香蜂花离体培养及其挥发油成分的GC-MS分析
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摘要
本试验以香蜂花(Melissa officinalis)带芽茎段为材料,比较系统地进行香蜂花茎段培养与快繁技术研究,摸索香蜂花试管苗工厂化生产模式的基本参数,并系统地研究了香蜂花试管苗玻璃化的影响因素;同时,以香蜂花种子、叶片和茎段为材料,进行愈伤组织培养与植株再生研究。最后,利用GC-MS技术分析香蜂花试管苗和盆栽实生苗的挥发油成分差异,以期为香蜂花离体培养建立有效途径,为香蜂花大规模工厂化试管育苗及种质资源保护提供科学依据。主要研究结果如下:
1.香蜂花带芽茎段的培养。以香蜂花带芽茎段为材料,研究了香蜂花带芽茎段培养和离体繁殖。结果表明:3月取香蜂花带芽茎段经过75%乙醇30s+0.1%HgCl_2 5 min处理为最佳取材时期和消毒方式;带芽茎段在MS+0.5mg/L 6-BA+0.1mg/L NAA培养基中不定芽诱导率达到100.0%;不定芽适宜增殖的培养基为MS+1.0 mg/L 6-BA+0.2 mg/L NAA+0.1mg/L IBA,增殖倍数达到4.6;最佳的生根培养基为1/2MS+0.5 mg/L IBA,生根率达到100.0%;适宜的移栽基质为1:1:1珍珠岩:蛭石:河沙,成活率为93.3%。
2.香蜂花试管苗工厂化生产模式的确定。香蜂花工厂化试管育苗主要技术参数:继代增殖系数4.5,每25 d继代1次,一年可增殖继代12代,生根率100.0%,移栽成活率93.3%。以此为参数,离体培养时污染率控制在5%以下,玻璃化苗控制在5%以下,理论上香蜂花1个芽1年继代12次既可增殖403.8万株,可以达到快繁的要求。
3.香蜂花试管苗玻璃化现象的研究。以香蜂花正常试管苗的带芽茎段为材料,针对6-BA浓度、温度、培养器皿、继代时间、蔗糖浓度和琼脂浓度等影响因素,研究了香蜂花带芽茎段培养过程中控制玻璃化现象的措施。结果表明:香蜂花正常试管苗的适宜培养的6-BA浓度为0.5-2.0 mg/L,30g/L蔗糖和7g/L琼脂的培养基中玻璃化率为最低。试管苗培养于玻璃瓶+塑料膜的培养容器内,置于25℃培养温度,30 d继代培养一次时可有效地控制玻璃化发生。
4.香蜂花愈伤组织的培养。以香蜂花种子、叶片、叶柄、茎段为材料,研究了香蜂花愈伤组织的培养。结果表明:香蜂花种子最佳的消毒方式为75%乙醇30 s+0.1%HgCl_2 5 min,于MS培养基中萌发率达到85.5%;愈伤组织最佳诱导培养基为MS+1.0 mg/L 6-BA+0.1 mg/L2,4-D,诱导率达97.1%;香蜂花不同取材部位(叶片,叶柄,茎段)的愈伤组织诱导率差异不显著,不同取材部位均可达到极高的愈伤诱导率;愈伤组织最佳增殖的培养基为MS+2mg/L 6-BA+0.01 mg/L NAA+0.2 mg/L 2,4-D,增殖率达到76.7%;在培养基MS+1.0 mg/L6-BA+0.1 mg/L NAA+0.1 mg/L 2,4-D上部分愈伤组织可以分化芽苗。
5.香蜂花试管苗挥发油成分的气相色谱-质谱(GC-MS)分析。以香蜂花试管苗为材料,利用GC-MS技术共分离出19种物质,主要成分为甲基庚烯酮、香茅醛、橙花醇、β-柠檬醛、香叶醇、α-柠檬醛、乙酸橙花酯、二丁基羟基甲苯、绿花白千层醇等。与香蜂花盆栽实生苗相比,两样本共测定出11种相同物质,虽然各成分的含量存在一些的差异,但其中主要物质成分橙花醇、β-柠檬醛、香叶醇和香叶醛在两样本中含量都很大,表明香蜂花试管苗可以应用于种苗快繁。
In this experiment,stems with buds were used as explants for the systematical studies on the tissue culture of stem segment and for further establishing the industrialized in vitro propagation model in Mellisa officinalis;and the systematical research on the vitrification in the culture of Mellisa officinali was also discussed.Leaves,stems and seeds were used as explants for the studies on the callus culture and plant regeneration.At the same time,GC-MS analysis was used for analyzing the difference of the volatile oil components of Mellisa officinalis between the plantlets in vitro and the seeding of potted planting.These studies would establish an effective way on tissue culture of Mellisa officinalis which could provide scientific evidence on industrial production of plantlets and protection of germplasm.The main results were as follows.
