Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration

Industrial Crops and Products 34 (2011) 943–951

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Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration Sweta Sharma a , Nitish Kumar a , Muppala P. Reddy a,b,∗ a b

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

a r t i c l e

i n f o

Article history: Received 15 April 2010 Received in revised form 18 February 2011 Accepted 24 February 2011 Available online 4 May 2011 Keywords: Age Genotype Hypocotyl Jatropha curcas Regeneration

a b s t r a c t Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. 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 efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca2+ concentrations to 0.22 g L−1 in the germination medium increased the 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 medium supplemented with 1.5 mg L−1 IAA (indole-3-acetic acid) and 0.5 mg L−1 BAP and 3.01–3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic 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-strength MS 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. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Jatropha curcas (Euphorbiaceae) has emerged as a promising renewable source for biodiesel (Ghosh et al., 2007) and will decrease the dependence on the depleting fossil fuels. It will also help in utilization of uncultivable abandoned land as it can grow on a wide variety of soils and is resistant to various environmental stresses (Francis et al., 2005). Genetic divergence studies conducted on J. curcas germplasm collected from different geographical regions of the globe indicated narrow genetic diversity,

∗ Corresponding author at: Plant Stress Genomics Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia. Tel.: +966 2808 2751; fax: +966 2802 0103. E-mail address: [email protected] (M.P. Reddy). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.02.017

a major limitation in genetic improvement of the species (Basha and Sujatha, 2007; Sudheer, 2008; Sun et al., 2008). In one of our study TDZ (Thidiazuron) was observed to induce somaclonal variations in the hypocotyl explants of J. curcas (Singh, 2009) which can be explored to increase genetic diversity. Regeneration from the various 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 few studies have been made on factors affecting the in vitro response which are known to influence organogenesis (Aneta et al., 1994; Josephina and van Staden, 1990; Kumar and Reddy, 2010; Kumar et al., 2010a,b; Singh et al., 2010; Wu et al., 2009). Efforts were therefore, made to study the factors like genotype, age, origin of explants and medium composition to optimize the regeneration protocol using TDZ for adventitious shoot regeneration in J. curcas hypocotyl segments. Though the genotype dependent regenera-

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tion is reported in J. curcas (da Camara Machado et al., 1997), no attempts were further made to study the intra-specific variations in the regeneration efficiency from hypocotyl segments. To determine if single regeneration protocol would allow regeneration in representatives from diverse areas of adaptation (provenance), four toxic (CSMCRI JC-1, CSMCRI JC-2, CSMCRI JC-3 and CSMCRI JC-4) and one non-toxic genotype (CSMCRI JC-NT) were compared for regeneration potential. A non toxic variety reported from Mexico has been included in the study due to its low or nil phorbol ester content which makes the oil/seed cake edible (Makkar and Becke, 1997), otherwise seeds are toxic and the pressed cake due to presence of phorbol esters is not useful as animal feed despite having the best protein composition (Makkar et al., 1998). Cultivation of the non-toxic variety of J. curcas can provide edible oil and seed cake for livestock and value addition to the crop. However, no serious attempts were made to perfect the micropropagation protocol in non-toxic variety (Kumar et al., 2010a,b) Further, the effect of TDZ on shoot bud induction from hypocotyl explants collected from both in vitro and ex vitro sources were evaluated. Age and origin of donor explant affects their physiological environment, which in turn affects their in vitro hormonal and mineral requirements (Ammirato, 1983). Therefore, to establish a relationship between these factors, effect of explant age, exogenously applied cytokinins (CKs) and the position of explant on regeneration efficiency was also included in our study. Ca2+ is reported to play an important role in vitro (Amzallag et al., 1992; Hepler and Wayne, 1985; Montoro et al., 1995) and modulates TDZ mediated response (Hosseini and Rashid, 2000; Jones et al., 2007; Mundhara and Rashid, 2006), hence role of Ca2+ on TDZ mediated regeneration in J. curcas was also included in our studied. Although TDZ has been used to regenerate adventitious shoots in many species, to our knowledge, this is the first study to report regeneration response from hypocotyls explants in different genotype and subsequent optimization of the protocol for J. curcas regeneration. 2. Materials and methods 2.1. Plant material and explant manipulation Seedlings were raised from seeds collected from selected high yielding genotypes of four toxic (CSMCRI JC-1, CSMCRI JC-2, CSMCRI JC-3, CSMCRI JC-4) and one non-toxic (CSMCRI JC-NT) from Central Salt and Marine Chemical Research Institute experimental plantations, Chorvadla (21◦ 75 N, 72◦ 14 E). Throughout the experiment, seeds collected from single plant of respective genotypes were used. For in vitro seedling germination, seeds were surface sterilized with 0.1% HgCl2 for 12 min and rinsed 5 times with sterile double distilled water. After sterilization, seeds were placed on liquid MS (Murashige and Skoog, 1962) basal medium supplemented with vitamins, 3% sucrose, 2 mg L−1 glycine, 1 mg L−1 biotin by the support of bridges. For studying the response of hypocotyl explant from ex vitro generated seedlings, seedlings were raised in Greenhouse and at the appropriate age seedlings were harvested. The explants were surface sterilized with 0.1% HgCl2 for 18 min and rinsed 6 times with sterile double distilled water. 2.2. Shoot bud induction To study the effect of genotype on regeneration response, the hypocotyl explants (both in vitro and ex vitro) from four toxic and one non-toxic genotype were cultured on the MS medium supplemented with varying concentrations of TDZ (0.05–3.0 mg L−1 ) (Fig. 2A). To investigate the effect of position of the hypocotyl segment with respect to cotyledonary node, explants were prepared by dividing hypocotyls into three parts: (1) apical (Ap), (2) middle

