CN101147467A - Method for improving induction rate of turf grass embryonic callus - Google Patents
Method for improving induction rate of turf grass embryonic callus Download PDFInfo
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- CN101147467A CN101147467A CNA2007100359283A CN200710035928A CN101147467A CN 101147467 A CN101147467 A CN 101147467A CN A2007100359283 A CNA2007100359283 A CN A2007100359283A CN 200710035928 A CN200710035928 A CN 200710035928A CN 101147467 A CN101147467 A CN 101147467A
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Abstract
The present invention relates to a method for raising lawn grass-embryonic callus induction rate. Said method includes the following steps: cutting off inembryonate end of mature seed, using half-seed with embryonic end as explant, inoculating it into the callus induction culture medium to induce callus. The described induction culture medium is characterized by that on the basis of MS basic culture medium 2,4-dichlorophenoxyacetic acid is added, placing the material in an environment with 25 plus or minus 2 deg.C, making dark culture, every about 20 days changing induction culture medium once, continuously making dark culture at 25 plus or minus 2 deg.C, when the callus is grown to that its diameter is 2-5mm, promptly cutting off glumal shell and embryonic bud, then making the callus be transferred onto secondary culture medium to make secondary culture, every about 20 days changing secondary culture medium once, making dark culture at 25 plus or minus 2 deg.C until the number of calli is not increased.
Description
Technical field
The present invention relates to improve the new method of induction rate of turf grass embryonic callus, particularly relate to the processing method of explant.
Background technology
Turfgrass (turfgrass) is meant the herbaceous plant that constitutes lawn vegetation, comprises that mainly grass family and a few sedge and some meet other plant of turfgrass characteristic, have 40 kinds of turfgrasss to be used for turf establishment at present approximately.Turfgrass have conserve water and soil, nature remodeling, function such as beautify the environment.Remove city forest network and garden landscape, the lawn is the source of the green life of urban size maximum.In order to satisfy the demand of varied turfgrass, the improvement of turfgrass has 50 years of development history.The character improvement of turfgrass mainly still adopts traditional breeding technique at present, has more time-consuming, effort, the big obvious weakness of difficulty, can not satisfy the needs of lawn development.And utilization genetically engineered plant technology very big shortening the breeding cycle is accelerated breeding process, and more on purpose acquisition has the new varieties of better proterties.
Inducing of embryo callus is the prerequisite and the basis of carrying out genetic engineering improvement plant trait.At first, embryo callus can produce somatic variation under the inducing of physics or chemical method, obtains the plant that makes a variation and improve by differentiation regeneration.Secondly, in all foreign gene receptor systems, the embryo callus receptor system is many with its quantity always, easily regeneration, and good stability forms and is first-selection; Embryo callus becomes the acceptor of the good genes of interest of external source, and differentiation regeneration obtains transfer-gen plant after the external source genetic transformation.Therefore, improve the inductivity of embryo callus, and on this basis, set up the high frequency embryonic callus induction, regenerating system seems particularly important.
Summary of the invention
Purpose of the present invention aims to provide a kind of foundation for turfgrass high frequency embryo callus regenerating system the basis is provided, and can improve the method for induction rate of turf grass embryonic callus.
The objective of the invention is to realize by following manner:
1) mature seed not being had a tip cut-off of embryo, is explant with the half granule seed of embryo end, is inoculated in the callus inducing medium evoked callus; Described inducing culture is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
2) material is positioned over 25 ± 2 ℃, dark culturing; Every about 20 days, change an inducing culture; 25 ± 2 ℃, dark culturing; Described fresh culture is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
When 3) treating that callus length is 2 to 5mm sizes to diameter, in time with clever shell and plumule excision, callus transferred to carry out successive transfer culture on the subculture medium, change 25 ± 2 ℃ of subculture mediums every about 20 days, number no longer increases dark culturing to going out more.
Described disinfect be with mature embryo behind 75% alcohol disinfecting 1min, with 0.1% mercuric chloride sterilization 15min, use aseptic water washing then 4～5 times again, dry.
The 2,4 dichlorophenoxyacetic acid concentration of described callus inducing medium is 2-8mg/l.
Also containing concentration in the described callus inducing medium is the caseinhydrolysate of 300mg/l and the proline of 500mg/l, perhaps contains the mannose that concentration is 25g/l.
Callus color and luster cadmium yellow graininess is for well.
Since utilization genetically engineered plant technology improved the turfgrass quality, the foundation of relevant its embryo callus regenerating system, the foundation of transformation receptor system had all had very extensive studies.But in the turfgrass of some kinds, there is the low problem of frequency of embryonic callus induction always, and, just mature seed there is not a tip cut-off of embryo, improve frequency of embryonic callus induction and yet there are no report.
