CN106171969B - A kind of clone's culture technique of dendrobium candidum embryo particle - Google Patents
A kind of clone's culture technique of dendrobium candidum embryo particle Download PDFInfo
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- CN106171969B CN106171969B CN201510227277.2A CN201510227277A CN106171969B CN 106171969 B CN106171969 B CN 106171969B CN 201510227277 A CN201510227277 A CN 201510227277A CN 106171969 B CN106171969 B CN 106171969B
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- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 5
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 5
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- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Mushroom Cultivation (AREA)
Abstract
The present invention discloses a kind of clone's culture technique of dendrobium candidum embryo particle, is mainly used in the large-scale production with medicinal, edible value high-quality dendrobium candidum protocorm.Patent of the present invention selects the ripe fruit pod of dendrobium candidum, the micro- material access clone's fluid nutrient medium of embryo is taken out after Aseptic sterilisation, low temperature light culture ventilate after a week, it is transferred at 27 DEG C of illumination and continues ventilation suspension culture three weeks, to embryo particle completion sprouting, expansion, turn green, the small protocorm of taper is formed, this can be as the germplasm materials of the medicinal protocorm large-scale production of dendrobium candidum.The present invention carries out clone's culture using dendrobium candidum protocorm Special ventilating suspension culture apparatus, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and real-time feed supplement, promote embryo fast-growth, entire cultivation cycle is only one month, compared with traditional culture technique needs 34 months, the production cycle is substantially reduced, and single batch production scale is big, yield is high, at low cost, it, can be with the genetic stability of effective guarantee plant and safety due to being to apply clone technology.
Description
Technical field
The invention belongs to plant cell engineering fields, and in particular to be a kind of dendrobium candidum embryo particle clone culture
Technology.
Background technology
Dendrobium candidum（Dendrobium officinale Kmuraet Migo）It is China's tradition rare traditional Chinese medicine, taste
Sweet, cold nature has promote the production of body fluid nourishing the stomach, nourishing Yin and clearing heat, moistening lung kidney-nourishing, the strong waist of improving eyesight and other effects.It is recorded, led according to National Pharmacopeia
It is dendrobium polysaccharide and dendrobine to want efficacy component.
The medicinal protocorm of dendrobium candidum is through liquid Clone formation bulb proembryo by the embryo particle in dendrobium candidum fruit pod
Body, the stage can make protocorm be enriched a large amount of nascent and secondary metabolism by nutrition adjustment and the induction of special culture item
The content of product, wherein dendrobine, erussian and polysaccharide is the highest stage in its entire growth course.It is demonstrate,proved through clinical research
This bright protocorms have the medicinal and health value of item fresh with commercially available dendrobium candidum and maple bucket on an equal basis.
Embryo particle refers to powdery in dendrobium candidum maturation fruit pod, tiny faint yellow particle, and each micro- material of embryo is all
Plant female parent all gene are carried, in the field environment, need to be combined with certain fungal elicitor could sprout, therefore natural
Germination rate is extremely low, in laboratory environments, can be formed directly in dendrobium candidum protocorm and iron sheet by way of cloning and inducing
Dendrobium plant.
At present, the traditional mode of production mode of protocorm is that the induction to dendrobium candidum explant leaf stem section is utilized to generate cellule
Group, then small cell cluster is carried out expanding numerous and continuous squamous subculture and is obtained, for stricti jurise, this protocorms should be known as class
Protocorm group, due to be from the induction of the cell of explant, and constantly squamous subculture and cause its hereditary capacity have randomness and
Compared with macromutation probability, it is impossible to maternal whole inhereditary features are fully retained, it cannot be with female parent complete one so as to cause its express spectra
Cause, for example the expression quantity such as polysaccharide, dendrobine are low or expression product changes for main active ingredient, make protocorms group medicinal or
It all cannot be guaranteed in edible value and safety.
In view of this, the present inventor furthers investigate in view of the foregoing drawbacks, there is this case generation then.Protocorm in the present invention is equal
For single embryo particle induced synthesis, protocorm does not form multiple irregular cell groups during proliferation, but grows hair
Larger plant embryoid is bred as, there are plant body characteristics.Each embryo particle is in the process of the medicinal protocorm of induced synthesis
In only carry out autologous growth development, without being divided into multiple variant cell groups, so as to which the heredity for completely remaining maternal is special
Property.
