牡丹体细胞胚间接再生途径研究
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摘要
本研究以牡丹品种‘洛阳红’、‘乌龙捧盛’、‘凤丹白’、‘珊瑚台’、‘胡红’等为材料,研究不同外植体类型及基本培养基类型、植物生长调节物质种类及浓度、不同光质比例及光照度等因素对牡丹愈伤组织诱导及增殖的影响,以期筛选出适宜于牡丹愈伤组织诱导、增殖的培养基配方及适宜的LED光质比例及光照度和适宜的CCFL光质比例,建立较为高效稳定的牡丹愈伤组织诱导体系。同时,以诱导出的愈伤组织为材料,研究不同基本培养基类型、植物生长调节物质种类及浓度、培养方式对愈伤组织丛诱导生芽及体细胞胚的影响,以期筛选出愈伤组织诱导丛生芽及体细胞的培养基配方及培养方式,建立牡丹体细胞胚间接再生途径,为解决牡丹试管苗生根困难提供一定的理论基础及技术参考。结果如下：
1.通过研究不同激素及浓度对牡丹品种‘洛阳红’愈伤组织诱导的影响,结果表明：5种不同的外植体类型中,叶柄各部分的整体愈伤组织诱导率均高于叶片及茎段组织；叶柄近茎端、中间端及近叶端中以近茎端的效果较好。单独使用6-BA即可获得愈伤组织；综合考虑愈伤组织诱导率及外植体褐变率情况,6-BA和2.4-D处理组合的愈伤组织诱导效应优于6-BA和NAA处理组合。在6-BA和2.4-D处理组合中,6-BA 1.0 mg·L-1+2.4-D1.0 mg·L-1处理是牡丹品种‘洛阳红’叶柄诱导愈伤组织的较好培养基。在牡丹愈伤组织增殖方面,NAA和6-BA处理组合优于NAA和KT处理,以NAA 1.0mg·L-1+6.BA 1.0 mg·L-1处理效果较好。
2.不同品种对牡丹愈伤组织诱导率的影响存在差异。牡丹品种‘胡红’的叶柄各部分及叶片的愈伤组织诱导率均高于‘乌龙捧盛’和‘洛阳红’,‘洛阳红’愈伤组织诱导效果最差。在不同外植体类型对牡丹愈伤组织诱导影响方面,‘乌龙捧盛’、‘洛阳红’及‘胡红’等三品种的叶柄愈伤组织诱导率均高于叶片,且叶柄的近茎端、中间端和近叶端的愈伤组织诱导率依次呈下降趋势；其中以‘胡红’的叶柄部位诱导率最高,达到62.2%。
3.在LED不同光质条件下,4种外植体类型中以叶柄中间端诱导愈伤组织效果最好；7种光质条件下以75%R+25%B处理的效果最好。黑暗处理及对照(PGF)处理下的牡丹愈伤组织诱导率均低于其他光质处理。在增殖方面,‘凤丹白’愈伤组织的增重及其增重倍率均在LED条件下以70%R+30%B处理最高,‘珊瑚台’愈伤组织增重效果在CCFL条件下以60%R+40%B处理最佳。而在一定LED光照度范围(30～80μmol·mol·s-1·m-2)内,牡丹品种‘凤丹白’愈伤组织增重及其增重倍率随着光照度的增强而整体呈先上升后下降的趋势。其中光照度为60μmol·s-1·m-2条件下的‘凤丹白’愈伤组织增重显著高于其他处理；但光照度较高时不利于愈伤组织的增殖。
4.两种培养方式下,‘凤丹白’愈伤组织的丛生芽诱导效果整体优于‘乌龙捧盛’固体培养条件下不同处理在以1/2 MS为基本培养基的丛生芽诱导效果整体优于MS培养基；综合丛生芽诱导率和愈伤组织玻璃化率、褐变率等影响因素,‘乌龙捧盛’愈伤组织的丛生芽诱导以MS+Ca2+-NAA 0.5 mg·L-1+6-BA 1.5 mg·L-1处理为最佳,‘凤丹白’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.1 mg·L-1+6-BA 0.5 mg·L-1处理效果最好。液体培养条件下,‘乌龙捧盛’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.5 mg·L-1+6-BA 1.0 mg·L-1处理为较佳,而‘凤丹白’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.1 mg·L-1+6-BA0.3 mg·L-1处理效果较好。同时发现愈伤组织的玻璃化率、褐变率与丛生芽诱导率之间具有一定的相关性。
5.两种培养方式下,‘凤丹白’愈伤组织的体细胞胚诱导效果均优于‘乌龙捧盛’在固体培养方式下,适宜于‘乌龙捧盛’愈伤组织诱导体胚的培养基为WPM>1/2MS>MS,适宜激素种类为6-BA>TDZ>KT;适宜于‘凤丹白’愈伤组织诱导体胚的培养基为1/2MS>WPM>MS,适宜激素种类为TDZ>6-BA>KT;综合考虑体胚诱导率、愈伤组织褐变率等因素,固体培养条件下‘乌龙捧盛’愈伤组织诱导体细胞胚的最佳培养基为WPM+6-BA 1.5 mg·L-1;‘凤丹白’愈伤组织诱导体细胞胚的最佳培养基为1/2MS+Ca2++TDZ 0.5 mg·L-1。液体培养条件下,‘乌龙捧盛’愈伤组织的体胚诱导效果以1/2MS+Ca2++TDZ 0.1 mg·L-1处理为最佳,‘凤丹白’愈伤组织的体胚诱导效果以1/2MS+Ca2++6-BA 1.5 mg·L-1处理下达到最高。同时发现愈伤组织褐变率与球形胚、子叶胚诱导率具有一定的相关性。
We investigated the in vitro callus induction and proliferation of tree peony (Paeonia suffruticosa) by the facts as explants types, basic medium types, plant growth regulator types and concentrations, light quality ratios and light intensity, five cultivars ('Luo Yang Hhong','Wu Long Peng Sheng','Feng Dan Bai','Hu Hong' and 'Shan Hu Tai') as the material, to select a suitable medium formula, light quality ratios of light-emitting diodes (LED) and cold cathode fluorescent lamps (CCFL) and LED illuminations for building a effective and stable callus induction system of tree peony. And we also studied the effect of basic medium types, plant growth regulator types and concentrations, culture methods on the adventitious buds and somatic embryos induction from callus in order to building a effective and stable somatic embryos induction system of tree peony. Results are as follows:
