山苍子无性繁殖技术的研究
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摘要
山苍子(Litsea cubeba),作为我国的能源树种,具有很高的经济开发价值。本试验以3个不同种源的山苍子为基本材料,通过组织培养各个环节的优化、诱导愈伤组织为起始材料,建立山苍子愈伤组织体系、山苍子扦插繁殖各个环节的优化,对我国优良的能源树种山苍子进行无性繁殖技术的研究,对于提高山苍子苗木质量、缩短育苗周期具有重要的现实意义,为山苍子无性繁殖技术提供理论和实践依据,同时也为木本植物的快繁技术提供参考和借鉴。主要研究结果如下:
1.山苍子组织培养
1.1山苍子外植体消毒处理
采用完全随机试验设计,筛选出0.1%升汞+20%次氯酸钠组合消毒效果最好,0.1%升汞+20%次氯酸钠组合消毒时间分别为10min+5min时,污染率最低,为最佳的消毒方案。
1.2山苍子初代培养基的筛选
采用正交试验设计,考虑不同激素种类和浓度对山苍子外植体初代培养的影响,筛选出初代培养基A5:MS+2.0mg/L BA+1.0mg/L KT+0.3mg/L IBA+蔗糖30g/L,A15:MS+2.5mg/LBA+0.4mg/L IBA+蔗糖30g/L。在初代培养基A5和A15中外植体萌芽快,苗绿,节间距较大,苗壮,增殖率高。
1.3山苍子防褐变处理
分别从外植体处理和在培养基中加入褐变抑制剂2个方面,进行了比较试验。外植体冷处理有一定的降低褐变率的效果,接种前外植体在5℃下冷处理15h的效果最好,褐变率降低了11.67%。接种前在1g/L PVP溶液中切割外植体也能减轻褐变,褐变率降低了10%。接种前在无菌水中切割外植体,褐变率降低了3.34%。
采用完全随机试验设计,在培养基中添加不同的褐变抑制剂,AC控制褐变的效果最好,在培养基中添加0.5g/L AC时,褐变率从对照的35%降低了18.89%,能够较好地控制山苍子外植体的褐变。
1.4不同种源和枝条部位对山苍子外植体增殖率的影响
采用完全随机试验设计,筛选出三明种源的枝条梢部在培养过程中,启动率最高为81.02%,增殖率最高,达到3.06。三明种源最优,其次为将乐种源,沙县种源培养的效果最差;不同枝条部位的外植体启动率和增殖率不同,梢部的启动率和增殖率最高,而后从上到下,启动率和增殖率也随之下降。
1.5不同采集月份对山苍子外植体污染率、启动率和增殖率的影响
不同月份采集的外植体,污染率、启动率和增殖情况不同,9月、11月是下半年较佳的培养时间,3月、4月的启动率和增殖率较高,是上半年较佳的培养时间。
1.6山苍子继代培养
采用正交试验设计,筛选出第一次继代的最适培养基为MS+1.0mg/L BA+0.5mg/LKT+0.4mg/L IBA+30g/L蔗糖。二次继代配方为MS+1.0mg/L BA+0.1mg/L KT+0.4mg/LIBA+30g/L蔗糖+0.5g/L AC。三次继代的最适培养基为MS+0.5mg/L BA+0.2mg/L IBA+20g/L蔗糖+0.5g/L AC。
1.7山苍子生根培养和移栽
采用正交试验设计,考虑不同生长素对山苍子无根苗生根的影响。筛选出最佳的生根培养基为1/2MS+0.8mg/L IBA+0.2mg/L IAA+20g/L蔗糖。移栽基质前期采用沙子,后期移到沙子:黄心土:泥炭土=1:1:1中。
2.山苍子愈伤诱导、继代和再生植株
采用3种不同的外植体(茎段、嫩叶、叶柄)为愈伤组织的诱导材料,建立山苍子愈伤组织体系。不同的外植体茎段、叶片、叶柄诱导愈伤组织的难易不同:茎段＞叶柄＞叶片,当年生嫩茎为山苍子诱导愈伤组织的理想材料。茎段诱导愈伤组织的最适配方为:MS+2.0mg/L2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L蔗糖。诱导山苍子叶片愈伤组织的最适配方为:MS+1.5mg/L 2,4-D+1.0mg/L BA+0.5mg/L KT+30g/L蔗糖。诱导叶柄愈伤组织的最适配方为:MS+1.5mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L蔗糖。山苍子继代的最适培养基为:MS+1.0mg/L 2,4-D+0.5mg/L BA+0.5mg/L IBA+30g/L蔗糖。最佳的诱导分化培养基为:MS+0.5mg/L IBA+0.5mg/L BA+0.5mg/L KT+30g/L蔗糖。
3.山苍子扦插繁殖
三个种源的平均扦插成活率的高低顺序为沙县＞将乐＞三明,平均生根率的高低顺序为沙县＞将乐＞三明,沙县种源的扦插材料较优。
河沙的扦插成活率、平均生根率最高,平均根数最多,平均根长最长,在扦插时选择河沙为基质的效果最佳。
不同插穗部分、生根促进剂、扦插处理对山苍子扦插成活率的影响不同,最佳组合为:根部萌发条+枝条上段+200mg/L L1生根粉浸泡30min+扦插时下端切成斜切口。
最佳穗条长度为14cm。穗条直径为0.3-0.4cm时,扦插成活率最高。
5月较适合移栽,之后随着月份的推移,外界环境的气温越来越高,成活率也随之下降。6月还可以移栽,最好在阴雨天进行,移栽后要注意水分管理。7、8月不适合移栽。
Litsea cubeba as China's energy species,have high economic value for development.The trial use three different provenances of Litsea cubeba for basic materials to study on asexual propagation techniques of the energy species,through opitimizing the process of tissue culture,establishing the callus system by inducting callus as the starting material and opitimizing the process of cutting propagation.Of great realistic significance to raising seedling quality and shorten the breeding cycle and providing theoretical and practical basis for Litsea Cubeba asexual reproduction technology,and also providing technical information and reference for the Propagation of woody plants.The main findings were as follows:
