Optimization Of Agrobacterium-mediated Transformation And Production Of Transgenic Plants With LFY And AP1 Genes In Citrus

Citrus is one of the most important ever green fruit crops in the world, which ranks the first in both acreage and yield. The conventional breeding program in citrus has long been hampered by such problems as polyembryony, apomixis, long juvenility, high heterozygosity and arthenogenesis. Transgenic technology, first applied to plant breeding in 1983, has developed rapidly, and now is indispensable for gene function analysis and plant genetic modification. The juvenile length is regulated by flowering genes, and ectopic expression of these genes in plants is helpful for altering the transition to flower. In this study, the Arabidopsis flowering identity genes LEAFY (LFY) and APETALA1 (AP1) were introduced into citrus embryogenic calluses or epicotyl segments by Agrobacterium tumefaciens, aiming at achieving transgenic citrus plants with shortened juvenile phase. Histochemical GUS assay, PCR and Southern blotting analysis were used to confirm the integration of transgenes. Real-time RT-PCR was used to analyze the expression of AP1 or LFY and endogenous flowering genes. Studies focused on the causation of diversities of transformation efficiency among different geotypes, the effect of AP1 or LFY on expression of endogenous flowering related genes, and the mechanism of early-flowering by ectopic expression of AP1. The main results are as follows:1. Citrus genotype and callus browning significantly influenced transformation efficiency. The transformation potentials of embryogenic calluses from 15 citrus genotypes were assessed. The results showed that, a) most resistant calluses regenerated from browning ones; b) genotypes were divided into four groups according to their browning degrees. Severe browning was observed in mandarins and its hybrids, browning in grapefruit and kumquat, and browning, weak or no browning in different sweet oranges, and no browning in microcitrus and the hybrid of Valencia and 'Meiwa' kumquat; c) transformation efficiency was higher in severe browning genotypes and lower in non-browning ones, which indicated that callus browning was related to transformation; d) the browning degree of calluses positively correlated with the total phenolic content which was genotype-dependent, while had nothing with PPO activity. To sum up, callus browning contributed to the effect of genotype on transformation efficiency, in Agrobacterium-mediated transformation of citrus calluses. The high frequency of transient GFP expression in genotype with high content phenolics also confirmed the conclusion above, when embryogenic calluses were transformed with pBIN-mGFP5.2. The Agrobacterium-mediated transformation of citrus embryogenic calluses was opitimized and five-step regeneration protocol was established, on the basis of previous researches. A total of 245 transgenic callus lines were achieved from 19 genotypes, of which 120 lines from 6 genotypes were regenerated into shoots and 34 lines were successfully transferred to the greenhouse. These included the following results.1) Transformation of Bingtang sweet orange (C. sinensis Osbeck.): a) Seven AP1 transgenic lines were acheived, and the conversion rate from embryo to shoot was 88％. Shoot-tip graft was adopted to regenerate whole plant due to the difficulty in rooting for shoots. Regenerated shoots were confirmed as transformants by GUS, PCR analysis. Single copy of AP1 integrated into the citrus genome, as certificated by Southern blotting analysis. Grafted plants grew rapidly at the initial stages, but later tended to be defoliating and welting, and finally died, while non-transformed plants grew well. b) Two LFY transgenic lines were obtained, of which only one regenerated into plant. Transgenic plant had a strong growth at the beginning, but later grew slowly with small leaves and muti-branches, compared to non-transformed controls. Transformants were confirmed by GUS and PCR analysis, and integrated 3 copies of NPTII certificated by Southern blotting analysis. The results of abnormal growth in transgenic plants indicated that the integration of transgenes (LFY or AP1) could affect the plant growth.2) Transformation of Ponkan(C. reticulata Blanco.): a) Nine LFY transgenic cell lines were achieved, four