CN105519416B - A kind of artificial breeding method of dendrobium devonianum symbiosis seedling - Google Patents
A kind of artificial breeding method of dendrobium devonianum symbiosis seedling Download PDFInfo
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- CN105519416B CN105519416B CN201510992749.3A CN201510992749A CN105519416B CN 105519416 B CN105519416 B CN 105519416B CN 201510992749 A CN201510992749 A CN 201510992749A CN 105519416 B CN105519416 B CN 105519416B
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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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
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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 discloses a kind of artificial breeding method of dendrobium devonianum symbiosis seedling.By glued membrane bacterium (Tulasnella sp.), CGMCC No.9551 are by amplifying fermented and cultured, filtering mycelium, which adds appropriate amounts of sterilized water and smashed with refiner, is made fungal suspension liquid, add dendrobium devonianum seeds and seed fungal suspension liquid is made, seedling medium is made with coconut palm chaff, leaf and bark, every square metre of matrix sprays 50~500ml seed fungal suspension liquid, and keeps the water content of seedling medium 50%~90%, can obtain a large amount of symbiosis seedling during to 45 days.The present invention can significantly shorten the seed seedling time, reduce seedling cost, disposably obtain larger amount of dendrobium devonianum seedling, and the survival rate for improving the speed of growth of symbiosis seedling and being transplanted in natural environment is higher than non-symbiosis germination seedling.The new way that dendrobium devonianum seeds obtain seedling is the method achieve, there are important real promotion and application to be worth.
Description
Technical field
The invention belongs to dendrobe cultivation technical field, is specifically related to one kind using fungal biodegradation dendrobium devonianum seeds with fast
Speed obtains the artificial breeding method of a large amount of dendrobium devonianum symbiosis seedlings.
Background technology
Dendrobium devonianum (Dendrobium devonianum) is used as traditional Chinese medicine, there is high medical value, and it is planted
Training scale is inferior to dendrobium candidum, or even turns into pillar industry in some regional (such as Yunnan Province Longling Counties).The stem of noble dendrobium at present
Main Cultivation pattern has the intensive cropping of enterprise and its " company+base+peasant household " pattern of use, also part small enterprise or
Peasant household uses the small-scale intensive cropping of simple frame, and a small amount of enterprise or the personal Ecology plantation carried out.Various cultivations
The source of seedling of pattern is mainly tissue-cultured seedling, and small part is Wild plant, rare symbiosis seedling.Main cause is that growing dendrobium seedlings are borrowed
Help tissue culture technology flow relatively stable, be easy to batch production large-scale production.Although dendrobe tissue culture seedling-raising technique has developed relative maturity,
But there are still cost height, cycle length, technical difficulty is larger to need special staff's operation, transplants under field conditions (factors)
Motility rate is relatively low, many defects such as later period growth and development is slow.Source of seedling turns into the bottleneck of dendrobe cultivation industry development, serious shadow
Health, the sustainable development of industry are rung.
Basic biological characteristics based on dendrobium devonianum：Seed is very tiny, and ripe seed does not have endosperm or cotyledon, only
There is the embryo of ateliosis, seed sprouting needs fully rely on specific symbiotic effects to obtain nutrients under field conditions (factors)
Matter.If substantial amounts of symbiosis seedling can be obtained by dendrobium devonianum seeds symbiotic germination technology, existing tissue culture can be overcome
The defects of nursery, seedling quantity can be not only improved, simplify seedling production process, shorten the seed seedling time, hence it is evident that reduce life
Produce cost, it is often more important that the survival rate after seedling is revert in natural environment and growth of seedling speed can be significantly improved, strengthened
The environmental suitability of seedling.But do not report successfully still in the prior art.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to providing one kind quickly obtains a large amount of dendrobium devonianum symbiosis kinds
The method of seedling, to shorten the dendrobium devonianum seedling fostering time, seedling fostering cost is reduced, improves seedling quantity, improves tooth valve stone
The environmental suitability of dry measure used in former times seedling.
The purpose of the present invention is achieved by the following technical programs.
