| Castanopsis hystrix Miq is an evergreen tree which belongs to Fagaceae Castanopsis, and it is also one of valuable local timer tree species in south China with many characteristics such as high-efficiency, multipurpose, fast growing and so on. Tissue culture technology is an important way in terms of rapid propagation and asexual promotion of superior individuals of Castanopsis hystrix Miq, but serious problem of browning has led to degeneration and death of the tissues. In this experiment, treatments like different sterilization time, drawing month, tenderness, genotype, light and temperature, and different anti-browning agents were applied to explore the browning-control methods in initial culture; Further, the effects of kinds and concentration of cytokinine, auxin and anti-browning agent were studied on browning-inhibition in proliferation culture.Tissue culture regeneration system was successfully established. The main conclusions were as follows:1.Effects of explant collection and treatment, and measures taken in buds induction on browning-control.(1) Surface disinfection of explants with alcohol increased the degree of browning of explants.(2) Drawing month of explants had no significant effect on the browning rate, while the induction rate, pollution rate were significantly affected. October was optimum with a frequence of 48.9%.(3) Tenderness of explants had a significant effect on inducing and browning rate. Browning rate increased with the extent of lignifications of branches strengthening. Synthesizing the growth conditions of axillary buds induced, the3th-5th stem segments with node in the middle part of semi-lignified branches were best for growth of shoots.(4) Genotypes of explants significantly affected inducing and browning rate, as well. HZ3 out of the six tested genotype achieved lower, browning rate(48.9%), and higher inducing rate(62.2%) at the same time. Axillary bud germinated fast and grew well.(5) Dark and cold treatment had no significant effect on the rate of browning.(6) As to the anti-browning agent, PVP(Polyvinyl pyrrolidone) and VC(Vitamin C) was better. In medium supplemented with VC(80 mg.l-1), axillary buds were strong and sprouted fast with inducing and browning rates of 47.8% and 33.3%, respectively; in medium containing PVP(1.0g.l-1), browning rate sharply decreased from 43.3% to 12.2%, while, the inducing rate was 56.7% and axillary bud grew well.Simply put, the optimal factor and level arrangement for controlling browning was that explants were firstly picked off from suitable tender branches in October, subsequently surface sterilized without ethanol pretreatment, eventually put in to modified-MS medium containing 1.0 mg.l-16-benzyladenine(6-BA), 0.05 mg.l-1 1-napthyl acetic acid(NAA) and 1.0 g.l-1 PVP or 80 mg.l-1 VC.2. Anti-browning measures in proliferation culture.(1) Kinds and concentrations of cytokinin had a significant effect on browning rate and multiplication of buds, the rate of 6-BA(55.6%) was lower than ZT, KT; medium containing1.0 mg.l-1 6-BA obtained higher multiplication rate of 2.14 than ZT, KT.(2) Kinds and concentrations of auxin had no significant effect on the rate of browning, however, significantly affected multiplication of shoots, NAA was better than 3-indole butyric acid(IBA), medium supplement with 0.05 mg.l-1 NAA achieved multiplication factor of 2.22.(3) Different concentrations of anti-browning agents had a very significant impaction the browning rate. Low concentration of Na2S2O3(0.1 g.l-1,0.5g.l-1), AC, PVP, VC could all reduce the degree of browning. Different anti-browning agents affected the proliferation greatly, and the proliferation effect of Na2S2O3 and AC was poor, with lower multiplication coefficient than control group(1.82) and buds grew badly; the whole proliferation effect of PVP was ordinary, PV(0.5 g.l-1and 1.0 g.l-1) was suitable with multiplication coefficient 1.88 and 1.92, respectively; Buds grew well; the effect of Vc on proliferation was the best, shoots grew obviously well. Vc(80 mg.l-1) achieved highest multiplication coefficient(2.27) and a browning rate of 31.1%, and plant lets were strong and flourishing.Modified MS containing 1.0 mg.l-16-BA, 0.05 mg.l-1 NAA and 80 mg.l-1VC was found to be better proliferation medium of superior individuals of Castanopsis hystrix. |
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