The Research For The Callus Induction And Physiological And Biochemical Characterization Of Dedifferentiation Of Hevea Brasiliensis M(u|¨)ll. Arg. Clone Reyan7-33-97Grafted Seedling Leaves

In order to expand the rubber tree explant sources and to establish efficient and stable rubber tree leaves in vitro regeneration system, annual bagged seedling leaves of Reyan7-33-97were used as explants to study on effects of leaves pretreatment,different disinfection time with0.1%HgCl2,different parts of detached leaves,different combinations of three kinds of phytohormone (6-BA,2,4-D,NAA),different feteh time on callus induction frequency, screen the best subculture medium.In the process of detached leaves(pretreated leaf blocks (with the main veins), untreated leaf blocks(with the main vein), pretreated leaf blocks (without the main vein) and untreated leaf blocks (without the main vein) dedifferentiation,this work mainly focused on the researeh of MDA contents,soluble sugars contents,POD enzyme activities and CAT enzyme activities,In order to have a preliminary understanding of dedifferentiation mechanism rubber leaves in vitro culture.The main results are as follows:1Study on callus culture from bagged seedling leaves of Hevea Brasiliensis Mull. Arg. Clone Reyan7-33-97leaves pretreated with liquid medium MS+3mg/L6-BA+0.5mg/L2,4-D+0.2mg/LNAA;disinfected with0.1%HgCl2for15min was the best disinfection method,its survival ratio can be2.47%;the optimum callus induction medium was MS+3mg/L6-BA+0.5mg/L2,4-D+0.2mg/LNAA+4%sucrose+3g/L phytagel,leaves (with principal vein) and no callus part of leaves (with principal vein) have already produced callus were cultured in this medium,induced callus successfully with bagged seedling leaves for the first time in China,their callus inducing ratio can amount to49.07%and42.22%;leaves (with principal vein) and no callus part of leaves (with principal vein) have already produced callus were the most suitable explants for rubber bagged seedlings leaves,callus induced by which showed tighten and yellow-green.2Influencing factors of callus induction of leaves of Hevea Brasiliensis Mull. Arg. Clone Reyan7-33-97MS was the best basic medium for lesves callus induction,pretreated leaves (with principal vein) and pretreated leaves (without principal vein) have already produced callus were cultured in this basic medium,their callus inducing ratio can amount to34.26%and12.04%.The leaves callus induction rate of the pH level5.8was the highest of all,callus induction rate of pretreated leaves (with principal vein) was amount to41.67%,callus induction rate of pretreated leaves (without principal vein) was14.81%,but there were no callus induced by pH4.6and7.0.A period of darkness for42days was the best for leaves in vitro culture.callus induction rate of pretreated leaves (with principal vein) was amount to36.11%,callus induction rate of pretreated leaves (without principal vein) was12.04%,no callus induced with no darkness or too short time darkness.The reverse side touching the medium was better for leaves. Grey green stage was the most suitble phenophase for leaves in vitro culture,callus induction rate of pretreated leaves (with principal vein) was amount to36.26%,callus induction rate of pretreated leaves (without principal vein) was10.19%.It was difficult to induce callus on budding and stable stages.June was the most suitble feteh time for leaves in vitro culture.callus induction rate of pretreated leaves (with principal vein) was amount to40.74%,callus induction rate of pretreated leaves (without principal vein) was12.96%,followed by March and September, it was the hardest to induce callus on December.3Study on biochemical and physiologica indexes of leaves dedifferentiation in vitro culture.The dynamic changes of MDA contents in leaf blocks of four treatment groups was decrease first then increase then decrease and then increase,Inflection point appeared at first14d,21d,42d,the MDA content of pretreated leaf blocks (with the main vein) were the lowest at the3pionts,8.33umol/g、11.76umol/g、5.26umol/g respectively. The dynamic changes of soluble sugars contents in leaf blocks of four treatment groups was showed down then up,Inflection point appeared at first42d,the soluble sugars content of pretreated leaf blocks (with the main vein) was33.57mmol/g,lowest of all. The dynamic changes of POD enzyme activities was increase first then decrease then increase and then decrease.two activity peaks appeared at first14d and42d,the POD enzyme activities of pretreated leaf blocks (with the main vein) were63.61u/g(FW)· min and87.82u/g(FW)· min respectively,highest of all.The dynamic changes of CAT enzyme activities was similar to the trend of changes of POD enzyme activities,two activity peaks appeared at first14d and42d,the CAT enzyme activities of pretreated leaf blocks (with the main vein) were43.65u/gFW/min and67.38u/gFW/min respectively,highest of all.