Study On The Breeding Of TYLCV Resistant Tomato Using Artificial Micro RNA Technology

Tomato（Lycopersicon esculentum Mill）has high nutritional value and is more and more loved by people,becoming one of the indispensable fruits and vegetables in people’s daily life.The growing area of tomatoes is growing,and the harm of diseases and insect pests is becoming more and more serious.Among them,the transmission of tomato yellow leaf curl virus（TYLCV）to plants by Bemisia tabaci is the most serious disease and insect pest,which can cause serious plants.Dwarfing,new leaf yellowing and curling,small fruits and deformity embolism,which seriously affect the growth of plants and make them basically lose their nutritional value and commercial value.In production,some tomato varieties have better traits and qualities than other varieties of tomatoes,but because they are not resistant to TYLCV in the later period,they face the risk of being eliminated.Therefore,the use of modern biotechnology to cultivate new varieties resistant to TYLCV has a major impact on the maintenance of high-quality varieties.At present,there is an anti-virus transgenic strategy of artificial micro RNA（amiificial micro RNA,amiRNA）interference technology,which artificially silences viral target genes through the mechanism of micro RNA（mi RNA）in vivo.The resulting,simultaneous application of agricultural production has fewer biosecurity issues.Therefore,this study intends to use amiRNA and tissue culture technology,first design amiRNA with target TYLCV CP and construct expression vectors,secondly establish an in vitro regeneration system with non-resistant TYLCV tomato’851’as the research material,and finally transform the amiRNA expression vector into tomato to establish in the genetic transformation system,antiviral transgenic tomatoes were obtained through resistance identification.Obtain the following research results:（1）An amiRNA expression vector that silences the TYLCV coat protein（CP）gene was constructed.On the WMD3 website,an amiRNA sequence for specifically silencing the TYLCV-CP gene was designed.Overlap PCR amplification was used to replace mi R319a in the precursor sequence of Arabidopsis mi R319a with amiRNA and the amiRNA expression vector p CPBI121-ami R-TYLCV was successfully constructed.（2）Established a highly efficient in vitro cultivation system for tomato material’851’.The culture conditions in vitro regeneration of tomato were screened by tissue culture and rapid propagation.The results showed that the tomato seeds were immersed for 3 h,dark cultured for 3days and then light cultured to obtain sterile seedlings with the best growth state;MS+2.0 mg/L6-BA is the basic medium.The cotyledonary nodes,hypocotyls and cotyledons are explants.The optimal IAA concentrations to induce adventitious bud formation are 0.2 mg/L,0.3 mg/L and 0.5mg/L,respectively,the best seedling age is:10 d,14 d and 8 d;the best adventitious bud rooting medium is MS+0.5 mg/L IAA.（3）Established amiRNA-mediated genetic transformation system for tomato resistance to TYLCV.Different concentrations of kanamycin were set up tomato anti-hormonal sensitivity test,and it was concluded that 25 mg/L kanamycin was the most suitable for the selection of resistant plants in genetic transformation.The hypocotyls of tomato were transformed by the leaf disc method and the orthogonal experiment method of L9（3⁴）.The optimal genetic system was selected after pre-cultivation for 3 days,Agrobacterium OD₆₀₀=0.6,infection for 10 minutes and co-culture for 2 days.（4）Identification of disease resistance of transgenic tomato plants.The transgenic plants of11 transformed lines were inoculated with the target virus TYLCV.It was found that 5 strains of transgenic plants had no symptoms of infection after challenge,and 6 strains showed mild symptoms of infection.Tomato has a certain resistance to the target virus TYLCV,thus verifying the feasibility and effectiveness of the amiRNA technology and ami R-TYLCV-mediated tomato genetic transformation system.