CN106718895A - A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm - Google Patents
A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm Download PDFInfo
- Publication number
- CN106718895A CN106718895A CN201611111371.2A CN201611111371A CN106718895A CN 106718895 A CN106718895 A CN 106718895A CN 201611111371 A CN201611111371 A CN 201611111371A CN 106718895 A CN106718895 A CN 106718895A
- Authority
- CN
- China
- Prior art keywords
- culture
- anther
- processing type
- hardening
- pepper
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- 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
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm, including：Processing type capsicum donor parents plant is under 26.4 ± 0.4 DEG C of temperature conditionss；Sporidiole of the selection in mid-late uninucleate stage occurs Anther Culture for inducing embryoid body；Bud was taken before 9 points of every morning, the 3d of Cold pretreatment 1 in 4 DEG C of refrigerators is placed in；It is alcohol-pickled under aseptic condition, then to sterilize, flower pesticide is taken out and is inoculated on inducing culture by aseptic water washing with tweezers from bud；Culture dish is inoculated with 8 buds；It is dispersed on inducing culture after aseptically taking out flower pesticide from bud；The embryoid that will be induced is gone on MS culture mediums, is transplanted when root is up to 4 5cm, needs to carry out hardening before transplanting.The present invention is easy to more effectively launch Anther Culture work, grasps materials bud morphological index, reduces operation link, it is ensured that Anther Culture induced efficiency.
Description
Technical field
The invention belongs to anther culture technique field, more particularly to a kind of quick initiative processing type hot pepper male sterile fertility
The method for recovering new germ plasm.
Background technology
Current Anther Culture has turned into the major technique of pepper breeding.Recent two decades are excellent peppery to be bred as some
Green pepper flower training kind, such as seaflower No. three (Li Chunling, 1990), Haifeng county 12 (punishment forever duckweed etc., 2002), (the punishment duckweed forever of Haifeng county 26
Deng 2003), Haifeng county 14 (Zhang Shugen etc., 2003), Haifeng county 25 (Zhang Shugen etc., 2008), Haifeng county 16 (punishment duckweed etc. forever,
2014), Haifeng county 1052 (Zhang Shugen etc., 2015) and Haifeng county 391 (Zhang Shugen etc., 2015) etc..By anther culture technique 1~2
The dliploid material of homozygosis is obtained by year, breeding process can be accelerated, shorten the time limit (Chen Xiao etc., 2003 needed for breeding；Yuan Li
Deng 2011).The accuracy of molecular marker assisted selection (molecular assisted selection, MAS) is high, by peppery
The influence of green pepper breeding time etc., is widely applied in pepper breeding.In recent years, researcher utilizes MAS and Anther Culture
The method that technology is combined is prepared for some good breeding materials (Wang Xingchun etc., 2004 in paddy rice；Yu Fengchi etc., 2010；Yuan
It is beautiful etc., 2011；Song Dingding, 2011).Processing type capsicum CMS Elite restorer lines are less in breeding practice, traditional breeding method seed selection processing
Type capsicum CMS restorers are time-consuming to take a lot of work.Therefore the method being combined using Anther Culture and MAS quickly formulates the peppery CMS of processing type
Fertility restorer material turns into breeder needs matter of utmost importance urgently to be resolved hurrily, but the method to be formulated in peppers specially for processing
In not yet relevant report.
In sum, processing type capsicum CMS Elite restorer lines are less in breeding practice, traditional breeding method seed selection processing type capsicum
CMS restorers are time-consuming to take a lot of work.
The content of the invention
It is an object of the invention to provide a kind of side of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm
Method, it is intended to which processing type capsicum CMS Elite restorer lines are less in solving breeding practice, CMS is extensive for traditional breeding method seed selection processing type capsicum
It is again the time-consuming problem taken a lot of work.
