Induction Of Nucellar Embryogenic Callus And Generation Of Somatic Hybrid By Protoplast Fusion In Citrus

China is one of the important origins of citrus,in which citrus has been planting for more than 4000 years with abundant citrus resources.Citrus embryogenic callus can not only be preserved in vitro,but also can be used to produce new germplasm through protoplast fusion and to establish regenerative gene editing system of citrus.Therefore,induct ion and preservat ion of embryogenic callus was important for the genet ic improvement of citrus by biotechnology.Protoplast fusion can effectively shorten the breeding period and achieve the recombination of nuclear and cytoplasmic to produce new germplasms,but the dynamics of organelles movement and inheritance during protoplast fusion is still unclear.In our study,embryogenic callus of several local citrus varieties were induced,to provide materials for citrus biotechnology;transgenic embryonic callus and plants which expressed mitochondrion-localized fluorescence protein were produced through Agrobacterium-mediated transformation,to provide materials for future research on cell engineering of citrus;and,new citrus somatic hybrids were produced by protoplast fusion,which provide valuable germ Lasm for citrus breeding.The main results are as follows:1.Embryogenic callus lines of five citrus spcies were induced from undeveloped ovules and filled seeds,with the same same route as regeneration of embryoid followed by propagation of embryogenic callus.which were Hong Kong kumquat,Ponkan(diploid),‘Guoqing No.1’ Sutsuma mandarin,‘Early gold’ sweet orange.Among them,the embryogenic callus of sweet orange shows strong embryogenic potential.2.Transgenic callus lines of ‘Guoqing No.1’ Sutsuma mandarin which express mitochondrion-localized GFP(G1-MTGB)and RFP(G1-MTRB)were produced reslepctively by Agrobacterium-mediated transformation,as well as the transgenic embryogenic callus lines of Hong Kong kumquat which express RFP in mitochondria.Twenty transgenic plants of early flowering lemon which express GFP in mitochondria were produced and transplanted to the greenhouse,including twelve trangene-positive plants and eight chimeras.3.Among the protoplast fusions of six combinations,regenerated plants were obtaied from two combinations,including 11 from ‘W.Murcott’ tangor + ‘Taoye’ sweet orange,seven from G1-MTGB + ‘Cutter’ Valencia sweet orange.Six embryoids were obtained from G1 + ‘Xiaoye’ tangerine.In addition,the effects of explant selection and culture environment on embryogenic callus inductiong of Citrus were discussed;and the potential application of embryogenic callus and genetic transformation materials which express different fluorescent protein in mitochondria was also prospected.