CN108753763A - A kind of selection of onion male sterile line - Google Patents
A kind of selection of onion male sterile line Download PDFInfo
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- CN108753763A CN108753763A CN201810596687.8A CN201810596687A CN108753763A CN 108753763 A CN108753763 A CN 108753763A CN 201810596687 A CN201810596687 A CN 201810596687A CN 108753763 A CN108753763 A CN 108753763A
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Abstract
The invention discloses a kind of selections of onion male sterile line.It is receptor that the selection, which is by donor, onion self-mating system of onion cytoplasmic male sterility system, carries out Asymmetric Protoplast Fusion, obtains onion male sterile line.It is demonstrated experimentally that selecting 5 onion male sterile lines using onion Alloplasmic male sterile system skiA and the red jade of middle life using method provided by the invention, this 5 onion male sterile lines show the form different from parents：Plant strain growth gesture is strong, middle sunshine, the crimson color of bulb crust, no pollen.The selection breeding cycle of onion male sterile line provided by the invention is short, has important application value.
Description
Technical field
The invention belongs to field of plant breeding, and in particular to a kind of selection of onion male sterile line.
Background technology
Onion (Alliumcepa) is a kind of important worldwide and health vegetables, in China the year-round supply of vegetables with
Occupy highly important status in the export trade.The onion in China, at 300,000 hectares or more, occupies the world the 2nd per annual planting area
Position, but because of reasons such as kinds, per unit area yield but comes after 20.Since onion breed breeding work in China's is started late, pay attention to not
Enough, the period of onion breed breeding is longer in addition, causes the varietal level of domestic onion self-fertile and international advanced country to have larger
Gap.For a long time, domestic cepa seed even some conventional varieties are monopolized or country of origin by external seeds company
Outside.The expensive and seed source of import seed is unstable, has aggravated the risk and market price fluctuations of China's onion production,
Cause main producing region seed price expensive, cost increase.Therefore, accelerate the selection and breeding of the onion improved seeds of China's autonomous innovation, it is right
The sustainable development of the foreign-exchange earning onion industry of China's export is pushed to be of great significance to.
The selection and breeding key of the onion improved seeds of autonomous innovation is the initiative of onion male sterile line.It is utilized at present
Onion cytoplasmic male sterility is mainly S type male sterility, total by the cytoplasmic factor (S) and core Recessive genes (ms) of infertility
With control, sterile line transformation is easier, and is widely used for preparing onion hybrid.But transformation process is complicated, the time is long.And
This cytoplasmic homogeneity brings prodigious potential risk to be especially very popular to disease and brings possibility to onion production.
Onion Alloplasmic male sterile system skiA is a preferable onion infertility source material.Not with S type onions male
It is to compare to educate, and the fertility of onion Alloplasmic male sterile system skiA is controlled by cytoplasm, is not controlled by karyogene, and transformation utilizes
It is simple and convenient；And onion Alloplasmic male sterile system skiA is totally different from the novel fine cytoplasm of S, T-type male sterile line
Male sterile line can effectively avoid the risk that cytoplasm homogeneity is come to onion industrial zone.However onion allo-plasm male is not
Educate be skiA be the long-day, bulb is yellow spherical, these are not all inconsistent with conventional breeding target.It is how that onion allo-plasm is male
Sterile line skiA sterility rapid transfers are excellent to Comprehensive Traits, meet the onion self-mating system of breeding objective for property, formulate out male
Sterile line is onion breeding main problem urgently to be resolved hurrily.Traditional method is continuous backcross 4-6 generations, but need 10 years or more, take
When it is laborious.
Invention content
The purpose of the present invention is quickly formulate onion male sterile line.
The present invention protects a kind of selection of onion male sterile line first, can be with onion cytoplasmic male sterility system
It is receptor for donor, onion self-mating system, carries out Asymmetric Protoplast Fusion, obtain onion male sterile line.
In above-mentioned selection, the male sterile line of the onion male sterile line concretely onion self-mating system.