(Mi) and (3) proximal (Pr). To investigate the influence of seedling age on shoot regeneration, hypocotyl explants were collected from 15, 30 and 45 days post-germination in vitro seedlings. Observation was recorded after 6 weeks of culture initiation. 2.3. Elongation, proliferation, rooting and acclimatization of shoot buds Further the induced shoot buds of all the genotypes were compared for elongation. Induced shoot buds from both toxic and non-toxic genotypes were sub-cultured on medium optimized in our laboratory (1 mg L−1 BAP and 2 mg L−1 Kn) (Singh, 2009). Shoots from different genotypes were individually separated and further used for propagation via axillary shoot proliferation on MS medium supplemented with 3% sucrose and combination of 0.5 mg L−1 BAP and 1.5 mg L−1 IAA (indole-3-acetic acid). The length of elongated shoots was recorded after 6 weeks of culture. Rooting percentage of the shoots was also compared among all the five genotypes using green and healthy elongated shoots with four to five nodes. Shoots were given pulse treatment for four days on half strength liquid MS medium supplemented with 2% sucrose and 3 mg L−1 IBA (indole-3-butyric acid), 1 mg L−1 IAA and 1 mg L−1 NAA (␣-naphthalene acetic acid) (Fig. 2D) and subsequently were transferred to ½MS medium amended with 2% sucrose and 0.25 mg L−1 activated charcoal (Fig. 2E). The efficiency of root induction for different genotypes was recorded after 4 weeks. Rooted shoots were carefully taken out of the medium and washed thoroughly with autoclaved distilled water to remove basal MS medium attached to the roots. The plantlets were transferred to polythene bags containing sterilized sand and soil (1:1) and wetted with 0.02% (w/v) carbendazole. The polythene bags were covered with transparent plastic bags to maintain humidity. After 1 week polythene bags were punched, thus decreasing the humidity gradually. After 3–4 weeks, the established plantlets were transplanted to poly-bags containing garden soil and farmyard manure and transferred to a greenhouse for further growth and the numbers of surviving 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 photoperiod with light intensity of 35–40 ␮mol m−2 s−1 (cool white fluorescent tubes). All the experiments were set up in completely randomized design and repeated three times with 25 replicates per treatment and one explant was cultured per test tube. Statistical difference among the means was analyzed by Duncan’s multiple range test using the SPSS ver 7.5 (Snedecor and Cochran, 1989). The results are expressed as the means ± SE of three independent 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. curcas was found to be genotype dependent (da Camara Machado et al., 1997). The morphogenic response was visible within 30 days of culture and ranged from direct shoot bud formation on the explant, to differentiation of shoots on the callus formed or some explants remaining undifferentiated after the formation of callus (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 the concentration of TDZ and at high concentration (3.0 mg L−1 ) callus formation was predominant, thus leading to the decreased

