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Industrial Crops and Products 34 (2011) 943– 951

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Industrial Crops and Products

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egeneration in Jatropha curcas: Factors affecting the efficiencyf in vitro regeneration

weta Sharmaa, Nitish Kumara, Muppala P. Reddya,b,∗

Discipline of Wasteland Research, Central Salt and Marine Chemical Research Institute (CSIR), G-B Marg, Bhavnagar 364002, IndiaPlant Stress Genomics Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 15 April 2010eceived in revised form 18 February 2011ccepted 24 February 2011vailable online 4 May 2011

eywords:geenotypeypocotyl

atropha curcasegeneration

a b s t r a c t

Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explantsof Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to begenotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was leastresponsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L−1

TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiencyas compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds couldbe induced from the distal end of explants in all the genotypes. The number of shoot buds formed andnot the number of explants responding to TDZ treatment were significantly affected by the position ofthe explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile andformed callus easily, whereas the regeneration response declined with increase in age of seedlings after30 days. Transient reduction of Ca2+ concentrations to 0.22 g L−1 in the germination medium increasedthe number of responding explants.

Induced shoot buds, upon transfer to MS medium containing 2 mg L−1 Kn (Kinetin) and 1 mg L−1 BAP(6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS mediumsupplemented with 1.5 mg L−1 IAA (indole-3-acetic acid) and 0.5 mg L−1 BAP and 3.01–3.91 cm elongationwas achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among thetoxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among thetoxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant

differences were observed. The elongated shoot could be rooted by pulse treatment on half-strengthMS medium supplemented with 2% sucrose, 3 mg L−1 IBA (indole-3-butyric acid), 1 mg L−1 IAA, 1 mg L−1

NAA (�-naphthalene acetic acid) and subsequent transfer on 0.25 mg L−1 activated charcoal medium. Therooted plants could be established in soil with more than 90% success. No significant differences wereobserved in rooting of shoots in the different toxic genotypes. However, rooting response was reducedin non-toxic genotype as compared to toxic genotypes.

. Introduction

Jatropha curcas (Euphorbiaceae) has emerged as a promis-ng renewable source for biodiesel (Ghosh et al., 2007) and willecrease the dependence on the depleting fossil fuels. It will alsoelp in utilization of uncultivable abandoned land as it can grown a wide variety of soils and is resistant to various environ-

ental stresses (Francis et al., 2005). Genetic divergence studies

onducted on J. curcas germplasm collected from different geo-raphical regions of the globe indicated narrow genetic diversity,

∗ Corresponding author at: Plant Stress Genomics Research Center, King Abdullahniversity of Science and Technology, Thuwal 23955-6900, Saudi Arabia.el.: +966 2808 2751; fax: +966 2802 0103.

E-mail address: reddymuppalareddy@gmail.com (M.P. Reddy).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.02.017

© 2011 Elsevier B.V. All rights reserved.

a major limitation in genetic improvement of the species (Bashaand Sujatha, 2007; Sudheer, 2008; Sun et al., 2008). In one of ourstudy TDZ (Thidiazuron) was observed to induce somaclonal varia-tions in the hypocotyl explants of J. curcas (Singh, 2009) which canbe explored to increase genetic diversity. Regeneration from thevarious explants of J. curcas is reported in the literature (Jha et al.,2007; Misra et al., 2010; Rajore and Batra, 2005; Sujatha and Mukta,1996; Sujatha et al., 2005; Wei et al., 2004). However, very fewstudies have been made on factors affecting the in vitro responsewhich are known to influence organogenesis (Aneta et al., 1994;Josephina and van Staden, 1990; Kumar and Reddy, 2010; Kumaret al., 2010a,b; Singh et al., 2010; Wu et al., 2009). Efforts were

therefore, made to study the factors like genotype, age, origin ofexplants and medium composition to optimize the regenerationprotocol using TDZ for adventitious shoot regeneration in J. curcashypocotyl segments. Though the genotype dependent regenera-

