CN105659979B - A kind of breeding method of bletilla striata fungal component - Google Patents

A kind of breeding method of bletilla striata fungal component Download PDF

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CN105659979B
CN105659979B CN201610030269.3A CN201610030269A CN105659979B CN 105659979 B CN105659979 B CN 105659979B CN 201610030269 A CN201610030269 A CN 201610030269A CN 105659979 B CN105659979 B CN 105659979B
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bletilla striata
fungal component
culture medium
days
culture
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CN105659979A (en
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寸建清
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Yunnan Lyuchen Biotechnology Development Co Ltd
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01CPLANTING; SOWING; FERTILISING
    • A01C1/00Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08Immunising seed
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00Cultivation of mushrooms
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00Cultivation of mushrooms
    • A01G18/10Mycorrhiza; Mycorrhizal associations
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor

Abstract

The present invention provides a kind of breeding methods of bletilla striata fungal component, it is characterized in that the disinfection treatment of the bletilla striata stem tuber root access PDA culture medium dark culture 7~8 days, after obtaining mycelium, the dark culture on the culture medium that sawdust and broad leaf tree leaf are prepared, purifying strain is obtained from the other end after 15~20 days, dark culture in RM improved culture medium after purifying strain transfer to sterilizing, the excellent species of mycelia stalwartness are obtained after 7~8 days, excellent species are by pedigree seed culture medium dark culture, obtain the original seed of bletilla striata fungal component within 25~30 days, original seed is accessed into dark culture in Cultivar culture medium, the cultivar of bletilla striata fungal component is obtained after 28~36 days.In use, after the cultivar of bletilla striata fungal component is embedded under soil at 15~20cm, then transplant bletilla striata seedling.Using the bletilla striata fungal component field planting bletilla striata of the invention, the shoot survival percent after transplanting improves 20% or more, and 40% or more yield volume increase, quality is significantly improved.

