Double haploids in ornamental crops

1. PRODUCTION AND UTILIZATIONPRODUCTION AND UTILIZATION
OF DOUBLE HAPLOIDS INOF DOUBLE HAPLOIDS IN
FLOWER CROPSFLOWER CROPS
K.Ravindra Kumar
Roll No. 10461
Division of Floriculture & Landscaping

2. Introduction
 Haploids - defined as the sporophyte plants that contain a
gametic chromosome number (n).
 Monohaploid: e.g. Rose n = x = 7
 Polyhaploid: e.g. French Marigold n = 2x = 24

3.  The history of DHs began with the observation of natural
sporophytic haploid in Datura stramonium L. reported by Bergner
in 1921.
 This was followed by similar discoveries in other plant species e.g.
Nicotiana tabacum and Triticum compactum
 First report on haploid production was published by Blakeslee et
al. (1922) in Datura stramonium.
 Guha and Maheshwari (1964, 1966) developed an in vitro anther
culture technique for the production of haploid Datura innoxia
plants.
 Haploids were reported in many other species, i.e. barley (Kasha
and Kao, 1970), tobacco (Burk et al., 1979), rice, maize, brassica.
History

4.  At present DHs production via anther culture has been reported
in more than 250 plant species belonging to 100 genera and 40
families. (V.K.Misra et al., 2014)
 The first in vitro induced haploid plants of female tissues origin
was achieved by San Noeum (1976) in barley.
 Commercial varieties developed through DH protocols -
reported for many crops and more than 290 varieties have
already been released (Ferrie and Caswell, 2011).
 Compared to agronomic species, there has been very little work
on DH in ornamental species despite the tremendous potential
benefits.

5. Methods to Induce DHs
In vivo occurrence of haploid plants
Spontaneous haploids
Hybridization
a) Intraspecific hybridization
b) Wide hybridization
Parthenogenesis
In vitro Methods
Haploids from male gametes
a) Anther culture
b) Pollen/Microspore culture
Haploids from female gametes
a) Ovary slice culture
b) Ovule culture

6. Spontaneous Haploids
Naturally occurring haploids in 71 species, representing 39
genera in 16 families of angiosperms. e.g. agropyron, alfalfa,
citrus, peach and trillium
Gymnosperms (Pohlheim, F. 1968)
e.g. Unique haploid from of Thuja plicata called Thuja gigantea
‘gracilis’ Beissn
But the exact source of haploids in these species, in terms of
their embryological origin, is often unknown (Dunwell 2010)
Cultivar development has utilized these naturally occurring
haploids.
e.g. ‘Kleine Liebling’ is a haploid cultivar of Pelargonium (Darker
1966), ‘Marglobe’ is a DH cultivar of tomato (Morrison 1932)

7. Haploids through hybridization
Intraspecific hybridization
Observed generation of haploid from diploid material (Haplopappus),
tetraploid material (Pathenium, Sorghum, Sisymbrium and alfalfa)
allopolyploid (Aegilotriticum)
In citrus haploid progeny observed in the interploidy cross between
diploid and triploids (Oiyama et.al. 1993)
Haploids of sugar beet were identified by crossing diploid male sterile
plants with green hypocotyls and tetraploid fodder beets homozygous for
red hypocotyls (Bosemark 1971)
There is a new method to produce haploid from the seed of plants by
manipulating a centromere-specific histone CENH3. When the cenh3
mutant expressing abnormal CENH3 is crossed to the wild type,
chromosomes from the mutant are eliminated, and then eventually the
haploids are produced. This method could be applied to any plant
because the CENH3 is universal in eukaryotes (Ravi and Chan, 2010)

8. In vivo DH induction in
Maize
CIMMYT
Step 1: Induction Cross Step 2: Haploid kernel identification with the
embryo & endosperm marker (R1-nj)
Step 3: Artificial genome doubling
0.06% Colchicine, 0.5% DMSO
solution for 8 h
Step 4: Recovery of seeds from D0
plants
Pollination with pollen of the same species (e.g. maize)
No in vitro culture is needed, since kernels containing haploid embryos display a normal
germination rate and lead to viable haploid seedlings.
Haploid embryos can be selected early in the breeding process, based on morphological
and physiological markers (Dominantly inherited purple coloration from inducer line).