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44 S. Sharma et al. / Industrial Cro

ion is reported in J. curcas (da Camara Machado et al., 1997), nottempts were further made to study the intra-specific variationsn the regeneration efficiency from hypocotyl segments. To deter-

ine if single regeneration protocol would allow regeneration inepresentatives from diverse areas of adaptation (provenance), fouroxic (CSMCRI JC-1, CSMCRI JC-2, CSMCRI JC-3 and CSMCRI JC-4)nd one non-toxic genotype (CSMCRI JC-NT) were compared foregeneration potential. A non toxic variety reported from Mexicoas been included in the study due to its low or nil phorbol esterontent which makes the oil/seed cake edible (Makkar and Becke,997), otherwise seeds are toxic and the pressed cake due to pres-nce of phorbol esters is not useful as animal feed despite havinghe best protein composition (Makkar et al., 1998). Cultivation ofhe non-toxic variety of J. curcas can provide edible oil and seedake for livestock and value addition to the crop. However, no seri-us attempts were made to perfect the micropropagation protocoln non-toxic variety (Kumar et al., 2010a,b) Further, the effect ofDZ on shoot bud induction from hypocotyl explants collected fromoth in vitro and ex vitro sources were evaluated. Age and originf donor explant affects their physiological environment, whichn turn affects their in vitro hormonal and mineral requirementsAmmirato, 1983). Therefore, to establish a relationship betweenhese factors, effect of explant age, exogenously applied cytokininsCKs) and the position of explant on regeneration efficiency waslso included in our study. Ca2+ is reported to play an important rolen vitro (Amzallag et al., 1992; Hepler and Wayne, 1985; Montorot al., 1995) and modulates TDZ mediated response (Hosseini andashid, 2000; Jones et al., 2007; Mundhara and Rashid, 2006), henceole of Ca2+ on TDZ mediated regeneration in J. curcas was alsoncluded in our studied. Although TDZ has been used to regener-te adventitious shoots in many species, to our knowledge, thiss the first study to report regeneration response from hypocotylsxplants in different genotype and subsequent optimization of therotocol for J. curcas regeneration.

. Materials and methods

.1. Plant material and explant manipulation

Seedlings were raised from seeds collected from selected highielding genotypes of four toxic (CSMCRI JC-1, CSMCRI JC-2, CSM-RI JC-3, CSMCRI JC-4) and one non-toxic (CSMCRI JC-NT) fromentral Salt and Marine Chemical Research Institute experimentallantations, Chorvadla (21◦75′N, 72◦14′E). Throughout the exper-

ment, seeds collected from single plant of respective genotypesere used. For in vitro seedling germination, seeds were surface

terilized with 0.1% HgCl2 for 12 min and rinsed 5 times with sterileouble distilled water. After sterilization, seeds were placed on liq-id MS (Murashige and Skoog, 1962) basal medium supplementedith vitamins, 3% sucrose, 2 mg L−1 glycine, 1 mg L−1 biotin by the

upport of bridges. For studying the response of hypocotyl explantrom ex vitro generated seedlings, seedlings were raised in Green-ouse and at the appropriate age seedlings were harvested. Thexplants were surface sterilized with 0.1% HgCl2 for 18 min andinsed 6 times with sterile double distilled water.

.2. Shoot bud induction

To study the effect of genotype on regeneration response, theypocotyl explants (both in vitro and ex vitro) from four toxic andne non-toxic genotype were cultured on the MS medium sup-

lemented with varying concentrations of TDZ (0.05–3.0 mg L−1)Fig. 2A). To investigate the effect of position of the hypocotyl seg-

ent with respect to cotyledonary node, explants were preparedy dividing hypocotyls into three parts: (1) apical (Ap), (2) middle

Products 34 (2011) 943– 951

(Mi) and (3) proximal (Pr). To investigate the influence of seedlingage on shoot regeneration, hypocotyl explants were collected from15, 30 and 45 days post-germination in vitro seedlings. Observationwas recorded after 6 weeks of culture initiation.

2.3. Elongation, proliferation, rooting and acclimatization ofshoot buds

Further the induced shoot buds of all the genotypes were com-pared for elongation. Induced shoot buds from both toxic andnon-toxic genotypes were sub-cultured on medium optimized inour laboratory (1 mg L−1 BAP and 2 mg L−1 Kn) (Singh, 2009). Shootsfrom different genotypes were individually separated and furtherused for propagation via axillary shoot proliferation on MS mediumsupplemented with 3% sucrose and combination of 0.5 mg L−1 BAPand 1.5 mg L−1 IAA (indole-3-acetic acid). The length of elongatedshoots was recorded after 6 weeks of culture.

Rooting percentage of the shoots was also compared amongall the five genotypes using green and healthy elongated shootswith four to five nodes. Shoots were given pulse treatment forfour days on half strength liquid MS medium supplemented with2% sucrose and 3 mg L−1 IBA (indole-3-butyric acid), 1 mg L−1 IAAand 1 mg L−1 NAA (�-naphthalene acetic acid) (Fig. 2D) and sub-sequently were transferred to ½MS medium amended with 2%sucrose and 0.25 mg L−1 activated charcoal (Fig. 2E). The efficiencyof root induction for different genotypes was recorded after 4weeks. Rooted shoots were carefully taken out of the medium andwashed thoroughly with autoclaved distilled water to remove basalMS medium attached to the roots. The plantlets were transferred topolythene bags containing sterilized sand and soil (1:1) and wettedwith 0.02% (w/v) carbendazole. The polythene bags were coveredwith transparent plastic bags to maintain humidity. After 1 weekpolythene bags were punched, thus decreasing the humidity gradu-ally. After 3–4 weeks, the established plantlets were transplanted topoly-bags containing garden soil and farmyard manure and trans-ferred to a greenhouse for further growth and the numbers ofsurviving plants were recorded after 6–8 weeks.