1.Tissue culture of Litsea cubeba
1.1 Litsea Cubeba explants disinfection
Using completely randomized experimental design,0.1%Mercuric chloride+20%Sodium hypochlorite was the best combination of disinfection.0.1%Mercuric chloride+20%Sodium hypochlorite disinfection combination of time of 10 rain +5 rain,have the lowest rate of pollution,was the best disinfection options.
1.2 Litsea Cubeba initial medium screening
Using orthogonal design,taking into the effect of different hormones and the concentration of Litsea Cubeba,Screening the initial medium A5:MS+2.0mg/L BA+1.0mg/L KT+0.4mg/L IBA+ Sucrose 30g/L,A15:MS+2.5mg/L BA+0.4mg/L IBA+ Sucrose 30g/L.In the initial medium A5 and A15 the explants budding fast,green seedlings,larger spacing,and the high rate of proliferation.
1.3 Anti-browning treatment of Litsea cubeba
From explants of handling and add some kinds of inhibitors in medium to have the comparison test.Explants of cold treatment to a certain extent the effect of reducing the rate of Browning, Browning reduced rate 11.67 percent.Cutting in the 1 g / L PVP,explants can also reduce Browning, Browning decreased by 10 percent.Cutting in the Sterile water,Browning reduced by 3.34 percent.
Using completely randomized experimental design,add different inhibitors into medium to have the comparison test.AC control the browning was the best of all,in the medium containing 0.5 g / L AC,browning decreased by 18.89 percent,and better able to control Litsea Cubeba explants browning.
1.4 Effect of different provenances and the site of branches on proliferation of Litsea cubeba
Using completely randomized experimental design,Sanming provenance of the Shoot branches have the highest launch rate(81.02%)and Proliferation rate(3.06).Sanming provenance was the optimal,Jiangle provenance was the second one and Shaxian provenance of the worst train,different branches parts of the explants have the different launch rate and the proliferation rate,the shoot have the highest launch rate and the proliferation,and then from top to bottom,the launch rate and the proliferation rate has decreased.
1.5 Effect of contamination rate、launch rate and proliferation on different months of Litsea cubeba
Collected in different months,the pollution rate and the rate of proliferation activated different, September and November was a better time in the second half of the training time,March,April s a better time in the first half of the training time,with the higher launch rate and proliferation.
1.6 The subculture of Litsea cubeba
Using orthogonal design,and selected the first subculture medium for the best was MS+1.0mg/L BA+0.5mg/L KT+0.4mg/L IBA+30g/L Sucrose.the second subculture medium was MS+1.0mg/L BA+0.1mg/L KT+0.4mg/L IBA+30g/L Sucrose +0.5g/L AC.the third subculture medium was MS+0.5mg/L BA+0.2mg/L IBA+20g/L Sucrose +0.5g/L AC.