Unless otherwise indicated, percentage of the present invention is mass percent.
A kind of artificial breeding method of dendrobium devonianum symbiosis seedling, comprises the following steps：
(1) collection field is naturally solid and ripe but the still uncracked dendrobium devonianum seeds of Fruit pod save backup；
(2) making of fungal suspension liquid：
The making of 2.1 culture mediums：PDA liquid medium is bottled with the amount of the volume of conical flask 10%, loads after sealing and goes out
Bacterium pot, in 121 DEG C, sterilize 20min under the conditions of 115KPa, standby；
2.2 by the activation culture glued membrane bacterium of 3~10 days (Tulasnella sp.), and CGMCC No.9551 are inoculated in standby
PDA liquid medium, in 23~28 DEG C, fermented and cultured 5~10 days under the conditions of 120~200rpm；
2.3 obtain mycelium with filtered through gauze zymotic fluids, with aseptic water washing mycelium 3~5 times and draining it is residual to remove
The glucose stayed, is put into refiner by the sterilized water of mycelium and 5~50 times of quality and smashes fungal suspension liquid is made；
(3) making of seed-fungal suspension liquid：Dendrobium devonianum seeds and fungal suspension liquid are mixed seed-fungi is made
Suspension, it is 20~200/ml to control great-hearted dendrobium devonianum seeds density in seed-fungal suspension liquid；
(4) seedling frame and seed plate prepare：
4.1 install the shade net that shade rate is 60~90% above seedbed；
4.2 by coconut palm chaff, bark and leaf respectively with clear water soak one week, then by coconut palm chaff account for matrix cumulative volume 50%~
95% ratio is well mixed, and in 121 DEG C, sterilize 20min under the conditions of 115KPa, as seedling medium；
Seedling medium is uniformly loaded seed plate, 1.5~2.0cm of thickness, then in stromal surface one piece of area of tiling by 4.3
Slightly larger than the nylon net cloth of seed plate to prevent seed from spilling seed plate, the aperture of nylon net cloth is 20~40 μm, on nylon net cloth
0.4~0.6cm of uniform fold a layer thickness seedling medium again, spray water to matrix saturation, it is standby；
(5) dendrobium devonianum seeds are sowed with fungi nursery symbiotic germination：
5.1 ratios that 50~500ml is sprayed according to every square metre of matrix uniformly spray the seed made-fungal suspension liquid
It is sprinkled upon on standby seed plate；
After 5.224 hours, atomized water is sprayed under the conditions of non-solar direct projection daily to keep the water content of seedling medium to exist
50%~90%；
When 5.3 seed symbiotic germinations were to 45 days, you can form a large amount of symbiosis seedlings.
Further, the condition of culture described in step (2.2) is preferably 28 DEG C, 150rpm, 7 days.
The quality for making sterilized water used in fungal suspension liquid described in step (2.3) is mycelial 10 times.
Dendrobium devonianum seeds density described in step (3) is 185/ml.
Shade net shade rate described in step (4.1) is 75%.
The volume ratio of coconut palm chaff, bark and leaf is 8 in seedling medium described in step (4.2):1:1.
The aperture of nylon net cloth described in step (4.3) is preferably 30 μm.
In step (5.1), every square metre of matrix sprinkling 300ml seed-fungal suspension liquid；
In step (5.2), the water content for keeping seedling medium is 70%~80%.
In step (5.2), the time of atomized water is sprayed for before 9 points of every morning or after 6 pm.
Compared with prior art, the present invention has advantages below：
1st, seedling cost can be greatly reduced compared to seed asepsis sprouting technology, the present invention, it is only necessary to prepare and expand numerous fungi
PDA liquid medium used and seedling medium can complete sapling multiplication process.Needed not move through in reproductive process repeatedly
Switching, without hardening, significantly shorten the seed seedling time.
2nd, same culture environment and under the conditions of, check experiment is that the dendrobium devonianum seeds that axenic germination is sowed can not
Sprouting forms seedling, and the germination rate for having Orchid Seeds under the natural conditions of document report field is no more than 0.03%, and applies
Germination rate greatly improves after the present invention, can obtain substantial amounts of symbiosis seedling in the shorter time.
