Studies On The Preliminary Mechanism For Induction And Formation Of Virus-free Corm Of Red Bud Taro

Research on virus-free taro corm induced in vitro can enhance the transplanting survival rate of virus-free taro seedling and reduce the differentiation of seedlings.Developing the studies on the physiological and molecular mechanisms related to the induction and formation of taro corm in vitro is of great significance for the development of taro industry.With taking the most famous and high-quality local cultivars of taro in Jiangxi province as experimental material,the detoxification culture of taro stem tip was carried out.After molecular detection,further explore the effects of different hormone ratio and culture conditions on the induction of virus-free seedlings.At the different stages(0 d,45 d,90 d and 135 d)of taro formation in vitro,internal morphology changes in taro corm were observed by paraffin tissue section,endogenous hormones change were determinate by enzyme-linked immunosorbent(ELISA),and use the transcriptome sequencing and qRT-PCR analysis toscreen and validate differentially expressed genes related to the formation of taro corm,with the hope to preliminarily clarify the induced formation mechanism of micro tube taro.The main research results are as follows:(1)Seedlings could be induced from 0.2～1.0mm taro buds inoculated in MS+2 mg/L 6-BA+0.5 mg/L NAA+3%sucrose medium.RT-PCR molecular detection showed that seedlings in vitro induced from stem tip under 0.3mm diameter in culture could not carry virus,such as DsMV,CMV.The optimal medium for the induction of secondary proliferation of virus-free seedlings was MS+3 mg/L 6-BA+0.5 mg/L NAA+3%sucrose,the optimal medium for the induction of virus-free taro corm was MS+1 mg/L 6-BA+0.5 mg/L NAA+8%sucrose,the optimal culture conditions were 25℃,4000 lx,14 h/d,the optimal matrix for transplanting and acclimation of virus-free seedlings and taro corm was peat:vermiculite(2:1),and the survival rate was 97.71%.The system of detoxification and rapid propagation of the stem tip of red-bud taro established in this study provided some technical support for the germplasm conservation and rejuvenation of species characteristics.(2)The anatomical observation of microtubule taro at different development stages showed that the number of parenchyma cells increased and the cells were closely arranged during the expansion of microtubule taro.Cell inclusions increased and starch grains gradually accumulated.The number of terminal buds increased and the cell size increased.The results of endogenous hormone content determination showed that the contents of abscisic acid(ABA),cytokinin(ZR,DHZR),auxin(IAA,IPA),gibberellin(GA3,GA4)and brassinolide(BR)were increased significantly with the expansion of taro crom in vitro,and the content of methyl jasmonate(JA-ME)was decreased gradually.It was speculated that auxin,cytokinin and gibberellin promoted cell division and tuber formation,abscisic acid promoted the change of the content of endogenous substances within cells,brassinolide enhanced the resistance of in vitro taro,methyl jasmonate inhibited the expansion of in vitro taro,and the formation of in vitro taro was regulated by a variety of hormones.(3)The transcriptome sequencing and bioinformatics analysis results show that the differentially expressed genes(DEGs)in vitro taro different development period is mainly enriched in phenylpropanoid biosynthesis,MAPK signal pathways,plant hormone signal transduction,starch and sucrose metabolism,etc.Among these DEGs,10 genes were significantly differentially expressed in relation to plant hormone signal transduction,4 genes were significantly differentially expressed in relation to starch and sucrose metabolic pathways,and 2 genes were involved in the regulation of tuber enlargement.Further expression of 16 significant differences during different periods of candidate genes in vitro taro qRT-PCR detection,the results show that TCH4,MYC2,NPR1,IAA,JAZ and GA20oxl were involved in plant hormone signal transduction genes,GN4,TPS,AMY were involved in starch and sucrose metabolic pathway genes,and StBel5 related to tuber bulking synthesis which were significantly related to the morphological changes and endogenous hormone change in the formation of the micro tube taro.In conclusion,these genes could be identified as important candidate genes associated with the formation of micro tube taro.