CN112913690A - Peony in-vitro regeneration method - Google Patents
Peony in-vitro regeneration method Download PDFInfo
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- CN112913690A CN112913690A CN202110121755.7A CN202110121755A CN112913690A CN 112913690 A CN112913690 A CN 112913690A CN 202110121755 A CN202110121755 A CN 202110121755A CN 112913690 A CN112913690 A CN 112913690A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a culture medium for peony in-vitro regeneration, which comprises a starting culture medium, a multiplication culture medium, a strong seedling culture medium and a rooting culture medium. The regeneration culture medium can overcome the problem of difficult start of the peony in vitro culture, induces the bottom bud point of the petiole to differentiate the lateral bud, and the germination rate of the lateral bud reaches 90 percent; under the culture period of 30 days, the value-added coefficient averagely reaches more than 5; can improve the air permeability of the matrix, effectively improve the condition that no root seedling base is necrotic in the rooting culture process, overcome the problem that the peony tissue culture seedling is difficult to root, and obtain the rooted plant.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to an isolated regeneration method of Chinese herbaceous peony, and especially relates to an isolated regeneration method of Chinese herbaceous peony.
Background
Paeonia lactiflora Pall is a perennial root herbaceous plant in Paeonia of Paeoniaceae, and is a famous ornamental flower and medicinal plant in China. Radix Paeoniae is the basic source of radix Paeoniae alba, and is prepared by decocting root in boiling water, removing outer skin or peeling, decocting, and sun drying. At present, the genuine producing areas of the white paeony root comprise Sichuan, Anhui and Zhejiang, wherein the Sichuan and Zhejiang white paeony root have the advantages of excellent quality, thick root, strong and high paeoniflorin content and the like. However, the paeonia lactiflora pall of the basic source plant of the paeonia lactiflora pall of Zhongjiang river only blooms and is not fruitful in the cultivation process, and the paeonia lactiflora pall is usually propagated by dividing plants, so that the propagation coefficient is low and the cost is high. With the development of the fresh cut flower market and the medicinal material market, the contradiction between the reproductive capacity of Zhongjiang peony and the dramatic increase of the planting area is increasingly prominent. Meanwhile, under the long-term plant division propagation of the white paeony roots, various fungi, bacteria and viruses in the bodies of the white paeony roots are accumulated, and the quality and the yield of the white paeony roots are seriously influenced. Therefore, finding a new breeding mode has important significance for the development of the Zhongjiang white paeony root industry.
The existing peony in vitro regeneration technology still has the technical problems of difficult differentiation, difficult rooting, serious browning and vitrification and the like, and the establishment of the Zhongjiang peony rapid propagation system has serious endophyte pollution besides the problems. At present, the research on the in vitro rapid propagation of Paeoniaceae plants is mostly focused on ornamental varieties, and the research on tissue culture of medicinal Paeonia lactiflora is less at home and abroad, particularly, the Chinese herbaceous peony is only discussed for establishing a sterile system at present, and a complete method for in vitro regeneration of the Chinese herbaceous peony is not established.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for peony in vitro regeneration, which comprises a starting culture medium, a multiplication culture medium, a strong seedling culture medium and a rooting culture medium;
the start culture medium is 1/2MS culture medium as basic culture medium, which is added with benzylpurine (6-BA) with final concentration of 2-5 mg/L and 0.2E0.8mg/L Gibberellin (GA)3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the enrichment medium is an improved 1/2MS culture medium as a basic culture medium, and is added with benzyl purine (6-BA) with the final concentration of 3-5 mg/L and Gibberellin (GA) with the final concentration of 0.8-1.0 mg/L3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the strong seedling culture medium is an improved 1/2MS culture medium as a basic culture medium, and is added with benzylpurine (6-BA) with the final concentration of 0.5-1 mg/L and Gibberellin (GA) with the final concentration of 0.2-0.8 mg/L3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the rooting culture medium is characterized in that 1/4MS culture medium is used as a basic culture medium, indolebutyric acid (IBA) with the final concentration of 0.3-0.8 mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.3-0.8 mg/L, sucrose with the final concentration of 7-13 g/L, perlite with the final concentration of 300-400 g/L and a vermiculite mixture are added, and the pH value is 6.0-6.5;
the modified 1/2MS medium is 1/2 CaCl in MS medium2·2H2The O concentration was adjusted to 660 mg/L.
