.8� 0.3 þþ2.3 � 0.2 6.2� 0.3 þ2.5 � 0.3 8.6� 0.1 þ2.8 � 0.2 10.2� 0.6 e

3.1 � 0.2 12.0� 0.8 þ2.5 � 0.3 11.3� 0.1 e

3.0 � 0.4 10.8� 0.3 þ2.2 � 0.3 12.0� 0.5 e

2.0 � 0.1 10.8� 0.4 þ

e cysteine.

ent.

Table 2 e Responses of in vitro-growing shoots of J. curcas after 40 d of incubation.

Treatments FW� SE(g shoot�1)

DW � SE(mg shoot�1)

*Mean ofnumber ofshoots � SE

*Mean of heightof shoots(cm) � SE

*Mean of numberof leaves

shoot�1 � SE

Canopy leaf area(cm2) � SE

Concentrationmg dm�3

Incubationperiod (d)

Control 20 0.52 � 0. 35.47 � 7.0 1.67 � 0.06 2.24 � 0.1 7.3 � 0.28 6.78 � 0.5

40 1.04 � 0. 51.33 � 8. 1.27 � 0.08 2.54 � 0. 6.2 � 0.34 4.13 � 0.4

40 GSHþ 10 AA 20 1.28 � 0.6 87.2 � 12.5 2.68 � 0.33 2.83 � 1.6 8.2 � 0.54 11.05 � 6.4

40 2.55 � 0.5 153.1 � 25.6 2.63 � 0.06 3.36 � 0.1 10.6 � 0.12 15.21 � 5.3

25 GSHþ 10 AA 20 0.97 � 0.6 69.6 � 3.8 3.52 � 0.63 3.33 � 1.9 8.4 � 0.48 9.11 � 5.3

40 1.74 � 0.6 132.3 � 42.5 3.67 � 0.23 3.44 � 0.1 11.6 � 0.78 21.05 � 6.4

25 GSHþ 40 Toco 20 0.99 � 0.6 72.4 � 3.8 2.82 � 0.33 3.50 � 2.0 9.2 � 0.56 12.35 � 7.1

40 2.27 � 0.2 153.0 � 10.0 3.00 � 0.58 3.20 � 0.1 12.6 � 0.12 15.26 � 7.1

25 GSHþ 10 Cyst 20 0.60 � 0.1 50.4 � 6.2 2.73 � 0.34 2.5 � 1.4 11.0 � 0.49 9.84 � 5.1

40 1.46 � 0.1 99.6 � 15.3 2.33 � 0.23 2.50 � 0.1 7.0 � 0.26 8.75 � 5.1

*Mean of 5 replicate shoots.

AA e ascorbic acid; GSH e glutathione reduced; Toco e tocopherol; Cyst e cysteine.

b i om a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 9 1863

of each) were used individually in the medium to remove

browning of in vitro-proliferating shoots and for their further

growth and development (Table 1). Later, on the basis of their

responses in culture these antioxidants were used in

combination. Four different combinations used were;

50 mg dm�3 glutathione red and 10 mg dm�3 ascorbic acid

(St1); 25 mg dm�3 glutathione red and 10 mg dm�3 ascorbic

acid (St2); 25 mg dm�3 glutathione red and 20 mg dm�3

tocopherol (St3); 25 mg dm�3 glutathione red and 10 mg dm�3

cysteine (St4). Different parameters were recorded after 40 d

of culture incubation and presented in Table 2 and Table 3.

Tocopherol was used from Evion� capsules (400 mg, Merck

Co. Ltd.) with a sterilized syringe and dissolved in 70%

alcohol. It was used directly in sterilized medium.

Half strength basal medium having the optimized

concentration and combination of antioxidants was used for

rooting of isolated shoots. Different concentrations of IBA

were used for root induction. During initial 10e15 d of hard-

ening, high humidity was maintained by covering the plants

with acclimatization hoods and irrigated with tap water.

For next 15 d, acclimatization hoods were raised slightly

decreasing the humidity slowly. Finally, the plants were then

grown under open field conditions.

