菘蓝组培快繁体系的建立及四倍体株系选育和品质鉴定
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摘要
菘蓝(Isatis indigotica Fortune)为十字花科(Brassicaceae)菘蓝属二年生草本植物,根称“板蓝根”,叶叫“大青叶”,是常用传统中药材。近年来,由于菘蓝品种长期只种不选,自留自用,产量和品质大大降低。因此培育高产、优质、遗传性状稳定的新品种是当务之急。植物多倍体具有生物量高、抗逆性强、药用活性成分含量增多等优点,为药用植物的开发与利用提供了一条新的途径。本研究围绕菘蓝多倍体株系的选育,建立了菘蓝组培快繁及栽培技术体系,利用秋水仙碱成功诱导、鉴定了16个同源四倍体,进行了遗传稳定性和品质评价分析,获得了优良的菘蓝四倍体育种材料。
(1)建立了菘蓝的无性快繁及栽培技术体系。外植体愈伤组织诱导的适宜外植体为下胚轴；诱导愈伤组织最佳培养基配方为MS+6-BA1.0mg/L+2,4-D0.8mg/L;诱导愈伤及分化最佳培养基配方为MS+6-B A2.0mg/L+NAA0.2mg/L;最佳生根培养基配方为1/2MS+NAA 0.2mg/L+IBA 0.2mg/L;最佳炼苗时间为4d,长势最佳的移栽基质为蛭石：珍珠岩=1：2；可明显促进菘蓝移栽苗的生长的营养液配方为Ca(NO3)2·4H2O 945mg/L+KNO3 607mg/L+NH4H2PO4 115mg/L+MgSO4·7H2O 493mg/L;菘蓝适宜的种植密度为行距30cm×株距10(或20)cm；华北地区适宜采收期10月15-20日。
(2)人工诱导、鉴定和筛选了16个菘蓝纯合同源四倍体。以90mg／L秋水仙碱加入培养基诱导28d,菘蓝染色体的加倍率可达28.30%；菘蓝四倍体试管苗特征：生长慢,芽粗短,胚轴膨大,叶片肥厚宽大皱褶,叶缘呈锯齿状,色泽深绿,叶脉粗；四倍体田间移栽苗特征：叶色深绿,叶片宽厚变形,叶面粗糙,叶缘锯齿状,整株生长势旺盛,分蘖能力强,冬性较强；四倍体叶部细胞显微结构特征：气孔巨大、保卫细胞的叶绿体数目增多；四倍体染色体倍性鉴定方法：根尖常规压片法和幼叶流式细胞仪法。
(3)用ISSR分子标记技术分析了菘蓝四倍体株系遗传稳定性。改良CTAB法是菘蓝DNA最佳的提取方法；ISSR反应的最佳体系为：25μl体系中含1×buffer2.5(不含MgCl2), MgCl2 2.0mmol·L-1、引物浓度为0.76μmol·L-1、dNTPs 190μmol·L、Taq DNA聚合酶1.5U；利用5个引物,用于3个菘蓝四倍体株系8-36代之间ISSR分子标记没有发现明显差异,在所检测的5个引物中,均未产生变异条带。
(4)综合评价了16个菘蓝四倍体株系的农艺性状、品质、抗逆性。获得的四倍体株系之间在叶片、根部性状及产量上存在一定差异；菘蓝叶片长度、最长干根长与产量间具有相关性；株系DB3、DB5、DB7、DB10以收获菘蓝叶为好,而株系DB3、DB7、DB10、DB16以收获菘蓝根为佳；90%以上的四倍体株系根中靛蓝、靛玉红含量均高于二倍体株系,而叶中靛蓝和靛玉红含量下降；四倍体株系根中的多糖含量均低于二倍体,70%以上四倍体株系叶中多糖含量高于二倍体,其中DB1、DB10、DB15根中多糖含量较高,DB6、DB13和DB15叶中多糖含量最高；测定了菘蓝四倍体株系的17种氨基酸含量,其中DB7、DB8根和叶中的精氨酸含量均高,可根、叶两用,DB1、DB7和DB12脯氨酸含量较高,可作抗旱材料,DB7、DB9和DB12叶中总氨基酸含量最高,可叶用；测定了PEG和NaCl胁迫处理后菘蓝四倍体中靛蓝和靛玉红的含量、SOD和POD活性、脯氨酸电导率的变化,发现DB3、DB5、DB16在抗旱性和提高有效成分上表现均优,DB2、DB5、DB6、DB9、DB10、DB12和DB14表现出较好的耐盐能力；分离并鉴定了菘蓝根腐病的病原菌包括茄腐镰刀菌、尖孢镰刀菌和禾谷丝核菌；通过根段回接法进行根腐病抗性鉴定,发现DB1、DB3、DB6、DB9、DB14抗病性较差,DB5和DB11抗根腐病能力较强；利用简化特尔裴法,选用产量、靛蓝含量、靛玉红含量、多糖含量、总氨基酸含量、抗旱性、抗盐性及抗根腐病能力8个指标,综合评价16个菘蓝四倍体株系,发现除DB2、DB4、DB5、DB6、DB13外均优于二倍体株系,其中DB3、DB10和DB16等3个四倍体株系均在80分以上,表现良好,可作为进一步繁殖研究的材料。
Isatis indigotica Fortune, a kind of biennial herb, belongs to Isatis genera, Cruciferae family. Both the isatis roots and indigowood leaves are used as traditional Chinese medicine. The quantity and quality of Isatis have reduced greatly in recent years because of planting without screening the excellent species. Therefore, there is an urgent demand to develop new species with better yields, qualities and genetic stability. The medicinal plant polyploidy posses manifold merits, such as improved biomass accumulation and pharmaceutical ingredients, as well as enhanced stress resistance which offers an alternative strategy for the development and utilization of traditional medicinal plants. Focused on the polyploidy breeding of isatis, in succession, the rapid propagation system of isatis was established, the isatis polyploidy was induced and identified, the inheritance stability was analyzed. Ultimately, the excellent tetraploid strains were screened. Following was the key results in this work.
