| Experiments of obtaining more favorous watermelon (Cirullus lanatus) were made with cultured cotyledons of homozygous Variety "No. 1 .HuoZhuo" by combining plant tissue culture with Agrobacterium-mediated transformation.In this research, establishment of high efficiency regeneration system, determination of parameters involved in Agrobacterium-mediated transformation, screening and characterization of transformant were systematically researched .The results indicated that:Success in establishment of high efficiency regeneration system was fundamental to the experiments. It concerned with two facets. One, Asep-ic, germination. It was closely related to shoot regeneration On condition that incubating 3~4h on 37癈, disinfecting 5min with 0.1%HgCl2, rinsing 4-5 times with sterile deionized-distilled water, then inserting in 0.8% Agar medium was economical, efficient and high qualitative way. All embryo germinated in 2 days, seed germination frequency was 97%. Neither infection nor malformation was observed. On the other hand, many factors influenced regeneration frequency such as explant varieties, distance from cotyledon petide, explant age, culture temperature, light time, explant position and hormone concentration. Cotyledons Which were away from cotyledon petide 0-1 mm and aged 3~4d(cotyledon colour turned green conpletely) were the best explants. Culture temperature 24-28 癈, light time 16h, explant light side towards up, the proporationable hormone concentration with MS+2.0mg/lBA+4.5mg/lAgNO3 were the optimum conditions, under which shoot regeneration frequency could reach 92%. More than 98% of the shoots were diploid exhibited the phenotype expected, after 1-2 months of culture, somatic embryos were coverted into plantlets on the medium.The Kanamycin was applied as a selectable marker at various concentrations. The evaluation of toxic damage indicated that lethalconcentrations of Kanamycin were > 75mg/l , It caused callus growth retardation and the lowest shoot regeneration frequency. Concentration of Carbanicilin was also defined. 500mg/l Carbanicilin could not only suppress Agrobacterium growth but also improve transformation frequency.By means of a series of cloning, a plant expression vector pGA643-INV was constructed with CaMVSSS promoter, Nos terminator and selectable Kanamycin resistance marker(NPTII). After that , the pGA643-INV was transferred into A. tumefaciens strain LBA4404 by freeze-thaw method. PCR Screening indicated that A. tumefaciens strain LBA4404 contained INV gene. The strain LBA4404 could be used as engineering bacteria.Five parameters were determinated in Agrobaeterium-mediated transformation. We designed a approriate protocol in which watermelon explants were precultured 2-3 d on MS medium, dipped for 9min in LBA4404 suspension(OD6oo ^ 0.4), co-cultivated with Agrobacterium for 5d on MS+2.0mg/lBA+4.5mg/lAgNO3 medium. Under conditions ,transformants grew well ^transformation frequency could reach to 0.97%, dwarfing was not found.Afterwards ,these explants were transferred to regeneration medium supplemented with SOOmg ?L"1 Carbanicilin and cultured 7d to suppress Agrobacterium before placed on selection medium .In the period of shoot development, transplant was indispensable at intervals of 15d.The Kanamycin-resistance shoot were obtained by the procedure as above The putative transformants were assayed by PCR analysis. Five transformants were observed the predicted band, but one trarsformant failed to appear the 41 Obp band. |
