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

作者：Abreu Tarazi M F; Leite L R; Dantas A C M; Guerra M P^*

来源：Journal of Horticultural Science & Biotechnology, 2009, 84(5): 513-518.

DOI：10.1080/14620316.2009.11512558

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. ne 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.

• 出版日期2009-9