CN101926259B - Orchid germchit propagating method - Google Patents
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- 239000003337 fertilizer Substances 0.000 claims description 3
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Abstract
一种春兰种苗繁殖方法,属于一种兰花育苗繁殖技术,尤其是一种组织培养与菌根真菌共生培养育苗的繁殖技术。本发明的一种春兰种苗繁殖方法,其种苗获得主要包括种子萌发、原球茎继代增殖和生根培养过程,其特征在于该繁殖方法在种苗获得后还进行生根苗的炼苗及与菌根真菌的菌根化过程。本发明采用春兰种子无菌萌发与菌根真菌共生的菌根化培育技术,既可繁殖优良品种,从种子实生苗中选择优良兰株,又能快速提高春兰繁育速度,克服纯组培苗生长缓慢,周期长,甚至不开花的缺点,对春兰的大面积产业化扩繁具有重要的利用价值。The invention relates to a spring orchid seedling propagation method, which belongs to orchid seedling breeding and propagation technology, in particular to a propagation technology of tissue culture and mycorrhizal fungus symbiotic culture and seedling cultivation. A kind of Chunlan seedling propagation method of the present invention, its seedling acquisition mainly comprises seed germination, protocorm subculture multiplication and rooting culture process, it is characterized in that this propagation method also carries out rooting seedling tempering after seedling obtains and with Mycorrhizal process of mycorrhizal fungi. The invention adopts the mycorrhizal cultivation technology of aseptic germination of Chunlan seeds and symbiosis of mycorrhizal fungi, which can not only propagate excellent varieties, select excellent orchid strains from seed seedlings, but also rapidly increase the breeding speed of Chunlan, and overcome the growth of pure tissue culture seedlings. The shortcomings of slowness, long cycle, and even no flowering have important utilization value for the large-scale industrial expansion of Chunlan.
Description
技术领域 technical field
本发明属于一种兰花育苗繁殖技术,尤其是一种组织培养与菌根真菌共生培养育苗的繁殖技术。The invention belongs to a technique for growing orchid seedlings, in particular to a technique for growing seedlings through symbiosis of tissue culture and mycorrhizal fungi.
背景技术 Background technique
兰花是指整个兰科植物(Orchidaceae)的总称。在我国,传统的“兰花”概念仅指产于我国的兰科、兰属(Cymbidium)植物,称国兰,地生,故又称地生兰。兰科植物是一类观赏价值和经济价值极高的珍贵植物。其中,云南兰科植物就有100属,530多种,为全国之冠。Orchid refers to the general term of the whole Orchidaceae. In my country, the traditional concept of "orchid" only refers to the Orchidaceae and Cymbidium plants produced in my country. Orchidaceae is a class of precious plants with high ornamental value and economic value. Among them, there are 100 genera and more than 530 species of orchids in Yunnan, ranking first in the country.
但是到目前为止,我国兰花开发与利用仍处于初级阶段,未能解决大量繁殖的问题,因此供需矛盾突出。一方面是由于经济利益的驱动,农民上山大量采挖野生兰花,野生兰花资源受到严重破坏,尤其是较名贵的墨兰(Cymbidium sinense)、春兰(Cymbidiumgoeringii)、寒兰(Cymbidium kanran)、春剑(Cymbidium longibracteatun)和连瓣兰(Cymbidiumlianpan)破坏最为严重。其次是森林面积大幅度减少,兰花生存环境的改变,不仅对野生兰花种群延续形成威胁,也使得许多种类兰花在一些地区消失。近年来,我国加强了野生植物资源的保护工作,我国第一部专门保护野生植物的行政法规《中华人民共和国野生植物保护条例》自1997年1月1日实施,根据该条例的规定,拟定并经审议通过了《国家重点保护野生植物名录》,兰科所有种均列在名录中。但是仅采用法律形式保护兰花资源还是远远不够的,在加强管理的同时,也应重视发展兰花的繁殖技术,才能根本解决保护兰花物种的目的。But so far, the development and utilization of orchids in my country are still in the primary stage, and the problem of mass reproduction has not been solved, so the contradiction between supply and demand is prominent. On the one hand, due to the drive of economic interests, farmers went to the mountains to dig a large number of wild orchids, and the resources of wild orchids were severely damaged, especially the more expensive Molan (Cymbidium sinense), Chunlan (Cymbidium goeringii), Hanlan (Cymbidium kanran), Chunjian (Cymbidium longibracteatun) and Cymbidium lianpan were the most damaged. Secondly, the forest area has been greatly reduced, and the changes in the living environment of orchids have not only threatened the continuation of wild orchid populations, but also caused many species of orchids to disappear in some areas. In recent years, my country has strengthened the protection of wild plant resources. my country's first administrative regulation dedicated to the protection of wild plants, the "Regulations of the People's Republic of China on the Protection of Wild Plants", came into effect on January 1, 1997. According to the regulations, the draft and After deliberation and approval of the "List of National Key Protected Wild Plants", all species of Orchidaceae are listed in the list. However, it is far from enough to protect orchid resources only by law. While strengthening management, we should also pay attention to the development of orchid reproduction technology, so as to fundamentally solve the purpose of protecting orchid species.
