CN111280068A - Peanut tissue culture seedling bud elongation culture medium and preparation method and application thereof - Google Patents
Peanut tissue culture seedling bud elongation culture medium and preparation method and application thereof Download PDFInfo
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- CN111280068A CN111280068A CN202010284118.7A CN202010284118A CN111280068A CN 111280068 A CN111280068 A CN 111280068A CN 202010284118 A CN202010284118 A CN 202010284118A CN 111280068 A CN111280068 A CN 111280068A
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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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Abstract
The invention provides a peanut tissue culture seedling bud elongation culture medium and a preparation method and application thereof, belonging to the technical field of crop production. The peanut tissue culture seedling bud elongation culture Medium comprises a mixed solution and a coagulant, wherein each 1L of the mixed solution comprises 4-4.8 g of MS Medium culture Medium, 25-35 g of sucrose, 0.2-0.3 mL of 15-25 mmol/L6-benzyladenine solution, 0.05-0.15 mL of 8-12 mmol/L gibberellin solution and water; the coagulating agent comprises Phytagel plant gel and/or agar. The peanut tissue culture seedling bud elongation culture medium provided by the invention has a good effect of promoting elongation of the peanut tissue culture seedling bud. After 1 month of bud elongation culture: the elongation rate of the cluster buds treated by the transgenosis can reach 4.11; the elongation rate of the cluster buds which are not subjected to transgenic treatment can reach 4.89.
Description
Technical Field
The invention belongs to the technical field of crop production, and particularly relates to a peanut tissue culture seedling bud elongation culture medium and a preparation method and application thereof.
Background
The peanut tissue culture has important significance for peanut variety improvement, new variety propagation, genetic transformation, germplasm preservation, resistant mutant screening and the like. Because the regeneration of peanuts is difficult and has genotype specificity, the research progress is slow, and particularly, a system which is efficient and stable and can continuously obtain regenerated plants needs to be further researched.
The elongation of the cluster buds in peanut tissue culture is directly related to the time from cluster bud induction to root induction and the transfer survival rate, and is a very key step. Peanut tissue culture has four critical periods in which the clumpy buds are extended approximately 1/4 times the growth cycle. The cluster buds just induced are small and have short internodes, and grow slowly in a bud induction culture medium, and can obtain normal plants only by being inoculated into a bud elongation culture medium for proliferation and elongation. If the elongation growth of buds can not be realized, the buds are easy to dry and die. Whether the bud can well elongate and grow directly determines the rooting condition of the tissue culture seedling. At present, the elongation of the clustered shoots of peanut tissue culture is difficult, the elongation speed is slow, the multiplication coefficient is low, and the elongation rate is low. Leaves are easy to yellow and fall off, the base tissues are browned, the seedlings die, the plant regeneration rate is low, and a set of efficient and stable cluster bud elongation method is lacked.
Disclosure of Invention
In view of the problems in the background art, the invention aims to provide a peanut tissue culture seedling bud elongation culture medium, a preparation method and application thereof, and the elongation of multiple buds of a peanut tissue culture seedling is improved.
The invention provides a peanut tissue culture seedling bud elongation culture Medium which comprises a mixed solution and a coagulant, wherein each 1L of the mixed solution comprises 4-4.8 g of MS Medium culture Medium, 25-35 g of cane sugar, 0.2-0.3 mL of 15-25 mmol/L6-benzyl adenine solution, 0.05-0.15 mL of 8-12 mmol/L gibberellin solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
Preferably, each 1L of the mixed solution comprises 4.4g of MS Medium Medium, 30g of sucrose, 0.25mL of a 20 mmol/L6-benzyladenine solution, 0.1mL of a 10mmol/L gibberellin solution, and water.
Preferably, 2-8 g of coagulant is added to 1L of the mixed solution.
Preferably, 2-4 g of Phytagel plant gel and 0.4-1.2 g of agar are added to 1L of the mixed solution.
The invention also provides a preparation method of the bud elongation culture medium, which comprises the following steps:
(1) mixing an MS Medium culture Medium, sucrose, a 6-benzyladenine solution and a gibberellin solution to obtain a basic mixture;
(2) mixing the basic mixture with water to obtain a mixed solution;
(3) and mixing the mixed solution with a coagulant, and sterilizing to obtain the bud elongation culture medium.
Preferably, after the basic mixture is obtained in the step (1), a reagent is used for adjusting the pH value of the basic mixture to be 5.6-6.0.
Preferably, the reagent is a 1mol/L NaOH solution.
