CN106538382A - A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe - Google Patents
A kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe Download PDFInfo
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- CN106538382A CN106538382A CN201610926203.2A CN201610926203A CN106538382A CN 106538382 A CN106538382 A CN 106538382A CN 201610926203 A CN201610926203 A CN 201610926203A CN 106538382 A CN106538382 A CN 106538382A
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- explant
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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
Abstract
The present invention relates to a kind of method for setting up eremochloa ophiuroides high-efficiency regeneration system as explant with young fringe, methods described includes selection and sterilization, the induction of calluss and propagation, the differentiation of calluss, the root induction of explant.Not only explant sterilization is simple for the method that the present invention is provided, Callus induction rate is high, easy to implement, and the cycle is short of the method wound healing induction, the Differentiation ration of adventitious buds height of gained calluss, rooting of vitro seedling are easy, it is adaptable to the modern biotechnology operation such as eremochloa ophiuroides germplasm innovation and gene genetic conversion.
Description
Technical field
The present invention relates to a kind of method that eremochloa ophiuroides isolated immature fringe approach sets up its high-efficiency regeneration system, belongs to plant biological
Technical field of research.
Background technology
Eremochloa ophiuroidesEremochloa ophiuroides(Munro.) Hack. ］ it is that grass family millet subfamily Eremochloa is more
Year life C4 herbaceous plant, and the excellent warm-season turf plant that uniquely can be used as turfgrass in the category.Lack except having in Southeast Asia
Amount distribution is outer, and eremochloa ophiuroides is distributed mainly on China Yangtze river basin and areas to the south, and China middle and south is its natural distributed center, is
That what is generally acknowledged both at home and abroad originates from the best turfgrass of China, therefore is assigned " Chinese turfgrass "（Chinese lawn grass）
Good reputation（Hanna et al. 1995）.Compared with other turfgrasss, the grass is few with barren-resistant, pest and disease damage and maintenance level is low
And it is famous, therefore and have " lazyboot's grass "（Lazybones grass）Title.Eremochloa ophiuroides can be widely used in garden lawn, have a rest
Lawn and the planting of slope protecting grassplot, are particularly suitable for water and soil conservation and large area landscape construction（Hanson et al. 1969；
Hanna 1995）, it is one of three big warm season turf of the world.But compared with other turfgrasss, eremochloa ophiuroides is shown to salt
It is sensitive, can not resist cold, drought resistance it is poor, these inborn defects are always and restrict its wide variety of bottleneck；Further, since eremochloa ophiuroides
Area of origin is single and distribution is relatively small, its hereditary basiies relatively narrower.Therefore, carry out eremochloa ophiuroides germ plasm resource innovation and
Genetic improvement is the fundamental way for overcoming its inherent shortcoming, and the premise and key of the effectively utilizes resources.
Germplasm innovation and agriculture bacillus mediated gene genetic transformation technology based on somatic mutation technology is on turfgrass
Application day contain.But low regeneration efficiency constrains its process in eremochloa ophiuroides breeding.Therefore explore it is high-frequency again
Raw approach seems of crucial importance.The selection of explant is the first link for setting up regenerating system.Although any tissue of plant
Can serve as in theory setting up the explant of regenerative system with organ, or even as the explant of gene transformation, but these
The dedifferentiation of explant and again differentiation capability, the potential trend of cellular omnipotency and competence degree have very big difference, even
The different parts of same histoorgan also have notable difference.Additionally, the optimal regeneration culture medium of searching is also most important, and hormone
Species, concentration and proportioning critical effect is played to the Growth and Differentiation of explant.
