CN106342680A

CN106342680A - Method suitable for cultivation of various genotype wheat microspores

Info

Publication number: CN106342680A
Application number: CN201510419370.3A
Authority: CN
Inventors: 夏兰琴; 杜文明; 孙永伟
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2015-07-16
Filing date: 2015-07-16
Publication date: 2017-01-25
Anticipated expiration: 2035-07-16
Also published as: CN106342680B

Abstract

The invention discloses a method suitable for cultivation of various genotype wheat microspores. A method for cultivating the wheat microspores to obtain regenerated plants, provided by the invention, comprises the following steps: (1) inoculating the microspores of to-be-cultivated wheat to an embryoid induction culture medium and performing induction culture to obtain microspore embryoids; (2) inoculating the microspore embryoids to a regeneration culture medium and performing regeneration culture to obtain regenerated seedlings; (3) inoculating the regenerated seedlings to a rooting culture medium and performing rooting culture to obtain regenerated plants of the to-be-cultivated wheat. The method is suitable for various genotype wheat varieties; furthermore, the embryoid induction ratio, the plant regeneration ratio, the green seedling ratio and the natural doubling ratio of the method are obviously higher than those of the traditional conventional microspore cultivation method. The method has important significance and application value for accelerating wheat haploid breeding and transgenic wheat commercialization progress.

Description

A kind of method being applied to the culture of Multi-genotype Wheat mitochondria

Technical field

The invention belongs to field of plant tissue culture, it is related to a kind of side being applied to the culture of Multi-genotype Wheat mitochondria Method, obtains method and the special culture media of regeneration plant particularly to a kind of culture Wheat mitochondria.

Background technology

Semen Tritici aestivi, as one of most important cereal crops, is the materials such as human protein, vitamin, fiber and magnesium, phosphorus Main picked-up source.Estimate, the world is little according to " world's food and Agricultural Sustainable Development " conference that 1997 hold Speed with 1.6% is increased by wheat demand, and the year two thousand twenty is up to 7.75 hundred million tons, 5.52 hundred million tons of increasings than 1993 Long 40%.China's the year two thousand twenty Semen Tritici aestivi demand will be about 1.4～1.5 hundred million tons, increases by 4600～5600 than current production rate Ten thousand tons, increase by 49～60%.Since the establishment of the nation, China has cultivated wheat breed more than 2000, realizes Cultivar replacing Regenerate 6～8 times, per unit area yield brings up to 350 kgs/acre from 43 kgs/acre, the effective supply for ensureing China's grain is made Tremendous contribution.But closely during the last ten years, wheat yield improves speed and eases up, and per unit area yield is in the state of hovering.In view of at present Cultivated Land Area Decrease and lasting population increase, and China's Wheat Production still faces very big challenge.

As self pollination crop, cross-breeding and wheat transgenic technology are considered as to improve the weight of wheat yield to Semen Tritici aestivi Want approach.But cross-breeding and transgenic line obtain homozygous lines needs continuous selfing many generations, per generation be required for through Evaluation and screening in large quantities, this process, in the case of 1 year generation, generally required for 5～6 years.In monoploid In breeding, by building dh colony, it is to avoid hybridize segregation phenomenon in cross-breeding, a generation obtains homozygous line, shortens Breeding cycle 3～4 years.Because each allele is that homozygosis is stable in dh system, thus offspring's choosing can be improved Select efficiency, beneficial to the initiative of new wheat germplasm.Wheat transgenic technology combines microspores culture, chooses transgenic t₀ Or t₁Carry out microspores culture for plant children's fringe, in 1～2 year, obtain transfer-gen plant homozygous line, shorten cycle 4～ 5 years.

Dh colony and transgenic homozygous strain can quickly be obtained by Wheat mitochondria culture.Build dh colony, from Hybridization group is fitted on the acquisition of stable pure lines, is equivalent to conventional breeding f₃Generation, general 3～4 years of breeding cycle.Transgenic Semen Tritici aestivi, after obtaining transfer-gen plant, can obtain homozygous line by microspores culture a generation.But Semen Tritici aestivi is little Spore cultivation has genotype-independent, and it is different with complexity that different genetic wheat varieties sporidiole embryoid produces frequency. Hu etc. carries out microspores culture to different genetic wheat varieties and their filial generation, finds Semen Tritici aestivi chris genotype tool There is preferable regeneration situation, but chris and sinton filial generation have sporidiole regeneration rate and be significantly higher than parent.

