Research On Test Tube Flowering, Seed Preservation And Hybrid Identification Of Dendrobium Candidum

Dendrobium officinale Kimura et Migo belongs to Dendrobium Sw.in Orchidaceae is traditional Chinese medicinal herb and mainly distributes in Jiangxi,Guangxi,Guizhou,Yunnan and Henan provinces.The wild resources of D.officinale have been greatly damaged due to the overexploit and difficulty in bloom,and hardly to fruitify which causing by the strict requirements for its habitat conditions.Therefore,it is necessary to seek an effective way to protect this indangered plant.This study including the optimization of cultivated conditions for flowering in vitro,the search of suitable methods for seeds storage and the identification of putative D.officinale hybrids and the maternal origin provided a theoretical basis for establishing rapid reproduction system,shortening the breeding cycle,prolonging seed longevity,and identification of germplasm.In order to explore the suitable conditions for flowering in vitro,D.officinale plantlets without roots were selected and treated with hormones BA,NAA,PP333,TDZ and low temperature in single-factor experiments and multi-factor orthogonal experiments.The results showed that the best conditions for flowering was:1/2MS+NAA 0.2 mg/L+BA 3.0 mg/L+TDZ 0.06 mg/L+PP333 0.4 mg/L,meanwhile supplemented with sugar 30.0 g/L,potato100.0 g/L,agar7.0 g/L,and under the 20 ? for 30 days.The frequency of floral buds formation could reach 100%and blossomed flowers were more than 80%.In order to research the preservation methods of D.officinale seeds,fresh seeds were dehydrated for different time.Then,the seeds with suitable water content were stored at normal temperature?4? and liquid nitrogen for 30 d?60 d?90 d,respectively.The seed vigor,germination rate and the morphological changes of seeds were recorded for the evaluation of the seed storage method.The results showed that the rate of seed germination was up to 94.15%when the moisture content of seed was 20.63%,the rate of germination reached to 90%when the moisture content of seed between 10%-20%.The best preservation method was drying seeds for 24 h and then storing at liquid nitrogen.Putative D.officinale hybrids collected from Huoshan County in Anhui province were selected as materials.The putative D.officinale hybrids and maternal origin were identified by reconstruction of the UPMGA tree based on nrDNA ITS and cpDNA psbK-psbI sequences.The alignment results revealed that ITS sequences of the putative hybrid species were nucleotide superposition of D.officinale and D.moniliforme in the 355 site,which indicated that the putative hybrid species came from the hybrids of D.officinale and D.moniliforme.The psbK-psbl sequences of putative hybrid species showed that plants MO1?M02?M04?M07?M09 had the same haplotype with D.offcinale while plants M03?M05?M06?M08?MO10 had the same haplotype with D.moniliforme,and the sequence similarity was 100%.According to the maternally inherited feature of angiosperms chloroplast genome,D.officinale was thematernal origin of plants MO1?M02?M04?M07?M09,D.monilifo was thematernal origin of plants M03?M05?M06?M08?MO10.