In Vitro Culture And Production Of Artificial Seeds In Acacia Leptocarpa And Acacia Fimbriata

In this experiment the tissue culture seedlings were used as materials in A.leptocarpa and the immature and mature seeds were used as explants in A.fimbriata for in vitro culture and artificial seeds production of the two Acacia species.The main contents of the experiment were as follows:â‘ establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa;â‘¡induction and regeneration of callus in A.leptocarpa;â‘¢establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata;â‘£production of artificial seeds in A. leptocarpa and A.fimbriata;⑤RAPD analyses of the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The main results were described as follows:1.Establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa. The tissue culture seedlings were used as materials to studying the proliferation,rooting and strong seedling and transplantation in A.leptocarpa.The results showed that the proliferative effect was the best on the MS medium supplemented with 0.3 mg·L^-1BA and 0.15 mg·L^-1NAA, the propagation coefficient was up to 3.75,and the rate of vitrification was also controlled on the lowest level(18.75%);the optimum medium of rooting and shoot growth was the 1/2MS supplemented with 0.3 mg·L^-1NAA and 0.5 g·L^-1AC and 100 g·L^-1coconut milk,the rooting rate was 97.56%,the growth vigor of tissue culture seedling was better;the surival rate was up to 93.33%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.2.Induction and regeneration of callus in A.leptocarpa.Tender stem segments of the tissue culture seedling were used as materials to callus induction in A.leptocarpa.The results showed that the MS medium supplemented with 0.1～0.15 mg·L^-1NAA and 1.0～2.0 mg·L^-1BA were faliciated to induce callus,rates of inducing callus were all up to 100%,and the quality of callus was better;the regeneration rate was up to 50%on the MS medium supplemented with 0.1 mg·L^-1NAA and 1.0 mg·L^-1BA,and the regeneration plantlets were all better in terms of growth vigor.3.The effect of different pre-treatments on the germination of the immature and mature seeds in A.fimbriata.The immature and mature seeds were used as explants to studying the aseptic germination in A.fimbriata.The results showed that the germination rate reached 100%when doing the incision treatment at the raphe far from embryo.Under the pre-treatment of the thick vitriol for 15 min on mature seeds in A caciafimbriata,the germination rate reached 100%.4.Establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata. The tissue culture seedlings germinated of seeds in viro were used as materials to studying the proliferation,rooting and transplantation in A.fimbriata.The results showed the best formula of proliferation was the MS medium supplemented with 0.1 mg·L^-1NAA and 0.3 mg·L^-1IBA and 0.3 mg·L^-16-BA and 30 g·L^-1sucrose and 6 g·L^-1agar,the propagation coefficient was 3.22;0.3 mg·L^-1IBA was faliciated to rooting culture,rooting rate reached 3.75;the surival rate was up to 90.48%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.5.Production of artificial seeds in A.leptocarpa and A.fimbriata.The shoot cuts of A. leptocarpa and A.fimbriata were used as encapsulating materials to study the non-embryo artificial seeds.The results showed that the 2.0%sodium alginate encapsulated 20 min was the best when using the method of calcium alginate to encapsulating the shoot cuts in A.leptocarpa, germination rate was 85.93%,plantlet formation rate was 79.69%;3.0%sodium alginate encapsulated 15 min was more suitable for gennination and regeneration of artificial seeds in A. fimbriata,germination rate was 85.71%;the calcium alginate hollow method was less than calcium alginate method in terms of germination;the rate of germination was improved 2.89%by adding 0.05 mg·L^-1NAA,0.1 mg·L^-1BA to artificial endosperm in A.leptocarpa;it was found that the double calcium alginate method,smearing glycerol,addition polyvinyl pyrrolidone were the better investing methods of water retention by studying dehydration and rehydration of artificial seeds on natural conditions;4℃was suitable for the storage of the artificial seeds in A. leptocarpa;the double calcium alginate methord was suitable for the storage of the artificial seeds in A.leptocarpa and A.fimbriata(germination rates were 39.58%and 30.3%after 4℃storage for 60 days);the investing method of additing polyvinyl pyrrolidone with 0.05%ALCl₃,0.05% MgCl₂,0.05%CoCl₂,0.2%carbendazim and 0.3%sodium benzoate was suitable for germination of the artificial seeds in A.leptocarpa under natural conditions,the mixture of sand and vermiculite and peat soil(2:1:1)was suitable for sowing,the germination of 60 days of artificial seeds was 25.56%.RAPD analyses in the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The tissue culture seedling,the plantlet regenerations from callus and the plantlet regenerations artificial seeds from the same plants were used as materials in A. leptocarpa.The result indicated that there were significant differences in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of callus were relatively unstable in heredity.There was no significant difference in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of artificial seeds were hgihly stable in heredity.As a whole,in this study the system of the high-effeciency regeneration in vitro was established in A.leptocarpa;Tender stem segments of the tissue culture seedling were first used to inducing callus and obtained regeneration in A.leptocarpa;the technique of high-frequency gerlnination of immature and mature seeds was developed in A.fimbriata;the technique of the production of artificial seeds was first developed in the two Acacia species.;and analyzed the hereditary stability of the regenerated plants of callus and the regenerated explants of artificial seeds by the method of RAPD,which would be of great importance for in vitro multiplication and artificial seeds application in Acacia.