CN114766356A - Rapid and efficient tobacco hairy root inducing and propagation method - Google Patents
Rapid and efficient tobacco hairy root inducing and propagation method Download PDFInfo
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- CN114766356A CN114766356A CN202210342239.1A CN202210342239A CN114766356A CN 114766356 A CN114766356 A CN 114766356A CN 202210342239 A CN202210342239 A CN 202210342239A CN 114766356 A CN114766356 A CN 114766356A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a rapid and efficient tobacco hairy root induction and propagation method, which comprises the following steps: infecting the explant of the tobacco soil-cultured seedling with an activated agrobacterium rhizogenes infection solution, inoculating the infected explant on a co-culture medium for dark culture for 24-48 h, transferring the dark-cultured explant to a hairy root induction culture medium for induction culture, and carrying out subculture and amplification culture on the hairy root generated by the induction culture, thereby obtaining a large amount of tobacco hairy roots. The invention provides a method for directly inducing hairy roots of soil-cultured seedlings, which is suitable for inducing the hairy roots of various tobacco varieties, has the advantages of convenient material acquisition, high inducing speed, stable heredity, cost saving and the like, and realizes the quick and efficient induction and propagation of the hairy roots of the soil-cultured seedlings of tobacco through directly inducing the soil-cultured seedlings of tobacco.
Description
Technical Field
The invention relates to a rapid and efficient tobacco hairy root induction and propagation method, and belongs to the technical field of agricultural application biology.
Background
The root system of tobacco is the basis of tobacco growth and quality, and at present, the research on the root system of tobacco is not in the future, but the root system of tobacco cannot be directly known in soil, and how to intuitively research the root system is a difficult point. The tobacco hairy roots are used as a bioreactor generated by artificial induction, the growth of the roots can be visually observed, and the research on the root system can be directly carried out.
At present, the induction of tobacco hairy roots 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 instruments and equipment are needed, the research and production are not facilitated, and the induction efficiency of the hairy roots is restricted. In recent years, related documents report that agrobacterium rhizogenes (A4, R1000 and R1601) can induce the rooting of tobacco sterile seedlings, but no related document reports that the method for directly inducing hairy roots by soil-cultured seedlings.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a rapid and efficient method for inducing and propagating tobacco hairy roots, realizes rapid and efficient induction of the tobacco hairy roots of soil-cultured seedlings by directly inducing tobacco soil-cultured seedlings, and is suitable for induction of the hairy roots of various tobacco varieties.
In order to solve the technical problems, the invention is realized by adopting the following technical scheme:
the invention provides a rapid and efficient tobacco hairy root induction and propagation method, 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 24-48 h;
transferring the dark cultured explants to a hairy root induction culture medium for induction culture;
subculture and amplification culture are carried out on the hairy roots generated by the induction culture.
As a preferred embodiment, the staining solution is prepared by centrifugation with 50mL of Agrobacterium rhizogenes with OD (600) of 0.8 and resuspension with an equal volume of MS liquid.
As a preferred embodiment, the Agrobacterium rhizogenes comprises any one of A4, R1000 and R1601.
As a preferable embodiment, the leaves of tobacco seedlings cultivated by soil are cut into 1-2 cm x 1-2 cm, added into an infection solution, infected for 5-10 min and then co-cultured.
As a preferred embodiment, the infected explants are transferred to a solid medium containing 50mg/mL of cephalosporin MS after dark culture is finished, and cultured for 2-3 weeks.
As a preferred embodiment, the induction medium components are as follows:
20 × macroelement mother liquor: NH (NH)4NO3 33.0g，KNO3 38.0g，MgSO4·7H2O 7.4g，KH2PO4 3.4g；
200 times microelement mother liquor: KI 0.166g, H3BO3 1.24g，MnSO4·4H2O 4.46g，ZnSO4·7H2O 1.72g，Na2MnO·2H2O 0.05g，CuSO4·5H2O 0.005g，CoCl2·6H2O 0.005g；
200 Xthe mother liquor of iron salt: FeSO4·7H2O 5.56g，EDTA-Na2·2H2O 7.46g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB6 0.1g，VB10.1g of glycine and 0.4g of glycine;
20 × calcium salt mother liquor: CaC12 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;
200X 5mL of organic mother liquor, and adjusting the pH value to 5.8-6.2.
