CN106508672A - Method for obtaining new salt-tolerant medicago variety through mutagenesis of loose embryogenic callus - Google Patents
Method for obtaining new salt-tolerant medicago variety through mutagenesis of loose embryogenic callus Download PDFInfo
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- CN106508672A CN106508672A CN201610848982.9A CN201610848982A CN106508672A CN 106508672 A CN106508672 A CN 106508672A CN 201610848982 A CN201610848982 A CN 201610848982A CN 106508672 A CN106508672 A CN 106508672A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a method for obtaining a new salt-tolerant medicago variety through mutagenesis of a loose embryogenic callus; the method comprises the following steps: (1) obtaining a medicago aseptic seedling; (2) inducing a medicago loose callus; (3) inducing a medicago embryogenic callus; (4) inducing the medicago loose embryogenic callus; (5) preparing a mutagenic material; (6) screening a salt-tolerant cell line; and (7) culturing a salt-tolerant mutant strain. The method comprises the main content that the medicago loose embryogenic callus is induced firstly, then the callus is subjected to in-vitro mutagenesis with a chemical mutagen, the medicago loose embryogenic callus after mutagenesis is transferred to a culture medium containing a certain concentration of salt and is subjected to directional screening, the salt-tolerant medicago mutant callus is obtained and is subjected to regeneration culture, and finally the salt-tolerant medicago mutant strain is obtained.
Description
Technical field
The invention belongs to tissue culture technology field, is related to one kind using Herba Medicaginiss Friable embryogenic callus in chemical mutagen
A kind of breeding method of salt-tolerant mutant is carried out under effect.
Background technology
Herba Medicaginiss (Medicago) are one of widest leguminous forages of most important, cultivated area in the world, due to its adaptation
The wide, grass yield of property is high, and the nutrient substance such as rich in proteins, vitamin and mineral, is described as " King of Pasture ".It is global
Alfalfa cultivation area about 3.22 × 107hm2, the U.S., Russia and Argentina account for 70%.Though China is alfalfa cultivation big country,
But from excluding agricultural production main body.In the U.S., Herba Medicaginiss are considered one of the fourth-largest crop, are only second to corn and soybean and Semen Tritici aestivi,
Cultivated area is about 8.5 × 106hm2.China alfalfa cultivation area about 3.77 × 106hm2, occupies first of all kinds of artificial pastures.Closely
The breeding of Herba Medicaginiss over year is mainly carried out using the method for conventional cross-breeding method and artificially breeding, with the expansion of alfalfa cultivation area
Greatly, market demand Herba Medicaginiss possess increasingly merit, and above seeding technique can not solve practical problems, therefore some
Breeder is transferred to sight on some modern breeding new techniques.In recent years by the method for gene transformation, cultivate
Go out some New alfalfa cultivars for possessing certain unique shape, but as Environment release is extremely strict, the short time can not enter market,
Therefore also only rest on laboratory stage.And adopt mutation breeding technologies obtain some specialized characters, but because of its efficiency
Low, hereditary stability is poor so that this technology is lain on the table always.Recent years, about the report using somatic mutagenesis breeding
It is more and more, also applied in the breeding research of Herba Medicaginiss.
It is very fast about the progress of plant salt endurance both at home and abroad, using induced-mutation technique combine with isolated culture into
Row crops salt-tolerant mutant screening work be made it is more, it is successful on the crops such as Radix Ipomoeae, Fructus Lycopersici esculenti at present, and
Report is seen in the salt tolerant breeding of Herba Medicaginiss seldom.With the reduction of land resource, some varieties in saline-alkali areas are also required to Planting Medicago sativa, because
The salt tolerant breeding of this Herba Medicaginis also gradually have received attention.Being combined with somatic cell Regeneration in Vitro technology using induced-mutation technique, it is fixed to carry out
To in breeding research, more using calluss as mutation object, and the form of calluss and physiological statuss are different, adopt
It is efficiency highest that Somatic hybridization is carried out with Friable embryogenic callus, and wherein reason is mainly shown as：One is mutation effect
Rate is high.Because Friable embryogenic callus lacuna is larger, between cell, affect less, the effect of mutagenic agent is more equal
Even, in calluss, each cell is affected by identical, and some cytosiies will not be caused because of inside and outside position effect
Power is larger, and some power of influence are less；Two be incompact callus regeneration mostly be single cell regeneration because iuntercellular contact compared with
Little, embryo is multiple to be born in individual cells, therefore the mutant that mutation is produced generally is homozygote, is used directly for breeding and grinds
Study carefully, and avoid chimeric genetic instability；Three when being that Friable embryogenic callus are screened, as iuntercellular is contacted
Less, each cell is acted on by identical screening pressure, such that it is able to precisely determine screening pressure, so as to improve mutation effect
Rate；Four is that Friable embryogenic callus segmentation is easy, can be easily controlled the size of calluss group, so that mutation
Operation becomes simple.Five is to adopt Friable embryogenic callus also as which is embryo characteristic, and physiological statuss are enlivened, and are held
Easily induce its hereditary material to change, there is in mutation corresponding advantage.Except above advantage, embryo callus may be used also
To be readily available regeneration plant, once obtaining the calluss of mutation, subsequently it is obtained with regenerating mutant, without
The induction of callus regeneration is carried out again, is that the time has been saved in breeding.Therefore the present invention adopts the loose type embryo callus subculture of Herba Medicaginiss
Setup action mutation object can obtain efficient induced mutation rate.
