CN102550413A - Rapid propagation method for tube seedlings of high-grade polygonatum cyrtonema hua - Google Patents
Rapid propagation method for tube seedlings of high-grade polygonatum cyrtonema hua Download PDFInfo
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Abstract
A rapid propagation method for tube seedlings of high-grade polygonatum cyrtonema hua relates to the tissue culture technology of plants. The formulas of culture mediums are as follows: an induction culture medium includes MS, 1.0 mg/L of 6-BA, and 0.5 mg/L of NAA, a proliferation culture medium for root tubers with buds include MS, 10 mg/L of 6-BA, 0.5 mg/L of NAA, and 60 g/L of sugar, and a proliferation culture medium for the bud body parts include MS, 1.0 mg/L of 6-BA, 0.5 mg/L of 2,4-D, and 60 g/L of sugar, a proliferation and seedling strengthening culture medium for the root tuber parts include MS, 1.0 mg/L of 6-BA, 0.5 mg/L of NAA, 0 to 50 mg/L of paclobutrazol, and 60 g/L of sugar, and a rooting culture medium for the root tuber parts include MS, 0.5 mg/L of 6-BA, 0.5 mg/L of NAA, and 0.5% of activated charcoal. The method comprises the steps as follows: the surface layers of the root tuber parts of the disinfected root tubers with buds are removed, the root tubers with buds are cut into root tubers and buds after being cultivated in the induction culture medium and the proliferation culture medium, the root tubers are transferred to enrichment culture and cultivated circularly for 3 to 6 generations, the buds are transferred to proliferation culture of the bud parts and are proliferated in a compound and circulating manner for 3 to 6 generations, and proliferation strong seedling culture and rooting culture are performed to final end products which are the root tubers with buds. The method is applicable to rapid propagation and scale production.
Description
Technical field:
The present invention relates to field of plant tissue culture technique, particularly high-quality David's-harp test-tube plantlet method for quickly breeding.
Background technology:
David's-harp (Polygonatum cyrtonema Hua) is a Liliaceae Polygonatum medicinal plant, and its rhizome is made Chinese medicine sealwort usefulness, contains compositions such as polysaccharide, amino acid, anthraquinone analog compound, has effects such as antibiotic, step-down, anti-ageing and treatment rheumatalgia.In the more southern areas of China cultivation is arranged, it mainly leans on the asexual reproduction and the seed of rhizome to breed, but because reproduction coefficient is low slow with long speed of seminal propagation cycle, a large amount of seedlings can't be provided, thereby establishing in large scale is restricted.In addition, long-term asexual reproduction is prone to cause germplasm character to degenerate, and affects the genetic stability of kind.Utilize method for tissue culture breeding David's-harp, can improve its moral character effectively, increase reproduction coefficient, shorten cultivation period.
At present; Sprout carries out about the report of the tissue culture of David's-harp and breeding fast is based on more, and the method step of group training system is: explant selection, preliminary treatment and sterilization → inducing culture cultivations → proliferated culture medium cultivation → propagation strong seedling culture base cultivation → root media cultivation.
For explant selection, sterilization; The wild sealwort rhizome that Xu Zhongchuan etc. 2006 will fetch is earlier washed earth off in clear water after; Use detergent immersion 30min again, running water flushing, again on the sterile working platform with the 0.15% mercuric chloride 10min that sterilizes; Sterilization deionized water washing 4 times is received on the inducing culture after cutting otch.Liu Hongmei etc. 2010 are after the sealwort rhizome is dug out from soil, and flush away earth cuts band bud rhizome with cutter and in the liquid detergent aqueous solution, soaks 30min, cleans afterwards, cleans with 75% ethanol, soaks 5min with liquid detergent again, the running water flushing; On the sterile working platform, adopt repetition 2.5% clorox 5min again, the sterilization deionized water is scrubbed 2 processes and is carried out for three times, uses the aseptic filter paper suck dry moisture at last, cuts the downright bad part of wound with cutter and inoculates inducing culture.Cultivate for inducing culture afterwards; Forefathers study and mainly concentrate on the not the same of medium component, and the inducing culture that Xu Zhongchuan etc. 2006 adopt is MS+6-BA4.0mg/L+NAA0.2mg/L, and Liu Hongmei etc. 2010 think 2; 4-D more helps the adventitious bud inducing of David's-harp than NAA; Yang Yuhong 2011 thinks 6-BA to inducing sealwort callus growth effect remarkable, 2, and 4-D, NAA and KT effect are not remarkable.
