CN106497971A - A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently - Google Patents
A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently Download PDFInfo
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- CN106497971A CN106497971A CN201710025503.8A CN201710025503A CN106497971A CN 106497971 A CN106497971 A CN 106497971A CN 201710025503 A CN201710025503 A CN 201710025503A CN 106497971 A CN106497971 A CN 106497971A
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- C—CHEMISTRY; METALLURGY
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to biological technical field, and in particular to a kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently.The method is using the Agrobacterium rhizogenes after the resuspended activation of Agrobacterium re-suspension liquid；Wound is manufactured at the nearly cotyledon end hypocotyls of the growth aseptic seedlings of Brassica napus L of 2 weeks；Wound is infected with Agrobacterium rhizogenes re-suspension liquid；Continue the seedling after aseptic culture infects, hairy root is produced in wound；After hypocotyls with hairy root are cut, culture obtains aseptic hairy root；The tip of a root of hairy root is cut culture in subculture medium and can obtain a large amount of hairy root.Agrobacterium rhizogenes used in the present invention can be wild type Agrobacterium rhizogenes, or proceed to the Agrobacterium rhizogenes with genes of interest.The hairy root that the induction of wild type Agrobacterium rhizogenes is produced can be used for the production of root physiological Study or secondary metabolites, and the hairy root that the Agrobacterium induction for carrying genes of interest is produced can be used for the functional study of the components such as specific gene or promoter.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of cabbage type rape hairy root induction and training rapidly and efficiently
Foster method.
Background technology
As cabbage type rape trophophase is long, gene genetic conversion is difficult, and the research of its gene function for a long time is subject to
Certain restriction, particularly localization of gene expression, the aspect such as checking for starting sub-feature, reverse geneticses research and some hypothesis
More by being limited.Part research can be quickly carried out in the nearer model plant arabidopsiss of sibship, but not jljl
The research conclusion of inter-species is not very reliable, because the research in model plant generally can not reappear the shadow of cellular environment completely
Ring.There is significant difference (Ron, M., et in expression pattern of the promoter that such as many roots are expressed in Fructus Lycopersici esculenti and arabidopsiss
al.Hairy root transformation using Agrobacterium rhizogenes as a tool for
exploring cell type-specific gene expression and function using tomato as a
model.Plant Physiology.Vol.166,pp.455–469,2014).Therefore, develop one kind easily and fast in Caulis et Folium Brassicae capitatae
The research method for carrying out gene function in type Brassica campestris L is very necessary.Hairy root and plant primary root that Agrobacterium rhizogenes induction is produced
Architectural feature consistent, but it is simple to grow fast, training method, and can make hairy root expression purpose by importing exogenous gene
Albumen, is the good carrier of the quick research of gene function.In addition, hairy root can also be used for some cabbage type rape secondary metabolites
Production source.
Content of the invention
Present invention aim at provide a kind of cabbage type rape hairy root root rapidly and efficiently and producing, cultivating and screening side
Method.
Technical solution of the present invention is comprised the following steps：
1) using the Agrobacterium rhizogenes after the resuspended activation of Agrobacterium re-suspension liquid；
2) wound is manufactured at the nearly cotyledon end hypocotyls of the growth aseptic seedlings of Brassica napus L of 2 weeks；
3) wound is infected with Agrobacterium rhizogenes re-suspension liquid；
4) continue the seedling after aseptic culture infects, hairy root is produced in wound；
5), after cut the hypocotyls with hairy root, cultivating in bacterium culture medium, obtaining aseptic hairy root；
6) tip of a root of hairy root is cut, is cultivated in subculture medium, a large amount of hairy root can be obtained for follow-up test.
It is characterized in that the step 1) in, LB liquid trainings of the Agrobacterium K599 using the corresponding antibiotic containing suitable concn
After supporting to saturation, take the centrifugation of 1ml bacterium solutions and remove supernatant, resuspended with 10ml Agrobacterium re-suspension liquids；
Preferably, the formula of the Agrobacterium re-suspension liquid is：4.4g/L MS powder, 20g/L sucrose, 200 μM of acetyl Flos Caryophyllis
Ketone (AS), 500mg/l polyvinylpyrrolidones, pH 5.8.
Step 2) in, aseptically, with sharp blade or scalpel by the aseptic Brassica Napus Seedling for growing 2 weeks away from son
Hypocotyls at leaf about 1cm scratch a wound for being about 0.5cm, but wound can not penetrate hypocotyls.