So far, about eremochloa ophiuroides tissue culture and regenerating system is studied for some time, but progress
And less.Zhanjiang Normal University and Agricultural University Of Hunan scholar 2004 with mature seed as explant, by 30 days or so
Inducing culture obtains Lax callus, by the successive transfer culture of continuous 60 days, obtains the fine and close tubercular embryo of yellow
Wound healing.With eremochloa ophiuroides mature seed as explant, Jing 2-3 week inductions and culture were it was observed that soft in 2008 for Agricultural University Of South China scholar
The appearance of soft aqueouss shape calluss, then a certain amount of embryo callus are just obtained through the continuation culture of 5 time-of-weeks,
The whole callus induction cycle is all up to 7-8.Institute of Botany discloses in June, 2008 " false thrifty
Careless the induction of the lateral bud calluss and plant regeneration method " patent, adopts the stem section with lateral bud to be explant material after 4 weeks
（28 days）Calluss are induced, so the calluss are relatively low in the plantlet differentiation rate in later stage.These prior arts are made a general survey of, is led to
Seed or the induction of the lateral bud calluss being crossed, then plant being divided into by calluss, whole process lasts longer, most needs 3
More than month, required time, man power and material are more, and Callus induction rate and its value-added coefficient are low.Therefore, the present invention passes through explant
The selection of body and hormone, it is intended to set up eremochloa ophiuroides fast and efficiently regenerating system, so as to the establishment for somatocyte allosome-changing and turn
Enter excellent gene and provide possible, to realizing the innovation and its drought resisting of eremochloa ophiuroides germ plasm resource, resisting the improvement of cold and salt tolerance
Deng.
The content of the invention
Technical problem
Technical assignment proposed by the present invention and the technical problem for solving are to overcome wound healing in existing eremochloa ophiuroides Techniques of in Vitro Culture
The induction duration length of tissue, induced efficiency are low, induce obtained calluss differentiation capability poor, are less suitable for advising for micropropagation
The defects such as modelling, a large amount of screenings of somatic mutants and agriculture bacillus mediated High-efficient Genetic Transformation.The present invention is begun with eremochloa ophiuroides
The young fringe at florescence is explant material, just can obtain graininess calluss in a short time by the regulation and control of exogenous hormone, it is established that
Suitable for the highly efficient regeneration technical system of scale micropropagation, germplasm innovation and gene genetic conversion.
Technical scheme
Invention broadly provides a kind of method for setting up eremochloa ophiuroides high-efficiency regeneration system as explant with young fringe, there is provided method
Not only explant sterilization is simple, Callus induction rate is high, easy to implement, and the cycle is short of the method wound healing induction, gained wound healing
The Differentiation ration of adventitious buds of tissue is high, rooting of vitro seedling is easy.Its technical scheme is as follows：
1）Material
The fresh and tender young fringe of sheath is not extracted in the collection in the eremochloa ophiuroides early flowering season out（Inflorescence）For explant material；
2）Culture medium
Minimal medium is the MS culture medium being made up of a great number of elements, trace element and vitamin, 30.0 g/L of additional saccharose, fine jade
8.0 g/L of fat；
Callus inducing medium MS1：Add 2,4-D, 0.1 mg/ of 2.0-4.0 mg/L in MS minimal mediums respectively
The BAP of L；
Subculture medium MS2：Add 2,4-D, the BAP of 0.1 mg/L of 1.5 mg/L in minimal medium；
Green plant regeneration culture medium MS3：Add KT, the NAA of 0.1 mg/L of 2.0 mg/L in minimal medium；
Rooting of vitro seedling culture medium MS4：Add the NAA of 0.6 mg/L, the KT of 0.2mg/L in minimal medium；
