CN114027199A - Peucedanum praeruptorum seedling breeding method - Google Patents
Peucedanum praeruptorum seedling breeding method Download PDFInfo
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- 241001254606 Peucedanum praeruptorum Species 0.000 title claims abstract description 39
- 238000009395 breeding Methods 0.000 title claims abstract description 28
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
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- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
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- 235000001671 coumarin Nutrition 0.000 description 2
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 2
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- OTAFHZMPRISVEM-UHFFFAOYSA-N chromone Chemical compound C1=CC=C2C(=O)C=COC2=C1 OTAFHZMPRISVEM-UHFFFAOYSA-N 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
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Classifications
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a breeding method of peucedanum praeruptorum seedlings, which comprises the following steps: the method comprises the following steps: selecting and processing explant, selecting fresh leaf without plant diseases and insect pests as explant, sterilizing with sterilizing agent, washing with sterile water, cutting off edge part of leaf, and cutting into 0.5-1.0cm pieces2Size and with veins; step two: inducing callus, namely inoculating the explant processed in the step one into an induction culture medium, and performing dark culture for 50 days to obtain callus; the culture conditions were: culturing at 25 + -2 deg.C in dark; step three: multiplication culture of callus, cutting the explant obtained in the second step into 0.5cm2Inoculating the obtained product into a proliferation culture medium, and culturing for 30 days to obtain a large amount of callus; step four: performing differentiation culture, cutting the callus obtained in step three into 0.5cm2The size of the test tube plantlet is that the test tube plantlet is inoculated in a differential culture medium and cultured for 30 days to obtain a sterile test tube plantlet; the culture conditions were: the temperature is 25 +/-2), the illumination time is 12h/d, and the illumination intensity is 1000-1500 LX.
Description
Technical Field
The invention belongs to the technical field of tissue culture breeding of peucedanum praeruptorum, and particularly relates to a breeding method of peucedanum praeruptorum seedlings.
Background
Radix peucedani is a perennial herb of decursia of Umbelliferae, the root can be used for medicine, the property of the medicine is bitter, pungent and slightly cold, the medicine enters lung channel, and the medicine has better functions of descending qi, reducing phlegm and dispelling wind and heat. Modern researches show that the root of radix Peucedani contains various coumarins, wherein the horn-shaped dihydropyranyl coumarins are the main effective components, and have various pharmacological effects of eliminating phlegm, relieving cough and asthma. The peucedanum praeruptorum has a medicinal history for thousands of years in China, and scholars at home and abroad carry out a great deal of research on chemical components and pharmacological action of the peucedanum praeruptorum. The peucedanum contains rich chemical components, and the main components found at present comprise coumarin, volatile oil, flavone, chromone, polyacetylene, lignin, styrene-acrylic derivative and the like. Radix Peucedani has pharmacological effects of eliminating phlegm, relieving cough, relieving asthma, resisting inflammation, relieving spasm, tranquilizing mind, etc., and radix Peucedani extractive solution has effects of resisting myocardial ischemia, protecting myocardium, improving cardiac function, etc., and petroleum ether extract has effects of dilating blood vessel and lowering blood pressure. At present, the peucedanum praeruptorum is mainly propagated through seeds and roots, the seed germination rate is low, the seedling emergence is irregular, the growth period is long, the root propagation coefficient is low, the seed property degradation is easily caused by long-term vegetative propagation, and the influence of seasons is large. Furthermore, the root bifurcation of the artificially cultivated peucedanum praeruptorum is serious, and the performance of the peucedanum praeruptorum is degraded and the quality is reduced along with the occurrence of variation. In addition, the peucedanum praeruptorum dunn cultivation product can generate bolting phenomenon, so that the quality is seriously reduced, and the sustainable development of peucedanum praeruptorum dunn is restricted.
