CN102630564A - Tissue culture and rapid propagation method of salt-tolerant field mint - Google Patents
Tissue culture and rapid propagation method of salt-tolerant field mint Download PDFInfo
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- CN102630564A CN102630564A CN2012100873920A CN201210087392A CN102630564A CN 102630564 A CN102630564 A CN 102630564A CN 2012100873920 A CN2012100873920 A CN 2012100873920A CN 201210087392 A CN201210087392 A CN 201210087392A CN 102630564 A CN102630564 A CN 102630564A
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- tissue culture
- salt tolerant
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- peppermint
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Abstract
The invention discloses a tissue culture and rapid propagation method of a salt-tolerant field mint and relates to a tissue culture and rapid propagation technology in the field of an agricultural biotechnology. The technical key points are as follows: the method comprises the following steps of: establishing an explant sterile system by utilizing a method for separately sterilizing stem sections in different growth periods; screening a callus induction culture medium; screening an adventitious bud propagation culture medium; screening a root-taking culture medium; screening a salt-tolerant variant; and carrying out acclimatization and transplanting method of tissue culture seedlings. Compared with the prior art, the method adopts the way for separately sterilizing different stem sections to completely disinfect and prevent disinfecting liquid from damaging the explants in the different growth periods; a culture medium formula in each culture phase is optimized, so that the salt-tolerant field mint is convenient for industrial production and has a high propagation coefficient; and the variants can be screened by oriented induction of the tissue culture phase so as to obtain a high-salt-tolerant product.
Description
Technical field
The present invention relates to the technology of the quick breeding by group culture of a kind of salt tolerant peppermint, the invention belongs to agricultural biological technical field.
Background technology
Soil salinization problem is a great problem of puzzlement agricultural production.Current, the irrigated land area is about 230M hm in the world
2, wherein suffer the soil of salt damage influence to account for 45M hm
2The dryland farming area is about 1500M hm
2, wherein suffer the soil of salt damage influence to account for 32M hm
2The saliferous degeneration area of arable soil is with 3 hm
2The speed increment of/min.Estimate according to FAO, in the world wide, annual because salt damage causes the area of productivity of land forfeiture to reach 0.25 ~ 0.50M hm
2Referring to Wang Xiaobin, pay close attention to water resource utilization and climatic variation eclipse effect (Chinese soil and fertilizer, 2009 (2): 80) to the soil salinization.With regard to China, at 100,000,000 hm
26,670,000 hm are arranged in the arable land
2Salinized soil, other has 0.346 hundred million hm
2Saline-alkali wasteland.Jia Lixia, NaC1 coerce influence (Inner Mongol grass cultivation, 2008,20 (1): 40-42) that alfalfa seed is sprouted.Along with the sharp increase and the industrial high speed development of China's population, arable area sharply descends, and simultaneously, unreasonable irrigation has caused the secondary salinization in a large amount of good farmlands again.Therefore, the large-area salination of development and utilization soil utilizes the salt-tolerant plant resource development salt lick ecological agriculture very necessary.
Salt tolerant peppermint involved in the present invention is that the Shandong Province academy of sciences sino-japanese friendship and biotechnology research center goes out through physiology such as leaf water content, osmotic potential, electrical conductivity and MDA content and the CAT enzymic activity reflection index screening that mensuration NaCl coerces the back peppermint, and it is 0.8% that tolerance NaCL coerces concentration.Be American mint (
Mentha piperitaL.) a kind of introduces a fine variety the research department available from Vegetable Research center, Beijing vegetables germ plasm resource and special vegetable.The salt tolerant peppermint is paid close attention to as the medicine reward dual purpose plant of salt tolerant alkali except that the general effect with peppermint gradually.Because American mint is the sterility intermediate form that fish mint and spearmint hybridization form, and is main with division propagation only.(referring to Chen Ying, plant soma somaclonal variation and breeding Nanjing: Jiangsu science tech publishing house, 1991:1-10).The salt tolerant peppermint is adopted vegetative propagation for a long time, is prone to virus disease takes place, and causes the decline of deterioration of variety and output, quality, causes salt tolerant peppermint germ plasm resource deficient.Therefore need a kind of tissue culture and rapid propagation method that is applicable to the salt tolerant peppermint of research and development.
Summary of the invention
The group culturation rapid propagating technology that the purpose of this invention is to provide a kind of salt tolerant peppermint.