Table 3 e Chlorophyll estimation in different treatments of an

Cultureincubation (d)

Parameters studies(mg g�1 FW)

C � SE

0 Total chlorophyll 0.66 � 0.12

Chlorophyll a 0.47 � 0.08

Chlorophyll b 0.20 � 0.02

Carotenoids 0.23 � 0.03

15 Total chlorophyll 0.48 � 0.06

Chlorophyll a 0.40 � 0.02

Chlorophyll b 0.08 � 0.01

Carotenoids 0.19 � 0.01

30 Total chlorophyll 0.40 � 0.02

Chlorophyll a 0.29 � 0.02

Chlorophyll b 0.11 � 0.01

Carotenoids 0.14 � 0.01

The pH of all the media was adjusted to 5.8 prior to the

addition of agar and autoclaved at 121 �C for 20 min. All the

media were solidified with 7 g dm�3 agar. Cultures were

incubated at 25� 2 �C under 16 h light and 8 h dark period.

Light at 60 mmol photon m�2 s�1 was supplied by fluorescent

tube lights fitted on culture racks.

2.3. Total phenolics

The content of the total phenolic in leaf extract was deter-

mined (according to Folin-Ciocalteu method) [23] following

the preparation of the caliberation curve where 1 cm3 aliquot

0.024, 0.075, 0.105 and 0.3 mg cm�3 ethanolic gallic acid solu-

tion was mixed with 5 cm3 Folin-Ciocalteu reagent (diluted 10

fold) and 4 cm3 sodium carbonate (75 g dm�3). The absorbance

at 765 nmwasmeasured after 1 h at 20 �C and the caliberation

curve was drawn. To the same reagent (Folin-Ciocalteu

reagent & sodium carbonate) 1 cm3 methanolic plant extract

(10 g dm�3) was mixed as described above and after 1 h the

absorbance was measured. All determinations were per-

formed in triplicates. Total phenolic content in plant meth-

anolic extracts in Gallic Acid Equivalents (GAE) was measured

by the formula:

tioxidants in J. curcas at different time intervals.

Treatments

St1 � SE St2 � SE St3 � SE St4 � SE

e e e e

e e e e

e e e e

e e e e

0.73 � 0.15 0.97 � 0.20 0.87 � 0.18 0.71 � 0.08

0.54 � 0.02 0.74 � 0.17 0.64 � 0.17 0.52 � 0.02

0.19 � 0.09 0.22 � 0.06 0.23 � 0.09 0.19 � 0.01

0.21 � 0.01 0.30 � 0.02 0.30 � 0.10 0.25 � 0.02

0.64 � 0.18 0.84 � 0.23 0.81 � 0.21 0.57 � 0.02

0.51 � 0.18 0.60 � 0.16 0.65 � 0.19 0.47 � 0.02

0.13 � 0.07 0.20 � 0.02 0.16 � 0.03 0.10 � 0.01

0.14 � 0.02 0.30 � 0.02 0.28 � 0.04 0.23 � 0.01

b i om a s s an d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 91864

C ¼ c,V=m

Where, C-total content of phenolic compounds, mg g�1 of

plant extract, in GAE; c-the concentration of gallic acid

deduced from the calibration curve, mg cm�3; V-volume of

extract, cm3; m-the DW of the plant material.

2.4. Estimation of chlorophyll and carotenoids

Fresh leaves (100mg) each from control, St1, St2, St3 and St4

treatments were crushed in 5 cm3 (80%v v�1) chilled acetone

preparedwith TDW. Following the standardmethod (according

to Arnon) [24], extract was centrifuged at 166.67 Hz for 10min.

Absorbanceofsupernatantwasreadat663,645,510and480 nm.

Total chlorophyll content along with its component i.e. chloro-

phyll a and chlorophyll b was calculated in mg g�1 FW using

following formulas (according to Machlachalan and Zalic) [25].

Total Chlorophyll ¼ ½20:2ðA645Þ þ 8:02ðA663Þ� � V1000� V

Chlorophyll a ¼ ½12:7ðA663Þ þ 2:63ðA645Þ� � V1000� V

Chlorophyll b ¼ ½22:9ðA645Þ � 4:68ðA663Þ� � V

1000� V

Carotenoid content was calculated in mg g�1 FW using

following formula (according to Duxbury and Yentsch) [26].