Furthermore, the start culture medium is 1/2MS culture medium as basic culture medium, and benzyl purine (6-BA) with final concentration of 2-3 mg/L and Gibberellin (GA) with final concentration of 0.5mg/L are added3) 8g/L of agar and 30g/L of cane sugar, wherein the pH value is 5.9-6.0; the proliferation culture medium is prepared from modified 1/2MS culture medium as basic culture medium, and added with benzylpurine (6-BA) with final concentration of 3mg/L and Gibberellin (GA) with final concentration of 0.8mg/L3) 8g/L of agar and 30g/L of cane sugar, and the pH value of the mixture is 5.9-6.0.
Furthermore, the strong seedling culture medium takes an improved 1/2MS culture medium as a basic culture medium, and is added with benzylpurine (6-BA) with the final concentration of 1mg/L and Gibberellin (GA) with the final concentration of 0.5mg/L3) 8g/L of agar and 30g/L of cane sugar, wherein the pH value is 5.9-6.0; the rooting culture medium is prepared by taking 1/4MS culture medium as a basic culture medium and adding indolebutyric acid (IBA) with the final concentration of 0.5mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.5mg/L, sucrose with the final concentration of 10g/L, perlite with the final concentration of 320g/L and vermiculite mixture, and the pH value of the rooting culture medium is 6.0-6.1.
Further, the ratio of perlite to vermiculite in the perlite and vermiculite mixture is 2: 1.
the invention also provides an isolated regeneration method of the Chinese herbaceous peony, which utilizes the culture medium for regeneration and comprises the following specific steps:
a. collecting peony sprouts, cleaning, sterilizing, cutting off stem tips and bottoms, inoculating stem segments into a starting culture medium for culture to obtain regenerated tissue culture seedlings;
b. taking the regeneration tissue culture seedling, cutting off lateral buds, inoculating into an enrichment medium for culture to obtain cluster buds;
c. dividing the cluster buds in the step b into single buds, and then transferring the single buds into a strong seedling culture medium for culture to obtain rootless seedlings;
d. and c, inoculating the rootless seedlings in the step c into a rooting culture medium to induce rooting, and obtaining peony regenerated plants.
Further, the peony sprouts in the step a are 3-5 cm unearthed sprouts with scales; and/or the length of the regeneration tissue culture seedling in the step b is 2-3 cm.
Further, the cleaning in the step a is to remove soil on the surfaces of the sprouts and remove browned scales; the cleaning is to soak the mixture by using a liquid detergent solution and clean the mixture by running water; the disinfection is carried out by soaking in mixed solution of carbendazim and sodium penicillin, shaking, washing with running water, soaking in alcohol and mercuric chloride, and washing with sterile water.
Further, the cleaning is to soak the fabric in a liquid detergent solution for 30min and then to wash the fabric for 2h by running water; the disinfection is carried out by soaking the raw materials in a carbendazim penicillin sodium mixed solution and oscillating for 3 hours, washing the raw materials with running water, soaking the raw materials with 75% alcohol for 6-10 seconds, washing the raw materials with sterile water for 3-5 times, soaking the raw materials with 0.1% mercuric chloride for 6-8 min, and washing the raw materials with sterile water for 5-8 times.
Furthermore, the concentration of the carbendazim in the carbendazim penicillin sodium mixed solution is 3-7 g/L, preferably 5g/L, and the concentration of the penicillin sodium is 0.15-0.25 g/L, preferably 0.2 g/L.
Further, the culturing in the step a is carried out for one week in the dark and then is carried out in the light,
further, the conditions of the culture are: the pH value is 5.9-6.0, the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark). Further, the peony is Zhongjiang peony.
The 1/2MS culture medium is a culture medium with half of macroelements in the MS culture medium formula and unchanged content of other components.
The 1/4MS culture medium is a culture medium with the macroelements reduced to 1/4 of the MS culture medium and the content of the other components unchanged.