9. Wide Hybridization
Interspecific or intergeneric crosses in which the pollinator
chromosomes are eliminated have been used successfully to produce
maternal haploids.
1: Barley DHs
Hordeum vulgare (cultivated) x H. bulbosum (wild )
(2n=2x=14) (female) (2n=2x=14) (male)
Kasha, K.J. and Kao, K.N., 1970
2 : Wheat , Triticale, Rye, Oat
Wheat (Triticum aestivum) x Maize (Zea mays L.)
Triticale (Triticosecale) x Maize (Z. mays L.)
Rye (Secale cereale) x Maize (Z. mays L.)
Oat (Avena sativa) x Maize (Z. may s L.)
Palmer, et. al., 2005
Embryo rescue is a necessary part of this technique as the endosperm
does not develop and therefore cannot provide nutrients for continued
development of the embryo

10. Parthenogenesis
In this method the egg cell developes into an embryo without
fertilization by the sperm nucleus.
This can be achieved by pollination with irradiated pollen or with
the addition of chemicals (Khush et. al., 1996)
There is generally a low frequency of haploid recovery and
therefore this method is not widely used for breeding purpose.
Ferrie Alison et.al. 2011

11. ANDROGENESIS
Androgenesis is defined as the
process of embryo development
from the male gametophytes
(i.e. microspores or anthers),
with the subsequent
regeneration of haploid and
doubled haploid plants from
these cells.

12. Half anther culture in Anthurium
PCTO, Budi Winarto et.al., 2012

13. Microspore culture – Zantedeschia aethiopica
80 pollen grains/drop
Medium
Microspores
Filter paper
Anthers
Pollen in hanging drops
Isolated microspore culture
Afri. J. Bio., Shimin Wang et.al., 2011

14. Gynogenesis is the culture of
unfertilized female gametophytes (i.e.
ovules, ovaries).
This method is used when plants do
not respond to androgenic methods,
there is a problem with regenerating
albino plants from anther culture, or
the donor plants are male sterile
Gynogenesis is usually less efficient
than androgenesis.
Used in plant families that do not
respond to androgenesis
- Liliaceae
- Compositae
GYNOGENESIS

15. Gynogenesis – Spathiphyllum wallisii
PCTO, Tom Eeckhaut et.al., 2001

16. Factors affecting haploid induction
Donor plant genotype
Physiological condition of donor plants & Pre-treatment
Culture medium composition
Physical factors during tissue culture (light, temperature)
Organic addenda/sugar/sugar alcohol
Developmental stage of gametes, microspores and ovules.
Other miscellaneous factors.

17. Stage and alternatives of microspores
J.Exp.Bot. Jose et.al., 2007

18. In vitro generated haploids in ornamental crops
POP, Ferrie
et.al.,2011

19. Identification of Haploids
Morphological observation
One year old seedlings of
Dendrocalamus latiflorus
a, C & d Anther
regenerated plants of 3x,
6x and 12x b) Seedling
sample 6x
In vitro cell Dev. Biol.
Guirong Qiao et al.,2013
Differences of leaf morphology a) Haploid, B) Diploid and C) Triploid in Anthurium andreanum cv. ‘Tropical
‘
PCTO, Winarto et.al., 2011
Acta.Hort. Gao et.al., 2011

20. Identification of Haploids cont.
Chloroplast number in stomatal guard cells
Anthurium
Chrysanthemum
Haploid, Tetraploid (4x) and Pentaploid (5x)
D) Haploid cell with 19, E) Diploid cell with 30, F) Triploid cell with 52 chloroplasts

21. Identification of Haploids cont.
Chromosomal count
&
Flow cytometry
Determination of ploidy level of regenerated
plants of Popular by flow cytometry (a-c) and
chromosome counting (d-f).
a and d plant displaying a haploid
histogram (2n=x=19)
b and e plant displaying a diploid
histogram (2n=2x=38)
c and f plant displaying a triploid histogram
(2n=3x=57)
PCTO, Ying Li., et. al., 2013

22. Identification of Haploids cont.
DNA finger printing
Anemone coronaria
Zantedeschia aethiopica
A section of AFLP banding patterns:
Lanes 1-4 are from 4 diploid plants from
anther culture, and 5-8 from 4 individual
donor plants. Arrows indicate polymorphic
bands
Scien. Hort. Xiyan Zhang et.al.,2011
RAPD profiles generated by primers
OPA18, OPA4, OPA17 and OPA18 from
DNA of the donor plant (DP) and three
regenerants (AP) of the family A,B and C,
respectively, M: x174 DNA (Hae IIIᶲ
fragment)
Plant Breed. Laura et.al., 2006

23. Double
haploids in
Crop
Improvement

24. Rapid technique
homozygous plants can be
achieved in one generation
(inbred lines)
Production of homozygous lines of
the cross pollinating species and
hybrids are highly desirable
(to avoid inbreeding depression)
The conventional method of
inbreeding is impractical for self-
incompatible, male sterile and tree
species.
Protocols already developed for
Albizza lebbek, Azadirachta indica,
Cassia siamea, Ceratonia siliqua,
Hevea brasiliensis, Peltophorum
pterocarpum, Populus sp.
Biotech. Advances. Priyanka et.al., 2008