1.Stem segment culture of Mellisa officinalis.Stems with buds were used as explants for the studies on tissue culture.The results showed that:the best season of taking explants was March.The best disinfection method for stems was 75%ethanol for 30 s +0.1%HgCl_2 5 min on MS medium.Adventitous buds induction rate was 100.0%on the medium:MS +0.5 mg/L 6-BA + 0.1 mg/L NAA.The most suitable medium for the multiplication of the adventitious buds was MS +1.0 mg/L 6-BA +0.2 mg/L NAA +0.1 mg/L IBA,the multiplication time was 4.6.The most suitable medium for rooting was 1/2 MS +0.5 mg/L IBA,rooting rate reached 100.0%.Substrate with perlite,vermiculite and sand by 1:1:1 was the optimal,the survival rate was 93.3%.
2.Establishment of the industrialized in vitro propagation model of Mellisa officinalis. The main technical parameters were as follows:the proliferation coefficient was up to 4.5,the subculture cycle was 25 days,and the subculture time was 12 times every year;the rooting rate was up to 100%,and the survival rate of transplanting was 93.3%,.According to these parameters, a bud could proliferate to 4038000 copies a year theoretically when contamination rate was less than 5%and vitrification rate was less than 5%.
3.Studies on the vitrification in the culture of Mellisa officinalis.Stems with buds of normal plantlets were used as explants,compared to different factors:6-BA,temperature,the containers,subculture time,and sucrose,studied on the control measures for the vitrification of Mellisa officinalis in vitro Culture.The results show that:when plantlets on the appropriate cultured medium with 0.5-2.0 mg/L 6-BA,30g/L sucrose and 7g/L agar,the rate of the vitrification could be lowest.When the temperature was 25℃,plantlets sealing with plastic film in glass bottles for 30 d subculture could be an effective control of the vitrification.
4.Callus culture of Mellisa officinalis.Seeds,leaves,petiole and stems of Mellisa officinalis were used as explants for the studies on callus culture.The results showed that:the most suitable disinfection method for seed was 75%ethanol for 30 s +0.1%HgCl_2 5 min in MS medium,and the germination rate was 85.5%.The most suitable medium for callus induction was MS+1.0 mg/L 6-BA+0.1 mg/L 2,4-D,callus induction rate was 97.1%;there were no significant difference of callus induction rate when callus induced from leaves,petiole and stems。The most suitable medium for the multiplication of the Callus was MS +2 mg/L 6-BA +0.01 mg/L NAA +0.2 mg/L 2,4-D,the growth rate reached 76.7%.The most suitable differentiation medium was MS +1.0 mg/L 6-BA +0.1 mg/L NAA +0.1 mg/L 2,4-D,differentiation rate was 3.75%.