1. By studying the effect of hormones types and concentrations on callus induction of Paeonia suffruticosa,'Luo Yang Hong', we found that the induction rates of callus from the parts of petiole were higher than leaf and stem's in five explants types; the induction rates of callus from the part neared the stem was better than other petiole parts which neared leaf or middle side. Callus of tree peony could be obtained by only using 6-BA, and effect of callus induction of 6-BA with 2,4-D treatments were better than 6-BA with NAA treatments by comprehensive considering with callus induction rate and explants browning rate.6-BA 1.0 mg·L-1 add 2,4-D 1.0 mg·L-1 treatment was the better one for Paeonia suffruticosa 'Luo Yang Hong' petiole to induce callus among the 6-BA with 2,4-D treatments, and NAA1.0 mg·L-1 add 6-BA 1.0 mg·L-1 treatment was best one for callus proliferation among the NAA with 6-BA treatments.
2. It had a significant difference on the rate of callus induction by Paeonia suffruticosa varieties. The callus induction rates from the petiole parts and leaf of Paeonia suffruticosa 'Hu Hong' were higher than Paeonia suffruticosa "Wu Long Peng Sheng' and 'Luo Yang Hong','Luo Yang Hong' was the worst. Callus induction rates from petiole of'Wu Long Peng Sheng','Luo Yang Hong' and 'Hu Hong' were all higher than leaf's, and it had a decreased tendency of callus induction rates from the petiole part neared stem, middle side to the part neared leaf, and the induction rate of petiole callus from 'Hong Hu' was highest (62.2%).
3. Effect of callus induction from the middle part of the petiole was the best one among the four explants types in different LED light quality conditions, and effect of callus induction under 75% R add 25% B treatment was also the best one among the seven light quality ratios. The rate of callus induction under dark treatment and control (PGF) treatments were less than others. In the proliferation, the increasing weight and increasing rate of Paeonia suffruticosa 'Feng Dan Bai' callus in the 70% R add 30% B treatment was the highest under LED conditions, and that of Paeonia suffruticosa 'Shan Hu Tai' in the 60% R add 40% B treatment was also the best one under CCFL conditions. When the illumination in a certain range from 30μmol·s-1·m-2 to 80μmol·s-1·m-2, it had a the overall increased first and then decreased trend of the increasing weight and increasing rate of Paeonia suffruticosa 'Feng Dan Bai' callus with increasing illumination, and that of Paeonia suffruticosa 'Feng Dan Bai' callus under the conditions of 60μmol·s-1·m-2 treatment were significantly higher than others; however, the higher illumination was not conducive to the callus proliferation.
