发根农杆菌介导的菘蓝（Isatis indigotica Fort.）遗传转化及植株再生体系的建立
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摘要
菘蓝(Isatis indigotica Fort．)不仅是常用的中药材，而且也是工业用染料的重要原料之一，用量极大，在全国范围内广为栽培。本文利用发根农杆菌Ri T-DNA建立并优化了四倍体菘蓝的遗传转化体系，并对再生植株的主要代谢产物靛蓝、靛玉红进行了含量检测，筛选了优良无性系，也为实现外源抗虫、抗病等基因的导入奠定了基础。
1 建立并筛选了四倍体菘蓝毛状根优良无性系
以四倍体菘蓝无菌苗的子叶为外植体，应用发根农杆菌A4和R1000成功地诱导出了毛状根，结果表明，A4菌株对菘蓝毛状根的诱导优于R1000，毛状根的诱导频率高达100％，得根率高达30以上；添加0.5mg／l的IAA能提高毛状根的诱导频率；光照对四倍体菘蓝毛状根的诱导极为重要，缺光条件下不能诱导出毛状根；提取毛状根DNA进行PCR分析，检测到了rolB、rolC基因，表明毛状根确系转化根。毛状根能在不含任何激素的MS固／液体培养基中快速生长，且表现出多分支，多根毛，失去向地性生长等典型毛状根特性；建立了TRA1,TRA2,TRA3,TRA4四个毛状根无性系。对四倍体毛状根和二倍体毛状根作了比较，四倍体菘蓝毛状根生长速度大于二倍体。
2 建立并优化了毛状根植株再生体系
通过三种途径可获得毛状根再生植株。
(1) 从毛状根上直接分化出不定芽和根。毛状根约一个月继代一次后能自发地分化出少量不定芽。90d后统计不定芽数量，平均每30个根段培养物上可获得21个不定芽；添加0.5mg／16-BA能显著地促进毛状根不定芽的分化，配合使用6-BA和NAA对毛状根不定芽的分化最为有效(MS+1.0mg／l NAA+0.5mg／l 6-BA)，90d后统计，平均每30个根段培养物上可获得67个不定芽。
(2) 通过毛状根愈伤组织途径再生植株。毛状根在不含激素的培养基上不能自发地
西南师范大学硕士学位论文 发根农杆菌介导的花蓝坦传转化及植株再生体系的建立
形成愈伤组织：2个D对毛状根愈伤组织诱导极为明显，6石 对愈伤组织的诱导作用不
明显，但2，4－D和 6EA配合使用对愈伤组织诱导有利。愈伤组织诱导最佳培养基为：
MS＋05mg／l个D＋05mg／l－BA，愈伤组织生长的最佳培养基为 MS＋0 ling／12，4－D＋
cling／16EA；愈伤组织能在不含激素的培养基上自发地分化出不定芽，但分化频率较低，
而 6EA对不定芽的分化作用较明显，配合使用 6习A和 NAA对芽的诱导及得芽率较好，
不定芽诱导的最佳培养基为 MS＋10mgil NAA＋05mg／lIA培养基。不定芽生根的最
佳培养基为 MS川 NAA＋05mg／l IAA。
门）诱导毛状根体细胞胚再生植株。通过正交实验筛选出了毛状根体细胞胚诱导的
最佳培养基为：MS＋05mg／l NAA＋0．sing／ KT＋30gil蔗糖。单独使用 NAA很难诱导出
体胚，单独使用KT或KT与NAA配合使用均能诱导出体胚，NAA、KT和蔗糖对体胚
诱导的影响作用依次是＊T＞＊＊A＞蔗糖。＊T、＊＊A浓度和蔗糖对体胚的发育均有较明
显的作用。对体胚的发育最好在cling／l的KT浓度下或不含激素的培养基中，蔗糖浓度
最好在 30g／l。
比较了三种途径再生的植株，由体胚再生的植株不仅耗时短，且获得再生植株量大，
再生植株成活率高，生长健壮，是毛状根植株再生体系的最佳途径；由毛状根直接再生
植株耗时较长，且获得再生植株少：而由毛状根愈伤组织再生的植株耗时虽不长，不定
芽数目较多，但植株纤细，成活率很低。
3 对再生植株进行了rol基因鉴定及靛蓝、靛玉红含量测定
通过对再生植株的PCR测定，三种途径再生的植株以及表型正常和不正常的植株核
基因组上均整合进了T－DNA片段；eLC分析表明，再生植株叶片中靛蓝、靛玉红含量
各为43333mg／gw和2门6＊＊ 叩w，分别为对照植株的1引倍和1叩倍：根中的靛
蓝、靛王红含量分别为 00666mg／gDW和 00143mg／gDW，分别是对照植株的2门倍和
122倍。表明通过应用发根农杆菌介导的遗传转化可获得次生代谢产物含量高而稳定的
优良无性系。
Indigoblue woad(/sato indigotica Fort.) is not only a largely used medicinal plant, but also an important industrial material, used as dyer's-weed in China. It is widely cultured in our country. In this paper, genetic transformation systems by Agrobacterhim rhizogems was established and optimized and high quality regenerating system was selected. The products of indigotin and indirubin in regenerating plantlets had been detected by HPLC. The main studies and results were reported as follows: 1 Establishment and selection of hairy root clones
