丛枝菌根与无糖培养对海棠组培苗生理效应研究
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摘要
植物组织培养是一项能获得大量同品质种苗的快速繁育技术，具有其它育苗方式无法比拟的优势，已成为植物良种繁育的重要手段。论文针对常规组织培养生长环境差、易污染、种苗玻璃化、驯化周期长等实际问题，以及植物无糖组培CO_2控制精度低、湿度难以控制、促苗手段缺乏等技术难题，从大型培养容器的设计、环境的精确控制、无糖暴露培养模式、PPFD与CO_2浓度相关性以及菌根促苗等关键技术入手，创建了以大型培养容器为核心的高品质种苗无糖培养技术体系。本研究主要内容与成果如下：
1．研制了装有新型CO_2施放装置的180L大型培养容器及其环境控制系统，通过对培养容器的改进，使操作更容易；同时，更大的垂直空间，提高了培养容器内气体浓度的缓冲性能；CO_2施放装置的设计与安装，为提高CO_2施放时的控制精度创造了条件；采用小流量控制、三通阀调节和PWM控制方式，实现对CO_2浓度的精确控制，控制精度达到±50μmol·mol~(-1)；采用覆膜技术和气体循环吸附除湿相结合的方式实现了对容器内相对湿度的自动控制，控制精度达到±2％。
2．采用自行设计的无糖培养容器和环境控制系统，建立了穴盘覆膜暴露培养新模式，改进了传统的密闭式小容器培养方法，实现了组培苗开放式培养，摆脱了许多传统组织培养的束缚，省略了组培苗驯化阶段，建立了新的技术体系，采用该模式培养的组培苗光合能力强、根系发达，植株的干物质积累多，组培苗生长均匀，品质高。
3．研究了PPFD与CO_2相互作用对组培苗光合作用的影响，明确了组培环境应处于适当的高CO_2浓度和与之相匹配的高PPFD，以使组培苗高效利用光能，促进组培苗的生长。
4．建立了生根阶段接种菌根真菌提高组培苗品质的方法。在生根阶段接种AM真菌，形成平衡的根际生物环境，促进了组培苗的生长发育。主要表现为：减少叶片的气孔阻力、增加叶绿素含量和CO_2的固定，提高寄主植物的光合速率，改善植株的生理状况及品质；而且，形成的菌根真菌—组培苗共生关系在移栽后仍然存在，并减轻了移栽对海棠组培苗造成的胁迫，缩短海棠组培苗的休眠时间7天。
5．研究并确立了在离体条件下建立丛枝菌根—组培苗共生体关键技术措施：
(1)降低培养基中营养离子浓度。对于圆叶海棠组培苗研究采用无激素无糖的1／4MS培养基，降低了培养基矿质离子浓度，为有益微生物生长提供一个低渗透压的生存环境。
(2)选择侵染率高效的菌株。本研究筛选了Gv菌根接种圆叶海棠组培苗能够提高组培苗的品质。
(3)适宜的接种量，本研究筛选了菌根接种圆叶海棠组培苗接种量为5 g·pot~(-1)的为最好。
(4)增加光照度和CO_2浓度能够提高组培苗的光合作用，促进组培苗光合产物的积累，减轻菌根真菌初期侵染寄主植物时的副作用。
Plant tissue culture is a widely used biological technique method for production of large number of genetically identical plantlets. It has many advantages over conventional propagation techniques, and has been developed to an important means of plantlet propagation. In this paper, due to the problems of the conventional tissue culture, such as: the unhealthy growth environment, the vulnerable pollution, the vitrification, the long dormant period, and the technical difficulties of the plant sugar-free tissue culture, such as: low CO_2 control precision, the high humidity, the lack of means to improve the plantlet quality and so on. Researches were undertaken on the large-scale vessel design, the environment accuracy control, the sugar-free exposition culture pattern, relevance of PPFD and CO_2 concentration, as well as the mycorrhiza technology to improve the plantlet and so on. The sugar-free culture technology system for high-quality plantlet was founded. The main achievement of this research as follows:
1. A novel 180 litres large-scale cultural vessel for sugar-free culture was designed. The vessel consisted of CO_2 release pipe (with holes in the pipe), environmental control device and gas exchange holes. This design made the operation to be easier; At the same time, a bigger vertical space enhanced the gas density cushion performance; the installment of CO_2 release pipe created the condition for high control precision; Through the small-flow control, the three-way-valve adjustment and the PWM, The control precision was up to±50μmol·mol~(-1); the membrane tectoria and the gas-cycle adsorption to eliminate the humidity were used to automatically control the relative humidity; the control precision was±2%.
