CN1813526A - Wheatgrass ear tissue culture regenerating method - Google Patents
Wheatgrass ear tissue culture regenerating method Download PDFInfo
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- CN1813526A CN1813526A CN 200610008712 CN200610008712A CN1813526A CN 1813526 A CN1813526 A CN 1813526A CN 200610008712 CN200610008712 CN 200610008712 CN 200610008712 A CN200610008712 A CN 200610008712A CN 1813526 A CN1813526 A CN 1813526A
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Abstract
The present invention discloses an agropyron cristatum young spike tissue culture regeneration method. Said method includes the following steps: (1), selecting material: selecting young spike of spike formation stage, then making treatment of explant, namely, using defatted cotton ball soaked in alcohol whose concentration is 75% to clean the leaf sheath covering young spike on the super clean bench to make surface disinfection; (2), cutting the treated young spike into small part, inoculating it on the culture medium for inducing callus to make culture; (3) transferring the callus onto secondary culture medium to make secondary culture; (4), inoculating the callus on the differentiation culture medium to induce differentiation; and (5), making secondary subculture on the above-mentioned secondary culture medium to make the plant be regenerated.
Description
Technical field:
The present invention relates to a kind of wheatgrass ear tissue culture regenerating method.
Background technology:
Agropyron (Agropyron Gaertn) plant is a gramineous forage grass, begins to cultivate in a organized way the research report of regeneration plant the external eighties in 20th century.Wheatgrass is long not familiar for many years clump herbage of life-span, be distributed widely in arid, semiarid grassland and desert steppe, resistance is stronger, turn green spring early, in withered and yellow evening in autumn, cauline leaf is tender, and is nutritious, good palatability is northwest drought semiarid zone improvement grassland and set up sown pasture and one of the important gramineous forage grass of ecological construction.Simultaneously, Agropyron except that being subjected to herbage scholar's the great attention as high quality forage, also has many excellent proterties that can be used for the wheat improvement as one of important wheat wild relative genus.People have obtained common wheat and Agropyron species hybrid and selfing and backcross progeny by methods such as embryo rescue, young fringe cultivations in recent years, and created wheat one wheatgrass alien addition line, the most important condition of development and use foreign gene under the wheat genetic background is provided.
Wheatgrass ear tissue culture regenerating method is also arranged at present, and this method is being chosen booting stage children fringe, carries out then adopting 75% alcohol disinfecting on the technology of processing of explant, uses 0.1%HgCl again
2With aseptic water washing method for several times, this cultivates the method for wheatgrass ear regeneration then in sterilization, because of disinfectant to the toxic effect of young fringe, particularly young tender young fringe, this is unfavorable for improving the efficient of tissue culture.The mercuric chloride that disinfectant is particularly commonly used not only has residual hazard, and also relatively more serious to the pollution of environment.
The object of the present invention is to provide a kind of healing rate of feasible young fringe and the wheatgrass ear tissue culture regenerating method that differentiation rate improves greatly.
Purpose of the present invention is implemented by following technical scheme: this method includes the following step: draw materials (1): choose children's fringe in booting stage, carry out the processing of explant then: get booting stage children fringe, on super-clean bench, be used in the rayon balls scouring of soaking in 75% the alcohol and wrap up the leaf sheath of young fringe and carry out surface sterilization.(2): will handle the young fringe in back and be cut into segment, and be seeded on the medium of evoked callus and cultivate; (3): again callus is transferred to successive transfer culture on the subculture medium; (4): callus is seeded in induces differentiation on the differential medium; (5): then again on the identical subculture medium of step (3) subculture at least once, plant regeneration.
The invention has the advantages that: the explant of handling with method of the present invention is seeded on identical the callus of induce medium and differential medium, its healing rate and differentiation rate can improve greatly, and healing rate and differentiation rate on average exceed 14.5% and 12.3% than original method.And environment is not polluted.
Embodiment:
Embodiment 1: wheatgrass ear tissue culture regenerating method, and this method includes the following step:
(1) draw materials: 4 parts of materials that experiment is selected for use are: Mongolian wheatgrass new lines (A.mongolicum Keng), illiteracy agricultural hybrid wheatgrass (A.cristatum x A.desertorumcv. ' Hycrest-Mengnong '), navigation channel wheatgrass (A.cristatumcv. ' Fairway '), the red wheatgrass of promise (A.desertorum cv. ' Nordan ') all are taken from Agricultural University of the Inner Mongol forage germplasm resources garden, and be 2～5 years breeding time.Get its booting stage (two rib phases) young fringe.Carry out the processing of explant then: get booting stage children fringe, on super-clean bench, be used in the rayon balls scouring of soaking in 75% the alcohol and wrap up the leaf sheath of young fringe and carry out surface sterilization.
(2): will handle back young fringe children fringe and strip out, be cut into the segment of 0.2～0.3cm, be seeded in the medium of evoked callus, 24～26 ℃ of dark 21d that cultivate observe callus induction rate (inducing the explant number of callus explant number/inoculation) and quality.
