CN105794639B - A kind of method of peppermint blade adventitious bud inducing and plant regeneration - Google Patents
A kind of method of peppermint blade adventitious bud inducing and plant regeneration Download PDFInfo
- Publication number
- CN105794639B CN105794639B CN201610182279.9A CN201610182279A CN105794639B CN 105794639 B CN105794639 B CN 105794639B CN 201610182279 A CN201610182279 A CN 201610182279A CN 105794639 B CN105794639 B CN 105794639B
- Authority
- CN
- China
- Prior art keywords
- adventitious bud
- peppermint
- culture
- agar
- blade
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to a kind of method that peppermint blade regeneration plant is obtained by peppermint leaf culture, belong to biological technical field.Using peppermint sterile test tube seedling leaf as material, the peppermint blade vitro Regeneration System that the present invention establishes is：The culture medium of callus induction and differentiation is the g/L agar of+30 g/L sucrose of MS+1.4 mg/L+0.2 mg/L NAA of TDZ+5.5；The culture medium of adventitious bud proliferation is the g/L agar of 30 g/L sucrose of MS+6 BA0.5 mg/L+IAA0.1 mg/L++5.5；The culture medium of adventitious bud rooting is the g/L agar of+20 g/L sucrose of MS+0.2 mg/L IBA+5.5.For inoculation position to remove the peppermint blade of blade edge and tip segment, the present invention establishes peppermint excised leaf regenerating system, and certain Research foundation is established for the genetic transformation of peppermint.
Description
Technical field
The present invention relates to a kind of method that peppermint blade regeneration plant is obtained by peppermint leaf culture, belong to biotechnology
Field.
Background technology
Peppermint (Mentha haplocalyx Briq.) is one kind of Labiatae (Labiatae) Mentha (MenthaL.)
Herbaceos perennial, also known as fish mint, rising Yang grass, jintan grass etc., complete stool has dense salubrious fragrance, and its aerial part is done
It can be used as medicine after dry, be the important Chinese herbal medicine in China and spice berry.In terms of medicine, peppermint can relieve summer heat, stomach of waking up opens spleen, only
Itch, relieve pain, medical value is high.Menthol in Peppermint essential oil is a kind of important spices, be mainly used in cigarette, cosmetics, toothpaste,
In chewing gum, perfumed soap, cold drink and medicine.
Peppermint research at present carry out it is more, its medical value and the basic research such as research in terms of extracting chemical composition with
And measure peppermint chemical composition content etc. is more ripe, also there is some relevant reports in terms of Plant Biotechnology, but
It is relatively fewer on peppermint blade regenerating system system research.Agriculture bacillus mediated leaf disk method genetic transfoumation is plant genetic
One of important method of Study on Transformation, and the regeneration plant viral level that excised leaf is formed after dedifferentiation is broken up again is extremely low
It is even virus-free.Using peppermint sterile test tube seedling leaf as material, by leaf culture evoking adventive bud, peppermint tooth in vitro is established
Piece regenerating system, certain Research foundation is established for peppermint genetic transformation.
The content of the invention
It is an object of the invention to provide a kind of method that peppermint blade regeneration plant is obtained by peppermint leaf culture.Mainly
It is that aseptic blade is inoculated into the MS minimal mediums for adding several hormons to carry out isolated regeneration culture, research is different dense
Influence of the hormone combinations to blade adventitious bud induction frequency is spent, and on this basis, carried out adventitious bud proliferation, taken root and transplant,
Further optimize peppermint regenerating system.
To reach the studies above purpose, the present invention provides following technical scheme：
The invention discloses a kind of method that peppermint blade regeneration plant is obtained by peppermint leaf culture, peppermint blade is again
Raw tissue culture medium (TCM), including callus induction and differentiation culture medium, adventitious bud proliferation culture medium, adventitious bud rooting culture
Base.The adventitious bud induction culture base is addition TDZ1.2~1.4mg/L, NAA0.2mg/ using MS culture mediums as minimal medium
L, sucrose 30g/L and agar 5.5g/L；The adventitious bud proliferation culture medium adds 6- using MS culture mediums as minimal medium
BA0.5mg/L, IAA0.1mg/L, sucrose 30g/L and agar 5.5g/L；The adventitious bud rooting culture medium is with MS culture mediums
IBA0.2mg/L, sucrose 20g/L and agar 5.5g/L are added for minimal medium.
