Use of Double Use of Double Haploid in Barley Haploid in Barley

ImprovementImprovement1- Introduction1- Introduction

- Terminology and Background of - Terminology and Background of HaploidsHaploids

2- Advantages and Limitations2- Advantages and Limitations

3- Haploid-Production Systems3- Haploid-Production Systems

A- Chromosome Elimination (Bulbosum A- Chromosome Elimination (Bulbosum Method)Method)

B- Androgenesis (Anther Culture)B- Androgenesis (Anther Culture)

C- Genogenesis (Ovary Culture)C- Genogenesis (Ovary Culture)

D- Haploid Initiation GeneD- Haploid Initiation Gene

4- Chromosome Doubling4- Chromosome Doubling

22 . .Terminology and Terminology and BackgroundBackground

HaploidHaploid: an individuals with the gametic: an individuals with the gametic chromosome chromosome number in its somatic cells number in its somatic cells

Doubled HaploidDoubled Haploid: an individual with the : an individual with the doubleddoubled chromosome number of chromosome number of the haploid the haploid

MonoploidMonoploid - - haploid derived from a diploid, haploid derived from a diploid, xx is is one genomic complement (n=x) as Barley one genomic complement (n=x) as Barley (n=x=7).(n=x=7).

PolyhaploidPolyhaploid - - haploid from a polyploid (haploid from a polyploid (nnxx)) , , asas wheat n = 3x = 21; durum n = 2x = 14 wheat n = 3x = 21; durum n = 2x = 14

Conventional Breeding Conventional Breeding SystemSystem

Year Population Processes Year Population Processes Initial Initial

1st1st crosses made crosses made AABB x AABB x aabb aabb

2nd2nd FF11 AaBb AaBb

3rd F3rd F2 2 1AABB 2AABb 1AAbb 2AaBB 1AABB 2AABb 1AAbb 2AaBB 4AaBb 2Aabb4AaBb 2Aabb

6.25% 1aaBB 2aaBb 6.25% 1aaBB 2aaBb 1aabb 1aabb

4th-7th F4th-7th F33-F-F66 Advance Progeny: pedigree Advance Progeny: pedigree selection method 8th-14th selection method 8th-14th Testing and Cultivar ReleaseTesting and Cultivar Release

Breeding Using Doubled Haploid Breeding Using Doubled Haploid SystemSystem

Year Population Processes Year Population Processes Initial Initial 1st1st crosses made crosses made AABB x aabb AABB x aabb

2nd2nd FF11 AaBb AaBb Haploid production Haploid production Cochicine treatment Cochicine treatment

Doubled Haploid (25%) AABBDoubled Haploid (25%) AABB AAbb AAbb aaBB aabb aaBB aabb

3rd Head Row Test 3rd Head Row Test

4th-10th Testing and 4th-10th Testing and Cultivar ReleaseCultivar Release

First First :: Homozygous lines can Homozygous lines can be obtained in the be obtained in the

shortest possible timeshortest possible time..

By extracting haploids from F1 hybrids of By extracting haploids from F1 hybrids of diploid barley and then doubling the diploid barley and then doubling the chromosome numbers , this saves at least chromosome numbers , this saves at least 3 to 4 generations of self-pollination, 3 to 4 generations of self-pollination, which are normally required when which are normally required when producing uniform lines for conventional producing uniform lines for conventional breeding of self-pollinated species.breeding of self-pollinated species.

Reduction of 3-5 years for cultivar release

SecondSecond:: selection is more reliable selection is more reliable and effective at the and effective at the DH than at the DH than at the diploid level diploid level . .

100% homozygosity of doubled haploid Using DH lines , we can evaluate quantitative Using DH lines , we can evaluate quantitative

characters knowing that all genes have been characters knowing that all genes have been fixed and that desirable alleles will not be fixed and that desirable alleles will not be lost due to segregation in later generations.lost due to segregation in later generations. The additive genetic variance of DH lines is always The additive genetic variance of DH lines is always

greater than the diploid lines ; therefore, the greater than the diploid lines ; therefore, the response to selection should beresponse to selection should be correspondingly correspondingly

greater in populations of DH lines than in greater in populations of DH lines than in diploid populations.diploid populations.