In above-mentioned selection, the Asymmetric Protoplast Fusion may include following steps：
(1) protoplast of the onion cytoplasmic male sterility system of core inactivation is obtained；
(2) protoplast of the onion self-mating system of cytoplasm inactivation is obtained；
(3) by the original of the onion self-mating system of the protoplast of the onion cytoplasmic male sterility system of core inactivation and cytoplasm inactivation
Raw plastid carries out protoplast fusion, culture.
In the step (1), the method for obtaining the protoplast of the onion cytoplasmic male sterility system of core inactivation can be：It takes
The protoplast of onion cytoplasmic male sterility system, radiation.
In the step (1), radiation can be ultraviolet radiation.The dose of radiation of the radiation can be 36-72J/cm2(such as
36J/cm2Or 72J/cm2)。
In the step (2), the method for obtaining the protoplast of the onion self-mating system of cytoplasm inactivation can be：Onion is taken to be selfed
The protoplast of system, the processing of iodine second phthalein amine.
In the step (2), iodine second phthalein amine processing concentration can be 0.1-0.4mmol/L (such as 0.1-0.3mmol/L,
0.3-0.4mmol/L, 0.1mmol/L, 0.3mmol/L or 0.4mmol/L).
In the step (3), the mode of protoplast fusion concretely electro' asion.
In any of the above-described selection, the onion cytoplasmic male sterility system can be onion allo-plasm male
Sterile line skiA.The onion self-mating system is raw red jade in onion kind.
Protection scope of the present invention is also belonged to using the onion male sterile line of any of the above-described selection selection and breeding.
The present invention also protects A1) or A2)：
A1) application of any of the above-described selection in onion breeding；
A2) applications of the onion Alloplasmic male sterile system skiA in selection and breeding onion male sterile line.
In above application, the onion breeding be using the onion male sterile line of any of the above-described method selection and breeding as
Parent is hybridized and/or is returned with other onion kinds or strain, obtains having other onion kinds or strain heredity
The onion new lines of background.
It is demonstrated experimentally that utilizing onion Alloplasmic male sterile system skiA and the red jade of middle life using method provided by the invention
Select 5 onion male sterile lines.This 5 onion male sterile lines show the form different from parents：Plant strain growth
Gesture is strong, middle sunshine, the crimson color of bulb crust, no pollen.The selection breeding week of onion male sterile line provided by the invention
Phase is short, has important application value.
Description of the drawings
Fig. 1 is to formulate onion male sterile line using Asymmetric Protoplast Fusion technology.
Fig. 2 is the Chromosome Analysis of somatic hybridization plant.
Fig. 3 is the Molecular Detection of somatic hybridization plant.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Experiment material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Onion Alloplasmic male sterile system skiA is to carry out distant hybridization using real Roripa green onion and onion, and one kind of creation is new
Heterologous onion male sterile line.The sterile line does not have anther, and without restoring gene, bulb size, blade etc. and maintainer are complete
Complete consistent, the yield and S types, T-type infertility of each bouquet production cenospecies are not significantly different, and have stronger disease resistance etc. excellent
Point；Long-day, spherical, crust brassy, storage tolerance, spherical index 0.99, moisture is low, pungent highly seasoned.Plant strain growth gesture
By force, 75~80cm of plant height, dark green leaf, wax powder are more.
The middle red jade of life be using precocious red jade of the purplish red skin onion of Japan be female parent, South Korea's Mid-late ripening Qarnet as paternal hybrid after pass through
4 generation systematic breedings form.Mid-early maturity, breeding time 240d or so；Plant strain growth gesture is medium, 65~70cm of plant height, blade light green color,
Wax powder is few；Bulb is thick oblate, spherical index 0.78, the crimson color of crust, and glossy, slightly sweet, commodity is Sino-Japan suitable for major part
It is planted according to area.