Taken cotyledons of autotetraploid indigoblue woad as explants and hairy roots induced successfully from them by Agrobaterium rhizogenes strains A4 and R1000. A4 had better root-inducing capability than R1000. The percentage of root induction by A4 was 100% and average root number per cotyledon was 30. Both IAA and light had significant effects on root-inducing efficiency. Percentage of root induction could be enhanced with MS medium supplemented with 0.5mg/l IAA. No root occurred without light. DNA of hairy roots for polymerase chain reaction (PCR) analysis was extracted. The presence of the rolB and rolC genes in the hairy roots was confirmed by PCR analysis. The positive result obtained in the amplification of the rolB and rolC in hairy roots could be attributed to successful transformation rather than to residual Agrobacterhim. These hairy roots grew well on MS agar medium or liquid medium withour growth regulator for over one year. They grew vigorously and produced numerous lateral branches. Four hairy root clones, namely TRA1, TRA2,TRA3 and TRA4, were established. Compared to hairy root system HRA3 which came from diploid
indigoblue woad(2n=14), hairy roots from autotetraploid indigoblue woad(2n=28) grew faster. It was shown that hairy root of autotetraploid indigoblue woad was better than that of diploid indigoblue woad. 2 Establishment and optimization of regenerating plantlet system from hairy roots.
(1) Differentiation of bud and root directly from hairy root. Hairy roots were subcultured every month. Twenty one adventitious buds from 30 root segment cultures were obtained after subculturing on medium lacking growth regulator for 90 days. The effects of hormones 6-BA and NAA on the adventitious bud differentiation were investigated and optimized. The results were showed that 6-BA had significant effect on bud differentiation and the optimized medium for adventitious bud differentiation was MS + l.0mg/l NAA + 0.5mg/L 6-BA. There were 67 adventitious buds occurred from average 30 hairy root segment cultures.
(2) Regenerating plantlets were obtained from calli, which derived of hairy roots. The results of bud induction showed that 2,4-D had the significant effects on callus formation and followed bud differentiation. No callus occurred from hairy roots on medium lacking 2,4-D. High concentration (l.0mg/1) of 2,4-D could induce much more calli, but it obstructed the growth of calli and followed buds differentiation. The result of callus induction showed that the optimum medium for callus induction was MS + 0.5mg/l 2,4-D + 0.5mg/l 6-BA and MS + 0. 1mg/1 2,4-D + 0. 1mg/l 6-BA for callus growth. Adventitious buds could be spontaneously formed from calli on MS medium lacking hormones. Despite of it, the bud-inducing frequency was low. High frequency of bud induction was found on MS medium supplemented with 6-BA The optimum medium for adventitious bud induction was MS + l.0mg/1 NAA + 0.5mg/l 6-BA, and MS + 1.5mg/l NAA + 0.5mg/l IAA for bud rooting.
