Use of fluorescein diacetate (FDA) to determine ploidy of in vitro watermelon shoots

Ploidy of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] shoots and plantlets was estimated by painting the lower epidermis of intact in vi tro-derived leaves with fluorescein diacetate (FDA) and observing fluoresce nce of guard cell chloroplasts with a microscope and UV light. Leaves from in vitro shoot-tip cultures of known diploid cultivars and tetraploid breed ing lines were used to establish the mean number of chloroplasts per guard cell pair. Leaves from diploid and tetraploid shoot cultures had 9.7 and 17 .8 chloroplasts per guard cell pair, respectively. This method then was use d to estimate the ploidy of shoots regenerated from cotyledon explants of t he diploid cultivar Minilee. Approximately 11% of the 188 regenerated shoot s were classified as tetraploid during in vitro culture. Putative tetraploi ds were transplanted to the field and self-pollinated. About 45% of tetrapl oids identified in vitro produced fruit and viable seed. Chloroplast counts of R-1 progeny were used to confirm their ploidy. All of the putative dipl oids were confirmed diploid and all putative tetraploids proved to be non-c himeric true breeding tetraploids.