Doubled Haploid Technology in

Maize breeding: Status and prospects

George Mahuku, Aida Kebede, Vanessa

Prigge, Leocadio Martinez

Outline

• Introduction to Doubled Haploid (DH) technology

• Advantages of DH lines in maize breeding

• Steps in DH line development

• CIMMYT’s experience in DH line generation

• Challenges

• On-going activities

Doubled Haploid (DH) lines – What

are they?

• Haploid: an individuals with the gametic

chromosome number (n) in its somatic cells.

• A Doubled Haploid: is a genotype formed when

haploid cells (n), i.e. egg or sperm cell undergo

chromosome doubling (2n).

• The resulting individual is completely homozygous.

Conventional vs DH Inbred Line

Development

• Produced by repeated generations of

selfing

• In each generation, heterozygosity

reduces by 50%

• Resulting inbred lines s are highly

homozygous but not 100%

• DH technique – a quicker method to

obtain 100% pure inbred lines

Generation S1 S2 S3 S4 S5 S6 S7

Homozygosity (%) 50 75 87.5 93.75 96.875 98.45 99.23

Months 6 12 18 24 30 36 42

Advantages of DH technique

in hybrid maize breeding

• Acceleration of inbred line development

• Evaluation of putative hybrids at the beginning of the

selection process

• Maximum additive variance available

• Reduction of masking effects which are caused by

residual heterozygosity

• Reduction of costs for nursery & maintenance breeding

work

• Simplyfied logistics

Schmidt 2004; Röber et al. 2005

Doubled haploids – a valuable tool for

research

• Establishment of DH mapping populations

– Improve the precision of genetic and mapping studies

– Analysis of linkage disequilibrium

– Analysis of haplotype/trait associations

• Accelerate gene pyramiding

• Evaluation, exploitation, and conservation of genetic

resources

– Extraction of individual gametes from heterozygous materials

transforming them into DH lines

– Detrimental effects are revealed to the full extent from the very

beginning

– Conservation of germplasm in form of reproducible DH lines

Methods for Producing haploids

• In vitro - Tissue Culture Techniques – Anther Culture (microspore culture)

– Highly complex & expensive

– Low plantlet regeneration rate which is dependent on genetic

background

– Greatly limited for application in breeding programs

• In vivo - Genetic induction – Widely used

– Involves use of inducer lines

– High frequency of haploid generation

– Simple to operate

– Relatively inexpensive

Two types of haploids

Cytoplasm Chromosome Importance

Paternal

haploids

Inducer

Donor

Effective for

converting high

combining seed parent

lines to isogenic CMS

analogues

Maternal

haploids

Donor

Donor

Rapid development of

completely

homozygous inbred

lines

DH-Donor/

Source germplasm (Female)

Inducer

(Pollinator)

Haploid

seedlings

Two

doubled haploid

(DH) plants

Two new DH lines

1

2

3

4

1) Induction of haploidy

2) Identification of haploids

3) Artificial chromosome

doubling

4) Self-pollination for seed

multiplication

Production of Maternal haploids

using in vivo method

Doubled haploid video in youtube

http://blog.cimmyt.org/?p=5880.

Materials: Haploid inducer

Heterozygous source

germplasm

Table 1. Inducers and their haploid induction rate (HIR)

Inducer HIR (%) Reference

Stock 6 2.3 Coe 1959

WS14 2.0 - 5.0

Lashermes & Beckert

1988

KEMS 6.3 Shatskaya et al. 1994

RWS 8 - 23 Röber et al. 2005

PK6 ~6 Barret et al. 2008

UH400 > 8

Prigge et al., in

preparation

R1-nj

color marker

Collect inducer pollen Pollinate

source germplasm

Haploid Induction

Harvesting induced ears Induction in yellow and white donors

DH-Donor

(colorless)

Inducer (purple embryo

& aleurone)

Haploid seed

- colorless embryo

- purple aleurone

Regular (diploid) F1

seed purple embryo

- purple aleurone

X

Haploid Kernel Identification Triploid endosperm (purple aleurone)

Diploid embryo (purple scutellum)

R1-nj color marker system

for identification of

haploids (Coe and Sarkar,

1964; Sarkar and Coe,

1966)

Haploid Kernel selection in CIMMYT

Haploid

(CAT 3) CAT 1

Regular F1

(CAT 2)

Step 3: Artificial genome doubling

• 0.06% colchicine, 0.5% DMSO solution; 8 hours

• Colchicine acts as mitotic inhibitor

Gayen et al. (1994) MNL 68:85

Germination of haploid seeds Cutting of coleoptile on 3 consecutive days Colchicine treatment over night

Planting into pots, recovery and

establishing of treated plants

Transplanting to the field

How does colchicine work?