Embodiment 1 formulates onion male sterile line using Asymmetric Protoplast Fusion technology
One, the acquisition of the protoplast of the protoplast of onion Alloplasmic male sterile system skiA and the red jade of middle life
According to document, (Chen Li, Liang Yi, Wang Liping, Tian Baohua onion callus isolation and purification of protoplast study China
Agronomy is notified to, 2011,27 (31)：Method separation described in 147-151.) and purifying onion Alloplasmic male sterile system
The protoplast of skiA.It is as follows：
1, the seed of the onion Alloplasmic male sterile system skiA of full stalwartness is taken, aseptic water washing 10min (mesh is first used
It is removal surface impurity), then 30s is impregnated with 75% (v/v) ethanol water, finally use 0.1%HgCl2Solution impregnates
10min, with aseptic water washing 5 times.
2, after completing step 1, the seed of onion Alloplasmic male sterile system skiA is taken, 1/2MS solids are inoculated in
On culture medium, 25 ± 2 DEG C of alternation of light and darkness culture (illumination 16h/ dark 8h) 20d obtain aseptic seedling.
3, after completing step 2, the plateau of the aseptic seedling is taken, is inoculated in containing 1.0mg/L 2,4-D and 1.0mg/L6-BA
MS solid mediums on, 25 ± 2 DEG C of alternation of light and darkness culture (illumination 16h/ dark 8h) 10d obtain callus.By the callus
Tissue is inoculated in again containing 1.0mg/L 2, on the MS solid mediums of 4-D and 1.0mg/L 6-BA, 25 ± 2 DEG C of alternation of light and darkness trainings
It supports (illumination 16h/ dark 8h) 12d, obtains embryo callus (see A in Fig. 1).
4, complete step 3 after, take the embryo callus, be inoculated in equipped with 25mL contain 1.0mg/L 2,4-D and
In the triangular flask (triangular flask specification is 100mL) of the MS fluid nutrient mediums of 1.0mg/L 6-BA, 25 DEG C, the dark training of 120rpm oscillations
3-5d is supported, suspension callus is obtained.
5, after completing step 4, the suspension callus is taken, by 1：10 (1g suspension callus：10mL enzymolysis liquids)
Ratio is added enzymolysis liquid (through 0.22 μm of aperture membrane filtration sterilizing), under dark condition, 28 DEG C, 25rpm oscillations 6h (purpose be into
Row enzymolysis), obtain protoplast.
The solute of enzymolysis liquid and its a concentration of 2.0% (m/v) cellulase Onozuka R-10,0.5% (m/v) macerozyme
Macerozyme R-10 and 0.1% (m/v) pectase Pectinase Y-23, solvent are CPW-0.6mol/L mannitol solutions
(contain CaCl2·2H2O 10mmol/L、KH2PO4 0.2mmol/L、KNO3 1.0mmol/L、MgSO4·7H2O 1.0mmol/L、
CuSO4·5H2The water of 15.37 μm of 0.1 μm of O 10 μm of ol/L, KI ol/L, 2-morpholine ethane sulfonic acid ol/L and 0.6mol/L mannitol
Solution, pH value 5.6), pH value is 5.8-6.0 (being adjusted with NaOH).
6, after completing step 5, the protoplast is taken, through double-layer stainless steel net (aperture is respectively 100 μm and 38.5 μm)
Filtering, obtains filtrate.The filtrate is placed in low speed centrifuge, 600r/min centrifuges 5min, abandons supernatant, collects precipitation.
7, after completing step 6, the precipitation is taken, is resuspended with 1mL CPW-0.6mol/L mannitol solutions, is then gently added
Enter and (contains CaCl equipped with 3mL CPW-25% sucrose2·2H2O 10mmol/L、KH2PO4 0.2mmol/L、KNO3 1.0mmol/L、
MgSO4·7H2O 1.0mmol/L、CuSO4·5H215.37 μm of 0.1 μm of O 10 μm of ol/L, KI ol/L, 2-morpholine ethane sulfonic acid ol/
The aqueous solution of L and 25% (m/v) sucrose, pH value 5.6) centrifuge tube (specification 10mL) in, 600r/min centrifuge 5min,
Collect milky protoplast band in the interface of solution.The protoplast band of collection is onion Alloplasmic male sterile
It is the protoplast of skiA (see B in Fig. 1).