In a preferred embodiment, the tobacco soil-culture seedlings are 4-week-old soil-culture seedlings.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a method for quickly and efficiently inducing and propagating hairy roots of tobacco, which comprises the following steps: infecting an explant of a tobacco soil-cultured seedling with an activated agrobacterium rhizogenes infection solution; inoculating the infected explants on a co-culture medium for dark culture for 24-48 h; transferring the dark-cultured explants to a rooting induction culture medium for induction culture; subculture and amplification culture are carried out on the hairy roots generated by the induction culture, and agrobacterium rhizogenes is used for inducing tobacco soil culture seedlings to generate the hairy roots.
2. According to the rapid and efficient tobacco hairy root induction and propagation method provided by the invention, researches show that an infection solution is prepared by centrifuging 0.8 agrobacterium rhizogenes with an OD value of 50mL (600) and carrying out resuspension by using an MS liquid with the same volume, when the OD value of the agrobacterium rhizogenes (A4, R1000 and R1601) is 0.8, hairy roots can be efficiently induced, the A4 agrobacterium rhizogenes has the highest induction efficiency and can reach 70% of induction rate, the induced hairy roots can be continuously produced, and the induced hairy roots can be used as a biochemical reactor to carry out related experimental research.
3. The method for quickly and efficiently inducing and propagating the tobacco hairy roots is suitable for inducing the hairy roots of various tobacco varieties, has the advantages of convenient material taking, high inducing speed, stable heredity, cost saving and the like, and realizes quick and efficient induction and propagation of the tobacco hairy roots of the soil-cultured seedlings by inducing the soil-cultured seedlings of the tobacco.
Drawings
FIG. 1 is a schematic diagram of the process of inducing an explant of a soil-cultured seedling by using a rapid and efficient method for inducing and propagating hairy roots of tobacco;
in the figure: A. tobacco soil culture; B. inoculating the pre-cultured wounded tobacco explant with agrobacterium rhizogenes; C. agrobacterium rhizogenes; D. hairy roots appear at the wound of the wounded tobacco explant; E. hairy roots were excised and cultured on MS solid cultures.
Detailed Description
The present invention will be further described below. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified.
Method for quickly and efficiently inducing and propagating hairy roots of tobacco
Example 1
The method comprises the following steps: the tobacco explants of the soil-cultured seedlings are infected by wild type A4 agrobacterium rhizogenes infection liquid.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 5min, wherein the invasion dye solution is prepared by centrifuging agrobacterium rhizogenes with an OD value of 50mL (600) of 0.8 and resuspending with an equal volume of MS liquid.
Step two: in this example, the infected explants were inoculated on a co-cultivation medium, MS medium, for 48h in dark at 25 ℃ and after 48h in dark, the explants differentiated to form hairy roots.
Step three: transferring the dark-cultured explants to a hairy root induction culture medium for induction culture, cutting off 3cm hairy roots, carrying out screening culture on the cut hairy roots and the cut hairy roots added with 50mg/mL cefMS solid culture medium, and screening out the hairy roots of the tobacco with high growth speed after 3 weeks.
Step four: subculturing and amplifying culturing the hairy roots generated by the induction culture to obtain a large amount of tobacco hairy roots.
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 ℃.
Example 2
The method comprises the following steps: and infecting the tobacco explants of the soil-cultured seedlings with wild type R1000 rooting agrobacterium infection liquid.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 10min, wherein the invasion dye solution is prepared by adopting 50mL of agrobacterium rhizogenes with OD value (600) of 0.8 for centrifugation and carrying out heavy suspension by using MS liquid with the same volume.
Step two: in this example, the infected explants were inoculated on co-cultivation medium, MS medium, for 24h at 25 ℃ in dark, and after 24 dark cultivation, the explants differentiated to form hairy roots.
Step three: transferring the dark-cultured explants to a hairy root induction culture medium for induction culture, cutting off 2cm hairy roots, carrying out screening culture on the cut hairy roots and the cut hairy roots added with 50mg/mL cefMS solid culture medium, and screening out the hairy roots of the tobacco with high growth speed after 2 weeks.
Step four: subculturing and amplifying culturing the hairy roots generated by the induction culture to obtain a large amount of tobacco hairy roots.
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, 3cm of cut hairy roots are added to the MS liquid culture medium for amplification culture, so that the hairy roots can grow faster, and the culture needs to be performed under a light source besides the environment with the temperature of 25 ℃.
Example 3
The method comprises the following steps: and infecting the tobacco explants of the soil-cultured seedlings with wild type R1601 agrobacterium rhizogenes infection liquid.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 10min, wherein the invasion dye solution is prepared by centrifuging agrobacterium rhizogenes with an OD value of 50mL (600) of 0.8 and resuspending with an equal volume of MS liquid.
Step two: in this example, the infected explants were inoculated on a co-cultivation medium, MS medium, for 48h in dark at 25 ℃ and after 48h in dark, the explants differentiated to form hairy roots.
