The Optimization For The Induction And Growth Of Hairy Root Of Cassia Obtusifolia L And Analysis Of Its Chemical Components

Due to the limitations of natural cultivation conditions, the planting techniques of the traditional Chinese medicinal plants have a great impact on both the quantity and the quality of Chinese herbal medicine. In the controlled and nonhormone culturing conditions, a large amount of the fast growing and genetically stable hairy-roots can be induced by Agrobacterium rhizogenes, and after batch culture of the hairy roots, secondary metabolites of comparable and even higher contents can be extracted from the hairy roots, compared with the original plants.Through the studies of the effects of different explants, different age of explants, different pre-culture time, co-culture time, degerming method, different kinds and concentrations of the antibiotics for the disinfection on the induction of hairy roots of Cassia obtusifolia L, the most suitable conditions for the induction and culture of the hairy-root of Cassia obtusifolia L were determined The cultured hairy root, aseptic seedling roots and Cassia semens were compared in the contents of anthraquinones, proteins, amino acids, crude fats, fatty acids, carbohydrates and mineral elements, in hope to provide the basis for improvement of the batch culture of hairy roots of Cassia obtusifolia L.The basic process of induction of hairy roots of Cassia obtusifolia L on MS culture medium:screening for plump seeds�'obtaining aseptic seedlings�'pre-culturing the explants�'activating Agrobacterium rhizogenes�'infecting explants by Agrobacterium rhizogenes�'co-culturing�'inducing and then degerming of the hairy-roots�'batch growing of the aseptic hairy roots.The methods for the measurement of the chemical components between the hairy root, seedling root and seed of Cassia obtusifolia L:anthraquinones was determined by a mixed alkaline solution of 5% sodium hydroxide plus 2% ammonium hydroxide; the soluble protein content was determined by Coomassie Brillant Blue. Free and hydrolytic amino acids was determined by the Hitachi L—8800 Amino Acid Analyzer; crude fats was evaluated by SZF-06A crude fat analyzer and fatty acids was determined by gas chromatography (SHIMUZU GC2100); reducing and total sugar was determined by 3,5-dinitrosalicylic acid (DNS); soluble sugar content was determined by anthrone-sulfuric acid colorimetry. The minerals were determined by atomic absorption spectrometer TA5-990.The main results are in the following.1. The optimum condition of cultivating the hairy root of Cassia obtusifolia LThe plunp seeds of Cassia obtusifolia L was first treated by soaking in concentrated sulfuric acid for 10 minutes, and then was cultured under aseptic conditions at 25℃,16h light (Ca.1800 Lx) in the MS medium for 7 days. The morphological lower parts of cotyledons of the sterile seedlings were used as explants and briefly soaked in the suspension of Agrobacterium rhizogenes of OD600=0.5 and immediately, co-cultured on MS medium for 2-3 days, and then 100mg/L carbenicillin was added on MS culture medium for sterilization. Under such conditions, the induction rate of hairy roots of Cassia obtusifolia L was the highest and the growth was optimum in subsequent batch culture.2. The comparison of the chemical components between the hairy root, seedling root and Cassia semensOn the basis of dry weight, the conjugated and free anthraquinones contents (0.112%,0.119%) of the hairy roots were lower than those of the seedling root (0.187%,0.232%) and of the seed (0.286%,0.629%); soluble protein content of the hairy root (40.96 mg/g) was higher than that of the seedling root (18.60 mg/g) but lower than that of the seed (130.38 mg/g); both the hydrolysate and free amino acid contents of the hairy root (23.562 g/100g,4878.800 mg/100g) were higher than those of the seedling root (14.212 g/100g,940.959 mg/100g) and Cassia semens (15.216 g/100g,240.514 mg/100g); total essential amino acids in hairy root was significantly higher than those of the seedling root and Cassia semens; crude fats in hairy root (1.91%) was lower than that of the seedling root (2.32%) and Cassia semens (15.69%); unsaturated fatty acids in hairy root was lower than those of the seedling root and Cassia semens; total sugar content of hairy root was 635.19 mg/g, lower than that of the seedling root(755.25 mg/g) and comparable with Cassia semens (619.32 mg/g); reducing and soluble sugar contents (58.88 mg/g,39.92 mg/g) of the hairy root were lower than those of the seedling root (84.46 mg/g,75.17 mg/g) but slight higher than those of Cassia semens (45.53 mg/g,34.76 mg/g); Mg content was the highest in the three materials tested, being 999.68,772.34,1221.563μg/g respectively in hairy roots, seedling roots and Cassia semens respectively; The contents of Mg and Ca in the hairy roots were higher than those in the seedling roots but lower than those in Cassia semens. The contents of Fe, Mn and Zn in hairy roots were significantly higher than those in the seedling roots and Cassia semens; the content of Cu in the hairy roots was comparable with that in the seedling roots but lower than that in Cassia semens.