s4.4 doubled haploid technology in maize breeding: status and prospects
DESCRIPTION
Presentacion de 11th Asian Maize Conference which took place in Beijing, China from November 7 – 11, 2011.TRANSCRIPT
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