(3) Regenerating plantlets were obtained via somatic embryo, which derived from hairy root. The effects on the induction of somatic embryos from hairy root were investigated using orthogonal test. The best somatic embryo induction was obtained on MS + 0.5mg/l NAA + 0.5mg/l KT + 30g/l sucrose. Among the three factors tested in our experiments, KT had the significant effect on the somatic embryo induction and its development, followed by NAA Sucrose had smallest effect on somatic embryo induction, but had the obviou
1 建立并筛选了四倍体菘蓝毛状根优良无性系
以四倍体菘蓝无菌苗的子叶为外植体，应用发根农杆菌A4和R1000成功地诱导出了毛状根，结果表明，A4菌株对菘蓝毛状根的诱导优于R1000，毛状根的诱导频率高达100％，得根率高达30以上；添加0.5mg／l的IAA能提高毛状根的诱导频率；光照对四倍体菘蓝毛状根的诱导极为重要，缺光条件下不能诱导出毛状根；提取毛状根DNA进行PCR分析，检测到了rolB、rolC基因，表明毛状根确系转化根。毛状根能在不含任何激素的MS固／液体培养基中快速生长，且表现出多分支，多根毛，失去向地性生长等典型毛状根特性；建立了TRA1,TRA2,TRA3,TRA4四个毛状根无性系。对四倍体毛状根和二倍体毛状根作了比较，四倍体菘蓝毛状根生长速度大于二倍体。
2 建立并优化了毛状根植株再生体系
通过三种途径可获得毛状根再生植株。
(1) 从毛状根上直接分化出不定芽和根。毛状根约一个月继代一次后能自发地分化出少量不定芽。90d后统计不定芽数量，平均每30个根段培养物上可获得21个不定芽；添加0.5mg／16-BA能显著地促进毛状根不定芽的分化，配合使用6-BA和NAA对毛状根不定芽的分化最为有效(MS+1.0mg／l NAA+0.5mg／l 6-BA)，90d后统计，平均每30个根段培养物上可获得67个不定芽。
(2) 通过毛状根愈伤组织途径再生植株。毛状根在不含激素的培养基上不能自发地
西南师范大学硕士学位论文 发根农杆菌介导的花蓝坦传转化及植株再生体系的建立
形成愈伤组织：2个D对毛状根愈伤组织诱导极为明显，6石 对愈伤组织的诱导作用不
明显，但2，4－D和 6EA配合使用对愈伤组织诱导有利。愈伤组织诱导最佳培养基为：
MS＋05mg／l个D＋05mg／l－BA，愈伤组织生长的最佳培养基为 MS＋0 ling／12，4－D＋
cling／16EA；愈伤组织能在不含激素的培养基上自发地分化出不定芽，但分化频率较低，
而 6EA对不定芽的分化作用较明显，配合使用 6习A和 NAA对芽的诱导及得芽率较好，
不定芽诱导的最佳培养基为 MS＋10mgil NAA＋05mg／lIA培养基。不定芽生根的最
佳培养基为 MS川 NAA＋05mg／l IAA。
门）诱导毛状根体细胞胚再生植株。通过正交实验筛选出了毛状根体细胞胚诱导的
最佳培养基为：MS＋05mg／l NAA＋0．sing／ KT＋30gil蔗糖。单独使用 NAA很难诱导出
体胚，单独使用KT或KT与NAA配合使用均能诱导出体胚，NAA、KT和蔗糖对体胚
诱导的影响作用依次是＊T＞＊＊A＞蔗糖。＊T、＊＊A浓度和蔗糖对体胚的发育均有较明
显的作用。对体胚的发育最好在cling／l的KT浓度下或不含激素的培养基中，蔗糖浓度
最好在 30g／l。
比较了三种途径再生的植株，由体胚再生的植株不仅耗时短，且获得再生植株量大，
再生植株成活率高，生长健壮，是毛状根植株再生体系的最佳途径；由毛状根直接再生
植株耗时较长，且获得再生植株少：而由毛状根愈伤组织再生的植株耗时虽不长，不定
芽数目较多，但植株纤细，成活率很低。
3 对再生植株进行了rol基因鉴定及靛蓝、靛玉红含量测定
通过对再生植株的PCR测定，三种途径再生的植株以及表型正常和不正常的植株核
基因组上均整合进了T－DNA片段；eLC分析表明，再生植株叶片中靛蓝、靛玉红含量
各为43333mg／gw和2门6＊＊ 叩w，分别为对照植株的1引倍和1叩倍：根中的靛
蓝、靛王红含量分别为 00666mg／gDW和 00143mg／gDW，分别是对照植株的2门倍和
122倍。表明通过应用发根农杆菌介导的遗传转化可获得次生代谢产物含量高而稳定的
优良无性系。
Indigoblue woad(/sato indigotica Fort.) is not only a largely used medicinal plant, but also an important industrial material, used as dyer's-weed in China. It is widely cultured in our country. In this paper, genetic transformation systems by Agrobacterhim rhizogems was established and optimized and high quality regenerating system was selected. The products of indigotin and indirubin in regenerating plantlets had been detected by HPLC. The main studies and results were reported as follows: 1 Establishment and selection of hairy root clones
Taken cotyledons of autotetraploid indigoblue woad as explants and hairy roots induced successfully from them by Agrobaterium rhizogenes strains A4 and R1000. A4 had better root-inducing capability than R1000. The percentage of root induction by A4 was 100% and average root number per cotyledon was 30. Both IAA and light had significant effects on root-inducing efficiency. Percentage of root induction could be enhanced with MS medium supplemented with 0.5mg/l IAA. No root