According to above-mentioned steps, onion Alloplasmic male sterile system skiA is replaced with into the red jade of middle life, other steps are not
Become, obtain the protoplast of the red jade of middle life (see C in Fig. 1).
Two, ultraviolet radiation
Radiating light source is superclean bench 30W ultraviolet lamps, and radiation intensity is about 1200 μ w/cm2。
1, the protoplast 2.0mL for taking the onion Alloplasmic male sterile system skiA that step 1 obtains, is laid in glass
Culture dish (a diameter of 60mm).
2, after completing step 1, the glass culture dish is placed at ultraviolet lamp vertical lower 10cm, is radiated.Radiation
Time is respectively that (corresponding dose of radiation is followed successively by 0,36,72,108 and 144J/cm by 0s, 30s, 60s, 90s and 120s2)。
3, after completing step 2, the glass culture dish is taken, the plasm of onion Alloplasmic male sterile system skiA is poured out
Body, 1000rpmin centrifuge 5min, collect precipitation.
4, after completing step 3, the precipitation is taken, is washed twice with electro' asion liquid, then electro' asion liquid used to dilute, obtained close
Degree is 5 × 105Dilution (the hereinafter referred to as sterile line of the protoplast of the onion Alloplasmic male sterile system skiA of a/mL
Protoplast dilution).
Electro' asion liquid：0.5mol/L containing mannitol and CaCl2The aqueous solution of 0.5mmol/L, pH value 5.7.
Three, protoplast vigor is detected
Detection protoplast vigor is dyed using fluorescein diacetate (Fluorescein diacetat, FDA).Experiment
It is averaged in triplicate, 5 visuals field of each repeated observation are as follows：
1, it takes 1mL FDA to store liquid (2mg FDA are dissolved in 1mL acetone), 50mLddH is added2O is diluted, and it is dilute to obtain FDA
Release liquid.
2,1 parts by volume protoplast (sterile line protoplast dilution) and 1 parts by volume FDA dilutions are mixed, is stood
5min is placed under fluorescence microscope (Leica DM2500) and observes, and the protoplast that can send out green fluorescence is as vibrant
Protoplast.Protoplast vigor in statistics sterile line protoplast dilution culture 7d (it is glimmering to send out green in dark field
The protoplast number of light accounts for the ratio of total protoplast number in same bright-field), the division frequency (under the same visual field of culture 7d
The protoplast number of division accounts for the ratio of total protoplast number) and culture 20d efficiency of plating (cell mass under the same visual field
Number accounts for the ratio of total protoplast number).
The experimental results showed that (table 1), influence of the ultraviolet light to protoplast is mainly manifested in protoplast vigor, division frequency
Rate and efficiency of plating.Protoplast vigor, division frequency and efficiency of plating are reduced with the increase of dose of radiation.Ultraviolet radiation causes
So that protoplast vigor is declined, and extend with radiated time, protoplast vigor declines notable：The protoplast and spoke not radiated
It is 36J/cm to penetrate dosage2Protoplast start respectively in 2d and 3d the 1st time division, later constantly division, 15d or so
Form cell mass；Dose of radiation is 72J/cm2Protoplast cuhnre 5d after just carry out first division, subsequently form a small amount of
Small cell cluster has no to form callus；Dose of radiation is 108J/cm2Protoplast early stage it is more normal, have a small amount of point
It splits, but is gradually crushed until dead after 10d；Dose of radiation is more than 108J/cm2Protoplast stop after first division life
It is long.1 × 10 is pressed to the protoplast through UV treatment5A/mL density cultures, radiating the protoplast of 30s-300s can carry out
Cell division, and embryo callus is ultimately formed, radiated time is more than the acellular division of protoplast of 400s.Therefore, it selects
Select 72J/cm2It is used for subsequent protoplast asymmetric fusion for ultraviolet light lethal dose.
