CN115053809A - Method for quickly and efficiently establishing tobacco regeneration system - Google Patents
Method for quickly and efficiently establishing tobacco regeneration system Download PDFInfo
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- CN115053809A CN115053809A CN202210682399.0A CN202210682399A CN115053809A CN 115053809 A CN115053809 A CN 115053809A CN 202210682399 A CN202210682399 A CN 202210682399A CN 115053809 A CN115053809 A CN 115053809A
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
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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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
- A01H6/823—Nicotiana, e.g. tobacco
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Abstract
The invention discloses a method for quickly and efficiently establishing a tobacco regeneration system, which comprises the following steps: infecting the explant of the tobacco soil-cultured seedling with an activated agrobacterium rhizogenes infection solution; inoculating the infected explants on a co-culture medium for dark culture for 24-48h, and differentiating the explants to form hairy roots; transferring the dark cultured explants to a rooting induction culture medium for induction culture, and screening out tobacco hairy roots with tobacco regeneration plants; carrying out subculture and amplification culture on the hairy roots generated by the induction culture to obtain regenerated plants; transplanting the obtained regeneration plant into nutrient soil for culturing to obtain the tobacco regeneration plant. The method is suitable for induction of the hairy roots of various tobacco varieties, has the advantages of convenience in material acquisition, high induction speed, stable heredity, cost saving and the like, and realizes quick and efficient induction and propagation of a tobacco regeneration system of soil-cultured seedlings by directly inducing the soil-cultured seedlings of tobacco.
Description
Technical Field
The invention relates to the technical field of tobacco regeneration, in particular to a method for quickly and efficiently establishing a tobacco regeneration system.
Background
The regeneration system of tobacco is the foundation for researching the growth and quality of tobacco, at present, the research on the regeneration system of tobacco is not much developed, but the related technology mostly adopts tobacco aseptic seedlings to form callus, dedifferentiation and redifferentiation are carried out to obtain the regeneration system of tobacco, and how to quickly and efficiently establish the regeneration plant of tobacco is a difficult point. The tobacco regeneration system can be used for researching the tobacco characteristics of the tobacco related genes, can visually evaluate the growth of the genes on the tobacco, and can more directly research the tobacco.
At present, the establishment of a tobacco regeneration system mostly adopts the form of aseptic seedlings. Among them, the related literature reports mostly adopt aseptic seedlings for treatment, the aseptic seedlings have high cost, the requirements on thallus pollution and tobacco seedling growth are high, related hormones are required for induction and culture, related instruments and equipment are required, the system is not favorable for building, and the building of a regeneration system is restricted. In recent years, related documents report that agrobacterium rhizogenes (a4, R1000, R1601) can induce other plant regeneration systems, but no related document reports that a method for directly inducing a regeneration system by soil-cultured seedlings.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for establishing a rapid and efficient tobacco regeneration system, which realizes rapid and efficient induction of the tobacco regeneration system of soil-cultured seedlings by directly inducing the soil-cultured seedlings of tobacco, and is suitable for induction of regeneration systems of various tobacco varieties.
The technical problem to be solved by the invention is realized by the following technical scheme:
a method for establishing a rapid and efficient tobacco regeneration system comprises the following steps:
step (1): infecting the explant of the tobacco soil-cultured seedling with an activated agrobacterium rhizogenes infection solution;
step (2): inoculating the infected explants on a co-culture medium for dark culture for 24-48h, and differentiating the explants to form hairy roots;
and (3): transferring the dark cultured explants to a rooting induction culture medium for induction culture, and screening out hairy roots of tobacco with tobacco regeneration plants;
and (4): carrying out subculture and amplification culture on the hairy roots generated by the induction culture to obtain regenerated plants;
and (5): transplanting the obtained regeneration plant into nutrient soil for culturing to obtain the tobacco regeneration plant.
Preferably, in the above technical scheme, the agrobacterium rhizogenes is a4, R1000 or R1601.
Preferably, in the above technical scheme, in the step (1), the infection solution is prepared by centrifuging agrobacterium rhizogenes with an OD value of 50mL (600) and 0.8, and resuspending the infection solution with an equal volume of MS liquid.
