Development of Viruses Resistance in some Plants via Genetic Engineering Approaches

Abstract

Melon and watennelon are among the most important vegetable crops of the Cucurbitaceae. Members ofthis family are susceptible to different kinds of viral diseases. This investigation was attempting to establish an efficient regeneration system for melon and watermelon and to produce transgenic plants resistant to ZYMV and WmCSV. Melon regeneration system of the melon cultivar Shahd El• Dokki was improved by adding 5 mg/l AgNO, to shoot formation medium (MR medium). This enhanced. the shoot regeneration frequency from 25% to 86%. A percentage of 75% of the regenerated shoots produced roots on MS medium containing 20 g/I NAA. An effective plant regeneration and a gene transfer systems via Agrobacterium tumefaciens were developed in watermelon local cultivars (Giza 1 and Giza 21) using cotyledons as explants. The shoot regeneration frequency was 49% and 47% for cvs. Giza I and Giza 21, respectively. The regenerated shoots were rooted on MS medium containing 40 g/l NAA A mutation by silencing the two possible initiator AUG codons was performed in ORF AC4 ofWmCSV. The mutated Ac 1 gene with the modified ORF AC4 was subcloned in binary vector pBin19 creating pBin/WA-11. Melon and watermelon cotyledons from 5-days-old seedlings were inoculated with Agrobacterium strain LBA4404 harboring one of the binary vectors pZYMV-Eg• cp, pBin/WA-12 or pBin/WA-11 containing ZYMV-cp, modified Acl or modified Acl/ Ac4 genes. Selection on regeneration medium supplemented with 125 mg/l kanamycin, revealed a number ofkanamycin-resistant plantlets. Integration of the transgenes in the genome of putatively transgenic plants and expression of these genes was verified by PCR and western blot analysis. The PCR results revealed that the number ofpositive melon plants transformed with plasmids pZYMV-Eg-cp, pBin/WA-12 and pBin/WA-11, were 12, 11 and 2 plants, respectively. Moreover, PCR results of putatively watermelon plants showed that three and one plants in cv. Giza I and Giza 21, respectively were positive to ZYMV-cp gene. Whereas, 28 and only 2 plants were positive to the Rep gene in the watermelon plants transformed with pBin/WA-12 and pBin/WA-11, respectively. Western blotting analysis of the transgenic plants confirmed the expression of CP in the three PCR positive watermelon plants cv. Giza 1. Whereas, the tested 5 Rep• PCR positive melon plants showed the Rep gene expression. While, in the watermelon 48 plants exhibited a weak Rep expression. Subsequently, the resistance to WmCSV was detected using the agroinoculation assay on the TO and Tl plants. A number of 29 watermelon plants out of 105 showed viral symptoms delay in the new shoots compared to the non-transformed plants.