Creation Of Early Flowering Lemon And Tetraploid Lemon New Germplasm With GFP Gene

As one of the important species of Citrus which ranks the number one in the world fruit trees, lemon has highly value not only on eating, but also on basic research. It is a very significant project on citrus molecular biology studying all the time to use the germplasm resources of short juvenile stage in order to accelerate the research progress. Fortunately, the lemon which has the trait of short juvenile stage is the excellent material of genetic transformation of citrus.In this study, the green fluorescent protein gene(GFP), was introduced into early flowering lemon and tetraploid Femminello lemon genome via Agrobacterium-mediated transformation, using etiolated epicotyl segment as plants, and transgenic plants of homozygous with GFP gene expressing steadily were obtained. The main experimental results are as follows:1. For transformation of early flowering lemon with GFP gene, 1699 etiolated epicotyl segments total were transformed partially and mean regeneration efficiency was 67.0%. Identification by fluorescence microscopy, 33 buds with GFP expressing were obtained after screening of culture, thus, transformation efficiency was 1.94%. Eight positive buds of first batch could not root by three months, induction of rhizogenesis and finally died after micro shoots grafting. The result shows that it is difficult to rooting for early flowering lemon. The positive buds obtained by the last four batch are during the phase of elongation.2. For transformation of tetraploid Femminello lemon with GFP gene, 5131 etiolated epicotyl segments into 9 batches were transformed and mean regeneration efficiency was 64.4%. Checked by fluorescence microscopy, there were 290 regeneration buds that had expression of GFP completely and transformation efficiency was 5.65%. 42 transgenic plants were obtained after rooting culture and rooting rate was 50%.Transformation efficiency and rooting rate of tetraploid Femminello lemon are much higher than other citrus varieties which are suitable for transgenic in the normal operation, so tetraploid citrus may become a new type of explant for efficient genetic transformation of citrus. Conversion rate of early flowering lemon is higher than the varieties which were used for transformation commonly in the laboratory, such as Hongkong Kumquat, trifoliate orange, Ponkan mandarin and so on. Under the premise of improving the rooting rate of early flowering lemon, it can be used as an excellent material for citrus research of functional genomes.