The content of the invention
It is an object of the invention in place of overcoming the deficiencies in the prior art, there is provided one kind utilizes Friable embryogenic callus
The method of mutagenic obtained salt tolerant New alfalfa cultivars.The present invention provides one efficiently, simply easily for Herba Medicaginiss somatic mutagenesis breeding
Capable new way.The method also has certain reference value for other plants carry out breeding of new variety using same procedure.
The present invention realizes that the technical scheme of purpose is as follows：
A kind of method of the mutagenic obtained salt tolerant New alfalfa cultivars of utilization Friable embryogenic callus, step are as follows
(1) Herba Medicaginiss aseptic seedling is obtained；
(2) the induction of Herba Medicaginiss loose callus
The hypocotyls of Herba Medicaginiss tissue cultured seedling are adopted for explant, in 1/2MS+0.5-1.0mg/L2.4-D+20g/L sucrose cultures
Induce on base；Condition of culture is：Temperature 24-25 DEG C, 24h illumination, intensity of illumination are 2000Lux, and 20-25d can obtain preferable
Softly calluss；
(3) the induction of Herba Medicaginiss embryo callus
By step (2) in the loose callus that induces separate from explant, be seeded in what is prepared in advance
Callus induction in the culture medium of MS+1-2mg/LKT+0.5mg/L2,4-D+30g/L sucrose；Condition of culture is：Temperature 22-
23 DEG C, 24h illumination, intensity of illumination are 2500Lux；In inoculation 14-16d follow-up generations, once used medium and condition of culture were constant；
Shift the harder calluss of quality in subculture as far as possible, after approximately passing through 3-5 subculture, calluss quality is hardened completely,
And in microscopy, calluss are substantially by small volume, spherical, Cytoplasm be dense, nucleus greatly and the cell containing multiple split coil methods
Constitute, now illustrate to have induced the embryo callus of Herba Medicaginiss；
(4) the induction of Herba Medicaginiss Friable embryogenic callus
By step (3) in the Herba Medicaginiss embryo callus that induce be divided into the fritter of a diameter of 3-5mm, be then inoculated with
To in the culture medium of MS+1mg/LKT+0.5mg/L2,4-D+30g/L sucrose；Cultivate at 20-22 DEG C, and in 7-10d subcultures one
Secondary, through 5-8 successive transfer culture, calluss occur significantly change in structure, tight when there is loosely organized, quality,
When microscopy is embryo callus, Herba Medicaginiss Friable embryogenic callus are now obtained；24h is adopted during culture calluss
Illumination, intensity of illumination are 2500Lux；
(5) the preparation of mutant materials
It is 2-3mm that the Herba Medicaginiss Friable embryogenic callus for inducing are divided into diameter with dissecting knife in super-clean bench
Fritter, be put into standby in culture dish；Separately by callus culture base (MS+1-0.5mg/LKT+1-0.5mg/L2,4-D+30g/
L sucrose) melt in microwave oven, 0.3% EMS when its temperature drops to 50 DEG C, is added, the triangle of 100ml is then dispensed into
In bottle, per bottle of culture medium for pouring 20ml into；Until calluss are inoculated in triangular flask after culture medium solidifying cultivated；Training
Foster condition is 24 DEG C, and 24h illumination, intensity of illumination are 2000Lux；
(6) the screening of salt tolerant cell line
After the mutagenic treatment of 20d, normal calluss will be grown and be transferred to containing 0.8-1.0%NaCl concentration
Salt Tolerance is carried out in culture medium, through the growth of 20d, growth is therefrom filtered out normally and is possessed the wound healing group of regeneration capacity
Knit；Condition of culture is 23-24 DEG C, and 24h illumination, intensity of illumination are 3000Lux；
(7) the culture of Salt tolerant mutant
The Salt-tolerant Callus for filtering out are transferred to into regeneration induction plant in the MS culture medium without hormone；In culture medium
Addition concentration is 0.8-1.0%NaCl, through the growth of 20-30d, you can obtain the salt tolerant Herba Medicaginiss regeneration plant of robust growth；
The condition of culture of this process is 24-25 DEG C, and 24h illumination, intensity of illumination are 3000Lux.
