Study On Browning Composition And Its Influencing Factors In Tissue Culture Of Cremastra Appenudiculata

In the current work,the protocorms of Cremastra appenudiculata were used to study the browning factors and control in the process of proliferation and differentiation.And obtained the effective measures to the browning of Cremastra appenudiculata.On this basis,the influencing factors of cluster bud induction of Cremastra appenudiculata were also explored.The main results were as follows:1、The protocorms of Cremastra appenudiculata at the stage of proliferation were used as experimental materials,the effects of main components contents、sucrose concentrations、thidiazuron（TDZ）concentrations、naphthaleneacetic acid（NAA）concentrations,culture conditions、browning inhibitors types and concentrations on browning of the protocorms were studied.The results showed the main factor which influenced the browning of the protocorms in the proliferation process was main components.NAA had less effect on the browning of the protocorms.The browning rate of protocorms was directly proportional to the content of main components;when the sucrose with its concentraton of 30 g·L^-1,it could reduce the protocorms browning;when the TDZ with its concentraton of 1.5 mg·L^-1,it was beneficial to reduce the browning rate of protocorms;the browning rate was lower under the treatment of 0.3 mg·L^-11 NAA;the browning could be reduced under the treatment of low light and low temperature;the glutathione（GSH）was the most ideal browning inhibitor in the process of proliferation.Based on the analysis of the browning rate and proliferation index of the protocorms,the suitable culture condition for protocorms in the proliferation process of Cremastra appenudiculata was 1/2 MS+sucrose 30 g·L^-1+TDZ 2.0 mg·L^-1+NAA 0.2mg·L^-1+GSH 25 mg·L^-1,then cultured at the temperature of（15±2）°C and the light intensity of 2000 lx.2、In this study,the protocorms of Cremastra appenudiculata at the stage of differentiation were used as experimental materials,the effects of main components contents、sucrose concentrations、TDZ concentrations、indole-3-acetic acid（IAA）concentrations、culture conditions、browning inhibitors types and concentrations on browning were studied.The results showed that the main factor influencing the browning of the protocorms at the stage of differentiation was main components,the IAA followed.The effects of main components and IAA on the browning of protocorms were extremely significant.However,the effects of sucrose and TDZ on the browning of protocorm were not significant.The browning rate was lower under 1/2 MS treatment;when the sucrose with its concentraton of30 g·L^-1,the browning rate of protocorms was lower;when the TDZ with its concentraton of1.5 mg·L^-1,it was beneficial to reduce the browning rate of protocorm;added 0.3 mg·L^-11 IAA was beneficial to control the browning of protocorm.High concentration of IAA could promote the production and accumulation of phenolic compounds.Suitable light intensity and temperature could reduce the degree of browning.At the stage of differentiation of protocorms,the sodium thiosulfate（Na₂S₂O₃）was the suitable browning inhibitor.Based on the analysis of the browning rate and the average number of buds,the suitable culture condition for protocorms was 1/2 MS+IAA 0.4 mg·L^-1+TDZ 1.0 mg·L^-1+sucrose 30 g·L^-1+15 mg·L^-11 Na₂S₂O₃,then cultured at（15±2）°C and 3000 lx in the differentiation process of Cremastra appenudiculata.3、In this work,the protocorms of Cremastra appenudiculata at the stage of proliferation were taken as materials,the effects of Na₂S₂O₃,GSH and polyvinyl pyrrolidone（PVP）on the induction of cluster buds of Cremastra appenudiculata were studied.And orthogonal test was used to study the effects of main components、TDZ、NAA and Na₂S₂O₃ on clustered buds induction of Cremastra appenudiculata.The results showed that Na₂S₂O₃ was optimal to induct clustered buds.Furthermore,the main components、TDZ、NAA and Na₂S₂O₃ all had significant effects on the induction of cluster buds of Cremastra appenudiculata,and main components had the greatest influence on the induction of clustered buds.The medium 1/4MS+TDZ 2.0 mg·L^-1+NAA 0.2 mg·L^-1+Na₂S₂O₃ 25 mg·L^-1+sucrose 30 g·L^-11 was suitable to induct the clustered buds of Cremastra appenudiculata,and the induction rate reached to 79.37%.