Establishment Of Genetic Traasforment System For Micro-Tom Tomato And Obtainment Of INH Transgenic Plants

Micro-Tom is a miniature dwarf tomato (Lycopersicon esculentum).This cultivar hasseveral unique features, such as a small size, a short life cycle and seed setting underfluorescent light. These features are similar to those of Arabidopsis.Consequently,Micro-Tom is considered to be a model cultivar for tomato functional genomics.Our laboratory had constructed recmbinant plasmid P1300-2A11-INH and transformedinto Agrobacterium EHA105. An efficient regeneration system of Micro-Tom has establishedby study the conditions of different explants and most appropriate medium. An orthogonalexperimental design was used to optimize the transformation, molecular identificationtransgenic plants, and established a high-throughput transformation protocol. The following ismajor results:1. The study investigated the effect of different conditions hormone combination,inoculation method and different explants on regeneration of development, screened the mostappropriate medium, and establish a efficient regeneration system. The results showed thatcotyledon is the optimum explant, compared with cross mid-vein cut mid-vein cut has betterregeneration frequency.2. An orthogonal experimental design was used to optimize the transformation ofcotyledon explants of Micro-Tom, and had been investigated the effect of four parameters onthe transformation frequency containing the concentration of bacterial suspension, time of dipin bacterial suspension, co-cultivation time and contcentration of carbenicillin, and attemptedto characterize these parameters effect of contamination rate, necrosis rate, mortality, cutsurface browning rate and undamaged rate. It showed that the optimal transformation protocolcomposition produced an Agrobacterium suspension at a concentration of0.5×108cells/mL(OD600=0.5) and an infection time of5min, one day of co-cultivation and500mg/L ofcarbenicillin,24mg/L of HygB.3. Under the above conditions, we obtained33plants of tomato of regeneration.19plantsof T0transgenic tomato have been identified by PCR, average transformation frequencies arearound3.9%.Seeding23plants of T1,22plants of T1transgenic tomato have been identifiedby PCR, average transformation frequencies are around95.6%.Currently, the gene oftransformation inherited stably.