Double Haploid Development and its Role in

Major Crops

Farah Naz
17-ARID-2921
M.Sc. (Hons) Agriculture, Plant breeding & Genetics,
Supervisor:
Dr. Mahmood-ul-Hassan
 CONTENTS

Introduction
Uses of DHs
Double haploid production
In Vitro Method
In Vivo Method
Identify DHs
Chromosome Doubling
Role of DHs in Major Crops
Achievements
Conclusion & Future Perspectives
INTRODUCTION
 Plant or cell with gametophytic
What is chromosome number {n}.
Haploid? meiosis results in haploidy

Introduction
A genotype formed when haploid cells
Double undergo chromosome doubling.
Haploid: Resulting individual is completely
homozygous
 Uses of Double Haploids

Overcome
Development of Development of Construction of
inbreeding
inbred lines cultivars genetic maps
depression

Production of Fixation of genetic

Gene Identification of
biotic and abiotic traits in each locus
tagging/location molecular markers
stress-resistant within one
genes for trait selection.
plants generation.
 Double Haploids Production

1- induction of 2- Doubling of
haploids chromosomes
In Vitro Methods In Vivo Methods
INTRASPECIFIC
GYNOGENESYS HYBRIDIZATION
INTERSPECIFIC
HYBRIDIZATION
ANDROGENESYS
POLLEN TREATMENT
 In vitro method

Anther culture:
 Development of haploid plantlets through in
vitro culture of immature anthers containing
haploid set (n) of chromosome microspores is
called anther culture.
 Referred as androgenesis
 Protocol
Collection of unopened flower buds

Surface sterilized with tween 80 and mercuric chloride

Anthers excised from flower buds and kept separately

Anthers in first meiotic division is selected by acetocarmine test

Inoculated in the medium containing glutamine, L-serine and inositol

Incubated the culture at 25˚C for 15 days. Here, anthers grow in to embryoids

Embryoids transfer to rooting medium, after 4-5 weeks the embryoids became plantlets

After aclimatization, transfer to green house

Interspecific Hybridization
Intraspecific hybridization
 Indirect approaches: morphological
markers, comparison between haploid
and donor plant

Identify
Direct method:
Haploids Cytological method- counting
chromosomes in root tip cells
Flow cytometry
 Chromosome Doubling
Haploids produced are showing poor plant vigour, short and sterile
since they cannot pair with homologous chromosome during meiosis.

Hence spontaneous or induced chromosome doubling is required to

recover their fertility and vigour.

In spontaneous doubling the haploids itself doubles their

chromosome during early developmental stages. E.g. Cereals, rice

In induced method chromosome doubling agents are used in early

stage of haploids e.g. maize
  Conventional methods for homozygous lines
production are cumbersome
 time-consuming, laborious
 it may take years to produce a pure line.
 The introduction of DH technique accelerated
Role of DHs in the breeding program.
major crops  Positive results have been obtained especially
with rice, wheat, potato, barley, maize,
asparagus, sunflower, brassica, tobacco, etc.
 rice and wheat are the best examples
 In wheat, the breeding cycle can be shortened
by three or four generations
 Achievements through DHs Technique
SOURCE MAIN ACHIEVEMENTS SPECIES
John et al. 2020 Haploids were obtained by Barley (H. bulbosum, H. vulgare)
pollination of diploid H. vulgare by
a wild diploid H. bulbosum through
complete loss of the H. bulbosum
genome.

Laurie and Bennett, 2019 Induction of haploids through Wheat, maize, pearl millet
intraspecific hybridization

Inoue et al. 2014 Induction of haploids through Pear and apple

anther culture

Ishii et al. 2015 Elimination of chromosomes in Triticeae, oat, pearl millet

interspecific hybrids due to violation
of the divergence of sister
chromatids in anaphase.
 Conclusion
Resulting plants are free from different social issues and legal
regulations associated with transgenic crops.

Maximizes genetic gains in breeding programs,

Shortens the time needed for cultivar development,

Reduces the cost of breeding programs.

Different multinational seed companies have adopted DH technology

for the wide-scale production of inbred lines.
 Future Perspectives

The identification of a mutation in the gene underlying the Stock6 haploid induction in maize
may make it possible to transfer this method to other cereals.

The development of novel techniques for haploid induction and the subsequent production of
DH plants thus hold huge potential for the management of genetic resources, germplasm
enhancement, and the development of novel plant populations.

Genomic selection, a recently emerging technology for predicting plant productivity without
phenotyping, which has the potential to increase selection efficiency when producing DH
lines, can be of significant help to breeders.
 References
 Aslam, M., Farid, B., Khakwani, K., Maqbool, M.A. and Zou, H. (2017). In vivo maternal
haploid seed production and chromosome doubling with different anti-microtubular agents in
maize. International Journal of Agriculture and Biology, 19, 114‒ 120.
 Ballesteros, J., Castillo, A.M., Cistué, L., García-Lamas, C., Martín, A. and Vallés, M.P.
(2015).Doubled haploid technique in durum wheat breading. In: Royo, C., M.M. Nachit, N. Di
Fonzo, J.L. Araus, W.H. Pfeiffer, and G.A. Slafer, (eds). Durum Wheat Breading, Current
Approaches and Future Strategies, Food Products Press, Binghamton, New York, pp 217-228.
 Bohanec, B., (2019). Ploidy determination using flow cytometry. In: Doubled Haploid
Production in Crop Plants: A Manual. Maluszynski, M., K.J., Kasha, B.P. Forster, and I.
Szarejko, pp. 397-403, Kluwer Academic Publishers, Dordrecht.
 Maqbool, M. A., Beshir, A., & Khokhar, E. S. (2020). Doubled haploids in maize:
Development, deployment, and challenges. Crop Science, 60(6), 2815-2840.
Thank you