CN107435079A - Utilize the method for the wide affine rice material of Anther Culture quick breeding - Google Patents
Utilize the method for the wide affine rice material of Anther Culture quick breeding Download PDFInfo
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Abstract
The invention belongs to biological field, the specific open method using the wide affine rice material of Anther Culture quick breeding.The shortcomings that applicant carries out Anther Culture using river in Zhejiang Province excellent 1540, and callus induction rate is up to 57.8%, and frequency of green plantlet differentiation reaches 43%, and the DH systems of acquisition are easier to overcome Xian round-grained rice to hand over Spikelet fertility low than the DH systems that other materials obtains.The method of the present invention can not only greatly shorten the breeding time limit, improve breeding efficiency, and the wide affine DH systems obtained using the inventive method can make typical long-grained nonglutinous rice, japonica rice and sterile line normal solid.The wide affine DH systems of homozygosis that the present invention obtains can hand over heterosis utilization to formulate new germ plasm resource directly as parent material for Xian round-grained rice.
Description
Technical field
The present invention relates to the method for the wide affine rice material of Anther Culture quick breeding, it is excellent to be mainly used in Indica-Japonica rice hybrid
It is snobbish to use and rice molecular marker-assisted breeding research field.
Background technology
Rice is divided to two Xian, round-grained rice subspecies, and the first generation of hybrid of subspecies has powerful hybrid vigour, between rice indica subspecies
The utilization of hybrid vigour is one of further important channel for improving rice yield.But the general setting percentage of subspecies indica and japonica hybrid is inclined
It is low, grain-filling degree is poor, these problems constrain the utilization of inter-subspecies hybrid rice.Rice wide compatibility gene S 5nXian round-grained rice can be overcome
Dysgenesia, normal fertile offspring can be produced with long-grained nonglutinous rice or the hybridization of japonica rice subspecies.Cultivate and utilize wide affine rice varieties
It is to promote Indica-Japonica rice hybrid advantage, further improve the task of top priority of rice yield.But known wide affine kind is mostly agriculture
The farm variety that skill character is not good enough, it is impossible to used directly as parent；And conventional breeding methods seed selection merit is wide affine
Kind is, it is necessary to hybridize, backcross transformation, time-consuming tediously long breeding cycle length.The men of breeding in recent years be bred as river in Zhejiang Province is excellent 12, Zhejiang is excellent 18,
Combined between the excellent indica and japonica subspecies of a collection of Comprehensive Traits such as spring excellent 84, further to have been established well using Xian round-grained rice Heterosis
Basis.Rice anther culture can be quickly sheerly, improve efficiency of selection, effectively shorten breeding cycle.But rice is spent
The callus induction rate and plantlet differentiation rate of medicine culture are larger by genotype effect, and have not yet to see and above pushed away using production
The excellent series of products in wide indica-japonica hybrid river in Zhejiang Province carries out Anther Culture and obtains homozygous DH (dihaploid) systems and further obtain with wide
Compatibility gene S 5nWide affine rice material report.
The content of the invention
The purpose of the present invention is to be directed to the present situation for lacking wide affine rice varieties in current China's Rice Production, there is provided is utilized
The method of the wide affine rice material of Anther Culture quick breeding, method is quick, effectively.
In order to achieve the above object, the present invention takes following technical measures：
Using the method for the wide affine rice material of Anther Culture quick breeding, comprise the steps：
1st, the acquisition of Anther Culture regeneration plant
(1) pre-process：Fresh paddy rice children's fringe, after 75% alcohol surface sterilization, young fringe is wrapped with preservative film, in 4 DEG C of refrigerators
Pretreatment 7-10 days；
(2) Fiber differentiation：After pretreatment terminates, unnecessary blade and leaf sheath are peelled off, leaves and takes and develops suitable band branch small ear, dress
Enter in sterile beaker, first with 75% alcohol surface sterilization 30s, then with 0.1% mercuric chloride solution soak 10-12min, and use sterilized water
Rinse 3-4 times, each 1-2min, finally blot small ear moisture with aseptic filter paper；On superclean bench at glume base portion 1/3
Filigree is cut, flower pesticide is shaken off to inducing culture, each triangular flask is about inoculated with 80-100 flower pesticide, 25-28 DEG C of light culture；
Described inducing culture is N6+2,4-D 2.0-3.0mg/L+KT 0.5-1.5mg/L+NAA2.0-3.0mg/L+
Sucrose 30-50g/L；
(3) differentiation is cultivated and taken root：Callus diameter is transferred into differential medium when reaching 2mm, 25-28 DEG C,
1000-1500Lx, 10-12h/d illumination cultivation are until be divided into green seedling；, will be green on superclean bench when green seedling grows to 5cm
Seedling, which is transferred on root media, is taken root；
Described differential medium is：MS+KT 2.0-3.0mg/L+6-BA2.0-3.0mg/L+NAA 0.2-0.8mg/L+
IAA0.2-0.8mg/L+ sucrose 30g/L；
The formula of described root media includes：1/2MS+NAA0.5-1.0mg/L+ activated carbon 0.8-1.0g/L+ sucrose
20g/L；
2nd, the acquisition of homozygous DH systems
By the good regeneration plant of root system development, crop field is transplanted after hardening, the plant individual plant in each callus source
Sowing, the seed next year kind that each individual plant obtains are investigated by Agronomic characteristic into strain, it is homozygous to choose Agronomic characteristic
Strain 10-30.With reference to indoor species test, the high yielding ability of the excellent setting percentage of economical character good DH systems 5-15 is chosen.
