Construction Of Canavalia Calli And Canavanine Formation In Calli

Canavanine, a structural analogue of arginine, is one of the predominant natural non-protein amino acids in the Leguminosae and Canavalia lineata. The structural analogy between L-canavanine and L-arginine is so marked that canavanine can be arginine instead of arginine tRNA synthetase mistaken for arginine and the synthesis of the protein .The protein have defective physiological functions, So it has good anti-tumor. The roles of canavanine as a nitrogen storing metabolite in Papilionaceae (Papilionnoidea), legumes (leguminosae), It has been proposed that canavanine as a defense chemical to predatory insects.It is considered that canavanine shows some toxic effectting on some pests.Therefor it could be think as a potential insecticide.The advantage of canavanine is low toxicity,no pollution, not damage the parasitoids pest from its mechanism of action, There are two sources of canavanine, One is the synthetic and another is extracted from plants. The plant extracts from tissue culture skills are generally low costcharging rate, less environmental pollution. There is still no reports of callus on the Canavalia lineate, This report study about induction to callus of beans seeds, and evaluation callus cells in fresh weight, dry weight, relative growth rate of cells and canavanine content, and the optimization of multi-factor and multi-level. Physiological and Biochemical process of Liquid Suspension Culture and on which the canavanine were determined, at the same time a new simple and rapid method of determination of canavanine. The main results were as followes:(1) This paper study optimum conditions about 2,4-dinitrofluorobenzene and L-canavanine reaction in pH9.5 borax buffer solution, for establishment of a new, fast and easy determination of L-canavanine method. The method used spectrophotometry determination 571nm absorbance. The results showed that L-canavanine with 2,4- dinitrofluorobenzene occurred color reaction at 50℃for 30 min with no light, forming a stable green complex with 1:1.The maximum absorption peak at 571 nm and the apparent molar absorption coefficientε=1.086×10~4L/(mol·cm), It obeys beer's law in 13.75-220.00μg/mL, the corresponding linear regression equation: y=0.0051x+0.0682; correlation coefficient: R2=0.9939.Recoveries were 96.60%-101.56%, relative standard deviation of 1.245% -1.768% range, thus establishing a simple new method for rapid determination L-canavanine.(2) Callus were induced with Canavalia ensiformis seeds, determinating a system of callus,and still training and proliferation, Canavanine was detectded in was measured in different colors callus and content of canavanine in different growth stages. The results were that it was induced the light green callus in the MS medium with beans seeds, and beans in the proliferation medium showed the characteristics of the callus growth with pattern of "S" curve. Found in the green callus canavanine content of up to 43.4620μg / g FW (fresh weight), second was light green callus, then white callus, the least browning of callus 21.6260μg /gFW . In Tissue Culture canavanine content was first increased and then decreased. To foster the highest levels during the 24d, the canavanine content 30.6750μg/g FW.(3) To study the optimum combinations of callus with polyfactors and polylevels by orthogonal design. Relative growth rate of cells, fresh weight, dry weight, and canavanine content was chosed as the experiment index. The results showed that: sucrose could effect the growth of callus and promote the synthesis of canavanine remarkably. For the growth of callus, the optimal culture medium for callus growth is: MS +3.0 mg/L6-BA +0.2 mg/LNAA +40g/L sucrose +7.8g/L agar, The relative growth of callus was 0.1016, dry weight was 1.1318g.flask-1 in the optimum medium conditions. For canavanine content, the optimal culture medium for callus growth is: MS +3.0 mg/L6-BA +0.8 mg/LNAA +20g/L sucrose +7.8g/L agar, the canavanine content is 63.2432μg/L FW and 617.5320μg/L DW.(4) With selecting of loose texture, growth state of uniform, dispersion of good calli as inoculation material, Suspension culture is established for study on phisiological variaton of suspension calli and canavanine formation in calli. The results showed that: The suspension cultures cell growth curves were like"S", the growth period of the cell is 18d; Intracellular and extracellular soluble proteins and cell activity was increased and then decreased, while pH of the cultured medium and extracellular peroxides activities of cultured cell were decreased at first, then increased gradually; and canavanine content was slightly decreased and then increased, later decreased. So the pH of the cultured medium and extracellular peroxidase activity were related to canavanine content.