CN1830242A - Method of obtaining plant mutant - Google Patents
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- CN1830242A CN1830242A CN 200510053483 CN200510053483A CN1830242A CN 1830242 A CN1830242 A CN 1830242A CN 200510053483 CN200510053483 CN 200510053483 CN 200510053483 A CN200510053483 A CN 200510053483A CN 1830242 A CN1830242 A CN 1830242A
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Abstract
A method for obtaining the mutant of plant includes such steps as taking the autotetraploid anther from plant and using it as explant, culturing in vitro to obtain the pollen plants, and screening from the pollen plants and their descendants to obtain the mutant.
Description
Technical field
The present invention relates to a kind of method that obtains plant mutant in the biological technical field.
Background technology
Mutant is the good experiment material of research plant genetic, gene location, clone and separate gene and expression of gene and function, also is the developing direction of current functional genome.
Making up the traditional method of mutant is spontaneous mutation and physics and chemistry mutagenesis.Spontaneous mutation provides very valuable breeding material for the mankind, as the discovery of paddy rice mutant of short stem, has opened the prelude of breeding wheat for semidwarfness.But the frequency of spontaneous mutation is very low, is difficult to carry out systematic collection.Physical mutagenesis (fast neutron, gamma-rays) easily obtains sudden change, and type is difficult to preserve but homology causes death, and the sudden change of generation mostly is deletion mutation, and fragment is bigger, often causes polygene deletion, separates and the comparatively difficulty of single-gene of isozygotying.Mutagenesis (EMS DES) also easily brings out sudden change, and mostly is point mutation, but because the quantity of uncontrollable point mutation, the mutant of a large amount of tool phenotypic variations may comprise a plurality of point mutation, need separate and identify, has increased the difficulty that functional gene is identified.
Along with development of molecular biology, paddy rice T-DNA, transposons, the rice mutant storehouse that methods such as retrotransposon insertion are set up provides an important resource platform for the research of paddy rice functional gene, but it is lower to obtain the frequency of visible phenotypic variation.Build in the process of storehouse, what transform that seedling has in test tube may also can cause lethal mutation because of the insertion of T-DNA, and its most of mutant material concentrates on plant height and Ye Seshang, especially at the report of fringe portion mutation type seldom, and has high input.Other approach such as somaclonal variation, anther culture though the single gene mutation of creation such as spontaneous mutation also has report, can not be used for producing a large amount of mutant.
Summary of the invention
The purpose of this invention is to provide a kind of method that obtains plant mutant.
The method of acquisition plant mutant provided by the present invention is to be explant with plant autotetraploid flower pesticide, by cultured in vitro, obtains pollen plant, and screening obtains mutant in described pollen plant and offspring plant thereof.
In order to strengthen the variation that may exist and to create new variation, described plant autotetraploid is preferably the autotetraploid hybrid.
Method of the present invention is particularly suitable for paddy rice.
Described paddy rice autotetraploid can be mutagenic obtained as follows by liploid plant: be the colchicine processing chitting piece (the long 0.5-1cm of bud) of 0.01-0.03% with mass percent concentration, time 2-3 days, clear water is cleaned bud, is planted in the sand table, preserves moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate big grain chimera (grain chimera, branch stalk chimera, three kinds of forms of fringe chimera), choosing is received large seed and is planted evaluation, represents then that for big grain this kind doubles success as complete stool, and the gained material is the tetraploid original seed.Former interspecific cross is the tetraploid hybrid.
Described cultured in vitro comprises evoked callus and callus differentiation two stages of regeneration plant; In the evoked callus stage, the inducing culture that is adopted is MS medium (additional inositol 500mg/L, 2.4-D2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar solidify), N6 medium (additional inositol 500mg/L, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar solidify) and C17 medium (additional inositol 500mg/L, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar solidify), at callus differentiation used differential medium of regeneration plant stage is MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, sucrose 3% and 8% agar solidify).
Described flower pesticide comes from the spike of rice that pollen grain (microspore) is in the monokaryon mid-term or the phase of keeping to the side.