Step 3) in, 1～20 μ l Agrobacterium rhizogenes re-suspension liquids are drawn with pipettor under the conditions of aseptic seedling drop in hypocotyls
Wound.
Step 2) and step 4) in condition of culture be photoperiod illumination 16h, dark 8h, 24 DEG C of ambient temperature, intensity of illumination
7000lux.
Generally the research of cabbage type rape gene or promoter is carried out in its homologous type plant Arabidopsis thaliana first, such as
Protein subcellular positioning, the physiological function for identifying albumen by arabidopsiss genetic transformation etc. are detected by arabidopsiss transient expression.
And using cabbage type rape as the material of the genetic transformation usual test period as more than 3 months and cumbersome, workload is big.
The present invention mediates the hairy root for producing as material with Agrobacterium rhizogenes, studies gene function and root physiology, can both ensure to study
In cellular environment, greatly save test period again.
Hairy root induction is generally used for secondary metabolites, and such as in Radix Ginseng, Radix Bupleuri etc., the production of the effective elements of the medicine, is also used for
To physiological function of plants, such as the physiological responses scheduling theory research of heavy metal pollution, root parasite.But at present in Wild cabbage type oil
The hairy root induction research carried out in dish is less, and its hairy root induced efficiency is relatively low, and (in ancestor's autumn dawn etc., Agrobacterium rhizogenes induce
The foundation of Semen sojae atricolor hairy root system. Hua Zhong Agriculture University's journal, 2012 (06):The 699-703 page .).The present invention is directed to this not
Foot, optimizes formula and infection condition that Agrobacterium infects liquid：Infected using live body rather than routine the side infected of explant
Method has prevented adventitious root generation substantially, greatly reduces the workload of screening electropositive radical.Polyvinyl pyrrole is added in liquid is infected
Alkanone effectively improves induced efficiency, makes induced efficiency reach 80% or so, and the hairy root that single plant produces is more.This
Outward, this method is easy to operate, without carrying out loaded down with trivial details explant preparation work, saves a large amount of manpowers and cost.Make in the present invention
Agrobacterium rhizogenes can be wild type Agrobacterium rhizogenes, or proceed to the Agrobacterium rhizogenes with genes of interest.Wild
The hairy root that the induction of type Agrobacterium rhizogenes is produced can be used for the production of root physiological Study or secondary metabolites, and with purposeful base
The hairy root that the Agrobacterium induction of cause is produced can be used for the functional study of the components such as specific gene or promoter.
Description of the drawings
Fig. 1 Agrobacterium rhyzogenesK599s infect the generation of live body cabbage type rape hypocotyls and hairy root：Cabbage type rape without
After vaccine hypocotyls are infected, a plurality of hairy root is produced.Arrow is for cutting out wound, and smears wound using the re-suspension liquid without Agrobacterium
The non-of mouth infects control.
Fig. 2 hairy root isolated culture：The cabbage type rape hypocotyls for bearing hairy root are cut, is trained in screening culture medium
The growth conditions of hairy root after supporting 2 weeks.
The tip of a root of hairy root is carried out result after GUS dyeing by Fig. 3.
Fig. 4 pCAMBIA-1303 binary expression vector collection of illustrative plates.
Specific embodiment
1 Agrobacterium is cultivated
Agrobacterium strain is the K599 root of hairs containing pCAMBIA-1303 binary expression vectors obtained using chemical transformation
Agrobacterium and the wild type K599 strains without binary expression vector.Wild type K599 strains are purchased from National
Collection of Plant Pathogenic Bacteria.PCAMBIA-1303 binary expression vector (Paula
M.Olhoft,Lex E.Flagel,David A.Somers.T-DNA locus structure in a large
population of soybean plants transformed using the Agrobacterium-mediated
Cotyledonary-node method.Plant Biotechnology Journal.2 (2004), pp.289 300), its figure
Spectrum such as Fig. 4.
By the K599 strains of -80 DEG C of preservations in LB+50mg/ml Rif (rifampicin)+100mg/l Kana (kanamycin)
Or line activation on the solid medium of LB+50mg/ml Rif (rifampicin), 28 DEG C of 36～48h of culture, picking single bacterium colony is in LB
+ 50mg/ml Rif (rifampicin)+100mg/l Kana (kanamycin) or LB+50mg/ml Rif (rifampicin) fluid medium
In, 200～220r/min, 28 DEG C of cultures 12～16h, bacterial concentration OD600>0.8 bacterium solution is used for further real as strain
Test.
