The Construction And Selection Of Aft Tomato EMS Mutant Population And Regeneration Systerm

Aft Tomato is a cultivated tomato that can synthesize anthocyanins on the fruit surface.Previous studies have shown that anthocyanin synthesis of Aft-type tomatoes is specifically induced by Blue+UV-B.In order to explore the mechanism of blue+UV-B-specific induction of Aft-type tomato anthocyanin synthesis and the genes that determine the trait,it is indispensable to construct mutants with missing target traits.For many decades,forward genetics has been a powerful technique to identify genes and mutations that underlie phenotypes of interest.In almost all biological systems,forward genetic screens follow the same basic principles.Organisms with well-defined genetic backgrounds are mutagenized using DNA damaging agents such as irradiation or chemical mutagens,which yields random mutants.The population of mutants is then screened for interesting mutant phenotypes..In this study,a chemical mutagen,EMS was used to construct Aft tomato mutant population.Anthocyanin biosynthesis related mutants were screened and analyzed.The main results were obtained:1.Anthocyanin content and anthocyanin biosynthesis genes expression analysis of wild type tomato AftAnthocyanin biosynthesis in Aft tomato is specific induced by Blue+UV-B light.The anthocyanins were determined by the treatment of Aft-type tomato fruits with different wavelengh of light.The results showed that Aft-type tomatoes synthesized a large amount of anthocyanins under UV-B + Blue.The mRNA level of Sl CHS,SlCHI,SlDFR,SlF3 H,SlANS,SlUFGT,SlMYB12,SlTTG1,SlEGL3 and SlPAP1 expressed highest under UV-B + Blue treatment.The expression of SlUVR8 and SlHY5 was higher under UV-B + Blue,whereas SlCRY1 was the highest in blue light,and SlCRY2 had the highest expression under UV-B.2.Construction of Aft tomato EMS mutant populationAlmost 45% seeds were germinated by 1.0% EMS treatment,the closest to LD50(Lethal Dose)among different EMS concentration treatment conditions.Then 0.5% EMS was used to induce mutagenesis in 10,000 and 12,000 Aft tomato seeds respectively.Through M2 generetion selection,we obvioused totally 472 mutants,which including 93 leaf mutants,84 flower phenotypic mutants,295 fruit shape mutants,94 fruit color mutants.And we successfully screened out a mutant at15-193 displayed deficient anthocyanin production in the epidermis of the fruit.3.Anthocyanin content and Anthocyanin biosynthesis related genes expression analysis in at15-193.With different light treatment,at15-193 nearly didn't synthesis and accumulate anthocyanins.SlCHS,expressing in the at15-193,was lower than WT under different light treatments.SlTTG1,SlEGL3,SlPAP1,which can form MBW complex to regulate the anthocyanin pathway genes expression,expressed lower in the at15-193 than wide type under Blue+UV-B light.Sl HY5 showed the same expression pattern as these regular genes,which indicated that it could be a mutantion of the promoter of SlCHS or a mutantion of upstream gene and thus inhibited the synthesis of anthocyanin in the mutant at15-193.4.The contruction of Aft tomato culture regeneretion systermWe used leaf disc method to analyze different hormone concentrations,affectting to plant regeneration and redifferentiation,and optimize the combination of different hormone concentrations.The results showed that the best medium for regeneration was MS+ZT 2.5 mg/L+IAA 0.1 mg/L and the best medium for redifferentiation was MS+ZT 2.5 mg/L+IAA 0.1 mg/L;the best root generation medium was MS+IBA 1.0 mg/L.