Haploid-diploid Protoplast Fusion And Ploidy Analysis Of Regenerated Callus Lines In Citrus

Citrus is one of the most commercially important fruit crops in both South China and the whole world. There are numerous Citrus germplasm resources in China. However, most of them are seedy thus affecting their economic traits and competitiveness in fresh fruit market. Somatic hybridization via protoplast fusion has become an important tool for ploidy manipulation in citrus because it can overcome some problems associated with conventional sexual hybridization including sexual incompatibility, nucellar embryogenesis and male or female sterility. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids. Comparation with sexual hybridization for triploid production, somatic hybridization using haploid + diploid is an efficient way for improving the breeding effiency. Herein, the first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Murcott’ tangor(C. reticulata Blanco × C. sinensis) suspension culture cells with haploid ‘Early Gold’ sweet orange(C.sinensis) protoplasts derived from microspores in the mid-late uninucleate stage. The second experiment was conducted in order to create allotetraploids by fusing diploid protoplasts of ‘Murcott’ tangor suspension culture cells with four diploid cultivars. The main research results are as following:1. Haploid + diploid protoplast fusion for triploid production. Protoplast fusion was carried out between haploid callus of ‘Early gold’ sweet orange and three diploid seedy cultivars e.g. ‘Cocktail’ grapefruit, ‘Murcott’ tangor and ‘Chenggubingtang’ tangerine. Among these three fusion combinations, the callus was regenerated when the diploid callus of ‘Murcott’ tangor was used as one of the fusion parents. Following the ploidy analysis using flow cytometry, the regenerated callus was proved to be a mixed polyploiy including haploid, diploid, triploid and tetraploid.2. Diploid + diploid protoplast fusion for tetraploid production. Symmetric protoplast fusion was carried out between ‘Murcott’ tangor as callus parent and three diploid seedy cultivars e.g. ‘Cocktail’ grapefruit, ‘Taoye’ sweet orange and ‘Nanchongbendi’ sweet orange as leaf parent. Among these conbinations, the combination ‘Murcott’ tangor + ‘Cock tail’ grapefruit has regenerated the callus. Flow cytometry analysis showed that the regenerated callus was a mixed polyploid of diploid and tetraploid.3. Comparing of the protoplast size between haploid and diploid of ‘Early Gold’ sweet orange. The average size of diploid protoplast is 16.3 μm, the standard deviation is 2.6. And the average size of diploid protoplast is 14.6 μm, the standard deviation is 2.8. It shows that diploid is bigger than haploid and grows more uniformly.