3rd, simultaneously, the symbiosis that Early seedling stage has been established with fungi, therefore the speed of growth of symbiosis seedling and transplanting
Survival rate into natural environment is higher than non-symbiosis germination seedling.
4th, the present invention realizes the new way that dendrobium devonianum seeds obtain seedling, has important real promotion and application valency
Value, brand-new thinking is provided for industry development.
The explanation of preservation biomaterial
The bacterial strain of the present invention, on 07 18th, 2014, it is general to be deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center (CGMCG)；The centre address：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, the Chinese Academy of Sciences is micro-
Biological study institute.The strain classification is named as glued membrane bacterium (Tulasnella sp.), and preserving number is CGMCC No.9551.
Embodiment
The present invention is described in further detail by the following examples, but embodiment is not to the technology of the present invention side
The limitation of case.
Embodiment 1：
1st, the acquisition and preservation of tooth valve germplasm
Collection field is naturally solid and ripe but still uncracked dendrobium devonianum seeds save backup.Dendrobium devonianum used
Seed picks up from the Wild population of Baoshan, Yunnan city Longling County.
2nd, the making of the suspension of fungi
The making of 2.1 culture mediums：PDA liquid medium Normal practice, in right amount.70 bottles are averagely distributed into every bottle of 50ml
500ml triangular flasks, bottleneck is wrapped up with newspaper after sealing, load autoclave in 121 DEG C, sterilize 20min under the conditions of 115KPa, standby
With.
2.2 by the activation culture bacterial strain of 7 days-glued membrane bacterium (Tulasnellasp.) CGMCC No.9551, are inoculated in above-mentioned
The triangular flask of the fluid nutrient medium containing 50ml of sterilizing, fermented and cultured 7 days under 28 DEG C, 150rpm condition of culture.
2.3 obtain mycelium and with aseptic water washing mycelium 4 times, each draining with the cultured zymotic fluids of filtered through gauze
To remove the glucose of residual.It is 1 in mass ratio:10 draining mycelium and sterilized water, which is put into refiner, to be smashed and fungi is made hangs
Supernatant liquid.
3rd, the making of seed-fungal suspension liquid
3.1 routinely TTC methods detect standby dendrobium devonianum seeds vigor, to confirm that it is vibrant that seed vitality has that it's too late
The ratio of seed, according to the ratio confirmation seed dosage of vibrant seed.Vigourous seed ratio is 79.6% in the present embodiment.
In one dendrobium devonianum Fruit pod about 30% seed is added the mixing of fungal suspension liquid by 3.2 is made seed-suspension.
200 μ l suspension, 6 free-hand film-makings are taken to detect seed number under the microscope, seed amount contained by every milliliter of calculating produces suspension
Seed density.This is made seed-fungal suspension liquid and amounts to 360ml altogether, and seed density is 147/ml.
3.3 prepare CK blank controls simultaneously：100ml 0.1% aqueous agar solution adds in a dendrobium devonianum Fruit pod about
Blank suspension is made in 20% seed, and seed density measures 6 times altogether, 375/ml of seed density.
4th, the preparation of simple seedling frame and seed plate
Shading treatment is done in the high spaces of about 2m above seedbed by 4.1 with the shade net that shade density is 75%, and floor space is about
15 square meters.
4.2 soak coconut palm chaff and the bark collected in the mountain forest near nursery, fallen leaves one week in advance, then by coconut palm
Chaff, bark and leaf volume 8：1：1 ratio is well mixed to make seedling medium, is transferred in container at 121 DEG C, 115KPa bars
Sterilize 20min under part, standby.
4.3 ready matrix are taken out, uniform flat to overlay on thickness about 1.5-2.0cm on seed plate (seed plate size is：It is long
× wide × height=53 × 26 × 6cm), the nylon for being now 30 μm in one piece of aperture slightly larger than seed plate area of stromal surface tiling
For screen cloth to prevent seed from spilling seed plate, then order is uniformly put down in nylon net cloth covers the identical base that a layer thickness is about 0.5cm
Matter, sprinkling running water are standby to water content of substrate saturation.
