CN102715089A - Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant - Google Patents
Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant Download PDFInfo
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Abstract
The invention relates to an establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides which is a symbiotic plant, and belongs to the field of biotechnology (plant tissues and Fungus cultivation). An overwhelming majority of amanita belongs to ectomycorrhizal fungi, can not be yet subjected to artificial cultivation at present, and can be hardly developped and utilized; and growth and development of sporocarps of the amanita has a close relationship with cyclobalanopsis glaucoids which is a symbiotic plant. The establishing method is characterized in that myceliums are induced through the sporocarps of amanita flavipes for reproduction; aseptic seedlings and potted seedlings are successfully obtained by using seeds of cyclobalanopsis glaucoides, collected in the field; reproduced myceliums are inoculated to the roots of the aseptic seedlings and the potted seedlings respectively; the result shows that inoculated seedlings are more exuberant than the non-inoculated seedlings and have more leaves, the leaves of inoculated seedlings are dark green, disease resistance of inoculated seedlings are reinforced; and therefore, the indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides is innovatively established, and a technological reserve is provided for future researches on symbiotic mechanism and mechanism generated in the sporocarps of amanita.
Description
Technical field
The present invention relates to the method for building up of a kind of yellow handle goose cream and the indoor symbiotic relation in Qinggang, aulophyte Yunnan, belong to biological technical field, specifically belong to Plant Tissue Breeding and macro fungi mycelium and cultivate category.
Background technology
Amanita (
Amanita) be under the jurisdiction of Basidiomycotina, Hymenomycetes, Agaricales, Amanita fuliginea section (
Amanitaceae), be in the poisonous macro fungi more special, more valuable one worldwidely blazon big genus.Major part kind in the Amanita belongs to famous hypertoxic bacterium, mainly contains amatoxin, and amatoxin has potential application aspect the new drugs such as research and development anti-bacteria and anti-virus, antitumor, sedation anesthesia, thereby becomes research focus in recent years.
The Amanita overwhelming majority belongs to the ectotrophic mycorrhiza fungi; Still can not carry out artificial cultivation at present; Be difficult to development and use; Growing of its fruit body is in close relations with aulophyte, so the research of the biology relation of Amanita fuliginea and its aulophyte, is the feasible way of further inquiring into Amanita fuliginea fruit body mechanism.
Yellow handle Amanita fuliginea (
Amanita flavipes Imai) be a dispersed species in the Amanita, in Wuding County, Yunnan Lion Rock distribute more, Qinggang, Yunnan of main and Fagaceae, Yuanjiang River evergreen chinquapin (
Castanopsis orthacantha) wait the broad-leaved tree symbiosis, Qinggang, Yunnan (
Cyclobalanopsis glaucoides) be Fagaceae (Fagaceae), Cyclobalanopsis (
CyclobalanopsisOerst.) plant; Can collect more Qinggang, Yunnan seed on the ground at the Lion Rock broad leaved forest; Therefore the present invention is a material with Qinggang, Yunnan seed and yellow handle goose ointment-like medicine for oral or plastering use entity; Successfully set up the indoor symbiotic relation in yellow handle goose cream and Qinggang, Yunnan, the Mechanism Study that takes place for symbiosis mechanism from now on and goose ointment-like medicine for oral or plastering use entity etc. provides technical reserve.
Summary of the invention
The objective of the invention is to, under indoor or controlled condition, set up a kind of simple and stable, mutually beneficial yellow handle goose cream and the symbiotic relation of Qinggang, Yunnan plant.
