CN101926259B - Orchid germchit propagating method - Google Patents

Abstract

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

A kind of spring orchid seedling propagation method

technical field

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.

Background technique

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.

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.

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.

Contents of the invention

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.

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:

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;

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.

Above-mentioned seed germination process mainly comprises the following steps:

a) selection of explants: select uncracked capsules of Chunlan as explants;

b) Seed treatment: after the aseptic treatment of Chunlan capsules, take out the powdery seeds for aseptic treatment;

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.

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.

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.

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.

Detailed ways

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) 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) 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) 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) 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) 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.

Claims (1)

1. a Chunlan seedling propagation method, its seedling obtains and mainly comprises seed germination, protocorm subculture propagation and rooting culture process, it is characterized in that this propagation method also carries out the seedling hardening of rooted seedling after seedling obtains and with bacteria The mycorrhization process of root fungi, specifically: (1) The seed germination process mainly includes the following steps: a) selection of explants: select uncracked capsules of Chunlan as explants; b) Seed treatment: after aseptic treatment of Chunlan capsules, the powdered seeds are taken out for aseptic treatment; c) Seed germination: the processed Chunlan seeds are evenly sown on the seed germination medium to cultivate and grow into protocorms; The medium formula used for seed germination 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, medium pH value 5.0-6.0; (2) The protocorm subculture 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 geometric Progressive 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, The pH value of the medium is 5.0-6.0; (3) The rooting culture process is to transfer the relatively underdeveloped seedlings of the root system to the rooting medium for cultivation, so that the rooting rate reaches more than 95%, and the plants grow vigorously. After 7 weeks, small seedlings of 5 to 7 cm are formed. The culture used The formula of the base is 1/2MS+NAA0.5mg/L+activated carbon 0.5g/L+sucrose 25g/L+agar 3g/L, and the pH value of the medium is 5.0-6.0; (4) The seedling hardening process is to take out the rooted seedlings after opening the bottle cap for 1 to 1.5 weeks under natural light conditions indoors, and clean the culture medium attached to the roots of the seedlings for subsequent use; (5) The process of mycorrhizalization of seedlings is to 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 which time water is sprayed on the leaves to promote the growth of new roots After 3 weeks, move it into the greenhouse and carry out normal water and fertilizer management. After 9.5 months, you can get mycorrhizal seedlings; It is obtained by adding a small amount of oak leaf tertiary bacteria mixed with Rhizoctonia sp.CLB111 bacteria solution to 4 parts of dry humus soil and 1/4 part of red soil.