CN106508673A - Method for directly obtaining plants through eggplant anther culture and medium - Google Patents
Method for directly obtaining plants through eggplant anther culture and medium Download PDFInfo
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- CN106508673A CN106508673A CN201610894010.3A CN201610894010A CN106508673A CN 106508673 A CN106508673 A CN 106508673A CN 201610894010 A CN201610894010 A CN 201610894010A CN 106508673 A CN106508673 A CN 106508673A
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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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for directly obtaining plant through eggplant anther cultivation as well as a medium thereof, which belong to the technical field of plant tissue culture technique. The method comprises the following steps: 1) selecting fresh buds; 2) disinfecting the surface of bud and acquiring the aseptic anther; 3) inoculating the aseptic anther in the late uninucleate stage in an anther culture solid medium, culturing the aseptic anther for 2-8 days at dark environment at the temperature of 33-37 DEG C, and performing illumination culture for 30-40 days at the temperature of 25-28 DEG C; and 4) transferring the cotyledonous embryoid to a healthy seedling solid medium for continuously culturing the materials, and growing to obtain the plant. The method has the following advantages that the method is simple and easy to carry out, the in-vitro culture of the anther can directly induce the embryoid to obtain the haploid plant, a condition that the callus formed by anther culture induction is transferred to a differentiation medium, and then the plant regeneration is induced can be avoided; and the chromosome ploidy mixing phenomenon due to participation of diploid somatic cells such as the anther wall in callus can be avoided.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of Fructus Solani melongenae Anther Culture directly obtains plant
Method and culture medium.
Background technology
Anther Culture is to obtain the important channel of haplobiont, can quickly obtain zygoid by the technology,
With shorten breeding cycle, improve efficiency of selection, overcome the advantages such as the sterility of distant hybrid, be also used as molecular marker and
Genetic map draws the good material of research.
Fructus Solani melongenae haplobiont is obtained by Anther Culture or microspore-isolated culture mainly three kinds of approach：1st, first dedifferentiation
Calluss are formed, then callus induction is differentiated to form embryoid, finally whole plant is developed into by embryoid；2nd, by inducing
The calluss of formation directly differentiate adventitious bud, and adventitious bud is developed again for complete plant；3rd, embryoid is directly obtained, is developed
Into whole plant.
Fructus Solani melongenae Research Work on Anther Culture is carried out relatively early, is trained by flower pesticide since Raina and Iyer is equal to reported first in 1973
Since foster Jing calluss approach obtains Fructus Solani melongenae haplobiont, Fructus Solani melongenae Anther Culture and microspores culture have reported success.
Although many Eggplant Varieties or cenospecies have obtained DH plant, being far from, it is efficient to have as another crop Nicotiana tabacum L. of Solanaceae
Monoploid regenerative system, so as to hinder the technology Fructus Solani melongenae production in large-scale application.
Fructus Solani melongenae Anther Culture forms calluss and calluss differentiation embryoid or indefinite through dedifferentiation mostly at present
In the two stages of bud, need to be placed in two kinds of different culture medium and cultivated, transit working amount is big, but the differentiation of calluss
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, it is impossible to fully meet the demand to haplobiont in production.Therefore, how to set up efficient, simple and direct embryoid induction body
System, obtains a large amount of haplobionts, is present Fructus Solani melongenae breeding problem demanding prompt solution.
The content of the invention
It is an object of the invention to disclose a kind of method that Fructus Solani melongenae Anther Culture directly obtains plant.
Another object of the present invention is the culture medium for disclosing the method for directly obtaining plant for Fructus Solani melongenae Anther Culture.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method that Fructus Solani melongenae Anther Culture directly obtains plant, comprises the steps：
(1), the acquisition of embryoid：The aseptic flower pesticide of mid-late uninucleate stage is inoculated in Anther Culture solid medium, 33
～37 DEG C of dark culturing 2～8 days, go to 25-28 DEG C of illumination cultivation 30-40 days, it is seen that embryoid is by sprouting in the anther wall for splitting
Issue；
The Anther Culture solid medium is consisted of：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 inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L salt
Sour sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (vitamin B6), 0.5mg/L Folic Acid, 0.05mg/L biotin,
800mg/L glutamine, 100mg/L serines, 30mg/L glutathion (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), regenerate the acquisition of haplobiont：When embryoid grows into the cotyledon type embryoid stage, it is forwarded to strong
Continue culture in seedling rooting solid medium, grow up to whole plant；
The strong seedling rooting solid medium is consisted of：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 inositol, 0.5mg/L nicotinic acid, 2mg/L glycine, 0.1mg/L
Hydrochloric acid sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (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 Fructus Solani melongenae Anther Culture described in above-mentioned technical proposal directly obtains plant, wherein, in methods described
Also comprise the steps before：
(1), the selection of fresh alabastrum：Alabastrum of the sporidiole in mid-late uninucleate stage is chosen, flower is determined by DAPI dyeing
Powder developmental stage is mid-late uninucleate stage；
(2), the surface sterilization of alabastrum, the acquisition of aseptic flower pesticide：Alabastrum with 75% alcohol-pickled 0.5-1 minutes, 5% time
Sodium chlorate solution soaks 3-5 minutes, aseptic water washing 3-5 time, aseptic filter paper suck dry moisture.
