Cold acclimation and cryopreservation of 'MM106' apple cultures

Abstract

Germplasm apple orchards are in danger of depletion and are often inaccessible due to land expropriation and political constraints. Since micropropagative methods are available for most apple cultivars and since methods of cryopreservation for some have been developed, this study was instigated to cryopreserve in vitro shoots to broaden the scope of technology available for conserving genetic resources. In vitro shoots of Malus domestica Borkh. cv 'MM106' were cold hardened to -19ºC in a simulation of autumnal hardening. When shoot-tips were excised from these cold-hardened cultures, and cryoprotected with 5% dimethylsulfoxide (DMSO) + 18.2% D-sorbitol, 60% of the shoot-tips survived freezing to -34°C. When cold-hardened shoots ( LT₅₀ of -19°C ) were incubated for an additional 7 days on MS media that contained 5 ppm of abscisic acid (ABA), the shoot-tips gained 7.5° cold resistance, but cold resistance was lost by longer incubation on media that contained ABA. Unhardened in vitro apple shots were severely damaged by preculturing on media with 5% DMSO + 5% glucose. The cold hardiness of unhardened shoots was unaffected by a 3 week incubation at 25°C on media that contained 5 to 50 ppm of ABA. Most shoot-tips and meristems, excised from cold hardened cultures, were consistently recovered only as callus, despite the use of different sized explants with several cryoprotectants and different freezing and thawing rates. More explants produced callus at -30 than at -40ºC. Less than 35% of explants produced callus after removal from liquid nitrogen. None of callused explants produced shoots. In this study, in vitro shoots of a woody species cold acclimated in response to a simulation of autumnal hardening, but the length of the hardening regime did not affect the level of cold hardiness. The shoots gained additional hardiness by a 7 day exposure to 5 ppm ABA. ABA did not become effective until after the 'MM106' apple shoots had been exposed to a cold-hardening regime.