CN101889548A - Cabbage haploid breeding method - Google Patents

Cabbage haploid breeding method Download PDF

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Publication number
CN101889548A
CN101889548A CN 201010119689 CN201010119689A CN101889548A CN 101889548 A CN101889548 A CN 101889548A CN 201010119689 CN201010119689 CN 201010119689 CN 201010119689 A CN201010119689 A CN 201010119689A CN 101889548 A CN101889548 A CN 101889548A
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CN
China
Prior art keywords
cabbage
breeding
haploid
monoploid
cabbage heart
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CN 201010119689
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Chinese (zh)
Inventor
徐代安
胡璇
易琼
张更鑫
吕绍庄
将启安
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SHENZHEN HUERMEI AGRICUTURAL BIOTECH CO Ltd
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SHENZHEN HUERMEI AGRICUTURAL BIOTECH CO Ltd
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Priority to CN 201010119689 priority Critical patent/CN101889548A/en
Publication of CN101889548A publication Critical patent/CN101889548A/en
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Abstract

The invention discloses a cabbage haploid breeding method, particularly a method for quickly breeding new cabbage variety, aiming at overcoming the defects that the traditional cabbage breeding method results in long process and low efficiency. The method utilizes the cabbage haploid culturing technology and the traditional male sterile line breeding technology to complete the quick breeding of the good and pure cabbage line. The method comprises the following steps of: preparing an explant, inducing the haploid, regenerating and culturing, transplanting to the field, doubling the chromosome, isolating and pollinating, sieving economic character, and subsequent breeding. The invention adopts the cabbage anther haploid breeding technology to breed and omits the insemination process, thereby avoiding generating heterozygous, simplifying the homozygous procedure of the heterozygous, greatly shortening the breeding period, and enhancing the breeding efficiency.

Description

The method of cabbage haploid breeding
Technical field:
The present invention relates to the cabbage heart breeding technique, refer to a kind of method of cabbage haploid breeding particularly, be used for the quick breeding new cabbage variety.
Background technology:
At present, mainly adopting strain in cabbage heart breeding field is that method for screening comes the seed selection cabbage heart 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 cabbage heart.Though being screening method, strain can select integral economic trait conventional new varieties of cabbage heart and hybrid combination preferably, but still there is following defective in this method: one, because the chromosome number of cabbage heart is 12 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 new cabbage variety seed selection; They are two years old, in the seed selection process of cabbage heart 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 cabbage heart 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 cabbage heart breeding technique process, provides a kind of method of quick breeding new cabbage variety, i.e. the method for cabbage haploid breeding.
For realizing this purpose, the present invention is on the basis of further investigation cabbage heart microspore development biological property, the method of cabbage haploid breeding has been proposed, this method is utilized cabbage heart flower pesticide monoploid culture technique, in conjunction with existing male-sterile line breeding technology, finish the quick breeding of the good pure lines of cabbage heart simultaneously.This method comprises the steps:
1) explant preparation: the complementary type breeding material stripping flower bud of selecting to meet breeding objective is castrated hybridization, obtains the filial generation of cabbage heart, sows its filial generation, treats 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 cabbage haploid inducing culture, be dark the cultivation 1-2 days under 28-32 ℃ 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 cabbage heart after monoploid embryoid greening sprouts on the medium, after this every 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 cabbage heart 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 pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of cabbage heart; Be used for the hybrid new breed seed selection, get the good pure lines of above-mentioned integral economic trait, artificial stripping flower bud is castrated the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability and measures, get the strong pure lines of specific combining ability, edge male sterile line nearly with it carries out many generations to be backcrossed, and selects corresponding male sterile line, the male sterile line of the above-mentioned fine combination of a large amount of breedings and the pure lines that are as male parent can obtain the new excellent Hybrid of cabbage heart.
In above-mentioned steps 2), the component of cabbage haploid inducing culture and proportioning are most important to the germ extraction rate of monoploid embryoid, show that through repeatedly testing the pH value that the cabbage haploid of better germ extraction rate inducing culture is arranged is 5.8～6.0, and component and proportioning are:
Component concentration mg/L
(NH4) 2SO 4 80.00-200.00
CaCl 2·2H 2O 500.00～1000.00
KNO 3 1800.00-3000.00
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
NaM 0O 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 100.00～1000.00
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 new cabbage variety, accelerates the speed that pure lines form greatly, thereby has 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
F 2For 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: beating the combined of * is 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, F 1For the rate of isozygotying after cultivating through monoploid is 1 -n=100%
4. cabbage heart has 12 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
The selfing F2 generation 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 cabbage heart 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 cabbage 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: 8:00-15:00 in afternoon wins bud morning, through alcohol-pickled 30 seconds of 70-75%, 0.1%HgCl 2Solution soaking 3～12 minutes is used aseptic water washing 3 times again, each 3-4 minute.After finishing above-mentioned surface sterilization handling procedure, cutting stripping flower pesticide is inoculated on the cabbage haploid inducing culture 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 cabbage haploid inducing culture of best germ extraction rate can be controlled between 5.8～6.0, and its best component and proportioning are:
Component concentration mg/I.
(NH4) 2SO 4 150.00
CaCl 2·2H 2O 750.00
KNO 3 2000.00
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
NaM 0O 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 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 23～27 ℃ the condition that the monoploid embryoid of being turned out is placed temperature, transferring to cabbage heart 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 cabbage heart 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 cabbage heart root media relaying supervention educates.Cabbage heart 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
C oCl 2·8H 7O 0.025
CuSO 4·5H 2O 0.025
H 3BO 4 8.00
KI 0.75
MnSO 4·H 2O 10.00
NaM 0O4·2H 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 cabbage heart medium of sprouting is added with 6-BA 0.2～1.0mg/L in addition;
The cabbage heart 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, take over the method for reason cabbage heart seedling and carry out field management.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% alkali lye, carry out artificial chromosome and double.
5) isolate pollination: long during in the seedbed when the monoploid regrowth in big field to 5～8 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.
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 pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of cabbage heart, and directly demonstration is promoted.Be used for the hybrid new breed seed selection, get the good pure lines of above-mentioned integral economic trait, manually shell flower bud and castrate the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability mensuration.Get the strong pure lines of specific combining ability that satisfy breeding objective, edge male sterile line 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 the pure lines that are as male parent can obtain the new excellent Hybrid of cabbage heart, are used for commercialization production.