Firstly the rapid propagation and culture technique system of isatis was established. It shows that hypocotyl is easier to induce callus by studies of inducing callus from explant of isatis and rooting of test-tube plantlet. The optimum medium recipe for inducing callus, differentiation and rooting is respectively MS+6-BA1.0mg/L+2,4-D0.8mg/L, MS+6-BA2.0mg/L+NAA0.2mg/L. and 1/2MS+NAA 0.2mg/L+IBA 0.2mg/L. The best time for hardening off is 4 days, and the best ground substance recipe for transplanting is vermiculite:perlite=1:2. The nutrient solution consisting of Ca(NO3)2 945mg/L+KNO3 607mg/L+(NH4)2HPO4 115mg/L+MgSO4 493mg/L can boost the growth of transplant significantly. The planting density of 30cm×10-20cm is suitable for isatis. The optimized harvest period is between October 15 and October 20.
Secondly, the 16 autoteraploid strains of isatis were screened by inducing and identifying poplyploid. It comes to the conclusion that the culture medium added with 90 mg/L colchicines for 28d is the best for polyploidy induction.
Compared to the corresponding plantlets of diploid, the test-tube plantlets and the transplants of polyploidy showed a lot of different situation:the seed germination was slowly, the seedling is stubby, also with incrassate hypocotyl; the leaves of differentiation seedlings are plumpy, with dark green color; the leaf veins are thick and obvious; the roots of the petiole expand; in comparison to the diploid, the leaves of polyploidy have more drape, the leafedge is zigzag. The transplants of polyploidy with vigorous growth are thick and strong, with strong tillering ability and winterness. It was found that the stomas of polyploidy were bigger significantly than diploid. Big stoma and the number of chloroplast in guard cell are important index for polyploidy identification.
Then the inheritance stability of isatis teraploid strains was analyzed with ISSR. The modified CTAB method is screened as the optimum method for extracting isatis DNA. The optimized ISSR reaction system is 1×buffer 2.5(not including MgCl2), MgCl2 2.0mmol·L-1,primer 0.76μmol·L-1, dNTPs 190μmol·L-1, Taq DNA polymerase 1.5U in 25μl system.5 polymorphic primers were chosen for ISSR analysis to explore the genetic stability of subcultrued test-tube plantlets from three tetraploid strains. Electrophoresis illustrated that there was no obvious difference within the plantlets and 5 polymorphic primers, which indicated that the sub-culture of isatis can keep genetic stability within this range.
