CN104304033B - A kind of method for tissue culture of butterfly beans - Google Patents
A kind of method for tissue culture of butterfly beans Download PDFInfo
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- CN104304033B CN104304033B CN201410628076.9A CN201410628076A CN104304033B CN 104304033 B CN104304033 B CN 104304033B CN 201410628076 A CN201410628076 A CN 201410628076A CN 104304033 B CN104304033 B CN 104304033B
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Abstract
The invention discloses a kind of method for tissue culture of butterfly beans, the method comprises the following steps: seed asepsis sprouting, Initial culture, Calli Differentiation, culture of rootage and acclimatization and transplants.Method of the present invention is used for the tissue cultures of butterfly beans, has that reproduction speed is fast, reproduction coefficient large, and raise up seed neat and consistent, the advantage that transplanting survival rate is high.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly a kind of method for tissue culture of butterfly beans.
Background technology
Butterfly beans (ClitoriaternateaL.), have another name called blue butterfly, Asian pigeonwings, blue pea, it is Papilionaceae (Papilionaceae) butterfly Macroptilium (Clitoria) plant, originate in the ground such as India, Indonesia, extensively cultivate in subtropical and tropical zones, the world.Butterfly beans have economic worth widely, and flower can extract natural dye; Tender folder edible; Root can be used as medicine; In addition, butterfly beans also can be modulated into hay, and as feed, butterfly beans are as flower garden ornamental plants especially in recent years, are widely used in Landscape Plants Design configuration.
Research at present about butterfly beans is few, and domestic research field relates generally to the simple description of some characteristics, and the research of seed germination and culture technique.The germination rate of butterfly beans seed is general lower, adopts conventional seed propagation method speed slow.Tissue culture technique has that reproduction speed is fast, reproduction coefficient is large, and raise up seed neat and consistent, can keep the merit of original kind, is not subject to the advantages such as the restriction in season, in the various ornamental plants of extensive use, has had not yet to see and be applied on butterfly beans.
Summary of the invention
The object of this invention is to provide a kind of method for tissue culture of butterfly beans, solve the butterfly beans problem that reproduction speed is slow because percentage of seedgermination is lower.Method of the present invention is used for butterfly beans Fast-propagation, and have that reproduction speed is fast, reproduction coefficient is large, raise up seed neat and consistent, the advantage that transplanting survival rate is high.
Technical scheme provided by the present invention is:
A method for tissue culture for butterfly beans, is characterized in that, the method comprises the following steps:
(1) seed asepsis sprouting: select full butterfly beans seed, through tap water, alcohol sterilizing, aseptic water washing, mercuric chloride sterilizing, aseptic water washing and vernalization process, then sow, seed germination goes out seedling;
(2) Initial culture; The hypocotyl of seedling after seed germination is cut the segment of growing into 5-10mm, is then seeded in modified MS medium, obtains callus;
(3) Calli Differentiation: the fritter callus of acquisition being cut into 0.5cm × 0.5cm, is then seeded in the modified MS medium containing 6-BA and NAA, obtains culturing young plants;
(4) culture of rootage: when culturing young plants grows to height about 2.0-4.0cm, transfers in the modified MS medium containing IBA or containing on the improvement 1/2MS medium of IBA, obtains plantlet in vitro of taking root;
(5) hardening and transplanting: plantlet in vitro of taking root is placed in room temperature lower refining seedling 1d, then except decap hardening 1-2d again, wash away residual media, and plantlet in vitro 50% carbendazol wettable powder dilution 800-1000 of taking root doubly or after 0.5% liquor potassic permanganate immersion 10min transplants in matrix.
Wherein, sowing described in step (1), is access in modified MS medium by seed or be sown in the sand that sterilization treatment keeps moistening.
Wherein, modified MS medium described in step (2): be that the macroelement in MS medium is increased to 20 times, with the anhydrous CaCl of 6.7g/L
2substitute the CaCl of 8.8g/L
22H
2o, is increased to 200 times by the trace element in MS medium, with the CuSO of 0.0032g/L
4substitute the CuSO of 0.005g/L
45H
2o, MnSO
44H
2the consumption of O changes into as 3.38g/L, and substitutes deionized water with running water, and sucrose is 20g/L, agar 3.5-4.0g/L, all the other compositions and consumption constant, pH value is 5.8-6.0.