Carotenoid ¼ ½7:6ðA480Þ � 2:63ðA510Þ� � V1000� V

2.5. Measurement of H2O2

Fresh leaves (500 mg) from in vitro regenerated J. curcas shoots

were taken each from control (without antioxidant), and from

different combinations of antioxidants (St1eSt4), at 15 and

30 d culture incubation, for studying H2O2 content. The H2O2

content of the leaves was measured spectrophotometrically

after reaction with potassium iodide (KI) [27]. The reaction

mixture consisted of 0.5 cm3 of 0.1% TCA (v v�1), leaf extract

supernatant, 0.5 cm3 of 10% potassium phosphate buffer

(w v�1, pH 7.8) and 2 cm3 reagent of 100% KI (w v�1), in fresh

double distilled water. The blank probe consisted of 0.1% TCA

(v v�1) in the absence of leaf extract. After 1 h of reaction in

darkness, the absorbance was measured at 390 nm. The

amount of hydrogen peroxidewas calculated using a standard

curve prepared with known concentrations of H2O2.

2.6. Enzyme extraction

Samples of leaves from in vitro cultures of different treatments

of antioxidants were collected at 15 and 30 d of culture incu-

bation and extracted in 10% potassium phosphate buffer

(w v�1, pH 7.8), containing 1% PVP (w v�1) and 0.5% Triton X

100 (v v�1) and 0.01% EDTA (w v�1) for enzymatic study. The

homogenisation was done using liquid nitrogen in the ratio of

1:2 (w v�1) of tissue to buffer for enzymatic activity. The

homogenate was centrifuged in a refrigerated centrifuge at

250 Hz for 30 min at 4 �C (Eppendorf). The supernatant was

collected, aliquoted and stored at �80 �C for subsequent

analysis. All operations were conducted at 0e4 �C.

2.7. Protein determination

Total protein of all the samples was determined (according to

Bradford) [28] using bovine serum albumin (BSA) as a stan-

dard. Enzyme activities and isoenzyme patterns were deter-

mined by themethods specific to each enzyme and isoenzyme

as described below. All enzyme assays were performed at

25 �C and activity was measured spectrophotometrically on

PerkinElmer, Lambda 35, UV/VIS Spectrometer.

2.8. Antioxidant enzyme activity

For measuring superoxide dismutase (SOD, EC 1.15.1.1)

activity (according to Nishikimi et al.) [29], the reaction

mixture contained 52% sodium pyrophosphate buffer (w v�1,

pH 8.3), 0.1% EDTA (w v�1), 0.018% PMSF (w v�1), 0.03% NBT

(w v�1) and 0.078% NADH (w v�1). Reaction was started by

addingNADH. Reactionmixturewas incubated at 30 �C for 90 s

and stopped by adding 1 cm3 glacial acetic acid and stirred

vigorously. Colour intensity of the chromogen in the reaction

mixture was measured at 560 nm against sodium pyrophos-

phate buffer. One unit of enzyme activity is defined as the

amount of enzyme required to inhibit the NBT reduction by

50%, under the assay conditions.

Total GPX (EC 1.11.1.7) activity was determined (according

to Hemeda and Klim) [30] monitoring the formation of tetra-

guaiacol from guaiacol. The reaction mixture contained 10%

sodiumphosphate buffer (w v�1, pH 5.8), 1% guiacol (v v�1) and

0.3% H2O2 (v v�1). Increase of absorbance due to oxidation of

guiacol was recorded at 470 nm every 15 s for 3 min.

For measuring APX (EC 1.11.1.11) activity (according to

Nakano and Asada) [31], the reaction mixture contained 5%

potassium phosphate buffer (w v�1, pH 7.0), 1.0% sodium

ascorbic acid (w v�1), 0.1% H2O2 (v v�1) and 0.1% EDTA (w v�1).

The reaction mixture started by addition of H2O2. The

decrease in absorbance was recorded at 290 nm at an interval

of 15 s up to 3 min per cycle period. The blank was determined

by the addition of phosphate buffer instead of extracts in the

reaction mixture.

For measuring the activity of GR (EC 1.6.4.2) (according to

Smith et al.) [32], the reaction mixture contained 20% potas-

sium phosphate buffer (w v�1, pH 7.5), containing 0.1% EDTA

(w v�1), 0.3% 5-50dithiobis (2-nitrobenzoic acid, w v�1) in 1.0%

potassium phosphate buffer (w v�1, pH 7.5), 0.2% NADPH

(w v�1) and 2.0% GSSG (w v�1). All the reagents were added

directly to cuvette and reaction was started by addition of

GSSG andwasmonitored for 5 min at 412 nm. Enzyme activity

was expressed as picomoles of GSSG reduced min�1 g�1 FW.

