| Salvia miltiorrhiza Bunge (Dan-shen) is a famious traditional Chinese medicine in our country. Modern pharmacological and clinical studies have demonstrated that the roots of S. miltiorrhiza possess very effective activities for treatment of cardiovascular and cerebrovascular diseases. Some compound drugs such as injection reagent,troche,capsule,pilula (which has applied for approvement by FDA as the therapeutic medicine 1997) mainly made of Dan-shen, were widely used to cure heart diseases, nephropathy, liver diseases, infection and so on. Whereas, some shortcomings including relative long growth period (more than two years), badly degenerate quality and high cost of production in traditional culture condition, limited the increasing market demanded for Dan-shen. The development of modern biotechn- ology provides one of promising ways to shorten culture period, improve the content of active components and resolve the problem of Dan-shen resources by transferring genes involved in the active components biosynthetic pathway into S. miltiorrhiza and culturing transgenic cell lines,tissues or regenerated plants in a large scale.This, however, significantly relies upon the detailed understanding of the transgenic system and the grope of clone condition of the rate-limiting enzymes on the pathway for active medicine components biosynthesis.In this paper, in order to establish and optimize genetic transform system of S. miltiorrhiza, several experiments such as the effect of callus inducement and differentiation based on different parts of leaf from S. miltiorrhiza, the fluencing factors on the induction frequency of hairy roots, the culture condition on hairy roots and the accumulation of active medicine components in vitro, the condition on regenerated plants and the reaction condition on cloning of genes encoding the rate-limiting enzymes involved in the active medicine components biosynthetic pathway of S. miltiorrhiza, were performed and the results were reported as follows:1) The regeneration system from leaf of S. miltiorrhiza was established. The results indicated that calli was induced with various frequency based on different parts of leaf and the highest callus inducement frequency could reach to 88.9% using the morphological underside of leaf as the explant followed by petiole with 80% and then morphological uperside of with 61.10%. Calli from different resoureces displayed various effects to shoot regeneration frequency and numbers among which calli induced from pestiole showed the highest differentiation frequency of 92.6% with the most average number of shoots reaching to 6.3. Compared to other three media (White, 1/2MS and MS), B5 was the most suitable medium to promote root regeneration. Adding some growth hormone to medium could improve the rooting frequence, average number of root and average length of root, and our results showed that the most suitable growth hormone was IBA with concentration of 1mg/L.2) The inducement condition of hairy roots from S. miltiorrhiza was optimized. The results indicated that remarkable difference was existent using different kinds of bacterium strains and explant types on hairy roots inducement. Compared with A4 and R1601,C58C1 showed much more inducement effect on hairy roots and used as in our subsequent experiment. Lamina was the most suitable explant among the three explants (lamina, petiole and stem) to induce hairy roots and the optimal duration of co-cultivation was about 3-4 days. Acetosyringo (As) of 400uM/L was the most beneficial to promote induct frequencies of hairy roots.3) The culture condition of hairy roots in vitro was investigated. The results indicated that 1/2MS was the as the most suitable culture medium and sucrose was the optimal carbon source. The growth of hairy roots in the medium containg inorganic ammonium salt as nitrogen source was significantly improved compared to that containg organic ammonia. Adding some growth hormone to medium could significantly accelerate the growth of hairy roots, among which 6-BA with the content of 0.5 mg/L exhibited higher promotion effect compared with other kinds of growth hormone.4) The effect of rare-earth element on pharmaceutical components in hairy roots of Dan-shen was determinated. The results indicated that the tanshinones content obviously increased and could reach to 1.800mg/g in the roots treated by rare-earth element with the concentration of 10mg/L, which increased 0.49 and 3.85 folds, respectively, compared with ordinary plant roots and hairy roots untreated. 5) The plantlet regeneration condition for hairy roots of S. miltiorrhiza was examined. The results indicated that different media showed notable difference on shoot differentiation from hairy roots of Dan-shen. Compared with the three other kinds of liquid medium, MS was the most suitable medium for shoot differentiation. The regeneration frequency of shoot could reach to 37.5% when hairy roots were cultured in MS liquid medium for 15 days.6) 3'-end of cDNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was cloned from S. miltiorrhiza and analysed. The length of fragment isolated was 622bp and bioinformatics analysis results indicated this fragment was a 3'end of HMGR from S. miltiorrhiza.In summary, a high frequency regeneration system from leaf of S. miltiorrhiza was successfully established, which provided technical support for genetic improvement of S. miltiorrhiza using Agrobacterium tumefaciens mediated transform- ation method. Different factors such as A. rhizogenes strains, explants type, and various concentration of bacterium infected, duration of co-cultivation and acetosyringe(As), were investigated for their influence on hairy root induction from S. miltiorrhiza, which provided another candidate approach for genetic improvement of S. miltiorrhiza. The in vitro culture conditions of hairy roots of S. miltiorrhiza were optimized, which was useful to produce pharmaceutical components of S. miltiorrhiza by ferment culcture technique in large scale. In addition, 3'end of HMGR was cloned from S. miltiorrhiza and the cloning of full-length cDNA was in process, which served the initiation step to molecular regulate pharmaceutical components biosynthetic pathway in the furture.
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