CN107873519A - A kind of method that asparagus zygoid is obtained using Anther Culture - Google Patents
A kind of method that asparagus zygoid is obtained using Anther Culture Download PDFInfo
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- CN107873519A CN107873519A CN201711338398.XA CN201711338398A CN107873519A CN 107873519 A CN107873519 A CN 107873519A CN 201711338398 A CN201711338398 A CN 201711338398A CN 107873519 A CN107873519 A CN 107873519A
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- zygoid
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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Abstract
The present invention provides a kind of method that asparagus zygoid is obtained using Anther Culture, comprises the following steps：The acquisition of asparagus flower pesticide, the pretreatment of asparagus flower pesticide, asparagus induction of anther callus, the differentiation of asparagus flower pesticide subculture, rooting of vitro seedling, haploid chromosomes double.The method of the present invention that asparagus zygoid is obtained using Anther Culture, the inductivity of the hard closely callus of quality of asparagus flower pesticide is 7.2 17%；The hard closely callus induction rate of quality of particularly kind " champion " is 15.8 more than 17%.The method of the present invention that asparagus zygoid is obtained using Anther Culture, monoploid differentiation rate are 2.2 7.2%, and the success rate that zygoid and polyploid are obtained by haploid induction is 45.86 49.73%, and wherein zygoid accounting is 1.6 2.9%.
Description
Technical field
The present invention relates to field of plant variety breeding technology, more particularly to one kind to pass through asparagus training approach and cultivate zygoid
Supermale strain and female plant.
Background technology
Asparagus scientific name asparagus（Asparagus officinalisL.）, tender stem quality is fine and smooth, and fiber softening is tasty, wind
Delicious, there is the fragranced of uniqueness.Containing abundant polysaccharide, flavone compound, saponin isoreactivity composition, asparagus has anti-swollen
Knurl, antifatigue, lowering blood pressure and blood fat, improve the effect such as immunologic function, be a kind of with high nutritive value health-care vegetable, by
One of world's " ten big famous dishes " is classified as, enjoys the laudatory title of " kings of vegetables " in the international market.
The approach of asparagus conventional variety seed selection is at present：The excellent staminiferous plant of first selection traits and female plant are hybridized；Selection
Strong hybrid vigour combination, then carries out 3-4 multiple spots field variety comparative test, is investigated by field Comprehensive Traits, finally selected
New varieties.Due to the dioecious particularity of asparagus, compared with androgynous crop, lack and be selfed this link, so as to
Bring up current asparagus kind and the characteristics of uniformity, stability difference, same kind difference individual plant, or even same individual plant difference bamboo shoot be present
All had differences between bar.
The asparagus Research Work on Anther Culture work that the country has reported at present is confined to influence induction of anther callus rate mostly
Factor and plant regeneration, and the identification of follow-up monoploid, haploid chromosomes double, the correlative study of supermale strain identification etc.
Less, also no successful incubation goes out the report of zygoid.Prior art carries out asparagus induction of anther callus and plant
Following technical problem be present in regeneration research：
（1）Asparagus Anther Culture it is inefficient, callus induction rate is low；
（2）Body cell serious interference, the pollen monoploid frequency of occurrences are low.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of side that asparagus zygoid is obtained using Anther Culture
Method, to realize following goal of the invention：
（1）Improve the inductivity of asparagus anther callus；
（2）Overcome the interference of body cell, improve the frequency that pollen monoploid occurs；
In order to solve the above technical problems, the technical scheme that the present invention takes is as follows：
A kind of method that asparagus zygoid is obtained using Anther Culture, it is characterised in that：Comprise the following steps：Asparagus flower pesticide
Acquisition, the pretreatment of asparagus flower pesticide, asparagus induction of anther callus, the differentiation of asparagus flower pesticide subculture, rooting of vitro seedling, Dan Bei
Autosome doubles.
It is the further improvement to above-mentioned technical proposal below：
The acquisition of the asparagus flower pesticide, annual 4-5 months gather the male flower inflorescence of asparagus difference individual plant, and bud length is in inflorescence
1-2mm, pollen are mid-late uninucleate stage.
The asparagus, kind are champion, Shandong asparagus No.1, Glan moral or green No. 1 of capital.
The pretreatment of the asparagus flower pesticide, asparagus bud is subjected to Cold pretreatment 2.5-3.5d in 3.5-4.5 DEG C of condition.
