The Preliminary Study Of Callus Induction And Genetic Transformation System Of Tea Plant

China is the origin of tea with abundant planting resources.At present,there are some problems in traditional breeding of tea trees,such as short flower period and high heterozygosity of genes,which makes it difficult to get high quality varieties and low popularization rate.Therefore,the utilization of genetic transformation technology is of great practical significance to tea breeding.In this study,the effects of different hormone ratios on callus induction of old leaves,tender leaves and stem segments of Anji white tea,Longjing 43tea and Fuding Dabai tea were studied,the suitable ratio of explants and hormones for callus induction was selected.Then,the contents of tea polyphenols,amino acids and caffeine in the explants and the activities of PPO,SOD,PAL,CAT and POD were determined.The explants suitable for tissue culture were further screened,and the genetic transformation system was established for preliminary discussion.The main results were as follows:1.In this experiment,the old leaves,tender leaves and stem segments of Anji white tea,Longjing 43 and Fuding Dabai tea were selected as the research materials.The callus was induced by adding 6-BA,2,4-D and NAA in MS basic medium to induce callus of Anji white tea,Longjing 43 and Fuding Dabai.Each period of the first generation is 30 days,browning rate and survival time of explants for 30 days,The time of callus formation,browning rate and survival time of explants were counted,and the suitable ratio of hormone concentration for callus induction and explants for tissue culture were selected.The results showed that the callus of Fuding Dabai was obtained in the medium of hormone ratio MS1,MS5 and MS7.The callus was successfully obtained in only 8 days under the induction of MS7:0.9mg/L 2,4-D+0.3mg/L NAA+0.3mg/L 6-BA,and the browning rate was almost 0 and the longest survival time was30 days.The optimum ratio of hormone for callus induction of Anji white tea was MS2:0.3mg/L2,4-D+0.6mg/L NAA+0.3mg/L 6-BA,browning rate was 22%and survival time was 26days.The best hormone ratio for Longjing 43 callus induction was MS6:0.6mg/L+2,4-d+0.9mg/L NAA+0.3mg/L 6-BA,the browning rate was 25%and the survival time was 30days.Among them,the browning rate of the tender leaves of Fuding Dabai was the lowest,the survival time was the longest,and the callus appeared the earliest.2.In order to ensure the accuracy of the experiment,the contents of tea polyphenols,amino acids and caffeine,and the enzyme activities of PPO,SOD,PAL,CAT and POD in the experimental materials were determined.The results showed that Fuding Dabai tender leaves had the characteristics of the least phenolic substances,high content of amino acids,the strongest antioxidant capacity and low activity of phenoloxidase.In tissue culture,browning rate was low,survival time was long and callus appeared earlier.Therefore,it is considered that Fuding Dabai tender leaf is a suitable explant for tissue culture.3.Tea plant CsACO1 gene was selected as a candidate gene to construct CsACO1-PBI121overexpression vector,and agrobacterium tumefaciens was used to infect the callus of Fuding Dabai leaves.By observing the pollution and browning rate of the infected callus,the optimum conditions for genetic transformation of tea callus were obtained as follows:dark culture for 2days,OD₆₀₀ concentration is 0.8,infection time for 15 minutes,spreading on the medium containing 100 mg/L kanamycin sodium salt.After 30 days of culture at 24℃under constant temperature and light,no pollution and browning occurred.PCR was used to identify the infected callus when it had obvious growth signs.According to the identification results,the transgenic callus was obtained.