【作者】 王进茂；

【导师】 杨敏生；

【作者基本信息】 河北农业大学 ， 森林培育， 2003， 硕士

【摘要】 为了满足我国对白桦品种改良的要求，为白桦的良种定向培育及基因工程育种奠定基础，本文利用组织培养技术研究了引进欧洲白桦优良用材无性系1／86、2／86、4倍体白桦4N和三倍体白桦G1的再生体系的建立。主要从外植体的选择、基本培养基的应用、外源激素的使用浓度、影响试管苗生根的主要因子以及试管苗的移栽等几个方面进行了探讨，确定了最佳再生途径。主要结果如下： 1 影响白桦无性系不定芽再生的主要因子 白桦再生不定芽的途径以间接途径为主，无菌试管苗的茎段、根、叶片均可做外植体诱导不定芽再生。不同的基因型，不定芽再生的难易程度不一样。无性系2／86比G1、4N容易诱导愈伤组织进而诱导不定芽发生。诱导无性系2／86茎段不定芽分化的适宜培养基为WPM+6-BA1.0+KT0.3+NAA0.01，根段为MCM，叶片为MS。不同外植体不定芽再生能力也不同，叶柄和茎段最容易诱导愈伤组织和不定芽分化，根较难诱导。 在细胞分裂素中6-BA的诱导效果好于ZT和TDZ，适宜浓度为1.0mg／L，高与低均影响不定芽的诱导率。另外叶片的成熟度也直接影响不定芽再生的频率，中龄叶不定芽的诱导率是成龄叶的4.5倍。暗培养不利于叶片不定芽的诱导。 2 不定芽的增殖培养 实验表明，白桦不定芽和嫩茎在增殖过程可分为增殖分化阶段与壮苗阶段。确定白桦无性系不定芽或嫩茎增殖的基本方式为：用丛生芽的方式进行继代培养，以获得最高的繁殖系数，通过控制培养基中细胞分裂素的浓度来完成对白桦试管苗的壮苗培养。 3 生根培养 影响白桦各无性系试管苗生根的因素是多方面的。白桦各无性系试管苗诱导生根的能力不同，按生根的速度、生根率按顺序排列为：2／86、7／86、4N、G1；基本培养基对白桦试管苗生根的诱导，以1／2MS效果最好；对于无性系4N，单独使用NAA比单独使用IBA和同时使用NAA与IBA更有利于试管苗不定根诱导，NAA的最适浓度为0.3mgL^-1，而诱导无性系1／86、2／86的试管苗生根，0.1mgL^-1NAA与0.1mgL^-1IBA配合使用效果最佳。 白桦试管苗木质化程度越高，其生根率反而有下降的趋势。1％～3％的蔗糖浓度对白桦试管苗生根率基本无影响，蔗糖浓度主要影响根的长度，浓度越高不定根越长。本试验认为诱导白桦试管苗生根采用20gL^-1的蔗糖浓度为宜。 4 白桦试管苗的移栽 白桦试管苗移栽采用腐殖土：细沙：园土（10：10：10）或腐殖土：园土（20：10）效果最佳。试管苗在生根培养基上培养的天数以及生根试管苗的生长状况对移栽成活率影响明显，试管苗不定根伸长最快的时期，移栽成活率最高。更多还原

【Abstract】 In order to support birch directional breeding in China and develop an efficient regeneration system for birch genetic transformation, tissue culture of birch (Betula pendula Rroth) superior clones from Germany was studied. In this paper, we screened the optimum regeneration system through studies conducted on explants selection, basal medium composition, hormones kinds and levels, rooting conditions and conditions which plantlets transferred to warm house. Main achievements were shown as follows: key factors affecting the regeneration of birch genotypesBirch mainly regenerates adventitious buds through indirect ways. Different types of explants from sterile plantlets, such as shoot segments, roots and leaves, was suitable for shoot regeneration, respectively. However, genotypes is a limited factor in the establishment of a complete regeneration system. The genotypes 2/86 could be dedifferentiated into callus and further differentiate into buds more easily than Gl, 4N. Moreover, different explants from clone 2/86 need different basal medium. WPM medium, MCM medium and MS medium was most suitable for shoot segments, root explants and leaves, respectively. In addition, the ability of adventitious bud regeneration induced from various explants showed different degrees. In this experiment, compared to root explants, the stem and leafstalk were most easily dedifferentiated and redifferentiatedThe inducing effect of 6-BA was better than that of ZT and TDZ. The better medium was MS macronutrients supplemented with 1.0 mg/L 6-BA+0.3 mg/L KT+0.01 mg/L NAA. The optium concentration of 6-BA was 1.0 mg/L, higher and less concentration of 6-BA could affect the percentage of adventitious bud induction. Moreover, the age of leaves directly influenced the adventitious bud regeneration. The inducing percentage of leaves of young ages is 4.5 time that of leaves in grown age. Multiplication of adventitious budThe results showed that the propagation course of birch adventitious bud and shoot tips could be divided into two stages: multiplication stage and fast-growing stage. In order to get the highest proliferation rate, we employed the method of subculturing rosette buds. At the same time, to ensure shoots grow healthy and strong, the level of CTK in the medium was regulated.Rooting cultureIt has been proposed that there were many factors affected birch clone seedling rooting in vitro. Genotype is an important factor which influence the ability of shoot rooting. The results demonstrateded that 2/86 had the highest rooting ability, while G1 had the lowest rooting ability among all tested materials. 1/2 MS medium was the most suitable for multiplied shoot rooting. For the clone 4N, only NAA was enough to induce multiplied shoot rooting and the suitable concentration of NAA was 0.3 mg/1. Whereas for the clone 1/86 or 2/86, 1/2 MS medium supplemented with 0.1 mg/1 NAA and 0.1 mg/1 IB A was optimum.Percentage of rooting is related to wooden level of Birch seedling, and the more the wooden, the less the percentage of rooting. The sucrose concentration between 1% and 3% haven’t apparently effect on rooting rate. But the high level of sucrose resulted in form of the longer root. These results indicated 20g/l sucrose concentration was suitable for shoot rooting. Plantlets transferred into warm houseBirch plantlets in vitro were finally transferred to soil with 1/3 humus soil+1/3 sand+1/3 stand soil or 2/3 humus soil +1/3 stand soil. In order to improve the rate of plantlets survival, we performed different conditions, for example, the days which plantlets cultured in rooting medium, growth status of the rooting plantlets. From our study, we found that the period of adventitious root extend quickly was linked to an increase in plantlets survival.更多还原