CN106508673B - A kind of eggplant Anther Culture directly obtains the method and culture medium of plant - Google Patents
A kind of eggplant Anther Culture directly obtains the method and culture medium of plant Download PDFInfo
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- CN106508673B CN106508673B CN201610894010.3A CN201610894010A CN106508673B CN 106508673 B CN106508673 B CN 106508673B CN 201610894010 A CN201610894010 A CN 201610894010A CN 106508673 B CN106508673 B CN 106508673B
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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
A kind of eggplant Anther Culture of the present invention directly obtains the method and culture medium of plant, belongs to field of plant tissue culture technique.The cultural method includes：(1), the selection of fresh bud；(2), the surface sterilization of bud, the acquisition of sterile anther；(3), the sterile anther of mid-late uninucleate stage is inoculated in Anther Culture solid medium, 33~37 DEG C of dark culturings 2~8 days, goes to 25 28 DEG C of illumination cultivations 30 40 days；(4), cotyledon type embryoid is gone in strong seedling rooting solid medium and continues to cultivate, grow up to intact plant.The cultural method of the present invention has the following advantages：Cultural method of the present invention is simple and practicable, obtains haplobiont by the direct inducing embryoid body of vitro anther culture, the callus for avoiding Anther Culture induced synthesis is forwarded to differential medium, then induces the process of plant regeneration；It also avoids mixing phenomenon by the ploidy that the diploids body cell such as anther wall participation callus forms and occurs.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of eggplant Anther Culture directly obtains plant
Method and culture medium.
Background technology
Anther Culture is to obtain the important channel of haplobiont, and zygoid can be quickly obtained by the technology,
With shorten breeding cycle, improve the advantages such as efficiency of selection, the sterility for overcoming distant hybrid, be also used as molecular labeling and
Genetic map draws the good material of research.
Obtaining eggplant haplobiont by Anther Culture or microspore-isolated culture, there are mainly three types of approach：1, first dedifferentiation
Callus is formed, then evoked callus is differentiated to form embryoid, finally develops into intact plant by embryoid；2, by inducing
The callus of formation directly differentiates adventitious bud, and adventitious bud is developed again for complete plant；3, embryoid is directly obtained, is developed
At intact plant.
The development of eggplant Research Work on Anther Culture is more early, reports within 1973 that passing through anther trains for the first time since Raina and Iyer is equal to
It has supported since obtaining eggplant haplobiont through callus approach, eggplant Anther Culture and microspores culture have reported success.
Although many Eggplant Varieties or cenospecies have obtained DH plant, it is far from having as another crop tobacco of Solanaceae efficient
Monoploid regenerative system, to hinder the technology eggplant production in large-scale application.
Eggplant Anther Culture passes through dedifferentiation formation callus mostly at present and callus breaks up embryoid or indefinite
The two stages of bud need to be placed on two different culture mediums and be cultivated, and transit working amount is big, but the differentiation of callus
Frequency is relatively low；Microspore-isolated culture can directly obtain embryoid, but method is relatively complicated, and the induction frequency of embryoid
It is low, the demand to haplobiont in production cannot be fully met.Therefore, efficient, simple and direct embryoid induction body how is established
System, obtains a large amount of haplobionts, is present eggplant breeding urgent problem to be solved.
Invention content
The method of plant is directly obtained it is an object of the invention to disclose a kind of eggplant Anther Culture
Another object of the present invention is to disclose the culture medium for the method for directly obtaining plant for eggplant Anther Culture.