All culture medium use potassium hydroxide and hydrochloric acid to adjust pH value to 5.8 before autoclaving；
3）Operational approach
Explant process：The selection eremochloa ophiuroides early flowering season does not extract fresh and tender, the healthy and strong young fringe of sheath out（Inflorescence）, first peel off outer layer leaf
Sheath, with the ethanol of 70-75% immersion 35-45 s on superclean bench, with aseptic water washing 2-3 time, then with mass concentration be
10% H2O2Sterilization 8-10min, afterwards with aseptic water washing 3-5 time, is finally placed in drained on aseptic filter paper；
Callus induction：The young fringe of Jing surface sterilizations first scratches bract with aseptic knife, grips out young fringe with sterilizing tweezers afterwards, cuts
It is inoculated in callus inducing medium MS1 into after the segment of 0.5-1.0 cm length, carries out wound healing inducing culture, 1 week or so
Just there are calluss；
The successive transfer culture of calluss：During wound healing inducing culture the 3rd week, therefrom select yellow, closely, graininess calluss,
And be transferred in calluss subculture medium MS2, carry out proliferation and subculture culture, and in culture carry out in 3-4 weeks second after
Culture；
Green plant regeneration and rooting of vitro seedling：MS3 in green plant regeneration culture medium is transferred to through the calluss of continuous 2 subcultures
In, differentiation and the test tube seedling grown cultures of adventitious bud are carried out, a subculture was changed per 2 weeks.After 4 weeks, regeneration plant goes to test tube
In seedling rooting culture medium MS4, root culture is carried out 2 weeks；
Seedling exercising and transplanting：The regeneration plant taken root when root is extended to 2.0 cm, ratoon growth to 5.0-6.0 cm, opening group
Seedlings cultivating bottle cap, carries out hardening treatment.After the completion of seedling exercising, regrowth is taken out, root culture medium is washed away, is transplanted in seedling medium,
After being placed in hot-house culture 2 weeks, Transplantation of Regenerated Plantlets is to greenhouse basin alms bowl or field；
During whole isolated culture, at 25 ± 1 DEG C, callus induction and proliferation and subculture culture are equal for culturing room's temperature control
Carry out under dark or scattered light, green plant regeneration, growth and rooting of vitro seedling culture are at 12h illumination is replaced with 12h dark
Reason, is carried out under 3000 Lux illumination conditions.
Beneficial effect：
The present invention is had the advantage that and good effect compared with prior art：
1. the present invention is using early flowering season children's fringe（Inflorescence）For explant, sterilizing operation is simple, easy to implement, and without using mercuric chloride
Deng toxic reagent, safety and environmental protection；
2. the method that the present invention is provided is greatly improved Callus induction rate, and short the time required to producing calluss, thrifty with existing vacation
Careless callus induction method is compared, and induced efficiency is obviously improved；
3. it is in yellow, tight, graininess to induce obtained calluss most of using the method for offer of the invention, and which breaks up
Rate is high, and is easy to take root, therefore obtains embryo callus and set up the efficiency of regenerating system and be greatly improved；
The present invention is suitable for the modern biotechnology operation such as eremochloa ophiuroides germplasm innovation and gene genetic conversion, while the invention is also fitted
Foundation and the follow-up theory about aspects such as germplasm innovation and character improvements for other turfgrass regenerating systems is ground with application
Study carefully.
Description of the drawings
Fig. 1 does not extract the fresh and tender young fringe of sheath out；
The young fringe explant of Fig. 2 just inoculations；
The calluss that Fig. 3 is initially induced；
The calluss of Fig. 4 successive transfer culture；
The test tube seedling of Fig. 5 wound healing differentiation；
Fig. 6 test tube seedlings are taken root.
