CN100356842C - Haploid culturing method for red-vegetable-bolt - Google Patents

Haploid culturing method for red-vegetable-bolt Download PDF

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CN100356842C
CN100356842C CNB2004100606107A CN200410060610A CN100356842C CN 100356842 C CN100356842 C CN 100356842C CN B2004100606107 A CNB2004100606107 A CN B2004100606107A CN 200410060610 A CN200410060610 A CN 200410060610A CN 100356842 C CN100356842 C CN 100356842C
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breeding
purple flowering
flowering stalk
purple
haploid
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CN1723767A (en
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梅时勇
邱正明
聂启军
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E-Shu Agriculture Science & Tech Co Ltd Hubei
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E-Shu Agriculture Science & Tech Co Ltd Hubei
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Abstract

The present invention discloses a method for culturing the haploid of purple flowering Chinese cabbage, which is a method for rapidly breeding a new rad flowering Chinese cabbage variety. The method mainly solves the defects of the long process and the low efficiency of the existing method for breeding purple flowering Chinese cabbage and carries out isolated culture to the anther of the purple flowering Chinese cabbage to obtain a haploid embryo body by the development of male gamete cells, and then regenerating culture, chromosome doubling and isolating pollination are carried out to the haploid embryo body so as to obtain a series of pure lines with different gene types; then, the field traits of the pure lines are compared to screen out the good pure lines to be used as conventional new variety lines or cross parents. The culturing method avoids the insemination process of purple flowering Chinese cabbage plants so as to avoid the generation of heterozygote, simplify the homozygous process of the heterozygote, largely shorten a breeding process and enhance breeding efficiency.

Description

The method of purple flowering stalk haploid breeding
Technical field
The present invention relates to the purple flowering stalk breeding technique, refer to a kind of method of purple flowering stalk haploid breeding particularly, be used for quick breeding purple flowering stalk new varieties.
Background technology
At present, mainly adopting strain in purple flowering stalk breeding field is that method for screening comes the seed selection purple flowering stalk newly to be sheerly.Strain is that screening method is the complementary type breeding material that meets breeding objective manually to be shelled flower bud castrate hybridization, obtains F 1Generation is a first-filial generation, again to this F 1In generation, carry out many for individual plant selfing, and the individual plant based material in per generation is carried out economic characters relatively, filters out good individual plant and carry out follow-on individual plant selfing, newly is sheerly thereby select satisfactory substantially purple flowering stalk.Though being screening method, strain can select integral economic trait conventional new varieties of purple flowering stalk and hybrid combination preferably, but still there is following defective in this method: one, because the chromosome number of purple flowering stalk is 10 pairs, under the situation of not considering to exchange with transposition reorganization factor, every chromosome can be regarded as a genetic linkage body, its self progeny's the rate of isozygotying only is 2 -10Promptly 1/1024, the overwhelming majority is heterozygote in its any one group of hill like this, and homozygote is very rare, and is difficult to differentiate homozygote and heterozygote, thereby has significantly increased the difficulty of purple flowering stalk breed breeding; They are two years old, in the seed selection process of purple flowering stalk kind, the most of heterozygosis degree of the individual plant that the field phenotype is good height, and most of high individual plant field phenotypes of degree of isozygotying are relatively poor, the plant that the degree of isozygotying like this is high is eliminated on the contrary easily, cause merit to be lost, be difficult to obtain the pure line cultivar of optimal set mould assembly; Its three, the inbreeding depression phenomenon of purple flowering stalk filial generation is very serious, the process that makes it breed pure line cultivar is very long, breeding work efficient is very low.
Summary of the invention
Purpose of the present invention is exactly the deficiency that will overcome the existing very long inefficiency of purple flowering stalk breeding technique process, and a kind of method of purple flowering stalk haploid breeding is provided, and is used for quick breeding purple flowering stalk new varieties.