Realize technical scheme of the present invention: utilize Qinggang, Fagaceae Yunnan plant seed; Aseptic seedling and potted plant seedling have successfully been obtained; Utilize yellow handle goose ointment-like medicine for oral or plastering use entity to induce mycelium; Mycelium is inoculated into aseptic seedling and potted plant shoot root portion respectively, the indoor symbiotic relation of having set up the two, realize that key step of the present invention is following:
(1) the inducing and cultivating of yellow handle Amanita fuliginea filament: open-airly pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture (potato 200.00g/L, glucose 20.00g/L, NH gently
4NO
30.30～0.40g/L, glutamic acid 0.28～0.32 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L; The control pH value is about 5.4) surface, cultivation temperature is 22～23 ℃, secretly cultivates 14～18 days; Begin to sprout few white hypha around the bacterium piece; 53～60 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, with enlarged culture base (potato 100.00～120.00g/L, glucose 9.00～12.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 80～100 order sub-sieves) 30.00～50.00g/L, habitat soil dry powder (crossing 80～100 order sub-sieves) 60.00～80.00g/L, peptone 3.00～5.00 g/L, NH
4NO
30.60～0.80g/L, 6-BA1.20～1.50mg/L, brewer's wort 200～230 ml/L, agar 6.00～8.00 g/L, the control pH value is about 5.4) carry out enlarged culture;
(2) cultivation of Qinggang, Yunnan aseptic seedling: in Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is met into germination medium (MS medium, sugar-free, 6-BA 1.00～1.50mg/L up; NAA 0.08～0.10mg/L, agar 7.50 g/L, pH are 6.80); 1 of every bottle graft kind, untainted brownization seed begins to sprout in 21～22 ℃ of down dark taking root after cultivating 8～10 days, changes under the illumination of 2000～2500Lux uniform temp over to and cultivates 5～8 days again; (inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, by 6 direction equidistance inoculations, the inoculation bottle seedling was cultivated 13～16 days down in 22～23 ℃ with 6 mycelia pieces of intercepting aseptic seedling in the blake bottle to carry out mycelium inoculation when growing the high seedling of 1.8～2.3cm; Mycelia is covered with blake bottle; High 3.0～the 3.5cm of seedling, 3～5 on leaf is inoculated seedling generally than not inoculating seedling; The thick shape of cane, the leaf look dark green, blade is many), set up the symbiotic relation of yellow handle Amanita fuliginea silk and aseptic seedling;
(3) cultivation of the potted plant seedling in Qinggang, Yunnan: habitat soil and rotted leaf that the field is fetched tanned by the sun 2～3 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 25～30 days seed sproutings treat to inoculate when seedling grew to 4～5 true leaves, high 4～6cm in 59～63 days, and inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (10～15 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 13～15 days, inoculation is once inoculated five times altogether; 118～122 days, the high 10.0～13.0cm of plant, 9～11 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and sets up the symbiotic relation of yellow handle Amanita fuliginea silk and potted plant seedling.
The invention has the beneficial effects as follows: successfully set up the syntaxial system in yellow handle Amanita fuliginea and Qinggang, Yunnan indoor (aseptic and potted plant) in two ways, its characteristics are following,
(1) with Qinggang, Yunnan seed, successfully obtained aseptic seedling and potted plant bacterium, the artificial culture method of Qinggang, two kinds of Yunnan plant is provided under controlled condition;
(2) two kinds of syntaxial systems setting up differ from one another, and replenish each other; Sterile system is because growth is fast; And be pure symbiont, can study the symbiosis situation of the two quickly and easily, potted plant seedling is because self-sow; More, can some experimental results under the aseptic condition directly be used in simulation or expansion experiment on the potted plant seedling near open-air former habitat;
(3) research method of symbiotic relation under mycorrhizal fungi and the plant experimental condition is provided, for other macro fungi and symbiosis Study on plants provide the method reference.
Embodiment
Following examples of implementation are to further specify of the present invention, are not limitations of the present invention.