3rd, the acquisition of wide affine rice material
For wide compatibility gene S 5nPrimer is designed, performing PCR electrophoresis is entered to the DH systems of selection, screening possesses the plant of the gene
Strain, is produced.
In the process described above, it is preferred that the kind of described rice arranges for rice river in Zhejiang Province major clique, and optimal selection is that river in Zhejiang Province is excellent
1540；
In the process described above, it is preferred that the acquisition methods of fresh paddy rice children's fringe are：Monokaryon is taken to lean on the afternoon of fine day
The young fringe of side phase, to ensure that young fringe is fresh, children's fringe stays 2 sections and 2-3 piece blades during sampling, is placed in the bucket for filling clear water.Take
Young fringe micro- Microscopic observation pollen mother cell developmental stage indoors, pollen development be in the young fringe pulvinus of mid-late uninucleate stage away from
8-10cm, now the glume width of young fringe connect maturescent size, glume is presented light green, and stamen elongation is up to the 1/ of glume
3-1/2；The young fringe that field is fetched, is trimmed again under lab, retains the young leaf of fringe 2 and 1 section, 75% alcohol surface sterilization
Afterwards, young fringe is wrapped with preservative film, is placed in 4 DEG C of refrigerators and pre-processes 7-10 days.
In schemes described above, it is preferred that for wide compatibility gene S 5nForward primer (F1) sequence of design be 5 '-
ATCAACCCATTTCCTTTCCT-3 ', reverse primer (R1) sequence are 5 '-ATACGCTCGATCGGATTAAC-3 '；
Further, to there is wide compatibility gene S 5nDH systems, 441bp fragment can be amplified, and do not contain wide affine base
Because of S5nKind, the fragment amplified be 577bp fragment.
Compared with prior art, the positive effect of the present invention is：
1st, the present invention obtains Anther Culture regeneration plant, can quickly obtain homozygosis by Rice anther culture technology
DH Doubled haploid lines.Rice DH systems can be used as breeding material, significant in rice breeding.
2nd, the present invention to excellent DH systems by carrying out wide compatibility gene S 5nPCR detection, it is extensively close can quickly to obtain rice
And material.Extensively affine rice material needs 5-7 to realize to the seed selection of traditional breeding method backcross transformation, and obtained using the present invention
Wide affine rice material can be achieved in the Second Year of Rice anther culture.The present invention can not only greatly shorten the breeding time limit,
Breeding efficiency is improved, and the wide affine DH systems obtained using the inventive method can make typical long-grained nonglutinous rice, japonica rice and sterile line just
It is often solid.The wide affine DH systems of homozygosis that the present invention obtains can hand over heterosis utilization wound directly as parent material for Xian round-grained rice
New germ plasm resource is made.
It is restraining factors that indica-japonica heterosis utilize that 3. the setting percentage of indica-japonica Hybrid progeny is relatively low.Indica-Japonica hybrid rice product
Kind river in Zhejiang Province excellent 1540 passed through in 2015 through the 3rd national the 6th meeting of Crop breed audit committee authorization, authorization numbering
Rice 2015040 is examined for state.The Seed-setting Rate of Varieties is normal, is widely applied at present in production.Excellent 1540 Anther Culture in river in Zhejiang Province
Callus induction and green plant regeneration are fast, and callus induction rate is up to 57.8%, and frequency of green plantlet differentiation reaches 43%.Using solid
Rate is normal, material of the wide variety of kind as Anther Culture in production, and the DH systems of acquisition are than DH that other materials obtains
The shortcomings that system is easier to overcome Xian round-grained rice to hand over Spikelet fertility low.