Step three: transferring the dark cultured explant to a hairy root induction culture medium for induction culture, cutting off 2-3cm hairy roots, adding 50mg/mL cefMS solid culture medium for screening culture, and screening out the tobacco hairy roots with high growth speed after 3 weeks.
Step four: subculture and amplification culture are carried out on the hairy roots generated by induction culture, and a large amount of tobacco hairy roots are obtained.
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, 3cm of cut hairy roots are added to the MS liquid culture medium for amplification culture, so that the hairy roots can grow faster, and the culture needs to be performed under a light source besides the environment with the temperature of 25 ℃.
Example 4
The method comprises the following steps: and infecting the tobacco explants of the soil-cultured seedlings with wild type A4 rooting agrobacterium infection solution.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 5min, wherein the invasion dye solution is prepared by adopting 50mL of agrobacterium rhizogenes with OD value (600) of 0.7 for centrifugation and carrying out heavy suspension by using MS liquid with the same volume.
Step two: in this example, the infected explants were inoculated on a co-cultivation medium, MS medium, for 24h in dark at 25 ℃ and after 24h in dark, the explants differentiated to form hairy roots.
Step three: transferring the dark cultured explants to a hairy root induction culture medium for induction culture, cutting off 3cm hairy roots, adding 50mg/mL cefMS solid culture medium for screening culture, and screening out the tobacco hairy roots with high growth speed after 3 weeks.
Step four: subculture and amplification culture are carried out on the hairy roots generated by induction culture, and a large amount of tobacco hairy roots are obtained.
In the embodiment, the tobacco hairy roots are placed on a 50mg/mL cefalex MS solid culture medium for subculture, and after 3 times of subculture, the detoxified tobacco hairy roots are obtained. It should be noted that, 3cm of cut hairy roots are added to the MS liquid culture medium for amplification culture, so that the hairy roots can grow faster, and the culture needs to be performed under a light source besides the environment with the temperature of 25 ℃.
Example 5
The method comprises the following steps: and infecting the tobacco explants of the soil-cultured seedlings with wild type A4 rooting agrobacterium infection solution.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 5min, wherein the invasion dye solution is prepared by adopting 50mL of agrobacterium rhizogenes with OD value (600) of 0.8 to centrifuge and resuspending with MS liquid of the same volume.
Step two: in this example, the infected explants were inoculated on a co-culture medium, MS medium, for 24h in the dark at 25 ℃ and the explants differentiated to form hairy roots after 24h in the dark culture.
Step three: transferring the dark-cultured explants to a hairy root induction culture medium for induction culture, cutting off 3cm hairy roots, carrying out screening culture on the cut hairy roots and the cut hairy roots added with 50mg/mL cefMS solid culture medium, and screening out the hairy roots of the tobacco with high growth speed after 3 weeks.
Step four: subculture and amplification culture are carried out on the hairy roots generated by induction culture, and a large amount of tobacco hairy roots are obtained.
In the embodiment, the tobacco hairy roots are placed on a 50mg/mL cefalex MS solid culture medium for subculture, and after 3 times of subculture, 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 ℃.
Example 6
The method comprises the following steps: the tobacco explants of the soil-cultured seedlings are infected by wild type A4 agrobacterium rhizogenes infection liquid.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 5min, wherein the invasion dye solution is prepared by adopting 50mL of agrobacterium rhizogenes with OD value (600) of 0.9 for centrifugation and carrying out heavy suspension by using MS liquid with the same volume.
Step two: in this example, the infected explants were inoculated on a co-cultivation medium, MS medium, for 24h in dark at 25 ℃ and after 24h in dark, the explants differentiated to form hairy roots.
Step three: transferring the dark-cultured explants to a hairy root induction culture medium for induction culture, cutting off 3cm hairy roots, carrying out screening culture on the cut hairy roots and the cut hairy roots added with 50mg/mL cefMS solid culture medium, and screening out the hairy roots of the tobacco with high growth speed after 2 weeks.
Step four: subculture and amplification culture are carried out on the hairy roots generated by induction culture, and a large amount of tobacco hairy roots are obtained.
In the embodiment, the tobacco hairy roots are placed on a 50mg/mL cefalex MS solid culture medium for subculture, and after 3 times of subculture, 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 ℃.
Example 7
The method comprises the following steps: the tobacco explants of the soil-cultured seedlings are infected by wild type A4 agrobacterium rhizogenes infection liquid.
