【作者】 喻晓敏；

【导师】 冯建灿；

【作者基本信息】 河南农业大学 ， 森林培育， 2009， 硕士

【摘要】 枣(Zizyphus jujuba Mill.)为鼠李科枣属植物,是我国第一大干果树种和最具代表性的民族果树之一。灰枣耐干旱、瘠薄和盐碱,抗干热风,结果早,是优良的鲜食和制干兼用型品种,但其也存在病虫害严重,落花落果严重等缺陷。离体叶片再生技术是果树细胞水平的诱变育种、变异筛选、嵌合体分离及基因转移等方面的基础,目前已有民勤小枣、沾化冬枣、骏枣、毛叶枣等十几个品种的枣叶片再生体系建立成功,但未见有关灰枣离体叶片再生体系建立的报道。本论文以灰枣为试材,分别通过愈伤组织分化不定芽、直接再生不定芽、不定根分化不定芽三种途径,研究各种因素如外植体条件、基本培养基类型、激素种类和浓度、光照条件、添加物等对灰枣不定芽再生过程的影响,优化各种条件,以期建立一个适合灰枣的高效、稳定再生体系。主要实验结论如下:(1)灰枣叶片愈伤组织的诱导宜选择组培苗叶片为外植体;苗茎中部叶片的愈伤组织诱导率高于茎顶部和下部的叶片愈伤组织诱导率,以第3-5片深绿色,平展,大而厚的叶片为佳;接种前不伤叶缘、垂直于主脉横切3-5刀,与纵切和不切相比,愈伤组织诱导率可提高到65.44%;接种方向对叶片愈伤组织的诱导影响不显著;暗培养可加速愈伤组织发生进程,适宜的暗培养时间为14d。叶片愈伤组织诱导的最适培养基为1/2MS附加6-BA0.5mg/L,2,4-D2.0 mg/L,AgNO3 0.5mg/L,愈伤组织诱导率可达到96.67%。由叶片诱导的愈伤组织,只有黄绿色,致密的愈伤组织在分化培养基上培养3代(5周转接一次,为一代)后才可分化出芽,将其切成0.5cm2大小接种于分化培养基,在MS附加6-BA3.0 mg/L和IBA0.2mg/L培养基上分化率为13.77%,且一个愈伤组织块上只能分化出一个芽;在MS附加ZT2.0 mg/L和IBA0.1mg/L培养基上能分化出丛生芽,分化率可达37.34%。(2)叶片直接再生不定芽的最适培养基为WPM﹢NAA0.2mg/L﹢TDZ0.5mg/L;最佳外植体为灰枣组培苗中部平展,大而厚叶片;不定芽多在近叶柄处发生,再生率比近叶尖处高59.74%;以近轴面接触培养基接种较以远轴面接触培养基接种的再生率高16.81%;提高蔗糖浓度到40g/L可降低再生芽的玻璃化程度;适当时间的暗培养可以提高灰枣叶片再生能力,以10d的暗培养处理最佳;添加AgNO30.5 mg/L叶片再生率可提高到82.25 %,平均每叶片再生芽数为2.64个。(3)灰枣组培苗叶片不定根诱导的最佳培养基为1/2MS附加NAA0.3mg/L和IBA 2.0mg/L,诱导率为92.95%,诱导的不定根乳白色,粗壮。将不定根切成0.5cm的小段接种于WPM附加TDZ1.0mg/L和NAA0.2mg/L的分化培养基上,分化率可达44.51%,平均每不定根再生芽数2.08个。(4)将分别通过愈伤组织再生、直接再生和不定根再生三种途径诱导的不定芽接种到继代增殖培养基MS﹢6-BA2.0mg/L﹢IBA 0.5mg/L﹢KT0.5mg/L,培养至35d,增殖系数为3.06。(5)将继代增殖的再生苗切成2cm左右的茎段接种在生根壮苗培养基上诱导生根,最佳的生根培养基为1/2MS﹢NAA0.5mg/L,培养40d的生根率达95.56%,再生苗基部仅有极少量的愈伤组织产生,发生的根粗壮,质量较好。更多还原