Wherein, the modified MS medium containing variable concentrations 6-BA and NAA described in step (3) is MS+6-BA0.1-1.0mg/L+NAA0.01-0.1mg/L.
Wherein, described improvement 1/2MS medium described in step (4): be on the basis of the modified MS medium described in step (2), macroelement is reduced to 1/2 in modified MS medium, sucrose is 10g/L, and all the other compositions and consumption and improvement MS are consistent; The described modified MS medium containing IBA is MS+IBA0.1-0.3mg/L; The described improvement 1/2MS medium containing IBA is 1/2MS+IBA0.1-0.3mg/L.
Wherein, plantlet in vitro of taking root described in step (4), for true leaf grows, main root about has 3-4 bar, and root is about the seedling into 2.5-4.5cm.
Wherein, transplant described in step (5), be transplant plantlet in vitro of taking root in the seedling culture hole plate of 50-72 hole, cave dish is of a size of 540mm × 280mm, in the dish of this cave, matrix is housed.
As preferably, described matrix is by peat by volume: perlite=2:1 formed, and its pH value is 6.0-7.0.
As preferably, described matrix needs to carry out disinfection with 800-1000 carbendazol wettable powder dilution doubly or 0.5% liquor potassic permanganate for 1 week before transplanting.
beneficial effect of the present invention
Method of the present invention is used for butterfly beans tissue cultures, and have that reproduction speed is fast, reproduction coefficient is large, raise up seed neat and consistent, the advantage that transplanting survival rate is high.
Embodiment
Below in conjunction with concrete example, the present invention is further detailed explanation.
embodiment 1:
On July 2nd, 2012, select full butterfly beans seed, after tap water, first use the alcohol sterilizing 40s of 75%, aseptic water washing 3 times, then use 0.1% mercuric chloride sterilizing 10min, carry out vernalization with after aseptic water washing 3 times.Vernalization 50 DEG C of sterile waters seed soaking 15min are placed on 24h under room temperature.Then to access in modified MS medium and to be placed in group training room, temperature 28 ± 2 DEG C, illumination 12h every day.
Through observing after a while, added up July 17: seed starts to sprout at 5d, and pollution rate is 0, and germination rate is 36.7%.The epicotyl of the seedling (cotyledon launches) after seed germination, hypocotyl and belt base of leaf section are cut the segment of growing into 5mm by July 25, are inoculated in respectively on the MS medium of improvement.
Through observing after a while, added up September 10: epicotyl, hypocotyl and belt base of leaf section all directly can induce callus from base portion in modified MS medium, callus starts to be formed at 15d, callus induction rate is 100%, callus quality is loose, and color is faint yellow.
The callus of acquisition is cut into 0.5cm × 0.5cm size by September 22, be inoculated into addition of hormon kind and concentration modified MS medium on, find that the Calli Differentiation ability of different parts is different, the Calli Differentiation ability order from high to low of epicotyl, hypocotyl and belt base of leaf section is: the callus differentiation rate of hypocotyl, epicotyl and belt base of leaf section is respectively 81%, 20% and 0%.The optimal medium of Calli Differentiation is: improvement MS+6-BA0.1mg/L+NAA0.02mg/L, and growth coefficient reaches 4.5, and callus becomes large in continuation simultaneously, and quality is loose, and the seedling growing way differentiated is good, healthy and strong.Calli Differentiation required time is about 25-30d.When plantlet in vitro grows to height about 2.0-3.5cm, transfer in the improved culture medium of 6-BA and NAA that addition of variable concentrations, result is: best root media is MS+IBA0.1mg/L, rooting rate 100%, seedling growing way is good, and main root mean is 3.5, main root average length 3.5cm, root system white, part is partially yellow, has a small amount of fibrous root.Rootage duration needs 10d.
By taking root, (true leaf grows good seedling, main root has 3, root is about as 4.5cm) be placed in room temperature lower refining seedling 1d, then except decap hardening 1d again, wash away residual media, and after seedling is soaked 10min with 1000 times of carbendazim, be transplanted into and (matrix volume ratio is peat: perlite=2:1, PH=7.0) in 50 hole seedling culture hole plates of matrix is housed.And irrigate normal root water, be placed in full exposure batch (-type) sprinkling irrigation system maintenance management.Daytime sprays 1 time at interval of 4h, gets final product normal growth after 7d, survival rate 100%.
embodiment 2:
On July 2nd, 2012, select full butterfly beans seed, after tap water, first use the alcohol sterilizing 30s of 70%, aseptic water washing 4 times, then use 0.1% mercuric chloride sterilizing 10min, carry out vernalization with after aseptic water washing 4 times.Vernalization 60 DEG C of sterile waters seed soaking 15min are placed on 24h under room temperature.Then be sown in the moistening sand of sterilizing (in Seed Germination, sand will keep moistening, but can not have ponding), and be placed in group training room, temperature 28 ± 2 DEG C, illumination 12h every day.
