CN110786245A - Peanut tissue culture seedling root induction culture medium and preparation method and application thereof - Google Patents
Peanut tissue culture seedling root induction culture medium and preparation method and application thereof Download PDFInfo
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- CN110786245A CN110786245A CN201911297169.7A CN201911297169A CN110786245A CN 110786245 A CN110786245 A CN 110786245A CN 201911297169 A CN201911297169 A CN 201911297169A CN 110786245 A CN110786245 A CN 110786245A
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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
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Abstract
The invention provides a peanut tissue culture seedling root induction culture medium and a preparation method and application thereof, belonging to the technical field of crop production. The peanut tissue culture seedling root induction culture Medium comprises a mixed solution and a coagulant, wherein each 1L of the mixed solution comprises 2-2.5 g of MS Medium culture Medium, 12-18 g of cane sugar, 0.8-1.2 mL of 4-6 mmol/L NAA solution, 200-300 mu L of 8-12 mmol/L IBA solution and water; the coagulating agent comprises Phytagel plant gel and/or agar. The peanut tissue culture seedling root induction culture medium provided by the invention has a good peanut tissue culture seedling rooting induction effect and is high in rooting rate. By using the root induction culture medium provided by the invention, the obtained tissue culture seedling is hardened, the transplanting survival rate is more than 95%, and the plump fruit rate is more than 90%.
Description
Technical Field
The invention belongs to the technical field of crop production, and particularly relates to a peanut tissue culture seedling root induction culture medium, and a preparation method and application thereof.
Background
Tissue culture is a highly efficient biotechnological approach. The key of peanut breeding and genetic transformation is to construct a peanut high-frequency plant regeneration system by using a tissue culture technology. The exploration and the improvement of a high-frequency plant regeneration system have important theoretical and practical application values.
The rooting efficiency of the tissue culture seedling is directly related to the survival rate and the growth condition of the transplanted transformed plant, and is an important circle influencing the genetic transformation effect. After the tissue culture seedling takes root, hardening and transplanting can be carried out. The hardening and transplanting of the peanut tissue culture seedlings are key links from successful popularization of peanut tissue culture to field planting. If indoor hardening of the tissue culture seedlings is not performed, the tissue culture seedlings are directly transited from a constant laboratory environment to a changing external natural environment, and the tissue culture seedlings are burnt and dehydrated to die easily due to sudden changes of temperature, humidity, illumination intensity and gas environment, so that the survival rate is very low.
At present, a set of efficient rooting, seedling hardening and transplanting methods is lacked in peanut tissue culture, the rooting rate is low, the number of roots is small, and the survival rate of later-stage transplanting is low.
Disclosure of Invention
In view of the problems in the background art, the invention aims to provide a peanut tissue culture seedling root induction culture medium, a preparation method and application thereof, and the rooting rate and the transplanting survival rate of the peanut tissue culture seedling are improved.
The invention provides a peanut tissue culture seedling root induction culture Medium which comprises a mixed solution and a coagulant, wherein each 1L of the mixed solution comprises 2-2.5 g of MS Medium culture Medium, 12-18 g of cane sugar, 0.8-1.2 mL of 4-6 mmol/L NAA solution, 200-300 mu L of 8-12 mmol/L IBA solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
Preferably, each 1L of the mixed solution comprises 2.2g of MS Medium Medium, 15g of sucrose, 1mL of 5mmol/L NAA solution, 250. mu.L of 10mmol/L IBA 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 1-3 g of agar are added to 1L of the mixed solution.
The invention provides a preparation method of the root induction culture medium, which comprises the following steps:
(1) mixing MS Medium culture Medium, sucrose, NAA solution and IBA solution to obtain 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 root induction 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.
The reagent is preferably 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 root induction culture medium in peanut tissue culture seedling planting.
Preferably, the application comprises the steps of tissue culture seedling root induction, seedling hardening and transplanting.
Has the advantages that: in order to solve the problems that the tissue culture seedling has short rooting and is difficult to survive in transplanting in peanut tissue culture, the invention provides an optimized peanut tissue culture seedling root induction culture medium by adjusting components such as coagulants, sucrose, auxin and the like in the culture medium.