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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 vitro explant). Table 1 Effect 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 seedlings of four toxic and a non-toxic genotype of J. curcas. TDZ (mg L−1 )

In vitro

In vivo

CSMCRI-JC1 0.05 0.1 0.5 1 1.5 3

34.7 68.3 78.2 87.5 91.9 16.1

± ± ± ± ± ±

2.5b 2.6c 1.6d 1.9e 1.8e 1.4a

CSMCRI-JC2 27.5 55.0 59.0 71.3 78.9 52.9

± ± ± ± ± ±

2.4a 1.9b 1.5b 3.0c 1.1c 3.1b

CSMCRI-JC3 31.0 62.5 70.0 77.1 82.0 50.0

± ± ± ± ± ±

5.0a 3.2c 2.2c,d 1.7d 2.2d 3.1b

CSMCRI-JC4

CSMCRI-JCNT

42.4 ± 76.5 ± 88.8 ± 92.9 ± 42.3 ± 27.2 ±

21.3 39.0 54.9 68.1 87.3 53.7

2.31b 3.0c 3.20d 2.10d 1.60b 2.4a

± ± ± ± ± ±

1.11a 2.2b 1.7c 2.10d 1.80e 3.3c

CSMCRI-JC1 0 0 9.2 ± 0.6a 13.2 ± 0.8b 19.0 ± 1.4c 0

CSMCRI-JC2 0 0 4.9 ± 0.8a 11.8 ± 0.9b 19.9 ± 1.5c 0

CSMCRI-JC3 0 0 5.6 ± 0.40a 13.8 ± 0.7b 14.6 ± 1.8b 0

ANOVA summary table Source

df

MS

F

TDZ S TDZ × S G TDZ × G S×G TDZ × S × G Error

5 1 5 4 20 4 20 120

9143.24 589.24 92.22 461.73 84.52 62.98 12.91 3.78

139.12* 79.81* 7.71* 117.81* 7.87* 11.87* 1.72NS

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).

CSMCRI-JC4 0 0 11.6 ± 0.7a 18.8 ± 0.9b 29.9 ± 1.4c 0

CSMCRI-JCNT 0 0 4.0 ± 0.6a 12 ± 0.9b 18.2 ± 1.2c 0

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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 BAP and 1.5 mg L−1 IAA. (D) Pulse treatment for root induction in liquid medium containing 3 mg L−1 IBA, 1 mg L−1 IAA and 1 mg L−1 NAA 1. (E) Rooting in charcoal containing medium. (F) Rooted plantlets in polybag.

response (Table 1). Best regeneration was observed at 0.5 mg L−1 TDZ in all the genotypes studied (Fig. 2B). Among four toxic and one non-toxic J. curcas, non-toxic genotype was observed to be least responsive (54.9 ± 1.7% response and 4.0 ± 0.6 shoots/explants). Among the four toxic genotypes CSMCRI-JC4 showed the best response (88.8 ± 3.2%) and 16.9 ± 1.1 (shoots/explant) followed by CSMCRI-JC1 (78.2 ± 1.6% response) and 14.9 ± 0.8 (shoots/explant). Least response was observed in the CSMCRI-JC2 (59.0 ± 1.5%) and 11.2 ± 0.3 (shoots/explant). 3.2. Effect of source of explant on shoot buds induction Significant differences in percentage response and the number of shoot buds per explant were observed between in vitro and ex vitro generated explants. Maximum response was observed in in vitro explants cultured on TDZ supplemented MS medium (Tables 1 and 2). In the ex vitro explants, regeneration efficiency and number of shoot buds per explant was very low as compared to the response showed by in vitro explants (Fig. 1D). Percentage response ranged from 92.9 ± 2.1 to 16.1 ± 1.4 (in vitro explants) and 29.9 ± 1.4 to 4.0 ± 0.6 (ex vitro explants) among all the four genotype of toxic and one non-toxic genotype. Number of shoot buds