Specific embodiment
（1）Experiment material
Take the fresh and tender young fringe that the eremochloa ophiuroides introduces a collection E092-1 early flowering season do not extract sheath out（Inflorescence）For explant material（Such as Fig. 1 institutes
Show）；
（2）Culture medium
Minimal medium is the MS culture medium being made up of a great number of elements, trace element and vitamin, and adds sucrose（Sigma）
30.0 g/L, agar（Japan）8.0 g/L；
Callus inducing medium MS1：Add the 2,4-D of 2.0 mg/L in MS minimal mediums（Sigma D-7299）Or
The 2,4-D of 4.0 mg/L（Sigma D-7299）Or 6.0 mg/L 2,4-D（Sigma D-7299）, 0.1 mg/L BAP
（Sigma B3408）；
Subculture medium MS2：Add the 2,4-D of 1.5 mg/L in minimal medium（Sigma D-7299）, 0.1 mg/L
BAP（Sigma B3408）；
Green plant regeneration culture medium MS3：Add the KT of 2.0 mg/L in minimal medium（Sigma K-3378）, 0.1 mg/L
NAA（Sigma N0640）, or the BAP of 2.0 mg/L（Sigma B3408）, 0.1 mg/L NAA（Sigma N0640）, or
The CPPU of 2.0 mg/L（Sigma C2791）, 0.1 mg/L NAA（Sigma N0640）；
Rooting of vitro seedling culture medium MS4：Add the NAA of 0.6 mg/L in minimal medium（Sigma N0640）、0.2 mg/
The KT of L；
All culture medium use potassium hydroxide and hydrochloric acid to adjust pH value to 5.8 before autoclaving；
（3）Operational approach
Explant process：The selection eremochloa ophiuroides E092-1 early flowering seasons do not extract fresh and tender, the healthy and strong young fringe of sheath out（Inflorescence）, first peel off outer
Layer sheath, with the ethanol of 70-75% immersion 35-45 s on superclean bench, with aseptic water washing 2-3 time, then with concentration be
10% H2O2Sterilization 8-10min, afterwards with aseptic water washing 3-5 time, is finally placed in drained on aseptic filter paper；
Callus induction：The young fringe of Jing surface sterilizations first scratches bract with aseptic knife, grips out young fringe with sterilizing tweezers afterwards, cuts
It is inoculated on the callus inducing medium MS1 containing hormon into after the segment of 0.5-1.0 cm length（As shown in Figure 2）,
It is placed in 25 ± 1 DEG C, cultivates under 12 h scattered lights/12h dark conditions, calluss just occurs in the 7th d（As shown in Figure 3）.Culture
After 21d, the inductivity of calluss is counted（As shown in table 1）.As can be seen from Table 1, can lure under different hormone concentrations
Lead calli induction, but inductivity under different gradients（Healing rate）Differ greatly.Wherein when 2,4-D concentration is 4.0
mg/L、BAP（6- benzyl purines）When concentration is 0.1 mg/L, the inductivity highest of calluss reaches 76.7 %.Therefore with
+ 8.0 g/L agar of+30.0 g/L sucrose of+4.0 mg/L 2 of MS ,+0.1 mg/L BAP of 4-D, PH=5.8 are for more
The optimal medium of injured tissue induction；
The induction of 1 different hormone combinations of table and concentrations on callus
The successive transfer culture of calluss：During wound healing 21 d of inducing culture, yellow, tight, graininess calluss are therefrom selected,
And be transferred in calluss subculture medium MS2, carry out proliferation and subculture culture（As shown in Figure 4）, culture period culture room temperature
It is that 12 h scattered light/12 h are dark to spend for 25 ± 1 DEG C, illumination condition, and when 21 d is cultivated carries out second subculture training
Support；
Green plant regeneration：Green plant regeneration culture medium MS3 containing hormon is transferred to through the calluss of continuous 2 subcultures
In, carry out differentiation and the test tube seedling grown cultures of adventitious bud, in incubation culturing room's temperature control at 25 ± 1 DEG C, illumination
Using 12 h illumination and 12 h dark alternate treatment, carry out under the conditions of 3000 Lux of light intensity, and change when 14 d and once train
Foster base（As shown in table 2）.Table 2 shows that eremochloa ophiuroides calluss can induce out indefinite on the division culture medium of hormon
Bud, but under hormon proportioning, Differentiation ration of adventitious buds and test tube seedling growth present significant difference.Swashing from KT+NAA
Most preferably, i.e., all calluss are successfully induced and differentiate adventitious bud for element combination, Induce aerosor and test tube seedling growth, and the
During 14 d, test tube seedling has grown and has extended to 2.0 cm（As shown in Figure 5）.Therefore the optimal medium of green plant regeneration is MS+2.0
+ 8.0 g/L agar of+30.0 g/L sucrose of+0.1 mg/L NAA of mg/L KT, PH=5.8；
Induction of 2 different hormone combinations of table to calluss adventitious bud is broken up
Rooting of vitro seedling：The regeneration plant in above-mentioned optimal green plant regeneration culture medium is gone to into rooting of vitro seedling culture medium after 4 weeks
On MS4, and the condition of culture consistent with green plant regeneration is kept, carry out 14 d of root culture（As shown in Figure 6）；
Seedling exercising and transplanting：The regeneration plant taken root when root is extended to 2.0 cm, ratoon growth to 5.0-6.0 cm, opening group
Seedlings cultivating bottle cap, carries out hardening treatment.After the completion of seedling exercising, regrowth is taken out, root culture medium is washed away, is transplanted in seedling medium,
After being placed in 14 d of hot-house culture, test tube plantlet is transplanted to greenhouse basin alms bowl.
