Construction Of High Frequency Regeneration System From Callus Of Alfalfa And Mechanism Of Main Elements On The Construction

This paper, orthogonal experimental design was used in conjunction with Single-factor experimental design to study the effect of different explants,which are from Suntory alfalfa, basic medium and exogenous growth regulators on callus induction and differentiation. The main results of the research were shown as follows:1. The optimal explants of alfalfa to induce callus was hypocotyls. The optimal basic medium was MS medium. The optimal combination of exogenous hormones was 2mg/L2,4-D+0.5mg/L6-BA+2.5mg/L KT+0.34 mg/L GA3.Callus induction rate is 100%.The structure of callus excellent.2. The optimal basic medium of alfalfa callus to differentiate was SH medium. The optimal combination of exogenous hormones, Exogenous KT, NAA, soluble sugar, Glycin, and Mg²⁺ was 1mg/L, 0.1mg/L, 10g/L, 4mg/L and 400mg/L, respectivelly. The optimal sucrose concentration was 10g/L. Callus differentiation rate is 61.82%.3. Exogenous NAA can reduce the soluble sugar content, promote the callus absorbing Fe²⁺ and Mg²⁺ from medium and improve the chlorophyll content of cell. Exogenous KT can promote the callus absorbing Fe²⁺ and Cu²⁺ from medium, and also improve the chlorophyll content of cell.4. Exogenous Glycin reduced the soluble sugar content, and improved contents of Fe²⁺, Cu²⁺ and Mg²⁺ in callus. As result that the callus differentiation rates were significantly increased. Exogenous Mg²⁺ increased the chlorophyll content of callus, which is the reason of increasing the callus differentiation rate of alfalfa by Exogenous Mg²⁺. These results suggested that the differentiation and morphogenesis of alfalfa callus were induced significantlly by optimal SH medium from the contents of exogenous growth regulators, soluble sugar, Glycin and Mg²⁺.