A method to increase regrowth of vitrified shoot tips of avocado (Persea americana Mill.): First critical step in developing a cryopreservation protocol

Conservation of avocado (Persea americana Mill.) diversity is traditionally restricted to field collections as is the case for most recalcitrant species. Cryopreservation combined with in vitro culture offers a method that is safe and cost-effective to conserve germplasm of avocado. To date, cryopreservation of Persea spp. has been limited to somatic embryos, which are heterogeneous. A protocol for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. Vitrification of plant material is an important step in a cryopreservation protocol. PVS2 is a commonly used cryoprotectant in such vitrification-based protocols and causes osmotic stress due to high molarity. Avocado is highly susceptible to osmotic stresses imposed by cryoprotectants. Obtaining high survival rate after vitrification treatment is a prerequisite in optimising a cryopreservation protocol. To prime avocado shoot tips to withstand cryoprotectants, in vitro donor plants of two avocado cultivars, ‘Velvick’ and ‘Reed’, were subjected to two different pre-treatment conditions; 1) high sucrose (0.3 M) or 2) low temperature (10 °C) incubation, over a 2-week period prior extraction of shoot tips. Extracted shoot tips were subjected to PVS2 treatments and evaluated for survival and regrowth before liquid nitrogen treatment. The study revealed that pre-treatment is effective in helping avocado shoot-tips to withstand PVS2 treatments and is cultivar-specific. Shoot tips pre-treated with high sucrose resulted in the best regrowth for cv ‘Velvick’ shoots (73 %), while for cv ‘Reed’ low temperature pre-treatment was found to be the best (80 %). These results indicate that high sucrose or low temperature pre-treatments of avocado shoot tips can be used to reduce the risk of cell damage caused by PVS2 in a cultivar-specific manner. The successful pre-treatment conditions can be used to prime avocado shoot tips for cryoprotectants and is critical to pave the way for the development of the first ever shoot-tip cryopreservation protocol for avocado