4. Effect of the adventitious buds induction from Paeonia suffruticosa 'Feng Dan Bai' callus was better than that of Paeonia suffruticosa 'Wu Long Peng Sheng' callus with two culture methods that was solid culture method or liquid culture method. And effect of different treatments on the adventitious buds induction from Paeonia suffruticosa callus based on 1/2 MS basic medium was better than that of MS medium under solid culture method, meanwhile, the induction rate of adventitious buds from Paeonia suffruticosa 'Wu Long Peng Sheng' was best one with MS add Ca2+(WPM) add NAA 0.5 mg·L-1 add 6-BA 1.5 mg·L-1 treatment, and that of Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add NAA 0.1 mg·L add 6-BA 0.5 mg·L-1 by considering the adventitious buds induction rate, glass-like rate and browning rate of callus. The treatment with 1/2MS add Ca2+(WPM) add NAA 0.5 mg·L-1 add 6-BA1.0 mg·L-1 was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce adventitious buds, and the best one for Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add NAA 0.1 mg·L-1 add 6-BA 0.3 mg·L-1 under liquid culture method. It was a certainly correlation among the rate of glass-like and browning on callus and the adventitious buds induction rate.
5. Effect of the somatic embryos induction from Paeonia suffruticosa 'Feng Dan Bai' callus was better than that of Paeonia suffruticosa 'Wu Long Peng Sheng' callus with two culture methods that was solid culture method or liquid culture method. And under solid culture method, WPM medium was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, followed by 1/2MS medium, and then was MS medium. The hormone of 6-BA was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, followed by TDZ, and then was KT. Effect of induction medium of somatic embryos for Paeonia suffruticosa 'Feng Dan Bai' was 1/2MS medium, WPM medium and MS medium, and effect of hormone on Paeonia suffruticosa 'Feng Dan Bai' somatic embryos induction was TDZ,6-BA and KT. And considering the somatic embryos induction rate and callus browning rate, the induction rate of somatic embryos from Paeonia suffruticosa 'Wu Long Peng Sheng' was best one with WPM add 6-BA 1.5 mg·L-1 treatment, and that of Paeonia suffruticosa 'Feng Dan Bai' was the treatment withl/2MS add Ca2+(WPM) add TDZ 0.5 mg·L-1 under solid culture method. The treatment with 1/2MS add Ca2+(WPM) add TDZ 0.1 mg·L-1 was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, and the best one for Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add 6-BA 1.5 mg·L-1 under liquid culture method. Meanwhile, It also had a certainly correlation among the callus browning rate and the induction rate of globular embryo and cotyledon embryo.
1.通过研究不同激素及浓度对牡丹品种‘洛阳红’愈伤组织诱导的影响,结果表明：5种不同的外植体类型中,叶柄各部分的整体愈伤组织诱导率均高于叶片及茎段组织；叶柄近茎端、中间端及近叶端中以近茎端的效果较好。单独使用6-BA即可获得愈伤组织；综合考虑愈伤组织诱导率及外植体褐变率情况,6-BA和2.4-D处理组合的愈伤组织诱导效应优于6-BA和NAA处理组合。在6-BA和2.4-D处理组合中,6-BA 1.0 mg·L-1+2.4-D1.0 mg·L-1处理是牡丹品种‘洛阳红’叶柄诱导愈伤组织的较好培养基。在牡丹愈伤组织增殖方面,NAA和6-BA处理组合优于NAA和KT处理,以NAA 1.0mg·L-1+6.BA 1.0 mg·L-1处理效果较好。
