| Oncidium Sharry Baby ’Sweet Fragrance’ is a cultivar of Oncidium,which has a rich chocolate creamy aroma when flowered and has high research value.The protocorm of the Oncidium Sharry Baby ’Sweet Fragrance’ is highly prone to differentiation when it is cultured,and it is not easy to maintain the protocorm in the subculture.Also the yield of plantlets is low.The peak period of Oncidium Sharry Baby cut flowers is concentrated in the high temperature and high humidity season which is extremely vulnerable to diseases,and hinders seriously the development of the Oncidium Sharry Baby industry.Therefore,it is imperative to carry out in vitro culture optimization and disease resistance research of Oncidium Sharry Baby ’Sweet Fragrance’.In this study,the protocols and conditions of Oncidiumin in vitro culture were optimized,and the medium suitable for rapid propagation of the Oncidium Sharry Baby’Sweet Fragrance’ was obtained.The transcriptome data of the Oncidium Sharry Baby ’Sweet Fragrance’ were used to clone the EDS1,PAD4 and PR1 genes,which are related to disease resistance.And functional verification and other analyses and experiments were performed.The main results were as follows:1 The optimization of in virtro rapid propagation conditions of PLBs in Oncidium Sharry BabyUsing the PLBs of Oncidium Sharry Baby as the experimental materials,the orthogonal design method was used to study the optimal medium on PLBs propagation and differentiation.The results revealed that the optimal medium was:MS+1.5 mg/L 6-BA+0.25 mg/L NAA+20 g/L banana-pulp.+25 g/L sucrose+6 g/L agar.The strength of influence for the hormones and banana-pulp on PLBs propagation was:NAA>6-BA>Banana-pulp.The optimal medium for differentiation was:1/2MS+0.3 mg/L NAA+0.5 mg/L KT+40 g/L Apple-pulp+25 g/L sucrose+6 g/L agar.The incidence of hormones and apple-pulp on PLBs differentiation was:KT>Apple-pulp>NAA.Furthermore,the alternative culture by hormones and banana-pulp reduced PLBs browning.2 Rooting culture in Oncidium Sharry BabyUsing the plantlets of Oncidium Sharry Baby as the experimental material,Meanwhile,apple-pulp,potato-pulp,and banana-pulp were used as natural additives for investigating their effects on the growth of Oncidium Sharry Baby plantlets.The results revealed that apple-pulp had a promoting effect on height and rooting number of plantlets,and contributed to the growth of pseudo bulbs.Potato-pulp had minor effect on height and rooting number and could help keeping the plantlets green.Banana-pulp played a significant role in promoting height of plantlets.The optimal medium for plantlets was:1/2MS+0.5 mg/L NAA+100 g/L apple-pulp+30 g/L sucrose+6 g/L agar.3 Transplantation and acclimatisation of the plantletsRooted plantlets were used as the materials to compare the efffects of transplantation methods and soil matrix contents on survival rates.The results revealed that the optimal scheme for transplantation is to open the lid after keeping the culture container for one week at room temperature in greenhouse.The plantlets were pricked out in the matrix with a ratio of4 grass charcoal soil:1 perlite for ninety days and then were transplanted to the matrix with a ratio of 3 bark:1 perlite matrix The survival was up to 100%.4 Cloning and bioinformatics analysis of EDS1,PAD4 and PR1 in Oncidium Sharry BabyThe RT-PCR combined with RACE method was used to clone the disease resistant genes from Oncidium sharry baby’Sweet Fragrance’.The complete cDNA sequence of PAD4 was 2 214 bp and the ORF was 1 894 bp,encoding 647 amino acids,including 65 bp of 5’ UTR,and 255 bp of 3’ UTR.We also obtained the ORF sequence of EDS1 and PR1,which was 1 863 bp and 474 bp,encoding 620 and 157 amino acids,respectively.Bioinformatics analysis showed that PAD4 and EDS1 protein belonged to alpha/beta hydrolase superfamily.It also showed that PR1 belonged to SRPBCC superfamily.They were not a secreted protein without signal peptide and highly conserved during the evolution of orchids.5 Determining the expression levels of EDS1,PAD4 and PR1 in Oncidium Sharry BabyThe healthy Oncidium was used as the materials to analyze the expression pattern of EDS1,PAD4 and PR1 genes by QPCR assay.It included the different plant tissues,pathogenic bacterium and the use of different hormone treatments.QPCR results showed that EDS1,PAD4 and PR1 expressed in all of the tissues and organs in Oncidium hybridum.For PAD4,the highest expression was found in pseudo-bulb,but for EDS1 and PR1 the highest expression was found in roots,suggesting that they all have tissue-specific expression.The results also showed that pathogens bacterium and different hormones could induce the expression of EDS1,PAD4 and PR1.The expression of PR1 in healthy plants was weak.After infection by pathogens,PR1 accumulated rapidly and the expression level increased greatly.It was speculated that EDS1,PAD4 and PR1 could play an important role in the disease resistance process of Oncidium.6 PR1 was transferred to Oncidium PLBsThe PR1 gene,as an important downstream gene in the SAR,can play an important role in plant disease resistance 35S:PR1 as over expressing vector.was constructed.Agrobacterium tumefaciens-medidted transformation was used to transfer PR1 gene into PLBs of Oncidium for transient expression.The results showed that the expression of PR1 in PLBs were highly increased.The PR1 gene was a marker gene for plant disease resistance.The success of the transient expression of PR1 in PLBs of Oncidium has paved the way for applying genetic engineering for the Oncidium disease resistant breeding programme. |
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