GSSG was replaced by water in the reaction mixture as blank.

2.9. Statistical analysis of data

Morphological changes were recorded by visual observation.

The values of data in case of in vitro shoots are presented as

mean� SE of five replicate cultures. All other analyses were

carried out in triplicate. Duncan’s multiple-range test was

used as a post hoc comparison of statistical significance

( p values<0.05). All statistical analyses were performed using

Statistical for Windows (version 5.1, StatSoft, Inc., Tulsa, OK).

b i om a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 9 1865

3. Results and discussion

3.1. Effects of antioxidants on growth of cultures

Shoot proliferation started in MSmedium having 15mg dm�3

L-glutamine and L-arginine supplemented with 0.5 mg dm�3

BA and 0.1 mg dm�3 IBA along with 10 mg dm�3 AdS, but leaf

margins started turning yellowish brown within 15 d of

culture incubation (Fig. 1a, Table 1). Later, whole leaves

necrosed within 30 d (Fig. 1b). Therefore, initially, two

concentrations of each of four antioxidants were incorpo-

rated in the basal media individually. With the addition of

antioxidants, overall growth of shoots was observed better

and the shoots remained fresh, green and healthy for

a longer period as against the shoots without antioxidants.

Regeneration of shoots was not affected with the addition or

lack of antioxidants in the medium. Lower concentrations of

all the antioxidants used were more effective to keep the

cultures fresh and green, as compared to higher concentra-

tions except GSH where shoots grew more vigorous than the

control but those were associated with callus formation

(Fig. 1c). Both the concentrations of GSH were almost equally

effective to keep the shoots fresh and green up to 25 d beyond

which shoots started browning. The shoots growing in lower

concentrations of antioxidants did not show associated

callus except AA. The AA (10 or 20 mg dm�3) was least

effective to remove browning as well as regeneration of

shoots in in vitro-growing cultures (Fig. 1d, Table 1). Similar

results had been observed by Zheng et al. [33] in peanut

regeneration, where they found the highest regeneration

with the addition of GSH, then tocopherol and least in the

Fig. 1 e Effect of antioxidants on shoot growth of Jatropha curcas;

browning and necrosis of leaves in control after 30 d of incubat

glutathione; (d) 20 mg dmL3 ascorbic acid; (e) 40 mg dmL3 toco

glutathione D 10 mg dmL3 ascorbic acid; (h) 25 mg dmL3 gluta

tocopherol D 10 mg dmL3 ascorbic acid; (j) 10 mg dmL3 cystein

presence of ascorbic acid. They explained that AA is an

unsuitable antioxidant in MS medium due to the stimulation

of oxidation in the presence of iron in MS media. In the

present communication, addition of tocopherol resulted in

sturdier shoots with more number of leaves (Fig. 1e),

whereas addition of cysteine did not prove beneficial to

protect the browning of cultures, although an average of 3.2

shoots regenerated in its lower concentration (Fig. 1f).

Similar to our results Grewal et al. [34] also found increased

regeneration with the addition of cysteine in Indica rice.

From this experiment, GSH was selected as the most effec-

tive antioxidant, as in its presence, the shoots remained

fresh, green and healthy for about 25 d. The role of gluta-

thione is already known in promoting callus and shoot

growth in Malus pumila [35], as stimulant of shoot organo-

genesis in Gladiolus hybridus [36], and also in controlling

hyperhydricity, tissue necrosis and increased plant regen-

eration in Ramonda myconi [37].

In the next experiment GSH was used with addition of

lower concentrations of AA, tocopherol and cysteine in

separate four treatments (St1eSt4). Data has been recorded

at 20 and 40 d and presented in Table 2. In the presence of

higher concentration of glutathione (40 mg dm�3) along

with AA (10 mg dm�3) in St1, shoots grew up to 3.36 cm of

height but necrosis of shoots started after 25 d of culture

incubation (Fig. 1g). However, lowering the concentration

of glutathione to 25 mg dm�3 in St2 treatment resulted in

growth of proliferating shoots with highest leaf canopy

area. Shoots remained fresh, green and regenerative up to

40 d of culture incubation without any subculture (Fig. 1h).