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 seedling were best responsive on 0.5 mg L−1 TDZ concentration. At this concentration efficiency of regeneration varied from 88.8 ± 3.2 to 54.9 ± 1.7 and number of shoot buds induced varied from 16.9 ± 1.1 to 11.2 ± 0.3 among the 5 different genotypes (Table 3). Explants collected from 15 days old seedling, irrespective of genotype, showed callus formation and no regeneration was observed in any of the explants. Percentage of responding explants significantly decreased on increasing the age of explants to 45 days and ranged from 25.7 ± 1.2 to 8.4 ± 1.4. Number of shoot buds formed was also reduced significantly. 3.4. Effect of position of explant on shoot bud induction The position of explant with respect to cotyledonary node did not 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-

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Table 2 Effect 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 30 days old seedling of four toxic and a non-toxic genotype of J. curcas. TDZ (mg L−1 )

0.05 0.1 0.5 1 1.5 3

In vitro

In vivo

CSMCRI-JC1

CSMCRI-JC2

± ± ± ± ± ±

± ± ± ± ± ±

6.8 11.4 14.9 25 28.1 2.2

b

0.9 0.8c 0.8d 0.9e 0.6e 0.8a

3.2 6.9 11.2 10.1 9.6 5.5

CSMCRI-JC3 a

0.60 0.8b 0.3d 0.4d 0.6c 0.7b

5.2 10.7 12.5 22.0 25.5 4.4

± ± ± ± ± ±

a

0.60 0.7b 0.5b 0.8c 1.10d 0.80a

CSMCRI-JC4

CSMCRI-JCNT

± ± ± ± ± ±

± ± ± ± ± ±

8.7 14.2 16.9 29.2 34.2 5.7

b

0.8 0.3b 1.1c 0.7d 0.9e 0.7a

3.4 5.9 13.5 10.3 14.1 7.8

a

0.4 0.7b 1.0e 1.6d 0.9e 0.7c

CSMCRI-JC1

CSMCRI-JC2

CSMCRI-JC3

CSMCRI-JC4

CSMCRI-JCNT

0 0 5.2 ± 0.7a 6.1 ± 0.8b 8.3 ± 1.5c Callus

0 0 4.8 ± 0.7a 7.0 ± 1.6b 7.4 ± 1.1b Callus

0 0 4.2 ± 0.4a 5.2 ± 1.1a 6.0 ± 0.8ab Callus

0 0 6.3 ± 0.6a 7.4 ± 0.5b 8.3 ± 0.7c Callus

0 0 2.9 ± 0.2a 4.0 ± 0.4b 5.7 ± 0.8b Callus

ANOVA summary table Source

df

MS

F

TDZ S TDZ × S G TDZ × G S×G TDZ × S × G Error

5 1 5 4 20 4 20 120

163.23 21.54 12.22 231.23 14.32 2.11 1.31 1.54

109.76* 9.11* 8.11* 173.11* 7.34* 1.67NS 0.54NS

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 3 Effect 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-toxic genotype of J. curcas at 0.5 mg L−1 TDZ concentration. Age (days)

15 30 45

% 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

0 78.2 ± 1.6b 19.1 ± 1.8a

0 59.0 ± 1.5b 8.4 ± 1.4a

0 70.0 ± 2.2b 14.9 ± 2.5a

0 88.8 ±3.20b 25.7 ± 1.2a

0 54.9 ± 1.7b 9.6 ± 1.9a

0 14.9 ± 0.8b 4.6 ± 1.7a

0 11.2 ± 0.3b 3.3 ± 0.6a

0 12.5 ± 0.5b 3.8 ± 1.1a

0 16.9 ± 1.1b 5.4 ± 1.7a

0 13.5 ± 1.0b 5.2 ± 1.4a

ANOVA summary table Source

% explants responded df

Age G Age × G Error

2 4 8 30

Total

44

No. of shoot buds/explants MS

F *

111.91 618.25 14.76 13.07

9.13 11.67* 3.58*

df

MS

F

2 4 8 30

193.08 57.06 6.75 5.19

7.78* 0.93NS 0.41NS

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 4 Effect 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 (30 days old seedlings) in four toxic and a non-toxic genotype of J. curcas at 0.5 mg L−1 TDZ concentration. Position