A generation is capable of by Wheat mitochondria culture and obtains pure lines, shorten in wheat breed cultivating process continuously certainly Hand over the time limit.Therefore, microspores culture is carried out to important wheat transgenic acceptor material and domestic central genetic breeding material Genotype screening, by microspores culture, quick obtain Semen Tritici aestivi dh colony and transgenic wheat homozygous line, for plus Fast Wheat Haploid breeding and the commercialization process of transgenic wheat, are of great significance and value.

Content of the invention

It is an object of the invention to provide a kind of method that culture Wheat mitochondria obtains regeneration plant.

The method that culture Wheat mitochondria provided by the present invention obtains regeneration plant, specifically mays include:

(1) sporidiole of Semen Tritici aestivi to be cultivated is inoculated in embryoid induction culture medium, carries out inducing culture, obtain little Spore embryoid；

(2) described sporidiole embryoid is inoculated in regeneration culture medium, carries out regeneration culture, obtain regrowth；

The solvent of described regeneration culture medium is water, and solute and concentration are as follows: kno₃1500mg/l；nh₄no₃250 mg/l；kh₂po₄200mg/l；cacl₂·2h₂o 450mg/l；mgso₄·7h₂o 350mg/l；ki 0.75mg/l； mnso₄15mg/l；h₃bo₃5mg/l；znso₄·7h₂o 7.5mg/l；cocl·6h₂o 0.025mg/l； cuso₄·5h₂o 0.025mg/l；na₂moo₄·2h₂o 0.25mg/l；feso₄·7h₂o 27.80mg/l；na₂edta 37.20mg/l；vb1 10mg/l；vb6 1mg/l；Nicotinic acid (acide nicotinique) 1mg/l；Inositol (myo-inositol)200mg/l；Calcium pantothenate (ca-pantothenate) 1.0mg/l；Ascorbic acid (ascorbic acid)1.0mg/l；Maltose 30000mg/l；2,4-d 0.10mg/l；Zeatin 5.0mg/l；cuso₄25 mg/l；ph5.70.

(3) described regrowth is inoculated in root media, carries out root culture, obtain described in Semen Tritici aestivi to be cultivated Regeneration plant.

In methods described step (1), the described sporidiole being inoculated in described embryoid induction culture medium can be from process Separate in the following wheat children tassel processing and obtain: by described wheat children tassel (with young tip of the spike away from boot leaf lower end 2～3cm It is preferred) it is placed in the cuso of 500mg/l₄In aqueous solution, 4 DEG C of lucifuges are processed 10 days.

Separately obtain described sporidiole from the described wheat children tassel that process is processed as above specifically to enter in accordance with the following steps OK: 1) will be with 5.25% (volume fraction) naclo sterilization 3 after described wheat children tassel being processed as above and cuts off the awn of wheat min；With sterile water wash (as cleaning 3 times, each 1min)；2) remove described wheat children tassel coetonium, collect little Flower is placed in sporidiole Extraction buffer, and in tissue mixer device, 6000g stirring 7s, repeats 3～5 times；With 300 Mesh filter screen filters and removes disrupting tissue；3) use a diameter of 100 μm of filter opening aseptic sieve filtration step 2) filtrate, Gained filtrate is centrifuged 5min in 4 DEG C of 100g；4) with the resuspended step 3 of described sporidiole Extraction buffer) centrifugation institute Must precipitate, be centrifuged 5min then at 4 DEG C of 100g；5) use the resuspended step 4 of npb99 buffer) centrifugation gained precipitation, It is centrifuged 5min then at 4 DEG C of 100g；6) use the resuspended step 5 of npb99 buffer described in 2ml) centrifugation gained precipitation； 7) take a centrifuge tube, add 2ml 30%percoll buffer (formula: solution: 121.33g/l Mannitol, 2.13g/l MES (mes), ph7.0, sucking filtration sterilizes；Solution: 2.13g/l MES (mes), Ph7.0, sucking filtration sterilizes；The solvent of above-mentioned 2 kinds of solution is water；Solution: 50ml percoll, sucking filtration sterilizes； Above-mentioned 3 kinds of solution are mixed according to the ratio that volume ratio i:ii:iii is 6:1:3), then by step 6) resuspended after Semen Tritici aestivi Sporidiole is gently laid in above described 30%percoll buffer, is centrifuged 5min in 4 DEG C of 100g, in centrifugation gained " white " band (i.e. monokaryon later stage or double-core early stage sporidiole) in the center of mixed liquor；8) draw Described " white " band, (centrifugation of this step can to add described npb99 buffer to be centrifuged 5min after 4 DEG C of 100g Repetitive operation is once), gained precipitation is purpose Wheat mitochondria.