1.7 The rooting culture and transplanting of Litsea cubeba
Using orthogonal design,taking into the effect of different hormones on the rooting of Litsea Cubeba,selected the best medium of the rooting culture was 1/2MS+0.8mg/L IBA+0.2mg/L IAA+20g/L Sucrose.Transplanting media was the sand in the first,and then transplant to the media (sand:yellow soil:peat soil =1:1:1)later.
2.Callus induction,subculture and regeneration of Litsea cubeba
Using three different explants(stems,leaves,petioles)to induce the callus,and established callus system of Litsea Cubeba.Different explants(stems,leaves,petioles)had different difficulty to induce the callus:stems＞petioles＞leaves.stems were the best material of all.The optimum medis of stem callus induction was MS+2.0mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L Sucrose.The optimum medis of leave callus induction was MS+1.5mg/L 2,4-D+1.0mg/L BA+0.5mg/L KT+30g/L Sucrose. The optimum medis of petioles callus induction was MS+1.5mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L Sucrose.The best media of the callus subculture was MS+1.0mg/L 2,4-D+0.5mg/L BA+0.5mg/L IBA+30g/L Sucrose.The best media of the callus differentiation was MS+0.5mg/L IBA+0.5mg/L BA+0.5mg/L KT+30g/L Sucrose.
3.The cutting propagation ofLitsea cubeba
Three kinds of provenances,the average survival rate of cutting the level of the order was Shaxian＞Jiangle＞Sanming,the average rooting rate of the order was Shaxian＞Jiangle＞Sanming,the materialof Shaxian provenance was the better for cutting.
Sand had the highest survival rate and average rooting rate,the most of the average number of roots,the longest average root length,sand had the best effect for cutting.
Dfferent cuttings part、the promotions of rooting,cutting treaments on the cutting of Litsea Cubeba had different survival rate.The best combination was germination of the roots+shoot of the branch+200mg/L Ll ABT Immersed 30rain+ Cutting into oblique incision at the bottom.
The best of the ear length was 14 cm.The diameter with 0.3-0.4 cm in cutting had the highest survival rate.
May was more suitable for transplanting,after the month,the temperature of the external environment was getting higher,the survival rate also decreased.June can be transplanted,and the best in the rainy days,after transplanting attention should be paid to water management.July and August were not suitable for transplanting.
1.山苍子组织培养
1.1山苍子外植体消毒处理
采用完全随机试验设计,筛选出0.1%升汞+20%次氯酸钠组合消毒效果最好,0.1%升汞+20%次氯酸钠组合消毒时间分别为10min+5min时,污染率最低,为最佳的消毒方案。
1.2山苍子初代培养基的筛选
采用正交试验设计,考虑不同激素种类和浓度对山苍子外植体初代培养的影响,筛选出初代培养基A5:MS+2.0mg/L BA+1.0mg/L KT+0.3mg/L IBA+蔗糖30g/L,A15:MS+2.5mg/LBA+0.4mg/L IBA+蔗糖30g/L。在初代培养基A5和A15中外植体萌芽快,苗绿,节间距较大,苗壮,增殖率高。
1.3山苍子防褐变处理
分别从外植体处理和在培养基中加入褐变抑制剂2个方面,进行了比较试验。外植体冷处理有一定的降低褐变率的效果,接种前外植体在5℃下冷处理15h的效果最好,褐变率降低了11.67%。接种前在1g/L PVP溶液中切割外植体也能减轻褐变,褐变率降低了10%。接种前在无菌水中切割外植体,褐变率降低了3.34%。
