Evaluation of in vitro rooting efficiency in the biodiesel plant, Jatropha curcas

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1 Evaluation of in vitro rooting efficiency in the biodiesel plant, Jatropha curcas Geetaa Singh, Sudheer Shetty Research and Development Division, Labland Biotech Private Limited, KRS Main Road, Mysore, Karnataka, , India; ABSTRACT Jatropha curcas L. is known to be a difficult-to-micropropagate crop. Even to-date a simple, repeatable and highly reproducible micropropagation process is not easily available. Poor in vitro rooting is a major obstacle for successful micropropagation of J. curcas. In the present study, the rooting efficiency of J. curcas was studied in the rooting media that have already been reported by various authors. Besides, a medium was standardized for improving the rooting efficiency of Labland s Jatropha selections. The challenging factors observed during the in vitro rooting process were recorded and suitable rooting media requirements and culture conditions for efficient rooting of J. curcas were standardized. Rooting hormones like IBA, IAA and NAA in different concentrations (0.5 to 2 mg/l), either singly or in combination, were attempted. Significant improvement for in vitro rooting was obtained in medium supplemented with IBA or IAA in various combinations. The implication of the study in mass multiplication of elite selections of J. curcas is discussed. Keywords: Jatropha curcas, in vitro rooting, indole acetic acid, indole butyric acid, naphthalene acetic acid Abbreviations: IAA - indole-3-acetic acid, IBA - indole-3-butyric acid, NAA - α-naphthalene acetic acid, GA3 - gibberellic acid, PG - phloroglucinol INTRODUCTION Jatropha curcas L. is a drought-resistant, multipurpose, photo-insensitive perennial plant belonging to family Euphorbiaceae [1]. Jatropha has received global attention due to its seeds, which contains 40 to 50% semi drying oil. It is considered as a potential source of non-edible fuel-producing plant [2,3] along with its traditionally known medicinal properties [4]. Jatropha biofuel contains more oxygen with a higher cetane value increasing the combustion quality; is clean, non-toxic and economical. It can be a good plantation material for eco-restoration in all types of wasted land and also serves as an important medicinal plant [5]. Seeds of Jatropha have a limited viability and can only be stored for 12 months after which its viability is reduced by 50%. The development of in vitro culture techniques offers the best route to develop tissue for germplasm preservation, rapid propagation, and crop improvement to increase productivity and profitability, from an established plantation. Adventitious root formation has a lot of commercial interest because there are many plant species that are difficult to root in vitro. In some plant species, adventitious root formation occurs without any treatment, while others require different growth regulators usually auxin. Auxins encourage root formation by breaking root apical dominance induced by cytokinin [6,7]. The plants growing from shoots/cuttings have the same genetic information as the mother plants and predictable yields can be obtained from plants whose genetics is recorded [8]. However, propagation by seeds without a genetic history will follow the biological variation rule. The yield prediction becomes easier when the yield potential of the mother plants is known. Therefore, proper screening of the potential mother plant is important. The superior plants Research Article, Biotechnol. Bioinf. Bioeng. 2012, 2(1): Society for Applied Biotechnology; pissn , eissn

