Studies On Genetic Transformation And Effects Of UV-B On Aroma Related Genes Expression In Tea Plant (Camellia Sinensis.)

Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the important crops for beverage, which belongs to the family Theacea. There are some characteristics that limit the genetic improvement of tea, such as long life cycle, self-incompatibility, high inbreeding depression, etc. The genetic transformation in Camellia sinensis was developed in recent years, however with limited successes because of difficult plant regeneration and low transformation efficiency. The advance in plant regeneration and genetic transformation in Camellia sinensis were reviewed and the author suggested that Agrobacterium rhizogenes mediated-transformation maybe a possible successful method in Camellia sinensis.L-theanine is a unique amino acid found exclusively in tea plant and certain species of genus Camellia, such as C. japonica and C sasanqua, constituting 1- 2% of the dry weight of tea leaves. It has recently received much attention due to its remarkable health benefits, such as promoting relaxation, reducing stress and anxiety, calming effect, regulating alpha brain activity, improving memory and learning ability and suppression of high blood pressure. Many attempts have been made to produce L-theanine from tea leaf commercially. However, most of them have remained inefficient, because of low yields, high cost, or highly complicated processes. L-theanine is biochemically synthesized in roots in tea plant, and therefore, the tea roots might be a good source for producing L-theanine. However, it is not feasible to get the roots from the tea plants growing in the fields because it will injure the plants. Hairy roots induced by A. rhizogenes may meet the demand of extracting L-theanine. Genetically transformed hairy roots obtained by infection of plant with A. rhizogenes offers a promising system for in vitro theanine production.Caffeine (1,3,7-trimethylxanthine), a purine alkaloid, is a key component of many popular drinks, most notably tea and coffee. A growing belief that the ingestion of caffeine can have adverse effects on health has resulted in an increased demand for decaffeinated beverages. Unpleasant short-term side effects from caffeine include palpitations, gastrointestinal disturbances, and anxiety. RNA interference (RNAi) is a double-stranded RNA (dsRNA)-induced gene-silencing phenomenon that is conserved among various organisms, including animals and plants. Gene silencing, not only by PTGS but also by the direct introduction of dsRNA, is known to spread systemically. This raises the possibility that when RNAi is induced in a particular tissue it might also suppress the target gene in other tissues where down-regulation is not desired. The author suggested that if hpRNA of TCS gene was integrated in hairy root, the interference effect would spread to the leave and shoots and inhibit TCS gene expression and caffeine biosynthesis.Tea aroma is one of the most important factors influencing the character and quality of made tea, especially in oolong and black tea. Fresh tea leaves are virtually odorless or slightly smell of green note, and most of floral aroma compounds are produced by endogenous enzymes during tea manufacturing process of withering, rolling, and fermentation. Among more than 500 identified compounds of tea aroma volatiles are known to contribute the floral aroma of oolong tea and black tea. It is well-known that most of the alcoholic tea aroma compounds are mainly present as glycosides in fresh tea leaves and are released by endogenous glycosidases during the manufacturing process of various tea products, especially of semi-fermented tea (oolong tea) and fermented tea (black tea). Many aroma glycosidic precursors have been isolated from tea plant; most of them were 6-primeverosides. Recently, there have been numerous reports on the effects of ultraviolet-B radiation (UV-B) on secondary metabolic products, but no report on the effects of UV-B on tea aroma formation is available on current literature.Genetic transformation mediated by A. rhizogenes and effects of UV-B on volatile release were investigated and the results were as follows:(1) A. rhizogenes-mediated high frequency hairy root induction and genetic transformation in tea plantAn efficient protocol for the establishment of transgenic hariy root infected by A. rhizogenes is reported using two wild type strains of A. rhizogenes, three different co-cultivation media and six different explants. The highest induction frequency was obtained from stem segments of aseptic plantlets infected by A. rhizogenes and co-cultivated on YMB solid media with 100 mmol/L AS for 2 d. The hairy roots grew rapidly on LG₀ medium without phytohormone and produced a plenty of branches and root hairs. PCR confirmed that rolA, rolB and rolC have inserted into the genomic DNA of hairy root induced by wild type A. rhizogenes. Hairy roots were also induced from explants of tea plant infected by A. rhizogenes 15834 carrying pCAMBIA2301 binary vector harboring Bt gene, transgenic root were confirmed by PCR and GUS histochemical staining that the Bt exogenous gene was inserted and expressed in the transformed hairy root.(2) Concentrations of theanine and catechins in hairy rootsHairy roots induced by A. rhizogenes wild strain 15834 grow well on LG₀ medium and half MS medium containing 1 mg/L IBA. However, content of theanine in the transformed hairy root (23.12 mg/g fresh weight) was higher than the non-transformed root (18.77 mg/g fresh weight). High contents of aspartic acid, glutamic acid, glutamine, histidine and arginine showed the same trend. Medium constitute also affected the contents of amino acid in hairy root clone, the content of theanine were reduced in two hairy root clones when they were cultivated on half MS with 1 mg/L IBA. Low content of simple catechins, catechin (C) and epicatechin (EC), were also detected in the hairy roots.(3) Construction of tea caffeine synthase gene RNAi vectorTea Caffeine synthase (TCS) is one of the key enzymes involved in caffieine biosynthsis in tea plant, which catalyses conversions of 7-methylxanthine to theobromine and theobromine to caffeine. Inhibition of TCS gene expression will lead to breeding low caffeine tea cultivars. The cDNA fragments of TCS gene were amplified by RT-PCR, and ligated into T-vector and RNAi vector pFGC5941 in reverse direction after double digestion with two pairs of restriction endonucleases. The insertion of two fragments, namely pFGC5941-TCS02 and pFGC5941-TCS03, into the RNAi vector were confirmed by PCR and DNA sequencing.(4) Effects of UV-B on aroma related genes expression in Camellia sinensisThe effects of ultraviolet-B radiation on tea aroma volatiles formation and the expression of two important genes involved volatile release were investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and gas chromatography- mass spectrometry (GC-MS). The results showed that the expressions ofβ-primeverosidase andβ-glucosidase genes were increased by UV-B treatment for 5 min in shoots, which in turn enhanced tea aroma formation and the contents of the main volatile compounds especially the alcohols compounds as phenylethyl alcohol and benzyl alcohol. The results suggested that supplement of short time treatment of UV-B is a simple and efficient method for improving tea aroma quality in manufacturing process.