Invention content
Present invention aims at a kind of clone's culture technique of dendrobium candidum embryo particle is provided, it is mainly used in medicine
With the large-scale production of the high-quality dendrobium candidum protocorm of, edible value.Patent of the present invention selects the mellow fruit of dendrobium candidum
Pod, takes out the micro- material access clone's fluid nutrient medium of embryo after Aseptic sterilisation, and ventilation low temperature light culture after a week, is transferred to illumination 27
Continue the culture three weeks that suspends of ventilating at DEG C, until embryo particle is completed to sprout, expands, turns green, form the small protocorm of taper, this can make
Germplasm materials for the medicinal protocorm large-scale production of dendrobium candidum.The present invention is suspended using dendrobium candidum protocorm Special ventilating
Culture apparatus carries out clone's culture, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and real-time feed supplement, promote
Into embryo fast-growth, entire cultivation cycle is only one month, compared with traditional culture technique needs 3-4 months, is substantially reduced
Production cycle, and single batch production scale is big, yield is high, at low cost, it, can be with effective guarantee plant due to being to apply clone technology
The genetic stability of body and safety.
In order to achieve the above objectives, solution of the invention is：
A kind of clone's culture technique of dendrobium candidum embryo particle, the technology select the dendrobium candidum mellow fruit of artificial growth
Pod, fruit pod is full, and dark green or green purple is harvested October to during November, first wrapped with dried towel, be put into have it is dry
The vial with cover of drying prescription, after handling 3 days at 4 DEG C of low temperature, taking-up knife blade cuts off the extra part of fruit pod, only stays 1
The carpopodium of~2cm long is weighed and is recorded.
Further, the fruit pod handled well is moved into sterilized superclean bench, first wipes fruit with cotton ball soaked in alcohol
Pod surface handles 10min in 10% sodium hypochlorite, and aseptic water washing 3 times is then immersed in 40s in 70% alcohol, aseptic water washing 3
It is secondary, it is placed in inoculation disk and cuts fruit pod bottom, be equably sown into the ventilation culture bottle equipped with clone's fluid nutrient medium, finish kind
Fruit pod take out, weigh and record, calculate and be sown into the grams of embryo particle.
Further, the ventilation culture bottle for finishing kind is first inserted in and be protected from light in bag, placed on culturing rack, connection ventilation pump is logical
Tolerance 5L/min, dissolved oxygen coefficient 2%, 20 DEG C of cultivation temperature carries out light culture 1 week, then removes and be protected from light bag, into illumination cultivation, leads to
Tolerance 10L/min, dissolved oxygen coefficient 2%, 27 DEG C, intensity of illumination 2500Lux of cultivation temperature are cultivated 4 weeks, are during which carried out week about
Fluid infusion, totally 3 times.
The formula of clone's Liquid Culture in the present invention is：Modified MS medium, wherein ammonium nitrate are replaced with ammonium sulfate, add
Enter amount to halve, calcium chloride addition halves；Plant growth regulator：6 benzyladenine, 0.02 deal；Osmolyte regulator, carbon source：
40 deal of sucrose；Amino acid supplements, nitrogen source：0.2 deal of acid hydrolyzed casein, natural organic additive：150 parts of potato juice
Amount, adds ultra-pure water to be configured to 1000 deals.
Feed supplement formula is：1/4 modified MS medium, single amount infused are the 1/3 of culture medium total amount, fluid infusion 3 times altogether.