The present invention is achieved in that a kind of method of processing type pepper anther culture, the processing type pepper anther
Cultural method is comprised the following steps：
Step one, pepper plant carries out pepper anther culture under 26.4 ± 0.4 DEG C of temperature conditionss；
Step 2, culture sporidiole of the selection in mid-late uninucleate stage occurs capsicum monoploid for inducing embryoid body；
Step 3, bud Cold pretreatment 1-3d in 4 DEG C of refrigerators；Afterwards aseptically will be for examination bud 70%
7min is soaked in 0.1% mercuric chloride again after 30s is soaked in alcohol, with aseptic water washing 3-4 times, is inoculated into MS+BA 0.5mg/L+2,
4-D 0.5mg/L+3% sucrose+agar 7g/L+AgNO3On the culture medium of 4mg/L+ activated carbons 0.4%, pH 5.8；Inoculum density
For diameter 100mm culture dishes are inoculated with 8 buds；
Step 4, inoculation flower pesticide is placed in 35 DEG C of constant incubators to the flower pesticide that will be inoculated with and processes 7d；Then on daytime
28 DEG C of temperature, night temperatures are to continue to cultivate in 20 DEG C of tissue culture room；
Step 5, selects the eugonic plant of the tip of a root, and the root 5-6 bars less than 2cm are taken from every plant, after distilled water rinsing,
3.5h is processed in saturation paracide solution, Ka Nuoshi fixers fix 2h, distilled water flushing 3-4 times, 0.2M HCl treatments
5min, distilled water flushing 3-4 times, 45% glacial acetic acid solution treatment 5min, the precious dyeing 20min of card, film-making, basis of microscopic observation,
Find and be in metaphase in cell division, the cell of Chromosome spread is counted；
Step 6, embryoid in the medium seedling when about 4-5cm is high, it is necessary to transplanted, need to cultivate flower before transplanting
Strain test tube seedling carries out hardening.
Further, the inducing culture is：MS+BA 0.5mg/L+2,4-D 0.5mg/L+3% sucrose+7g/L agar+
AgNO3On the culture medium of 4mg/L+ activated carbons 0.4%, pH5.8；Root media is：MS+0.2mg/L NAA+ sucrose 3%+ fine jades
On the culture medium of fat 0.7%, pH5.8.
Further, the hardening includes：
(1) a bottle high light hardening is closed：The blake bottle of Anther-culture test tube seedling is moved on into outdoor carry out high light and close a bottle hardening 7d；
(2) high light hardening is opened：Blake bottle sealed membrane is opened, hardening 5-7d is opened under natural light；
(3) transplanting of Anther-culture test tube seedling：Test tube seedling is removed with tweezers are careful from culture medium, wash clean root
Afterwards in immigration matrix；Cover plastic sheeting and keep humidity；Anther-culture first moves to illumination under natural light, then opens adaptation humidity；
(4) open country condition hardening：Seedling is grown under the conditions of can be moved to after young leaves, new root open country, suitable before being colonized
Answering property is taken exercise；When growing 2-3 piece young leaves, you can field planting.
Further, the matrix of the transplanting is humus according to volume ratio：Vermiculite=4:1.
Another object of the present invention is to provide one kind capsicum list is carried out using the processing type pepper anther culture method
Times body culture.
It is a kind of using processing type pepper anther culture technology initiative processing type another object of the present invention is to provide
Capsicum CMS restorer materials.
Further, the construction method of the processing type capsicum cytoplasmic male sterility restorer is comprised the following steps：
(1)F2Colony is formulated by restorer 812 and the hybridization of Parents 940；With molecular marker assisted selection in F2Group
The individual plant containing Restore gene Rf is screened in body carries out Anther Culture；
(2) using the CRF-SCAR label screenings F for screening2The individual plant containing Restore gene carries out Anther Culture in colony,
And then with the Marker Identification spend training regeneration strain DH systems whether containing Restore gene Rf, CRF-SCAR upstream primer sequence be：5′-
GTACACACCACTCGTCGCTCCT-3 ', downstream primer sequence is：5′-TTCTTGGGTCCCTTTCTTCCAA-3′；
(3) Pepper Leaves DNA is extracted using CTAB methods；Containing 5 × PCR buffer solutions 4 μ L, 25mmol/LMgCl2Solution
The μ L of 1.2 μ L, dNTP solution 1.6, each 2.0 μ L of 33ng/ μ L upstream and downstream primers, 2U/ μ LTaq polymerases 0.5 μ L, ddH2The μ L of O 7 and
The μ L of template DNA 1.0；
(4) 0.1%HgCL is used afterwards with 70% alcohol-pickled 30s on the table2Sterilization 7min, rinsed with sterile water 4
It is secondary, then it is placed on aseptic filter paper and drains the water；Aseptically it is inoculated in embryoid induction culture medium, 9mm × 9mm culture dishes
6 flower pesticide of bud are connect, 37 DEG C of high temperature pre-process 5d.
