Plant Tissue Cult. 11(1) : 55-64, 2001(June)

Isolation and Culture of Mesophyll Protoplasts from Tea (Camellia sinensis L.)

M. T. K. Gunasekare and P. K. Evans¹

Plant Breeding Division, tea Research Institute of Sri Lanka, Talawakelle, Sri Lanka

Key words: Isolation, Culture, Mesophyll protoplasts, Camellia sinensis

Abastract

Protoplasts were successfully isolated from the second and leaf from the apex of actively growing shoots of Glasshouse-grown tea (Camellia sinensis L.) plants. After plasmolysis in CPW11M solution for 1 h, high yields (4.14 ? 10⁶ protoplasts per g fresh wt) of viable protoplasts (79% viability) were obtained in an enzyme mixture containing 1% (w/v) cellulase ?Onozuka? R-10 and 0.05% (w/v) pectolyase dissolved in CPW11m solution. Protoplast yield and viability were affected by the composition of the enzyme mixture (i.e. concentration and type of enzymes) used. As a source of pectinase, pectolyase was found to be more suitable than macerozyme R-10. Better survival of the protoplasts were embedded in the semi-solid medium containing agarose than in the liquid medium. Compared to KM8p medium MS basal medium was found more suitable. Cultured protoplasts could not survive in the latter medium although both media were supplemented with same growth regulator composition. Although sustained division of protoplasts was not observed under the present culture conditions, competence of the cultured protoplasts was indicated by early cell division and prolonged survival in culture.

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