Study On Browning Mechanism Of Explants During Tissue Culture Of Juglans Mandshurica

Juglans mandshurica is one of the three valuable hardwood species in northeast China with high economic and medicinal value.At present,seedling propagation is the main method in forestry production,but the seeds of J.mandshurica have the characteristics of deep dormancy,which leads to its low germination rate and can’t meet the production requirements.Therefore,a large number of tissue culture seedlings with consistent growth traits can be obtained in a short time by tissue culture and rapid propagation in vitro.However,in the process of tissue culture of J.mandshurica,the explants are prone to browning,which leads to the decline of physiological functions of explants,which has become a bottleneck restricting the rapid propagation of J.mandshurica in vitro to obtain regenerated plants.Therefore,in this study,J.mandshurica stem segments with axillary buds or terminal buds were used as explants,and the explants were browned in the process of in vitro culture.The dynamic changes of enzymes in physiological metabolism level of stem segments before and after browning and the changes of cell morphology and structure of explants were discussed from physiological and cytological perspectives.At the same time,the explants with different browning degrees were selected for transcriptome sequencing,and the genes related to browning of explants were screened,and then cloned and expressed.The research results laid a solid foundation for the establishment of in vitro rapid propagation and plant regeneration system of J.mandshurica.The main results are as follows:1.When budded stems of J.mandshurica were cultured in vitro,0.1%Hg Cl₂had the best disinfection effect for 10 min,and Na₂S₂O₃（1.0,2.0,3.0,4.0,5.0）mg/L and2%PVP（1.0,2.0,3.0,4.0）mg/L were added to the primary culture medium.After and VC（1.0,2.0,3.0,4.0,5.0）mg/L anti-browning agent,the browning degree of explants decreased significantly,and 3 mg/L AC and 5 mg/LNa₂S₂O₃were the best for J.mandshurica in vitro culture.It was found that the activities of polyphenol oxidase（PPO）,peroxidase（POD）,malondialdehyde（MDA）and superoxide dismutase（SOD）in browning explants were significantly greater than those in non browning explants,and PPO activities showed a trend of increasing first because of the increase of substrate concentration and contact opportunities with substrates caused by mechanical damage.With the gradual consumption of substrates and the increase of products,PPO activities began to show a downward trend,which indicated that PPO was closely related to browning of explants.The protective enzymes POD and SOD showed a trend of increasing at first and then decreasing.The activities of POD and SOD increased because of adversity,and the balance of active oxygen was destroyed with the extension of culture time,which inhibited the activities of POD and SOD and showed a downward trend.MDA always showed an upward trend,indicating that membrane peroxidation increased,the regional distribution of phenols and enzymes was destroyed,and the browning of explants was aggravated.Freehand sections showed that after the browning of cells,the cell structure was destroyed and a lot of brown substances were deposited in the cells.2.Through transcriptome sequencing of J.mandshurica stem segments with different browning degrees,16398 non-redundant unigene with an average length of1439.30bp were obtained.All unigenes were compared to the protein database by Blastx,and it was noted that the number of unigenes in Nr database was the largest（45002,51.83%）,followed by egg NOG database（43270,49.83%）,KEGG database has the least number of comments（21716,25.01%）;There are 18,382,6,942 and15452 differentially expressed genes in CKvs M,CKvs T and Tvs M,respectively.The up-regulated genes in CKvs M and Tvs M are much higher than down-regulated genes.GO functional classification notes CK and M groups,mainly with 3639 functions,enriched to 124 pathway in KEGG;A total of 3723 functional annotations were obtained in CK and T groups,and 123 pathways were enriched in KEGG while 3,398functional annotations were obtained in T and M groups,and 124 pathways were enriched in Kegg.Among them,the synthesis of phenylpropane and anthocyanin played a key role in browning of J.mandshurica explants,and 37 genes related to browning were obtained.Fifteen key genes related to browning（PAL,PAL1,CCR,HCT,POD1,POD4,POD12,POD42,POD51,CAD1,CAD9,BGLU42,UGT9,UGT1）were randomly selected from differentially expressed genes,and the q RT-PCR results showed that their expression levels were similar to those of transcriptome results.3.The pathway of benzene synthesis plays an important role in the browning process of J.mandshurica tissue culture.Using transcriptome data,the important genes of benzene propane synthesis,HCT and POD4,were screened out and successfully cloned,named J.mandshurica HCT and Jm POD4,with lengths of 873 bp and 417 bp,respectively,which were closely related to Julans regia through phylogenetic analysis.The expression of Jm HCT in J.mandshurica tissues with different browning degrees was high on the 9th day.When the explants were damaged mechanically,the substrate content increased,so the expression of Jm HCT increased first,and the substrate phenols were continuously consumed,so it showed a downward trend later.The expression of Jm POD4 was the highest on the 6th day,which was consistent with the results of enzyme activity determination.The increase of Jm POD4 in the early stage was caused by stress caused by mechanical damage.With the accumulation of reactive oxygen species and products,the activity of Jm POD4 was inhibited,so the expression of Jm POD4 increased first and then decreased.To sum up,the external morphology,internal cell structure,physiology and biochemistry and internal gene expression of J.mandshurica responded to browning stress.With the deepening of browning degree,the external color of explants gradually changed from green to dark brown;The internal enzymes PPO,POD and SOD increased first and then decreased,while MDA always increased,and the cell structure was destroyed.The genes were differentially enriched to the synthesis of phenylpropane and anthocyanin in two pathways related to browning,and a total of37 genes related to browning were obtained.Adding anti-browning agent to the culture medium is an effective way to reduce browning.Anti-browning agent can effectively reduce PPO activity and increase oxygen dissolution,thus reducing browning.