2. The sugar-free culture box with environmental control system independently designed was adopted, the sugar-free exposed culture pattern in the membrana-tectoria plate were proposed for the first time, which improved the traditional method using the small container, and broke away from many of the traditional constraints of plant tissue culture. The open-vitro cultivation became true, which omitted from the acclimation phase, and established a new technical system. Using this pattern, the plantlets had higher photosynthesis, the developed root, the more dry matter, the growing unanimity, and the better quality.
3. The relevance of PPFD and CO_2 concentration to the photosynthesis of plantlets was studied, it was showed that when the high CO_2 concentration was matched with high PPFD the photosynthetic rate of plantlets in vitro would be promoted effectively, and the plantlets was vigorous and healthy.
4. An integrated inoculation technology to enhance of the growth of the plantlets had been established in rooting stage in sugar-free culture in vitro, which formed the balanced rhizosphere biology environment, and promoted the growth of the plantlets, such as: reduced leaf's stoma resistance, increased chlorophyll content and CO_2 fixation, enhanced the photosynthesis rate of the host plantlets, and improved physiological condition and the quality of the plantlets; Moreover, this kind of paragenesis relations still existed after transplanted, which reduced environment stress after transplanted, and shortened the dormant period of crabapple, the sprout time of new leaf was ahead of time 7 days than those of non-mycorrhizal explants.
5. The key technology of mycorrhizal inoculation with AM fungus on the photosynthesis of plantlets in vitro was:
(1) Reducing the nutrition ion concentration in the culture medium. 1/4 hormone-free and sugar-free MS medium was used to culture Malus pruniolia var ringo plantlets, which reduced mineral ion concentration, and provided a low-osmolality living environment for microbial growth.
(2) Choosing species of highly colonization. The better selection of mycorrhizal inoculation species of Malus pruniolia var ringo plantlet in vitro was Gv.
(3) Using suitable amount of inoculums. The better selection of mycorrhizal inoculation amount in vitro of Malus pruniolia var ringo plantlet was 5 g pot~(-1).
(4) Increasing the light intensity and CO_2 concentration in plant tissue culture can improve the plantlets' photosynthesis, accelerate accumulation of photosynthetic products, and reduce the side effects of AM initial infection to the host plantlets
1．研制了装有新型CO_2施放装置的180L大型培养容器及其环境控制系统，通过对培养容器的改进，使操作更容易；同时，更大的垂直空间，提高了培养容器内气体浓度的缓冲性能；CO_2施放装置的设计与安装，为提高CO_2施放时的控制精度创造了条件；采用小流量控制、三通阀调节和PWM控制方式，实现对CO_2浓度的精确控制，控制精度达到±50μmol·mol~(-1)；采用覆膜技术和气体循环吸附除湿相结合的方式实现了对容器内相对湿度的自动控制，控制精度达到±2％。
2．采用自行设计的无糖培养容器和环境控制系统，建立了穴盘覆膜暴露培养新模式，改进了传统的密闭式小容器培养方法，实现了组培苗开放式培养，摆脱了许多传统组织培养的束缚，省略了组培苗驯化阶段，建立了新的技术体系，采用该模式培养的组培苗光合能力强、根系发达，植株的干物质积累多，组培苗生长均匀，品质高。
3．研究了PPFD与CO_2相互作用对组培苗光合作用的影响，明确了组培环境应处于适当的高CO_2浓度和与之相匹配的高PPFD，以使组培苗高效利用光能，促进组培苗的生长。
4．建立了生根阶段接种菌根真菌提高组培苗品质的方法。在生根阶段接种AM真菌，形成平衡的根际生物环境，促进了组培苗的生长发育。主要表现为：减少叶片的气孔阻力、增加叶绿素含量和CO_2的固定，提高寄主植物的光合速率，改善植株的生理状况及品质；而且，形成的菌根真菌—组培苗共生关系在移栽后仍然存在，并减轻了移栽对海棠组培苗造成的胁迫，缩短海棠组培苗的休眠时间7天。
5．研究并确立了在离体条件下建立丛枝菌根—组培苗共生体关键技术措施：
(1)降低培养基中营养离子浓度。对于圆叶海棠组培苗研究采用无激素无糖的1／4MS培养基，降低了培养基矿质离子浓度，为有益微生物生长提供一个低渗透压的生存环境。
(2)选择侵染率高效的菌株。本研究筛选了Gv菌根接种圆叶海棠组培苗能够提高组培苗的品质。
(3)适宜的接种量，本研究筛选了菌根接种圆叶海棠组培苗接种量为5 g·pot~(-1)的为最好。
(4)增加光照度和CO_2浓度能够提高组培苗的光合作用，促进组培苗光合产物的积累，减轻菌根真菌初期侵染寄主植物时的副作用。
