Aflp Analysis On The Genetic Relationship Of Spring Dendrobium Cultivars (or Varities)and Transformation For ’Sanya’

Dendrobium is the biggest genus within Orchidaceae. There are more than1500wild ancestors in the world which distribute mainly in the tropical&subtropical areas in which more than70species of Dendrobium originated from China. Spring Dendrobium is a group of Dendrobium representing blooming in spring, and is also one of the top grade pot flowers which are very popular in the international orchid market. In order to create more valuable new cultivars of Spring Dendrobium, the related breeding technilogy needed to be solved both in production and research. However, the research on genetic relationship between species or cultivars by molecular markers, constracting the highly efficient and stable transformation system, and getting the new cultivars consistent with the purpose gene by transgenic breeding technology, are still limited.For the first time, this study was intended to determine genetic relatedness of popular pot ornamental Sping Dendrobium cultivars and varieties materials using AFLP markers with near-infrared fluorescence-labeled primers, providing a reference for the parent selection in the breeding of Spring Dendrobium. Then,’Sanya’ as an important commercial cultivar was selected for the transgenic explant. The in vitro regeneration system of it by PLBs was studied. An efficient method of sonication-assisted-Agrobacterium-mediated transformation (SAAT) was developed for it. Agrobacterium tumefaciens (LBA4404) harboring ASACC gene or CHS gene was used to transform PLBs explants of’Sanya’. Our main research results as follows:1. Using modified CTAB method, before carrying out nuclear lysis buffer into the nuclear separation, can be successfully extracted high-quality gDNA. The ratios of OD260nm/OD280n of the genomic DNA extracted by the modified CTAB methods were respectively1.85and1.73. The comprehensive results from electrophoresis, ratios of OD260nm and OD280nm and Fluorescent-AFLP amplification suggested that the modified CTAB can produce high quality genomic DNA while the conventional CTAB and Genomic DNA Kit methods are not suitable for the DNA extraction from Spring Dendrobium;2. The genetic relationship of30Spring Dendrobium cultivars (or varieties) were analyzed using amplified fragment length polymorphism (AFLP) markers with near-infrared fluorescence-labeled primers. Eight EcoRI+3bases/MseI+3bases primer set combinations were used in this investigation. Each selected primer set generated113-158scorable fragments. A total of1102AFLP fragments were detected, of which778were polymorphic (70.6%). An un-weighted pair-group method of the arithmetic averages (UPGMA), principal coordinate analysis (PCOA), and bootstrap analysis were used to analyze the genetic relationships. The30cultivars or varieties were separated into five clusters. Cluster Ⅰ contains6varieties materials that are either from Senlan No.l to Senlan No.6with Jaccard’s similarity coefficients ranging from0.70to0.80. All of these6varieties materials came from Taiwan, and were derived from somaclonal variants or sports; Just3cultivars are positioned in cluster Ⅱ ranging from0.71to0.77, and also origined from Taiwan; Cluster Ⅲ has13cultivars(or varieties), Jaccard’s similarity coefficients varied from0.69to0.84. Seven cultivars(or varieties) from Senlan No.15to’Snowboy Romance’were situated in cluster IV with Jaccard’s similarity ranging from0.68to0.83; Only’Santana Canary’was positioned in cluster V.3. The regeneration and proliferation system in vitro of’Sanya’and’China Doll’by PLBs was studied. At first, the plantlet in vitro were obtained from explants, then, PLBs were induced by basal stem without bud of the plantlet as the explants, which were pretreated with40KHz Ultrosonic Wave for10mines, then were respectively cultured in1/2MS+6-BA0.2mg/L solid culture medium. The induction rates of two cultivars were10.5%and9%, while the control did not induce the formation of PLBs. And then were respectively cultured in MS+6-BA0.2mg/L liquid medium for PLBs proliferation. At last, proliferation rates of PLBs of’Sanya’and’China Doll’as4.57and4.31times per month in liquid medium. Cultured by1/2MS+6-BA1.0mg/L+NAA0.2mg/L solid medium for buds culture could get the greatest propagation rate as2.90and2.66. There are distinct different to induced rate of PLBs among different Spring Dendrobium cultivars.4. An efficient and reproducible transformation method of sonication-assisted-Agrobacterium-mediated transformation (SAAT) was developed for’Sanya’. assisted with Ultrasonic Wave40KMz for10min and inculture for60min. By using a series of co-cultivation, filteated on the1/2MS medium supplemented with200mg/L Km, shoot initiation and root inducing media,63positive lines of trans-CHS gene and64lines of trans-ASACC gene were recovered. Analysis of positive materals by GUS、PCR and Southern hybridization confirmed that,20of trans-CHS gene and10of trans-ASA CC gene positive plants were transgenic lines. The highest transformation efficiency respectively was1.17%and1.04%. cDNA analysis of the trans-CHS and trans-ASACC lines by Real-time RT-PCR show that their comparative quantification of NPTⅡ gene were high distinctly compare with control. B3of the trans-ASACC gene lines and A1of the trans-CHS gene lines had highest comparative quantification among these transgenic lines.