Developing a cryopreservation protocol for avocado (Persea americana Mill.) apical shoot tips using different antioxidants

Avocado seeds are highly heterozygous and recalcitrant, therefore, not suitable for conservation purposes. Presently, avocado germplasm is stored in field collections, which are constantly exposed to abiotic and biotic stresses. Moreover, size of gene pool, number of replications and quality of maintenance are largely constrained by local environmental conditions, space and funding. Cryopreservation offers a necessary, complimentary method that is safe, cost-effective and allows for long-term storage of Persea spp. Currently there are no cryopreservation protocols for storing in vitro shoot tips which are clonal to the mother tree, thus advantageous over somatic embryos generated from immature seeds which are heterozygous. This study optimised early stages of an in vitro system for shoot tip preservation of ‘Reed’ avocado. Fresh shoot tips were dissected (2 mm in size) from clonally propagated adult material and were treated with varying concentrations of different antioxidants (ascorbic acid, PVP, citric acid and melatonin) aiming to reduce browning. Once optimal antioxidant treatment was selected, shoot tips were then pre-cultured overnight in liquid medium containing 0.3 M sucrose and antioxidant. After pre-culture, tips were loaded (2 M glycerol + 0.4 M sucrose) for 20 min and treated with PVS2 at 0°C for 20 min. Results for 1) shoot health, 2) stunted growth, 3) dead shoots and 4) browning were recorded. Ascorbic acid at 100 and 250 mg L‑1 reduced browning of avocado shoot tips. These preliminary trials are revealing the best options for survival.