Through observing after a while, added up July 17: seed starts to sprout at 7d, and pollution rate is 0, and germination rate is 27.27%.The epicotyl of the seedling (cotyledon launches) after seed germination, hypocotyl and belt base of leaf section are cut the segment of growing into 10mm by July 25, are inoculated in respectively on the MS medium of improvement.
Through observing after a while, added up September 10: epicotyl, hypocotyl and belt base of leaf section all directly can induce callus from base portion in modified MS medium, callus starts to be formed at 15d, callus induction rate is 100%, callus quality is loose, and color is faint yellow.
The callus of acquisition is cut into 0.5cm × 0.5cm size by September 22, be inoculated into addition of hormon kind and concentration modified MS medium on, find that the Calli Differentiation ability of different parts is different, the Calli Differentiation ability order from high to low of epicotyl, hypocotyl and belt base of leaf section is: the phenylacetic acid of hypocotyl, epicotyl and belt base of leaf section is respectively 70%, 25% and 0%.The optimal medium of Calli Differentiation is: improvement MS+6-BA1.0mg/L+NAA0.1mg/L, and growth coefficient reaches 4.0, and callus becomes large in continuation simultaneously, and quality is loose, and the seedling growing way differentiated is good, healthy and strong.Calli Differentiation required time is about 25-30d.When plantlet in vitro grows to height about 2.0-3.5cm, transfer in the improved culture medium of 6-BA and NAA that addition of variable concentrations.Found that: best root media is 1/2MS+IBA0.2mg/L, rooting rate 100%, and seedling growing way is good, main root mean is 3.9, main root average length 4.1cm, root system white, and part is partially yellow, has a small amount of fibrous root.Rootage duration needs 12d.
By taking root, (true leaf grows good seedling, main root about has 3, root is about as 3.5cm) be placed in room temperature lower refining seedling 1d, then except decap hardening 1d again, wash away residual media, and after seedling is soaked 10min with 0.5% liquor potassic permanganate, be transplanted into and (matrix is peat according to volume ratio: perlite=2:1 is formed, PH=6.5) in 50 hole seedling culture hole plates of matrix is housed.And irrigate normal root water, be placed in full exposure batch (-type) sprinkling irrigation system maintenance management.Daytime sprays 1 time at interval of 4h, gets final product normal growth after 10d, survival rate 100%.
Formula involved in above-described embodiment is as follows:
Pollution rate (%)=pollution number/inoculation number × 100%;
Seed germination rate (%)=sprouting number/inoculation number × 100%;
Callus induction rate (%)=generation callus number/inoculation number × 100%;
Phenylacetic acid (%)=generation Calli Differentiation number/inoculation number × 100%;
Growth coefficient=(propagation number+inoculation number)/inoculation number;
Rooting rate (%)=number of taking root/inoculation number × 100%;
Quantity/total quantity × 100% of survival rate (%)=survive.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (3)
1. a method for tissue culture for butterfly beans, is characterized in that, the method comprises the following steps:
(1) seed asepsis sprouting: select full butterfly beans seed, through tap water, alcohol sterilizing, aseptic water washing, mercuric chloride sterilizing, aseptic water washing and vernalization process, then sow, seed germination goes out seedling;
(2) Initial culture; The hypocotyl of seedling after seed germination is cut the segment of growing into 5-10mm, is then seeded in modified MS medium, obtains callus;
(3) Calli Differentiation: the fritter callus of acquisition being cut into 0.5cm × 0.5cm, is then seeded in the modified MS medium containing 6-BA and NAA, obtains culturing young plants;
(4) culture of rootage: when culturing young plants grows to high 2.0-4.0cm, transfers in the modified MS medium containing IBA or containing on the improvement 1/2MS medium of IBA, obtains plantlet in vitro of taking root;
