Agrobacterium-MEDIATED GENETIC TRANSFORMATION OF SEAWEED Kappaphycus alvarezii USING Gα GENE AND CALLUS CULTURES

摘要

Cottonii seaweed ( Kappaphycus alvarezii Doty) is one of the most important commercial sources of carrageenans which are widely used in the pharmaceuticals and the food industry. A problem in the cultivation of this seaweed is the ice-ice disease, which is caused by extreme changes in environmental conditions such as temperature and seawater salinity. Gene transformation to produce Cottonii seaweed transgenics that are tolerant to environmental stress is potential solution to this problem. Gα gene encodes for the heterotrimeric G protein α subunit is a gene that plays a role in tolerance to biotic and abiotic environmental stresses. This study aimed to: (a) introduce the Gα gene into callus cells of K. alvarezii and regenerate transformed callus cells to transgenic plantlets; (b) determine the appropriate concentration of acetosyringone and Agrobac terium tumefaciens strain for gene transfer into the callus of K. alvarezii . Callus cells of K. alvarezii were transformed using Agrobacterium tumefaciens strains LBA4404 and EHA101 carrying the expression vector pGWB502- Gα with a CaMV-35S promoter. The calli and A. tumefaciens were co-cultivated in several concentrations of acetosyringone (20, 40, 60 mg/L). Regeneration of transformed callus cells into transgenic plantlets was successfully performed using the somatic embryogenesis technique. The result showed the highest percentage of putative transgenic micropropagule formation occurred with 20-40 mg/L acetosyringone. Polymerase chain reaction (PCR) analysis on twenty transgenic plantlets indicated that the Gα gene was successfully introduced into the genomic DNA of all of them. The transformation using EHA101 produced more transgenic plantlets that were morphologically normal and grew faster in aerated culture than using LBA4404. This study concluded that the appropriate method of gene transfer into K. alvarezii callus was co-cultivation with A. tumefaciens EHA101 in 20-40 mg/L concentration of acetosyringone.