CN103828705A - Cultivation method of genuine salvia - Google Patents
Cultivation method of genuine salvia Download PDFInfo
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- CN103828705A CN103828705A CN201410013083.8A CN201410013083A CN103828705A CN 103828705 A CN103828705 A CN 103828705A CN 201410013083 A CN201410013083 A CN 201410013083A CN 103828705 A CN103828705 A CN 103828705A
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Abstract
The invention relates to a cultivation method of genuine salvia, which comprises the following steps of: establishment of a salvia germplasm resource garden, selection of representative genuine salvia germplasm resources, identification of genuine salvia genome karyotype structure, determination of genuine salvia DNA bar codes, artificial mutation to obtain autotetraploid, sexual hybridization to obtain triploid salvia permanent hybrids, detoxification treatment, tissue culture seedling cloning and proliferation, bottle seedling transplanting, transplanted seedling management, new species comparison and testing, determination of the medicinal material yield and the main drug effective components, and genuine salvia new species determination and species identification. The genuine salvia obtained in the invention is, so far, the only population that has two great natural advantages: the polyploid advantage and the heterosis, and the advantages are giant and irreplaceable. Therefore, the method of the invention is an effective method for cultivating genuine traditional Chinese medicine with strong biotic stress and abiotic stress capability by using traditional Chinese medicine genuine functional genomes and feature genes and based on inherent genes of genuine traditional Chinese medicine.
Description
Technical field
The present invention relates to the breeding method of new variety of plant, particularly the breeding method of the super genuine red sage root of a kind of green.
Background technology
The red sage root (
salvia miltiorrhizabunge) be in China's traditional medicine application the earliest, one of Chinese medicine the most widely, be famous promoting blood flow and remove blood stasis drug, be widely used in clinically treating the disease such as coronary heart diseases and angina pectoris, ishemic stroke.Along with China's aging population, angiocardiopathy becomes the principal disease of harm humans health, and the incidence of disease rises year by year, and pharmaceutical requirements amount is increased.The herbal mixture that China produces taking the red sage root as raw material at present has kind more than 100, and there is family more than 100 in the enterprise relating to, huge to the demand of high-quality red rooted salvia.But China goes back the genuine red sage root cultivar that neither one is unified so far.In recent years, agricultural community, for meeting the green revolution for the third time, proposes to cultivate again the objective of the struggle of " green super hybridization rice " after Yuan Longping " super hybridization rice ".Under this inspiration, author proposes to cultivate the objective of the struggle of " the green super genuine red sage root ".
In fact, compared with agriculture germ plasm resource, China's Chinese medicine germ plasm resource is abundanter, has more advantage to cultivating green super species.Traditional Chinese medicine is long at China's applicating history, the a collection of medicinal plant with unique function is throughout the country distributing, they are the special populations that form through the accumulation of long-term natural selection and artificial selection, having characteristic functional genome, is to cultivate the most valuable gene pool of green super genuine traditional Chinese medicine.A lot of genuine traditional Chinese medicines are all that long term growth is under extremely special natural environmental condition, accumulate the gene that the various external worlds can be resisted coerce ability, therefore, excavate as soon as possible, save from damage and utilize the specific function genome in genuine traditional Chinese medicine material, not only favourable to cultivating green super genuine traditional Chinese medicine, China's modernization of Chinese medicine development is also had to leading action.
Summary of the invention
The object of this invention is to provide the breeding method of the super genuine red sage root of a kind of green, it is genuine functional genome and the gene intrinsic according to the genuine traditional Chinese medicine red sage root itself, opposing adventive and abiotic coercing, reach increase root yield, improve the target of main effective ingredient content, realize and not spraying insecticide, do not execute or the object of Shaoshi chemical fertilizer.
The breeding method that the object of this invention is to provide the super genuine traditional Chinese medicine red sage root of a kind of green comprises the following steps:
First be to collect national wild Salvia miltiorrhiza Bge, set up Chinese Salvia miltiorrhiza Bge garden.
