Doubled Haploid Technology inMaize breeding: Status and prospects   George Mahuku, Aida Kebede, Vanessa   Prigge, Leocadio...
Outline• Introduction to Doubled Haploid (DH) technology• Advantages of DH lines in maize breeding• Steps in DH line devel...
Doubled Haploid (DH) lines – What           are they?• Haploid: an individuals with the gametic  chromosome number (n) in ...
Conventional vs DH Inbred Line             Development• Produced by repeated generations of  selfing• In each generation, ...
Advantages of DH technique              in hybrid maize breeding •    Acceleration of inbred line development •    Evaluat...
Doubled haploids – a valuable tool for               research• Establishment of DH mapping populations   – Improve the pre...
Methods for Producing haploids• In vitro - Tissue Culture Techniques   – Anther Culture (microspore culture)   – Highly co...
Two types of haploids           Cytoplasm Chromosome Importance                                Effective for              ...
Production of Maternal haploids           using in vivo method                                        11) Induction of hap...
Haploid Induction                                 Table 1. Inducers and their haploid induction rate (HIR)  Materials:   ...
Induction in yellowHarvesting induced ears   and white donors
Haploid Kernel Identification     Triploid endosperm     (purple aleurone)                                  DH-Donor      ...
Haploid Kernel selection in CIMMYT                          Regular F1   Haploid                  CAT 1   (CAT 2)      (CA...
Step 3: Artificial genome doubling  • 0.06% colchicine, 0.5% DMSO solution; 8 hours                              Gayen et ...
How does colchicine work?• Colchicine is an alhkloid produced by Colchicum  autumnale• It works as mitotic inhibitor: by b...
Chromosome doubling agents• Colchicine is commonly used as doubling agent          α and β          Tubulin• Nitric Oxide ...
Step 4: Self-pollinationElimination of “false“plants• vigor & tillering• stalk color• endosperm & embryo color
DH lines express uniformity within the     line and diversity among the lines!Cycle   DH                   Conventional1  ...
DH line generation at CIMMYT-           progress
CIMMYT GMP started its        involvement in DH in 2007• University of Hohenheim provided temperate inducer  genotypes and...
Tropically adapted inducer line         development          New tropical          Inducer lines        Induction rate    ...
Topically adapted Inducer lines        TAIL           Temperate        Line           Inducer
Progress in Haploid kernel induction            Hand Pollination                                 Isolation Block Hand Poll...
Harvest from Isolation block                                Harvest of 2011                                inductions•Movi...
Optimizing Agronomic    Management
Lack of flowering or synchronization•   Lack of synchronization•   Good female flowers (stigmas)•   Little or no pollen•  ...
Insect pest problem -Ear worms                     • Cipermetrina  • Heliothis spp.
Mechanization
Progress: DH line development                                       Goal : 5000 DH lines/year                             ...
Number of lines                        150                              200                                    250        ...
D1 seeds per line                  350                  300                  250Number of lines                  200      ...
On-going activities• Continue to optimize the DH production protocols• Develop a detailed protocol on how to develop DH  l...
DH Group in Agua Fria
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S4.4 Doubled Haploid Technology in Maize breeding: Status and prospects

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

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S4.4 Doubled Haploid Technology in Maize breeding: Status and prospects

  1. 1. Doubled Haploid Technology inMaize breeding: Status and prospects George Mahuku, Aida Kebede, Vanessa Prigge, Leocadio Martinez
  2. 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. 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. 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. 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 logisticsSchmidt 2004; Röber et al. 2005
  6. 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. 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. 8. Two types of haploids Cytoplasm Chromosome Importance Effective for converting highPaternal Inducer Donor combining seed parenthaploids lines to isogenic CMS analogues Rapid development ofMaternal Donor Donor completelyhaploids homozygous inbred lines
  9. 9. Production of Maternal haploids using in vivo method 11) Induction of haploidy 22) Identification of haploids DH-Donor/ Source germplasm (Female) Inducer (Pollinator)3) Artificial chromosome 3 Haploid seedlings doubling Two doubled haploid4) Self-pollination for seed (DH) plants multiplication 4Doubled haploid video in youtubehttp://blog.cimmyt.org/?p=5880. Two new DH lines
  10. 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 germplasmR1-njcolor marker
  11. 11. Induction in yellowHarvesting induced ears and white donors
  12. 12. Haploid Kernel Identification Triploid endosperm (purple aleurone) DH-Donor Inducer (purple embryo (colorless) & aleurone) X Diploid embryo (purple scutellum)R1-nj color marker systemfor identification ofhaploids (Coe and Sarkar, Haploid seed Regular (diploid) F1 - colorless embryo seed purple embryo1964; Sarkar and Coe, - purple aleurone - purple aleurone1966)
  13. 13. Haploid Kernel selection in CIMMYT Regular F1 Haploid CAT 1 (CAT 2) (CAT 3)
  14. 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 nightTransplanting to the field Planting into pots, recovery and establishing of treated plants
  15. 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. 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. 17. Step 4: Self-pollinationElimination of “false“plants• vigor & tillering• stalk color• endosperm & embryo color
  18. 18. DH lines express uniformity within the line and diversity among the lines!Cycle DH Conventional1 Generate F1 Generate F12 Cross F1 x inducer Generate F23 Treat & self (D0) Generate F2:34 Self & generate Generate F3:4 D15 Generate F4:56 Generate F5:6
  19. 19. DH line generation at CIMMYT- progress
  20. 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. 21. Tropically adapted inducer line development New tropical Inducer lines Induction rate ≥10% Temperate inducer
  22. 22. Topically adapted Inducer lines TAIL Temperate Line Inducer
  23. 23. Progress in Haploid kernel induction Hand Pollination Isolation Block Hand Pollination Inducer Donor Inducer
  24. 24. Harvest from Isolation block Harvest of 2011 inductions•Moving into production phase•Increase the number ofinductions to 150 sourcepopulations
  25. 25. Optimizing Agronomic Management
  26. 26. Lack of flowering or synchronization• Lack of synchronization• Good female flowers (stigmas)• Little or no pollen• Use of shading
  27. 27. Insect pest problem -Ear worms • Cipermetrina • Heliothis spp.
  28. 28. Mechanization
  29. 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. 30. Number of lines 150 200 250 300 350 100 0 50POP 1POP 2POP 3POP 4POP 5POP 6POP 7POP 8POP 9POP 10POP 11POP 12POP 13POP 14POP 15POP 16POP 17POP 18POP 19POP 20POP 21POP 22POP 23POP 24POP 25POP 26POP 27POP 28POP 29POP 30 DH lines / populationPOP 31POP 32POP 33POP 34POP 35POP 36POP 37POP 38POP 39
  31. 31. D1 seeds per line 350 300 250Number 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. 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. 33. DH Group in Agua Fria

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