采用完全随机试验设计,在培养基中添加不同的褐变抑制剂,AC控制褐变的效果最好,在培养基中添加0.5g/L AC时,褐变率从对照的35%降低了18.89%,能够较好地控制山苍子外植体的褐变。
1.4不同种源和枝条部位对山苍子外植体增殖率的影响
采用完全随机试验设计,筛选出三明种源的枝条梢部在培养过程中,启动率最高为81.02%,增殖率最高,达到3.06。三明种源最优,其次为将乐种源,沙县种源培养的效果最差;不同枝条部位的外植体启动率和增殖率不同,梢部的启动率和增殖率最高,而后从上到下,启动率和增殖率也随之下降。
1.5不同采集月份对山苍子外植体污染率、启动率和增殖率的影响
不同月份采集的外植体,污染率、启动率和增殖情况不同,9月、11月是下半年较佳的培养时间,3月、4月的启动率和增殖率较高,是上半年较佳的培养时间。
1.6山苍子继代培养
采用正交试验设计,筛选出第一次继代的最适培养基为MS+1.0mg/L BA+0.5mg/LKT+0.4mg/L IBA+30g/L蔗糖。二次继代配方为MS+1.0mg/L BA+0.1mg/L KT+0.4mg/LIBA+30g/L蔗糖+0.5g/L AC。三次继代的最适培养基为MS+0.5mg/L BA+0.2mg/L IBA+20g/L蔗糖+0.5g/L AC。
1.7山苍子生根培养和移栽
采用正交试验设计,考虑不同生长素对山苍子无根苗生根的影响。筛选出最佳的生根培养基为1/2MS+0.8mg/L IBA+0.2mg/L IAA+20g/L蔗糖。移栽基质前期采用沙子,后期移到沙子:黄心土:泥炭土=1:1:1中。
2.山苍子愈伤诱导、继代和再生植株
采用3种不同的外植体(茎段、嫩叶、叶柄)为愈伤组织的诱导材料,建立山苍子愈伤组织体系。不同的外植体茎段、叶片、叶柄诱导愈伤组织的难易不同:茎段＞叶柄＞叶片,当年生嫩茎为山苍子诱导愈伤组织的理想材料。茎段诱导愈伤组织的最适配方为:MS+2.0mg/L2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L蔗糖。诱导山苍子叶片愈伤组织的最适配方为:MS+1.5mg/L 2,4-D+1.0mg/L BA+0.5mg/L KT+30g/L蔗糖。诱导叶柄愈伤组织的最适配方为:MS+1.5mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L蔗糖。山苍子继代的最适培养基为:MS+1.0mg/L 2,4-D+0.5mg/L BA+0.5mg/L IBA+30g/L蔗糖。最佳的诱导分化培养基为:MS+0.5mg/L IBA+0.5mg/L BA+0.5mg/L KT+30g/L蔗糖。
3.山苍子扦插繁殖
三个种源的平均扦插成活率的高低顺序为沙县＞将乐＞三明,平均生根率的高低顺序为沙县＞将乐＞三明,沙县种源的扦插材料较优。
河沙的扦插成活率、平均生根率最高,平均根数最多,平均根长最长,在扦插时选择河沙为基质的效果最佳。
不同插穗部分、生根促进剂、扦插处理对山苍子扦插成活率的影响不同,最佳组合为:根部萌发条+枝条上段+200mg/L L1生根粉浸泡30min+扦插时下端切成斜切口。
最佳穗条长度为14cm。穗条直径为0.3-0.4cm时,扦插成活率最高。
5月较适合移栽,之后随着月份的推移,外界环境的气温越来越高,成活率也随之下降。6月还可以移栽,最好在阴雨天进行,移栽后要注意水分管理。7、8月不适合移栽。
Litsea cubeba as China's energy species,have high economic value for development.The trial use three different provenances of Litsea cubeba for basic materials to study on asexual propagation techniques of the energy species,through opitimizing the process of tissue culture,establishing the callus system by inducting callus as the starting material and opitimizing the process of cutting propagation.Of great realistic significance to raising seedling quality and shorten the breeding cycle and providing theoretical and practical basis for Litsea Cubeba asexual reproduction technology,and also providing technical information and reference for the Propagation of woody plants.The main findings were as follows:
1.Tissue culture of Litsea cubeba
1.1 Litsea Cubeba explants disinfection
Using completely randomized experimental design,0.1%Mercuric chloride+20%Sodium hypochlorite was the best combination of disinfection.0.1%Mercuric chloride+20%Sodium hypochlorite disinfection combination of time of 10 rain +5 rain,have the lowest rate of pollution,was the best disinfection options.
1.2 Litsea Cubeba initial medium screening
Using orthogonal design,taking into the effect of different hormones and the concentration of Litsea Cubeba,Screening the initial medium A5:MS+2.0mg/L BA+1.0mg/L KT+0.4mg/L IBA+ Sucrose 30g/L,A15:MS+2.5mg/L BA+0.4mg/L IBA+ Sucrose 30g/L.In the initial medium A5 and A15 the explants budding fast,green seedlings,larger spacing,and the high rate of proliferation.