Specific implementation process of the present invention is:
1. a tip cut-off that mature seed is not had embryo is an explant with the half granule seed of embryo end, behind 75% alcohol disinfecting 1min, with 0.1% mercuric chloride sterilization 15min, uses aseptic water washing then 4 to 5 times again, is seeded on the inducing culture after drying; Inducing culture is to have added caseinhydrolysate, proline, mannose and 2 on MS minimal medium basis, and 4-D changes once fresh inducing culture every about 20 days;
2. when treating that callus length is 2 to 5mm sizes to diameter, in time clever shell and plumule are excised, callus is transferred to carried out successive transfer culture on the subculture medium, every about 20 days, change once fresh subculture medium.The composition of subculture medium is identical with inducing culture.Induce and the successive transfer culture of callus carry out under dark condition, and all the temperature of incubation all is controlled at about 25 ± 2 ℃.
3. certainly after the inoculation, observe material every day, per three heaven-made once records, the record content comprises that callus goes out more the time at first, goes out more number, callus color and luster and form; 25 ± 2 ℃, number no longer increases dark culturing to going out more, writes down at last that callus goes out more number in every triangular flask.
Callus induction rate=(inducing the explant sum of the explant number/inoculation of callus) * 100%
The concentration of the caseinhydrolysate of using in said process is 300mg/l; The concentration of proline is 500mg/l; The concentration of mannose is 25g/l; 2, the concentration of 4-D is 2 to 8mg/l.
Embodiment
The new method of the frequency of embryonic callus induction of embodiment 1, raising kentucky blue grass
(1) from kentucky blue grass excellence, Bahrain, three kinds in Kentucky, select full, ripe seed separated into two parts, a part is left intact and is made as control group, and the tip cut-off that a part will not have embryo is made as experimental group.Half granule seed with whole seed and embryo end is an explant respectively, behind 75% alcohol disinfecting 1min, again with 0.1% mercuric chloride sterilization 15min, use aseptic water washing then 4 to 5 times, be seeded on the inducing culture respectively after drying, 25 explants of inoculation in every triangular flask, every kind of explant inoculation 10-12 bottle.Inducing culture is to have added caseinhydrolysate, proline, mannose and 2 on MS minimal medium basis, and 4-D changes a fresh culture every about 20 days.
When (2) treating that callus length is 2 to 5mm sizes to diameter, in time clever shell and plumule are excised, callus is transferred on the subculture medium (identical with inducing culture) carried out successive transfer culture, every about 20 days, change a fresh culture.The composition of subculture medium is identical with inducing culture.Induce and the successive transfer culture of callus carry out under dark condition, and all the temperature of incubation all is controlled at 25 ± 2 ℃.
(3) certainly after the inoculation, observe material every day, per three heaven-made once records, the record content comprises that callus goes out more the time at first, goes out more number, callus color and luster and form; 25 ± 2 ℃, number no longer increases dark culturing to going out more, writes down respectively at last that callus goes out more number in the every triangular flask of control group and experimental group.
Experimental result shows: with control material relatively, treated seed goes out more that the time shortened about 10 days, inductivity rises to 78.7% by 39.29%.By variance analysis, its inductivity difference is (table 1) extremely significantly.
Table 1 is removed the influence of endosperm to callus induction
Table 1 Effects of excising endosperm for callus’inductiviry
| Kind Variety | Processing mode Treatment method | Inoculation number Planted numbers | Go out more number Callus numbers | Healing rate (%) Percentage of callus | P-valu e |
| Excellent Bahrain Kentucky | Half rice grain seed Excising endosperm whole seed Reserving endosperm half rice grain seed Excising endosperm whole seed Reserving endosperm half rice grain seed Excising endosperm whole seed Reserving endosperm | 300 240 240 270 240 300 | 237 95 47 22 167 83 | 78.7 39.29 19.22 8.15 69.46 27.67 | 0.5984 23 0.8162 06 0.0373 18 |
The new method of the frequency of embryonic callus induction of embodiment 2, raising Festuca Arundinacea
(1) from Festuca Arundinacea hunting dog No. five, boil wave, three kinds in belligerent No. two Kentucky, select full, ripe seed separated into two parts, a part is left intact and is made as control group, and the tip cut-off that a part will not have embryo is made as experimental group.Half granule seed with whole seed and embryo end is an explant respectively, behind 75% alcohol disinfecting 1min, again with 0.1% mercuric chloride sterilization 15min, use aseptic water washing then 4 to 5 times, be seeded in respectively after drying on the inducing culture, 25 explants of inoculation in every triangular flask, every kind of explant is inoculated 8 bottles.Inducing culture is to have added caseinhydrolysate, proline, mannose and 2 on MS minimal medium basis, and 4-D changes a fresh culture every about 20 days.