With 0.15% mercuric chloride sterilization 10min, the deionized water of sterilizing washs 4 times or on the sterile working platform, adopts repetition 2.5% clorox 5min on the sterile working platform, and the sterilization deionized water is scrubbed 2 processes and carried out the disinfection technology of all conduct routines at present for three times.
Cultivate for proliferated culture medium, Xu Zhongchuan etc. 2006 are with after inducing gained clump bud to be cut into one clump of 2-3 strain (being 2-3 sprout), are inoculated in that serial proliferated culture medium breeds; On the medium component; Xu Hongmei etc. 2003 think hormone combinations TDZ 1.5mg/L+2, and 4-D1.0mg/L is the most favourable to root of Rhizoma Polygonati stem eye propagation, and Xu Zhongchuan etc. 2006 think that hormone combinations 6-BA4.0mg/L+NAA0.2mg/L can not only make the bud fast breeding; And the comprehensive quality of adventitious bud proliferation is good, does not have lopsided blade and produces.For the strengthening seedling and rooting cultivation stage, Xu Zhongchuan etc. 2006 are with differentiation clump bud inoculation medium MS+6-BA1.0mg/L+NAA0.2mg/L, with the clump bud separately, get healthy and strong single rhizome bud behind the 30d, are inoculated on the MS+NAA0.5mg/L root media.In a word, existing research is confined to utilize sprout to cultivate, and is slower for the growth rate of later stage seedling, can't satisfy the large-scale production of high-quality sealwort seedling.
Summary of the invention:
The object of the invention is based on above-mentioned technical background; Adopted the preliminary treatment before the sterilization; In conjunction with having studied the influence that sugar concentration and growth regulator paclobutrazol concentration in the medium are partly bred David's-harp piece root portion and sprout; So that the culture media composition prescription and the application thereof of David's-harp piece root propagation to be provided, high-quality David's-harp test-tube plantlet method for quickly breeding is provided further.Satisfy the large-scale production of high-quality sealwort seedling.
The present invention solves the technical scheme that its technical problem is taked: comprise culture medium preparation; The step of propagation method is: explant selection, preliminary treatment and sterilization → inducing culture cultivation → proliferated culture medium cultivation → propagation strong seedling culture base cultivation → root media is cultivated; Each condition of culture of organizing the training stage is: 25 ± 2 ℃ of temperature, light application time 12h/d, and induction period adopts scattered light, propagation, the strong sprout and the stage of taking root intensity of illumination 2000Ix; It is characterized in that:
(1) culture medium preparation:
Various culture medium prescriptions are:
(I) inducing culture MS+1.0mg/L 6-BA+0.5mg/L NAA;
(II) the proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L NAA+ sugar 60g/L of band sprout tuber root;
(III) sprout part proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L 2,4-D+ sugar 60g/L;
(IV) piece root portion propagation strong seedling culture base MS+1.0mg/L 6-BA+0.5mg/L NAA+ paclobutrazol 0-50mg/L+ sugar 60g/L;
(V) piece root portion root media MS+0.5mg/L 6-BA+0.5mg/L NAA+0.5% active carbon; Above I-V medium pH is all between 5.6-5.7;
(2) step of propagation method is:
1. explant selection, preliminary treatment and sterilization: material selection then 9, October; Choose the piece root of David's-harp band bud in fine day; After it is cleaned up, place the thiophanate methyl of 1/700 concentration to soak 30min and take out, place normal temperature condition to dry; Carry out disinfection with the piece root of conventional disinfection technology afterwards, sterile water is blotted subsequent use with aseptic filter paper at last the band bud;
2. inducing culture is cultivated: the piece root of the band bud that will disinfect cuts open piece root portion top layer with scalpel; Only stay main bud to add the piece root of 0.8-1.2cm3, insert on (I) number inducing culture and carry out inducing culture, after 40-50 days; Induce the young shoot that makes new advances, the piece root portion is expanded;
3. proliferated culture medium is cultivated:
(a), the proliferated culture medium that will induce piece root switching (II) number band sprout tuber root of sprouting carries out enrichment culture, after 30-40 days, the piece root portion is expanded, new sprout is emerged;
