CN112450074A - Method for establishing tissue culture and rapid propagation system of panax japonicus leaves - Google Patents
Method for establishing tissue culture and rapid propagation system of panax japonicus leaves Download PDFInfo
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- CN112450074A CN112450074A CN202011424527.9A CN202011424527A CN112450074A CN 112450074 A CN112450074 A CN 112450074A CN 202011424527 A CN202011424527 A CN 202011424527A CN 112450074 A CN112450074 A CN 112450074A
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- 241000168720 Panax japonicus Species 0.000 title claims abstract description 30
- 235000003174 Panax japonicus Nutrition 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 20
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 22
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims abstract description 19
- 235000008434 ginseng Nutrition 0.000 claims abstract description 19
- 239000008280 blood Substances 0.000 claims abstract description 18
- 210000004369 blood Anatomy 0.000 claims abstract description 18
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 11
- 230000006698 induction Effects 0.000 claims abstract description 9
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- 241000208340 Araliaceae Species 0.000 claims abstract 7
- 239000001963 growth medium Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 15
- 239000002689 soil Substances 0.000 claims description 14
- 239000011159 matrix material Substances 0.000 claims description 13
- 229920001817 Agar Polymers 0.000 claims description 12
- 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 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- 241000196324 Embryophyta Species 0.000 claims description 9
- 230000004069 differentiation Effects 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000010451 perlite Substances 0.000 claims description 6
- 235000019362 perlite Nutrition 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 239000010455 vermiculite Substances 0.000 claims description 6
- 235000019354 vermiculite Nutrition 0.000 claims description 6
- 229910052902 vermiculite Inorganic materials 0.000 claims description 6
- 239000012883 rooting culture medium Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000000645 desinfectant Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 2
- 240000005373 Panax quinquefolius Species 0.000 claims 3
- 239000012882 rooting medium Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000001954 sterilising effect Effects 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 235000002791 Panax Nutrition 0.000 abstract 4
- 241000208343 Panax Species 0.000 abstract 4
- 230000009261 transgenic effect Effects 0.000 abstract 1
- 240000004371 Panax ginseng Species 0.000 description 13
- 244000173853 Sanguisorba officinalis Species 0.000 description 10
- 235000008282 Sanguisorba officinalis Nutrition 0.000 description 10
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 235000002789 Panax ginseng Nutrition 0.000 description 1
- 241001072909 Salvia Species 0.000 description 1
- 235000017276 Salvia Nutrition 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
-
- 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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- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for establishing a tissue culture and rapid propagation system of panax japonicus leaves, which comprises the following steps: s1: sterilizing leaves of a panax japonicus explant; s2: inducing the callus of the blood ginseng leaf; s3: multiplication culture of the callus of the blood ginseng leaf; s4: differentiating the callus buds of the blood ginseng leaves; s5: rooting culture; s6: and (5) transplanting the tissue culture seedlings of the panax japonicus. The method is adopted to establish a rapid propagation system of the panax sanguinea, the blade of the panax sanguinea is taken as an explant, the induction rate of the blade of the panax sanguinea reaches 87%, the multiplication coefficient reaches 4.6, and the transplanting survival rate of tissue culture seedlings reaches 95%, so that the method can improve the transgenic and genetic improvement of the panax sanguinea and provide technical support; also provides important basis for the commercial production and the medicinal value development of the blood ginseng.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine plant tissue culture, and particularly relates to a method for establishing a tissue culture and rapid propagation system of panax japonicus leaves.
Background
Sang Shen, also known as Dan Shen and Zi Dan Shen, is a plant of Salvia of Labiatae, and its dried root and rhizome have high medicinal value, and is first recorded in Shen nong Ben Cao Jing, and has the effects of promoting blood circulation, removing blood stasis, relieving pain, nourishing blood, tranquilizing, etc. The blood ginseng is taken as a medicine or food in all countries of the world, and has certain curative effects in the aspects of blood supply, inflammation diminishing, bacteriostasis, antioxidation, tumor resistance and the like.
