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Key Message: Drastic changes in soil water content can activate the short-term high expression of key enzyme-encoding genes involved in secondary metabolite synthesis thereby increasing the content of secondary metabolites. Bupleurum chinense DC. is a traditional medicinal herb that is famous for its abundant saikosaponins. In the current study, the effects of drought-re-watering-drought on the photosynthesis physiology and biosynthesis of saikosaponins were investigated in 1-year-old B. chinense. The results showed that alterations in soil moisture altered the photosynthesis physiological process of B. chinense. The dry weight and fresh weight of the roots, photosynthesis capacity, chlorophyll fluorescence parameters, and SOD, POD and CAT activities were significantly reduced, and the contents of SP, soluble sugars, PRO and MDA increased. There were strong correlations between different physiological stress indices. All indices promoted and restricted each other, responded to soil moisture changes synergistically, maintained plant homeostasis and guaranteed normal biological activities. It was found that RW and RD_1 were the key stages of the water-control experiment affecting the expression of saikosaponin-related genes. At these two stages, the expression of multiple genes was affected by changes in soil moisture, with their expression levels reaching several-fold higher than those at the previous stage. We noticed that the expression of saikosaponin synthesis genes (which were rapidly upregulated at the RW and RD_1 stages) did not coincide with the rapid accumulation of saikosaponins (at the RD-2 stage), which were found to correspond to each other at the later stages of the water-control experiment. This finding indicates that there is a time lag between gene expression and the final product synthesis. Rapid changes in the external environment (RW to RD_1) have a short-term promoting effect on gene expression. This study reveals that short-term stress regulation may be an effective way to improve the quality of medicinal materials.

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.

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and β-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.