The MS culture medium is one of the most common culture media in plant tissues, and the formula of the MS culture medium comprises the following components:
compared with the prior art, the invention has the following remarkable effects:
1) by adopting the start culture medium, the problem of difficult start of isolated culture of the Chinese herbaceous peony can be solved, the bottom bud point of the petiole is induced to differentiate the lateral bud, and the germination rate of the lateral bud reaches almost 100%;
2) the multiplication culture is carried out by adopting the multiplication culture medium of the invention, the multiplication coefficient can reach more than 5 on average in a culture period of 30 days, and the obtained cluster buds are robust and have no necrotic symptom at the stem tips.
3) By adopting the rooting culture medium, the air permeability of the matrix can be improved, the condition that no root seedling base is necrotic in the rooting culture process is effectively improved, the problem that the peony tissue culture seedling is difficult to root is solved, and a rooting plant is obtained.
The invention establishes a complete peony in-vitro regeneration system for the first time, and the in-vitro regeneration method can effectively reduce the pollution condition of the explant in the in-vitro culture process and improve the survival rate of the explant; when the culture is started, the browning of the explant in the culture process can be effectively reduced, and the survival rate of the start culture is improved; during the increment culture and the strong seedling culture, the stem tip necrosis rate of the tissue culture seedling at the stage is reduced to zero.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials, reagents and equipment used in the embodiment of the invention are all known products; wherein 1/2MS culture medium, 1/4MS culture medium is obtained by commercial purchase or prepared according to the existing method, and the improved 1/2MS culture medium is prepared according to the following method:
1. preparing a macroelement mother solution: 8250mg NH was accurately weighed4NO3、8000mg KNO3、6600mg CaCl2·2H2O、1850mg MgSO4·7H2O、850mg KH2PO4Dissolving completely with small amount of distilled water, mixing and dissolving, and metering to 1L;
2. preparing a microelement mother solution: accurately weigh 8.3mg KI and 62mg H3BO3、223mg MnSO4·4H2O、86mg ZnSO4·7H2O、2.5mg Na2MoO4·2H2O、0.25mg CuSO4·5H2O、0.25mg CoCl2·6H2O, dissolving the components completely by using a small amount of distilled water respectively, then mixing and dissolving the components, and finally fixing the volume to 100 mL;
3. preparing a ferric salt mother solution: 278mg of FeSO was accurately weighed4·7H2O、373mg Na2-EDTA·2H2Dissolving the O in small amount of distilled water while heating under stirring, mixing the two solutions, adjusting pH to 5.5, and diluting to 1L;
4. preparing an organic substance mother solution: accurately weighing 1g of inositol, 5mg of nicotinic acid, 5mg of pyridoxine hydrochloride (vitamin B6), 1mg of thiamine hydrochloride (vitamin B1) and 20mg/L of glycine, respectively and completely dissolving with a small amount of distilled water, then mixing and dissolving the components, and finally fixing the volume to 100 mL;
5. respectively mixing the prepared macroelement mother liquor, microelement mother liquor, ferric salt mother liquor and organic substance mother liquor, and obtaining the improved 1/2MS culture medium with the following element concentrations:
macroelements: 825mg/L NH4NO3、950mg/L KNO3、660mg/L CaCl2·2H2O、185mg/L MgSO4·7H2O、85mg/L KH2PO4；
Trace elements: 0.83mg/L KI, 6.2mg/L H3BO3、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·6H2O；
Iron salt: 27.8mg/L FeSO4·7H2O、37.3mg/L Na2-EDTA·2H2O；
Organic matter: 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride (vitamin B6), 0.1mg/L thiamine hydrochloride (vitamin B1), 2.0mg/L glycine.
Example 1 in vitro regeneration of Zhongjiang peony
Preparing a culture medium:
starting a culture medium: 1/2MS culture medium, 2-3 mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Proliferation culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 3mg/L benzylpurine (6-BA), 0.8mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Strong seedling culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 1mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Rooting culture medium: 1/4MS culture medium, 0.5mg/L indolebutyric acid (IBA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L sucrose, 320g/L perlite and vermiculite mixture (perlite: vermiculite: 2: 1), and the pH value of the culture medium is 6.0-6.1.
In-vitro regeneration cultivation:
step 1, selecting and processing materials: selecting 3-5 cm old Chinese-Jiangjiang peony sprouts with scales, slightly brushing soil on the surfaces of the sprouts by using a soft brush, cleaning the browned scales, soaking for 30min by using a detergent solution, and washing with running water.