Further, the embryoid induction culture medium is MS+BA 0.5mg/L+2,4-D 0.5mg/L+3% sucrose+agar
7g/L+AgNO34mg/L+ activated carbons 0.4%.
The method of the quick initiative processing type hot pepper male sterile fertility restorer new germ plasm that the present invention is provided, formulates processing type
Capsicum (CMS) restorer material, Restore gene donor processing type capsicum restorer 812 and Chongqing City complementary using advantage proterties
The Elite inbred 940 of academy of agricultural sciences's seed selection in recent years makees parent, to its F2Carry out Anther Culture；By molecular marker assisted selection pair
Hua Pei regeneration strain carries out the screening of Restore gene Rf, is quickly introduced Rf using Anther Culture and molecular marker assisted selection
In self-mating system 940, acceleration prepares processing type capsicum CMS fertility restorer resources；It is easy to capsicum researcher more effectively to launch
Pepper anther culture works, and grasps materials bud morphological index, reduces operation link, it is ensured that Anther Culture Efficiency.The present invention is
Processing type capsicum cytoplasmic male sterilty (CMS) restorer material quickly is prepared, using molecular marker assisted selection and Anther Culture
The Restore gene Rf of the restorer 812 that the method being combined will be recovered completely imports self-mating system 940, prepares new containing Restore gene
Germplasm；In 5 flower two generations seedling system (DH) of training to wherein containing Rf, carry out test cross with sterile line 83-3A, as a result show, tester strain is equal
Performance recovers, and the recovery strain rate of tester strain is 100%.The present invention combines Anther Culture and molecular mark can
To reach the purpose of quick efficient breeding；Quickly to prepare processing type capsicum CMS restorer materials, aid in selecting using molecular labeling
The Restore gene Rf for selecting the restorer 812 that the method being combined with Anther Culture will be recovered completely imports self-mating system 940, and preparation contains
The new germ plasm of Restore gene；In 5 flower two generations seedling system (DH) of training to wherein containing Rf, carry out test cross with sterile line 83-3A, as a result table
Bright, tester strain shows recovery, and the recovery strain rate of tester strain is 100%.The present invention explanation combine Anther Culture and
Molecular mark can reach the purpose that excellent breeding resources are fast and effeciently formulated for breeding practice.
Brief description of the drawings
Fig. 1 is processing type pepper anther culture method flow diagram provided in an embodiment of the present invention.
Fig. 2 is processing type capsicum F provided in an embodiment of the present invention2The Markers for Detection schematic diagram of colony；
In figure:M:DL5000；1:Restorer 812；2:Sterile line 83-3A；3~18:Processing type capsicum F2 individual plants.
Fig. 3 is the amplification schematic diagram during CRF-SCAR provided in an embodiment of the present invention is marked at processing type capsicum DH systems；
In figure:M:DM2000；1:Sterile line 83-3A；2:Restorer 812；3~8:DH systems number.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail below in conjunction with the accompanying drawings.
As shown in figure 1, the cultural method of processing type pepper anther provided in an embodiment of the present invention is comprised the following steps：
S101：Pepper plant carries out pepper anther culture under 26.4 ± 0.4 DEG C of temperature conditionss；
S102：Culture sporidiole of the selection in mid-late uninucleate stage occurs capsicum monoploid for inducing embryoid body；
S103：Bud Cold pretreatment 1-3d in 4 DEG C of refrigerators；Afterwards aseptically will be for examination bud in 70% wine
7min is soaked in 0.1% mercuric chloride again after 30s is soaked in essence, with aseptic water washing 3-4 times, is inoculated into MS+BA0.5mg/L+2,4-D
0.5mg/L+3% sucrose+agar 7g/L+AgNO34mg/L+ activated carbons 0.4%, pH5.8；Inoculum density is cultivated for diameter 100mm
Ware is inoculated with 8 buds；
S104：Inoculation flower pesticide is placed in 35 DEG C of constant incubators to the flower pesticide that will be inoculated with and processes 7d；Then in temperature on daytime
28 DEG C of degree, night temperatures are to continue to cultivate in 20 DEG C of tissue culture room；
S105, selects the eugonic plant of the tip of a root, and the root 5-6 bars less than 2cm are taken from every plant, after distilled water rinsing, satisfies
With process 3.5h in paracide solution, Ka Nuoshi fixers fix 2h, distilled water flushing 3-4 times, 0.2M HCl treatment 5min,
Distilled water flushing 3-4 times, 45% glacial acetic acid solution treatment 5min, the precious dyeing 20min of card, film-making, basis of microscopic observation, at searching
In metaphase in cell division, the cell of Chromosome spread is counted；
S106：Embryoid in the medium seedling when about 4-5cm is high, it is necessary to transplanted, needed to Anther-culture before transplanting
Test tube seedling carries out hardening.
