CN107094630A - Callus breaks up culture process of sprouting in red sage root tissue cultures - Google Patents
Callus breaks up culture process of sprouting in red sage root tissue cultures Download PDFInfo
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- CN107094630A CN107094630A CN201710498166.4A CN201710498166A CN107094630A CN 107094630 A CN107094630 A CN 107094630A CN 201710498166 A CN201710498166 A CN 201710498166A CN 107094630 A CN107094630 A CN 107094630A
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- callus
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- red sage
- sage root
- sprouting
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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
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Break up culture process of sprouting the invention discloses callus in red sage root tissue cultures, comprise the following steps：1) preparation of culture medium；2) selection and culture of explant；3) selection of callus；4) processing of callus；5) inoculation of callus；6) induction of clump bud；The technique is mainly optimized and improved to the medium component during callus differentiation is sprouted in tissue cultures, is reduced the usage amount of hormone and is used species, using single hormonal components, can obtain high breeding coefficient, and bud growing way is vigorous, and is adapted to later stage culture.
Description
Technical field
Break up culture process of sprouting the present invention relates to callus in a kind of red sage root tissue cultures.
Background technology
The red sage root (Salvia miltiorrhiza Bunge) is that Lamiaceae Salvia belongs to herbaceos perennial, You Mingzi
The red sage root, blood ginseng, clovershrub etc., all parts of the country are distributed, and are the primary raw material of the traditional common medicine of China and a variety of Chinese patent drugs, tool
Have promoting blood circulation, inducing meastruation to relieve menalgia, relieving restlessness that clears away heart-fire, coronary artery dilator, improve myocardial ischemia situation, reduce blood pressure, calm the nerves it is with all worries set aside,
The effects such as hypoglycemic and antibacterial.Its active component mainly has two major classes：Fat-soluble diterpene-kind compound and two is water miscible many
Poly- liposoluble ingredient.The red sage root has for the medicine of main component on the market at present：Compound danshen dripping pills, Fufang Danshen Pian, red sage root glue
Capsule etc..
The red sage root is as the important source material of pharmaceuticals industry, the huge market demand, and traditional seminal propagation and root division is equal
In the presence of certain defect.Salvia seeds storage period is short, and germination rate is low；And root division coefficient is low, and it is easily carried pest and disease damage.Cause
This red sage root is faced with variety deterioration, the problems such as yield declines.And and utilize plant tissue culture technique, not only breeding coefficient is high,
The merit of original kind can be kept again, be to solve red sage root face variety deterioration, the important channel for the problems such as yield declines.
Plant tissue culture technique is the totipotency according to cell, utilizes in vitro tissue, organ or cell, raw plastid
Deng, by sterile working, appropriate culture medium and with the intact plant that culture obtains regeneration is carried out under suitable external condition
Process.Callus is produced from each organ in incubation, callus passes through again is differentiated to form Multiple Buds again, and then
Form intact plant.And callus be differentiated to form Multiple Buds process be red sage root tissue culture procedures essential step, decide group
The height of breeding coefficient during training.
The content of the invention
For above-mentioned problem, the present invention is intended to provide callus breaks up culture process of sprouting in red sage root tissue cultures,
The technique is mainly optimized and improved to the medium component during callus differentiation is sprouted in tissue cultures, reduces hormone
Usage amount and species is used, using single hormonal components, high breeding coefficient can be obtained, and bud growing way is vigorous, and after being adapted to
Phase cultivates.
To achieve these goals, the technical solution adopted in the present invention is as follows：
Callus breaks up culture process of sprouting in red sage root tissue cultures, comprises the following steps：
1) preparation of culture medium
A, hormone mother liquor preparation：6-BA 0.1g are weighed, draw molten with 5mL 1M NaOH, then 100mL is settled to pure water,
As concentration is 1.0mg/mL 6-BA mother liquors；
B, the sterilizing of hormone suction filtration：Put filter membrane into suction filtration head, autoclaving brings super-clean bench into, IBA mother liquor suction filtrations are gone out
Bacterium, it is standby；
C, preparation culture medium basis：MS+30g/L sucrose+5.26g/L agar, pH5.8；Autoclaving；Bring into ultra-clean
Platform is standby；
D, addition hormone：Absorption 6-BA is robbed with sterile liquid relief to be added in minimal medium, make its dense on super-clean bench
Spend for 1.5mg/L, mixing；
E, packing：The culture medium for having added hormone is dispensed into sterile tissue culture bottle, every bottle of 30~40mL, composition is：
MS+1.5mg/L 6-BA+30g/L sucrose+5.26g/L agar, pH5.8；
2) selection and culture of explant：Using red sage root blade as explant, after sterilization, it is cut into 0.5*0.5cm sizes and is placed in
Cultivated in callus culture medium；
3) selection of callus：Explant callus induction 28-30 days, selects glossiness callus；
4) processing of callus；
5) inoculation of callus；
6) induction of clump bud：Callus after inoculation is positioned over the induction that clump bud is carried out on the tissue culture frame of tissue culture room, cultivates bar
Part：27 ± 0.5 DEG C of cultivation temperature, light application time 12h, light intensity 3000Lx, T5 plant culture fluorescent tube.