5th, carry out in Chinese Academy of Sciences's Xishuangbanna tropical botanical garden seedling nursery 06~July in 2015 dendrobium devonianum seeds with
Experiment, 23 DEG C~31 DEG C of temperature on average are sprouted in mycosymbiosis.
5.1 seed-fungal suspension the liquid for taking 40ml to make uniformly are sprayed at watering can on the seed plate equipped with matrix.Together
When, sprinkling agar-seed suspension liquid is placed apart from more than symbiosis nursery seed plate 50m to prevent true as control experiment, control seed plate
Bacterium pollutes.
From 5.2 second days, appropriate atomized water is sprayed to keep artificial substratum before 9 points of every morning or after 6 pm
Humidity.
Start to pay attention to the observation developmental state of protocorm, quantity at 5.330 days and carry out experimental record.It was found that during protocorm
Take part Experiment sample to take back laboratory and microexamination is carried out to it：Mycorrhizal morphology in protocorm, structure, hypha body color etc.
Deng.
6th, data analysis
Seed-mycosymbiosis is sprouted finds there is substantial amounts of protocorm and seedling when testing 45 days, or even has protocorm primary
Separate living tissue is further differentiated to form two panels leaf, and now the protocorm quantity of each seed plate is shown in Table 1.The blank control of bacterium is not connect
Protocorm has been not observed in group or seedling is formed.The protocorm formation rate 4.95 ± 3.48% (n=9) of inoculating strain is notable
Handled higher than blank control.Interpretation of result shows that dendrobium devonianum seeds, which only connect bacterium processing, to be sprouted, and form protocorm and children
Seedling.Relative to blank test, mycosymbiosis Germination Technique of the invention has remarkable effect to promoting dendrobium devonianum seeds to sprout.
1 each experiment process of table and control experiment protocorm quantity
Table 1 The protcorm number of each seedling plate and control test
plate
Embodiment 2
Embodiment 1 is repeated, there is following difference：
Build a simple seedling in 01~02 month 2015 vacant lot in Xishuangbanna tropical botanical garden scientific research center of the Chinese Academy of Sciences
Bed, shading treatment is done into the high spaces of about 1m above seedbed with the shade net that shade density is 75%, the seedbed floor space about 9 is flat
Rice.13 DEG C~24 DEG C of temperature on average.
Seed-fungal suspension the liquid for taking 100ml to make uniformly is sprayed at watering can on the seed plate equipped with matrix, is suspended
The seed density of liquid is 185/ml, sows 4 seed plates altogether.Meanwhile agar-seed suspension liquid is sprayed as control experiment, seed
Density is 320/ml, and control seed plate is placed apart from more than symbiosis nursery seed plate 30m to prevent fungal contamination, sows 2 seed plates altogether.Its
Remaining condition of culture is consistent.
Seed-mycosymbiosis is sprouted finds there is substantial amounts of protocorm and seedling when testing about 50 days, or even has part protocorm
Stem separate living tissue is further differentiated to form two panels leaf, and the protocorm quantity of each seed plate is shown in Table 2.The protocorm shape of inoculating strain
It is significantly higher than control treatment into rate 5.34 ± 1.80% (n=4).Interpretation of result shows that dendrobium devonianum seeds only connect bacterium processing
It could sprout, form protocorm and seedling.Relative to check experiment, mycosymbiosis Germination Technique of the invention is to promoting tooth valve stone
Dry measure used in former times seed, which is sprouted, has remarkable effect.
2 each experiment process of table and control experiment protocorm quantity
Table 2 The protcorm number of each seedling plate and control test
plate in summer
The present invention sprouts under the conditions of open environment to dendrobium devonianum seeds and mycosymbiosis has carried out research, successfully obtains
Substantial amounts of symbiosis seedling was obtained, (is respectively although average germination rate is relatively low：4.95 ± 3.48% (n=9) and 5.34 ± 1.80%
(n=4) and each seed plate sprouts protocorm quantity heterogeneity, still, in view of dendrobium devonianum Fruit pod seed content pole is up to tens thousand of
To hundreds thousand of (once measured value is about 18.3 ten thousand in this laboratory) and this method sowing, operating process is simple, cost is cheap and nothing
Many advantages, such as needing hardening, therefore this sows obtained germination rate still important application value, is cultivated for dendrobium devonianum
The acquisition of industry seedling provides feasible method.
