Presentacion de 11th Asian Maize Conference which took place in Beijing, China from November 7 – 11, 2011.

1. Doubled Haploid Technology in
Maize breeding: Status and prospects
George Mahuku, Aida Kebede, Vanessa
Prigge, Leocadio Martinez

2. 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

3. 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.

4. 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

5. 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

6. 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

7. 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

8. Two types of haploids
Cytoplasm Chromosome Importance
Effective for
converting high
Paternal Inducer Donor combining seed parent
haploids lines to isogenic CMS
analogues
Rapid development of
Maternal Donor Donor completely
haploids homozygous inbred
lines

9. Production of Maternal haploids
using in vivo method
1
1) Induction of haploidy
2
2) Identification of haploids DH-Donor/
Source germplasm (Female)
Inducer
(Pollinator)
3) Artificial chromosome 3
Haploid
seedlings
doubling
Two
doubled haploid
4) Self-pollination for seed (DH) plants
multiplication 4
Doubled haploid video in youtube
http://blog.cimmyt.org/?p=5880.
Two new DH lines

10. Haploid Induction
Table 1. Inducers and their haploid induction rate (HIR)
 Materials:
Inducer HIR (%) Reference
Stock 6 2.3 Coe 1959
 Haploid inducer WS14 2.0 - 5.0
Lashermes & Beckert
1988
 Heterozygous source KEMS
RWS
6.3
8 - 23
Shatskaya et al. 1994
Röber et al. 2005
germplasm PK6 ~6 Barret et al. 2008
Prigge et al., in
UH400 >8 preparation
Collect inducer pollen Pollinate
source germplasm
R1-nj
color marker

11. Induction in yellow
Harvesting induced ears and white donors

12. Haploid Kernel Identification
Triploid endosperm
(purple aleurone)
DH-Donor Inducer (purple embryo
(colorless) & aleurone)
X
Diploid embryo
(purple scutellum)
R1-nj color marker system
for identification of
haploids (Coe and Sarkar, Haploid seed Regular (diploid) F1
- colorless embryo seed purple embryo
1964; Sarkar and Coe, - purple aleurone - purple aleurone
1966)

13. Haploid Kernel selection in CIMMYT
Regular F1 Haploid
CAT 1 (CAT 2) (CAT 3)

14. Step 3: Artificial genome doubling
• 0.06% colchicine, 0.5% DMSO solution; 8 hours Gayen et al. (1994) MNL 68:85
• Colchicine acts as mitotic inhibitor
Germination of haploid seeds Cutting of coleoptile on 3 consecutive days Colchicine treatment over night
Transplanting to the field
Planting into pots, recovery and
establishing of treated plants

15. 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)

16. Chromosome doubling agents
• Colchicine is commonly used as doubling agent
α and β
Tubulin
• Nitric Oxide (Kato and Geiger, 2002)
• Microtubule binding herbicides
• Caused chromosomal doubling of root tip cells (Hantzschel
and Weber, 2010)

17. Step 4: Self-pollination
Elimination of “false“plants
• vigor & tillering
• stalk color
• endosperm & embryo color

18. 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 Generate F3:4
D1
5 Generate F4:5
6 Generate F5:6

19. DH line generation at CIMMYT-
progress

20. 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

21. Tropically adapted inducer line
development
New tropical
Inducer lines
Induction rate
≥10%
Temperate inducer

22. Topically adapted Inducer lines
TAIL Temperate
Line Inducer

23. Progress in Haploid kernel induction
Hand Pollination
Isolation Block
Hand Pollination
Inducer Donor Inducer

24. Harvest from Isolation block
Harvest of 2011
inductions
•Moving into production phase
•Increase the number of
inductions to 150 source
populations

25. Optimizing Agronomic
Management

26. Lack of flowering or synchronization
• Lack of synchronization
• Good female flowers (stigmas)
• Little or no pollen
• Use of shading

27. Insect pest problem -Ear worms
• Cipermetrina
• Heliothis spp.

28. Mechanization

29. Progress: DH line development
Goal : 5000 DH lines/year
• 4350 DH lines generated in
2010/2011
• >10 000 DH lines in 2012
Cycle DH Conventional
1 Generate F1 Generate F1
2 Cross F1 x Generate F2
(LPS C7-F180-3-1-1-1-BBB / CML-449 ) inducer
3 Treat & self (D0) Generate F2:3
• Challenges
• Agronomic management 4 Generate F3:4
• Haploid seed identification
• Chromosome doubling 5 Generate F4:5
6 Generate F5:6

30. Number of lines
150
200
250
300
350
100
0
50
POP 1
POP 2
POP 3
POP 4
POP 5
POP 6
POP 7
POP 8
POP 9
POP 10
POP 11
POP 12
POP 13
POP 14
POP 15
POP 16
POP 17
POP 18
POP 19
POP 20
POP 21
POP 22
POP 23
POP 24
POP 25
POP 26
POP 27
POP 28
POP 29
POP 30
DH lines / population
POP 31
POP 32
POP 33
POP 34
POP 35
POP 36
POP 37
POP 38
POP 39

31. D1 seeds per line
350
300
250
Number of lines
200
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 to 21 to 51 to100>
20 50 100
# Quantity of seed

32. 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

33. DH Group in Agua Fria