In recent years, the demand of the blood ginseng is continuously increased, and the market demand cannot be met by traditional breeding. The traditional breeding period is long, the propagation coefficient is low, the propagation coefficient is increased in a short time by applying a tissue culture technology, and the method is beneficial to the efficient commercial production of good varieties of the panax japonicus.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for establishing a tissue culture and rapid propagation system of a panax japonicus leaf, and the method is used for establishing a sterile rapid propagation system of the panax japonicus leaf.
In order to achieve the purpose, the method for establishing the tissue culture and rapid propagation system of the leaves of the panax japonicus comprises the following steps:
s1: selecting a panax japonicus leaf with the average length of 3cm and the width of 2cm as an explant, firstly soaking the panax japonicus leaf in 75% ethanol for 10s, then flushing with double distilled H2O for 3 times, then soaking in 2% NaClO disinfectant for 8min, and finally flushing with double distilled H2O for 4-5 times to obtain a sterile panax japonicus leaf;
s2: the sterile panax japonicus leaves obtained in the step S1 are cut into a plurality of parts by using a sterile scalpel, inoculated into an MS induction culture medium with the pH value of 5.8, and subjected to dark culture for 14d at the culture temperature of 25 +/-1 ℃, and the cut panax japonicus leaves form callus;
s3: transplanting the sterile panax japonicus leaves obtained in the step S2 into an MS proliferation culture medium with the pH value of 5.8, and culturing for 20d under the condition that the illumination intensity threshold is 4800-6000 lx and the illumination culture time is 16 h/d;
s4: transplanting the sterile panax japonicus leaves obtained in the step S3 into a bud differentiation medium with a pH value of 5.8, and culturing and inducing germination under the conditions that the illumination intensity threshold is 4800-6000 lx and the illumination culture is 16 h/d;
s5: cutting the sterile black ginseng leaves obtained in the step S4 into individual plants by using a sterile knife, transplanting the individual plants into a rooting culture medium with the pH value of 5.8, and culturing for 30 days under the conditions that the culture temperature is 25 +/-1 ℃ and the illumination culture time is 16 h/d;
s6, transplanting the rooting seedlings of the sanguinea root obtained in the step S5 into a flowerpot filled with nutrient matrix soil, wherein the greenhouse temperature is 25 ℃.
Preferably, the MS induction culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.1mg/L NAA;
preferably, the MS multiplication culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.5mg/L NAA;
preferably, the bud differentiation medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 0.5mg/L6-BA and 0.1mg/L NAA;
preferably, the rooting culture medium is MS minimal medium added with 3.5g/L agar, 15g/L sucrose and 2.215g/L MS;
preferably, the nutrient matrix soil comprises matrix soil, vermiculite and perlite, and the ratio of the matrix soil to the vermiculite to the perlite is 3: 1.
The method for establishing the tissue culture and rapid propagation system of the leaves of the panax japonicus has the following beneficial effects:
because of the medicinal plants of the panax japonicus, the traditional propagation method has long period and slow propagation speed, and the propagation period is shortened by establishing a tissue culture rapid propagation system of the panax japonicus; the invention takes the leaves of the panax japonicus as the explant, and the induction rate reaches more than 87 percent, the multiplication coefficient reaches 4.6, and the survival rate of the transplantation reaches more than 95 percent through the steps of callus induction, multiplication, bud differentiation, rooting culture, transplantation and the like. Can be used for the commercial production of the sang Shen, better exerts the medicinal value thereof and provides a foundation for the follow-up sang Shen system research and genetic improvement work.
Drawings
FIG. 1 is a flow chart of a method for establishing a tissue culture and rapid propagation system of a leaf of Panax schinseng.
Detailed Description
The present invention will be further described with reference to the following specific embodiments and accompanying drawings to assist in understanding the contents of the invention.