The root induction culture Medium provided by the invention comprises a mixed solution and a coagulant, wherein each 1L of the mixed solution comprises 2-2.5 g of MS Medium culture Medium, 12-18 g of sucrose, 0.8-1.2 mL of 4-6 mmol/L NAA solution, 200-300 mu L of 8-12 mmol/L IBA solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
Wherein the agar is extracted from seaweed such as Eucheuma Gelatinosum, mainly comprises agarose and agar pectin, and has the advantages of high transparency, good water-retaining property, no toxicity and stable structure after the agar forms gel; phytagel plant gel is a substitute of agar secreted from pseudomonas, is a mixture of glucuronic acid, rhamnose and glucose, has the characteristics of colorlessness, transparency and high toughness, and has a remarkable anti-browning effect; the sucrose has a certain effect in plant tissue differentiation, influences the growth and germination of plant roots and can obviously improve the rooting effect of tissue culture seedlings; IBA and NAA are auxin for inducing plant to root.
In the root induction culture medium provided by the invention, all components are matched for use and mutually promoted, so that the root induction culture medium has a good peanut tissue culture seedling rooting induction effect, can greatly promote the rooting rate of the peanut tissue culture seedling, and improves the later stage transplanting survival rate. By using the root induction culture medium provided by the invention, the obtained tissue culture seedling is hardened, the transplanting survival rate is more than 95%, and the plump fruit rate is more than 90%.
Detailed Description
The invention provides a peanut tissue culture seedling root induction culture Medium which comprises a mixed solution and a coagulant, wherein the mixed solution comprises an MS Medium culture Medium, cane sugar, NAA, IBA and water; the coagulating agent comprises Phytagel plant gel and/or agar.
In the invention, the content of MS Medium culture Medium in each 1L of mixed solution is 2-2.5 g, preferably 2.2 g; the content of sucrose in each 1L of mixed solution is 12-18 g, preferably 15 g; the content of the NAA solution in each 1L of the mixed solution is 0.8-1.2 mL, preferably 1 mL; the concentration of the NAA solution is 4-6 mmol/L, preferably 5 mmol/L; the content of IBA solution in each 1L of mixed solution is 200-300 mu L, preferably 250 mu L; the concentration of the IBA solution is 8-12 mmol/L, 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 a more specific embodiment of the invention, the MS Medium is purchased from the company MDBio, Inc, Lot: 1270401.
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 1-3 g of agar are added into 1L of the mixed solution; more preferably, 3g of Phytagel plant gel and 2g 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 root induction culture medium provided by the invention, all components are matched for use and are mutually promoted, so that the root induction culture medium has a good peanut tissue culture seedling rooting induction effect and can greatly promote the rooting rate of the peanut tissue culture seedling.
The invention provides a preparation method of the root induction culture medium, which comprises the following steps:
(1) mixing MS Medium culture Medium, sucrose, NAA solution and IBA solution to obtain 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 root induction culture medium
According to the invention, an MS Medium culture Medium, cane sugar, an NAA solution and an IBA solution are mixed according to the proportion of the root induction culture Medium to obtain a basic mixture. 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 adjusted pH value is more suitable for the growth of the peanut tissue culture seedlings in the invention.
After the pH value is adjusted, the basic mixture is mixed with water to obtain a mixed solution. In the invention, the mixing in the step preferably adopts a constant volume device, the basic mixture is firstly added into a volumetric flask, and then water is added to the volumetric flask according to the proportion of the root induction culture medium.
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 mixture is preferably sterilized at high temperature and high pressure. 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 ℃. After sterilization, obtaining a sterilized material; the sterilized material is the root induction culture medium.
According to the invention, after the sterilized material is cooled, cefotaxime and/or hygromycin are preferably added to the sterilized material. 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 cefotaxime acts to remove bacterial contamination. 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 hygromycin is used for screening the transgenic tissue culture seedlings, and is not added into the non-transgenic tissue culture seedlings. In the present invention, the hygromycin is used for screening transgenic peanut positive seedlings. 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. In a more specific embodiment of the invention, the cefotaxime and hygromycin are purchased from Solarbil, ltno. 513b041.
The root induction culture medium prepared by the invention is preferably subpackaged 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 root induction culture medium in peanut tissue culture seedling planting. The application preferably comprises the steps of tissue culture seedling root induction, seedling hardening and transplanting. The seedling hardening aims at inducing the stem and leaf of the tissue culture seedling to protect tissues and recovering the water-gas regulation function of pore opening and closing, and ensures higher survival rate after transplanting.
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
Transferring the transgenic cluster seedlings of the peanut variety No. 26 to a root induction culture medium when the seedlings grow to 3-5 cm. At the time of transfer, the callus was cut off, leaving only clumpy seedlings. If the yellow leaves exist, the yellow leaves are cut off by a knife to avoid bacterial contamination.
