double haploids in plant breeding

1. Double Haploids

2. What is Double Haploid?
• A doubled haploid (DH) is a genotype formed
when haploid cells undergo chromosome
doubling.

3. What is Haploid?
Plant or cell with gametophytic chromosome number {n}.
meiosis results in haploidy : Haploid organism/cell is the result of normal meiosis, in
which chromosome number gets halved. So, be it diploid, tetraploid, hexaploid, etc
E.g.: A hexaploid organism has 42 chromosomes; When its meiocytes undergo meiosis,
each of the four resultant cells will have 21 chromosomes each and will be
called haploid cells.
If it's tetraploid with 42 chromosomes, result of meiosis will still remain 21.
So, basically, haploid is term used for half the number of chromosomes an organism
has, as a result of meiosis.

4. Monoploids
• Monoploid : is basic set of chromosomes
in an organism{x}
• If the original plant was diploid {2n}, the
haploid cells are monoploid {x}
• Therefore n=x
• If a hexaploid organism has 42 chromosomes,
that means its diploid cell had 14
chromosomes and thus, its monoploid will be
7. Remember, this is different from haploid.

5. Difference b/w haploid and monoploid
haploid monoploid
haploid is (cytology) of a cell having a
single set of unpaired chromosomes, such
as a gamete
monoploid is (genetics) having a single
set of chromosomes
Denoted by “n” Denoted by “x”
If an organism is diploid, it's monoploidy and haploidy will be same.

6. Difference b/w double haploids and
dihaploids?
double haploids dihaploids
Double haploid organism is produced by
doubling chromosomes in a haploid cell.
E.g: If a diploid organism has 14
chromosomes, it's haploid cells would
naturally contain 7 chromosomes. If that
haploid cell is subjected to chromosome
duplication, resultant organism would
be double haploid.
Dihaploid organism is produced when
haploid cells of tetraploid, hexaploid, etc.
organisms are subjected to chromosome
duplication.
E.g. If a hexaploid organism has 42
chromosomes, it's haploid cells will have 21
chromosomes and it's dihaploid cells will
have 42 chromosomes, where 21
chromosomes will be same as other 21.
Note : resultant organism would be
homozygous for every gene
Note : this organism might be
heterozygous, as genes may have different
alleles in it's haploid cells itself. Of course,
it's a hexaploid.
Double haploid is strictly used for diploid organisms while dihaploid can be used for
tetraploid, hexaploid, etc. organisms

7. History
• 1922- Blackslee et al., reported first
spontaneous haploid in Datura stramonium.
• 1964: Guha and Maheshwari, first haploid via
anthers of Datura inoxia

8. Traditional breeding / DH Breeding
Traditional breeding DH Breeding
After 7 yrs homozygosity attained is 98.4% After 2yrs homozygosity attained is 100%
Hetrosis cannot be fixed Hetrosis can be fixed
New cultivar is formed in 8-10 yrs New cultivar is formed in 3-5 yrs
Recessive mutant identification is difficult Recessive mutant identification is easy
Is not cost effective expensive
Temporary mapping population Permanent mapping population achieved

9. Application of DH
• Homozygosity Achieved Is 100%
– In SP crops used directly as cultivar
– In CP crops used as inbreds
• Used to identify recessive mutants
• Used as permanent mapping population
• Used to identify molecular markers for trait
selection
• Used in QTL analysis
• Constructions of genetic maps

10. Disadvantages of DH
Frequency of haploid production is very low
Highly genotype dependent
Highly unpredictable
Costly
Chemicals used are harmful

11. Methods for haploid production
• Chromosome elimination
• Bulbosome method
• Parthenogesis: pseudogamy, semigamy, apogamy
• Inducer based approach
In vivo method
• Androgenesis: anthers
• Gynogenesis: ovary, egg, ovule
In vitro method

12. How are haploids?
smaller
Lower plant vigour
Sterile {as unable to pair during meiotic pairing}
Note : in order to use these Haploids in breeding programes and propogate them
through seeds , their fertility has to be retained by either spontaneously or induced
chromosome doubling

13. Haploid techniques
Induction of
maternal
haploids
In situ
induction
In vitro
induction
Induction of
paternal
haploids
Androgenesis

14. In situ induction of maternal haploid
It can initiated
by pollination:
pollens from
Same species
{maize}
Irradiated pollen
Wild relatives
{barley, potato}
Unrelated
species {wheat}