兰花的繁殖技术目前主要有两种,分株和组织培养。传统的分株繁殖速度慢,繁殖率低,周期长,且易伤母株,种子在自然条件下发芽率极低,很难满足规模化生产的需要。而组织培养,移栽无菌苗成活率低,生长发育缓慢,开花迟,甚至不开花,原因主要是缺少与之共生的菌根真菌。真菌为兰花提供生长繁殖所必需的营养物质。兰科植物与菌根真菌形成菌根是自然界的一种普遍现象,所以菌根真菌为兰科植物提供营养进行生长发育是解决人工栽培兰科植物的关键,也是我国兰花资源与利用保护所面临的主要问题。There are two main types of orchid propagation techniques, division and tissue culture. Traditional ramets have slow propagation speed, low reproduction rate, long cycle, and are easy to damage the mother plant. The germination rate of seeds is extremely low under natural conditions, which is difficult to meet the needs of large-scale production. In tissue culture, the survival rate of transplanted aseptic seedlings is low, the growth and development are slow, flowering is delayed, or even no flowering, the main reason is the lack of symbiotic mycorrhizal fungi. Fungi provide orchids with the nutrients they need to grow and reproduce. The formation of mycorrhizae between orchids and mycorrhizal fungi is a common phenomenon in nature. Therefore, mycorrhizal fungi provide nutrition for orchids for growth and development. main problem.
发明内容 Contents of the invention
本发明所要解决的就是仅靠分株和组织培养繁殖春兰不能够满足我国兰花植物资源保护的问题。本发明从野生春兰根中分离内生真菌,使其与春兰组培苗共生培养,筛选出能与春兰组培苗有效共生并能明显促进其生长发育的有效菌根真菌,对春兰组培苗进行菌根化培育,从根本上克服了试管苗繁殖,缺乏菌根真菌,移栽无菌苗成活率低,生长缓慢,开花迟缓,花小甚至不开花的缺点。What the present invention aims to solve is the problem that only relying on ramets and tissue culture to propagate Chunlan cannot satisfy the protection of orchid plant resources in my country. The present invention separates endophytic fungi from wild Chunlan roots, makes them symbiotically cultivated with Chunlan tissue-cultured seedlings, and screens out effective mycorrhizal fungi that can effectively symbiose with Chunlan tissue-cultured seedlings and can obviously promote their growth and development. Mycorrhizal cultivation fundamentally overcomes the shortcomings of test-tube seedling propagation, lack of mycorrhizal fungi, low survival rate of transplanted aseptic seedlings, slow growth, slow flowering, small flowers or even no flowering.