2.4. Culture condition and statistical analysis

The cultures were maintained at 25 ± 2◦ C under a 16-h photo-period with light intensity of 35–40 �mol m−2 s−1 (cool whitefluorescent tubes). All the experiments were set up in completelyrandomized design and repeated three times with 25 replicates pertreatment and one explant was cultured per test tube. Statisticaldifference among the means was analyzed by Duncan’s multiplerange test using the SPSS ver 7.5 (Snedecor and Cochran, 1989).The results are expressed as the means ± SE of three indepen-dent experiments. Data were also subjected to analysis of variance(ANOVA).

3. Results

3.1. Effect of genotype and explant on shoot bud induction

In agreement with the previous reports, regeneration in J. cur-cas was found to be genotype dependent (da Camara Machadoet al., 1997). The morphogenic response was visible within 30days of culture and ranged from direct shoot bud formation onthe explant, to differentiation of shoots on the callus formed orsome explants remaining undifferentiated after the formation ofcallus (Fig. 1A–C). Swelling at the base of hypocotyl segment

was also observed, irrespective of the hormone concentration.Number of explants responding was directly proportional to theconcentration of TDZ and at high concentration (3.0 mg L−1) cal-lus formation was predominant, thus leading to the decreased

S. Sharma et al. / Industrial Crops and Products 34 (2011) 943– 951 945

Fig. 1. Shoot bud induction in hypocotyl explants at different TDZ concentrations: (A) 0.1 mg L−1; (B)1.5 mg L−1; (C) 3.0 mg L−1 (in vitro explants) and (D) 0.5 mg L−1 (ex vitroexplant).

Table 1Effect of different concentrations of thidiazuron (TDZ), source (S) [in vitro or in vivo] and genotype (G) on percentage response from hypocotyl explants of 30 days old seedlingsof four toxic and a non-toxic genotype of J. curcas.

TDZ (mg L−1) In vitro In vivo

CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JCNT CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JCNT

0.05 34.7 ± 2.5b 27.5 ± 2.4a 31.0 ± 5.0a 42.4 ± 2.31b 21.3 ± 1.11a 0 0 0 0 00.1 68.3 ± 2.6c 55.0 ± 1.9b 62.5 ± 3.2c 76.5 ± 3.0c 39.0 ± 2.2b 0 0 0 0 00.5 78.2 ± 1.6d 59.0 ± 1.5b 70.0 ± 2.2c,d 88.8 ± 3.20d 54.9 ± 1.7c 9.2 ± 0.6a 4.9 ± 0.8a 5.6 ± 0.40a 11.6 ± 0.7a 4.0 ± 0.6a

1 87.5 ± 1.9e 71.3 ± 3.0c 77.1 ± 1.7d 92.9 ± 2.10d 68.1 ± 2.10d 13.2 ± 0.8b 11.8 ± 0.9b 13.8 ± 0.7b 18.8 ± 0.9b 12 ± 0.9b

1.5 91.9 ± 1.8e 78.9 ± 1.1c 82.0 ± 2.2d 42.3 ± 1.60b 87.3 ± 1.80e 19.0 ± 1.4c 19.9 ± 1.5c 14.6 ± 1.8b 29.9 ± 1.4c 18.2 ± 1.2c

3 16.1 ± 1.4a 52.9 ± 3.1b 50.0 ± 3.1b 27.2 ± 2.4a 53.7 ± 3.3c 0 0 0 0 0

ANOVA summary table

Source df MS F

TDZ 5 9143.24 139.12*

S 1 589.24 79.81*

TDZ × S 5 92.22 7.71*

G 4 461.73 117.81*

TDZ × G 20 84.52 7.87*

S × G 4 62.98 11.87*

TDZ × S × G 20 12.91 1.72NSError 120 3.78

Total 179

Mean in each column followed by same letters are not significantly different according to DMRT at = 0.05. NS: Not significant.* Significant at 5% probability level (F test).

