CN105794639A - Mint leaf adventitious bud induction and plant regeneration method - Google Patents
Mint leaf adventitious bud induction and plant regeneration method Download PDFInfo
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- CN105794639A CN105794639A CN201610182279.9A CN201610182279A CN105794639A CN 105794639 A CN105794639 A CN 105794639A CN 201610182279 A CN201610182279 A CN 201610182279A CN 105794639 A CN105794639 A CN 105794639A
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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
- A01H4/008—Methods for regeneration to complete plants
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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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Abstract
The invention relates to a mint leaf adventitious bud induction and plant regeneration method, and belongs to the technical field of biologics.With mint sterile tube seedling leaves as the material, according to an established mint leaf in-vitro regeneration system, a culture medium of healing tissue induction and differentiation includes MS, 1.4 mg/L TDZ, 0.2 mg/L NAA, 30 g/L cane sugar and 5.5 g/L agar, a culture medium of adventitious bud proliferation includes MS, 0.5 mg/L 6-BA, 0.1 mg/L IAA, 30 g/L cane sugar and 5.5 g/L agar, and a culture medium of adventitious bud rooting includes MS, 0.2 mg/L IBA, 20 g/L cane sugar and 5.5 g/L agar.The inoculation parts are mint leaves without leaf edges or leaf tips, and a certain research foundation is laid for mint genetic transformation by means of the established mint leaf in-vitro regeneration system.
Description
Technical field
The present invention relates to a kind of method obtaining Herba Menthae blade regeneration plant by Herba Menthae leaf culture, belong to biological technical field.
Background technology
Herba Menthae (Mentha haplocalyx Briq.) is a kind of herbaceos perennial of Labiatae (Labiatae) Mentha (MenthaL.), have another name called water mint, yang invigorating grass, people's Adiantum monochlamys Eaton. etc., Herb has dense salubrious fragrance, its aerial parts can be used as medicine after drying, is the important Chinese herbal medicine of China and spice berry.In terms of medicine, Herba Menthae can be relieved summer heat, stomach of waking up opens spleen, antipruritic, pain relieving, and medical value is high.Menthol in Peppermint essential oil is a kind of important spice, is mainly used in medicated cigarette, cosmetics, toothpaste, chewing gum, fancy soap, refreshment drink and medicine.
Herba Menthae research at present is carried out more, its medical value and the basic research such as research extracted in terms of chemical composition and in terms of measuring Herba Menthae chemical composition content etc. the most ripe, also some relevant reports are had in terms of Plant Biotechnology, but relatively fewer about Herba Menthae blade regenerating system systematic study.Agriculture bacillus mediated leaf disk method genetic transfoumation is one of important method of Genetic Transformation in Higher Plants research, and the regeneration plant viral level that excised leaf is formed after dedifferentiation breaks up again is extremely low the most virus-free.With Herba Menthae sterile test tube seedling leaf as material, by leaf culture evoking adventive bud, establish Herba Menthae excised leaf regenerating system, establish certain Research foundation for Herba Menthae genetic transformation.
Summary of the invention
It is an object of the invention to provide a kind of method obtaining Herba Menthae blade regeneration plant by Herba Menthae leaf culture.Mainly aseptic blade inoculation is carried out isolated regeneration culture in the MS minimal medium add several hormons, the research variable concentrations hormone combinations impact on blade adventitious bud induction frequency, and on this basis, carry out adventitious bud proliferation, taken root and transplant, optimize Herba Menthae regenerating system further.
For reaching the studies above purpose, the present invention provides following technical scheme:
The invention discloses a kind of method obtaining Herba Menthae blade regeneration plant by Herba Menthae leaf culture, the tissue culture medium (TCM) of Herba Menthae leaf regeneration, including callus induction and differentiation culture medium, adventitious bud proliferation culture medium, adventitious bud rooting culture medium.Described adventitious bud induction culture base is with MS culture medium as minimal medium, adds TDZ1.2～1.4mg/L, NAA0.2mg/L, sucrose 30g/L and agar 5.5g/L；Described adventitious bud proliferation culture medium, with MS culture medium as minimal medium, adds 6-BA0.5mg/L, IAA0.1mg/L, sucrose 30g/L and agar 5.5g/L；Described adventitious bud rooting culture medium is to add IBA0.2mg/L, sucrose 20g/L and agar 5.5g/L with MS culture medium for minimal medium.