Finally, the economical character, quality and stress resistance were evaluated. By comparison of economical characters between different strains, the results show that tetraploid and diploid exist difference, moreover, the indigowoad leaf and isatis root output are different between different tetraploid strains. Different strains can be choosed according to different harvest target. If the indigowoad leaf is needed, DB3, DB5, DB7 and DB10 are the best; if the isatis root is wanted, DB3, DB7, DB10 and DB16 are the best.
Though comparing the content of indigotin and indirubin in different strains, more than 90% of 16 tetraploid strains in root contain high content than that in diploid. While in the leaves, the content of both indigotin and indirubin is just the opposite.
As respect to the content of polysaccharide in isatis root, tetraploid is lower than diploid, among the strains determined, DB1, DB10 and DB15, contained more polysaccharide. While in the indigowoad leaves, the content of polysaccharide of 70% of the 16 strains is higher than that of diploid. The content in DB6, DB13 and DB15 is the highest.
Amino acid analysis showed that the content of arginine both in indigowoad leaves and isatis roots of strain DB7 and DB8 is very high; The DB1, DB7 and DB12 strains contain high praline content, which can be drought resisting materials. As respect to the content of total amino acid, the strains of DB7, DB9 and DB12 are the best.
PEG and NaCl were used as stress to screen the stress resistant abilities based on their physiology index such as the activity of SOD, POD, the praline content and electrical conductivity as well as the content of important metabolites such as indigotin and indirubin. It illustrated that DB3, DB5 and DB6 were the best strains based on all indexes. As far as the saltresistance is concerned, DB2, DB5, DB6, DB8, DB10, DB12 and DB14 showed an advantage over other strains, but further studies was needed to confirm.
The pathogenic bacteria were obtained from disease plants, and identified as F. solan, F.oxysporum and R.cerealis respectively. The disease resistance of tetraploid strains was compared by the means of root segment inoculation. Most strains appeared the disease symptom from the third day. By the seventh day, the disease rates of DB1, DB3, DB6, DB9 and DB14 get higher, while that of DB5 and DB11 were lower, and they possess high resistance to root rot disease.
On the base of the comprehensive evaluation of 16 tetraploid strains of isatis, all of which, except DB2, DB4, DB5, DB6 and DB13 are superior to the diploid. Among them, DB3, DB10 and DB16 are the optimum and can be further studied as reproduction materials.
(1)建立了菘蓝的无性快繁及栽培技术体系。外植体愈伤组织诱导的适宜外植体为下胚轴；诱导愈伤组织最佳培养基配方为MS+6-BA1.0mg/L+2,4-D0.8mg/L;诱导愈伤及分化最佳培养基配方为MS+6-B A2.0mg/L+NAA0.2mg/L;最佳生根培养基配方为1/2MS+NAA 0.2mg/L+IBA 0.2mg/L;最佳炼苗时间为4d,长势最佳的移栽基质为蛭石：珍珠岩=1：2；可明显促进菘蓝移栽苗的生长的营养液配方为Ca(NO3)2·4H2O 945mg/L+KNO3 607mg/L+NH4H2PO4 115mg/L+MgSO4·7H2O 493mg/L;菘蓝适宜的种植密度为行距30cm×株距10(或20)cm；华北地区适宜采收期10月15-20日。