Third:Third: the homozygosity of DH lines the homozygosity of DH lines is reached without the selection is reached without the selection pressure of a field environmentpressure of a field environment..

can be repeated anytimecan be used in different Lab

can be used by different researchers

Because of this, one could set up facilities Because of this, one could set up facilities at one location to produce new lines for at one location to produce new lines for

evaluation in any part of the worldevaluation in any part of the world..

FourthFourth:: DH lines can be used for DH lines can be used for quantitative genetic studies if quantitative genetic studies if they are a random sample of gametes they are a random sample of gametes from F from F11 hybrids hybrids..

A. Detection of Gene InteractionA. Detection of Gene Interaction

B. Estimation of Genetic variancesB. Estimation of Genetic variances

C. Detection of LinkageC. Detection of Linkage

D. Estimation of the Number of D. Estimation of the Number of GenesGenes

E. location of polygenes E. location of polygenes

FifthFifth:: haploids have a value in haploids have a value in mutation breeding:mutation breeding:

(1) (1) Both recessive and dominant mutations Both recessive and dominant mutations will be expressed. will be expressed.

(2) (2) Selection on the basis of gamete Selection on the basis of gamete frequency as haploid plants dose not frequency as haploid plants dose not require large population compared require large population compared

with diploidwith diploid..

(3) (3) Any mutations associated with Any mutations associated with deleterious effects should be selected deleterious effects should be selected

against during haploid production. against during haploid production.

SixthSixth,, the population size required the population size required to obtain a desired to obtain a desired genotyoe from haploids is genotyoe from haploids is samller than that from an F samller than that from an F22 populationpopulation

LimitationsLimitations Limited numbers of DH lines could be Limited numbers of DH lines could be

produced, therefore, the number of produced, therefore, the number of crosses one can utilize per year is crosses one can utilize per year is limited.limited.

Both the bulbosum method and anther Both the bulbosum method and anther culture techniques require controlled-culture techniques require controlled-environment growth facilities and environment growth facilities and skilled technical personnel.skilled technical personnel.

Haploid-Production SystemsHaploid-Production Systems Barley haploids can arise spontaneously or by Barley haploids can arise spontaneously or by

chemical inductionchemical induction. . Because the frequency of such spontaneous Because the frequency of such spontaneous

haploids is very low and unreliable, they have haploids is very low and unreliable, they have little value for breeding.little value for breeding.

Based on the derivations of haploids, Based on the derivations of haploids, haploid-production systems for breeding haploid-production systems for breeding barley can be classified into four categoriesbarley can be classified into four categories : : chromosome elimination (chromosome elimination (bulbosum methodbulbosum method), ), androgenesis (androgenesis (anther cultureanther culture), genogenesis ), genogenesis ((ovary cultureovary culture), and ), and haploid initiation genehaploid initiation gene..

A- Chromosome Elimination (Bulbosum A- Chromosome Elimination (Bulbosum Method) and Embryo RescueMethod) and Embryo Rescue

The phenomenon of chromosome The phenomenon of chromosome elimination was discovered elimination was discovered

in interspecific cross between in interspecific cross between ((Hordeum vulgareHordeum vulgare x x H. bulbosumH. bulbosum ). ).

For long time , barley breeders have shown For long time , barley breeders have shown interest in crossing interest in crossing Hordeum vulgareHordeum vulgare x x H. H.

bulbosum bulbosum in the hope of transferring in the hope of transferring desirable characters from the wild species desirable characters from the wild species

to cultivated barley. A few hybrids had been to cultivated barley. A few hybrids had been obtained from this cross but the success obtained from this cross but the success

rate was low .rate was low .