The purpose of the present invention is what is be achieved through the following technical solutions：
A kind of method that eggplant Anther Culture directly obtains plant, includes the following steps：
(1), the acquisition of embryoid：The sterile anther of mid-late uninucleate stage is inoculated in Anther Culture solid medium, 33
~37 DEG C of dark culturings 2~8 days, go to 25-28 DEG C of illumination cultivation 30-40 days, it is seen that embryoid in the anther wall that splits by sprouting
It issues；
The group of the Anther Culture solid medium becomes：950mg/L KNO3、825mg/L NH4NO3、332.02mg/L
CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L
H3BO3、16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L
CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 5mg/L niacin, 2mg/L glycine, 0.5mg/L salt
Sour sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.5mg/L folic acid, 0.05mg/L biotins,
800mg/L glutamine, 100mg/L serines, 30mg/L glutathione (GSH), 0.1~1mg/L KT, 0.1~1mg/L
IAA, 0.01~0.05mg/L NAA, 0.1%~0.3% activated carbon, 3%~6% sucrose and 0.6%~0.8% agar；Solid
The pH value of culture medium is 5.8；
(2), the acquisition of haplobiont is regenerated：When embryoid grows into the cotyledon type embryoid stage, it is forwarded to strong
Continue to cultivate in seedling rooting solid medium, grows up to intact plant；
The group of the strong seedling rooting solid medium becomes：1900mg/L KNO3、1650mg/L NH4NO3、332.02mg/
L CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L
H3BO3、16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L
CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 0.5mg/L niacin, 2mg/L glycine, 0.1mg/L
Hydrochloric acid sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.01~0.1mg/L KT, 0.1~0.5mg/L
IBA, 3% sucrose and 0.8% agar；The pH value of strong seedling rooting solid medium is 5.8.
The method that a kind of eggplant Anther Culture described in above-mentioned technical proposal directly obtains plant, wherein in the method
Further include following step before：
(1), the selection of fresh bud：The bud that microspore is in mid-late uninucleate stage is chosen, is dyed by DAPI and determines flower
Powder developmental stage is mid-late uninucleate stage；
(2), the surface sterilization of bud, the acquisition of sterile anther：75% alcohol of bud impregnates 0.5-1 minutes, 5% time
Sodium chlorate solution impregnates 3-5 minutes, aseptic water washing 3-5 times, aseptic filter paper suck dry moisture.
The culture medium of sterile anther is cultivated in cultural method described in above-mentioned technical proposal, wherein the sterile Anther Culture
The group of solid medium becomes：950mg/L KNO3、825mg/L NH4NO3、332.02mg/L CaCl2、180.54mg/L
MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L H3BO3、16.9mg/L
MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·5H2O、
0.025mg/L CoCl2·6H2O, 100mg/L inositols, 5mg/L niacin, 2mg/L glycine, 0.5mg/L hydrochloric acid sulphur ammonia (vitamins
B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.5mg/L folic acid, 0.05mg/L biotins, 800mg/L glutamine,
100mg/L serines, 30mg/L glutathione (GSH), 0.1~1mg/L KT, 0.1~1mg/L IAA, 0.01~0.05mg/L
NAA, 0.1%~0.3% activated carbon, 3%~6% sucrose and 0.6%~0.8% agar；The pH value of solid medium is 5.8.
The culture medium of seedling rooting is good in cultural method described in above-mentioned technical proposal, wherein the strong seedling rooting solid culture
The group of base becomes：1900mg/L KNO3、1650mg/L NH4NO3、332.02mg/L CaCl2、180.54mg/L MgSO4、
170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L H3BO3、16.9mg/L MnSO4·H2O、
8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·5H2O、0.025mg/L
CoCl2·6H2O, 100mg/L inositols, 0.5mg/L niacin, 2mg/L glycine, 0.1mg/L hydrochloric acid sulphur ammonia (vitamin B1),
0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.01~0.1mg/L KT, 0.1~0.5mg/L IBA, 3% sucrose and 0.8%
Agar；The pH value of strong seedling rooting solid medium is 5.8.
The invention has the advantages that：
(1), cultural method step of the present invention is few, simple and practicable, and embryo shape is directly induced by vitro anther culture
Body obtains haplobiont.Since the method for the invention used is without callus process, so there is no callus point
Change problem, the callus for avoiding Anther Culture induced synthesis are forwarded to differential medium, then induced synthesis regeneration plant
Process；It also avoids mixing phenomenon by the ploidy that the diploids body cell such as anther wall participation callus forms and occurs.
(2), the method that uses of the present invention has that the haplobiont regeneration period is short, induction frequency is high, time saving and energy saving etc. excellent
Point.Overcome genotype barrier simultaneously, the Anther Culture of different genotype purple and green circle eggplant, long eggplant all obtains higher
Embryoid induction rate.
(3), use microspores culture that can directly obtain embryoid in the prior art, although not by diploids such as anther walls
The interference of cell, but culture technique difficulty is not larger, easy to operate, and embryoid induction rate is relatively low, generally all 10% hereinafter,
And method using the present invention, embryoid induction frequency is generally in 20%-40%.
