| The enzymes involved in the biosynthetic pathways of tea polyphenols, such ascatechin, flavonoid and proanthocyanidin, have been basically clear, but the functions ofthe structural gene and the regulator gene have not been proved effectively. The maindifficulty is to establish a stable transgenic system of tea plant, and the cycle of currentregeneration system is long with low efficiency. Thus, it is still a very urgent task that howto improve the efficiency of regeneration system and achieve an instantaneous genetictransformation system. In addition, transgenic system of model plant could be used toverify the functions of related genes in tea. The main results obtained were as follows:1. A regeneration system of tea plantIn this paper, the regeneration system of callus in tea including five steps (callusinduction, embryogenic callus proliferation, adventitious bud induction, adventitiousproliferation and rooting in vitro) was established. It consists of two proliferation processes,callus proliferation and adventitious bud proliferation.Main steps are as follows: seeds of tea plant (Camellia sinensis) in early Septemberwere picked as explants, then, they were disinfected and cultured in vitro on B5mediumsupplemented with0.5mg/L2,4-D and0.1mg/L KT. The callus was induced in two weeksand subcultured for four years steadily. The callus was subcultured on MS mediumsupplemented with0.1mg/L IBA and2.0mg/L6-BA. The shoot induction rate was65.71%after being cultured for four weeks. The shoots were cultured on MS mediumsupplemented with0.1mg/L IBA and0.01mg/L TDZ. The large number of cluster budswas induced in four weeks, which were then cultivated to strong plantlets on MS mediumsupplemented with0.2mg/L IBA. The seedlings with height above5cm were selected andtransplanted in soil, and the survival rate of it was above80.8%. The whole culture periodwas about twenty-five weeks.2. The study of transient expression system in tea callus based on instantaneousgenetic transformation systemFour conditions concerning the infection of Agrobacterium tumefaciens EHA105including the OD value of Agrobacterium, infection time, co-culture time and the dosageof acetosyringone were optimized by SPSS17.0software. The results indicated with callusfrom Nong kang zao as material, GUS as reporter gene, when the OD value ofAgrobacterium is0.6, the infection time is30minutes, the dosage of AS is9.8mg/L andthe time of co-culture is5days, the infection rate can reach34.15%. The infection rate ofcallus from Long jing43#is obviously higher than that from Nong kang zao. The analysis of tissue localization indicated that the exogenous gene can be expressed in tea plant, butthe expression rate decreased with the passage of time.3. Identifying functions of related genes in biosynthetic pathway of flavonoid in teaplant by tobacco genetic transformation systemIn this paper, the identification of functions of related genes in biosynthetic pathwayof flavonoid in tea plant was studied in the tobacco genetic transformation system based onherbicide screening. It was found that the color change of transgenic tobacco flower caneffectively and easily identify the functions of related genes in flavonoid biosyntheticpathway in tea plant. But it should be noticed that the anthocyanin content of wild tobaccowas relatively low before blooming, the highest in the very day of full blooming, then,decreased gradually in the following days. Temperature would affect the synthesis ofanthocyanin, the higher the temperature, the lighter the flower color.Agrobacterium EHA105with target gene CsANR was used to infect tobacco G28.The transgenic tobacco with ANR from tea plant was obtained after bacteria free, screeningof glufosinate-ammonium herbicide and regeneration of tobacco. It was found that theflower color of tobacco with CsANR transgenic gene was obviously lighter than that ofwild tobacco. The result of detection and analysis showed that the anthocyanin content oftobacco with CsANR transgenic gene reduced, whereas the content of procyanidineincreased distinctly stained by DMACA.4. Identifying functions of related genes in biosynthetic pathway of flavonoid in teaplant by Arabidopsis thaliana transformation systemThe cultivation pattern of Arabidopsis thaliana was studied in this paper. It is foundthat the cultivation of Arabidopsis thaliana was affected by environmental factors, such astemperature, humidity, light intensity and ventilation.Agrobacterium C58C1with target gene ANR was chosen to infect Arabidopsis pap1mutant. The phenotype of T1generation was observed after screening T0generation seedby medium with glufosinate-ammonium. It was found that the color of leaves and stems ofpap1mutant was red compared with wild type. However, the color of purple decreasedobviously in leaves and stems of pap1infected by Agrobacterium with CsANR at thegrowth stage of two leaves. Meanwhile, the content of anthocyanins reduced, but theincrease of the procyanidine content detected by DMACA was not found in thisexperiment. |
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