Hordeum vulgare Hordeum Hordeum vulgare Hordeum

bulbosumbulbosum

Kasha and Kao (1970) pointed out Kasha and Kao (1970) pointed out that a high frequency of haploids that a high frequency of haploids

could be obtained from this could be obtained from this cross using various lines of cross using various lines of diploid diploid Hordeum vulgare Hordeum vulgare as female as female

parentparent.. In addition to the In addition to the H. vulgareH. vulgare x x H. H.

bulbosum bulbosum cross, chromosome elimination cross, chromosome elimination and haploid plants have been obtained and haploid plants have been obtained from many other interspecific and from many other interspecific and intergeneric crossesintergeneric crosses (( barley x maize , barley x maize , barley x Italian ryegrassbarley x Italian ryegrass )). . However, the However, the frequencies are too low to be of interest frequencies are too low to be of interest as a barley breeding systems.as a barley breeding systems.

Bulbosum MethodBulbosum Method Day 0Day 0 – – emasculationemasculation Day 2Day 2 - - pollination with pollination with H. bulbosumH. bulbosum pollen pollen Day 3 (to 5)Day 3 (to 5) - - 40% of the embryonic cells are 40% of the embryonic cells are

haploid, endosperm abortion occurs, GA3 haploid, endosperm abortion occurs, GA3 treatment enhances retention of floretstreatment enhances retention of florets

Day 11Day 11 - - 94% of the embryonic cells are 94% of the embryonic cells are haploidhaploid

Day 14 (to 16)Day 14 (to 16) - - embryos are dissected embryos are dissected and cultured in the dark at 18 to 22 C, and cultured in the dark at 18 to 22 C, embryos develop in vitroembryos develop in vitro

Day 22 (to 28)Day 22 (to 28) - - embryos are transferred to embryos are transferred to light for seedling developmentlight for seedling development

Day 50Day 50 – – plantsplants

Double fertilization dose occur and Double fertilization dose occur and chromosome elimination proceeds chromosome elimination proceeds gradually in both the embryo and gradually in both the embryo and

endosperm .endosperm .

By 8-10 days post fertilization, most By 8-10 days post fertilization, most dividing cells in the embryo are dividing cells in the embryo are

haploidhaploid.. The preferential loss of bulbosum The preferential loss of bulbosum

chromosomes has been chromosomes has been demonstrated cytologically and by demonstrated cytologically and by gene markers.gene markers.

Chromosome elimination process in embryos Chromosome elimination process in embryos derived fromderived from

H. vulgareH. vulgare x x H. bulbosumH. bulbosum

Days after Days after pollinationpollination

cultivarcultivar No. ofNo. of

EmbryEmbryoo

ScoredScored

No. of cells containingNo. of cells containing

ChromosomeChromosome

77 88 99 1010 1111 1212 1313 1414

TotalTotal MeanMean

ChromosomChromosomee

numbernumber

33Kanto Nijo Kanto Nijo 2525

Yoshikei 15Yoshikei 15

1515

1515 1515 22 00 1616 22 2424 00 117117

1212 11 00 99 00 1313 11 124124 176176

160160 12.512.5

13.013.0 55Kanto Nijo Kanto Nijo

2525

Yoshikei 15Yoshikei 15

1313

1313 2626 1010 33 2020 11 44 00 1515

2020 88 00 1515 00 1111 33 2020 7979

7777 9.69.6

10.510.5 77Kanto Nijo Kanto Nijo

2525

Yoshikei 15Yoshikei 15

1010

1010 5454

55555454

5555 7.07.0

7.07.0 99Kanto Nijo Kanto Nijo

2525

Yoshikei 15Yoshikei 15

1010

1010 5656

5151 5656

5151 7.07.0

7.07.0

1111Kanto Nijo Kanto Nijo 2525

Yoshikei 15Yoshikei 15

88

88 3535

3333 3535

3333 7.07.0

7.07.0

Up toUp to 50-60 % of the embryos cultured 50-60 % of the embryos cultured have given rise to mature have given rise to mature haploid plants. More than 90 % haploid plants. More than 90 % of these mature haploid of of these mature haploid of H.vulgareH.vulgare and the remainder are diploid and the remainder are diploid hybrids that can be readily hybrids that can be readily distinguished on the basis of their distinguished on the basis of their decumbent growth habit and decumbent growth habit and other other bulbosumbulbosum- like - like

characteristicscharacteristics.. Thus, chromosome checks are not required to Thus, chromosome checks are not required to identify haploids. Using identify haploids. Using this method , one technician can produce 50-this method , one technician can produce 50-100 haploids/week, or under ideal conditions 100 haploids/week, or under ideal conditions and with specific genotypes, up to 300 and with specific genotypes, up to 300 haploids/week .haploids/week .