of which regenerated into plants. PCR analysis confirmed the integration of transgenes in 3 lines, and one line was further certificated by Southern blotting. b) One hundred and thirteen AP1 trangenic cell lines were achieved, 61 lines of which regenerated into shoots, and 104 plants from 29 lines were transferred to the greenhouse. All transplanted plants were detected by GUS and PCR analysis, and 93％of which were confirmed as transformant. Most transgenes (AP1, NPTII and GUS) integrated into citrus genome with 1 to 2 copies by Southern blotting analysis. Different from non-transformed controls, transgenic plants had a dominant growth in axillary meristems, which resulted in 2 to 4 branches per plant. Real-time RT-PCR analysis in transgenic tissues showed various accumulation levels of AP1 RNA in different transgenic tissues, with the highest expression level in shoot, followed by root, and then callus and embryo. The expression levels of endogenous flowering genes like CiAP1, CiLFY, CiFT and CiTFL in transgenic tissues were up-regulated or down-regulated due to the different expression levels of AP1.3) AP1 transgenic plants were also acheived from calluses of other citrus genotypes, including Valencia and Succari sweet orange, Chazhigan mandarin, and the hybrid of Gailiangcheng orange×Weizhang Satsuma mandarin.3. Transformation of epicotyl segments were performed in four genotypes. Fourty-two GUS positive lines of from 3 genotypes were obtained, 21 lines of which were regenerated into whole plants and transferred to the greenhouse. 18 lines were confirmed by PCR analysis. Among transgenic plants, one 'Meiwa' kumquat plant transformed with AP1 exhibited early-flowering. Over-expression of AP1 or LFY affected the expression of endogenous flowering genes, like CiLFY, CiFT, CiTFL, and CiAP1, as revealed by real-time RT-PCR. These results were elucidated as follows.1) Transformation of AP1 in 'Meiwa' kumquat(Fortunella crossifolia Swing.): Nineteen GUS positive shoots were achieved from 1, 021 explants, with a transformation efficiency of 1.86％. Eight GUS positive plants were transferred to the greenhouse, 6 of which were confirmed as transformants by PCR analysis. Southern blotting analysis indicated that the transgenes integrated into citrus genome with 1 to 5 copies. No differences were observed on the growth between transgenic plants and non-transformed controls. One transgenic plant(J3) flowered 11 months after transfer to the greenhouse, while it needs 3 years for wild type to bolssom, this indicated that ectopic expression of AP1 promoted early-flowering in 'Meiwa' kumquat. Expression levels of AP1 in transgenic lines were positively correlated with its copy number revealed by real-time RT-PCR analysis, while was irrelative to the precocity. The promotion of flowering by over-expression of AP1 was due to the up-regulation of CiLFY and CiFT and down-regulation of CiTFL, independent of CiAP1.2) Transformation of LFY in Bingtang sweet orange(Citrus sinensis Osbeck.): The ultimate transformation efficiency of this genotype was 8.88％. One GUS positive plant transformed with AP1 and 12 GUS positive plants transformed with LFY were achieved. All GUS positive plants, except one with transformed with LFY, were positive by PCR analysis. One LFY transgenic plant B1 was further confirmed by Southern blotting analysis with single copy of transgene integration. Real-time RT-PCR analysis showed that various accumulation levels of LFY RNA in different transgenic lines, and the expression level was higher in the shoot than in the root. The expression of endogenous CLAP1, CiLFY and CiFT was up-regulated or suppressed in different transgenic lines, while CiTFL was suppressed in all transgenic lines. These results indicated that LFY affected the expression of endogenous flowering genes.3) Transformation of AP1 in Hongkang kumquat: Eight resistant shoots regenerated from 50 explants, 3 of which was GUS positive, but all of them showed weak growth and failed to regenerate into whole plant in the end.4. Aspects favorite for improving the efficiency of Agrobacterium-mediated transformation in citrus, the copy number of T-DNA integrated into citrus genome, the necessity of construction of transgenic plant populations, and the potential usage of flowering genes in shortening juvenility were also discussed.