| In this paper, the tissue culture and clone selection of E.dunnii, a cold-resisting species, was systematically studied. The experiment materials was collected from E.dunnii seedlings. In the course of culturing the whole plant by bud induction, the effects of BA, KT, ZT, PP333, AC, media (abundant element, microelement and organic compound), saccharose, tempetature and illumination on initial culture, clump shoots induction culture and rooting culture was studied. And very great progress was maked in the facet of rooting culture, solving the hard problem of rooting in breeding E.dunnii via tissue culture, and several fine clones were found out. The experiment was valued by extending the utilization of E.dunnii through asexual propagation.The results were as follows.1. Initial culture: The optimal explant were the half-lignification bud stem section that was located from third to six in the tress of E.dunnii seedlings, and the optimal length of the bud stem section was 1 cm. The best disinfection method was established via orthogonal tests and single factor tests as follows: (1) the bud was laundered by bluent about 2 min and rinsed by clean water about 30 min;(2) the time of 70% alcohol disinfection was 5 second;(3) the time of 0.1% HgCl disinfection was 30 second, the optimal initial media:l/2MS+BA0.2-1.0mg L-1 +NAA0.05-0.5 mg L-1 +saccharose3%. The optimal culture temperature: 24-28C. The optimal illumination: 2000-3000Lx.2. Clump shoots induction culture: the optimal clump bud induction media: amended MS+ BA0.5mg L-1+ NAA0.2 mg L-1+saccharose3%. As the number of clump shoots induction culture was increased, the concentration of BA and NAA needed to be reduced gradually, at the end, the concentration of BA was about 0.2mg L-1,and the concentration of NAA was about 0.1 mg L-1. The periods of clump shoots induction culture was different in different season: the periods of clump shoots induction culture was 15-25 day in summer, 25-35 day in spring and in autumn, 40-50 day in winter. The ratio of monthly multiplications was 3-6, the vitrification phenomena of clump shoots was seldom happened, and efficiency seedlings were easily rooted besease of the optimal length between two bud burls.3. Clump shoots subculture: On the ground of the basic media: amended MS+ BA0.2mg L-1+NAA0.1 mg L-1+saccharose3% 11 experiment was tested: (1)add to AC 0.01%, 0.03%, 0.06%, 0.1% respectively, at the same time, improving the concentration of BA to 0.5mg L-1 and NAA to 0.2 mg L-1 (2) add to VH 1mg L-1, 2 mg L-1 3 mg L-1 respectively; (3) amended MS (organic compoung and Fe2+ 1.5 double), add to saccharose 3%, 4% 5% respectively. The results were as follows: AC restrained the growth of clump shoots, and VH had little effects on clump shoots subculture. BA and NAA were absolutely necessary for the growth and multiplication of clump shoot, otherwise, the clump shoots would be perished quickly. In order to cutting down the costs of producing seedlings, the clump shoots subculture or efficiency seedlings induction could be leaved out.4. Rooting culture: The effects of abundant element, saccharose, IBA, NAA, IAA, BA concentration and number of clump shoots induction culture, clone and illumination on rooting culture were prominent. The optimal rooting media: S+ saccharose 2%+ IBA 3mg L-1 the dosage of each abundant element was different for different clone in rooting culture. Two-step rooting method was adopted, the efficiency seedlings was cultured for 7-11 d without illumination in rooting media with IBA 3mg/L first, and then cultured in rooting media without IBA in natural light. The rooting ratio could reach to 85% for some fine clone, however, the rooting ratio was different for different clone and different number of clump shoots induction.5. Fine clone selection: In the selected fifty half-year E.dunnii seedlings, shoot forming frequency, shoot forming index and shoot growth speed in the initial culture, ratio of monthly multiplication and number of efficiency seedlings in clump shoo... |
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