越橘离体叶片再生体系的建立及其影响因素的研究
摘要
本试验以越橘四大种类(矮丛越橘、半高丛越橘、高丛越橘、兔眼越橘)，14个越橘品种(‘园蓝’、‘日出’、‘塞埃罗’、‘都克’、‘伯克利’、‘埃利奥特’、‘蓝丰’、‘泽西’、‘乔治亚姆’、‘瑞维尔’、‘夏普蓝’、‘北陆’、‘美登’、‘芬蒂’)为材料，用叶片作为外植体进行离体再生培养，并对影响越橘离体再生的因素进行了初步的研究。
通过试验发现，影响越橘离体再生的因素可以从内因和外因两个方面进行探讨。内因主要是基因型，它是越橘离体再生很重要的限制因素；外因指激素种类及其浓度、培养基类型、蔗糖浓度、pH值、暗培养时间等因子。在从多外在因子中，培养基类型、激素种类及其浓度、暗培养时间是影响越橘离体再生的重要因素。
不同基因型的品种在再生能力上有很大的差别。供试的各品种中，‘日出’、‘塞埃罗’、‘乔治亚姆’、‘芬蒂’的再生能力很强，在适宜的培养基再生频率可达95％以上。‘埃利奥特’、‘北陆’、‘都克’、‘伯克利’再生能力次之，在适宜的培养基上再生频率可达85％以上，单叶片平均不定芽数达25个。‘泽西’、‘蓝丰’、‘美登’的再生能力很低，再生频率接近零。这三个品种的再生培养条件仍须继续摸索。对多种培养基进行研究得出结论：WPM培养基对越橘离体再生最为有利，各品种以其为基本培养基再生频率高于其他类型的培养基。试验中采用的细胞分裂素，包括ZT、TDZ、CPPU、2ip、BA，其中ZT、CPPU对越橘离体再生的效果最好。CPPU处理中品种再生频率最高可达100％，单叶片平均不定芽数最高可达41个。多数应试品种的再生对CPPU的反应良好，再生频率和平均不定芽数均高于ZT处理。如南高丛品种‘乔治亚姆’在ZT处理中的再生频率最高只有42.3％，单叶片平均不定芽数为6.8个；但在CPPU处理中再生率最高可达100％，但叶片平均不定芽数达最高可达20个。TDZ和2ip只对个别品种的再生有效果，如‘日出’、‘塞埃罗’、‘伯克利’、‘埃利奥特’，再生频率最高只有69％，不定芽数明显低于ZT、CPPU的处理。BA对越橘离体叶片的再生没有效果。此外暗培养对越橘叶片的再生也有很大的影响，虽然有些品种，如‘日出’和‘塞埃罗’，在不经暗处理的情况下再生率仍能达到85％以上，但不定芽的质量和数量远不如经过暗处理的。试验中多数品种都需要暗处理，才能获得较高的再生
频率和不定芽数。还有一些因子如蔗糖浓度、pH、外植体的摆放方向等，对越橘叶片
的再生影响不显著。
A procedure to regenerate shoots in vitro from leaf sections of fourteen blueberries genotypes, 'Gardenblue', 'Sunrise', 'Sierra', 'Duke', 'Berkeley', 'Elliot', 'Bluecrop', 'Jersey', 'Georgia Gem', 'Reveil', 'Sharpblue', 'Northland', 'Blomidon', 'Fundy', in Woody Plant Medium was established, and influencing factors such as genotype, medium, hormone, sugar concentration, pH, were also studies.
Cultivars with different genotype had different regeneration ability. Among the fourteen genotypes tested , 'Sunrise', 'Sierra', 'Georgia Gem' and 'Fundy' had the strongest ability in shoot regeneration with shoot induction frequency of 95% and mean number 20 shoots per leaf. Hormone types and concentration in medium were the most important factors influencing shoot regeneration from leaf segments of blueberry. The effect of Zeatin and CPPU on leaf segments regeneration was better than other hormones tested in experiment. Cytoinin TDZ or 2ip had effect on some cultivars. For example, regeneration percent of Sunrise obtained from 2ip medium was 59.4% or 69%, which was fewer than that from Zeatin or CPPU medium. Medium supplemented with the cytokinin CPPU was compared with medium supplemented with Zeatin. Use of 1.0 mg/L ~ 2.0 mg/L CPPU resulted in the higher regeneration rate and larger number of shoots per leaf section than 5.0 mg/L Zeatin did. Such as Georgia Gem , its highest regeneration percent on CPPU m
edium was 100%, while on the Zeatin medium was 42.3%. Consequently, we concluded that the cytokinin CPPU was more effective than either of the cytokinins, Zeatin, 2ip or TDZ, in promoting shoot regeneration from leaf sections of blueberry. Dark treatment was also important factor in blueberry leaf section regeneration. Ten days dark treatment after leaf sections inoculated on the medium promised the best adventitious buds and highest regeneration rate. And shorter or longer than ten days would decrees shoot regeneration rate. In addition, sugar concentration and pH had effect on blueberry leaf sections regeneration. Medium with 20mg/L sugar was optimum of leaf regeneration , produced
more shoots than any other concentrations of sugar. The optimal pH of medium was 5.4. The shoot regeneration rate of blueberry would be decreasing with pH of medium increasing. For example, the shoot regeneration rate of Georgia Gem on pH 5.4 medium was 97%, while on pH 6.5 medium the shoot regeneration was 10%. The temperature during dark treatment influenced shoot regeneration too. The optimal temperature of low bush blueberry was 20 ± 2 ℃ , for high bush blueberry was 25 ±2 ℃. The optimal temperature of rabbiteye was higher than 25 ± 2 ℃., but lower than 30℃.