Preferably, in the above technical scheme, in the step (2), the co-culture medium is an MS medium, and the culture temperature is 25 ℃.
Preferably, in the above technical scheme, in the step (3), the hairy root induction culture medium is an MS solid culture medium added with antibiotics, and the culture time is 2-3 weeks.
Preferably, in the above technical scheme, in the step (3), the antibiotic is cephalosporin with an addition amount of 50 mg/mL.
Preferably, in the above technical solution, in the step (3),
the components of the induction medium are as follows:
20 × macroelement mother liquor: NH 4 NO 3 33.0g，KNO 3 38.0g，MgSO 4 ·7H 2 O 7.4g，KH 2 PO 4 3.4g；
200 times microelement mother liquor: KI 0.166g, H 3 BO 3 1.24g，MnSO 4 ·4H 2 O 4.46g，ZnSO 4 ·7H 2 O 1.72g，Na 2 MnO·2H 2 O 0.05g，CuSO 4 ·5H 2 O 0.005g，CoCl 2 ·6H 2 O 0.005g；
200 times ferric salt mother liquor: FeSO 4 ·7H 2 O 5.56g，EDTA-Na 2 ·2H 2 O 7.46g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB 6 0.1g，VB 1 0.1g of glycine and 0.4g of glycine;
20 × calcium salt mother liquor: CaC1 2 6.64g。
The preparation method of the induction culture medium comprises the following steps:
the components are weighed, dissolved by deionized water and fixed to 1L, and then sterilized at 121 ℃ for 15min for standby.
Preferably, in the above technical scheme, in the step (4), the culture medium for amplification culture is an MS liquid culture medium.
Preferably, in the above technical scheme, the tobacco soil-cultured seedlings are 3-4 weeks old soil-cultured seedlings.
A tobacco regeneration plant obtained by a method established according to a rapid and efficient tobacco regeneration system.
The technical scheme of the invention has the following beneficial effects:
the method for quickly and efficiently regenerating the tobacco can induce the tobacco soil-cultured seedlings to generate the hairy roots by utilizing the agrobacterium rhizogenes (A4, R1000 and R1601), is suitable for inducing the hairy roots of various tobacco varieties, has the advantages of convenient material taking, high induction speed, stable heredity, cost saving and the like, and realizes quick and efficient induction of the hairy roots of the tobacco soil-cultured seedlings by directly inducing the tobacco soil-cultured seedlings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a schematic diagram of the induction process of the fast and efficient tobacco regeneration method of the present invention on the soil-cultured seedling explant. Wherein: A. culturing tobacco seedlings by soil culture; B. wounded tobacco explants inoculated with agrobacterium rhizogenes; C. agrobacterium rhizogenes; D. after 2-3 weeks, hairy roots appear at the wound; E. cutting off hairy roots, culturing on MS solid culture, and growing regenerated plants on the hairy roots; F. the regenerated plants are cut off and grown on the MS solid; G. and when the plant height is 1cm, transplanting the plant into nutrient soil to obtain a tobacco regeneration plant.
FIG. 2 shows the result of PCR detection of VirD gene in the regeneration system. Wherein: m is Marker, CK uninfected tobacco (negative control), P plasmid positive control; 1. 2 and 3 are tobacco regeneration plants with positive detection.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
Example 1
A method for establishing a rapid and efficient tobacco regeneration system (see fig. 1), comprising the following steps:
step (1): the tobacco explants of the soil-cultured seedlings are infected by wild type A4 agrobacterium rhizogenes infection liquid.
Specifically, the explant of 4-week-old tobacco soil-cultured seedlings is cut into 1cm × 1cm leaves, i.e., the tobacco soil-cultured seedlings leaves are added into an invasion dye solution and infected for 5 min.
The staining solution was prepared by centrifugation with 50mL of 0.8 Agrobacterium rhizogenes at OD 600 and resuspension with an equal volume of MS liquid.
Step (2): the infected explants were inoculated on the co-cultured medium at 25 ℃ for 48h in the dark.
The co-culture medium was: MS medium, after 48h dark culture, at this point the explants differentiated to form hairy roots.
And (3): transferring the dark cultured explant to a hairy root induction culture medium for induction culture, cutting off a hairy root of 3cm, adding a cephalosporin MS solid culture medium of 50mg/mL for screening culture, and screening the hairy root of the tobacco with a tobacco regeneration plant after 3-4 weeks.