Step 2, material disinfection: and (3) sterilizing the sprouts subjected to the step (1) by adopting a two-step sterilization method. Firstly, soaking the mixture in a carbendazim and penicillin sodium solution (the concentration of the carbendazim in the solution is 5g/L, and the concentration of the penicillin sodium in the solution is 0.2g/L), placing the mixture in a shaking table for shaking and disinfection for 3 hours, and then washing the mixture for 1 hour by running water. And step two, putting the buds subjected to the step one on a super-clean workbench, soaking and disinfecting for 6s by using 75% alcohol, washing for 4 times by using sterile water, soaking and disinfecting for 7min by using 0.1% mercuric chloride, and washing for 8 times by using the sterile water.
Step 3, starting culture: cutting off the stem tip and the bottom of the sterilized bud, reserving a stem section with a bud point, inoculating the stem section into a starting culture medium, carrying out dark culture for one week, and carrying out illumination culture to obtain a lateral bud; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
And 4, propagation culture: cutting off lateral buds when the lateral buds grow to 2-3 cm, transferring the cut lateral buds to a multiplication culture medium for multiplication culture to obtain cluster buds; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 5, strong seedling culture: dividing cluster buds into single buds, and inoculating the single buds into a strong seedling culture medium for culture to obtain healthy Chinese Jiangjiang peony tissue culture rootless seedlings; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 6, rooting culture: transferring the tissue culture seedling after strong seedling culture to an improved rooting culture medium to induce rooting to obtain a radix paeoniae rubra rooting plant; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Example 2
Preparing a culture medium:
starting a culture medium: 1/2MS culture medium, 2-3 mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Proliferation culture medium: 1/2MS culture medium, 3mg/L benzylpurine (6-BA), 0.8mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Strong seedling culture medium: 1/2MS culture medium, 1mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Rooting culture medium: 1/4MS culture medium, 0.5mg/L indolebutyric acid (IBA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L sucrose, 320g/L perlite and vermiculite mixture (perlite: vermiculite: 2: 1), wherein the pH value of the culture medium is 6.0-6.1.
In-vitro regeneration cultivation:
step 1, selecting and processing materials: selecting 3-5 cm old Chinese-Jiangjiang peony sprouts with scales, slightly brushing soil on the surfaces of the sprouts by using a soft brush, cleaning the browned scales, soaking for 30min by using a detergent solution, and washing with running water.
Step 2, material disinfection: and (3) sterilizing the sprouts subjected to the step (1) by adopting a two-step sterilization method. Firstly, soaking the mixture in a carbendazim and penicillin sodium solution (the concentration of the carbendazim in the solution is 5g/L, and the concentration of the penicillin sodium in the solution is 0.2g/L), placing the mixture in a shaking table for shaking and disinfection for 3 hours, and then washing the mixture for 1 hour by running water. And step two, putting the buds subjected to the step one on a super-clean workbench, soaking and disinfecting for 6s by using 75% alcohol, washing for 4 times by using sterile water, soaking and disinfecting for 7min by using 0.1% mercuric chloride, and washing for 8 times by using the sterile water.
Step 3, starting culture: cutting off the stem tip and the bottom of the sterilized bud, reserving a stem section with a bud point, inoculating the stem section into a starting culture medium, carrying out dark culture for one week, and carrying out illumination culture to obtain a lateral bud; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
And 4, propagation culture: cutting off lateral buds when the lateral buds grow to 2-3 cm, transferring the cut lateral buds to a multiplication culture medium for multiplication culture to obtain cluster buds; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 5, strong seedling culture: dividing cluster buds into single buds, and inoculating the single buds into a strong seedling culture medium for culture to obtain healthy Chinese Jiangjiang peony tissue culture rootless seedlings; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 6, rooting culture: transferring the tissue culture seedling after strong seedling culture to an improved rooting culture medium to induce rooting to obtain a radix paeoniae rubra rooting plant; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Example 3
Preparing a culture medium:
starting a culture medium: 1/2MS culture medium, 2-3 mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Proliferation culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 3mg/L benzylpurine (6-BA), 0.8mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Strong seedling culture medium: 1/2MS culture medium, 1mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Rooting culture medium: 1/4MS culture medium, 0.5mg/L indolebutyric acid (IBA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L sucrose, 320g/L perlite and vermiculite mixture (perlite: vermiculite: 2: 1), wherein the pH value of the culture medium is 6.0-6.1.
