The Use Of The PMI/mannose Selection System To Recover Transgenic Sweet Orange Plants (Citrus Sinensis Osb. 'Xuegan')

Citrus is one of the most important fruit crops in the world, it holds an important position in the fruit industry in China. However, long juvenility, polyembryony, high genetic heterozygosity and arthenogenesis have been impeding the traditional breeding in citrus. Fortunately, genetic transformation technique is now providing us a new, fast and direct strategy for genetic improve of citrus cultivars.It is well-known that transformation technology provides a powerful tool for plant genetic improvement. And we all know that the current protocol of transformation can deliver the gene to only a minor fraction of the treated cells, while the majority of the cells remain untransformed and need to be eliminated by selection procedures usually using the antibiotic or herbicide resistant genes as selectable markers. But our inadequate knowledge of just how antibiotic or herbicide resistant genes may affect the environment and human health, which has brought widespread public concern and limited the using of transgenic crops. The best way to solve this problem is the use of the safe marker genes such as manA gene which was from E. coli encoding phosphomannose isomerase (PMI).In this study, high efficiency in vitro regeneration system of sweet orange 'Xuegan' was developed using epicotyl segments from in vitro seedlings as explants. An expression vextor was constructed with a safe marker gene, 6-phosphomannose isomerase (PMI) gene, Agrobacterium-mediated transformation of 'Xuegan'was established initially using PMI gene as selection marker. The main results are as follows:1) High efficiency in vitro regeneration system of sweet orange 'Xuegan' was developed using epicotyl segments from in vitro seedlings as explants. Epicotyls were cutted longitudinally and cultured in the dark first, on the MS basal medium with 3 mg·L^-1 BA and 30 g·L^-1 sugar, 100% adventitious shoot induction and an average of 33．8 shoots per explant were obtained. The base of the adventitious shoots was treated with 1000 mg·L^-1 IBA for 10 min, then placed onto the filter papers which ends dipped into liquid medium with 1/4 MT (macro-inorganic salts), 1/2 MT (other inorganic salts and vitamins) and 1 g·L^-1 sugar. 100% rooting and an average of 2．5 roots per shoot were achieved. Plantlets 100% adapted well to soil conditions.2) Three plant expression vectors were constructed.Ⅰ: pBIPMI, PMI gene (manA) was cloned into plant expression vector pBI121 which GUS gene was digested.Ⅱ: pC1301PMI, PMI gene (manA) as marker gene was cloned into plant expression vector pC1301 which htp marker gene was digested.Ⅲ: pC1301PMI-LFY, the flower-meristem-identity gene LFY from Arabidopsis thaliana was cloned into plant expression vector pC1301PMI which contained safe marker PMI gene (manA).3) The establishment of transformation system of 'Xuegan' using PMI gene as selection marker. The best selection pressure was produced on medium containing 25 g·L^-1 mannose and 5 g·L^-1 sucrose. The results showed that epicoytl segments precultured in medium with 1 mg/L 2,4-D for 3 hours enhanced regeneration frequency. 30 minutes inoculation, 4 days co-culture improved the transformation efficiency. The resistant shoots detected by CPR analyse.