(2)人工诱导、鉴定和筛选了16个菘蓝纯合同源四倍体。以90mg／L秋水仙碱加入培养基诱导28d,菘蓝染色体的加倍率可达28.30%；菘蓝四倍体试管苗特征：生长慢,芽粗短,胚轴膨大,叶片肥厚宽大皱褶,叶缘呈锯齿状,色泽深绿,叶脉粗；四倍体田间移栽苗特征：叶色深绿,叶片宽厚变形,叶面粗糙,叶缘锯齿状,整株生长势旺盛,分蘖能力强,冬性较强；四倍体叶部细胞显微结构特征：气孔巨大、保卫细胞的叶绿体数目增多；四倍体染色体倍性鉴定方法：根尖常规压片法和幼叶流式细胞仪法。
(3)用ISSR分子标记技术分析了菘蓝四倍体株系遗传稳定性。改良CTAB法是菘蓝DNA最佳的提取方法；ISSR反应的最佳体系为：25μl体系中含1×buffer2.5(不含MgCl2), MgCl2 2.0mmol·L-1、引物浓度为0.76μmol·L-1、dNTPs 190μmol·L、Taq DNA聚合酶1.5U；利用5个引物,用于3个菘蓝四倍体株系8-36代之间ISSR分子标记没有发现明显差异,在所检测的5个引物中,均未产生变异条带。
(4)综合评价了16个菘蓝四倍体株系的农艺性状、品质、抗逆性。获得的四倍体株系之间在叶片、根部性状及产量上存在一定差异；菘蓝叶片长度、最长干根长与产量间具有相关性；株系DB3、DB5、DB7、DB10以收获菘蓝叶为好,而株系DB3、DB7、DB10、DB16以收获菘蓝根为佳；90%以上的四倍体株系根中靛蓝、靛玉红含量均高于二倍体株系,而叶中靛蓝和靛玉红含量下降；四倍体株系根中的多糖含量均低于二倍体,70%以上四倍体株系叶中多糖含量高于二倍体,其中DB1、DB10、DB15根中多糖含量较高,DB6、DB13和DB15叶中多糖含量最高；测定了菘蓝四倍体株系的17种氨基酸含量,其中DB7、DB8根和叶中的精氨酸含量均高,可根、叶两用,DB1、DB7和DB12脯氨酸含量较高,可作抗旱材料,DB7、DB9和DB12叶中总氨基酸含量最高,可叶用；测定了PEG和NaCl胁迫处理后菘蓝四倍体中靛蓝和靛玉红的含量、SOD和POD活性、脯氨酸电导率的变化,发现DB3、DB5、DB16在抗旱性和提高有效成分上表现均优,DB2、DB5、DB6、DB9、DB10、DB12和DB14表现出较好的耐盐能力；分离并鉴定了菘蓝根腐病的病原菌包括茄腐镰刀菌、尖孢镰刀菌和禾谷丝核菌；通过根段回接法进行根腐病抗性鉴定,发现DB1、DB3、DB6、DB9、DB14抗病性较差,DB5和DB11抗根腐病能力较强；利用简化特尔裴法,选用产量、靛蓝含量、靛玉红含量、多糖含量、总氨基酸含量、抗旱性、抗盐性及抗根腐病能力8个指标,综合评价16个菘蓝四倍体株系,发现除DB2、DB4、DB5、DB6、DB13外均优于二倍体株系,其中DB3、DB10和DB16等3个四倍体株系均在80分以上,表现良好,可作为进一步繁殖研究的材料。
Isatis indigotica Fortune, a kind of biennial herb, belongs to Isatis genera, Cruciferae family. Both the isatis roots and indigowood leaves are used as traditional Chinese medicine. The quantity and quality of Isatis have reduced greatly in recent years because of planting without screening the excellent species. Therefore, there is an urgent demand to develop new species with better yields, qualities and genetic stability. The medicinal plant polyploidy posses manifold merits, such as improved biomass accumulation and pharmaceutical ingredients, as well as enhanced stress resistance which offers an alternative strategy for the development and utilization of traditional medicinal plants. Focused on the polyploidy breeding of isatis, in succession, the rapid propagation system of isatis was established, the isatis polyploidy was induced and identified, the inheritance stability was analyzed. Ultimately, the excellent tetraploid strains were screened. Following was the key results in this work.
Firstly the rapid propagation and culture technique system of isatis was established. It shows that hypocotyl is easier to induce callus by studies of inducing callus from explant of isatis and rooting of test-tube plantlet. The optimum medium recipe for inducing callus, differentiation and rooting is respectively MS+6-BA1.0mg/L+2,4-D0.8mg/L, MS+6-BA2.0mg/L+NAA0.2mg/L. and 1/2MS+NAA 0.2mg/L+IBA 0.2mg/L. The best time for hardening off is 4 days, and the best ground substance recipe for transplanting is vermiculite:perlite=1:2. The nutrient solution consisting of Ca(NO3)2 945mg/L+KNO3 607mg/L+(NH4)2HPO4 115mg/L+MgSO4 493mg/L can boost the growth of transplant significantly. The planting density of 30cm×10-20cm is suitable for isatis. The optimized harvest period is between October 15 and October 20.