The root induction culture medium is prepared from the following reagents: MS Medium (MS Medium, MDBio Inc.) 2.2g, sucrose 15g, 5mM NAA (Naphthaleneacetic acid) 1mL, 10mM IBA (indolebutyric acid) 250. mu.L, pH adjusted to 5.8 using 1N NaOH, and water was added to the solution to make a volume of 1L. Then 3g of Phytagel (plant gel) and 2g of agar are added. Autoclaved at 100kpa at 121 ℃ for 30 minutes. After sterilization was complete, it was cooled for 40 minutes, and then 500. mu.L of 500mM cefotaxime and 1mL hygromycin (50mg/mL) were added. The prepared culture medium can be subpackaged into different volumes according to requirements for storage, placed in a freezer at 4 ℃ and used by microwave heating at the later stage.
Transferring the selected 42 experimental materials of the cluster seedlings to a root induction culture medium, and obtaining the result: rooting in 2-3 weeks with a rooting rate of 100%. In 1 month of growth on the root induction culture medium, 97 percent of tissue culture seedlings have more than 3 roots and the root length is more than 2 cm. The grown adventitious roots are thick and long, and the growth state of capillary roots is good.
Example 2
The peanut tissue culture seedlings with more than two internodes, normal leaf color, more than 3 roots and more than 2cm root length obtained in example 1 were acclimatized.
Stirring matrix (mixture of 7/10 Cui Yun nutritive soil, 1/5 sandy soil and 1/10 coarse-grained vermiculite) with sterile water until water does not leak, wrapping with sterilizing bag, and sterilizing. Spreading a layer of newspaper on the bottom of small flowerpot (5 cm long, 5cm wide, 7cm high, with water-permeable hole at the bottom), separately filling into sterilized matrix, and compacting. The clumped seedlings were removed from the medium using forceps. The residual medium on the roots was rinsed clean with sterile water. Pricking holes in the center of the flowerpot by using tweezers, and putting the plantlets in the flowerpot. The substrate was lightly covered and slightly compacted. A small amount of 0.4g/L Hoagland medium nutrient solution was poured. The small flowerpot is sleeved in a plastic bag and put into an illumination incubator (humidity is 75-80%, illumination intensity is 3000Lx, and temperature is 25 ℃). Growing for 2 weeks without pouring sterile water and nutrient solution. After two weeks, elongation or growth of the upper half of the clumped seedlings was observed, and hardening of seedlings was started. Taking out peanut seedlings from the illumination incubator in the daytime, putting the peanut seedlings into room temperature, opening the plastic bag and hardening the seedlings. Sealing the plastic bag at night, and placing the plastic bag into an incubator. Water was poured every two days. After 4 days, it is ready for transplantation into the greenhouse. One day before transplanting, the seedlings are still placed at room temperature in the daytime, and sufficient water is poured at night.
Stirring matrix (mixture of 7/10 Cui Yun nutritive soil, 1/5 sandy soil and 1/10 coarse granular vermiculite) with sterile water until water does not leak, wrapping with sterilizing bag, sterilizing, and packaging into large flowerpot (diameter of 40cm, height of 35cm, and bottom with water-permeable hole). When transplanting, digging a hole in the center of the large flowerpot substrate, taking out the whole plant with soil from the small flowerpot, putting the plant in the center of the hole, covering the substrate, and compacting. 3 small sticks are inserted into the big flowerpot, and a plastic bag with 2-angle cut openings at the top end is sleeved on the big flowerpot. After 3 days of transplantation, the plants were watered by infiltration from the bottom and carbendazim (final concentration 0.01g/ML) was added to the water. And then watered every 3 days. After transplanting, 40 peanut seedlings (95%) survive and normally bloom, each peanut seedling has 8-10 fruits on average, and the plumpness rate is about 90%.
Example 3
And transferring the cluster seedlings of the peanut variety No. 33 to a root induction culture medium when the cluster seedlings grow to 3-5 cm. At the time of transfer, the callus was cut off, leaving only clumpy seedlings. If the yellow leaves exist, the yellow leaves are cut off by a knife to avoid bacterial contamination.
The root induction culture medium is prepared from the following reagents: MS Medium (MS Medium, MDBio Inc.) 2.2g, sucrose 15g, 5mM NAA (Naphthylacetic acid) 1ML, 10mM IBA (indolebutyric acid) 250. mu.L, pH adjusted to 5.8 using 1N NaOH, and water was added to the solution to make 1L. Then 3g of Phytagel (plant gel) and 2g of agar are added. Autoclaved at 100kpa at 121 ℃ for 30 minutes. After sterilization was complete, it was cooled for 40 minutes and then 500. mu.L of 500mM cefotaxime was added. The prepared culture medium can be subpackaged into different volumes according to requirements for storage, placed in a freezer at 4 ℃ and used by microwave heating at the later stage.