Ap Mi Pr

% 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

81.5 ± 2.1 78.2 ± 1.6a 77.3 ± 1.9a

61.6 ± 1.4 59.0 ± 1.5a 60.3 ± 1.4a

75.1 ± 1.9 70.0 ± 2.2a 74.3 ± 2.8a

90.2 ± 4.4 88.8 ± 3.20a 88.1 ± 1.6a

55.1 ± 3.2 54.9 ± 1.7a 49.5 ± 1.7a

21.9 ± 1.5 14.9 ± 0.8b 5.3 ± 0.10a

18.0 ± 0.5 11.2 ± 0.3b 2.1 ± 0.8a

19.6 ± 1.3 12.5 ± 0.5b 2.9 ± 0.1a

25.6 ± 1.0 16.9 ± 1.1b 1.50 ± 0.4a

19.6 ± 1.5c 13.5 ± 1.0b 3.6 ± 1.1a

a

a

a

a

a

c

c

c

c

ANOVA summary table Source

% explants responded

No. of shoot buds/explant

df

MS

F

df

MS

F

P G P×G Error

2 4 8 30

11.11 907.49 3.70 11.03

1.13NS 9.03* 0.51NS

2 4 8 30

93.08 87.06 9.78 8.13

4.01* 1.03NS 0.42NS

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).

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Table 5 Effect of different concentrations of thidiazuron (TDZ) (in shoot bud induction medium) and Ca2+ (in germination medium) on the shoot bud induction from hypocotyl explants of J. curcas. TDZ (mg L−1 )

Ca2+ (mg L−1 ) 0

0.05 0.1 0.5 1 1.5 3

0.22

0 0 0 0 0 0

50.30 79.0 93.3 96.30 48.30 49.30

0.44 ± ± ± ± ± ±

a

1.50 3.60b 3.10c 2.50c 1.50a 1.50a

0.88

42.30 76.30 88.30 93.3 42.0 27.70

± ± ± ± ± ±

b

2.50 1.50c 2.30d 3.10d 2.0b 1.20a

9.0 13.70 19.0 20.30 17.0 15.30

± ± ± ± ± ±

1.0a 1.50b 1.0c 1.50c 1.0c 1.50b

ANOVA summary table Source

df

MS

F

TDZ Ca2+ TDZ × Ca2+ Error

5 3 15 48

113.61 987.49 13.70 12.33

2.13* 11.03* 9.53*

Total

71

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

ever, the position of the explant on the seedling axis significantly affected the response of the explants for number of shoot buds formed (Table 4). Regeneration potential of explants increased with an increase in distance from the root and explant adjacent to cotyledonary node showed the highest response (number of shoot buds). In CSMCRI-JCNT number of shoot buds increased from 3.6 ± 1.1 in Pr segment to 19.6 ± 1.5 in Ap segment. 3.5. Effect of Ca2+ concentrations in the germination medium on shoot buds induction Use of 0.5 mg L−1 and 1.0 mg L−1 TDZ resulted in best response at all the Ca2+ concentrations studied. The hypocotyl explants from the seedlings raised in medium containing half strength Ca2+ concentration (0.22 g mL−1 ), when cultured on the TDZ supplemented medium containing 0.44 g mL−1 Ca2+ (standard MS) showed increased response. This transient decrease in the Ca2+ amount resulted in increased number of responding explants from 88.3 ± 2.3 to 93.3 ± 3.1 and 93.3 ± 3.1 to 96.3 ± 2.5 at 0.5 mg L−1 and 1 mg L−1 TDZ, respectively (Table 5). Absence of Ca2+ in the germination medium led to 100% mortality of explants. Increase in Ca2+ concentration to 0.88 g mL−1 in the germination medium, resulted in reduced response of explants on TDZ supplemented medium. 3.6. Elongation, proliferation, rooting and acclimatization of shoots For further elongation of the TDZ induced shoot buds, the explants were transferred to the sub-culture medium containing 1 mg L−1 BAP and 2 mg L−1 Kn. Among all the toxic genotypes tested, CSMCRI-JC2 showed least response (Table 6). Length of the shoot was observed to be least in the non-toxic genotype (CSMCRI-JCNT). These shoots when kept for further proliferation and elongation maximum proliferation was obtained on 0.5 mg L−1 BAP and 1.5 mg L−1 IAA and no significant genotype specific response was observed for proliferation among the toxic genotypes except CSMCRI-JC4 which showed highest number of shoot formation (Fig. 2C). However, elongation among toxic genotypes was least in CSMCRI-JC2, while others genotypes showed no significant difference in the shoot length and non-toxic genotype showed minimum number of shoots and elongation (Table 7). No significant differences were observed, either in percentage of rooting or number of roots per explants among the toxic geno-

