Research On The Cryopreservation Methods Of Six Resources In Actinidia Arguta Including ’Kuilv’ And ’Lvwang’

Actinidia arguta is one of the precious cold-resistant fruit trees in our country,and is an important germplasm resource for kiwifruit variety improvement.A.arguta is an emerging precious fruit and its fruit is rich in Vc and nutrients.At present,wild resources of A.arguta have been severely damaged and its natural gene library has been threatened,while conventional methods such as off-site preservation and tissue culture are vulnerable to environmental and human influences,with limited conservation capacity and they large resources utilization.Therefore,it is of great significance to explore new medium-and long-term conservation methods.Cryopreservation is currently the most ideal and widely used method for long-term preservation of germplasm resources.In this study,the shoot tips of the tissue culture seedlings,dormant buds and pollen of A.arguta were used as the test materials.The establishment of a simple and efficient cryopreservation system for A.arguta germplasm resources,with a view to providing a reference for A.arguta production,breeding and resource conservation.1.Established cryopreservation system for dormant buds of A.arguta by vitrification.The best sterilization method for’kuilv’single bud stem segment is to peeled off the skin,dipped it in 75%alcohol for 30 s and washed with sterile water for 4 to 5 times,then sterilized with 0.1%HgCl₂ for 30min and washed with sterile water for 4 to 5 times.Peelled off dormant buds after sterilization and precultured in liquid MS containing 0.3 mol·L^-11 Sucrose+1 mol·L^-11 glycerol for 2 days with shaking.Treated with 2 mol·L^-11 glycerin+0.4 mol·L^-11 sucrose+MS loading solution for 20 min at room temperature,using plant vitrification protection solution PVS₂（30%glycerol+15%ethylene glycol+15%dimethyl sulfoxide+0.4 mol·L^-11 sucrose+MS）for 120 minutes at 0℃Replaced with fresh PVS₂,and quickly put into liquid nitrogen for freezing.After 24 h,took it out,immediately immersed it in a38℃water bath for 2 min,and then washed it with the unloading solution containing 1.2 mol·L^-1sucrose and MS for 30 min,and changed the unloading solution every 10 min..After absorbing sloutions with the sterile filter paper,put the dormant shoots on MS+2 mg·L^-11 6-BA+0.02 mg·L^-1NAA recovery medium and cultured in the dark for 3 days,and then transferred to normal light for cultivation,with a survival rate of 86.30%.The ploidy of regenerated plants was identified by flow cytometry,and no obvious changes were found.SRAP molecular marker method was used to identify the genetic stability of the regenerated plants,and no mutation bands were found.2.Established the cryopreservation system of A.arguta tissue culture seedling shoot tips by droplet-vitrification method.Under sterile conditions,excised the 2-3 mm shoot tips of‘kuilv’and precultured in liquid MS containing 0.3 mol·L^-11 sucrose for 2 d with shaking,loaded at room temperature for 40 min,dehydrated with PVS₂ at 0℃for 40 to 60 min.Transferred the shoot tips into the PVS₂ droplets on the aluminum foil strip,and put it directly into liquid nitrogen for storage.Freezed for 24 h,immediately took out and added 40℃preheated unloading solution,waited for the droplets on the aluminum foil strips to fell off and changed to fresh unloading solution to wash for 30 min.Sucked the tips dry with sterile filter paper and put on recovery medium(MS+2 mg·L^-11 6-BA+0.02 mg·L^-1NAA).After dark culture for 3 days,it is transferred to light culture,and the survival rate of the shoot tips can reach more than 41.11%.3.Using the pollen of A.arguta"Lvwang"as the test material,the optimal medium for pollen germination in vitro is 5%sucrose+0.04%H₃BO₃+1%agar,and different concentrations of Ca（NO₃）₂are added to the culture medium to inhibit pollen germination;cultured at 20℃in the dark,pollen had the highest germination rate.The pollen of five germplasms of A.arguta’Lvwang’,’A14-5-2’,’B6-1-1’,’T7-3-3’and’T7-6-3’stored in different low temperature conditions,the germination rate of pollen is highest at-80℃,followed by-20℃,and worst at 4℃.The pollen of’Lvwang’,’A14-5-2’and’T7-6-3’were stored at-80℃for 12 months,the pollen germination rate was not significantly different from that before storage,and the germination rate was all above 84.96%.