Flowering And Its Mechanism Of Dendrobium Officinale Kimura Et Migo In Vitro

The flowering conditions of Dendrobium officinale Kimura et Migo is optimized by applying tissue culture and some technologies of molecular biology.The main purpose is getting more capsules through flowering and pollination of test-tube plantlet to provide material foundation for subsequent embryo and relevant experiments.Higher plants with flower formation were obtained,the quality of flower and seedling were determined through the single factor experiment of 5 different hormones.The flowering conditions of D.officinale is optimized and the flower formation is advanced through the plant tissue culture;In the transformation process of D.officinale from nutritional growth to reproductive growth,the cell section image of the most representative shoot tip and cell development change chart were observed by applying paraffin section technology;The gene sequences related to flowering was cloned by applying RACE technology;the method of bioinformatics was adopted to take preliminary analysis towards some basic features of sequence which include length,subcellular localization,isoelectric point,secondary structure and etc.;the Quantitative real-time reverse transcription-PCR(RT-qPCR)was adopted to analyze the expression pattern of these genes under the different tissues and hormon conditions,optimization condition was combined to make further analysis on the promoting condition relation between the genes and hormones.ABA(Abscisic Acid)can promote the in vitro flowering of D.officinale when it is cultivated in flowering medium for 15 to 20 days,the germination quantity and germination rate increase significantly when it is transferred into inducing mediums contains 0.2 mg·L-1 NAA and 0.2 mg·L-1 6-BA.The most appropriate days for cultivation is 20 days when the Spermidine HCL concentration is 0.1 mg·L-1,and 15 days when the Spermidine HCL concentration is 1.5 mg·L-1.The frequency of floral bud induction reaches the highest as 24%when PP333(Paclobrazlo)concentration is 0.6 mg·L-1,and it reaches the highest as 13.3%when ethephon concentration is 1.0 mg·L-1,its lethal concentration is about 2.5mg·L-1.At the same time,the stems of D.officinale turned yellow,Leaves wither,and eventually die after being induced for 30 days.TDZ(Thidiazuron)can shorten vegetative period for 2 to 3 years.Ethephon treatment can accelerate the transformation from vegetative growth to reproductive growth.The root has depressant effect on flower bud differentiation of D.officinale.The complete cds sequences of genes related to flowering is obtained based on transcriptome data and application of RACE technology.Some genic basic natures were obtained by applying the method of bioinformatics.The amino acid sequence length is from 237bp to 2958bp,the molecular weight is from 19304.06ku to 245735.14ku,and the G+C content of all sequences is between 0.44 and 0.54.It is found that gene has different expressions in different tissues and different hormones by applying RT-qPCR.Gene is treated by hormone ABA,spermidine HCL and TDZ,and the result is consistent with the experiment result of tissue culture optimization.The flowering rate of D.officinale would reach the relatively high peak when it applies pre-treatment for 15 days or so;The expression quantity of most genes reaches the highest around 20 days by applying hormone spermidine HCL treatment;The expression quantity of most genes reaches the highest around 18 days by applying hormone TDZ treatment.No.4 gene Flowering Locus T,No.5 gene Flowering time control protein FCA,No.12 Flowering time control protein FPA,No.14 gene Flowering time control protein FCA,and No.16 gene LEAFY expression pattern were special,can be screened as flower-specific genes.These experimental results lay the foundation for the subsequent research of molecular mechanism.