(b), the piece undercut of the band bud that will breed becomes piece root portion and sprout part, on the proliferated culture medium of piece root portion switching (II) number band sprout tuber root, after 30-40 days, expands once more, new sprout is emerged once more; The piece root of the new propagation band bud that grows; The piece root repetitive cycling of new propagation band bud this (b) is the sprout that scales off for the piece root that produces end-product band sprout in the last reign of a dynasty and intermediate product branch of said process enrichment culture 3-6 step by step, and the piece root of end-product band sprout can get into and bred the follow-up propagation strong seedling culture base incubation step of piece root portion the last reign of a dynasty;
(c), the intermediate product sprout that (b) middle step by step circulation step cutting is got off is partly transferred on (III) number sprout part proliferated culture medium; After 30-40 days; Sprout part base portion piece root expands; Then return again (b) step by step with (c) step by step said process carry out 3-6 and breed for combined-circulation, produce the piece root of end-product band sprout in the last reign of a dynasty at last;
4. breeding the strong seedling culture base cultivates: will pass through (b) circulate step by step and through (b) (c) step by step the combined-circulation enrichment culture produce the last reign of a dynasty end-product band sprout piece root piece root portion switching (IV) number piece root portion propagation strong seedling culture base carry out strong seedling culture; After 30-40 days; The piece root expands once more, and new sprout is emerged;
5. root media is cultivated: will through step 4. the piece root portion in strong sprout be inoculated on (V) number piece root portion root media, after 20-30 days, piece root portion base portion grows domestication and the transplanting that root system can carry out tissue cultivating seedling.
The invention has the advantages that:
1, the David's-harp test-tube plantlet that proposes is cultivated and is cut the circulation incubation for new bud and piece root; The big quality of David's-harp piece root manure that obtains at last is all good, and rooting rate reaches 100%, is beneficial to follow-up field production, effectively shortens growth cycle.
2, method adopts astigmatism to cultivate because the piece root of band bud has carried out the preliminary treatment of the thiophanate methyl of 1/700 concentration in the inducing culture stage, reduces the sterilization pollution rate of inducing effectively, promotes the differentiation and the growth of tissue cultivating seedling.
3, sugar and MS medium that the David's-harp test-tube plantlet MS medium that proposes adds 60g/L concentration add the piece root proliferate that certain density paclobutrazol can effectively promote David's-harp; Solved the problem of nutrient distribution in the David's-harp enrichment culture process; In addition, certain density paclobutrazol can also promote the formation of bud.
Description of drawings:
Accompanying drawing is cultivated schematic flow sheet for the present invention.
Embodiment:
In step (c) combined-circulation propagation step by step that 3. proliferated culture medium is cultivated; Turn out by (III) number sprout part proliferated culture medium each in generation the band sprout piece root; Except that being with the last reign of a dynasty sprout piece root directly as the last reign of a dynasty finished product, all the other previous generations all get into (b) (c) circulation step by step.
Embodiment 1: this David's-harp tissue culture quick propagation culturing method in the present embodiment 1, carry out as follows:
(1) culture medium preparation:
Culture medium prescription: (I) inducing culture MS+1.0mg/L 6-BA+0.5mg/L NAA; (II) the proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L NAA+ sugar 60g/L of band sprout tuber root; (III) sprout part proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L 2,4-D+ sugar 60g/L; (IV) piece root portion propagation strong seedling culture base MS+1.0mg/L 6-BA+0.5mg/L NAA+ paclobutrazol 0-50mg/L+ sugar 60g/L; (V) piece root portion root media MS+0.5mg/L 6-BA+0.5mg/L NAA+0.5% active carbon; Above I-V medium pH is all between 5.6-5.7.