And, the method that (1) step obtains Herba Medicaginiss aseptic seedling is as follows：
Full alfalfa seed is chosen, 30-40s is soaked in 70% ethanol, then the antiformin using 40% is water-soluble
Liquid carries out surface sterilization, and disinfecting time is 10-15Min；Seed sterilized water after disinfecting rinses 5 times repeatedly, every time
10Min is washed, then sterilized seed is inoculated in 1/2MS culture medium the culture for carrying out aseptic seedling, trained at a temperature of 25 DEG C
Foster 7d is obtained with the tissue cultured seedling of robust growth.
The advantage and good effect of the application is as follows：
The present invention to the effect that first induces Herba Medicaginiss Friable embryogenic callus, then with chemical mutagen to wound healing group
Knitting carries out in vitro mutagenesis, and the Herba Medicaginiss Friable embryogenic callus after mutation are transferred to the culture medium containing finite concentration salt
On carry out directed screening, so as to obtain salt tolerant Herba Medicaginiss be mutated calluss, through regeneration culture, finally give Herba Medicaginiss salt tolerant dash forward
Mutant.
Description of the drawings
Fig. 1 is Herba Medicaginiss Friable embryogenic callus；
It is Herba Medicaginiss incompact callus that Fig. 2 is left side, and right side is mutated calluss for the Herba Medicaginiss that Salt Tolerance goes out；
Fig. 3 is Herba Medicaginiss salt tolerant regeneration plant.
Specific embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
Example 1
A kind of method of the mutagenic obtained salt tolerant New alfalfa cultivars of utilization Friable embryogenic callus, step are as follows：This reality
Apply example the mutant that salt tolerant " favors " Herba Medicaginiss is obtained using said method screening
(1) the preparation of Herba Medicaginiss aseptic seedling
Choose full " favor " alfalfa seed, 30s soaked in 70% ethanol, then using 40% antiformin water
Solution carries out surface sterilization, and disinfecting time is 15Min.Seed sterilized water after disinfecting is rinsed 5 times repeatedly, is washed every time
10Min, then sterilized seed is inoculated in 1/2MS culture medium the culture for carrying out aseptic seedling.Cultivate at a temperature of 25 DEG C
7d is obtained with the tissue cultured seedling of robust growth.
(2) the induction of Herba Medicaginiss loose callus
The hypocotyls of " favor " Herba Medicaginiss tissue cultured seedling are adopted for explant, is trained in 1/2MS+1.0mg/L2.4-D+20g/L sucrose
Induce on foster base.Condition of culture is：24 DEG C of temperature, 24h illumination, intensity of illumination are 2000Lux.20d can obtain preferably pine
Soft-type calluss.
(3) the induction of Herba Medicaginiss embryo callus
By step (2) in the loose callus that induces separate from explant, be carefully seeded in advance
Callus induction in the culture medium of the MS+1mg/LKT+0.5mg/L2,4-D+30g/L sucrose for preparing.Condition of culture is：Temperature
23 DEG C, 24h illumination, intensity of illumination are 2500Lux.In inoculation 14d follow-up generations, once used medium and condition of culture were constant.
Shift quality harder calluss during subculture as far as possible, after approximately passing through 5 subcultures, calluss quality is hardened completely, and
During microscopy, calluss are substantially by small volume, spherical, Cytoplasm be dense, nucleus greatly and the cellularity containing multiple split coil methods,
Now illustrate to have induced the embryo callus of Herba Medicaginiss.