Compared with the prior art, the present invention carries out clone's training using dendrobium candidum protocorm Special ventilating suspension culture apparatus
It supports, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and real-time feed supplement, promote embryo fast-growth, it is whole
A cultivation cycle is only 35 days, compared with traditional culture technique needs 3-4 months, substantially reduces the production cycle, and single batch is given birth to
Production scale is big, yield is high, at low cost, can be with the genetic stability and peace of effective guarantee plant due to being to apply clone technology
Quan Xing.The present invention produces the small protocorm obtained can be as best kind of material of the medicinal protocorm large-scale production of dendrobium candidum
Material, this can quickly propel iron sheet to realize that the factorial praluction of the medicinal protocorm of dendrobium candidum provides technology and raw material support
The development process of stem of noble dendrobium industrialization, with science and technology come ensure product safety and effectiveness and by effectively reduce production cost and
Retail cost, more people to be allowed consume can to play civil celestial grass --- the dendrobium candidum once exclusively enjoyed only for imperial family.
Specific embodiment
In order to further explain the technical solution of the present invention, implement with reference to the embodiment of the present invention and existing solid culture
Example, the present invention will be described in detail for agitated submerged culture implementation.
Embodiment one：This case is invented
The dendrobium candidum maturation fruit pod of artificial growth is selected, fruit pod is full, dark green or green purple, is harvested October 15,
It is first wrapped with dried towel, is put into the vial with cover for having drier, it, will with knife blade after being handled 3 days at 4 DEG C of low temperature
The extra part excision of fruit pod, only stays the carpopodium of 1.5cm long, weighs and record, obtain 1.63g.
The fruit pod handled well is moved into sterilized superclean bench, first wipes fruit pod surface with cotton ball soaked in alcohol,
10 ~ 20min is handled in 10% sodium hypochlorite, aseptic water washing 3 times is then immersed in 40smin in 70% alcohol, aseptic water washing 3
It is secondary, it is placed in inoculation disk and cuts fruit pod bottom, be equably sown into the ventilation culture bottle equipped with clone's fluid nutrient medium, finish kind
Fruit pod take out, weigh and record, obtain 0.93g, it is 0.7g to calculate and be sown into the grams of embryo particle.
The ventilation culture bottle for finishing kind is first inserted in and is protected from light in bag, is placed on culturing rack, connection ventilation pump, ventilatory capacity 5L/
Min, dissolved oxygen coefficient 2%, 20 DEG C of cultivation temperature carries out light culture 1 week, then removes and be protected from light bag, into illumination cultivation, ventilatory capacity 10L/
Min, dissolved oxygen coefficient 2%, 25 DEG C, intensity of illumination 2500Lux of cultivation temperature cultivate 4 weeks, during which carry out a fluid infusion week about,
Totally 3 times.
Small protocorm is taken out after culture, appearance is recorded as：Yellow green, form is full, and taper is spherical, and bulb is larger；
After drain well, weigh to obtain 81.23g, and it is 115.7 to calculate proliferation times.
The formula of clone's Liquid Culture of the present embodiment is：Modified MS medium, wherein ammonium nitrate are replaced with ammonium sulfate, add
Enter amount to halve, calcium chloride addition halves；Plant growth regulator：6 benzyladenine 0.2mL；Osmolyte regulator, carbon source：Sugarcane
Sugared 40g；Amino acid supplements, nitrogen source：Acid hydrolyzed casein 0.2g, natural organic additive：Potato juice 150g, adds ultra-pure water
It is settled to 1L.
Feed supplement formula is：1/4 modified MS medium, single amount infused are the 350mL of culture medium total amount, and common fluid infusion is three times.
Embodiment two：Solid culture
The dendrobium candidum maturation fruit pod of artificial growth is selected, fruit pod is full, dark green or green purple, is harvested October 15,
It is first wrapped with dried towel, is put into the vial with cover for having drier, it, will with knife blade after being handled 3 days at 4 DEG C of low temperature
The extra part excision of fruit pod, only stays the carpopodium of 1.5cm long, weighs and record, obtain 1.45g.The fruit pod handled well is moved into
Through in sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, 10 ~ 20min, nothing are handled in 10% sodium hypochlorite
Bacterium water rinses 3 times, is then immersed in 40smin in 70% alcohol, and aseptic water washing 3 times is placed in inoculation disk and cuts fruit pod bottom,
It is sown into evenly in the tissue culture bottle equipped with solid medium, the fruit pod for finishing kind is taken out, and weighs and records, obtain 0.81g, calculating is sown into
The grams of embryo particle is 0.64g.