In-vitro regeneration cultivation:
step 1, selecting and processing materials: selecting 3-5 cm old Chinese-Jiangjiang peony sprouts with scales, slightly brushing soil on the surfaces of the sprouts by using a soft brush, cleaning the browned scales, soaking for 30min by using a detergent solution, and washing with running water.
Step 2, material disinfection: and (3) sterilizing the sprouts subjected to the step (1) by adopting a two-step sterilization method. Firstly, soaking the mixture in a carbendazim and penicillin sodium solution (the concentration of the carbendazim in the solution is 5g/L, and the concentration of the penicillin sodium in the solution is 0.2g/L), placing the mixture in a shaking table for shaking and disinfection for 3 hours, and then washing the mixture for 1 hour by running water. And step two, putting the buds subjected to the step one on a super-clean workbench, soaking and disinfecting for 6s by using 75% alcohol, washing for 4 times by using sterile water, soaking and disinfecting for 7min by using 0.1% mercuric chloride, and washing for 8 times by using the sterile water.
Step 3, starting culture: cutting off the stem tip and the bottom of the sterilized bud, reserving a stem section with a bud point, inoculating the stem section into a starting culture medium, carrying out dark culture for one week, and carrying out illumination culture to obtain a lateral bud; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
And 4, propagation culture: cutting off lateral buds when the lateral buds grow to 2-3 cm, transferring the cut lateral buds to a multiplication culture medium for multiplication culture to obtain cluster buds; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 5, strong seedling culture: dividing cluster buds into single buds, and inoculating the single buds into a strong seedling culture medium for culture to obtain healthy Chinese Jiangjiang peony tissue culture rootless seedlings; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 6, rooting culture: transferring the tissue culture seedling after strong seedling culture to an improved rooting culture medium to induce rooting to obtain a radix paeoniae rubra rooting plant; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Example 4
Preparing a culture medium:
starting a culture medium: 1/2MS culture medium, 2-3 mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Proliferation culture medium: 1/2MS culture medium, 3mg/L benzylpurine (6-BA), 0.8mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Strong seedling culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 1mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Rooting culture medium: 1/4MS culture medium, 0.5mg/L indolebutyric acid (IBA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L sucrose, 320g/L perlite and vermiculite mixture (perlite: vermiculite: 2: 1), wherein the pH value of the culture medium is 6.0-6.1.
In-vitro regeneration cultivation:
step 1, selecting and processing materials: selecting 3-5 cm old Chinese-Jiangjiang peony sprouts with scales, slightly brushing soil on the surfaces of the sprouts by using a soft brush, cleaning the browned scales, soaking for 30min by using a detergent solution, and washing with running water.
Step 2, material disinfection: and (3) sterilizing the sprouts subjected to the step (1) by adopting a two-step sterilization method. Firstly, soaking the mixture in a carbendazim and penicillin sodium solution (the concentration of the carbendazim in the solution is 5g/L, and the concentration of the penicillin sodium in the solution is 0.2g/L), placing the mixture in a shaking table for shaking and disinfection for 3 hours, and then washing the mixture for 1 hour by running water. And step two, putting the buds subjected to the step one on a super-clean workbench, soaking and disinfecting for 6s by using 75% alcohol, washing for 4 times by using sterile water, soaking and disinfecting for 7min by using 0.1% mercuric chloride, and washing for 8 times by using the sterile water.
