Give an account on double haploid and its applications in plant breeding. Also discuss the production methods of haploid.

Doubled haploid technology is a cornerstone of modern plant breeding, offering unparalleled efficiency in developing homozygous lines. Its applications span various aspects of crop improvement, while its production relies on sophisticated in vitro and in vivo techniques.

Applications of Double Haploids in Plant Breeding

The ability to rapidly achieve complete homozygosity makes double haploids extremely valuable across numerous plant breeding applications:

• Accelerated Breeding Programs: DH technology significantly shortens the breeding cycle. Conventional inbreeding takes 6-8 generations to achieve near homozygosity, whereas DH achieves 100% homozygosity in just one generation (or 2-3 years) [1, 3, 12]. This accelerates the development and release of new cultivars.
• Hybrid Development: DH lines serve as excellent parental lines for hybrid breeding, enabling the fixation of hybrid performance in homozygous lines. This avoids challenges associated with hybrid seed production and allows for precise selection of desirable traits [1, 14].
• Genetic Mapping and Quantitative Trait Loci (QTL) Analysis: DH populations are ideal for genetic mapping due to their simple 1:1 segregation ratio, facilitating the identification of markers linked to useful traits. This speeds up the development of genetic maps for various crops like barley, rapeseed, rice, wheat, and pepper [3, 17].
• Gene Discovery and Functional Genomics: The complete homozygosity of DH lines simplifies genetic analysis, making it easier to identify and study genes responsible for specific traits, thus advancing functional genomics research [17].
• Tolerance to Inbreeding Depression: In species susceptible to inbreeding depression, DH technology provides a viable method to develop inbred lines, which might otherwise be difficult to achieve through traditional self-pollination [2].
• Overcoming Self-Incompatibility: For self-incompatible species, dioecious species, or those with long life cycles (e.g., trees, ornamentals), DH technology offers new alternatives for developing homozygous lines and seed propagation where traditional breeding is impractical [2, 3, 14, 18].
• Mutation Breeding: DH lines can be effectively used in mutation breeding programs, allowing for the rapid identification and fixation of desired mutations in a homozygous state.
• Development of Substitution and Addition Lines: DH technology assists in creating specific genetic lines useful for advanced genetic studies and trait introgression [18].

Production Methods of Haploids

Haploid plants, containing a single set of chromosomes (n), are the precursors to doubled haploids. Their production typically involves inducing gametic cells or other haploid tissues to develop into a whole plant, followed by chromosome doubling. Production methods are broadly categorized into in vitro and in vivo approaches.

1. In Vitro Approaches (Tissue Culture-based)

These methods involve culturing isolated plant parts in controlled sterile conditions:

• Androgenesis (Male Gamete Culture): This is the most common and effective method, involving the culture of male gametophytic cells (microspores or immature pollen) or entire anthers [2, 4, 5, 7].
  • Anther Culture: Intact anthers containing immature pollen are excised and cultured on a nutrient medium. The pollen cells are induced to switch their developmental pathway from forming gametes to forming embryos or callus, which then regenerate into haploid plants [7, 16]. This method was pioneered by Guha and Maheshwari (1964) in Datura innoxia [10].
  • Pollen (Microspore) Culture: Pollen grains are extracted from anthers, filtered, and cultured directly. This often leads to a more uniform haploid population as somatic cells from the anther wall are excluded, reducing the chance of diploid or aneuploid regeneration [5, 14, 16].
• Gynogenesis (Female Gamete Culture): This involves culturing unfertilized ovaries or ovules to develop into haploid plants [4, 5, 7].
  • Ovary/Ovule Culture: Unpollinated ovaries or ovules are cultured on a suitable medium. The unfertilized egg cell or other cells within the embryo sac can be induced to form an embryo, leading to a haploid plant. This method is often used when anther culture is unsuccessful or less efficient for a particular species [5, 9].

2. In Vivo Approaches (Whole Plant-based)

These methods involve manipulations within the living plant to induce haploidy:

• Distant Hybridization (Chromosome Elimination): This involves crossing two distantly related species or genera. During early embryonic development, the chromosomes of one parent are preferentially eliminated, leading to the formation of a haploid embryo derived from the other parent [2, 4, 6, 9, 14].
  • Wheat x Maize System: A classic example where wheat is pollinated with maize. The maize chromosomes are eliminated during embryo development, yielding haploid wheat embryos that are then rescued and grown [2, 14].
• Haploid Inducer Lines: Certain genotypes, known as haploid inducers, when used as pollinators, can trigger the formation of haploid embryos from the egg cell of the maternal parent without fertilization [6, 8, 10]. This system is well-established in maize and barley and is becoming increasingly important with recent advancements in cloning inducer genes and genome editing [8, 10].
• Irradiation Effects: Pollen treated with ionizing (e.g., X-rays, gamma-rays) or non-ionizing radiation can be used for pollination. The radiation damages the paternal genome, which is then eliminated, leading to haploid development from the unfertilized egg cell [4, 9].
• Chemical Treatments: Application of certain chemicals (e.g., chloramphenicol, nitrous oxide) can induce chromosomal elimination in somatic cells, potentially leading to haploid formation [4, 9].

Following haploid plant regeneration, chromosome doubling is essential to produce fertile doubled haploids. This is often achieved spontaneously or induced using mitotic inhibitors like colchicine, which prevents spindle fiber formation during mitosis, leading to a doubling of the chromosome number [5, 11].