946 S. Sharma et al. / Industrial Crops and Products 34 (2011) 943– 951

Fig. 2. (A) Hypocotyl explant. (B) Shoot bud induction in 0.5 mg L−1 TDZ concentration. (C) Shoot buds elongation and proliferation in medium containing 0.5 mg L−1 BAPa ining

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nd 1.5 mg L−1 IAA. (D) Pulse treatment for root induction in liquid medium contaedium. (F) Rooted plantlets in polybag.

esponse (Table 1). Best regeneration was observed at 0.5 mg L−1

DZ in all the genotypes studied (Fig. 2B). Among four toxic and oneon-toxic J. curcas, non-toxic genotype was observed to be leastesponsive (54.9 ± 1.7% response and 4.0 ± 0.6 shoots/explants).mong the four toxic genotypes CSMCRI-JC4 showed the bestesponse (88.8 ± 3.2%) and 16.9 ± 1.1 (shoots/explant) followed bySMCRI-JC1 (78.2 ± 1.6% response) and 14.9 ± 0.8 (shoots/explant).east response was observed in the CSMCRI-JC2 (59.0 ± 1.5%) and1.2 ± 0.3 (shoots/explant).

.2. Effect of source of explant on shoot buds induction

Significant differences in percentage response and the num-er of shoot buds per explant were observed between in vitrond ex vitro generated explants. Maximum response was observedn in vitro explants cultured on TDZ supplemented MS mediumTables 1 and 2). In the ex vitro explants, regeneration efficiencynd number of shoot buds per explant was very low as compared

o the response showed by in vitro explants (Fig. 1D). Percentageesponse ranged from 92.9 ± 2.1 to 16.1 ± 1.4 (in vitro explants) and9.9 ± 1.4 to 4.0 ± 0.6 (ex vitro explants) among all the four geno-ype of toxic and one non-toxic genotype. Number of shoot buds

3 mg L−1 IBA, 1 mg L−1 IAA and 1 mg L−1 NAA 1. (E) Rooting in charcoal containing

per explant also varied from 34.2 ± 0.9 to 2.2 ± 0.8 (in vitro explants)and 8.3 ± 0.7 to 2.9 ± 0.2 (ex vitro explants).

3.3. Effect of age of explant on shoot bud induction

Explants collected from 30 days old in vitro raised seedlingwere best responsive on 0.5 mg L−1 TDZ concentration. At thisconcentration efficiency of regeneration varied from 88.8 ± 3.2 to54.9 ± 1.7 and number of shoot buds induced varied from 16.9 ± 1.1to 11.2 ± 0.3 among the 5 different genotypes (Table 3). Explantscollected from 15 days old seedling, irrespective of genotype,showed callus formation and no regeneration was observed in anyof the explants. Percentage of responding explants significantlydecreased on increasing the age of explants to 45 days and rangedfrom 25.7 ± 1.2 to 8.4 ± 1.4. Number of shoot buds formed was alsoreduced significantly.

3.4. Effect of position of explant on shoot bud induction

The position of explant with respect to cotyledonary node didnot significantly affect the number of explants responding to TDZ(Ap, 90.2 ± 4.4; Mi, 88.8 ± 3.20; Pr, 88.1 ± 1.6 CSMCRI-JC4). How-

S. Sharma et al. / Industrial Crops and Products 34 (2011) 943– 951 947

Table 2Effect of different concentrations of thidiazuron (TDZ), source (S) [in vitro or in vivo] and genotype (G) on the number of shoot buds induced per hypocotyls explants of 30days old seedling of four toxic and a non-toxic genotype of J. curcas.

TDZ (mg L−1) In vitro In vivo

CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JCNT CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JCNT

0.05 6.8 ± 0.9b 3.2 ± 0.60a 5.2 ± 0.60a 8.7 ± 0.8b 3.4 ± 0.4a 0 0 0 0 00.1 11.4 ± 0.8c 6.9 ± 0.8b 10.7 ± 0.7b 14.2 ± 0.3b 5.9 ± 0.7b 0 0 0 0 00.5 14.9 ± 0.8d 11.2 ± 0.3d 12.5 ± 0.5b 16.9 ± 1.1c 13.5 ± 1.0e 5.2 ± 0.7a 4.8 ± 0.7a 4.2 ± 0.4a 6.3 ± 0.6a 2.9 ± 0.2a

1 25 ± 0.9e 10.1 ± 0.4d 22.0 ± 0.8c 29.2 ± 0.7d 10.3 ± 1.6d 6.1 ± 0.8b 7.0 ± 1.6b 5.2 ± 1.1a 7.4 ± 0.5b 4.0 ± 0.4b