(3) the piece root induction of David's-harp band bud is cultivated: will cut open piece root portion top layer with scalpel with the piece root of the band bud of disinfecting, and only stay main bud to add 0.8-1.2cm
3The piece root, insert on (I) number inducing culture and carry out inducing culture, after 40-50 days, induce the young shoot that makes new advances, the piece root portion is expanded;
(4) David's-harp has induced the enrichment culture of the piece root of sprouting: piece root switching (II) number proliferated culture medium that (a) will induce sprouting carries out enrichment culture, and 30-40 days, the piece root portion was expanded, and new sprout is emerged.The piece undercut of the band bud that (b) will breed becomes the sprout part of piece root portion and band bud, piece root portion switching (II) number proliferated culture medium, and 30-40 days, piece root piecemeal piece root expanded once more, and new sprout is emerged once more.Piece root portion circulation (b) process enrichment culture 3-6 of new propagation band bud is for the strong seedling culture that can breed the piece root portion.
(5) David's-harp has bred the strong seedling culture of the piece root portion of separation: piece root portion switching (IV) number medium of band bud of newly expanding in the propagation that will circulate 3-6 generation carries out strong seedling culture, and after 30-40 days, the piece root expands once more, and new sprout is emerged.
(6) culture of rootage of David's-harp piece root portion: will partly be inoculated on (V) number root media through the piece root portion in step (5) strong sprout, after 20-30 days, piece root portion base portion grows domestication and the transplanting that root system can carry out tissue cultivating seedling.
Each condition of culture of organizing the training stage is: 25 ± 2 ℃ of temperature, light application time 12h/d, induction period adopts scattered light, propagation, the strong sprout and the stage of taking root intensity of illumination 2000Ix.
Embodiment 2: this David's-harp tissue culture quick propagation culturing method in the present embodiment 2, carry out as follows:
(1) culture medium preparation:
Culture medium prescription: (I) inducing culture MS+1.0mg/L 6-BA+0.5mg/L NAA; (II) the proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L NAA+ sugar 60g/L of band sprout tuber root; (III) sprout part proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L 2,4-D+ sugar 60g/L; (IV) piece root portion propagation strong seedling culture base MS+1.0mg/L 6-BA+0.5mg/L NAA+ paclobutrazol 0-50mg/L+ sugar 60g/L; (V) piece root portion root media MS+0.5mg/L 6-BA+0.5mg/L NAA+0.5% active carbon; Above I-V medium pH is all between 5.6-5.7.
(2) explant selection, preliminary treatment and sterilization: material selection coming year 9, October, choose the piece root of David's-harp band bud in fine day, after it is cleaned up; Place the thiophanate methyl of 1/700 concentration to soak 30min; Take out, place normal temperature condition, dry; Carry out disinfection with the piece root of conventional technology afterwards, sterile water is blotted subsequent use with aseptic filter paper at last the band bud.
(3) the piece root induction of David's-harp band bud is cultivated: will cut open piece root portion top layer with scalpel with the piece root of the band bud of disinfecting, and only stay main bud to add 0.8-1.2cm
3The piece root, insert on (I) number inducing culture and carry out inducing culture, after 40-50 days, induce the young shoot that makes new advances, the piece root portion is expanded;
(4) David's-harp has induced the enrichment culture of the piece root of sprouting: piece root switching (II) number proliferated culture medium that (a) will induce sprouting carries out enrichment culture, and after 30-40 days, the piece root portion is expanded, and new sprout is emerged.The piece undercut of the band bud that (b) will breed becomes piece root portion and sprout part, on piece root portion switching (II) number proliferated culture medium, after 30-40 days, expands once more, and new sprout is emerged once more.Piece root portion circulation (b) process enrichment culture 3-6 of new propagation band bud is for the strong seedling culture that can breed the piece root portion; (c) on sprout was partly transferred (III) number sprout proliferated culture medium, after 30-40 days, sprout part base portion piece root expanded; Piece root (II) number proliferated culture medium of transferring once more of band bud of will expanding carries out enrichment culture; After 30-40 days, the piece root portion is expanded, and new sprout is emerged.The piece undercut of the band bud that (d) will breed becomes the sprout part of piece root portion and band bud, on piece root portion switching (II) number proliferated culture medium, after 30-40 days, expands once more, and new sprout is emerged once more.(e) the piece root portion of new propagation band bud circulation (d) process enrichment culture 3-6 is for the strong seedling culture that can breed the piece root portion.And the new sprout that separates partly circulate (c) (d) (e) process cultivate 3-6 generation, the new piece of propagation root portion is carried out follow-up strong seedling culture, new sprout partly adopts conventional method to carry out strengthening seedling and rooting.