To those skilled in the art, technical scheme that can be as described above and design concept, other various phases are made
The change answered or deformation, and it is all these it is corresponding change and deformation should all belong to the claims in the present invention protection domain it
It is interior.
Claims (7)
1. a kind of method that eremochloa ophiuroides high-efficiency regeneration system is set up as explant with young fringe, it is characterised in that comprise the following steps：
（1）The selection eremochloa ophiuroides early flowering season does not extract the fresh and tender young fringe of sheath out（Inflorescence）, peel off after outermost layer sheath in ultra-clean work
Disinfection in platform, then the bract outside young fringe is peelled off, obtain aseptic young fringe；
（2）Aseptic young fringe explant is inoculated on callus inducing medium MS1, inducing culture is carried out and is obtained wound healing group
Knit；
（3）Calluss are peeled off from explant, is forwarded on subculture medium MS2, is carried out proliferation and subculture culture；
（4）Calluss after successive transfer culture are transferred in green plant regeneration culture medium MS3 and are cultivated, the differentiation for carrying out adventitious bud is lured
Lead, obtain test tube seedling；
（5）The test tube seedling of differentiation is transferred on root media MS4, is cultivated to taking root；
（6）The regeneration plant taken root is carried out into hardening treatment；
（7）After seedling exercising terminates, regrowth is taken out, root culture medium is washed away, is transplanted in seedling medium, be placed in hot-house culture.
2. a kind of method that eremochloa ophiuroides highly efficient regeneration body is set up as explant with young fringe according to claim 1, its feature
It is：Step（1）In the method for young fringe disinfection to not going out sheath be, first with the ethanol that volume content is 70-75%
Bract surface sterilization 35-45 s to the young fringe of parcel, with aseptic water washing 2-3 time, then are 10% H with concentration2O2Sterilization 8-
10min, afterwards with aseptic water washing 3-5 time, is finally placed in drained on aseptic filter paper.
3. a kind of method that eremochloa ophiuroides highly efficient regeneration body is set up as explant with young fringe according to claim 1, its feature
It is：Step（2）In explant used be the aseptic young fringe cut-out that 0.5-1.0 cm length is cut into sterile scissors；Calluss are lured
Leading culture medium MS1 includes：MS culture medium, the 2,4-D of 2-4 mg/L, the BAP of 0.1 mg/L, the sucrose of 30.0 g/L, 8.0
The agar of g/L, culture medium adjust pH value before autoclaving to 5.8；Explant is induced in callus inducing medium MS1
Incubation time is that 2-3 is all, and during isolated culture, culture room temperature is maintained at 25 ± 1 DEG C, enters under complete darkness or scattered light
OK.