Wherein, the solvent of described sporidiole Extraction buffer is water, and solute and concentration are as follows: kno₃1900mg/l； nh₄no₃165mg/l；kh₂po₄170mg/l；cacl₂·2h₂o 440mg/l；mgso₄·7h₂o 370mg/l； feso₄·7h₂o 27.8mg/l；na₂edta 37.2mg/l；D- Mannitol 72866mg/l；MES (mes)150mg/l；ph7.0.

The solvent of described npb99 buffer is water, and solute and concentration are as follows: kno₃1415mg/l；(nh₄)₂so₄232 mg/l；kh₂po₄200mg/l；cacl₂·2h₂o 83mg/l；mgso₄·7h₂o 93mg/l；ki 0.4mg/l； mnso₄5mg/l；h₃bo₃5mg/l；znso₄·7h₂o 5mg/l；cocl·6h₂o 0.0125mg/l； cuso₄·5h₂o 0.0125mg/l；na₂moo₄·2h₂o 0.0125mg/l；feso₄·7h₂o 27.8mg/l； na₂edta 37.2mg/l；vb1 5mg/l；vb6 0.5mg/l；Nicotinic acid (acide nicotinique) 0.5mg/l； Inositol (myo-inositol) 50mg/l；L-Glutamine 500mg/l；Glutathione 0.615mg/l；Maltose 90 g/l；2,4-d 0.2mg/l；6-Furfurylaminopurine (kinetin) 0.2mg/l；Cefotaxime (cefotaxime) 100 mg/l；ph7.0.

In the step (1) of methods described, during carrying out described inducing culture, in the training of described embryoid induction The ovary of Semen Tritici aestivi to be cultivated described in coming from is added in foster base；3 are added in embryoid induction culture medium described in every ml Individual described ovary.

In the step (1) of methods described, described sporidiole is inoculated in after described embryoid induction culture medium, institute State content in described embryoid induction culture medium for the sporidiole and be about 2 × 10⁵Individual/ml.

In the step (1) of methods described, described inducing culturing condition is cultivated to described microspore embryoid for 28 DEG C of lucifuges During a diameter of 1～2mm of shape body only (4～6 weeks).

In the step (2) of methods described, the condition of described regeneration culture is 16h illumination/8h dark, 23 DEG C of trainings Support and only (4～5 weeks) grow to during 2～3cm to described regrowth.

In the step (3) of methods described, the condition of described root culture is 16h illumination/8h dark, 23 DEG C of trainings Support 3～4 weeks.

In the process, the solvent of described embryoid induction culture medium is water, and solute and concentration are as follows: kno₃1415 mg/l；(nh4)₂so₄232mg/l；kh₂po₄200mg/l；cacl₂·2h₂o 83mg/l；mgso₄·7h₂o 93 mg/l；ki 0.4mg/l；mnso₄5mg/l；h₃bo₃5mg/l；znso₄·7h₂o 5mg/l；cocl·6h₂o 0.0125mg/l；cuso₄·5h₂o 0.0125mg/l；na₂moo₄·2h₂o 0.0125mg/l；feso₄·7h₂o 27.8 mg/l；na₂edta 37.2mg/l；vb1 5mg/l；vb6 0.5mg/l；Nicotinic acid (acide nicotinique) 0.5 mg/l；Inositol (myo-inositol) 50mg/l；L-Glutamine 500mg/l；Glutathione 0.615mg/l； Ficoll (ficoll-400) 100 000mg/l；Maltose 90000mg/l；2,4-d 0.2mg/l；Bran amidopurin (kinetin)0.2mg/l；Cefotaxime (cefotaxime) 100mg/l；ph7.0.

Described root media is by 1/2ms a great number of elements, 1/2ms trace element, 1/2ms organic principle, 30g/l Sucrose and 0.6mg/l naa composition, specifically its formula is as follows: solvent is water, and solute and concentration are as follows: kno₃ 950mg/l；nh₄no₃825mg/l；kh₂po₄85mg/l；cacl₂·2h₂o 220mg/l；mgso₄·7h₂o 185 mg/l；ki 0.415mg/l；mnso₄11.15mg/l；h₃bo₃3.10mg/l；znso₄·7h₂o 4.30mg/l； cocl·6h₂o 0.0125mg/l；cuso₄·5h₂o 0.0125mg/l；na₂moo₄·2h₂o 0.125mg/l； feso₄·7h₂o 13.9mg/l；na₂edta 18.65mg/l；Inositol (myo-inositol) 50mg/l；vb1 0.05 mg/l；vb6 0.25mg/l；Nicotinic acid (acide nicotinique) 0.25mg/l；naa 0.6mg/l；Sucrose 30g/l；Plant gel (pytagel) 3g/l；ph 5.70.