(5) David's-harp has bred the strong seedling culture of piece root portion and the sprout part of separation: piece root portion switching (IV) number medium of band bud of newly expanding in the propagation that will circulate 3-6 generation carries out strong seedling culture, and after 30-40 days, the piece root expands once more, and new sprout is emerged.
(6) culture of rootage of David's-harp piece root portion: will partly be inoculated on (V) number root media through the piece root portion in step (5) strong sprout, after 20-30 days, piece root portion base portion grows domestication and the transplanting that root system can carry out tissue cultivating seedling.
Each condition of culture of organizing the training stage is: temperature 25 ± 2C, light application time 12h/d, induction period adopts scattered light, propagation, the strong sprout and the stage of taking root intensity of illumination 2000Ix.
The applicant discovers the David's-harp explant that is used to induce owing to be under the face of land for a long time, and its rhizome covers earth outward, and mushroom grows, and induces if directly carry out disinfection, and can cause in the group training process seriously pollutedly, so explant sterilization pre-treatment is most important.Simultaneously; Research finds that also the piece root size and the quality of David's-harp propagation test-tube plantlet are the key influence factors of later stage field production; Therefore study on the basis forefathers; Experiment is inquired into the propagation of David's-harp, has mainly studied in the medium polysaccharide concentration and growth regulator paclobutrazol to the influence of David's-harp propagation.Polysaccharide is serving as the effect that provides the energy and steady seepage to press as the important supplies of carbon source in the tissue culture in tissue culture, its interpolation concentration in medium is particularly important.The growth regulator paclobutrazol is the triazole type plant growth regulator of succeeding in developing the eighties, and it is synthetic to suppress endogenous gibberellins, has the plant growing of delaying; Suppress the cane elongation; Shorten internode, promote that plant tillers, promotes flower bud differentiation, increase the plant stress-resistance performance, improve effects such as output.It had series report to use in recent years on the various plants tissue culture, and Xi Mengli etc. 2000 think and alternately add paclobutrazol 0 and 0.2mg/L at the flameray gerbera proliferated culture medium, can make the sprout growth coefficient high and the propagation bud is healthy and strong.Chen Chuanhong etc. 2006 think in ginger test-tube plantlet expansion breeding culture medium; Additional 1.5mg/L paclobutrazol can make test-tube plantlet propagation carry out synchronously with strong sprout; Improve transplanting survival rate greatly; And in the medium of MS+8.0-10.0mg/L paclobutrazol, the quality saving phase of ginger test-tube plantlet can extend to 4-5 month.Xue Han blue or green 2007; Liu Huaxia 2009 is reported in the flower lily group training medium and adds paclobutrazol; Can promote the reproductive growth of flower lily group training, promote bud propagation, adventitious embryo generation, bud formation and bulb to form, strengthen lily resistance, improve protoplast cold resistance etc.
