[International Journal of Plant Biology 2014; 5:5448] [page 33]

Optimized in vitro plantregeneration of the biodieselplant Jatropha curcas L.:the effects of using seedsat different stages of maturityas starting materialsSillma Rampadarath, DaneshwarPuchooa, Mala Ranghoo-SanmukhiyaFaculty of Agriculture, University ofMauritius, Rduit, MauritiusAbstract

Jatropha curcas L. is an important biofuelplant that can be regenerated in vitro usingseeds. The fruits of J. curcas do not reachmaturity at the same time although they are onthe same bunch. This study was undertaken toevaluate the effect of seeds, from fruits at dif-ferent stages of maturity, on in vitro plantletformation. Callus was induced on Murashigeand Skoog medium using different concentra-tions of 2,4 dichlorophenoxyacetic acid (2,4-D)and 6 benzylaminopurine (BAP). 2,4D at aconcentration of 3.0 mg/L and BAP at 1.0 mg/Lwere found to be optimum for callus formation.In vitro plantlets obtained on BAP were small-er with thicker hypocotyl and thicker roots. Onthe other hand, seeds from mature fruits gavebetter results for in vitro plantlet regenerationon 1.0 mg/L BAP without any rooting medium.A combination of sand and soil were used foracclimatization. Best results were obtainedwhen sand and soil were used in equalamounts. Results also showed that seed matu-rity is an important factor for in vitro plantregeneration of J. curcas and that root forma-tion in vitro does not require additional plantgrowth regulator substance.

Introduction Plants that contain high levels of secondary

metabolites are difficult to mass propagatethrough tissue culture.1 Jatropha curcas L.commonly known as physic nut belongs to theEuphorbiaceae family and has various desir-able characteristics that make the plant verypromising for the agro-energy market.2 Thebiofuel industry is being subjected to contro-versies including food insecurity due to diver-gence of staple crops towards biofuel produc-tion.3 The choice of proper feedstock is there-fore crucial. Lipid from non-edible feedstocksis popular because they do not compete withthe food market.4 Also, the prohibitive cost of

edible oils prevents their use in Biodieselpreparation, but nonedible oils are affordablefor biodiesel production.5 Much attention isbeing given to the non-edible J. curcas. The oilcontent of J. curcas seeds is made up of almost60% fatty acid that has a close resemblance tothe edible oils, which makes the oil a good sub-stitute for biodiesel fuel.5 However, scarcity ofseeds, delay of rooting with vegetative cuttingsand poor seed germination paved way for amajor need of micropropagation of J. curcas.6-8

Furthermore, to ensure a disease free explantsembryo explants culture is preferred.8 Therehave been few studies done to date on the invitro culture of J. curcas.9 In vitro regenerationof plantlets is essential for mass propagationby either organogenesis or somatic embryoge-nesis and it requires good starting materials.Hence, the aim of the study was to identify themost appropriate stage of maturity of the seedsand the most appropriate plant growth regula-tors (PGRs) combinations for plantlet regener-ation and the most appropriate substrate(s)combinations for hardening of the plantlets.

Materials and MethodsPlant materialsSeeds from fruits of Jatropha curcas L. at

three maturity stages were used as explants(immature - green color 30 days (d) after polli-nation; mature - yellow color 40d after pollina-tion; fully mature-brown/black color - 90d afterpollination) in this study. Surface sterilization and cultureestablishmentFor surface sterilization, the seeds were

first washed for 10 min with lauric acid; 40%(v/v) under running tap water. The seeds werethen soaked for 24h in 0.5 mg/L of benomyl and0.5 mg/L ciprofloxacin. This was followed byrinsing in sterile distilled water and thentransferring to ethanol [70% (v/v)] for 6 min.The seeds were rinsed and transferred in asodium hypochlorite solution [40% (w/v)] for20 min. Following another rinse, the seedswere dipped into mercuric chloride [0.02%(w/v)] for a further 6 min. After a last rinse,the seed coats were cracked and the embryoswere inoculated on the specific media. The pHof the media was adjusted to 5.8 before addi-tion of agar and autoclaved at 15 psi at 121Cfor 20 min. Cultures were maintained ingrowth room at 232oC and 16/8h light/darkphotoperiod under light intensity of 1000 lux. Callus inductionThe embryos of the immature, mature and

fully mature seeds were inoculated onMurashige and Skoog medium (MS).10 After

7d, the hypocotyl (11 cm) was dissected andinoculated on MS medium supplemented withdifferent concentrations and combinations of2,4 dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The effect of thedifferent combinations of PGRs on the weightand morphology of the calli were observed(Table 1).In vitro plant regenerationIn this part of the study the concentration of

