In-vivo Haploid Technique for Developing Double Haploid Plants in Maize


Double haploid are the true-breeding homozygous lines produced in one generation. Nowadays, double haploid (DH) technology is being used for the rapid development of inbred lines in two to three generations whereas, conventionally inbred lines were developed after 6 to 8 generations of selfing.

Commercially feasible technique is used for the in-vivo haploid production using haploid inducers. Its foundation was laid by Coe (1959) when described “Stock-6” (a line with high haploid frequency i.e., 2.3 per cent). It ultimately enhances the breeding efficiency.

DH lines are created by crossing F1 or F2 plants with haploid inducers. These haploid inducers can be maternal or paternal. When inducers used as pollen source parents, haploid embryo produced will receive the genome of seed-bearing parent only and thus, called maternal haploid inducers, but when inducers are used as seed bearing parent then haploid embryo will receive the nuclear genome of paternal donor parents, it is called as paternal haploid inducers. Paternal inducers are used to introduce Cytoplasmic male sterility (CMS) to inbred lines.


1. Paternal haploid end induction-based development

  • Pollinate paternal inducer (female parent) to inbred lithene (male parent) for the converting it to CMS background

  • Select the haploid seeds from seed bearing plant

  • Sow the haploid seeds and again cross the haploid plants with the inbred line for restoring the diploid state of resulting embryos.

2. In-vivo maternal haploid induction-based DH development

  • Cross the source population as female parent with pollen of haploid inducer

  • Identify the haploid kernels from the diploid ones using anthocyanin colour marker referred as R1-Navajo (R1-nj) (expresses in aleurone as well as embryo in haploid inducer)

  • Sow the haploid kernels of maize in field, after germination apply colchicine or any other chromosome doubling agent during the seedling stage. Plants obtained are known as D0

  • Self-pollinate the D0 to obtain D1 (double haploid) plants. These DH plant can be further use in breeding programmes

 Molecular basis:

  1. Maternal inducers: A frameshift mutation in gene MATRILINEAL(MTL)/ZEA MAYS PHOSPHOLIPASE A1 (ZmPLA1)/NOT LIKE DAD (NLD), coding for a pollen-specific phospholipase causes haploid induction in maternal inducers.

  2. Paternal Inducer: Indeterminate gametophyte(ig) gene is responsible for haploid induction as a single gene trait.

Benefits of DH in maize breeding

  • Development of completely homozygous plant in 2-generation only

  • Enable greater efficiency and precision of selection, especially when used in combination with molecular marker

  • Allow rapid gene pyramiding for favorable alleles influencing maize productivity and stress resistance

  • Efforts to maintain the lines reduce

  • Provides opportunities for undertaking marker-trait association studies, marker-based gene introgression, functional genomics, molecular cytogenetics, and genetic engineering

Future Perspectives:

Implementation of DH technology requires new skill both in production of Dh plants and its implementation in breeding programmes. To scale up the DH production by institutions in maize growing countries, there are some important issues which need to be considered namely, for DH production trained scientific and technical personnel is required, haploid maize plant derived in vivo are often weak and prone the environmental stresses and breeding efficiency and genetic gain can be enhanced when DH technology is combined with marker assisted selection and year-round nurseries.


Trentin, H.U., Frei, U.K. and Lubberstedt, T. (2020). Breeding maize maternal haploid inducers, Plants (Basel), 9(5): 614.

Parsana, B.M., Chaikam, V and Mahuku, G (2012). Doubled Haploid Technology in Maize Breeding: Theory and Practice. Mexico, D.F.: CIMMYT.


Kiran*1, Deepak Kumar1, Ganesh Kumar Koli1 and Deepak Kumar2

1Department of Genetics and Plant breeding, CCS HAU, Hisar

2Agronomy Section, ICAR-NDRI, Karnal


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