Preferably, the sterilization in the step (3) adopts high-temperature high-pressure sterilization; the sterilization pressure is 90-110 kpa; the sterilization temperature is 118-125 ℃.
Preferably, after the sterilization in the step (3), adding cefotaxime and/or hygromycin to the sterilized materials; the addition amount of the cefotaxime is as follows: adding 400-600 mu L of 400-600 mol/L cefotaxime into every 1L of sterilized material; the addition amount of the hygromycin is as follows: and adding 0.5-1.5 mL of hygromycin of 40-60 mg/mL into each 1L of sterilized material.
The invention also provides application of the bud elongation culture medium in peanut tissue culture seedling planting.
Has the advantages that: in order to solve the problems of difficult elongation, slow elongation speed, low multiplication coefficient, low elongation rate and the like of the peanut tissue culture cluster buds in the peanut tissue culture, the invention provides an optimized peanut tissue culture seedling bud elongation culture medium by adjusting components such as coagulants, sucrose, auxin and the like in the culture medium. Wherein, the 6-benzyl adenine plays the role of cytokinin, and the addition of the 6-benzyl adenine in a differentiation medium is beneficial to adventitious bud elongation and plant regeneration. Gibberellin is used for promoting cell elongation, rapidly promoting elongation of differentiated cluster buds and shortening elongation time. 6-benzyladenine and gibberellin are combined for use, so that the synergistic effect is achieved.
In the bud elongation culture medium provided by the invention, all components are matched for use and mutually promoted, so that the bud elongation culture medium has a good effect of promoting the bud elongation of the peanut tissue culture seedling. After 1 month of bud elongation culture: the elongation rate of the cluster buds treated by the transgenosis can reach 4.11; the elongation rate of the cluster buds which are not subjected to transgenic treatment can reach 4.89. The bud elongation culture medium provided by the invention can effectively shorten the elongation period and has good application prospect in tissue rapid propagation.
Detailed Description
The invention provides a peanut tissue culture seedling bud elongation culture Medium which comprises a mixed solution and a coagulant, wherein the mixed solution comprises an MS Medium culture Medium, cane sugar, a 6-benzyladenine solution, a gibberellin solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
In the invention, the content of the MS Medium culture Medium in each 1L of the mixed solution is 4-4.8 g, preferably 4.4 g; the content of sucrose in each 1L of mixed solution is 25-35 g, preferably 30 g; the content of the 6-benzyladenine solution in each 1L of the mixed solution is 0.2-0.3 mL, preferably 0.25 mL; the concentration of the 6-benzyladenine solution is 15-25 mmol/L, preferably 20 mmol/L; the content of the gibberellin solution in each 1L of mixed solution is 0.05-0.15 mL, and 0.1mL is preferred; the concentration of the gibberellin solution is 8-12 mmol/L, and preferably 10 mmol/L; the sources of the above components are not particularly limited in the present invention, and any products conventionally commercially available in the art may be used.
In the present invention, the coagulating agent comprises Phytagel and/or agar, preferably Phytagel and agar. In the invention, 2-8 g of coagulant is preferably added into every 1L of mixed solution; more preferably, 2-4 g of Phytagel plant gel and 0.4-1.2 g of agar are added into each 1L of mixed solution; more preferably, about 3g of Phytagel plant gel and about 1g of agar are added to 1L of the mixed solution. The sources of the above-mentioned Phytagel plant gel or agar are not particularly limited in the present invention, and any of those commercially available products which are conventional in the art can be used.
In the bud elongation culture medium provided by the invention, all components are matched for use and are mutually promoted, so that the bud elongation culture medium has a good peanut tissue culture seedling bud elongation effect, and can greatly promote the elongation of cluster buds of peanut tissue culture seedlings.
The invention provides a preparation method of the bud elongation culture medium, which comprises the following steps:
(1) mixing an MS Medium culture Medium, sucrose, a 6-benzyladenine solution and a gibberellin solution to obtain a basic mixture;
(2) mixing the basic mixture with water to obtain a mixed solution;
(3) mixing the mixed solution with a coagulant, and sterilizing to obtain a bud elongation culture medium
According to the invention, an MS Medium culture Medium, sucrose, a 6-benzyladenine solution and a gibberellin solution are mixed according to the proportion of the bud elongation culture Medium to obtain a basic mixture. Before mixing, the present invention preferably filter-sterilizes the 6-benzyladenine solution and the gibberellin solution; filtering, sterilizing, and storing in plastic tube at-20 deg.C or 4 deg.C. The present invention does not specifically limit the mixing method in this step, and any conventional method in the art may be used.