25. Shortens the breeding cycle
Normally, in a
hybridization programme
evaluation of lines is
possible only after 4-5
years of pedigree
breeding and it takes
another 4-5 years to
release a new variety.
By anther culture of F1
hybrids the various
genotypes of gametes can
be fixed and evaluated in
the first generation

26. Double haploidy with marker assisted selection

27. New plant breeding techniques (NPBT)
Homozygous parental lines of a
selected heterozygous plant are
reproduced.
The genes involved in the meiotic
recombination process are silenced
through transgenesis.
Consequently, nonrecombined haploid
lines are obtained from the
heterozygous plant and their
chromosomes are doubled through the
double-haploid technique.
The doubled haploids obtained are
screened to find a pair that, would
reconstitute the original heterozygous
plants.
Only nontransgenic plants are
selected, thus the offspring of the
selected parental lines would not carry
any additional genomic charge.

28. Recessive mutants
Haploids are extremely useful for detecting recessive mutants which
may not express themselves in the heterozygous diploid background
and therefore can be easily lost.
Different spate colors produced from flowered plants derived from anther culture of Anthurium. a) spate and
spadix color of A.andreanum cv. Tropical as the donor plant. B-h Spate and spadix color variation of regenerants

29. Other advantages of Double Haploids (DH)
DHs can be represent as new variety (self-pollinated crops)
Induction of mutations
DH lines are also valuable tools in marker-trait association studies,
molecular marker-assisted or genomic selection – based breeding and
functional genomics
Useful in cytogenetic research
Genetic engineering
Useful in development of chromosome substitution lines.
QTL mapping, genetic and physical mapping

30. Objective:
To develop comprehensive anther culture
method for double haploid production in
Primula.
Case study - 1

31. Introduction
Primula is an ornamental plant popular for its early spring blooms and
diversity of colors.
The species exhibits desirable horticultural traits such as long
flowering season, plentiful flowers and a pleasant fragrance.
Producing homozygous lines by conventional methods in this species
is time consuming and difficult, because the species is self-
incompatible.
Anther culture has been successfully applied in many field crops, but
its use is still limited in ornamentals and not yet reported in Primula.
In this study special attention was given to several factors influencing
androgenesis, such as the correlation of bud morphology and size,
microspore developmental stages, plant growth regulator
concentrations.

32. Materials and Methods
Plant material :
200 glass house raised Baby prime rose plants were used as anther donor plants
Determination of microspore development stage
The relationship between the flower bud morphology, bud size, microspore
developmental stage and viability was determined firstly.
Preparation of induction media and induction of callus
Preparation of differentiation media and plant Regeneration
Plantlet rooting and transplantation
Abbrevi
ated
number
PGR (mg/l) No. of
anthers
cultured
BAP 2,4-D
I1 1.0 0.5 603
I2 1.0 1.0 600
I3 1.0 1.5 596
I4 1.0 2.0 598
I5 2.0 0.5 600
I6 2.0 1.0 595
I7 2.0 1.5 605
I8 2.0 2.0 602
Abbreviat
ed
number
PGR (mg/l) No. of calli
cultured
BAP NAA
D1 0.1 0.01 60
D2 0.2 0.01 58
D3 0.5 0.01 56
D4 1.0 0.1 57
D5 1.0 0.2 60
D6 1.0 0.5 56
D7 2.0 0.1 58
D8 2.0 0.2 60
D9 2.0 0.5 59
Abbreviated
number
PGR
(mg/l)
IBA
R1 0.0
R2 0.05
R3 0.1
R4 0.2

33. i) Flow cytometry :
 Leaf samples of in vitro-grown plants derived from anther culture
were collected
 And added to Lysis buffer to release the nuclei.
 The extract was stained with 2 µl DAPI.
 Analysis of nuclei was conducted using a Partec CA II flow
cytometer.
 Tissue from a diploid plant of P.forbesii was used as an internal
standard.
 The ploidy level of each plant was determined based on a
comparison between the fluorescence peak value of the tested
plants with that of the diploid control.
ii) Cytological analysis:
 Actively growing root tips (5-8) approximately 2 mm in length
were used for cytological analysis
 Samples were stained with Carbol fuchsin solution and observed
under microscope (100 x)
Determination of ploidy level

34. Results:

35. Callus induction of P.forbesii via anther culture

36. Shoot regeneration of P.forbesii via anther cultureShoot regeneration of P.forbesii via anther culture

37. Comparison of regenerated plants with different ploidy levels in cultureComparison of regenerated plants with different ploidy levels in culture
Morphological Flow cytometry histograms Chromosomal count

39. Inference
Identified buds of 4.0 – 5.0 mm long for in vitro culturing.
The callus induction rate was significantly high in the media containing
1.0 mg/l BAP and 0.5 mg/l 2,4 – D.
Adventitious buds were observed only on shoot induction medium
with 0.2 mg/l BAP and 0.01 mg/l NAA.
MS without PGRs was suitable for rooting of baby primrose plantlets.
Regenerated plants in this study showed multiple ploidy levels viz.
haploid, diploid, triploid, tetraploid, hexaploid and mixoploid. Only 2%
of plants observed as haploids.
Reasons might be spontaneous, repeated chromosome doubling to
generate polyploid plants include nuclear fusion during early
microspore divisions, endomitosis, endoreduplication or multipolar
mitosis during the callus phase.

