HIGH FREQUENCY PLANT REGENERATION FROM CALLUS CULTURES DERIVED FROM PRIMORDIAL LEAVES OF ADULT JAPANESE PERSIMMON AND PROTOPLAST ISOLATION FROM THOSE CALLUS LINES

1. Callus cultures were initiated from leaf primordia of Japanese persimmon (Diospyros kaki L. cv. Jiro) on MS(½N) medium containing 10 μM zeatin and 1 μM IAA. After several subcultures, the calli were subdivided and cultured on modified MS medium, with NH₄NO₃ and/or KNO₃ modified to ½ – 2 strength of normal MS medium containing fructose, glucose, or sucrose at 0 – 5 %. These media were supplemented with 10 μM zeatin and 0.1 uM IAA to induce adventitious buds. The best regeneration occurred on the normal MS containing 3 % sucrose with 88 % of the calli regenerating adventitious buds.
2. Callus cultures of cv. Jiro described above were used to develop efficient techniques for protoplast isolation. Isolation efficiency was influenced by the enzyme composition, osmotic pressure of the digestion mixture, and the age of callus. The best yield of protoplasts was obtained using callus 14 days after subculture, plasmolized in CPW medium (pH 5.6) containing 0.7 M mannitol prior to maceration in CPW medium containing 0.5 % Celluase RS, 0.05 % Macerozyme R-10, 1% PVP-10, 5 mM MES, and 0.7 M mannitol. Over 2x10⁶ protoplasts per gram fresh weight of callus were produced with the viability being more than 70 %. When the obtained protoplasts were cultured in KM8p agarose medium containing 2 mM glutamine, 1 μM zeatin, and 1 μM NAA, microcalli consisted of 10 to 20 cellls formed after 3 weeks of culture.

Download Adobe Acrobat Reader (free software to read PDF files)