CN103039361B - Method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings - Google Patents
Method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings Download PDFInfo
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- CN103039361B CN103039361B CN2013100108613A CN201310010861A CN103039361B CN 103039361 B CN103039361 B CN 103039361B CN 2013100108613 A CN2013100108613 A CN 2013100108613A CN 201310010861 A CN201310010861 A CN 201310010861A CN 103039361 B CN103039361 B CN 103039361B
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Abstract
The invention discloses a method for directly inducing cluster buds of milletia speciosa champ seeds and quickly breeding seedlings. According to the method, cluster buds of milletia speciosa champ seeds are directly induced by an explant; and the method for breeding seedlings comprises explant sterilization, primary induction, secondary multiplication, rooting and transplanting to obtain milletia speciosa champ seedlings. The method disclosed by the invention has the advantages of simplicity and convenience in operation, low production cost, fast multiplication and high survival rate of rooting and transplanting, facilitates large-scale industrial production and application, can quickly and continuously obtain a great quantity of high-quality milletia speciosa champ test-tube seedlings, and has high practicability.
Description
Technical field
The invention belongs to agricultural biological technical field, specifically a kind of ox energetically the direct induced bundle of seed sprout and the method for fast breeding seedling.
Background technology
Ox is pulse family Millettia plant beautiful millettia (Millettia speciosa Champ.) energetically, has another name called potato, beautiful millettia root energetically, begins to be stated from " the sward property of medicine is standby to be wanted ", is the local medicinal material that " Guangxi traditional Chinese medicine standard " (nineteen ninety version) recorded.Ox is used as medicine with root energetically, distinguishes the flavor of sweet, flat.Return lung, kidney channel.Have qi-restoratives moistening lung, the strong active effect of muscle, it has curative effect preferably to chronic diseases such as lumbar muscle strain, rheumatic arthritis, cough due to deficiency of the lung, pulmonary tuberculosis, chronic bronchitis, chronic hepatitiss clinical proof.The Zhuang pain in waist and lower extremities that is usually used among the people, send out prosperous (treating rheumatic ostealgia), pulmonary tuberculosis, chronic hepatitis, the treatment of chronic gastritis.
Modern study is found, ox contains the compositions such as polysaccharide, alkaloid, coumarin and various trace elements energetically, wherein the Niu great Li polysaccharide is its main active, have and regulate immune system, anti-oxidant, anti-inflammatory, antitumor activity, especially the anti-tumor aspect curative effect is clear and definite, and it has good result for the treatment of to nasopharyngeal carcinoma, oophoroma, lung cancer and colon cancer current clinical studies show.The Niu great Li polysaccharide is also desirable immunopotentiator simultaneously; Alkaloid and coumarin have protect the liver, eliminate the phlegm, the effect of antibechic, analgesia, calmness, spasmolysis, and normal cell is not all had to toxic and side effect, aspect antitumor, antiviral, the anti-ageing medicine of waiting for a long time of exploitation, special advantage is being arranged, become and extracted the fresh target that enterprise competitively develops, market demand is huge.From 20 century 70s, be processed into the Chinese patent drugs such as loins-strengthening and kidney-invigorating bolus, building-up body capsule, osmanthus dragon ointment, Huoluozhitong pills, powerful ZHUIFENGTOUGU WAN, muscle relaxing waist strengthening ball, intelligence development health brain ball, antirheumatic liquid by pharmacy corporation as primary raw material, in national extensive use.In recent years, Niu great Li becomes again the fresh target that extraction enterprise competitively develops, and extracts the compositions such as polysaccharide, maackiain.
