Biotechnology in horticulture

1. Haploids & their applications

2. Definition
 The term haploid refers to those plants which possess
a gametophytic number of chromosomes (n) in their
sporophytes.

3. History
- In1953, Tulecke obtained haploid callus (but no
plants) derived from Ginkgo biloba.
- In1964, Guha and Maheshwari reported direct
development of embryosfrom microspores of
Datura innoxia.
- In1967, Bourgin & Nitsch obtained complete
haploid plants of Nicotiana tabacum.

4. Overview of floral organs

5. Haploid Plant Formation
• In vitro methods:
– Anther culture (androgenesis) - production of
haploid plants from microspores
• Anther culture for production of haploids
reported in about 250 species
• Solanaceae, Cruciferae, Gramineae,
Ranunculaceae most common
– Ovule culture (gynogenesis) - production of
haploid plants from unfertilized egg cell.

6. Androgenesis
• Haploid plant derived from male gametophyte of an
angiosperm plant (most common method in vitro).
• Haploids can be obtained by the culture of excised
anthers & culture of isolated pollen.

7. Reproductive floral organs: male
• Stamen – male floral organ,
consists of:
-Anther – A typical anther
shows 2 anther lobes & each
lobe possesses 2
microsporangia or pollen
sacs.
-Filament – stalk-like structure
that holds anther
-Pollen – immature male
gametophyte

9. Microsporogenesis/microgametogenesis
leading to haploid embryo formation
• Haploid embryo formation based on continued
divisions of the vegetative or generative cells -
embryos are derived from continued proliferation of
either of these cells rather than pollen formation.
• Haploid embryo formation based on symmetric
division of the microspore - rather than asymmetric
division that leads to pollen formation, most common
path to haploidy.

10. Normal pollen development
• Pollen mother cells are in anther primordia
• First phase - meiosis - pollen mother cell (PMC)
• A tetrad forms from each PMC
• Second phase - microspores released from tetrads
• Third phase - microspores mature into pollen grains -
first pollen mitosis
• Second pollen mitosis, maybe after germination
• Generative and vegetative cells formed

11. Pathways to Androgenesis

12. Culture medium
• Anther culture –
- essential micro- and macronutrients, sucrose and
vitamins; bicellular pollen types require 2 to 4% and
tricellular types 6 to 12% sucrose.
• Microspore/pollen culture –
- bi-cellular pollen types only - basal components +
glutamine, serine and elevated levels of inositol.

13. Culture medium
• Hormone dependency as follows:
• Hormone independent group - embryos directly from
the microspore , predominantly bi-cellular pollen
types, e.g. tobacco
• Hormone dependent group - bi- or tri-cellular pollen
types and plants are regenerated through a callus
intermediary, typically requires auxin and, in some
instances cytokinin, e.g. grasses.

14. Growth Regulators
• Pollen embryogenesis requires low levels of auxins,
cytokinins and giberellins.
• Wheat anthers cultured on a medium having 2,4-D
produce callus while those kept on a medium
supplemented with coconut milk give rise to
embryos.

15. Factors affecting the development of
haploid plants in vitro
• Anther stage - most responsive cells for haploid
embryo formation are those between the tetrad stage
of microsporogenesis to just past the first pollen
mitosis.
• Donor plant or anther pretreatment – enhances
haploid embryo formation.
• Cold pretreatment of anthers - either pre- or post-
culture treatment (3 to 5 oC for 2 to 4 days).

16. Similar nuclei
3 to 5°C
Microspore
Embryo
3 to 5°C
Generative
Vegetative
Cold Treatment (3 to 5°C) Enhances Symmetric Division
of Microspores or Division of Vegetative Nuclei

17. Cold Pretreatment of Anthers Enhances the
Embryogenic Response
Cold treatment imposed prior to the first pollen mitosis
increases the frequency of symmetric divisions of the
microspore leading to embryo formation, control – room
temperature.
Tobacco Datura
0
20
40
60
80
100
5°C
3°C
C
C
%
Anthers
Producing
Embryos

18. Factors influencing androgenesis
– Genotype of donor plants
– Anther wall factors
– Culture medium and culture density
– Stage of microspore or pollen development
– Effect of temperature and/or light
– Physiological status of donor plant

19. In vitro morphogenesis of pollens
Direct androgenesis
(Normal embryogenesis
occurs as in the plants of
family Solanaceae and
Brassicaceae)
Indirect androgenesis
(Callus formation
occures as in barley,
wheat and coffee)

20. aj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y. Yamada (eds.), Handbook of Plant Cell Culture. Volume 1. Techniques for
pagation and Breeding. MacMillan, New York. p. 228-287.
Production of haploid plants & diploidization

21. Flower buds
Anther
Microspore mother cells
Meiosis
Tetrads of microspores (Pollen grains)
Uninucleated pollens
Culture medium
Callus (haploid)
Embryogenesis
Plantlet (n)
Mutagens
0.5% Colchicine
For 24-48 hrs
Binucleate (n)
Fusion (2n)
Plantlet (2n)

22. Gynogenic Haploids
• Haploid plant derived from megaspore or
female gametophyte of an angiosperm plant .
• Haploids can be obtained by the culture of
excised ovary and ovule.

23. Gynogenic Haploids
• In vitro culture of unpollinated ovaries and ovules
represents an alternative for the production of haploid
plants in species for which anther culture has given
unsatisfactory results.
• Used in plant families that do not respond to
androgenesis
– Liliaceae
– Compositae
• The first report on the induction of gynogenic
haploids was in Barley by San Noeum (1976)

24. Culture medium
• The normal White or MS or N6 inorganic salt
media supplemented with growth substances
are used.
• Sucrose as a carbon source is essential

25. Factors influencing gynogenesis
– Genotype of donor plants
– Growth conditions of the donor plant
– Stage of harvest of ovule
– Embryo-sac stage
– Culture medium
– Physical factors

26. Value of Haploids in Breeding
• Haploids are very valuable in plant breeding for
several reasons
– Since they carry only one allele of each gene,
mutations and recessive characteristics are
expressed in the plant.
– Plants with lethal genes are eliminated from the
gene pool.
– Can produce homozygous diploid or polyploid
plants - valuable in breeding
– Shorten the time for inbreeding for production
of superior hybrids genotypes.

27. Agricultural applications for
haploids
• Rapid generation of homozygous genotypes after
chromosome doubling.
• Reduce time for variety development, e.g. 10 to 6
years or less.
• Homozygous recombinant line can be developed in
one generation instead of after numerous backcross
generations.
• Selection for recessive traits in recombinant lines is
more efficient since these are not masked by the
effects of dominant alleles.