Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration
ABSTRACT 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 (α-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.Highlights► Efficient and reproducible in vitro propagation protocol in Jatropha curcas via hypocotyls explants was developed. ► Regeneration was found to be genotype dependent. ► In vitro source of explants led to higher regeneration efficiency as compared to ex vitro source. ► Number of shoot buds induced were affected by the explants position on seedling axis. ► Age of seedling also influenced the regeneration potential which decreased with increase in age. ► Transient decrease of Ca2+ concentrations in germination medium increased percentage response.
- SourceAvailable from: Laura Yesenia Solís-Ramos[Show abstract] [Hide abstract]
ABSTRACT: Establecimiento de suspensiones celulares de dos especies Jatropha (Euphorbiaceae) de Costa Rica. J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were suc-cessfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initi-ated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated. Rev. Biol. Trop. 61 (3): 1095-1107. Epub 2013 September 01.
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ABSTRACT: In order to establish a highly efficient and sustainable regeneration system, we systematically researched the key factors affecting direct shoot regeneration from Jatropha curcas leaves that were collected from Hainan (HN1-1), Lijiang (LJ3-1), and Yuxi (YX2-12) provinces in China. The L9(34) orthogonal test of thidiazuron (TDZ), kinetin (Kn), and gibberellic acid (GA3) were studied, and the explant type, growth age, and cultivar of leaves were subsequently investigated. Simultaneously, the combinations of plant growth regulators (PGRs) promoting shoot bud proliferation, elongation, and root establishment were examined. The results showed that the best medium for shoot bud induction was Murashige and Skoog (MS) medium supplemented with 1.0 mg/L TDZ, 0.5 mg/L Kn, and 0.5 mg/L GA3. TDZ was the key PGR, while Kn and GA3 played an important role in shoot bud elongation and the number of shoots per leaf disk, respectively. The induced shoot buds proliferated and readily elongated in MS medium with 0.3 mg/L 6-benzylaminopurine and 0.01 mg/L indole-3-butyric acid (IBA) and established roots in half-strength MS medium supplemented with 2.0 mg/L IBA. Using the previously described methods, the third to fifth leaves were found to be the best explant source for shoot bud induction, with a high induction rate, large shoot numbers per disk, excellent proliferation, and consistent rooting. With the use of this regeneration system, the shoot bud induction rate increased from the reported rate of 53.5% to more than 90% using different explants and cultivars, and the shoot number per leaf disk (shoot length ≥ 0.5 cm) increased from 1.6 to 3.5. Thus, this optimized regeneration system will effectively promote the propagation and genetic transformation of J. curcas.In Vitro Cellular & Developmental Biology - Plant 11/2013; · 1.14 Impact Factor
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ABSTRACT: In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0mg L(-1)) and BA (1.5mg L(-1)) was found to be the most effective (<90%) in callus induction. Two concentrations (1.5, 2.0mg L(-1)) of the IBA produced>80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (<90%) was observed on IBA (1.5mg L(-1)). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91mg/g-DW) and flavonoid content (7.38mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe(2+) chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.Comptes rendus biologies 01/2014; 337(1):19-28. · 1.71 Impact Factor