Preferably, step (2) " selection and culture of the explant " condition of culture：Cultivation temperature (27 ± 0.5 DEG C),
Light application time 12h, light intensity 3000Lx, T5 plant cultivate fluorescent tube.
Preferably, step (3) " selection of the callus " growth selection is vigorous, green or the glossiness callus of yellow green
Bring super-clean bench into standby, and bottle mouth position is wiped with 75% cotton ball soaked in alcohol.
Preferably, the step (4) " processing of callus " is from tissue culture with sterile tweezers by well-grown callus
Tweezer rises in bottle, is placed on sterile inoculation disk, browning part in callus is removed with sterile scissors or scalpel, by well-grown
Part be cut into 0.5cm × 0.5cm sizes.
Preferably, the step (5) " inoculation of callus " is by 0.5cm × 0.5cm callus tweezers with sterile tweezers
Rise, be positioned on MS solid mediums, medium component is MS+ 1.5mg/L6-BA+30g/L sucrose+5.26g/L agar,
PH5.8, autoclaving.
The beneficial effects of the invention are as follows：Advantage of the invention is that Corticosteroids are reduced, can be with using single hormonal components
High breeding coefficient is obtained, and bud growing way is vigorous, and it is adapted to later stage culture；The present invention is using MS cultures as minimal medium, and addition is suitable
When the 6-BA callus inductions of concentration are divided into clump bud.
Embodiment
In order that one of ordinary skill in the art is better understood on technical scheme, with reference to embodiment
Technical scheme is further described.
Embodiment：Callus breaks up culture process of sprouting in red sage root tissue cultures, comprises the following steps：
1) preparation of culture medium
A, hormone mother liquor preparation：6-BA 0.1g are weighed, draw molten with 5mL 1M NaOH, then 100mL is settled to pure water,
As concentration is 1.0mg/mL 6-BA mother liquors；
B, the sterilizing of hormone suction filtration：Put filter membrane into suction filtration head, autoclaving brings super-clean bench into, IBA mother liquor suction filtrations are gone out
Bacterium, it is standby；
C, preparation culture medium basis：MS+30g/L sucrose+5.26g/L agar, pH5.8；Autoclaving；Bring into ultra-clean
Platform is standby；
D, addition hormone：Absorption 6-BA is robbed with sterile liquid relief to be added in minimal medium, make its dense on super-clean bench
Spend for 1.5mg/L, mixing；
E, packing：The culture medium for having added hormone is dispensed into sterile tissue culture bottle, every bottle of 30~40mL, composition is：
MS+1.5mg/L 6-BA+30g/L sucrose+5.26g/L agar, pH5.8；
2) selection and culture of explant：Using red sage root blade as explant, after sterilization, it is cut into 0.5*0.5cm sizes and is placed in
Cultivated in callus culture medium, condition of culture：27 ± 0.5 DEG C of cultivation temperature, light application time 12h, the training of light intensity 3000Lx, T5 plant
Support fluorescent tube；
3) selection of callus：Explant callus induction 1 month or so, growth selection is vigorous, and green or yellow green are glossy
Callus to bring super-clean bench into standby, and wipe bottle mouth position with 75% cotton ball soaked in alcohol；
4) processing of callus：With sterile tweezers by well-grown callus from tweezer in tissue culture bottle, be placed in sterile connect
In discharge plate, browning part in callus is removed with sterile scissors or scalpel, by well-grown part be cut into 0.5cm ×
0.5cm sizes；
5) inoculation of callus：0.5cm × 0.5cm callus tweezer is risen with sterile tweezers, is positioned on MS solid mediums,
Medium component is MS+1.5mg/L 6-BA+30g/L sucrose+5.26g/L agar, pH5.8, autoclaving；
6) induction of clump bud：Callus after inoculation is positioned over the induction that clump bud is carried out on the tissue culture frame of tissue culture room, after 30 days
Start to count quantity, the growing state that clump bud is induced；Condition of culture：Cultivation temperature (27 ± 0.5 DEG C), light application time 12h, light intensity
3000Lx, T5 plant cultivate fluorescent tube.
