牡丹组织培养技术的研究
摘要
以牡丹的鳞芽、叶片和叶柄为外植体,采用正交试验设计方法,探讨不同消毒剂及消毒时间对外植体的影响;研究不同基本培养基类型、不同生长调节物质、不同附加物质和不同环境因子在牡丹鳞芽诱导、增殖和生根中的作用,找出不同培养过程中的主要影响因子以及有效浓度范围,并进一步对生根试管苗进行了炼苗及移栽研究;同时以牡丹的叶片及叶柄为外植体,探讨愈伤组织诱导与分化过程中的主要影响因子及其有效浓度范围,对由叶柄愈伤组织分化的不定芽进行生根诱导,优化愈伤组织诱导与分化的研究技术。试验结果如下:
1.采用不同消毒剂和消毒时间进行消毒处理,结果表明:适合牡丹鳞芽的消毒剂为0.1%HgCl2,最佳消毒时间为8min;叶柄最佳消毒时间为5min;叶片则以3min为佳。
2.以牡丹品种“大胡红”、“乌龙捧盛”和“金阁”的鳞芽为材料,研究不同基本培养基类型和不同品种对鳞芽诱导的影响,结果表明:1/2MS为最佳基本培养基,牡丹品种“金阁”诱导率最高。
3.以牡丹品种“金阁”鳞芽为外植体,研究不同生长调节物质和不同浓度AC对鳞芽诱导的影响,结果表明:不同因素对牡丹鳞芽诱导率的影响不同,各因素的影响程度依次为:6-BA > NAA > AC >IAA;1/2MS+6-BA 1.0mg/L+NAA 0.2mg/L+IAA 0.3mg/L+AC 3.0g/L为牡丹鳞芽诱导的最佳培养基。
4.以牡丹品种“金阁”鳞芽为外植体,研究不同基本培养基类型和不同生长调节物质对鳞芽增殖的影响,结果表明:各个因素对鳞芽增殖率的影响为:NAA >基本培养基类型> 6-BA > NAA;培养基1/2MS+6-BA 1.0mg/L+NAA 0.5mg/L+GA3 0.2mg/L有利于牡丹鳞芽的增殖;另外,不同继代周期对牡丹鳞芽增殖倍数也有影响,以继代30d为宜。
5.以牡丹品种“大胡红”、“乌龙捧盛”和“金阁”的叶片和叶柄为外植体,比较不同品种和不同外植体类型对愈伤组织诱导的影响,结果表明:同一品种,叶柄愈伤组织诱导率较叶片高;牡丹品种“金阁”愈伤诱导率最高,为64.50%。
6.以牡丹品种“金阁”的叶片和叶柄为外植体,研究不同基本培养基类型对牡丹愈伤组织诱导率的影响,结果表明:适宜牡丹叶片愈伤诱导的基本培养基为1/2MS;WPM为叶柄愈伤诱导的最佳基本培养基。
7.以牡丹品种“金阁”的叶片和叶柄为外植体,研究不同生长调节物质和不同浓度CH对愈伤诱导的影响,结果表明:NAA、2,4-D和6-BA是影响叶片愈伤诱导率的主要因素;适合叶片愈伤诱导的最佳培养基为:1/2MS +2,4–D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+CH 300mg/L;叶柄愈伤诱导最佳培养基:WPM+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+TDZ 0.5mg/L+CH 300mg/L。
8.研究不同生长调节物质和不同浓度CH对愈伤诱导的影响,结果表明:NAA、2,4-D和6-BA是影响叶片愈伤诱导率的主要因素;适合叶片愈伤诱导的最佳培养基为:1/2MS +2,4-D2.0mg/L+6-BA1.0mg/L+NAA 1.0mg/L+CH 300mg/L;叶柄愈伤诱导最佳培养基:WPM+2,4-D2.0mg/L+6-BA1.0mg/L+NAA 1.0mg/L+TDZ 0.5mg/L+CH 300mg/L。
9.以牡丹品种“金阁”试管苗为材料,研究不同基本培养基的类型、不同生长调节物质、不同蔗糖浓度和不同暗处理时间对牡丹试管苗生根的影响,结果表明:各因素对于生根率的影响程度依次为:基本培养基类型> IAA> IBA >蔗糖浓度>暗处理时间;各因素对于平均根长的影响程度依次是:基本培养基类型>暗处理时间> IAA >蔗糖浓度> IBA;培养基WPM+IBA1.0 mg/L+IAA 0.5mg/L+蔗糖20g/L,暗培养5d有利于牡丹试管苗的诱导;培养基WPM+IBA 1.0mg/L+IAA 0.5mg/L+蔗糖40 g/L,暗培养10d有利于牡丹试管苗根的生长。另外,以叶柄愈伤组织诱导出的分化芽为材料进行生根诱导研究,发现IBA 2.0mg/L有利于根的诱导。
10.以生长健壮的牡丹生根试管苗为材料,进行炼苗及移栽试验,结果表明:在温度15~20℃、湿度85%~90%条件下,牡丹试管苗的最佳炼苗时间为12d;在珍珠岩:蛭石(1:1)基质中移栽成活率最高,达到50%。
The research took buds, leaves and leafstalks of different species of peony ( Paeonia Suffruticosa ) as explants to probe into the influence of different disinfectors and sterilized time on explants by Orthogonal design; Different basic medias, regulators, accessories and environmental factors were studied for their effects on buds of peony in the course of inducement, proliferation and inducing root, the main influence factors and effective concentration scale were analyzed. Further more to study the training and transplant of rooting peony; At the same time, the leaves and leafstalks of peony were used as explants in order to get the main influence factors and effective concentration scale in the callus inducement and differentiation, then adventitious buds from callus of leafstalk were induced root, to optimize the technique of callus inducement and differentiation. The results of experiments as follows:
1. The buds of peony were used as explants, to study the effect of different disinfectors and sterilized time on explants. The results showed that: the optimal disinfector and sterilized time for bud are 0.1%HgCl2 and 8min, the suitable time for leaf and leafstalk is 3min and 5min.