In step s 106：
Hardening can be divided into 4 Walk to be carried out：(1) a bottle high light hardening is closed：The blake bottle of Anther-culture test tube seedling is moved on into outdoor to enter
Row high light closes a bottle hardening 7d；(2) high light hardening is opened：Blake bottle sealed membrane is opened, hardening 5-7d is opened under natural light.
(3) transplanting of Anther-culture test tube seedling：Test tube seedling is removed with tweezers are careful from culture medium, and wash clean moves into base behind root
In matter.Cover plastic sheeting and keep humidity.Anther-culture first moves to next section of temporal adaptation illumination of natural light, then to open adaptation wet
Degree, can make seedlings root flourishing, and transplanting survival rate is up to more than 90%.(4) open country condition hardening：After seedling grows young leaves, new root
Under the conditions of can be moved to open country, the adaptive training before being colonized.When growing 2-3 piece young leaves, you can field planting.
Transplanting medium is humus：Vermiculite=4:1 (volume ratio), first next section of natural light is moved to before transplanting by Anther-culture
Temporal adaptation illumination, then open lid adaptation humidity, it is to avoid occur wilting phenomenon during transplanting, it is progressively grown adaptation certainly
The epidermal structure of right environment, young leaves and new root.Culture medium is cleaned in warm water, after moving into matrix, root water is poured.
Application effect of the invention is explained in detail with reference to specific embodiment.
Embodiment 1
1 result and analysis
The screening of 1.1 Anther Culture donor plants
With processing type capsicum restorer 812 as male parent, Elite inbred 940 is female parent, using molecular marker assisted selection
F is built with hybridization technique2Colony, then screens the individual plant containing Restore gene Rf with molecular labeling CRF-SCAR, and this experiment is altogether
16 individual plants are filtered out containing Rf, with its as Anther Culture material to be tested (Fig. 2).
The acquisition and identification of 1.2 flower training regeneration plants
The 5-7 months in 2014 are in key lab of academy of agricultural sciences of Chongqing City biotech research center adverse circumstance agricultural research Chongqing City
16 individual plants containing Restore gene Rf are inoculated with 130 wares altogether, about 3200 pieces of flower pesticide obtain embryoid 34, inductivity 1.06%.
Wherein 21 plants of seedling, planting percent is 61.76%, but after doubling then only 6 parts receive seed, fix tentatively entitled DH-1, DH-2,
DH-3, DH-4, DH-5, DH-6, next year tie up to academy of agricultural sciences of Chongqing City base and plant according to seedling.
Marked with CRF-SCAR and 6 DH systems plant genomic DNAs are entered with performing PCR amplification identification, except DH-1 is not amplified
Specific band, shows as band missing, and without Restore gene Rf, it is 870bp's that remaining 5 DH system can amplify stripe size
Specific band, (Fig. 3) consistent with the banding pattern of restorer 812.
1.3 flower training regeneration plants are to the restorative of 83-3A
Make maternal with 83-3A sterile lines within 2015, male parent is made with 6 DH systems, each combined flower of test cross trains the recovery of DH systems
Property.Summer in 2016 plants resulting test cross generation seed kind in academy of agricultural sciences of Chongqing City base, investigates its pollen fertility (table
1).Except 83-3A × DH-1 cross combination tester strains do not recover strain, the anther culture descendant tester strain of remaining cross combination
Having can recover strain, and the recovery strain rate of tester strain is 100% (table 1).It is consistent with molecular marker assisted selection result.