Step 3, starting culture: cutting off the stem tip and the bottom of the sterilized bud, reserving a stem section with a bud point, inoculating the stem section into a starting culture medium, carrying out dark culture for one week, and carrying out illumination culture to obtain a lateral bud; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
And 4, propagation culture: cutting off lateral buds when the lateral buds grow to 2-3 cm, transferring the cut lateral buds to a multiplication culture medium for multiplication culture to obtain cluster buds; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 5, strong seedling culture: dividing cluster buds into single buds, and inoculating the single buds into a strong seedling culture medium for culture to obtain healthy Chinese Jiangjiang peony tissue culture rootless seedlings; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 6, rooting culture: transferring the tissue culture seedling after strong seedling culture to an improved rooting culture medium to induce rooting to obtain a radix paeoniae rubra rooting plant; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Example 5
Preparing a culture medium:
starting a culture medium: 1/2MS culture medium, 2-3 mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Proliferation culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 3mg/L benzylpurine (6-BA), 0.8mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Strong seedling culture medium: modified 1/2MS Medium (i.e. CaCl in 1/2MS Medium)2·2H2O was adjusted to 660mg/L), 1mg/L benzylpurine (6-BA), 0.5mg/L Gibberellin (GA)3) 8g/L agar and 30g/L cane sugar, and the pH value of the culture medium is 5.9-6.0.
Rooting culture medium: 1/4MS culture medium, 0.5mg/L indolebutyric acid (IBA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L sucrose, 8g/L agar and 10g/L sucrose, wherein the pH value of the culture medium is 6.0-6.1.
In-vitro regeneration cultivation:
step 1, selecting and processing materials: selecting 3-5 cm old Chinese-Jiangjiang peony sprouts with scales, slightly brushing soil on the surfaces of the sprouts by using a soft brush, cleaning the browned scales, soaking for 30min by using a detergent solution, and washing with running water.
Step 2, material disinfection: and (3) sterilizing the sprouts subjected to the step (1) by adopting a two-step sterilization method. Firstly, soaking the mixture in a carbendazim and penicillin sodium solution (the concentration of the carbendazim in the solution is 5g/L, and the concentration of the penicillin sodium in the solution is 0.2g/L), placing the mixture in a shaking table for shaking and disinfection for 3 hours, and then washing the mixture for 1 hour by running water. And step two, putting the buds subjected to the step one on a super-clean workbench, soaking and disinfecting for 6s by using 75% alcohol, washing for 4 times by using sterile water, soaking and disinfecting for 7min by using 0.1% mercuric chloride, and washing for 8 times by using the sterile water.
Step 3, starting culture: cutting off the stem tip and the bottom of the sterilized bud, reserving a stem section with a bud point, inoculating the stem section into a starting culture medium, carrying out dark culture for one week, and carrying out illumination culture to obtain a lateral bud; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
And 4, propagation culture: cutting off lateral buds when the lateral buds grow to 2-3 cm, transferring the cut lateral buds to a multiplication culture medium for multiplication culture to obtain cluster buds; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 5, strong seedling culture: dividing cluster buds into single buds, and inoculating the single buds into a strong seedling culture medium for culture to obtain healthy Chinese Jiangjiang peony tissue culture rootless seedlings; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
Step 6, rooting culture: transferring the tissue culture seedling after strong seedling culture to an improved rooting culture medium to induce rooting to obtain a radix paeoniae rubra rooting plant; the culture conditions were: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
The advantageous effects of the present invention are described below by way of test examples.
Test example 1
Selecting the sprouts of the rooted plants of the Chinese Jiangpeony obtained by the cultivation in the embodiments 1-6 respectively to carry out isolated culture, wherein the culture results are as follows:
(first) culture results of example 1
The lateral buds are obtained by starting culture, the survival rate of the starting culture is 55 percent (death is pollution and browning), and the germination rate of the lateral buds is 100 percent; culturing the regenerated tissue culture seedling in a multiplication culture medium to obtain cluster buds, wherein the multiplication coefficient is 10.5; after the plants are divided, the seedlings of the Chinese herbaceous peony are cultured in a strong seedling culture medium to obtain healthy rootless seedlings, and the rootless seedlings are inoculated into a rooting culture medium to be cultured to obtain rooting tissue culture seedlings with strong root systems and lateral roots.
(II) culture results of example 2
The lateral bud is obtained by starting culture, the survival rate of the starting culture is 65 percent (death is pollution and browning), and the germination rate of the lateral bud is 100 percent. The regenerated tissue culture seedling is cultured in a multiplication culture medium to obtain cluster buds, and the multiplication coefficient is 6.3. The stem tip necrosis symptom begins to appear in the second week of the enrichment culture and gradually worsens, and the stem tip necrosis rate reaches 90 percent in the enrichment culture period of one month; after the plants are divided, the seedlings of the Chinese herbaceous peony are cultured in a strong seedling culture medium, the necrosis of the stem tip continuously increases to 100 percent, 45 percent of the stem tip is necrotic in the whole plant, and the rooting culture cannot be carried out.