2 brassica napus prepare
Cabbage type rape variety " raising oil 9 " seed is preserved for this laboratory.The kind is the common varieties of plant in Jiangsu Province,
Can be in each seed shop purchase in Jiangsu Province.
Routinely for " raising oil 9 ", (75% ethanol disinfection 2min's seed disinfection method, 50% sodium hypochlorite disappear for seed disinfection
Malicious 15min), condition of culture be illumination 16h, dark 8h, 24 DEG C, illumination 7000lux.14 days are cultivated in culture bottle as induction
The acceptor material of hairy root.
The induction of 3 hairy root
The K599 bacterium solutions for drawing incubated overnight press 1:200 are seeded to LB+50mg/ml Rif (rifampicin)+100mg/l
16h is cultivated in Kana (kanamycin) or LB+50mg/ml Rif (rifampicin) fluid medium, to OD600>2.0.Take 1ml bacterium
Liquid is centrifuged, resuspended stand-by with 10ml re-suspension liquids.The growth aseptic Brassica Napus Seedling of 2 weeks is used at the hypocotyls away from cotyledon about 1cm
Scalpel carefully scratches a wound for being about 0.5cm (being careful not to penetrate hypocotyls), draws 5 μ l Agrobacteriums weight with pipettor
Suspension Deca is in wound.Seedling after infecting continues to cultivate under the same conditions.As shown in figure 1,10～15 days after infecting
When, a plurality of hairy root is successfully induced in wound, and the wound infected without Agrobacterium K599 shown in arrow does not produce hairy
Root.
4. the culture of hairy root
About 10～20 days after infecting, when the hairy root that grows thickly is born from from infecting, length there is hairy root with scalpel
Hypocotyls cut from each about 0.5cm of wound upper and lower sides, be placed in except bacterium culture medium (4.4g/l MS powder, 30g/l sucrose,
The cephamycin of 300mg/l, Carbenicillin or Ticarcillin/Clavulanate Acid, pH 5.8) in 24 DEG C of dark culturing 15～20 days,.Subsequently, cut
The hairy root of about 1cm length is transferred in new culture medium.New degerming culture need to be proceeded to therebetween in time if there is Agrobacterium bacterium colony
Base.As shown in Fig. 2 having the hypocotyls of hairy root after on bacterium culture medium in the length for cutting, hairy root is mushroomed out, and nothing
The typical gravitropism growth of adventitious root.
5. hairy root successive transfer culture
After cultivating 2 times in bacterium culture medium, hairy root is transferred to equipped with 50ml subculture mediums (MS containing 4.4g/l
Powder, 30g/l sucrose are cultivated in pH 250ml triangular flasks 5.8).Condition of culture is：Under dark, 100rpm, 24 DEG C.
6.GUS is dyeed
Normal hairy root will be grown to cut at the tip of a root about 1cm, 5ml GUS dyeing liquor (1 × PBS, 0.5M is placed in
EDTA, 50mM K3[Fe(CN)6], 50mM K4[Fe(CN)6], 10%TritonX-100,20mg/ml X-Gluc) in, 37 DEG C
Lower dyeing 6h.As shown in figure 3, being that the induction of the Agrobacterium rhyzogenesK599 containing pCAMBIA-1303 binary expression vectors is produced in A figures
Raw hairy root, B are that wild type K599 induces the hairy root for producing.In A, most hairy root is dyed to indigo plant as seen from the figure
Color, illustrates to proceed in most hairy root and express genes of interest.
7. result is counted
To the Agrobacterium rhyzogenesK599 containing binary expression vector (K599+pCAMBIA-1303) and wild type K599
(K599) hairy root that induction is produced is counted, as a result such as table 1.
1 hairy root induction result of table is counted
Claims (6)
1. a kind of hairy root induction of cabbage type rape rapidly and efficiently and cultural method, it is characterised in that comprise the following steps：
1) using the Agrobacterium rhizogenes after the resuspended activation of Agrobacterium re-suspension liquid；
2) wound is manufactured at the nearly cotyledon end hypocotyls of the growth aseptic seedlings of Brassica napus L of 2 weeks；
3) wound is infected with Agrobacterium rhizogenes re-suspension liquid；
4) continue the seedling after aseptic culture infects, hairy root is produced in wound；
5), after cut the hypocotyls with hairy root, cultivating in bacterium culture medium, obtaining aseptic hairy root；
6) tip of a root of hairy root is cut, cultivates in subculture medium, a large amount of hairy root can be obtained.