The present invention is an explant with salt tolerant peppermint stem section, adopts different puberty stem sections to separate disinfectant method, its cultured in vitro and plant regeneration condition is studied, for the breed improvement of salt tolerant peppermint has been established important basis with breeding fast.
The tissue culture and rapid propagation method of salt tolerant peppermint of the present invention comprises the screening that explant aseptic process, callus of induce are cultivated, indefinite bud rises in value cultivation, culture of rootage, salt tolerant variant, domestication and each step of transplanting of tissue cultivating seedling; Its technical essential comprises: adopt the stem section of different growing to separate the method for sterilizing, set up the explant aseptic strain; The screening of callus of induce medium; The screening of indefinite bud increment medium; The screening of root media; The screening of salt tolerant variant; The domestication of tissue cultivating seedling and transplanting method.
Concrete operations step and process conditions are following:
1. the foundation of explant aseptic strain
From the nursery, get eugonic mint plants, defoliation stays stem and bud point, after flowing water washes 20 min; The filter paper suck dry moisture is pruned explant with operating scissors, intercepting 3cm long shoot section; Then the stem section (stem apex, middle part stem section, base portion stem section) of different growing is separated, subsequent use.On the superclean bench with 75 % alcoholic solutions sterilizations 10s～60s, 0.1 % mercuric chloride solution sterilization, 8～15 min after; With aseptic water washing 4～5 times; With operating scissors explant is pruned again after blotting water; Remove two ends blackout part, then it is seeded in the 1/2MS medium, place illumination box to cultivate.Observe the pollution condition of plant after 3 days, statistics pollution rate (table 1).
Confirm that through test peppermint stem section adopts 75 % alcohol, 30～60s+0.1 % mercuric chloride solution sterilization, 8～10min, can the pollution rate of explant be controlled at below 1%, and thimerosal is harmless to the stem section of different growing.
2. orthogonal experiment L4 (2 is adopted in the screening of callus of induce medium
3) screening.Choose the long healthy and strong aseptic seedling of 3cm, place medium, carry out inducing of callus.After cultivating 30 d, investigation callus of induce rate (table 2, table 3).
Callus of induce rate=stem section callus forms stem section number * 100% of number/inoculation.
Confirm that through test NAA is the key factor of salt tolerant peppermint callus induction ability, its optimum concentration scope 0.1 ~ 0.3 mgL
-1
3. the screening of indefinite bud increment medium.
Get the long healthy and strong aseptic seedling of 3cm, be inoculated in the 2/3MS medium of variable concentrations 6-BA and NAA proportioning, and add 30 gL
-1Sucrose and 4 gL
-1GEL carries out adventitious bud inducing.After cultivating 50d (during switching 1 time), investigation value-added coefficient (table 4).Stem hop count * 100% of indefinite bud value-added coefficient=indefinite bud sum/inoculation.
The proliferated culture medium of confirming the salt tolerant peppermint through test is 2/3MS, 2.0 ~ 6.0 mgL
-16-BA, 0.1 ~ 0.3mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0.
4. the screening of root media
Getting the long stalwartness of 2 cm does not have offspring, is inoculated in the 1/2MS medium of variable concentrations 6-BA and NAA proportioning, and adds 30 gL
-1Sucrose and 4 gL
-1GEL carries out root induction.After cultivating for two weeks, statistics growth increment (plant height), quantity of taking root (radical) and root growth amount (root is long) are carried out comparative analysis (table 5) behind 21 d.
Confirm that through test the culture of rootage based formulas is 1/2MS, 0.1 ~ 0.3mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0.Need not add 6-BA.
5. the screening of salt tolerant variant
Getting the long stalwartness of 2 cm does not have offspring, is inoculated into (weight %) in the root media that adds 0.1%, 0.3%, 0.5%, 0.8%, 1.0% sodium chloride respectively, cultivates three all " Invest, Then Investigate "s situation (table 6) of taking root.
According to salt tolerant mutagenic and breeding result, confirm that the weight of interpolation sodium chloride is 0.5%～0.8% of root media weight.
6. the domestication of tissue cultivating seedling and transplanting
There is the tissue culture bottle of the seedling of taking root in the laboratory, to open bottle cap with long, adds the sodium chloride (adding with 1% solution form usually) of root media weight 0.5～0.8%, refining seedling 2～3 days; Carefully take out with tweezers; Flush away root medium is planted in the dish of the cave of carrying substrates, is positioned over half shady place; Attention is watered, and keeps matrix moistening.After 7 days, connect matrix and be transplanted to together in the various composts, note simultaneously watering three all " Invest, Then Investigate " peppermint survival rates (table 7).
