Haploids & their applications

Definition
 The term haploid refers to those plants which possess
a gametophytic number of chromosomes (n) in their s
porophytes.
 History
- In1953, Tulecke obtained haploid callus (but no
plants) derived from Ginkgo biloba.
- In1964, Guha and Maheshwari reported direct de
velopment of embryos from microspores of Datur
a innoxia.
- In1967, Bourgin & Nitsch obtained complete hapl
oid plants of Nicotiana tabacum.
Overview of floral organs
 Haploid Plant Formation

• In vitro methods:
– Anther culture (androgenesis) - production of hap
loid plants from microspores
• Anther culture for production of haploids reporte
d in about 250 species
• Solanaceae, Cruciferae, Gramineae, Ranunculac
eae most common
– Ovule culture (gynogenesis) - production of haplo
id plants from unfertilized egg cell.
 Androgenesis
• Haploid plant derived from male gametophyte of an a
ngiosperm plant (most common method in vitro).
• Haploids can be obtained by the culture of excised ant
hers & culture of isolated pollen.
 Reproductive floral organs: male
• Stamen – male floral organ, c
onsists of:
-Anther – A typical anther sho
ws 2 anther lobes & each lob
e possesses 2 microsporangia
or pollen sacs.
-Filament – stalk-like structure
that holds anther
-Pollen – immature male gamet
ophyte
 Production of haploid plants & diploidization
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 P
agation and Breeding. MacMillan, New York. p. 228-287.
 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
Microsporogenesis/microgametogenesis l
eading to haploid embryo formation
• Haploid embryo formation based on continued divisio
ns 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 divisi

on of the microspore - rather than asymmetric divisio
n that leads to pollen formation, most common path t
o haploidy.
Pathways to Androgenesis
 Culture medium
• Anther culture –
- essential micro- and macronutrients, sucrose and vita
mins; bicellular pollen types require 2 to 4% and trice
llular types 6 to 12% sucrose.
• Microspore/pollen culture –
- bi-cellular pollen types only - basal components + glu
tamine, serine and elevated levels of inositol.
 Culture medium
• Hormone dependency as follows:
• Hormone independent group - embryos directly from
the microspore , predominantly bi-cellular pollen type
s, e.g. tobacco

• Hormone dependent group - bi- or tri-cellular pollen t

ypes and plants are regenerated through a callus inter
mediary, typically requires auxin and, in some instanc
es cytokinin, e.g. grasses.
 Growth Regulators
• Pollen embryogenesis requires low levels of auxins, c
ytokinins and giberellins.

• Wheat anthers cultured on a medium having 2,4-D pr

oduce callus while those kept on a medium suppleme
nted with coconut milk give rise to embryos.
Factors affecting the development of hap
loid plants in vitro

• Donor plant or anther pretreatment – enhances haploi

d embryo formation.
• Cold pretreatment of anthers - either pre- or post-cult
ure treatment (3 to 5 oC for 2 to 4 days). Cold Treatm
ent (3 to 5°C) Enhances Symmetric Division of Mic
rospores or Division of Vegetative Nuclei
Cold Pretreatment of Anthers Enhances the Embry
ogenic Response
Cold treatment imposed prior to the first pollen mitosis in
creases the frequency of symmetric divisions of the microsp
ore leading to embryo formation, control – room temperature
.
100 3°C

80
Producing Embryos

5°C C
% Anthers

60

40
C
20

0
Tobacco Datura
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
 Flower buds

Anther

Microspore mother cells

Meiosis
Tetrads of microspores (Pollen grains)

Uninucleated pollens

Culture medium

Callus (haploid)

Embryogenesis

Plantlet (n)
 Gynogenic Haploids
• Haploid plant derived from megaspore or fema
le gametophyte of an angiosperm plant .
• Haploids can be obtained by the culture of exci
sed ovary and ovule.
 Gynogenic Haploids
• In vitro culture of unpollinated ovaries and ovules rep
resents an alternative for the production of haploid pl
ants in species for which anther culture has given uns
atisfactory results.
• Used in plant families that do not respond to androge
nesis
– Liliaceae
– Compositae
• The first report on the induction of gynogenic haploid
s was in Barley by San Noeum (1976)
 Culture medium
• The normal White or MS or N6 inorganic salt
media supplemented with growth substances a
re used.
• Sucrose as a carbon source is essential
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
 Value of Haploids in Breeding

• Haploids are very valuable in plant breeding for se

veral reasons
– Since they carry only one allele of each gene, m
utations and recessive characteristics are express
ed in the plant.
– Plants with lethal genes are eliminated from the
gene pool.
– Can produce homozygous diploid or polyploid p
lants - valuable in breeding
– Shorten the time for inbreeding for production o
f superior hybrids genotypes.
Agricultural applications for haploids
• Rapid generation of homozygous genotypes after chr
omosome doubling.
• Reduce time for variety development, e.g. 10 to 6 yea
rs or less.
• Homozygous recombinant line can be developed in o
ne generation instead of after numerous backcross ge
nerations.
• Selection for recessive traits in recombinant lines is m
ore efficient since these are not masked by the effects
of dominant alleles.