1) callus induction and differentiation：In superclean bench, the test tube seedling leaf of squamous subculture is cut into about
(leaf back contact culture medium) is carried out not in the blade access callus induction and differentiation culture medium of 0.5cm × 0.5cm sizes
Normal bud Fiber differentiation；
2) adventitious bud proliferation：The adventitious bud that peppermint blade edge is grown is transferred and increased in adventitious bud proliferation culture medium
Grow；
3) adventitious bud rooting：The adventitious bud seedling of propagation is inoculated into culture of rootage in the root media；
4) test tube seedling hardening and transplanting：When seedling base portion grows adventitious root, when length is up to 1~2cm, cleaned after 2~3d of hardening
Root agar is transplanted to volume ratio as 7:Cultivated in the Nutrition Soil of 3 rural area soil-organic fertilizer.
Above-mentioned each Medium's PH Value is 5.8~6.0.
Above-mentioned culture room temperature (25 ± 1) DEG C, 1800~2000lx of intensity of illumination, light application time 12h/d.
The advantages and positive effects of the present invention：Peppermint blade callus induction and differentiation is merged into a step by the present invention
It is rapid to complete, i.e., once complete callus induction and the differentiation of adventitious bud and propagation is taken root seedling, simplify conventional plant blade
The step of regenerating system, mint plants Tissue Culture Regeneration System is optimized, certain research base is established for peppermint genetic transformation
Plinth.
Brief description of the drawings
The present invention provides the following drawings and illustrated：
Fig. 1 is inoculated and cultured 20d rear blades；
The adventitious bud that Fig. 2 breaks up after being leaf culture 60d；
Fig. 3 is the adventitious bud of single explant differentiation；
Fig. 4 is the adventitious bud of propagation；
Fig. 5 is adventitious bud rooting；
Fig. 6 is the regeneration plant after transplanting 60d.
Embodiment
The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings：
25~30d of leaf age that test material in the embodiment of the present invention preserves for Anhui Science and Technology College's gardening tissue culture room
Peppermint test tube seedling.Condition of culture in tests below：Cultivate room temperature (25 ± 1) DEG C, 1800~2000lx of intensity of illumination, illumination
Time 12h/d.
1) Callus of Leaf induction and the screening of differential medium：In superclean bench, blade is placed on sterile filter
Blade edge and tip portion are removed with scalpel on paper, is cut into about 0.5cm × 0.5cm sizes access adventitious bud induction culture base
In.Adventitious bud induction culture base used in experiment using MS add 0.2mg/L NAA as minimal medium, addition various concentrations TDZ and
6-BA (each combination is shown in Table 1), sucrose 30g/L, agar 5.5g/L, pH value are 5.8~6.0, in 121 DEG C of sterilizings of high-pressure sterilizing pot
20min.Every group of 7 bottles of repetition, every bottle connects 6 blades.As shown in Figure 1.
Result of the test is shown in Table 1, the results showed that, hormone kind has considerable influence to peppermint blade adventitious bud induction frequency, culture
There is callus to be formed when adding TDZ, 6-BA and NAA in base respectively, but the combination for adding 6-BA does not induce adventitious bud, and after
Phase blade browning is serious.Addition TDZ concentration has adventitious bud generation between 0.6~1.6mg/L, and adventitious bud upgrowth situation is as schemed
Shown in 2, Fig. 3 is the adventitious bud of single explant differentiation, but when TDZ concentration reaches 1.6mg/L, adventitious bud vitrifying is more serious,
And inductivity drops to 10% again, therefore, consolidated statement 1 is understood, be adapted to the culture medium of blade adventitious bud inducing for MS+TDZ1.2~
1.4mg/L+NAA0.2mg/L combination.
The various concentrations plant growth regulator of table 1 combines the influence to peppermint blade adventitious bud inducing
2) adventitious bud proliferation：Peppermint adventitious bud is transferred in proliferated culture medium and carries out Multiplying culture.Experiment propagation used
Culture medium has 3 kinds, i.e. B1：MS+6-BA0.5mg/L+IAA0.1mg/L；B2：MS+6-BA1.0mg/L+IAA0.1mg/L；B3：MS
+6-BA2.0mg/L+IAA0.1mg/L；30d after inoculation, as shown in figure 4, as can be seen from Table 2, processing B1 adventitious bud proliferations times
Though number is low with processing B3 compared with processing B2, test tube seedling robust growth, and handles B2 and B3 test tube seedlings and vitrification phenomenon, seedling occur
It is weaker, it is unfavorable for later stage culture of rootage, therefore, the culture medium for being adapted to peppermint blade adventitious bud proliferation is MS+6-BA0.5mg/L+
IAA0.1mg/L is combined.
