Seeds were taken from the fruit of Japanese persimmon 'Jiro' at weekly intervals from July 27 until August 17 in 1990. After removing seed coats and embryos, endosperms were cultured on MS (1/2N) medium containing 10 mu M zeatin, 0-10 mu M IAA, and 500 mgul - 1 casein hydrolysate. The highest frequency of callus formation was obtained with the endosperms sampled on August 11 when they were cultured on MS (1/2N) medium supplemented with 10 mu M zeatin, 10 mu M IAA and 500 mgul - 1 casein hydrolysate. Under this condition, 24% of the endosperms formed calli. These calli were subcultured on MS (1/2N) medium supplemented with 10 mu M zeatin and 1 mu M IAA. After five to seven subcultures, they were transferred onto regeneration medium which consisted of MS medium supplemented with 10 mu M zeatin and 0.1 mu M IAA. Five of seven callus strains formed adventitious buds on this medium, with the percentages of adventitious bud formation ranging from 10 to 37%. More than two thirds of the adventitious buds developed into shoots on MS medium containing 5 mu M zeatin and 1 mu M IBA. For rooting, the basal ends of these shoots were quickly dipped in 1.5 mM IBA 50% aqueous ethanol solution and cultured on 1/2MS (1/2N) medium. About half of them rooted on the medium after 40 days of culture.
As opposed to our initial expectance, flow cytometric determination of the nuclear DNA content and the chromosome count of the root tip cells revealed that only dodecaploid and hexaploid plants were regenerated from the endosperm-derived calli but not nonaploid. All the plantlets regenerated from three of five callus strains were dodecaploid (2n = 180), while those from the other two callus strains were hexaploid (2n=90). Although there was no isozyme variation among the plantlets regenerated from the same callus strain, differences were observed among the plantlets derived from different callus strains in at least one of the three enzyme systems tested. Also differences were observed in at least two enzyme systems between 'Jiro' and the regenerants from endosperms.