1.5 28.1 ± 0.6e 9.6 ± 0.6c 25.5 ± 1.10d 34.2 ± 0.9e 14.1 ± 0.9e 8.3 ± 1.5c 7.4 ± 1.1b 6.0 ± 0.8ab 8.3 ± 0.7c 5.7 ± 0.8b

3 2.2 ± 0.8a 5.5 ± 0.7b 4.4 ± 0.80a 5.7 ± 0.7a 7.8 ± 0.7c Callus Callus Callus Callus Callus

ANOVA summary table

Source df MS F

TDZ 5 163.23 109.76*

S 1 21.54 9.11*

TDZ × S 5 12.22 8.11*

G 4 231.23 173.11*

TDZ × G 20 14.32 7.34*

S × G 4 2.11 1.67NSTDZ × S × G 20 1.31 0.54NSError 120 1.54

Total 179

Mean in each column followed by same letters are not significantly different according to DMRT at = 0.05. NS: Not significant.* Significant at 5% probability level (F test).

Table 3Effect of age of seedling (age) and genotype (G) on the percentage of response of explants and the number of shoot buds from in vitro hypocotyl of four toxic and a non-toxicgenotype of J. curcas at 0.5 mg L−1 TDZ concentration.

Age (days) % explants responded No. of shoot buds/explants

CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JC NT CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JC NT

15 0 0 0 0 0 0 0 0 0 030 78.2 ± 1.6b 59.0 ± 1.5b 70.0 ± 2.2b 88.8 ±3.20b 54.9 ± 1.7b 14.9 ± 0.8b 11.2 ± 0.3b 12.5 ± 0.5b 16.9 ± 1.1b 13.5 ± 1.0b

45 19.1 ± 1.8a 8.4 ± 1.4a 14.9 ± 2.5a 25.7 ± 1.2a 9.6 ± 1.9a 4.6 ± 1.7a 3.3 ± 0.6a 3.8 ± 1.1a 5.4 ± 1.7a 5.2 ± 1.4a

ANOVA summary table

Source % explants responded No. of shoot buds/explants

df MS F df MS F

Age 2 111.91 9.13* 2 193.08 7.78*

G 4 618.25 11.67* 4 57.06 0.93NSAge × G 8 14.76 3.58* 8 6.75 0.41NSError 30 13.07 30 5.19

Total 44 44

Mean in each column followed by same letters are not significantly different according to DMRT at = 0.05. NS: Not significant.* Significant at 5% probability level (F test).

Table 4Effect of genotype and position of explant on the seedlings axis on the percentage of response of explants and the number of shoot buds formed per hypocotyl explant (30days old seedlings) in four toxic and a non-toxic genotype of J. curcas at 0.5 mg L−1 TDZ concentration.

Position % explants responded No. of shoot buds/explant

CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JC NT CSMCRI-JC1 CSMCRI-JC2 CSMCRI-JC3 CSMCRI-JC4 CSMCRI-JC NT

Ap 81.5 ± 2.1a 61.6 ± 1.4a 75.1 ± 1.9a 90.2 ± 4.4a 55.1 ± 3.2a 21.9 ± 1.5c 18.0 ± 0.5c 19.6 ± 1.3c 25.6 ± 1.0c 19.6 ± 1.5c

Mi 78.2 ± 1.6a 59.0 ± 1.5a 70.0 ± 2.2a 88.8 ± 3.20a 54.9 ± 1.7a 14.9 ± 0.8b 11.2 ± 0.3b 12.5 ± 0.5b 16.9 ± 1.1b 13.5 ± 1.0b

Pr 77.3 ± 1.9a 60.3 ± 1.4a 74.3 ± 2.8a 88.1 ± 1.6a 49.5 ± 1.7a 5.3 ± 0.10a 2.1 ± 0.8a 2.9 ± 0.1a 1.50 ± 0.4a 3.6 ± 1.1a

ANOVA summary table

Source % explants responded No. of shoot buds/explant

df MS F df MS F

P 2 11.11 1.13NS 2 93.08 4.01*

G 4 907.49 9.03* 4 87.06 1.03NSP × G 8 3.70 0.51NS 8 9.78 0.42NSError 30 11.03 30 8.13

Total 44 44

Ap, apical; Mi, middle; Pr, proximal. Mean in each column followed by same letters are not significantly different according to DMRT at = 0.05. NS: Not significant.* Significant at 5% probability level (F test).

948 S. Sharma et al. / Industrial Crops and Products 34 (2011) 943– 951

Table 5Effect of different concentrations of thidiazuron (TDZ) (in shoot bud induction medium) and Ca2+ (in germination medium) on the shoot bud induction from hypocotylexplants of J. curcas.

