树莓离体快繁体系建立及叶片不定芽诱导研究
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摘要
本研究分别以树莓‘Chilliwack'、‘Dinkum'、‘Kotata'和‘Shawnee'的顶芽和茎段为外植体,通过初代培养、继代增殖和生根培养,建立起树莓离体快繁体系。在此基础上,通过正交试验设计并结合方差分析和多重比较,探讨了基因型、基本培养基、不同种类的生长素和细胞分裂素、暗培养以及AgNO_3对树莓叶片不定芽再生的影响。试验结果如下:
(1)外植体建立阶段:四个品种中‘Kotata'的外植体成活率最高,为48.29%,其次是‘Shawnee'(41.73%),这两个品种的最高成活率都出现在MS+BA2.0mg/L+IBA0.2mg/L+GA_30.5mg/L(2#)上。‘Chilliwack'和‘Dinkum'最高成活率分别为31.1%和21.53%,且最高成活率都出现在MS+BA1.0mg/L+NAA0.1mg/L(1#)上。说明以MS为基本培养基附加BA(1.0mg/L～2.0mg/L)和较低浓度的IBA(0.2mg/L)的培养基有利于树莓无菌材料的建立。
(2)增殖阶段:MS+BA1.0mg/L+NAA0.1mg/L对‘Chilliwack'而言是最合适的增殖培养基。1/2MS+BA1.0mg/L+IBA0.4mg/L是‘Dinkum‘最合适的增殖培养基。对‘Kotata'和‘Shawnee'来说,MS+BA2.0mg/L+IBA0.2mg/L是最佳的增殖培养基。
(3)生根阶段:‘Kotata'的生根效果优于其它三个品种;1/4MS的生根效果明显地好于其它三个培养基;蔗糖是生根培养基中必需的;生长素和AC在生根前期是必需的,但在后期可有可无。
(4)在所选定的四个树莓品种进行叶片不定芽诱导时发现,‘Chilliwack'表现出最高的再生力,‘Kotata'次之,‘Dinkum'和‘Shawnee'没有不定芽发生。‘Chilliwack'在所选用的32个组合中,在NN69+BA1.0mg/L+NAA0.2mg/L组合上,不定芽诱导率最高(49.67%),且质量最好。‘Kotata'在NN69+BA2.0mg/L+IBA0.2mg/L组合上,不定芽诱导率达12.13%,质量最好。
(5)通过对TDZ和BA两种细胞分裂素在树莓叶片不定芽再生上的诱导结果进行多重比较和方差分析发现,BA的诱导效果优于TDZ。
(6)通过比较QL、WPM、NN69、MS四种基本培养基对树莓叶片不定芽再生的影响后发现,高盐浓度、高N含量的MS基本培养基有利于不定芽的再生,‘Chilliwack'叶片不定芽再生率为64.13%,‘Kotata'的再生率为23.21%,较NN69上的再生率分别提高15.44%和11.07%。其余两种基本培养基都会导致‘Chilliwack'和‘Kotata'叶片不定芽再生率的明显降低。
(7)暗培养对树莓叶片不定芽诱导有显著的促进效果。暗培养14d可将‘Chilliwack'不定芽再生率由62.00%提高到83.32%,‘Kotata'不定芽再生率提高了11.87%,但随着暗培养时间延长,叶片不定芽再生率下降。
(8) 0.5mg/L～2.0mg/LAgNO_3的加入抑制了‘Chilliwack'和‘Kotata'叶片不定芽的再生,且随着浓度的增加,对不定芽的再生抑制作用越明显,‘Chilliwack'不定芽再生率由0.0mg/LAgNO_3时的61.17%下降到2.0mg/LAgNO_3时的9.98%。‘Kotata'叶片的再生率也由0.0mg/LAgNO_3时的22.10%下降到2.0mg/LAgNO_3时的7.42%。但AgNO_3改变了树莓不定芽的再生途径,以直接从叶片再生不定芽的方式为主。
Four varieties of raspberry such as 'Chilliwack'、'Dinkum'、'Kotata' and 'Shawnee' whose shoot tips and stem segments were used as material to set up the non-bacillus propagation system through initial induction, subculture proliferation and rooting cultured. By means of the orthogonal experiment design, analysis of variance and multiple comparisons, the factors including genotypes, basic media, plant growth regulators, dark periods and AgNO_3 concentrations were analyzed and the ideal regeneration system of raspberry by leaf explants was established. The results were as follows:
(1)The stage of explants building: 'Kotata' whose rate of shoot survival was 48.29% was the highest rate shoot regeneration of breed among the four varieties, 'Shawnee' followed(41.73%). MS supplemented with 1.0mg/L BA, 0.2mg/L NAA and 0.5mg/L GA3 was the best combination to both 'Kotata' and 'Shawnee' .The highest rates of shoot survival of 'Chilliwack' and 'Dinkum' were 31.1% and 21.53% each and the best combination for the two varieties was MS supplemented with 1.0mg/L BA and 0.1mg/L NAA. It could get the conclusion that MS supplemented with 1.0～2.0mg/L BA and 0.1mg/L NAA was better to set up raspberry non-bacillus propagation system.