The combination of 25 mg dm�3 GSHþ 40 mg dm�3

tocopherol (St3) also proved beneficial as a maximum

(a) yellowing of leaves in control after 15 d of incubation; (b)

ion; (cej) cultures after 30 d of incubation, (c) 50 mg dmL3

pherol; (f) 10 mg dmL3 cysteine; (g) 50 mg dmL3

thione D 10 mg dmL3 ascorbic acid; (i) 40 mg dmL3

e D 10 mg dmL3 ascorbic acid.

Fig. 2 e Propagation of J. curcas; (a) in vitro-proliferation of shoots; (b) in vitro rooting; (c) 4-wk-old in vitro-raised plants in pots.

Fig. 3 e Estimation of total phenolic compounds in in vitro

shoots of J. curcas at 0, 15 and 30th d of culture incubation.

b i om a s s an d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 91866

number of 12.6 leaves could be developed in this treatment,

but the shoots formed started browning after 35 d of

culture incubation (Fig. 1i). In the presence of cysteine

(10 mg dm�3, St4) best regeneration of shoots was observed

but browning started after 23 d of culture incubation

(Fig. 1j). The importance of AA has been shown in

controlling lethal browning and disease incidence of tissue

culture shoots in Cavendish banana [38]. Tocopherol was

reported beneficial for stimulated shoot organogenesis in G.

hybridus [36], while cysteine in Dactylis glomerata [39]. On

the basis of our results obtained, St2 (25 mg dm�3

GSHþ 10 mg dm�3 AA) was selected as the best treatment

for biomass accumulation, where shoots remained fresh,

green, healthy and regenerative up to 40 d. After excision of

isolated grown-up shoots, the remaining regenerative

small shoots had been subcultured into fresh medium and

like this it became a repository of shoots for a long-term

period.

Fig. 4 e Estimation of H2O2 concentration in in vitro shoots

of J. curcas at different time intervals.

b i om a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 9 1867

Grown-up shoots proliferating in St2 treatment (Fig. 2a)

were isolated and used for rooting. Half strength St2 medium

without any growth supplement was used as the basal

medium for rooting. Eighty percent rooting could be achieved

in the presence of 0.5 mg dm�3 IBA within 3 wk of culture

incubation (Fig. 2b). The rooted shoots/plantlets were trans-

ferred directly to the pots filled with sterilized soilrite. After

Fig. 5 e Antioxidant enzyme activity in J. curcas shoots after 30

GPX activity; and (d) GR enzyme activity. *Values followed by th

Duncan’s multiple-range test at 5% level.

4 wk, plants were shifted to net house, where they grew nor-

mally (Fig. 2c).

3.2. Effects of total phenolics, chlorophyll content andH2O2 concentration on growth of proliferating shoots

In the beginning, healthy shoots were inoculated in the

control, which remained green and healthy up to 15 d. After

15 d, leaves started yellowing resulting into necrosis of shoots

within 30 d. Different parameters, like, chlorophyll content,

total phenolics, and H2O2 concentration were studied at 0, 15

and 30 d intervals and data is presented in Table 3 and in Figs.

2 and 3. The growth behavior of shoots in control plants could

be comparable to the decreasing concentration of total chlo-

rophyll content or increasing concentration of phenolic

compounds and H2O2 from 0, 15 and 30 d. Addition of anti-

oxidants either individually or in combination, increased the

total chlorophyll content (Table 3) and decreased the total

phenolics and H2O2 concentration resulting in fresh, green

and healthy growth of the shoots (Figs. 3 and 4, respectively).

