Development of sago suspension culture system and optimization of transformation system for sago palm (metroxylon sagu rottb.) cultures

Evra Raunie, Ibrahim (2013) Development of sago suspension culture system and optimization of transformation system for sago palm (metroxylon sagu rottb.) cultures. Masters thesis, Universiti Malaysia Sarawak, (UNIMAS).

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Abstract

There are issues such as long maturation period, variable starch quality and lack of quality planting material that makes investors have less interest in commercial scale sago planting. Genetic transformation system for sago palm has the potential to improve the characteristic which would maximized yield per Ha and subsequently attract more investors. Therefore this study was aimed to optimized sago genetic transformation system in order to improve the characteristic of existing sago palm and to develop a suspension culture system to support mass production of sago clonal planting material. This study consists of development of sago palm suspension culture, determination of minimal inhibitory concentration of Basta™ on sago, development of sago transformation system via Agrobacterium and gene gun. The suspension culture of sago palm was successfully propagated using 4.5-15 gram of inoculum in every litre of media containing 0.18-1.8 mg/l NAA and 1-1.5 mg/l 2, 4-D. The propagated cells are ready for transformation after 3 months cycle. The minimal inhibitory concentration of Basta™ for sago palm was determined at 30 mg/l. The genetic transformation of sago target tissues derived from suspension culture was conducted using two methods; the Agrobacterium tumefaciens strain LB4404 containing plasmid vector pGSA1131 and the Biorad™ Helios Gene Gun System. The putative transgenic embryoids were selected through Basta™ selection process using media containing minimum concentration of Basta™ at 30 mg/l. The presence of bar and gus genes in selected regenerated callus were verified by PCR amplification, GUS staining analysis and dot blot analysis. From this study it is proven that iv the most suitable plant material for transformation is the embryogenic callus (D0E/EC) compared to embryoid stage (D1 and D2) and early formation of plantlets (D3) for both methods. As for the high sugar treatment during and after infection, the wounding using gold particle bombardment and the sonication treatment on the target cells had also proven able to increase the efficiency of the transformation via Agrobacterium. The transformation using Helios Gene Gun showed a higher transformation rates when target tissues at embryogenic stage (D0E/EC) were bombarded with 280psi of helium pressure at 6 to 8 cm of distance and with the number of firing once and twice of bombardment.

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