Described flower pesticide before inoculation through following low temperature preliminary treatment: 8-10 ℃ of low temperature preliminary treatment 7-15 days.
The condition of culture in described evoked callus stage is dark cultivation, 28 ℃.
The condition of culture in described callus differentiation regeneration plant stage is illumination 10-12 hour every day, 28 ℃.
Described pollen plant offspring plant is pollen plant selfing 1-4 generation.
The present invention utilizes the autotetraploid plant to have four identical chromosome sets, has 4 identical allelomorph on the same site, and its hereditary pattern is more than the dliploid complexity.Under the situation of pair of alleles difference, tetraploid plant has 5 kinds of genotype, A
4, A
3A, A
2a
2, Aa
3, a
4, the frequency of appearance is 1: 8: 18: 8: 1, promptly the apparent recessive shape segregation ratio of a pair of Gene Handling was 35: 1 on the tetraploid level, and dliploid is 3: 1.Promptly in the tetraploid colony of limited bulk and random mating, compare with dliploid, tetraploid is higher because of the balance heterozygosity that suddenlys change and random drift causes, and it is slower that heterozygosity is lost, especially occur isozygotying and cause death and can not be by selfing or the solid sudden change situation that goes down to posterity of artificial pollination under, in colony, accumulate variation frequency by selfing.Behind a certain gene point mutation, recessive mutation often is difficult for expressing on the tetraploid level, by the anther cultural times method that subtracts, can obtain to have two times of heterozygotes of recessive mutation, at H
2And the know-why that the recessive mutation proterties will be showed among the self progeny, with tetraploid paddy rice indica-japonica hybrid is material, pass through anther culture, induce pollen plant regeneration, in pollen plant and offspring thereof, directly screen mutant, and it is carried out chromosome cytological Identification (chromosome number 2n=24), the phenotype economical character is observed and genetic analysis, filter out the mutant of inheritance stability, particularly obtained mutant such as dominant dwarf, dense cluster type small ear, different shape grain husk are sterile fully, grain ear type, wheatear type.
Method of the present invention has the following advantages: 1, method of the present invention is when obtaining homozygous mutation system, obtain and its corresponding equipotential system and its heterozygosis system of isozygotying again more conveniently, this is very beneficial for mutant is carried out genetic analysis, gene clone and judge the research of this gene function.2, method of the present invention is for the mutant that isozygotys and cause death and can not produce the offspring, can from the heterozygosis progeny population, isolate this mutant plant continuously by certain mode, can not cause losing of lethal mutation because can not get progeny seed, than be more convenient for the causing death research of type sudden change of other method that produces mutant, and the type of causing death to suddenly change often be the sudden change of important gene.3, method of the present invention is compared with existing method, and the phenotype mutant character is obvious, and is easy to identify; The mutant that obtains mostly is the less fringe portion mutant of report; Mutant isozygotys stable fast, is mostly genetic mutant; The material that produces mutantion line has mutantion line, equipotential system, heterozygosis system simultaneously, is easy to use.Can avoid physics and chemistry mutagenesis and T-DNA insertion deadly type mutant that produces and the problem that is difficult to go down to posterity.4, the rice mutant material of the present invention's acquisition all is successively from H
2To H
6Separate in the normal group of hill body in generation, the variant of all genetic researches, be single-gene recessive mutation (except " No. 2 short " dominant mutant of short stem), if heterozygosis involves continuous selfing in a criminal case, very easily obtain stable isogenic line, this will be very favourable to follow-up molecular cloning, avoid in physics and chemistry mutagenesis, it is many to relate to the site, shortcomings such as separation and purifying single-gene difficulty.
Method of the present invention is easy, quick, economical, efficient, and the plant or the dliploid mutagenesis of general autotetraploid are that (grain, vegetables, flowers) can utilize this method to screen its corresponding mutant behind the tetraploid, have broad application prospects.
Description of drawings
Fig. 1 is high-frequency regeneration polygerm body A87203
Fig. 2 is a H4198 dense cluster type small ear mutant
Fig. 3 is the complete sterile mutant of the different shape grain husk of H3990
Fig. 4 is the short variant of H2558
Fig. 5 is the only stalk variant of H2558
Fig. 6 is " No. 2 short " dominant mutant of short stem
Embodiment
Experimental technique among the following embodiment is conventional method if no special instructions.
