| Shatianyou (Citrus grandis var. shatinyu Hout)is a famous cultivar citrus in ourcountry with a long planting history. Benefit from its various styles and strongadaptive faculty, it has been widely planted. The local annual output is around 1.6million tons, and been exported to south East Asia. However, each fruit counts aboutmore than 120 seeds, its goods does not share a large pie in international market dueto the large seeds content; in addition, the bulkiness body and long virgin issue makesthe production cost at a relatively high level. Considering that, seedless anddowngrading properties becoming important subjects for research in Shatianyou sincethat short and small body increase plant density, improve the productivity, and thenreduce the production cost.In this work, high frequency regeneration and transformationsystems were established based on study of the factors affecting in Shatianyouregeneration efficiency and gene transfer efficiency, and the transgenic plants wereconfirmed by PCR analysis. The main research work and results were as follows:1.Regeneration system for seedling epicotyl segments of Shatianyou which usedas explants p was established,the experiment results showed that the optimal mediumcompositions for adventitious buds regeneration was MS+3mg·L-16-BA. Theadventitious bud differentiation rate of different explants types was epicotyl segments(95％)＞hypocotyls segments (85％)＞cotyledons (12.5％)＞root segments(7.8％).2.Kanamycin sensitive test showed that the critical kanamycinsensitive concentration for epicotyl segments was 75mg·L-1. Effects of Cefotaxime(Cef) and Carbenicillin (Cb) on regeneration of adventitious buds from epicotylsegments explants were investigated. The results indicated that regeneration ofadventitious buds was suppressed when Cef or Cb concentration was over 500mg·L-1, and their ability of regeneration would decrease as the antibiotics concentrationincreased in the culture medium. Cef was more effective than Cb in killingAgrobacteria, 500mg·L-1 was suggested as the suitable concentration to control thepropagation of Agrobacteria.3.Several crucial factors influencing the transformation efficiency were studied.It was found that pre-culture of explants for 2 days before infection, Agrobacteriainfective density OD600 0.5, infect 10 minutes, cocultivation for 2～4 days afterinfection would be favorable for the transformation. Cocultivation mediumsupplemented with 200μmol·L-1 acetosyringone was not able to increase thetransformation frequency.4.The transformed buds could be obtained after the co-cultured epicotylsegments put on selective medium (MS+3mg·L-16-BA+75mg·L-1Kanamycin+500mg·L-1Cefotaxime) for 56 days, then, they were micro-graftedonto seedlings of Carrizo citrange grown in vitro and one month later re-grafted ongreenhouse grown rootstocks. Now, there are 31 transgenic DefH9-iaaM5 geneplants and 9 transgenic Rol ABC gene plants are growing well in the greenhouse,the ratio of Genetic transformation could reach to 1.73％and 3.19％confirmed byPCR analysis.
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