CN109644875A - A kind of method of celery microspore callus differentiation and regeneration plant - Google Patents
A kind of method of celery microspore callus differentiation and regeneration plant Download PDFInfo
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- CN109644875A CN109644875A CN201910131211.1A CN201910131211A CN109644875A CN 109644875 A CN109644875 A CN 109644875A CN 201910131211 A CN201910131211 A CN 201910131211A CN 109644875 A CN109644875 A CN 109644875A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of methods of celery microspore callus differentiation and regeneration plant, belong to vegetable breeding technical field.Techniqueflow of the invention includes that first celery microspore callus is dried, make its part dehydration, differential medium is prepared according still further to specific formula, callus is inoculated on differential medium and is cultivated under appropriate conditions, culture acquisition regeneration seedling on strengthening seedling and rooting culture medium is transferred to after differentiating seedling, regeneration seedling grows up to regeneration plant by rooting culture, then carries out Methods of Ploidy Identification.The present invention on the basis of studying before, a set of system for obtaining monoploid and double haploid by Organ Differentiation, strengthening seedling and rooting, rooting culture from celery microspore callus is established, the researchs such as research, gene cloning and transgenosis is drawn for celery haploid breeding and genetic map and ideal material is provided.
Description
Technical field
This hair is related to a kind of method of celery microspore callus differentiation and regeneration plant, belongs to vegetable cultivation technical field.
Background technique
Celery belongs to umbelliferae, originates in Mediterranean littoral, and Han dynasty is passed to China, plants away from the present existing more than 2000 years
Train history.Celery also contains celery rich in protein, carbohydrate, apiolin, vitamin, calcium, phosphorus, iron, sodium etc. in base of leaf
The effective components such as dish glycosides, majudin and volatile oil have and improve a poor appetite, stomach invigorating, anti-cancer, lowering blood pressure and blood fat, prevent and treat and move
The effect of pulse atherosclerosis also has certain auxiliary dietary function to neurasthenia, menstrual disorder, gout, muscle cramp.Celery
Or high fiber food is conducive to defecation commonly using that can wriggle with stimulating gastrointestinal, and the enteral digested effect of celery can generate
The substance of a kind of lignin or enteral rouge, this substance is a kind of antioxidant, can play the work of skin whitening, anti-aging
With.Chinese medicine thinks that celery has calming liver and clearing heat, expelling wind and removing dampness, and relieving restlessness is reduced swelling, cooling blood and hemostasis, promoting the dispersing function of the lung of detoxifying, stomach invigorating benefit blood,
Gut purge is convenient, moistening lung to arrest cough, reduces blood pressure, effect of brain tonic calmness.With the improvement of people ' s living standards with health care consciousness
Constantly enhancing, celery is increasingly by the favor of the majority of consumers.
After the 1990s, with the agricultural restructuring of China each department, celery planting scale is expanded rapidly, especially
It is that most in the past 10 years, celery production in various regions all obtains stable development.Currently, China's celery production scale is always held at 550,000
hm2Left and right, height occupy first place in the world.China region is wide, and the weather conditions, planting habit and consumption habit between different regions are poor
It is different obvious, therefore the more and more abundant multiplicity of demand to celery kind.Increase as celery cultivated area expands with plantation stubble,
Pest and disease damage caused by continuous cropping is got worse, and the limitation of microclimate conditions and the anomalous variation of natural climate be more when facility cultivation
Pest and disease damage occurrence degree is weighed.On the other hand, China is increasingly stringenter vegetable safety production control, to pest control measure
It is required that increasingly standardizing, these factors increase celery prevention and control of plant diseases, pest control difficulty obviously.It is contemplated that high-quality from now on, high yield,
The promotion rate of disease-resistant type celery kind will be further speeded up, and the market occupancy volume of the celery kind with region characteristic
It will suitably increase, and will be once further reduced in the celery kind market occupancy volume of various regions popularity.