Second, from morphology, cell biology (chromosome), molecular biology different aspects, China's Salvia miltiorrhiza Bge is studied, choose representative genuine Salvia miltiorrhiza Bge, and from genome chromosome structure and DNA molecular bar codes technique, the genuine red sage root is identified.
The 3rd, the genuine red sage root of dliploid with hidden indicium proterties and one are there is to the genuine red sage root of dliploid of dominant marker's proterties, carry out respectively induced mutations and become autotetraploid (4x contains 4 identical genuine red sage root functional genomes).
The 4th, triploid red sage root hybridization transformation, to there is respectively hidden indicium proterties (as spending in vain) and there is dominant marker's proterties tetraploid red sage root of (as pale reddish brown), respectively with the genuine red sage root (2x of the dliploid with relative mark property, contain 2 identical genuine red sage root functional genomes) carry out sexual hybridization (4x ♀ × 2x ♂ or 2x ♀ × 4x ♂), obtain triploid red sage root permanent hybrid (3x contains 3 identical genuine red sage root functional genomes).
The 5th, detoxification treatment: taking triploid red sage root cross-pollinated seed seedling stem apex or newborn spire as explant, carry out detoxification treatment with 75% alcohol and 0.1% mercuric chloride.
The 6th, group training seedling clonal expansion: the good explant of detoxification treatment is seeded on MS+6BA1.5mg/L medium, 14 hours/day photoperiods, luminous intensity 3000Lux, under 20-25 DEG C of conditions of room temperature, carry out illumination cultivation, treat that inoculation material cut surface produces Multiple Buds, proceed on 1/2MS+6BA1mg/L medium, carry out clonal expansion.
The 7th, bottle transplantation of seedlings, in the time of the long 1-2 centimetre of the new root of bottle seedling, carries out a bottle transplantation of seedlings, transplants and within first 2 days, first opens bottle cap white silk seedling.
The 8th, transplanted seedling management, the seedling just having shifted out is wanted half shading, keeps the humidity of 45-55%, and the survival rate of strengthening management can reach more than 90%.
The 9th, new varieties rating test, new varieties rating test requires to carry out routinely.
The tenth, root yield, drug main are wanted active constituent content measuring: according to Pharmacopoeia of the People's Republic of China regulation, tanshinone IIA, tanshin polyphenolic acid B and Cryptotanshinone are measured.
The 11, green super genuine red sage root new varieties are assert: press said procedure, according to the red rooted salvia output obtaining and main effective ingredient content, it is comprehensive good with biological character to cultivate, the super genuine red sage root new varieties of green (medicine village output and the comprehensive raising of main effective ingredient content are more than 50%) that antibiont and abiotic stress ability are strong.
The 12, green super genuine red sage root new varieties qualification: by genome Chromosome Identification, can determine that the super genuine red sage root of the green newly cultivating is a triploid red sage root (2n=3x=24) with 3 homologous genes groups, can determine the source of the genuine red sage root functional genome being fixed by genuine red sage root parent mark property and DNA bar code.
The red sage root new varieties that select by the above program of the present invention, different from conventional sexual hybridization breeding, it is by known genuine red sage root functional genome and all characterizing genes thereof, being fixed up by " forever " with triploid red sage root form, triploid can not go normal sexual reproduction, in " space ", upper and other species have formed reproduction isolation, do not have sexual progeny to separate, it is " permanent F1 " permanent hybrid, uniquely up to now to there is the large advantage of natural world two, that is: polyploid advantage and heterotic population, its Heterosis goes out huge property and irreplaceability.Therefore, the present invention utilizes the genuine functional genome of Chinese medicine and characterizing gene thereof, according to the intrinsic gene of genuine traditional Chinese medicine itself, cultivates the effective ways to biology and the strong super genuine traditional Chinese medicine of green of abiotic stress ability.