Influence of the 1. ultraviolet radiation dosage of table to onion protoplast
Note：Data statistics is average ± standard deviation.Significance of difference level is that P≤0.05. letters a, b, c, d, e are indicated
The protoplast vigor level of difference of difference UV doses on the same day.
Before protoplast asymmetric fusion, the protoplast of onion Alloplasmic male sterile system skiA is carried out such as successively
Lower processing：(1) the protoplast 2.0mL for taking the onion Alloplasmic male sterile system skiA that step 1 obtains, is laid in glass
Culture dish (a diameter of 60mm)；(2) after completing step (1), the glass culture dish is placed at ultraviolet lamp vertical lower 10cm,
Radiating 60s, (corresponding dose of radiation is 72J/cm2)；(3) after completing step (2), take the protoplast, 1000rpmin from
Heart 5min collects precipitation；(4) after completing step (3), the precipitation is taken, is washed twice with electro' asion liquid, then use electro' asion liquid
Dilution, it is 5 × 10 to obtain density5The protoplast working solution of the sterile line of a/mL.
Four, iodine second phthalein amine (IOA) is handled
The solute of IOA mother liquors and its a concentration of 5mmol/L IOA, solvent are CPW solution, and pH value is 5.8 (with NaOH tune
Section).IOA mother liquors need filtration sterilization.CPW solution：Containing CaCl2·2H2O 10mmol/L、KH2PO4 0.2mmol/L、KNO3
1.0mmol/L、MgSO4·7H2O 1.0mmol/L、CuSO4·5H20.1 μm of O 10 μm of ol/L, KI ol/L and 2- morpholine second sulphurs
The aqueous solution of 15.37 μm of ol/L of acid.
1, IOA mother liquors are taken, ddH is added2O is diluted, and obtains the dilution 1, a concentration of of a concentration of 0.1mmol/L
The dilution 4 of the dilution 2 of 0.4mmol/L, the dilution 3 of a concentration of 0.8mmol/L or a concentration of 1.2mmol/L.
2, after completing step 1, take centrifuge tube, be added dilution (dilution 1, dilution 2, dilution 3 or dilution 4) and
The protoplast for the red jade of middle life that step 1 obtains, room temperature processing 15min (period gently shakes centrifuge tube every 5min taking-ups,
In order to avoid protoplast deposition influences treatment effect).
3, it after completing step 2, takes centrifuge tube, 1000rpmin to centrifuge 5min, collects precipitation.
4, after completing step 3, the precipitation is taken, is washed twice with electro' asion liquid, then electro' asion liquid used to dilute, obtained close
Degree is 5 × 105The protoplast dilution (raw red beautiful protoplast dilution in hereinafter referred to as) of the red jade of middle life of a/mL.
Five, protoplast vigor is detected
Detection protoplast vigor is dyed using fluorescein diacetate (Fluorescein diacetat, FDA).Experiment
It is averaged in triplicate, 5 visuals field of each repeated observation are as follows：
1, it takes 1mL FDA to store liquid (2mg FDA are dissolved in 1mL acetone), 50mLddH is added2O is diluted, and it is dilute to obtain FDA
Release liquid.
2,1 parts by volume protoplast (the red beautiful protoplast dilution of middle life) and 1 parts by volume FDA dilutions are mixed, it is quiet
It sets 5min and is placed under fluorescence microscope (Leica DM2500) and observe, the protoplast that can send out green fluorescence is to have work
The protoplast of power.Protoplast vigor in statistics sterile line protoplast dilution culture 7d (sends out green in dark field
The protoplast number of fluorescence accounts for the ratio of total protoplast number in same bright-field), culture 7d division frequency (the same visual field
The protoplast number of lower division accounts for the ratio of total protoplast number) and culture 20d efficiency of plating (cell mass under the same visual field
Number account for total protoplast number ratio).