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

Length of shoots

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

2.70 2.40 2.80 2.80 2.20

± ± ± ± ±

0.70c 0.40b 0.31c 0.20c 0.30a

ANOVA summary table Source

df

MS

F

Genotypes Error

4 15

4.07 0.67

5.1*

Total

19

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

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

Number of shoots

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

6.60 6.73 6.60 7.25 5.58

± ± ± ± ±

Length of shoots

0.39b 0.40b 0.20b 0.35c 0.39a

2.74 2.15 2.93 2.75 2.08

± ± ± ± ±

0.12b 0.15a 0.25b 0.19b 0.30a

ANOVA summary table Number of shoots Source Genotypes Error

df 4 15

Total

19

MS 5.11 0.97

Length of shoots F 5.22*

df 4 15

MS 2.07 0.63

F 3.1*

19

Mean in each column followed by same letters are not significantly different according 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 regenerated plants (Fig. 2F).

S. Sharma et al. / Industrial Crops and Products 34 (2011) 943–951 Table 8 Rooting percentage in shoots obtained from four toxic and a non-toxic genotype on hormone free half strength solid MS charcoal medium after four days pulse treatment with 3.0 mg L−1 IBA, 1.0 mg L−1 IAA and 1.0 mg L−1 NAA containing half strength liquid MS medium in J. curcas. Genotype

Rooting percentage

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

60.9 62.6 62.4 61.8 45.8

± ± ± ± ±

2.30b 2.40b 3.20b 3.30b 2.40a

ANOVA summary table Source

df

MS

F

Genotypes Error

4 15

211 3.67

23.1*

Total

19

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

4. Discussion In the present study, shoot bud induction could be achieved directly from in vitro and ex vitro hypocotyl explant of toxic and non-toxic genotypes of J. curcas on MS basal medium supplemented with TDZ (N-phenyl-N-1,2,3-thidiazol-5yl urea), a phenylurea compound. TDZ is reported to play a major role in the induction of adventitious shoot regeneration by organogenesis, especially from hypocotyl and has proved to be a potent cytokinin for regeneration in J. curcas (Kumar et al., 2010a,b; Reddy et al., 2008; Sujatha et al., 2005). It was observed that high TDZ concentrations promoted massive callus production on all explant types. The number of explants responding to TDZ treatment increased with increasing TDZ concentrations. The main factors found to affect regeneration in our study were the genotype, source, age, position of explants and medium composition. Genotype/cultivar is one of the most important factors affecting regeneration (Conde et al., 2007; Feyissa et al., 2005; Landi and Mezzetti, 2006; Reichert et al., 2003; Rodrıguez et al., 2008). As the regeneration in J. curcas was reported to be genotype dependent, it was considered important to screen different genotypes of J. curcas for their regeneration potential from hypocotyl explant. In our study, it was observed that all the four genotypes of toxic and one non-toxic genotype showed differences in percentage of shoot bud induction and number of shoot buds per explant. This differential behavior can be related to different mechanisms for control of the endogenous PGRs metabolism and/or contents. Cells within the same plant can have different endogenous levels of plant growth regulators (Pellegrineschi, 1997; Schween and Schwenkel, 2003) and variation in receptor affinity (Minocha, 1987), thus it is reasonable to expect that in vitro response will vary with genotype (Bhaskaran and Smith, 1990). Similar results were reported in Mulberry (Morus) species (Chitra and Padmaja, 2005), Hagenia abyssinica (Feyissa et al., 2005) and Fragaria (Landi and Mezzetti, 2006). Henry et al. (1994) reported that genotypic differences with respect to embryogenesis and regeneration result from quantitative or qualitative genetic differences. The type of explant is very important factor in establishing an efficient regeneration system. Explant used from in vitro sources were observed to have a higher rate of regeneration efficiency and more number of shoot buds as compared to ex vitro explant in both toxic and a non-toxic genotypes. This may be due to different level of endogenous PGRs or can be related to different mechanisms of control of the endogenous PGRs metabolism. Similar results were