The breeding method of the super genuine traditional Chinese medicine red sage root of green provided by the invention new varieties, different from conventional sexual hybridization breeding in the past, so-called green Chinese medicine (red sage root) is not execute or Shaoshi chemical fertilizer, do not spray insecticide, it is according to genuine traditional Chinese medicine (red sage root) self intrinsic feature functionality genome and gene completely, and opposing adventive and abiotic stress, reach raising root yield, increase effective ingredient content, promote Chinese medicine sustainability to produce.It is one by chemical fertilizer, and two by agricultural chemicals, and three by alien gene, by intrinsic feature functionality genome and the gene of genuine traditional Chinese medicine self, realizes green health industrialization of Chinese medicine completely.The so-called super red sage root, neither be outer carry out heterologous gene, it is according to the main evolutionary path of higher plant----genome polyploidization combines with hybridization, and the genuine functional genome of more than 3 homology is condensed together, and fully demonstrates the dose effect of genuine red sage root characterizing gene group.Isolate by reproduction, form a permanent hybrid, by polyploid advantage with hybrid vigour is permanent is saved, and be fixed in a kind, become and there are the unique two large advantage-hybrid vigours that have of natural world and the population of polyploid advantage up to now.Therefore, the breeding method of the super genuine red sage root of green provided by the invention, is to excavate and utilize the important channel of genuine traditional Chinese medicine functional genome of China, to China's Chinese medicine rearing new variety with sustainable development is significant and using value.
Brief description of the drawings
Fig. 1: Chinese Salvia miltiorrhiza Bge garden.
Fig. 2: red sage root genome chromosome structure: (a) 2x, (b) 3x, (c) 4x.
Fig. 3-1: the red sage root is dominant, hidden indicium proterties: (a) pale reddish brown, (b) spend in vain.
Fig. 3-2: genuine red sage root coding triplet DNA bar code.
Fig. 4: green super genuine red sage root field planting growth potential.
Fig. 5: green super genuine red sage root new varieties root medicinal material.
Embodiment:
The present invention can be embodied from following example, but it can not impose any restrictions the present invention.
The breeding method of the super genuine red sage root of green provided by the invention comprises the following steps:
(1) set up Chinese Salvia miltiorrhiza Bge garden, collect national wild Salvia miltiorrhiza Bge;
(2) from morphology, cell biology, molecular biology, chemistry (drug ingedient mensuration) aspect, China's Salvia miltiorrhiza Bge is studied, chosen representative genuine Salvia miltiorrhiza Bge.
(3) the genome chromosome structure of the genuine Salvia miltiorrhiza Bge of choosing is identified.
(4) utilize AFLP molecular marking technique (Wang Bing etc.: CHINA JOURNAL OF CHINESE MATERIA MEDICA 32(19) 2007.10)) determine genuine red sage root DNA bar code.
(5) triploid hydrid transformation: will there is hidden indicium proterties and the tetraploid red sage root with dominant marker's proterties respectively, respectively with the genuine red sage root (2x of the dliploid with relative mark property, contain 2 identical genuine red sage root functional genomes) hybridize (4x ♀ × 2x ♂ or 2x ♀ × 4x ♂), obtain triploid red sage root permanent hybrid (3x contains 3 identical genuine red sage root functional genomes).
(6) detoxification treatment: taking triploid red sage root cross-pollinated seed seedling stem apex or newborn spire as explant, carry out detoxification treatment with 75% alcohol and 0.1% mercuric chloride.
(7) group training seedling clonal expansion, is seeded in 1/2MS+6BA1 by explant complete detoxification treatment.On 5mg/L medium, 14 hours/day photoperiods, luminous intensity 3000Lux, carried out illumination cultivation under 20-25 DEG C of conditions of room temperature, treated that inoculation material cut surface produces Multiple Buds, proceeded on 1/2MS+6BA1mg/L medium, carried out expanding propagation.
(8) bottle transplantation of seedlings: in the time that the new root of bottle seedling is grown 2-3 centimeters, carry out a bottle transplantation of seedlings, transplant and first open bottle cap white silk seedling for first 2 days;
(9) transplanted seedling management: the bottle seedling just having shifted out is wanted half shading, keeps the humidity of 50% left and right, and the survival rate of strengthening management can reach more than 90%.
(10) new varieties rating test: new varieties rating test requires to carry out routinely.