Instance one:
Open-air pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture (potato 200.00g/L, glucose 20.00g/L, NH gently
4NO
30.30g/L, glutamic acid 0.32 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L; The control pH value is about 5.4) surface, cultivation temperature is 22～23 ℃, secretly cultivates 14 days; Begin to sprout few white hypha around the bacterium piece; 60 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, with enlarged culture base (potato 100.00g/L, glucose 10.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 80 order sub-sieves) 30.00g/L, habitat soil dry powder (crossing 80 order sub-sieves) 70.00g/L, peptone 3.00g/L, NH
4NO
30.80g/L, 6-BA1.50mg/L, brewer's wort 200ml/L, agar 6.00 g/L, the control pH value is about 5.4) carry out enlarged culture;
In Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is met into germination medium (MS medium, sugar-free, 6-BA 1.50mg/L up; NAA0.10mg/L, agar 7.50 g/L, pH are 6.80); 1 of every bottle graft kind, untainted brownization seed begins to sprout in 21～22 ℃ of down dark taking root after cultivating 8 days, changes under the illumination of 2000～2500Lux uniform temp over to and cultivates 5 days again; (inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, by 6 direction equidistance inoculations, the inoculation bottle seedling was cultivated 13 days down in 22～23 ℃ with 6 mycelia pieces of intercepting aseptic seedling in the blake bottle to carry out mycelium inoculation when growing the high seedling of about 2.0cm; Mycelia is covered with blake bottle; The high about 3.0cm of seedling, 4～5 on leaf is inoculated seedling generally than not inoculating seedling; The thick shape of cane, the leaf look dark green, blade is many), set up the symbiotic relation of yellow handle Amanita fuliginea silk and aseptic seedling;
Habitat soil and rotted leaf that the field is fetched tanned by the sun 2 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 25 days seed sproutings treat to inoculate when 60 days seedlings grow to 4～5 true leaves, high about 4cm, and inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (10 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 13 days, inoculation is once inoculated five times altogether; 118 days, the high about 10.0cm of plant, 9～10 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and sets up the symbiotic relation of yellow handle Amanita fuliginea silk and potted plant seedling.
Instance two:
Open-air pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture (potato 200.00g/L, glucose 20.00g/L, NH gently
4NO
30.30g/L, glutamic acid 0.32 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L; The control pH value is about 5.4) surface, cultivation temperature is 22～23 ℃, secretly cultivates 14 days; Begin to sprout few white hypha around the bacterium piece; 60 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, with enlarged culture base (potato 100.00g/L, glucose 10.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 80 order sub-sieves) 30.00g/L, habitat soil dry powder (crossing 80 order sub-sieves) 70.00g/L, peptone 3.00 g/L, NH
4NO
30.80g/L, 6-BA1.50mg/L, brewer's wort 200ml/L, agar 6.00g/L, the control pH value is about 5.4) carry out enlarged culture;
In Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is met into germination medium (MS medium, sugar-free, 6-BA 1.50mg/L up; NAA 0.10mg/L, agar 7.50 g/L, pH are 6.80); 1 of every bottle graft kind, untainted brownization seed begins to sprout in 21～22 ℃ of down dark taking root after cultivating 10 days, changes under the illumination of 2000～2500Lux uniform temp over to and cultivates 8 days again; (inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, by 6 direction equidistance inoculations, the inoculation bottle seedling was cultivated 16 days down in 22～23 ℃ with 6 mycelia pieces of intercepting aseptic seedling in the blake bottle to carry out mycelium inoculation when growing the high seedling of about 1.8cm; Mycelia is covered with blake bottle; The high approximately 3.5cm of seedling, 3～4 on leaf is inoculated seedling generally than not inoculating seedling; The thick shape of cane, the leaf look dark green, blade is many), set up the symbiotic relation of yellow handle Amanita fuliginea silk and aseptic seedling;
Habitat soil and rotted leaf that the field is fetched tanned by the sun 3 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 30 days seed sproutings treat to inoculate when 63 days seedlings grow to 4～5 true leaves, high about 6cm, and inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (15 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 15 days, inoculation is once inoculated five times altogether; 122 days, the high approximately 13.0cm of plant, 10～11 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and sets up the symbiotic relation of yellow handle Amanita fuliginea silk and potted plant seedling.
