Study On Mechanism Of Tissue Culture And Germplasm Conservation In Cephalotaxus Fortunei

Cephalataxus fortunei Hook.f. was an ancient relict plant, which was an evergreen and species endemic to China, then which could be used for timber, industrial, ornamental, anticancer. Due to natural and man-made and so on reasons,natural resources was lacked in Cephalataxus fortunei, how to solve the Cephalataxus fortunei's resources became the key to its development and utilization. The use of plant tissue culture seedlings which established Cephalataxus fortunei rapid propagation technique was a very promising solution. In this experiment, it's investigated that tissue culture on bud induction mechanism,subculture mechanism, rooting, callus induction mechanism in Cephalataxus fortunei for the study of hormones and shoot induction rate, subculture coefficient, rooting rate and callus induction. As well as it's researched that the subculture of algebra's effect on the proliferation rate. In addition to combine with micro-cuttings and tissue culture for simplifing procedures for tissue culture and reducing the cost of tissue culture. This study based on the work of previous studies then to improve and perfect.Aim was to establish vitro culture systemthen of Cephalataxus fortunei, and to provide technical support for tissue factory of Cephalataxus fortunei. The major findings were as follows:(1) Best disinfectant combination of vaccination in Cephalataxus fortunei was 75% alcohol disinfection of lmin,0.1% HgCl2 disinfection of 9min,2% NaClO disinfection of 5min, in that time which reached the lowest point of contamination, the lowest of browning rate, the highest of survival rate. The effect of compound disinfectant was more effective than single disinfectant. To disinfect stem immediately after collection was the best. Pretreatment did not improve the disinfection effect of stem. Cephalataxus fortunei stem of indoor cultivation was disinfected easier. New stem of germination of seedlings was disinfected easier, and survival rate was high. In each season, stem of spring was most likely disinfected. Pollution of stem collected in sun was lower than the rate of stem pollution in rain.(2) In the induction time, the induction rate was highest with the medium WPM+IBA2.0mg·L-1+6-BA3.0mg·L-1+CM100mg·L-1 induced, buds were also the largest number, maximum induction rate was 4.22. The bud growed best with SH+IBA0.5mg·L-1+6-BA3.0mg·L-1, the induction rate was up to 3.85. The most favorable combination of various factors was WPM+IBA0.5 mg·L-1+6- BA3.0 mg·L-1+CM100mg·L-1. The relationship of various components in between primary and secondary factors was the basic medium>IBA>6-BA>natural additives. WPM and the DCR were suitable for basic medium of tissue culture of Cephalataxus fortunei. The effect to add organic compounds on the induction was not obvious.To add the IBA and 6-BA was conducive to concentration of endogenous hormones improvement of IAA and ZT, but endogenous hormones ABA and GA3 decreased. ABA and GA3 slightly anti-correlated. the buds grown slower when GA3 was high. (3) In the proliferation of bud, these two combinations WPM+6-BA3.0mg·L-1+ NAA0.3mg·L-1, WPM+6-BA2.0mg·L-1+IBA 0.3mg·L-1 caused proliferation rate which was the highest, reaching 4.2 and 4.3. Different culture mediums on proliferation of explants were significantly affected. To add the NAA,IBA and 6-BA was conducive to improvement of endogenous hormones IAA and ZT, between ABA and GA3 had slightly antagonistic effect. When GA3 was higher, shoot growth was slower. Preliminary determinating among ZT/IAA=4.4-4.6 was better. With the increase of subculture number, the multiplication coefficient was following. When the multiplication factor increases to 6, the multiplication coefficient generation was maximum, even up to 12.After generations of 6, as the generation increased, the multiplication coefficient decreased.(4) In the seedlings culture, WPM+IBA0.1mg·L-1+GA30.1mg·L-1 was the best combination. To initially determinate when ZT/IAA is in value of 4,the plant grew better. With GA3 was higher, the plant significantly grew well.(5) In the root culture, the impact on the rooting rate of primary and secondary relationships: IBA>sucrose>NAA>basic medium. With 1/2WPM+IBA1.0mg·L-1+NAA0.1mg·L-1+sugarl0g·L-the induced rate was 73.3%. With the use of rooting powder, the 1/2WPM+ABT15.0mg·L-1 made rooting rate of 80%, and the plants were growing very well. In rooting process, ZT/IAA produced significant changed, the values were less than 1. To initially determinate that the value of 0.25-0.45 of ZT/IAA was most appropriate. ABA content increased and GA3 content decreased at this time.(6) In the micro-cutting, after the treatment the cutting of Cephalataxus fortunei begun to take root in the first 30d, compared to other methods of rooting of Cephalataxus fortunei which was nearly a month ahead of schedule. Different solution treatments on the roots of Cephalataxus fortunei played a decisive role. Experiment showed that in condition of choosing current year without terminal bud, WPM+ABT15mg·L-1 100-fold diluted as culture medium, fine sand as the cutting medium, the rooting rate could achieve the best combination, the rooting rate could be achieve 85%.(7) In callus tissue culture, combination of MS+NAA 3.0 mg·L-1+6-BA0.1 mg·L-1 was the best, at that time start time of callus was the shortest, averaged 8.3d, differentiation rate was highest, reaching 93.3%. Cultivated callus grew faster, callus had been increased significantly. Compared with training before, the endogenous hormone content of ZT of callus increased. The addition of NAA,2,4-D,6-BA increased endogenous hormones ZT and IAA, but also led to decline of GA3. Callus subculture took the most appropriate subculture number of 4-5. The callus growth showed the phenomenon of decline when the subculture number was more than 5.(8) In the cryopreservation of callus, the most favorable combination of factors was in the sugar concentration of 1.2mol·L-1 as the conditions of pre-culture, with 15% dimethyl sulfoxide, sorbitol of 0.1mol·L-1,5% polyethylene glycol,10% sucrose,20% glycerol as antifreeze, and then slow freezing and slower solution program. Sorbitol in antifreeze ingredient on cryopreservation of Callus of Cephalataxus fortunei had a significant effect. Sorbitol of 0.1mol·L-1 as a component of antifreeze on callus cryopreservation of Cephalataxus fortunei was better.