Studies On The Transformation Of Antisense ACS Gene Mediated By Agrobacterium Tumefaciens And In Vitro Flowering In Dendrobium

In this experiment,a high-efficiency and systematic transgenic procedure mediated by Agrobacterium tumefaciens with antisense ACS gene was developed from protocorm-like bodies(PLBs)in Dendrobium,and preliminary studies on in vitro flowering from plantlets without roots were also carried out.The main results were described as follows:1 The plant regeneration system of Dendrobium was established.The studies on the efficiently transgenic establishment of the regeneration from stems,leaves and seeds(4 kinds of colors in flower)were carried out.The results showed that the rate of PLBs induced from stems and leaves cultured on the KC medium supplemented with 0.2 mg/L 6-BA and 1.5 mg/L NAA,the KC mudium supplemented with 4.0 mg/L 6-BA and 1.0 mg/L NAA was 35.57%and 11.99%, respectively;the best multiplication medium of PLBs was the KC medium supplemented with 2.0 mg/L 6-BA and 0.5 mg/L NAA,and the rate of multiplication was 8.02;PLBs cultured on the 1/2MS medium supplemented with 0.5 mg/L 6-BA differentiated to plantles;plantles cultured on the 1/2 MS medium supplemented with 1.0 mg/L NAA rooted best.2 The transgenic system of protocorm-like bodies was established and optimized.The transgenic system of the subtle green PLBs was established by adjusting factors such as different hormone concentrations,different kinds of sugar and different concentrations of sugar.The culture of the subtle green PLBs was optimized by adjusting pH,argar concentrations,natural additives, basal medium,light intensity,medium forms during the growth of PLBs.The results showed that the subtle green PLBs was induced on the KC medium supplemented with 2.0 mg/L 6-BA and 0.5 mg/L NAA,the best cultural way was 20.0 g/L sugar,no natural additive,5.4 pH,5.8 g/L agar,KC medium,solid cultural way and 1 500 lx light intensity.It was good for transgenic systems to use PLBs with incised growing point as the explants.The PLBs were very sensitive to kanamycin,and PLBs of Dendrobium could be killed with kanamycin concentration as high as 150.0 mg/L,while the concentration of 50.0 mg/L for kanamycin which proved to be restricted rooting of transgenic plantlets was used for selecting transgenic plantlets.3 The introduction of antisense ACS gene to Dendrobium used in Agrobacterium-mediated transformation was obtained.The best transient expression of GUS was obtained under the conditions as follows:the PLBs which were not precultured but pretreated with 0.3 mol/L mannitol for 6 h;and the Agrobacterium suspension was with OD₆₀₀value of 0.8 and 20.0 g/L sugar,the samples were infected for 30 mins,and then they were transferred onto the KC medium (pH 5.4)supplemented with 2.0 mg/L 6-BA and 0.5 mg/L NAA for co-cultivation for 5 days at 25℃in the dark..4 A whole technical system of Dendrobium resistant PLBs and transgenic plant regeneration was selected and examed.The indirect method of selecting resistant PLBs was that after co-culture, the PLBs were cultured on the KC medium supplemented with 2.0 mg/L 6-BA and 0.5 mg/L NAA and 100.0 mg/L Cef.for 20 d,then selected on the KC medium supplemented with 2.0 mg/L 6-BA and 0.5 mg/L NAA and 150.0 mg/L Km.for three month with the gradual enhancement of Km. concentration,transferred on the 1/2MS medium supplemented with 0.5 mg/L 6-BA when PLBs were differentiated,rooted on the 1/2MS medium supplemented with 1.0 mg/L NAA and 50.0 mg/L Km.and 100.0 mg/LCef.At last,13 resistant plantlets were obtained,the transgenic rate was 12.15%,GUS histochemical assay and PCR assays proved that the gus gene was integrated into the genome of these resistant plantlets.There was no difference between transgenic plantlets and no-transgenic plantlets.Transgenic plantlets transferred after 2 months were survival.5 A basic research of in vitro flowering of plantlets in Dendrobium.The studies researched the best medium of in vitro flowering of plantlets without roots for 2 months and the effect of the basal media,6-BA and sugar on in vitro flowering.The result showed that the KC₂ medium supplemented with 5.0 mg/L 6-BA and 40.0 g/L sugar was the best medium for in vitro flowering after culturing 150 d,the rate was 25.0%,these flowers abnormally blossomed with normal flower bud;the factors of the basic media,6-BA and sugar affecting in vitro flowering was 6-BA＞the basal media＞sugar concentration.As a whole,in this experiment the efficient regeneration of plantlets in Dendrobium was established;the transgenic system of the subtle green PLBs was abtained;the stable transformation of antisense ACS gene mediated by Agrobacterium tumefaciens to PLBs in Dendrobium was established;the method and time selected the transgenic PLBs were developed; the technique of the high transformation rate by enhancing sugar concentration without AS was founded;the method of in vitro flowering in Dendrobium was developed.These results would be of great importance for the new method of the prolonging life of cut flowers and the technique of the transgenic research in Dendrobium.