Androgenesis (Anther Androgenesis (Anther Culture)Culture)

Androgenesis –Androgenesis – haploid plant derived haploid plant derived from male gamete, from male gamete,

most common method most common method in vitroin vitro

Anther and microspore (pollen) Anther and microspore (pollen) culture culture -- haploid plants are derived haploid plants are derived from microspores (pollen) cultured from microspores (pollen) cultured individually or in anthers individually or in anthers

History:

In barley, Clapham (1971) first reported the In barley, Clapham (1971) first reported the induction of callus from cultured anthers , induction of callus from cultured anthers ,

and in (1973) the production of haploid plantsand in (1973) the production of haploid plants

Subsequently, significant progress has Subsequently, significant progress has been made in haploid production .been made in haploid production .

If reliable and productive anther culture If reliable and productive anther culture techniques became available, this system techniques became available, this system could have greater potential than the bulbosum could have greater potential than the bulbosum method for use in barley breeding.method for use in barley breeding.

The callus can arise by one The callus can arise by one of the following three of the following three

pathwayspathways::

Division of the vegetative cell while the Division of the vegetative cell while the generative cell is non functionalgenerative cell is non functional

Division of the microspores in which a Division of the microspores in which a generative cell is not formedgenerative cell is not formed

Division involving both the vegetative Division involving both the vegetative cell and the generative cellcell and the generative cell

VegetativeGenerative

Vegetative

Generativ

e

Similar nuclei

3 to 5°C

Microspore

Embryo

3 to 5°C

Factors affecting the development of haploid plants in vitro

Anther stage - most responsive cells for haploid embryo formation are those between the tetrad stage of microsporogenesis to just past the first pollen mitosis.

Donor plant or anther pretreatment – enhances haploid embryo formation

Actively growing plants and the first set of flowers are most responsive

Cold pretreatment of anthers - either pre- or post-culture treatment (3 to 5 oC for 2 to 4 days), symmetric rather than asymmetric division of the microspore nuclei or division of the vegetative nucleus

Although these results are Although these results are most encouraging, the most encouraging, the following areas require following areas require

further researchfurther research: : the strong genotypic response to the strong genotypic response to

culture condition culture condition the relatively high frequencies of the relatively high frequencies of

albino plantsalbino plants the variation in chromosome numbers the variation in chromosome numbers

of plants derived from callus of plants derived from callus establishing the stability of establishing the stability of

microspore-derived plants in microspore-derived plants in subsequent generation subsequent generation

Genogenesis (Ovary Genogenesis (Ovary Culture)Culture)

All plants obtained have been green and All plants obtained have been green and haploid, but the frequencies were very haploid, but the frequencies were very low , ranging from 0.2 to 1.4% of the low , ranging from 0.2 to 1.4% of the ovaries culture .ovaries culture .

There has been an influence of genotype , There has been an influence of genotype , and perhaps as in the case of and perhaps as in the case of parthenogenesis , some stimulus will be parthenogenesis , some stimulus will be required to increase haploid frequencies .required to increase haploid frequencies .

At present , haploids from ovary culture At present , haploids from ovary culture are not interest for breeding barley . are not interest for breeding barley .

Parthenogenesis - from unfertilized

egg

Apogamy - from other cells of the mega-

gametophyte

Parthenogenesis and Apogamy

Haploid Initiation Gene Haploid Initiation Gene

Hagberg and Hagberg (1980 ) discovered Hagberg and Hagberg (1980 ) discovered this mutant gene accidentally in a this mutant gene accidentally in a cytological study.cytological study.