The culture medium prescription that utilized callus differentiation most at present is sprouted is mostly using MS cultures as minimal medium, combination
Add several hormones such as 6-BA, 2,4-D, NAA；And after improvement, only need to add a kind of hormones of 6-BA, you can reach optimal bud
Differentiated result.
Advantage of the invention is that reducing Corticosteroids, using single hormonal components, high breeding coefficient, and bud can be obtained
Growing way is vigorous, and is adapted to later stage culture.The present invention adds the 6-BA callus inductions of debita spissitudo using MS cultures as minimal medium
It is divided into clump bud；6-BA is the plant growth regulator of broad spectrum, multi purpose, with the function of promoting the differentiation of undifferentiated tissue；2、
4-D and NAA are usually used in the induction of callus, 2,4-D be a kind of benzene oxidatoin class plant growth regulator, NAA is a kind of organic naphthalenes
Plant growth regulator, both physiologically actives are all higher, and cell differentiation can be all stimulated at low concentrations；Generally plant
A small amount of hormone can be inherently produced in vivo, and has been added in callus Induction Process NAA and 2,4-D, therefore more
Accumulated in wound, if artificial again add, concentration can too high, the generation opposite effect, can be that callus is undifferentiated or even browning；Cause
This, we need to only add a certain amount of 6-BA can rapid induction callus differentiation clump bud.
During specific experiment, the present invention is using red sage root blade as explant, in suitable culture medium, suitable culture bar
Callus is induced under part, growth selection is vigorous, quality is close, color is the callus of yellow green, is cut into 0.5*0.5cm2Fritter is put
In on bud differential medium.In single use 6-BA, it has been found that the startup of bud differentiation when 6-BA concentration is less than 1.5mg/L
Time is long, and inductivity increases with increasing for concentration, and when concentration is 1.5mg/L, inductivity is high, and starts the time
It is short, and during higher than 1.5mg/L, although the startup time is also shorter, but differentiation rate is relatively low, and vitrification phenomenon occurs.
Table 1：Various concentrations 6-BA breaks up the influence sprouted to red sage root callus
Table 1:Various concentrations 6-BA breaks up the influence sprouted to red sage root callus
When being used alone 2,4-D, concentration is less than the bud for having part during 1.0mg/L and starts differentiation, during higher than 1.0mg/L, only
There is a small amount of callus to be differentiated to form bud.Being differentiated to form for bud in callus may be suppressed by illustrating 2,4-D of high concentration.
Table 2：Various concentrations 2,4-D breaks up the influence sprouted to red sage root callus
Note:+ poor, ++ preferably, +++ it is vigorous
When NAA is used alone, small part callus has the differentiation of bud when concentration is less than 1.0mg/L, and callus brown occurs not
Determine root, during higher than 1.0mg/L, inductivity starts to reduce, when concentration is 2.5mg/L, almost no bud is differentiated to form, and is gone out
Existing substantial amounts of brown adventitious root.The generation of adventitious root can be promoted by illustrating the NAA of high concentration, but be detrimental to the formation of bud.
Table 3：Various concentrations NAA breaks up the influence sprouted to red sage root callus
In addition, these three hormones are used in mixed way, the situation of observation bud differentiation.As long as by experiment it was found that addition
The suitable 6-BA of concentration, the inductivity of callus is all higher, and to be used in mixed way inductivity relatively low by 2,4-D and NAA.
From the simplification of experimental procedure and it is cost-effective from the aspect of, we select that a kind of hormones of 6-BA are used alone, and can be optimal
Effect.
