Study on the cryopreservation of dormant buds by vitrification in Actinidia arguta ‘Kuilü’

Abstract:【Objective】Actinidia arguta is a precious resource of cold-resistant fruit tree in China. It is extremely resistant, and rich in nutrientsand vitamin C, so it is of important nutritional and economic values. A. arguta is one of the important utilization values in the Actinidia and an important germplasm resource for variety improvement. Due to the increasing commercialization and large-scale production of A. arguta, resulting in a single variety and the disappearance of many excellent genetic resources, the research on ultra-low temperature preservation technology for kiwifruit germplasm resources is of great significance. The tara vine‘Kuilü’is an excellent variety that has been bred by the Institute of Special Wild Economic Animals and Plants of the Chinese Academy of Agricultural Sciences for more than 10 years. Using‘Kuilü’as a test material, the objective of this project is to study the ultra-low temperature preservation method, so as to provide a reference for the long-term preservation of kiwifruit germplasm resources.【Methods】The‘Kuilü’dormant branches were cut into single-bud stem sections with a length of about 2 cm. Firstly, the single-bud stem sections were peeled, then soaked in 75% alcohol for 30 s, and finally, sterilized by shaking with 0.1% HgCl2 for 30 min. The dormant buds were peeled for ultra-low temperature preservation by vitrification. Basic procedure of dormant bud vitrification cryo- preservation was as follows: the dormant buds soaked in preculture fluid containing 0.3 mol·L-1 sucrose + 1 mol · L- 1 glycerol + MS were shaken and cultured for 2 d, and thereafter they were transferred into 2 mL cryopreservation tubes under sterile conditions, with 10 buds per tube, and repeated 3 times. Load- ing was made at room temperature for 20 min with loading solution (2 mol·L-1 glycerol + 0.4 mol·L-1sucrose + MS), dehydration was conducted with PVS2 for 120 min at 0 °C, and then immediately put in- to liquid nitrogen to freeze for more than 24 h after they were changed to fresh PVS2. The tubs were tak- en out, and thawed immediately in 38 °C water bath for 2 min. Then they were washed 3 times for 10 min, each time using unloading solution including 1.2 mol·L-1 sucrose and MS, and the sterile filter paper was blotted and inoculated to MS + 2 mg·L-1 6-BA + 0.02 mg·L-1 NAA recovery medium, the tubs were cultured for 3 d in the dark, and then placed under light. Single factor experiment was performed on this basis. There were 9 kinds of preculture medium, 0.3 mol·L-1 sucrose + MS, 0.3 mol·L-1 sucrose + 1 mol·L-1 glycerol + MS, 0.3 mol·L-1 sucrose + 2 mol·L-1 glycerol + MS, 0.5 mol·L-1 sucrose + MS, 0.5 mol·L-1 sucrose + 1 mol·L-1 glycerol + MS, 0.5 mol·L-1 sucrose + 2 mol·L-1 glycerol + MS, 0.7 mol·L-1 su- crose + MS, 0.7 mol·L-1 sucrose + 1 mol·L-1 glycerol + MS, 0.7 mol·L-1 sucrose + 2 mol·L-1 glycerol + MS; the pre-cultivation time was 1, 2, 3, 4, 5 or 6 d; the loading time was divided into 10, 20, 30, 40, 50, 60 min; there were four kinds of plant vitrification solution, respectively: PVS1 (0.5 mol·L-1 Sorbitol + 22% glycerol + 15% ethylene glycol + 15% polyethylene glycol + 7% dimethyl sulfoxide + MS), PVS2(30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 mol · L- 1 sucrose + MS), PVS3(50% glycerol + 50% sucrose + MS), PVS4 (35% glycerol + 20% ethylene glycol + 0.6 mol·L-1 sucrose + MS); PVS2 dehydration time was divided into 30, 60, 90 , 120, 150, 180, 240 or 300 min; MS was used as the basic recovery medium, and 6-BA and NAA of different concentrations were added. Flow cytom- etry was used to identify ploidy of plants that were directly developed from dormant buds and regenerat- ed plants that derived from dormant buds after cryopreservation.【Results】The results showed that the optimum treating conditions were: the dormant buds were shaken and cultured for 2 days in 0.3 mol·L-1sucrose + 1 mol·L-1 glycerol preculture fluid, treated with the loading solution for 20 min at room tem- perature, and PVS2 dehydrated for 120 min at 0 °C, and quickly put into liquid nitrogen (LN) after they were changed to fresh PVS2. It was recommended to freeze the buds in LN for more than 24 hours, im- mediately take them out and transfer them in 38 °C water bath for 2 min. The unloading solution con- taining 1.2 mol·L-1 sucrose and MS was used to wash the buds for 30 minutes, and then they were blot- teddryusingthesterilefilterpaperandinoculatedontoMS+2mg·L-16-BA+0.02mg·L-1 NAArecov- ery medium. The survival rate of dormant buds reached 86.30%. The ploidy of 23 regenerative plants was identified by flow cytometry, and there was no significant difference in ploidy among plants that developed directly from dormant buds.【Conclusion】The dormant buds of A. arguta‘Kuilü’had a higher survival rate after cryopreservation by vitrification method, and an efficient‘Kuilü’dormant bud ultra- low temperature storage system has been successfully established, which may provide a theoretical ba- sis for the ultra-low temperature preservation of A. arguta.