2. the hairy root induction and cultural method of cabbage type rape according to claim 1, it is characterised in that the step 1)
In, Agrobacterium K599 uses the LB liquid cultures of the corresponding antibiotic containing suitable concn to saturation, takes the centrifugation of 1ml bacterium solutions and goes
Clearly, resuspended with 10ml Agrobacterium re-suspension liquids.
3. according to claim 1 and 2 cabbage type rape hairy root induction and cultural method, it is characterised in that the agriculture bar
The formula of bacterium re-suspension liquid is：4.4g/L MS powder, 20g/L sucrose, 200 μM of acetosyringones (AS), 500mg/l polyethylene pyrroles
Pyrrolidone, pH 5.8.
4. the hairy root induction and cultural method of cabbage type rape according to claim 1, it is characterised in that the step 2)
In, aseptically, under the aseptic Brassica Napus Seedling of 2 weeks will be grown at cotyledon about 1cm with sharp blade or scalpel
A wound for being about 0.5cm scratched by plumular axis, but wound can not penetrate hypocotyls.
5. the hairy root induction and cultural method of cabbage type rape according to claim 1, it is characterised in that the step 3)
In, 1～20 μ l Agrobacterium rhizogenes re-suspension liquids are drawn with pipettor under the conditions of aseptic seedling drop in hypocotylar wound.
6. the hairy root induction and cultural method of cabbage type rape according to claim 1, it is characterised in that the step 2)
With step 4) in condition of culture be photoperiod illumination 16h, dark 8h, 24 DEG C of ambient temperature, intensity of illumination 7000lux.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107119070A (en) * | 2017-05-18 | 2017-09-01 | 中国医学科学院药用植物研究所 | It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency |
| CN107164502A (en) * | 2017-06-15 | 2017-09-15 | 西南大学 | The molecular labeling being closely related and its application are whether there is with cabbage leaves epidermal hair |
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
| CN107926700A (en) * | 2017-11-22 | 2018-04-20 | 西北农林科技大学 | A kind of method using DH plants of root regeneration plant of wild cabbage |
| CN108359672A (en) * | 2017-12-18 | 2018-08-03 | 湖南科技大学 | A kind of method of quick clone mustard type rape seed coat gene TT2CDS sequences |
| CN110317825A (en) * | 2019-04-29 | 2019-10-11 | 聊城大学 | A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes |
| CN113652446A (en) * | 2021-09-22 | 2021-11-16 | 内蒙古农业大学 | Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia |
| CN114271141A (en) * | 2021-12-24 | 2022-04-05 | 中国农业科学院都市农业研究所 | Technology for inducing aerial rooting and breeding of taxus chinensis by utilizing agrobacterium rhizogenes |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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| CN107119070A (en) * | 2017-05-18 | 2017-09-01 | 中国医学科学院药用植物研究所 | It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency |
| CN107164502B (en) * | 2017-06-15 | 2020-09-08 | 西南大学 | Molecular marker closely related to existence of skin hair of cabbage leaf and application thereof |
| CN107164502A (en) * | 2017-06-15 | 2017-09-15 | 西南大学 | The molecular labeling being closely related and its application are whether there is with cabbage leaves epidermal hair |
| CN107926700A (en) * | 2017-11-22 | 2018-04-20 | 西北农林科技大学 | A kind of method using DH plants of root regeneration plant of wild cabbage |
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
| CN108359672A (en) * | 2017-12-18 | 2018-08-03 | 湖南科技大学 | A kind of method of quick clone mustard type rape seed coat gene TT2CDS sequences |
| CN110317825A (en) * | 2019-04-29 | 2019-10-11 | 聊城大学 | A kind of hairy root induction method for transformation of soybean mediated with agrobacterium rhizogenes |
| CN110317825B (en) * | 2019-04-29 | 2023-03-31 | 聊城大学 | Agrobacterium rhizogenes-mediated soybean hairy root induced transformation method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN113652446A (en) * | 2021-09-22 | 2021-11-16 | 内蒙古农业大学 | Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermedia |
| CN114271141A (en) * | 2021-12-24 | 2022-04-05 | 中国农业科学院都市农业研究所 | Technology for inducing aerial rooting and breeding of taxus chinensis by utilizing agrobacterium rhizogenes |
| CN114271141B (en) * | 2021-12-24 | 2023-02-07 | 中国农业科学院都市农业研究所 | Technology for inducing aerial rooting and breeding of taxus chinensis by utilizing agrobacterium rhizogenes |
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