Research On The Optimization Technology Of Preparation And Fusion In Kiwifruit Protoplast

Actinidia is one of the most successful domesticated cash crops in the 20 th century.It is widely cultivated in the world because of its high nutritional value and economic value.However,most varieties of kiwifruit have poor stress resistance.As kiwifruit is a perennial vine plant with long childhood and different male and female plants,conventional cross breeding efficiency is low.Protoplast fusion technology can combine the advantages of male and female parents and has polyploid advantage and heterosis.However,due to its high difficulty in operation,low repeatability and high cost,it is rarely used in kiwifruit breeding.In this thesis,by exploring the effects of enzymatic hydrolysis time,enzyme concentration and osmotic pressure on protoplast separation,and the effects of interface method and precipitation method on the efficiency of protoplast purification,an efficient protoplast transformation and fusion system was studied.The main research results are as follows:(1)Plant tissue culture was performed on three typical kiwifruit varieties: Kiwifruit chinensis,Kiwifruit woolflower and Kiwifruit jujube.Leaves with less phenolic substances and callus with soft texture and high activity were obtained.Then,callus induction was optimized by using callus proliferation medium instead of suspension culture in traditional methods to reduce the dependence on experimental equipment and other conditions.By optimizing the enzymatic hydrolysis system,the leaves and callus can be put into the same system for enzymatic hydrolysis,avoiding the use of MES and pectinase,and greatly reducing the complexity of enzymatic hydrolysis of protoplast and the cost of protoplast fusion.(2)Protoplasts were obtained by enzymolysis,and the concentration of mannitol,enzyme concentration and enzymatic hydrolysis time were optimized.Finally,the optimal enzyme concentration of enzymatic hydrolysis of callus was 2% cellulase and 2% segregation enzyme,the optimal concentration of mannitol was 0.8M,and the optimal enzymatic hydrolysis time was 8 h.The concentration of callus protoplasts was 4.7×104±9.2×102 /g and the activity of FW was 69.6%±3%.The optimal enzymatic concentration of protoplasts was 2% cellulase and 3% macerase,the optimal concentration of mannitol was 0.8M,and the optimal enzymatic duration was 8 h.The protoplast concentration was 2.1×105±1.0×104FW /g,and the activity was 82.%±3%.(3)By comparing the purification efficiency of 6 kiwi materials by interface method and precipitation method,it was found that the protoplasts purified by interface method were more abundant,more active and less cell debris.Finally,the best purification method of leaf protoplasts and callus protoplasts was interface method.(4)The above optimized enzymatic hydrolysis and purification system was used to obtain high-quality protoplasts,and protoplast fusion was carried out by high Ca2+-high p H-PEG method,and finally 9 kinds of different cells with proliferation ability were obtained.(5)The empty 35S::GFP plasmids were transformed into protoplasts instantaneously by PEG mediated method,so that GFP fluorescence protein could be expressed in protoplasts.In this study,by optimizing protoplast fusion technology,the protoplast fusion between different varieties of kiwifruit was achieved,providing more options for future peach breeding of rhesus monkey,and providing theoretical basis for somatic cell hybridization breeding of kiwifruit.The optimized system increases the reproducibility of protoplasmic preparation and fusion and reduces the cost of experiment.In addition,the characteristics of transient expression of GFP fluorescence protein by protoplasts were used to rapidly locate the origin and target genes of kiwifruit.