2.不同品种对牡丹愈伤组织诱导率的影响存在差异。牡丹品种‘胡红’的叶柄各部分及叶片的愈伤组织诱导率均高于‘乌龙捧盛’和‘洛阳红’,‘洛阳红’愈伤组织诱导效果最差。在不同外植体类型对牡丹愈伤组织诱导影响方面,‘乌龙捧盛’、‘洛阳红’及‘胡红’等三品种的叶柄愈伤组织诱导率均高于叶片,且叶柄的近茎端、中间端和近叶端的愈伤组织诱导率依次呈下降趋势；其中以‘胡红’的叶柄部位诱导率最高,达到62.2%。
3.在LED不同光质条件下,4种外植体类型中以叶柄中间端诱导愈伤组织效果最好；7种光质条件下以75%R+25%B处理的效果最好。黑暗处理及对照(PGF)处理下的牡丹愈伤组织诱导率均低于其他光质处理。在增殖方面,‘凤丹白’愈伤组织的增重及其增重倍率均在LED条件下以70%R+30%B处理最高,‘珊瑚台’愈伤组织增重效果在CCFL条件下以60%R+40%B处理最佳。而在一定LED光照度范围(30～80μmol·mol·s-1·m-2)内,牡丹品种‘凤丹白’愈伤组织增重及其增重倍率随着光照度的增强而整体呈先上升后下降的趋势。其中光照度为60μmol·s-1·m-2条件下的‘凤丹白’愈伤组织增重显著高于其他处理；但光照度较高时不利于愈伤组织的增殖。
4.两种培养方式下,‘凤丹白’愈伤组织的丛生芽诱导效果整体优于‘乌龙捧盛’固体培养条件下不同处理在以1/2 MS为基本培养基的丛生芽诱导效果整体优于MS培养基；综合丛生芽诱导率和愈伤组织玻璃化率、褐变率等影响因素,‘乌龙捧盛’愈伤组织的丛生芽诱导以MS+Ca2+-NAA 0.5 mg·L-1+6-BA 1.5 mg·L-1处理为最佳,‘凤丹白’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.1 mg·L-1+6-BA 0.5 mg·L-1处理效果最好。液体培养条件下,‘乌龙捧盛’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.5 mg·L-1+6-BA 1.0 mg·L-1处理为较佳,而‘凤丹白’愈伤组织的丛生芽诱导率以1/2MS+Ca2++NAA 0.1 mg·L-1+6-BA0.3 mg·L-1处理效果较好。同时发现愈伤组织的玻璃化率、褐变率与丛生芽诱导率之间具有一定的相关性。
5.两种培养方式下,‘凤丹白’愈伤组织的体细胞胚诱导效果均优于‘乌龙捧盛’在固体培养方式下,适宜于‘乌龙捧盛’愈伤组织诱导体胚的培养基为WPM>1/2MS>MS,适宜激素种类为6-BA>TDZ>KT;适宜于‘凤丹白’愈伤组织诱导体胚的培养基为1/2MS>WPM>MS,适宜激素种类为TDZ>6-BA>KT;综合考虑体胚诱导率、愈伤组织褐变率等因素,固体培养条件下‘乌龙捧盛’愈伤组织诱导体细胞胚的最佳培养基为WPM+6-BA 1.5 mg·L-1;‘凤丹白’愈伤组织诱导体细胞胚的最佳培养基为1/2MS+Ca2++TDZ 0.5 mg·L-1。液体培养条件下,‘乌龙捧盛’愈伤组织的体胚诱导效果以1/2MS+Ca2++TDZ 0.1 mg·L-1处理为最佳,‘凤丹白’愈伤组织的体胚诱导效果以1/2MS+Ca2++6-BA 1.5 mg·L-1处理下达到最高。同时发现愈伤组织褐变率与球形胚、子叶胚诱导率具有一定的相关性。
We investigated the in vitro callus induction and proliferation of tree peony (Paeonia suffruticosa) by the facts as explants types, basic medium types, plant growth regulator types and concentrations, light quality ratios and light intensity, five cultivars ('Luo Yang Hhong','Wu Long Peng Sheng','Feng Dan Bai','Hu Hong' and 'Shan Hu Tai') as the material, to select a suitable medium formula, light quality ratios of light-emitting diodes (LED) and cold cathode fluorescent lamps (CCFL) and LED illuminations for building a effective and stable callus induction system of tree peony. And we also studied the effect of basic medium types, plant growth regulator types and concentrations, culture methods on the adventitious buds and somatic embryos induction from callus in order to building a effective and stable somatic embryos induction system of tree peony. Results are as follows:
1. By studying the effect of hormones types and concentrations on callus induction of Paeonia suffruticosa,'Luo Yang Hong', we found that the induction rates of callus from the parts of petiole were higher than leaf and stem's in five explants types; the induction rates of callus from the part neared the stem was better than other petiole parts which neared leaf or middle side. Callus of tree peony could be obtained by only using 6-BA, and effect of callus induction of 6-BA with 2,4-D treatments were better than 6-BA with NAA treatments by comprehensive considering with callus induction rate and explants browning rate.6-BA 1.0 mg·L-1 add 2,4-D 1.0 mg·L-1 treatment was the better one for Paeonia suffruticosa 'Luo Yang Hong' petiole to induce callus among the 6-BA with 2,4-D treatments, and NAA1.0 mg·L-1 add 6-BA 1.0 mg·L-1 treatment was best one for callus proliferation among the NAA with 6-BA treatments.