In terms of celery genetic breeding and breeding of new variety, the U.S. is chronically at rank first, such as obtains in China
" the texts and pictures drawing " planted to large area is exactly 1 excellent variety released U.S.'s nineteen eighty-three.And China's celery breeding is started late,
Previous main research work concentrates on the collection of resource, purifying utilizes, and breeding technique is confined to the introduction of external kind more, tames and dociles
Change and selection cross.Between the more than ten years, China's researcher also more than 10 celery excellent variety of successful incubation and launches city
, but selection cross remain breeding process slowly, the breeding time limit is very long and filial generation will appear asking for trait segregation
Topic.Haploid breeding technology is a kind of biotechnology breeding means, and there is extremely early stablize to separate offspring, shorten the breeding time limit, letter
The advantages that changing breeding program.It is so far, only beautiful although haploid breeding technology has just been born early in decades ago
A small amount of plant such as rice, Chinese cabbage, rape has mature haploid breeding technology.To find out its cause, haploid breeding technology and plant
Object genotype is closely related.In addition to genotype, haploid breeding technology will also be by condition of culture, medium component, hormone kind
The influence of the series of factors such as class and proportion, other processing modes, it is relatively independent between these impact factors and mutually interconnect
System, only in all impact factors all in OK range, being possible to succeed.
Obtaining haploid main path includes Anther Culture and microspores culture.Research work of this R&D team in early period
In work, celery microspore induced medium has successfully been found out and method that celery microspore induces callus,
On this basis, we continue to study, establish it is a set of from celery microspore callus by Organ Differentiation, strengthening seedling and rooting,
Rooting culture obtains the system of monoploid and double haploid, grinds for celery haploid breeding from now on and genetic map drafting
Study carefully, the researchs such as gene cloning and transgenosis provide ideal material, be of great significance for accelerating celery breeding process.
Summary of the invention
The object of the present invention is to provide a kind of methods of celery microspore callus differentiation and regeneration plant, to set up celery
The integral framework of microspores culture acquisition regeneration plant.
The present invention is achieved by the following technical solutions:
A kind of method of celery microspore callus differentiation and regeneration plant, which is characterized in that the techniqueflow of this method is as follows:
(1) callus is dried
By the callus that celery microspore induces be put into paving have three layers aseptic filter paper culture dish in, be placed in 23 ~ 25 DEG C, it is black
Dry 2 ~ 3d, makes callus sectors dehydration under dark environment；
(2) differential medium is prepared according to formula as below
A great number of elements: NH4NO3930 ~ 980mg/L, KNO3 1550 ~ 1750mg/L, KH2PO4104 ~ 110mg/L, K2HPO4 65~
73mg/L, MgSO4·7H2O 245 ~ 255mg/L, Ca(NO3)2204~212mg/L；
Microelement: KI 0.77 ~ 0.81mg/L, H3BO34.3 ~ 4.9mg/L, MnSO4·4H2O 20 ~ 23mg/L, ZnSO4·
7H2O 8.5 ~ 9.0mg/L, Na2MoO4·2H2O 0.21 ~ 0.25mg/L, CuSO4·5H2O 15 ~ 18mg/L, CoCl2·6H2O
0.02~0.03mg/L；
Molysite complex compound: 28.6 ~ 30.2mg/L of ferrous glycine；
Conventional organic principle: 95 ~ 105mg/L of inositol, 1.2 ~ 1.4mg/L of niacin, 1.5 ~ 2.5mg/L of puridoxine hydrochloride, hydrochloric acid sulphur
6.6 ~ 7.0mg/L of amine element；
Carbon source and coagulator: 12 ~ 15g/L of cellobiose, 22 ~ 26g/L of sucrose, 4 ~ 6g/L of plant gel；