本发明的一种春兰种苗繁殖方法,其种苗获得主要包括种子萌发、原球茎继代增殖和生根培养过程,其特征在于该繁殖方法在种苗获得后还进行生根苗的炼苗及苗木菌根化过程,具体为:A kind of Chunlan seedling propagation method of the present invention, its seedling acquisition mainly comprises seed germination, protocorm subculture multiplication and rooting culture process, is characterized in that this propagation method also carries out rooting seedling hardening and seedling stock after seedling is obtained Mycorrhization process, specifically:
炼苗:将生根苗在室内自然光照条件下开瓶盖过渡1~1.5周后取出,洗净沾附在小苗根部的培养基备用;Seedling hardening: take out the rooted seedlings after opening the bottle cap for 1 to 1.5 weeks under natural light indoors, and wash the medium attached to the roots of the seedlings for later use;
苗木菌根化:将取出的小苗用含有菌根真菌的基质包裹种植于穴盘中,在适度温度下,置于阴凉通风处栽培,期间向叶部喷洒水,促进新根生长,3周后移入温室中进行正常水、肥、药管理,9.5月后即可得菌根化种苗;所述的基质是按重量份分别将1/4份泥炭、1/4份珍珠岩、1/4份干腐质土和1/4份红土中加入拌有菌液(Rhizoctonia sp.CLB111)的栎树叶三级菌种少许而得。Mycorrhizalization of seedlings: wrap the taken out seedlings with a matrix containing mycorrhizal fungi and plant them in plug trays, and cultivate them in a cool and ventilated place at a moderate temperature. During this period, spray water on the leaves to promote the growth of new roots. After 3 weeks Move into the greenhouse and carry out normal water, fertilizer, medicine management, can get the mycorrhized seedling after 9.5 months; Described substrate is respectively by weight 1/4 part peat, 1/4 part perlite, 1/4 part It is obtained by adding a small amount of oak leaf tertiary bacteria mixed with bacterial solution (Rhizoctonia sp.CLB111) to 1 part of dry humus soil and 1/4 part of red soil.
上述的种子萌发过程主要包括以下步骤:Above-mentioned seed germination process mainly comprises the following steps:
a)外植体的选择:选择春兰未裂开的蒴果为外植体;a) selection of explants: select uncracked capsules of Chunlan as explants;
b)种子处理:将春兰蒴果无菌处理后取出粉沫状种子无菌处理;b) Seed treatment: after the aseptic treatment of Chunlan capsules, take out the powdery seeds for aseptic treatment;
c)种子萌发:将经处理的春兰种子均匀播种在种子萌发培养基上培养生长为原球茎;所使用的培养基配方为:1/2MS+NAA0.5~1.0mg/L+IBA2~3mg/L+100ml/L椰乳+甘露醇1g/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。c) Seed germination: sow the treated Chunlan seeds evenly on the seed germination medium to cultivate and grow into protocorms; the medium formula used is: 1/2MS+NAA0.5~1.0mg/L+IBA2~3mg/ L + 100ml/L coconut milk + mannitol 1g/L + activated carbon 0.5g/L + sucrose 25g/L + agar 3g/L, the pH value of the medium is 5.0-6.0.
上述的原球茎继代增殖过程是将初代培养的原球茎和芽分别在培养基上继代增殖培养50~60天,长成新芽或原球茎,以后不断切割原球茎继代,即可几何级数增长;所使用的培养基配方为:1/2MS+NAA0.1~0.5mg/L+6-BA0.5~0.8mg/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。The above-mentioned protocorm subculture propagation process is to subculture the primary cultured protocorms and buds on the culture medium for 50 to 60 days, grow into new shoots or protocorms, and then continue to cut the protocorms for subsequent generations, which can be geometrically graded. growth; the medium formula used is: 1/2MS+NAA0.1~0.5mg/L+6-BA0.5~0.8mg/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L, culture The base pH value is 5.0-6.0.
上述的生根培养过程是将较大的根系不发达的苗转入生根培养基上培养,使生根率达95%以上,植株生长旺盛,7周后形成5~7cm的小苗;所使用的培养基配方为:1/2MS+NAA0.5mg/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。The above-mentioned rooting culture process is to transfer larger seedlings with underdeveloped root systems to the rooting medium for cultivation, so that the rooting rate reaches more than 95%, the plant grows vigorously, and 5-7cm seedlings are formed after 7 weeks; the medium used The formula is: 1/2MS+NAA0.5mg/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L, the pH value of the medium is 5.0-6.0.