The test cross anther culture descendant of table 1 is to the restorative of 83-3A
1.4 3 Elite restorer lines for utilizing anther culture technique seed selection
5 Anther-culture DH systems in base are planted, strain variable rate technology economical character is consistent, has shown this 5 parts of materials
Homozygosis stabilization.Species test (table 2) is sampled after carrying out Agronomic characteristic investigation and maturation.Comprehensive Agronomic characteristic and molecular labeling
Assisted Selection, selects 3 good DH systems restorers of economical character, is the restorer recovered completely.Wherein DH-3 plant
Plant type it is compact, fruit surface smooth, mid-early maturity；DH-5 fruits are straight, fruit surface smooth, resistance；DH-6 plant hang many, fruit surface smooth,
It is more precocious.
The economical character trait expression of 25 restorers of table
2 at past more than 20 years, and Anther Culture obtains relatively broad application in pepper breeding.Use Anther Culture skill
Art prepares processing type capsicum Elite restorer line, can select restorer in 2~3 years, the 5-7 of the conventional method that compares,
Save human and material resources and financial resources that screening restorer needs.This and the result in K Type Wheat male sterile restoring line seed selection
Unanimously.Anther Culture donor parents are all selection F in major part research1Generation.Through Anther Culture obtain regrowth system difference compared with
Greatly, this is not inconsistent with the breeding objective for needing to cultivate proterties stabilization.
In this experiment, the F for building flower training donor parents2Colony restorer parent 812 is the recovery for recovering completely
System, and donor parents F is trained to flower in advance with the method for CRF-SCAR molecular marker assisted selections2Colony carries out Restore gene sieve
Choosing, flower training donor parents used are the test cross offspring of the plant containing Rf, molecular marker assisted selection and 6 DH systems and 83-3A
Fertility shows that it is restorer that 6 DH systems of seed selection have chosen 5 DH systems.With reference to Agronomic characteristic, it is believed that selected 3
Individual DH systems COMPARISON OF CHARACTERS is excellent, can be prepared for Combination nova as breeding material.It can be seen that, by molecular marker assisted selection
With the method that Anther Culture is combined, new processing type capsicum restorer material can be efficiently prepared.
Therefore, it is considered herein that the method being combined with Anther Culture using molecular marker assisted selection, can quickly be sieved
Genes of interest is selected, the seedling system of seed selection can be made to more conform to breeding objective again.Breeder can be special excellent as preparing
One important means of different breeding material.
3 materials and methods
3.1 materials and design
F used2Colony is formulated by restorer 812 and the hybridization of Parents 940.812 is by Agricultural in Chongqing science
The Elite restorer line that institute vegetable or flower research institute capsicum room prepares for many years, fruit shape is longhorn shape；940 be room of the present invention for many years
The Elite inbred of seed selection, fruit shape is thin cattle horn shape.With molecular marker assisted selection in F2Screening contains Restore gene in colony
The individual plant of Rf carries out Anther Culture.
3.2 molecular labelings
Using the CRF-SCAR label screenings F for having screened2The individual plant containing Restore gene carries out Anther Culture in colony, enters
And spend training regeneration strain DH systems whether to contain Restore gene Rf with the Marker Identification, CRF-SCAR upstream primer sequences are：5′-
GTACACACCACTCGTCGCTCCT-3 ', downstream primer sequence is：5′-TTCTTGGGTCCCTTTCTTCCAA-3′.
3.3 Markers for Detection
In the present invention, Pepper Leaves DNA is extracted using CTAB methods.Containing 5 × PCR buffer solutions 4 μ L, 25mmol/
LMgCl2(ancient cooking vessel state gives birth to for the μ L of 1.2 μ L, dNTP solution of solution 1.6, each 2.0 μ L of 33ng/ μ L upstream and downstream primers, 2U/ μ LTaq polymerases
Thing) 0.5 μ L, ddH2The μ L of the O 7 and μ L of template DNA 1.0.
