Plant Breeding and Genetics

2. Indian
Agricultural
Research
Institute
New
Delhi,
India
Department of Plant Breeding and Genetics
S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner
Credit Seminar on
Doubled Haploid Technology for Line
Development in Maize
Major Advisor
Dr. D.K. Gothwal
Professor
Seminar Incharge
Dr. S.S. Rajput
Assistant Professor
Presented by
Giradhari Lal Yadav
Ph.D. Scholar (PBG)

3. Indian
Agricultural
Research
Institute
New
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India
Contents
 Introduction
 History of maize doubled haploids
 Types and techniques of doubled haploids
 Selection of individual for haploid induction
 Induction of haploids
 Identification of haploids
 Chromosome doubling in the haploids
 Seed production from D0 nursery
 Case study
 Advantages of doubled haploids
 Disadvantages of doubled haploids
 Applications of DHs in plant breeding
 Conclusions

4. Indian
Agricultural
Research
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New
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India
Introduction
Doubled haploid: A “doubled haploid” (DH) is a genotype
formed when haploid (n) cells successfully undergo either
spontaneous or artificially induced chromosome doubling.

5. Indian
Agricultural
Research
Institute
New
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India
History of maize doubled haploids
 Chase(1949): spontaneous haploid production 0.1% HIR
and chromosome doubling
 Coe(1959): Identified a line having higher haploid
production and designated as “STOCK 6” with 2-3% HIR
 Rober et al., (2005): RWS line with 8.1% HIR frequency
based on R-nj marker
 Li et al., (2009): CAUHOI line with 2% HIR frequency,
based on kernel oil content

6. Indian
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In vitro In vivo/ in situ
Paternal
Anther Culture and
Pollen Culture
Inducer line used as female
and Donor parent used as
male
Maternal Ovule culture
Inducer line used as male
and Donor parent used as
female
Techniques
Different techniques of Doubled Haploids
production in maize
Types

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(A) In vitro methods of doubled
haploid production
 These methods use tissue culture techniques for the production
of haploids.
 In vitro methods have shown little promise to reliably produce
the large numbers of DH lines required by maize breeding
programs.
 In vitro methods had very limited success due to non
responsiveness of many maize genotypes.
 These methods require a good laboratory and skilled staff.
 In vitro methods are of two types:
1. Androgenesis: includes pollen culture and anther culture
2. Gynogenesis: includes ovule culture and ovary culture

8. Indian
Agricultural
Research
Institute
New
Delhi,
India (1) Anther Culture/ Pollen Culture
(In Vitro Androgenesis)
 Anon (1975) first reported the successful anther culture of
maize.
 The successful application of anther culture techniques in
maize breeding is largely dependent on the androgenic
responses of the genotypes and on the frequency of induced or
spontaneous genome doubling in plants of microspore origin.
 Intensive studies have been carried out to improve the culture
conditions, leading to greater androgenic response, but most of
the genotypes responsive to anther culture have been found in
non-commercial maize germplasm.

9. Indian
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Research
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India Anther Culture/ Pollen Culture
(In Vitro Androgenesis)
Collect Anther/ pollen
Plating of
anthers/pollen
Proliferating
anthers/ pollen
Callus
Formation
Haploid plantlets
Colchicine treatment
DH Plant
Embryo development

10. Indian
Agricultural
Research
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New
Delhi,
India (2) Ovary/Ovule Culture
(In Vitro Gynogenesis)
 In vitro gynogenesis so far has been reported only by
two groups i.e. Ao et al. (1982); Truong-Andre &
Demarly (1984). So more research will be required to
evaluate the potential of this method for maize
 The induction of ovule responses is strongly genotype-
dependent.
 The frequency of response with permissive genotypes
ranged from 3.4 to 12%. Only 6 responses were found
from 175 cultured ovules (Ao et al. 1982)
 Truong-Andre and Demarly (1984) obtained 6 plants
from 317 cultured ovules.

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Merits & Demerits
Merits:
It is useful in production of doubled haploids where
plant is male sterile.
This method is useful in embryo rescue technique.
Demerits:
It is highly genotype dependent.
This method gives less number of haploid plants as
compared to anther / pollen culture.