H
1The regeneration pollen plant that expression is obtained by flower pesticide, H
2Expression H
1The seed that produces for selfing reaches by the plant that it grew up to H
3Expression H
2The seed that produces for selfing reaches by the plant that it grew up to, and the rest may be inferred, H
4, H
5... represent the selfing third generation, the 4th generation etc. respectively.
M
1Expression screening obtain mutant plant, M
2Expression M
1The seed that produces for selfing reaches by the plant that it grew up to M
3Expression M
2The seed that produces for selfing reaches by the plant that it grew up to, and the rest may be inferred, M
4, M
5... represent the selfing third generation, the 4th generation etc. respectively.
The acquisition of embodiment 1, high-frequency regeneration polygerm body A87203
1, the acquisition of paddy rice autotetraploid
Be the material that sets out with paddy rice liploid variety water source 52, river farming 422, Yin Fang (3 liploid varieties), with mass percent concentration is that 0.02% colchicine is handled chitting piece (the long 0.5cm of bud), 2 days time, clean with clear water, be planted in the sand table, preserve moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate big grain chimera (grain chimera), choosing is received large seed and is planted evaluation, obtains river farming 422 tetraploid original seeds, water source 52 tetraploid original seeds and silver-colored mill tetraploid original seed respectively.With the agricultural 422 tetraploid original seeds in the river that obtains is male parent, and water source 52 tetraploid original seeds are that female parent is hybridized, the autotetraploid hybrid F that obtains
1Again with silver-colored mill tetraploid original seed is female parent, and river farming 422 tetraploid original seeds are the hybrid F that male parent obtains
1Reestablish diplomatic relations, obtain autotetraploid H3774.
2, anther culture
The spike of rice that selfing F4 generation of the autotetraploid H3774 of step 1 is in pollen grain monokaryon mid-term 10 ℃ leave standstill carried out the low temperature preliminary treatment in 8 days after; with 2 fringes totally 280 flower pesticide (140 flower pesticide/strains) insert C17 medium (additional inositol 500mg/L; 2.4-D 2mg/L; lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT0.5mg/L; 8% agar solidifies); under 28 ℃ of conditions, the dark cultivation forms callus.Callus is inserted differential medium MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, the curing of sucrose 3%, 8% agar), and illumination in 12 hours, is cultivated and was induced pollen plant regeneration in one month by 28 ℃.Observe through phenotype, find to have a bud clump of callus differentiation, have fast growth, multiplication capacity is strong, the characteristics that synchronism is good, after this bud clump is cut apart and successive transfer culture (identical with differentiation culture based formulas and condition of culture), set up clone A87203 (Fig. 1).The bud clump of clone A87203 was cultivated 1 month, and the bud number can be bred 150～200 times, and high reaches more than 350 times.The occurring mode of bud is to sprout on the bud.The bud clump is shredded, and renewed vaccination is on MS dedifferentiation medium (additional inositol 500mg/L, 2.4-D2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L, the curing of 8% agar), and callus can increase weight about 5.0 times.Callus is gone to the MS differential medium, nearly half callus lines breaks up the clump that sprouts again, still has the characteristic of high-frequency regeneration bud again.This material is more than the 10 year time of subculture, still keeps extremely strong multiplication capacity.20 ℃, the condition of culture of illumination in 10 hours every day can make it keep the indefinite bud state, and 30 ℃, the condition of culture of illumination in 12 hours produces a large amount of millions of complete regenerated plant H rapidly
1(pollen plant).Regeneration plant H
1Plant in the field, 31 strains growth is normal, 6 unusual pollen plants of plant forms is carried out the pollen mother cell chromosomes number observe.9:00-11:00 gets the young fringe that pollen plant is in meiosis stage and puts the Kano fixer 24h of new preparation the morning, puts 70% alcohol again in 4 ℃ of preservations.Before the microscopy small ear is put 4% siderotil mordant dyeing 3-5 minute, picking flower pesticide, aceto-camine dyeing, compressing tablet.The result shows that paramorph 6 strain chromosome numbers are the trisome strain of 2n=2x+1=25.
