Agrobacterium-mediated transformation of Jatropha curcas leaf explants with a fungal chitinase gene

Abstract

Jatropha curcas L. oil has been shown to be suitable for the production of biodiesel. However, this species has not been domesticated yet. Genetic breeding through conventional methods is time consuming and costly, hence, genetic transformation could contribute positively to the improvement of interesting traits. Although in vitro regeneration and stable genetic transformation has been pursued for several years, variation in transformation efficiency remains strongly genotype-dependent and indicates that protocols optimization is still needed. Thus, this study was carried out to introduce a chitinase gene from the Trichoderma viride fungus into the genome of a J. curcas superior genotype by inoculating leaf explants with Agrobacterium tumefaciens EHA 105 strain in axenic conditions. Some key parameters such as pre-culture period and antibiotic doses were optimized with 500 mg.L-1 cefotaxime and 100 mg.L-1 kanamycin concentrations being suitable for A. tumefaciens inhibition and explant selection, respectively. The best transformation efficiency (50%) was obtained when leaf explants were incubated on a culture medium promoting shoot regeneration at 15 days before the induction of the transformation process. Plants where chitinase gene amplicons could be detected were assessed for transgene copy number and expression levels by quantitative real-time polymerase chain reaction (PCR). One, two and three copies of the introduced gene were confirmed in nine transgenic lines with two of them that were assessed for gene expression and showed quantitative variation for this variable. These results bring valuable information for further gene insertions in breeding programs of J. curcas for fungal disease resistance.

Key words: Agrobacterium tumefaciens, pre-culture time, kanamycin, quantitative real-time polymerase chain reaction (PCR), transgene copy number.