Establishment Of In Vitro Regeneration And Genetic Transformation System Of Actinidia Arguta

Acitinidia arguta was native to China.Not only it have a unique fruit flavor and is convenient to eat,but also the plant has strong resistance to stress and was an excellent rootstock material.So,it has broad market prospects and development and utilization value.Soft jujube kiwifruit is a dioecious plant with a complicated genetic background.Conventional hybrid breeding has problems of long cycle and low efficiency and is difficult to obtain high-quality cultivars.Modern biotechnology creates a new path for kiwifruit breeding.The high-frequency and stable plant regeneration system is the basis of genetic transformation.However,so far,there have been few reports on regeneration of kiwifruit at home and abroad,and these regeneration systems have many problems such as low regeneration rate and unstable regeneration system.Moreover,the genetic transformation parameters of kiwifruit are quite different,the transformation rate is low,and a complete genetic transformation system is lacking.Based on these research situations,the high-frequency regeneration system of kiwifruit leaves and stems was established,and the genetic transformation of kiwifruit leaves was studied used leaves as receptor materials in this experiment.The main results were as follows:1.The leaves of‘Wiki’,‘Purple Sadova’and‘Red September’were used as explants to induce the adventitious buds.Studies showed that:Among them,the regeneration rate and cluster bud effect of‘Purple Sadova’were the highest among the three varieties.Therefore,it was chosen as the follow-up research material.2.Establish an in vitro regeneration system using the leaves and stems of‘Purple Sadova’as explants.The results showed that,in the callus induction and multiplication,the combination of 6-BA+2,4-D was better than 6-BA+NAA,the average fresh weight of callus induced by leaves and stem segments on 6-BA+2,4-D medium was significantly higher than that on 6-BA+NAA medium,and the best medium of callus induction was MS+1.0 mg/L 6-BA+0.3 mg/L 2,4-D.Light intensity had an inhibitory effect on callus multippication,and callus wass suitable for cultivation in dark environment.The best condition for adventitious bud regeneration of‘Purple Sadova’leaves was that the base of leaf was darkly cultured on MS+3.0 mg/L ZT+0.5mg/L IAA medium.After 30 days,it was transferred to light culture,and the regeneration rate was 80.00%and the average number of adventitious buds was 16.The adventitious buds had the best multiplication effect on the medium of MS+1.0mg/L 6-BA+0.3 mg/L NAA+0.1 mg/L GA₃.The best rooting medium was 1/2MS+0.7 mg/L IBA.3.The sensitivity of the leaves to Kan and Hyg antibiotics was compared.The results showed that the leaves were more resistant to Kan,while it was more sensitive to Hyg.When Hyg was 10 mg/L,no callus grew.from the leaves,and some leaves began to brown and die.So,10 mg/L Hyg was selected as the critical concentration for resistance screening.4.Transform the leaves of‘Purple Sadova’with Agrobacterium EHA105 and analyze the GUS staining to obtain the optimal parameters of the transformation system.The results showed that Pre-cultivation for 4 days,OD₆₀₀ 0.4～0.6,infecting for 20～30 min,adding 100μmol/L AS to the Agrobacterium culture medium and co-culture medium,and co-cultivation for 3 d were the optimal condition for genetic transformation of‘Purple Sadova’leaves,and the genetic transformation rate was as high as 21.00%.At the stage of resistance screening,it was difficult for resistant callus to redifferentiate.Therefore,only the resistant callus was obtained from the1600 explants tested,and no transgenic shoots were obtained.GUS staining showed the resistant callus was stained blue.