4. a kind of method that eremochloa ophiuroides highly efficient regeneration body is set up as explant with young fringe according to claim 1, its feature
It is：Step（3）Middle subculture medium MS2 includes：Add in MS culture medium the 2,4-D of 1.5 mg/L, the BAP of 0.1 mg/L,
The agar of the sucrose of 30.0 g/L, 8.0 g/L, culture medium PH are 5.8；The enrichment culture time is that 3-4 is all, continuous subculture 2 times；After
Carry out under the conditions of 25 ± 1 DEG C of room temperature of culture, dark or scattered light for enrichment culture.
5. a kind of method that eremochloa ophiuroides highly efficient regeneration body is set up as explant with young fringe according to claim 1, its feature
It is：Step（4）Middle calluss incubation time in green plant regeneration culture medium MS3 is that 3-4 is all；Green plant regeneration culture medium MS3
Including：Add KT, the NAA of 0.1 mg/L of 2.0 mg/L, and sucrose, the fine jade of 8.0 g/L of 30.0 g/L in MS culture medium
Fat, culture medium PH are 5.8；Induce aerosor breaks up with test tube seedling growth course, and culture room temperature is maintained at 25 ± 1 DEG C, 12h
Illumination and 12h dark alternate treatments, under 3000Lux illumination conditions, change a subculture per 10-14d.
6. a kind of method that eremochloa ophiuroides highly efficient regeneration body is set up as explant with young fringe according to claim 1, its feature
It is：Step（5）Middle root media MS4 includes：Add in MS culture medium the NAA of 0.6 mg/L, the KT of 0.2 mg/L, 30.0
The agar of the sucrose of g/L, 8.0 g/L, culture medium PH are 5.8, completely the same during the condition of culture of culturing room and green plant regeneration,
It is 2 weeks to cultivate to the time taken root.
7. a kind of abductive approach for setting up eremochloa ophiuroides highly efficient regeneration body with young fringe as explant according to claim 1, its
It is characterised by：Step（6）In plant root length to be regenerated to about 2 cm, regrowth when growing to about 5-6 cm, open tissue cultured seedling bottle cap,
Carry out hardening treatment.
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| CN107996400A (en) * | 2017-11-30 | 2018-05-08 | 广西壮族自治区中国科学院广西植物研究所 | A kind of method that its adventitious bud is induced using sage inflorescence as explant |
| CN108849528A (en) * | 2018-08-24 | 2018-11-23 | 江苏省中国科学院植物研究所 | A method of obtaining eremochloa ophiuroides mutant |
| CN111826381A (en) * | 2020-04-13 | 2020-10-27 | 江苏省中国科学院植物研究所 | Centipede grass root promoting gene EoSINAT5, plant expression vector and application thereof |
| CN117327712A (en) * | 2023-10-24 | 2024-01-02 | 江苏省中国科学院植物研究所 | Method for regulating and controlling root growth of eremochloa ophiuroides through transferring EoBBR gene |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107996400A (en) * | 2017-11-30 | 2018-05-08 | 广西壮族自治区中国科学院广西植物研究所 | A kind of method that its adventitious bud is induced using sage inflorescence as explant |
| CN107996400B (en) * | 2017-11-30 | 2021-04-16 | 广西壮族自治区中国科学院广西植物研究所 | Method for inducing adventitious buds of salvia miltiorrhiza by taking anthurium andraeanum inflorescence as explant |
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| CN111826381A (en) * | 2020-04-13 | 2020-10-27 | 江苏省中国科学院植物研究所 | Centipede grass root promoting gene EoSINAT5, plant expression vector and application thereof |
| CN111826381B (en) * | 2020-04-13 | 2021-09-17 | 江苏省中国科学院植物研究所 | Centipede grass root promoting gene EoSINAT5, plant expression vector and application thereof |
| CN117327712A (en) * | 2023-10-24 | 2024-01-02 | 江苏省中国科学院植物研究所 | Method for regulating and controlling root growth of eremochloa ophiuroides through transferring EoBBR gene |
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| CN106538382B (en) | 2020-08-14 |
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