The described regeneration plant that methods described also includes step (3) is obtained carries out Methods of Ploidy Identification, to single times The step that body plant carries out artificial doubling.The method of described artificial doubling is processed for Colchicine.

It is a further object to provide a kind of culture medium obtaining regeneration plant for cultivating Wheat mitochondria.

The culture medium obtaining regeneration plant for cultivating Wheat mitochondria provided by the present invention, for as follows (a) or (b):

(a) described regeneration culture medium；

B () is made up of described embryoid induction culture medium, described regeneration culture medium and described root media.

Described culture medium obtains the application in regeneration plant in culture Wheat mitochondria and falls within protection scope of the present invention.

In the present invention, described Semen Tritici aestivi can be Multi-genotype Semen Tritici aestivi, such as Zheng wheat 7698, all wheats 22, short by anti-58, Polling 987, bobwhite, fielder, cadenza, florida or section's agriculture 199.

Culture Wheat mitochondria provided by the present invention obtains the method for regeneration plant it is adaptable to Multi-genotype Semen Tritici aestivi product Kind, and notable using the embryoid induction number of every fringe, regrowth number, green Seedling number and Natural doubling rate during the method Higher than traditional conventional microspores culture method.The present invention is for the business accelerating Wheat Haploid breeding and transgenic wheat Industry process, is of great significance and value.

Brief description

Fig. 1 isolates and purifies for Wheat mitochondria and incubation.A. wheat children tassel is chosen；B-c. wheat children tassel go awns and Sterilization；D-f. active Wheat mitochondria extracting and developing process；G. monokaryon later stage Wheat mitochondria；H. the little spore of Semen Tritici aestivi Sub- embryoid induction.

Fig. 2 is different genetic wheat varieties sporidiole embryoid induction situation.A. section's agriculture 199；B. short by anti-58； c.bobwhite；d.cadenza；e.fielder；f.florida；G. polling 987；H. all wheats 22；I. Zheng wheat 7689.

Fig. 3 is different genetic wheat varieties sporidiole embryoid regeneration induction seedling.A. section's agriculture 199；B. short by anti-58； c.bobwhite；d.cadenza；e.fielder；f.florida；G. polling 987；H. all wheats 22；I. Zheng wheat 7698.

Fig. 4 regenerates situation cartogram for different genetic wheat varieties sporidiole.A. embryoid induction regeneration situation；B. regenerate Seedling statistical conditions；C. Albino Seedling statistical conditions；D. green Seedling regenerates statistical conditions.

Fig. 5 is Wheat mitochondria regrowth Ploidy detection.A and c.fielder wild-type leaves pore opening and dyeing Body mesh number detects；B and d.fielder microspores culture regeneration plant pore and chromosome number detection.

Fig. 6 is different genetic wheat varieties sporule regeneration plant Natural double frequency statistics.

Specific embodiment

Experimental technique used in following embodiments if no special instructions, is conventional method.

Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.

In following embodiments, involved wheat breed has: Zheng wheat 7698, all wheats 22, short by anti-58, polling 987, Bobwhite, fielder, cadenza, florida, section's agriculture 199.

Zheng wheat 7698: be recorded in " Tian Wei, Zhang Shenju, Guo Zhensheng etc. super wheat Zheng wheat 7698 growth promoter characteristic And its matching technique study. Henan Agricultural Sciences, 07 phase in 2011 " literary composition, the public can obtain at applicant, but It is only limitted to repeat the present invention.

All wheats 22: be recorded in " Ni Yongjing, Ren Dechao, Ge Jun etc. straw-returning and nitrogenous fertilizer are joined and are applied to ' all wheats 22 ' Filling rate and the impact of yield. Chinese agriculture is circulated a notice of, 24 phases in 2013 " literary composition, the public can obtain at applicant , but be only limitted to repeat the present invention.

Short by anti-58: be recorded in " research of short anti-58 progeny variation of Zhang Xitai .nan3 mutation Semen Tritici aestivi and ssr analysis. make Thing is studied, 03 phase in 2011 " literary composition, the public can obtain at applicant, but is only limitted to repeat the present invention.