通过试验发现，影响越橘离体再生的因素可以从内因和外因两个方面进行探讨。内因主要是基因型，它是越橘离体再生很重要的限制因素；外因指激素种类及其浓度、培养基类型、蔗糖浓度、pH值、暗培养时间等因子。在从多外在因子中，培养基类型、激素种类及其浓度、暗培养时间是影响越橘离体再生的重要因素。
不同基因型的品种在再生能力上有很大的差别。供试的各品种中，‘日出’、‘塞埃罗’、‘乔治亚姆’、‘芬蒂’的再生能力很强，在适宜的培养基再生频率可达95％以上。‘埃利奥特’、‘北陆’、‘都克’、‘伯克利’再生能力次之，在适宜的培养基上再生频率可达85％以上，单叶片平均不定芽数达25个。‘泽西’、‘蓝丰’、‘美登’的再生能力很低，再生频率接近零。这三个品种的再生培养条件仍须继续摸索。对多种培养基进行研究得出结论：WPM培养基对越橘离体再生最为有利，各品种以其为基本培养基再生频率高于其他类型的培养基。试验中采用的细胞分裂素，包括ZT、TDZ、CPPU、2ip、BA，其中ZT、CPPU对越橘离体再生的效果最好。CPPU处理中品种再生频率最高可达100％，单叶片平均不定芽数最高可达41个。多数应试品种的再生对CPPU的反应良好，再生频率和平均不定芽数均高于ZT处理。如南高丛品种‘乔治亚姆’在ZT处理中的再生频率最高只有42.3％，单叶片平均不定芽数为6.8个；但在CPPU处理中再生率最高可达100％，但叶片平均不定芽数达最高可达20个。TDZ和2ip只对个别品种的再生有效果，如‘日出’、‘塞埃罗’、‘伯克利’、‘埃利奥特’，再生频率最高只有69％，不定芽数明显低于ZT、CPPU的处理。BA对越橘离体叶片的再生没有效果。此外暗培养对越橘叶片的再生也有很大的影响，虽然有些品种，如‘日出’和‘塞埃罗’，在不经暗处理的情况下再生率仍能达到85％以上，但不定芽的质量和数量远不如经过暗处理的。试验中多数品种都需要暗处理，才能获得较高的再生
频率和不定芽数。还有一些因子如蔗糖浓度、pH、外植体的摆放方向等，对越橘叶片
的再生影响不显著。
A procedure to regenerate shoots in vitro from leaf sections of fourteen blueberries genotypes, 'Gardenblue', 'Sunrise', 'Sierra', 'Duke', 'Berkeley', 'Elliot', 'Bluecrop', 'Jersey', 'Georgia Gem', 'Reveil', 'Sharpblue', 'Northland', 'Blomidon', 'Fundy', in Woody Plant Medium was established, and influencing factors such as genotype, medium, hormone, sugar concentration, pH, were also studies.
Cultivars with different genotype had different regeneration ability. Among the fourteen genotypes tested , 'Sunrise', 'Sierra', 'Georgia Gem' and 'Fundy' had the strongest ability in shoot regeneration with shoot induction frequency of 95% and mean number 20 shoots per leaf. Hormone types and concentration in medium were the most important factors influencing shoot regeneration from leaf segments of blueberry. The effect of Zeatin and CPPU on leaf segments regeneration was better than other hormones tested in experiment. Cytoinin TDZ or 2ip had effect on some cultivars. For example, regeneration percent of Sunrise obtained from 2ip medium was 59.4% or 69%, which was fewer than that from Zeatin or CPPU medium. Medium supplemented with the cytokinin CPPU was compared with medium supplemented with Zeatin. Use of 1.0 mg/L ~ 2.0 mg/L CPPU resulted in the higher regeneration rate and larger number of shoots per leaf section than 5.0 mg/L Zeatin did. Such as Georgia Gem , its highest regeneration percent on CPPU m
edium was 100%, while on the Zeatin medium was 42.3%. Consequently, we concluded that the cytokinin CPPU was more effective than either of the cytokinins, Zeatin, 2ip or TDZ, in promoting shoot regeneration from leaf sections of blueberry. Dark treatment was also important factor in blueberry leaf section regeneration. Ten days dark treatment after leaf sections inoculated on the medium promised the best adventitious buds and highest regeneration rate. And shorter or longer than ten days would decrees shoot regeneration rate. In addition, sugar concentration and pH had effect on blueberry leaf sections regeneration. Medium with 20mg/L sugar was optimum of leaf regeneration , produced
more shoots than any other concentrations of sugar. The optimal pH of medium was 5.4. The shoot regeneration rate of blueberry would be decreasing with pH of medium increasing. For example, the shoot regeneration rate of Georgia Gem on pH 5.4 medium was 97%, while on pH 6.5 medium the shoot regeneration was 10%. The temperature during dark treatment influenced shoot regeneration too. The optimal temperature of low bush blueberry was 20 ± 2 ℃ , for high bush blueberry was 25 ±2 ℃. The optimal temperature of rabbiteye was higher than 25 ± 2 ℃., but lower than 30℃.
引文
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