3.4 anther culture methods
Spring in 2014 is taken in monokaryon before Chongqing Agricultural Academy of Science proving ground, at 9 points in the morning on target individual plant
The keep to the side bud of phase is placed in ice chest, takes back laboratory.With 70% alcohol-pickled 30s on superclean bench, afterwards with 0.1%
HgCL2Then sterilization 7min, rinsed with sterile water 4 times is placed on aseptic filter paper and drains the water.Aseptically it is inoculated in embryoid
Inducing culture (MS+BA 0.5mg/L+2,4-D 0.5mg/L+3% sucrose+agar 7g/L+AgNO34mg/L+ activated carbons
0.4%), 9mm × 9mm culture dishes connect 6 flower pesticide of bud, and 37 DEG C of high temperature pre-process 5d.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (8)
1. a kind of cultural method of processing type pepper anther, it is characterised in that the cultural method bag of the processing type pepper anther
Include following steps：
Step one, pepper plant carries out pepper anther culture under 26.4 ± 0.4 DEG C of temperature conditionss；
Step 2, culture sporidiole of the selection in mid-late uninucleate stage occurs capsicum monoploid for inducing embryoid body；
Step 3, bud Cold pretreatment 1-3d in 4 DEG C of refrigerators；Afterwards aseptically will be for examination bud in 70% alcohol
7min is soaked in 0.1% mercuric chloride again after middle immersion 30s, with aseptic water washing 3-4 times, is inoculated into MS+BA 0.5mg/L+2,4-D
0.5mg/L+3% sucrose+agar 7g/L+AgNO3On the culture medium of 4mg/L+ activated carbons 0.4%, pH5.8；Inoculum density is straight
Footpath 100mm culture dishes are inoculated with 8 buds；
Step 4, will be inoculated with the culture dish of flower pesticide and has been placed in 35 DEG C of constant incubators and processed 7d；Then in 28 DEG C of day temperature,
Night temperatures are to continue to cultivate in 20 DEG C of tissue culture room；
Step 5, selects the eugonic plant of the tip of a root, and the root 5-6 bars less than 2cm are taken from every plant, after distilled water rinsing, saturation
3.5h is processed in paracide solution, Ka Nuoshi fixers fix 2h, and distilled water flushing 3-4 times, 0.2M HCl treatment 5min steam
Distilled water is rinsed 3-4 times, and 45% glacial acetic acid solution treatment 5min, the precious dyeing 20min of card, film-making, basis of microscopic observation, searching is in
Metaphase in cell division, the cell of Chromosome spread is counted；
Step 6, embryoid in the medium seedling to 4-5cm it is high when, it is necessary to transplanted, need to try Anther-culture before transplanting
Guan Miao carries out hardening.
2. the cultural method of processing type pepper anther as claimed in claim 1, it is characterised in that the inducing culture is：
MS+BA 0.5mg/L+2,4-D 0.5mg/L+3% sucrose+agar 7g/L+AgNO3The culture medium of 4mg/L+ activated carbons 0.4%
On, pH 5.8；Root media is：On the culture medium of MS+NAA 0.2mg/L+ sucrose 3%+ agar 0.7%, pH 5.8.
3. the cultural method of processing type pepper anther as claimed in claim 1, it is characterised in that the hardening includes：
(1) a bottle high light hardening is closed：The blake bottle of Anther-culture test tube seedling is moved on into outdoor carry out high light and close a bottle hardening 7d；
(2) high light hardening is opened：Blake bottle sealed membrane is opened, hardening 5-7d is opened under natural light；
(3) transplanting of Anther-culture test tube seedling：Test tube seedling is removed with tweezers are careful from culture medium, is moved behind wash clean root
In entering matrix；Cover plastic sheeting and keep humidity；Anther-culture first moves to illumination under natural light, then opens adaptation humidity；
(4) open country condition hardening：Seedling is grown under the conditions of can be moved to after young leaves, new root open country, the adaptability before being colonized
Take exercise；When growing 2-3 piece young leaves, you can field planting.
4. the cultural method of processing type pepper anther as claimed in claim 1, it is characterised in that the matrix of the transplanting according to
Volume ratio is humus：Vermiculite=4:1.
5. the method for processing type pepper anther culture described in a kind of utilization claim 1-4 any one.
6. a kind of processing type capsicum cytoplasmic male sterility of processing type pepper anther culture described in utilization claim 5 is recovered
System.
