CN107736248A - A kind of tissue culture method of scythian lamb rhizome spore - Google Patents
A kind of tissue culture method of scythian lamb rhizome spore Download PDFInfo
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- CN107736248A CN107736248A CN201711123136.1A CN201711123136A CN107736248A CN 107736248 A CN107736248 A CN 107736248A CN 201711123136 A CN201711123136 A CN 201711123136A CN 107736248 A CN107736248 A CN 107736248A
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- spore
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- sporangium
- scythian lamb
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
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Abstract
The invention discloses a kind of tissue culture method of scythian lamb rhizome spore, pluck the accessory pinna with sporangiorus on wild Cibotium barometz plant, collect sporangium, sterilized and rinsed with 0.1% mercuric chloride solution, be collected in after drying in sterile bag, then by sterile bag after 26 DEG C of preservations 340 380 days, break sporangium and collect spore, and the alcohol disinfecting with 70% 75% and flushing, then centrifuged and rinsed with 2 5% hypochlorite disinfectant, spore suspension is then made；Spore suspension coating inoculating spores germination medium is subjected to sprouting culture, sporinite culture medium is forwarded to after original foliage is grown and carries out gametophyte conversion culture；After gametophyte grows the 3rd leaf, hardening culture in nutrition cave is transferred to, finally carries out field transplanting.The scythian lamb rhizome Spore cultivation method of the present invention, operating process is simple, and seedling early growth is neat, and plant vitality is strong, and survival rate is high；It is adapted to promote the use of.
Description
Technical field
The present invention relates to field of plant growing technology, in particular to a kind of scythian lamb rhizome spore tissue culture mating system.
Background technology
Scythian lamb rhizome (Cibotium barometz (L.) J.Sm.) is fern Dicksoniaceae plant,《Dictionary of medicinal plant》In
Record：Scythian lamb rhizome is born in acid soil at foot of the hill limes marginis, or sylvan life the moon.It is distributed Southwestern China, south, the southeast and Henan, lake
The ground such as north, main product Sichuan, Fujian, Zhejiang.In addition, the ground such as Guangxi, Guangdong, Guizhou, Jiangxi, Hubei also produces.
It is Chinese medicine rhizoma cibotii that scythian lamb rhizome, which dries plucked rhizome, first recorded in《Sheng Nong's herbal classic》, go through version《Chinese Pharmacopoeia》
Record, have filling liver kidney, strengthening the bones and muscles, relax channels and collaterals, dehumidifying pain and diuresis function, for treat waist and leg ache, it is numb in every limb,
The diseases such as hemiplegia, leukorrhea seminal emission, metrorrhagia；Animal experiment proves that the young pilose antler of rhizoma cibotii to scar tissue, liver, spleen damage
The evil traumatic hemorrhage such as property and extraction has preferable anastalsis, and its effect is rapid compared with gelfoam etc., meanwhile, rhizoma cibotii young pilose antler is seemingly
It can gradually be assimilated by tissue, also there are increased platelets counts；Modern study is found：Scythian lamb rhizome extract has suppression
Make a variety of cancer cells, anti-inflammatory, antibacterial and other effects.
Due to a variety of Chinese patent drugs containing rhizoma cibotii of domestic pharmacy corporation, the demand to rhizoma cibotii is increased, is caused among the people crazy
Mad excavation, wild resource is day by day rare, and the seedling breeding of artificial growth rhizoma cibotii is complicated in addition, uses root-like stock cutting section propagation nursery
Cost is high, cycle length, is not suitable for promoting；And spore artificial environment breeding coefficient is low；Be not suitable for propagation in scale.