2 can be multiplied by vegetative cuttings or by adopting the techniques of tissue culture. Tissue cultured Jatropha plants obtained from elite selections has always been in demand for various requirements like plantation development, seed production, research and development activities. Tissue culture of J. curcas allows production of uniform quality, true-to-type, disease-free plants on a commercial scale. The present study has been focused on the significant improvement of in vitro rooting efficiency to overcome the challenging issues limiting the extensive application of techniques of micropropagation to mass propagate J. curcas. In addition, the rooting efficiency of J. curcas in already reported rooting media has been evaluated and all the challenging aspects observed during the in vitro rooting process have been recorded in order to standardize suitable in vitro media requirements and culture conditions for successful rooting of J. curcas. In present study, shoots obtained from the nodal explants of elite selections with a genetic history have been used. MATERIAL AND METHODS Nodal and shoot tip explants from the 5 year-old adult elite selections of Jatropha curcas grown in the germplasm center of Labland Biotech were subjected to techniques of micropropagation consisting of initiation, multiplication and shooting in appropriate media combination. Healthy shoots measuring 4-5 cm with 6-10 leaves each were selected to evaluate the rooting efficiency. Media coded from S 01 to S 09 (Table 1) was prepared for evaluation of rooting efficiency in the research articles already published. Half strength and full strength MS basal [9] media supplemented with IAA, NAA, IBA, PG and GA3 was used individually and in combination in these preparations. The ph of the media was adjusted to 5.7 before gelling with agar and autoclaved at 121ºC at 15 lbs pressure for 20 minutes. In the present study, half strength MS media supplemented with IAA, NAA and IBA was prepared as described earlier and has been coded from S 10 to S 21 (Table 1). Elongated shoots (3-4cm long) regenerated from mature in vitro explants were excised singly and were transferred to the rooting medium indicated in table 1. Cultures were maintained in a culture room under cool white fluorescent light (2000 lux) with photoperiod of 16 h light and 8 h darkness at 25±1ºC. The cultures were observed at the end of each week. For each of the medium under study, 25 replicates were maintained and the experiment was repeated thrice. The average rooting percentage of all the three experiment was considered for evaluation. All the observations were recorded after 3 weeks of culture but before the end of 4 weeks. RESULTS AND DISCUSSION Evaluation of rooting efficiency in the previously reported media Nine different earlier published articles were evaluated in the present study. Among the nine evaluated media, the results of the report described by Datta et al. [4] could be reproduced with some similarities. While Datta et al. [4] reported 52% rooting in medium containing 0.2 mg/l IBA, the rooting outcome in the present study was only 33.3%. Apart from the result of Datta [4], the reproducibility of rooting was repeated to some extent with the report of Sardana [10]. Sardana used 3 mg/l each of GA3 and IAA to obtain rooted plants. However, they have not indicated the percentage rooting in the report. Using the same formulation in the present study, we could get 25% rooting. A very low percentage of rooting was obtained in the present study with formulations described by Kalimuthu (16.67%) [11], Li (16.67%) [12], Deore and Johnson (9.1%) [13] and Thepsamran (8.3%) [8]. In the present study, rooting could not be reproduced for formulations described by Sujatha and Mukta [14], Rajore [15], Shrivastava and Banerjee [16], where auxin free medium or medium containing NAA and NAA/IBA have been used respectively. 592