12. Indian
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(B) In vivo methods of doubled
haploid production
In vivo methods of DH production in maize is
relatively easier and widely used.
In this method special genetic stocks are used for
haploid production which are known as Haploid
Induction Lines (HILs)
 These methods do not require any sophisticated
laboratory.
There are two types of doubled haploids-
(1) Paternal doubled haploids
(2) Maternal doubled haploids

13. Indian
Agricultural
Research
Institute
New
Delhi,
India (1) Paternal Doubled Haploids
(In Situ Androgenesis)
 Reported by Kermicle in 1969.
 It is due to mutant gene “ig1” (indeterminate gametophyte).
 Homozygous ig1 mutants show several embryological
abnormalities such as development of egg cell without nuclei.
cc
(HIL)
Note: line W 23 (which carries the ig = indeterminate gametophyte
mutation; Kermicle, 1969) is used as the female parent.
CC
(Donor Source)
x
Haploid
(C)
Chromosome Doubling
DH Plant
(CC)

14. Indian
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India Transfer of CMS using paternal doubled haploids
(HIL)
X
(Normal diploid)
Chromosome Doubling
cc
S
CC
F1 C
S
CC
S
F
DH CMS line

15. Indian
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India Merits and Demerits of Paternal Doubled
Haploids
Merits:
 These are helpful in transferring cytoplasmic male
sterility (CMS) to a different genetic background
within a short period of time (within 2-3 generations)
 Demerits:
• Low frequency of haploid induction i.e. 1-2%.
• Such DHs contain cytoplasm of the inducer and
chromosomes from the donor parent, so this system is
not very attractive to derive inbred lines for breeding.

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(2) Maternal Doubled Haploids
(In Situ Gynogenesis)
 It is very common in maize breeding.
 In this method HIL is used as male parent.
 The first inducer recorded by Coe (1959) called
“Stock 6,” produced maternal haploids at a frequency
of 2–3%.
CC
(Donor Source)
cc
(HIL)
x
Haploid
(C)
Chromosome Doubling
DH Plant
(CC)

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Mechanism of maternal haploid induction
The exact mechanism of maternal haploid induction
have not yet been clearly understood.
The regular double fertilization is distorted after
pollination with the pollen of a haploid inducer line
One sperm cell fuses with the central cell but the
other sperm cell does not fuse with the egg cell. But a
fertilized and dividing central cell stimulates the
unfertilized haploid egg cell to develop into a haploid
embryo (Chase, 1969).

18. Indian
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Steps involved in doubled haploid
Production
1. Selection of individual for haploid induction
2. Induction of haploids
3. Identification of haploids
4. Chromosome doubling in the haploids
5. Seed production from D0 nursery

19. Indian
Agricultural
Research
Institute
New
Delhi,
India (1) Selection of individual for haploid
induction
 Individual plant can be selected by two ways-
a) From a cross between two parents
b) From heterozygous population.
Selection of individual also depends on the breeding objective:
 For inbred development best plant is selected from a
population or from a cross, as a donor and only superior
haploid is developed as inbred after chromosome doubling.
 For development of mapping population F1 from contrast
parents is used as a donor and all haploids are used to make
DH population.

20. Indian
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(2) Induction of haploids
CC
(Donor Source)
cc
(HIL)
X
Haploid
(C)
(2-3% haploid seeds)
Note: In F1 there will be 3 types of seed-
F1
2n 2n
n
3n 2n
3n
Normal Haploid Lethal

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(3) Identification of haploids
 Following methods are used for haploid
identification-
a) Anthocyanin color marker, R1-nj (R1-navajo)
system
b) Purple root and purple stem marker system
c) Transgenic method
d) High oil marker method

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a) R1-nj (Navajo) marker
 Inducer lines carry dominant “R1-nj allele” which codes for
anthocyanin pigment which give purple coloration in seed.
r1r1r1
X
R1 - -
r1r1
R1R1
R1r1r1
r1r1r1
r1r1 r1
Haploids
Out crossed or
Selfed seed
Donor
Source
HIL

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R1-nj….Cont….
Merits:
 It identifies haploids at seed level, hence no need to grow
diploid seed.
Demerits:
 Marker expression can be inhibited by anthocyanin inhibitor
genes like C1-I
 It is a manual method of haploid identification.
 If source germplasm has “R1-nj” allele, then this method can’t
be used.
 Intensity of color expression depends on moisture content of
seed.

24. Indian
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India b) Purple root and purple stem marker
system
 Two genes Pl1 (Purple1) & B1 (Booster1) involved in this
system, which can impart purple or red color to the plant tissues.
 Pl1 (Purple1) conditions sunlight independent purple
pigmentation in plant tissues, and B1 (Booster1) conditions
sunlight dependent purple pigmentation in most of the above
ground plant tissues (Coe, 1994).
 HILs like MHI and Procera have this system along with R1-nj.
Diploid seedlings: Purple roots and stems
Haploid seedlings: No coloration

25. Indian
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Purple root…Cont….
Merits:
 It can be used in a wide array of maize germplasm as root
color phenotype is very rare in maize germplasm.
 It is recommended when the R1-nj marker is not effective.
Demerits:
 It demands germination of large numbers of induced seeds,
which is labor intensive.
 Expression of the B1 and Pl1 genes are affected by plant
growth conditions, especially sunlight and temperature.

