Genetic transformation of Jatropha curcas L. by Agrobacterium tumefaciens-mediated with type 2 metallothionein gene from Melastoma malabathricum

Abstract

The main reason why we use marginal land for non-food crops like jatropha is the limited potential land to cultivate it. The condition of marginal land with low fertility, low pH and high content of heavy-metal can caused inhibit the growth and decreasing production of crop. The tolerance of plants to metal toxicity can be genetically improved by introducing and expressing a gene associated with the metal tolerance. One of these genes is MaMt2, a gene coding type 2 metallothionein that isolated from Melastoma affine. Metallothionein is a cystein-rich protein so it can binding various types of metal. The objective of this research was to obtain increase resistance of jatropha to heavy-metal by integrated MaMt2 gene to jatropha and molecular analysis from transgenic plant. Genetic transformation was performed by using Agrobacterium tumefaciens strain LBA4404 which containing pIG6-SMt2 plasmid. pIG6-SMt2 plasmid contains of MaMt2 gene, hptII gene (hygromycin phosphotransferase) under ubiquitin promoter and NOS (nopaline synthase) terminator. Cotyledon from 2 weeks-old of jatropha cultivar IP-2P was used. The non-transformed explants regenerated 14 days in the non selective regeneration medium while the transformed ones regenerated 18 days in the selective regeneration medium. The average of regenerated shoot each explant was 2.1 for non-transformed explants and 1.6 for transformed ones. Putative transgenic shoots were selected in two step by using medium containing 1.5 mg l-1 and 2.5 mg l-1 of hygromycin successively. This research resulted 25 putative transgenic shoots. Molecular analysis was performed to determine the integration of MaMt2 gene under control for ubiquitin promoter in transgenic shoots by PCR. PCR was carried out by using with UbiQF-NosTR and SMt2F-NosTR combination primers. PCR of transgenic shoots resulted 1160 bp and 526 bp fragments respectively. Molecular analysis showed that three of 15 putative transgenic shoots are transgenic shoots containing MaMt2 gene under control of ubiquitin promoter.