Transferring 50 selected experimental materials of the cluster seedlings to a root induction culture medium, and obtaining the result: rooting in 2-3 weeks with a rooting rate of 100%. 99 percent of tissue culture seedlings grow on the root induction culture medium within 1 month, the number of the roots is more than 3, and the root length is more than 2 cm. The grown adventitious roots are thick and long, and the growth state of capillary roots is good.
Example 4
The peanut tissue culture seedlings with more than two internodes, normal leaf color, more than 3 roots and more than 2cm root length obtained in example 3 were acclimatized.
Stirring matrix (mixture of 7/10 Cui Yun nutritive soil, 1/5 sandy soil and 1/10 coarse-grained vermiculite) with sterile water until water does not leak, wrapping with sterilizing bag, and sterilizing. Spreading a layer of newspaper on the bottom of small flowerpot (5 cm long, 5cm wide, 7cm high, with water-permeable hole at the bottom), separately filling into sterilized matrix, and compacting. The clumped seedlings were removed from the medium using forceps. The residual medium on the roots was rinsed clean with sterile water. Pricking holes in the center of the flowerpot by using tweezers, and putting the plantlets in the flowerpot. The substrate was lightly covered and slightly compacted. A small amount of 0.4g/L Hoagland medium nutrient solution was poured. The small flowerpot is sleeved in a plastic bag and put into a light incubator (humidity is 75-80%, light intensity is 3000Lx, temperature is 25 ℃). Growing for 2 weeks without pouring sterile water and nutrient solution. After two weeks, elongation or growth of the upper half of the clumped seedlings was observed, and hardening of seedlings was started. Taking out peanut seedlings from the illumination incubator in the daytime, putting the peanut seedlings into room temperature, opening the plastic bag and hardening the seedlings. Sealing the plastic bag at night, and placing the plastic bag into an incubator. Water was poured every two days. After 4 days, it is ready for transplantation into the greenhouse. One day before transplanting, the seedlings are still placed at room temperature in the daytime, and sufficient water is poured at night.
Stirring matrix (mixture of 7/10 Cui Yun nutritive soil, 1/5 sandy soil and 1/10 coarse granular vermiculite) with sterile water until water does not leak, wrapping with sterilizing bag, sterilizing, and packaging into large flowerpot (diameter of 40cm, height of 35cm, and bottom with water-permeable hole). When transplanting, digging a hole in the center of the large flowerpot substrate, taking out the whole plant with soil from the small flowerpot, putting the plant in the center of the hole, covering the substrate, and compacting. 3 small sticks are inserted into the big flowerpot, and a plastic bag with 2-angle cut openings at the top end is sleeved on the big flowerpot. After 3 days of transplantation, the plants were watered by infiltration from the bottom and carbendazim (final concentration 0.01g/ML) was added to the water. And then watered every 3 days. After transplanting, 49 peanut seedlings (98%) survive and normally bloom, each peanut seedling has 7-9 fruits on average, and the plumpness rate is about 91%.
The embodiments show that the peanut tissue culture seedling root induction culture medium provided by the invention has a good peanut tissue culture seedling rooting induction effect and a high rooting rate. By using the root induction culture medium provided by the invention, the obtained tissue culture seedling is hardened, the transplanting survival rate is more than 95%, and the plump fruit rate is more than 90%.
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. A peanut tissue culture seedling root induction culture Medium comprises a mixed solution and a coagulant, and is characterized in that every 1L of the mixed solution comprises 2-2.5 g of MS Medium culture Medium, 12-18 g of cane sugar, 0.8-1.2 mL of 4-6 mmol/L NAA solution, 200-300 mu L of 8-12 mmol/L IBA solution and water; the coagulating agent comprises Phytagel plant gel and/or agar.
2. The root induction medium according to claim 1, wherein each 1L of the mixed solution comprises 2.2g of MSMedium medium, 15g of sucrose, 1mL of NAA solution of 5mmol/L, 250 μ L of IBA solution of 10mmol/L and water.
3. The root-induction 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 root induction medium according to claim 3, wherein 2 to 4g of Phytagel plant gel and 1 to 3g of agar are added to 1L of the mixed solution.
5. The method for preparing a root induction medium according to any one of claims 1 to 4, comprising the steps of:
(1) mixing MS Medium culture Medium, sucrose, NAA solution and IBA solution to obtain 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 root induction 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 root induction culture medium of any one of claims 1 to 4 and the root induction culture medium prepared by the preparation method of any one of claims 5 to 9 are applied to peanut tissue culture seedling planting.
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