Except being undertaken by the requirement of general conventional new varieties rating test, the super genuine traditional Chinese medicine red sage root of green provided by the invention, only execute 2000 kgs/acre of farm organic fertilizers, whole process is not executed or Shaoshi chemical fertilizer, do not spray insecticide, the genuine functional genome that relies on the green super red sage root itself to have completely, to the resistivity of biology and abiotic stress.
(11) root yield, drug main are wanted active constituent content measuring: according to Pharmacopoeia of the People's Republic of China regulation, tanshinone IIA, tanshin polyphenolic acid B and Cryptotanshinone are measured.
(12) green super genuine red sage root new varieties are assert: press said procedure, can cultivate comprehensive organism proterties good, antibiont and abiotic stress ability are strong, root yield and the main high super genuine red sage root new varieties of green (comprehensively improving more than 50%) of effective ingredient content.
By genome Chromosome Identification, can determine that the super genuine red sage root of the green newly cultivating is a triploid red sage root (2n=3x=24) with 3 homologous genes groups, can determine the source of the genuine red sage root functional genome being fixed by genuine red sage root parent mark property and DNA bar code.
Carry out according to above-mentioned step, within 2004-2012 years, gathered 36 different Geographical population reds sage root from the whole nation 12 provinces, cities, and set up Chinese Salvia miltiorrhiza Bge garden (Fig. 1) in Ji County, In The North of Tianjin mountain area.
Through the research from morphology, chemistry, Celluar and Molecular Biology different aspects in 2005-2007 years, choosing No. 33, No. 1, No. 135 germ plasm resources was the genuine Salvia miltiorrhiza Bge of China.
Apply subsequently AFLP molecular marking technique, determine these 3 genuine red sage root DNA bar codes with coding triplet method (3 primer methods), and using " pale reddish brown " and " spending in vain " as dominant and recessive morphological characters mark, subsequently No. 33 (spending in vain) become to autotetraploid (2n=4x=32) with No. 1 (pale reddish brown) genuine red sage root induced mutations, then carry out sexual hybridization with the dliploid red sage root of relative mark property, obtain autotriploid red sage root permanent hybrid (2n=3x=24).For example: to spend the Tetraploid of recessive character in vain, no matter be to do male parent or female parent, its F1 triploid is pale reddish brown, spends 4x ♀ × pale reddish brown 2x ♂ → 3x in vain pale reddish brown that is:, or pale reddish brown 2x ♀ × to spend 4x ♂ → 3x in vain pale reddish brown.Like this, by pale reddish brown with spend mark property in vain and DNA bar code just can know its genome is from which genuine red sage root parent.Table 1 is its content of employing high effective liquid chromatography for measuring, the main effective ingredient measurement result of the green super genuine red sage root.
Table 1:
The steady tanshinone mg/g of the heavy Kg/ strain tanshinone IIA mg/g salvia miltiorrhiza bge I mg/g of the thick root of the long root of code name root number/strain root tanshin polyphenolic acid B mg/g
RY3 50.25 57.50 18.20 2.420 6.389 1.0795 2.046 62.992
Explain: Pharmacopoeia of the People's Republic of China version regulation in 2005: tanshinone IIA content is 0.2%, and danshinolic acid content is 3%.