Along with the continuous increase of Niu great Li demand, wild resource is exhausted, and seriously supply falls short of demand for raw material.In order to solve imbalance between supply and demand, people attempt adopting tame method to enlarge the medicine source, have found that there is some successful research reports at present.Aspect seed germination, find to have by statistics 2 pieces of reports (ending on November 22nd, 2012) both at home and abroad, yellow autumn silver philosophy carries out germination test and the different substrates seed germination experiment of different seed treatment in same illumination box, seed sprouting best results after result of study shows to process with Seed soaking, average germination percentage reaches 61.25%; The percentage of seedgermination of processing with river sand is the highest, and germinate when needed ask also shorter.Yao Shao the lady in the moons etc. are by measuring Niu great Li seed morphology, thousand kernel weight and water content, the germination rate of research under different seed soaking times, different light, condition of different temperatures, and the further correlation between them relatively, compare germination rate, the germination index under different germinating beds are cultivated, the difference of start bud grain number and germination number of days simultaneously, result shows on the duplex paper bed, seed soaking 24h, 25 ℃, illumination condition, the sprouting of optimum Niu great Li seed.Organizing aspect cultivation, finding by statistics has 5 pieces of reports (ending on November 22nd, 2012) both at home and abroad, wherein take the stem section as explant for 4 pieces, and take fruit pod (seed) for 1 piece is explant.Through relatively, the pollution rate that the stem section of take is explant is than take the height that seed is explant, and seedling organizes the training cycle long through callus, easily causes variation.Wang Zhunian etc. be take mellow fruit pod (seed) and are carried out tissue-culturing quick-propagation Niu great Li as explant, the seed germination medium is 1/2MS, the propagation of bud and subculture medium are MS+6.BA2.0mg/L (same under unit)+NAA0.5, root media is 1/2MS+IBA1.0, the aseptic seedling segment of seed germination is inoculated on proliferated culture medium, form indefinite bud (easily causing variation) through callus, the average growth coefficient of 40d reaches 5.0.Culture of rootage 20d, rooting rate reaches 80%.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art provide a kind of ox energetically the direct induced bundle of seed sprout and the method for fast breeding seedling.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
A kind of ox direct induced bundle of seed energetically sprouts and the method for fast breeding seedling, and the Niu great Li seed is that the direct induced bundle of explant is sprouted, the method for breeding seedling comprise explant sterilization, just generation induce, shoot proliferation, take root, the transplanting process:
1. explant sterilization process
Get the ripe black seed of Niu great Li, be that 0.05% liquid detergent solution soaks 10min by concentration expressed in percentage by volume, then rinse 15min through flowing water.Use 0.1%HgCl on superclean bench
2solution sterilization 16min, then sway washing 4 times with sterile water, then after with aseptic blotting paper, moisture being blotted, be seeded to just for inducing culture.
2. just for Induction Process
The good seed of step (1) sterilizing is seeded to just for inducing culture MS+3.0mgL
-16-BA+1.5mgL
-1kT+0.2mgL
-1nAA+500mgL
-1carrying out seed germination and inducing clumping bud on PVP cultivates; Incubation time<the 25d of this process, inductivity can reach 100%.
The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes.Condition of culture is (26 ± 2) ℃, intensity of illumination 2000Lx, light application time 10h/d;
3. shoot proliferation process
The bud clump that step (2) is just cut containing 2-3 bud for the Multiple Buds of inducing cultivation to obtain is transferred to subculture medium MS+2.0mgL
-16-BA+0.5mgL
-1kT+0.2mgL
-1tDZ+0.2mgL
-1breed cultivation in NAA, the cycle is 20-25d, and the propagation multiple is 9.68/20d.
The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes.Condition of culture is (26 ± 2) ℃, intensity of illumination 2000Lx, light application time 10h/d.
4. rooting process
More than shoot proliferation bud seedling grows to 2cm, be cut into single bud from base portion, be transferred to root media 1/2MS+2.5mgL
-1iBA+1.5mgL
-1root induction in IAA, cultivate 7d and start the former base of long root, obtains a bottle seedling after 25d, the hardening of can uncapping.
The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes.Condition of culture is (26 ± 2) ℃, intensity of illumination 2000Lx, light application time 10h/d;
5. transplanting process
The bottle seedling that step (4) is taken root is moved the hardening canopy to, open bottle cap hardening 4d, then with tweezers, the bottle seedling is taken out, wash away the medium that sticks to root with clear water, be transplanted on thin river sand, after transplanting, in 10d, keep relative moisture 95% left and right, temperature is controlled at 23-25 ℃ daytime, night, 15-18 ℃, do not have the condition of direct light, transplanting survival rate 84.07%.The cup plantation can be gone up after transplanting 15d, after 30d, field production can be carried out.
Advantage of the present invention: a whole set of method provided by the invention is easy and simple to handle, and production cost is low, and growth rate is fast, the rooting and transplant survival rate is high, be convenient to large-scale industrial production application, can be in a large number, fast, continue the high-quality ox of acquisition test-tube plantlet energetically, practical.This technology directly induces Multiple Buds by the Niu great Li seed, under the just effect for inducing culture, seed germination and inducing clumping bud carry out simultaneously, from existing bibliographical information first to induce seed asepsis sprouting to cut the method that stem section induced bundle sprouts different again, greatly shortened the cycle of group training, compare Multiple Buds with existing research report and obtain higher propagation multiple, and inducing clumping bud is without the callus stage, greatly reduce the possibility of variation, be conducive to keep the merit of seedling.
Embodiment
Below in conjunction with specific implementation method, the invention will be further described.