For realizing this purpose, the present invention is on the basis of further investigation purple flowering stalk microspore development biological property, a kind of method of new purple flowering stalk haploid breeding has been proposed, this method is utilized purple flowering stalk monoploid culture technique, in conjunction with existing male-sterile line breeding technology, finish quick breeding simultaneously as the good pure lines of purple flowering stalk of male sterility maintainer line, male parent system or conventional new lines.This method comprises the steps:
1) explant preparation: be used for conventional new varieties or hybrid new breed male parent and be seed selection, selection meets the complementary type breeding material stripping flower bud of breeding objective and castrates hybridization, obtain the filial generation of purple flowering stalk, sow its filial generation, treat to get after bolting is bloomed its flower pesticide as explant; Be used for male sterile line and be the hybrid new breed female parent and be seed selection, selection meets the existing male sterility maintainer line of breeding objective and castrates hybridization with corresponding complementary type breeding material stripping flower bud, obtain the filial generation of purple flowering stalk, sow its filial generation, treat to get after bolting is bloomed its flower pesticide as explant;
2) haploid induction: will be inoculated in as the flower pesticide of explant on the purple flowering stalk haploid induction medium, be dark the cultivation 1～2 day under 33～38 ℃ the condition in temperature, then it is changed over to temperature and is secretly be cultured to the monoploid embryoid under 20～28 ℃ the condition and occur till;
3) regeneration is cultivated: it is that light is cultivated under 20～28 ℃ the condition that the monoploid embryoid of being turned out is placed temperature, transferring to purple flowering stalk after monoploid embryoid greening sprouts on the medium, after this per 25～30 days subcultures once, the monoploid regrowth that will differentiate eye when sliding samming was lower than 28 ℃ when five days is transferred to purple flowering stalk root media relaying supervention and educates;
4) transplant and chromosome doubling in the land for growing field crops: the monoploid regrowth that root system development is good moves into field production, when growing 3～5 true leaves, get its blade lower epidermis microscopy, determine its ploidy, the individual plant that spontaneously doubled haploid does not take place is carried out artificial chromosome double;
5) isolate pollination: behind the monoploid regrowth bolting in big field,, divide individual plant to gather seed with its minute individual plant bagging selfing;
6) economic characters screening: the seed that selfing is obtained to the individual plant bagging, respectively take a morsel in breeding objective sowing in season, observe and filter out the good pure lines of integral economic trait;
7) the later stage procedure of breeding: be used for conventional breeding of new variety, get the good recovery type pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of purple flowering stalk; Be used for the hybrid new breed seed selection, get good maintenance of above-mentioned integral economic trait and male parent type pure lines, artificial stripping flower bud is castrated the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability and measures, for the strong combination of specific combining ability, the maintenance pure lines edge male sterile line of getting wherein nearly with it carries out how generation backcrosses, and selects corresponding male sterile line, the male sterile line of the above-mentioned fine combination of a large amount of breedings and male parent system can obtain the new excellent Hybrid of purple flowering stalk.
