| Apple is the largest cultivated fruit tree in China and occupies an important position in the development of agricultural economy.But at present,our country apple variety type is onefold.In order to further increase the economic income of Chinese fruit farmers and improve the competitiveness of Chinese apple industry in the international market,it is necessary to vigorously promote and produce new apple varieties with high quality.The method of bud mutation selection plays a very high role in the selection of new apple varieties,and Fuji varieties are the main force of bud selection in China.The material L7 used in this experiment is produced by Fuji bud mutation.It has the advantages of easy flower formation,high fruit setting rate,large and correct fruit,fast coloring,etc.,but L7 carries rust virus in the field.In order to promote the application of L7 in production,this experiment aims to establish L7 rapid propagation system and remove rust fruit virus to obtain L7 non-toxic seedlings as soon as possible.However,in the process of L7 plant tissue culture,it was found that it was difficult for L7 plantlets to enter into the normal growth state in the early stage of L7 plant tissue culture,which was characterized by poor growth state such as no increase of plants height,excessive growth of large leaf width,large callus and vitrification.It took a long time for the plantlets to be transformed into normal plantlets.Therefore,the transcriptome analysis of L7 plantlets before and after the state transformation was carried out in this experiment.The main results are as follows:1.Selection of suitable disinfection methods.In this experiment,L7 explants were sterilized with 0.1%mercuric chloride for 10 minutes at different sampling time in January,April and June.The results showed that the most suitable month for L7 explants sampling was April.The L7 explants were disinfected with 0.1%mercuric chloride and 5%sodium hypochlorite and treated with 5,10 and 15 minutes of different disinfecting time.The results showed that when the disinfection time was 10 minutes,the two disinfectants had the best disinfection effect on L7 explants,but the disinfection effect of 0.1%mercuric chloride was slightly better than that of 5%sodium hypochlorite.Considering the harm of mercuric chloride to human body and environment,5%sodium hypochlorite was preferred to disinfect L7 explants.2.Selection of suitable subculture conditions.By comparing the subculture effects of 1/2MS,MS and C17 on L7 plantlets,the basic medium suitable for L7 plantlets growth was MS medium.L7 tissue culture seedlings were inoculated on MS medium supplemented with 6-BA of 0.5mg/L,1mg/L,1.5mg/L,2mg/L and NAA of 0.05mg/L,0.1mg/L,0.15mg/L and 0.2mg/L for subculture.the suitable hormone ratio for the subculture proliferation of L7 plantlets was 1.5mg/L 6-BA and 0.1mg/L NAA.In order to obtain a large number of L7 effective shoots for rooting,the inoculated L7 plantlets were pre-dark cultured for 5,10,15 and 20 days,20-25 days,20-30 days,20-35 days,25-30 days,25-35 days of late dark culture as well as the whole process of light training,dark training.the optimal dark culture time for L7 tissue cultured seedlings to be subculture is 15 days of early dark culture.3.Selection of suitable rooting conditions.In this experiment,1 1 2MS was used as the basic rooting medium,and 0.5mg/L,1mg/L,2mg/L,3mg/L IAA,0.05mg/l,0.1mg/L,0.2mg/L,0.3mg/L NAA and 0.12mg/L,0.3mg/L,0.6mg/L,1mg/L IBA were added to induce the adventitious roots of L7 plantlets.The results showed that the rooting effect of NAA was better than that of IAA and IBA,among which IBA was the worst,NAA was the most suitable hormone for rooting of L7 plantlets,and its optimal concentration was 0.2mg/L.In order to further improve the rooting effect of L7 tissue culture seedlings,dark culture and light culture were carried out for 2,4,6 and 12 days respectively,and the optimal dark culture time was 6 days.4.Heat treatment virus-free test.Under the condition of dark culture,the inoculated L7 plantlets were cultured for one week at room temperature,and then treated with variable temperature heat treatment for 25,30,35,40 and 45 days respectively.The results showed that the growth effect of L7 plantlets was the best after heat treatment for 30 days,and the suitable heat treatment time was 35 days by detecting the removal effect of L7 stem tip rust fruit virus.5.Study on virus-free of Exogenous Additives.The L7 plantlets were inoculated in the MS medium with different concentrations of ribavirin(0 mg/L,5 mg/L,10 mg/L,15 mg/L,20 mg/L)and melatonin(5 ?M,10 ?M,15 ?M,20?M),Under the condition of dark culture,the L7 plantlets were cultured at room temperature for one week,and then at variable temperature for 35 days.The results showed that the L7 plantlets with the concentration of melatonin of 25 ?M had the best growth condition and detoxification effect.6.Analysis of transcriptome data before and after the growth state transformation of L7 plantlets.Based on the Illumina sequencing platform,we sequenced the transcriptome of L7 tissue culture plantlets with poor growth state at the initial stage of tissue culture and L7 tissue culture plantlets transformed into normal growth state after 5-6 months of subculture.We found that there were 1189 differentially expressed genes before and after the growth state transformation of L7 tissue culture plantlets,of which 635 have abnormal expression levels higher than normal seedlings,and 554 have abnormal expression levels lower than normal seedlings.After GO functional analysis of these differential genes,it was found that there was no significant enrichment of the differential genes in the biological process.In the cell components,ribonucleoprotein complex and ribosome had the highest enrichment degree.133 differential genes were enriched equally,and 134 differential genes were enriched equally in the structural components and structural molecular activities of ribosome.After further analysis of the enrichment of KEGG,it was found that the most significant enrichment pathway was ribosome,which enriched 68 differential genes.The functional analysis of KEGG and GO showed that the differential expression genes before and after the transformation of growth state of L7 plantlet were all enriched in the pathway related to ribosome,which indicated that the transformation of growth state of L7 plantlet was closely related to ribosome. |
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