| Dendrobium belongs to Orchidaceae, which has both high ornamental value and highmedicinal value. In this experiment, the PLBs of Dendrobium nobile and in vitro plantlets ofDendrobium huoshanense were used as the materials for optimization of in vitro cultureconditions including optimization of in vitro culture conditions of Dendrobium nobile andDendrobium huoshanense and in vitro flowering conditions of Dendrobium nobile), and furtherstudies on microRNA isolation and identification as well as quantitative expression by qPCR werealso conducted in the in vitro cultures in Dendrobium nobile. The main results were as follows:1The optimization of in vitro culture conditions in Dendrobium①Optimization of in vitro culture conditions in Dendrobium nobile.The PLBs of Dendrobium nobile were used as the materials. Optimization of in vitro cultureconditions was conducted by the single factor and orthogonal experimental design. a). The singlefactor experiment design was used for the experiments of the proliferation of PLBs and the growthof plantlets in Dendrobium nobile."Hormone addition and no hormone addition alternatingculture" was applied to the proliferation of PLBs and the growth of in vitro plantlets inDendrobium nobile. The results showed that the PLBs were first cultured on the medium with nohormone addition for30d, then were further subcultured on the same medium for another30d,which was the best for the proliferation of PLBs; the plantlets were first cultured on the mediumwith hormone addition for30d, then were further subcultured on the same medium for another30d, which was the best for the proliferation of plantlets. b). The orthogonal experimental designwas used for optimization of in vitro culture conditions. The effects of different concentrations of6-BA, IBA and coconut water on the proliferation of PLBs were compared in Dendrobium nobile,and the results showed that the best proliferation for PLBs occurred on KC medium supplementedwith1.5mg/L6-BA,0.5mg/L IBA and100ml/L coconut water. The effects of differentconcentrations of IBA, NAA and potato homogenate for growth of plantlets were also analysed inDendrobium nobile, and the result showed that the best rooting and growth for plantlets occurred on1/2MS medium supplemented with1.0mg/L IBA,1.0mg/L NAA and100g/L potatohomogenate.②Optimization of in vitro culture conditions in Dendrobium huoshanense.The plantlets of Dendrobium huoshanense were used as the materials. This study was toinvestigate the effects of different concentrations of organic additives on the growth of in vitroplantlets. The media were supplemented with homogenates of banana, and potato at variousconcentrations. Results showed that50g/L homogenates of potato and100g/L homogenates ofbanana were the best for the growth of the plantlets. The orthogonal experimental design was usedfor optimization of in vitro culture conditions. The effects of the basal media, IBA and NAA ongrowth of plantlets were compared and the result showed that the best growth for plantletsoccurred on KC medium supplemented with0.05mg/L IBA and0.1mg/L NAA.③Optimization of in vitro flowering conditions in Dendrobium nobile.This study was to investigate the effect of different color genotypes on in vitro flowering inDendrobium nobile. The results showed that the rose color was the highest frequencey of floralbud induction, and the rate was up to38.67%. Then, the effects of different phosphorusconcentrations on in vitro flowering of Dendrobium nobile(Rose color) were analysed and theresult showed that when the phosphorus concentration increased by5times, the frequency offloral bud induction was the highest with the rate of32.14%.2Isolation and identification of small RNA in vitro cultures by Solexa sequencingin Dendrobium nobileThe Solexa sequencing technique was used to separate and identify small RNA from in vitrocultures (Samples of PLBs, differentiation plantlets, rooting plantlets, inducing plantlets,inflorescence buds and flowering plantlets) in Dendrobium nobile. The results showed that thetotal reads of19,665,726, including high quality reads of19,301,162were obtained from the smallRNA library from in vitro cultures in Dendrobium nobile. After treatment of the high quality reads,19,228,819clean reads were obtained, which contained6,021,550unique sequences. Thedistribution proportion of the sequence's length of sRNA was different, ranging from18nt to35nt,and among them24nt being the predominant (39.46%). The expression patterns of differentunique sRNAs during in vitro culture varied drastically, and most of them were at the lowexpression levels.33~4highly conservative miRNAs were identified, and belong to many families.6novel miRNA candidates were also identified. de-miR1/2/3/6with single locus and de-miR4/de-miR5with multiple loci. About2167targets of104conservative miRNAs werepredicted, and65targets of4novel miRNAs were also predicted. Besides, function annotations ofthe predicted targeting genes of specific miRNAs were conducted based on the GO database andthe KEGG pathway database.3The analysis of the quantitative expression of small RNAs by qPCR in theprocess of the in vitro culture in Dendrobium nobileIn this study, the qPCR was used to analyse the expression models of25de-miRNAs. Theresults showed that the internal control gene was de-miR535, which was suitable for the analysisof the quantitative expression of small RNAs in the process of in vitro culture in Dendrobiumnobile.