【Abstract】 Jujube (Zizyphus jujuba Mill.) is one of the most important and representative fruit trees in China.‘Huizao’is a cultivated variety of jujube in China that has the longest planting history, the largest orchard area, and highest annual production. Since‘Huizao’has small flowers, artificial emasculation is difficult. High embryo abortion rate and the low rate of the fruit set in this perennial woody fruit tree make improvement of quality by conventional inter-specific cross breeding quite difficult. Leaves of regeneration technology is the basis of mutation breeding on the level of cell, mutation screening, and isolation of chimeric gene transfer and so on. From then on, shoot regeneration systems from leaf explants have been achieved in several Z. jujuba cultivars, such as‘Mingqinxiazao’,‘Zhanhuadongzao’,‘Junzao’and‘Maoyezao’. As of yet there is no reliable regeneration procedure applicable to‘Huizao’, one of the most prominent high quality varieties of jujube.Critical factors affect the frequency of leaf regeneration in jujube, such as basal medium, concentration and combination of plant growth regulators, leaf maturity, leaf position on the shoot, orientation of explant contacting medium, and incubation conditions. These factors are investigated in this study, and our objective was to obtain an efficient and stable regeneration system using leaf segments from‘Huizao’.The main conclusions are as follows:(1)The leaves of tissue culture plantlets were the optimal explant source for callus induction of Z. jujuba‘Huizao’. The callus induction frequency of leaves from the central stem of plantlet is higher than the ones which from the lower part and the upper part. The optimal explant is the leaves on the three to five of the stem, which are flat, large and thick. Cut 3-5 knives vertical to the main vein not to hurt the leaf margin before vaccination, the callus induction frequency can be increased to 65.44% compared to parallel to the main vein and no cuts. Effect of explant orientation on callus induction frequency was not significant. Incubation length of pre-culture in darkness can accelerate the process of callus formation, appropriate time in darkness is 14 days. The optimal callus induction medium is 1/2MS supplemented with 0.5mg/L 6-BA and 2.0 mg/L 2,4-D, 0.5mg/L AgNO3, callus induction frequency can achieve to 96.67%. Only the yellow-green, dense callus cultured in differentiation medium on the 3rd generation can differentiate regeneration shoots. Cut them into 0.5cm2 size and then incubate on the differentiation medium. The differentiation frequency of callus on MS supplemented with 6-BA 3.0 mg/L and IBA 0.2mg/L is 13.77%, and one callus can only differentiate one shoot. The differentiation frequency of callus on MS supplemented with ZT 2.0mg/L and IBA 0.1mg/L was 37.34%.(2)WPM with 0.5mg/L TDZ plus 0.2mg/L NAA was effective for leaf explant regeneration of‘Huizao’. The optimal explant is the young leaves on the middle of the stem, which are flat, large and thick. While more adventitious buds initials developed at the petiole portion of the midrib and surrounding area, regeneration capacity increased 59.74% from the tip towards the base of the leaf. The frequency of regeneration of‘Huizao’was 16.81% higher when the adaxial surface of the explants was in contact with the medium compared to the abaxial surface of the explants was in contact with the medium. The concentration of sucrose in 40g/L can reduce the hyperhydric degree of adventitious buds. A 10-day dark of inoculated can increase the frequency of regeneration. The frequency of regeneration of leaf explants increased 14.14% with the addition of 0.5 mg/L AgNO3 to the medium and the average number of shoots per explant can reach to 2.64 piece when compared to that of the control without AgNO3.(3)1/2MS supplemented with 0.3mg/L NAA and 2.0mg/L IBA was effective for adventitious roots induced by leaf explant of‘Huizao’, the frequency can reach to 92.95%, the adventitious roots induced are white, thick. Cut adventitious roots into 0.5cm,then inoculate on WPM supplemented with 1.0mg/L TDZ and 0.2mg/L NAA, the differentiation frequency can reach to 44.51%, average number of adventitious buds were 2.08 piece.(4)Inoculate the adventitious buds through three ways above on the proliferation medium which contain MS plus 2.0mg/L 6-BA, 0.5mg/L IBA and 0.5mg/L KT, cultured for 35 days, multiplication coefficient were about 3.0.(5)Cut the proliferation of the regeneration buds into 2.0cm,then inoculate on the root induction medium, the best rooting medium was 1/2MS plus 0.5mg/L NAA. After 40 days culture, rooting frequence can achieve to 95.56%. Only a small number of callus at the base of the plantlet, the occurrence of the root were thick, had good quality.更多还原