After obtaining the base mixture, the present invention preferably adjusts the pH of the base mixture with an agent. In the present invention, the pH adjusting agent is preferably a NaOH solution, and the concentration of the NaOH solution is preferably 1 mol/L. The pH value of the basic mixture after the pH value is adjusted is preferably 5.6-6.0, and more preferably 5.8.
The present invention mixes the base mixture with water to obtain a mixed solution. After the mixed solution is obtained, the mixed solution is mixed with the coagulant. The present invention does not specifically limit the mixing method in this step, and any conventional method in the art may be used.
After the mixed solution is mixed with the coagulant, the invention preferably adopts a high-temperature and high-pressure mode for sterilization. In the invention, the pressure for sterilization is preferably 90-110 kpa, more preferably 100 kpa; the sterilization temperature is preferably 118-125 ℃, and more preferably 121 ℃. The sterilization time is 20-40 min, preferably 30 min. After sterilization, obtaining a sterilized material; the sterilized material is the bud elongation culture medium.
According to the invention, after the sterilized material is cooled, cefotaxime and/or hygromycin are preferably added to the sterilized material, and more preferably, cefotaxime and hygromycin are added. In the invention, the cooling time is preferably 30-50 min, and more preferably 40-45 min. In the present invention, the addition amount of cefotaxime is preferably: 400-600 mu L of 400-600 mol/L cefotaxime is added into every 1L of sterilization material, and more preferably: 500 mu L of 500mol/L cefotaxime is added into every 1L of sterilized materials; the addition amount of hygromycin is preferably as follows: adding 0.5-1.5 mL of hygromycin of 40-60 mg/mL into each 1L of sterilized material, and preferably: 1mL of 50mg/mL hygromycin was added per 1L of sterilized material. The sources of cefotaxime and hygromycin are not particularly limited in the present invention, and any product conventionally commercially available in the art may be used.
The bud elongation culture medium prepared by the invention is preferably divided into different volumes for storage according to requirements. The preparation and storage temperature is preferably 2-6 ℃, and more preferably 4 ℃. When the microwave heating device is to be used, the microwave heating mode is preferably adopted to restore the use temperature.
The invention also provides application of the bud elongation culture medium in peanut tissue culture seedling planting. The invention is not particularly limited with respect to the specific operational details of the application.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A stem tip explant of peanut variety No. 26 cultured for 14 days is taken, is infected by agrobacterium and then is inoculated on a bud induction culture medium to induce cluster buds, when the cluster buds grow to about 0.5-1cm, the cluster buds are transferred to a bud elongation culture medium to grow, and the cluster buds are subjected to elongation and multiplication culture. Subcultured every 2 weeks. During the first subculture, the bud clusters are not cut as much as possible, and only the browned tissues at the bottom of the bud clusters need to be cleaned. The browning phenomenon is that the polyphenol oxidase is activated in plant tissues, so that the cell metabolism is changed, and the phenolic substances are oxidized to generate brown quinone substances which gradually diffuse into a culture medium to poison the cultured materials, so that the browning death of the cultured materials is caused. When subculture is carried out subsequently, the cluster buds growing better can be divided into single plants for culture, and some cluster buds which are not formed can be divided into several bud clusters for culture. The partially browned tissue and the yellowing leaves were excised, and the bottom callus was still retained. Clumpy buds that are too small are not amenable to cutting, with minimal damage to the clumpy buds. When the cluster buds are extended to 3-5cm and the shoots are well differentiated, cutting off the callus, only leaving the cluster seedlings, and transferring the cluster seedlings to a root induction culture medium for rooting culture.
Induced 80 clumpy shoot calli were transferred to shoot elongation medium, with results: the multiple shoots proliferate and elongate within 1 month, the number of the proliferated multiple shoots is 625, and the proliferation coefficient is 7.81; the number of elongation cluster buds (bud height is more than or equal to 2cm) is 329, and the elongation is 4.11. The cluster buds have the advantages of high multiplication coefficient, high elongation, quick elongation, vigorous growth, strong bud bodies, obvious differentiation of stem leaves, thick stems, light green and glossy leaf colors, more effective buds, quick seedling formation and high regeneration frequency.