①The analysis of expression models of12de-miRNAs related to floweringThe trend of the expression of differentiated seedlings, bud induction period, floweringperiod for de-miR156g/156i/156j.1/156j.2was sharp decline, compared with contrast, the overalllevel of expression was below0.14, almost unexpressed, which suggested that their target genesshould be at the high expression, and it should promote all the further stages' development; inaddition, it also promote the transformation from vegetative growth to reproductive growth. Thetrend of de-miR159b.1/159b.2expression was up-down-up-down-up, which was identicalin theprocess of in vitro culture in Dendrobium nobile. We conjectured that the de-miR159b.1/159b.2'slow transcription level at the stages of rooting, induction and alabastrum, was beneficial toflowering of Dendrobium nobile, but during the flowering period the expression level rose, evenhigher than that of the contrast, that might be a certain relationship with deformity of someflowers. The trend of de-miR166f/166g/166n's expression was alike, lower than that of thecontrast, which might be closely related to formation of tissues and organs, even the change ofeach stage during the development. de-miR167c/167j was also belonged to the same gene family,but their expression models were different, which showed that different members of themiRNA family had different regulating modes. After analysis, we found that de-miR167c mightpromote the process from growing to flowering development. The flowering of Dendrobiumnobile plantlets might be inhibited because of the overexpression of de-miR167j. And theexpression levels at the stages of PLB, differentiation, alabastrum and flowering were fluctuating,which lead to the fluctuating of the corresponding target genes. Because of that situation, themorphological structure of Dendrobium nobile might be changed, also the phenomenon ofpseudobulb inflated, the morphological structure of leaves, the occurrence of deformity flowers would happen. The trend of de-miR169a expression was down-down-up-down-up during the invitro culture in Dendrobium nobile, which might inhibit the process of induction period andflower bud formatin, and also might delay the bud induction time and lead to floweringabnormally.②The analysis of expression models of10de-miRNAs during the6developmentalstagesAt the stage of vegetative growth, the trend of de-miR162b's expression was declined, thelowest expression was at the satge of rooting, then was at the satge of reproductive growth; afterthe stage of bud induction, the expression started to rising, the stage of flowering came to the peak,which suggest that de-miR162b promote the differentiation of PLBs and the growth of plantlets,but had the opposite effect on bud induction and flowering. de-miR168a might promote thedifferentiation of PLBs and the growth of plantlets, at the stage of bud induction, the trend of itsexpression rose, but lower than that of the constant, suggested that de-miR168a have a certaineffect on leaf modality. de-miR171c/394/408/528belonged to different gene families, but theirexpression trend was alike. After analysis, we suggested that de-miR171c/394/408/528promotethe process of the whole growth and development, which not only promoted the differentiation ofPLBs, vegetative propagation,and rooting, but also had a certain related to flowering, controlingtarget genes and promoting flowering. de-miR396b/397b belonged to two gene families, theirexpression trends were the same, the last5stages were ovrexpressed, and the stage of inductionreached the peak. It was suggested that de-miR396b/397b promote the maintenance of PLBs,inhibit the the differentiation of PLBs and inhibit the transformation from vegetative growth toreproductive growth. In addition, the expressions of de-miR390/529a at the last5stages werelower than that of the contrast, but the relative expression was fluctuating, which suggested thatde-miR390/529a have some effect on growth and development.③The analysis of potential specific miRNAs(de-miR1and de-miR3) during the in vitrocultureThe internal control gene was de-miR535, and PLB was used as the contrast, defined as1U,for analysing the expression modes of the potential specific miRNAs. The result showed thatde-miR1might have a certain inhibiting effect, but it would promote seeding formation, rootingand would promote the transformation from vegetative growth to reproductive growth to a certainextent. de-miR1had low-level expression during induction and formation of the flower buds. After analysing the expression mode of de-miR3, we found that de-miR3had low-level expressionduring the in vitro culture in Dendrobium nobile, the targeting gene had high-level expression,which suggested that it have a promoting effect on advancing the time of flowering and also havea closely relationship with morphogenesis, environmental signal responses and flowering. |
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