5.GC-MS analysis of essential oil constituents of Melissa officinalis in vitro culture. Mellisa officinalis in vitro were used as the materials,using GC-MS separated 19 kinds of substances.Main composition were 6-Methyl-5-heptene-2-one,citronellal,neryl alcohol,β-citral, geraniol,1,3,4-Trimethyl-3-cyclohexen-1-carboxaldehyde,α-citral,nerol acetate,butylated hydroxytoluene,viridiflorol and so on.Compared to normal plants of Melissa officinalis there were 11 kind of same material in two samples together,although the contents existenced some differences,all of the main substances neryl alcohol,β-citral,geraniol,α-citral and nerol acetate have great content in the two samples.
1.香蜂花带芽茎段的培养。以香蜂花带芽茎段为材料,研究了香蜂花带芽茎段培养和离体繁殖。结果表明:3月取香蜂花带芽茎段经过75%乙醇30s+0.1%HgCl_2 5 min处理为最佳取材时期和消毒方式;带芽茎段在MS+0.5mg/L 6-BA+0.1mg/L NAA培养基中不定芽诱导率达到100.0%;不定芽适宜增殖的培养基为MS+1.0 mg/L 6-BA+0.2 mg/L NAA+0.1mg/L IBA,增殖倍数达到4.6;最佳的生根培养基为1/2MS+0.5 mg/L IBA,生根率达到100.0%;适宜的移栽基质为1:1:1珍珠岩:蛭石:河沙,成活率为93.3%。
2.香蜂花试管苗工厂化生产模式的确定。香蜂花工厂化试管育苗主要技术参数:继代增殖系数4.5,每25 d继代1次,一年可增殖继代12代,生根率100.0%,移栽成活率93.3%。以此为参数,离体培养时污染率控制在5%以下,玻璃化苗控制在5%以下,理论上香蜂花1个芽1年继代12次既可增殖403.8万株,可以达到快繁的要求。
3.香蜂花试管苗玻璃化现象的研究。以香蜂花正常试管苗的带芽茎段为材料,针对6-BA浓度、温度、培养器皿、继代时间、蔗糖浓度和琼脂浓度等影响因素,研究了香蜂花带芽茎段培养过程中控制玻璃化现象的措施。结果表明:香蜂花正常试管苗的适宜培养的6-BA浓度为0.5-2.0 mg/L,30g/L蔗糖和7g/L琼脂的培养基中玻璃化率为最低。试管苗培养于玻璃瓶+塑料膜的培养容器内,置于25℃培养温度,30 d继代培养一次时可有效地控制玻璃化发生。
4.香蜂花愈伤组织的培养。以香蜂花种子、叶片、叶柄、茎段为材料,研究了香蜂花愈伤组织的培养。结果表明:香蜂花种子最佳的消毒方式为75%乙醇30 s+0.1%HgCl_2 5 min,于MS培养基中萌发率达到85.5%;愈伤组织最佳诱导培养基为MS+1.0 mg/L 6-BA+0.1 mg/L2,4-D,诱导率达97.1%;香蜂花不同取材部位(叶片,叶柄,茎段)的愈伤组织诱导率差异不显著,不同取材部位均可达到极高的愈伤诱导率;愈伤组织最佳增殖的培养基为MS+2mg/L 6-BA+0.01 mg/L NAA+0.2 mg/L 2,4-D,增殖率达到76.7%;在培养基MS+1.0 mg/L6-BA+0.1 mg/L NAA+0.1 mg/L 2,4-D上部分愈伤组织可以分化芽苗。
5.香蜂花试管苗挥发油成分的气相色谱-质谱(GC-MS)分析。以香蜂花试管苗为材料,利用GC-MS技术共分离出19种物质,主要成分为甲基庚烯酮、香茅醛、橙花醇、β-柠檬醛、香叶醇、α-柠檬醛、乙酸橙花酯、二丁基羟基甲苯、绿花白千层醇等。与香蜂花盆栽实生苗相比,两样本共测定出11种相同物质,虽然各成分的含量存在一些的差异,但其中主要物质成分橙花醇、β-柠檬醛、香叶醇和香叶醛在两样本中含量都很大,表明香蜂花试管苗可以应用于种苗快繁。
In this experiment,stems with buds were used as explants for the systematical studies on the tissue culture of stem segment and for further establishing the industrialized in vitro propagation model in Mellisa officinalis;and the systematical research on the vitrification in the culture of Mellisa officinali was also discussed.Leaves,stems and seeds were used as explants for the studies on the callus culture and plant regeneration.At the same time,GC-MS analysis was used for analyzing the difference of the volatile oil components of Mellisa officinalis between the plantlets in vitro and the seeding of potted planting.These studies would establish an effective way on tissue culture of Mellisa officinalis which could provide scientific evidence on industrial production of plantlets and protection of germplasm.The main results were as follows.