Claims (10)
1. a kind of artificial breeding method of dendrobium devonianum symbiosis seedling, comprises the following steps：
(1) collection field is naturally solid and ripe but the still uncracked dendrobium devonianum seeds of Fruit pod save backup；
(2) making of fungal suspension liquid：
The making of 2.1 culture mediums：PDA liquid medium is bottled with the amount of the volume of conical flask 10%, autoclave is loaded after sealing,
In 121 DEG C, sterilize 20min under the conditions of 115KPa, standby；
2.2 by the activation culture glued membrane bacterium of 3~10 days (Tulasnella sp.), and CGMCC No.9551 are inoculated in standby PDA
Fluid nutrient medium, in 23~28 DEG C, fermented and cultured 5~10 days under the conditions of 120~200rpm；
2.3 obtain mycelium with filtered through gauze zymotic fluids, with aseptic water washing mycelium 3~5 times and draining to remove residual
Glucose, is put into refiner by the sterilized water of mycelium and 5~50 times of quality and smashes fungal suspension liquid is made；
(3) making of seed-fungal suspension liquid：Dendrobium devonianum seeds and fungal suspension liquid are mixed seed-fungal suspension is made
Liquid, it is 20~200/ml to control great-hearted dendrobium devonianum seeds density in seed-fungal suspension liquid；
(4) seedling frame and seed plate prepare：
4.1 install the shade net that shade rate is 60~90% above seedbed；
4.2 soak coconut palm chaff, bark and leaf one week with clear water respectively, then account for matrix cumulative volume 50%~95% by coconut palm chaff
Ratio is well mixed, and in 121 DEG C, sterilize 20min under the conditions of 115KPa, as seedling medium；
Seedling medium is uniformly loaded seed plate by 4.3, and 1.5~2.0cm of thickness is then bigger in stromal surface one piece of area of tiling
In the nylon net cloth of seed plate to prevent seed from spilling seed plate, the aperture of nylon net cloth is 20~40 μm, equal again on nylon net cloth
0.4~0.6cm of even covering a layer thickness seedling medium, sprays water to matrix saturation, standby；
(5) dendrobium devonianum seeds are sowed with fungi nursery symbiotic germination：
The seed made-fungal suspension liquid is uniformly sprayed at by 5.1 ratios that 50~500ml is sprayed according to every square metre of matrix
On standby seed plate；
After 5.2 24 hours, atomized water is sprayed under the conditions of non-solar direct projection daily to keep the water content of seedling medium 50%
~90%；
When 5.3 seed symbiotic germinations were to 45 days, you can form symbiosis seedling.
2. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (2.2)
Described in condition of culture be 28 DEG C, 150rpm, 7 days.
3. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (2.3)
Described in make fungal suspension liquid used in sterilized water quality be mycelial 10 times.
4. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：In step (3)
Described dendrobium devonianum seeds density is 185/ml.
5. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (4.1)
Described in shade net shade rate be 75%.
6. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (4.2)
Described in seedling medium in coconut palm chaff, bark and leaf volume ratio be 8:1:1.
7. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (4.3)
Described in nylon net cloth aperture be 30 μm.
8. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (5.1)
In, every square metre of matrix sprinkling 300ml seed-fungal suspension liquid.
9. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (5.2)
In, the water content for keeping seedling medium is 70%~80%.
10. the artificial breeding method of dendrobium devonianum symbiosis seedling according to claim 1, it is characterised in that：Step (5.2)
In, the time of atomized water is sprayed for before 9 points of every morning or after 6 pm.
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| CN103468588A (en) * | 2013-09-17 | 2013-12-25 | 中国科学院西双版纳热带植物园 | Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds |
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