Targeted Cybridization To Transfer Sterile Cytoplasm And Related Basic Research In Citrus

Cytoplasmic male sterility (CMS) was known to be controlled by mitochondrialgenome in higher plants (Kuamr and Cocking, 1987). Satsuma mandarin (Citrus unshiuMarc.) was a CMS type, and citrus symmetric fusion can produce diploid cybridspossessing nuclear DNA from mesophyll parent and mtDNA from callus one. Therefore,transfer of CMS from Satsuma mandarin to seedy citrus cultivars provides a novelstrategy for the production of potential seedless diploid fruits. Herein, the symmetricfusion technique was adopted to create cybfid plants for further seedless citrus breedingby transferring CMS from a CMS cultivar Citrus unshiu Marc. cv. Guoqing No. 1 intotraditional seedy citrus cultivars. On the other hand, it is possible to study thephenomenon of somatic hybrid vigor, screen somatic hybrids or cybrids at an early stageand study the regeneration mechanisms of cybrids by means of protoplast fusion withtransgenic GFP (green fluorescent protein) citrus as one parent. In this study, we also aimto clarify some related basic mechanisms of citrus protoplast fusion remaining unclearlyexplained, to give a correct guide to citrus cell fusion without GFP marker in the future,and create a large quantity of hybrid plants in vitro by conducting protoplast fusion withtransgenic GFP citrus as callus parent or mesophyll one. The main results of this researchare as follows:1. Protoplast isolation, culture and embryoid induction were carried out fortransgenic GFP Guoqing No.1 embryogenic callus. Purified transgenic GFP GuoqingNo.1 Satsuma protoplasts all expressed strong GFP, and the viability reached to about 87％.First cell division was observed about 12-14 d after protoplast cuture, and visible colonieswere formed after 40 d of protoplast culture and many callus clusters with a diameter of1-2 mm occurred after 50-60 d culture. Some calli were transferred to differentembryoid-induction medium, and all calli still remained in a callus stage and weresurrounded with regenerated fresh calli even after 6 months of culture.2. The symmetric fusion technique was adopted to transfer CMS from Guoqing No.1Satsuma into two traditional seedy citrus cultivars in China, 'Shatian' pummelo (C.grandis (L) Osbeck) and 'Bingtang' orange (C. sinensis (L) Osbeck), and 'Page' tangelo(C. reticulata Blanco×C. paradisi Macf. ). Flow cytometry analysis showed that 19plants recovered from Guoqing No.1 Satsuma+'Bingtang' orange, 17 of 35 plants regenerated from Guoqing No.1 Satsuma+'Shatian' pummelo and 14 plants fromGuoqing No.1 Satsuma+'Page' tangelo were all diploids, and the rest from Guoqing No.1Satsurna+'Shatian' pummelo were tetraploids. By SSR and CAPS analysis, we found thatthe diploid products from the three combinations were true cybrids with nuclear DNAfrom their corresponding leaf parent and mtDNA from Guoqing No.1 Satsuma, and thetetraploid plants from Guoqing No.1 Satsurna+'Shatian' pummelo were allotetraploidhybrids with recombination of nucear DNA at TAA15 locus. For cpDNA segregationpattern, cpSSR analysis of the plants recovered from the three combinations showed theircpDNA derived from either of their corresponding parents, and co-existance of cpDNAalso occurred in this study, especially for regenerated hybrid embryoids from GuoqingNo.1 Satsuma+'Bingtang' orange, which possessed cpDNA from both parents. Thisdemonstrated that we succeeded in introducing CMS from Guoqing No.1 Satsumamandarin into the three citrus cultivars simply by symmetric fusion.3. Protoplast fusion was performed via electrofusion between embryogenicsuspension-derived protoplasts of transgenic GFP Guoqing No.1 Satsuma callus andmesophyll protoplasts of calamondin (C. microcarpa Bunge), and three embryoidsexpressing GFP under UV light were obtained 60 d after protoplast culture. The threeembryoids were not considered as diploid cybrids but true tetraploid somatic hybrids,based on the fact 1) citrus heterokaryons are generally more vigorous and have highercapacity for embryogenesis compared with unfused and homo-fused embryogenic callusprotoplasts; 2) the callus line of Guoqing No.1 Satsuma has lost the embryogenesiscapacity; and 3) citrus diploid cybrids produced by symmetric fusion always possessnuclear genome of mesophyll parent, and calamondin without GFP gene was used as leafparent in this study. Subsequent flow cytometry, SSR and CAPS analysis of oneregenerated callus mass and three resulting plants validated this supposition, i.e. the calluswas derived from transgenic Guoqing No. 1 Satsuma callus protoplasts, and the three plantswere true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parentsand cytoplasmic DNA from callus parent.4. Mesophyll protoplasts of 'Valencia' orange (C. sinensis (L.) Osbeck) plantsexpressing GFP created previously were fused with protoplasts isolated from threeembryogenic callus lines including Guoqing No.1 Satsuma, 'Murcott' tangor (C. reticulataBlanco×C. sinensis (L.) Osbeck)and Microcitrus (Microcitrus papuana). Ploidy analysisand SSR characterization showed that the embryoids regenerated from intergenericcombination Microcitrus+transgenic GFP 'Valencia' orange were allotetraploid somatic hybrids, the plants recovered from interspecific combination Guoqing No.1 Satsuma +transgenic GFP 'Valencia' orange were diploid cybrids, and the plants regenerated fromthe combination with tangor as one parent consisted of both diploid cybrids andallotetraploid hybrids. We also observed the phenomenons of hybrid regenerationadvantage in somatic cells through intuitionistic and spatio-temporal experiments underUV light.5. Homo-electrofusion was conducted between 'Valencia' orange and transgenicGFP 'Valencia' orange to identify whether homo-mitochondrial genome plays animportant role in triggerring the division of mesophyll protoplast. We successfullyobtained one plant and one embryoid, both of which had GFP expression. Wehypothesised that the regeneration of this plant might be due to present of mtDNA fromembryogenic callus in the leaf parent, which triggered the divison of transgenic GFP'Valencia' orange (leaf parent) protoplast and at last led to regeneration of a whole plant.6. Many vigorous plantles were recovered from 'Bingtang' orange+calamondin and'Murcott' tangor+Hirado Buntan Pink pummelo (C. grandis (L.) Osbeck). Subsequentploidy analysis and molecular characterization revealled they were allotetraploid somatichybrids.The practical prospectives of transferring CMS from Satsuma mandarin to seedycitrus cultivars via symmetric fusion, the somatic hybrid vigor and screening somatichybrid cells at an early stage, the potential of transgenic GFP citrus callus as suspensionparent in citrus somatic cybridization, the factors influencing the formation of diploidcybrids, and the inheritance patterns of organelle genomes were also discussed in thepresent study.