On assessing the different combinations of antioxidants,

the addition of glutathione (40 mg dm�3) along with AA

(10 mg dm�3, St1), total phenolics as well as H2O2 concentra-

tion, was decreased lower than the control. The concentration

of total phenolics as well as H2O2 was further decreased when

a lower concentration of glutathione (25 mg dm�3) was used.

d of culture incubation; (a) SOD activity; (b) APX activity; (c)

e same letters are not significantly different according to

Fig. 6 e APX isoenzymes in J. curcas shoots in different

treatments of antioxidants (from L to R-C, St1, St2, St3 and

St4) after 30 d of culture incubation.

b i om a s s an d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 91868

The results are compatible with the data presented in Table 3

showing higher chlorophyll content in St2 than St1. Total

chlorophyll content was more after 15 d than in 30 d, in all the

treatments including the control. Addition of tocopherol to

the medium again resulted in decrease of total phenolics as

well as H2O2 than the control but the decrease was less than

St1 and St2, while total chlorophyll content (higher but not

the highest) resulted in greening and better growth of leaves/

shoots. However, the addition of cysteine, could not sustain

good growth, freshness and green colour of the shoots due to

higher phenolic content and higher H2O2 concentration

which caused stress to the regenerating shoots. Total chlo-

rophyll content was also the least in St4 resulting in the

yellowing of shoots.

3.3. Antioxidant enzyme activity

High SOD activity increases concentration of H2O2 in the

tissue which triggers programmed cell death ultimately

causing browning and tissue necrosis. In J. curcas shoots, SOD

activity was found very high in the control which subse-

quently increases H2O2 content that could possibly lead to

oxidative damage, a ‘negative’ consequence of its presence

and a common response to many environmental stresses

leading to shoot browning/necrosis. Addition of different

antioxidants reduces SOD activity resulting in fresh, green

and healthy shoots. St2 treatment showing the lowest SOD

activity was found as the optimum treatment for the growth

of the cultures (Fig. 5a). Similar results of decreased SOD

activity have been reported inducing shoot organogenesis in

gladiolus [36]. It has been reported in chrysanthemum that

a decrease in the activity of a number of antioxidant enzymes

that normally prevent the buildup of free radicals can be

accounted for senescence in plants [19].

APX, GR and GPX decreased in treatments St1 and St2 as

compared to control (Fig. 5b and d). APX plays a key role in the

ascorbateeglutathione cycle by reducing H2O2 to water using

the reducing power of ascorbate and producing mono-

dehydroascorbate (MDHA). Since >90% of total leaf APX

activity is localized in the chloroplast [40], this activity can

represent the best estimation of chloroplast-based detoxifi-

cation of ROS via the Mehlerperoxidase pathway as sink for

reducing equivalents. Native PAGE is also supporting the

activity data, and a single band was clearly visible in all the

treatments (Figs. 5b and 6). Since it has already been observed

that chlorophyll content was highly reduced in control indi-

cating chloroplast degradation under necrotic condition

potentially increasing the probability of generating ROS

(Table 3). According to Marchner and Possingham oxidative

stress resulted in a reduction in the number of chloroplasts

[41]. GPX is involved in the removal of hydrogen peroxide and

organic peroxides [42] showing the increased activity in St1

and St2 as compared to control (Fig. 5c). Similarly, GR is

involved in the recycling of reduced glutathione to maintain

a constant intracellular concentration of GSH [43]. Elevated

concentrations of GSH are associatedwith increased oxidative

stress tolerance which is found true in our study (Fig. 5d).

The finding that control shoots had significantly lower

chlorophyll and higher phenolic contents than healthy shoots

which led us to conclude that control shoots after 30 d of

culture caused a substantial decline in photosynthesis

because the antioxidant enzyme system was swamped by

a massive accumulation of H2O2, the production of which is

favoured when metabolic pathways are down-regulated. The

resulting imbalance between H2O2 production and antioxi-

dants defences resulted in oxidative damage of the photo-

synthetic machinery in the chloroplasts resulting in tissue

browning/necrosis. However, supplementation of antioxi-

dants like GSH/AA resulting in increase antioxidant metabo-

lism in determining the ability of the plants to survive in

oxidative stress, an up regulation of these enzymes would

help to reduce the built up of H2O2.

The problem of necrosis is the main constraint in the large

scale propagation of J. curcas to obtain quality planting mate-

rial including higher biomass in long-term culture. In the

present study, we have manipulated the problem by using

different antioxidants and their combinations in the medium

so as to obtain long-term proliferating cultures of J. curcas

which remained fresh, green, healthy and regenerative up to

40 d of culture incubation.

Acknowledgements

The authors are thankful to the Council of Scientific and

Industrial Research, New Delhi, Govt. of India, for providing

the financial support to carry out this work.

b i om a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 1 8 6 1e1 8 6 9 1869

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