Development and commercialization of PRSV [papaya ringspot virus] resistant GM [genetically modified] papaya for fresh fruit and papain production

A total of 7 T sub4 lines (142.3, 132.2, 124.3, 132.1, 152.1×148.1, 152.5×148.1 and 163.3) resistant to PRSV consisting of 73 individual plant progenies were characterized morphologically and evaluated for fruit characteristics. These lines have morphological and fruit characteristics that are similar to the non-transgenic 'Davao Solo' Control. Seven T sub1 lines (142.3, 132.2, 124.3, 132.1, 152.1×148.1, 152.5×148.1 and 163.3) consisting of 73 individual plants were self or sib-pollinated giving a total of 669 self-pollinated fruits and 113 sib-pollinated fruits. A total of 6,631 zygotic embryos were isolated from immature 'Davao Solo' papaya seeds for the induction of somatic embryos. From these, 2454 somatic embryos were developed. Nine hundred forty six (946) somatic embryos were transformed using replicate gene, while 1,098 somatic embryos were transformed using transformed using CP plus inverted repeats gene. Regeneration of transformed calli using the various gene constructs is in progress. Seventy T sub1 progeny plants out of 73 that were planted in soil in the BL2 screenhouse were found to be resistant to PRSV. Twenty-three (23) T sub1 lines were identified to be resistant to PRSV. Fifteen T sub2 lines were screened for resistance to PRSV. Out of these, 5 T sub2 lines including 132.2-9.13, 132.3-17.12, 142.3-15.23, 132.2-1.13 and (152.5×148.1-8.11) were resistant to PRSV. A total of 215 PRSV isolates were collected from various provinces in Luzon. Five isolates (Cavite, Bulacan, Marinduque, Quezon and Isabela) produced local lesions on its diagnostic host, (tenopodium quinoa while chlorotic symptoms were produced on its propagatine host, Zucchini grey. Five host plants (eggplants, cotton, okra, zucchini and papaya) and at least 500 aphids were raised and used for transmission studies. A Transmission Parameter Combination (TPC) of 5 minutes (AFT) - 5 minutes (IFT) - 10 aphids per plant was found effective in transmitting PRSV 2 to 3 weeks after inoculation in 'Davao Solo'. DNA of 30 progeny plants from 18 T sub1 lines extracted for use in the segregation analysis of the Cp transgene. In addition, Southern analysis were also done. Plasmid DNA of pMON 65306 was isolated using alkaline lysis method. The CP gene insert (900 bp) in this plasmid was amplified by PCR. A Kanamycin spray using 300 mg/l was identified to be effective in screening for nptll expressing transgenic plants was done. The three transgenic events planted in the Confined Trial Site at CSC-IPB were evaluated for morphological characteristics. The transgenic events are not morphologically different from the non-transgenic control. A total of 14 individual plants belonging to 3 transgenic events were selected. The plants had vigorous growth, normal canopy size, big stem diameter and they produced flowers and fruits despite the heavy virus inoculation pressure. A total of 19 T sub2 clones belonging to 3 transgenic lines were successfully micropropagated. The self-pollination of the grafted clones was done. A total of 17 PRSV isolates obtained from Luzon showed positive reaction to the PRSV-P antiserum in indirect ELISA test. Three (30) transgenic lines (132-2.9-13.27, 132-2.17-12.39 and 124-3.9-2.17) that were assessed for their reactions to 3 Luzon PRSV isolates showed two types of resistance: a) symptomless appearance and b) few to several stem streaks only. T sub3 lines of papaya in the confined field showed homozygosity for the CP transgene based on PCR analysis. Fourteen (14) out of 15 plants of Event 132 had the 318 bp inset. Twelve (12) out of 13 plants of Event 124 are positive for the Cp fragment. Based on Southern Blot analysis, the transgenic events are positive for a high mollecular weight CP insect.