3, Genetic identification
To the normal H of 31 strain forms
1Pollen plant individual plant results, numbering, H
2In generation, is later on all by the plantation of strain system.In self-generation subsequently, 5 kinds of phenotypic variation strains have appearred, and the cytological observation of pollen mother cell shows that variant is normal dliploid (2n=24).Fact proved that it is a good mutant variation source.
The acquisition of embodiment 2, dense cluster type small ear mutant H4198
1, the acquisition of paddy rice autotetraploid
Accounting for and be the material that sets out with paddy rice liploid variety Yin Fang, three cun, short son, is 0.01% colchicine processing chitting piece (the long 0.6cm of bud) with mass percent concentration, and 2 days time, clear water is cleaned bud, is planted in the sand table, preserves moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate big grain chimera (branch stalk chimera), choosing is received large seed and is planted evaluation, obtains silver-colored mill tetraploid original seed, and three cun tetraploid original seeds and short son account for the tetraploid original seed.With silver-colored mill tetraploid original seed is female parent, and three cun tetraploid original seeds are that male parent is hybridized, the autotetraploid hybrid F that obtains
1, be female parent with it again, it is that male parent is hybridized that short son accounts for the tetraploid original seed, obtains autotetraploid H4198.
2, anther culture
Selfing F with autotetraploid H4198
3Seville orange flower powder monokaryon keep to the side the phase flower pesticide 10 ℃ leave standstill carried out the low temperature preliminary treatment in 8 days after; with 10 fringes totally 800 flower pesticide (80 flower pesticide/strains) insert MS medium (additional inositol 500mg/L; 2.4-D 2mg/L; lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L; 8% agar solidifies); under 28 ℃ of conditions, the dark cultivation forms callus.Callus is inserted differential medium MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, the curing of sucrose 3%, 8% agar), and illumination in 11 hours, is cultivated and was formed complete regenerated plant H in one month by 28 ℃
1(pollen plant).The normal regeneration plant H of form
1Plant in the field, the unusual pollen plant of plant forms is carried out the pollen mother cell chromosomes number observe.9:00-11:00 gets the young fringe that pollen plant is in meiosis stage and puts the Kano fixer 24h of new preparation the morning, puts 70% alcohol again in 4 ℃ of preservations.Before the microscopy small ear is put 4% siderotil mordant dyeing 3-5 minute, picking flower pesticide, aceto-camine dyeing, compressing tablet.The result shows chromosome number 2n=2x=24, is normal dliploid.
To the normal H of form
1Pollen plant individual plant results, numbering, H
2In generation, is later on all by the plantation of strain system.At H
2, H
3, H
4, H
5With, H
6In generation, carried out the selection of individual plant and strain system, and the variant that guarantees gained all separates from normal dliploid strain and gets.The result is at H
6In generation, obtain 65 strains, wherein 10 strain small ear strain (M
1), with this 10 strain small ear strain called after dense cluster type small ear mutant H4198 (Fig. 2).This 10 strain small ear strain and contrast (H
6Normal strain in generation) compare, the mutant plant height reduces by 14.5%; Spike length reduces by 35.5%; Reduce by 23.4% between first segment, what do not change but the 2nd to the 5th internode is compared with contrast; Panicle number per hill increases by 32.6% on the contrary; Primary tiller stalk number reduces by 16.1% (being bottom low level branch stalk); Secondary branch stalk number reduces by 71.8%; Number of grain per ear reduces by 56.6%, and luffing is between 44～103; Grain length reduces by 13.9%, but wide not the comparing with contrast of grain changes.In appearance, tassel is little and upright, the granule density height, and the fringe type is bar-shaped, does not change because of the field growing environment change, and it is the fringe portion heredity that relates to all yield traitses of rare report.