TDZ (mg L−1) Ca2+ (mg L−1)

0 0.22 0.44 0.88

0.05 0 50.30 ± 1.50a 42.30 ± 2.50b 9.0 ± 1.0a

0.1 0 79.0 ± 3.60b 76.30 ± 1.50c 13.70 ± 1.50b

0.5 0 93.3 ± 3.10c 88.30 ± 2.30d 19.0 ± 1.0c

1 0 96.30 ± 2.50c 93.3 ± 3.10d 20.30 ± 1.50c

1.5 0 48.30 ± 1.50a 42.0 ± 2.0b 17.0 ± 1.0c

3 0 49.30 ± 1.50a 27.70 ± 1.20a 15.30 ± 1.50b

ANOVA summary table

Source df MS F

TDZ 5 113.61 2.13*

Ca2+ 3 987.49 11.03*

TDZ × Ca2+ 15 13.70 9.53*

Error 48 12.33

Total 71

M ing to DMRT at = 0.05.

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Table 6Effect of 1.0 mg L−1 BAP and 2 mg L−1 Kn on elongation of induced shoot buds in fourtoxic and a non-toxic genotype of J. curcas.

Genotype Length of shoots

CSMCRI-JC1 2.70 ± 0.70c

CSMCRI-JC2 2.40 ± 0.40b

CSMCRI-JC3 2.80 ± 0.31c

CSMCRI-JC4 2.80 ± 0.20c

CSMCRI-JCNT 2.20 ± 0.30a

ANOVA summary table

Source df MS F

Genotypes 4 4.07 5.1*

Error 15 0.67

Total 19

Mean in each column followed by same letters are not significantly different accord-ing to DMRT at = 0.05.

* Significant at 5% probability level (F test).

Table 7Effect of 0.5 mg L−1 BAP and 1.5 mg L−1 IAA on further proliferation and elongationfrom the elongated shoot buds in four toxic and one non-toxic genotype of J. curcas.

Genotype Number of shoots Length of shoots

CSMCRI-JC1 6.60 ± 0.39b 2.74 ± 0.12b

CSMCRI-JC2 6.73 ± 0.40b 2.15 ± 0.15a

CSMCRI-JC3 6.60 ± 0.20b 2.93 ± 0.25b

CSMCRI-JC4 7.25 ± 0.35c 2.75 ± 0.19b

CSMCRI-JCNT 5.58 ± 0.39a 2.08 ± 0.30a

ANOVA summary table

Number of shoots Length of shoots

Source df MS F df MS FGenotypes 4 5.11 5.22* 4 2.07 3.1*

Error 15 0.97 15 0.63

Total 19 19

Mean in each column followed by same letters are not significantly different accord-

ean in each column followed by same letters are not significantly different accord* Significant at 5% probability level (F test).

ver, the position of the explant on the seedling axis significantlyffected the response of the explants for number of shoot budsormed (Table 4). Regeneration potential of explants increased withn increase in distance from the root and explant adjacent to cotyle-onary node showed the highest response (number of shoot buds).

n CSMCRI-JCNT number of shoot buds increased from 3.6 ± 1.1 inr segment to 19.6 ± 1.5 in Ap segment.

.5. Effect of Ca2+ concentrations in the germination medium onhoot buds induction

Use of 0.5 mg L−1 and 1.0 mg L−1 TDZ resulted in best responset all the Ca2+ concentrations studied. The hypocotyl explantsrom the seedlings raised in medium containing half strengtha2+ concentration (0.22 g mL−1), when cultured on the TDZ sup-lemented medium containing 0.44 g mL−1 Ca2+ (standard MS)howed increased response. This transient decrease in the Ca2+

mount resulted in increased number of responding explants from8.3 ± 2.3 to 93.3 ± 3.1 and 93.3 ± 3.1 to 96.3 ± 2.5 at 0.5 mg L−1 and

mg L−1 TDZ, respectively (Table 5). Absence of Ca2+ in the germi-ation medium led to 100% mortality of explants. Increase in Ca2+

oncentration to 0.88 g mL−1 in the germination medium, resultedn reduced response of explants on TDZ supplemented medium.

.6. Elongation, proliferation, rooting and acclimatization ofhoots

For further elongation of the TDZ induced shoot buds, thexplants were transferred to the sub-culture medium containing

mg L−1 BAP and 2 mg L−1 Kn. Among all the toxic genotypes tested,SMCRI-JC2 showed least response (Table 6). Length of the shootas observed to be least in the non-toxic genotype (CSMCRI-JCNT).

hese shoots when kept for further proliferation and elonga-ion maximum proliferation was obtained on 0.5 mg L−1 BAP and.5 mg L−1 IAA and no significant genotype specific response wasbserved for proliferation among the toxic genotypes exceptSMCRI-JC4 which showed highest number of shoot formationFig. 2C). However, elongation among toxic genotypes was least inSMCRI-JC2, while others genotypes showed no significant differ-

nce in the shoot length and non-toxic genotype showed minimumumber of shoots and elongation (Table 7).