In above-mentioned steps 2) in, the component and the proportioning of purple flowering stalk haploid induction medium are most important to the germ extraction rate of monoploid embryoid, show that through repeatedly testing it is 5.8～6.0 that the pH value of the purple flowering stalk of better germ extraction rate haploid induction medium is arranged, component and proportioning are:
Component concentration mg/L
(NH 4) 2SO 4 80.00～300.00
CaCl 2·2H 2O 300.00～1200.00
KNO 3 1200.00～1800.00
KH 2PO 4 150.00～1900.00
MgSO 4·7H 2O 185.00～740.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 3 10.00
KI 0.75
MnSO 4·4H 2O 10.00
ZnSO 4·4H 2O 1.25
NaMoO 4 0.25
Inositol 0.00～100.00
Nicotinic acid 1.00
Folic acid 1.00
VB 1 2.00～10.00
VB 6 1.00
Glycine 1.00
Vitamin h 1.00
Serine 30.00
Glutamine 10.00～500.00
Glutathione 5.00
Active carbon 500.00
Sucrose 100000.00
Agar-agar powder 8000.00
The invention has the advantages that: compare with the existing conventional hybridization breeding technique system that obtains new pure lines, the present invention has avoided the generation of heterozygote in the process of seed selection purple flowering stalk new varieties, accelerate the speed that pure lines form greatly, thereby realized the purpose of rapid breeding.We can carry out following theory analysis at this point:
1. for pair of alleles, its heterozygote separation case under selfing and monoploid condition of culture is as follows:
(1) selfing separates
F 1For Aa
↓
F 1For gametophyte separation case 1A: 1a
F 2(homozygote accounts for 50%=2 to combined separation case 1AA:2Aa:1aa of generation -1)
Gametophyte genotype number is 2 1=2
(2) the Gene Isolation situation after monoploid is cultivated
F 1For Aa
↓
F 1For gametophyte separation case 1A: 1a
(homozygote accounts for 100%=1 to monoploid culture separation case 1AA:1aa -1)
Gametophyte genotype number is 2 1=2
2. for the allelomorph of two pairs of independent inheritances, its heterozygote separation case under selfing and monoploid condition of culture is as follows:
(1) selfing separates
F 1For AaBb
↓
F 1For gametophyte separation case 1AB:1Ab:1aB:1ab
F2 is for combined separation case
AB Ab aB ab
AB AABB * AABb AaBB AaBb
Ab AABb AAbb * AaBb Aabb
aB AaBB AaBb aaBB * aaBb
ab AaBb Aabb aaBb aabb *
Wherein: homozygote accounts for 4/16=1/4=2 -2, gametophyte genotype number is 2 2=4 annotate: beat *Combined be homozygote
(2) the Gene Isolation situation after monoploid is cultivated
F 1For AaBb
↓
F 1Gametophytic separation case 1AB:1Ab:1aB:1ab of generation
The separation case of monoploid culture
1AABB：1AAbb：1aaBB：1aabb
Wherein homozygote accounts for 100%=1 -2, gametophyte genotype number is 2 2=4
3. for the allelomorph of n to independent inheritance, its extreme heterozygote F 1The combined number of gametophyte that produces for selfing is 2 n, F 1F for the selfing generation 2For the rate of isozygotying is 2 -n, F1 is 1 for the rate of isozygotying after cultivating through monoploid -n=100%
4. purple flowering stalk has 10 pairs of chromosomes, under the situation of not considering to exchange with transposition reorganization factor, every chromosome can be regarded as a genetic linkage body, and a hybrid combination is under selfing and monoploid condition of culture, and its separation case is as follows:
(1) selfing separates
Gametophyte genotype number is 2 10=1024
Selfing F 2For the rate of isozygotying is 2 -10=1/1024
(2) under the monoploid condition of culture
Gametophyte genotype number is 2 10=1024
The double haploid aggregate rate of isozygotying is 1 -10=100%
In sum, adopt the breeding of purple flowering stalk flower pesticide monoploid culture technique to avoid the insemination process, thereby avoided the appearance of heterozygote, simplified the program of isozygotying of heterozygote, greatly shortened breeding cycle, improved the efficient of breeding work.
Embodiment
Be described in further detail below in conjunction with the method for specific embodiment to purple flowering stalk haploid breeding of the present invention, this method comprises the steps:
1) explant preparation: according to the difference of breeding objective, also difference to some extent in the preparation of explant: be used for conventional new varieties or hybrid new breed male parent and be seed selection, selection meets the complementary type breeding material stripping flower bud of breeding objective and castrates hybridization, obtain first-filial generation, its first-filial generation of interval sowing treats to get after bolting is bloomed its flower pesticide as explant.Be used for male sterile line and be the hybrid new breed female parent and be seed selection, the existing male sterility maintainer line of selecting to meet breeding objective is castrated hybridization with corresponding complementary type breeding material stripping flower bud, obtains first-filial generation.This first-filial generation is after planting carried out selfing get second filial generation.With this second filial generation after planting the branch individual plant selfing must hybridize three generations's strain system, with time-division individual plant and the other paired test cross of sterile strain, with the male sterile conservation rate of checking that corresponding hybridization three generations strain is.The hybridization three generations strain system of interval sowing conservation rate 100% treats to get after bolting is bloomed its flower pesticide as explant.