The asparagus induction of anther callus, inducing culture are MS+1.0mg/L 6-BA+1.25mg/L NAA, sugarcane
Sugared concentration is 5.0 %, and agar is 0.7 %, pH 5.8.
The asparagus induction of anther callus, in（26±1）DEG C dark condition under carry out light culture 2 weeks, Ran Hou
（26±1）DEG C, after intensity of illumination cultivates 20d under conditions of 1000lx, the light application time 16h/d, start callus occur.
The asparagus flower pesticide subculture differentiation, differentiation and bud formation culture medium is MS+0.5mg/L 6-BA+0.2mg/L NAA, culture
In base the concentration of sucrose be 4.0%, agar 0.7%, pH 5.8.
Asparagus flower pesticide subculture differentiation, at 26 ± 1 DEG C, intensity of illumination is 2000 lx, light application time 16h/d condition
Lower culture 30d.
The rooting of vitro seedling, root media are：MS+2.0mg/LNAA+1.0mg/L IBA+0.01mg/L KT, training
Support and contain sucrose 3.5%, agar 0.5% in base, pH5.8, condition of culture is 25-28 DEG C, daily illumination 16h.
The haploid chromosomes are doubled, and asparagus monoploid test tube seedling, minimal medium MS+ are handled using colchicine
The mg/L of 6-BA0.3 mg/L+NAA 0.1, the % of sucrose 3.0, agar 0.7 %, pH5.8, colchicine concentration are added in culture medium
For 0.05mg/L, after cultivating 15d, switch through and culture 15d is carried out on the minimal medium for being incubated at and be not added with colchicine.
By adopting the above-described technical solution, the present invention reach have the technical effect that：
（1）The method of the present invention that asparagus zygoid is obtained using Anther Culture, the quality of asparagus flower pesticide are hard close
The inductivity of callus be 7.2-17%；The hard closely callus induction rate of the quality of particularly kind ' champion ' is
More than 15.8-17%.
（2）The method of the present invention that asparagus zygoid is obtained using Anther Culture, monoploid differentiation rate is 2.2-
7.2%, the success rate that zygoid and polyploid are obtained by haploid induction is 45.86-49.73%, wherein zygoid
Accounting is 1.6-2.9%.
（3）The method of the present invention that asparagus zygoid is obtained using Anther Culture, bud ratio 113.2-
125.4%, growth coefficient 2.5-4.9.
（4）The method of the present invention that asparagus zygoid is obtained using Anther Culture, Anther Culture test tube seedling are had
Effect rooting rate is 65.2-72.3%.
（5）The method of the invention, using diploid asparagus Anther Culture, haplobiont is filtered out, dyed body adds
Zygoid germplasm is obtained after times, a this method generation can be homozygous, shortens 3-5 compared with conventional breeding.
The present invention above-mentioned practical problem with reference to existing for current asparagus breeding, it is main to carry out flower pesticide training using to excellent staminiferous plant
Support, obtain excellent asparagus monoploid；Then using chromosome doubling is carried out under colchicin indoors condition of tissue culture, obtain excellent
Homozygous asparagus germ plasm resource；The mirror of sex and ploidy is carried out to the germ plasm resource of acquisition finally by molecular labeling and microscopy technology
It is fixed, so as to finally obtain excellent zygoid female plant（mm）, diploid staminiferous plant（MM, hereinafter referred to as " supermale strain "）Provided Deng germplasm
Source.
（6）The characters such as asparagus new varieties Shooting stage that the present invention cultivates, the diameter of bamboo shoot, packet header tight ness rating, plant height
It is stable, consistent.
（7）The asparagus that the present invention cultivates, yield reach more than 1800 kgs/acre, improve 20% or so, to promoting China
The stable development of asparagus production is significant.
Embodiment
With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that to one of ordinary skill in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention
Protect scope.
A kind of method that asparagus zygoid is obtained using Anther Culture of embodiment 1
Major programme is as follows：
1st, asparagus Anther Culture step
The acquisition of 1.1 asparagus flower pesticide
The annual 4-5 mornings in month 8:00-10:00 obtains the male flower inflorescence of 20 plants of different individual plants from field, is marked respectively, reed
Bamboo shoot staminiferous plant inflorescence selects bud of the length for 1-2mm or so after fetching, microscopy shows that now pollen is mid-late uninucleate stage.
The Asparagus Plants kind is ' champion '.
