CN111183899A - Method for rapidly inducing safflower hairy roots - Google Patents
Method for rapidly inducing safflower hairy roots Download PDFInfo
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- CN111183899A CN111183899A CN202010057269.9A CN202010057269A CN111183899A CN 111183899 A CN111183899 A CN 111183899A CN 202010057269 A CN202010057269 A CN 202010057269A CN 111183899 A CN111183899 A CN 111183899A
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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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- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a method for rapidly inducing safflower hairy roots. Respectively infecting 3-4 weeks of aseptic seedlings with an infection solution A and an infection solution B for 8-15 minutes, then carrying out co-culture for 24-72 hours, transferring to screening culture for culture, cutting off rapidly-growing hairy roots from explants after 10-25 days, transferring to a subculture medium, transferring to a liquid medium after 15 days for amplification and shaking culture, and obtaining a large amount of hairy roots after 30-45 days; the infection liquid A is wild agrobacterium rhizogenes K599, and the infection liquid B is K599 agrobacterium rhizogenes transferred with GUS reporter genes. The method has the advantages of rapid induction of the safflower hairy roots, suitability for the induction of the hairy roots of various safflower varieties, convenient material acquisition, strong applicability and rapid induction speed.
Description
Technical Field
The invention relates to a method for inducing safflower hairy roots, in particular to a method for rapidly inducing safflower hairy roots.
Background
Carthami flos is dry flower of Carthamus tinctorius L of Compositae, and is called grass Carthamus tinctorius L, Woodward Variegatus (L.) Druce, and radix seu folium Linderae Strychnifoliae. It is pungent and warm in property, enters heart and liver meridians, and is a good herb for activating blood and dredging meridians, removing blood stasis and relieving pain. The chemical components of safflower mainly comprise flavonoids, alkaloids, polyacetylenes, spermidine, lignans, sesquiterpenes, organic acids, sterols, alkyl glycols, polysaccharides and other components. The compounds in safflower have wide pharmacological activity, not only have certain effect on cardiovascular and cerebrovascular systems, nervous systems, immune systems and the like of human, but also have various physiological activities of anti-inflammation, analgesia, anti-tumor, antioxidation and the like. The safflower oil is edible oil which is recognized in the world and has the functions of eating, health care and beauty treatment.
Just as safflower has so many chemical components and functions of treatment and health care, it has been more and more widely used. Research shows that in the method for gene function verification of safflower, arabidopsis thaliana or callus induction regeneration is generally adopted, the growth cycle is long, and the transformation efficiency is low. The hairy root has the advantages of short growth cycle, convenient culture, easy transformation, high transformation rate and the like, and particularly, the successful induction and related application of safflower in the hairy root are not reported at home and abroad at present.
At present, only 1 safflower plant is accepted in the world in China, but the introduction and breeding work of safflower varieties in China is late, and the biotechnological application and development of safflower are slow, so that the induction and gene transformation efficiency of safflower hairy roots is very necessary.
Disclosure of Invention
The invention aims to provide a method for rapidly inducing safflower hairy roots. The method has the advantages of rapid induction of the safflower hairy roots, suitability for induction of the hairy roots of various safflower varieties, convenient material acquisition, strong applicability and rapid induction speed.
At present, no report for constructing a genetic system of the hairy roots of the safflower exists, and the invention fills the blank of research in the field. The method can be used for quickly obtaining the hairy root system, and provides good help for quickly developing gene function verification research on safflower.
The technical scheme of the invention is as follows: a method for inducing safflower hairy root rapidly, it is 3-4 weeks aseptic seedling, infect liquid A and infect liquid B to infect 8-15 minutes separately, then transfer to and screen and cultivate after co-culturing for 24-72 hours, cut the hairy root growing rapidly from the explant after 10 days-25 days, transfer to the subculture medium, transfer to the liquid culture medium after 15 days and enlarge and shake and cultivate, get a large amount of hairy roots after 30-45 days; the infection liquid A is wild agrobacterium rhizogenes K599, and the infection liquid B is K599 agrobacterium rhizogenes transferred with GUS reporter genes.
In the method for rapidly inducing hairy roots of safflower, the infection solution A is obtained by centrifuging 50ml OD value (600)0.4-1.2 agrobacterium K599 and resuspending 0.5-2.0 times volume of MS liquid.
In the method for rapidly inducing the hairy roots of the safflower, the infection liquid B is obtained by centrifuging 50ml of the Agrobacterium K599 with a GUS reporter gene with an OD value of 600 of 0.4-1.2 and then carrying out heavy suspension by using MS liquid with 0.5-2.0 times of the volume of the MS liquid.