By the tissue culture bottle that finish kind, place darkroom and cultivate one week, 20 DEG C of cultivation temperature, then into illumination cultivation, cultivation temperature
It 25 DEG C, intensity of illumination 2500Lux, cultivates 8 weeks.
Small protocorm is taken out after culture, appearance is recorded as：Surface layer bulb is yellow green, and bottom bulb is yellow-white,
Bulb is smaller, is elliptic conic shape；Weigh to obtain 25.12g, and it is 39.2 to calculate proliferation times.
The formula of the fixed culture of the present embodiment is：Modified MS medium, wherein ammonium nitrate are replaced with ammonium sulfate, addition
Halve, calcium chloride addition halves；Plant growth regulator：6 benzyladenine 0.2mL；Osmolyte regulator, carbon source：Sucrose
40g；Amino acid supplements, nitrogen source：Acid hydrolyzed casein 0.2g, natural organic additive：Potato juice 150g, 8 grams of agar, adds
Ultra-pure water is settled to 1L.
Embodiment three：Agitated submerged culture
The dendrobium candidum maturation fruit pod of artificial growth is selected, fruit pod is full, dark green or green purple, is harvested October 15,
It is first wrapped with dried towel, is put into the vial with cover for having drier, it, will with knife blade after being handled 3 days at 4 DEG C of low temperature
The extra part excision of fruit pod, only stays the carpopodium of 1.5cm long, weighs and record, obtain 1.65g.The fruit pod handled well is moved into
Through in sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, 10 ~ 20min, nothing are handled in 10% sodium hypochlorite
Bacterium water rinses 3 times, is then immersed in 40smin in 70% alcohol, and aseptic water washing 3 times is placed in inoculation disk and cuts fruit pod bottom,
It is sown into evenly in the tissue culture bottle equipped with fluid nutrient medium, the fruit pod for finishing kind is taken out, and weighs and records, obtain 0.86g, calculating is sown into
The grams of embryo particle is 0.79g.
The tissue culture bottle of kind will be finished, place light culture one week, 100 turns/min of rotating speed in shaken cultivation case, cultivation temperature 20
DEG C, then into illumination cultivation, 25 DEG C of cultivation temperature, rotating speed 150 turns/min, intensity of illumination 2500Lux are cultivated 4 weeks.
Small protocorm is taken out after culture, appearance is recorded as：Bulb is yellow green, and bulb is smaller, there is hyalinization；
Weigh to obtain 48.31g, and it is 54.8 to calculate proliferation times.
The formula of the fixed culture of the present embodiment is：Modified MS medium, wherein ammonium nitrate are replaced with ammonium sulfate, addition
Halve, calcium chloride addition halves；Plant growth regulator：6 benzyladenine 0.2mL；Osmolyte regulator, carbon source：Sucrose
40g；Amino acid supplements, nitrogen source：Acid hydrolyzed casein 0.2g, natural organic additive：Potato juice 150g, adds ultra-pure water to determine
Hold to 1L.
To sum up, as shown in table 1, it is of the invention compared with existing solid tissue culture technology and agitated submerged culture technology, increase
It grows coefficient to improve significantly, protocorm quality is good.
The parameter comparison of 1 three kinds of Protocorm technologies of table
| Technical solution | Inoculum concentration | Cultivation cycle | Appearance describes | It is proliferated weight | Proliferation times |
| This case | 0.70g | 35 days | Green, form is full, and taper is spherical, and bulb is larger | 81.23g | 115.7 |
| Solid culture | 0.64g | 35 days | Surface layer bulb is yellow green, and bottom bulb is yellow-white, and bulb is smaller, is elliptic conic shape | 25.12g | 39.2 |
| Agitated submerged culture | 0.79g | 35 days | Bulb is yellow green, and bulb is smaller, there is hyalinization | 48.31g | 54.8 |
Claims (5)
1. a kind of clonal culture protocol of dendrobium candidum embryo particle, which is characterized in that including the following contents：1. select iron sheet stone
Embryo particle in dry measure used in former times maturation fruit pod is as inoculation material；Lead to 2. dendrobium candidum embryo particle is accessed in clone's fluid nutrient medium
Gas suspension culture；3. light culture of first ventilating after completing inoculation, which is transferred to after a week at 27 DEG C of illumination, continues ventilation suspension culture three weeks；
4. since incubation time to culture terminal procedure in carry out a feed operation week about；5. it cultivates complete to embryo particle
Sprout, expansion, turn green, form taper bottom set, the content 2. in the formula of clone's Liquid Culture be：Improve MS cultures
Base, wherein ammonium nitrate are replaced with ammonium sulfate, and addition halves, and calcium chloride addition halves；Plant growth regulator：6- benzyls
0.02 deal of adenine；Osmolyte regulator, carbon source：40 deal of sucrose；Amino acid supplements, nitrogen source：0.2 part of acid hydrolyzed casein
Amount, natural organic additive：150 deal of potato juice, adds ultra-pure water to be configured to 1000 deals.