Polling 987: be recorded in " Yang Li. the selection-breeding of wheat breed polling 987 and popularization. the Chinese Academy of Agricultural Sciences, plant Plant, 2006, Master's thesis " literary composition, the public can obtain at applicant, but is only limitted to repeat the present invention.

Bobwhite and Ke Nong 199: be all recorded in " Liu Yang. common wheat new varieties Shan Nong 33 tissue culture's high frequency is again The foundation of raw body system. Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology, Crop Genetic Breeding, 2014, master " literary composition, the public can be from application Obtain at people, but be only limitted to repeat the present invention.

Fielder: be recorded in " Wang Xiaomin. Semen Tritici aestivi is anti-with the defence of active oxygen and defensin gene in strip rust bacteria Interaction Should and the identification of disease-resistant related gene and functional verification. Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology, Plant Pathology, 2010, doctor discusses Literary composition " one literary composition, the public can obtain at applicant, but is only limitted to repeat the present invention.

Cadenza: be recorded in " white rainbow. the qtl of Semen Tritici aestivi amount of nitrogen sucking, plant height, yield and Root Traits at Seedling Stage is fixed Position. Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology, Plant Nutrition, 2014, thesis for the doctorate " literary composition, the public can obtain at applicant, But it is only limitted to repeat the present invention.

Florida: be recorded in " Liu Yang, Wang Genping, Wang Chaojie etc. common wheat new varieties Shan agriculture 33 immature embryo is high The foundation of frequency regenerating system. northwest agricultural journal, 02 phase in 2015 " literary composition, the public can obtain at applicant, but It is only limitted to repeat the present invention.

In following embodiments, involved various culture medium and buffer formulation are as follows:

1st, sporidiole Extraction buffer

Formula is as shown in table 1.Solvent is water, and in table, each concentration of component is in Wheat mitochondria Extraction buffer Final concentration.

Table 1 Wheat mitochondria Extraction buffer formula (ph7.0)

Composition	mg/l
		A great number of elements
kno₃	1900
		nh₄no₃	165
kh₂po₄	170
		cacl₂·2h₂o	440
mgso₄·7h₂o	370
		Iron salt
feso₄·7h₂o	27.8
		na₂edta	37.2
Other
		d-mannitol	72866
mes	150

2nd, npb99 buffer

Formula is as shown in table 2.Solvent is water, and in table, each concentration of component is the final concentration in npb99 buffer.

The formula (ph7.0) of table 2 npb99 buffer

Composition	mg/l
		A great number of elements
kno₃	1415
		(nh4)₂so₄	232
kh₂po₄	200
		cacl₂·2h₂o	83
mgso₄·7h₂o	93
		Trace element
ki	0.4
		mnso₄	5
h₃bo₃	5
		znso₄·7h₂o	5
cocl·6h₂o	0.0125
		cuso₄·5h₂o	0.0125
na₂moo₄·2h₂o	0.0125
		Iron salt

feso₄·7h₂o	27.8
		na₂edta	37.2
Vitamin
		thiamine-hcl(vb1)	5
pyridoxine-hcl(vb6)	0.5
		acide nicotinique	0.5
myo-inositol	50
		glutamine	500
glutithione	0.615
		Other
maltose	90000
		2,4-d	0.2
kinetin	0.2
		cefotaxime	100
ph	7.0

3rd, embryoid induction culture medium

Formula is as shown in table 3.Solvent is water, and the end that in table, each concentration of component is in embryoid induction culture medium is dense Degree.

The formula (ph7.0) of table 3 embryoid induction culture medium

Composition	mg/l
		A great number of elements
kno₃	1415
		(nh4)₂so₄	232
kh₂po₄	200
		cacl₂·2h₂o	83
mgso₄·7h₂o	93
		Trace element
ki	0.4
		mnso₄	5
h₃bo₃	5
		znso₄·7h₂o	5

cocl·6h₂o	0.0125
		cuso₄·5h₂o	0.0125
na₂moo₄·2h₂o	0.0125
		Iron salt
feso₄·7h₂o	27.8
		na₂edta	37.2
Vitamin
		thiamine-hcl(vb1)	5
pyridoxine-hcl(vb6)	0.5
		acide nicotinique	0.5
myo-inositol	50
		glutamine	500
glutithione	0.615
		Other
ficoll-400	100000
		maltose	90000
2,4-d	0.2
		kinetin	0.2
cefotaxime	100
		ph	7.0

4th, regeneration culture medium

Formula is as shown in table 4.Solvent is water, and in table, each concentration of component is the final concentration in regeneration culture medium.