3 Table 1. Composition of rooting media used for evaluation of in vitro rooting efficiency of J. curcas L. Media Basal salt Plant growth regulators Concentration (mg/l) S 01 ½ MS PG + IBA S 02 ½ MS IBA 0.3 S 03 MS IBA 0.1 S 04 ½ MS IBA + NAA S 05 MS - - S 06 ½ MS NAA 5.0 S 07 MS IAA 0.1 S 08 MS IBA 0.2 S 09 MS GA3 + IAA S 10 ½ MS IAA 0.5 S 11 ½ MS IAA 1.0 S 12 ½ MS IAA 1.5 S 13 ½ MS IAA 2.0 S 14 ½ MS NAA 0.5 S 15 ½ MS NAA 1.0 S 16 ½ MS NAA 1.5 S 17 ½ MS NAA 2.0 S 18 ½ MS IBA 0.5 S 19 ½ MS IBA 1.0 S 20 ½ MS IBA 1.5 S 21 ½ MS IBA 2.0 Hence, the result of this evaluation indicates that there is very less percentage of reproducibility of rooting efficiency in J. curcas. None of the nine selected articles evaluated in the present study have indicated responses like leaf dropping, shoot drying, yellowing of leaves or profuse callusing as seen in the present study with the same formulations (Figure 1A-C). This could be attributed to many factors such as type of explant used (hypocotyls, petiole, leaf, shoot tip and nodal explant), explant source and the age. Further, the shoots in the present study for comparison have been used from field grown adult plants of 5 to 6 yrs age while the articles under evaluation have used explants from juvenile sources. Explant taken from juvenile plant tissue particularly that produced from seedlings is usually highly responsive. Evaluation of rooting efficiency in newly formulated rooting media The rooting response and further growth performance of in vitro raised shoots was found to be highly variable ranging from 8 to 50% in all the newly formulated media (S 10 to S 21) in the present study. The response of the shoots in each of the rooting hormone tried is elaborated below. Response of shoots in the medium amended with IAA In the present study, IAA at concentrations of 0.5, 1.0, 1.5 and 2.0 mg/l was amended in ½ MS basal medium. Rooting efficiency at different level (25 to 50%) was achieved in all 4 different concentrations tried. Highest rooting (50 %) was obtained in the medium with 2 mg/l IAA. At lower concentrations (0.5 to 1.5 mg/l) up to 33% rooting was obtained. Leaf dropping, shoot mortality and callus formation was observed at all concentrations of IAA which was maximum in medium containing 0.5 mg/l IAA (Figure 2A). Although severe leaf dropping occurred in the 1 st 593

4 week in the medium with 2 mg/l IAA, the shoots recovered fully with new leaf regeneration by 3 rd week and all the rooted shoots were in good condition (Figure 2B,C). Some amount of callus was observed in spite of good shoots and roots in each of the rooted plantlet. Sardana [10] has reported the use of IAA (3 mg/l) along with GA3 (3 mg/l) and Kalimuthu [11] (0.1 mg/l) but have not reported the percentage of rooting efficiency. A B C Figure 1. Response of J. curcas shoots to media formulations already reported. A: Leaf dropping and shoot drying without root formation in S 01 medium containing 100 mg/l + IBA 1 mg/l; B: Profuse callusing, shoot drying, without root formation in S 04 medium containing IBA 3 mg/l 3 mg/l + NAA 3 mg/l; C: Yellowing of leaves, shoot drying without root formation in S 09 medium containing GA3 3 mg/l + IAA 3 mg/l. A B C Figure 2. A: Response of J. curcas shoot in medium containing IAA (0.5 mg/l) showing leaf drop, shoot mortality and callus formation; B,C: Rooted shoots of J. curcas in medium containing IAA (2 mg/l) with good root and fresh leaf regeneration. Response of shoots in the medium amended with NAA In the present study, NAA at concentrations of 0.5, 1.0, 1.5 and 2.0 mg/l was amended in ½ MS medium and the rooting response and shoot quality was found to be poor. In NAA concentrations consisting 0.5, 1.0 and 1.5 mg/l, there was no rooting at all. In medium with 2 mg/l NAA, 16.7 % rooting was obtained. The healthy shoots in the respective medium appeared poor by the end of 2 weeks of culture (Figure 3A,B). No rooting was seen in medium with 0.5 to 1.5 mg/l NAA. 16.7% rooting was achieved with 2mg/L NAA by 4th week of culture. Severe leaf dropping, shoot mortality and profuse callusing was observed at all concentrations of NAA. Successful rooting with NAA has been reported by Rajore [15] who has used 5mg/L NAA and obtained 60 to 80 % rooting. 594

