Direct Organogenesis and Plantlet Multiplication from Leaf Explants of in Vitro-Grown Shoots of Apple {Malm domestica Borkh.) cv. 'Golden Delicious' and 'MM111' Rootstock

摘要

The aim of the present study was to develop an efficient direct shoot formation system for apple {Malus domestica Borkh.) cv. 'Golden Delicious' and 'MM111' rootstock as a prerequisite for genetic transformation with antifungal genes and also as a method for rapid clonal multiplication. Adventitious shoot formation from leaf pieces of 'Golden Delicious' and 'MM111' was achieved using leaves from in vtfro-grown shoots. Optimum conditions for 'direct' shoot organogenesis resulted in 92 and 90% of the explants producing one or more shoot per explant with high regeneration rate of 4 and 4.1 in 'Golden Delicious' and 'MM111', respectively on MS basal medium containing 1.0 g/1 MES (morpholino ethanesulfonic acid), 2.0 mg/1 TDZ, with 0.2 mg/1 NAA. Organogenesis did not occur on media without cytokinins. The organogenic capacity of leaf pieces was dependent on the leaf maturity and the origin of the leaf piece with the youngest light green expanding leaves being more regenerative than the older ones. Middle leaf segments were more responsive than the upper or lower part of the leaf. Adventitious shoots originated from both cut areas and from surfaces of the wounded leaf explants. Shoot multiplication was achieved on media consisting of MS media supplemented with B5 vitamins, 1.0 g/1 MES, 30 g/1 sucrose, 1 mg/1 BAP, 0.3 mg/1 IBA, 0.2 mg/1 GA_3 and 6 g/1 agar and were subcultured every 4 weeks. In vitro rooting was achieved easily by transferring 2-3 cm long shoot tips to rooting MS basal medium supplemented with 1.0 mg/1 indole-3-butyric acid (IBA). Multiplied plants were successfully acclimatized and cultivated in the field under natural conditions to evaluate their phenotypic uniformity and field performance.