Use of Double

Haploid in Barley
Improvement
Introduction-1
Terminology and Background of -
Haploids
Advantages and Limitations- 2
Haploid-Production Systems- 3
A- Chromosome Elimination (Bulbosum
(Method
(B- Androgenesis (Anther Culture
(C- Genogenesis (Ovary Culture
D- Haploid Initiation Gene
 Terminology and. 2
Background
Haploid: an individuals with the gametic chromosome
number in its
somatic cells
Doubled Haploid: an individual with the doubled
chromosome number of the haploid

Monoploid - haploid derived from a diploid, x is one

genomic complement (n=x) as Barley (n=x=7).

Polyhaploid - haploid from a polyploid (n> x) , as

wheat n = 3x = 21; durum n = 2x = 14
Year Population Processes
Initial

1st crosses made AABB x

aabb

2nd F1 AaBb

3rd F2 1AABB 2AABb 1AAbb 2AaBB

4AaBb 2Aabb
6.25% 1aaBB 2aaBb
1aabb
Year Population Processes
Initial 1st crosses made
AABB x aabb

2nd F1 AaBb
Haploid production
Cochicine treatment
Doubled Haploid (25%) AABB AAbb
aaBB aabb

3rd Head Row Test

 First : Homozygous lines can be
obtained in the
.shortest possible time
 By extracting haploids from F1 hybrids of diploid barley
and then doubling the chromosome numbers , this saves
at least 3 to 4 generations of self-pollination, which are
normally required when producing uniform lines for
conventional breeding of self-pollinated species.
 Reduction of 3-5 years for cultivar release
 Second: selection is more reliable
and effective at the
DH than at the
. diploid level
 100% homozygosity of doubled haploid
 Using DH lines , we can evaluate quantitative
characters knowing that all genes have been
fixed and that desirable alleles will not be lost
due to segregation in later generations.
 The additive genetic variance of DH lines is always
greater than the diploid lines ; therefore, the response
to selection should be correspondingly greater in
populations of DH lines than in diploid
populations.
Third: the homozygosity of DH lines
is reached without the selection
.pressure of a field environment

can be repeated anytime

can be used in different Lab
can be used by different researchers

Because of this, one could set up facilities

at one location to produce new lines for
evaluation in any part of the world.

Fourth: DH lines can be used for
quantitative genetic studies if
they are a random sample of gametes
. from F1 hybrids

A. Detection of Gene Interaction

B. Estimation of Genetic variances
C. Detection of Linkage
D. Estimation of the Number of Genes
E. location of polygenes
  Fifth: haploids have a value in
mutation breeding:

(1) Both recessive and dominant mutations

will be expressed.
(2) Selection on the basis of gamete
frequency as haploid plants dose not
require large population compared with
diploid.
(3) Any mutations associated with
deleterious effects should be selected
against during haploid production.
Sixth, the population size
required to obtain a
desired genotyoe from haploids is
samller than that from
an F2 population
 Limitations
 Limited numbers of DH lines could be
produced, therefore, the number of
crosses one can utilize per year is
limited.

 Both the bulbosum method and anther

culture techniques require controlled-
environment growth facilities and skilled
technical personnel.
 Haploid-Production Systems
 Barley haploids can arise spontaneously or by
chemical induction.
 Because the frequency of such spontaneous
haploids is very low and unreliable, they have
little value for breeding.
 Based on the derivations of haploids, haploid-
production systems for breeding barley can be
classified into four categories : chromosome
elimination (bulbosum method), androgenesis
(anther culture), genogenesis (ovary culture),
and haploid initiation gene.
 A- Chromosome Elimination (Bulbosum
Method) and Embryo Rescue
 The phenomenon of chromosome
elimination was discovered
in interspecific cross between
(Hordeum vulgare x H. bulbosum ).

 For long time , barley breeders have

shown interest in crossing Hordeum
vulgare x H. bulbosum in the hope of
transferring desirable characters from the
wild species to cultivated barley. A few
hybrids had been obtained from this cross
but the success rate was low .
Hordeum vulgare Hordeum
bulbosum
Kasha and Kao (1970) pointed out that
a high frequency of haploids could
be obtained from this cross using
various lines of diploid
.Hordeum vulgare as female parent
 In addition to the H. vulgare x H. bulbosum
cross, chromosome elimination and haploid
plants have been obtained from many
other interspecific and intergeneric crosses
( barley x maize , barley x Italian ryegrass ).
However, the frequencies are too low to be
of interest as a barley breeding systems.
 Bulbosum Method
 Day 0 – emasculation
 Day 2 - pollination with H. bulbosum pollen
 Day 3 (to 5) - 40% of the embryonic cells are haploid,
endosperm abortion occurs, GA3 treatment
enhances retention of florets
 Day 11 - 94% of the embryonic cells are haploid
Day 14 (to 16) - embryos are dissected and
cultured in the dark at 18 to 22 C, embryos
develop in vitro
Day 22 (to 28) - embryos are transferred to light
for seedling development
Day 50 – plants
Double fertilization dose occur and
chromosome elimination proceeds
gradually in both the embryo and
endosperm .