1.3 Anti-browning treatment of Litsea cubeba
From explants of handling and add some kinds of inhibitors in medium to have the comparison test.Explants of cold treatment to a certain extent the effect of reducing the rate of Browning, Browning reduced rate 11.67 percent.Cutting in the 1 g / L PVP,explants can also reduce Browning, Browning decreased by 10 percent.Cutting in the Sterile water,Browning reduced by 3.34 percent.
Using completely randomized experimental design,add different inhibitors into medium to have the comparison test.AC control the browning was the best of all,in the medium containing 0.5 g / L AC,browning decreased by 18.89 percent,and better able to control Litsea Cubeba explants browning.
1.4 Effect of different provenances and the site of branches on proliferation of Litsea cubeba
Using completely randomized experimental design,Sanming provenance of the Shoot branches have the highest launch rate(81.02%)and Proliferation rate(3.06).Sanming provenance was the optimal,Jiangle provenance was the second one and Shaxian provenance of the worst train,different branches parts of the explants have the different launch rate and the proliferation rate,the shoot have the highest launch rate and the proliferation,and then from top to bottom,the launch rate and the proliferation rate has decreased.
1.5 Effect of contamination rate、launch rate and proliferation on different months of Litsea cubeba
Collected in different months,the pollution rate and the rate of proliferation activated different, September and November was a better time in the second half of the training time,March,April s a better time in the first half of the training time,with the higher launch rate and proliferation.
1.6 The subculture of Litsea cubeba
Using orthogonal design,and selected the first subculture medium for the best was MS+1.0mg/L BA+0.5mg/L KT+0.4mg/L IBA+30g/L Sucrose.the second subculture medium was MS+1.0mg/L BA+0.1mg/L KT+0.4mg/L IBA+30g/L Sucrose +0.5g/L AC.the third subculture medium was MS+0.5mg/L BA+0.2mg/L IBA+20g/L Sucrose +0.5g/L AC.
1.7 The rooting culture and transplanting of Litsea cubeba
Using orthogonal design,taking into the effect of different hormones on the rooting of Litsea Cubeba,selected the best medium of the rooting culture was 1/2MS+0.8mg/L IBA+0.2mg/L IAA+20g/L Sucrose.Transplanting media was the sand in the first,and then transplant to the media (sand:yellow soil:peat soil =1:1:1)later.
2.Callus induction,subculture and regeneration of Litsea cubeba
Using three different explants(stems,leaves,petioles)to induce the callus,and established callus system of Litsea Cubeba.Different explants(stems,leaves,petioles)had different difficulty to induce the callus:stems＞petioles＞leaves.stems were the best material of all.The optimum medis of stem callus induction was MS+2.0mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L Sucrose.The optimum medis of leave callus induction was MS+1.5mg/L 2,4-D+1.0mg/L BA+0.5mg/L KT+30g/L Sucrose. The optimum medis of petioles callus induction was MS+1.5mg/L 2,4-D+1.0mg/L BA+1.0mg/L KT+30g/L Sucrose.The best media of the callus subculture was MS+1.0mg/L 2,4-D+0.5mg/L BA+0.5mg/L IBA+30g/L Sucrose.The best media of the callus differentiation was MS+0.5mg/L IBA+0.5mg/L BA+0.5mg/L KT+30g/L Sucrose.
3.The cutting propagation ofLitsea cubeba
Three kinds of provenances,the average survival rate of cutting the level of the order was Shaxian＞Jiangle＞Sanming,the average rooting rate of the order was Shaxian＞Jiangle＞Sanming,the materialof Shaxian provenance was the better for cutting.
Sand had the highest survival rate and average rooting rate,the most of the average number of roots,the longest average root length,sand had the best effect for cutting.
Dfferent cuttings part、the promotions of rooting,cutting treaments on the cutting of Litsea Cubeba had different survival rate.The best combination was germination of the roots+shoot of the branch+200mg/L Ll ABT Immersed 30rain+ Cutting into oblique incision at the bottom.
The best of the ear length was 14 cm.The diameter with 0.3-0.4 cm in cutting had the highest survival rate.
May was more suitable for transplanting,after the month,the temperature of the external environment was getting higher,the survival rate also decreased.June can be transplanted,and the best in the rainy days,after transplanting attention should be paid to water management.July and August were not suitable for transplanting.
引文
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