IN VITRO PROPAGATION OF APPLE

CATHERINE ANN LUNDERGAN, Purdue University

Abstract

In vitro culture of apple (Malus domestica Borkh.) was investigated with 2 major objectives: (1) to develop a micropropagation system and (2) to investigate the feasibility of long term germplasm storage in vitro. The micropropagation system involved multiplying shoots in vitro and grafting those shoots onto in vitro-grown seedlings with an established root system or onto stems of rootstock clones which would be induced to form roots. The system was investigated in 5 stages: (1) production of seedling rootstocks in vitro, (2) production of scions in vitro, (3) graft union formation, (4) scion growth after grafting, and (5) establishment of in vitro-grown seedlings in the greenhouse. Seedlings could be successfully cultured in vitro after stratification at 4(DEGREES)C for 6 weeks. Seeds from which all seed coats were removed were placed in a medium containing Murashige & Skoog salts, 0.1 mg/liter thiamine(.)HCl, 0.5 mg/liter pyridoxine(.)HCl, 0.5 mg/liter nicotinic acid, 2.0 mg/liter glycine, 30 g/liter sucrose, and 10 g/liter Bacto agar (BCM). The inclusion of sucrose in the medium was found to be necessary for the development of secondary roots. Seedlings grown in vitro could be successfully established in greenhouse conditions by acclimating with a mist system. Shoot tips cultured in BCM + 5 mg/liter BA gave maximum shoot proliferation but the shoots and leaves were dwarfed. Shoot tips could be stimulated to grow tall by placing them in BCM + 5 mg/liter 2iP or in BCM + 1 mg/liter BA and 1 mg/liter IBA. Shoot tips grown in these media exhibited an apical dominance effect with 1 tall shoot and reduced secondary bud break. Shoot proliferation and growth were unaffected by the addition of GA or inositol to the medium. In vitro grafting was achieved by inserting a scion, cut to a wedge, either in a diagonal slice in the hypocotyl of a seedling with an established root system or in a longitudinal cleft in a stem piece of an unrooted Malling rootstock clone. Roots were induced on M 7 and M 106 by dipping the basal ends of stem pieces in 50% ethanol containing, 2,500 or 10,000 mg/liter IBA. By combining the grafting and rooting operations a grafted plan to a size-controlling rootstock could be produced in vitro. 'Golden Delicious' scions proliferated in BCM + 5 mg/liter BA appeared to form a graft union with seedling rootstocks but did not grow. However, the transfer of proliferated shoots to a pregrafting media of either BCM + 5 mg/liter 2iP or BCM + 1 mg/liter BA and 1 mg/liter IBA promoted growth of the grafted scions. The "dwarfing effect" of BA on excised shoots was apparently carried over to grafted scions. Grant union formation was unaffected by the presence of inositol in the pregrafting medium but was stimulated by a postgrafting medium of BCM + 1 mg/liter BA and 1 mg/liter IBA as compared to BCM alone. Graft union formation was greater with 18-day-old seedlings than with 39- or 52-day-old seedlings. Apple shoot tips cultured in BCM + 5 mg/liter BA survived 9 months storage at 1(DEGREES)C or 4(DEGREES)C in this medium and proliferated new shoots when returned to 26(DEGREES)C. This suggests that in vitro-grown apple shoots may be amenable to long term storage at low temperatures.

Degree

Ph.D.

Subject Area

Livestock

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