15. Procedure
pollination Fertilization of
egg
Hybrid
developed
Paternal
chromosome
eliminated in early
embryogenesis
Egg is not fertilized
Haploid
embryo
developed
Polar nuclei
pollinated forming
endosperm

16. Pollination
pollen of same
species
Majority of regular hybrids formed
Small proportion of haploid maternal
embryo with normal triploid
endosperm formed
Irradiated pollen
Irradiated pollen unable o fertilize egg cells
Eg. Sunflower, nicotiana
Disadvantages: need efficient
emasculation
Too laborius
Dose of irradiation
•If low: generative nucleus partly damage
hence maintain the capacity to fertilize egg
cells
•If dose increased: decrease the no. of
developed embryo but obtained
regenerants are mostly haploids
Wide
hybridization
Very effective
Involves
interspecific and
intergeneric
pollination
Bulbosome
method

17. Bulbosome method
• First ever used induction method to produce large no. of haploids
• In Barley: wide hybridization between barley and wild relative
Hordium
vulgare
• Female
• 2n=2x=14
H.
Bulbosome
• Male
• 2n=2x=14
Hybrid
embryo
{chromosom
e of both
parents }

18. chrromosome of wild relative is
preferentially eliminated from
developing embryo.
Due to failure of endosperm
development, haploid embryo is
formed.
This embryo is extracted and grown
invitro
During embryogenesis:

19. Bulbosome method: wheat X maize
Hybrid grown in vitro: because endosperm fails to
develop in such seeds
Maize chromosome eliminated after hybrid embryo
formed to get wheat haploid
After pollination

20. In vitro induction of maternal haploids
Used where androgenesis and pollination failed
Used in many species like cucumber, onion, sugarbeat,
squash, sunflower, wheat, barley
In vitro Culturing :
• Ovule
• Placenta attached ovule
• Ovaries/whole flower bud

21. Induction of Paternal Haploids:
Androgenesis
Androgenesis :
Direct: embryo Indirect: callus
Used in crops like barley, wheat, maize, rice, triticale, rye, tobacco,
rapeseed and other brassica
Haploids are produced by using male gametophyte:
Anther Microspore

22. Direct Androgenesis
Similar to zygotic embryogenesis
At globular stage: most of the embryo released from
pollen call wall
After 4-5 weeks:
•cotyledons unfolds
•Plantlet emerged from anthers
Eg. Mustard and tobacco

23. Indirect Androgenesis
At globular stage:
irregular and
asynchronous
division occurs
callus formed Organogenesis Haploid plant
Eg: cereals

24. Major drawbacks: Androgenesis
Highly genotype
dependent
• gametophytic to saprophytic.
Relies on ability of
microspore and
mature pollen grain to
covert their pathway :
• Woody plants
• Leguminous plants
• A. thaliana
Recalcitrance of some
important agriculture
species:

25. Factors affecting Androgenesis
Development stage of male gamete
Anther/ microspore stage:
•uninucleated or binucleated
Stress treatment: arrests the microspore
in gamatophytic pathway and triggers
embryogeneis by promoting cell divison
and formation of embryo
Temperature pretreatment
Sucrose and nitrogen starvation
Osmotic stress
Irradiation
Colchicine
Auxin
Water stress

26. Factors affecting haploid induction
Genotype of donor plant
• temperature
• humidity
Physiological conditions
• Late uninucleated
• Early binicleate
Developmental stage of
microspore
• Cold
• Hot
Pretreatment
Culture medium composition
• Light
• temperature
Physical factors

27. Chromosome Doubling: Why?
Haploid plants:
reduced vigour
Smaller
One set of
chromosome- no
meiosis- no seed set.

28. Colchicine
Antimicrobial drug
Extracted from: “Colchicum autumnale”
It binds to tubulin and inhibits the microtubule polymerization
Highly toxic
Used in millimolar cocentration
Other options:
• Oryzaline
• Trifluraline
• Pronamide
• APM{amiprophosmethyle}

29. Maintenance of DH
SP crops: normally selfed and maintained
• Treating with co2 rich environment
• Gibberlic acid
• Nacl, urea, ammonium sulfate used on stigmas
CP crops: for strongly self incompatible crops various
techniques been used:
Clonally propagated: micropropagation