The culture medium of aseptic flower pesticide is cultivated in cultural method described in above-mentioned technical proposal, wherein, the aseptic Anther Culture
Solid medium is consisted of：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 inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L hydrochloric acid sulfur ammonia (vitamin
B1), 0.5mg/L Pyridoxin hydrochloride (vitamin B6), 0.5mg/L Folic Acid, 0.05mg/L biotin, 800mg/L glutamine,
100mg/L serines, 30mg/L glutathion (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, wherein, the strong seedling rooting solid culture is good in cultural method described in above-mentioned technical proposal
Base is consisted of：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 inositol, 0.5mg/L nicotinic acid, 2mg/L glycine, 0.1mg/L hydrochloric acid sulfur ammonia (vitamin B1),
0.5mg/L Pyridoxin hydrochloride (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, directly induces embryo shape by vitro anther culture
Body obtains haplobiont.As the method for present invention employing is without calluss process, so there are no calluss point
Change problem, it is to avoid the calluss of Anther Culture induced synthesis are forwarded to division culture medium, then induced synthesis regeneration plants
Process；The ploidy that it also avoid being participated in Callus formation and being occurred by the somatic cell of the diploid such as anther wall mixes phenomenon.
(2), the method that the present invention is adopted have that the haplobiont regeneration period is short, induction frequency is high, it is time saving and energy saving etc. excellent
Point.Genotype barrier overcome simultaneously, different genotype purple and green circle eggplant, the Anther Culture of long eggplant all obtain higher
Embryoid induction rate.
(3) embryoid can be directly obtained using microspores culture in prior art, although not by diploids such as anther walls
The interference of cell, but culture technique difficulty is larger, not easy to operate, and also embryoid induction rate is relatively low, typically all below 10%,
And the method for the present invention is adopted, embryoid induction frequency is typically in 20%-40%.
Specific embodiment：
For readily appreciating technical scheme, a kind of Fructus Solani melongenae flower pesticide of the invention is trained below in conjunction with concrete test example
The foster method and culture medium for directly obtaining plant is further described.
Embodiment 1：Hosta ventricosa piece purple justifies the Anther Culture of eggplant：
Between the 5-6 months in 2015, alabastrum, pollen Jing DAPI (4,6- diamidines -2-phenylindone) dyeing is fetched from planting greenhouse
Confirmation developmental stage is mid-late uninucleate stage.75% ethanol of alabastrum Jing 1 minute, 5% surface sterilization of sodium hypochlorite 5 minutes, sterilized water
Rinse 3 times, aseptic filter paper suck dry moisture.Carefully flower pesticide is taken out with pincet, be placed in Anther Culture solid medium, 37
DEG C dark culturing goes to after 3 days；30 days or so, naked eyes visible white embryoid was from the flower pesticide for splitting
Sprout out in wall；
Upper continued growth in strong seedling rooting solid medium is gone to when embryoid differentiates the little cotyledon of two panels, is carried out
Strong sprout and take root.1130 flower pesticide common properties go out embryoid 446, and embryoid induction rate is 39.5%；
Wherein, aseptic Anther Culture solid medium is consisted of：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 inositol, 5mg/L nicotinic acid, 2mg/L glycine,
0.5mg/L hydrochloric acid sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (vitamin B6), 0.5mg/L Folic Acid, 0.05mg/L lifes
Thing element, 800mg/L glutamine, 100mg/L serines, 30mg/L glutathion (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, it is good for consisting of for seedling rooting solid medium：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 inositol, 0.5mg/L nicotinic acid, the sweet ammonia of 2mg/L
Acid, 0.1mg/L hydrochloric acid sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (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 is carried out respectively to the Fructus Solani melongenae of 13 kinds of different genotypes between 2013-2016
Test, wherein to 4 Fructus Solani melongenae cenospecies (A, C, G, J) carried out repeat experiment, as a result as shown in table 2, in table 2 cenospecies A in
Carry out within 2014 and 2015 repeating experiment；Cenospecies C has carried out repeating experiment in 2015 and 2016；Cenospecies G in
Carry out within 2014 and 2015 repeating experiment；Cenospecies J has carried out repeating experiment in 2015 and 2016：
The anther cultural embryoid induction result of 2 different genotype Fructus Solani melongenae of table
As shown in Table 2, using the method for the present invention, embryoid induction frequency is typically in 20%-40%.