1.Stem segment culture of Mellisa officinalis.Stems with buds were used as explants for the studies on tissue culture.The results showed that:the best season of taking explants was March.The best disinfection method for stems was 75%ethanol for 30 s +0.1%HgCl_2 5 min on MS medium.Adventitous buds induction rate was 100.0%on the medium:MS +0.5 mg/L 6-BA + 0.1 mg/L NAA.The most suitable medium for the multiplication of the adventitious buds was MS +1.0 mg/L 6-BA +0.2 mg/L NAA +0.1 mg/L IBA,the multiplication time was 4.6.The most suitable medium for rooting was 1/2 MS +0.5 mg/L IBA,rooting rate reached 100.0%.Substrate with perlite,vermiculite and sand by 1:1:1 was the optimal,the survival rate was 93.3%.
2.Establishment of the industrialized in vitro propagation model of Mellisa officinalis. The main technical parameters were as follows:the proliferation coefficient was up to 4.5,the subculture cycle was 25 days,and the subculture time was 12 times every year;the rooting rate was up to 100%,and the survival rate of transplanting was 93.3%,.According to these parameters, a bud could proliferate to 4038000 copies a year theoretically when contamination rate was less than 5%and vitrification rate was less than 5%.
3.Studies on the vitrification in the culture of Mellisa officinalis.Stems with buds of normal plantlets were used as explants,compared to different factors:6-BA,temperature,the containers,subculture time,and sucrose,studied on the control measures for the vitrification of Mellisa officinalis in vitro Culture.The results show that:when plantlets on the appropriate cultured medium with 0.5-2.0 mg/L 6-BA,30g/L sucrose and 7g/L agar,the rate of the vitrification could be lowest.When the temperature was 25℃,plantlets sealing with plastic film in glass bottles for 30 d subculture could be an effective control of the vitrification.
4.Callus culture of Mellisa officinalis.Seeds,leaves,petiole and stems of Mellisa officinalis were used as explants for the studies on callus culture.The results showed that:the most suitable disinfection method for seed was 75%ethanol for 30 s +0.1%HgCl_2 5 min in MS medium,and the germination rate was 85.5%.The most suitable medium for callus induction was MS+1.0 mg/L 6-BA+0.1 mg/L 2,4-D,callus induction rate was 97.1%;there were no significant difference of callus induction rate when callus induced from leaves,petiole and stems。The most suitable medium for the multiplication of the Callus was MS +2 mg/L 6-BA +0.01 mg/L NAA +0.2 mg/L 2,4-D,the growth rate reached 76.7%.The most suitable differentiation medium was MS +1.0 mg/L 6-BA +0.1 mg/L NAA +0.1 mg/L 2,4-D,differentiation rate was 3.75%.
5.GC-MS analysis of essential oil constituents of Melissa officinalis in vitro culture. Mellisa officinalis in vitro were used as the materials,using GC-MS separated 19 kinds of substances.Main composition were 6-Methyl-5-heptene-2-one,citronellal,neryl alcohol,β-citral, geraniol,1,3,4-Trimethyl-3-cyclohexen-1-carboxaldehyde,α-citral,nerol acetate,butylated hydroxytoluene,viridiflorol and so on.Compared to normal plants of Melissa officinalis there were 11 kind of same material in two samples together,although the contents existenced some differences,all of the main substances neryl alcohol,β-citral,geraniol,α-citral and nerol acetate have great content in the two samples.
引文
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