| Cassia obtusifolia L belongs to the genus of Cassia and the family of Leguminosae. It is distributed in Africa, America and Asia. In China it is mainly distributed in the South of the Yangtze River. Cassia obtusifolia L is an important medicinal material. It has many therapeutic functions. Its excellent functions in the immune and digestive system, in clearing heat from liver and improving vision, in resisting bacteria and tumor, in health-protection, etc. have been confirmed pharmacologically in recent years. So Cassia obtusifolia L is widely used in both clinics and daily life.Presently Cassia obtusifolia L used in our country mainly comes from the artificial cultivation. Because of the continuous decrease of its wild resources and decrease in its quality due to artificial cultivation, the yield of Cassia Semens is declining, which brings great difficulty to its clinical and daily life requirements. Therefore, it is necessary to find new ways to increase yield and quality.In this study, first, cassia seeds were treated with concentrated sulfuric acid (80%) for different time to help the seed embryos to break through the seed coat and surface wax layer. The seed germination rate was estimated, and early seedling growth was measured, in a hope to find the best seed treatment method. And then, culture media with different ratio of plant hormones for callus inducing, adventitious budding and rooting were investigated with plant tissue culture technique, using stem segments, buds and stem segment with buds as explants, in order to determine the best culture medium for each purpose and obtain their clones.Our previous successful induction of hairy roots of Cassia obtusifolia by Agrobacterium rhizogenes and its culture has opened a new pathway to produce hairy roots of Cassia obtusifolia by biotechnology. It has been also reported that compared with seedling roots, the hairy roots of Cassia obtusifolia had both advantages and disadvantages nutritionally. There is room for further improving its culture. So far, the anatomical and physiological features of the hairy roots of Cassia obtusifolia were unclear. In this research, stereomicroscopic and light microscopic observation and comparison of morphology, the paraffin sections stained with safranine and fast green and polysaccharide-periodate-Schiff (PAS) reaction were applied to identify the differences in morphology, anatomy and the distribution of starch granules of the hairy roots and the seedling roots. The enzyme assay kits were also used to identify features of the antioxidant enzyme system in hairy roots of Cassia obtusifolia.The main results are in the following.1. The Optimum Sulfuric Acid Treatment of Seeds for Improving Germination and Growth of Cassia obtusifolia SeedlingsIt was discovered that plump cassia seed grains treated with80%strong sulfuric acid for50min significantly improved the germination rate and remarkably promoted early seedling growth. Seed germination was concentrated in1-2d, accounts for90%; the germination is relatively uniform, and germination index, vigor index, plant height and fresh weight per seedling all were the highest among the treatments.2. Establishment of Clones of Cassia obtusifolia by Tissue CultureThe stems, buds and the stems with a bud of Cassia obtusifolia were used as explants to first induce calluses and then induce them to differentiate adventitious buds and roots. And thus their respective clones were obtained. It was discovered that the combination of6-BA and NAA could promote callus induction and differentiation of adventitious buds from calluses; NAA could induce the differentiation of roots from budded calluses. According to the experiments, MS supplemented with6-BA1.Omg/L and NAAb0.4mg/L was the optimum medium for inducing calluses from different explants. MS supplemented with6-BA0.75mg/L and NAA0.12mg/L was the optimum medium for promoting the differentiation of adventitious buds. Buds and the stems with a bud both could continue to grow in callus inducing medium and form multiple buds and MS supplemented with NAA O.lmg/L could promote rooting of the multiple buds. The transplanting survival rate and the planting survival rate of the plantlets from stem induction and differentiation are80%and90%respectively.The transplanting and planting survival rat from buds and stems with a bud are92%and95.4%respectively.3. Anatomic Features of Hairy Roots of Cassia obtusifoliaIt was found that the hairy roots were more uniform in thickness, their root-tips were thicker and more robust, and had more root hairs than the seedling roots; the epidermal cells of the hairy roots were smaller, more closely and irregularly arranged; the cortex cells were more closely and irregularly arranged; the endodermal cells were more closely arranged with smaller intercellular space and no apparent Casparian strips; the protoxylem of the hairy root was diarch, while that of the seedling root was tetrarch; the cambial cells of the hairy roots were smaller, more closely arranged and had more layers of cells; the root cap of the hairy roots had almost no starch granules, whereas that of the seedling roots had quite a lot. Considerable differences existed in both the morphology and anatomy of the hairy roots and seedling roots of Cassia obtusifolia L. This result laid an excellent basis for the further investigation on the cytological mechanism of hairy rooting, physiochemical and gene expression features as well as the molecular mechanism concerned with the unlimited and ageotropical growth of hairy roots of Cassia obtusifolia L.4. Features of the Antioxidant Enzyme Systems and Zymogram of the Hairy Roots of Cassia obtusifoliaComparative studies on the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione peroxidase (GSH-Px) in hairy seedling roots, and on the zymograms of SOD and POD as well as on the contents of some reactive oxygen species were conducted. The results shows that SOD, POD, CAT and GSH-PX activity was significantly different in hairy roots from seedling roots. The SOD, CAT and GSH-Px activity in hairy roots were all decreased, while POD activity was increased. SOD activity in hairy roots was about1/3that in seedling roots, and the activity of both CAT and GSH-PX was about1/2that in the seedling roots. There are no specific band differences of the SOD and POD zymograms from the hairy and seedling roots. Hairy roots contained more O2-, though it contained less H2O2compared with seedling roots. Generally speaking, the antioxidant enzyme system of hairy roots was relatively weak, it could not effectively scavenge the damaging reactive oxygen species, and this may be the reason that hairy roots is more prone to browning during its growth.All the above research results has laid a necessary basis for further studies on the mechanism of hairy rooting, the physicochemical properties, gene expression profile and the utilization of the hairy roots technique of Cassia obtusifolia. |
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