occurred without light. DNA of hairy roots for polymerase chain reaction (PCR) analysis was extracted. The presence of the rolB and rolC genes in the hairy roots was confirmed by PCR analysis. The positive result obtained in the amplification of the rolB and rolC in hairy roots could be attributed to successful transformation rather than to residual Agrobacterhim. These hairy roots grew well on MS agar medium or liquid medium withour growth regulator for over one year. They grew vigorously and produced numerous lateral branches. Four hairy root clones, namely TRA1, TRA2,TRA3 and TRA4, were established. Compared to hairy root system HRA3 which came from diploid
indigoblue woad(2n=14), hairy roots from autotetraploid indigoblue woad(2n=28) grew faster. It was shown that hairy root of autotetraploid indigoblue woad was better than that of diploid indigoblue woad. 2 Establishment and optimization of regenerating plantlet system from hairy roots.
(1) Differentiation of bud and root directly from hairy root. Hairy roots were subcultured every month. Twenty one adventitious buds from 30 root segment cultures were obtained after subculturing on medium lacking growth regulator for 90 days. The effects of hormones 6-BA and NAA on the adventitious bud differentiation were investigated and optimized. The results were showed that 6-BA had significant effect on bud differentiation and the optimized medium for adventitious bud differentiation was MS + l.0mg/l NAA + 0.5mg/L 6-BA. There were 67 adventitious buds occurred from average 30 hairy root segment cultures.
(2) Regenerating plantlets were obtained from calli, which derived of hairy roots. The results of bud induction showed that 2,4-D had the significant effects on callus formation and followed bud differentiation. No callus occurred from hairy roots on medium lacking 2,4-D. High concentration (l.0mg/1) of 2,4-D could induce much more calli, but it obstructed the growth of calli and followed buds differentiation. The result of callus induction showed that the optimum medium for callus induction was MS + 0.5mg/l 2,4-D + 0.5mg/l 6-BA and MS + 0. 1mg/1 2,4-D + 0. 1mg/l 6-BA for callus growth. Adventitious buds could be spontaneously formed from calli on MS medium lacking hormones. Despite of it, the bud-inducing frequency was low. High frequency of bud induction was found on MS medium supplemented with 6-BA The optimum medium for adventitious bud induction was MS + l.0mg/1 NAA + 0.5mg/l 6-BA, and MS + 1.5mg/l NAA + 0.5mg/l IAA for bud rooting.
(3) Regenerating plantlets were obtained via somatic embryo, which derived from hairy root. The effects on the induction of somatic embryos from hairy root were investigated using orthogonal test. The best somatic embryo induction was obtained on MS + 0.5mg/l NAA + 0.5mg/l KT + 30g/l sucrose. Among the three factors tested in our experiments, KT had the significant effect on the somatic embryo induction and its development, followed by NAA Sucrose had smallest effect on somatic embryo induction, but had the obviou
引文
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