| Gentiana macrophylla Pall, is an important traditional Chinese herb for the use of its dried roots as large-leaved gentian, a kind of traditional Chinese medicine. Large-leaved gentian was one of the most common herbal medicines to cure chronic liver disease by herbalist doctors in China thousands years ago. Modern pharmacology research has revealed that the large-leaved gentian contains kinds of effective compounds. The State Council of the People's Republic of China has listed large-leaved gentian into one of the protective wild herbs to prohibit the illegal collection due to the shortage of wild resources. According to the recent market survey, the supply sources are reduced seriously and the price of gentian is rising up continuously. The study of somatic embryogenesis of Gentiana macrophylla Pall, can not only alleviate the pressure of resources shortage but is also beneficial to breeding and genetic engineering.Using Gentiana macrophylla Pall, as an experiment material, we have studied the morghogenesis of its somatic embryo and established its plant regeneration system. Histological studies has confirmed the somatic embryogenic nature of the process as well. The research results show:1. Callus induction: 95% leaf and hypocotyls can produce callus on the MS medium with some concentration BA and NAA, which affects observably the induction of callus compared with BA, KT and IB A. Callus proliferates 15 times on the medium including NAA and BA or 2,4-D. Callus culture in dark condition lowers the process of aging.2. Sterile seedling proliferating culture: Sterile seedling increases 4.30 times on the medium with 1 mg.L-1 NAA and 1 mg.L-1 BA. 20% seedling can form bud-cluster and the multiplication multiple is 2-3 times.3. Adventitious root induction: After 4 weeks, hypocotyls, young leaves can produce white adventitious roots with fine hair. These roots can induce somatic embryo.4. Somatic embryogenesis: NAA and 2,4-D help callus form light yellow, frail embryogenic callus. Embryogenic callus proliferate 4.5 times on the medium with 0.5 mg.L-1 or 2 mg.L-1 NAA and 2,4-D and it can maintain the function ofsomatic embryogenesis. After 4 weeks of culture on MS medium supplemented with 0.5 mg.L-1 NAA and 0.5 mg.L-1 BAP, 66.7% of the globular embryos develop into mature ones. Up to 90% of normal mature embryos root on MS medium without any plant growth regulators.5. Effective factors of somatic embryogenesis: (1) the effect of explants: leaf, hypocotyl, adventitious root and callus all can induce somatic embryogenesis. (2) the effect of plant growth regulators: NAA promotes induction of embryogenic callus and somatic embryo; BA restrains the formation of somatic embryo; callus induces somatic embryogenesis on the MS medium with 0.5 mg.l-1 NAA and 0.5 mg.l-1 2,4-D; 30% adventitious roots produces somatic embryo on the MS medium with 2 mg.l-1 NAA and 2 mg.l-1 2,4-D. There are, however, many abnormal embryos. On the medium with 3 mg.L-1 NAA and 0.1 mg.L-1 BA 13% adventitious roots induce embryogenic callus, and then complete regeneration plantlet formed through somatic embryo. And (3) the effect of somatic embryo origin: somatic embryo, which is produced on the medium with 3 mg.l-1 NAA+3 mg.l-1 IBA+1 mg.l-1 KT. This could develop better mature somatic embryo than somatic embryo produced on the medium with 0.5 mg.l-1 BA+4 mg.l-1 NAA+ 0.3 mg.l-1 KT.6. Histological study: It is concluded that the development of somatic embryo of Gentiana macrophylla Pall, has the similar process with zygotic embryo through continuous histological study. They all experience the stages of proembryoid, globular embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo. Based on the observation of the experimental result, we assume that the origin of somatic embryo of Gentiana macrophylla Pall is from the single-cell.7. Ultrastructure study: The main ultrastructure difference of somatic embryo and other tissue (leaf, adventitious root and callus) is the cell inclusion. The cell of somatic embryo has dense cy...
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