A kind of ox direct induced bundle of seed energetically sprouts and the implementation method of fast breeding seedling:
1. explant is selected and sterilization process
Get the ripe black seed of Niu great Li, be that 0.05% liquid detergent solution soaks 10min by concentration expressed in percentage by volume, then rinse 15min through flowing water.Use 0.1%HgCl on superclean bench
2solution sterilization 16min, then sway washing 4 times with sterile water, then after with aseptic blotting paper, moisture being blotted, be seeded to just for inducing culture.The pollution rate of this sterilizing methods is 13.33%, and survival rate is 100.00%.
If the fruit pod is not ftractureed, get ripe black fruit pod, soak 10min with 0.05% liquid detergent solution, scrub surface to remove surperficial pubescence and dirt with brush in immersion process, then rinse 15min through flowing water.Use 0.1%HgCl on superclean bench
2solution sterilization 20min, then sway washing 3 times with sterile water, then with aseptic blotting paper, moisture is blotted to rear rip cutting and cut the fruit pod open, takes out seed and be seeded to just for inducing culture.This sterilizing methods is pollution-free, and survival rate is 100.00%.
2. just for Induction Process
By sterilizing, good seed is seeded to just for inducing culture MS+3.0mgL
-16-BA+1.5mgL
-1kT+0.2mgL
-1nAA+500mgL
-1carry out seed germination and inducing clumping bud on PVP and cultivate, at medium supplemented 30g/L sucrose and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes.Condition of culture is (26 ± 2) ℃, intensity of illumination 2000Lx, light application time 10h/d, inductivity 100%.Cultivate 25d, seed germination and while induce the Multiple Buds with 4-5 budlet at the hypocotyl place, inducing clumping bud rate 100%.Under the medium effect of this formula, seed germination and inducing clumping bud carry out simultaneously, from existing bibliographical information first to induce seed asepsis sprouting to cut the method that stem section induced bundle sprouts different again.This method has shortened the required time of inducing clumping bud greatly, the bud fast growth, and without vitrification phenomenon, and bud has greatly reduced the possibility of variation without the callus process.In addition, added PVP in medium and also solved the problem of planting brownization of skin, shortened the required time of seed germination and improved germination rate, added after PVP seed and show money or valuables one carries unintentionally and only needs the 7d left and right.
3. shoot proliferation process
The bud clump that the Multiple Buds of inducing cultivation to obtain first generation cuts containing 2-3 bud is transferred to subculture medium MS+2.0mgL
-16-BA+0.5mgL
-1kT+0.2mgL
-1tDZ+0.2mgL
-1breed cultivation in NAA, at medium supplemented 30g/L sucrose and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes.Condition of culture is (26 ± 2) ℃, intensity of illumination 2000Lx, light application time 10h/d, and the propagation multiple is 9.68/20d, and the Multiple Buds growth is fast, without vitrification phenomenon.
4. rooting process
More than shoot proliferation bud seedling grows to 2cm, be cut into single bud from base portion, be transferred to root media 1/2MS+2.5mgL
-1iBA+1.5mgL
-1root induction in IAA, cultivate 7d and start the former base of long root, the hardening of can uncapping after 25d.Rooting rate is more than 90%.
5. acclimatization and transplants process
The bottle seedling of taking root is moved to the hardening canopy, open bottle cap hardening 4d, then with tweezers, the bottle seedling is taken out, wash away the medium that sticks to root with clear water, be transplanted on thin river sand, after transplanting, in 10d, keep relative moisture 95% left and right, temperature is controlled at 23-25 ℃ daytime, night, 15-18 ℃, do not have the condition of direct light, transplanting survival rate 84.07%.The cup plantation can be gone up after transplanting 15d, after 30d, field production can be carried out.