The experimental results showed that (table 2) finds that protoplast is lived after raw red beautiful protoplast in various concentration IOA processing
Power is reduced as concentration for the treatment of increases.Processed protoplast first division is more late than untreated protoplast, point
It splits frequency and efficiency of plating wants low.Untreated protoplast is through cultivating renewable plant；It is primary with 0.1mmol/L IOA processing
Plastid can also regenerate plant；And when a concentration of 0.4mmol/L of IOA, protoplast can regenerate callus, but fail regeneration plant；
When concentration reaches 0.8mmol/L, protoplast does not re-form raw callus；After being handled with higher 1.2mmol/L concentration
The gradual browning of protoplast, it is in irregular shape until rupture.Therefore, after selecting 0.4mmol/L to be used for for the minimum deactivating concentrations of IOA
Continuous protoplast asymmetric fusion.
Before protoplast asymmetric fusion, the protoplast of the red jade of middle life is handled as follows：(1) IOA mother liquors are taken, are added
Enter ddH2O is diluted, and obtains the dilution of a concentration of 0.4mmol/L；(2) after completing step (1), centrifuge tube is taken, is added dilute
It releases liquid and the protoplast of the red jade of middle life that step 1 obtains, room temperature handles 15min；(3) after completing step (2), centrifuge tube is taken,
1000rpmin centrifuges 5min, collects precipitation；(4) after completing step (3), the precipitation is taken, is washed twice with electro' asion liquid, so
Electro' asion liquid is used to dilute afterwards, it is 5 × 10 to obtain density5The protoplast working solution of the red jade of middle life of a/mL.
Influences of the table 2.IOA to onion protoplast
Note：Data statistics is average ± standard deviation.Significance of difference level is that P≤0.05. letters a, b, c, d, e are indicated
The protoplast vigor level of difference of difference UV doses on the same day.
Six, the acquisition of protoplast asymmetric fusion and regeneration plant
1, the red jade of middle life of the protoplast working solution and the preparation of 1mL step 5 of the sterile line of 1mL step 3 preparation is taken
Protoplast working solution, mixing, then carries out electro' asion.Electrofusion parameter is as follows：Ac electric field strength is 60V/cm, and the time is
60s；DC electric field intensity is 1500V/cm, 50 μ s of burst length, pulse number 5 times, each pulse spacing 1s.
2, the mixed liquor of step 1 is taken into, first stands 20min (purpose is for ease of spheroidization), then washed with electro' asion liquid
Twice, fusion protoplast culture is obtained.
3, it takes and fills sorbierite containing 0.3mol/L and 0.1mg/L 2, the glass culture dish (rule of the MS fluid nutrient mediums of 4-D
Lattice are 15mm × 60mm), fusion protoplast culture is added, obtains cultivating system.In the cultivating system, protoplast it is close
Degree is 5 × 105A/mL.
4, after completing step 3, the cultivating system, 27 DEG C of light culture 30d are taken.
Experimental result is as follows：Cultivate 5h, protoplast fusion (D and E in Fig. 1)；6d, cell start division (in Fig. 1
F)；30d forms macroscopic embryoid or embryo callus subculture (G in Fig. 1).
5, after completing step 4, embryoid or embryo callus subculture is taken, the MS fluid nutrient medium cultures of the sorbierite containing 0.1M are transferred to
Two weeks or so (decompression culture), is then transferred on MS solid mediums with suction pipe absorption, carries out liquid and consolidate Double layer culture.
6, after the fluid nutrient medium of step 5 at the middle and upper levels is dried, embryoid or embryo callus subculture are transferred to containing 2,4-
Squamous subculture on the MS solid mediums of D1.0mg/L and 6-BA 1.0mg/L, when embryoid or embryo callus subculture become graininess embryo
When property callus (vivid ecru), the MS solid mediums of 2.0mg/L containing 6-BA and NAA 1.0mg/L are transferred to
Upper culture, until differentiating seed plantlet.
7, the seed plantlet that step 6 differentiates is transferred to culture 25d (purposes on the 1/2MS culture mediums containing 0.1% activated carbon
To take root), obtain vegetative seedling (H in Fig. 1).Vegetative seedling is transplanted in the seed plate for filling fresh frog stone, routine culture obtains
To regeneration plant (I in Fig. 1).