Embodiment
Technical scheme of the present invention, it is the ordinary skill in the art if not otherwise specified；The reagent or material,
If not otherwise specified, commercial channel is derived from.
Embodiment 1：
Illustrated below so that wide variety of Xian round-grained rice in current production hands over combination river in Zhejiang Province excellent 1540 as an example, other rice
River in Zhejiang Province major clique row can also complete the present invention, reach the effect of high inductivity and differentiation rate.
1st, the acquisition of Anther Culture regeneration plant
(1), rice material is planted：Spring in 2015 is enough to obtain in interval sowing river in Zhejiang Province, Wuhan excellent 1540, field-transplanting
The suitable flower pesticide of developmental stage.Specific interval sowing and transplanting time are shown in Table 1.
The material seedtime of table 1
| The first phase | The second phase | The third phase | The fourth phase | The fifth phase | 6th phase | 7th phase | |
| Sowing | March 27 | April 7 | April 30 | May 10 | May 25 | June 10 | June 25 |
| Transplanting | May 10 | May 15 | May 25 | June 5 | June 18 | July 10 | July 20 |
(2) draw materials：From in July, 2015, the river in Zhejiang Province excellent 1540 of interval sowing sequentially enters boot stage.List is taken the afternoon of fine day
Core keeps to the side the young fringe of phase, and to ensure that young fringe is fresh, children's fringe stays 2 sections and 2-3 piece blades during sampling, is placed in the bucket for filling clear water.
Micro- Microscopic observation pollen mother cell developmental stage, excellent 1540 Varieties in Pollen Morphology in the river in Zhejiang Province that makes discovery from observation are female indoors for the young fringe taken
Cell development is in the young fringe pulvinus of mid-late uninucleate stage away from 8-10cm, and now the glume width of young fringe has connect maturescent big
Small, light green, 1/3-1/2 of the stamen elongation up to glume is presented in glume.
(3) pre-process：The young fringe that field is fetched, is trimmed again under lab, retains the young leaf of fringe 2 and 1 section, 75% (body
Product ratio) alcohol surface sterilization after, wrap young fringe with preservative film, be placed in 4 DEG C of refrigerators and pre-process 7 days.
(4) Fiber differentiation：After pretreatment terminates, unnecessary blade and leaf sheath are peelled off, leaves and takes and develops suitable band branch small ear, dress
Enter in sterile beaker, first with 75% alcohol surface sterilization 30s, then with 0.1% (mass ratio) mercuric chloride solution soak 10min, it is sterile
Water rinses 3-4 times, each 1min, finally blots small ear moisture with aseptic filter paper.On superclean bench at glume base portion 1/3
Filigree is cut, flower pesticide is shaken off to inducing culture, each triangular flask is inoculated with 80-100 flower pesticide, 28 DEG C of light cultures 20 days three
Start to induce callus in the bottle of angle, callus diameter is up to 2mm within 25 days.Callus induction rate is 57.8%.(5) divide
Change culture and take root：The callus that diameter is reached to 2mm is transferred to differential medium on superclean bench, 28 DEG C,
1500Lx, 12h/d illumination cultivation, begin with 7 days green bud point differentiation, plantlet differentiation rate 43%.Differentiation 20 days green seedlings of culture
When growing to 5cm, green seedling is transferred on root media on superclean bench and taken root, green seedling can grow prosperity within 15 days
Root system.
Described inducing culture is：N6+2,4-D 2.5mg/L+KT1.0mg/L+NAA2.5mg/L+ sucrose 50g/L；Point
Changing culture medium is：MS+KT2mg/L+6-BA2mg/L+NAA0.5mg/L+IAA0.5mg/L+ sucrose 30g/L；Root media is：
1/2MS+NAA0.5mg/L+ activated carbon 0.8g/L+ sucrose 20g/L.
2nd, the acquisition of homozygous DH systems
Winter in 2015 takes the good regeneration plant of root system development to Hainan Datian transplanting, each callus after hardening
Tissue-derived plant individual plant sowing.The seed kind in 2016 that each individual plant obtains is investigated by Agronomic characteristic into strain,
Choose Agronomic characteristic homozygous lines 25.With reference to indoor species test, it is good to choose the high yielding ability of the excellent setting percentage of economical character
12, DH systems, DH systems numbering and indoor species test are shown in Table 2.