(2) The stage of proliferation: for 'Chilliwack', shoot proliferation best on MS supplemented with BA1.0mg/L and NAA0.1mg/L. For 'Dinkum', shoot proliferation best on 1/2MS supplemented with BA1.0mg/L and IBA0.4mg/L. For 'Kotata' and 'Shawnee', MS supplemented with BA2.0mg/L and IBA0.2mg/L was the best combination.
(3)The stage of rooting: the effect of rooting of 'Kotata' was better than the other three varieties. 1/4MS was the best among the four rooting basic mediums. Sugar was necessary for rooting. Auxin and AC were also necessary in the earlier stage of rooting, but not necessary in the later.
(4)According to the observation of the shoot regeneration of leaves in four varieties of raspberry, 'Chilliwack' showed highest rate of regeneration, 'Kotata' followed, but there was on buds regenerated in 'Dinkum' and 'Shawnee'. During the 32 combinations, NN69 supplemented with BA1.0mg/L and NAA0.2mg/L was best for 'Chilliwack' with regeneration rate of 49.67%. For 'Kotata', shoot regeneration on NN69 supplemented with BA2.0mg/L and IBA0.2mg/L with12.13%.
(5)According to the analysis of variance and multiple comparisons, two kinds of cytokines such as TDZ and BA had different effects in shoot regeneration of leaves in raspberry. The result showed that BA is better than TDZ when it referred to leaves culture.
(6) Compared with the effect of four basic mediums to buds regeneration of leaves, it found that MS was better to bud regeneration for its higher concentration of salt and N ( the rate of bud regeneration of 'Chilliwack was 64.13% and the rate of bud regeneration of 'Kotata' was 23.21%). However, the other three basic mediums all lead to rate of bud regeneration of 'Chilliwack' and 'Kotata' descend.
(7) Dark culture could improve the rate of shoot regeneration of leaves in raspberry. The rate of shoot regeneration of leaves in 'Chilliwack' rose from 62.00% to 83.32% when deal with dark culture 14 days, so does 'Kotata'. But the rate of regeneration of leaves would descend with the period of dark culture extending.
(8) Adding 0.5mg/L～2.0mg/LAgNO_3 to basic medium can cause rate of shoot regeneration of leaves in 'Chilliwack' and 'Kotata' decrease and the higher concentration added, the more negative effect indicated. When the concentration of AgNO_3 reached 2.0mg/L, the rate of shoot regeneration of leaves in Chilliwack' was only 9.98% which descended 51.19 percents compared with the check. Similarly, the rate of shoot regeneration of leaves in Chilliwack' decrease from 22.10% to 7.42%. At the same time, AgNO_3 could change the method of regeneration from callus bringing buds to generating buds directly.
(1)外植体建立阶段:四个品种中‘Kotata'的外植体成活率最高,为48.29%,其次是‘Shawnee'(41.73%),这两个品种的最高成活率都出现在MS+BA2.0mg/L+IBA0.2mg/L+GA_30.5mg/L(2#)上。‘Chilliwack'和‘Dinkum'最高成活率分别为31.1%和21.53%,且最高成活率都出现在MS+BA1.0mg/L+NAA0.1mg/L(1#)上。说明以MS为基本培养基附加BA(1.0mg/L～2.0mg/L)和较低浓度的IBA(0.2mg/L)的培养基有利于树莓无菌材料的建立。
(2)增殖阶段:MS+BA1.0mg/L+NAA0.1mg/L对‘Chilliwack'而言是最合适的增殖培养基。1/2MS+BA1.0mg/L+IBA0.4mg/L是‘Dinkum‘最合适的增殖培养基。对‘Kotata'和‘Shawnee'来说,MS+BA2.0mg/L+IBA0.2mg/L是最佳的增殖培养基。
(3)生根阶段:‘Kotata'的生根效果优于其它三个品种;1/4MS的生根效果明显地好于其它三个培养基;蔗糖是生根培养基中必需的;生长素和AC在生根前期是必需的,但在后期可有可无。
(4)在所选定的四个树莓品种进行叶片不定芽诱导时发现,‘Chilliwack'表现出最高的再生力,‘Kotata'次之,‘Dinkum'和‘Shawnee'没有不定芽发生。‘Chilliwack'在所选用的32个组合中,在NN69+BA1.0mg/L+NAA0.2mg/L组合上,不定芽诱导率最高(49.67%),且质量最好。‘Kotata'在NN69+BA2.0mg/L+IBA0.2mg/L组合上,不定芽诱导率达12.13%,质量最好。
(5)通过对TDZ和BA两种细胞分裂素在树莓叶片不定芽再生上的诱导结果进行多重比较和方差分析发现,BA的诱导效果优于TDZ。
(6)通过比较QL、WPM、NN69、MS四种基本培养基对树莓叶片不定芽再生的影响后发现,高盐浓度、高N含量的MS基本培养基有利于不定芽的再生,‘Chilliwack'叶片不定芽再生率为64.13%,‘Kotata'的再生率为23.21%,较NN69上的再生率分别提高15.44%和11.07%。其余两种基本培养基都会导致‘Chilliwack'和‘Kotata'叶片不定芽再生率的明显降低。
(7)暗培养对树莓叶片不定芽诱导有显著的促进效果。暗培养14d可将‘Chilliwack'不定芽再生率由62.00%提高到83.32%,‘Kotata'不定芽再生率提高了11.87%,但随着暗培养时间延长,叶片不定芽再生率下降。
(8) 0.5mg/L～2.0mg/LAgNO_3的加入抑制了‘Chilliwack'和‘Kotata'叶片不定芽的再生,且随着浓度的增加,对不定芽的再生抑制作用越明显,‘Chilliwack'不定芽再生率由0.0mg/LAgNO_3时的61.17%下降到2.0mg/LAgNO_3时的9.98%。‘Kotata'叶片的再生率也由0.0mg/LAgNO_3时的22.10%下降到2.0mg/LAgNO_3时的7.42%。但AgNO_3改变了树莓不定芽的再生途径,以直接从叶片再生不定芽的方式为主。