Secondly, the 16 autoteraploid strains of isatis were screened by inducing and identifying poplyploid. It comes to the conclusion that the culture medium added with 90 mg/L colchicines for 28d is the best for polyploidy induction.
Compared to the corresponding plantlets of diploid, the test-tube plantlets and the transplants of polyploidy showed a lot of different situation:the seed germination was slowly, the seedling is stubby, also with incrassate hypocotyl; the leaves of differentiation seedlings are plumpy, with dark green color; the leaf veins are thick and obvious; the roots of the petiole expand; in comparison to the diploid, the leaves of polyploidy have more drape, the leafedge is zigzag. The transplants of polyploidy with vigorous growth are thick and strong, with strong tillering ability and winterness. It was found that the stomas of polyploidy were bigger significantly than diploid. Big stoma and the number of chloroplast in guard cell are important index for polyploidy identification.
Then the inheritance stability of isatis teraploid strains was analyzed with ISSR. The modified CTAB method is screened as the optimum method for extracting isatis DNA. The optimized ISSR reaction system is 1×buffer 2.5(not including MgCl2), MgCl2 2.0mmol·L-1,primer 0.76μmol·L-1, dNTPs 190μmol·L-1, Taq DNA polymerase 1.5U in 25μl system.5 polymorphic primers were chosen for ISSR analysis to explore the genetic stability of subcultrued test-tube plantlets from three tetraploid strains. Electrophoresis illustrated that there was no obvious difference within the plantlets and 5 polymorphic primers, which indicated that the sub-culture of isatis can keep genetic stability within this range.
Finally, the economical character, quality and stress resistance were evaluated. By comparison of economical characters between different strains, the results show that tetraploid and diploid exist difference, moreover, the indigowoad leaf and isatis root output are different between different tetraploid strains. Different strains can be choosed according to different harvest target. If the indigowoad leaf is needed, DB3, DB5, DB7 and DB10 are the best; if the isatis root is wanted, DB3, DB7, DB10 and DB16 are the best.
Though comparing the content of indigotin and indirubin in different strains, more than 90% of 16 tetraploid strains in root contain high content than that in diploid. While in the leaves, the content of both indigotin and indirubin is just the opposite.
As respect to the content of polysaccharide in isatis root, tetraploid is lower than diploid, among the strains determined, DB1, DB10 and DB15, contained more polysaccharide. While in the indigowoad leaves, the content of polysaccharide of 70% of the 16 strains is higher than that of diploid. The content in DB6, DB13 and DB15 is the highest.
Amino acid analysis showed that the content of arginine both in indigowoad leaves and isatis roots of strain DB7 and DB8 is very high; The DB1, DB7 and DB12 strains contain high praline content, which can be drought resisting materials. As respect to the content of total amino acid, the strains of DB7, DB9 and DB12 are the best.
PEG and NaCl were used as stress to screen the stress resistant abilities based on their physiology index such as the activity of SOD, POD, the praline content and electrical conductivity as well as the content of important metabolites such as indigotin and indirubin. It illustrated that DB3, DB5 and DB6 were the best strains based on all indexes. As far as the saltresistance is concerned, DB2, DB5, DB6, DB8, DB10, DB12 and DB14 showed an advantage over other strains, but further studies was needed to confirm.
The pathogenic bacteria were obtained from disease plants, and identified as F. solan, F.oxysporum and R.cerealis respectively. The disease resistance of tetraploid strains was compared by the means of root segment inoculation. Most strains appeared the disease symptom from the third day. By the seventh day, the disease rates of DB1, DB3, DB6, DB9 and DB14 get higher, while that of DB5 and DB11 were lower, and they possess high resistance to root rot disease.
On the base of the comprehensive evaluation of 16 tetraploid strains of isatis, all of which, except DB2, DB4, DB5, DB6 and DB13 are superior to the diploid. Among them, DB3, DB10 and DB16 are the optimum and can be further studied as reproduction materials.
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