Partial regeneration plant shows the form different from parents：Plant strain growth gesture is strong, middle sunshine, the crimson color of bulb crust
(L in Fig. 1), no pollen (K is inflorescence in Fig. 1), at the beginning of these morphologically show the regeneration plant of the form different from parents
Step is accredited as somatic hybridization plant.The inflorescence of onion Alloplasmic male sterile system skiA is shown in J in Fig. 1.
According to above-mentioned steps, " the protoplast working solution of sterile line prepared by step 3 " in step 1 is replaced with into electricity
Liquid is merged, other steps are constant (as a contrast).It is unable to get regeneration plant.
According to above-mentioned steps, " the protoplast working solution of the red jade of middle life prepared by step 5 " in step 1 is replaced with
Electro' asion liquid, other steps are constant (as a contrast).Also it is unable to get regeneration plant.
Seven, Chromosome Analysis
1, taking onion, (onion Alloplasmic male sterile system skiA, the red beautiful or step 6 of middle life are initially identified as body cell
Hybrid plant) tip of a root, it is placed in the paracide solution of saturation, room temperature pre-processes 4h.
2, after completing step 1, the root top of onion is transferred to Kano fixer (3 parts by volume absolute ethyl alcohols and 1 parts by volume
Glacial acetic acid mixes), 4 DEG C of fixations are for 24 hours.
3, after completing step 2, the root top of onion is taken first wash with distilled water to add the HCl/water of a concentration of 6mol/L
Solution, 65 DEG C of water-bath 10min finally dye 5min, tabletting with 5% (m/v) carbolfuchsin solution.
The chromosome number of root top of onion is observed under an optical microscope.
The result shows that onion Alloplasmic male sterile system skiA (A in Fig. 2), the red beautiful or part steps six of middle life are preliminary
The chromosome number for being accredited as the tip of a root of somatic hybridization plant (B in Fig. 2) is 16, is diploid.
In the somatic hybridization plant of step 6 Preliminary Identification, root tip chromosomes number is shown as 16 on a cellular level
I.e. be accredited as somatic hybridization plant.These somatic hybridization plant are identical with the ploidy of its parents.
Eight, Molecular Detection
Onion to be measured is parents and 5 plants of somatic hybridization plant.Parents are respectively onion Alloplasmic male sterile system
SkiA and the red jade of middle life.
1, RAPD analyzes the nuclear genome of onion to be measured
Genomic DNA is extracted from onion blade to be measured using CTAB methods, then according to document (Cui Cheng, Xu Qijiang, Cui
High scholar, the 04th phases of Li Jing roc onion RAPD analysis of genetic diversity gardening journals .2006) described in method progress RAPD
Analysis.
Reaction system is 25 μ L, by the genomic DNA (about 40ng) of onion to be measured, 2.5 μ L 10 × PCR Buffer (Mg2+
Plus), (concentration of dATP, dTTP, dGTP and dCTP are 2.5mmolL to 1.4 μ L dNTP-1), 1.0 μ L RAPD primers
(10mmol/L), 0.25 μ L LA Taq enzymes (a concentration of 5U/ μ L) and distilled water composition.
Response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 36 DEG C of annealing 30s, 72 DEG C of extension 90s, 40 recycle；
72 DEG C of extension 7min.
RAPD analyses carry out in PE-9700 type PCR instruments.Amplified production (contains ethidium bromide in 0.8% Ago-Gel
0.5 μ g/mL) in detached with the electrophoresis of 5V/cm, through full automatic gel imaging system Syngene GeneGenius observations,
It takes pictures.
When using SBSA-19：5 '-CAAACGTCGG-3 ' or SBSB20：5 '-GGACCCTTAC-3 ' carry out RAPD analyses
When, obviously (see A in Fig. 3, swimming lane 1 is DNA marker to the AFLP system difference of amphophile cell hydridization plant, and swimming lane 2 is
The middle red jade of life, swimming lane 3 are onion Alloplasmic male sterile system skiA, and swimming lane 4-8 is somatic hybridization onion plant).