The plant tissue culture technology is widely applied to the aspects of plant in-vitro rapid propagation, seedling detoxification, cell mutant screening, gene (transgenic) breeding, genetic resource preservation and the like. Correspondingly, the plant tissue culture technology is applied to the seedling breeding of the peucedanum praeruptorum so as to effectively improve the propagation coefficient, shorten the propagation period, be free from the limitation of seasons, maintain the excellent properties of the original variety, be beneficial to breeding new excellent varieties and obtain the high-quality peucedanum praeruptorum seedlings by the transgenic technology. Currently, there are few reports on the tissue culture of peucedanum praeruptorum.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide the method for breeding the seedlings of the peucedanum praeruptorum, the operation is simple and convenient, the induction rate is high and can reach 80%, the multiplication coefficient is high, the differentiation rate is high and can reach 300%, the limitation of seasons on seedling breeding is overcome, the obtained regeneration plants effectively retain the excellent characters of the female parent, and the method is an effective way for breeding the seedlings of the peucedanum praeruptorum.
In order to achieve the purpose, the breeding method of the peucedanum root seedlings provided by the invention comprises the following steps:
the method comprises the following steps: selection and treatment of explants
Selecting fresh leaves without plant diseases and insect pests as explants, removing petioles, pretreating, sterilizing by a sterilizing agent, washing by sterile water, cutting off the edge part of the leaves, and cutting into leaves with veins and the size of 0.5-1.0cm 2;
step two: induction of callus
Inoculating the explant treated in the first step into an induction culture medium, and performing dark culture for 50 days to obtain a callus; the culture conditions were: culturing at 25 + -2 deg.C in dark;
step three: callus proliferation culture
Cutting the explant obtained in step two into 0.5cm2Inoculating the obtained product into a proliferation culture medium, and culturing for 30 days to obtain a large amount of callus; the culture conditions were: culturing at 25 + -2 deg.C in dark;
step four: differential culture
Cutting the callus obtained in step three into 0.5cm2The size of the test tube plantlet is that the test tube plantlet is inoculated in a differential culture medium and cultured for 30 days to obtain a sterile test tube plantlet; the culture conditions were: the temperature is 25 +/-2), the illumination time is 12h/d, and the illumination intensity is 1000-1500 LX.
Preferably, the induction culture medium takes NB culture medium as a basic culture medium, and is added with 6-BA with the concentration of 0.5-1.0mg/L, 2,4-D with the concentration of 1.0-3.0mg/L, sucrose with the concentration of 30g/L and agar with the concentration of 7.0g/L, and the PH value is 5.8-6.2;
preferably, the enrichment medium takes NB medium as a basic medium, the concentration of 6-BA is 0.5-2.0mg/L, the concentration of 2,4-D is 1.0-3.0mg/L, the concentration of sucrose is 30g/L and the concentration of agar is 7.0g/L, and the pH value is 5.8-6.2;
preferably, the differentiation medium takes an MS culture medium as a basic culture medium, the concentration of 6-BA is 1.0-3.0mg/L, NAA and is 0.5-2.0mg/L, the concentration of 2,4-D is 0.1-0.5mg/L, the concentration of sucrose is 30g/L and the concentration of agar is 7.0g/L, and the pH value is 5.8-6.2;
preferably, in the first step, the sterilizing agent comprises 75% alcohol and 0.1% mercury bichloride solution, and the sterilizing time of the 75% alcohol and the 0.1% mercury bichloride solution is 30s and 8min respectively;
preferably, in the second step, the processed explant is cut off at the leaf margin, and the leaf is laid on the induction culture medium with the leaf back facing upwards;
preferably, in the third step, the explant is cut into 0.5cm2 size and inoculated on proliferation medium;
preferably, in the fourth step, the callus is cut into 0.5cm2 size and inoculated into differentiation medium.
The breeding method of the peucedanum praeruptorum seedling provided by the invention has the following beneficial effects:
1. the invention uses the fresh leaves of the peucedanum praeruptorum as the explant to induce the callus, establishes a set of stable plant regeneration system of the peucedanum praeruptorum, the obtained tissue culture seedling has excellent female parent character, can be used for seedling production and breeding all the year round, overcomes the defects of variety degeneration, breeding season limitation and the like of the peucedanum praeruptorum caused by root division and cutting propagation, realizes the regeneration of the tissue culture seedling of the peucedanum praeruptorum and effectively carries out mass breeding.