7. the construction method of processing type capsicum cytoplasmic male sterility restorer as claimed in claim 6, it is characterised in that institute
The construction method for stating processing type capsicum cytoplasmic male sterility restorer is comprised the following steps：
(1)F2Colony is formulated by restorer 812 and the hybridization of Parents 940；With molecular marker assisted selection in F2In colony
Individual plant of the screening containing Restore gene Rf carries out Anther Culture；
(2) using the CRF-SCAR label screenings F for screening2The individual plant containing Restore gene carries out Anther Culture, Jin Eryong in colony
Marker Identification flower training regeneration strain DH systems whether be containing Restore gene Rf, CRF-SCAR upstream primer sequence：5′-
GTACACACCACTCGTCGCTCCT-3 ', downstream primer sequence is：5′-TTCTTGGGTCCCTTTCTTCCAA-3′；
(3) Pepper Leaves DNA is extracted using CTAB methods；Containing the μ L of 5 × PCR buffer solutions 4,25mmol/L MgCl2The μ of solution 1.2
Each 2.0 μ L of the μ L of L, dNTP solution 1.6,33ng/ μ L upstream and downstream primers, 2U/ μ L Taq polymerases 0.5 μ L, ddH2The μ L of O 7 and template
DNA 1.0μL；
(4) 0.1%HgCL is used afterwards with 70% alcohol-pickled 30s on superclean bench2Sterilization 7min, rinsed with sterile water 4 times,
Then it is placed on aseptic filter paper and drains the water；Embryoid induction culture medium is aseptically inoculated in, diameter 100mm culture dishes connect
8 buds, 37 DEG C of high temperature pre-process 5d.
8. the construction method of processing type capsicum cytoplasmic male sterility restorer as claimed in claim 7, it is characterised in that institute
Embryoid induction culture medium is stated for MS+BA 0.5mg/L+2,4-D 0.5mg/L+3% sucrose+agar 7g/L+AgNO3 4mg/L+
Activated carbon 0.4%, pH 5.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111371.2A CN106718895A (en) | 2016-12-06 | 2016-12-06 | A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111371.2A CN106718895A (en) | 2016-12-06 | 2016-12-06 | A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106718895A true CN106718895A (en) | 2017-05-31 |
Family
ID=58874692
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611111371.2A Pending CN106718895A (en) | 2016-12-06 | 2016-12-06 | A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106718895A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108990801A (en) * | 2018-09-05 | 2018-12-14 | 重庆市农业科学院 | A kind of selection of processing type capsicum blight-resistant new germ plasm |
| CN109156336A (en) * | 2018-08-20 | 2019-01-08 | 青岛农业大学 | A kind of selection of High color values and slow sweet tea pigment green pepper restorer of fading |
| CN115669402A (en) * | 2022-09-16 | 2023-02-03 | 北京林业大学 | Method and equipment for high-temperature restoration of fertility of lily distant hybrid |
| CN115812589A (en) * | 2022-11-24 | 2023-03-21 | 重庆市农业科学院 | Culture method for obtaining embryoid by culturing gene type anther of processed pepper with low possibility of embryo emergence |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1836496A (en) * | 2006-04-03 | 2006-09-27 | 西北农林科技大学 | Method for obtaining chilli embryoid by microspore induction |
| CN101622960A (en) * | 2009-08-14 | 2010-01-13 | 江苏省农业科学院 | Method for fast obtaining regenerated plant for cayenne pepper anther culture |
| CN103355158A (en) * | 2013-07-19 | 2013-10-23 | 镇江瑞繁农艺有限公司 | Breeding method of processing type Capsicum annuum L. species |
-
2016
- 2016-12-06 CN CN201611111371.2A patent/CN106718895A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1836496A (en) * | 2006-04-03 | 2006-09-27 | 西北农林科技大学 | Method for obtaining chilli embryoid by microspore induction |
| CN101622960A (en) * | 2009-08-14 | 2010-01-13 | 江苏省农业科学院 | Method for fast obtaining regenerated plant for cayenne pepper anther culture |
| CN103355158A (en) * | 2013-07-19 | 2013-10-23 | 镇江瑞繁农艺有限公司 | Breeding method of processing type Capsicum annuum L. species |
Non-Patent Citations (5)
| Title |
|---|
| 刘进平等: "《植物细胞工程简明教程》", 28 February 2005 * |