Instance three:
Open-air pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture (potato 200.00g/L, glucose 20.00g/L, NH gently
4NO
30.40g/L, glutamic acid 0.28 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L; The control pH value is about 5.4) surface, cultivation temperature is 22～23 ℃, secretly cultivates 16 days; Begin to sprout few white hypha around the bacterium piece; 55 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, with enlarged culture base (potato 120.00g/L, glucose 10.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 100 order sub-sieves) 50.00g/L, habitat soil dry powder (crossing 100 order sub-sieves) 60.00g/L, peptone 5.00 g/L, NH
4NO
30.60g/L, 6-BA1.20mg/L, brewer's wort 230 ml/L, agar 8.00 g/L, the control pH value is about 5.4) carry out enlarged culture;
In Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is met into germination medium (MS medium, sugar-free, 6-BA 1.00mg/L up; NAA 0.08 mg/L, agar 7.50 g/L, pH are 6.80); 1 of every bottle graft kind, untainted brownization seed begins to sprout in 21～22 ℃ of down dark taking root after cultivating 9 days, changes under the illumination of 2000～2500Lux uniform temp over to and cultivates 7 days again; (inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, by 6 direction equidistance inoculations, the inoculation bottle seedling was cultivated 15 days down in 22～23 ℃ with 6 mycelia pieces of intercepting aseptic seedling in the blake bottle to carry out mycelium inoculation when growing the high seedling of about 2.3cm; Mycelia is covered with blake bottle; The high approximately 3.3cm of seedling, 3～4 on leaf is inoculated seedling generally than not inoculating seedling; The thick shape of cane, the leaf look dark green, blade is many), set up the symbiotic relation of yellow handle Amanita fuliginea silk and aseptic seedling;
Habitat soil and rotted leaf that the field is fetched tanned by the sun 3 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 27 days seed sproutings treat to inoculate when 59 days seedlings grow to 4～5 true leaves, high about 5cm, and inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (13 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 14 days, inoculation is once inoculated five times altogether; 120 days, the high approximately 12.0cm of plant, 9～10 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and sets up the symbiotic relation of yellow handle Amanita fuliginea silk and potted plant seedling.
Instance four:
Open-air pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture (potato 200.00g/L, glucose 20.00g/L, NH gently
4NO
30.40g/L, glutamic acid 0.28 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L; The control pH value is about 5.4) surface, cultivation temperature is 22～23 ℃, secretly cultivates 16 days; Begin to sprout few white hypha around the bacterium piece; 55 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, with enlarged culture base (potato 120.00g/L, glucose 10.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 100 order sub-sieves) 50.00g/L, habitat soil dry powder (crossing 100 order sub-sieves) 60.00g/L, peptone 5.00 g/L, NH
4NO
30.60g/L, 6-BA1.20mg/L, brewer's wort 230 ml/L, agar 8.00 g/L, the control pH value is about 5.4) carry out enlarged culture;
In Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is met into germination medium (MS medium, sugar-free, 6-BA 1.00mg/L up; NAA 0.08 mg/L, agar 7.50 g/L, pH are 6.80); 1 of every bottle graft kind, untainted brownization seed begins to sprout in 21～22 ℃ of down dark taking root after cultivating 8 days, changes under the illumination of 2000～2500Lux uniform temp over to and cultivates 6 days again; (inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, by 6 direction equidistance inoculations, the inoculation bottle seedling was cultivated 14 days down in 22～23 ℃ with 6 mycelia pieces of intercepting aseptic seedling in the blake bottle to carry out mycelium inoculation when growing the high seedling of about 2.1cm; Mycelia is covered with blake bottle; The high approximately 3.1cm of seedling, 4～5 on leaf is inoculated seedling generally than not inoculating seedling; The thick shape of cane, the leaf look dark green, blade is many), set up the symbiotic relation of yellow handle Amanita fuliginea silk and aseptic seedling;
Habitat soil and rotted leaf that the field is fetched tanned by the sun 3 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 28 days seed sproutings treat to inoculate when 61 days seedlings grow to 4～5 true leaves, high about 5cm, and inoculation method is: with having expanded the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (12 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 15 days, inoculation is once inoculated five times altogether; 121 days, the high approximately 11.0cm of plant, 10～11 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and sets up the symbiotic relation of yellow handle Amanita fuliginea silk and potted plant seedling.
