Abstract
The number of vegetatively propagated species conserved by cryopreservation is increasing thanks to the development of vitrification-based methods in recent years. Although the most economically important horticultural crop in New Zealand is kiwifruit (Actinidia spp.), there is currently no cryopreserved collection of its genetic resources. The field collections of kiwifruit are limited to a few countries and are at a high risk of genetic erosion due to diseases such as Pseudomonas syringae pv. actinidiae. An alternative method of ex situ conservation is through in vitro collections but these are prone to contamination and somaclonal variation, and require maintenance through subculture. Cryopreservation is considered the only method available for the long-term preservation of clonally propagated species while maintaining their genetic fidelity. As cryopreservation outcomes can be species and genotype-specific, understanding biochemical and cell ultrastructural changes during cryopreservation is important for the successful development of protocols for the establishment of a kiwifruit germplasm collection in cryo-storage. Among different vitrification-based cryopreservation techniques, droplet vitrification is becoming the preferred method for many species, as it ensures fast freezing and thawing rates that are vital for vitrification of tissue, but it has not been tested in kiwifruit yet. This research aimed to develop a droplet vitrification protocol for kiwifruit using A. chinensis ‘Hort16A’ as a model, by optimising different pre-treatments to enhance the survival after cryopreservation and assessing the levels of enzymatic and non-enzymatic oxidative markers as well as quantification of oxidative damage and ultrastructural changes. Cold acclimation of donor plants at 4 °C for 2 weeks followed by daily increasing sucrose preculture (0.25, 0.5, 0.75 and 1.0 M) of 1-2 mm shoot tips and supplementation of ascorbic acid (0.4 mM) in all media throughout the procedure registered 40 % regeneration after cryopreservation against 0 % regeneration in shoot tips without any pre-treatment. One-hour treatment of shoot tips in a modified vitrification solution at 0 °C is another important step in the protocol. All the antioxidant markers showed a significant increase in pretreated shoot tips compared to non-pretreated shoot tips throughout the steps, especially 24 h after recovery from cryopreservation. Oxidative damage markers, lipid peroxides and protein carbonyls were significantly lower in pretreated shoot tips than in non-pretreated shoot tips after cryopreservation. suggesting better protection as a result of pre-treatment and ascorbic acid supplementation. Transmission electron microscopic images of the ultrastructure after cryopreservation showed damage in cell organelles of all cells of non-pretreated shoot tips whereas, some cells in pretreated shoot tips had structural integrity confirming the results of oxidative marker assays and recovery after cryopreservation. These observations show that pre-treatment is necessary to ensure survival after droplet vitrification of Actinidia species. Testing the applicability of this protocol to other A. chinensis genotypes and then to other Actinidia species, and further research to improve it would pave the way for the establishment of a cryopreserved Actinidia collection.