The haploid The haploid Initiation GeneInitiation Gene ( ( haphap ) ) appears to be a single gene that controls appears to be a single gene that controls the abortion or the survival of abnormal the abortion or the survival of abnormal embryos and endosperms, and especially embryos and endosperms, and especially favors the formation and survival of favors the formation and survival of haploids.haploids.

The original mutant line produced The original mutant line produced 11-14% haploids in its progeny when a 11-14% haploids in its progeny when a plant homozygous for theplant homozygous for the haphap gene gene

was used as the female parent inwas used as the female parent in crosses with other cultivarscrosses with other cultivars

8% of the progeny were maternal 8% of the progeny were maternal haploids: in contrast, their haploids: in contrast, their reciprocal crosses produced no reciprocal crosses produced no haploidshaploids..

Thus the Thus the haphap locus acted through locus acted through the maternal tissue, either the maternal tissue, either toto

--prevent fertilization of the egg cell prevent fertilization of the egg cell nucleus nucleus

-stimulate the egg cell nucleus to -stimulate the egg cell nucleus to divide prematurely,divide prematurely,

- possibly to allow the haploid or - possibly to allow the haploid or unbalanced embryos to develop unbalanced embryos to develop normallynormally

The FThe F22 progeny of the progeny of the crosses betweencrosses between

Homozygous mutant parent x Homozygous mutant parent x Normal parent Normal parent hap/hap hap/hap x +/+ x +/+ or or its reciprocal crosses its reciprocal crosses included between 2 and included between 2 and 3% haploids3% haploids Indicting that Indicting that

the the hap/hap hap/hap gene was partially gene was partially dominantdominant..

The haploid plants, when their The haploid plants, when their chromosome numbers were chromosome numbers were doubled , gave ratio of 34 doubled , gave ratio of 34

hap/haphap/hap : 14 +/+ , a significant : 14 +/+ , a significant deviation from the expected 1:1 deviation from the expected 1:1

ratioratio. . Suggesting that the maternal Suggesting that the maternal genotype had an important influence genotype had an important influence on the formation and survival of on the formation and survival of haploid embryos .haploid embryos .

The FThe F33 progeny lines often had 30- progeny lines often had 30-40% viable haploid plants.40% viable haploid plants.

The Limitations of this The Limitations of this methodmethod

The viability and the vigor of haploids The viability and the vigor of haploids differed from cross to cross . differed from cross to cross .

Many Many hap/haphap/hap seed did not seed did not germinate because of embryo germinate because of embryo abortion.abortion.

The mechanism of this haploid-The mechanism of this haploid-product system and the reason for product system and the reason for embryo abortion are not clear . embryo abortion are not clear .

Chromosome Doubling Chromosome Doubling

The spontaneous doubling rate of The spontaneous doubling rate of barley haploids ranges from 1 to barley haploids ranges from 1 to

3% .3% . this rate is too low for barley this rate is too low for barley breeders and geneticists to breeders and geneticists to

achieve an efficient and rapid achieve an efficient and rapid transformation of sterile , haploid transformation of sterile , haploid materials into fertile homozygous materials into fertile homozygous

lineslines..

Consequently, artificial Consequently, artificial means have to be used to means have to be used to

doubling chromosome doubling chromosome number is number is spindle inhibition spindle inhibition

by colchicinesby colchicines.. It doesn’t affect

chromosome replication, but does

prolong the time for mitosis

Normal versus colchicinecolchicine Mitosis

Barley haploids can be Barley haploids can be treated with colchicines treated with colchicines at various growth stages. at various growth stages.

Three-leaf stage Three-leaf stage Two tiller stage Two tiller stage Three tiller stageThree tiller stage

Colchicine Treatment• Colchicine solution: 1g colchicine

powder (1%), 20 ml dimethylsulphoxide

(DMSO), and 10 drop of Tween 20 per liter

• Post - treatment after vernalization

– take more time but may save more haploid plants

– vernalize green plants right after regeneration

– transfer haploid plants to vermiculite after 6-8 weeks– treat them with colchicine

solution when plants become healthy

Before Colchcinie Treatment

After Colchicine Treatment