The pretreatment of 1.2 asparagus flower pesticide
Asparagus bud is placed on 4 DEG C of refrigerators and carries out Cold pretreatment 2.5-3.5d, by bud first with 75 % on superclean bench
Alcoholic solution soaks 10s, with aseptic water washing 3 times, 0.2 % mercuric chloride 15 min of molten immersion, with aseptic water washing 6-7 times.
1.3 asparagus induction of anther callus
Flower pesticide is stripped on superclean bench and is inoculated in inducing culture, induction of anther callus culture medium is MS+1.0mg/L
6-BA+1.25mg/L NAA, sucrose concentration are 5.0 %, and agar is 0.7 %, pH 5.8.Every bottle of inoculation outward appearance is unbroken
30 pieces of flower pesticide, blake bottle is placed in after inoculation（26±1）DEG C dark condition under carry out light culture 2 weeks, Ran Hou（26±1）℃、
After intensity of illumination cultivates 20d under conditions of 1000 lx, light application time 16h/d, start callus occur, callus has
Two kinds of forms, one kind is the hard close callus of quality, and another kind is yellow, loose, flocculence callus；Afterwards
Continue operation is using the hard close callus of quality, the inductivity of the hard closely callus of the quality
15.8%。
1.4 asparagus flower pesticide subcultures break up
The bud broken up by callus, which is seeded on differentiation and bud formation culture medium, to be broken up,（26±1）DEG C, intensity of illumination is
Cultivated under conditions of 2000 lx, light application time 16h/d, bud ratio, growth coefficient and growth conditions are determined after 30d.
The differentiation and bud formation culture medium is MS+0.5mg/L 6-BA+0.2mg/L NAA, and the concentration of sucrose is in culture medium
4.0%th, agar 0.7%, pH 5.8.
The bud ratio is 125.4%, and growth coefficient 4.9, sprout is sturdy, and growth is normal.
1.5 Anther Culture rooting of vitro seedling
Using shoot proliferation culture formed normal tender shoots stem apex as material of taking root, root media using MS as minimal medium,
Sucrose 3.5%, agar 0.5%, pH5.8, add NAA, IBA, KT of various concentrations, and the culture medium suitably taken root is：MS+2.0mg/
LNAA+1.0mg/L IBA+0.01mg/L KT, sucrose 3.5%, agar 0.5%, pH5.8, condition of culture are（26±1）DEG C, daily
Illumination 16h, effective rooting rate is up to 72.3 %.
1.6 asparagus Anther Culture regeneration plant Ploidy Identifications
Ploidy Identification is carried out to asparagus Anther Culture regeneration plant using root-tip squashing method.As a result show, have monoploid, two times
Body, tetraploid and aneuploid etc., wherein monoploid differentiation rate are 4.5%, and diploid differentiation rate is 68.5%, aneuploid differentiation
Rate is 18.2%, and tetraploid differentiation rate is 8.8%.
2nd, haploid chromosomes double step
Zygoid and polyploid, minimal medium MS can be obtained using colchicine processing asparagus monoploid test tube seedling
The mg/L of+6-BA0.3 mg/L+NAA 0.1, the % of sucrose 3.0, agar 0.7 %, pH5.8, colchicine concentration are added in culture medium
For 0.05mg/L, after cultivating 15d, switch through and cultivated on the minimal medium for being incubated at and be not added with colchicine, after 15d, it is lured
Success rate is led as 49.73%.
3rd, asparagus new germ plasm sex and Ploidy Identification
Using molecular labeling and cytobiology technology, the identification of sex and ploidy is carried out to the Asparagus Plants of acquisition.Choose institute
The germ plasm resources such as the zygoid that needs, tetraploid, aneuploid, the wherein accounting of zygoid are 1.76%.
The result of the test of remaining the several asparagus kind of embodiment 2
Using the method that asparagus zygoid is obtained using Anther Culture described in embodiment 1, change the kind of asparagus, distinguish
Using Shandong asparagus No.1, Glan moral, green No. 1 of capital.
TableInduction of anther callus rate（%）
Above-mentioned callus is the hard close callus of quality.
TableThe bud ratio and growth coefficient of different cultivars
TableEffective rooting rate of different cultivars（%）
TableDifferent cultivars breaks up monoploid, diploid, tetraploid and the aneuploid situation of green bud（%）
TableThe haploid chromosomes induction success rate of different cultivars（%）
The different cultivars haploid chromosomes of table 6 double result table
Influence of the different flower pesticide acquisition times of embodiment 3 to callus induction rate
Using the method described in embodiment 1, only change the flower pesticide in step " acquisitions of 1.1 asparagus flower pesticide "
Acquisition time and the kind of asparagus, are tested.