And (4): and (4) carrying out subculture and amplification culture (MS liquid culture medium) on the regenerated plant generated by the induction culture to obtain a regenerated plant.
And (5): transplanting the regenerated plant generated by the induction culture into nutrient soil to obtain a regenerated plant.
The VirD gene PCR detection of the regeneration system plant shows that the regeneration plant has been detoxified (as shown in figure 2).
In the embodiment, the tobacco hairy roots are placed on a 50mg/mL cefaloxime MS solid culture medium for subculture, and after subculture is carried out for 2-3 times, the detoxified tobacco hairy roots are obtained. It should be noted that, when 3cm of the hairy root is cut off and added to the MS liquid medium for amplification culture, the hairy root can grow faster, and the culture needs to be performed under a light source besides the environment with the temperature of 25 ℃.
It will be appreciated by those skilled in the art that the tobacco seedlings may also be selected for 3 weeks of age, and the invention is not limited thereto.
The induction medium is composed as follows:
20 × macroelement mother liquor: NH (NH) 4 NO 3 33.0g，KNO 3 38.0g，MgSO 4 ·7H 2 O 7.4g，KH 2 PO4 3.4g；
200 times microelement mother liquor: KI 0.166g, H 3 BO3 1.24g，MnSO4·4H 2 O 4.46g，ZnSO 4 ·7H 2 O 1.72g，Na 2 MnO·2H 2 O 0.05g，CuSO 4 ·5H 2 O 0.005g，CoCl 2 ·6H 2 O 0.005g；
200 times ferric salt mother liquor: FeSO 4 ·7H 2 O 5.56g，EDTA-Na 2 ·2H 2 O 7.46g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB 6 0.1g，VB 1 0.1g of glycine and 0.4g of glycine;
20 × calcium salt mother liquor: CaC1 2 6.64g；
The preparation method of the induction culture medium comprises the following steps:
weighing the components, dissolving the components by using deionized water, fixing the volume to 1L, and then sterilizing the components for 15min at a high temperature of 121 ℃ for later use;
preparing an MS liquid culture medium:
20 multiplied by 50mL of macroelement mother liquor and 200 multiplied by 5mL of microelement mother liquor;
200 times 5mL of ferric salt mother liquor and 20 times 50mL of Ca salt mother liquor;
200 times of 5mL of organic mother liquor, adjusting the pH value to 5.8-6.2, and fixing the volume to a 1L volumetric flask.
In this example, the MS solid medium (1L) was prepared as follows:
on the basis of the MS liquid medium, 3g of plant gel was added, and it is understood that the plant gel was added after the pH of the MS liquid medium was adjusted to 5.8.
And (3) inducing results:
examples 1 to 3 of the present invention (the methods of example 2 and example 3 are the same as example 1 except that the species of Agrobacterium rhizogenes are different) were all the results of directly inducing soil-cultured seedlings of tobacco by centrifugation of 0.8 Agrobacterium rhizogenes (A4, R1000 or R1601) at an OD value of 50mL (600). Wherein, the total infected strains of different strains are 100 strains, and the induction results are shown in table 1.
TABLE 1 results of Agrobacterium rhizogenes direct induction of tobacco soil-cultured seedlings in examples 1-3
| Bacterial strain | Total infection number/plant | Number/plant of hairy root | Induction rate/% |
| A4 | 100 | 39 | 39 |
| R1000 | 100 | 11 | 11 |
| R1601 | 100 | 5 | 5 |
According to the table 1, the induction rate of directly inducing tobacco soil culture seedlings by agrobacterium rhizogenes A4 reaches 39%, and the induction effect is good.
Therefore, examples 4 to 8 of the present invention are the results of the test of inducing and expanding the propagation of tobacco seedlings cultivated in soil by using A4 Agrobacterium rhizogenes at different OD values (600). The total number of infected A4 Agrobacterium rhizogenes strains was 100 strains at different OD values (600), and the induction results are shown in Table 2.