Claims (3)

1. the method for cabbage haploid breeding, it is characterized in that: this method comprises the steps:
1) explant preparation: the complementary type breeding material stripping flower bud of selecting to meet breeding objective is castrated hybridization, obtains the filial generation of cabbage heart, sows its filial generation, treats 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 cabbage haploid inducing culture, be dark the cultivation 1-2 days under 28-32 ℃ 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 cabbage heart after monoploid embryoid greening sprouts on the medium, after this every 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 cabbage heart 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 pure lines of above-mentioned integral economic trait, expanding propagation can obtain the new quality product kind of cabbage heart; Be used for the hybrid new breed seed selection, get the good pure lines of above-mentioned integral economic trait, artificial stripping flower bud is castrated the preparing hybrid combination, the filial generation of obtaining after planting carries out combining ability and measures, get the strong pure lines of specific combining ability, edge male sterile line nearly with it carries out many generations to be backcrossed, and selects corresponding male sterile line, the male sterile line of the above-mentioned fine combination of a large amount of breedings and the pure lines that are as male parent can obtain the new excellent Hybrid of cabbage heart;
2. the method for cabbage haploid breeding according to claim 1, it is characterized in that: in above-mentioned steps 2), the component of cabbage haploid inducing culture and proportioning are most important to the germ extraction rate of monoploid embryoid, show that through repeatedly testing the pH value that the cabbage haploid of better germ extraction rate inducing culture is arranged is 5.8～6.0, component and proportioning are:
Component concentration mg/L
(NH4) 2SO 4 80.00-200.00
CaCl 2·2H 2O 500.00～1000.00
KNO 3 1800.00-3000.00
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
NaM 0O 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 100.00～1000.00
Glutathione 5.00
Active carbon 500.00
Sucrose 100000.00
Agar-agar powder 8000.00
3. the method for cabbage haploid breeding according to claim 1 is characterized in that: in above-mentioned steps 3), cabbage heart 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
C 0Cl 2·8H 7O 0.025
CuSO 4·5H 2O 0.025
H 3BO 4 8.00
KI 0.75
MnSO 4·H 2O 10.00
NaM 0O4·2H 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 cabbage heart medium of sprouting is added with 6-BA 0.2～1.0mg/L in addition;
The cabbage heart root media is added with NAA 0.2～1.0mg/L in addition.
CN 201010119689 2010-03-05 2010-03-05 Cabbage haploid breeding method Pending CN101889548A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104686343A (en) * 2015-02-22 2015-06-10 梁彩英 In-vitro culture method for brassica capestris pollen
CN110731271A (en) * 2019-12-02 2020-01-31 海南省农业科学院蔬菜研究所 method for increasing embryoid output number of flowering cabbage isolated microspore culture embryoid
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

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN1723767A (en) * 2004-07-22 2006-01-25 湖北鄂蔬农业科技有限公司 Haploid culturing method for red-vegetable-bolt

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Publication number Priority date Publication date Assignee Title
CN1723767A (en) * 2004-07-22 2006-01-25 湖北鄂蔬农业科技有限公司 Haploid culturing method for red-vegetable-bolt

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Title
《农业生物技术学报》 20031231 顾宏辉等 菜苔小袍子培养及再生植株的倍性鉴定 第572-576页 1-3 第11卷, 第6期 2 *
《生物技术通报》 20081231 朱允华等 菜薹花药培养诱导胚状体的研究 第136-139页 1-3 , 第2期 2 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104686343A (en) * 2015-02-22 2015-06-10 梁彩英 In-vitro culture method for brassica capestris pollen
CN110731271A (en) * 2019-12-02 2020-01-31 海南省农业科学院蔬菜研究所 method for increasing embryoid output number of flowering cabbage isolated microspore culture embryoid
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

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Open date: 20101124