The formula (ph5.70) of table 4 regeneration culture medium

Composition	mg/l
		A great number of elements
kno₃	1500
		nh₄no₃	250
kh₂po₄	200
		cacl₂·2h₂o	450
mgso₄·7h₂o	350
		Trace element
ki	0.75

mnso₄	15
		h₃bo₃	5.0
znso₄·7h₂o	7.5
		cocl·6h₂o	0.025
cuso₄·5h₂o	0.025
		na₂moo₄·2h₂o	0.25
Iron salt
		feso₄·7h₂o	27.8
na₂edta	37.2
		Vitamin
thiamine-hcl(vb1)	10
		pyridoxine-hcl(vb6)	1
acide nicotinique	1
		myo-inositol	200
ca-pantothenate	1
		ascorbic acid	1
Other
		maltose	30000
2,4-d	0.1
		zeatin	5
cuso₄	25
		ph	5.70

5th, root media

Formula is as shown in table 5.By 1/2ms a great number of elements, 1/2ms trace element, 1/2ms organic principle, 30g/l Sucrose and 0.6mg/l naa composition.Solvent is water, and in table, each concentration of component is the final concentration in root media.

The formula (ph5.70) of table 5 root media

Embodiment 1, culture Multi-genotype Wheat mitochondria obtain regeneration plant

For trying wheat breed: include producing kind and the transgenic acceptor of upper spread.Wherein, in production The kind of spread has Zheng wheat 7698, all wheats 22, short anti-58 and polling 987；Transgenic acceptor has Bobwhite, fielder, cadenza, florida and Ke Nong 199.

1st, plant each in greenhouse or field for examination wheat breed.

2nd, in the wheat children tassel phase, choose 8 young fringes, be preferred away from boot leaf lower end 2～3cm with young tip of the spike, now little Spore development period is monokaryon middle and late stage and double-core early stage (as shown in a and g in Fig. 1), 4 DEG C of low temperature cuso₄Molten Liquid (500mg/l) pretreatment children's fringe (immersion) 10 days.

3rd, take the pretreated wheat children tassel of step 2, cut off the awn of wheat, disappeared with 5.25% (volume fraction) naclo Malicious 3min；With sterile water wash 3 times (as shown in Figure 1).Jar is put on ice for pre-cooling, and adds little spore Sub- Extraction buffer (table 1)；On aseptic paper, removal wheat children tassel is coetonium, collects little Hua in pre-cooling jar.

4th, 7s is stirred with 6000g, repeat 3～5 times；Extracted slow with the sporidiole after 300 mesh sieve net filtration stirrings Rush liquid in pre-cooling beaker, remove disrupting tissue, obtain filtrate 1.With a diameter of 100 μm of aseptic sieved filter of filter opening Filtrate 1, in 50ml sterile centrifugation tube, obtains filtrate 2.By filtrate 2 at 4 DEG C, 100g is centrifuged 5min, receives Collection sporidiole.Lightly slowly outwell supernatant, retain precipitation；With the new resuspended precipitation of sporidiole Extraction buffer, 100g centrifugation 5min (as shown in Figure 1) again.

5th, outwell supernatant, precipitation transferred in 15ml centrifuge tube, add 15ml npb99 buffer (table 2), 4 DEG C of 100g are centrifuged 5min.Outwell supernatant, add the resuspended precipitation of 2ml npb99 buffer.In a new centrifugation 2ml 30%percoll buffer (formula: solution: 121.325g/l Mannitol, 2.133g/l 2- is added in pipe Morpholino b acid (mes), ph7.0, sucking filtration sterilizes；Solution: 2.132g/l MES (mes), ph7.0, Sucking filtration sterilizes；Solution: 50ml percoll, sucking filtration sterilizes；The solvent of above-mentioned 3 kinds of solution is water；By above-mentioned 3 Kind of solution mixes according to the ratio that volume ratio i:ii:iii is 6:1:3), will be resuspended after precipitation be gently laid on 30%percoll On buffer, 4 DEG C of 100g are centrifuged 5min, " white " band (as Fig. 1 in mixed liquor center Shown).

6th, draw the sporidiole at " white " pillar location, transfer in a new 15ml centrifuge tube, use npb99 Buffer solution is once.4 DEG C of 100g centrifugation 5min collect sporidiole, outwell supernatant, add appropriate npb99 The resuspended sporidiole of buffer (as shown in Figure 1).