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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 the caullogenesis in J. curcas (Fig. 1). Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas beyond 30 days, the regeneration response decreased with increase in age of seedlings. Similar results were obtained in Saussurea obvallata where 10–15-day-old seedlings were more regenerative (58%) than younger (5 days) seedlings, and significantly higher than explants from an older (20 days) source (Dhar and Joshi, 2005). It has been reported that younger explants exhibit greater morphogenic potential as compared to older explants (Welander, 1988; Yepes and Aldwinckle, 1994), as they might have more metabolically active cells with hormonal, nutritional and other physiological conditions that are responsible for increased organogenesis (Famiani et al., 1994). Experiments with different position of explants collected from in vitro raised seedling indicated that the regeneration capacity of hypocotyl segments increased with the increasing distance from the root. Hypocotyl segments near the cotyledonary node were more responsive than the segments near the root. This may be due to an inhibitory role of the root meristem of main root and laterals present on the end of the hypocotyl. Similar results were obtained by Kameya and Widholm (1981) in Glycine canescens and Fari and Czako (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 different hormones is a high external Ca2+ concentration or rise in the level of this cation in the tissue (Trewavas, 1999). TDZ mediated response has been reported to be influenced by the Ca2+ (Hosseini and Rashid, 2000; Yip and Yang, 1986). We observed increase in number of responding explants when transient Ca2+ stress was given and our results are in agreement with Mundhara and Rashid (2006). In this investigation, the induced shoot buds were elongated on the medium containing reduced concentration of cytokinins (BAP and Kn). For shoot elongation and proliferation responses, no significant differences were noted among the genotype studied except in CSMCRI-JC2 and CSMCRI-JCNT. This indicated that shoot elongation and proliferation may not be much effected by the genotype. In the present investigation it was observed that compact shoot buds were induced at high concentration of TDZ (1–2 mg L−1 ) due to which shoot elongation was inhibited in subsequent culture. Lower levels of TDZ induced relatively fewer shoot buds which developed 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 culture. When these shoots were excised other surrounding shoots elongated within 7–10 days and could be harvested after another 2 weeks. Similar observations were reported in other species (Sharma and Amla, 1998; Tawfik and Noga, 2001) suggesting a possible apical dominance effect. Therefore, separation of explants into pieces containing individual shoot buds, before transferring to the shoot proliferation and elongation medium, may enhance shoot regeneration efficiency. Significant differences observed in rooting percentage between toxic and non-toxic genotypes may be due to level of endogenous PGRs, or can be related to different mechanisms of control of the endogenous PGRs metabolism and/or contents. Shoots that did not responded to rooting treatment were observed to root after a time lag, i.e. after a month. The possible reason may be decrease 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 when CKs concentration is sufficiently high to initiate shoot proliferation. A lower concentration of CKs may be necessary to form roots