本发明通过调整激素浓度、配比和根菌真菌的共生培养,能有效利用兰科植物菌根共生促进生长的特点,缩短兰科植物组培苗的繁殖周期,成活率达到了95%以上。本发明采用春兰种子无菌萌发与菌根真菌共生的菌根化培育技术,既可繁殖优良品种,从种子实生苗中选择优良兰株,又能快速提高春兰繁育速度,克服纯组培苗生长缓慢,周期长,甚至不开花的缺点,对春兰的大面积产业化扩繁具有重要的利用价值。The invention can effectively utilize the characteristics of orchidaceae mycorrhizal symbiosis to promote growth by adjusting hormone concentration, ratio and symbiotic culture of rhizophytes, shorten the reproduction cycle of orchidaceae tissue culture seedlings, and the survival rate reaches more than 95%. The invention adopts the mycorrhizal cultivation technology of aseptic germination of Chunlan seeds and symbiosis of mycorrhizal fungi, which can not only propagate excellent varieties, select excellent orchid strains from seed seedlings, but also rapidly increase the breeding speed of Chunlan, and overcome the growth of pure tissue culture seedlings. The shortcomings of slowness, long cycle, and even no flowering have important utilization value for the large-scale industrial expansion of Chunlan.
具体实施方式Detailed ways
实施例1:选用2003年在云南省保山的优良单株上采得的春兰种子,于2004年6月份在西南林学院的温室大棚内进行炼苗,该繁殖方法春兰种苗成活率达到了95%以上,具体的种苗繁殖过程如下:Embodiment 1: select the Chunlan seed that was collected on the excellent single plant of Baoshan, Yunnan Province in 2003, and in June, 2004, carry out hardening in the greenhouse of Southwest Forestry College, and the survival rate of this breeding method Chunlan seedling has reached 95%. More than %, the specific seedling propagation process is as follows:
(1)种子萌发:将野外成熟180天的蒴果采回,在4度冰箱种冷藏2周,用流水冲洗20分钟取春兰未裂开的蒴果为外植体。在70~75%的酒精中表面消毒30~50秒,无菌水漂洗一次,又用2%的次氯酸钠消毒20分钟,用无菌水冲洗4~5次,再以0.1%的升汞溶液消毒10~15分钟,最后用无菌水冲洗5次。将洗净的成熟蒴果置于灭菌滤纸上吸干水分,把消毒的蒴果置于已灭菌的培养皿中,用无菌手术刀纵向切开春兰蒴果;再用镊子取出春兰粉沫状种子放入预先准备好的无菌小滤纸袋内,浸入0.15~0.2mol/L的NaOH溶液中处理8~10分钟取出,在无菌水中冲洗4~5次,每次换水前用灭过菌的干滤纸挤压滤纸袋吸干水分;然后把经处理的春兰种子均匀地播种在种子萌发培养基上,暗培养2周后转入每天光照12~14小时,光照强度1000~1500lux,温度25℃。培养160~180天即可生长为原球茎。其培养基的配方为1/2MS+NAA0.5~1.0mg/L+IBA2-3mg/L+100ml/L椰乳+甘露醇1g/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。(1) Seed germination: the capsules matured in the field for 180 days were harvested, planted in a 4-degree refrigerator for 2 weeks, rinsed with running water for 20 minutes, and uncracked capsules of Chunlan were taken as explants. Disinfect the surface in 70-75% alcohol for 30-50 seconds, rinse with sterile water once, then disinfect with 2% sodium hypochlorite for 20 minutes, rinse with sterile water for 4-5 times, and then disinfect with 0.1% mercury chloride solution 10 to 15 minutes, and finally rinsed with sterile water 5 times. Put the washed mature capsules on sterilized filter paper to absorb the water, put the sterilized capsules in a sterilized petri dish, cut the Chunlan capsules longitudinally with a sterile scalpel; then use tweezers to take out the Chunlan powdery seeds Put it into a pre-prepared sterile small filter paper bag, soak it in 0.15-0.2mol/L NaOH solution for 8-10 minutes, take it out, rinse it in sterile water for 4-5 times, and use sterile water before changing the water each time. Then the treated Chunlan seeds were evenly sown on the seed germination medium, and after 2 weeks of dark cultivation, they were transferred to 12-14 hours of light every day, with light intensity of 1000-1500lux and temperature of 25 ℃. After 160-180 days of cultivation, it can grow into a protocorm. The formula of the culture medium is 1/2MS+NAA0.5~1.0mg/L+IBA2-3mg/L+100ml/L coconut milk+mannitol 1g/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L , the pH value of the medium is 5.0-6.0.