Table 2：The excellent indoor species test table of DH systems
3rd, the acquisition of wide affine rice material is excellent to above-mentioned 12 economical characters, and the good excellent homozygous DH systems of yielding ability enter
Row wide compatibility gene S 5nPCR detection.Forward primer (F1) sequence of the genetic test be 5 '-
ATCAACCCATTTCCTTTCCT-3 ', reverse primer (R1)) sequence is 5 '-ATACGCTCGATCGGATTAAC-3 ', it is wide to having
Compatibility gene S 5nDH systems, 441bp fragment can be amplified, and do not contain wide compatibility gene S 5nKind, the fragment amplified
For 577bp fragment.
The PCR amplification system is：10 × contain Mg2+PCR buffer solutions 1.5 μ l, 2.5mmol.L-1dNTP 1.2 μ l, Taq
Archaeal dna polymerase 1U, DH systems genomic DNA template 1 μ l, 10 μm of ol.L-1 μ l of forward primer F1 0.2,10 μm of ol.L-1 it is anti-
To primer R1 0.2 μ l, ddH2O supplies the μ l of volume 15.
The PCR reaction conditions are：94 DEG C of pre-degenerations 4min, 94 DEG C of denaturation 40s, 50 DEG C of renaturation 30s, 72 DEG C of extension 30s,
Totally 30 circulations；Then 72 DEG C of extension 5min.
The method of the detection amplified production banding pattern is to carry out 1.0% agarose gel electrophoresis, then ultraviolet gel imaging
System takes pictures.
It is 441bp fragments so to filter out amplified fragments, and economical character is excellent, and 5 good DH systems of yielding ability, are us
The wide affine rice material that quick breeding goes out.
4th, wide compatibility identifies that the winter in 2016 chooses 3 and contains wide compatibility gene S 5nDH systems 2016DH689,
2016DH751,2016DH915 respectively with long-grained nonglutinous rice China Resources 2, japonica rice Hubei Province is fragrant No. 2, long-grained nonglutinous rice R007, sterile line 144S, sterile line
Y58S hybridizes, and prepares 15 cross combinations altogether.Spring in 2017 plants 15 cross combination F in Wuhan1, each combined when ripe
Random to take 5 plants, every plant takes 3 fringes to carry out species test to setting percentage, and counts the average of setting percentage.Species test the results are shown in Table 3, by table 3
It can be seen that contain wide compatibility gene S 5nDH systems 2016DH689,2016DH751,2016DH915 can make long-grained nonglutinous rice, japonica rice and not
It is normal solid to educate.
Sequence table
<110>Grain Crop Institute of Hubei Academy of Agricultural Sciences
<120>Utilize the method for the wide affine rice material of Anther Culture quick breeding
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atcaacccat ttcctttcct 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atacgctcga tcggattaac 20
Claims (5)
1. using the method for the wide affine rice material of Anther Culture quick breeding, comprise the steps：
1.1st, the acquisition of Anther Culture regeneration plant
（1）Pretreatment：Fresh paddy rice children's fringe, after 75% alcohol surface sterilization, young fringe is wrapped with preservative film, is pre-processed in 4 DEG C of refrigerators
7-10 days；
（2）Fiber differentiation：After pretreatment terminates, unnecessary blade and leaf sheath are peelled off, leaves and takes and develops suitable band branch small ear, loads nothing
In bacterium beaker, first with 75% alcohol surface sterilization 30s, then with 0.1% mercuric chloride solution 10-12 min are soaked, and use aseptic water washing
3-4 times, each 1-2min, finally blot small ear moisture with aseptic filter paper；Cut on superclean bench at glume base portion 1/3
Filigree, flower pesticide is shaken off to inducing culture, each triangular flask is inoculated with 80-100 flower pesticide, 25-28 DEG C of light culture；
Described inducing culture is N6+2,4-D 2.0-3.0mg/L+KT 0.5-1.5mg/L+NAA2.0-3.0 mg/L+
Sucrose 30-50g/L；
（3）Differentiation is cultivated and taken root：Callus diameter is transferred into differential medium, 25-28 DEG C, 1000- when reaching 2mm
1500Lx, 10-12h/d illumination cultivation are until be divided into green seedling；When green seedling grows to 5cm, green seedling is turned on superclean bench
Move on on root media and taken root；
Described differential medium is：MS+KT 2.0-3.0 mg/L +6-BA2.0-3.0mg/L +NAA 0.2-0.8 mg/L
The g/L of+IAA0.2-0.8mg/L+sucrose 30；
Described root media is：The g/L of 1/2MS+NAA0.5-1.0 mg/L+ activated carbon 0.8-1.0 g/L+ sucrose 20；
1.2nd, the acquisition of homozygous DH systems
By the good regeneration plant of root system development, crop field is transplanted after hardening, and the plant individual plant in each callus source is received
Kind, the seed next year kind that each individual plant obtains is investigated by Agronomic characteristic into strain, chooses Agronomic characteristic homozygous strain
It is 10-30；With reference to indoor species test, the high yielding ability of the excellent setting percentage of economical character good DH systems 5-15 is chosen；
1.3rd, the acquisition of wide affine rice material
For wide compatibility gene S 5nPrimer is designed, performing PCR detection is entered to the DH systems of selection, screening possesses the plant of the gene, i.e.,
.