Elongation rate is the number of the cluster buds (the bud height is more than or equal to 2 cm)/the number of the inoculated calluses
Multiplication coefficient is the number of clumpy buds after multiplication/number of inoculated callus
The bud elongation culture medium is prepared from the following reagents: MS Medium (MS Medium, MDBio, Inc, Lot:1270401)4.4g, sucrose 30g, 20 mmol/L6-BA (6-benzylaminopurine, 6-benzyladenine, Sigma, Lot: WXBB5831V)250uL, 10mmol/L GA3(Gibberellic acid, gibberellin, Sigma, Lot: BCBR 3974) 100uL, adjusting the pH to 5.8 by using 1mol/L NaOH, and adding water to a constant volume of 1L. Then 3g of Phytagel (plant gel, sigma, Lot: WXBC9170V) and 1g of agar are added. 100kpa, 121 ℃, autoclaved for 30 minutes. After sterilization was complete and cooling was carried out for 40 minutes, 500. mu.L of 500mmol/L cefotaxime sodium salt and 1mL hygromycin (50mg/mL) were added. The prepared culture medium is subpackaged into glass culture dishes with the diameter of 125 mm.
The cefotaxime sodium salt has the function of removing bacterial contamination. Preparation method of 500mmol/L mother liquor of cefotaxime sodium salt (cefotaxime sodium: MDBio, Inc, Lot: Z7060501): 11.93g cefotaxime sodium salt, dissolved in 50mL water, filter sterilized. The mixture was dispensed into sterile 1.5mL EP tubes and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
The hygromycin is used for screening the transgenic tissue culture seedlings, and is not added into the non-transgenic tissue culture seedlings. 50mg/ml hygromycin (hygromycin B) mother liquor: the concentration at the time of purchase was 50mg/ml, and filtration sterilization was required at the time of use (Solarbio Co., Lot: 513B 041).
Preparation method of 20 mmol/L6-BA (6-benzylaminopurine, 6-benzyladenine, Sigma, Lot: WXBB5831V) mother liquor: 0.11g of 6-BA, 25mL of water, was dissolved in 1mL of 1mol/L NaOH, and the solution was subjected to filtration sterilization. The mixture was divided into portions and put into sterile 15mL plastic tubes and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
10mmol/L GA3(Gibberellic acid, gibberellin, Sigma, Lot: BCBR 3974) mother liquor preparation method: 0.1732g GA3The resulting solution was dissolved in 5mL of 100% ethanol, and water was added to the solution to a volume of 50 mL. And (5) filtering and sterilizing. Put into a sterile 50mL plastic tube and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
Example 2
Inoculating the stem tip explant of peanut variety No. 23 cultured for 14 days on a bud induction culture medium, inducing cluster buds, transferring the cluster buds to a bud elongation culture medium for growth when the cluster buds grow to about 0.5-1cm, and performing elongation and multiplication culture on the cluster buds. Subcultured every 2 weeks. During the first subculture, the bud clusters are not cut as much as possible, and only the browned tissues at the bottom of the bud clusters need to be cleaned. The browning phenomenon is that the polyphenol oxidase is activated in plant tissues, so that the cell metabolism is changed, and the phenolic substances are oxidized to generate brown quinone substances which gradually diffuse into a culture medium to poison the cultured materials, so that the browning death of the cultured materials is caused. When subculture is carried out subsequently, the cluster buds growing better can be divided into single plants for culture, and some cluster buds which are not formed can be divided into several bud clusters for culture. The partially browned tissue and the yellowing leaves were excised, and the bottom callus was still retained. Clumpy buds that are too small are not amenable to cutting, with minimal damage to the clumpy buds. When the cluster buds are extended to 3-5cm and the shoots are well differentiated, cutting off the callus, only leaving the cluster seedlings, and transferring the cluster seedlings to a root induction culture medium for rooting culture.
Induced 85 clumpy shoot calli were transferred to shoot elongation medium, with results: proliferating and extending the cluster buds within 1 month, wherein the number of the proliferated cluster buds is 698, and the proliferation coefficient is 8.21; the number of the extended cluster buds (the height of the buds is more than or equal to 2cm) is 416, and the elongation is 4.89. The cluster buds have the advantages of high multiplication coefficient, high elongation, quick elongation, vigorous growth, strong bud bodies, obvious differentiation of stem leaves, thick stems, light green and glossy leaf colors, more effective buds, quick seedling formation and high regeneration frequency.