3, Genetic identification
4 small ear strain (M of plantation
1Generation) M
2The offspring all is the small ear strain, 55 big fringe strain M
2Among the offspring, the strain that has is that pure big fringe strain is, the strain that has is the mixing strain system of big fringe strain and small ear strain.Once more this material is carried out genetic test, planted 100 (M of strain system altogether
3), wherein 3 is that 16 is that 81 mixing strains from the previous generation are from the big fringe of the previous generation from previous generation's small ear.Its result and M
2Generation the same be the small ear strain from the pure of small ear system, be big fringe strain from the pure of pure big fringe system, and large and small fringe the mixings strain that then occurs again that mixes strain system from the previous generation is that (56) and pure big fringe strain are (25) two kinds.The ratio that mixes strain system and big fringe strain coefficient is 56: 25=2.24: 1 ≈ 2: 1.56 are mixed that the ratio of big fringe strain number and small ear strain number is 1359 in the strains system: 428=3.18: 1 ≈ 3: 1, χ 2 tests show, meet the proterties law of segregation of a pair of Gene Handling.The small ear proterties belongs to recessive mutation.
The acquisition of embodiment 3, the complete sterile mutant of different shape grain husk
1, the acquisition of paddy rice autotetraploid
Be the material that sets out purple No. 2 with paddy rice liploid variety Ze Jin and dragon, is 0.03% colchicine processing chitting piece (the long 0.6cm of bud) with mass percent concentration, and 2 days time, clear water is cleaned bud, is planted in the sand table, preserves moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate big grain chimera (branch stalk chimera), choosing is received large seed and is planted evaluation, obtains the purple No. 2 autotetraploid original seeds of damp bright and beautiful autotetraploid original seed and dragon.With the bright and beautiful tetraploid original seed in pool is female parent, and the purple No. 2 tetraploid original seeds of dragon are that male parent is hybridized, and obtain autotetraploid H3990.
2, anther culture
Selfing F with autotetraploid H3990
4The keep to the side flower pesticide of phase of seville orange flower powder monokaryon; 10 ℃ leave standstill carried out the low temperature preliminary treatment in 10 days after; 390 flower pesticide of 5 strain autotetraploids (78 flower pesticide/strains) are inserted MS medium (additional inositol 500mg/L, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L; sucrose 6%; KT0.5mg/L, 8% agar solidifies), under 28 ℃ of conditions; the dark cultivation forms callus.Callus is inserted differential medium MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, the curing of sucrose 3%, 8% agar), and illumination in 12 hours is cultivated for 28 ℃ and was formed complete regenerated plant H in 25 days
1(pollen plant).Regeneration plant H
3Plant in the field, 22 strains growth is normal, and 7 strains are that holandry is sterile.(7, ratio M1) is 3.14: 1 (approximate 3: 1) with sterile strain in solid strain (22).Results and the normal strain of species test 19 strains then, the luffing of ripening rate is big (38.1%～87.1%, average 69.2%).Above-mentioned sterile strain is tillered many, and tassel is big, and clever shell has structure variation, and there is flower pesticide the inside, is flower pesticide abortion type male sterile (Fig. 3).
3, Genetic identification
(the H for strain system is all planted in the normal strain of above-mentioned 19 strains
4), wherein 7 strains are the normal strain of isozygotying of sterile strain system not occur, the mixed stocker of solid strain and sterile strain appears again in 12 strain systems.Mixing strain coefficient is 12 with the ratio of pure solid strain coefficient: 7=1.7: 1 ≈ 2: 1, in mixing strain system, solid strain number is 306 with the ratio of sterile strain number: 91=3.36: 1 ≈ 3: 1, and χ 2 tests show, meet the proterties law of segregation of a pair of Gene Handling, sterile proterties belongs to recessive mutation.
