Development and Selection for Homozygous Transgenic Papaya Seedling

Somatic embryos of papaya lines were genetically engineered via Agrobacterium tumefaciens to contain the coat protein gene of the Virgin Islands strain of Papaya Ringspot Virus (PRSV). Regenerated plants, R0, had resistance to PRSV. Plants from the R1 generation segregated for resistance. Self-pollinations were conducted to develop inbred homozygous PRSV-resistant papaya lines. A quick efficient method for the screening and selection of transgenic papaya seedlings was needed to identify homozygous transgenic papaya lines. Transgenic lines of papaya also contained the neomycin phosphotransferase gene which was used for in vitro selection of transgenic papaya embryos. Papaya R2 seedlings were sprayed twice at 10 and 17 days with kanamycin in combination with dimethyl sulfoxide (DMSO). Non-transgenic papaya seedlings developed yellow spots within four days. DMSO enhanced the effect of kanamycin-susceptible plants, but, did not itself induce the formation of yellow spots in the transgenic lines. Following the success of the initial trial, seedlings of eleven transgenic R3 papaya lines were screened using 1,000 mg/L kanamycin with 1 ml/L DMSO. Three papaya lines produced no yellowing following two applications of kanamycin and DMSO, indicating homozygous lines for resistance. Through the use of kanamycin and DMSO, homozygous transgenic papaya lines can be successfully identified in the greenhouse at the seedling stage.