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

Abstract

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.