Description

A kind of breeding method of bletilla striata fungal component
Technical field
The present invention relates to a kind of breeding methods of bletilla striata fungal component.
Background technique
The bletilla striata [Bletilla striata (Thunb.) Reichb. f.] also known as bletilla are orchid.The bletilla striata Pseudobulb can be used as medicine, the tonifying lung that can stop blooding, myogenic pain relieving, profit muscle promoting the circulation of qi.First recorded in《Sheng Nong's herbal classic》:" main pain is swollen, malignant sore, loses Perverse trend in the dead flesh of subcutaneous ulcer, impairment of yin, stomach ".《Compendium of Materia Medica》It also indicates that:" bletilla striata holds back Qi, Shen phlegm, stops blooding, the medicine for the pain that disappears.This medicine matter Very viscous greasy, property pole inducing astrigency, nationality hardship gas are cold, are apt to stop up blood stasis into lung channel because of heat and form disease person, grind that oral, body is hard holds back dirty, packing with this Damaged, bitterly swollen to disappear, routed to hold in the palm, dead flesh can be gone, and purulence blood can be clean, had and comforted early raw new magical effect ".Past, the bletilla striata was mainly by open country Raw breeding, but in recent years since the demand of the bletilla striata is continuously increased, wild plant resource sharply declines, and the bletilla striata has become rare be on the point of Danger species, have been included in《Endangered animal and plant international trade pact》It is protected.
In order to meet the needs of market is to the bletilla striata, people start to plant the bletilla striata using artificial cultivation method in recent years.But mesh It is the breeding of bletilla striata seedling that preceding research is more, the fertilizer practice research after not finding bletilla seed transplantation of seedlings to crop field, generally Using the fertilizing method of conventional crop.Because there are nutriment exchanges, mainly mycorhiza between orchid and mycorrhizal fungi Inorganic salts and organic compound needed for fungi supplies orchid, orchidaceal plant mycorrhizal fungi are different from other mycorrhizal fungis It is that they can not obtain organic nutrient from the root of host, it is necessary to be obtained from ambient enviroment, it is small to be broken down into glucose etc. After molecule carbohydrate, it is absorbed and utilized for orchid root system.The bletilla striata is orchid, if routinely after plantation to crop field The fertilizing method of crops applies fertilizer, because lacking bletilla striata mycorrhizal fungi, it is difficult to which the nutritional need for absorbing and meeting bletilla striata growth causes Bletilla seed shoot survival percent is low, bletilla striata low output and of poor quality.
Summary of the invention
The purpose of the present invention is in view of the above shortcomings, providing a kind of breeding method of bletilla striata fungal component, cultivate After bletilla striata fungal component is applied to bletilla seed transplantation of seedlings crop field, increase the bletilla striata fungal component quantity in crop field, optimize the environment of bletilla striata growth, Bletilla seed shoot survival percent is improved, bletilla striata yield and quality is improved.
The present invention includes that the extraction of bletilla striata fungal component, purification, rejuvenation, protospecies breeding, cultivar expand the following steps such as numerous:
1, bletilla striata stem tuber root is disinfected:Fresh wild bletilla striata stem tuber root clear water is taken to rinse 3~5 removing silt particles miscellaneous Matter after impregnating 15~25min with washing powder water, with aseptic water washing 4~5 times, after then impregnating 25~30s with 75% ethyl alcohol, is used Aseptic water washing 3~5 times, with 0.1~0.2%HgC12Aseptic water washing 5~6 times after 6~8min of immersion, blotted with aseptic filter paper Moisture;
2, the extraction of bletilla striata fungal component:By the bletilla striata stem tuber root after step 1 disinfection treatment with through disappearing on superclean bench The scissors of poison is cut into long 0.2~0.5cm segment, accesses in the PDA culture medium after sterilizing, dark culture at a temperature of 24~26 DEG C, Obtain the bletilla striata fungal component for covering with entire 3~4cm long of media surface within 7~8 days after inoculation;
3, the purification of bletilla striata fungal component:Test tube is clipped into bottom, manages what loading sawdust and broad leaf tree leaf among interior were prepared Culture medium, after both ends add tampon to sterilize, after the mycelium that access step 2 obtains from one end, dark culture at a temperature of 24~26 DEG C, The other end is extended to sawdust depths after mycelia material feeding, obtains purifying strain from the other end after 15~20 days;
4, the rejuvenation of bletilla striata fungal component:In RM improved culture medium after the purification strain transfer to sterilizing that step 3 is obtained, Dark culture at a temperature of 24~26 DEG C, obtains the excellent species of mycelia stalwartness after 7~8 days;
5, the protospecies breeding of bletilla striata fungal component:After pedigree seed culture medium to be packed into the 2/3 of glass culture bottle volume, tampon is covered The rejuvenation strain that step 4 obtains is accessed after sterilizing, dark culture at a temperature of 24~26 DEG C, mycelia grows to bottom of bottle after 25~30 days, The original seed of bletilla striata fungal component is obtained, the mass percent of pedigree seed culture medium is:Weed tree sawdust 39%, broad leaf tree leaf 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1%, material water quality ratio is 1:1.3~1.5;