Elongation rate is the number of the cluster buds (the bud height is more than or equal to 2 cm)/the number of the inoculated calluses
Multiplication coefficient is the number of clumpy buds after multiplication/number of inoculated callus
The bud elongation culture medium is prepared from the following reagents: MS Medium (MS Medium, MDBio, Inc, Lot:1270401)4.4g, sucrose 30g, 20 mmol/L6-BA (6-benzylaminopurine, 6-benzyladenine, Sigma, Lot: WXBB5831V)250uL, 10mmol/L GA3(Gibberellic acid, gibberellin, Sigma, Lot: BCBR3974V)100uL, pH adjusted to 5.8 using 1mol/L NaOH, and water was added to make a volume of 1L. Then 3g of Phytagel (plant gel, sigma, Lot: WXBC9170V) and 1g of agar are added. 100kpa, 121 ℃, autoclaved for 30 minutes. After sterilization was complete and cooling was carried out for 40 minutes, 500. mu.L of 500mmol/L cefotaxime sodium salt was added. The prepared culture medium is subpackaged into glass culture dishes with the diameter of 125 mm.
The cefotaxime sodium salt has the function of removing bacterial contamination. Preparation method of 500mmol/L mother liquor of cefotaxime sodium salt (cefotaxime sodium: MDBio, Inc, Lot: Z7060501): 11.93g cefotaxime sodium salt, dissolved in 50mL water, filter sterilized. The mixture was dispensed into sterile 1.5mL EP tubes and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
Preparation method of 20 mmol/L6-BA (6-benzylaminopurine, 6-benzyladenine, Sigma, Lot: WXBB5831V) mother liquor: 0.11g of 6-BA, 25mL of water, was dissolved in 1mL of 1mol/L NaOH, and the solution was subjected to filtration sterilization. The mixture was divided into portions and put into sterile 15mL plastic tubes and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
10mmol/L GA3(Gibberellic acid, gibberellin, Sigma, Lot: BCBR 3974) mother liquor preparation method: 0.1732g GA3The resulting solution was dissolved in 5mL of 100% ethanol, and water was added to the solution to a volume of 50 mL. And (5) filtering and sterilizing. Put into a sterile 50mL plastic tube and frozen at-20 ℃. Or can be placed in a refrigerator at 4 deg.C, and taken out for use when needed.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The peanut tissue culture seedling bud elongation culture medium comprises a mixed solution and a coagulant, and is characterized in that every 1L of the mixed solution comprises 4-4.8 g of MSMedium culture medium, 25-35 g of cane sugar, 0.2-0.3 mL of 15-25 mmol/L6-benzyl adenine solution, 0.05-0.15 mL of 8-12 mmol/L gibberellin solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
2. The shoot elongation medium of claim 1, wherein each 1L of the mixed solution comprises 4.4g of MSMedium medium, 30g of sucrose, 0.25mL of a 20mmol/L solution of 6-benzyladenine, 0.1mL of a 10mmol/L solution of gibberellin, and water.
3. The shoot elongation culture medium according to claim 1 or 2, wherein 2 to 8g of the coagulant is added to 1L of the mixed solution.
4. The shoot elongation medium according to claim 3, wherein 2 to 4g of Phytagel plant gel and 0.4 to 1.2g of agar are added to 1L of the mixed solution.
5. The method for producing a shoot elongation medium according to any one of claims 1 to 4, comprising the steps of:
(1) mixing an MSMedium culture medium, sucrose, a 6-benzyladenine solution and a gibberellin solution to obtain a basic mixture;
(2) mixing the basic mixture with water to obtain a mixed solution;
(3) and mixing the mixed solution with a coagulant, and sterilizing to obtain the bud elongation culture medium.
6. The method according to claim 5, wherein the pH of the base mixture obtained in step (1) is adjusted to 5.6 to 6.0 with a reagent.
7. The method according to claim 6, wherein the reagent is a 1mol/L NaOH solution.
8. The method according to claim 5, wherein the sterilization in the step (3) is performed by autoclaving; the sterilization pressure is 90-110 kpa; the sterilization temperature is 118-125 ℃.
9. The process according to claim 5, wherein after the sterilization in the step (3), cefotaxime and/or hygromycin are added to the sterilized materials; the addition amount of the cefotaxime is as follows: adding 400-600 mu L of 400-600 mol/L cefotaxime into every 1L of sterilized material; the addition amount of the hygromycin is as follows: and adding 0.5-1.5 mL of hygromycin of 40-60 mg/mL into each 1L of sterilized material.
10. The bud elongation culture medium of any one of claims 1 to 4 and the bud elongation culture medium prepared by the preparation method of any one of claims 5 to 9 are applied to planting of peanut tissue culture seedlings.
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