Specific implementation mode：
To make technical scheme of the present invention be easy to understand, a kind of eggplant anther of the present invention is trained below in conjunction with specific test example
It supports the method for directly obtaining plant and culture medium is further described.
Embodiment 1：Hosta ventricosa piece purple justifies the Anther Culture of eggplant：
Between the 5-6 months in 2015, bud is fetched from planting greenhouse, pollen is dyed through DAPI (4,6- diamidines -2-phenylindone)
Confirmation developmental stage is mid-late uninucleate stage.Bud is through 75% alcohol 1 minute, 5% surface sterilization of sodium hypochlorite 5 minutes, sterile water
It rinses 3 times, aseptic filter paper suck dry moisture.Carefully anther is taken out with pincet, is placed in Anther Culture solid medium, 37
DEG C dark culturing, which is gone to after 3 days under 25 DEG C of illumination, to be continued to cultivate；30 days or so, naked eyes visible white embryoid was from the anther to split
It sprouts and comes out in wall；
Upper continued growth in strong seedling rooting solid medium is gone to when embryoid differentiates the small cotyledon of two panels, is carried out
It strong sprout and takes root.1130 anther common properties go out embryoid 446, and embryoid induction rate is 39.5%；
Wherein, the group of sterile Anther Culture solid medium becomes：950mg/L KNO3、825mg/L NH4NO3、
332.02mg/L CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L
KI、6.2mg/L H3BO3、16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、
0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 5mg/L niacin, 2mg/L glycine,
0.5mg/L hydrochloric acid sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.5mg/L folic acid, 0.05mg/L lifes
Object element, 800mg/L glutamine, 100mg/L serines, 30mg/L glutathione (GSH), 0.1~1mg/L KT, 0.1~
1mg/L IAA, 0.01~0.05mg/L NAA, 0.1%~0.3% activated carbon, 3%~6% sucrose and 0.6%~0.8% fine jade
Fat；The pH value of solid medium is 5.8；
Wherein, being good for the group of seedling rooting solid medium becomes：1900mg/L KNO3、1650mg/L NH4NO3、
332.02mg/L CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L
KI、6.2mg/L H3BO3、16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、
0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 0.5mg/L niacin, the sweet ammonia of 2mg/L
Acid, 0.1mg/L hydrochloric acid sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.01~0.1mg/L KT, 0.1
~0.5mg/L IBA, 3% sucrose and 0.8% agar；The pH value of strong seedling rooting solid medium is 5.8.
Embodiment 2：
According to the implementation process of embodiment 1, reality has been carried out respectively to the eggplant of 13 kinds of different genotypes between 2013-2016
Test, wherein to 4 eggplant cenospecies (A, C, G, J) carried out repeat test, the results are shown in Table 2, in table 2 cenospecies A in
It has carried out within 2014 and 2015 repeating to test；Cenospecies C has carried out repeating to test in 2015 and 2016；Cenospecies G in
It has carried out within 2014 and 2015 repeating to test；Cenospecies J has carried out repeating to test in 2015 and 2016：
The embryoid induction result of 2 different genotype eggplant Anther Culture of table
As shown in Table 2, method using the present invention, embryoid induction frequency is generally in 20%-40%.
The above, only presently preferred embodiments of the present invention, not to the present invention make in any form with substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made are the equivalent embodiment of the present invention；Meanwhile it is all according to
According to the substantial technological of the present invention to the variation, modification and evolution of any equivalent variations made by above example, this is still fallen within
In the range of the technical solution of invention.