(4) the induction of Herba Medicaginiss Friable embryogenic callus
" favor " Herba Medicaginiss embryo callus that (3) step is induced are divided into the fritter of a diameter of 5mm, are then connect
Plant in the culture medium of MS+1mg/LKT+0.5mg/L2,4-D+30g/L sucrose.Cultivate at 22 DEG C, and, in 7d subcultures once
Through 8 successive transfer culture, calluss occur significantly change in structure, and when there is loosely organized, quality closely, microscopy is
During embryo callus, Herba Medicaginiss Friable embryogenic callus are now obtained.24h illumination is adopted during culture calluss,
Intensity of illumination is 2500Lux.
(5) the preparation of mutant materials
" favor " the Herba Medicaginiss Friable embryogenic callus for inducing are divided into into diameter with dissecting knife in super-clean bench
For the fritter of 3mm, it is put into standby in culture dish；Separately by callus culture base (MS+1mg/LKT+0.5mg/L2,4-D+30g/L
Sucrose) melt in microwave oven, when its temperature drops to 50 DEG C, add 0.3%.EMS, be then dispensed into the triangle of 100ml
In bottle, per bottle of culture medium for pouring 20ml into.Until calluss are inoculated in triangular flask after culture medium solidifying cultivated.Training
Foster condition is 24 DEG C, and 24h illumination, intensity of illumination are 2000Lux.
(6) the screening of salt tolerant cell line
After the mutagenic treatment of 20d, normal calluss will be grown and be transferred to the culture containing 0.8%NaCl concentration
Salt Tolerance is carried out on base, through the growth of 20d, growth is therefrom filtered out normally and is possessed the calluss of regeneration capacity.Training
Foster condition is 24 DEG C, and 24h illumination, intensity of illumination are 3000Lux.
(7) the culture of Salt tolerant mutant
The Salt-tolerant Callus for filtering out are transferred to into regeneration induction plant in the MS culture medium without hormone.In culture medium
Addition concentration is 0.8%NaCl, through the growth of 30d, you can the salt tolerant for obtaining robust growth " favors " Herba Medicaginiss regeneration plant.This
The condition of culture of process is 25 DEG C, and 24h illumination, intensity of illumination are 3000Lux.
Claims (2)
1. the method for the mutagenic obtained salt tolerant New alfalfa cultivars of a kind of utilization Friable embryogenic callus, it is characterised in that：Step
It is as follows
(1) Herba Medicaginiss aseptic seedling is obtained；
(2) the induction of Herba Medicaginiss loose callus
The hypocotyls of Herba Medicaginiss tissue cultured seedling are adopted for explant, in 1/2MS+0.5-1.0mg/L2.4-D+20g/L sucrose medium
Induction；Condition of culture is：Temperature 24-25 DEG C, 24h illumination, intensity of illumination are 2000Lux, and 20-25d can obtain preferably loose
Soft-type calluss；
(3) the induction of Herba Medicaginiss embryo callus
By step (2) in the loose callus that induces separate from explant, be seeded in the MS+ for preparing in advance
Callus induction in the culture medium of 1-2mg/LKT+0.5mg/L2,4-D+30g/L sucrose；Condition of culture is：Temperature 22-23
DEG C, 24h illumination, intensity of illumination are 2500Lux；In inoculation 14-16d follow-up generations, once used medium and condition of culture were constant；
Shift quality harder calluss during subculture as far as possible, after approximately passing through 3-5 subculture, calluss quality is hardened completely, and
In microscopy, calluss are substantially by small volume, spherical, Cytoplasm be dense, nucleus greatly and the cell structure containing multiple split coil methods
Into now illustrating to have induced the embryo callus of Herba Medicaginiss；
(4) the induction of Herba Medicaginiss Friable embryogenic callus
By step (3) in the Herba Medicaginiss embryo callus that induce be divided into the fritter of a diameter of 3-5mm, be then inoculated into MS+
In the culture medium of 1mg/LKT+0.5mg/L2,4-D+30g/L sucrose；Cultivate at 20-22 DEG C, and, Jing. in 7-10d subcultures once
5-8 successive transfer culture is crossed, calluss occur significantly change in structure, when there is loosely organized, quality closely, microscopy is
During embryo callus, Herba Medicaginiss Friable embryogenic callus are now obtained；24h illumination is adopted during culture calluss,