Claims (5)
1. the method for building up of the indoor symbiotic relation of yellow handle goose cream and Qinggang, aulophyte Yunnan is characterized by, utilize yellow handle goose cream (
Amanita flavipes) fruit body induces mycelium, mycelium expanded numerous; Utilize field acquisition Qinggang, Yunnan (
Cyclobalanopsis glaucoides) seed culture aseptic seedling and potted plant seedling, being inoculated into aseptic seedling and potted plant shoot root portion respectively with expanding numerous mycelium, the indoor symbiotic relation of setting up mainly comprises the following steps:
(1) the inducing and cultivating of yellow handle Amanita fuliginea filament: open-airly pluck that good, the no insect pest of growth, not parachute-opening or parachute-opening fully, stem are more sturdy, the cap mature sporophore of plumpness; Fruit body is taken back the laboratory, on super-clean bench, use cotton ball soaked in alcohol, dab positions such as cap surface and stem; Remove surperficial silt or soil; Fruit body is divided into four from cap middle part one, gets the interior tissue piece of stem and cap junction, be cut into about 0.08～0.16cm with scalpel
2Fritter; The small tissue blocks of above cutting is embedded test tube slant inducing culture primary surface gently, and cultivation temperature is 22～23 ℃, secretly cultivates 14～18 days; Begin to sprout few white hypha around the bacterium piece; 53～60 days, piece of tissue many places protuberances, the surface grows bulk, dense fine hair shape white hypha; The above mycelia of inducing is changed in the culture dish, carry out enlarged culture with the enlarged culture base;
(2) cultivation of Qinggang, Yunnan aseptic seedling: in Wuding County, Yunnan Lion Rock broad leaved forest, gather Qinggang, Yunnan seed; Seed is put to clear water, cleaned silt and dirt, remove and swim in the mashed kind on the water surface; With the seed natural seasoning of selecting, reject planting skin, select the seed of no obvious bacterial plaque in the cotyledon; Seed after selecting is positioned in 75% ethanolic solution, rocks and soak 30 s, take out seed, with sterile water wash 3 times; Seed is positioned over 0.1% HgCl
2In the solution, rock and soak 10 min, take out seed, with sterile water wash 6～8 times; Seeds treated is positioned over aseptic filter paper upward filtration solid carbon dioxide branch; Seed bud embryo is connect into germination medium up; 1 of every bottle graft kind; Untainted brownization seed is taken root after 8～10 days in 21～22 ℃ of dark down cultivations and is begun to sprout; Change under the illumination of 2000～2500Lux uniform temp over to and cultivated again 5～8 days, carry out mycelium inoculation when growing the high seedling of 1.8～2.3cm;
(3) cultivation of the potted plant seedling in Qinggang, Yunnan: habitat soil and rotted leaf that the field is fetched tanned by the sun 2～3 days in the sun, were positioned in the flowerpot; The disinfection seed that above filter paper filter solid carbon dioxide divides is imbedded in the flowerpot, and the about 3～4cm of planting depth waters to passing through; Watered primary water in per three days, if soil table overdrying waters every other day; Place outdoor natural light shine down in flowerpot after the 9:00 morning, about 7:00 it moved to indoor heat insulating evening; About 25～30 days seed sproutings treat to inoculate when seedling grew to 4～5 true leaves, high 4～6cm in 59～63 days.
2. the method for building up of yellow handle goose cream according to claim 1 and the indoor symbiotic relation in Qinggang, aulophyte Yunnan is characterized in that the inducing culture in the step (1) is: potato 200.00g/L, glucose 20.00g/L, NH
4NO
30.30～0.40g/L, glutamic acid 0.28～0.32 g/L, V
B10.10mg/L, brewer's wort 150 ml/L, agar 11.00g/L, the control pH value is about 5.4; The enlarged culture base is: potato 100.00～120.00g/L, glucose 9.00～12.00g/L, Qinggang, Yunnan rotted leaf dry powder (crossing 80～100 order sub-sieves) 30.00～50.00g/L, habitat soil dry powder (crossing 80～100 order sub-sieves) 60.00～80.00g/L, peptone 3.00～5.00 g/L, NH
4NO
30.60～0.80g/L, 6-BA1.20～1.50mg/L, brewer's wort 200～230 ml/L, agar 6.00～8.00 g/L, the control pH value is about 5.4.