(III) culture results of example 3
The lateral bud is obtained by starting culture, the survival rate of the starting culture is 40 percent (death is pollution and browning), and the germination rate of the lateral bud is 100 percent. Culturing the regenerated tissue culture seedling in a multiplication culture medium to obtain cluster buds, wherein the multiplication coefficient is 10; after the plants are divided, the seedlings of the Chinese herbaceous peony are cultured in a strong seedling culture medium, the tissue culture seedlings gradually have stem tip necrosis, the necrosis rate reaches over 85 percent in a one-month culture period, and the rest seedlings show that stem leaves are light brown, soft and water-stain-shaped. The rootless seedlings are inoculated into a rooting culture medium for culture and can not root.
(fourth) culture results of example 4
Culturing in a starting culture medium to obtain a regeneration tissue culture seedling, wherein the survival rate of the starting culture is 55.5% (death is pollution and browning), and the germination rate of lateral buds is 100%. The regenerated tissue culture seedling is cultured in a multiplication culture medium to obtain cluster buds, and the multiplication coefficient is 5.5. The stem tip necrosis symptom begins to appear in the second week of the enrichment culture, and gradually worsens, and the stem tip necrosis rate is 86% in the enrichment culture period of one month; the seedlings with stem tip necrosis are cultivated in a strong seedling culture medium after the stem tip necrosis is divided, stem tip necrosis symptoms are gradually improved and rejuvenated, healthy rootless seedlings are obtained after the cultivation time is prolonged, the rootless seedlings are inoculated into a rooting culture medium for cultivation, the rooting rate reaches 50.1%, and the root system is strong and vigorous.
(V) culture results of example 5
The lateral bud is obtained by starting culture, the survival rate of the starting culture is 55 percent (death is pollution and browning), and the germination rate of the lateral bud is 100 percent. Culturing the regenerated tissue culture seedling in a multiplication culture medium to obtain cluster buds, wherein the multiplication coefficient is 11.5; after the plants are divided, the paeonia lactiflora pall seedlings are cultured in a strong seedling culture medium to obtain healthy rootless seedlings, the rootless seedlings are inoculated into a rooting culture medium for culture, and after a period of culture, the parts of the seedling bases, which are contacted with the culture medium, are in a browning and necrosis state and cannot root.
In conclusion, the peony in-vitro regeneration method can effectively reduce the browning of the explant in the culture process and improve the survival rate of the start culture when the start culture is carried out; during the increment culture and the strong seedling culture, the stem tip necrosis rate of the tissue culture seedling is reduced to zero; in the process of peony in vitro culture, the pollution condition of the explant is effectively reduced, the survival rate of the explant is improved, and the method has popularization and application values.
Claims (11)
1. A culture medium for peony in-vitro regeneration is characterized in that: the method comprises a starting culture medium, an enrichment culture medium, a strong seedling culture medium and a rooting culture medium;
the start culture medium is 1/2MS culture medium as basic culture medium, and added with benzylpurine (6-BA) with final concentration of 2-5 mg/L and Gibberellin (GA) with final concentration of 0.2-0.8 mg/L3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the enrichment medium is an improved 1/2MS culture medium as a basic culture medium, and is added with benzyl purine (6-BA) with the final concentration of 3-5 mg/L and Gibberellin (GA) with the final concentration of 0.8-1.0 mg/L3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the strong seedling culture medium is an improved 1/2MS culture mediumThe basal culture medium is added with benzylpurine (6-BA) with final concentration of 0.5-1 mg/L and Gibberellin (GA) with final concentration of 0.2-0.8 mg/L3) 5-10 g/L of agar and 25-35 g/L of cane sugar, wherein the pH value is 5.5-6.0;
the rooting culture medium is characterized in that 1/4MS culture medium is used as a basic culture medium, indolebutyric acid (IBA) with the final concentration of 0.3-0.8 mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.3-0.8 mg/L, sucrose with the final concentration of 7-13 g/L, perlite with the final concentration of 300-400 g/L and a vermiculite mixture are added, and the pH value is 6.0-6.5;
the modified 1/2MS culture medium is prepared by adding 1/2MS culture medium CaCl2·2H2The O concentration was adjusted to 660 mg/L.
