Establishment Of Composite Plants And UV-B Elicitation Of Flavour Metabolites In Tea Plant

Tea?Camellia?is a commercially important plantation crop whose diversely processed products are consumed across the world as a nonalcoholic beverage.Besides its unique flavor,tea beverage is also considered a health drink due to its nutritional and rich medicinal components.In recent years,use of biotechnology techniques in improving crop production and crop quality?concentrations of minerals,nutrients,and specialized metabolites?has been on the rise.DNA delivery through Agrobacterium-mediated transformation has been tremendously used for the production of transgenic plants to improve the agricultural and nutritional traits.However,genetic transformation of tea plants remains a challenge up to date.One of the problems for tea genetic transformation through tissue cultural regeneration is oxidative browning and necrosis of tea explants,which is often associated with the exuded phenolic compounds and microbial contamination from the explants.In this study,different antibiotics and 2-aminoindane-2-phosphonic acid?AIP?,a known inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase?PAL?,were used to arrest necrosis and browning of tea explant.Different concentrations of these compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron.Our data revealed the effectiveness of 2?M of AIP in arresting callus browning,significantly reducing EGC abundance and enhancing callus induction and growth.Moreover,elimination of the surface and endophytic microbes associated with explants of C.var.sinensis was effective with the use of 150 mg/L of timentin and 30 mg/L gentamycin.Our study indicated that the inhibition of PAL using AIP combined with the two tested antibiotics could advance research in tea genetic manipulation and tissue culture.Among the different of Agrobacterium rhizogenes strains tested?ATCC15834,LBA9204,A4 and their engineered strains containing p BI121 plasmid?,we observed that A.rhizogenes A4 and A4 harboring p BI121 to be most effective in hairy root induction compared to other strains tested using tobacco as a model plant.A.rhizogenes A4 harboring p BI121 was used further to generate tea composite plants due to high infection rate of 87%in tobacco.We established a fast,simple and high efficient protocol for generation of composite tea plants with 88.3%of infected plants producing hairy roots.Molecular assay of roots from antibiotic selected plants showed amplification of rol B C and gus confirming the integration of A.rhizogenes T-DNA region into the root system.From our data,it was clear that hairy root generation in tea can be achieved using A.rhizogenes.With this method,composite tea plants containing wild-type shoots with transgenic roots can be developed for gene functional characterization as well as specialized metabolites production and root-shoot interaction research in plants.To understand the induction effect of UV-B exposure on tea leaf flavor compounds,we investigated the impact of UV-B exposure(at 35?W cm^-2 for 0,0.5,2,and 8 hr)on tea transcriptional and metabolic alterations in tea shoots,specifically related to the production of tea terpenoid volatiles and polyphenols.Out of 34,737unigenes,a total of 18,081 differentially expressed genes?DEGs?due to UV-B treatments were identified.In the UVR8-signal transduction pathway,The UV-B receptor gene,UVR8 was suppressed at both short and long exposure of UV-B with genes downstream differentially expressed.Specialized metabolite profiling revealed the correlated alterations in the abundances of catechins and some volatile terpenoids in all the treatments with significant accumulation of specialized metabolites at 0.5 hr treatment.A significant increase in specialized metabolites at 0.5 hr treatment and no significant alteration observed at longer UVB treatment suggested that shorter exposure to UV-B led to different display in gene expression and accumulation of specialized metabolites in tea shoots in response to UV-B stress.Taken together,our results indicated that the UV-B treatment applied in this study differentially altered the UVR8-signal transduction,flavonoid and terpenoid pathways at transcriptional and metabolic levels in tea plants.Our results show strong potential for UV-B application in flavor improvement in tea at the industrial level.