(5) hardening and transplanting: plantlet in vitro of taking root is placed in room temperature lower refining seedling 1d, then except decap hardening 1-2d again, wash away residual media, and will take root plantlet in vitro 50% carbendazol wettable powder dilution 800-1000 times or 0.5% liquor potassic permanganate are transplanted in matrix after soaking 10min;
Described modified MS medium: be that the macroelement in MS medium is increased to 20 times, with the anhydrous CaCl of 6.7g/L
2substitute the CaCl of 8.8g/L
22H
2o, is increased to 200 times by the trace element in MS medium, with the CuSO of 0.0032g/L
4substitute the CuSO of 0.005g/L
45H
2o, MnSO
44H
2the consumption of O changes 3.38g/L into, and substitutes deionized water with running water, and sucrose is 20g/L, agar 3.5-4.0g/L, all the other compositions and consumption constant, pH value is 5.8-6.0;
Sowing described in step (1), is access in modified MS medium by seed or be sown in the sand that sterilization treatment keeps moistening;
The modified MS medium containing 6-BA and NAA described in step (3) is improvement MS+6-BA0.1-1.0mg/L+NAA0.01-0.1mg/L;
Improvement 1/2MS medium described in step (4): be on the basis of the modified MS medium described in step (2), macroelement is reduced to 1/2 in modified MS medium, sucrose is 10g/L, and all the other compositions and consumption and improvement MS are consistent; The described modified MS medium containing IBA is improvement MS+IBA0.1-0.3mg/L; The described improvement 1/2MS medium containing IBA is improvement 1/2MS+IBA0.1-0.3mg/L;
Transplant described in step (5), be transplant plantlet in vitro of taking root in the seedling culture hole plate of 50-72 hole, cave dish is of a size of 540mm × 280mm, in the dish of this cave, matrix is housed; Described matrix is by peat by volume: perlite=2:1 formed, and its pH value is 6.0-7.0.
2. the method for tissue culture of butterfly beans as claimed in claim 1, it is characterized in that, plantlet in vitro of taking root described in step (4), for true leaf grows, main root has 3-4 bar, the long seedling for 2.5-4.5cm of root.
3. the method for tissue culture of butterfly beans as claimed in claim 1, is characterized in that, described matrix needs to carry out disinfection with 800-1000 carbendazol wettable powder dilution doubly or 0.5% liquor potassic permanganate for 1 week before transplanting.
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| CN1155907A (en) * | 1994-06-10 | 1997-07-30 | 丹尼斯科有限公司 | Transformation of guar beans |
| CN1187933A (en) * | 1997-01-13 | 1998-07-22 | 丁庆 | Cultivation method for crassula perforate, cotyledon malacophylla, monochoria vaginalis fordia cauliflora |
| CN103493663A (en) * | 2013-09-26 | 2014-01-08 | 霍振中 | Method for cultivating kidney beans in early stage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011025840A1 (en) * | 2009-08-25 | 2011-03-03 | Targeted Growth, Inc. | Modified transgene encoding a growth and/or development related protein in plants |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1155907A (en) * | 1994-06-10 | 1997-07-30 | 丹尼斯科有限公司 | Transformation of guar beans |
| CN1187933A (en) * | 1997-01-13 | 1998-07-22 | 丁庆 | Cultivation method for crassula perforate, cotyledon malacophylla, monochoria vaginalis fordia cauliflora |
| CN103493663A (en) * | 2013-09-26 | 2014-01-08 | 霍振中 | Method for cultivating kidney beans in early stage |
Non-Patent Citations (4)
| Title |
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| Effect of cytokinins and auxins on micropropagation of Clitoria ternatea L;GYANA RANJAN ROUT;《BIOL. LETT.》;20041231;第41卷(第1期);第21-26页，尤其是第24页第15-2行 * |
| Influencing micropropagation in Clitoria ternatea L.through the manipulation of TDZ levels and use of different explant types;Seemab Mukhtar et al.;《Physiol Mol Biol Plants 》;20121231;第18卷(第4期);第381–386页，尤其是第382页左栏最后1段-右栏第6段 * |
| MICROPROPAGATION OF CLITORIA TERNATEA LINN. (FABACEAE) –AN IMPORTANT MEDICINAL PLANT;G. R. ROUT;《In Vitro Cell. Dev. Biol.—Plant》;20050831;第41卷;第516–519页 * |
| 光温对蝶豆种子萌发的影响;闫海霞等;《南方农业学报》;20121231;第43卷(第12期);第2015-2019页 * |
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