No significant differences were observed, either in percentagef rooting or number of roots per explants among the toxic geno-

ing to DMRT at = 0.05.* Significant at 5% probability level (F test).

types (Table 8). However, non-toxic genotype showed low rooting

response. After 6–8 weeks, approximately 90% of plants survived.No morphological abnormalities were observed in regeneratedplants (Fig. 2F).

S. Sharma et al. / Industrial Crops and

Table 8Rooting percentage in shoots obtained from four toxic and a non-toxic genotypeon hormone free half strength solid MS charcoal medium after four days pulsetreatment with 3.0 mg L−1 IBA, 1.0 mg L−1 IAA and 1.0 mg L−1 NAA containing halfstrength liquid MS medium in J. curcas.

Genotype Rooting percentage

CSMCRI-JC1 60.9 ± 2.30b

CSMCRI-JC2 62.6 ± 2.40b

CSMCRI-JC3 62.4 ± 3.20b

CSMCRI-JC4 61.8 ± 3.30b

CSMCRI-JCNT 45.8 ± 2.40a

ANOVA summary table

Source df MS F

Genotypes 4 211 23.1*

Error 15 3.67

Total 19

Mean in each column followed by same letters are not significantly different accord-ing to DMRT at = 0.05.

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* Significant at 5% probability level (F test).

. Discussion

In the present study, shoot bud induction could be achievedirectly from in vitro and ex vitro hypocotyl explant of toxic andon-toxic genotypes of J. curcas on MS basal medium supplementedith TDZ (N-phenyl-N-1,2,3-thidiazol-5yl urea), a phenylurea

ompound. TDZ is reported to play a major role in the induction ofdventitious shoot regeneration by organogenesis, especially fromypocotyl and has proved to be a potent cytokinin for regenera-ion in J. curcas (Kumar et al., 2010a,b; Reddy et al., 2008; Sujathat al., 2005). It was observed that high TDZ concentrations pro-oted massive callus production on all explant types. The number

f explants responding to TDZ treatment increased with increasingDZ concentrations. The main factors found to affect regenerationn our study were the genotype, source, age, position of explantsnd medium composition.

Genotype/cultivar is one of the most important factors affect-ng regeneration (Conde et al., 2007; Feyissa et al., 2005; Landi and

ezzetti, 2006; Reichert et al., 2003; Rodrıguez et al., 2008). Ashe regeneration in J. curcas was reported to be genotype depen-ent, it was considered important to screen different genotypes of

. curcas for their regeneration potential from hypocotyl explant.n our study, it was observed that all the four genotypes of toxicnd one non-toxic genotype showed differences in percentage ofhoot bud induction and number of shoot buds per explant. Thisifferential behavior can be related to different mechanisms forontrol of the endogenous PGRs metabolism and/or contents. Cellsithin the same plant can have different endogenous levels of plant

rowth regulators (Pellegrineschi, 1997; Schween and Schwenkel,003) and variation in receptor affinity (Minocha, 1987), thus it iseasonable to expect that in vitro response will vary with geno-ype (Bhaskaran and Smith, 1990). Similar results were reportedn Mulberry (Morus) species (Chitra and Padmaja, 2005), Hageniabyssinica (Feyissa et al., 2005) and Fragaria (Landi and Mezzetti,006). Henry et al. (1994) reported that genotypic differences withespect to embryogenesis and regeneration result from quantita-ive or qualitative genetic differences.

The type of explant is very important factor in establishing anfficient regeneration system. Explant used from in vitro sourcesere observed to have a higher rate of regeneration efficiency and

ore number of shoot buds as compared to ex vitro explant in both

oxic and a non-toxic genotypes. This may be due to different levelf endogenous PGRs or can be related to different mechanisms ofontrol of the endogenous PGRs metabolism. Similar results were

Products 34 (2011) 943– 951 949

observed in Paulownia tomentosa Steud. (Ozaslan et al., 2005) and J.curcas (Reddy et al., 2008).

Our results indicated that the age of the explants also affect thecaullogenesis in J. curcas (Fig. 1). Explants from younger seedlings(≤15 days) were still juvenile and formed callus easily, whereasbeyond 30 days, the regeneration response decreased with increasein age of seedlings. Similar results were obtained in Saussureaobvallata where 10–15-day-old seedlings were more regenerative(58%) than younger (5 days) seedlings, and significantly higherthan explants from an older (20 days) source (Dhar and Joshi,2005). It has been reported that younger explants exhibit greatermorphogenic potential as compared to older explants (Welander,1988; Yepes and Aldwinckle, 1994), as they might have moremetabolically active cells with hormonal, nutritional and otherphysiological conditions that are responsible for increased organo-genesis (Famiani et al., 1994).