1) callus induction and differentiation: in superclean bench, by the test tube seedling leaf of successive transfer culture, it is cut into (leaf back contact culture medium) in the blade access callus induction and differentiation culture medium of about 0.5cm × 0.5cm size and carries out adventitious bud induction culture；
2) adventitious bud proliferation: breed in adventitious bud proliferation culture medium that the adventitious bud that Herba Menthae blade edge grows is transferred；
3) adventitious bud rooting: the adventitious bud seedling of propagation is inoculated into root culture in described root media；
4) test tube Seedling seedling exercising and transplanting: when Seedling base portion grows adventitious root, and length reaches 1～2cm, seedling exercising 2～3d afterwash root agar is transplanted in the Nutrition Soil of rural area soil-fertilizer that volume ratio is 7:3 cultivate.
Above-mentioned each Medium's PH Value is 5.8～6.0.
Above-mentioned culturing room temperature (25 ± 1) DEG C, intensity of illumination 1800～2000lx, light application time 12h/d.
Advantages of the present invention and good effect: Herba Menthae blade callus induction and differentiation is merged into a step and completed by the present invention, the most once complete callus induction and the differentiation of adventitious bud and propagation is taken root seedling, simplify the step of conventional plant leaf regeneration system, optimize mint plants Tissue Culture Regeneration System, establish certain Research foundation for Herba Menthae genetic transformation.
Accompanying drawing explanation
The present invention provides the following drawings to illustrate:
Fig. 1 is inoculated and cultured 20d rear blade；
Fig. 2 is the adventitious bud of differentiation after leaf culture 60d；
Fig. 3 is the adventitious bud of single outer implant differentiation；
Fig. 4 is the adventitious bud of propagation；
Fig. 5 is adventitious bud rooting；
Fig. 6 is the regeneration plant after transplanting 60d.
Detailed description of the invention
Below in conjunction with the accompanying drawings the preferred embodiments of the present invention are described:
Test material in the embodiment of the present invention is the Herba Menthae test tube Seedling of the leaf age 25～30d that Anhui Science and Technology College's gardening tissue culture room preserves.Condition of culture in tests below: culturing room's temperature (25 ± 1) DEG C, intensity of illumination 1800～2000lx, light application time 12h/d.
1) Callus of Leaf induction and the screening of division culture medium: in superclean bench, is placed on blade on aseptic filter paper and removes blade edge and tip portion with dissecting knife, is cut into about 0.5cm × 0.5cm size and accesses in adventitious bud induction culture base.Test adventitious bud induction culture base used adds the NAA of 0.2mg/L as minimal medium with MS, add variable concentrations TDZ and 6-BA (each combination is shown in Table 1), sucrose 30g/L, agar 5.5g/L, pH value is 5.8～6.0, in 121 DEG C of sterilizing 20min of high-pressure sterilizing pot.Often group repeats 7 bottles, 6 blades of every bottle graft.As schemed shown in A.
Result of the test is shown in Table 1, result shows, hormone kind has considerable influence to Herba Menthae blade adventitious bud induction frequency, all has wound healing to be formed when adding TDZ, 6-BA and NAA in culture medium respectively, but the combination adding 6-BA does not induce adventitious bud, and anaphase blade brownization is serious.Adding TDZ concentration all has adventitious bud to produce between 0.6～1.6mg/L, adventitious bud upgrowth situation is as shown in panelb, figure C is the adventitious bud of single outer implant differentiation, but when TDZ concentration reaches 1.6mg/L, adventitious bud vitrification is more serious, and inductivity drops to again 10%, therefore, consolidated statement 1 understands, and the culture medium being suitable for blade Induce aerosor is MS+TDZ1.2～1.4mg/L+NAA0.2mg/L combination.
The table 1 variable concentrations plant growth regulator combination impact on Herba Menthae blade Induce aerosor
2) adventitious bud proliferation: Herba Menthae adventitious bud proceeds to carry out in proliferated culture medium enrichment culture.Proliferated culture medium used by test 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 schemed shown in D, as can be seen from Table 2, though it is low compared with processing B2 and processing B3 to process B1 adventitious bud proliferation multiple, but test tube Seedling robust growth, and processes B2 and B3 test tube Seedling vitrification phenomenon occurs, Seedling is more weak, being unfavorable for later stage root culture, therefore, the culture medium being suitable for Herba Menthae blade adventitious bud proliferation is MS+6-BA0.5mg/L+IAA0.1mg/L combination.