The selection of the adventitious bud proliferation culture medium of table 2
3) adventitious bud rooting：Adventitious bud after propagation is inoculated into following three culture mediums and carries out culture of rootage.Experiment
Root media used has 3 kinds, i.e. C1：MS+IBA0.2mg/L+ sucrose 20g/L+ agar 5.5g/L；C2：MS+IBA0.5mg/
L+ sucrose 20g/L+ agar 5.5g/L；C3：MS+IBA1.0mg/L+ sucrose 20g/L+ agar 5.5g/L；30d is cultivated, can by table 3
Know, processing C1 is that MS+IBA0.2mg/L+ sucrose 20g/L+ agar 5.5g/L rooting efficiencies are best, and root of hair is early, and number of averagely taking root reaches
To 12.12, average root length reaches 4.52cm, and rooting rate 97.62%, test tube seedling growing way is good, as shown in Figure 5.Therefore, it is adapted to thin
The culture medium of lotus-leaf plate adventitious bud rooting combines for MS+IBA0.2mg/L+ sucrose 20g/L+ agar 5.5g/L.
The selection of the root media of table 3
4) regeneration plant hardening and transplanting：After 15~20d being grown in root media, during root long about 1~2cm, hardening 2
Root agar is cleaned after~3d and is transplanted to volume ratio as 7:Cultivated in the Nutrition Soil of 3 rural area soil-organic fertilizer, count survival rate
To 98%, plant strain growth is normal, and potted plant rear 60d regrowth is as shown in Figure 6.
Claims (3)
1. a kind of method of peppermint blade adventitious bud inducing and plant regeneration, it is characterised in that：Comprise the following steps：
(1) callus induction and differentiation：In superclean bench, by the test tube seedling leaf of squamous subculture, be cut into about 0.5cm ×
Cultivated in the blade access callus induction and differentiation culture medium of 0.5cm sizes, culture medium is using MS culture mediums as base
Basal culture medium, addition TDZ1.2~1.4mg/L, NAA0.2mg/L, sucrose 30g/L and agar 5.5g/L；
(2) adventitious bud proliferation：The adventitious bud that peppermint blade edge is grown is transferred and bred in adventitious bud proliferation culture medium；
Culture medium is using MS culture mediums as minimal medium, addition 6-BA0.5mg/L, IAA0.1mg/L, sucrose 30g/L and agar
5.5g/L；
(3) adventitious bud rooting：The adventitious bud seedling of propagation is inoculated into culture of rootage in root media；Adventitious bud rooting culture
Base is addition IBA0.2mg/L, sucrose 20g/L and agar 5.5g/L using MS culture mediums as minimal medium；
(4) test tube seedling hardening and transplanting：When seedling base portion grows adventitious root, when length is up to 1~2cm, root is cleaned after 2~3d of hardening
It is 7 that agar, which is transplanted to rural area soil with organic fertilizer volume ratio,:Cultivated in 3 Nutrition Soil.
2. the method for peppermint blade adventitious bud inducing as claimed in claim 1 and plant regeneration, wherein step (1)~step
(4) in, each Medium's PH Value is 5.8~6.0.