The medium of evoked callus is:
Improvement MS+2,4-D 2.0mg/L, sucrose 3%, agar 0.7%; PH5.8～6.0,117 ℃, autoclaving under the 17min;
(3): callus is transferred on the subculture medium successive transfer culture 2 times, subculture is spaced apart 20d again, and condition of culture is 24～26 ℃ of dark cultivations,
The callus subculture medium:
Improvement MS+2,4-D 2.0mg/L+6-BA 0.2mg/L, sucrose 3%, agar 0.7%, PH5.8～6.0,117 ℃, autoclaving under the 17min;
(4): callus is seeded in induces differentiation on the differential medium: select state preferably embryo callus change differential medium over to, 26 ℃ of following 24h illumination cultivation;
The callus differential medium:
MS+KT3.0mg/L+NAA0.5mg/L, sucrose 3%, agar 0.7%, PH5.8～6.0,117 ℃, autoclaving under the 17min;
(5): then twice of subculture on the identical subculture medium of step (3) again, subculture is once on same medium every about 21d, statistics callus differentiation situation behind 35～42d, the seedling ramp in 7～14d that differentiates green bud is a plantlet, and with very thin root shape thing, change over to when treating seedling length to 3～4cm and just produce root in the root media after the week, form complete plantlet;
Root media:
1/2MS+NAA0.1mg/L, sucrose 3%, agar 0.7%; PH5.8～6.0; 117 ℃, autoclaving under the 17min,
1/2MS medium wherein: MS minimal medium macroelement reduces by half, and all the other are constant
Modified MS medium: (unit: mg/L)
Macroelement: NH
4NO
3956, KH
2PO
41160, MgSO
47H
2O 370, CaCl
22H
2O96.
Organic element: inositol 100, VB
12, nicotinic acid 1, VB
60.5, glycine 2.
Molysite and trace element are with the MS minimal medium.
Embodiment 2: present existing wheatgrass ear tissue culture regenerating method, draw materials, and as embodiment 1, handle: get children's fringe in booting stage, peel off young fringe, 75% alcohol disinfecting 30s uses 0.1%HgCl again
2Sterilization 2～4min, then for several times with aseptic water washing.Following method is all as described in the embodiment 1.
Embodiment 3: contrast the influence of two kinds of methods to the wheatgrass tissue culture regeneration by embodiment 1 and embodiment 2, as table 1.
Table 1
| Kind | The inoculation number | Healing rate | Differentiation rate | |
| Embodiment 2 methods | The red wheatgrass of navigation channel wheatgrass promise Mongolia wheatgrass new lines is covered agricultural hybrid wheatgrass | 400 400 400 400 | 69.6 64.3 57.5 73.2 | 54.3 52.1 49.0 57.6 |
| Embodiment 1 method | The red wheatgrass of navigation channel wheatgrass promise Mongolia wheatgrass new lines is covered agricultural hybrid wheatgrass | 400 400 400 400 | 79.8 77.5 75.4 90.1 | 65.2 63.5 61.7 72.8 |
As can be seen from Table 1, the explant of handling with two kinds of methods is seeded on identical the callus of induce medium and differential medium, but healing rate and differentiation rate exist than big-difference, and healing rate of the inventive method and differentiation rate on average exceed 14.5% and 12.3% than the method for prior art.
Conclusion:
The physiological status of explant and development degree are the key factors that influences the cultured in vitro reaction.This experiment is not being used thimerosal (mercuric chloride in the disinfecting of explant, clorox etc.), clean the leaf sheath of the young fringe of parcel and carry out surface sterilization but be used in the rayon balls that soaked in 75% the alcohol, alleviated the toxic action of disinfectant widely to young fringe, particularly young tender young fringe, this helps improving the efficient of tissue culture.The mercuric chloride that disinfectant is particularly commonly used not only has residual hazard, and also relatively more serious to the pollution of environment.
Claims (1)
1, wheatgrass ear tissue culture regenerating method, this method includes the following step: draw materials (1): choose children's fringe in booting stage, carry out the processing of explant then; (2): will handle the young fringe in back and be cut into segment, and be seeded on the medium of evoked callus and cultivate; (3): again callus is transferred to successive transfer culture on the subculture medium; (4): callus is seeded in induces differentiation on the differential medium; (5): then again on the identical subculture medium of step (3) subculture once, plant regeneration; It is characterized in that described drawing materials: choose booting stage children fringe, carry out the processing of explant then: get children's fringe in booting stage, on super-clean bench, be used in the rayon balls scouring of soaking in 75% the alcohol and wrap up the leaf sheath of young fringe and carry out surface sterilization.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610008712 CN1813526A (en) | 2006-01-28 | 2006-01-28 | Wheatgrass ear tissue culture regenerating method |
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| CN 200610008712 CN1813526A (en) | 2006-01-28 | 2006-01-28 | Wheatgrass ear tissue culture regenerating method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104273035A (en) * | 2014-10-24 | 2015-01-14 | 中国科学院新疆生态与地理研究所 | In-vitro cultivation method of gramineous plants |
| CN107637213A (en) * | 2017-10-16 | 2018-01-30 | 河北科技师范学院 | A kind of method for improving wheatgrass germination percentage and planting percent |
| CN110384045A (en) * | 2019-09-03 | 2019-10-29 | 南通大学 | The preservation of wheatgrass leaf source sterilizable material and method for resuscitation |
-
2006
- 2006-01-28 CN CN 200610008712 patent/CN1813526A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104273035A (en) * | 2014-10-24 | 2015-01-14 | 中国科学院新疆生态与地理研究所 | In-vitro cultivation method of gramineous plants |
| CN104273035B (en) * | 2014-10-24 | 2016-08-24 | 中国科学院新疆生态与地理研究所 | A kind of grass in-vitro culture method |
| CN107637213A (en) * | 2017-10-16 | 2018-01-30 | 河北科技师范学院 | A kind of method for improving wheatgrass germination percentage and planting percent |
| CN110384045A (en) * | 2019-09-03 | 2019-10-29 | 南通大学 | The preservation of wheatgrass leaf source sterilizable material and method for resuscitation |
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