Claims (6)
1. a breeding method for the genuine red sage root, is characterized in that comprising the following steps:
1) collect national wild Salvia miltiorrhiza Bge and set up Salvia miltiorrhiza Bge garden, set up the Salvia miltiorrhiza Bge garden with representative of China's property;
2) study from morphology, cell biology (Chromosome level), molecular biology horizontal centring state Salvia miltiorrhiza Bge, choose representative genuine Salvia miltiorrhiza Bge;
3) the genome chromosome structure of the genuine Salvia miltiorrhiza Bge of choosing is identified;
4) utilize AFLP molecular marking technique to determine genuine red sage root DNA bar code;
5) the genuine red sage root of dliploid with hidden indicium proterties and one are there is to the genuine red sage root of dliploid (2x) of dominant marker's proterties, carry out respectively induced mutations and become autotetraploid: 4x, contains 4 identical genuine red sage root functional genomes;
6) triploid red sage root hybridization transformation, to there is respectively hidden indicium proterties (spending in vain) and there is the tetraploid red sage root of dominant marker's proterties (pale reddish brown), respectively with the genuine red sage root of the dliploid with relativity mark property, 2x, contain 2 identical genuine red sage root functional genomes, hybridize transformation: 4x ♀ × 2x ♂ or 2x ♀ × 4x ♂, obtain and have the triploid red sage root permanent hybrid of mark property: 3x, contains 3 identical genuine red sage root functional genomes;
7) detoxification treatment: getting triploid red sage root cross-pollinated seed seedling stem apex or newborn spire is explant, carries out detoxification treatment with 75% alcohol and 0.1% mercuric chloride;
8) group training seedling clone is numerous soon: material complete detoxification treatment is seeded on 1/2MS+6BA1.5mg/L medium, 14 hours/day photoperiods, luminous intensity 3000Lux, under 20-25 DEG C of conditions of room temperature, cultivate, treat that material cut surface produces Multiple Buds, proceeds to containing carrying out expanding propagation on 1/2MS+6BA1mg/L medium;
9) bottle transplantation of seedlings, in the time that the new root of bottle seedling is grown 2-3 centimeters, carries out a bottle transplantation of seedlings, transplants and within first 2 days, opens bottle cap white silk seedling;
10) transplanted seedling management, the bottle seedling just having shifted out is wanted half shading, keeps the humidity of 50% left and right, and the survival rate of strengthening management can reach more than 90%;
11) new varieties rating test, new varieties rating test requires to carry out routinely;
12) root yield, drug main are wanted active constituent content measuring, according to Pharmacopoeia of the People's Republic of China regulation, tanshinone IIA, tanshin polyphenolic acid B and Cryptotanshinone are measured;
13) genuine red sage root new varieties are assert: press said procedure, according to the red rooted salvia output obtaining and main effective ingredient content, can cultivate comprehensive organism proterties good, antibiont and abiotic stress ability are strong, improve more than 50% genuine red sage root new varieties comprehensively.
2. according to breeding method claimed in claim 1, it is characterized in that the expanding propagation described in step 8) adopts 5-10 seedlings of every bottle graft kind.
3. according to breeding method claimed in claim 1, it is characterized in that the bottle transplantation of seedlings described in step 9) is to take out a bottle seedling with the long son of taking the photograph, clean medium, directly transplanting is in the prior heap fermentation of process good mill aunt mud and sand 3:1 mixotrophism alms bowl.
4. a breeding method for the genuine traditional Chinese medicine red sage root, is characterized in that comprising the following steps:
1) collect national wild Salvia miltiorrhiza Bge, set up representative Salvia miltiorrhiza Bge garden;
2) from morphology, cell biology (Chromosome level), molecular biology level, Salvia miltiorrhiza Bge is studied, chosen representative genuine Salvia miltiorrhiza Bge;
3) the genome chromosome structure of the genuine Salvia miltiorrhiza Bge of choosing is identified;
4) utilize AFLP molecular marking technique to determine genuine red sage root DNA bar code;
5) by the genuine red sage root of dliploid (2x) that there is hidden indicium proterties and and have dominant marker's proterties, carry out respectively induced mutations and become autotetraploid: 4x, contains 4 identical genuine red sage root functional genomes;
6) triploid red sage root hybridization transformation, to there is respectively hidden indicium proterties (as: spending in vain) and there is the tetraploid red sage root of dominant marker's proterties (as: pale reddish brown), (be 2x with the genuine red sage root of the dliploid with relative mark property respectively, contain 2 identical genuine red sage root functional genomes) hybridize transformation: 4x ♀ × 2x ♂ or 2x ♀ × 4x ♂, acquisition has the triploid red sage root permanent hybrid of mark property: 3x, contains 3 identical genuine red sage root functional genomes;
7) detoxification treatment: taking triploid red sage root cross-pollinated seed seedling stem apex or newborn spire as explant, carry out detoxification treatment with 75% alcohol and 0.1% mercuric chloride;
8) group training seedling clone is numerous soon: explant complete detoxification treatment is seeded on 1/2MS+6BA1.5mg/L medium, 14 hours/day photoperiods, luminous intensity 3000Lux, under 20-25 DEG C of conditions of room temperature, cultivate, treat that material cut surface produces Multiple Buds, proceed on 1/2MS+6BA1mg/L medium, carry out expanding propagation, can inoculate 5-10 seedlings for every bottle;
9) bottle transplantation of seedlings: in the time of the long 1-2 centimetre of the new root of bottle seedling, carry out a bottle transplantation of seedlings, transplant and within first 2 days, first open bottle cap white silk seedling, with the long son taking-up bottle seedling of taking the photograph, clean medium, directly transplant in the mill aunt mud and sand 3:1 mixotrophism alms bowl good through prior heap fermentation.