Continue to 9-10 months at the Weifang Prefecture asparagus florescence, therefore different months are gathered in the 2010-2015 4-9 months
Flower pesticide, have studied the induction of anther callus rate of different acquisition time, the results showed that the inductivity highest of 4-5 month flower pesticide, 6
Month takes second place, and what is be inoculated with after July is all relatively low.Four strains used in experiment show this rule（Table 7）.
The induction of anther callus rate of the different times of table 7（%）
Above-mentioned callus refers to the hard close callus of quality
Influence of the number of days of the Cold pretreatment of embodiment 4 to Callus Types and inductivity
Using the method described in embodiment 1, change the acquisition time that part is flower pesticide, the acquisition time of flower pesticide is June 8, together
When change time of Cold pretreatment in step " pretreatments of 1.2 asparagus flower pesticide ", carry out analysis experiment.
The Cold pretreatment of different time is carried out before the inoculation of asparagus flower pesticide under the conditions of 4 DEG C, the results showed that, flower pesticide is not by
After the processing of number of days, the callus induction rate significant difference of different disposal time.Handle 3d best results, callus
Inductivity reached peak；5d, 7d are handled, part bud yellow, influences the induction of callus（Table 8）.
Influence of the Cold pretreatment of table 8 to induction of anther callus rate
The screening of the callus differentiation and bud formation condition of embodiment 5
Using the method described in embodiment 1, only change the group of differentiation and bud formation culture medium in step " differentiation of 1.4 asparagus flower pesticide subcultures "
Into if 3 processing, respectively MS+0.25mg/L 6BA+0.1mg/L NAA, MS+0.5mg/L 6BA+0.2mg/L NAA and MS
+ 0.75mg/L 6BA+0.3mg/L NAA, 20 buds of each processing inoculation, are repeated 3 times,（26±1）DEG C, intensity of illumination is
Cultivated under conditions of 2000 lx, light application time 16h/d.The growth coefficient of each processing is investigated after 30d.
The bud broken up by callus, which is seeded on different culture media, can germinate axillary bud, can also pass through base portion incision shape
Break up and sprout into callus.Bud ratio, the growth coefficient of different disposal are shown in Table 9.As can be seen from Table 9, in MS+
Bud ratio and growth coefficient highest on 0.75mg/L 6BA+0.3mg/L NAA culture mediums, although sprout is thick on this culture medium
It is strong, but produce substantial amounts of lopsided bud；Take second place on MS+0.5mg/L 6BA+0.2mg/L NAA culture mediums, on this culture medium
Sprout it is sturdy, growth is normal；And it is minimum on MS+0.25mg/L 6BA+0.1mg/L NAA culture mediums, on this culture medium
Sprout is elongated, and growing way is weak.Final choice MS+0.5mg/L 6BA+0.2mg/L NAA are the culture medium of suitable shoot proliferation.
Influence of the different disposal of table 9 to shoot proliferation
The selection of the root media of embodiment 6
Using the method described in embodiment 1, only change the concentration of IBA in root media, by NAA2.0mg/L and KT0.01mg/
L optium concentration respectively with IBA with 0.1,0.5,1.0,1.5,2.0,2.5, seven concentration such as 3.0mg/L give birth to asparagus test tube seedling
Root induction interacts experiment, and the situation of taking root of test tube seedling is shown in Table 10.As shown in Table 10, rooting of vitro seedling rate is in certain scope
Interior to be raised with the increase of IBA concentration, with highest during 1.0mg/L, effective rooting rate is up to 72.3 %, with other concentration levels
The difference of effective rooting rate reaches the level of signifiance.
The hormon of table 10 is with the influence for comparing rooting of vitro seedling
Being found from table 10, the quality of test tube seedling root is improved when three kinds of auxin is used in combination, and the quantity of lopsided root is reduced,
Effective radical showed increased of every plant of rooting tube plantlet, so as to improve effective rooting rate, finally obtains suitable root media：
MS+2.0mg/LNAA+1.0mg/L IBA+0.01mg/L KT。
The zygoid inductive condition Selection experiment of embodiment 7
Using the method for embodiment 1, during changing chromosome doubling, the concentration and incubation time of colchicine, using culture
Base method, minimal medium are the mg/L of MS+6-BA0.3 mg/L+NAA 0.1, the % of sucrose 3.0, agar 0.7 %, pH5.8.Culture
The concentration that colchicine is added in base is respectively 0.01mg/L, 0.05mg/L, 0.1mg/L.After inoculated and cultured 5d, 10d and 15d,
Switch through and cultivated on the minimal medium for being incubated at and be not added with colchicine, after 15d carry out survival rate investigation stem apex survival rate with
The raising of processing time and colchicine concentration and reduce, and double raising of the rate with processing time and colchicine concentration
And improve, it is optimal with colchicine concentration 0.05 mg/L, 15d culture, it is 49.73% that it, which induces success rate,.