The selection of table 2 adventitious bud proliferation culture medium
3) adventitious bud rooting: the adventitious bud after propagation is inoculated in three below culture medium and carries out root culture.Root media used by test 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；Cultivating 30d, as shown in Table 3, process C1 i.e. MS+IBA0.2mg/L+ sucrose 20g/L+ agar 5.5g/L rooting efficiency best, early, number of averagely taking root reaches 12.12 to root of hair, and average root length reaches 4.52cm, rooting rate 97.62%, and test tube Seedling growing way is good, as shown in figure E.Therefore, the culture medium being suitable for Herba Menthae blade adventitious bud rooting is MS+IBA0.2mg/L+ sucrose 20g/L+ agar 5.5g/L combination.
The selection of table 3 root media
4) regeneration plant seedling exercising and transplanting: after growing 15～20d in root media, during root length about 1～2cm, seedling exercising 2～3d afterwash root agar is transplanted in the Nutrition Soil of rural area soil-fertilizer that volume ratio is 7:3 cultivate, statistics survival rate is to 98%, plant strain growth is normal, and the regrowth of potted plant rear 60d is as shown in figure F.
Claims (4)
1. a Herba Menthae blade Induce aerosor and the method for plant regeneration, it is characterised in that: comprise the following steps:
Callus induction and differentiation: culture medium is with MS culture medium as minimal medium, adds TDZ1.2 ~ 1.4
Mg/L, NAA0.2 mg/L, sucrose 30 g/L and agar 5.5 g/L；
Adventitious bud proliferation: culture medium, with MS culture medium as minimal medium, adds 6-BA0.5 mg/L, IAA0.1 mg/L, sucrose 30 g/L and agar 5.5 g/L；
Adventitious bud rooting: described adventitious bud rooting culture medium is to add IBA0.2 with MS culture medium for minimal medium
Mg/L, sucrose 20 g/L and agar 5.5 g/L；
Regeneration plant seedling exercising and transplanting: gained regeneration test tube seedling is transplanted in the Nutrition Soil of rural area soil-fertilizer that volume ratio is 7:3 and cultivates.
2. use the Herba Menthae blade Induce aerosor described in claim 1 and the method for plant regeneration, comprise the following steps:
1) callus induction and differentiation: in superclean bench, by the test tube seedling leaf of successive transfer culture, is cut into (leaf back contact culture medium) in the blade access callus induction and differentiation culture medium of about 0.5 cm × 0.5 cm size and cultivates；
2) adventitious bud proliferation: breed in adventitious bud proliferation culture medium that the adventitious bud that Herba Menthae blade edge grows is transferred；
3) adventitious bud rooting: the adventitious bud seedling of propagation is inoculated into root culture in described root media；
4) test tube Seedling seedling exercising and transplanting: when Seedling base portion grows adventitious root, when length reaches 1 ~ 2 cm, seedling exercising 2 ~ 3 d afterwash root agar is transplanted in the Nutrition Soil of rural area soil-fertilizer that volume ratio is 7:3 cultivate.
3., in step 1) ~ step 4) described in, each Medium's PH Value is 5.8 ~ 6.0.
4. in step 1) ~ step 4) described in, culturing room's temperature (25 ± 1) DEG C, intensity of illumination 1800 ~ 2000 lx, cultivates under conditions of light application time 12 h/d.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106561454A (en) * | 2016-10-25 | 2017-04-19 | 王克彬 | Method for cultivating common sage herb improved variety |
| 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 |
| CN114027188A (en) * | 2021-11-02 | 2022-02-11 | 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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2016
- 2016-03-28 CN CN201610182279.9A patent/CN105794639B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| 于盱: "两个薄荷品种叶片愈伤组织的诱导及增殖", 《植物资源与环境学报》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106561454A (en) * | 2016-10-25 | 2017-04-19 | 王克彬 | Method for cultivating common sage herb improved variety |
| 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 |
| CN114027188A (en) * | 2021-11-02 | 2022-02-11 | 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 |
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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 |
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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 |