PGRs used were kept constant (1.0 mg/L). BasalMS medium containing 3% (w/v) sucrose and0.8% (w/v) agar was supplemented with either2, 4-D, BAP or kinetin (K) alone and with com-binations of either 2,4D & BAP, 2,4D & K or BAP& K. Shoot lengths were measured every 7dfrom the day of inoculation and morphologicaldifferences of the developed plantlets were alsoobserved. The lengths of the plants shoots weremeasured at 7, 14 and 21d after transplantationusing a Vernier calliper.Acclimatization After 21d healthy plantlets with well devel-

oped roots were thoroughly washed under run-ning tap water and transplanted in a small lab-

International Journal of Plant Biology 2014; volume 5:5448Correspondence: Daneshwar Puchooa, Faculty ofAgriculture, University of Mauritius, Reduit,Moka, Mauritius.Tel.: +230.454.1041 - Fax: +230.465.5743.E-mail: sudeshp@uom.ac.mu

Key words: seed maturity, plantlets, plant growthregulator substance, Jatropha curcas L.

Contributions: the authors contributed equally.

Acknowledgements: the authors are grateful tothe staff of the Botany and BiotechnologyLaboratories of the Faculty of Agriculture,University of Mauritius, in particular, Mrs DBhookun-Seeruttun, Mrs A. Sobhee and Mr LJunye. Sincere thanks to Dr. Sujatha M, Dr. S. Baland Mr S. Seeruttun for their help and guidance.We are also grateful to the University ofMauritius authorities for providing the fund.

Funding: this paper was partially funded by theUniversity of Mauritius.

Received for publication: 24 April 2014.Revision received: 19 May 2014.Accepted for publication: 19 May 2014.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

Copyright S. Rampadarath et al., 2014Licensee PAGEPress srl, ItalyInternational Journal of Plant Biology 2014; 5:5448doi:10.4081/pb.2014.5448

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based greenhouse containing vermiculite asgrowing medium for hardening. The humiditylevel was kept at around 75% by spraying regu-larly. The plantlets were kept under this condi-tion for a period of 2 weeks and then progres-sively exposed to increasing light intensity. Theplantlets were afterwards transferred in potscontaining soil and sand mixed at differentratios. The mixtures of sand and soil used were1:1, 1:2 and 2:1. The plants were, again acclima-tized for two weeks and then grown under fieldconditions. The shoot lengths were measured atday 15, 30 and 45 after transplantation.Statistical analysisA completely randomized design was used in

the study. Results were determined as mean standard deviation. Normality tests for therecorded data were also performed. For the threedifferent stages of seed maturity (5replicates/PGRs concentration combinations),one way ANOVA tables (LSD at 5%) were gener-ated to test differences between mean parame-ters of seed and embryo morphology, the effect ofthe different concentration combinations ofPGRs on the following characteristics: i) meancallus weight at three different time intervals(15, 30 and 45d), ii) mean shoot length for thein vitro plant regeneration at 7, 14 and 21d timeintervals and iii) the effect of three differentsoil/sand mixtures ratios on shoot length of theplantlets regenerated in vitro. For the latter,there were 10 replicates per treatment.Minitab (Minitab Inc., State College, PA, USA)16.2.4 and Microsoft Excel (Microsoft Co.,Redmond, WA, USA) 2010 were used for the sta-tistical analyses, and tables and graphs output.

Results Seeds and embryos characterization The immature, mature and fully mature

seeds and embryos were physically character-ized. All the parameters (weight, length anddiameter) for both the seeds and embryos of dif-ferent maturity stages varied significantly(P

[International Journal of Plant Biology 2014; 5:5448] [page 35]

the plantlets selected for acclimatization hadwell developed roots. The only difference wasthat the roots formed on BAP medium wasshorter and thicker compared to those on MSonly or MS supplemented with K.

