High frequency plant regeneration from callus cultures derived from primordial leaves of adult Japanese persimmon and protoplast isolation from those callus lines

Tao, R.; Yonemori, K.; Sugiura, A.

Acta Horticulturae 300: 251-254


ISSN/ISBN: 0567-7572
Accession: 002398236

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Callus cultures of Diospyros kaki cv. Jiro were initiated on half-strength MS medium containing 10 micro M zeatin and 1 micro M IAA. After several subcultures, calluses were subdivided and cultured on MS medium containing NH4NO3 and/or KNO3, modified to half to twice the strength of normal MS medium. Media contained fructose, glucose or sucrose at 0-5% and were supplemented with 10 micro M zeatin and 0.1 micro M IAA to induce adventitious buds. The best regeneration (88%) was seen on normal MS containing 3% sucrose. Efficiency of protoplast isolation from the callus was influenced by enzyme composition, osmotic pressure of the digestion mixture and age of callus. The best yield 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% Cellulase RS, 0.05% Macerozyme R-10, 1% PVP-10, 5 mM MES and 0.7 M mannitol. Over 2 x 106 protoplasts/g callus fresh weight were produced; viability was >70%. When protoplasts were cultured in KM8p agarose medium containing 2 mM glutamine, 1 micro M zeatin and 1 micro M NAA, after 3 weeks microcalluses consisted of 10-20 cells.