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Isolation and culture of protoplasts from anthers of apple (Malus pumila Mill.) rootstock ‘M9’

Abreu-Tarazi, M. F., Leite, L. R., Dantas, A. C. M., Guerra, M. P.
Journal of horticultural science & biotechnology 2009 v.84 no.5 pp. 513-518
Malus pumila, agarose, anthers, apples, biotechnology, callus, culture media, endo-1,4-beta-glucanase, horticulture, liquids, protoplasts, rootstocks, viability
This report describes a reliable method for callus induction, isolation, and the culture of anther-derived protoplasts from the apple rootstock, ‘M9’, and provides information on the development of a protoplast-to-plant regeneration system. Callus induction occurred in anthers when cultivated on Nitsch and Nitsch (NN) or on AT3 media. Suspension cells were established from friable, anther-derived callus formed on NN basal medium. Protoplasts were isolated from callus and anther-derived suspension cells.The highest viability value for freshly isolated protoplasts (62.67 ± 1.53%) was achieved using a 4 h digestion period with suspension cells on Kao and Michayluk (KM) basal medium diluted in an enzyme solution primarily composed of [1% (w/v)] cellulase ‘Onozuka’ RS in combination with [1% (w/v)] macerase and [2% (w/v)] pectolyse Y-23. Protoplasts were cultured using three methods: agarose bead, hanging drop, or liquid culture. The first protoplast division was observed after 8 d of cultivation. The liquid culture method led to sustained division of callus and suspension-derived protoplasts. Micro-colonies were formed within 21 d, and microcalli were observed after 28 d of cultivation.