Additional adding ingredient: 0.7 ~ 0.9mg/L of phytondione, 1.3 ~ 1.5mg/L of riboflavin, 3.3 ~ 3.7mg/L of nucleotide are left
Revolve 0.22 ~ 0.25mg/L of carnitine, 1.1 ~ 1.5mg/L of polyvinylpyrrolidone, 2.5 ~ 2.7mg/L of soybean lecithin, lanthanum sulfate
0.06 ~ 0.1mg/L, N- nitroso-N- ethyl 0.06 ~ 0.08mg/L of urethane, 13 ~ 17mg/L of dithiothreitol (DTT)；
Plant extraction liquid: 14 ~ 18mL/L of Radix Astragali extractive solution, 15 ~ 20mL/L of root of kudzu vine extract；
Plant growth regulator: 6-BA 2.8 ~ 3.2 mg/L, 2-(3,4- dichlorophenoxy) triethylamine 0.9 ~ 1.3mg/L, 5- nitre
0.3 ~ 0.5mg/L of base guaiacol sodium；PH is adjusted between 5.6 ~ 6.0 after preparing；
(3) callus differentiation culture
Callus after drying process is inoculated on above-mentioned differential medium and carries out Fluctuation temperature culture, cultivation temperature is set as light
According to when 24 ~ 26 DEG C, 20 ~ 22 DEG C when dark, light application time be 10 ~ 12h/d, interlunation be 12 ~ 14h/d, illumination use green light
Light source, intensity are 1500 ~ 2000Lx, and every 20d or so is transferred to fresh differential medium and makees squamous subculture, visible callus after 30 ~ 40d
Tissue starts gradually to break up, the visible adventitious bud sprouted or seedling of growing thickly after 50 ~ 60d；
(4) strengthening seedling and rooting culture
The long seedling single plant to 3 ~ 42 ~ 3cm high, tool true leaves is cut and is transferred in strengthening seedling and rooting culture medium, in cultivation temperature
23 ~ 27 DEG C, 14 ~ 16h/d of light application time, 8 ~ 10h/d of interlunation, under conditions of 2600 ~ 3000Lx of intensity of illumination, culture 20 ~
The regeneration seedling with complete root system can be formed after 30d；
(5) rooting culture
When regenerate seedling root system it is long to 3 ~ 4cm long when, open hardening 1 week in culturing room, then seedling taken from culture bottle
Out, it moves into the flowerpot for filling matrix, buckles polybag moisturizing, give natural lighting, transplanted after seedling extracts young leaves out
It is colonized to big Tanaka；
(6) regeneration plant Methods of Ploidy Identification
Carry out Methods of Ploidy Identification to the regeneration plant after transplant survival using tip of a root decoration method: the children for choosing 1cm or so is tender
The tip of a root handles 3h with 8 ~ oxyquinoline solution of 0.002 mol/L, be then placed in Kano solution (dehydrated alcohol: glacial acetic acid=3:
1) processing for 24 hours, is put into enzymolysis liquid after 3 times wash with distilled water, digests 1h in 37 DEG C of water-baths in；Then the tip of a root is placed on load
On slide, dyed with carbolfuchsin solution, the ploidy of each regeneration plant of observation analysis, normal with celery under the microscope
Ploidy plant is compared.
The optimization formula of differential medium described in step (2) is as follows:
A great number of elements: NH4NO3955mg/L, KNO3 1650mg/L, KH2PO4107mg/L, K2HPO4 69mg/L, MgSO4·
7H2O 250mg/L, Ca(NO3)2208mg/L；
Microelement: KI 0.79mg/L, H3BO34.6mg/L, MnSO4·4H2O 21.5mg/L, ZnSO4·7H2O 8.75mg/
L, Na2MoO4·2H2O 0.23mg/L, CuSO4·5H2O 16.5mg/L, CoCl2·6H2O 0.025mg/L；
Molysite complex compound: ferrous glycine 29.4mg/L；
Conventional organic principle: inositol 100mg/L, niacin 1.3mg/L, puridoxine hydrochloride 2.0mg/L, thiamine hydrochloride 6.8mg/L；
Carbon source and coagulator: cellobiose 13.5g/L, sucrose 24g/L, plant gel 5g/L；
Additional adding ingredient: phytondione 0.8mg/L, riboflavin 1.4mg/L, nucleotide 3.5mg/L, L-carnitine
0.24mg/L, polyvinylpyrrolidone 1.3mg/L, soybean lecithin 2.6mg/L, lanthanum sulfate 0.08mg/L, N- nitroso-N- second
Base urethane 0.07mg/L, dithiothreitol (DTT) 15mg/L；
Plant extraction liquid: Radix Astragali extractive solution 16mL/L, root of kudzu vine extract 17.5mL/L；
Plant growth regulator: 6-BA 3.0mg/L, 2-(3,4- dichlorophenoxy) triethylamine 1.1mg/L, 5- nitro guaiaci lignum
Phenol sodium 0.4mg/L.