Table 4：The influence that the differentiation of different hormone combinations factor pair red sage root callus is sprouted
Table 5：Callus induction breaks up cultivation results after process optimization of sprouting
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (5)
1. callus breaks up culture process of sprouting in red sage root tissue cultures, it is characterised in that comprise the following steps：
1) preparation of culture medium
A, hormone mother liquor preparation：6-BA 0.1g are weighed, draw molten with 5mL 1M NaOH, then 100mL is settled to pure water, are
Concentration is 1.0mg/mL 6-BA mother liquors；
B, the sterilizing of hormone suction filtration：Put filter membrane into suction filtration head, autoclaving brings super-clean bench into, IBA mother liquors suction filtration is sterilized, it is standby
With；
C, preparation culture medium basis：MS+30g/L sucrose+5.26g/L agar, pH5.8；Autoclaving；Bring super-clean bench into standby
With；
D, addition hormone：Absorption 6-BA is robbed with sterile liquid relief on super-clean bench to be added in minimal medium, makes its concentration be
1.5mg/L, is mixed；
E, packing：The culture medium for having added hormone is dispensed into sterile tissue culture bottle, every bottle of 30~40mL, composition is：MS+
1.5mg/L 6-BA+30g/L sucrose+5.26g/L agar, pH5.8；
2) selection and culture of explant：Using red sage root blade as explant, after sterilization, it is cut into 0.5*0.5cm sizes and is placed in callus
Cultivated in culture medium；
3) selection of callus：Explant callus induction 28-30 days, selects glossiness callus；
4) processing of callus；
5) inoculation of callus；
6) induction of clump bud：Callus after inoculation is positioned over the induction that clump bud is carried out on the tissue culture frame of tissue culture room, condition of culture：Training
Support 27 ± 0.5 DEG C of temperature, light application time 12h, light intensity 3000Lx, T5 plant culture fluorescent tube.
2. callus breaks up culture process of sprouting in red sage root tissue cultures according to claim 1, it is characterised in that：The step
Suddenly (2) " selection and culture of explant " condition of culture：Cultivation temperature (27 ± 0.5 DEG C), light application time 12h, light intensity 3000Lx,
T5 plants cultivate fluorescent tube.
3. callus breaks up culture process of sprouting in red sage root tissue cultures according to claim 1, it is characterised in that：The step
Suddenly (3) " selection of callus " growth selection is vigorous, and it is standby that green or the glossiness callus of yellow green bring super-clean bench into, and uses 75%
Cotton ball soaked in alcohol wipes bottle mouth position.
4. callus breaks up culture process of sprouting in red sage root tissue cultures according to claim 1, it is characterised in that：The step
Suddenly (4) " processing of callus " be with sterile tweezers by well-grown callus from tweezer in tissue culture bottle, be placed in sterile inoculation
On disk, browning part in callus is removed with sterile scissors or scalpel, 0.5cm × 0.5cm is cut into well-grown part
Size.
5. callus breaks up culture process of sprouting in red sage root tissue cultures according to claim 1, it is characterised in that：The step
Suddenly (5) " inoculation of callus " is to be played 0.5cm × 0.5cm callus tweezer with sterile tweezers, is positioned on MS solid mediums, is trained
It is MS+1.5mg/L 6-BA+30g/L sucrose+5.26g/L agar, pH5.8, autoclaving to support based component.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739405A (en) * | 2018-07-07 | 2018-11-06 | 云南澈川生物科技有限公司 | A kind of tissue cultures and in-vitro regeneration method of panax japonicus majoris |
| CN114982639A (en) * | 2022-07-01 | 2022-09-02 | 四川农业大学 | Efficient breeding method for salvia miltiorrhiza tissue culture seedlings |
-
2017
- 2017-06-26 CN CN201710498166.4A patent/CN107094630A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 李胜等: "《植物组织培养》", 31 July 2015, 中国林业出版社 * |
| 王维婷等: "丹参组织培养及其再生体系的建立与优化", 《现代中药研究与实践》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739405A (en) * | 2018-07-07 | 2018-11-06 | 云南澈川生物科技有限公司 | A kind of tissue cultures and in-vitro regeneration method of panax japonicus majoris |
| CN108739405B (en) * | 2018-07-07 | 2023-01-20 | 云南省农业科学院药用植物研究所 | Tissue culture and in-vitro regeneration method of rhizoma panacis majoris |
| CN114982639A (en) * | 2022-07-01 | 2022-09-02 | 四川农业大学 | Efficient breeding method for salvia miltiorrhiza tissue culture seedlings |
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Inventor after: Chao Xiaofeng Inventor after: Sheng Yelong Inventor before: Chao Xiaofeng Inventor before: Sheng Yilong |
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Effective date of registration: 20180730 Address after: 212000 29 buildings of No. 99 road fifteen, Ting Mao Jing, Zhenjiang New District, Jiangsu. Applicant after: Zhejiang Deer Biological Product Research Institute Co., Ltd. Address before: 213139 village, Gu Cun, Meng he Town, Xinbei District, Changzhou, Jiangsu Applicant before: Changzhou Meng Shuangfeng Chinese herbal medicine science and Technology Co., Ltd. |
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