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(de Fossard, 1978; Gaspar and Coumans, 1987). CKs in the rooting medium are known to be inhibitory for rooting (Ruzicka et al., 2009; Werner et al., 2003). Whereas, Nemeth (1979) reported its positive response in some fruit trees. Regeneration has been extensively used for the rapid multiplication of many plant species. However, its more widespread use is restricted often by the high percentage of plants lost or damaged when transferred to ex vitro conditions. The shoots were thoroughly washed with distilled autoclaved water so that the attached sugar enriched media is removed thereby reducing the chances of fungal and bacterial contamination and thus, ultimately mortality. Humidity was gradually reduced. The acclimatization of the rooted shoots was accomplished and approximately 90% of the plants were successfully transferred to polybags in greenhouse. To the best our knowledge, this is the first study to report a regeneration response from hypocotyl explants in different genotypes along with non-toxic genotype and subsequently optimized the protocol for J. curcas regeneration. Acknowledgements Authors are thankful to K.G. Vijay Anand for his help. We are grateful to Dr. P.K. Ghosh Director, CSMCRI for his encouragement and Centre for Scientific and Industrial Research, New Delhi, India for financial support (SRF) to pursue this work. The authors gratefully acknowledge Prof. K. Becker, Department of Aquaculture Systems and Animal Nutrition, University of Hohenheim, Stuttgart, Germany for providing Mexican non-toxic J. curcas seeds. References Ammirato, P.V., 1983. Embryogenesis. In: Evans, D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. I. Macmillan, New York. Amzallag, G.N., Lerner, H.R., Poljakoff-Mayber, A., 1992. Interaction between mineral nutrient, cytokinin and gibberellic acid during growth of Sorghum at high NaCl salinity. J. Exp. Bot. 43, 81–87. Aneta, I., Margarita, V., Plamen, D., Atanas, A., Henri, A.V.O., 1994. Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata. Physiol. Plant. 92, 85–89. Basha, S.D., Sujatha, M., 2007. Inter and intra-population variability of J. curcas (L.) characterized by RAPD and ISSR markers and development of populationspecific SCAR markers. Euphytica 56, 375–386. Bhaskaran, S., Smith, R.H., 1990. Genotypic differences in morphogenesis may be due to differences in endogenous hormone levels. Regeneration in cereal tissue culture: a Review. Crop Sci. 30, 1328–1337. Bollmark, M.M., Kubat, B., Eliasson, L., 1988. Variation in endogenous cytokinin content during adventitious root formation in Pea cuttings. J. Plant Physiol. 132, 262–265. Chitra, D.S., Padmaja, G., 2005. Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine. Sci. Hortic. 106, 593–602. Conde, P., Alexandra, S., Armando, C., Conceic, S., 2007. A protocol for Ulmus minor mill. Micropropagation and acclimatization. Plant Cell Tissue Org. Cult. 92, 113–119. da Camara Machado, A., Frick, N.S., Kremen, R., Katinger, H., da Camara, M.M.L., 1997. Biotechnological approaches to the improvement of Jatropha curcas. In: Proceedings of the International Symposium on Jatropha , Nicaragua, p. 15. de Fossard, R.A., 1978. Tissue culture of Eucalyptus Ficifolia. In: Muell, F. (Ed.), Proceedings of Symposium on Plant Tissue Culture. Science Press, Peking, China, pp. 425–438. Dhar, U., Joshi, M., 2005. Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew (Asteraceae): effect of explant type, age and plant growth regulators. Plant Cell Rep. 24, 195–200. Famiani, F., Ferradini, N., Staffolani, P., Standari, A., 1994. Effect of leaf excision time and age BA concentration and dark treatments on in vitro shoot regeneration of M. 26 Apple rootstock. J. Hortic. Sci. 69, 679–685. Fari, M., Czako, M., 1981. Relationship between position and morphogenetic response of pepper hypocotyl explants cultured in vitro. Sci. Hortic. 15, 207–213. Feyissa, T., Welander, M., Negash, L., 2005. In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants. Plant Cell Rep. 24, 392–400. Francis, G., Edingger, R., Becker, K., 2005. A concept for simultaneous wasteland reclamation, fuel production, and socio-economic development in degraded areas in India Need, potential and perspectives of Jatropha plantations. Nat. Resour. Forum 29, 12–24. Gaspar, T.H., Coumans, M., 1987. Root formation. In: Bonga, J.M., Durzan, D.J. (Eds.), Cell and Tissue Culture in Forestry, vol. 2.

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