[Expression analyses of BcUGT3 and BcUGT6, and their in vitro expression in Escherichia coli].

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.

[Advances in studies on classification of Bupleurum].

This article introduced the herbal medicine studies on Bupleurum in recent years, focused on the classification just like morphological and chemical classification, microscopic characteristics and molecular biology classification for Bupleurum. Identification combined with a variety of classification is the most effective method for Bupleurum. Due to the short supply of Bupleurum in the current market the current Bupleurum classification studies should focuses on combining pharmacological research to expand the Bupleurum's herbal sources.

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization.

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

Hybrid inflorescences derived from gamma-fusion of Arabidopsis thaliana with Bupleurum scorzonerifolium.

In our early experiments, a variety of Bupleurum scorzonerifolium-like somatic hybrid plants were obtained from protoplast fusion between Arabidopsis thaliana and UV-treated/untreated B. scorzonerifolium. To compare the effects of UV and γ-ray irradiation on the B. scorzonerifolium partner and obtain Arabidopsis-like hybrids, we designed a novel combination of somatic hybridization between A. thaliana and B. scorzonerifolium. Before protoplast isolation and fusion, the suspension cells of B. scorzonerifolium were irradiated by gamma ray ((60)Co, 50 Gy with 1.3 Gy min(-1)). Both parental protoplasts lost regeneration capacity, but over 100 somatic hybrids restored the capacity and developed to Arabidopsis-like inflorescences and flowers with some characteristics of B. scorzonerifolium. Some hybrid flowers showed yellow sepal, petal, or carpel, whose color was similar to the petal of B. scorzonerifolium; the others had silique of Arabidopsis with angularity of B. scorzonerifolium, and their parts possessed five stamens, the same as B. scorzonerifolium. Cytological analysis showed that three hybrids had Arabidopsis-like karyotypes. Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR) profiles revealed that both parental fragments were amplified from these hybrids. These results indicated chromatin introgression from B. scorzonerifolium to A. thaliana, which may be related to the complementation of hybrid inflorescence and flower generation.

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization.

Swertia tetraptera Maxim. is an important source of secoiridoid glucosides. To produce these pharmacologically valuable compounds heterologously in somatic hybrid cell lines, S. tetraptera protoplasts were irradiated with various doses of UV light and fused with protoplasts from a long-term cell line of Bupleurum scorzonerifolium Willd. This recipient was chosen as the cell line is cytogenetically stable and fast growing; furthermore, protoplasts isolated from the cell line are readily regenerable. From a set of 86 putative hybrid calli, only two were able to regenerate viable green plants. The hybridity of the 19 of the 86 selections was revealed by a combined isozyme and RAPD analysis, supported by a karyotypic study based on genomic in situ hybridization (GISH). Clone I-3 contained 0.014% swertiamarin while the regenerants had 0.069% swertiamarin and 0.409% gentiopicroside while the III-4 plants contained only 0.015% gentiopicroside.

[Characteristics and abortion cytological pathways of male sterility of Bupleurum chinense].

OBJECTIVE: To study the characteristics of male sterility of Bupleurum chinense and further explore the developmental period and reason of abortion for the male sterile plants. METHOD: The morphological characteristics of B. chinense male sterile and normal plants were investigated and compared. The anther development process and pollen viability of two types of plants were examined by microscopic assay. RESULT AND CONCLUSION: The shapes and sizes of anther and filament were different between the male sterile and the normal plants. For the male sterile plant's, the filament size was no more than 1/2 of that of normal plants and the anthers were shriveled, failed to dehisce and pollinate naturally, and the pollen grains in the anthers had no vitality. Other morphological characteristics were similar between two types of plants. The main reason leading to male sterility of B. chinense was the abnormal development of tapetum cells with two circumstances. The one is that the tapetum cells degraded early during the period of microsporocyte phase to tetrad phase and the other is that the tapetum cells proliferated with delayed degradation in the tetrad to uninucleate phase.

[Somatic hybridization between carrot and Bupleurum scorzonerifolium and esteras isoenzyme analysis].

OBJECTIVE: To transfer the effective elements of Bupleurum scorzonerifolium into carrot, and provide theoretical data for the exploitation, improvement and selection of the germplasm of Chinese medicinal plants. METHOD: The protoplasta of Bupleurum scorzonerifolium irradiated by ultraviolet light (UV) at an intensity of 300 microW.(cm2)-1 for 0, 1, 2 min respectively were fused with those of carrot Fisch by PEG method. The regenerated clones, derived form a single fused cell, were examined for their hybrid nature by phenotype and Esterase isoenzyme analysis. RESULT: Nine clones were identified as the somatic hybrids between B. scorzonerifolium and carrot. CONCLUSION: This provides a firm foundation for the further analysis of the main active components saikosaponin of somatic hybrids and the screening out of high-medicine-content hybrid cell lines.