蒲苇组培再生体系建立及其越夏适应性研究
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摘要
蒲苇(Cortaderia selloana)系禾本科蒲苇属植物,是一种多年生草本植物。在最近几年,随着城市绿化种植品种的丰富,观赏草类植物越来越受到园林爱好者和设计者们的青睐。作为一种著名的观赏草,蒲苇株型优美,常绿,叶呈线形自然下垂,花序成纺锤状圆锥花序,观赏性很强可保持至春季。本文以粉红蒲苇为材料,研究了蒲苇愈伤组织的诱导和植株再生的条件以及其在重庆地区干旱炎热的夏季条件下的生理生化变化和适应性。这为蒲苇的遗传转化奠定了基础;为蒲苇的基因工程育种提供了前提;有利于品种改良和新品种的开发:同时,也给其它一些禾本科观赏草的组织培养、引种适应性和园林应用性提供参考。
本文首先对蒲苇组织培养的外植体进行了选择。实验结果表明,蒲苇的成熟种子能成功诱导出愈伤组织并能进一步分化再生成完整的植株,而蒲苇幼嫩的叶片和茎段却未能诱导出愈伤组织,也没有能分化出再生植株。
同时对蒲苇组织培养外植体的消毒方式进行了探索。结果显示,蒲苇种子的消毒灭菌方式为:75%酒精处理20s+0.1%氯化汞处理10min,能达到发芽率高和污染率低的最佳组合;蒲苇嫩叶的消毒方式为:75%酒精处理30s+0.1%氯化汞处理10min;而幼嫩茎段的消毒方式为:75%酒精处理40s+0.1%氯化汞处理12min。
蒲苇成熟种子愈伤组织诱导最佳培养基选择了MS+2,4-D3.0mg/L+6-BA0.3mg/L,在这种培养成份组合下,其愈伤组织诱导率可以达到78%,且形态整齐,体积较大,呈乳白色分散颗粒状。
将愈伤组织进行再分化培养,发现MS+6-BA2.0mg/L+NAA0.1mg/L为最佳培养成份组合;幼苗继代培养时则需要MS+6-BA1.5m个/L+NAA0.2mg/L,此时幼苗生长较快,且高度一致,整体健壮繁茂。
蒲苇再生植株生根培养中,活性炭对生根起了决定性的作用,另外NAA的加入促进了根的生成,缩短了生根时间,并增强了生根质量。因而蒲苇生根培养的培养基成分组合为1/2MS+NAA0.8mg/L+6-BA0.2mg/L+(0.03%)AC。
对蒲苇盆栽植株的越夏适应性研究表明,在高温干旱的双逆境胁迫下,蒲苇植株通过各种生理反应,胁迫初期能积极响应抵御逆境的伤害,随着胁迫的加深则出现无法适应的现象,最终导致死亡,其具体变化如下:形态上,蒲苇植株叶片经历了从绿色自然舒展到白色干枯卷曲的过程,表现出蒲苇对干热胁迫的一定适应性反应。同时,叶绿素的含量呈现先升高后降低并总是高于对照水平的趋势。
干热胁迫下,蒲苇叶片细胞膜系统遭到伤害,使得膜脂过氧化产物MDA持续上升;同时,膜透性也随之增大,相对电导率也呈现一直上升的趋势。
随着胁迫的持续进行,蒲苇叶片抗氧化系统也随之活跃起来。抗氧化酶SOD、POD活性一直都高于对照水平,且呈现上升的趋势,以此来抵御干热胁迫产生的氧自由基对蒲苇植株的伤害。
干热胁迫下,蒲苇叶片可溶性物质含量的变化呈现出一定的相关性,这显示了蒲苇在耐热抗旱下的一些适应措施。可溶性糖、可溶性蛋白以及脯氨酸含量在蒲苇受干热胁迫后都出现了上升趋势,并在一定程度上具有相互转化的可能性,为蒲苇抵御干热胁迫制造有利条件。
Cortaderia selloana is belong to Gramineae and it is a herb perennial. In recent years, with the development of landscaping, ornamental grasses had been paid attention more and more by designers and garden lovers.As a famous ornamental grasses, Cortaderia selloana has beautiful ideotype, evergreen and linear natural drop leaves, cone-shaped spindle inflorescence and the inflorescence can maintain a strong view to the spring. In this paper, we take Cortaderia selloana pink for materials. We study on the conditions of callus induction and plant regeneration system and then on the physiological and biochemical reactions of hot and drought stress in the hot summer conditions in Chongqing.