2) haploid induction: 8:00～afternoon 15:00 wins bud morning, through 70～75% alcohol-pickled 30 seconds, 0.1%HgCl 2Solution soaking 3～12 minutes is used aseptic water washing 3 times again, each 3～4 minutes.After finishing above-mentioned surface sterilization handling procedure, cutting stripping flower pesticide is inoculated on the purple flowering stalk haploid induction medium as explant, be dark the cultivation 1 day under 35 ℃ the condition in temperature, then it changed over to temperature and be secretly to be cultured under 25 ℃ the condition and the monoploid embryoid occurs.The pH value that satisfies the purple flowering stalk haploid induction medium of best germ extraction rate can be controlled between 5.8～6.0, and its best component and proportioning are:
Component concentration mg/L
(NH 4) 2SO 4 200.00
CaCl 2·2H 2O 750.00
KNO 3 1600.00
KH 2PO 4 180.00
MgSO 4·7H 2O 370.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 3 10.00
KI 0.75
MnSO 4·4H 2O 10.00
ZnSO 4·4H 2O 1.25
NaMoO 4 0.25
Inositol 60.00
Nicotinic acid 1.00
Folic acid 1.00
VB 1 10.00
VB 6 1.00
Glycine 1.00
Vitamin h 1.00
Serine 30.00
Glutamine 400.00
Glutathione 5.00
Active carbon 500.00
Sucrose 100000.00
Agar-agar powder 8000.00
3) regeneration is cultivated: it is that light is cultivated under 26～28 ℃ the condition that the monoploid embryoid of being turned out is placed temperature, transferring to purple flowering stalk after monoploid embryoid greening sprouts on the medium, go out embryo for single medicine is in heaps, once discovery, at first disperse to cultivate, transfer to purple flowering stalk and sprout on the medium under the situation of not greening, otherwise its nutrient absorption is bad, the growth of embryoid will be subjected to bigger influence.After this per 25 days subcultures once if occur the clump bud on certain embryoid, then will cut subculture.When sliding samming was lower than 28 ℃ when five days, the monoploid regrowth that differentiates eye is transferred to purple flowering stalk root media relaying supervention educates.Purple flowering stalk is sprouted, the pH value of root media generally is controlled between 5.5～5.6, and it to the component and the proportioning of sprouting, rooting effect is preferable is:
Component concentration mg/L
NH 4NO 3 825.00
KNO 3 950.00
CaCl 2·2H 2O 110.00～440.00
KH 2PO 4 170.00
MgSO 4·7H 2O 185.00～370.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 4 8.00
KI 0.75
MnSO 4·H 2O 10.00
NaMoO 4·2H 2 0.25
ZnSO 4·H 2O 1.25
Inositol 25.00～100.00
Nicotinic acid 1.00
VB 1 10.00
VB 6 1.00
Active carbon 500.00
Sucrose 2000.00
Agar-agar 8000.00
Wherein: the purple flowering stalk medium of sprouting is added with 6-BA 0.2～1.0mg/L in addition
The purple flowering stalk root media is added with NAA 0.2～1.0mg/L in addition
4) transplant and chromosome doubling in the land for growing field crops: the monoploid regrowth that root system development is good shifts out culturing room, throw off blake bottle and seal film, placed the room temperature lower refining seedling 48 hours, take out the monoploid regrowth then, clean the medium that adheres on the root system, heel in booth, carry out with canopy film and sunshade net on the big ceiling.Cover little shed after having heeled in again, and pour feelings according to the ambient temperature situation and cover plastic sheeting for farm use or sunshade net, and strengthen ventilation day by day.Throw off little shed after one week, carry out field management by the method for management purple flowering stalk seedling.When it survives when growing 3～5 true leaves, get its blade lower epidermis microscopy, determine its ploidy, to the individual plant of spontaneously doubled haploid does not take place at the regeneration cultivation stage, handled its growing point 48 hours with 0.4% colchicine alkali lye, carry out artificial chromosome and double.