TABLE 2 test of different OD values (600) for tobacco soil-cultured seedling induction and propagation
| OD value (600) | Total infection number/plant | Number/plant of hairy root | Induction rate/% |
| 1.0 | 100 | 27 | 27 |
| 0.9 | 100 | 35 | 35 |
| 0.8 | 100 | 39 | 39 |
| 0.7 | 100 | 36 | 36 |
| 0.6 | 100 | 35 | 35 |
Through research, the infection liquid is prepared by centrifuging 50mL of Agrobacterium rhizogenes with OD value (600) of 0.8 and resuspending with an equal volume of MS liquid, the Agrobacterium rhizogenes A4 can efficiently induce hairy roots when the OD value is 0.8, the Agrobacterium rhizogenes A4 induction efficiency is the highest and can reach 70% induction rate, the induced hairy roots can be continuously produced and used as a biochemical reactor for relevant experimental research.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.
Claims (10)
1. A method for establishing a rapid and efficient tobacco regeneration system is characterized by comprising the following steps:
step (1): infecting an explant of a tobacco soil-cultured seedling with an activated agrobacterium rhizogenes infection solution;
step (2): inoculating the infected explants on a co-culture medium for dark culture for 24-48h, and differentiating the explants to form hairy roots;
and (3): transferring the dark cultured explants to a rooting induction culture medium for induction culture, and screening out tobacco hairy roots with tobacco regeneration plants;
and (4): carrying out subculture and amplification culture on the hairy roots generated by the induction culture to obtain regenerated plants;
and (5): transplanting the obtained regeneration plant into nutrient soil for culturing to obtain the tobacco regeneration plant.
2. The method for establishing the rapid and efficient tobacco regeneration system according to claim 1, wherein in the step (1), the agrobacterium rhizogenes is A4, R1000 or R1601.
3. The method for establishing the rapid and efficient tobacco regeneration system according to claim 1, wherein in the step (1), the infection solution is prepared by centrifuging 50mL of Agrobacterium rhizogenes with OD value of 600 and 0.8 and resuspending with an equal volume of MS liquid.
4. The method for establishing a rapid and efficient tobacco regeneration system according to claim 1, wherein in the step (2), the co-culture medium is MS culture medium, and the culture temperature is 25 ℃.
5. The method for establishing a rapid and efficient tobacco regeneration system according to claim 1, wherein in the step (3), the hairy root induction culture medium is MS solid culture medium added with antibiotics, and the culture time is 2-3 weeks.
6. The method for establishing the rapid and efficient tobacco regeneration system according to claim 5, wherein in the step (3), the antibiotic is cephalosporin with an addition amount of 50 mg/mL.
7. The method for establishing a rapid and efficient tobacco regeneration system according to claim 5, wherein in the step (3),
the induction medium components were as follows:
20 × macroelement mother liquor: NH (NH) 4 NO 3 33.0g，KNO 3 38.0g，MgSO 4 ·7H 2 O 7.4g，KH 2 PO 4 3.4g；
200 × mother liquor of trace elements: KI 0.166g, H 3 BO 3 1.24g，MnSO 4 ·4H 2 O 4.46g，ZnSO 4 ·7H 2 O 1.72g，Na 2 MnO·2H 2 O 0.05g，CuSO 4 ·5H 2 O 0.005g，CoCl 2 ·6H 2 O 0.005g；
200 times ferric salt mother liquor: FeSO 4 ·7H 2 O 5.56g，EDTA-Na 2 ·2H 2 O 7.46g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB 6 0.1g，VB 1 0.1g of glycine and 0.4g of glycine;
20 × calcium salt mother liquor: CaC1 2 6.64g。
The preparation method of the induction culture medium comprises the following steps:
the components are weighed, dissolved by deionized water and subjected to constant volume of 1L, and then sterilized at the high temperature of 121 ℃ for 15min for later use.
8. The method for establishing a rapid and efficient tobacco regeneration system according to claim 1, wherein in the step (4), the culture medium for amplification culture is MS liquid culture medium.
9. The method for establishing the rapid and efficient tobacco regeneration system according to claim 1, wherein the tobacco soil-culture seedlings are 3-4 weeks old soil-culture seedlings.
10. The regenerated tobacco plant obtained by the method established by the rapid and efficient tobacco regeneration system according to any one of claims 1 to 9.
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2022
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