Unless specifically indicated, the ratio that the present invention uses, is mass ratio, the percentage of use, is quality percentage
Than.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., it should be included in the present invention's
Within protection domain.
Claims (10)
- A kind of 1. method that asparagus zygoid is obtained using Anther Culture, it is characterised in that：Comprise the following steps：Asparagus The acquisition of medicine, the pretreatment of asparagus flower pesticide, asparagus induction of anther callus, the differentiation of asparagus flower pesticide subculture, rooting of vitro seedling, list Times Autosome doubles.
- A kind of 2. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： The acquisition of the asparagus flower pesticide, gather the male flower inflorescence of asparagus difference individual plant annual 4-5 months, bud length is 1- in inflorescence 2mm, pollen are mid-late uninucleate stage.
- A kind of 3. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： The asparagus, kind are champion, Shandong asparagus No.1, Glan moral or green No. 1 of capital.
- A kind of 4. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： The pretreatment of the asparagus flower pesticide, asparagus bud is subjected to Cold pretreatment 2.5-3.5d in 3.5-4.5 DEG C of condition.
- A kind of 5. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： The asparagus induction of anther callus, inducing culture are MS+1.0mg/L 6-BA+1.25mg/L NAA, and sucrose concentration is 5.0 %, agar are 0.7 %, pH 5.8.
- A kind of 6. method that asparagus zygoid is obtained using Anther Culture according to claim 5, it is characterised in that： The asparagus induction of anther callus, in（26±1）DEG C dark condition under carry out light culture 2 weeks, Ran Hou（26±1） DEG C, after intensity of illumination cultivates 20d under conditions of 1000lx, the light application time 16h/d, start callus occur.
- A kind of 7. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： Asparagus flower pesticide subculture differentiation, differentiation and bud formation culture medium are MS+0.5mg/L 6-BA+0.2mg/L NAA, sucrose in culture medium Concentration be 4.0%, agar 0.7%, pH 5.8.
- A kind of 8. method that asparagus zygoid is obtained using Anther Culture according to claim 7, it is characterised in that： The asparagus flower pesticide subculture differentiation, at 26 ± 1 DEG C, intensity of illumination is 2000 lx, is cultivated under conditions of light application time 16h/d 30d。
- A kind of 9. method that asparagus zygoid is obtained using Anther Culture according to claim 1, it is characterised in that： The rooting of vitro seedling, root media are：MS+2.0mg/LNAA+1.0mg/L IBA+0.01mg/L KT, contain in culture medium Sucrose 3.5%, agar 0.5%, pH5.8, condition of culture are 25-28 DEG C, daily illumination 16h.
- 10. a kind of method that asparagus zygoid is obtained using Anther Culture according to claim 1, its feature are existed In：The haploid chromosomes are doubled, and asparagus monoploid test tube seedling, minimal medium MS+6- are handled using colchicine The mg/L of BA0.3 mg/L+NAA 0.1, the % of sucrose 3.0, agar 0.7 %, pH5.8, addition colchicine concentration is in culture medium 0.05mg/L, after cultivating 15d, switch through and culture 15d is carried out on the minimal medium for being incubated at and be not added with colchicine.
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| CN110036913A (en) * | 2019-06-03 | 2019-07-23 | 四川省农业科学院经济作物育种栽培研究所 | The method for reducing asparagus Anther Culture pollution rate |
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| CN106718866A (en) * | 2017-03-21 | 2017-05-31 | 河北省农林科学院经济作物研究所 | A kind of asparagus all-male breeding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106718866A (en) * | 2017-03-21 | 2017-05-31 | 河北省农林科学院经济作物研究所 | A kind of asparagus all-male breeding method |
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| 林宗铿: "芦笋花药培养技术研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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| CN110036913A (en) * | 2019-06-03 | 2019-07-23 | 四川省农业科学院经济作物育种栽培研究所 | The method for reducing asparagus Anther Culture pollution rate |
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