Intensity of illumination is 2500Lux；
(5) the preparation of mutant materials
It is the little of 2-3mm that the Herba Medicaginiss Friable embryogenic callus for inducing are divided into diameter with dissecting knife in super-clean bench
Block, is put into standby in culture dish；Separately by callus culture base (MS+1-0.5mg/LKT+1-0.5mg/L2,4-D+30g/L sugarcanes
Sugar) melt in microwave oven, 0.3% EMS when its temperature drops to 50 DEG C, is added, the triangular flask of 100ml is then dispensed into
In, per bottle of culture medium for pouring 20ml into；Until calluss are inoculated in triangular flask after culture medium solidifying cultivated；Culture
Condition is 24 DEG C, and 24h illumination, intensity of illumination are 2000Lux；
(6) the screening of salt tolerant cell line
After the mutagenic treatment of 20d, normal calluss will be grown and be transferred to the culture containing 0.8-1.0%NaCl concentration
Salt Tolerance is carried out on base, through the growth of 20d, growth is therefrom filtered out normally and is possessed the calluss of regeneration capacity；Training
Foster condition is 23-24 DEG C, and 24h illumination, intensity of illumination are 3000Lux；
(7) the culture of Salt tolerant mutant
The Salt-tolerant Callus for filtering out are transferred to into regeneration induction plant in the MS culture medium without hormone；Add in culture medium
Concentration is 0.8-1.0%NaCl, through the growth of 20-30d, you can obtain the salt tolerant Herba Medicaginiss regeneration plant of robust growth；This mistake
The condition of culture of journey is 24-25 DEG C, and 24h illumination, intensity of illumination are 3000Lux.
2. the method for the mutagenic obtained salt tolerant New alfalfa cultivars of utilization Friable embryogenic callus according to claim 1,
It is characterized in that：The method that (1) step obtains Herba Medicaginiss aseptic seedling is as follows：
Full alfalfa seed is chosen, 30-40s is soaked in 70% ethanol, is then entered using 40% antiformin aqueous solution
Row surface sterilization, disinfecting time are 10-15Min；Seed sterilized water after disinfecting is rinsed 5 times repeatedly, is washed every time
10Min, then sterilized seed is inoculated in 1/2MS culture medium the culture for carrying out aseptic seedling, is cultivated at a temperature of 25 DEG C
7d is obtained with the tissue cultured seedling of robust growth.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN107094622A (en) * | 2017-04-18 | 2017-08-29 | 天津农学院 | A kind of method for inducing raspberry Callus formation globular embryo |
| CN108419669A (en) * | 2018-02-28 | 2018-08-21 | 山东农业大学 | A kind of EMS directed mutagenesis, the method and device for screening salt tolerant alfalfa |
| CN110663551A (en) * | 2019-11-05 | 2020-01-10 | 天津农学院 | Method for cultivating stress-resistant novel cauliflower variety |
| CN110663550A (en) * | 2019-11-05 | 2020-01-10 | 天津农学院 | Induction method and application of loose embryonic callus of silk cotton wood |
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| AU2012208997B1 (en) * | 2012-07-30 | 2013-09-19 | Dlf Usa Inc. | An alfalfa variety named magnum salt |
| CN104719162A (en) * | 2015-03-25 | 2015-06-24 | 河北省农林科学院滨海农业研究所 | Method for screening dandelion salt-tolerance mutants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU2012208997B1 (en) * | 2012-07-30 | 2013-09-19 | Dlf Usa Inc. | An alfalfa variety named magnum salt |
| CN104719162A (en) * | 2015-03-25 | 2015-06-24 | 河北省农林科学院滨海农业研究所 | Method for screening dandelion salt-tolerance mutants |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN107094622A (en) * | 2017-04-18 | 2017-08-29 | 天津农学院 | A kind of method for inducing raspberry Callus formation globular embryo |
| CN108419669A (en) * | 2018-02-28 | 2018-08-21 | 山东农业大学 | A kind of EMS directed mutagenesis, the method and device for screening salt tolerant alfalfa |
| CN108419669B (en) * | 2018-02-28 | 2023-11-17 | 山东农业大学 | Method and device for EMS (enhanced message service) directional mutagenesis and screening salt-tolerant alfalfa |
| CN110663551A (en) * | 2019-11-05 | 2020-01-10 | 天津农学院 | Method for cultivating stress-resistant novel cauliflower variety |
| CN110663550A (en) * | 2019-11-05 | 2020-01-10 | 天津农学院 | Induction method and application of loose embryonic callus of silk cotton wood |
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