Cryopreservation of apple (Malus domestica 'Benoni') dormant buds using two-step freezing method

Apple is the most important temperate fruit in India. The top apple producing states are Jammu and Kashmir (J&K), Himachal Pradesh (HP) and Uttarakhand (UK). The apple germplasm is mainly conserved in the field genebanks at the ICAR-National Bureau of Plant Genetic Resource’s Regional Station, Shimla (HP) and Bhowali (UK); ICAR-Central Institute of Temperate Horticulture, Srinagar (J&K); ICAR-Indian Agricultural Research Institute’s Regional Station, Shimla (HP); Dr. Y.S. Parmar University of Horticulture and Forestry, Solan (HP); Govind Ballabh Pant University of Agriculture and Technology, Pantnagar (UK); Sher-e-Kashmir University of Agricultural Science and Technology, Srinagar (J&K). To have a backup, some accessions are being maintained under in vitro genebank at New Delhi. For long-term conservation of apple accessions, cryopreservation is the only method. Dormant buds of apple (Malus domestica ‘Benoni’) were cryopreserved by two-step freezing method. Apple twigs were collected from Srinagar at the sub-freezing temperature. Twigs were stored at 5-6°C until use. The buds were scooped from the twigs and desiccated on silica gel. Then the buds were subjected to gradual low temperature treatments up to
-30°C before submerging into liquid nitrogen. Programmable freezer was not used for freezing treatments. Post-thaw recovery (in vitro) was about 20%. The average rate of shoot formation was about 80%. This protocol of two-step freezing method was successfully applied to other three apple cultivars with the post-thaw in vitro recovery about 11-40%. This method appears to be a promising technique for cryopreserving dormant buds from field grown apple trees.