As shown in FIG. 1, the method for establishing a tissue culture and rapid propagation system of a leaf of panax japonicus provided by the invention comprises the following steps: s1: sterilizing the leaves of the sanguisorba officinalis explant, selecting the sanguisorba officinalis leaves which have the average length of 3cm and the width of 2cm and grow healthily and uniformly as the explant, removing soil stains, firstly soaking the sanguisorba officinalis leaves for 10s by using 75% ethanol, then washing the sanguisorba officinalis leaves for 3 times by using double distilled H2O, then soaking the sanguisorba officinalis leaves for 8min by using 2% NaClO disinfectant, finally washing the sanguisorba officinalis leaves for 4-5 times by using double distilled H2O, placing the sanguisorba officinalis leaves in a sterile filter paper culture dish paved with 3 layers, and draining the water on the surfaces of the leaves to obtain the sterile sanguisorba officinalis.
S2: inducing the callus of the blood ginseng leaves, cutting a plurality of parts of the sterile blood ginseng leaves obtained in the step S1 by using a sterile scalpel, inoculating the cut sterile blood ginseng leaves into an MS induction culture medium with the pH value of 5.8, and carrying out dark culture for 14 days at the culture temperature of 25 +/-1 ℃, wherein the cut blood ginseng leaves form the callus, namely the leaves grow the callus which is emerald green compact tissue; wherein the MS induction culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.1mg/L NAA.
S3: and (3) carrying out multiplication culture on the blood ginseng leaf callus, transplanting the sterile blood ginseng leaf obtained in the step S2 into an MS multiplication culture medium with the pH value of 5.8, and culturing for 20d under the conditions that the illumination intensity threshold value is 4800-60001 x and the illumination culture is 16 h/d: wherein the MS multiplication culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.5mg/L NAA.
S4: bud differentiation is carried out on the callus of the blood ginseng leaves, the sterile blood ginseng leaves in the step S3 are transplanted into a bud differentiation culture medium with the pH value of 5.8, and the bud differentiation culture medium is cultured and induced to bud under the condition that the illumination intensity threshold value is 4800-60001 x and the illumination culture time is 16 h/d; wherein the bud differentiation culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 0.5mg/L6-BA and 0.1mg/L NAA.
S5: rooting culture, namely cutting the sterile black ginseng leaves obtained in the step S4 into single plants by using a sterile knife, transplanting the single plants into a rooting culture medium with the pH value of 5.8, and culturing for 30d under the conditions that the culture temperature is 25 +/-1 ℃ and the illumination culture time is 16 h/d; wherein the rooting culture medium is MS minimal medium added with 3.5g/L agar, 15g/L sucrose and 2.215g/L MS.
S6: transplanting the tissue culture seedlings of the sanguisorba officinalis kuntze, namely transplanting the rooting seedlings of the sanguisorba officinalis kuntze obtained in the step S5 into a flowerpot filled with nutrient matrix soil, wherein the temperature of the greenhouse is 25 ℃; wherein the nutrient matrix soil comprises matrix soil, vermiculite and perlite, and the ratio of the matrix soil to the vermiculite to the perlite is 3: 1.
The culture medium, culture dish and scalpel are sterilized by high temperature sterilization pot at 121 deg.C for 20 min. The fresh medium needs to be replaced 1 time every 15 days in each tissue culture stage, and 2% of vitamin C is added into the medium for treatment, so that the browning problem is relieved. The PH value of the tissue culture seedling transplanting soil matrix is alkalescent, the humidity is 55-65%, more than 1 flower is sprayed every week, and the dilution is 2000 times, so that the balanced growth of the sanguinea potted plant is facilitated.
