钟花樱离体繁殖技术体系研究
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摘要
钟花樱(Cerasus campanulata)具有冠型美,色彩艳丽,花期长等特点,是优良的园林观赏树种,但是钟花樱自身结实量少,采种期短,采种困难,且扦插繁殖以嫩枝扦插为主,受限制较多,致使钟花樱可用资源极少,处于渐危状态。本研究对钟花樱离体繁殖技术体系进行了探索,分别对外植体的表面灭菌、启动培养、继代增殖培养、生根培养以及炼苗移栽等方面进行了研究,着重探讨了组织培养过程中最佳培养基配方及培养程序,为以后钟花樱的遗传转化及品种改良等研究奠定技术基础,并为钟花樱的组培快繁提供了技术理论依据,具有重要的经济和科学价值。主要研究结果如下:
1钟花樱不同外植体材料消毒的难易程度相差较大。顶芽消毒最易,叶消毒最难,茎段的消毒效果居中。顶芽的消毒以70%酒精处理30-45s后,再以0.1%HgCl_2溶液灭菌5min的处理效果最好,存活率达74.2%;茎段灭菌以70%酒精处理1min后,再以0.1%HgCl_2溶液灭菌7min的处理效果最好,存活率达65.5%;叶的灭菌较为困难,消毒效果最好的为70%酒精处理30-45s后,再以0.1%HgCl_2溶液灭菌8min的处理,但其污染率也高达60.7%,存活率仅为14.3%。
2启动培养试验结果表明:
(1)在附加不同浓度NAA的MS培养基上,钟花樱嫩枝均可诱导愈伤组织分化,但未能诱导出芽。NAA浓度对外植体启动速度有极显著影响,当NAA浓度为0.1mg·L~(-1)时,外植体启动速度最快,平均启动速度为10.71d。
(2)以MS为基本培养基,6-BA浓度0.1-5.0mg·L~(-1)均可诱导愈伤组织分化和芽体分化。6-BA浓度对钟花樱嫩枝的启动培养速度、愈伤组织分化率和芽诱导率均有极显著影响。当6-BA浓度为5.0mg·L~(-1)时,外植体启动速度最快,平均启动速度仅为11.07d:愈伤组织分化率也最大,为89.47%;当6-BA浓度为4.0mag·L~(-1)时,芽诱导率达到最大,为52.60%。
(3)在附加不同浓度6-BA、NAA的MS、改良WH、改良MS培养基上,各处理均有不同程度的愈伤组织形成。统计分析结果表明:基本培养基对愈伤组织分化率影响显著,6-BA浓度对愈伤组织分化率影响极显著,NAA浓度对愈伤组织分化率无显著影响:基本培养基和6-BA浓度对芽诱导率和平均芽数量均有显著性的影响。适合钟花樱嫩枝启动培养的培养基组合为:MS+6-BA2.0mg·L~(-1)+NAA 0.1mg·L~(-1),愈伤组织分化率较低,为38.89%:芽诱导率和平均芽数量均最大,分别为94.44%、3.78。
3在增殖培养试验中,6-BA浓度对增殖系数有极显著影响,NAA浓度对增殖系数有显著影响。6-BA、NAA、蔗糖三者对钟花樱增殖培养效果影响的主次关系为:6-BA＞NAA＞蔗糖。MS+6-BA3.0mg·L~(-1)+NAA 0.01mg·L~(-1)+蔗糖30g·L~(-1)组合,增殖系数最高达7.44。
4 MS+NAA 0.1mg·L~(-1)+GA_3 0.3mg·L~(-1)较有利于壮苗培养。经过壮苗培养,芽长势好,木质化程度高,有利于提高生根率和移栽成活率。MS+NAA 0.1 mg·L~(-1)+GA_3 0.3mg·L~(-1)较有利于壮苗培养。
5生根培养试验结果表明:
(1)基本培养基、IBA和IAA三者对生根率的影响的主次关系为:IBA＞IAA＞基本培养基。基本培养基、IBA浓度、IAA浓度对生根率均有极显著性影响。处理:1/2 MS+IBA 1.0mg·L~(-1)+IAA0.5mg·L~(-1)+蔗糖20g·L~(-1)的生根率最高,达82.35%,且植株长势好,生根快,根长3-4cm。
(2)ABT_1、ABT_3各浓度均可诱导生根,以处理:1/2 MS+ABT_1 1.5mg·L~(-1)+蔗糖20g·L~(-1)生根率最高,达93.05%;其次为:1/2MS+ABT_3 3.0mg·L~(-1)蔗糖20g·L~(-1),生根率达71.30%,但植株长势较差。
6炼苗移栽:不同炼苗方式对组培苗成活率有极显著影响。将生根瓶苗移离培养室,常温条件下闭瓶锻炼6d,开瓶锻炼6d,组培苗平均成活率最高,为93.81%。不同基质处理对移栽成活率有极显著影响。最有利于移栽的基质为蛭石:珍珠岩=1:1,平均成活率高达94.07%。
Cerasus campanulata is a good species among ornamental plants.It has the characteristics of beautiful shape,flamboyant color,long period of blooming and so on.However,the resource application of Cerasus campanulata is limited to low bearing,short and difficult seed collection and restricted cutting and propagation.This paper studied the in-vitro propagation of Cerasus campanulata,including sterilization of explants,initiation,proliferation and rooting culture and domestication and transplantation. These studies aimed to find out the best composition of medium and propagation procedure,which would lay a foundation for genetic transformation and variety improvement and application of other biotechnology of Cerasus campanulata,and also offer a technique theory for its tissue culture propagation, so this study had an important economic and scientific worth.The main results were as follows:
1 There were significant differences between sterilization of explants.The apical buds were disinfected easily,the leaves were difficult and the stems were in the middle.The best sterilization procedure of apical buds was 0.1%HgCl_2 for 5 minutes after 70%alcohol for 30-45 seconds,its survival rate was 74.2%;the best sterilization procedure of stems was 0.1%HgCl_2 for 7 minutes after 70%alcohol for 1 minute,its survival rate could reach 65.5%;the best sterilization procedure of leaves was 0.1%HgCl_2 for 8 minutes after 70%alcohol for 30-45 seconds,but its infected rate was up to 60.7%and its survival rate was only 14.3%.