7th, add 3.3ml embryoid induction culture medium (table 3) in little culture dish, draw 200 μ l sporidioles and suspend Liquid, in each culture dish, adds 3 ovary/ml embryoid induction culture medium (same cultures in each culture dish Sporidiole in ware and ovary come from same wheat breed).With sealing each little culture dish of film phonograph seal, it is placed into big training In foster ware, place the culture dish added with sterilized water at big culture dish center, 28 DEG C of lucifuges cultivate 4～6 weeks (as Fig. 1 Shown), grow to 1～2mm to embryoid diameter.

8th, (different genetic wheat varieties sporidiole wound healing induces statistics different genetic wheat varieties sporidiole embryoid induction situation Situation is as shown in Figure 2).

Find Zheng wheat 7698 sporidiole embryoid induction situation preferably, " embryoid number/fringe " numerical value is descending is followed successively by Zheng wheat 7698 > cadenza > all wheats 22 > section's agriculture 199 > fielder > florida > short by anti-58 > bobwhite > polling 987, Significance difference analysis are as shown in table 6 and Fig. 4.

9th, the embryoid of a diameter of 1～2mm is transferred in regeneration culture medium (table 4) and cultivate, 23 DEG C, 16h light According to/8h dark culturing 4～6 weeks, Seedling to be regenerated grew to 2～3cm, and regrowth is shifted in root media (table 5), Culture 3～4 weeks, obtains regeneration plant (different genetic wheat varieties sporidiole embryoid regeneration induction seedling situation such as Fig. 3 Shown).

10th, statistics different genetic wheat varieties every small ear sporidiole regrowth number, Albino Seedling number and green Seedling number.

" regrowth number/fringe " numerical value is descending is followed successively by: cadenza > Zheng wheat 7698 > all wheats 22 > fielder > short anti- 58 > section's agriculture 199 > florida > bobwhite > polling 987." Albino Seedling number/fringe " numerical value is descending is followed successively by: Zheng Mai 7698 > all wheats 22 > cadenza > fielder > short anti-58 > section agricultures 199 > florida > bobwhite > polling 987." green Seedling Number/fringe " numerical value is descending to be followed successively by: cadenza > Zheng wheat 7698 > all wheats 22 > fielder > section agriculture 199 > florida > Short anti-58 > bobwhite > polling 987 (as shown in tables 6 and Fig. 4).

11st, by Transplantation of Regenerated Plantlets in soil, blade middle part detection pore opening and the detection dyeing of the part tip of a root are taken Body number, primarily determines that regrowth ploidy (as shown in Figure 5).Process haplobiont with Colchicine, carry out artificial Double.Continue culture gained regeneration plant to ripe, obtain the seed after maturation.

12nd, count the Natural doubling rate of different genetic wheat varieties sporule regeneration plant.

As shown in fig. 6, Natural doubling rate is followed successively by: bobwhite > polling 987 > fielder > section agriculture 199 > cadenza > All wheats 22 > florida > short anti-58 > Zheng wheats 7698.

Table 6 different genetic wheat varieties microspores culture, regeneration index of correlation statistics

Genotype	Embryoid number/fringe	Regrowth number/fringe	Albino Seedling number/fringe	Green Seedling number/fringe
					Section's agriculture 199	88.35±5.51	20.67±2.08	15.67±3.06	5±1.05
Short by anti-58	55.45±7.02^**	22±2.65	17.45±2.08	4.67±1.53
					bobwhite	34.67±7.00^**	10.34±1.53^**	6.67±1.15	3.67±0.58
candenza	489±13.53^**	110.45±10.12	38.77±4.16^**	71.67±7.64^**
					fielder	77.33±8.02	30.67±4.16	18.67±4.933	11.35±1.53
florida	59.67±6.43^*	13.67±3.79	8.67±3.05	4.67±0.58
					Polling 987	33.67±4.51^**	8.55±1.53	4.67±1.52^*	3.67±0.57
All wheats 22	262.67±13.20^**	81±8.19^**	59.58±5.51^**	21.45±3.21^**
					Zheng wheat 7698	545.50±17.62^**	95.55±7.51^**	72.84±4.16^**	22.67±3.79^**

Claims

1. a kind of method that culture Wheat mitochondria obtains regeneration plant, comprising:

The solvent of described regeneration culture medium is water, and solute and concentration are as follows: kno₃1500mg/l；nh₄no₃250 mg/l；kh₂po₄200mg/l；cacl₂·2h₂o 450mg/l；mgso₄·7h₂o 350mg/l；ki 0.75mg/l； mnso₄15mg/l；h₃bo₃5mg/l；znso₄·7h₂o 7.5mg/l；cocl·6h₂o 0.025mg/l； cuso₄·5h₂o 0.025mg/l；na₂moo₄·2h₂o 0.25mg/l；feso₄·7h₂o 27.80mg/l；na₂edta 37.20mg/l；vb1 10mg/l；vb6 1mg/l；Nicotinic acid 1mg/l；Inositol 200mg/l；Calcium pantothenate 1.0mg/l； Ascorbic acid 1.0mg/l；Maltose 30000mg/l；2,4-d 0.10mg/l；Zeatin 5.0mg/l；cuso₄25 mg/l；ph5.70；

2. method according to claim 1 it is characterised in that: in step (1), be inoculated in described embryoid The described sporidiole of inducing culture obtains from separation through the young fringe of Semen Tritici aestivi to be cultivated described in following process: The young fringe of described Semen Tritici aestivi to be cultivated is placed in the cuso that concentration is 500mg/l₄In aqueous solution, 4 DEG C are processed 10 days.

3. method according to claim 1 and 2 it is characterised in that: in step (1), carry out described induction During culture, also add in the described embryoid induction culture medium being vaccinated with described sporidiole described in coming from The ovary of Semen Tritici aestivi to be cultivated.

4. according to described method arbitrary in claim 1-3 it is characterised in that: in step (1), described induction The condition of culture is to stop when 28 DEG C of lucifuges cultivate a diameter of 1～2mm to described sporidiole embryoid.

5. according to described method arbitrary in claim 1-4 it is characterised in that: in step (2), described regeneration The condition of culture is 23 DEG C, and 16h illumination/8h dark culturing to described regrowth grows to stops during 2～3cm.

6. according to described method arbitrary in claim 1-5 it is characterised in that: in step (3), described take root The condition of culture is 23 DEG C, 16h illumination/8h dark culturing 3～4 weeks.

7. according to described method arbitrary in claim 1-6 it is characterised in that: described embryoid induction culture medium Solvent is water, and solute and concentration are as follows: kno₃1415mg/l；(nh₄)₂so₄232mg/l；kh₂po₄200mg/l； cacl₂·2h₂o 83mg/l；mgso₄·7h₂o 93mg/l；ki 0.4mg/l；mnso₄5mg/l；h₃bo₃5 mg/l；znso₄·7h₂o 5mg/l；cocl·6h₂o 0.0125mg/l；cuso₄·5h₂o 0.0125mg/l； na₂moo₄·2h₂o 0.0125mg/l；feso₄·7h₂o 27.8mg/l；na₂edta 37.2mg/l；vb1 5mg/l； vb6 0.5mg/l；Nicotinic acid 0.5mg/l；Inositol 50mg/l；L-Glutamine 500mg/l；Glutathione 0.615 mg/l；Ficoll 100 000mg/l；Maltose 90000mg/l；2,4-d 0.2mg/l；Bran amidopurin 0.2 mg/l；Cefotaxime 100mg/l；ph7.0；

The solvent of described root media is water, and solute and concentration are as follows: kno₃950mg/l；nh₄no₃825 mg/l；kh₂po₄85mg/l；cacl₂·2h₂o 220mg/l；mgso₄·7h₂o 185mg/l；ki 0.415mg/l； mnso₄11.15mg/l；h₃bo₃3.1mg/l；znso₄·7h₂o 4.3mg/l；cocl·6h₂o 0.0125mg/l； cuso₄·5h₂o 0.0125mg/l；na₂moo₄·2h₂o 0.125mg/l；feso₄·7h₂o 13.9mg/l；na₂edta 18.65mg/l；Inositol (myo-inositol) 50mg/l；vb1 0.05mg/l；vb6 0.25mg/l；Nicotinic acid (acide nicotinique)0.25mg/l；naa 0.6mg/l；Sucrose 30g/l；Plant gel 3g/l；ph 5.70.

8. according to described method arbitrary in claim 1-7 it is characterised in that: methods described is also included to step (3) The described regeneration plant obtaining carries out Methods of Ploidy Identification, the step carrying out artificial doubling to haplobiont.

9. it is used for cultivating the culture medium that Wheat mitochondria obtains regeneration plant, for as follows (a) or (b):

Regeneration culture medium described in (a) claim 1；

(b) regeneration culture described in the embryoid induction culture medium described in claim 7, claim 1 Root media composition described in base and claim 8.

10. culture medium described in claim 9 obtains the application in regeneration plant in culture Wheat mitochondria.