2. The invention takes the new leaves about 1 week after the leaves are developed in the current year as the explant material, ensures the quality of the test material, induces and generates callus, and can obtain a large amount of callus after 50 days.
3. In the process of cultivating the explant, the invention adopts the concentration ratios of hormones such as different cytokinins 6-BA and auxin NAA, 2,4-D and the like at different stages of the explant growth, which is beneficial to the growth and development of the explant and ensures the quality of the test-tube plantlet of the peucedanum praeruptorum.
4. The differentiation medium has high differentiation rate, and greatly improves the propagation coefficient of the peucedanum praeruptorum.
Detailed Description
The present invention will be further described with reference to specific examples to assist understanding of the invention.
The invention provides a breeding method of peucedanum praeruptorum seedlings, which comprises the following steps:
the method comprises the following steps: selection and treatment of explants
Selecting new leaves (about 1 week after leaf expansion) without diseases and insect pests as explants in 5-6 months of the year, removing petioles, soaking and washing for about 10min with detergent, and washing for 30min with running water for later use. Sterilizing with 75% alcohol for 30s, sterilizing with 0.1% mercuric chloride solution for 8min, washing with sterile water for 5 times, drying with sterile filter paper, cutting off leaf edge, and cutting into pieces of 0.5-1.0cm2 with leaf vein on each explant.
Step two: induction of callus
The processed explant is flatly laid on an induction culture medium with the leaf back upward, and is cultured in dark for 50 days to induce callus, wherein the induction rate can reach 80%.
The induction culture conditions are as follows: culturing at 25 + -2 deg.C in dark.
The induction culture medium is as follows: NB +6-BA 0.5mg/L +2, 4-D2.0 mg/L + sucrose 30g/L + agar 7 g/L.
Step three: proliferation culture
Selecting well-grown callus, cutting into about 0.5cm2 size, spreading in proliferation culture medium, and culturing for 30 days to obtain large amount of callus.
The culture conditions were: culturing at 25 + -2 deg.C in dark.
The proliferation culture medium is: NB +6-BA 0.5mg/L +2, 4-D1.0 mg/L + sucrose 30g/L + agar 7 g/L.
Step four: differential culture
Selecting well-grown callus, cutting into about 0.5cm2 size, spreading in proliferation culture medium, and culturing for 30 days to obtain a large amount of sterile test-tube plantlets.
The culture conditions were: the temperature is 25 +/-2), the illumination time is 12h/d, and the illumination intensity is 1000-1500 Lx.
The differentiation culture medium is as follows: MS +6-BA 2.0mg/L + NAA 1.0mg/L +2, 4-D0.3 mg/L + sucrose 30g/L + agar 7.0 g/L.
The invention uses the fresh leaves of the peucedanum praeruptorum as the explant to induce the callus, establishes a set of stable plant regeneration system of the peucedanum praeruptorum, the obtained tissue culture seedling has excellent female parent character, can be used for seedling production and breeding all the year round, overcomes the defects of variety degeneration, breeding season limitation and the like of the peucedanum praeruptorum caused by root division and cutting propagation, realizes the regeneration of the tissue culture seedling of the peucedanum praeruptorum and effectively carries out mass breeding.
The invention takes the new leaves about 1 week after the leaves are developed in the current year as the explant material, ensures the quality of the test material, induces and generates callus, and can obtain a large amount of callus after 50 days.
In the process of cultivating the explant, the invention adopts the concentration ratios of hormones such as different cytokinins 6-BA and auxin NAA, 2,4-D and the like at different stages of the explant growth, which is beneficial to the growth and development of the explant and ensures the quality of the test-tube plantlet of the peucedanum praeruptorum.
The differentiation medium has high differentiation rate, and greatly improves the propagation coefficient of the peucedanum praeruptorum.
The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.