2 The results of initiation culture showed that:
(1)Using young branches of Cerasus campanulata as explants,the callus could be induced on MS medium with different concentration of NAA,but the shoot could not.The concentration of NAA had greatly significant effect on the initial culture speed.When the concentration of NAA was 0.1mg·L~(-1),the initial culture speed reached the fastest,and the average speed was 10.71 days.
(2)The callus and shoot both could be induced on MS medium adding different concentration of 6-BA 0.1-5.0mg·L~(-1).The concentration of 6-BA had greatly significant effect on the initial culture speed, differentiation rate of callus and shoot induction rate.When the concentration of 6-BA was 5.0mg·L~(-1),the initiation culture speed of explants and differentiation rate of callus achieved their maximum,respectively for 11.07days and 89.47%;while it was 4.0mg·L~(-1),the shoot induction rate was up to the highest with 52.60%.
(3)All treatments had more or less callus formation on the MS,modified WH and modified MS medium containing different concentration of NAA and 6-BA.The statistical analysis showed that basal medium and 6-BA had significant influence on differentiation rate of callus.However,NAA had nothing to do with the speed.They also had obvious effect on shoot induction rate and average number of shoots. The optimal medium for initiation culture was MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1),which was in favor of growth.The differentiation rate of callus was lower and decreased to 38.89%.At the same time,the shoot induction rate and average number of shoots both reached maximum separately at 94.44%and 3.78.
3 In the proliferation culture,the concentration of 6-BA and NAA significantly affected multiplication coefficient.According to the range analysis,the sequence of influential factor of proliferation culture was found to be 6-BA＞NAA＞sucrose.The best combination of medium was MS+6-BA 3.0mg·L~(-1)+NAA 0.01mg·L~(-1)+sucrose 30g·L~(-1),with which the multiplication coefficient was up to 7.44.
4 The suitable medium to promote growth was MS+NAA 0.1 mg·L~(-1)+GA_3 0.3mg·L~(-1).After strong seedling culture,the buds had good growth vigor and higher lignified degree,and the rooting rate and survival rate had been promoted too.
5 The results of rooting culture showed that:
(1)The effects of the three factors on the rooting rate were IBA＞IAA＞basic medium,and they all had significantly influence.The medium of the highest rooting rate was 1/2 MS+IBA 1.0mg·L~(-1)+IAA 0.5mg·L~(-1)+sucrose 20g·L~(-1)with 82.35%rooting ratio.On this medium,the plantlets had good growth vigor, accelerated rooting speed and the root length was 3-4cm.
(2)The concentration of ABT_1 and ABT_3 could all induce rooting.The treatment of 1/2MS+ABT_1 1.5mg·L~(-1)+sucrose 20g·L~(-1)could achieve the highest rooting rate of 93.05%.The following was 1/2MS+ABT_3 3.0mg·L~(-1)+sucrose 20g·L~(-1)with 71.30%rooting ratio,but the growth vigor was poor.