Short variant of embodiment 4, H2558 and the solely acquisition of stalk variant
1, the acquisition of paddy rice autotetraploid
With paddy rice liploid variety gold, short son account for, silver-colored mill (3 dliploid original seeds) be the material that sets out, with mass percent concentration is that 0.01% colchicine is handled chitting piece (the long 0.5cm of bud), and 2 days time, clear water is cleaned bud, be planted in the sand table, preserve moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate grain chimera (grain chimera) greatly, choosing is received large seed and is planted evaluation, and acquisition gold tetraploid original seed, short son account for tetraploid original seed, silver-colored mill tetraploid original seed.With gold tetraploid original seed is female parent, and is that to account for the tetraploid original seed be the F that male parent obtains for female parent, short son with silver-colored mill tetraploid original seed
1Reestablish diplomatic relations, obtain autotetraploid H2558.
2, anther culture
Selfing F with autotetraploid H2558
4The flower pesticide in seville orange flower powder monokaryon mid-term 10 ℃ leave standstill carried out low temperature treatment in 8 days after; with 15 fringes totally 1000 flower pesticide (67 flower pesticide/strains) insert N6 medium (additional inositol 500mg/L; 2.4-D 2mg/L; lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L; 8% agar solidifies); under 28 ℃ of conditions, the dark cultivation forms callus.Callus is inserted differential medium MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, the curing of sucrose 3%, 8% agar), illumination in 10 hours, 28 ℃, cultivate and formed a bud in month, obtain complete regenerated plant H
1(pollen plant).Regeneration plant H
1Plant in the field, 100% plant strain growth is normal, to the normal H of 65 strain forms
1Pollen plant individual plant results, numbering, H
2In generation, is later on all by the plantation of strain system.At 65 H
2In the strain system, wherein 5 strain systems have isolated high, short two kinds of plant.High stalk plant height 95～100cm, mutant (M1) plant height is 30cm only, and stem is thicker, and it is many to tiller, and grain number per spike is many, and sword-like leave is short wide, and the plant type compactness is with the short variant of this mutant called after H2558 (Fig. 4).
At H
3In strain system, only stalk variant (Fig. 5) has appearred, and it shows stable under the situation of isozygotying, and can isolate only stalk variant constantly in the heterozygosis strain.
3, the Genetic identification of the short variant of H2558
H
3(M
2), H
4(M
3) type of short stem in generation then is of short stem entirely; High stalk type in the pure high stalk strain system then is high stalk entirely; High stalk strain in separated strain high, the of short stem system, height appears again in the offspring, short strain mixing strain system with pure high stalk strain is, the ratio of its strain coefficient is 1.51: 1 (approximate 2: 1), high strain number in the mixing strain system is 3.3: 1 (approximate 3: 1) with the ratio of short strain number, meet the pair of alleles law of segregation, dwarf gene is recessive mutation.
The acquisition of embodiment 5, " No. 2 short " dominant mutant of short stem
1, the acquisition of paddy rice autotetraploid
Be the material that sets out with paddy rice liploid variety water source 52, river farming 422, Yin Fang (3 dliploid original seeds), with mass percent concentration is that 0.02% colchicine is handled chitting piece (the long 0.5cm of bud), and 2 days time, clear water is cleaned bud, be planted in the sand table, preserve moisture.Recover 2 weeks of growth, be transplanted to the seedling bed.5 leaf after dates are transplanted to the land for growing field crops.When ripe, differentiate big grain chimera (grain chimera), choosing is received large seed and is planted evaluation, obtains water source 52 tetraploid original seeds, river farming 422 tetraploid original seeds and silver-colored mill tetraploid original seed.With river farming 422 tetraploid original seeds is male parent, and water source 52 tetraploid original seeds are that female parent is hybridized, and obtain autotetraploid hybrid F
1Again with silver-colored mill tetraploid original seed serves as maternal and the agricultural 422 tetraploid original seeds in river are the hybrid F that male parent obtains
1Reestablish diplomatic relations, obtain autotetraploid H9251.
