[Optimization of tomato genetic transformation, kanamycin-resistant screening and seed selection].
OBJECTIVE
To optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene (MAG) and truncated major surface antigen 1 (tSAG1) of Toxoplasma gondii as the target insert genes.


METHODS
Tomato high-frequency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone (AS) supplements. Three concentrations of kanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations.


CONCLUSIONS
Cotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98% (59/60) using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number of transgenic buds increased significantly at the appropriate temperature condition (23-/+1 degrees celsius;), and AS of 100 micromol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35% (28/81) using the medium ZB2 under (23-/+1) degrees celsius;. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% (31/65). 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.
Ying
Cai-yun
CY
Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. yingcyhb@163.com
Huang
Xiao-qin
XQ
Guo
Yu-qi
YQ
Zhong
Li-li
LL
Liu
Yan
Y
Li
Shi-lun
SL
Gu
Xiao-min
XM
Zhou
Xiao-hong
XH
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D004351:Drug Resistance; D007612:Kanamycin / Q000494:pharmacology; D018551:Solanum lycopersicum / Q000187:drug effects / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics; D012639:Seeds / Q000187:drug effects / Q000235:genetics*; D014122:Toxoplasma / Q000235:genetics; D014170:Transformation, Genetic
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma; D007612:Kanamycin