The content of the invention
It is an object of the invention to provide a kind of scythian lamb rhizome spore tissue culture mating system, the fast of scythian lamb rhizome can be realized
Speed is bred, and realizes large-scale planting.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：A kind of culture side of scythian lamb rhizome spore
Method, comprise the following steps：
(1) collection of spore：The accessory pinna with sporangiorus on wild Cibotium barometz plant is plucked, is put naturally cloudy in clean bag
It is dry, the removal of impurity is gone after sporangium comes off naturally, collects sporangium, after 0.1% mercuric chloride solution effect 4-6 minutes, then is used
Aseptic water washing 3-5 times, it is collected in after drying in sterile bag；
(2) spore pre-processes：The sterile bag containing sporangium that step (1) obtains is placed in 2-6 DEG C of preservation 340-380
My god, break sporangium and collect spore, and centrifuged after sterilizing the 20-40 seconds to spore with 70%-75% alcohol, aseptic water washing 2-
3 times, then spore sterilization 4-10 minutes are centrifuged with 2-5% sodium hypochlorite, then with aseptic water washing 2-3 times；Collect sterilization
Spore adds sterile saline and spore suspension is made；
(3) inoculation and culture：Take 0.05-0.1ml to be seeded to the spore suspension that step (2) obtains to train equipped with spore germination
In the tissue culture tank for supporting base, coating makes spore suspension be uniformly distributed in media surface, and sprouting culture is carried out in illumination box,
22-28 DEG C of temperature, daily 2000-2500Lx illumination 10-12 hours；After original foliage is grown, original foliage is seeded to containing spore
In the culture tank of daughter culture medium, gametophyte conversion culture, 22-28 DEG C of temperature, daily 2000- are carried out in illumination box
2500Lx illumination 10-12 hours；
(4) acclimatization and transplantses：After gametophyte grows the 3rd leaf, gametophyte root media transfer is rinsed well to containing
In the nutrition cave of Nutrition Soil, daily spraying 1-2 time keep humidity, cultivated under the conditions of room temperature and natural lighting 15-30 days it is laggard
Row field transplanting；
Further technical scheme, the spore germination culture medium is by 1/2 modified MS medium, 1-3% sucrose, 0.6-
0.8% agar forms；
Further technical scheme, the sporinite culture medium is by improveing MS2 culture mediums, 1-3% sucrose, 0.6-
0.8% agar is formed, and the improvement MS2 culture mediums are made up of modified MS medium, potato extract solution and melissyl alcohol；
Further technical scheme, the composition improved in the modified MS medium are：MgSO4·7H2O is adjusted to 180-
200mg/L；CaCl2·2H2O is adjusted to 200-220mg/L；KNO3600-700mg/L is adjusted to, (NH)4NO3It is adjusted to 500-
600mg/L；KH2PO4It is adjusted to 170-200mg/L；Increase (NH)2HPO4350-400mg/L；
Further technical scheme, the spore germination culture medium also include sterile 6-BA0.02-0.1mg/L and sterile
IAA0.02-0.1mg/L, the sporinite culture medium also include sterile GA30.02-0.1mg/L and sterile 6-BA0.02-
0.1mg/L；
Further technical scheme, described to sprout culture for 60-90 days, the gamete conversion culture is 45-90 days.
Compared with prior art, the beneficial effects of the invention are as follows：The scythian lamb rhizome Spore cultivation method of the present invention, was operated
Journey is simple, using spore as raw material, does not injure the parent of wild plant, does not influence the growth of plant, explant dosage is few, breeds into
This is low；The seedling that a large amount of physiological ages are consistent, growth is neat can be obtained by culture, be adapted to scale breeding and cultivation；And this
Method follows nature growth rhythm, the scythian lamb rhizome seedling obtained in a manner of zoogamy, greatly reduces vegetative propagation not
Evitable virus or other diseases infection, plant vitality is strong, robust growth, and survival rate is high；Avoid simultaneously a large amount of wild
The excavation of scythian lamb rhizome resource, country is protected to treasure fern resource, beneficial to the protection of wild scythian lamb rhizome.
Embodiment
Technical scheme is described in detail below in conjunction with embodiment, the embodiment is the excellent of the present invention
Embodiment is selected, the present invention is merely to illustrate and limits the scope of the present invention, it will be understood by those skilled in the art that based on the present invention's
Technical scheme, the other technologies scheme obtained on the premise of no creativeness both fall within protection scope of the present invention.Embodiment
The middle unreceipted production firm person of agents useful for same or instrument, it is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of cultural method of scythian lamb rhizome spore, comprises the following steps：
(1) collection of spore：The accessory pinna with sporangiorus on wild Cibotium barometz plant is plucked, is put naturally cloudy in clean bag
It is dry, the removal of impurity is gone after sporangium comes off naturally, collects sporangium, after being acted on 5 minutes with 0.1% mercuric chloride solution, then with nothing
Bacterium water rinses 5 times, is collected in after drying in sterile bag；
(2) spore pre-processes：The sterile bag containing sporangium that step (1) obtains is placed in 2-6 DEG C of preservation 360 days, broken
Open sporangium and collect spore, and spore sterilization is centrifuged after 30 seconds with 75% alcohol, aseptic water washing 3 times, then with 5%
Sodium hypochlorite is sterilized 4 minutes to spore and centrifuged, then with aseptic water washing 3 times；The spore for collecting sterilization adds sterile saline
Spore suspension is made；
(3) inoculation and culture：0.1ml is taken to be seeded to equipped with spore germination culture medium the spore suspension that step (2) obtains
Tissue culture tank in, coating spore suspension is uniformly distributed in media surface, sprouting culture, temperature are carried out in illumination box
22 DEG C, daily 2000-2500Lx illumination 10 hours；After original foliage is grown, original foliage is seeded to containing sporinite culture medium
Culture tank in, in illumination box carry out gametophyte conversion culture, 22 DEG C of temperature, daily 2000-2500Lx illumination 10 are small
When；
(4) acclimatization and transplantses：After gametophyte grows the 3rd leaf, gametophyte root media transfer is rinsed well to containing
In the nutrition cave of Nutrition Soil, spraying daily keeps humidity to cultivate 15- under the conditions of room temperature and natural lighting in 70%-72% 1 time
Field transplanting is carried out after 30 days.