3. the method for building up of yellow handle goose cream according to claim 1 and the indoor symbiotic relation in Qinggang, aulophyte Yunnan; It is characterized in that Qinggang, the Yunnan seed germination medium in the step (2) is: the MS medium; Sugar-free, 6-BA 1.00～1.50mg/L, NAA 0.08～0.10mg/L; Agar 7.50 g/L, pH are 6.80.
4. the method for building up of yellow handle goose cream according to claim 1 and the indoor symbiotic relation in Qinggang, aulophyte Yunnan; It is characterized in that the mycelium inoculation method in the step (2) is: with expanding of the card punch intercepting of numerous mycelium in the culture dish of front with 1 cm diameter; 6 mycelia pieces of intercepting are inoculated by 6 direction equidistance around the aseptic seedling in the blake bottle, and the inoculation bottle seedling was cultivated 13～16 days down in 22～23 ℃, and mycelia is covered with blake bottle; High 3.0～the 3.5cm of seedling; 3～5 on leaf is inoculated seedling generally than not inoculating seedling, and the thick shape of cane, the leaf look dark green, blade is many (does not inoculate the high cane seedling that seedling forms 8.0～9.0cm; Stem comes into leaves few), show the symbiotic relation of having set up yellow handle Amanita fuliginea silk and aseptic seedling.
5. the method for building up of yellow handle goose cream according to claim 1 and the indoor symbiotic relation in Qinggang, aulophyte Yunnan is characterized in that the mycelium inoculation method in the step (3) is: with expanding the card punch intercepting of numerous mycelium with 1 cm diameter in the culture dish of front, the soil in the flowerpot is opened gently expose root; The mycelia piece of intercepting around root inoculation (10～15 of every seedling inoculations), is covered earth and rotted leaf, place outdoor illumination better, the air cleaner place cultivates; Every at a distance from 13～15 days, inoculation is once inoculated five times altogether; 118～122 days, the high 10.0～13.0cm of plant, 9～11 on leaf; The inoculation seedling is not than inoculating seedling, and plant and root growth are healthy and strong, and the number of sheets is many; Leaf is dark green, and disease resistance strengthens, and shows the symbiotic relation of having set up yellow handle Amanita fuliginea silk and potted plant seedling.
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| CN103667084A (en) * | 2013-12-19 | 2014-03-26 | 云南大学 | Method for co-culturing amanita subfrostiana mycelium by using mycorrhizal fungus and saprobic fungus |
| CN105052499A (en) * | 2015-08-20 | 2015-11-18 | 云南大学 | Method for quickly establishing phialophora fungus and crop mutualism system |
| CN106171515A (en) * | 2016-07-08 | 2016-12-07 | 云南大学 | A kind of Applying Ectomycorrhizal Fungi and the symbiosis method for building up of pine genus plant |
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| CN103667084A (en) * | 2013-12-19 | 2014-03-26 | 云南大学 | Method for co-culturing amanita subfrostiana mycelium by using mycorrhizal fungus and saprobic fungus |
| CN103667084B (en) * | 2013-12-19 | 2015-07-08 | 云南大学 | Method for co-culturing amanita subfrostiana mycelium by using mycorrhizal fungus and saprobic fungus |
| CN105052499A (en) * | 2015-08-20 | 2015-11-18 | 云南大学 | Method for quickly establishing phialophora fungus and crop mutualism system |
| CN106171515A (en) * | 2016-07-08 | 2016-12-07 | 云南大学 | A kind of Applying Ectomycorrhizal Fungi and the symbiosis method for building up of pine genus plant |
| CN106171515B (en) * | 2016-07-08 | 2019-03-19 | 云南大学 | A kind of symbiosis method for building up of Applying Ectomycorrhizal Fungi and pine genus plant |
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