Significant differences in shoot length(P

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combinations BAP/K and 2,4D/K with MSmedium gave very good shoot development.The length of shoots obtained on MS mediumsupplemented with BAP/K were 10.01.3 cmand 7.90.7 cm, and supplemented with2,4D/K were 8.61.5 cm and 8.00.5 cmfor mature and fully mature seeds respective-ly at 21d. Acclimatization and transplantationAfter 21 d the fully developed plantlets from

the mature seeds were transferred to thegreen house (Figure 1N) containing the ver-miculite medium. However, the plantlets fromK and MS media wilted and died even thoughhigh initial humidity was maintained in thegreenhouse. 86% of the plantlets that survivedwere those from BAP medium (Figure 1P).After acclimatization, the plants were trans-

ferred on three soil and sand mixture ratio.The longest shoot length and good plantletsdevelopment growing on 1:1 ratio of sand andsoil were observed (Figure 1O,P). New plantswere regenerated from in vitro plantlets of J.curcas with new leaves on well-developed longand strong stem (Figure 1Q).Significant differences in shoot length

(P

[International Journal of Plant Biology 2014; 5:5448] [page 37]

Seed maturity also played an important fac-tor on callus weight. The increase in callusweight was higher during the period between15-30d. Similar observation was also made byChaudhary.13 They also reported that 2,4D,even at a low concentration gave very goodresults on the callus formation. This study con-firms that at 1.0 mg/L, callogenesis was occur-ring. Although similar explants were used,Chaudhary and co-workers observed thatincreasing the amount of 2,4D formed brown-ish callus, but no brownish callus was obtainedin the present study.13Plant regeneration

J. curcas is a xenogamic plant and it has ahigh degree of segregation, therefore breedingtakes a long period of time to be achieved.There is also the problem of degeneration ofhybrid embryos formed by the plant due toinadequate endosperm development. Hence, invitro embryo rescue can be a very useful tech-nique. However, the success of embryo rescuetechnique relies greatly on the maturationstage of the embryo and composition of theculture medium. Therefore, in this study theregeneration of in vitro plantlets using PGRs2,4D, an auxin, and K and BAP as cytokinin hasbeen demonstrated. The seven media usedgave positive formation of shoots but morpho-logical differences of the shoots obtained wereobserved. MS medium without PGRs alsorevealed to be a good medium since healthyplantlet were regenerated within 21d. Despitethe fact that the MS generated plantlets did notsurvive acclimatization the result obtained canbe used to explore further for the possibilitiesof the presence of, endogenous auxin andcytokinin present in the J. curcas L, embryo.14

It is known that rooting medium is impor-tant for tissue culture plantlets but in thisstudy no specific rooting medium wasrequired. Plantlets generated with good rootformation from mature seeds were directlytransferred for acclimatization. However, onlyplantlets regenerated on medium supplement-ed with BAP survived. These plantlets had astronger root architecture compared to thosegrown on MS medium or on MS medium sup-plemented with K. In vitro root formation hadalso been observed by Sujatha and Muktawhereby within 8-10d 88% of rooting wasrecorded.15

Although Warakagoda and Subasinghe usedseeds at different stages of maturity for in vitro

plant regeneration, extended study on differentcombination of PGRs have not been demon-strated.16 In vitro regeneration of plantlet isessential for mass propagation through eitherorganogenesis or somatic embryogenesis and itrequires good starting materials. The resultsshowed that fully mature seeds can be used asexplants for induction of callus. Although theseseeds are at a dehiscence stage and the embryolighter in weight, it is still viable and active invitro. The concentration of 2,4D and BAP can bevaried depending on the type of callus requiredeither friable or compact. A rapid method of in vitro embryo rescue

was also recorded. Fully regenerated plantletswere obtained within 21 d of culture initiationon medium supplemented with BAP (1.0mg/L). The plantlets were successfully accli-matized on a substrate consisting of sand andsoil in the ratio of 1:1.

ConclusionsSeeds from all three stages of fruit maturity

responded positively to callus induction and invitro plant regeneration. Different callus mor-phology can be obtained by varying the 2,4Dand BAP concentration. It was also shown thatselecting the appropriate starting material isimportant in achieving the desired results.BAP only can be used at 1 mg/L without anyrooting medium for in vitro plantlet regenera-tion within 21d. Further studies are requiredto optimize the findings presented here as tis-sue culture remains an important propagationtool for Jatropha curcas L.

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4. Deore CA, Johnson ST. High frequencyplant regeneration from leaf disc culturesof Jatropha curcas L.: an importantbiodiesel plant. Plant Biotechnol Rep 2008;2:7-11

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12. Attaya AS, Geelen D, Belal El-Fatah H.Progress in Jatropha curcas tissue culture.Am-Eurasian J Sustain Agric 2012; 6:6-13.

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