The above, only presently preferred embodiments of the present invention not makees any formal and substantial limit to the present invention
System, all those skilled in the art, in the range of without departing from technical solution of the present invention, when using disclosed above skill
Art content, and a little change, modification and the equivalent variations for developing made, are the Equivalent embodiments of the present invention；Meanwhile, it is all according to
The change of any equivalent variations made to above example according to the substantial technological of the present invention, modification and differentiation, still fall within this
In the range of the technical scheme of invention.
Claims (4)
1. a kind of method that Fructus Solani melongenae Anther Culture directly obtains plant, comprises the steps：
(1), the acquisition of embryoid：The aseptic flower pesticide 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 is by sprouting in the anther wall for splitting
Come；
The Anther Culture solid medium is consisted of：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 inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L salt
Sour sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (vitamin B6), 0.5mg/L Folic Acid, 0.05mg/L biotin,
800mg/L glutamine, 100mg/L serines, 30mg/L glutathion (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), regenerate the acquisition of haplobiont：When embryoid grows into the cotyledon type embryoid stage, strong Seedling life is forwarded to
Continue culture in root solid medium, grow up to whole plant；
The strong seedling rooting solid medium is consisted of：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 inositol, 0.5mg/L nicotinic acid, 2mg/L glycine, 0.1mg/L
Hydrochloric acid sulfur ammonia (vitamin B1), 0.5mg/L Pyridoxin hydrochloride (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 Fructus Solani melongenae Anther Culture according to claim 1 directly obtains plant, it is characterised in that described
Also comprise the steps before method：
(1), the selection of fresh alabastrum：Alabastrum of the sporidiole in mid-late uninucleate stage is chosen, determines that pollen is sent out by DAPI dyeing
Period is educated for mid-late uninucleate stage；
(2), the surface sterilization of alabastrum, the acquisition of aseptic flower pesticide：Alabastrum 75% alcohol-pickled 0.5-1 minutes, 5% hypochlorous acid
Sodium solution soaks 3-5 minutes, aseptic water washing 3-5 time, aseptic filter paper suck dry moisture.
3. the culture medium of aseptic flower pesticide is cultivated in cultural method described in claim 1 or 2, it is characterised in that：The aseptic flower
Medicine culture solid medium is consisted of：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 inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L hydrochloric acid sulfur ammonia
(vitamin B1), 0.5mg/L Pyridoxin hydrochloride (vitamin B6), 0.5mg/L Folic Acid, 0.05mg/L biotin, 800mg/L paddy ammonia
Amide, 100mg/L serines, 30mg/L glutathion (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
It is worth for 5.8.
4. the culture medium of seedling rooting is good in cultural method described in claim 1 or 2, it is characterised in that：The strong seedling rooting is solid
Body culture medium is consisted of：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 inositol, 0.5mg/L nicotinic acid, 2mg/L glycine, (the dimension life of 0.1mg/L hydrochloric acid sulfur ammonia
Plain B1), 0.5mg/L Pyridoxin hydrochloride (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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109089885A (en) * | 2018-09-03 | 2018-12-28 | 江苏高航农业科技有限公司 | A kind of induction cherry and tomato anther obtains the cultural method of regeneration plant |
| CN111374048A (en) * | 2020-04-28 | 2020-07-07 | 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 |
-
2016
- 2016-10-14 CN CN201610894010.3A patent/CN106508673B/en active Active
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| 刘独臣等: ""茄子花药培养诱导胚状体成苗"", 《西南农业学报》 * |
| 潘羽丰: ""茄子（Solanum melongena L.）花药培养诱导单倍体植株的研究"", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109089885A (en) * | 2018-09-03 | 2018-12-28 | 江苏高航农业科技有限公司 | A kind of induction cherry and tomato anther obtains the cultural method of regeneration plant |
| CN111374048A (en) * | 2020-04-28 | 2020-07-07 | 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 |
| 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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| Publication number | Publication date |
|---|---|
| CN106508673B (en) | 2018-07-24 |
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