Soil salt content is that 0.5% regional survival rate reaches more than 95% to peppermint tissue cultivating seedling after salt tolerant screening in the Dongying City Kenli County; At Dongying City China industry new material Co., Ltd soil salt content is that 1.0% regional survival rate reaches more than 50%.
Advantage compared with prior art of the present invention is: different stipes are sterilization separately, not only thorough disinfection but also avoid the explant of thimerosal infringement different growing; Make the salt tolerant peppermint be convenient to batch production production, reproduction coefficient is high; Directed mutagenesis through the group training stage can screen variant, obtains high salt tolerant kind.
Embodiment
Following examples are to further specify of the present invention, but the present invention is not limited thereto.
The method that the foundation of embodiment 1. explant aseptic strains adopts the stem section of different growing separately to sterilize
From the nursery, get eugonic mint plants, defoliation stays stem and bud point, after flowing water washes 20 min; The filter paper suck dry moisture is pruned explant with operating scissors, intercepting 3cm long shoot section; Then the stem section (stem apex, middle part stem section, base portion stem section) of different growing is separated, subsequent use.On the superclean bench with 75 % alcoholic solutions sterilizations 10s～60s, 0.1 % mercuric chloride solution sterilization, 8～15 min after; With aseptic water washing 4～5 times; With operating scissors explant is pruned again after blotting water; Remove two ends blackout part, then it is seeded in the 1/2MS medium, place illumination box to cultivate.Observe the pollution condition of plant after 3 days, the statistics pollution rate.
Can know that by table 1 peppermint stem section adopts 75 % alcohol, 30～60s+0.1 % mercuric chloride solution sterilization, 8～10min, can the pollution rate of explant be controlled at below 1%; Can find out from the concrete data of stem apex, middle part stem section, base portion stem section, adopt the stem section of different growing to separate the method for sterilizing, can guarantee that thimerosal is harmless to the stem section of different growing.
Embodiment 2: the screening of group training prescription
2.1 the screening of callus of induce medium (seeing table 2, table 3).
Adopt orthogonal experiment L4 (2
3) screening.Choose the long healthy and strong aseptic seedling of 3cm, place medium, carry out inducing of callus.After cultivating 30 d, investigation callus of induce rate.Callus of induce rate=stem section callus forms stem section number * 100% of number/inoculation.
Can know that by table 3 the orthogonal optimum seeking prescription is A
(1,2)B
1C
2, i.e. 1/2MS or 2/3MS, 1.0 mgL
-16-BA, 0.2 mgL
-1NAA, 30 gL
-1Sucrose and 4 gL
-1GEL.Can be known that by the R value NAA (C) is a key factor, 6-BA (B) also is than key factor, and MS (A) is influence less factor, i.e. influence factor C>B>A.Therefore, confirm that through this test NAA is the key factor of salt tolerant peppermint callus induction ability, its optimum concentration is 0.2 mgL
-1, the callus of induce rate reaches more than 92%.
2.2 the screening (seeing table 4) of indefinite bud increment medium.
Get the long healthy and strong aseptic seedling of 3cm, be inoculated in the 2/3MS medium of variable concentrations 6-BA and NAA proportioning, and add 30 gL
-1Sucrose and 4 gL
-1GEL carries out adventitious bud inducing.After cultivating 50d (during switching 1 time), the investigation value-added coefficient.Stem hop count * 100% of indefinite bud value-added coefficient=indefinite bud sum/inoculation.
Can know by table 4, at NAA (0.2 mgL
-1) concentration is identical, 6-BA concentration is less than 6.0 mgL
-1Situation under, the growth coefficient of indefinite bud increases (prescription B except) with the increase of 6-BA concentration.Data show, prescription G to increase coefficient the highest, reach 19.23, and with prescription A, B, C, D, there is significant difference in E between H.Therefore, confirm that through this test the proliferated culture medium of salt tolerant peppermint is 2/3MS, 5.0 mgL
-16-BA, 0.2 mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0.
