Morphological and molecular analysis of jatropha curcas l. related to aluminum stress as a preliminary study to develop transgenic plant

Abstract

As a response to a progressive price increase of fossil based oil in the world, various initiatives to develop a reliable source of renewable energy become a major global attention. One potentially promising option is biofuel since these are derived from biomass. Biofuel crops should be planted on land that is considered marginal. A crop that is often cited as ideal for growing on marginal land including acid soil with high Al solubility, is the physic nut, Jatropha curcas. However, the major problem of the plant cultivation in acid soil is the decreasing productivity. Jatropha plants are not, by nature, tolerant to Al stress. A promising approach to overcome this limitation is to develop a transgenic Jatropha plant that can withstand to severe Alstress. Thus it is possible to use TaALMT (Triticum aestivum Al-activated malate transporter 1) gene which encodes the member of a novel membrane protein family that functions as an anion channel to mediate malate efflux from plant roots cells in the presence of extracellular Al3+. Investigating the physiology and molecular basis of Al-stress tolerance during seedling development is an essential prerequisite before developing Al-tolerant Jatropha plant by genetic engineering. Among six populations of Jatropha curcas L., IP-2P population could be categorized as slightly tolerant to Al stress, but it had uniform seed viability, and high productivity character which became the reason to use it as a plant material to develop transgenic Jatropha plant. For preliminary study, J. curcas IP-2P was choosen to be used for gene expression analysis by Quantitative Real-Time Polymerase Chain Reaction (qRT PCR). To normalize mRNA expression in the gene expression analysis we used actin gene. Because there is no information of actin gene nucleotide sequence from J. curcas we isolated Jatropha actin (JcACT) by PCR. Three cDNA of actin genes from J. curcas L. IP-2P had been isolated. The analysis revealed that Al stress induced ALMT transcript accumulation both in roots and leaves of non transgenic J. curcas IP-2P. Low pH treatment alone could also induce transcript accumulation both in the root apices and leaf but at lower rates than that observed for Al. This result indicated that response of Jatropha seedlings were not specific to Al stress but also low-pH stress. The TaALMT gene was successfully introduced into Jatropha curcas L. IP-2P via Agrobacterium tumefaciens-mediated transformation. Two putative transformant shoots were regenerated following selection on the bispyribac-containing medium. The presence of the transgene in the genome of transgenic plants was confirmed by PCR. The results indicated that the exogenous TaALMT1 gene was successfully integrated into the genome of J. curcas IP-2P plants.