Claims (1)
- An ox energetically the direct induced bundle of seed sprout and the method for fast breeding seedling, it is characterized in that, ox seed energetically is that the direct induced bundle of explant is sprouted, the method for breeding seedling comprise explant sterilization, just generation induce, shoot proliferation, take root, the transplanting process:(1) explant sterilization processGet the ripe black seed of Niu great Li, be that 0.05% liquid detergent solution soaks 10min by concentration expressed in percentage by volume, then rinse 15min through flowing water, use 0.1%HgCl on superclean bench 2solution sterilization 16min, then sway washing 4 times with sterile water, then after with aseptic blotting paper, moisture being blotted, be seeded to just for inducing culture;(2) just for Induction ProcessThe good seed of step (1) sterilizing is seeded to just for inducing culture MS+3.0mgL -16-BA+1.5mgL -1kT+0.2mgL -1nAA+500mgL -1carry out seed germination and inducing clumping bud on PVP and cultivate, the incubation time<25d of this process;The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes, condition of culture was 26 ± 2 ℃, intensity of illumination 2000Lx, light application time 10h/d;(3) shoot proliferation processThe bud clump that step (2) is just cut containing 2-3 bud for the Multiple Buds of inducing cultivation to obtain is transferred to subculture medium MS+2.0mgL -16-BA+0.5mgL -1kT+0.2mgL -1tDZ+0.2mgL -1breed cultivation in NAA, the cycle is 20-25d, and the propagation multiple is 9.68/20d;The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes, condition of culture was 26 ± 2 ℃, intensity of illumination 2000Lx, light application time 10h/d;(4) rooting processMore than shoot proliferation bud seedling grows to 2cm, be cut into single bud from base portion, be transferred to root media 1/2MS+2.5mgL -1iBA+1.5mgL -1root induction in IAA, cultivate 7d and start the former base of long root, obtains a bottle seedling after 25d, the hardening of can uncapping;The additional 30g/L sucrose of described medium and 5g/L agar, pH6.0, after preparing and packaging, in 121 ℃ of sterilizings 20 minutes, condition of culture was 26 ± 2 ℃, intensity of illumination 2000Lx, light application time 10h/d;(5) transplanting processThe bottle seedling that step (4) is taken root is moved the hardening canopy to, open bottle cap hardening 4d, then with tweezers, the bottle seedling is taken out, wash away the medium that sticks to root with clear water, be transplanted on thin river sand, keep relative moisture 95% after transplanting in 10d, temperature is controlled at 23-25 ℃ daytime, night 15-18 ℃, the condition that there is no direct light, transplanting survival rate 84.07%, can go up the cup plantation after transplanting 15d, carry out field production after 30d.
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| CN103749029B (en) * | 2013-12-29 | 2015-07-15 | 四川师范大学 | Seed germination method for idesia polycarpa seedling culture |
| CN104372020B (en) * | 2014-11-17 | 2017-06-16 | 广西壮族自治区药用植物园 | A kind of method for tissue culture of beautiful millettia root hairy root induction and propagation |
| CN104429971B (en) * | 2014-12-18 | 2017-01-11 | 广州市鲸力生物科技有限公司 | Tissue culture seedling-raising method of millettia fordii dunn |
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| CN104521764B (en) * | 2015-01-26 | 2016-07-06 | 广西壮族自治区药用植物园 | A kind of method of knot potato in Millettia Speciosa tissue cultured seedling bottle |
| CN104604691B (en) * | 2015-02-11 | 2016-07-27 | 湖南省农业生物资源利用研究所 | A kind of Millettia dielsiana tissue culture and rapid propagation method |
| CN104737905B (en) * | 2015-02-14 | 2016-08-17 | 涂湘炎 | The method that a kind of ox tissue cultures energetically is taken root |
| CN106962188A (en) * | 2017-03-01 | 2017-07-21 | 防城港市防城区蚕种场 | Beautiful millettia root seed directly induces the method from bud and fast breeding seedling |
| CN107135944A (en) * | 2017-05-04 | 2017-09-08 | 南宁市辉雄中药材农民专业合作社 | A kind of beautiful millettia root root tissues culture medium and preparation method thereof |
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| CN107896890A (en) * | 2017-12-08 | 2018-04-13 | 桂林亦元生现代生物技术有限公司 | The method for culturing seedlings and implantation methods of a kind of beautiful millettia root |
| CN108142297A (en) * | 2018-01-30 | 2018-06-12 | 东兰县委荣村伟造林下药材种植合作社 | A kind of method for tissue culture for improving beautiful millettia root kind shoot survival percent |
| CN110521594A (en) * | 2019-07-22 | 2019-12-03 | 佛山市粤山生物科技有限公司 | A kind of cultural method improving beautiful millettia root rooting rate |
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| JPS63313579A (en) * | 1987-06-16 | 1988-12-21 | Kikkoman Corp | Preparation of callus originated from protoplast of leguminous plant |
| JPH07184494A (en) * | 1993-12-27 | 1995-07-25 | Nissan Chem Ind Ltd | Plant cell embryo and germination stimulation |
| CN101124891A (en) * | 2006-08-15 | 2008-02-20 | 钦州市中医药研究所 | Method for propagating tissue cultured millettia specisoa champ seedling |
| CN101395991B (en) * | 2008-10-24 | 2010-09-01 | 中国热带农业科学院热带作物品种资源研究所 | Method for propagating Millettia speciosa Champ. seedlings using cottage |
| CN101379950B (en) * | 2008-10-24 | 2010-12-08 | 中国热带农业科学院热带作物品种资源研究所 | Method for quickly breeding millettia speciosa champ germchit using test tube micro cuttage |
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