10) transplanted seedling management: the seedling just having shifted out is wanted half shading, keeps the humidity of 45-55%, strengthens management according to a conventional method;
11) new varieties rating test: new varieties rating test requires to carry out routinely;
12) root yield, drug main are wanted active constituent content measuring: according to Pharmacopoeia of the People's Republic of China regulation, tanshinone IIA, tanshin polyphenolic acid B and Cryptotanshinone are measured;
13) genuine red sage root new varieties are assert: press said procedure, the red rooted salvia output that foundation obtains and main effective ingredient content, cultivate comprehensive and biological character is good, the genuine red sage root new varieties that antibiont and abiotic stress ability are strong.
5. according to breeding method claimed in claim 4, it is characterized in that hidden indicium proterties described in step 5) is for spending in vain, dominant marker's proterties is pale reddish brown.
6. according to breeding method claimed in claim 4, it is characterized in that the triploid red sage root hybridization transformation described in step 6) is: will spend the genuine red sage root in vain and become autotetraploid with pale reddish brown genuine red sage root induced mutations: 2n=4x=32, then carry out sexual hybridization with the dliploid red sage root of relative mark property, obtain autotriploid red sage root permanent hybrid: 2n=3x=24, to spend the Tetraploid of recessive character in vain, no matter be to do male parent or female parent, its F1 triploid is pale reddish brown:
Spend 4x ♀ × pale reddish brown 2x ♂ → 3x in vain pale reddish brown, or pale reddish brown 2x ♀ × spend 4x ♂ → 3x in vain is pale reddish brown.
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| CN107094617A (en) * | 2017-04-24 | 2017-08-29 | 陈瑞阳 | Genuine red sage root first-filial generation use of advantage and its producing method for seed |
| CN108403827A (en) * | 2018-05-29 | 2018-08-17 | 陈瑞阳 | A kind of compound Danshen Root pedicure Chinese medicine composition and preparation method thereof |
| CN109964602A (en) * | 2019-04-19 | 2019-07-05 | 山东省农业科学院农产品研究所 | A kind of Radix Salviae Miltiorrhizae method for culturing and transplanting seedlings and transplantation device |
| CN112946113A (en) * | 2021-02-01 | 2021-06-11 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107043819A (en) * | 2017-04-17 | 2017-08-15 | 云南农业大学 | The bletilla prediction of heterosis method and its application that important phenotypic character is selected are combined based on psbA trnH sequence analyses |
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| CN108403827A (en) * | 2018-05-29 | 2018-08-17 | 陈瑞阳 | A kind of compound Danshen Root pedicure Chinese medicine composition and preparation method thereof |
| CN109964602A (en) * | 2019-04-19 | 2019-07-05 | 山东省农业科学院农产品研究所 | A kind of Radix Salviae Miltiorrhizae method for culturing and transplanting seedlings and transplantation device |
| CN109964602B (en) * | 2019-04-19 | 2021-10-26 | 山东省农业科学院农产品研究所 | Salvia miltiorrhiza seedling transplanting method and transplanting device |
| CN112946113A (en) * | 2021-02-01 | 2021-06-11 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
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