6, the cultivar of bletilla striata fungal component expands numerous:Cultivar culture medium is packed into high density low pressure polyethylene polybag and is gone out Bacterium, accesses the original seed for the bletilla striata fungal component that step 5 obtains after cooling, dark culture at a temperature of 2~23 DEG C obtains after 28~36 days The mass percent of the cultivar of bletilla striata fungal component, Cultivar culture medium is:Weed tree sawdust 30%, broad leaf tree leaf 48%, wheat bran skin 10%, corn flour 10%, sucrose 1%, land plaster 1%, material water quality ratio is 1:1.3~1.5.
In use, after first the cultivar for the bletilla striata fungal component that step 5 obtains is embedded under soil at 15~20cm, then transplant white Splendid achnatherum seedling, measure routinely carry out field management.In Lijiang County In Yunnan Province city, Gucheng District Kingsoft office made by continuous 3 years Shown with experiment using the bletilla striata fungal component field planting bletilla striata of the invention, than not using bletilla striata fungal component crop field of the invention The plantation bletilla striata is compared, and the shoot survival percent after transplanting improves 20% or more, and 40% or more yield volume increase, quality is significantly improved.
Specific embodiment
The present invention includes the extraction, purification, rejuvenation, protospecies breeding, numerous, the specific embodiment party of cultivar expansion of bletilla striata fungal component Formula is:
1, bletilla striata stem tuber root is disinfected:Fresh wild bletilla striata stem tuber root clear water is taken to rinse 3~5 removing silt particles miscellaneous Matter, after impregnating 15~25min with washing powder water, with aseptic water washing 4~5 times, after then impregnating 25~30s with 75% ethyl alcohol, nothing Bacterium water rinses 3~5 times, with 0.1~0.2%HgC12Aseptic water washing 5~6 times after 6~8min of immersion, water is blotted with aseptic filter paper Point;
2, the extraction of bletilla striata fungal component:By the bletilla striata stem tuber root after step 1 disinfection treatment with through disappearing on superclean bench The scissors of poison is cut into long 0.2~0.5cm segment, accesses in the PDA culture medium after sterilizing, dark culture at a temperature of 24~26 DEG C, 2 See that white flocculence mycelia grows after~3 days, mycelia is long to 3~4cm long after 5~6 days, is grown within 7~8 days after inoculation The bletilla striata fungal component of full entire 3~4cm long of media surface;PDA culture medium is:Potato(Peeling)200 grams, glucose 20 Gram, 18~20 grams of agar, 1000 milliliters of water;Through 120~125 DEG C of temperature, 1.1~1.2Kgf/cm of pressure2It is lower sterilizing 25~ 30min accesses bletilla striata stem tuber root segment material after cooling;
3, the purification of bletilla striata fungal component:Test tube is clipped into bottom, manages what loading sawdust and broad leaf tree leaf among interior were prepared The mass percent of culture medium, culture medium is:Weed tree sawdust 60%, broad leaf tree leaf 30%, wheat bran 8%, sucrose 1%, land plaster 1%, material Water quality ratio is 1:1.3~1.6, after both ends add tampon, in 128~130 DEG C of temperature, 1.5~1.6Kgf/cm of pressure2Lower sterilizing 120~150min, after cooling after the mycelium that one end accesses that step 3 obtains, dark culture at a temperature of 24~26 DEG C, mycelia is eaten The other end is extended to sawdust depths after material, the other end obtains purifying strain after 15~20 days, carries out using sawdust bacteriological filtration method Filter, purification, to achieve the purpose that the bletilla striata fungal component strain of high-purity;
4, the rejuvenation of bletilla striata fungal component:In RM improved culture medium after the purification strain transfer to sterilizing that step 3 is obtained, Dark culture at a temperature of 24~26 DEG C is observed on the 3rd~4 day it is observed that the 2nd day can be seen white hypha growth, bacterium colony growth For circular, aerial hyphae is undeveloped, and mycelia can cover with media surface after 7~8 days, at this point, can cultivate growth it is vigorous, The excellent species of mycelia stalwartness, are observed through surface, and the positive back side of mycelia is white to pale red, RM improved culture medium formula in test tube For:2 grams of peptone, 20 grams of glucose, 0.46 gram of potassium dihydrogen phosphate, 1 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, agar 15~20 Gram, 200 grams of potato, 1000 milliliters of distilled water, through 120~125 DEG C of temperature, 1.1~1.2Kgf/cm of pressure2It is lower sterilizing 25~ 30min accesses purification strain after cooling;
5, the protospecies breeding of bletilla striata fungal component:After pedigree seed culture medium to be packed into the 2/3 of glass culture bottle volume, tampon is covered Afterwards in 128~130 DEG C of temperature, 1.5~1.6Kgf/cm of pressure2120~150min of lower sterilizing accesses what step 4 obtained after cooling Rejuvenation strain, dark culture at a temperature of 24~26 DEG C, mycelia grows to bottom of bottle after 25~30 days, obtains the original seed of bletilla striata fungal component, The mass percent of pedigree seed culture medium is:Weed tree sawdust 39%, broad leaf tree leaf 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1% expect water Mass ratio is 1:1.3~1.5;
6, the cultivar of bletilla striata fungal component expands numerous:Cultivar culture medium is packed into high density low pressure polyethylene polybag, Culture medium be attached to volume 2/3 after cover non-cotton cover and sterilize 18~22 hours under 95~100 DEG C of normal pressures of temperature, cooling is followed by The original seed for entering the bletilla striata fungal component that step 5 obtains, dark culture at a temperature of 20~23 DEG C, after 25~30 days, mycelia can be covered with Bacterium bag;After-mature cultivation 3~6 days again at this time, can be used to bletilla striata seedling and mix bacteria cultivation, the mass percent of Cultivar culture medium is: Weed tree sawdust 30%, broad leaf tree leaf 48%, wheat bran skin 10%, corn flour 10%, sucrose 1%, land plaster 1%, material water quality ratio are 1:1.3 ~1.5.

