Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt

Bananas and plantains are the fourth most important crop in the world as well as Malaysia. In recent years, their production has been seriously threatened by Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense fungus, and it is one of the most important destructive diseases of banana (Musa spp.). Genetic engineering offers the greatest opportunity to the breeders to increase tolerance to Fusarium wilt. Hence, this study aims to produce a new banana cultivar tolerant to F. oxysporum f.sp. cubense race 4 through genetic engineering. Male inflorescence of Pisang Berangan (AAA), Pisang Nangka (AAB), Pisang Rastali (AAB) and Pisang Abu (ABB) as a potential explant for rapid micropropagation were used for in vitro propagation study. Considering the importance of cooking banana in the diet of the local people, Pisang Nangka (AAB), a cooking banana, was chosen for the genetic engineering study. Single cauliflower-like bodies cluster induced from male flowers, in MS medium containing 8 mg/L of BAP, was used as target tissue for gene transfer. Hygromycin was applied as a selection agent since plasmids used in this study contain the hygromycin resistance gene. Physical and biological parameters which affect DNA delivery into the Pisang Nangka (AAB) single cauliflower-like bodies’ have been optimised using gfp as reporter gene. The optimised bombardment parameters for the ‘CLBs’ clusters was 1100 psi, 9 cm target distance, 28 mmHg, 1 μm gold particle size, two times bombardment, 60 μg/μL amount of the gold particles per bombardment, 1.5 μg/μL DNA per bombardment and three days pre-culture prior to the bombardment. Rice thaumatin-like protein gene was cloned in pCambia 1304 binary vector by NcoI and PmlI restriction enzymes. Transformation of cloned rice thaumatin-like protein (tlp) gene was performed using particle bombardment. Integration of transgene was assessed by PCR amplification of tlp gene using its specific primer. Genomic southern blot hybridization confirmed the incorporation of the tlp gene in the host genome. RT-PCR revealed the expression of transgene in leaf tissue as well as root tissue in transformants.The 28 day old spores of F. oxysporum f.sp. cubense (Race 4) with 66% germinating capacity were used to estimate the transgenic Pisang Nangka (AAB) plantlets tolerance against Fusarium wilt. The percentage of disease incidence occurred in control plantlets was 89.1% after four weeks of infection while it was 29.4 % in transgenic plants. The results demonstrated that expression of rice thaumatin-like protein in transgenic banana plants enhanced resistanceto Fusarium wilt significantly.

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