Radix Astragali extractive solution described in step (2) the preparation method comprises the following steps: weigh appropriate Radix Astragali, ground after 30 DEG C of forced air dryings,
It is added in the distilled water of 20 times of volumes and impregnates 2h, 100 DEG C of decoction 30min are filtered, and residue adds the distilled water 100 of 20 times of volumes again
DEG C 30min, filtering are decocted, rotary evaporation is concentrated into the 1/10 of original volume, spare after 115 DEG C of sterilizing 15min；Root of kudzu vine extract
Preparation method is identical as Radix Astragali extractive solution.
Green-light source described in step (3) is issued by one-color fluorescence lamp, 490 ~ 590nm of wave-length coverage, rated power 40W.
The formula of strengthening seedling and rooting culture medium described in step (4) are as follows: 1/2MS culture medium+caseinhydrolysate 50mg/L+ pyridine
Alcohol 0.5mg/L+IBA1.0mg/L.
The ingredient of matrix described in step (5) are as follows: 5 parts of peat soil, 5 parts of vermiculite, 2 parts of fulvic acid, 3 parts of powdered rice hulls, oyster mushroom
1 part of mushroom bran.
The ingredient of enzymolysis liquid described in step (6) are as follows: cellulase 4%, zytase 2%, pectase 1%, lemon acid buffering
Liquid 93%.
Celery normal ploidy plant chromosome number described in step (6) is 2n=22.
The beneficial effect comprise that:
1) celery microspore callus atomization is optimized in the present invention, and the differential medium of use is in conventional training
Support and improved on the basis of base, optimize nutrition content and proportion, increase polyvinylpyrrolidone, soybean lecithin,
Lanthanum sulfate, N- nitroso-N- ethyl urethane, dithiothreitol (DTT), astragalus extraction, root of kudzu vine extract, 2-(3,4- dichlorophenoxy)
Triethylamine, 5- nitroguaiacol sodium salt etc. have the substance for improving callus differentiation frequency；Callus is additionally used simultaneously
It is dried, the means of green light source irradiation further increase callus differentiation frequency.
2) present invention establishes celery microspores culture regenerating system, successfully obtains regeneration plant and transplant survival, passes through
Methods of Ploidy Identification confirmation obtains celery monoploid and double haploid, provides for the haploid breeding work of celery
Basic material is of great significance to celery breeding of new variety.
Specific embodiment
In order to make the technical solution of the present invention easy to understand, below in conjunction with specific test example to a kind of celery microspore of the present invention
The method of callus differentiation and regeneration plant is further described.
Embodiment 1
The test of celery microspore callus differentiation and regeneration plant is carried out according to technical solution provided by the invention, test material is this
The celery kind texts and pictures that R&D team's early-stage study obtains are drawn and the odd No. 1 microspore callus of saliva, the process of the test are as follows:
(1) callus is dried
By the callus that celery microspore induces be put into paving have three layers aseptic filter paper culture dish in, be placed in 24 DEG C, it is dark
Dry 2 ~ 3d, makes callus sectors dehydration under environment；
(2) differential medium is prepared according to formula as below
A great number of elements: NH4NO3955mg/L, KNO3 1650mg/L, KH2PO4107mg/L, K2HPO4 69mg/L, MgSO4·
7H2O 250mg/L, Ca(NO3)2208mg/L；
Microelement: KI 0.79mg/L, H3BO34.6mg/L, MnSO4·4H2O 21.5mg/L, ZnSO4·7H2O 8.75mg/
L, Na2MoO4·2H2O 0.23mg/L, CuSO4·5H2O 16.5mg/L, CoCl2·6H2O 0.025mg/L；
Molysite complex compound: ferrous glycine 29.4mg/L；
Conventional organic principle: inositol 100mg/L, niacin 1.3mg/L, puridoxine hydrochloride 2.0mg/L, thiamine hydrochloride 6.8mg/L；
Carbon source and coagulator: cellobiose 13.5g/L, sucrose 24g/L, plant gel 5g/L；
Additional adding ingredient: phytondione 0.8mg/L, riboflavin 1.4mg/L, nucleotide 3.5mg/L, L-carnitine
0.24mg/L, polyvinylpyrrolidone 1.3mg/L, soybean lecithin 2.6mg/L, lanthanum sulfate 0.08mg/L, N- nitroso-N- second
Base urethane 0.07mg/L, dithiothreitol (DTT) 15mg/L；
Plant extraction liquid: Radix Astragali extractive solution 16mL/L, root of kudzu vine extract 17.5mL/L；Radix Astragali extractive solution is the preparation method comprises the following steps: weigh
Appropriate Radix Astragali grinds after 30 DEG C of forced air dryings, is added in the distilled water of 20 times of volumes and impregnates 2h, 100 DEG C of decoction 30min, filtering,
Residue adds 100 DEG C of decoction 30min of distilled water of 20 times of volumes again, and filtering, rotary evaporation is concentrated into the 1/10 of original volume, and 115 DEG C
It is spare after sterilizing 15min；The preparation method of root of kudzu vine extract is identical as Radix Astragali extractive solution；
Plant growth regulator: 6-BA 3.0mg/L, 2-(3,4- dichlorophenoxy) triethylamine 1.1mg/L, 5- nitro guaiaci lignum
Phenol sodium 0.4mg/L；PH is adjusted 5.8 after preparing.
