Study On Higher Asymmetric Somatic Hybrids Between Arabidopsis And Bupleurum Scorzonerifolium

Plant somatic hybridization method can transfer large amount of genomic DNA or alternatively organelle DNA between sexually incompatible species. This technique not only allows bypassing normal crossfertilization, but also increases our ability to investigate the mechanism of chromosome elimination between remote species. In our former research, B. scorzonerifolium was considered as an interesting donor parent to produce hybrids that could enable its gene flow to the A. thaliana gene pool, so as to target the important genes in relation to the effect content of secondary metabolites. However, the genetic constitution of the hybrids obtained was unexpected. Instead of generating A thaliana hybrids carrying chromosome fragment(s) of B. scorzonerifolium, we found the reverse result. The somatic hybrid clones remained a whole set of chromosomes of B. scorzonerifolium and the chromosomal exclusion / recombination took place in the fusant. In order to explore the mechanism of UV-resistance of B. scorzonerifolium partner and A. thaliana chromatin was introgressed into the nuclear genomes of B. scorzonerifolium in the somatic hybrids, although B. scorzonerifolium protoplasts were irradiated by UV (Wang et al., 2005). The higher asymmetric somatic hybridization between A. thaliana and B. scorzonerifolium was opratcd in this experiment. The concentration of reactive oxygen species (ROS) was analysis using Laser Scanning Confocal Microscope (Carl Zeiss, LSM510) by (Dichlorodihydrofluorescein diacetate , DCHF-DA) enmarking the cells of A. thaliana/B. scorzonerifolium. The concentration of total flavonoids of biparents was compared before and after UV treatment respectively. Based on the somatic hybridization between A. thaliana (+) B. scorzonerifolium ( γ -ray treatment, 5kGy), a lot of abnormal anthotaxy/ flowerage was obtained. The morphologic dissection and paraffin method were used to analysis these regenerated abnormal anthotaxy/ flowerage.In this research, the following experiments were operated: Protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Protoplasts of B. scorzonerifolium were irradiated by UV (380μW/cm~2, 5min) prior to fusion, then mixed at a rate of 1:1 and fused with PEG-method. Total 80 microcalli were gained from P5 liquid medium, 49 somatic clones could proliferate into calli. Seventeen clones were identified as somatic hybrids by morphogical and molecular (RAPD and SSR) analysis, the hybrid frequency was about 34.7% in this investigation. Among 17 somatic hybrids, 8 clones can regenerated into buds/shoots and 5 clones can differentiated into abundant of plants, the other clones died during subculturing on IB medium. The regenerate frequency was about 29.4%.Out of the 17 hybrids, those capable of differentiating to buds /shoots (No. 1,24 27, 62,67,70,76,82) and plants (No. 37,52,54,55,61) were chosen for RAPD analysis. Among 10 primers used, OPP-09, OPA-8 and OPG-10, gave partial characteristic band(s) of both parents (P type) and new band(s). Seven wheat cp-SSR and ten nuclear genomic SSR primers of A. thaliana were used to analysis 17 hybrids that could regenerated into buds/shoots or plants. The main genomes of somatic hybrids were resembled to that B. scorzonerifolium partner, and genetic recombination and introgression were occurred in the hybrids. Since the A. thaliana and B. scorzonerifolium chromosomes are significantly different in size and morphology, we can safely state that the selected cell lines cany whole B. scorzonerifolium chromosomes or scarcely had 11 chromosomes. It is noted that the small size of intact chromosomes/chromosomal fragmentitions of A thaliana was not existed in the hybrids.The content of ROS increased dramatically in the protoplasts of A. thaliana/B. scorzonerifolium after UV irradiation which detected by laser scanning confocal microscope.The concentration of in both parents also increased remarkably after UV treatment. So we deduced that B. scorzonerifolium could protect itself being injuried by absorbing UV or quenching ROS.From asymmetric hybridization between A. thaliana /B. scorzonerifolium (γ -ray treatment, 5kGy), we obtained a great many abnomal anthotaxy/ flowerage hybrid plants from No.21 and No.23. Combining the chromosome analysis and molecular assay, we deduced that the balance of floristic development in the somatic hybrids was disturbed by B. scorzonerifolium DNA introgressing or recombinating into genomes of A. thaliana partner.