When (2) treating that callus length is 2 to 5mm sizes to diameter, in time clever shell and plumule are excised, callus is transferred on the subculture medium (identical with inducing culture) carried out successive transfer culture, every about 20 days, change a fresh culture.The composition of subculture medium is identical with inducing culture.Induce and the successive transfer culture of callus carry out under dark condition, and all the temperature of incubation all is controlled at 25 ± 2 ℃.
(3) certainly after the inoculation, observe material every day, per three heaven-made once records, the record content comprises that callus goes out more the time at first, goes out more number, callus color and luster and form; 25 ± 2 ℃, number no longer increases dark culturing to going out more, writes down respectively at last that callus goes out more number in the every triangular flask of control group and experimental group.
Experimental result shows: with control material relatively, treated seed goes out more that the time shortened about 10 days, average inductivity rises to 69.17% by 52%.By variance analysis, its inductivity difference is (table 1-8) extremely significantly.
Table 1-8 removes the influence of endosperm to callus induction
Table 1-8 Effects of excising endosperm for callus’inductiviry
| Kind Variety | Processing mode Treatment method | Inoculation number Planted numbers | Go out more number Callus numbers | Healing rate (%) Percentage of callus | P-value |
| No. five Huntdog V of hunting dog belligerent No. two Crossfire II of unrestrained Finelawn that boil | Half rice grain seed Excising endosperm whole seed Reserving endosperm half rice grain seed Excising endosperm whole seed Reserving endosperm half rice grain seed Excising endosperm whole seed Reserving endosperm | 100 100 80 100 100 100 | 74 57 46 38 76 61 | 74 57 57.5 38 76 61 | 0.001615 0.000543 0.016298 |
Claims (6)
1. improve the method for induction rate of turf grass embryonic callus, it is characterized in that this method may further comprise the steps:
1) mature seed not being had a tip cut-off of embryo, is explant with the half granule seed of embryo end, is inoculated in the callus inducing medium evoked callus; Described inducing culture is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
2) material is positioned over 25 ± 2 ℃, dark culturing; Every about 20 days, change an inducing culture; 25 ± 2 ℃, dark culturing;
When 3) treating that callus length is 2 to 5mm sizes to diameter, in time with clever shell and plumule excision, callus transferred to carry out successive transfer culture on the subculture medium, change 25 ± 2 ℃ of subculture mediums every about 20 days, number no longer increases dark culturing to going out more.
2. improve the method for induction rate of turf grass embryonic callus according to claim 1, it is characterized in that the composition of described subculture medium is identical with inducing culture.
3. the method for raising induction rate of turf grass embryonic callus according to claim 1, it is characterized in that: described mature embryo is behind 75% alcohol disinfecting 1min, with 0.1% mercuric chloride sterilization 15min, use aseptic water washing then 4～5 times again, be seeded on the inducing culture after drying.
4. the method for raising induction rate of turf grass embryonic callus according to claim 1 is characterized in that: the 2,4 dichlorophenoxyacetic acid concentration of described callus inducing medium is 2-8mg/l.
5. the method for raising induction rate of turf grass embryonic callus according to claim 1 and 2, it is characterized in that: also containing concentration in the described callus inducing medium is the caseinhydrolysate of 300mg/l and the proline of 500mg/l, perhaps contains the mannose that concentration is 25g/l.
6. the method for raising induction rate of turf grass embryonic callus according to claim 1 is characterized in that: callus color and luster cadmium yellow graininess is for well.
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| CNA2007100359283A CN101147467A (en) | 2007-10-18 | 2007-10-18 | Method for improving induction rate of turf grass embryonic callus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103188606A (en) * | 2011-12-30 | 2013-07-03 | 北大方正集团有限公司 | Electronic information providing method based on positions and device |
| CN104686360A (en) * | 2015-03-16 | 2015-06-10 | 甘肃农业大学 | Method for rapidly inducing embryonic callus of halogeton |
| CN109644870A (en) * | 2018-12-30 | 2019-04-19 | 信阳农林学院 | A kind of method of turf grass species subgroup training explant disinfection |
-
2007
- 2007-10-18 CN CNA2007100359283A patent/CN101147467A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103188606A (en) * | 2011-12-30 | 2013-07-03 | 北大方正集团有限公司 | Electronic information providing method based on positions and device |
| CN104686360A (en) * | 2015-03-16 | 2015-06-10 | 甘肃农业大学 | Method for rapidly inducing embryonic callus of halogeton |
| CN104686360B (en) * | 2015-03-16 | 2016-08-24 | 甘肃农业大学 | The method of rapid induction salt SHENGCAO embryo callus |
| CN109644870A (en) * | 2018-12-30 | 2019-04-19 | 信阳农林学院 | A kind of method of turf grass species subgroup training explant disinfection |
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