Embodiment 2
A kind of cultural method of scythian lamb rhizome spore, comprises the following steps：
(1) collection of spore：The accessory pinna with sporangiorus on wild Cibotium barometz plant is plucked, is put naturally cloudy in clean bag
It is dry, the removal of impurity is gone after sporangium comes off naturally, collects sporangium, after being acted on 4 minutes with 0.1% mercuric chloride solution, then with nothing
Bacterium water rinses 3 times, is collected in after drying in sterile bag；
(2) spore pre-processes：The sterile bag containing sporangium that step (1) obtains is placed in 4 DEG C of preservations 340 days, broken
Sporangium collects spore, and is centrifuged after being sterilized 30 seconds to spore with 70% alcohol, aseptic water washing 3 times, then with 2% it is secondary
Sodium chlorate is sterilized 10 minutes to spore and centrifuged, then with aseptic water washing 3 times；The spore for collecting sterilization adds sterile saline system
Into spore suspension；
(3) inoculation and culture：0.05ml is taken to be seeded to equipped with spore germination culture medium the spore suspension that step (2) obtains
Tissue culture tank in, coating spore suspension is uniformly distributed in media surface, sprouting culture, temperature are carried out in illumination box
25 DEG C, daily 2000-2500Lx illumination 12 hours；After original foliage is grown, original foliage is seeded to containing sporinite culture medium
Culture tank in, in illumination box carry out gametophyte conversion culture, 25 DEG C of temperature, daily 2000-2500Lx illumination 12 are small
When；
(4) acclimatization and transplantses：After gametophyte grows the 3rd leaf, gametophyte root media transfer is rinsed well to containing
In the nutrition cave of Nutrition Soil, spraying daily keeps humidity to cultivate 15- under the conditions of room temperature and natural lighting in 70%-72% 2 times
Field transplanting is carried out after 30 days.
Embodiment 3
A kind of cultural method of scythian lamb rhizome spore, comprises the following steps：
(1) collection of spore：The accessory pinna with sporangiorus on wild Cibotium barometz plant is plucked, is put naturally cloudy in clean bag
It is dry, the removal of impurity is gone after sporangium comes off naturally, collects sporangium, after being acted on 6 minutes with 0.1% mercuric chloride solution, then with nothing
Bacterium water rinses 5 times, is collected in after drying in sterile bag；
(2) spore pre-processes：The sterile bag containing sporangium that step (1) obtains is placed in 6 DEG C of preservations 380 days, broken
Sporangium collects spore, and is centrifuged after being sterilized 20 seconds to spore with 75% alcohol, aseptic water washing 3 times, then with 3% it is secondary
Sodium chlorate is sterilized 7 minutes to spore and centrifuged, then with aseptic water washing 3 times；The spore for collecting sterilization adds sterile saline system
Into spore suspension；
(3) inoculation and culture：0.07ml is taken to be seeded to equipped with spore germination culture medium the spore suspension that step (2) obtains
Tissue culture tank in, coating spore suspension is uniformly distributed in media surface, sprouting culture, temperature are carried out in illumination box
28 DEG C, daily 2000-2500Lx illumination 10 hours；After original foliage is grown, original foliage is seeded to containing sporinite culture medium
Culture tank in, in illumination box carry out gametophyte conversion culture, 28 DEG C of temperature, daily 2000-2500Lx illumination 12 are small
When；
(4) acclimatization and transplantses：After gametophyte grows the 3rd leaf, gametophyte root media transfer is rinsed well to containing
In the nutrition cave of Nutrition Soil, spraying daily keeps humidity to cultivate 15- under the conditions of room temperature and natural lighting in 70%-72% 2 times
Field transplanting is carried out after 30 days.
The spore germination culture medium is made up of 1/2 modified MS medium, 1-3% sucrose, 0.6-0.8% agar；
The sporinite culture medium is formed by improveing MS2 culture mediums, 1-3% sucrose, 0.6-0.8% agar, described to change
Good MS2 culture mediums are made up of modified MS medium, potato extract solution and melissyl alcohol；
The composition improved in the modified MS medium is：MgSO4·7H2O is adjusted to 180-200mg/L；CaCl2·
2H2O is adjusted to 200-220mg/L；KNO3600-700mg/L is adjusted to, (NH)4NO3It is adjusted to 500-600mg/L；KH2PO4Adjust
Whole is 170-200mg/L；Increase (NH)2HPO4350-400mg/L；
The spore germination culture medium also includes sterile 6-BA0.02-0.1mg/L and sterile IAA0.02-0.1mg/L, institute
Stating sporinite culture medium also includes sterile GA30.02-0.1mg/L and sterile 6-BA0.02-0.1mg/L；It is described sprouting culture be
60-90 days, the gamete conversion culture was 45-90 days.