26. Indian
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India c) Transgenic method
 In this method, Transgenic HILs are used which carry
herbicide tolerance gene.
 At seedling stage screening is done by herbicide
application.
 Haploids are herbicide susceptible and diploids are
herbicide tolerant.
 In order to identify haploids herbicide is applied on
small portion of seedling.
 The application of transgenic haploid inducers may
not be possible in many countries due to restrictions
on the use of transgenics.

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d) High oil marker method
 This method can facilitate mechanical screening by using NMR
(Nuclear Magnetic Resonance) technique.
 Haploids seeds and diploid seeds naturally show differences in
their average kernel oil content with haploids showing 0.6–
0.8% less oil than the diploids, as >85% oil accumulates in the
seed embryo (Rotarenco et al. 2007; Melchinger et al. 2014).
 Several high oil inducers such as CAUHOI (7.8% Oil content),
UH600 (10.8% OC) and UH601 (11.7% OC) has been
developed.
2n n
3n 3n
Diploid
(High oil content)
Haploid
(low oil content)

28. Indian
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High oil….Cont……
Merits:
 It identifies haploids at seed level.
 This method can be automated.
 It can be applicable in all germplasm including land races and
wild relatives of maize.
Demerits:
 Requires inducer with high oil content for clear difference
between haploids and diploids.
 Requires high cost for establishment of NMR-based
automation platform.

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(4) Chromosome doubling in the haploids
 A commonly used chromosome doubling agent is colchicine,
which is a water-soluble alkaloid produced from the bulbs of
Colchicum autumnale.
 Colchicine binds to tubulins and prevents the formation of
spindle microtubules during the metaphase stage of mitosis.
 4-5 days old seedlings are used for colchicine treatment.
 A solution with 0.04% colchicine and 0.5% DMSO is used for
chromosomal doubling.
 Seedlings are kept in the colchicine tank for 12 hours.
 Seedlings are washed at least three times by distilled water.

30. Indian
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Steps involved in chromosome doubling
(E)
Recovery of
treated D0
seedlings in a
greenhouse
(A)
Germination
of putative
haploid seed
(D0 seed) on
paper towels.
(B)
Cutting of the
coleoptile tip of
D0 seedlings to
facilitate better
penetration of
colchicine.
(C)
Placement of
coleoptile cut
D0 seedling into
mesh bags.
(D)
Treatment of
D0 seedlings
with colchicine
in an iron tank.

31. Indian
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India (5) Seed production from D0 nursery
 Colchicine treated seedlings are very weak, hence need to be
handled with care under controlled condition.
 In D0 nursery there is limited number of pollen production.
 There will be fertile and sterile pollens, so fertile pollens are
used for selfing
 During selfing “false” diploid should be discarded to avoid
contamination.
 Self pollinated ear should be harvested after physiological
maturity.
 Each D0 derived seed will represent distinct DH line.
 In D1 nursery seed is multiplied by selfing and DH lines are
developed.

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Case study
Objectives :
(1) To study the differences between haploids and diploid seedlings
in terms of radicle length (RL), coleoptile length (CL), and
number of lateral seminal roots (NLSR) during early growth
stages
(2) To validate the use of such traits for haploid identification in
populations with complete inhibition of Navajo marker
(3) To determine if seedling traits can be effectively used for early
identification of false positives.

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Results & Conclusion
 Significant differences could be noted among the diploids and
haploids for RL, CL, NLSR, thickness of coleoptile, thickness
of radicle and presence of root hairs upon visual inspection

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Advantages of doubled haploids
 Development of complete homozygous line in 2-3
generations
 Perfect fulfillment with DUS criteria for variety
protection
 Require less time and labor for development of new
breeding lines
 Higher frequency of desirable homozygous plant
 DH can be developed from any material like F1, F2
and Landraces

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Disadvantages of doubled haploids
 The main disadvantage with the DH population is that
selection cannot be imposed on the population.
 Another disadvantage associated with the double
haploidy is the cost involved in establishing tissue
culture and growth facilities.
 The over-usage of doubled haploidy may reduce
genetic variation in breeding germplasm.

37. Indian
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 Mapping quantitative trait loci
 Backcross breeding
 Genetic maps
 Genetic studies
 Genomics
 Elite crossing
 Cultivar development

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Conclusions
 Technological advances have now provided DH
protocols for most plant genera.
 The number of species amenable to doubled haploidy
has reached a staggering 250 in just a few decades.
 Response efficiency has also improved with gradual
removal of species from recalcitrant category. Hence
it will provide greater efficiency of plant breeding