2. It had a significant difference on the rate of callus induction by Paeonia suffruticosa varieties. The callus induction rates from the petiole parts and leaf of Paeonia suffruticosa 'Hu Hong' were higher than Paeonia suffruticosa "Wu Long Peng Sheng' and 'Luo Yang Hong','Luo Yang Hong' was the worst. Callus induction rates from petiole of'Wu Long Peng Sheng','Luo Yang Hong' and 'Hu Hong' were all higher than leaf's, and it had a decreased tendency of callus induction rates from the petiole part neared stem, middle side to the part neared leaf, and the induction rate of petiole callus from 'Hong Hu' was highest (62.2%).
3. Effect of callus induction from the middle part of the petiole was the best one among the four explants types in different LED light quality conditions, and effect of callus induction under 75% R add 25% B treatment was also the best one among the seven light quality ratios. The rate of callus induction under dark treatment and control (PGF) treatments were less than others. In the proliferation, the increasing weight and increasing rate of Paeonia suffruticosa 'Feng Dan Bai' callus in the 70% R add 30% B treatment was the highest under LED conditions, and that of Paeonia suffruticosa 'Shan Hu Tai' in the 60% R add 40% B treatment was also the best one under CCFL conditions. When the illumination in a certain range from 30μmol·s-1·m-2 to 80μmol·s-1·m-2, it had a the overall increased first and then decreased trend of the increasing weight and increasing rate of Paeonia suffruticosa 'Feng Dan Bai' callus with increasing illumination, and that of Paeonia suffruticosa 'Feng Dan Bai' callus under the conditions of 60μmol·s-1·m-2 treatment were significantly higher than others; however, the higher illumination was not conducive to the callus proliferation.
4. Effect of the adventitious buds induction from Paeonia suffruticosa 'Feng Dan Bai' callus was better than that of Paeonia suffruticosa 'Wu Long Peng Sheng' callus with two culture methods that was solid culture method or liquid culture method. And effect of different treatments on the adventitious buds induction from Paeonia suffruticosa callus based on 1/2 MS basic medium was better than that of MS medium under solid culture method, meanwhile, the induction rate of adventitious buds from Paeonia suffruticosa 'Wu Long Peng Sheng' was best one with MS add Ca2+(WPM) add NAA 0.5 mg·L-1 add 6-BA 1.5 mg·L-1 treatment, and that of Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add NAA 0.1 mg·L add 6-BA 0.5 mg·L-1 by considering the adventitious buds induction rate, glass-like rate and browning rate of callus. The treatment with 1/2MS add Ca2+(WPM) add NAA 0.5 mg·L-1 add 6-BA1.0 mg·L-1 was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce adventitious buds, and the best one for Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add NAA 0.1 mg·L-1 add 6-BA 0.3 mg·L-1 under liquid culture method. It was a certainly correlation among the rate of glass-like and browning on callus and the adventitious buds induction rate.
5. Effect of the somatic embryos induction from Paeonia suffruticosa 'Feng Dan Bai' callus was better than that of Paeonia suffruticosa 'Wu Long Peng Sheng' callus with two culture methods that was solid culture method or liquid culture method. And under solid culture method, WPM medium was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, followed by 1/2MS medium, and then was MS medium. The hormone of 6-BA was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, followed by TDZ, and then was KT. Effect of induction medium of somatic embryos for Paeonia suffruticosa 'Feng Dan Bai' was 1/2MS medium, WPM medium and MS medium, and effect of hormone on Paeonia suffruticosa 'Feng Dan Bai' somatic embryos induction was TDZ,6-BA and KT. And considering the somatic embryos induction rate and callus browning rate, the induction rate of somatic embryos from Paeonia suffruticosa 'Wu Long Peng Sheng' was best one with WPM add 6-BA 1.5 mg·L-1 treatment, and that of Paeonia suffruticosa 'Feng Dan Bai' was the treatment withl/2MS add Ca2+(WPM) add TDZ 0.5 mg·L-1 under solid culture method. The treatment with 1/2MS add Ca2+(WPM) add TDZ 0.1 mg·L-1 was the best one for Paeonia suffruticosa 'Wu Long Peng Sheng' callus to induce somatic embryos, and the best one for Paeonia suffruticosa 'Feng Dan Bai' was the treatment with 1/2MS add Ca2+(WPM) add 6-BA 1.5 mg·L-1 under liquid culture method. Meanwhile, It also had a certainly correlation among the callus browning rate and the induction rate of globular embryo and cotyledon embryo.
引文
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