Doubled Haploid (DH) Technique in Plant Breeding

1. Introduction

Plant breeding aims to create superior crop varieties with traits such as higher yield, resistance to diseases, and tolerance to stresses. Traditionally, homozygosity is achieved after 6–8 generations of selfing, which is time-consuming. The Doubled Haploid (DH) technique overcomes this limitation by producing homozygous lines in a single generation. Haploid plants (n) are first obtained and then treated with chromosome-doubling agents to restore fertility, resulting in doubled haploid (2n) plants. These plants are genetically uniform, stable, and directly usable in crop improvement.

2. Historical Background

• 1922: Blakeslee reported haploids in Datura stramonium.
• 1964: Guha and Maheshwari regenerated haploid plants via anther culture in Datura innoxia.
• Later, techniques extended to cereals (wheat, barley, rice) and dicots (tobacco, Brassica).
• Development of haploid inducer lines in maize enabled large-scale use in commercial breeding.

3. Concept of Doubled Haploids

A haploid plant (n) carries a single chromosome set, usually from gametophytic cells. By doubling its chromosomes artificially, a doubled haploid (2n) is created. Since haploids are hemizygous at every locus, DH plants become 100% homozygous after doubling, providing pure lines in just one generation.

4. Methods of Doubled Haploid Production

4.1. Androgenesis (Anther and Microspore Culture)

The most widely used method, involving the male gametophyte.

• Anther culture: Immature anthers are cultured; microspores switch from gametophytic to sporophytic development.
• Isolated microspore culture: Microspores are directly cultured, widely used in Brassica, barley, wheat.

4.2. Gynogenesis (Ovule and Ovary Culture)

Unfertilized ovules or ovaries are cultured in vitro to regenerate haploids. Useful for species where androgenesis is inefficient. Examples: Onion, sugar beet, sunflower.

4.3. Wide Hybridization with Chromosome Elimination

Interspecific or intergeneric crosses are made; chromosomes of one parent are selectively eliminated. The resulting embryo is haploid of the other parent. Example: Wheat × maize crosses yield haploid wheat.

4.4. In Vivo Haploid Induction

Involves special haploid inducer lines. When crossed, they produce haploid embryos. Example: Maize (Stock 6 inducer line). This method is simple, scalable, and highly used in commercial breeding.

5. Chromosome Doubling

Haploids are sterile and must undergo chromosome doubling to regain fertility. Methods include:

• Chemical methods: Colchicine, oryzalin, trifluralin.
• Physical methods: Nitrous oxide treatment.
• Spontaneous doubling: Occurs naturally in some species.

6. Applications of DH Technique in Plant Breeding

• Rapid homozygosity: Pure lines in one generation.
• Hybrid breeding: Development of parental inbred lines.
• Genetic mapping: DH lines are ideal for QTL mapping and genome sequencing.
• Marker-assisted breeding: Fixing desirable alleles rapidly.
• Mutation studies: Recessive mutations expressed directly.
• Disease resistance: Quick fixation of resistance genes.

7. Advantages of DH Technique

• Provides complete homozygosity in one generation.
• Shortens breeding cycles and reduces cost.
• Uniform lines for experiments and multi-location trials.
• Facilitates selection of recessive traits.

8. Limitations

• Species- and genotype-dependent response.
• Low efficiency in some crops (e.g., legumes).
• Requires tissue culture expertise and infrastructure.
• Possible somaclonal variation and abnormalities from chemicals.

9. Examples of Successful Applications

• Cereals: Wheat, barley, rice, maize.
• Oilseeds: Rapeseed, mustard, sunflower.
• Vegetables: Onion, pepper, cucumber.
• Tobacco: First species successfully regenerated from anther culture.

10. Future Prospects

The future of DH technology lies in integration with CRISPR-Cas genome editing, universal haploid inducer lines, and speed breeding. Research continues to expand its utility in pulses, horticultural crops, and functional genomics, making DH a cornerstone of next-generation crop improvement.

11. Conclusion

The Doubled Haploid (DH) technique is a milestone in modern plant breeding, enabling rapid production of pure, stable lines. It has wide applications in hybrid development, genetic studies, and molecular breeding. Although limited by species-specific response and technical requirements, continuous innovations promise to expand its use and impact in global crop improvement.