Specifically, the explant of a tobacco soil-cultured seedling of 4 weeks old is cut into 1 × 1cm leaves, namely the leaves of the tobacco soil-cultured seedling are added into an invasion dye solution to infect for 5min, wherein the invasion dye solution is prepared by adopting 50mL of Agrobacterium rhizogenes with OD value (600) of 1.0 for centrifugation and carrying out heavy suspension by using MS liquid with the same volume.
Step two: in this example, the infected explants were inoculated on a co-culture medium, MS medium, for 24h in the dark at 25 ℃ and the explants differentiated to form hairy roots after 24h in the dark culture.
Step three: transferring the dark-cultured explants to a hairy root induction culture medium for induction culture, cutting off 3cm hairy roots, carrying out screening culture on the cut hairy roots and the cut hairy roots added with 50mg/mL cefMS solid culture medium, and screening out the hairy roots of the tobacco with high growth speed after 3 weeks.
Step four: subculture and amplification culture are carried out on the hairy roots generated by induction culture, and a large amount of tobacco hairy roots are obtained.
In the embodiment, the tobacco hairy roots are placed on a 50mg/mL cefalex MS solid culture medium for subculture, and after 3 times of subculture, the detoxified tobacco hairy roots are obtained. It should be noted that, 3cm of cut hairy roots are added to the MS liquid culture medium for amplification culture, so that the hairy roots 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.
In addition, the induction medium is composed of:
20 × macroelement mother liquor: NH4NO3 33.0g，KNO3 38.0g，MgSO4·7H2O 7.4g，KH2PO4 3.4g；
200 × mother liquor of trace elements: KI 0.166g, H3BO3 1.24g，MnSO4·4H2O 4.46g，ZnSO4·7H2O 1.72g，Na2MnO·2H2O 0.05g，CuSO4·5H2O 0.005g，CoCl2·6H2O 0.005g；
200 times ferric salt mother liquor: FeSO4·7H2O 5.56g，EDTA-Na2·2H2O 7.46g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB6 0.1g，VB10.1g, 0.4g of glycine;
20 × calcium salt mother liquor: CaC12 6.64g；
Specifically, the preparation method of the induction 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 at the high temperature of 121 ℃ for 15min 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 iron salt mother liquor and 20 times 50mL of Ca salt mother liquor;
200 Xorganic mother liquor 5mL, adjust pH to 5.8-6.2.
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.
Second, inducing the results
Examples 1 to 3 of the present invention are the results of directly inducing soil-cultured tobacco seedlings by using 50mL of Agrobacterium rhizogenes with OD value of 600 of 0.8 to carry out centrifugation and Agrobacterium rhizogenes A4, R1000 and R1601. 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 (A4, R1000, R1601) directly inducing tobacco seedlings grown on soil in examples 1 to 3
| Strain | Total infection number/plant | Number/plant of hairy root | Induction rate/% |
| A4 | 100 | 77 | 77 |
| R1000 | 100 | 76 | 76 |
| R1601 | 100 | 60 | 60 |
According to the table 1, the inductivity of the agrobacterium rhizogenes A4, R1000 and R1601 directly inducing tobacco soil culture seedlings reaches more than 60%, and the induction effect is good.
Embodiments 4 to 7 of the invention are test results of inducing and expanding propagation of tobacco soil-cultured seedlings by adopting 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 results of the induction and propagation of tobacco seedlings cultivated in soil at different OD values (600)
| OD value (600) | Total infection number/plant | Number/plant of hairy root | Induction rate/% |
| 1.0 | 100 | 57 | 57 |
| 0.9 | 100 | 60 | 60 |
| 0.80 | 100 | 76 | 76 |
| 0.7 | 100 | 50 | 54 |
According to 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 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.
Therefore, the method for inducing and propagating the tobacco hairy roots quickly and efficiently can induce the tobacco soil culture 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 convenience in material obtaining, high induction speed, stable heredity, cost saving and the like, and realizes quick and efficient induction of the tobacco hairy roots of the soil culture seedlings by directly inducing the tobacco soil culture seedlings.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A rapid and efficient tobacco hairy root induction and propagation method is characterized by comprising 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 24-48 h;
transferring the dark-cultured explants to a rooting induction culture medium for induction culture;
subculture and amplification culture are carried out on the hairy roots generated by the induction culture.
2. The method for rapid and efficient induction and propagation of tobacco hairy roots according to claim 1, wherein the staining solution is prepared by using 50mL of Agrobacterium rhizogenes with OD value (600) of 0.8 for centrifugation and resuspending with an equal volume of MS liquid.
3. The method for rapid and efficient tobacco hairy root induction and expansion according to claim 1, wherein the agrobacterium rhizogenes comprises any one of a4, R1000 and R1601.
