Development of sweet potatoes varieties resistant to sweet potatoes virus disease complex (SPUD) through Agrobacterium-mediated transformation:phase 2 - a proof of concept

Sweet potato is an important cash crop in the Philippines and an emerging industrial crop for value-added foods, feeds and starch products aside from being a traditional food of high nutritive value. However, production is greatly hampered by pests and diseases, particularly viruses causing tremendous losses in yield and quality of the crop. Considerable efforts were directed towards the virus disease management like pathogen tested planting materials, rouging and genetic transformation. The use of Agrobacterium-mediated transformation system in genetic transformation has been of utmost importance in clonally propagated crops such as sweet potato and has been used in this study. Three gene constructs from two SPFMV [sweet potato feathery mottle virus] strains, the Russet Crack (RC) and Common strain (C) were subcontracted to a private genetic engineering laboratory through the initiative and financial support of ISAAA. The presence of gene inserts was validated through restriction enzyme digestion and polymerase chain reaction (PCR). An optimized regeneration protocol has been established and used in transforming the two local sweet potato varieties, (VSP6 and NSIC SP 31). The four stages on the production and maintenance of virus free mother plants, calli induction, embryogenic calli induction and regeneration has been done with success. More than 3000 embryogenic calli have been produced and are being maintained for Agrobacterium transformation. The use of meristem as explants gave higher percentage of calli and shoots produced than leaf explants. Embryogenic calli from V.S.P. 6 shoot meristems showed higher regeneration rate than NSIC SP 31 embryogenic calli. For Kanamycin sensitivity, the untransformed sweetpotato embryogenic calli were 100% killed at 200 mg/L giving an optimal Kanamycin concentration at 100 mg/L. Three batches of experiments on the transformation of the two local sweet potato varieties were done using the Agrobacterium strain ABI harbouring the CP gene of SPFMV-Sapang (RC) isolate, driven by an E 35S promoter in a SSFLA S01.0398 binary vector. The first batch of transformation was tried in liquid suspension in both the coculture and Kanamycin selection then placed back in basal medium for regeneration. Selection for Kanamycin resulted to a moderate percentage of calli that survived the 100 mg/L Kanamycin concentrations. The second batch of transformation was however, hampered by bacterial and fungal growth contaminations. The killing of the Agrobacterium in the transformed calli also became a problem. For the third batch of transformation, calli and bacteria were co-cultured in MS medium supplemented with 20 mg/L acetosyringone, 2 mg/L 2, 4D, 3% (w/v) sucrose and 0.3% phytagel for 3 days in the dark. The cultures were then washed in 500 mg/L carbenicillin and selected in MS medium supplemented with 100 mg/L Kanamycin, 2 mg/L 2, 4D, 0.3% sucrose and 0.3% phytagel with carbenicillin for 2 weeks. The calli were again washed with distilled water supplemented with 500 mg/L carbenicillin blotted dry and were being sub-cultured every two weeks for two months in same medium. Majority of the calli turned brown 2 weeks after culturing in Kanamycin selection medium and died subsequently a week after. Surviving calli were cleared with 500 mg/L carbenicillin and again subcultured in MS proliferating medium with 500 mg/L Kanamycin, 2, 4D and carbenicillin. Proliferating calli turned yellowish in color, as observed in both transformed and untransformed control. These were subjected to regeneration after two months using MS supplemented with 4 mg/L ABA, 1 mg/L GA, 500 mg/L carbenicillin and 0.3% phytagel. Some of the untransformed calli in 25 mg/L Kanamycin were observed to regenerate faster than untransformed calli with 100 mg/L Kanamycin. Transformed calli of both sweet potato varieties are still being observed for regeneration. Confirmation for transformants will be done using molecular methods.