2. The culture medium according to claim 1, wherein: the start culture medium is 1/2MS culture medium as basic culture medium, and added with benzylpurine (6-BA) with final concentration of 2-3 mg/L and Gibberellin (GA) with final concentration of 0.5mg/L3) 8g/L of agar and 30g/L of cane sugar, wherein the pH value is 5.9-6.0; the proliferation culture medium is prepared from modified 1/2MS culture medium as basic culture medium, and added with benzylpurine (6-BA) with final concentration of 3mg/L and Gibberellin (GA) with final concentration of 0.8mg/L3) 8g/L of agar and 30g/L of cane sugar, and the pH value of the mixture is 5.9-6.0.
3. The culture medium according to claim 1, wherein: the strong seedling culture medium takes an improved 1/2MS culture medium as a basic culture medium, and is added with benzylpurine (6-BA) with the final concentration of 1mg/L and Gibberellin (GA) with the final concentration of 0.5mg/L3) 8g/L of agar and 30g/L of cane sugar, wherein the pH value is 5.9-6.0; the rooting culture medium is prepared by taking 1/4MS culture medium as a basic culture medium and adding indolebutyric acid (IBA) with the final concentration of 0.5mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0.5mg/L, sucrose with the final concentration of 10g/L, perlite with the final concentration of 320g/L and vermiculite mixture, and the pH value of the rooting culture medium is 6.0-6.1.
4. The culture medium according to claim 1 or 3, wherein: in the mixture of the perlite and the vermiculite, the ratio of the perlite to the vermiculite is 2: 1.
5. an isolated regeneration method of Chinese herbaceous peony is characterized in that: the method is to regenerate the culture medium of any one of claims 1 to 4, and comprises the following specific steps:
a. collecting peony sprouts, cleaning, sterilizing, cutting off stem tips and bottoms, inoculating stem segments into a starting culture medium for culture to obtain regenerated tissue culture seedlings;
b. taking the regeneration tissue culture seedling, cutting off lateral buds, inoculating into an enrichment medium for culture to obtain cluster buds;
c. dividing the cluster buds in the step b into single buds, and then transferring the single buds into a strong seedling culture medium for culture to obtain rootless seedlings;
d. and c, inoculating the rootless seedlings in the step c into a rooting culture medium to induce rooting, and obtaining peony regenerated plants.
6. The culture medium according to claim 5, wherein: the peony sprouts in the step a are 3-5 cm unearthed sprouts with scales; and/or the length of the regeneration tissue culture seedling in the step b is 2-3 cm.
7. The regeneration method according to claim 5, wherein: the cleaning of the step a is to remove soil on the surfaces of the sprouts and remove browned scales; the cleaning is to soak the mixture by using a liquid detergent solution and clean the mixture by running water; the disinfection is carried out by soaking in mixed solution of carbendazim and sodium penicillin, shaking, washing with running water, soaking in alcohol and mercuric chloride, and washing with sterile water.
8. The regeneration method according to claim 7, characterized in that: the cleaning is to soak the mixture for 30min by using a liquid detergent solution and then to wash the mixture for 2h by using running water; the disinfection is to soak the mixture with carbendazim penicillin sodium solution and shake the mixture for 3 hours, after the mixture is washed by running water, the mixture is soaked in 75% alcohol for 6 to 10 seconds, washed by sterile water for 3 to 5 times, and finally soaked in 0.1% mercuric chloride for 6 to 8min, and washed by sterile water for 5 to 8 times; the concentration of the carbendazim in the carbendazim penicillin sodium mixed solution is 3-7 g/L, preferably 5g/L, and the concentration of the penicillin sodium is 0.15-0.25 g/L, preferably 0.2 g/L.
9. The regeneration method according to claim 5, wherein: the culture in the step a is dark culture for one week and then illumination culture.
10. The regeneration method according to claim 5 or 9, characterized in that: the culture conditions are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 Lux, and the illumination period is 14h (light)/10 h (dark).
11. The regeneration method according to any one of claims 5 to 10, characterized in that: the radix Paeoniae is radix Paeoniae of Zhongjiang river.
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