5) isolate pollination: long during in the seedbed when the monoploid regrowth in big field to 8～10 true leaves, move into the solarium of reserving seed for planting, conventional field management with its minute individual plant bagging selfing, divides individual plant to gather seed behind the bolting.The monoploid regrowth that is used for the male sterility maintainer line seed selection also should be its nearly edge male sterile line plant of same solarium's equivalent field planting, and the paired test cross of bagging when blooming is to measure its conservation rate.
6) economic characters screening: the seed that selfing is obtained to the individual plant bagging, respectively get 1/3rd in breeding objective sowing in season, observe and filter out the good pure lines of integral economic trait.
7) the later stage procedure of breeding: be used for conventional breeding of new variety, get the good recovery type pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of purple flowering stalk, directly demonstration popularization.Be used for the hybrid new breed seed selection, get the good maintenance of above-mentioned integral economic trait and male parent type pure lines, manually shell flower bud and castrate the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability mensuration.For the strong combination of the specific combining ability that satisfies breeding objective, the maintenance pure lines edge male sterile line of getting wherein nearly with it carries out backcrossing of 5～8 generations, selects corresponding male sterile line.The male sterile line of the above-mentioned fine combination of a large amount of breedings and male parent system can obtain the new excellent Hybrid of purple flowering stalk, are used for commercialization production.

Claims (2)

1. the method for a purple flowering stalk haploid breeding, it is characterized in that: this method comprises the steps:
1) explant preparation: be used for conventional new varieties or hybrid new breed male parent and be seed selection, selection meets the complementary type breeding material stripping flower bud of breeding objective and castrates hybridization, obtain the filial generation of purple flowering stalk, sow its filial generation, treat to get after bolting is bloomed its flower pesticide as explant; Be used for male sterile line and be the hybrid new breed female parent and be seed selection, selection meets the existing male sterility maintainer line of breeding objective and castrates hybridization with corresponding complementary type breeding material stripping flower bud, obtain the filial generation of purple flowering stalk, sow its filial generation, treat to get after bolting is bloomed its flower pesticide as explant;
2) haploid induction: will be inoculated in as the flower pesticide of explant on the purple flowering stalk haploid induction medium, in temperature dark the cultivation 1～2 day under 33～38 ℃ the condition, then it is changed over to temperature and is secretly be cultured to the monoploid embryoid under 20～28 ℃ the condition and occur till, wherein the pH value of purple flowering stalk haploid induction medium is 5.8～6.0, and its component and proportioning are:
Component concentration mg/L
(NH 4) 2SO 4 80.00～300.00
CaCl 2·2H 2O 300.00～1200.00
KNO 3 1200.00～1800.00
KH 2PO 4 150.00～1900.00
MgSO 4·7H 2O 185.00～740.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 3 10.00
K1 0.75
MnSO 4·4H 2O 10.00
ZnSO 4·4H 2O 1.25
NaMoO 4 0.25
Inositol 0.00～100.00
Nicotinic acid 1.00
Folic acid 1.00
VB 1 2.00～10.00
VB 6 1.00
Glycine 1.00
Vitamin h 1.00
Serine 30.00
Glutamine 10.00～500.00
Glutathione 5.00
Active carbon 500.00
Sucrose 100000.00
Agar-agar powder 8000.00