4. The method for rapid and efficient induction and propagation of tobacco hairy roots according to claim 1, wherein leaves of tobacco soil-cultured seedlings are cut into 1-2 cm x 1-2 cm, added into an infection solution, infected for 5-10 min and then co-cultured.
5. The method for rapid and efficient induction and propagation of tobacco hairy roots according to claim 1, wherein the infected explants are transferred to an MS solid medium containing 50mg/mL of cephalo after dark culture is finished, and are cultured for 2-3 weeks.
6. The method for rapid and efficient induction and propagation of tobacco hairy roots according to claim 1, wherein the induction medium comprises the following components:
20 × macroelement mother liquor: NH (NH)4NO3 33.0 g，KNO3 38.0 g，MgSO4·7H2O 7.4 g，KH2PO4 3.4 g；
200 times microelement mother liquor: KI 0.166g, H3BO3 1.24 g，MnSO4·4H2O 4.46 g，ZnSO4·7H2O 1.72 g，Na2MnO·2H2O 0.05 g，CuSO4·5H2O 0.005 g，CoCl2·6H2O 0.005 g；
200 times ferric salt mother liquor: FeSO4·7H2O 5.56 g，EDTA-Na2·2H2O 7.46 g；
200 × organic mother liquor: inositol 20.0g, nicotinic acid 0.1g and VB6 0.1 g，VB10.1g of glycine and 0.4g of glycine;
20 × calcium salt mother liquor: CaC12 6.64 g；
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 times 50mL of macroelement mother liquor and 200 times 5mL of microelement mother liquor;
200 times 5mL of ferric salt mother liquor and 20 times 50mL of Ca salt mother liquor;
200 times 5mL of organic mother liquor, and adjusting the pH value to 5.8-6.2.
7. The method for rapid and efficient tobacco hairy root induction and propagation according to claim 1, wherein the tobacco soil-cultured seedling is a 4-week-old soil-cultured seedling.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994020606A1 (en) * | 1993-03-05 | 1994-09-15 | Celex Laboratories Inc. | Plant tissue culture method for taxol production |
| CN101121941A (en) * | 2007-03-26 | 2008-02-13 | 吉林师范大学 | Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system |
| CN101985633A (en) * | 2010-11-26 | 2011-03-16 | 毛清黎 | High-frequency induction and genetic transformation method for pear hairy roots of Agrobacterium rhizogene-mediated balsam |
| CN103695460A (en) * | 2013-12-06 | 2014-04-02 | 中国科学院西北高原生物研究所 | Method for obtaining plant hairy roots with high anthocyanin content |
| CN109315290A (en) * | 2018-11-14 | 2019-02-12 | 云南中烟工业有限责任公司 | A method of induction Hongda tobacco Hairy root and plant regeneration |
| CN111269931A (en) * | 2019-12-31 | 2020-06-12 | 贵州大学 | Agrobacterium rhizogenes-mediated flower tobacco hairy root induction transformation method |
-
2022
- 2022-04-02 CN CN202210342239.1A patent/CN114766356A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994020606A1 (en) * | 1993-03-05 | 1994-09-15 | Celex Laboratories Inc. | Plant tissue culture method for taxol production |
| CN101121941A (en) * | 2007-03-26 | 2008-02-13 | 吉林师范大学 | Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system |
| CN101985633A (en) * | 2010-11-26 | 2011-03-16 | 毛清黎 | High-frequency induction and genetic transformation method for pear hairy roots of Agrobacterium rhizogene-mediated balsam |
| CN103695460A (en) * | 2013-12-06 | 2014-04-02 | 中国科学院西北高原生物研究所 | Method for obtaining plant hairy roots with high anthocyanin content |
| CN109315290A (en) * | 2018-11-14 | 2019-02-12 | 云南中烟工业有限责任公司 | A method of induction Hongda tobacco Hairy root and plant regeneration |
| CN111269931A (en) * | 2019-12-31 | 2020-06-12 | 贵州大学 | Agrobacterium rhizogenes-mediated flower tobacco hairy root induction transformation method |
Non-Patent Citations (3)
| Title |
|---|
| 侯萌萌等: "发根农杆菌对不同烟草品种发状根诱导和培养的影响", 《贵州农业科学》 * |
| 吴英杰等: "农杆菌介导的烟草瞬时表达试验条件优化", 《东北林业大学学报》 * |
| 黄丽萍等: "发根农杆菌菌株和烟草品种对毛状根诱导率的影响", 《山地农业生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116636458A (en) * | 2023-03-24 | 2023-08-25 | 郑州轻工业大学 | Regeneration method of tobacco soil culture seedlings |
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