Plant tissue culture is a widely used biological technique method for production of large number of genetically identical plantlets. It has many advantages over conventional propagation techniques, and has been developed to an important means of plantlet propagation. In this paper, due to the problems of the conventional tissue culture, such as: the unhealthy growth environment, the vulnerable pollution, the vitrification, the long dormant period, and the technical difficulties of the plant sugar-free tissue culture, such as: low CO_2 control precision, the high humidity, the lack of means to improve the plantlet quality and so on. Researches were undertaken on the large-scale vessel design, the environment accuracy control, the sugar-free exposition culture pattern, relevance of PPFD and CO_2 concentration, as well as the mycorrhiza technology to improve the plantlet and so on. The sugar-free culture technology system for high-quality plantlet was founded. The main achievement of this research as follows:
1. A novel 180 litres large-scale cultural vessel for sugar-free culture was designed. The vessel consisted of CO_2 release pipe (with holes in the pipe), environmental control device and gas exchange holes. This design made the operation to be easier; At the same time, a bigger vertical space enhanced the gas density cushion performance; the installment of CO_2 release pipe created the condition for high control precision; Through the small-flow control, the three-way-valve adjustment and the PWM, The control precision was up to±50μmol·mol~(-1); the membrane tectoria and the gas-cycle adsorption to eliminate the humidity were used to automatically control the relative humidity; the control precision was±2%.
2. The sugar-free culture box with environmental control system independently designed was adopted, the sugar-free exposed culture pattern in the membrana-tectoria plate were proposed for the first time, which improved the traditional method using the small container, and broke away from many of the traditional constraints of plant tissue culture. The open-vitro cultivation became true, which omitted from the acclimation phase, and established a new technical system. Using this pattern, the plantlets had higher photosynthesis, the developed root, the more dry matter, the growing unanimity, and the better quality.
3. The relevance of PPFD and CO_2 concentration to the photosynthesis of plantlets was studied, it was showed that when the high CO_2 concentration was matched with high PPFD the photosynthetic rate of plantlets in vitro would be promoted effectively, and the plantlets was vigorous and healthy.
4. An integrated inoculation technology to enhance of the growth of the plantlets had been established in rooting stage in sugar-free culture in vitro, which formed the balanced rhizosphere biology environment, and promoted the growth of the plantlets, such as: reduced leaf's stoma resistance, increased chlorophyll content and CO_2 fixation, enhanced the photosynthesis rate of the host plantlets, and improved physiological condition and the quality of the plantlets; Moreover, this kind of paragenesis relations still existed after transplanted, which reduced environment stress after transplanted, and shortened the dormant period of crabapple, the sprout time of new leaf was ahead of time 7 days than those of non-mycorrhizal explants.
5. The key technology of mycorrhizal inoculation with AM fungus on the photosynthesis of plantlets in vitro was:
(1) Reducing the nutrition ion concentration in the culture medium. 1/4 hormone-free and sugar-free MS medium was used to culture Malus pruniolia var ringo plantlets, which reduced mineral ion concentration, and provided a low-osmolality living environment for microbial growth.
(2) Choosing species of highly colonization. The better selection of mycorrhizal inoculation species of Malus pruniolia var ringo plantlet in vitro was Gv.
(3) Using suitable amount of inoculums. The better selection of mycorrhizal inoculation amount in vitro of Malus pruniolia var ringo plantlet was 5 g pot~(-1).
(4) Increasing the light intensity and CO_2 concentration in plant tissue culture can improve the plantlets' photosynthesis, accelerate accumulation of photosynthetic products, and reduce the side effects of AM initial infection to the host plantlets
引文
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