Claims (4)
1. a kind of method that eggplant Anther Culture directly obtains plant, includes the following steps：
(1), the acquisition of embryoid：The sterile anther of mid-late uninucleate stage is inoculated in Anther Culture solid medium, 33~37
DEG C dark culturing 2~8 days, goes to 25-28 DEG C of illumination cultivation 30-40 days, it is seen that embryoid in the anther wall that splits by sprouting
Come；
The group of the Anther Culture solid medium becomes：950mg/L KNO3、825mg/L NH4NO3、332.02mg/L
CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L
H3BO3、16.9mg/LMnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L
CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 5mg/L niacin, 2mg/L glycine, 0.5mg/L salt
Sour sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.5mg/L folic acid, 0.05mg/L biotins,
800mg/L glutamine, 100mg/L serines, 30mg/L glutathione (GSH), 0.1~1mg/L KT, 0.1~1mg/L
IAA, 0.01~0.05mg/L NAA, 0.1%~0.3% activated carbon, 3%~6% sucrose and 0.6%~0.8% agar；Solid
The pH value of culture medium is 5.8；
(2), the acquisition of haplobiont is regenerated：When embryoid grows into the cotyledon type embryoid stage, it is forwarded to strong seedling life
Continue to cultivate in root solid medium, grows up to intact plant；
The group of the strong seedling rooting solid medium becomes：1900mg/L KNO3、1650mg/L NH4NO3、332.02mg/L
CaCl2、180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L
H3BO3、16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L
CuSO4·5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 0.5mg/L niacin, 2mg/L glycine, 0.1mg/L
Hydrochloric acid sulphur ammonia (vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.01~0.1mg/L KT, 0.1~0.5mg/L
IBA, 3% sucrose and 0.8% agar；The pH value of strong seedling rooting solid medium is 5.8.
2. the method that a kind of eggplant Anther Culture according to claim 1 directly obtains plant, which is characterized in that described
Further include following step before method：
(1), the selection of fresh bud：The bud that microspore is in mid-late uninucleate stage is chosen, is dyed by DAPI and determines pollen hair
It is mid-late uninucleate stage to educate period；
(2), the surface sterilization of bud, the acquisition of sterile anther：Bud is impregnated 0.5-1 minutes with 75% alcohol, 5% hypochlorous acid
Sodium solution impregnates 3-5 minutes, aseptic water washing 3-5 times, aseptic filter paper suck dry moisture.
3. cultivating the culture medium of sterile anther in cultural method described in claims 1 or 2, it is characterised in that：The sterile flower
The group of medicine culture solid medium becomes：950mg/L KNO3、825mg/L NH4NO3、332.02mg/L CaCl2、
180.54mg/L MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L H3BO3、
16.9mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·
5H2O、0.025mg/L CoCl2·6H2O, 100mg/L inositols, 5mg/L niacin, 2mg/L glycine, 0.5mg/L hydrochloric acid sulphur ammonia
(vitamin B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.5mg/L folic acid, 0.05mg/L biotins, 800mg/L paddy ammonia
Amide, 100mg/L serines, 30mg/L glutathione (GSH), 0.1~1mg/L KT, 0.1~1mg/L IAA, 0.01~
0.05mg/L NAA, 0.1%~0.3% activated carbon, 3%~6% sucrose and 0.6%~0.8% agar；The pH of solid medium
Value is 5.8.
4. being good for the culture medium of seedling rooting in cultural method described in claims 1 or 2, it is characterised in that：The strong seedling rooting is solid
The group of body culture medium becomes：1900mg/L KNO3、1650mg/L NH4NO3、332.02mg/L CaCl2、180.54mg/L
MgSO4、170mg/L KH2PO4、36.7mg/L FeNaEDTA、0.83mg/L KI、6.2mg/L H3BO3、16.9mg/L
MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L CuSO4·5H2O、
0.025mg/L CoCl2·6H2O, 100mg/L inositols, 0.5mg/L niacin, 2mg/L glycine, (the dimension life of 0.1mg/L hydrochloric acid sulphur ammonia
Plain B1), 0.5mg/L pyridoxine hydrochlorides (vitamin B6), 0.01~0.1mg/L KT, 0.1~0.5mg/L IBA, 3% sucrose and
0.8% agar；The pH value of strong seedling rooting solid medium is 5.8.
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| CN111374048B (en) * | 2020-04-28 | 2022-03-08 | 北京市海淀区植物组织培养技术实验室 | Chromosome doubling method of pepper or eggplant haploid plant |
| CN116806694A (en) * | 2023-08-16 | 2023-09-29 | 金陵科技学院 | Eggplant doubled haploid anther culture method |
| CN116998405A (en) * | 2023-08-23 | 2023-11-07 | 金陵科技学院 | Method for improving embryoid seedling rate of eggplant anther culture |
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