2.3 the screening of root media (seeing table 5)
Getting the long stalwartness of 2 cm does not have offspring, is inoculated in the 1/2MS medium of variable concentrations 6-BA and NAA proportioning, and adds 30 gL
-1Sucrose and 4 gL
-1GEL carries out root induction.After cultivating for two weeks, statistics growth increment (plant height), quantity of taking root (radical) and root growth amount (root is long) are carried out comparative analysis behind 21 d.
Can know that by table 5 the salt tolerant peppermint lures the root effect different in the medium of variable concentrations 6-BA and NAA proportioning.Comprehensive growth increment (plant height), quantity of taking root (radical) and root growth amount (root is long) are not added the more favourable salt tolerant peppermint of 6-BA and are taken root, like prescription 1., and 8., significant difference 9. and between other prescription.When 6-BA concentration was 0 mgL-1, luring the root effect was not to increase with NAA concentration.As luring root during 3 weeks, growth increment is to successively decrease with the increase of concentration, and 1., 8., 9. number prescription differences is remarkable; The quantity of taking root is maximum is 9. number prescription; The root growth amount is maximum is 1. number prescription.Consider that root media should be an important indicator with the quantity of taking root, so confirm that 9. number medium is an optimum formula, i.e. 1/2MS, 0.2 mgL-1NAA, 30 gL-, 1 sucrose, 4 gL-, 1 GEL, PH6.0.
Embodiment 3: the screening of salt tolerant variant (seeing table 6)
Getting the long stalwartness of 2 cm does not have offspring, is inoculated in the root media that adds percetage by weight 0.1%, 0.3%, 0.5%, 0.8%, 1.0% sodium chloride respectively three all " Invest, Then Investigate "s situation of taking root.
Can know that by table 6 salinity of in root media, adding below 0.5% does not influence normally taking root of salt tolerant peppermint.But when salinity reaches 0.8% when above, rooting rate drops to 45%, and the quantity of root obviously tails off, and the length of root obviously shortens.
Show that in the salt tolerant mutagenic and breeding process, the preferred scope of application of sodium chloride is 0.5%～0.8%.
Embodiment 4: the domestication of tissue cultivating seedling and transplanting
There is the tissue culture bottle of the seedling of taking root in the laboratory, to open bottle cap with long,, adds the sodium chloride of root media weight 0.8% with 1% solution form; Refining seedling 2～3 days carefully takes out flush away root medium with tweezers; Plant in the dish of the cave of carrying substrates; Be positioned over half shady place, note watering, keep matrix moistening.After 7 days, connect matrix and be transplanted to together in the various composts, note simultaneously watering three all " Invest, Then Investigate " peppermint survival rates.
Use the medium that the present invention screened and cultivate step by step and the tissue cultivating seedling of salt tolerant domestication and the transplanting result contrast of using the tissue cultivating seedling of prior art medium and training method, see table 7.
No matter can be known by table 7, be that tissue cultivating seedling that medium is cultivated step by step and salt tolerant is tamed that the present invention screens and the survival rate of using the tissue cultivating seedling of prior art medium and training method all reduce with the increase of sodium chloride content.When sodium chloride content reached 1.0% (weight %) in compost, the survival rate of the tissue cultivating seedling that prior art obtained in the soil of nursery was 21.76%, and the survival rate in Dongying China industry green wood soil is merely 15.00%; The survival rate of tissue cultivating seedling in the soil of nursery that the medium that the present invention screened is cultivated step by step and the salt tolerant domestication is obtained is 77.78%, and the survival rate in Dongying China industry green wood soil is 52.00%.The tissue cultivating seedling that the present invention obtained is that 1.0% o'clock survival rate is more than 3.5 times of tissue cultivating seedling that prior art obtains at sodium chloride content.
Claims (10)
1. the tissue culture and rapid propagation method of a salt tolerant peppermint; Comprise the screening that explant aseptic process, callus of induce are cultivated, indefinite bud rises in value cultivation, culture of rootage, salt tolerant variant, domestication and each step of transplanting of tissue cultivating seedling; It is characterized in that: adopt the stem section of different growing to separate the method for sterilizing, set up the explant sterile system; The screening of callus of induce medium; The screening of indefinite bud increment medium; The screening of root media; The screening of salt tolerant variant; The domestication of tissue cultivating seedling and transplanting method.