(2)原球茎继代增殖:将初代培养的原球茎和芽分别在培养基上继代增殖培养,原球茎增殖速度快,增殖效果好,50~60天长成新芽或原球茎,以后不断切割原球茎继代,即可几何级数增长。其培养基的配方为1/2MS+NAA0.1~0.5mg/L+6-BA0.5~0.8mg/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。(2) Subculture propagation of protocorms: Protocorms and buds cultivated in the first generation are subcultured and cultured respectively on the culture medium. The protocorms proliferate quickly and have good multiplication effect. They grow into new buds or protocorms in 50 to 60 days, and they will be cut continuously in the future. Protocorm subculture can grow geometrically. The formula of the medium is 1/2MS+NAA0.1~0.5mg/L+6-BA0.5~0.8mg/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L, and the pH value of the medium is 5.0 ~6.0.
(3)生根培养:将较大的根系不发达的苗转入生根培养基上培养,植株生长旺盛,7周后形成5~7cm的小苗,根系发达。其培养基的配方为1/2MS+NAA0.5mg/L+活性碳0.5g/L+蔗糖25g/L+琼脂3g/L,培养基PH值为5.0~6.0。(3) Rooting culture: Transfer the larger seedlings with underdeveloped root systems to the rooting medium for cultivation. The plants grow vigorously, and 5-7 cm seedlings are formed after 7 weeks with well-developed root systems. The formula of the culture medium is 1/2MS+NAA0.5mg/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L, and the pH value of the culture medium is 5.0-6.0.
(4)炼苗:将8~10厘米高的生根苗在室内自然光照条件下开瓶盖过渡1~1.5周后,取出洗净沾附在小苗根部的培养基备用。(4) Seedling hardening: After the rooted seedlings with a height of 8 to 10 cm are opened under natural light conditions indoors for 1 to 1.5 weeks, the culture medium attached to the roots of the seedlings is taken out and washed for subsequent use.
(5)苗木菌根化:将取出的苗用含有菌根真菌的基质包裹种植于穴盘中,管护中保持适度温度,置于阴凉通风处栽培,期间只需叶部喷洒水,有利于新根生长,3周后移入温室中,进行正常水、肥管理,成活率可达95%以上,9.5月后即可得菌根化种苗。其基质是按重量份分别将1/4份泥炭、1/4份珍珠岩、1/4份干腐质土和1/4份红土中加入拌有菌液(Rhizoctonia sp.CLB111)的栎树叶三级菌种少许而得。(5) Mycorrhizalization of seedlings: wrap the taken out seedlings with a matrix containing mycorrhizal fungi and plant them in plug trays, maintain a moderate temperature in the management and protection, and place them in a cool and ventilated place for cultivation. During this period, only the leaves need to be sprayed with water, which is beneficial The new roots grow, and after 3 weeks, move them into the greenhouse, and carry out normal water and fertilizer management. The survival rate can reach more than 95%, and the mycorrhizal seedlings can be obtained after 9.5 months. The substrate is to add 1/4 part of peat, 1/4 part of perlite, 1/4 part of dry humus soil and 1/4 part of red soil into oak leaves mixed with bacterial liquid (Rhizoctonia sp.CLB111) in parts by weight. A little bit of the third-class strains are obtained.
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