Claims (8)
1. A breeding method of peucedanum praeruptorum seedlings is characterized by comprising the following steps:
the method comprises the following steps: selection and treatment of explants
Selecting fresh leaves without plant diseases and insect pests as explants, removing petioles, pretreating, sterilizing by a sterilizing agent, washing by sterile water, cutting off the edge part of the leaves, and cutting into leaves with veins and the size of 0.5-1.0cm 2;
step two: induction of callus
Inoculating the explant treated in the first step into an induction culture medium, and performing dark culture for 50 days to obtain a callus; the culture conditions were: culturing at 25 + -2 deg.C in dark;
step three: callus proliferation culture
Cutting the explant obtained in step two into 0.5cm2Inoculating the obtained product into a proliferation culture medium, and culturing for 30 days to obtain a large amount of callus; the culture conditions were: culturing at 25 + -2 deg.C in dark;
step four: differential culture
Cutting the callus obtained in step three into 0.5cm2The size of the test tube plantlet is that the test tube plantlet is inoculated in a differential culture medium and cultured for 30 days to obtain a sterile test tube plantlet; the culture conditions were: the temperature is 25 +/-2), the illumination time is 12h/d, and the illumination intensity is 1000-1500 LX.
2. The method for breeding peucedanum praeruptorum dunn seedlings as claimed in claim 1, wherein the inducing medium is NB medium as basic medium, 6-BA is added at a concentration of 0.5-1.0mg/L, 2,4-D is added at a concentration of 1.0-3.0mg/L, sucrose is added at a concentration of 30g/L, agar is added at a concentration of 7.0g/L, and pH is 5.8-6.2.
3. The method for breeding peucedanum praeruptorum dunn seedlings according to claim 1, wherein the propagation medium uses NB medium as a basic medium, 6-BA is added at a concentration of 0.5-2.0mg/L, 2,4-D is added at a concentration of 1.0-3.0mg/L, sucrose is added at a concentration of 30g/L, agar is added at a concentration of 7.0g/L, and pH is 5.8-6.2.
4. The method for breeding peucedanum praeruptorum dunn as claimed in claim 1, wherein the differentiation medium is MS medium as basic medium, and is added with 6-BA at a concentration of 1.0-3.0mg/L, NAA at a concentration of 0.5-2.0mg/L, 2,4-D at a concentration of 0.1-0.5mg/L, sucrose at 30g/L and agar at 7.0g/L, and pH at 5.8-6.2.
5. The breeding method of peucedanum praeruptorum dunn of claim 1, wherein in the first step, the sterilizing agent comprises 75% alcohol and 0.1% mercuric chloride solution, and the sterilizing time of the 75% alcohol and 0.1% mercuric chloride solution is 30s and 8min respectively.
6. The method for breeding the seedlings of peucedanum praeruptorum dunn of claim 1, wherein in the second step, the processed explant is cut off at the leaf margin, and the leaf is laid on the induction medium with the leaf back facing upwards.
7. The method for breeding the seedlings of peucedanum praeruptorum dunn of claim 1, wherein in the third step, the explant is cut into 0.5cm2 size and inoculated on a proliferation medium.
8. The method for breeding peucedanum praeruptorum seedling as claimed in claim 1, wherein in step four, the callus is cut into 0.5cm2 size and inoculated into differentiation medium.
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Cited By (2)
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| CN115380830A (en) * | 2022-10-08 | 2022-11-25 | 浙江大学 | Propagation method of Peucedanum praeruptorum dunn adventitious bud |
| CN115777536A (en) * | 2022-11-30 | 2023-03-14 | 中南民族大学 | Method for establishing efficient regeneration system by utilizing stems of peucedanum praeruptorum dunn |
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| CN106613171A (en) * | 2016-11-28 | 2017-05-10 | 巫溪县祥发农业开发有限公司 | Planting method for peucedanum praeruptorum dunn |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115380830A (en) * | 2022-10-08 | 2022-11-25 | 浙江大学 | Propagation method of Peucedanum praeruptorum dunn adventitious bud |
| CN115777536A (en) * | 2022-11-30 | 2023-03-14 | 中南民族大学 | Method for establishing efficient regeneration system by utilizing stems of peucedanum praeruptorum dunn |
| CN115777536B (en) * | 2022-11-30 | 2023-08-18 | 中南民族大学 | Method for establishing efficient regeneration system by utilizing stems of peucedanum praeruptorum dunn |
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Application publication date: 20220211 |