3) regeneration is cultivated: it is that light is cultivated under 20～28 ℃ the condition that the monoploid embryoid of being turned out is placed temperature, transferring to purple flowering stalk after monoploid embryoid greening sprouts on the medium, after this per 25～30 days subcultures once, the monoploid regrowth that will differentiate eye when sliding samming was lower than 28 ℃ when five days is transferred to purple flowering stalk root media relaying supervention and educates, wherein purple flowering stalk is sprouted, the pH value of root media is 5.5～5.6, and its component and proportioning are:
Component concentration mg/L
NH 4NO 3 825.00
KNO3 950.00
CaCl 2·2H 2O 110.00～440.00
KH 2PO 4 170.00
MgSO 4·7H 2O 185.00～370.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 4 8.00
KI 0.75
MnSO 4·H 2O 10.00
NaMoO 4·2H 2 0.25
ZnSO 4·H 2O 1.25
Inositol 25.00～100.00
Nicotinic acid 1.00
VB 1 10.00
VB 6 1.00
Active carbon 500.00
Sucrose 2000.00
Agar-agar 8000.00
Wherein: the purple flowering stalk medium of sprouting is added with 6-BA 0.2～1.0mg/L in addition
The purple flowering stalk root media is added with NAA 0.2～1.0mg/L in addition
4) transplant and chromosome doubling in the land for growing field crops: the monoploid regrowth that root system development is good moves into field production, when growing 3～5 true leaves, get its blade lower epidermis microscopy, determine its ploidy, the individual plant that spontaneously doubled haploid does not take place is carried out artificial chromosome double;
5) isolate pollination: behind the monoploid regrowth bolting in big field,, divide individual plant to gather seed with its minute individual plant bagging selfing;
6) economic characters screening: the seed that selfing is obtained to the individual plant bagging, respectively take a morsel in breeding objective sowing in season, observe and filter out the good pure lines of integral economic trait;
7) the later stage procedure of breeding: be used for conventional breeding of new variety, get the good recovery type pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of purple flowering stalk; Be used for the hybrid new breed seed selection, get good maintenance of above-mentioned integral economic trait and male parent type pure lines, artificial stripping flower bud is castrated the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability and measures, for the strong combination of specific combining ability, the maintenance pure lines edge male sterile line of getting wherein nearly with it carries out how generation backcrosses, and selects corresponding male sterile line, the male sterile line of the above-mentioned fine combination of a large amount of breedings and male parent system can obtain the new excellent Hybrid of purple flowering stalk.
2. the method for purple flowering stalk haploid breeding according to claim 1 is characterized in that: said step 2), the pH value of purple flowering stalk haploid induction medium is 5.8～6.0, and its component and proportioning are:
Component concentration mg/L
(NH 4) 2SO 4 200.00
CaCl 2·2H 2O 750.00
KNO 3 1600.00
KH 2PO 4 180.00
MgSO 4·7H 2O 370.00
FeSO 4·7H 2O 27.80
Na 2-EDTA 37.30
CoCl 2·8H 2O 0.025
CuSO 4·5H 2O 0.025
H 3BO 3 10.00
KI 0.75
MnSO 4·4H 2O 10.00
ZnSO 4·4H 2O 1.25
NaMoO 4 0.25
Inositol 60.00
Nicotinic acid 1.00
Folic acid 1.00
VB 1 10.00
VB 6 1.00
Glycine 1.00
Vitamin h 1.00
Serine 30.00
Glutamine 400.00
Glutathione 5.00
Active carbon 500.00
Sucrose 100000.00
Agar-agar powder 8000.00
CNB2004100606107A 2004-07-22 2004-07-22 Haploid culturing method for red-vegetable-bolt Expired - Fee Related CN100356842C (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101889548A (en) * 2010-03-05 2010-11-24 深圳市湖尔美农业生物科技有限公司 Cabbage haploid breeding method
CN102986523B (en) * 2012-11-30 2014-01-15 江苏太湖地区农业科学研究所 Breeding method of novel germplasm resource of green tender flower stalk
CN114303943A (en) * 2022-01-20 2022-04-12 贵州省园艺研究所(贵州省园艺工程技术研究中心) Haploid breeding device and breeding method for red flowering cabbage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
红菜薹雄性不育系的选育 晏儒来等.长江蔬菜，第9期 2000 *
菜薹部分初级三体的选育与细胞学鉴定 张成合等.中国农业科学，第36卷第6期 2003 *

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