(3) callus differentiation culture
Callus after drying process is inoculated on above-mentioned differential medium and carries out Fluctuation temperature culture, cultivation temperature is set as light
According to when 25 DEG C, 21 DEG C, light application time 11h/d, interlunation 13h/d when dark, illumination use green-light source by monochrome
Fluorescent lamp issues, 490 ~ 590nm of wave-length coverage, rated power 40W, and intensity 1800Lx, every 20d or so are transferred to fresh differentiation training
Feeding base makees squamous subculture, and visible callus starts gradually to break up after 30 ~ 40d, the visible adventitious bud sprouted or clump after 50 ~ 60d
Raw seedling；Count the phenylacetic acid (differentiation rate=differentiate adventitious bud or grow thickly of texts and pictures drawing and odd No. 1 two kind of saliva
The callus number of seedling/inoculation callus sum).
(4) strengthening seedling and rooting culture
The long seedling single plant to 3 ~ 42 ~ 3cm high, tool true leaves is cut and is transferred in strengthening seedling and rooting culture medium (ingredient 1/2MS
Culture medium+caseinhydrolysate 50mg/L+ pyridine alcohol 0.5mg/L+IBA1.0mg/L), in 25 DEG C of cultivation temperature, light application time 15h/
D, it is small that the regeneration with complete root system can be formed under conditions of interlunation 9h/d, intensity of illumination 2800Lx, after 20 ~ 30d of culture
Seedling.
(5) rooting culture
When regenerate seedling root system it is long to 3 ~ 4cm long when, open hardening 1 week in culturing room, then seedling taken from culture bottle
Out, the flower for filling matrix (ingredient is 5 parts of peat soil, 5 parts of vermiculite, 2 parts of fulvic acid, 3 parts of powdered rice hulls, 1 part of oyster mushroom mushroom bran) is moved into
In alms bowl, polybag moisturizing is buckled, natural lighting is given, big Tanaka's field planting is transplanted to after seedling extracts young leaves out.
Embodiment 2(is dried the influence broken up to celery microspore callus)
According to the operation that the method that embodiment 1 provides, cancellation step (1) callus are dried, celery microspore is induced
Callus out is directly inoculated on differential medium, carries out differentiation culture at identical conditions, is counted texts and pictures respectively and is drawn
With the phenylacetic acid of odd No. 1 two kind of saliva, the results are shown in Table 1.
As it can be seen from table 1 callus is first dried, texts and pictures are drawn and the callus of odd No. 1 two kind of saliva
4.7% and 5.4% has been respectively increased in tissue differentiation rate, illustrates to be dried the differentiation frequency that celery microspore callus can be improved.
Callus after drying process is in dehydration starvation, it may be more advantageous to after being inoculated on differential medium to nutrition
Substance and plant growth regulator fully absorb, while causing a series of physiology, the variation on biochemistry, as drawn under Water deficit
The raising etc. of certain signaling molecule contents is played, and then improves the differentiation rate of callus.
The influence that embodiment 3(differential medium and plant growth regulator break up celery microspore callus)
According to the method that embodiment 1 provides, the differential medium in step (2) and (3) is replaced with into following a few breedings and is routinely trained
Base is supported, and addition 6-BA 3.0mg/L is as plant growth regulator: W14 culture medium, MS culture medium, B5 medium, N6 training
Feeding base, C17 culture medium carry out differentiation culture at identical conditions, count being cured for odd No. 1 two kinds of texts and pictures drawing and saliva respectively
Injured tissue differentiation rate, the results are shown in Table 2.