2. according to the method for claim 1, the kind of described rice is rice river in Zhejiang Province major clique row indica-japonica hybrid rice varieties.
3. according to the method for claim 1, the acquisition methods of described fresh paddy rice children's fringe are：List is taken the afternoon of fine day
Core keeps to the side the young fringe of phase, and to ensure that young fringe is fresh, children's fringe stays 2 sections and 2-3 piece blades during sampling, is placed in the bucket for filling clear water；
Micro- Microscopic observation pollen mother cell developmental stage, pollen development are in the children of mid-late uninucleate stage to the young fringe taken indoors
Fringe pulvinus has connect maturescent size away from 8-10cm, now the glume width of young fringe, and light green is presented in glume, and stamen elongation reaches grain husk
The 1/3-1/2 of shell；The young fringe that field is fetched, is trimmed again under lab, retains the young leaf of fringe 2 and 1 section, 75% alcohol surface
After sterilization, young fringe is wrapped with preservative film, is placed in 4 DEG C of refrigerators and pre-processes 7-10 days.
4. according to the method for claim 1, for wide compatibility gene S 5nThe forward primer sequence of design be 5 '-
ATCAACCCATTTCCTTTCCT-3 ',-ATACGCTCGATCGGATTAAC -3 ' of reverse primer sequences 5 '.
5. according to the method for claim 1, the kind of described rice is river in Zhejiang Province excellent 1540.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108207617A (en) * | 2018-01-25 | 2018-06-29 | 湖北省农业科学院粮食作物研究所 | A kind of quick method using Indica-Japonica rice hybrid advantage |
| CN108651281A (en) * | 2018-04-17 | 2018-10-16 | 湖北省农业科学院粮食作物研究所 | A kind of flower training rice young panicle long-distance transport method |
| CN108739400A (en) * | 2018-06-22 | 2018-11-06 | 安徽袁粮水稻产业有限公司 | A kind of efficient anther culture of indica rice differential medium |
| CN110754365A (en) * | 2019-12-04 | 2020-02-07 | 河北省农林科学院滨海农业研究所 | Method for strengthening and controlling seedlings in rice anther culture process |
| CN111567400A (en) * | 2020-06-29 | 2020-08-25 | 湖北省农业科学院粮食作物研究所 | Method for efficiently breeding high-yield high-quality fragrant long-grain japonica rice |
| CN112385545A (en) * | 2020-12-04 | 2021-02-23 | 黑龙江省农业科学院绥化分院 | Culture method of rice anther callus |
| CN114711108A (en) * | 2022-04-01 | 2022-07-08 | 上海市农业科学院 | Method for rapidly determining development period of rice microspore |
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| CN110754365A (en) * | 2019-12-04 | 2020-02-07 | 河北省农林科学院滨海农业研究所 | Method for strengthening and controlling seedlings in rice anther culture process |
| CN111567400A (en) * | 2020-06-29 | 2020-08-25 | 湖北省农业科学院粮食作物研究所 | Method for efficiently breeding high-yield high-quality fragrant long-grain japonica rice |
| CN112385545A (en) * | 2020-12-04 | 2021-02-23 | 黑龙江省农业科学院绥化分院 | Culture method of rice anther callus |
| CN114711108A (en) * | 2022-04-01 | 2022-07-08 | 上海市农业科学院 | Method for rapidly determining development period of rice microspore |
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