Claims (6)
1. a kind of tissue culture method of scythian lamb rhizome spore, it is characterised in that comprise the following steps：
(1) collection of spore：The accessory pinna with sporangiorus on wild Cibotium barometz plant is plucked, puts in clean bag and dries in the shade naturally,
Go the removal of impurity after sporangium comes off naturally, collect sporangium, after 0.1% mercuric chloride solution effect 4-6 minutes, then with sterile
Water rinses 3-5 times, is collected in after drying in sterile bag；
(2) spore pre-processes：The sterile bag containing sporangium that step (1) obtains is placed in 2-6 DEG C of preservation 340-380 days, broken
Open sporangium and collect spore, and centrifuged after sterilizing the 20-40 seconds to spore with 70%-75% alcohol, aseptic water washing 2-3 times,
Spore sterilization 4-10 minutes are centrifuged with 2-5% sodium hypochlorite again, then with aseptic water washing 2-3 times；Collect the spore of sterilization
Add sterile saline and spore suspension is made；
(3) inoculation and culture：0.05-0.1ml is taken to be seeded to equipped with spore germination culture medium the spore suspension that step (2) obtains
Tissue culture tank in, coating spore suspension is uniformly distributed in media surface, sprouting culture, temperature are carried out in illumination box
22-28 DEG C, daily 2000-2500Lx illumination 10-12 hours；After original foliage is grown, original foliage is seeded to containing sporinite
In the culture tank of culture medium, gametophyte conversion culture, 22-28 DEG C of temperature, daily 2000-2500Lx are carried out in illumination box
Illumination 10-12 hours；
(4) acclimatization and transplantses：After gametophyte grows the 3rd leaf, gametophyte root media transfer is rinsed well to containing nutritious
In the nutrition cave of soil, 1-2 holding humidity of spraying daily, field is carried out after being cultivated 15-30 days under the conditions of room temperature and natural lighting
Between transplant.
A kind of 2. tissue culture method of scythian lamb rhizome spore according to claim 1, it is characterised in that the spore germination training
Base is supported to be made up of 1/2 modified MS medium, 1-3% sucrose, 0.6-0.8% agar.
A kind of 3. tissue culture method of scythian lamb rhizome spore according to claim 1, it is characterised in that the sporinite culture
Base is formed by improveing MS2 culture mediums, 1-3% sucrose, 0.6-0.8% agar, and the improvement MS2 culture mediums are trained by improveing MS
Support base, potato extract solution and melissyl alcohol composition.
A kind of 4. tissue culture method of scythian lamb rhizome spore according to Claims 2 or 3, it is characterised in that the improvement MS
The composition improved in culture medium is：MgSO47H2O is adjusted to 180-200mg/L；CaCl22H2O is adjusted to 200-220mg/
L；KNO3 is adjusted to 600-700mg/L, and (NH) 4NO3 is adjusted to 500-600mg/L；KH2PO4 is adjusted to 170-200mg/L；Increase
Add (NH) 2HPO4 350-400mg/L.
A kind of 5. tissue culture method of scythian lamb rhizome spore according to claim 4, it is characterised in that the spore germination training
Foster base also includes sterile 6-BA0.02-0.1mg/L and sterile IAA0.02-0.1mg/L, and the sporinite culture medium also includes nothing
Bacterium GA30.02-0.1mg/L and sterile 6-BA0.02-0.1mg/L.
A kind of 6. tissue culture method of scythian lamb rhizome spore according to claim 1, it is characterised in that it is described sprouting culture be
60-90 days, the gamete conversion culture was 45-90 days.
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| CN201711123136.1A CN107736248A (en) | 2017-11-14 | 2017-11-14 | A kind of tissue culture method of scythian lamb rhizome spore |
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| CN201711123136.1A CN107736248A (en) | 2017-11-14 | 2017-11-14 | A kind of tissue culture method of scythian lamb rhizome spore |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109699495A (en) * | 2019-02-12 | 2019-05-03 | 云南农业大学 | A method of improving scythian lamb rhizome spore germination rate |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
-
2017
- 2017-11-14 CN CN201711123136.1A patent/CN107736248A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109699495A (en) * | 2019-02-12 | 2019-05-03 | 云南农业大学 | A method of improving scythian lamb rhizome spore germination rate |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
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