Transformation Of Green Fluorescent Protein Gene (gfp) Into Citrus Species And Transgenic Plants Regeneration

Citrus ranks the number one in the world fruit production. Obtaining "high quality, multi-resistances, ripening all year round" cultivars via genetic improvement is the main objective of citrus researchers. However, the conventional genetic breeding is time-consuming and low efficient because of nucellar polyembryony, pollen/ovule sterility, genetic heterozygosity, wide sexual incompatibility and long juvenility of most citrus cultivars. Fortunately, genetic transformation technique is now providing us an "new, fast and direct" strategy to citrus genetic improvement.In this study, the transformation of green fluorescent protein gene (gfp) into citrus calli and epicotyl segments mediated by Agrobacterium was conducted. The transformants expressing fgp from 14 cultivars were achieved. Some of them have been applied to cell fusion and programmed cell death (PCD) research. All these transformants will facilitate the study of transformation mechanism in the coming year. The main results are as follows:1) Transformed callus of 6 cultivars, transgenic embryoids of 5 cultivars, and transgenic plantlets of 3 cultivars were obtained respectively.2) Totally 93 transgenic lines from different transformation events were produced. Further genetic analysis (Southern blot) of them would reveal the mechanism of transgene integration in citrus.3) The transgenic callus of Satsuma mandarin cultivar Guoqing No. 1 (G|) expressing GFP brightly and stably, has become a live tool in cell fusion and PCD research in citrus.4) The role and accuracy of antibiotic and gfp gene in transformant selection were compared. It was shown that selecting by gfp gene was faster, more convenient and reliable.5) The determinants affecting Agrobacterium-mediated transformation and regeneration in citrus were evaluated. It indicated that:① The interaction between Agrobacterium and citrus cells is the prophasic factor. The following measurements would make this interaction favorable to transformation efficiency: infecting vigorous, yellow-colored suspension cells in their grain condition with newly electroporated and subcultured Agrobacterium, shaking the mixture for a while to make them fully contact with each other, then keeping them still for about 20 min to ensure Agrobacterium attach onto the hostcell wall; Making artificial wounds in callus precocultivation or applying AS in cocultivation medium would favor T-DNA transfer into plant cells.(D The survival and regeneration of transformed cells is the premise of successful transformation. Commonly, newly induced embyogenic callus has high potential of embyogenesis ability within 3 years' subculture. After cocultivation, employing cefotaxime to restrain Agrobacterium, culturing callus culture on renew medium without select pressure for one or two weeks, then culturing the infected callus on selection medium by dividing into small groups and timely screening and subculturing the transgenic materials to fresh mediums without antibiotic would help to refresh and regenerate transformed callus.6) GFP gene as a vital marker was used to screen and monitor the transformation of Bingtang sweet orange (Citrus sinensis (L.) Osbeck) epicotyl segments at various developing stages. It suggested that culturing the segments in dark after co-cultivation for 2 — 4 weeks resulted in more transgenic plantlets regenerated, which mostly located in the cambium ring of the segments' cut. Untransformed plantlets survived by the production of transformants nearby or because of agrobacterium persistence. It therefore suggested that selection by antibiotics be imprecise.7) GFP expression analysis throughout the regeneration of transformants revealed green fluorescent intensity differences among different parts of the plantlet as well as different developmental stages. Green fluorescence is blightest at the earlier developmental stages such as callus, early embryiod and etiolation tissues and burgeons. While at the subsequent stages, green fluorescence turned to light green and unconspicuous because of chlorophyll accumulation mask and physiological changes along with repetitive subculturing.8) Chimeras were also observed in epicotyl transformation via fluorescence microscope. In one of these chimeras, the transformed sector came from the cambial callus, while the untransformed sector came from the fibrovascular tissue of epicotyl explant. This could be an evidence of the viewpoint that the competent cells to Agrobacterium-med'iated transformation lies in the regenerated calli from cambium ring.9) Selecting the fastly reproduced transgenic materials for PCR detection and southern blot analysis. The result revealed that GFP detection is as effective as the PCR analysis. Southern blot analysis is still underway.