Analysis of organogenic competence of cotyledons of Jatropha curcas and their in vitro histological behavior

Abstract

Using cut pieces cotyledons from germinating zygotic embryos of Jatropha curcas, we monitored the series of anatomical events leading to the generation of shoot through organogenesis by histological analysis. 14 days old cotyledons that were pre-cultured in a half strength Murashige and Skoog (MS) media supplemented with 100 mg/L myoinositol and 10 mg/L thiamine HCl, were cultured in organogenic competence induction media (CIM) comprising of MS salts with 1.5 mg/L benzyl adenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) and incubated for the induction of organogenic competence. Following their sequential transfer to shoot induction media (containing MS + 1.5 mg/L BA, 0.05 mg/L IBA and 0.5 mg/L GA₃); shoot elongation media (containing MS + 0.3 mg/L BA) and rooting media (containing half strength MS + IBA at different concentrations), we selected individual explants and subjected them to histological analysis in order to study the morphological changes occurring during organogenesis. Our findings show that to induce organogenesis using cut pieces of cotyledons, these tissues must be harvested at least by the 14th day following germination. Optimal organogenic competence was attained after 21 days incubation period in the dark where most of the explants showed evidence of protruding shoots surrounded by calli with various morphological features. Up to 53.91% of the total explants cultured in this study produced well defined shoots with each explant producing an average number of 1.5 shoots bearing two to eight leaves. We recorded the highest percentage of root formation, which stood at 27.50% when the shoots were cultured in a rooting media containing 0.3 mg/L IBA. Histological analysis of the different events occurring during the process of organogenesis suggest that the protuberances arising from parenchymatous cells and perhaps bundle sheath forming meristematic centres acquiring organogenic competence have a multicellular origin, indicating that the regeneration process takes place through direct organogenesis.

Key words: Jatropha curcas, organogenesis, auxins, histological analysis.