5 This report however, does not mention any thing about callus formation, shoot drying and leaf dropping and the results of the present study are contrary to the other published reports. Response of shoots in the medium amended with IBA In the present study, IBA at concentrations of 0.5, 1.0, 1.5, 2.0 mg/l was amended in ½ MS medium. Rooting response seemed to be good when compared to IAA and NAA medium. Rooting was seen at all concentrations of IBA (0.5 to 2mg/L). Highest rooting (50%) was obtained in 1.5mg/L IBA (Figure 4A,B) and lowest rooting was observed in 0.5 mg/l and 2mg/L IBA. Shoot drying was observed to an extent of 16.7% in the 3 rd week. Some amount of callus was also observed. It is encouraging to note that rooting with IBA has been reported by Datta [4], Deore and Johnson [13] and Li [12] who have used 0.1 to 1.0 mg/l IBA and obtained 52 to 97% rooting. This suggests that the results of the present study corroborate with the reports mentioned above. A Figure 3. A: Freshly inoculated healthy shoots of J. curcas in medium containing NAA (2.0, 1.5, 1.0 and 0.5 mg/l respectively); B: Response of J. curcas shoots in the respective medium after 2 to 3 weeks of incubation, severe leaf drop, shoot mortality and profuse callusing can be seen. B A B Figure 4. A,B: Response of J. curcas shoot in medium containing IBA (1.5 mg/l) with good root system. 595

6 CONCLUSION The results presented in this article constitute the first systematic study of its kind on the rooting and associated growth responses of J. curcas and provide useful information on physiological and biochemical requirements that are involved in the micropropagation of Jatropha plantlets. This indicates that apart from plant growth regulators, a number of other factors like chemical (macro/micro nutrients), physical (ph, temperature, light), biological (explant age, type, time of collection, period of culture) etc. could be directly or indirectly influencing the repeatability of rooting response in J. curcas which needs to be evaluated separately. This opens avenues for further research and the difficulty often experienced in growing and manipulating older tissue could be frustrating, because it is usually desirable to propagate mature plants of proven worth. It can be summarized from the present study that the in vitro rooting of J. curcas shoots obtained from adult explants can be achieved in ½ MS basal media amended with rooting hormones like IBA, IAA and NAA. Significant rooting (50%) was obtained in medium amended with IBA (1.5 mg/l) or IAA (2 mg/l). Rooting efficiency was poor in medium containing NAA. Irrespective of the kind of rooting hormone used, whether the roots were produced or not, undesirable growth responses such as leaf dropping, yellowing of shoots, shoot mortality and callus formation were observed. This clearly indicates that efficiency of rooting in J. curcas from adult tissues may not be controlled by plant growth regulators alone. REFERENCES [1] Kumar A, Sharma S. Ind. Crops Prod. 2008, 28:1-10. [2] Roy S. In: Plant Biotechnology - Recent Advances, Trivedi PC (ed), Panima Publishing Corporation, New Delhi, 2000, [3] Openshaw K. Biomass and Bioenergy 2000, 19:1-15. [4] Datta MM, Mukherjee P, Ghosh B, Jha TB. Curr. Sci. 2007, 93: [5] Prabakaran AJ, Sujatha M. Genet. Resour. Crop Evol. 1999, 46: [6] Kochhar S, Kochhar VK, Singh SP, Katiyar RS, et al. Curr. Sci. 2005, 89: [7] Noor-Camellia NA, Thohirah LA, NAP Abdullah, Mohd Khidir O. Res. J. Agri. Biol. Sci. 2009, 5: [8] Thepsamran N, Thepsithar C, Thongpukdee A. J. Hort. Sci. Biotech. 2008, 83: [9] Murashige T, Skoog F. Phys. Plant. 1962, 15: [10] Sardana J, Batra A, Sharma R. Adv. Plant Sci. 1998, 11: [11] Kalimuthu K, Paulsamy S, Senthilkumar R, Sathya M. Plant Tiss. Cult. Biotech. 2007, 17: [12] Li M, Li H, Jiang H, Pan X, et al. Plant Cell Tiss. Org. Cult. 2008, 92: [13] Deore AC, Johnson TS. Plant Biotech. Rep. 2008, 2:7-11. [14] Sujatha M, Mukta N. Plant Cell Tiss. Org. Cult. 1996, 44: [15] Rajore S, Sardana J, Batra A. J. Plant Biol. 2002, 29: [16] Shrivastava S, Banerjee M. Int. J. Integ. Biol. 2008, 3: