CN105659979A - Method for cultivating symbiotic bacteria of Bletilla striata - Google Patents
Method for cultivating symbiotic bacteria of Bletilla striata Download PDFInfo
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- CN105659979A CN105659979A CN201610030269.3A CN201610030269A CN105659979A CN 105659979 A CN105659979 A CN 105659979A CN 201610030269 A CN201610030269 A CN 201610030269A CN 105659979 A CN105659979 A CN 105659979A
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- bletillae
- pseudobulbus bletillae
- rhizoma bletillae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention provides a method for cultivating symbiotic bacteria of Bletilla striata. The method is characterized in that disinfection treatment is carried out for a stem tuber root of Bletilla striata, and the disinfected stem tuber root is inoculated on a PDA medium for dark culture of 7-8 days; after mycelium is obtained, dark culture is carried out for mycelium on a medium which is prepared by wood chips and broadleaf tree leaves; after 15-20 days, a purified bacterial strain is obtained from the other end; the purified bacterial strain is inoculated on a disinfected RM improved medium for dark culture, after 7-8 days, an excellent bacterial strain with healthy mycelium is obtained; the excellent bacterial strain is cultivated on an original species medium for dark culture, after 25-30 days, the original species of the symbiotic bacteria of Bletilla striata is obtained; the original species is inoculated on a cultivated species medium for dark culture, after 28-36 days, the cultivated species of the symbiotic bacteria of Bletilla striata is obtained. During usage, the cultivated species of the symbiotic bacteria of Bletilla striata is imbedded at the place 15-20cm below soil, and bletilla striata seedlings are transplanted. Bletilla striata is planted in the field with the symbiotic bacteria of Bletilla striata, survival rate of transplanted seedlings is increased by more than 20%, yield is increased by more than 40%, and quality is obviously improved.
Description
Technical field
The present invention relates to the breeding method of a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component.
Background technology
The Pseudobulbus Bletillae (Rhizoma Bletillae) [Bletillastriata (Thunb.) Reichb.f.], has another name called Pseudobulbus Bletillae (Rhizoma Bletillae), for orchid. Pseudobulbus Bletillae (Rhizoma Bletillae) pseudobulb can be used as medicine, tonifying the lung of stopping blooding, granulation promoting pain relieving, profit muscle circulation of qi promoting. Begin to be loaded in Shennong's Herbal: " main pain is swollen, malignant boil, lose pathogen in cellulitis, the dead flesh of impairment of YIN, stomach ". Compendium of Material Medica also indicates that: " Pseudobulbus Bletillae (Rhizoma Bletillae), astringing QI, expectorant, stop blooding, the medicine of the pain that disappears also. This medicine matter is very viscous greasy, and property pole astringent therapy, nationality hardship gas are cold, are apt to into lung meridian, stops up blood stasis because of heat and forms disease person, with this grind oral, body hard hold back dirty, packing is damaged, pain is swollen can disappear, and routed holds in the palm, dead flesh can go, and pus and blood can be clean, have comfort early tissue regeneration promoting magical effect also ". The past Pseudobulbus Bletillae (Rhizoma Bletillae) is mainly by wild breeding, but in recent years owing to the demand of the Pseudobulbus Bletillae (Rhizoma Bletillae) is continuously increased, wild plant resource sharply declines, and the Pseudobulbus Bletillae (Rhizoma Bletillae) has become rare and endangered species, now lists " endangered animal and plant international trade pact " in and is protected.
In order to meet the market demand to the Pseudobulbus Bletillae (Rhizoma Bletillae), people begin with the artificial cultivation method plantation Pseudobulbus Bletillae (Rhizoma Bletillae) in recent years. But at present what research was more is the breeding of Pseudobulbus Bletillae (Rhizoma Bletillae) seedling, it does not have find that bletilla seed transplantation of seedlings is to the fertilizer practice research behind land for growing field crops, is generally adopted the fertilizing method of conventional crop. Because there is nutrient substance exchange between orchid and mycorrhizal fungi, mainly mycorrhizal fungi supplies the inorganic salt needed for orchid and organic compound, orchidaceal plant mycorrhizal fungi and other mycorrhizal fungi cannot obtain organic nutrient from the root of host the difference is that them, must obtain from surrounding, after being broken down into the little molecular carbon hydrates such as glucose, utilize for orchid Root Absorption. The Pseudobulbus Bletillae (Rhizoma Bletillae) is orchid, if plantation fertilizing method fertilising of crops routinely behind land for growing field crops, because of lacking Pseudobulbus Bletillae (Rhizoma Bletillae) mycorrhizal fungi, it is difficult to absorb and meet the nutritional need that the Pseudobulbus Bletillae (Rhizoma Bletillae) grows, causes that bletilla seed shoot survival percent is low, the Pseudobulbus Bletillae (Rhizoma Bletillae) yields poorly and of poor quality.
Summary of the invention
It is an object of the invention to for above-mentioned weak point, the breeding method of a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component is provided, after the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component cultivated is applied to bletilla seed transplantation of seedlings land for growing field crops, increase the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component quantity in land for growing field crops, optimize the environment of Pseudobulbus Bletillae (Rhizoma Bletillae) growth, improve bletilla seed shoot survival percent, improve Pseudobulbus Bletillae (Rhizoma Bletillae) yield and quality.
The present invention includes the following steps such as the extraction of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, purification, rejuvenation, protospecies breeding, cultigen expanding propagation:
1, Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root is disinfected: takes fresh wild Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root clear water and rinses 3～5 removing sediment contaminations, after detergent water soaking 15～25min, with aseptic water washing 4～5 times, then with after 75% soak with ethanol 25～30s, with aseptic water washing 3～5 times, with 0.1～0.2%HgC12After immersion 6～8min, aseptic water washing 5～6 times, uses aseptic filter paper suck dry moisture;
2, the extraction of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: the Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root after step 1 being disinfected is cut into long 0.2～0.5cm segment with sterilized shears on superclean bench, access in the PDA culture medium after sterilizing, light culture at 24～26 DEG C of temperature, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component covering with whole media surface 3～4cm length in after inoculation 7～8 days;
3, the purification of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: test tube is clipped bottom, wood flour and the culture medium of broad leaf tree leaves preparation is loaded in the middle of in pipe, after two ends add tampon sterilizing, after accessing, from one end, the mycelium that step 2 obtains, light culture at 24～26 DEG C of temperature, extend to the other end to wood flour depths after mycelia material feeding, after 15～20 days, obtain purification strain from the other end;
4, the rejuvenation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: what step 3 obtained purifies strain transfer to the RM improved culture medium after sterilizing, light culture at 24～26 DEG C of temperature obtains the excellent species that mycelia is healthy and strong after 7～8 days;
5, the protospecies breeding of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: after pedigree seed culture medium is loaded the 2/3 of glass culture bottle volume, the rejuvenation strain that step 4 obtains is accessed after covering tampon sterilizing, light culture at 24～26 DEG C of temperature, at the bottom of mycelia grows to bottle after 25～30 days, obtains the original seed of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, the mass percent of pedigree seed culture medium is: weed tree sawdust 39%, broad leaf tree leaves 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1%, material water quality ratio is for 1:1.3～1.5;
6, the cultigen expanding propagation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: Cultivar culture medium is loaded sterilizing in high density low-pressure polyethylene plastic bag, the original seed of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component that step 5 obtains is accessed after cooling, light culture at 2～23 DEG C of temperature, obtains the cultigen of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component after 28～36 days, the mass percent of Cultivar culture medium is: weed tree sawdust 30%, broad leaf tree leaves 48%, Testa Tritici skin 10%, Semen Maydis powder 10%, sucrose 1%, Gypsum Fibrosum powder 1%, material water quality ratio is for 1:1.3～1.5.
During use, after the cultigen of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component first step 5 obtained is embedded under soil 15～20cm place, then transplanting Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling, measure routinely carries out field management. Experiments show that through the use of continuous 3 years in Kingsoft, Gucheng District, Lijiang County In Yunnan Province city office, use the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component field planting Pseudobulbus Bletillae (Rhizoma Bletillae) of the present invention, ratio does not use the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component field planting Pseudobulbus Bletillae (Rhizoma Bletillae) of the present invention to compare, shoot survival percent after transplanting improves more than 20%, yield volume increase more than 40%, quality is significantly improved.
Detailed description of the invention
The present invention includes the extraction of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, purification, rejuvenation, protospecies breeding, cultigen expanding propagation, and its detailed description of the invention is:
1, Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root is disinfected: takes fresh wild Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root clear water and rinses 3～5 removing sediment contaminations, after detergent water soaking 15～25min, with aseptic water washing 4～5 times, then with after 75% soak with ethanol 25～30s, aseptic water washing 3～5 times, with 0.1～0.2%HgC12After immersion 6～8min, aseptic water washing 5～6 times, uses aseptic filter paper suck dry moisture;
2, the extraction of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: the Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root after step 1 being disinfected is cut into long 0.2～0.5cm segment with sterilized shears on superclean bench, access in the PDA culture medium after sterilizing, light culture at 24～26 DEG C of temperature, namely see after 2～3 days that white flocculence mycelia grows, after 5～6 days, mycelia is long to 3～4cm length, within after inoculation 7～8 days, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component covering with whole media surface 3～4cm length; PDA culture medium is: Rhizoma Solani tuber osi (peeling) 200 grams, glucose 20 grams, 18～20 grams of agar, 1000 milliliters of water; Through temperature 120～125 DEG C, pressure 1.1～1.2Kgf/cm2Lower sterilizing 25～30min, accesses Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root segment material after cooling;
3, the purification of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: test tube is clipped bottom, wood flour and the culture medium of broad leaf tree leaves preparation is loaded in the middle of in pipe, the mass percent of culture medium is: weed tree sawdust 60%, broad leaf tree leaves 30%, wheat bran 8%, sucrose 1%, Gypsum Fibrosum powder 1%, material water quality ratio is for 1:1.3～1.6, after two ends add tampon, at temperature 128～130 DEG C, pressure 1.5～1.6Kgf/cm2Lower sterilizing 120～150min, after accessing, from one end, the mycelium that step 3 obtains after cooling, light culture at 24～26 DEG C of temperature, the other end is extended to wood flour depths after mycelia material feeding, after 15～20 days, the other end obtains purification strain, wood flour bacteriological filtration method is adopted to be filtered, purify, thus reaching the purpose of highly purified Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component strain;
4, the rejuvenation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: what step 3 obtained purifies strain transfer to the RM improved culture medium after sterilizing, light culture at 24～26 DEG C of temperature, it be observed that the 2nd day namely it can be seen that white hypha grows, within 3rd～4 day, observe, colony growth is circular, aerial hyphae is undeveloped, after 7～8 days, mycelia just can cover with media surface, now, growth can be cultivated vigorous, the excellent species that mycelia is healthy and strong, through surface observation, in test tube, the positive back side of mycelia is white to pale red, RM improved culture medium formula is: peptone 2 grams, glucose 20 grams, potassium dihydrogen phosphate 0.46 gram, dipotassium hydrogen phosphate 1 gram, 0.5 gram of magnesium sulfate, 15～20 grams of agar, Rhizoma Solani tuber osi 200 grams, distilled water 1000 milliliters, through temperature 120～125 DEG C, pressure 1.1～1.2Kgf/cm2Lower sterilizing 25～30min, accesses after cooling and purifies strain;
5, the protospecies breeding of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: after pedigree seed culture medium loads the 2/3 of glass culture bottle volume, covers after tampon at temperature 128～130 DEG C, pressure 1.5～1.6Kgf/cm2Lower sterilizing 120～150min, the rejuvenation strain that step 4 obtains is accessed after cooling, light culture at 24～26 DEG C of temperature, at the bottom of mycelia grows to bottle after 25～30 days, obtains the original seed of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, the mass percent of pedigree seed culture medium is: weed tree sawdust 39%, broad leaf tree leaves 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1%, material water quality ratio is for 1:1.3～1.5;
6, the cultigen expanding propagation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: Cultivar culture medium is loaded in high density low-pressure polyethylene plastic bag, culture medium installs to non-cotton cover sterilizing 18～22 hours under 95～100 DEG C of normal pressures of temperature on 2/3 bonnet of volume, the original seed of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component that step 5 obtains is accessed after cooling, light culture at 20～23 DEG C of temperature, after 25～30 days, mycelia can cover with bacterium bag; Now After-mature cultivation 3～6 days again, namely can be used for Pseudobulbus Bletillae (Rhizoma Bletillae) Seedling and mix bacteria cultivation, and the mass percent of Cultivar culture medium is: weed tree sawdust 30%, broad leaf tree leaves 48%, Testa Tritici skin 10%, Semen Maydis powder 10%, sucrose 1%, Gypsum Fibrosum powder 1%, and material water quality ratio is for 1:1.3～1.5.
Claims (3)
1. a breeding method for Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, is characterized in that comprising the steps:
(1) Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root is disinfected: takes fresh wild Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root clear water and rinses 3～5 removing sediment contaminations, after detergent water soaking 15～25min, with aseptic water washing 4～5 times, then with after 75% soak with ethanol 25～30s, with aseptic water washing 3～5 times, with 0.1～0.2%HgC12After immersion 6～8min, aseptic water washing 5～6 times, uses aseptic filter paper suck dry moisture;
(2) extraction of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: the Pseudobulbus Bletillae (Rhizoma Bletillae) tuber root after step (1) being disinfected is cut into long 0.2～0.5cm segment with sterilized shears on superclean bench, access in the PDA culture medium after sterilizing, light culture at 24～26 DEG C of temperature, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component covering with whole media surface 3～4cm length in after inoculation 7～8 days;
(3) purification of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: test tube is clipped bottom, wood flour and the culture medium of broad leaf tree leaves preparation is loaded in the middle of in pipe, after two ends add tampon sterilizing, after accessing, from one end, the mycelium that step (2) obtains, light culture at 24～26 DEG C of temperature, extend to the other end to wood flour depths after mycelia material feeding, after 15～20 days, obtain purification strain from the other end;
(4) rejuvenation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: what step (3) obtained purifies strain transfer to the RM improved culture medium after sterilizing, light culture at 24～26 DEG C of temperature obtains the excellent species that mycelia is healthy and strong after 7～8 days;
(5) protospecies breeding of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: after pedigree seed culture medium is loaded the 2/3 of glass culture bottle volume, the rejuvenation strain that step (4) obtains is accessed after covering tampon sterilizing, light culture at 24～26 DEG C of temperature, at the bottom of mycelia grows to bottle after 25～30 days, obtains the original seed of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component, the mass percent of pedigree seed culture medium is: weed tree sawdust 39%, broad leaf tree leaves 39%, wheat bran 20%, sucrose 1%, calcium carbonate 1%, material water quality ratio is for 1:1.3～1.5;
(6) the cultigen expanding propagation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component: Cultivar culture medium is loaded sterilizing in high density low-pressure polyethylene plastic bag, the original seed of the Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component that step (5) obtains is accessed after cooling, light culture at 2～23 DEG C of temperature, obtains the cultigen of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component after 28～36 days, the mass percent of Cultivar culture medium is: weed tree sawdust 30%, broad leaf tree leaves 48%, Testa Tritici skin 10%, Semen Maydis powder 10%, sucrose 1%, Gypsum Fibrosum powder 1%, material water quality ratio is for 1:1.3～1.5.
2. the breeding method of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component according to claim 1, is characterized in that the mass percent of the purification culture medium of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component is: weed tree sawdust 60%, broad leaf tree leaves 30%, wheat bran 8%, sucrose 1%, Gypsum Fibrosum powder 1%, and material water quality ratio is for 1:1.3～1.6.
3. the breeding method of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component according to claim 1, it is characterized in that the RM improved culture medium formula of the rejuvenation of Pseudobulbus Bletillae (Rhizoma Bletillae) fungal component is: peptone 2 grams, glucose 20 grams, potassium dihydrogen phosphate 0.46 gram, dipotassium hydrogen phosphate 1 gram, 0.5 gram of magnesium sulfate, 15～20 grams of agar, Rhizoma Solani tuber osi 200 grams, distilled water 1000 milliliters.
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| CN110338047A (en) * | 2019-06-28 | 2019-10-18 | 长江大学 | A kind of bletilla striata seeds imitate wild efficient direct sowing and seedling method |
| 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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| 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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