By 8-10 days post fertilization, most

dividing cells in the embryo are
.haploid
 The preferential loss of bulbosum
chromosomes has been
demonstrated cytologically and by
gene markers.
 Chromosome elimination process in embryos
derived from
H. vulgare x H. bulbosum
Days after No. of No. of cells containing Mean
pollination Total Chromosome
Chromosome
cultivar Embryo
 14 13 12 11 10 9 8 7
number
Scored

3 Kanto Nijo 15 117 0 24 2 16 0 2 15 176 12.5

25
15 124 1 13 0 9 0 1 12 160 13.0
Yoshikei 15
5 Kanto Nijo 13 15 0 4 1 20 3 10 26 79 9.6
25
13 20 3 11 0 15 0 8 20 77 10.5
Yoshikei 15
7 Kanto Nijo 10 54 54 7.0
25 55
10 55 7.0
Yoshikei 15
9 Kanto Nijo 10 56 56 7.0
25 7.0
10 51 51
Yoshikei 15
11 Kanto Nijo 8 35 35 7.0
25 7.0
8 33 33
Up to 50-60 % of the embryos cultured
have given rise to mature
haploid plants. More than 90 %
of these mature haploid of H.vulgare
and the remainder are diploid
hybrids that can be readily
distinguished on the basis of their
decumbent growth habit and
other bulbosum- like
.characteristics
Thus, chromosome checks are not required to
identify haploids. Using this
method , one technician can produce 50-100
haploids/week, or under ideal conditions and with
specific genotypes, up to 300 haploids/week .
 Androgenesis (Anther
(Culture
Androgenesis – haploid plant derived
from male gamete,
most common method in vitro

Anther and microspore (pollen)

culture - haploid plants are derived
from microspores (pollen) cultured
individually or in anthers
 :History
In barley, Clapham (1971) first reported the
induction of callus from cultured
anthers ,
and in (1973) the production of haploid plants

 Subsequently, significant progress has

been made in haploid production .
 If reliable and productive anther culture
techniques became available, this system could
have greater potential than the bulbosum
method for use in barley breeding.
 The callus can arise by one
of the following three
:pathways
 Division of the vegetative cell while the
generative cell is non functional
 Division of the microspores in which a
generative cell is not formed
 Division involving both the vegetative
cell and the generative cell
Vegetative
Generative
Vegetat 3 to 5°C

ive Microspore

Similar nuclei
Generativ
e
3 to 5°C

Embryo
 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
Actively growing plants and the first set of
flowers are most responsive
Cold pretreatment of anthers - either pre- or
post-culture treatment (3 to 5 oC for 2 to 4
days), symmetric rather than asymmetric
division of the microspore nuclei or division of
the vegetative nucleus
 Although these results are
most encouraging, the
following areas require
:further research
 the strong genotypic response to culture
condition
 the relatively high frequencies of albino
plants
 the variation in chromosome numbers of
plants derived from callus
 establishing the stability of microspore-
derived plants in subsequent generation
 Genogenesis (Ovary
(Culture
 All plants obtained have been green and haploid,
but the frequencies were very low , ranging from
0.2 to 1.4% of the ovaries culture .
 There has been an influence of genotype , and
perhaps as in the case of parthenogenesis , some
stimulus will be required to increase haploid
frequencies .
 At present , haploids from ovary culture are not
interest for breeding barley .
 Parthenogenesis - from
unfertilized
egg

 Apogamy - from other cells of the

mega-
gametophyte
Parthenogenesis and Apogamy
 Haploid Initiation Gene
 Hagberg and Hagberg (1980 ) discovered this
mutant gene accidentally in a cytological study.

 The haploid Initiation Gene ( hap ) appears to

be a single gene that controls the abortion or
the survival of abnormal embryos and
endosperms, and especially favors the
formation and survival of haploids.
The original mutant line produced
11-14% haploids in its progeny when a
plant homozygous for the hap gene
was used as the female parent in
crosses with other cultivars

8% of the progeny were maternal

haploids: in contrast, their
reciprocal crosses produced no
.haploids
Thus the hap locus acted through
the maternal tissue, either
to
 -prevent fertilization of the egg cell
nucleus
 -stimulate the egg cell nucleus to
divide prematurely,
 - possibly to allow the haploid or
unbalanced embryos to develop
normally
 The F2 progeny of the
crosses between
Homozygous mutant parent x Normal
parent hap/hap x +/+
or its
reciprocal crosses
included between 2 and 3%
haploids Indicting that the
hap/hap gene was partially
dominant.
 The haploid plants, when their
chromosome numbers were
doubled , gave ratio of 34
hap/hap : 14 +/+ , a significant
deviation from the expected 1:1
. ratio
 Suggesting that the maternal
Suggesting that the maternal
genotype had an important influence
on the formation and survival of
haploid embryos .
 The F3 progeny lines often had 30-
40% viable haploid plants.
 The Limitations of this
method
 The viability and the vigor of haploids
differed from cross to cross .
 Many hap/hap seed did not
germinate because of embryo
abortion.
 The mechanism of this haploid-
product system and the reason for
embryo abortion are not clear .
Chromosome Doubling

The spontaneous doubling rate of

barley haploids ranges from 1 to
3% .
this rate is too low for barley
breeders and geneticists to
achieve an efficient and rapid
transformation of sterile , haploid
materials into fertile homozygous
.lines
 Consequently, artificial
means have to be used to
doubling chromosome
number is spindle inhibition
by colchicines.
It doesn’t affect
chromosome replication, but
does
prolong the time for mitosis
Normal versus colchicine
Mitosis
 Barley haploids can be
treated with colchicines
at various growth
stages.
Three-leaf stage
Two tiller stage
Three tiller stage
 powder (1%), 20 ml
dimethylsulphoxide
(DMSO), and 10 drop of Tween 20
per liter
• Post - treatment after
vernalization
– take more time but may save
more haploid plants
– vernalize green plants right after
regeneration
– transfer haploid plants to
vermiculite after 6-8 weeks
– treat them with colchicine
Before Colchcinie Treatment
After Colchicine Treatment