2, cpSSR analyzes the cytoplasmic skeleton of onion to be measured
Total DNA is extracted from onion blade to be measured using CTAB methods, then according to document (Cheng YJ, Guo WW, and
Deng XX.cpSSR：a new tool to analyze chloroplast genome of Citrus somatic
Hybrids.Acta Botanica Sinca, 2003,45 (8)) described in method carry out cpSSR analyses.CpSSR primers are
CP40, by 5 '-TAATTTGATTCTTCGTCGC-3 ' and 5 '-GATGTAGCCAAGTGGATCA-3 ' is formed.
Reaction system is 25 μ L, by the total DNA (about 40ng) of onion to be measured, 3.0 μ L 10 × PCR Buffer, 3.0 μ L
Mg2+(20mmol/L), (concentration of dATP, dTTP, dGTP and dCTP are 2.5mmolL to 3.0 μ L dNTP-1)、2.0μL
CP40 (a concentration of 50pmol/L), 0.4 μ L Taq enzymes (a concentration of 5U/ μ L) and distilled water composition.
Response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 90s, 45 recycle；
72 DEG C of extension 10min.
Amplified production is detached in 0.8% Ago-Gel (the 0.5 μ g/mL containing ethidium bromide) with the electrophoresis of 5V/cm,
Through full automatic gel imaging system Syngene GeneGenius observations, take pictures.
As a result see that (swimming lane 1-5 is somatic hybridization onion plant to B in Fig. 3, and swimming lane 6 is the red jade of middle life, and swimming lane 7 is onion
Alloplasmic male sterile system skiA).When carrying out cpSSR analyses using CP40, parents respectively generate a characteristic strip, and body cell
Hybrid plant only has the characteristic bands of onion Alloplasmic male sterile system skiA.
By above-mentioned steps, somatic hybridization plant is the onion male sterile line formulated.
Claims (10)
1. a kind of selection of onion male sterile line, for using onion cytoplasmic male sterility system as donor, onion self-mating system
For receptor, Asymmetric Protoplast Fusion is carried out, onion male sterile line is obtained.
2. selection as described in claim 1, it is characterised in that：The Asymmetric Protoplast Fusion includes following step
Suddenly：
(1) protoplast of the onion cytoplasmic male sterility system of core inactivation is obtained；
(2) protoplast of the onion self-mating system of cytoplasm inactivation is obtained；
(3) by the plasm of the onion self-mating system of the protoplast of the onion cytoplasmic male sterility system of core inactivation and cytoplasm inactivation
Body carries out protoplast fusion, culture.
3. selection as claimed in claim 2, it is characterised in that：
In the step (1), the method for obtaining the protoplast of the onion cytoplasmic male sterility system of core inactivation is：Take onion thin
The protoplast of cytoplasm male sterility line, radiation；
In the step (2), the method for obtaining the protoplast of the onion self-mating system of cytoplasm inactivation is：Take the original of onion self-mating system
Raw plastid, the processing of iodine second phthalein amine.
4. selection as claimed in claim 3, it is characterised in that：In the step (1), radiate as ultraviolet radiation.
5. selection as described in claim 3 or 4, it is characterised in that：The dose of radiation of the radiation is 36-72J/cm2。
6. selection as claimed in claim 3, it is characterised in that：In the step (2), iodine second phthalein amine is handled a concentration of
0.1-0.4mmol/L。
7. selection as claimed in claim 2, it is characterised in that：In the step (3), the mode of protoplast fusion is
Electro' asion.
8. the selection as described in claim 1 to 7 is any, it is characterised in that：The onion cytoplasmic male sterility system is
Onion Alloplasmic male sterile system skiA；The onion self-mating system is raw red jade in onion kind.
9. using the onion male sterile line of any selection selection and breeding of claim 1 to 8.
10.A1) or A2)：
A1) application of any selection of claim 1 to 8 in onion breeding；
A2) applications of the onion Alloplasmic male sterile system skiA in selection and breeding onion male sterile line.
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