2. The buds of cv.‘Da Hu Hong’, cv.‘Wu Long Peng Sheng’and cv.‘Jin Ge’of peony were used as explants, to study the effect of different basic medias and species on bud inducing. The results showed that: the best medium is 1/2MS; the best percent of bud inducing is cv.‘Jin Ge’.
3. Different regulators and AC were studied in the course of bud inducing of cv.‘Jin Ge’. The results showed that: the impact of different factor on bud inducing is different, 6-BA > NAA > AC > IAA; the best percent of bud inducing was obtained with medium 1/2MS + 6-BA 1.0mg/L+ NAA 0.2mg/L+IAA 0.3mg/L+AC 3.0g/L.
4. The buds of cv.‘Jin Ge’were used as explants, to study the effect of different basic medias and regulators on bud proliferation. The results showed that: the effect of factor on the proliferation of bud: NAA>basic medium>6-BA>IAA; the best percent of bud proliferation was obtained with medium 1/2MS+6-BA 1.0mg/L+NAA 0.5mg/L+GA3 0.2mg/L. In addition, different cycle of subculture had different impact on bud proliferation; the optimal cycle of subculture is thirty days.
5. The leaves and leafstalks of different cv.‘Da Hu Hong’, cv.‘Wu Long Peng Sheng’and cv.‘Jin Ge’were used as explants, to study the effect of different species and types on percent of callus inducing. The results showed that: cv.‘Jin Ge’had the best percent of callus inducing in the different species; leafstalk was better than leaf in the callus inducing.
6. Different basic medias were studied in the percent of callus inducing of cv.‘Jin Ge’. The results showed that: the optimal medium for leaf and leafstalk callus inducing are 1/2MS and WPM.
7. The leaves and leafstalks of cv.‘Jin Ge’were used as explants, to study the effect of different regulators and CH on the callus inducing. The results showed that: NAA, 2,4-D and 6-BA are the main effective factors in callus inducing by leaf, the optimal medium is 1/2MS+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+CH 300mg/L; 6-BA and 2,4-D are the main effective factors in callus inducing by leafstalk, the optimal medium is: WPM+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0 mg/L+TDZ 0.5mg/L+CH 300mg/L.
8. Different combination of regulators was studied in the callus differentiation of cv.‘Jin Ge’. The results showed that: the leafstalk had higher percent of callus differentiation than leaf in the same cultural case; 2,4-D 0.25mg/L+TDZ 0.5mg/L is helpful to the callus differentiation; In addition, different cycles of subculture had different effect on callus differentiation, the optimal turning is 30d after inoculating.
9. Different basic medias, regulators, sugar concentration and time of dark treatment were studied in the course of root inducing by cv.‘Jin Ge’test-tube plantlet. The results showed that: the effect of factor on the percent of root inducing: basic medium>IAA>IBA>sugar concentration>time of dark treatment; the effect of factor on the average length of root: basic medium>time of dark treatment >IAA>sugar concentration >IBA; the best medium for root inducing is: WPM+IBA 1.0mg/L+IAA 0.5 mg/L +sugar 20 g/L + dark culture 5d; the best medium for root length is: WPM +IBA 1.0mg/L+IAA 0.5mg/L+ sugar 40g/L+dark culture 10d. In addition, they were used to study on the percent of root inducing, IBA 2.0mg/L is hopeful to the root inducing by the adventitious buds from leafstalk callus of cv.‘Jin Ge’.
