Study On The Elimination And Fragmentation Of Exogenous Chromosomes In The Somatic Hybrids Of Bupleurum Scorzonerifolium

Bupleurum scorzonerifolium Willd is a traditional Chinese medicine herb. Somatic hybridization containing B. scorzonerifolium as one partner gave an interesting result in different combinations, including Arabidopsis thaliana (L.) Heynh. (+) B. scorzonerifolium and Festuca arundinacea Schreb (tall fescue) (+) B. scorzonerifolium. Both symmetric and asymmetric fusions showed that a whole set of chromosomes of B. scorzonerifolium existed in the somatic hybrids, but the chromosomes of other parents were exclusion and fragmentation. In order to explore the mechanism of UV-resistance in B. scorzonerifolium and of chromosome exclusion and introgression in asymmetric somatic hybridization, we compared the biochemical characters of B. scorzonerifolium with common wheat. A possible reason for chromosome elimination and fragmentation of fusion receptor was discussed in this study.The main results in this research are as follows.1. An improved protocol for the establishment of embryonic suspension of Arabidopsis thalianaOptimization of experimental material and medium for establishment of embryonic cell suspension of A. thaliana is a basis and prerequisite for the somatic hybridization of A. thaliana. In this experiment, seeding-derived calli were subcultured in D1 or D2 media for about eight weeks,' and then transferred to P7 medium to maintain the genetic stabilization of the embryonic calli. This method, being fast and reliable, can be used toproduce a number of protoplasts and provide suitable material for somatic hybridization.2. Introgression of chromosome fragments of Arabidopsis thaliana to chromosome of Bupleurum scorzonerifolium induced by UVThe protoplasts of Arabidopsis thaliana treated with 7.5mM of IOA (iodoacetoamide) were fused with those of Bupleurum scorzonerifolium Willd. The protoplasts of B. scorzonerifolium were irradiated with UV at an intensity of 380 μW/cm2 for 30s, 60s, 90s, and 120s respectively before fusion. In cell clones produced in all the fusion combinations, only those from combination VI (UV 120s) differentiated to plantlets. The cell lines and plantlets were identified as somatic hybrids by analysis of chromosome size and number, RAPD, 5SrDNA spacer sequence respectively. Genomic in situ hybridization (GISH) revealed that there was a complete set of chromosomes of donor (B. scorzonerifolium) in all hybrids with a few intact chromosomes and chromosome segment(s) of recipient (A. thaliana) in the hybrid cell lines from combinations Ⅰ-Ⅴ or only chromosomal segment(s) in the regenerated cell lines from combination Ⅵ. The numbers of chromosomal fragments from A. thaliana translocated or inserted to donor chromosomes increased with UV dose enhanced. The UV-tolerance of B. scorzonerifolium and indirect effect of UV on the exclusion and introgression of receptor (A. thaliana) chromosomes in the hybrid cells was discussed.3. Chromosome elimination in the symmetric fusion between and Bupleurum scorzonerifoliumBesides the indirect effect of UV on receptor, whether other factors impact on the chromosome exclusion is necessary to be investigated. Thus, symmetric somatic hybridization was conducted. In this study, we performed symmetric fusion between A. thaliana and B. scorzonerifolium using PEG method. B. scorzonerifolium-like hybrids were obtained via A.thaliana chromosome elimination. It is likely caused by an incompatible reaction between remotely related parents that result in the asynchronous division of the parent chromosomes and chromosome reconstruction, or relative with the specific machanism, which expel exogenous meterial in B. scorzonerifolium. The hybrid calli have lower regeneration ability than that from asymmetric fusion of the same parents, although most of the hybrids contain a complete of B. scorzonerifolium chromosome and some of A. thaliana DNA segments. We infer from this result that the machanism of chromosome exclusion in this test is different from UV-induced recipient chromosome elimination in former experiment.4. Chromosome elimination in the symmetr...