Four varieties of raspberry such as 'Chilliwack'、'Dinkum'、'Kotata' and 'Shawnee' whose shoot tips and stem segments were used as material to set up the non-bacillus propagation system through initial induction, subculture proliferation and rooting cultured. By means of the orthogonal experiment design, analysis of variance and multiple comparisons, the factors including genotypes, basic media, plant growth regulators, dark periods and AgNO_3 concentrations were analyzed and the ideal regeneration system of raspberry by leaf explants was established. The results were as follows:
(1)The stage of explants building: 'Kotata' whose rate of shoot survival was 48.29% was the highest rate shoot regeneration of breed among the four varieties, 'Shawnee' followed(41.73%). MS supplemented with 1.0mg/L BA, 0.2mg/L NAA and 0.5mg/L GA3 was the best combination to both 'Kotata' and 'Shawnee' .The highest rates of shoot survival of 'Chilliwack' and 'Dinkum' were 31.1% and 21.53% each and the best combination for the two varieties was MS supplemented with 1.0mg/L BA and 0.1mg/L NAA. It could get the conclusion that MS supplemented with 1.0～2.0mg/L BA and 0.1mg/L NAA was better to set up raspberry non-bacillus propagation system.
(2) The stage of proliferation: for 'Chilliwack', shoot proliferation best on MS supplemented with BA1.0mg/L and NAA0.1mg/L. For 'Dinkum', shoot proliferation best on 1/2MS supplemented with BA1.0mg/L and IBA0.4mg/L. For 'Kotata' and 'Shawnee', MS supplemented with BA2.0mg/L and IBA0.2mg/L was the best combination.
(3)The stage of rooting: the effect of rooting of 'Kotata' was better than the other three varieties. 1/4MS was the best among the four rooting basic mediums. Sugar was necessary for rooting. Auxin and AC were also necessary in the earlier stage of rooting, but not necessary in the later.
(4)According to the observation of the shoot regeneration of leaves in four varieties of raspberry, 'Chilliwack' showed highest rate of regeneration, 'Kotata' followed, but there was on buds regenerated in 'Dinkum' and 'Shawnee'. During the 32 combinations, NN69 supplemented with BA1.0mg/L and NAA0.2mg/L was best for 'Chilliwack' with regeneration rate of 49.67%. For 'Kotata', shoot regeneration on NN69 supplemented with BA2.0mg/L and IBA0.2mg/L with12.13%.
(5)According to the analysis of variance and multiple comparisons, two kinds of cytokines such as TDZ and BA had different effects in shoot regeneration of leaves in raspberry. The result showed that BA is better than TDZ when it referred to leaves culture.
(6) Compared with the effect of four basic mediums to buds regeneration of leaves, it found that MS was better to bud regeneration for its higher concentration of salt and N ( the rate of bud regeneration of 'Chilliwack was 64.13% and the rate of bud regeneration of 'Kotata' was 23.21%). However, the other three basic mediums all lead to rate of bud regeneration of 'Chilliwack' and 'Kotata' descend.
(7) Dark culture could improve the rate of shoot regeneration of leaves in raspberry. The rate of shoot regeneration of leaves in 'Chilliwack' rose from 62.00% to 83.32% when deal with dark culture 14 days, so does 'Kotata'. But the rate of regeneration of leaves would descend with the period of dark culture extending.
(8) Adding 0.5mg/L～2.0mg/LAgNO_3 to basic medium can cause rate of shoot regeneration of leaves in 'Chilliwack' and 'Kotata' decrease and the higher concentration added, the more negative effect indicated. When the concentration of AgNO_3 reached 2.0mg/L, the rate of shoot regeneration of leaves in Chilliwack' was only 9.98% which descended 51.19 percents compared with the check. Similarly, the rate of shoot regeneration of leaves in Chilliwack' decrease from 22.10% to 7.42%. At the same time, AgNO_3 could change the method of regeneration from callus bringing buds to generating buds directly.
引文
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