From table 2 it can be seen that texts and pictures are drawn and the differentiation rate of odd No. 1 two kind of saliva on differential medium of the invention
32.4% and 45.5% are respectively reached, the phenylacetic acid being all remarkably higher than on other differential mediums illustrates of the invention
Differential medium is more advantageous to the differentiation of celery microspore callus.Differential medium of the invention optimize a great number of elements,
The content and proportion of microelement, molysite complex compound, conventional organic principle etc., make it more suitable for celery microspore callus
Proliferation, Differentiation, additional adding ingredient, plant extraction liquid and 6-BA, 2-(3,4- dichlorophenoxy) triethylamine, 5- nitro be cured
The wooden phenol sodium is created as plant growth regulator, these ingredients all have positive effect to the growth and development of callus, Organ Differentiation
It answers, achievees the purpose that improve phenylacetic acid jointly.
The influence that embodiment 4(light source breaks up celery microspore callus)
According to the method that embodiment 1 provides, the light source used in step (3) is replaced with following several: white light, feux rouges, blue light,
Yellow light, light source are issued by one-color fluorescence lamp, wavelengths of white light 410 ~ 690nm of range, 600 ~ 900nm of red wavelength range, blue light wave
Long 410 ~ 540nm of range, yellow wavelengths 520 ~ 650nm of range, rated power is 40W, is broken up at identical conditions
Culture counts the phenylacetic acid of texts and pictures drawing and odd No. 1 two kind of saliva respectively, and the results are shown in Table 3.
From table 3 it can be seen that texts and pictures are drawn and the differentiation of odd No. 1 two kind of saliva using green light of the invention as light source
Rate highest illustrates the differentiation that celery microspore callus is more advantageous to using green light as light source.Plant tissue is in growth and development
It will receive the adjustment effect of light in the process, the most suitable light source of different plants is different.White light is that Plant Tissue Breeding longest is used
Light source, and we in experiments it is found that, celery microspore callus differentiation incubation in apply green light source, obtain
Effect it is more preferable than white light, it may be possible to the adjustment effect that green light source breaks up celery callus proliferation is stronger.
Embodiment 5(regeneration plant Methods of Ploidy Identification)
Texts and pictures draw 67 plants of regeneration plant, and saliva is No. 1 99 plants of regeneration plant odd, are contaminated using tip of a root decoration method these regeneration plants
Colour solid Ploidy Identification: choosing the tender tip of a root of children of 1cm or so, handles 3h with 8 ~ oxyquinoline solution of 0.002 mol/L, then puts
Enter in Kano solution (dehydrated alcohol: glacial acetic acid=3:1) processing for 24 hours, be put into after 3 times wash with distilled water enzymolysis liquid (ingredient are as follows:
Cellulase 4%, zytase 2%, pectase 1%, citrate buffer solution 93%) in, 1h is digested in 37 DEG C of water-baths；Then by root
Point is placed on glass slide, is dyed with carbolfuchsin solution, under the microscope the ploidy of each regeneration plant of observation analysis, with
Celery normal ploidy plant is compared, and the results are shown in Table 4.
