Development Of Cryopreservation Protocol For Actinidia Spp. And Assessments Of Genetic Stability In Cryo-derived Plants

Kiwifruit belongs to the family Actinidiaceae and the genus Actinidia.Availability of and easy access to diverse germplasm are necessary for plant genetic improvements in both traditional and biotechnological programs.Cryopreservation is at the present time the most effective method for long-term preservation of plant germplasm.There is still a lack of a simple,efficient and widely applicable cryopreservation method for kiwifruit germplasm,which greatly limits the establishment of cryo-banks of genetic resources of Actinidia spp.Meanwhile,evaluations of root formation and vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants when cultured in vitro and grown in soil provide valuable information on the use of cryopreservation for setting-up cryo-banks of plants.The main results obtained in this study are as followings:1.A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’(Actinidia chinensis var.deliciosa).This droplet-vitrification was applied to five cultivars and three rootstocks belonging to five Actinidia species including A.chinensis var.deliciosa,A.chinensis var.chinensis,A.macrosperma,A.polygama and A.valvata.The highest(68.3%)and lowest(22.5%)shoot regrowth were obtained in A.macrosperma and A.chinensis var.chinensis ‘Jinmi’,respectively,with an average of 46.4% shoot regrowth obtained across the eight genotypes.2.No significant differences were found in root formation and shoot growth between the in vitro-derived plantlets(the control)and cryo-derived ones when cultured in vitro.No significant differences were detected in survival and vegetative growth between the in vitro-derived plants(the control)and cryo-derived ones after re-establishment in greenhouse conditions.3.Inter-simple sequence repeat(ISSR)and amplified fragment length polymorphism(AFLP)did not detect any polymorphic bands in the cryo-derived regenerants when cultured in vitro and after re-establishment in greenhouse conditions.These data indicate rooting ability,vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation.4.Methylation sensitive amplification polymorphism(MSAP)detected 12.8% and 1.6% DNA methylation in cryo-derived samples when cultured in vitro and after re-established in greenhouse conditions,respectively.The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia.Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.