From this study ,we laid the foundation of the genetic transformation of Cortaderia selloana; provide a prerequisite for genetic engineering breeding of Cortaderia selloana.lt is conducive to improved varieties and the development of new varieties. At the same time, it provide a reference application for the other grasses on tissue culture and landscape.
First we select the explants of tissue culture. The results show that the mature seed can induct callus successfully and the callus can be differentiation and re-generation to complete plants. But the young leaves and stem can not induce callus, nor to divide the regeneration.
At the same time, we explore the disinfection methods with the tissue culture. The results showed that the sterilization for seed is the 75% alcohol to deal with 20 s and the 0.1% mercuric chloride to deal with 10 min, this can achieve high germination rate and low pollution rate; the leaves disinfection methods as follows: 75% alcohol to deal with 30 s and 0.1% mercuric chloride to deal with 10 min; and the young stem segments of disinfection for: 75% alcohol to deal with 40s and 0.1% mercuric chloride processing 12min.
We choose the best medium for callus induction from mature seeds is the MS and 2, 4-D 3.0 mg/L and 6- BA 0.3mg / L. In this combination of nutrients, the rate of callus induction is 78%.The callus shape is large and tidy and it is milky white and dispersion granule.
We cultured callus re-differentiation and then found that the best combination of training components is MS and 6- BA 2.0mg / L and NAA0.1 mg / L. And the subculture needs MS and 6-BA 1.5mg / L and NAA 0.2mg / L. In this Condition the seedling growth fast and highly and the entirety is healthy and strong.
In the rooting culture the activated carbon play a decisive role on roots. And the addition of NAA promoted root formation. It shortens the time and enhances the quality. So the best medium composition for rooting culture is 1/2MS and NAA0.8 mg / L and 6-BA 0.2 mg / L and 0.03%AC.
The study on adaptation in summer of potted plants showns thatin the dual stress of high temperature and drought stress through a variety of physiological responses Cortaderia selloana can resist the initial positive response to adversity injury. With the stress-depth appeared it is unable to adapt to the phenomenon, which eventually led to the death. The specific changes are as follows: First the leaves experience the process which from green to white and dry naturally to curly stretch. It shows the adaptive responses on the hot and dry stress. And At the same time, chlorophyll content increased at first and after the lower level is always higher than the trend of the control.
Under the stress the membrane system of leaf was injured, making the product of lipid peroxidation MDA continued to rise. At the same time, membrane permeability also increases. The relative conductivity has been also shown an upward trend.
Under the stress the activity of SOD and POD has always above the contrast level. And the upward trend to withstand dry heat stress produced by oxygen free radicals on injured plants.
Under the stress the content of soluble sugar soluble protein and proline has emerged in an upward trend. And they will transfer mutually.
本文首先对蒲苇组织培养的外植体进行了选择。实验结果表明,蒲苇的成熟种子能成功诱导出愈伤组织并能进一步分化再生成完整的植株,而蒲苇幼嫩的叶片和茎段却未能诱导出愈伤组织,也没有能分化出再生植株。
同时对蒲苇组织培养外植体的消毒方式进行了探索。结果显示,蒲苇种子的消毒灭菌方式为:75%酒精处理20s+0.1%氯化汞处理10min,能达到发芽率高和污染率低的最佳组合;蒲苇嫩叶的消毒方式为:75%酒精处理30s+0.1%氯化汞处理10min;而幼嫩茎段的消毒方式为:75%酒精处理40s+0.1%氯化汞处理12min。
蒲苇成熟种子愈伤组织诱导最佳培养基选择了MS+2,4-D3.0mg/L+6-BA0.3mg/L,在这种培养成份组合下,其愈伤组织诱导率可以达到78%,且形态整齐,体积较大,呈乳白色分散颗粒状。
将愈伤组织进行再分化培养,发现MS+6-BA2.0mg/L+NAA0.1mg/L为最佳培养成份组合;幼苗继代培养时则需要MS+6-BA1.5m个/L+NAA0.2mg/L,此时幼苗生长较快,且高度一致,整体健壮繁茂。
蒲苇再生植株生根培养中,活性炭对生根起了决定性的作用,另外NAA的加入促进了根的生成,缩短了生根时间,并增强了生根质量。因而蒲苇生根培养的培养基成分组合为1/2MS+NAA0.8mg/L+6-BA0.2mg/L+(0.03%)AC。
对蒲苇盆栽植株的越夏适应性研究表明,在高温干旱的双逆境胁迫下,蒲苇植株通过各种生理反应,胁迫初期能积极响应抵御逆境的伤害,随着胁迫的加深则出现无法适应的现象,最终导致死亡,其具体变化如下:形态上,蒲苇植株叶片经历了从绿色自然舒展到白色干枯卷曲的过程,表现出蒲苇对干热胁迫的一定适应性反应。同时,叶绿素的含量呈现先升高后降低并总是高于对照水平的趋势。
干热胁迫下,蒲苇叶片细胞膜系统遭到伤害,使得膜脂过氧化产物MDA持续上升;同时,膜透性也随之增大,相对电导率也呈现一直上升的趋势。
随着胁迫的持续进行,蒲苇叶片抗氧化系统也随之活跃起来。抗氧化酶SOD、POD活性一直都高于对照水平,且呈现上升的趋势,以此来抵御干热胁迫产生的氧自由基对蒲苇植株的伤害。