6 Acclimatization and transplantation
Different methods of training seedling had significantly effect on the survival rate of tissue culture seedlings.At first,the rooting seedling was moved from the culture chamber,placed under the condition of normal temperature for six days,and then the lid was opened.After six days,the average survival rate reached the highest of 93.81%.Different transplanting mediums had significant effect on the survival rate of seedlings.The optimal transplanting substrate was the perlite and vermiculate which mixed with 1:1,in which the average survival rate was up to 94.07%.
1钟花樱不同外植体材料消毒的难易程度相差较大。顶芽消毒最易,叶消毒最难,茎段的消毒效果居中。顶芽的消毒以70%酒精处理30-45s后,再以0.1%HgCl_2溶液灭菌5min的处理效果最好,存活率达74.2%;茎段灭菌以70%酒精处理1min后,再以0.1%HgCl_2溶液灭菌7min的处理效果最好,存活率达65.5%;叶的灭菌较为困难,消毒效果最好的为70%酒精处理30-45s后,再以0.1%HgCl_2溶液灭菌8min的处理,但其污染率也高达60.7%,存活率仅为14.3%。
2启动培养试验结果表明:
(1)在附加不同浓度NAA的MS培养基上,钟花樱嫩枝均可诱导愈伤组织分化,但未能诱导出芽。NAA浓度对外植体启动速度有极显著影响,当NAA浓度为0.1mg·L~(-1)时,外植体启动速度最快,平均启动速度为10.71d。
(2)以MS为基本培养基,6-BA浓度0.1-5.0mg·L~(-1)均可诱导愈伤组织分化和芽体分化。6-BA浓度对钟花樱嫩枝的启动培养速度、愈伤组织分化率和芽诱导率均有极显著影响。当6-BA浓度为5.0mg·L~(-1)时,外植体启动速度最快,平均启动速度仅为11.07d:愈伤组织分化率也最大,为89.47%;当6-BA浓度为4.0mag·L~(-1)时,芽诱导率达到最大,为52.60%。
(3)在附加不同浓度6-BA、NAA的MS、改良WH、改良MS培养基上,各处理均有不同程度的愈伤组织形成。统计分析结果表明:基本培养基对愈伤组织分化率影响显著,6-BA浓度对愈伤组织分化率影响极显著,NAA浓度对愈伤组织分化率无显著影响:基本培养基和6-BA浓度对芽诱导率和平均芽数量均有显著性的影响。适合钟花樱嫩枝启动培养的培养基组合为:MS+6-BA2.0mg·L~(-1)+NAA 0.1mg·L~(-1),愈伤组织分化率较低,为38.89%:芽诱导率和平均芽数量均最大,分别为94.44%、3.78。
3在增殖培养试验中,6-BA浓度对增殖系数有极显著影响,NAA浓度对增殖系数有显著影响。6-BA、NAA、蔗糖三者对钟花樱增殖培养效果影响的主次关系为:6-BA＞NAA＞蔗糖。MS+6-BA3.0mg·L~(-1)+NAA 0.01mg·L~(-1)+蔗糖30g·L~(-1)组合,增殖系数最高达7.44。
4 MS+NAA 0.1mg·L~(-1)+GA_3 0.3mg·L~(-1)较有利于壮苗培养。经过壮苗培养,芽长势好,木质化程度高,有利于提高生根率和移栽成活率。MS+NAA 0.1 mg·L~(-1)+GA_3 0.3mg·L~(-1)较有利于壮苗培养。
5生根培养试验结果表明:
(1)基本培养基、IBA和IAA三者对生根率的影响的主次关系为:IBA＞IAA＞基本培养基。基本培养基、IBA浓度、IAA浓度对生根率均有极显著性影响。处理:1/2 MS+IBA 1.0mg·L~(-1)+IAA0.5mg·L~(-1)+蔗糖20g·L~(-1)的生根率最高,达82.35%,且植株长势好,生根快,根长3-4cm。
(2)ABT_1、ABT_3各浓度均可诱导生根,以处理:1/2 MS+ABT_1 1.5mg·L~(-1)+蔗糖20g·L~(-1)生根率最高,达93.05%;其次为:1/2MS+ABT_3 3.0mg·L~(-1)蔗糖20g·L~(-1),生根率达71.30%,但植株长势较差。
6炼苗移栽:不同炼苗方式对组培苗成活率有极显著影响。将生根瓶苗移离培养室,常温条件下闭瓶锻炼6d,开瓶锻炼6d,组培苗平均成活率最高,为93.81%。不同基质处理对移栽成活率有极显著影响。最有利于移栽的基质为蛭石:珍珠岩=1:1,平均成活率高达94.07%。
Cerasus campanulata is a good species among ornamental plants.It has the characteristics of beautiful shape,flamboyant color,long period of blooming and so on.However,the resource application of Cerasus campanulata is limited to low bearing,short and difficult seed collection and restricted cutting and propagation.This paper studied the in-vitro propagation of Cerasus campanulata,including sterilization of explants,initiation,proliferation and rooting culture and domestication and transplantation. These studies aimed to find out the best composition of medium and propagation procedure,which would lay a foundation for genetic transformation and variety improvement and application of other biotechnology of Cerasus campanulata,and also offer a technique theory for its tissue culture propagation, so this study had an important economic and scientific worth.The main results were as follows:
1 There were significant differences between sterilization of explants.The apical buds were disinfected easily,the leaves were difficult and the stems were in the middle.The best sterilization procedure of apical buds was 0.1%HgCl_2 for 5 minutes after 70%alcohol for 30-45 seconds,its survival rate was 74.2%;the best sterilization procedure of stems was 0.1%HgCl_2 for 7 minutes after 70%alcohol for 1 minute,its survival rate could reach 65.5%;the best sterilization procedure of leaves was 0.1%HgCl_2 for 8 minutes after 70%alcohol for 30-45 seconds,but its infected rate was up to 60.7%and its survival rate was only 14.3%.
