Studies On Culture Of Embryogenic Suspension Cells Of Sisal H.11648 And Transformation Of Hevein Gene

Sisal is the most important tropical fiber crop in the world,with wide application value and development prospects.However,due to the fact that the main varieties of sisal in China are single,they are vulnerable to various diseases,resulting in its fibre yield reduction and serious economic losses.Therefore,it is urgent to cultivate sisal resistant varieties.Since sisal is a perennial plant it usually grows for more than 10 years before flowering.Therefore,the conventional breeding cycle is long and the efficiency is low,so that it has not bred better varieties than the H.11648 for more than 60 years.The rapid development of cell biology provides a new technical approach for sisal cell engineering breeding.Through the operation of sisal cells,cell clusters or protoplasts,a suspension cell culture system can be established,and then through induction screening or transgenic.Therefore,screening resistant sisal lines,creating new disease-resistant germplasm.and finally cultivating resistant varieties can greatly shorten the breeding cycle and improve breeding efficiency.In this study,H.11648 hybrid sisal sterile seedlings were used to establish sisal embryogenic suspension cell culture and regeneration system.On this basis,the suspension cells or cell clusters of sisal are used as receptors for genetic transformation.The expression vector(pCAMBIA3301-pUC57-Hevein)was introduced into Agrobacterium EH A 105 by "freeze-thaw method",exploring various factors affecting Agrobacterium infecting sisal suspension cells,screening genetic transformation conditions provides a new way to cultivate resistant varieties of sisal.The main findings are as follows:1.The optimization of experimental conditions for sisal callus induction.MS was determined to be the basic medium by using the stems.leaves,and shoot tips(growth points)of sisal sterile seedlings as explants.It was found from this ecperiment that young leaves were the most suitable explant material for sisal callus induction.When sisal leaves were inoculated on MS+2.0 mg/L 2,4-D+1.0 mg/L 6-BA medium for induction,after 1?2 times of subculture,a large number of homogeneous embryogenic callus were obtained.2.Establishment of sisal suspension cell system.Cell culture was carried out by inoculating embryogenic callus of sisal into medium(MS+2,4-D 1.5 mg/L+6-BA 4.0 mg/L+350 mg/L casein hydrolysis.30 g/L sucrose,pH 5.8).It was found that the growth of sisal cells was consistent with the "S" type,and the optimal subculture time was 7 days.In the early growth stage,the pH value of sisal suspension cells decreased.In the logarithmic growth stage,the pH value increased slowly,and tended to be flat in the later stage.3.Suspension cell regenerationtypes:By studying three methods,it was found that liquid suspension culture was the most suitable regeneration method for sisal suspension cells.The planting rate of regeneration by this culture method was 0.193%,the regeneration period was about 28 days,and the number of differentiation was relatively large.The cells on the plate could be treated with low temperature for 2 h,which could significantly increase the regeneration rate of the suspended cells.The optimal differentiation formulation to determine the regenerative callus was MS+NAA 0.5 mg/L+6-BA 2.0 mg/L.The regenerated seedlings were rooted on sisal rooting medium(1/2 MS+0.1 IAA+30 g/L sucrose+6 g/L carrageenan),after 21 days or so of culture,removed and washed the seedlings and transplanted them into the 1:1 medium of coconut bran and sand for seedling refining.4.To optimize the transformation factors of embryogenic suspension cells of sisal.The results showed that the optimal bacterial concentration of Agrobacterium used for infestation was OD600=0.5?0.6,the infiltration time was 30 min,the co-culture was 3 d,and the concentration of acetosyringone(AS)was 200 ?mol/L.5.Screening of resistant callus and acquisition of transgenic plants.Through the combination of antibacterial ability test and impact on callus growth,this experiment decided to use 300 mg/L Car for screening.Started the culture in a medium containing 5 mg/L PPT for one week,then transfered to a medium containing 10 mg/L PPT for one week,and finally increaseed the PPT to 15 mg/L.Then the transformed seedlings were verified by PCR and RT-PCR.