2, anther culture
Selfing F with autotetraploid H9251
4The flower pesticide in seville orange flower powder monokaryon mid-term 10 ℃ leave standstill carried out low temperature treatment in 8 days after; with 2 fringes totally 280 flower pesticide (140 flower pesticide/strains) insert C17 medium (additional inositol 500mg/L; 2.4-D 2mg/L; lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L; 8% agar solidifies); under 28 ℃ of conditions, the dark cultivation forms callus.Callus is inserted differential medium MS (additional NAA 0.5mg/L, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, the curing of sucrose 3%, 8% agar), and illumination in 12 hours, is cultivated and was formed bud in one month by 28 ℃.Regeneration plant H
1Plant in the field, to the normal H of form
1Pollen plant individual plant results, numbering, H
2In generation, is later on all by the plantation of strain system.At H
3(M1) in the strain system (code name 986083), height occurred, short two kinds of individual plants are called short bar individual plant " No. 2 short " (Fig. 6, the right side plant is " No. 2 short " individual plant among the figure, the left side plant is high stalk individual plant).Plant three strain (M of system of short stem
2), height has appearred again, and (strain is 9963021,14 short: 6 height in short separation; Strain is 9963022,4 short: 7 height; Strain is 9963023,17 short: 8 height), all in all, 37 is short: 21 height, of short stem is most.M
317 strains of short stem for plantation are that though wherein 7 strains systems do not isolate high stalk strain, individual plant has only the 1-2 strain in every system, is not enough to illustrate that it is to occur that pure strain of short stem is arranged.All the other 10 strain systems all isolate high stalk strain.Separated strain is that plant adds up to 186 of short stem and 123 high stalks, the plant height luffing of strain of short stem is at 38～67cm, be continuous distribution, the plant height distribution 97～111cm of high stalk strain, wherein strain is 046078 to be 27 short: 10 height, 046540 is 43 short: 24 height, there is not transitional type between the of short stem and high stalk strain, and judge this complete dominance mutant for single-gene control of short stem.
Claims (10)
1, a kind of method that obtains plant mutant is to be explant with plant autotetraploid flower pesticide, by cultured in vitro, obtains pollen plant, and screening obtains mutant in described pollen plant and offspring plant thereof.
2, method according to claim 1 is characterized in that: described plant autotetraploid is the autotetraploid hybrid.
3, method according to claim 1 and 2 is characterized in that: described plant is a paddy rice.
4, method according to claim 3 is characterized in that: described paddy rice autotetraploid can be mutagenic obtained as follows by liploid plant: with mass percent concentration is that the colchicine of 0.01-0.03% was handled chitting piece 2-3 days.
5, method according to claim 3 is characterized in that: described cultured in vitro comprises evoked callus and callus differentiation two stages of regeneration plant; In the evoked callus stage, the inducing culture that is adopted is the additional inositol 500mg/L of MS medium, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar or N6 medium add inositol 500mg/L, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar or C17 medium add inositol 500mg/L, 2.4-D 2mg/L, lactoalbumin hydrolysate 400mg/L, sucrose 6%, KT 0.5mg/L and 8% agar; The differential medium used in the callus differentiation regeneration plant stage is the additional NAA 0.5mg/L of MS medium, KT 2mg/L, lactoalbumin hydrolysate 800mg/L, inositol 500mg/L, sucrose 3% and 8% agar.
6, method according to claim 5 is characterized in that: the condition of culture in described evoked callus stage is cultivated 28 ℃ for dark.
7, method according to claim 5 is characterized in that: the condition of culture in described callus differentiation regeneration plant stage is illumination 10-12 hour every day, 28 ℃.
8, method according to claim 3 is characterized in that: described flower pesticide comes from the spike of rice of the pollen grain monokaryon mid-term or the phase of keeping to the side.
9, method according to claim 3 is characterized in that: described flower pesticide before inoculation through following low temperature preliminary treatment: 8-10 ℃ of low temperature preliminary treatment 7-15 days.
10, method according to claim 3 is characterized in that: described pollen plant offspring plant is pollen plant selfing 1-4 generation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510053483 CN1830242A (en) | 2005-03-11 | 2005-03-11 | Method of obtaining plant mutant |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2018536399A (en) * | 2016-09-29 | 2018-12-13 | 武漢多倍体生物科技有限公司 | Polyploid rice 2 system recovery system and its breeding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2018536399A (en) * | 2016-09-29 | 2018-12-13 | 武漢多倍体生物科技有限公司 | Polyploid rice 2 system recovery system and its breeding method |
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