Compared with the prior art, the invention has the following beneficial effects:
1. the transformation efficiency can be identified by transferring GUS genes in hairy roots and carrying out GUS staining on the genes, and the method has very important significance for utilizing the hairy roots of the safflower. The safflower explant is induced by agrobacterium rhizogenes, wherein the agrobacterium rhizogenes strain is a key substance. K599 is one of agrobacterium rhizogenes, and the plant rhizogenesis can be induced by the literature report. We studied the induction time, concentration, culture time and the like of K599 Agrobacterium rhizogenes on safflower, constructed transformed K599 Agrobacterium rhizogenes with GUS reporter genes, and performed GUS staining on transformed hairy roots. Experimental research shows that wild type and transformed K599 agrobacterium rhizogenes can efficiently induce hairy roots to safflower when OD value is within 0.4-1.2, and the safflower with GUS gene can be dyed into blue.
2. Starting with the approaches of K599 agrobacterium rhizogenes, biology of transformation efficiency and the like, the invention deeply researches the induction conditions and transformation efficiency of the hairy roots and finds that the strain selection and OD value thereof, the co-culture time and the induction part are key factors in the induction process of the hairy roots of the safflower. The method is applied to the induction of safflower hairy roots, constructs transformed K599 agrobacterium rhizogenes with GUS reporter genes, and performs GUS staining on the transformed hairy roots and is used for researching the transformation efficiency. The invention uses chemical staining method to stain GUS, so that the transformation efficiency can be simply, conveniently and intuitively explored. And the method has the advantages of simple operation, high analysis speed and low price of used reagents.
The principle of "dyeing red flower hairy roots infected with B-infection solution and cultured with GUS" in examples 1-3 to stain with color and find blue "is that GUS (β -glucuronidase, β -D-glucuronidase) gene is a reporter gene commonly used at present, and its expression product β -Glucuronidase (GUS) is a hydrolase which catalyzes the hydrolysis of many β -glucoside ester substances, and it can decompose 5-bromo-4-chloro-3-indole- β -glucoside acid ester (X-gluc. htm' target ═ blank > X-gluc) into blue substances, and its detection method is simple, rapid, sensitive, stable, and has low background activity.
Experiments prove that:
in examples 1 to 3 of the present invention, 40 explants were infected, and the number of hair roots and induction rate of hypocotyls and cotyledons were counted as shown in table 1:
table 1 examples 1-3 table of explant infections
In conclusion, the method has the advantages of rapid induction of the safflower hairy roots, suitability for induction of the hairy roots of various safflower varieties, convenience in material acquisition, strong applicability and rapid induction speed.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1:
a method for rapidly inducing safflower hairy roots, comprising the steps of:
(1) and (3) immersing a dye solution A: 50ml OD value (600) 0.4-0.6 Agrobacterium K599 centrifugal, use 2.0 times volume MS liquid heavy suspension to get and mix for subsequent use.
(2) And (3) immersing a dye solution B: 50ml OD value (600) 0.4-0.6 Agrobacterium K599 with GUS reporter gene for centrifugation, using 2.0 times volume MS liquid to carry out heavy suspension, and mixing evenly for standby.
(3) Taking two bottles of 15-20-day-old safflower aseptic seedlings.
(4) One group was added to 50ml of the infection solution A, and both hypocotyls and cotyledons were completely immersed in the liquid for 10 minutes of infection. The other set was added to 50ml of the staining solution B.
(5) Adding into MS culture medium at 25 deg.C, and dark culturing for about 24 h.
(6) After the dark culture is finished, transferring MS solid medium containing 200mg/ml of cephalosporin for 10-25 days, and then differentiating hypocotyls and cotyledons to form hairy roots.
(7) Shearing off 2-4CM hairy root, adding 100mg/ml solid medium of cephalosporin MS, and screening and culturing. After 30 days, the hairy roots of the safflower with high growth speed are screened out and subcultured on an MS solid culture medium containing 50mg/ml of cephalosporin. Subculturing for 2-3 times to obtain detoxified hairy root of Carthami flos.
(8) Cutting off 2-4CM hairy root, and adding MS liquid culture medium for amplification culture. Thereby obtaining the safflower hairy roots which grow fast and have more lateral root branches.
(9) GUS staining of the hairy roots of the safflower infected and cultured by the B infection solution can dye colors, and the color turns blue.
Example 2:
a method for rapidly inducing safflower hairy roots, comprising the steps of:
(1) and (3) immersing a dye solution A: centrifugation was performed at 50ml OD (600)0.8 Agrobacterium K599 and resuspension was performed with an equal volume of MS fluid to obtain a uniform mixture.