2 The results of initiation culture showed that:
(1)Using young branches of Cerasus campanulata as explants,the callus could be induced on MS medium with different concentration of NAA,but the shoot could not.The concentration of NAA had greatly significant effect on the initial culture speed.When the concentration of NAA was 0.1mg·L~(-1),the initial culture speed reached the fastest,and the average speed was 10.71 days.
(2)The callus and shoot both could be induced on MS medium adding different concentration of 6-BA 0.1-5.0mg·L~(-1).The concentration of 6-BA had greatly significant effect on the initial culture speed, differentiation rate of callus and shoot induction rate.When the concentration of 6-BA was 5.0mg·L~(-1),the initiation culture speed of explants and differentiation rate of callus achieved their maximum,respectively for 11.07days and 89.47%;while it was 4.0mg·L~(-1),the shoot induction rate was up to the highest with 52.60%.
(3)All treatments had more or less callus formation on the MS,modified WH and modified MS medium containing different concentration of NAA and 6-BA.The statistical analysis showed that basal medium and 6-BA had significant influence on differentiation rate of callus.However,NAA had nothing to do with the speed.They also had obvious effect on shoot induction rate and average number of shoots. The optimal medium for initiation culture was MS+6-BA 2.0mg·L~(-1)+NAA 0.1mg·L~(-1),which was in favor of growth.The differentiation rate of callus was lower and decreased to 38.89%.At the same time,the shoot induction rate and average number of shoots both reached maximum separately at 94.44%and 3.78.
3 In the proliferation culture,the concentration of 6-BA and NAA significantly affected multiplication coefficient.According to the range analysis,the sequence of influential factor of proliferation culture was found to be 6-BA＞NAA＞sucrose.The best combination of medium was MS+6-BA 3.0mg·L~(-1)+NAA 0.01mg·L~(-1)+sucrose 30g·L~(-1),with which the multiplication coefficient was up to 7.44.
4 The suitable medium to promote growth was MS+NAA 0.1 mg·L~(-1)+GA_3 0.3mg·L~(-1).After strong seedling culture,the buds had good growth vigor and higher lignified degree,and the rooting rate and survival rate had been promoted too.
5 The results of rooting culture showed that:
(1)The effects of the three factors on the rooting rate were IBA＞IAA＞basic medium,and they all had significantly influence.The medium of the highest rooting rate was 1/2 MS+IBA 1.0mg·L~(-1)+IAA 0.5mg·L~(-1)+sucrose 20g·L~(-1)with 82.35%rooting ratio.On this medium,the plantlets had good growth vigor, accelerated rooting speed and the root length was 3-4cm.
(2)The concentration of ABT_1 and ABT_3 could all induce rooting.The treatment of 1/2MS+ABT_1 1.5mg·L~(-1)+sucrose 20g·L~(-1)could achieve the highest rooting rate of 93.05%.The following was 1/2MS+ABT_3 3.0mg·L~(-1)+sucrose 20g·L~(-1)with 71.30%rooting ratio,but the growth vigor was poor.
6 Acclimatization and transplantation
Different methods of training seedling had significantly effect on the survival rate of tissue culture seedlings.At first,the rooting seedling was moved from the culture chamber,placed under the condition of normal temperature for six days,and then the lid was opened.After six days,the average survival rate reached the highest of 93.81%.Different transplanting mediums had significant effect on the survival rate of seedlings.The optimal transplanting substrate was the perlite and vermiculate which mixed with 1:1,in which the average survival rate was up to 94.07%.
引文
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