The Use of Sucrose Pre-culture for Cryopreservation of Avocado (Persea americana Mill.) Cultivar ‘Velvick’

Chris O’Brien, Jayeni Hiti Bandaralage, Raquel Folgado, Sean Lahmeyer, Alice Hayward, Neena Mitter Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Australia The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA., United States

Australia’s avocado industry is worth over ~$557 million and produces more than 77,000t. The genetic diversity of the avocado genus remains largely unexplored but a vast resource (approximately 90 species/>3000 varieties) for crop improvement. Persea spp. are traditionally restricted to ex-situ conservation due to high heterozygosity and recalcitrance nature of seeds. Field banks are vulnerable to abiotic and biotic stresses. Thus, size of the gene pool, replications and quality are restrained by environmental conditions, space and funding. Cryopreservation is a safe and cost-effective method of germplasm conservation. Preserving shoot-tips enables conservation of exact gene pool of interest. Cryo-protocols that use vitrification solutions usually cause high mortality. Avocado is highly susceptible to osmotic stresses upon vitrification. To date there are no reports of survival/regrowth of in vitro cryopreserved shoot tips. This study aimed to optimize sucrose pre-culture to sustain shoot tip survival/regrowth after cryopreservation. In vitro shoots of cultivar ‘Velvick’ were pre-cultured for 2 weeks on 0.3 M sucrose and 100 mg/L Ascorbic acid containing media to be compared with normal sucrose concentration of 0.09 M. For cryo-treatments shoots were dissected to obtain 1 x 1 mm shoot-tips. When treated with PVS2 and evaluated for survival, pre-cultured shoots displayed higher survival 83%, higher regrowth 73% with vigorous green cultures and appeared morphologically normal. In contrast non-precultured shoots-tips recorded 70% survival, 23% regrowth resulted in stunted yellow-brown and less vigorous cultures. Very interestingly, after liquid nitrogen treatment pre-cultured shoot-tips showed 60% survival while no sucrose treatment lead to 0% survival. The cryo-preserved shoots developed into green proliferating clumps after 8 weeks in culture. This result is the first report of successful survival and proliferation of cryopreserved shoots of avocado and further optimizations may lead to development of a high efficiency cryopreservation protocol of the species.