干热胁迫下,蒲苇叶片可溶性物质含量的变化呈现出一定的相关性,这显示了蒲苇在耐热抗旱下的一些适应措施。可溶性糖、可溶性蛋白以及脯氨酸含量在蒲苇受干热胁迫后都出现了上升趋势,并在一定程度上具有相互转化的可能性,为蒲苇抵御干热胁迫制造有利条件。
Cortaderia selloana is belong to Gramineae and it is a herb perennial. In recent years, with the development of landscaping, ornamental grasses had been paid attention more and more by designers and garden lovers.As a famous ornamental grasses, Cortaderia selloana has beautiful ideotype, evergreen and linear natural drop leaves, cone-shaped spindle inflorescence and the inflorescence can maintain a strong view to the spring. In this paper, we take Cortaderia selloana pink for materials. We study on the conditions of callus induction and plant regeneration system and then on the physiological and biochemical reactions of hot and drought stress in the hot summer conditions in Chongqing.
From this study ,we laid the foundation of the genetic transformation of Cortaderia selloana; provide a prerequisite for genetic engineering breeding of Cortaderia selloana.lt is conducive to improved varieties and the development of new varieties. At the same time, it provide a reference application for the other grasses on tissue culture and landscape.
First we select the explants of tissue culture. The results show that the mature seed can induct callus successfully and the callus can be differentiation and re-generation to complete plants. But the young leaves and stem can not induce callus, nor to divide the regeneration.
At the same time, we explore the disinfection methods with the tissue culture. The results showed that the sterilization for seed is the 75% alcohol to deal with 20 s and the 0.1% mercuric chloride to deal with 10 min, this can achieve high germination rate and low pollution rate; the leaves disinfection methods as follows: 75% alcohol to deal with 30 s and 0.1% mercuric chloride to deal with 10 min; and the young stem segments of disinfection for: 75% alcohol to deal with 40s and 0.1% mercuric chloride processing 12min.
We choose the best medium for callus induction from mature seeds is the MS and 2, 4-D 3.0 mg/L and 6- BA 0.3mg / L. In this combination of nutrients, the rate of callus induction is 78%.The callus shape is large and tidy and it is milky white and dispersion granule.
We cultured callus re-differentiation and then found that the best combination of training components is MS and 6- BA 2.0mg / L and NAA0.1 mg / L. And the subculture needs MS and 6-BA 1.5mg / L and NAA 0.2mg / L. In this Condition the seedling growth fast and highly and the entirety is healthy and strong.
In the rooting culture the activated carbon play a decisive role on roots. And the addition of NAA promoted root formation. It shortens the time and enhances the quality. So the best medium composition for rooting culture is 1/2MS and NAA0.8 mg / L and 6-BA 0.2 mg / L and 0.03%AC.
The study on adaptation in summer of potted plants showns thatin the dual stress of high temperature and drought stress through a variety of physiological responses Cortaderia selloana can resist the initial positive response to adversity injury. With the stress-depth appeared it is unable to adapt to the phenomenon, which eventually led to the death. The specific changes are as follows: First the leaves experience the process which from green to white and dry naturally to curly stretch. It shows the adaptive responses on the hot and dry stress. And At the same time, chlorophyll content increased at first and after the lower level is always higher than the trend of the control.
Under the stress the membrane system of leaf was injured, making the product of lipid peroxidation MDA continued to rise. At the same time, membrane permeability also increases. The relative conductivity has been also shown an upward trend.
Under the stress the activity of SOD and POD has always above the contrast level. And the upward trend to withstand dry heat stress produced by oxygen free radicals on injured plants.
Under the stress the content of soluble sugar soluble protein and proline has emerged in an upward trend. And they will transfer mutually.
引文
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