3. the method for peppermint blade adventitious bud inducing as claimed in claim 1 and plant regeneration, wherein step (1)~step
(4) in, room temperature (25 ± 1) DEG C is cultivated, is cultivated under conditions of 1800~2000lx of intensity of illumination, light application time 12h/d.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610182279.9A CN105794639B (en) | 2016-03-28 | 2016-03-28 | A kind of method of peppermint blade adventitious bud inducing and plant regeneration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610182279.9A CN105794639B (en) | 2016-03-28 | 2016-03-28 | A kind of method of peppermint blade adventitious bud inducing and plant regeneration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105794639A CN105794639A (en) | 2016-07-27 |
| CN105794639B true CN105794639B (en) | 2017-12-15 |
Family
ID=56453987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610182279.9A Active CN105794639B (en) | 2016-03-28 | 2016-03-28 | A kind of method of peppermint blade adventitious bud inducing and plant regeneration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105794639B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106561454B (en) * | 2016-10-25 | 2018-10-02 | 王克彬 | A kind of method of toad grass stock breeding |
| CN107197673A (en) * | 2017-06-19 | 2017-09-26 | 贵州山乡原生态农业开发有限公司 | The cultural method and nutrient solution of a kind of peppermint |
| CN114027188B (en) * | 2021-11-02 | 2023-04-07 | 江苏省中国科学院植物研究所 | Culture medium for obtaining iris interspecific hybridization progeny by utilizing immature embryos and application of culture medium |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102783419A (en) * | 2012-08-23 | 2012-11-21 | 江苏省中国科学院植物研究所 | Efficient regeneration system method for mint internode stems |
-
2016
- 2016-03-28 CN CN201610182279.9A patent/CN105794639B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105794639A (en) | 2016-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101507415B (en) | In-vitro culture method of antlerpilose grass | |
| CN107094625B (en) | Tissue culture seedling breeding method for taxus mairei | |
| CN106718934B (en) | A kind of Rhizoma Atractylodis Macrocephalae regenerating system directly breaking up adventitious bud using plumular axis and radicle | |
| CN105794639B (en) | A kind of method of peppermint blade adventitious bud inducing and plant regeneration | |
| CN105010140A (en) | Culture media for promoting induction and rooting of cluster buds of dendrobium candidum and culture method by using rare earth elements | |
| CN102907318A (en) | Rapid propagation of notoginseng regenerated plant by using bioreactor to cultivate notoginseng somatic embryos | |
| CN114946657B (en) | Hispid fig tissue culture method | |
| CN112273232A (en) | Curcuma wenyujin detoxification seedling culture method | |
| KR100973839B1 (en) | Method for mass propagation of callus and method for mass division of Iris odaesanensis flower utilizing leaf segments of Iris odaesanensis | |
| CN106538382B (en) | Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants | |
| CN101953300B (en) | Tissue culture method for Curcuma wenyujin No.1 | |
| CN105475129A (en) | Tissue-culture rapid propagation method for arundina graminifolia | |
| Kaur et al. | Optimization of potting mixture for hardening of in vitro raised plants of Tylophora indica to ensure high survival percentage | |
| CN103070078A (en) | Rapid propagation method for performing tissue culture by using taro stem tip | |
| CN106973796A (en) | A kind of tissue cultivating and seedling method of Idesia polycarpa | |
| CN105746352A (en) | Tissue culture method of succulent Haworthia emelyae v.comptoniana 'KYODAI AKASEN' HO1 | |
| CN111869569B (en) | Culture system for in vitro culture of hedychium japonicum flowers and application thereof | |
| CN107278562A (en) | A kind of cuttage and seedling culture method of tea tree | |
| CN108142284A (en) | A kind of tissue culture and rapid propagation method of five leaflets maple | |
| CN103299902B (en) | Method for carrying out tissue culture on chinaberry seedlings | |
| CN102090336B (en) | Propagation method for Asarum forbesiiMaxim tissue culture | |
| CN112616675B (en) | Tissue culture and rapid propagation method for Zingiber dance | |
| CN105815214B (en) | A kind of blade seedling rapid propagation method of Ornithogalum caudatum | |
| CN101595845B (en) | Method for embryo culture in vitro and plant regeneration of euscaphis konishii hayata | |
| CN101120654B (en) | Tissue culturing method for chia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Shu Yingjie Inventor after: Zhou Yuli Inventor after: Hu Nengbing Inventor after: Zhang Xueping Inventor after: Sui Yihu Inventor after: Wang Hong Inventor before: Zhou Yuli Inventor before: Shu Yingjie Inventor before: Hu Nengbing Inventor before: Zhang Xueping Inventor before: Sui Yihu Inventor before: Wang Hong |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230110 Address after: 233100 Intersection of Renai Road and Shejitan Road, Fucheng Town, Fengyang County, Chuzhou City, Anhui Province Patentee after: Fengyang County Economic Development Investment Co.,Ltd. Address before: 5404, West Area, Anhui University of Science and Technology, No. 9, Donghua Road, Fengyang County, Chuzhou City, Anhui Province, 233100 Patentee before: ANHUI SCIENCE AND TECHNOLOGY University |