• Colchicine is an alhkloid produced by Colchicum

autumnale

• It works as mitotic inhibitor: by binding to tubulin

during mitosis it inhibits spindle formation so that

the cell cannot split into two daughter cells

Haploid Doubled

haploid

(diploid)

Chromosome doubling agents

α and β

Tubulin

• Nitric Oxide (Kato and Geiger, 2002)

• Microtubule binding herbicides

• Caused chromosomal doubling of root tip cells (Hantzschel

and Weber, 2010)

• Colchicine is commonly used as doubling agent

Step 4: Self-pollination

Elimination of “false“plants

• vigor & tillering

• stalk color

• endosperm & embryo color

DH lines express uniformity within the

line and diversity among the lines!

Cycle DH Conventional

1 Generate F1 Generate F1

2 Cross F1 x inducer Generate F2

3 Treat & self (D0) Generate F2:3

4 Self & generate

D1

Generate F3:4

5 Generate F4:5

6 Generate F5:6

DH line generation at CIMMYT-

progress

CIMMYT GMP started its

involvement in DH in 2007

• University of Hohenheim provided temperate inducer

genotypes and technical support

• Various aspects under investigation:

– Development of tropical adapted inducer lines

– Induction rate of temperate inducers in tropical

environments

– Novel marker system for haploid kernel identification

– Optimization of agronomic management to increase

success rate of DH line development

Temperate inducer

Tropically adapted inducer line

development

New tropical

Inducer lines

Induction rate

≥10%

Topically adapted Inducer lines

TAIL

Line

Temperate

Inducer

Progress in Haploid kernel induction

Hand Pollination

Isolation Block

Inducer Inducer Donor

Hand Pollination

Harvest from Isolation block

Harvest of 2011

inductions

•Moving into production phase

•Increase the number of

inductions to 150 source

populations

Optimizing Agronomic

Management

Lack of flowering or synchronization

• Lack of synchronization

• Good female flowers (stigmas)

• Little or no pollen

• Use of shading

Insect pest problem -Ear worms

• Cipermetrina

• Heliothis spp.

Mechanization

Progress: DH line development

Cycle DH Conventional

1 Generate F1 Generate F1

2 Cross F1 x

inducer

Generate F2

3 Treat & self (D0) Generate F2:3

4 Generate F3:4

5 Generate F4:5

6 Generate F5:6

Goal : 5000 DH lines/year

• 4350 DH lines generated in

2010/2011

• >10 000 DH lines in 2012

• Challenges

• Agronomic management

• Haploid seed identification

• Chromosome doubling

(LPS C7-F180-3-1-1-1-BBB / CML-449 )

DH lines / population

0

50

100

150

200

250

300

350

PO

P 1

PO

P 2

PO

P 3

PO

P 4

PO

P 5

PO

P 6

PO

P 7

PO

P 8

PO

P 9

PO

P 1

0

PO

P 1

1

PO

P 1

2

PO

P 1

3

PO

P 1

4

PO

P 1

5

PO

P 1

6

PO

P 1

7

PO

P 1

8

PO

P 1

9

PO

P 2

0

PO

P 2

1

PO

P 2

2

PO

P 2

3

PO

P 2

4

PO

P 2

5

PO

P 2

6

PO

P 2

7

PO

P 2

8

PO

P 2

9

PO

P 3

0

PO

P 3

1

PO

P 3

2

PO

P 3

3

PO

P 3

4

PO

P 3

5

PO

P 3

6

PO

P 3

7

PO

P 3

8

PO

P 3

9

Nu

mb

er o

f li

nes

D1 seeds per line

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10 11 to

20

21 to

50

51 to

100

100>

# Quantity of seed

Nu

mb

er o

f li

nes

On-going activities

• Continue to optimize the DH production protocols

• Develop a detailed protocol on how to develop DH

lines

• Finalize development of a tropically adapted inducer

line

• Look for new haploid seed identification phenotypic

marker

• Develop alternative chromosome doubling agents

• Training partners in DH techniques

DH Group in Agua Fria