| 李怡斐等: "加工型辣椒细胞质雄性不育育性基因分子标记及辅助育种", 《分子植物育种》 * |
| 李怡斐等: "辣椒花药培养及其在遗传育种上的应用", 《辽宁农业科学》 * |
| 杨春文等: "分子标记技术在辣椒研究中的应用进展", 《中国农学通报》 * |
| 静一等: "加工型辣椒花药培养技术研究", 《中国农学通报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109156336A (en) * | 2018-08-20 | 2019-01-08 | 青岛农业大学 | A kind of selection of High color values and slow sweet tea pigment green pepper restorer of fading |
| CN108990801A (en) * | 2018-09-05 | 2018-12-14 | 重庆市农业科学院 | A kind of selection of processing type capsicum blight-resistant new germ plasm |
| CN115669402A (en) * | 2022-09-16 | 2023-02-03 | 北京林业大学 | Method and equipment for high-temperature restoration of fertility of lily distant hybrid |
| CN115669402B (en) * | 2022-09-16 | 2023-06-20 | 北京林业大学 | Method and equipment for recovering lily distant hybrid fertility at high temperature |
| CN115812589A (en) * | 2022-11-24 | 2023-03-21 | 重庆市农业科学院 | Culture method for obtaining embryoid by culturing gene type anther of processed pepper with low possibility of embryo emergence |
| CN115812589B (en) * | 2022-11-24 | 2023-12-19 | 重庆市农业科学院 | Culture method for obtaining embryoid by culturing genotype anther with difficulty in embryo emergence of processed capsicum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Manipulation of ploidy for kiwifruit breeding: in vitro chromosome doubling in diploid Actinidia chinensis Planch. | |
| CN106718895A (en) | A kind of method of quick initiative processing type hot pepper male sterile fertility restorer new germ plasm | |
| CN104871964B (en) | A kind of method improving wild rice and cultivated rice distant hybridization embryo crash breeding efficiency | |
| CN107435079A (en) | Utilize the method for the wide affine rice material of Anther Culture quick breeding | |
| CN104255443B (en) | A kind of polymerization is containing the seed selection method for rice of anti-three kinds of disease genes | |
| CN103651111B (en) | Pickle and purple cabbage trigenomic species allohexaploid vegetable germplasm and acquisition method | |
| CN105532450A (en) | Hybrid paper mulberry industrial tissue culture and breeding method | |
| CN1994065B (en) | Method for improving seed selection of watermelon polyploid | |
| CN111876336A (en) | Mucuna fungus and application thereof in promoting germination of paphiopedilum brandisil seeds to form seedlings | |
| CN104782482B (en) | Stable high-efficient method for ex-vivo preservation and growth recovery of strawberry germplasm resource | |
| CN1413448A (en) | Quick screening method of alkali resistance rice breeding material | |
| CN103782910B (en) | The method of lily Unfertilized Ovaries cultured in vitro | |
| CN108949899A (en) | The identification method of Physiological Race of Phytophthora infestans and its application | |
| CN102124950B (en) | Method for obtaining haploid of cultivated dendranthema morifolium | |
| CN115119748B (en) | Method for obtaining water horn homozygote through in vitro culture | |
| CN104026017B (en) | The breeding method of Semen Maydiss haplobiont | |
| CN106148530A (en) | With pepper anther chloroaniline because of closely linked molecular marker GSSR187 and application thereof | |
| Yan et al. | Acquisition of triploid germplasms by controlled hybridisation between diploid and tetraploid in Chinese jujube | |
| CN109006456A (en) | A kind of selection of pimento genic male sterility dual purpose | |
| KR101887221B1 (en) | Method of mass propagation of bamboo by in vitro culture | |
| CN103782902A (en) | Method for creating Chinese cabbage mutant by means of 60Co-gamma ray mutagenesis and microspore culture | |
| CN103583367B (en) | Quick breeding method for novel triploid salvia miltiorrhiza detoxification varieties | |
| CN109042332B (en) | Tissue culture and rapid propagation method of tripterygium wilfordii | |
| CN105340732A (en) | Chromosome doubling method for wheat haploid test-tube plants | |
| CN107094626B (en) | Contaminate the tissue culture and rapid propagation method of well Ji wild cherry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
| RJ01 | Rejection of invention patent application after publication |