2. separately the method for sterilization is for getting eugonic mint plants for the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1, the stem section that it is characterized in that said different growing, and defoliation stays stem and bud point; After flowing water washes 20 min; The filter paper suck dry moisture is pruned explant with operating scissors, intercepting 3cm long shoot section; Then with the stem section of different growing be stem apex, middle part stem section, base portion stem section separately; Adopt 75 % alcohol, 30～60s+0.1 % mercuric chloride solution sterilization, 8～10min, with aseptic water washing 4～5 times, blot water after pruning subsequent use.
3. the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1 is characterized in that in the said callus of induce medium that NAA is the key factor of salt tolerant peppermint callus induction ability, its optimum concentration scope 0.1 ~ 0.3 mgL
-1
4. the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1 is characterized in that the formula range of said indefinite bud increment medium is following: 2/3MS, 2.0 ~ 6.0 mgL
-16-BA, 0.1 ~ 0.3mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0.
5. the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1 is characterized in that the formula range of said root media is following; 1/2MS, 0.1 ~ 0.3mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0; Need not add 6-BA.
6. the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1 is characterized in that in the screening of said salt tolerant variant, and the weight of adding sodium chloride is 0.5～0.8 % of root media weight.
7. the tissue culture and rapid propagation method of salt tolerant peppermint as claimed in claim 1 is characterized in that the mode of operation of domestication and transplanting method of said tissue cultivating seedling is following: have the tissue culture bottle of the seedling of taking root in the laboratory, to open bottle cap with long, add the sodium chloride of root media weight 0.5～0.8% with 1% solution form; Refining seedling 2～3 days carefully takes out flush away root medium with tweezers; Plant in the dish of the cave of carrying substrates; Be positioned over half shady place, note watering, keep matrix moistening; After 7 days, connect matrix and be transplanted to together in the various composts.
8. like the tissue culture and rapid propagation method of claim 1 or 3 described salt tolerant peppermints, it is characterized in that the prescription of said callus of induce medium is following: 1/2MS or 2/3MS, 1.0 mgL
-16-BA, 0.2 mgL
-1NAA, 30 gL
-1Sucrose and 4 gL
-1GEL.
9. like the tissue culture and rapid propagation method of claim 1 or 4 described salt tolerant peppermints, it is characterized in that the prescription of said indefinite bud increment medium is following: 2/3MS, 5.0 mgL
-16-BA, 0.2 mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0.
10. like the tissue culture and rapid propagation method of claim 1 or 5 described salt tolerant peppermints, it is characterized in that the prescription of said root media is following: i.e. 1/2MS, 0.2 mgL
-1NAA, 30 gL
-1Sucrose, 4 gL
-1GEL, PH6.0; Do not add 6-BA.
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Cited By (4)
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| CN103563743A (en) * | 2012-08-07 | 2014-02-12 | 高山林 | Commercial crop meristem culture virus-free novel technology |
| CN103583360A (en) * | 2013-10-29 | 2014-02-19 | 浙江万里学院 | Method for improving Abelia seedling salt tolerance by oriented induction |
| CN106665089A (en) * | 2017-01-03 | 2017-05-17 | 山东省黄河三角洲可持续发展研究院 | Tissue re-culture replacement interplanting method in mint planting in saline-alkali soil |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563743A (en) * | 2012-08-07 | 2014-02-12 | 高山林 | Commercial crop meristem culture virus-free novel technology |
| CN103583360A (en) * | 2013-10-29 | 2014-02-19 | 浙江万里学院 | Method for improving Abelia seedling salt tolerance by oriented induction |
| CN103583360B (en) * | 2013-10-29 | 2016-04-13 | 浙江万里学院 | A kind of directional induction improves the method for Abelia biflora nursery stock salt resistance |
| CN106665089A (en) * | 2017-01-03 | 2017-05-17 | 山东省黄河三角洲可持续发展研究院 | Tissue re-culture replacement interplanting method in mint planting in saline-alkali soil |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
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Address after: 250000 28789 Licheng Road, Licheng District, Ji'nan, Shandong, 28789 Patentee after: ECOLOGY INSTITUTE OF SHANDONG ACADEMY OF SCIENCES (THE SINO-JAPANESE FRIENDSHIP BIOTECHNOLOGY RESEARCH CENTER, SHANDONG ACADEMY OF SCIENCES) Address before: 250013 No. 19, ASTRI Road, Lixia District, Shandong, Ji'nan Patentee before: Biotechnology Center of Shandong Academy of Sciences |
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