Experiments with different position of explants collected fromin vitro raised seedling indicated that the regeneration capacity ofhypocotyl segments increased with the increasing distance fromthe root. Hypocotyl segments near the cotyledonary node weremore responsive than the segments near the root. This may be dueto an inhibitory role of the root meristem of main root and lateralspresent on the end of the hypocotyl. Similar results were obtainedby Kameya and Widholm (1981) in Glycine canescens and Fari andCzako (1981) in Capsicum annuum. However, Okubo et al. (1991),reported highest response in basal segments in Antirrhinum majus.

A common requirement for the response of plants cells to dif-ferent hormones is a high external Ca2+ concentration or rise in thelevel of this cation in the tissue (Trewavas, 1999). TDZ mediatedresponse has been reported to be influenced by the Ca2+ (Hosseiniand Rashid, 2000; Yip and Yang, 1986). We observed increase innumber of responding explants when transient Ca2+ stress wasgiven and our results are in agreement with Mundhara and Rashid(2006).

In this investigation, the induced shoot buds were elongated onthe medium containing reduced concentration of cytokinins (BAPand Kn). For shoot elongation and proliferation responses, no sig-nificant differences were noted among the genotype studied exceptin CSMCRI-JC2 and CSMCRI-JCNT. This indicated that shoot elonga-tion and proliferation may not be much effected by the genotype.In the present investigation it was observed that compact shootbuds were induced at high concentration of TDZ (1–2 mg L−1) due towhich shoot elongation was inhibited in subsequent culture. Lowerlevels of TDZ induced relatively fewer shoot buds which devel-oped rapidly into shoots in subsequent culture. Nielsen et al. (1993)reported similar results in Miscanthus sinensis.

One to two shoots grew faster during first two weeks of the cul-ture. When these shoots were excised other surrounding shootselongated within 7–10 days and could be harvested after another2 weeks. Similar observations were reported in other species(Sharma and Amla, 1998; Tawfik and Noga, 2001) suggesting apossible apical dominance effect. Therefore, separation of explantsinto pieces containing individual shoot buds, before transferring tothe shoot proliferation and elongation medium, may enhance shootregeneration efficiency.

Significant differences observed in rooting percentage betweentoxic and non-toxic genotypes may be due to level of endoge-nous PGRs, or can be related to different mechanisms of controlof the endogenous PGRs metabolism and/or contents. Shoots thatdid not responded to rooting treatment were observed to rootafter a time lag, i.e. after a month. The possible reason may bedecrease in endogenous CKs content with time. Endogenous CKs

levels are reported to have a role in adventitious root production(Bollmark et al., 1988). Root formation is usually inhibited whenCKs concentration is sufficiently high to initiate shoot prolifera-tion. A lower concentration of CKs may be necessary to form roots

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50 S. Sharma et al. / Industrial Cro

de Fossard, 1978; Gaspar and Coumans, 1987). CKs in the root-ng medium are known to be inhibitory for rooting (Ruzicka et al.,009; Werner et al., 2003). Whereas, Nemeth (1979) reported itsositive response in some fruit trees.

Regeneration has been extensively used for the rapid multipli-ation of many plant species. However, its more widespread use isestricted often by the high percentage of plants lost or damagedhen transferred to ex vitro conditions. The shoots were thor-

ughly washed with distilled autoclaved water so that the attachedugar enriched media is removed thereby reducing the chances ofungal and bacterial contamination and thus, ultimately mortal-ty. Humidity was gradually reduced. The acclimatization of theooted shoots was accomplished and approximately 90% of thelants were successfully transferred to polybags in greenhouse. Tohe best our knowledge, this is the first study to report a regenera-ion response from hypocotyl explants in different genotypes alongith non-toxic genotype and subsequently optimized the protocol

or J. curcas regeneration.

cknowledgements

Authors are thankful to K.G. Vijay Anand for his help. We arerateful to Dr. P.K. Ghosh Director, CSMCRI for his encourage-ent and Centre for Scientific and Industrial Research, New Delhi,

ndia for financial support (SRF) to pursue this work. The authorsratefully acknowledge Prof. K. Becker, Department of Aquacultureystems and Animal Nutrition, University of Hohenheim, Stuttgart,ermany for providing Mexican non-toxic J. curcas seeds.

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