10. The test-tube plantlets were used as explants, to study training and transplant. The results showed that: the optimal training time is 12d in the temperature of 15~20℃and the moisture of 85%~90%; the best percent of transplant survival is the combination of perlite and vermiculite (1:1).
1.采用不同消毒剂和消毒时间进行消毒处理,结果表明:适合牡丹鳞芽的消毒剂为0.1%HgCl2,最佳消毒时间为8min;叶柄最佳消毒时间为5min;叶片则以3min为佳。
2.以牡丹品种“大胡红”、“乌龙捧盛”和“金阁”的鳞芽为材料,研究不同基本培养基类型和不同品种对鳞芽诱导的影响,结果表明:1/2MS为最佳基本培养基,牡丹品种“金阁”诱导率最高。
3.以牡丹品种“金阁”鳞芽为外植体,研究不同生长调节物质和不同浓度AC对鳞芽诱导的影响,结果表明:不同因素对牡丹鳞芽诱导率的影响不同,各因素的影响程度依次为:6-BA > NAA > AC >IAA;1/2MS+6-BA 1.0mg/L+NAA 0.2mg/L+IAA 0.3mg/L+AC 3.0g/L为牡丹鳞芽诱导的最佳培养基。
4.以牡丹品种“金阁”鳞芽为外植体,研究不同基本培养基类型和不同生长调节物质对鳞芽增殖的影响,结果表明:各个因素对鳞芽增殖率的影响为:NAA >基本培养基类型> 6-BA > NAA;培养基1/2MS+6-BA 1.0mg/L+NAA 0.5mg/L+GA3 0.2mg/L有利于牡丹鳞芽的增殖;另外,不同继代周期对牡丹鳞芽增殖倍数也有影响,以继代30d为宜。
5.以牡丹品种“大胡红”、“乌龙捧盛”和“金阁”的叶片和叶柄为外植体,比较不同品种和不同外植体类型对愈伤组织诱导的影响,结果表明:同一品种,叶柄愈伤组织诱导率较叶片高;牡丹品种“金阁”愈伤诱导率最高,为64.50%。
6.以牡丹品种“金阁”的叶片和叶柄为外植体,研究不同基本培养基类型对牡丹愈伤组织诱导率的影响,结果表明:适宜牡丹叶片愈伤诱导的基本培养基为1/2MS;WPM为叶柄愈伤诱导的最佳基本培养基。
7.以牡丹品种“金阁”的叶片和叶柄为外植体,研究不同生长调节物质和不同浓度CH对愈伤诱导的影响,结果表明:NAA、2,4-D和6-BA是影响叶片愈伤诱导率的主要因素;适合叶片愈伤诱导的最佳培养基为:1/2MS +2,4–D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+CH 300mg/L;叶柄愈伤诱导最佳培养基:WPM+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+TDZ 0.5mg/L+CH 300mg/L。
8.研究不同生长调节物质和不同浓度CH对愈伤诱导的影响,结果表明:NAA、2,4-D和6-BA是影响叶片愈伤诱导率的主要因素;适合叶片愈伤诱导的最佳培养基为:1/2MS +2,4-D2.0mg/L+6-BA1.0mg/L+NAA 1.0mg/L+CH 300mg/L;叶柄愈伤诱导最佳培养基:WPM+2,4-D2.0mg/L+6-BA1.0mg/L+NAA 1.0mg/L+TDZ 0.5mg/L+CH 300mg/L。
9.以牡丹品种“金阁”试管苗为材料,研究不同基本培养基的类型、不同生长调节物质、不同蔗糖浓度和不同暗处理时间对牡丹试管苗生根的影响,结果表明:各因素对于生根率的影响程度依次为:基本培养基类型> IAA> IBA >蔗糖浓度>暗处理时间;各因素对于平均根长的影响程度依次是:基本培养基类型>暗处理时间> IAA >蔗糖浓度> IBA;培养基WPM+IBA1.0 mg/L+IAA 0.5mg/L+蔗糖20g/L,暗培养5d有利于牡丹试管苗的诱导;培养基WPM+IBA 1.0mg/L+IAA 0.5mg/L+蔗糖40 g/L,暗培养10d有利于牡丹试管苗根的生长。另外,以叶柄愈伤组织诱导出的分化芽为材料进行生根诱导研究,发现IBA 2.0mg/L有利于根的诱导。
10.以生长健壮的牡丹生根试管苗为材料,进行炼苗及移栽试验,结果表明:在温度15~20℃、湿度85%~90%条件下,牡丹试管苗的最佳炼苗时间为12d;在珍珠岩:蛭石(1:1)基质中移栽成活率最高,达到50%。
The research took buds, leaves and leafstalks of different species of peony ( Paeonia Suffruticosa ) as explants to probe into the influence of different disinfectors and sterilized time on explants by Orthogonal design; Different basic medias, regulators, accessories and environmental factors were studied for their effects on buds of peony in the course of inducement, proliferation and inducing root, the main influence factors and effective concentration scale were analyzed. Further more to study the training and transplant of rooting peony; At the same time, the leaves and leafstalks of peony were used as explants in order to get the main influence factors and effective concentration scale in the callus inducement and differentiation, then adventitious buds from callus of leafstalk were induced root, to optimize the technique of callus inducement and differentiation. The results of experiments as follows:
1. The buds of peony were used as explants, to study the effect of different disinfectors and sterilized time on explants. The results showed that: the optimal disinfector and sterilized time for bud are 0.1%HgCl2 and 8min, the suitable time for leaf and leafstalk is 3min and 5min.