Celery is diplont, and normal chromosomal number is 2n=22, and haploid chromosome number is n=11.It can from table 4
To find out, texts and pictures are drawn in the 67 plants of regeneration plants generated, 51 plants of monoploid, are accounted for 76.1%, 14 plants of diploid, are accounted for 20.9%, saliva surprise 1
Number generate 99 plants of regeneration plants in, 59 plants of monoploid, account for 59.6%, 37 plants of diploid, account for 37.4%, it is seen that different genotype it
Between regeneration plant times sex ratio difference.Since the callus that the present invention uses is got by celery microspore Fiber differentiation, base
Originally the interference of the body cells such as anther wall can be excluded, so the liploid plant obtained is that during the cultivation process, monoploid passes through
Double haploid caused by Natural double.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (8)
1. a kind of method of celery microspore callus differentiation and regeneration plant, which is characterized in that the techniqueflow of this method is as follows:
(1) callus is dried
By the callus that celery microspore induces be put into paving have three layers aseptic filter paper culture dish in, be placed in 23 ~ 25 DEG C, it is black
Dry 2 ~ 3d, makes callus sectors dehydration under dark environment；
(2) differential medium is prepared according to formula as below
A great number of elements: NH4NO3930 ~ 980mg/L, KNO3 1550 ~ 1750mg/L, KH2PO4104 ~ 110mg/L, K2HPO4 65~
73mg/L, MgSO4·7H2O 245 ~ 255mg/L, Ca(NO3)2204~212mg/L；
Microelement: KI 0.77 ~ 0.81mg/L, H3BO34.3 ~ 4.9mg/L, MnSO4·4H2O 20 ~ 23mg/L, ZnSO4·
7H2O 8.5 ~ 9.0mg/L, Na2MoO4·2H2O 0.21 ~ 0.25mg/L, CuSO4·5H2O 15 ~ 18mg/L, CoCl2·6H2O
0.02~0.03mg/L；
Molysite complex compound: 28.6 ~ 30.2mg/L of ferrous glycine；
Conventional organic principle: 95 ~ 105mg/L of inositol, 1.2 ~ 1.4mg/L of niacin, 1.5 ~ 2.5mg/L of puridoxine hydrochloride, hydrochloric acid sulphur
6.6 ~ 7.0mg/L of amine element；
Carbon source and coagulator: 12 ~ 15g/L of cellobiose, 22 ~ 26g/L of sucrose, 4 ~ 6g/L of plant gel；
Additional adding ingredient: 0.7 ~ 0.9mg/L of phytondione, 1.3 ~ 1.5mg/L of riboflavin, 3.3 ~ 3.7mg/L of nucleotide are left
Revolve 0.22 ~ 0.25mg/L of carnitine, 1.1 ~ 1.5mg/L of polyvinylpyrrolidone, 2.5 ~ 2.7mg/L of soybean lecithin, lanthanum sulfate
0.06 ~ 0.1mg/L, N- nitroso-N- ethyl 0.06 ~ 0.08mg/L of urethane, 13 ~ 17mg/L of dithiothreitol (DTT)；
Plant extraction liquid: 14 ~ 18mL/L of Radix Astragali extractive solution, 15 ~ 20mL/L of root of kudzu vine extract；
Plant growth regulator: 6-BA 2.8 ~ 3.2 mg/L, 2-(3,4- dichlorophenoxy) triethylamine 0.9 ~ 1.3mg/L, 5- nitre
0.3 ~ 0.5mg/L of base guaiacol sodium；PH is adjusted between 5.6 ~ 6.0 after preparing；
(3) callus differentiation culture
Callus after drying process is inoculated on above-mentioned differential medium and carries out Fluctuation temperature culture, cultivation temperature is set as light
According to when 24 ~ 26 DEG C, 20 ~ 22 DEG C when dark, light application time be 10 ~ 12h/d, interlunation be 12 ~ 14h/d, illumination use green light
Light source, intensity are 1500 ~ 2000Lx, and every 20d or so is transferred to fresh differential medium and makees squamous subculture, visible callus after 30 ~ 40d
Tissue starts gradually to break up, the visible adventitious bud sprouted or seedling of growing thickly after 50 ~ 60d；
(4) strengthening seedling and rooting culture
The long seedling single plant to 3 ~ 42 ~ 3cm high, tool true leaves is cut and is transferred in strengthening seedling and rooting culture medium, in cultivation temperature
23 ~ 27 DEG C, 14 ~ 16h/d of light application time, 8 ~ 10h/d of interlunation, under conditions of 2600 ~ 3000Lx of intensity of illumination, culture 20 ~
The regeneration seedling with complete root system can be formed after 30d；
(5) rooting culture
When regenerate seedling root system it is long to 3 ~ 4cm long when, open hardening 1 week in culturing room, then seedling taken from culture bottle
Out, it moves into the flowerpot for filling matrix, buckles polybag moisturizing, give natural lighting, transplanted after seedling extracts young leaves out
It is colonized to big Tanaka；
(6) regeneration plant Methods of Ploidy Identification
Carry out Methods of Ploidy Identification to the regeneration plant after transplant survival using tip of a root decoration method: the children for choosing 1cm or so is tender
The tip of a root handles 3h with 8 ~ oxyquinoline solution of 0.002 mol/L, be then placed in Kano solution (dehydrated alcohol: glacial acetic acid=3:
1) processing for 24 hours, is put into enzymolysis liquid after 3 times wash with distilled water, digests 1h in 37 DEG C of water-baths in；Then the tip of a root is placed on load
On slide, dyed with carbolfuchsin solution, the ploidy of each regeneration plant of observation analysis, normal with celery under the microscope
Ploidy plant is compared.
2. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(2) optimization formula of differential medium described in is as follows:
A great number of elements: NH4NO3955mg/L, KNO3 1650mg/L, KH2PO4107mg/L, K2HPO4 69mg/L, MgSO4·
7H2O 250mg/L, Ca(NO3)2208mg/L；
Microelement: KI 0.79mg/L, H3BO34.6mg/L, MnSO4·4H2O 21.5mg/L, ZnSO4·7H2O 8.75mg/
L, Na2MoO4·2H2O 0.23mg/L, CuSO4·5H2O 16.5mg/L, CoCl2·6H2O 0.025mg/L；
Molysite complex compound: ferrous glycine 29.4mg/L；
Conventional organic principle: inositol 100mg/L, niacin 1.3mg/L, puridoxine hydrochloride 2.0mg/L, thiamine hydrochloride 6.8mg/L；
Carbon source and coagulator: cellobiose 13.5g/L, sucrose 24g/L, plant gel 5g/L；
Additional adding ingredient: phytondione 0.8mg/L, riboflavin 1.4mg/L, nucleotide 3.5mg/L, L-carnitine
0.24mg/L, polyvinylpyrrolidone 1.3mg/L, soybean lecithin 2.6mg/L, lanthanum sulfate 0.08mg/L, N- nitroso-N- second
Base urethane 0.07mg/L, dithiothreitol (DTT) 15mg/L；
Plant extraction liquid: Radix Astragali extractive solution 16mL/L, root of kudzu vine extract 17.5mL/L；
Plant growth regulator: 6-BA 3.0mg/L, 2-(3,4- dichlorophenoxy) triethylamine 1.1mg/L, 5- nitro guaiaci lignum
Phenol sodium 0.4mg/L.
3. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(2) Radix Astragali extractive solution described in the preparation method comprises the following steps: weigh appropriate Radix Astragali, ground after 30 DEG C of forced air dryings, 20 times of volumes be added
Distilled water in impregnate 2h, 100 DEG C of decoction 30min, filter, residue adds 100 DEG C of decoction 30min of distilled water of 20 times of volumes again,
Filtering, rotary evaporation are concentrated into the 1/10 of original volume, spare after 115 DEG C of sterilizing 15min；The preparation method and Huang of root of kudzu vine extract
Stilbene extracting solution is identical.
4. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(3) green-light source described in is issued by one-color fluorescence lamp, 490 ~ 590nm of wave-length coverage, rated power 40W.
5. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(4) formula of strengthening seedling and rooting culture medium described in are as follows: 1/2MS culture medium+caseinhydrolysate 50mg/L+ pyridine alcohol 0.5mg/L+
IBA1.0mg/L。
6. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(5) ingredient of matrix described in are as follows: 5 parts of peat soil, 5 parts of vermiculite, 2 parts of fulvic acid, 3 parts of powdered rice hulls, 1 part of oyster mushroom mushroom bran.
7. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(6) ingredient of enzymolysis liquid described in are as follows: cellulase 4%, zytase 2%, pectase 1%, citrate buffer solution 93%.
8. a kind of method of celery microspore callus differentiation and regeneration plant according to claim 1, which is characterized in that step
(6) the plant chromosome number of celery normal ploidy described in is 2n=22.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111528101A (en) * | 2020-06-18 | 2020-08-14 | 陈滋倩 | Differentiation culture medium for carnation anther culture |
| CN113881698A (en) * | 2021-10-29 | 2022-01-04 | 上海市农业科学院 | Method for transforming barley microspore callus by utilizing agrobacterium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111528101A (en) * | 2020-06-18 | 2020-08-14 | 陈滋倩 | Differentiation culture medium for carnation anther culture |
| CN113881698A (en) * | 2021-10-29 | 2022-01-04 | 上海市农业科学院 | Method for transforming barley microspore callus by utilizing agrobacterium |
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