Claims (1)
1. high-quality David's-harp test-tube plantlet method for quickly breeding comprises culture medium preparation; The step of propagation method is: explant selection, preliminary treatment and sterilization → inducing culture cultivation → proliferated culture medium cultivation → propagation strong seedling culture base cultivation → root media is cultivated; Each condition of culture of organizing the training stage is: 25 ± 2 ℃ of temperature, light application time 12h/d, and induction period adopts scattered light, propagation, the strong sprout and the stage of taking root intensity of illumination 2000Ix; It is characterized in that:
(1) culture medium preparation:
Various culture medium prescriptions are:
(I) inducing culture MS+1.0mg/L 6-BA+0.5mg/L NAA;
(II) the proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L NAA+ sugar 60g/L of band sprout tuber root;
(III) sprout part proliferated culture medium MS+1.0mg/L 6-BA+0.5mg/L 2,4-D+ sugar 60g/L;
(IV) piece root portion propagation strong seedling culture base MS+1.0mg/L 6-BA+0.5mg/L NAA+ paclobutrazol 0-50mg/L+ sugar 60g/L;
(V) piece root portion root media MS+0.5mg/L 6-BA+0.5mg/L NAA+0.5% active carbon; Above I-V medium pH is all between 5.6-5.7;
(2) step of propagation method is:
1. explant selection, preliminary treatment and sterilization: material selection then 9, October; Choose the piece root of David's-harp band bud in fine day; After it is cleaned up, place the thiophanate methyl of 1/700 concentration to soak 30min and take out, place normal temperature condition to dry; Carry out disinfection with the piece root of conventional disinfection technology afterwards, sterile water is blotted subsequent use with aseptic filter paper at last the band bud;
2. inducing culture is cultivated: the piece root of the band bud that will disinfect cuts open piece root portion top layer with scalpel; Only stay main bud to add the piece root of 0.8-1.2cm3, insert on (I) number inducing culture and carry out inducing culture, after 40-50 days; Induce the young shoot that makes new advances, the piece root portion is expanded;
3. proliferated culture medium is cultivated:
(a), the proliferated culture medium that will induce piece root switching (II) number band sprout tuber root of sprouting carries out enrichment culture, after 30-40 days, the piece root portion is expanded, new sprout is emerged;
(b), the piece undercut of the band bud that will breed becomes piece root portion and sprout part, on the proliferated culture medium of piece root portion switching (II) number band sprout tuber root, after 30-40 days, expands once more, new sprout is emerged once more; The piece root of the new propagation band bud that grows; The piece root repetitive cycling of new propagation band bud this (b) is the sprout that scales off for the piece root that produces end-product band sprout in the last reign of a dynasty and intermediate product branch of said process enrichment culture 3-6 step by step, and the piece root of end-product band sprout can get into and bred the follow-up propagation strong seedling culture base incubation step of piece root portion the last reign of a dynasty;
(c), the intermediate product sprout that (b) middle step by step circulation step cutting is got off is partly transferred on (III) number sprout part proliferated culture medium; After 30-40 days; Sprout part base portion piece root expands; Then return again (b) step by step with (c) step by step said process carry out 3-6 and breed for combined-circulation, produce the piece root of end-product band sprout in the last reign of a dynasty at last;
4. breeding the strong seedling culture base cultivates: will pass through (b) circulate step by step and through (b) (c) step by step the combined-circulation enrichment culture produce the last reign of a dynasty end-product band sprout piece root piece root portion switching (IV) number piece root portion propagation strong seedling culture base carry out strong seedling culture; After 30-40 days; The piece root expands once more, and new sprout is emerged;
5. root media is cultivated: will through step 4. the piece root portion in strong sprout be inoculated on (V) number piece root portion root media, after 20-30 days, piece root portion base portion grows domestication and the transplanting that root system can carry out tissue cultivating seedling.
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| CN111183902A (en) * | 2020-02-26 | 2020-05-22 | 四川农业大学 | Tissue culture method for polygonatum sibiricum |
| CN111512965A (en) * | 2020-05-28 | 2020-08-11 | 福建省南平市农业科学研究所 | High-efficiency seedling raising method for polygonatum cyrtonema |
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| CN116530415A (en) * | 2023-05-30 | 2023-08-04 | 贵州省植物园 | Circularly operated polygonatum cyrtonema tissue culture breeding method |
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| CN102771395A (en) * | 2012-08-13 | 2012-11-14 | 安徽农业大学 | Tissue culture method for Polygonaturm sibiricum Redoute |
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