A TRANSGENIC APPROACH FOR DETERMINING SEX OF PAPAYA SEEDLINGS

Abstract

Sex segregation in gynodioecious papayas necessitates expensive and inefficient over-planting of indistinguishable hermaphrodite and female seedlings, followed by subsequent removal of surplus plants at flowering time, to ensure uniform stands of economically valuable hermaphrodite trees. Our objective is to produce transgenic papayas with marker genes linked to the sex-determining region, so that growers can distinguish the sex of seedlings at planting time by marker phenotypes. Transgene insertion is random, but genes controlling papaya sex reside in a relatively large non-recombining region of chromosome 1 amounting to 1-2% of the entire genome, so about 150 independent transformations are needed for 95% probability of sex linkage. We have used both particle bombardment and Agrobacterium inoculation to transform Hawaii cultivars with constructs containing a fluorescent yellow protein gene (Eyfp) and/or glufosinate resistance gene (bar). Sex-linked insertions will permit selection of glufosinate-resistant hermaphrodites or elimination of fluorescent yellow females. The papaya regeneration protocol of Fitch and Manshardt (1990), based on embryogenic callus derived from immature zygotes, was improved to promote more normal development by lowering 2,4-D concentration in the callus-induction medium, by venting headspace gasses during germination of somatic embryos and shoot growth, and by rooting shoots under photo-auxotrophic conditions. Nearly 500 putative transgenic lines representing independent transformation events were selected after 4 to 5 months of culture on glufosinate-containing media. Initial PCR assays on 40 lines obtained by particle bombardment indicated that 72.5% carried the bar transgene, however, no expression of Eyfp was detected by epifluorescence microscopy. Transformation success was independent of the transgene construct used (CAMBIA3300 vs. E3410) or the genotype of targeted papaya tissues (Kapoho, Sunrise, line D), but bombarded tissues responded better to glufosinate selection after a 2-week tissue recovery interval on non-selective medium. Success of Agrobacterium transformation was not affected by strain differences (LBA4404 vs. EHA105), but duration of the inoculation period interacted significantly with duration of co-cultivation. Transgene expression assays to confirm functionality are in progress, and screening for lines with sex-linked insertions will be conducted utilizing inverse PCR to sequence the DNA in regions flanking insertion sites and matching them with sequences in the publicly available papaya genome database to locate the sites on chromosomes.