Claims (2)

1. a kind of breeding method of bletilla striata fungal component, steps are as follows:
(1)The disinfection treatment of bletilla striata stem tuber root:Fresh wild bletilla striata stem tuber root clear water is taken to rinse 3~5 removing silt particles miscellaneous Matter after impregnating 15~25min with washing powder water, with aseptic water washing 4~5 times, after then impregnating 25~30s with 75% ethyl alcohol, is used Aseptic water washing 3~5 times, with 0.1~0.2%HgC12Aseptic water washing 5~6 times after 6~8min of immersion, blotted with aseptic filter paper Moisture;
(2)The extraction of bletilla striata fungal component:By step(1)Bletilla striata stem tuber root after disinfection treatment is on superclean bench with sterilized Scissors be cut into long 0.2~0.5cm segment, in the PDA culture medium after access sterilizing, dark culture at a temperature of 24~26 DEG C is connect Obtain the bletilla striata fungal component for covering with entire 3~4cm long of media surface within 7~8 days after kind;
It is characterized in that also having the following steps:
(3)The purification of bletilla striata fungal component:Test tube is clipped into bottom, manages the interior intermediate culture for being packed into sawdust and broad leaf tree leaf and preparing Base after both ends add tampon to sterilize, accesses step from one end(2)After extracting obtained mycelium, secretly trained at a temperature of 24~26 DEG C It supports, extends to the other end to sawdust depths after mycelia material feeding, obtain purifying strain from the other end after 15~20 days;
(4)The rejuvenation of bletilla striata fungal component:By step(3)In RM improved culture medium after obtained purification strain transfer to sterilizing, Dark culture at a temperature of 24~26 DEG C, obtains the excellent species of mycelia stalwartness after 7~8 days;The rejuvenation is improved with RM and is cultivated Based formulas is:2 grams of peptone, 20 grams of glucose, 0.46 gram of potassium dihydrogen phosphate, 1 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, agar 15~20 grams, 200 grams of potato, 1000 milliliters of distilled water;
(5)The protospecies breeding of bletilla striata fungal component:After pedigree seed culture medium to be packed into the 2/3 of glass culture bottle volume, covers tampon and go out Step is accessed after bacterium(4)Obtained rejuvenation strain, dark culture at a temperature of 24~26 DEG C, mycelia grows to bottom of bottle after 25~30 days, The original seed of bletilla striata fungal component is obtained, the mass percent of pedigree seed culture medium is:Weed tree sawdust 39%, broad leaf tree leaf 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1%, material water quality ratio is 1:1.3~1.5;
(6)The cultivar of bletilla striata fungal component expands numerous:Cultivar culture medium is packed into high density low pressure polyethylene polybag and is sterilized, Step is accessed after cooling(5)The original seed of obtained bletilla striata fungal component, dark culture at a temperature of 2~23 DEG C, obtains after 28~36 days The mass percent of the cultivar of bletilla striata fungal component, Cultivar culture medium is:Weed tree sawdust 30%, broad leaf tree leaf 48%, wheat bran skin 10%, corn flour 10%, sucrose 1%, land plaster 1%, material water quality ratio is 1:1.3~1.5.
2. the breeding method of bletilla striata fungal component according to claim 1, it is characterized in that the purification culture medium of bletilla striata fungal component Mass percent be:Weed tree sawdust 60%, broad leaf tree leaf 30%, wheat bran 8%, sucrose 1%, land plaster 1%, material water quality ratio are 1: 1.3~1.6.
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CN110338047B (en) * 2019-06-28 2021-09-17 长江大学 Wild-imitating efficient direct seeding seedling method for bletilla striata seeds
CN113207590A (en) * 2021-04-29 2021-08-06 南京工业大学大丰海洋产业研究院 Bletilla direct seeding and seedling raising method utilizing symbiotic bacteria

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CN105103924A (en) * 2015-09-25 2015-12-02 马龙县兴裕农业生物科技开发有限公司 High-yield cultivation method for pollution-free bletilla striata

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