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 (6)
1. A method for establishing a tissue culture and rapid propagation system of a panax japonicus leaf is characterized by comprising the following steps:
s1: selecting a panax japonicus leaf with the average length of 3cm and the width of 2cm as an explant, firstly soaking the panax japonicus leaf in 75% ethanol for 10s, then flushing with double distilled H2O for 3 times, then soaking in 2% NaClO disinfectant for 8min, and finally flushing with double distilled H2O for 4-5 times to obtain a sterile panax japonicus leaf;
s2: the sterile panax japonicus leaves obtained in the step S1 are cut into a plurality of parts by using a sterile scalpel, inoculated into an MS induction culture medium with the pH value of 5.8, and subjected to dark culture for 14d at the culture temperature of 25 +/-1 ℃, and the cut panax japonicus leaves form callus;
s3: transplanting the sterile blood ginseng leaves obtained in the step S2 into an MS proliferation culture medium with the pH value of 5.8, and culturing for 20d under the conditions that the illumination intensity threshold value is 4800-60001 x and the illumination culture time is 16 h/d;
s4: transplanting the sterile blood ginseng leaves obtained in the step S3 into a bud differentiation culture medium with the pH value of 5.8, and culturing and inducing the leaves to bud under the conditions that the illumination intensity threshold value is 4800-60001 x and the illumination culture is 16 h/d;
s5: cutting the sterile black ginseng leaves obtained in the step S4 into individual plants by using a sterile knife, transplanting the individual plants into a rooting culture medium with the pH value of 5.8, and culturing for 30 days under the conditions that the culture temperature is 25 +/-1 ℃ and the illumination culture time is 16 h/d;
s6, transplanting the rooting seedlings of the sanguinea root obtained in the step S5 into a flowerpot filled with nutrient matrix soil, wherein the greenhouse temperature is 25 ℃.
2. The method for establishing the tissue culture and rapid propagation system of the leaves of the panax quinquefolius as claimed in claim 1, wherein the MS induction culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.1mg/L NAA.
3. The method for establishing the tissue culture and rapid propagation system of the leaves of the panax quinquefolius as claimed in claim 1, wherein the MS multiplication culture medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 2 mg/L6-BA and 0.5mg/L NAA.
4. The method for establishing the tissue culture and rapid propagation system of the leaves of the panax japonicus as claimed in claim L, wherein the bud differentiation medium is MS minimal medium added with 3g/L agar, 30g/L sucrose, 4.43g/L MS, 0.5mg/L6-BA and 0.1mg/L NAA.
5. The method for establishing the tissue culture and rapid propagation system of the leaves of the panax quinquefolius as claimed in claim 1, wherein the rooting medium is MS minimal medium supplemented with 3.5g/L agar, 15g/L sucrose and 2.215g/L MS.
6. The method for establishing the tissue culture and rapid propagation system of the leaves of the panax japonicus as claimed in claim 1, wherein the nutrient matrix soil comprises matrix soil, vermiculite and perlite, and the ratio of the matrix soil to the vermiculite to the perlite is 3: 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114145230A (en) * | 2021-12-09 | 2022-03-08 | 四川省农业科学院经济作物育种栽培研究所 | Propagation method for inducing salvia miltiorrhiza to generate cluster bud seedlings |
Citations (1)
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|---|---|---|---|---|
| CN102428875A (en) * | 2011-12-29 | 2012-05-02 | 四川农业大学 | Tissue culture and rapid propagation method of Szechuan salvia miltiorrhiza bunge |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428875A (en) * | 2011-12-29 | 2012-05-02 | 四川农业大学 | Tissue culture and rapid propagation method of Szechuan salvia miltiorrhiza bunge |
Non-Patent Citations (3)
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| 何金莲: "丹参组织培养综述", 《乡村科技》 * |
| 解晓红等: "丹参组培快繁技术研究", 《中药材》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114145230A (en) * | 2021-12-09 | 2022-03-08 | 四川省农业科学院经济作物育种栽培研究所 | Propagation method for inducing salvia miltiorrhiza to generate cluster bud seedlings |
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Application publication date: 20210309 |