A kind of 2. clonal culture protocol of dendrobium candidum embryo particle as described in claim 1, which is characterized in that the content
1. first with cotton ball soaked in alcohol wiping fruit pod surface in, 10 ~ 20min is handled in 5 ~ 10% sodium hypochlorite, aseptic water washing 3 ~ 5 times, then
30s ~ 1min in 70 ~ 75% alcohol is immersed, aseptic water washing 3 ~ 5 times is placed in inoculation disk and cuts fruit pod bottom, is equably sown into
In ventilation culture bottle equipped with clone's fluid nutrient medium.
A kind of 3. clone's culture technique of dendrobium candidum embryo particle as described in claim 1, which is characterized in that the content
3. the parameter of middle ventilation light culture is：Ventilatory capacity 5L/min, dissolved oxygen coefficient 2%, 20 DEG C of cultivation temperature are inserted in be protected from light in bag and are placed in
Tissue culture frame starts to cultivate；The parameter of illumination cultivation is：Ventilatory capacity 10L/min, dissolved oxygen coefficient 2%, 27 DEG C of cultivation temperature, illumination is strong
2500Lux is spent, puts and is cultivated under illumination cultivation frame.
4. the clonal culture protocol of a kind of dendrobium candidum embryo particle as described in claim 1 or 3, which is characterized in that described
Content 4. in feed supplement formula be：1/4 modified MS medium, single amount infused are the 1/3 of culture medium total amount, and common fluid infusion is three times.
A kind of 5. clonal culture protocol of dendrobium candidum embryo particle as described in claim 1, which is characterized in that the content
5. middle embryo particle after the culture of four weeks, can be sprouted, be expanded, turning green completely, cone of the size in 0.2-0.5mm is formed
The small protocorm of shape, and using this small protocorm as the germplasm materials of the medicinal protocorm of dendrobium candidum.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101268759A (en) * | 2008-04-16 | 2008-09-24 | 中国林业科学研究院林业研究所 | Dendrobium orchid primary seed embryo culture method |
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| CN103563741A (en) * | 2012-07-27 | 2014-02-12 | 宁波德健生物科技有限公司 | Aseptic detoxicant culturing method of cracked Dendrobium officinale capsule embryos |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101268759A (en) * | 2008-04-16 | 2008-09-24 | 中国林业科学研究院林业研究所 | Dendrobium orchid primary seed embryo culture method |
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| CN103563741A (en) * | 2012-07-27 | 2014-02-12 | 宁波德健生物科技有限公司 | Aseptic detoxicant culturing method of cracked Dendrobium officinale capsule embryos |
Non-Patent Citations (3)
| Title |
|---|
| "铁皮石斛类原球茎生物反应器培养体系构建;徐步青;《中国优秀硕士学位论文全文数据库 农业科技辑》;20121215(第12期);摘要 * |
| Effects of light quality on growth and development of protocorm-like bodies of Dendrobium officinale in vitro;Yuan Lin et.al.;《PLANT CELL TISSUE AND ORGAN CULTURE》;20101117;第105卷;第329-335页 * |
| 铁皮石斛种胚原球茎液体悬浮培养研究;魏小勇;《中国现代应用药学杂志》;20051231;第22卷(第6期);第471-473页 * |
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