Ploidy of in vitro watermelon plantlets was estimated by painting the lower epidermis of leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from shoot-tip cultures of known diploid and tetraploid cultivars were used to establish the mean number of chloroplasts per guard cell pair for in vitro plantlets. Leaves from diploid and tetraploid plantlets had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method was used to estimate ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 10.6% of regenerated shoots were classified as tetraploid while still in vitro. Putative tetraploids were transplanted to the field and self-pollinated. A majority of polyploids identified in vitro were true breeding, nonchimeric tetraploids. This study demonstrate that FDA can be used to estimate ploidy of in vitro shoots of watermelon prior to acclimatization and transfer of plants to the greenhouse or field.
Michael E. Compton, J.W. Harris, and D.J. Gray
Michael E. Compton, D.J. Gray, and G.W. Elmstrom
Tetraploid individuals were identified among plants regenerated from cotyledons of diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultured in vitro. Tetraploid and diploid plants were distinguished by counting the number of chloroplast per guard cell pair. The mean number of chloroplasts was 19 and 11 for tetraploid and diploid plants, respectively. Self-fertile tetraploids were obtained from the diploid cultivars Mickylee, Jubilee II and Royal Sweet. `Dixielee' and `Minilee' tetraploids failed to set fruit. Progeny obtained from self-fertile tetraploids were crossed with diploid pollinators to produce triploid hybrid seed. All triploid plants produced seedless fruit that was superior or equal to fruit produced by currently available triploid hybrids. This demonstrates that tissue culture can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.
Sadanand A. Dhekney, Zhijian T. Li, Michael E. Compton, and Dennis J. Gray
Stamens and pistils from mature grapevines and leaves from in vitro micropropagation cultures were used to optimize parameters influencing somatic embryogenesis in Vitis. Embryogenic competence was dependent on species/variety, explant type and developmental stage, medium composition, and growth regulator concentration. Of varieties evaluated, a greater number produced embryogenic cultures from stamens and pistils (26) compared with leaves (six). Among the different stamen and pistil stages, Stage II and III explants produced the maximum embryogenic response regardless of genotype and medium composition. Of seven culture media tested, the highest embryogenic response was recorded from varieties cultured on MSI (18) and PIV (16) media. Experiments annually repeated over 3 to 10 years demonstrated reproducible results. Highly reliable protocols for somatic embryogenesis were obtained for 29 Vitis species and varieties, including 18 Vitis vinifera varieties, Vitis riparia, Vitis rupestris, Vitis champinii, and eight Vitis hybrids. Embryogenic cultures were maintained on X6 medium for a period of 6 months to 2 years depending on the variety and used in studies involving genetic transformation and transgenic plant regeneration.