2. The buds of cv.‘Da Hu Hong’, cv.‘Wu Long Peng Sheng’and cv.‘Jin Ge’of peony were used as explants, to study the effect of different basic medias and species on bud inducing. The results showed that: the best medium is 1/2MS; the best percent of bud inducing is cv.‘Jin Ge’.
3. Different regulators and AC were studied in the course of bud inducing of cv.‘Jin Ge’. The results showed that: the impact of different factor on bud inducing is different, 6-BA > NAA > AC > IAA; the best percent of bud inducing was obtained with medium 1/2MS + 6-BA 1.0mg/L+ NAA 0.2mg/L+IAA 0.3mg/L+AC 3.0g/L.
4. The buds of cv.‘Jin Ge’were used as explants, to study the effect of different basic medias and regulators on bud proliferation. The results showed that: the effect of factor on the proliferation of bud: NAA>basic medium>6-BA>IAA; the best percent of bud proliferation was obtained with medium 1/2MS+6-BA 1.0mg/L+NAA 0.5mg/L+GA3 0.2mg/L. In addition, different cycle of subculture had different impact on bud proliferation; the optimal cycle of subculture is thirty days.
5. The leaves and leafstalks of different cv.‘Da Hu Hong’, cv.‘Wu Long Peng Sheng’and cv.‘Jin Ge’were used as explants, to study the effect of different species and types on percent of callus inducing. The results showed that: cv.‘Jin Ge’had the best percent of callus inducing in the different species; leafstalk was better than leaf in the callus inducing.
6. Different basic medias were studied in the percent of callus inducing of cv.‘Jin Ge’. The results showed that: the optimal medium for leaf and leafstalk callus inducing are 1/2MS and WPM.
7. The leaves and leafstalks of cv.‘Jin Ge’were used as explants, to study the effect of different regulators and CH on the callus inducing. The results showed that: NAA, 2,4-D and 6-BA are the main effective factors in callus inducing by leaf, the optimal medium is 1/2MS+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0mg/L+CH 300mg/L; 6-BA and 2,4-D are the main effective factors in callus inducing by leafstalk, the optimal medium is: WPM+2,4-D 2.0mg/L+6-BA 1.0mg/L+NAA 1.0 mg/L+TDZ 0.5mg/L+CH 300mg/L.
8. Different combination of regulators was studied in the callus differentiation of cv.‘Jin Ge’. The results showed that: the leafstalk had higher percent of callus differentiation than leaf in the same cultural case; 2,4-D 0.25mg/L+TDZ 0.5mg/L is helpful to the callus differentiation; In addition, different cycles of subculture had different effect on callus differentiation, the optimal turning is 30d after inoculating.
9. Different basic medias, regulators, sugar concentration and time of dark treatment were studied in the course of root inducing by cv.‘Jin Ge’test-tube plantlet. The results showed that: the effect of factor on the percent of root inducing: basic medium>IAA>IBA>sugar concentration>time of dark treatment; the effect of factor on the average length of root: basic medium>time of dark treatment >IAA>sugar concentration >IBA; the best medium for root inducing is: WPM+IBA 1.0mg/L+IAA 0.5 mg/L +sugar 20 g/L + dark culture 5d; the best medium for root length is: WPM +IBA 1.0mg/L+IAA 0.5mg/L+ sugar 40g/L+dark culture 10d. In addition, they were used to study on the percent of root inducing, IBA 2.0mg/L is hopeful to the root inducing by the adventitious buds from leafstalk callus of cv.‘Jin Ge’.
10. The test-tube plantlets were used as explants, to study training and transplant. The results showed that: the optimal training time is 12d in the temperature of 15~20℃and the moisture of 85%~90%; the best percent of transplant survival is the combination of perlite and vermiculite (1:1).
引文
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