(2) And (3) immersing a dye solution B: 50ml OD value (600)0.8 Agrobacterium K599 with GUS reporter gene for centrifugation and equal volume MS liquid for heavy suspension to obtain uniform mixture.
(3) Taking two bottles of safflower aseptic seedlings with age of about 25-30 days.
(4) One group was added to 50ml of the infection solution A, and both hypocotyls and cotyledons were completely immersed in the liquid for 15 minutes of infection. The other set was added to 50ml of the staining solution B.
(5) Adding into MS culture medium at 25 deg.C, and dark culturing for about 48 h.
(6) After the dark culture is finished, transferring MS solid medium containing 200mg/ml of cephalosporin for 10-25 days, and then differentiating hypocotyls and cotyledons to form hairy roots.
(7) Shearing off 2-4CM hairy root, adding 100mg/ml solid medium of cephalosporin MS, and screening and culturing. After two weeks, the hairy roots of the safflower with high growth speed are screened out and subcultured on an MS solid culture medium containing 50mg/ml of cephalosporin. Subculturing for 2-3 times to obtain detoxified hairy root of Carthami flos.
(8) Cutting off 2-4CM hairy root, and adding MS liquid culture medium for amplification culture. Thereby obtaining the safflower hairy roots which grow fast and have more lateral root branches.
(9) GUS staining of NILA infected and cultured with the B-infection solution enables staining and bluing to be found.
Example 3:
a method for rapidly inducing safflower hairy roots, comprising the steps of:
(1) and (3) immersing a dye solution A: centrifugation was performed by 50ml OD (600)1.0 Agrobacterium K599, and resuspension was performed by 0.5 times MS liquid to obtain a uniform mixture.
(2) And (3) immersing a dye solution B: 50ml OD (600) about 1.0 Agrobacterium K599 with GUS reporter gene for centrifugation and 0.5 volume MS liquid heavy suspension to mix.
(3) Taking two bottles of 35-42-day-old safflower aseptic seedlings.
(4) One group was added to 50ml of the infection solution A, and both hypocotyls and cotyledons were completely immersed in the liquid for 15 minutes of infection. The other set was added to 50ml of the staining solution B.
(5) Adding into MS culture medium at 25 deg.C, and dark culturing for about 72 h.
(6) After the dark culture is finished, transferring MS solid medium containing 200mg/ml of cephalosporin for 10-25 days, and then differentiating hypocotyls and cotyledons to form hairy roots.
(7) Shearing off 2-4CM hairy root, adding 100mg/ml solid medium of cephalosporin MS, and screening and culturing. After two weeks, the hairy roots of the safflower with high growth speed are screened out and subcultured on an MS solid culture medium containing 50mg/ml of cephalosporin. Subculturing for 2-3 times to obtain detoxified hairy root of Carthami flos.
(8) Cutting off 2-4CM hairy root, and adding MS liquid culture medium for amplification culture. Thereby obtaining the safflower hairy roots which grow fast and have more lateral root branches.
(9) GUS staining of the hairy roots of the safflower infected and cultured by the B infection solution can dye colors, and the color turns blue.
Claims (3)
1. A method for rapidly inducing safflower hairy roots is characterized in that: respectively infecting 3-4 weeks of aseptic seedlings with an infection solution A and an infection solution B for 8-15 minutes, then carrying out co-culture for 24-72 hours, transferring to screening culture for culture, cutting off rapidly-growing hairy roots from explants after 10-25 days, transferring to a subculture medium, transferring to a liquid medium after 15 days for amplification and shaking culture, and obtaining a large amount of hairy roots after 30-45 days; the infection liquid A is wild agrobacterium rhizogenes K599, and the infection liquid B is K599 agrobacterium rhizogenes transferred with GUS reporter genes.
2. The method for rapidly inducing hairy roots of safflower according to claim 1, wherein: the infection liquid A is obtained by centrifuging 50ml OD value (600)0.4-1.2 agrobacterium K599 and resuspending 0.5-2.0 times volume MS liquid.
3. The method for rapidly inducing hairy roots of safflower according to claim 1, wherein: the infection liquid B is obtained by centrifuging 50ml of Agrobacterium K599 with an OD value (600) of 0.4-1.2 and GUS reporter gene and carrying out heavy suspension by using MS liquid with 0.5-2.0 times volume.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111937749A (en) * | 2020-08-25 | 2020-11-17 | 贵州大学 | High-efficiency induction and culture method of uncaria hairy roots |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN111937749B (en) * | 2020-08-25 | 2021-11-19 | 贵州大学 | High-efficiency induction and culture method of uncaria hairy roots |
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