Transgenic Banana Rastali (AAB) with beta-1, 3-glucanase Gene for Tolerance to Fusarium Wilt Race 1 Disease via Agrobacterium-medi&ted Transformation System

摘要

A soybean endo beta-1, 3-glucanase gene (pRUKla-Eg) multiplied in Agrobactenum strain LBA 4404, was simultaneously introduced into single buds of in vitro grown banana cultivar, Rastali (AAB). Plasmid pROKla-Eg contained a neomycin phosphotransferasegene (npt 11) as the selectable marker to identify the transformants. Treatment A contained kanamycin at 100 mg L~(-1) and treatment B contained geneticin G-418 at 50 mg L~(-1) in both MS medium supplemented 5 mg L~(-1) of BAP together with 2.7 g of gelrite agar. Single buds derived from multiple bud clumps (Mbcs), were the target explants for transformation. An assay was performed to identify the minimum concentration required for two antibiotics (carbenicillin and cefotaxime) that is most effectiveagainst Agrobacterium strains, LBA 4404 and the effect on tissue regeneration capacity. Even though the transformation frequency based on kanamycin selection medium (treatment A) is higher, but there is no transformant could be confirmed based on PCR andSouthern blot analyses, as compared using geneticin G-418 selection medium (treatment B). These results suggested that using geneticin G-418 as selection agent is preferably than kanamycin due to lower concentration required to allow for the small numbers of putative transgenic cells in a large population of non-transformed ones to undergo multiplication and also reduced the occurrence of escapes. The transgenic banana plantlets were inoculated with 2xl06 spores mL~! conidial suspension of Fusarium oxysporum f. sp. cubense (Race 1) to evaluate the degree of tolerance and to investigate the effectiveness of the bioassay system as a potential tool for early screening. Assay of protein extract from the transgenic plantlets showed an significantly increased in beta-1, 3-glucanase enzymes activity over the untransformed plantlets. The present of Agrobacterium-mediaied transformation reported here is suitable for using tiny meristem tissues to obtain fungal disease tolerant or resistant banana through genetic engineering.