Studies On Protoplast Fusion And Inheritance Of Nuclear And Cytoplasmic Components Of Somatic Hybrids In Citrus

Somatic hybridization via protoplast fusion is a new alternative for circumventing some reproductive barriers in citrus traditional breeding, such as sexual incompatibility, female and/or male sterility and polyembryony, and it can also realize novel combinations of nuclear and/or cytoplasmic genomes. For transferring the CMS trait of Satsuma mandarin to seedy cultivars and obtaining novel seedless germplasms of Citrus, "cytoplast-protoplast" fusion and asymmetric fusions were carried out in this research. Furthermore, nuclear and cytoplasmic compositions of the somatic hybrid plants, which were previously obtained by protoplast fusion in our laboratory, were analyzed in this study. The main results of this research are as follows:1. Cytoplasts isolation of Citrus was conducted, and the efficient system of Cytoplasts isolation of Citrus was established. Cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin (Guoqing No. 1, cv. Citrus unshiu) via ultra-centrifugation in a discontinuous mannitol-sucrose gradient. Effects of different centrifugation speeds,timex temperature and concentrations and volumes of solutions on isolation of cytoplast were compared, the results showed that best conditions for fraction of cytoplast were obtained with a running time of 20 min at 38,000 r/min in 25 ℃ and 15% sucrose, and a fraction containing 90% cytoplast was obtained. The average size of isolated cytoplasts was 23.2 μm. The viability of cytoplasts ranged from 75 to 85%, as shown by FDA staining. When the isolated cytoplasts were cultured with solid embedding method, they burst 48 h after the culture.2. Cytoplast-protoplast fusion was carried out between cytoplasts of Satsuma mandarin and protoplasts of seedy cultivars, which led to regeneration of calli or embryoids in two combinations. In the fusion combination between Guoqing No.1 (C. unshiu) and Nova tangelo (C. reticulata × C. paradise), the regenerated calli did not develop after cultured in embryoid-induction medium (2% glycerin medium). While, in the fusion combination between Guoqing No.1 (C. unshiu) and Murcott tangor (C. reticulata ×C. sinensis) 44 regenerated calli were cultured on 2% glycerin medium, only 3...