40. Case study - 2
Objective:
1.To study the effect of cold pre-treatment on unfertilized
ovule culture.
2.To investigate the ability of embryo like structure (ELS)
production through gynogenesis and reproducibility of the
haploids and DHs through this technique.

41. Two Japanese Gentian species, G.triflora and G.scabra are one of the
most important plants for cut-flower and pot plant use in Japan.
Production of homozygous lines are indispensable for F1 hybrid
breeding, but it is difficult In this species owing to their intense
inbreeding depression.
Efficiency of embryogenesis in anther culture was quite low and large
genotypic variation was observed (Mostly triploids). More over this
method is not effective for G.scabra.
Unfertilized ovule culture has more advantageous than anther culture
from the point of view of embryogenic efficiency, limited influence of
donor plant genotype and a high frequency of haploids production.
Cold pretreatment of flower buds or inflorescences before culture has
been reported to enhance the frequency of embryogenesis in several
species.
Introduction

42. Plant materials: 43 genotypes of gentians consisted of 12 cultivars and 31 lines
of G.triflora (2n = 26), G.scabra (2n = 26), G.triflora var. japonica f. montana (2n
= 26) and their hybrids.
Cold pretreatment: Four genotypes (G.triflora cv. Ashiro-no-Aki, G.scabra line
17-260, G.triflora x G.scabra lines 17-386 and 17-260) were tested.Anthers
removed from each flower, inflorescences were stored at low temperature (40
C)
for 3,7 and 14 days in dark.
Unfertilized ovule culture and plant regeneration: Ovules excised from a pistil
were cultured in the medium of ½ NLN containing 0.8 % agar-solidified and 10
% sucrose.
The ELSs developed from ovules were transferred to modified agar (1.0%) –
solidified MS medium with concentration of major salts reduced by 50% (1/2
MS) and supplemented with 3% sucrose and 1.0 mg/l GA3.
Materials and Methods

43. Flow cytometry:
Young leaf samples were
analyzed by using Partec CyFlow
PA and Cell Lab Quanta SC.
Chromosome doubling
treatment:
Chromosome doubling of
haploids was performed as
described by Morgan et al.
(2003). Shoots containing axillary
buds were cut and subcultured to
propagation medium (Pr)
containing 50 µM oryzalin for
1,2,3 and 4 weeks and elongated
axillary shoots were transferred
to Propagation medium without
BA after 6 weeks.
a) Unfertilized ovules in an ovary. b) Ovules cultured on ½
NLN soild medium c) ELSs emerged from ovules after 50 days
of culture d) A magnified ELS in c plate
Determination of ploidy level

44. Cold pretreatment produced more ELSs than non-
treatment in 3 genotypes except for line 17-386. In
particular 7 and 14 days tended to exhibit a higher
response.
e, f : A plantlet regenerated from ELS
Results

47. W14 and W15 genes are allelic and
encode proteins related to the α/β
hydrolase fold superfamily.
Donor plants of 17-386 and 17-488 carried
both alleles W14/W15 (heterozygous),
whereas regenerated diploid plants
carried one of them (homozygous)
On the other hand, strain 17-260 carried
only W14 allele (homozygous). Therefore,
for strain 17-260, the FLS, which is
flavonoid biosynthetic genes, was used.
FLS amplified two bands (heterozygous),
in the 17-260 strain. When 43 diploid
plants were examined, 41 plants showed a
single band: however, 2 plants of Go963
and Go990 had the same two bands as the
donor plant.
W14 and W15 genes are allelic and
encode proteins related to the α/β
hydrolase fold superfamily.
Donor plants of 17-386 and 17-488 carried
both alleles W14/W15 (heterozygous),
whereas regenerated diploid plants
carried one of them (homozygous)
On the other hand, strain 17-260 carried
only W14 allele (homozygous). Therefore,
for strain 17-260, the FLS, which is
flavonoid biosynthetic genes, was used.
FLS amplified two bands (heterozygous),
in the 17-260 strain. When 43 diploid
plants were examined, 41 plants showed a
single band: however, 2 plants of Go963
and Go990 had the same two bands as the
donor plant.

48. Cold pretreatment for 1-2 weeks at 40
C found to be beneficial
In the present study using 43 genotypes 40 genotypes produced ELSs
Though variations are present, unfertilized ovule culture was affected less by
genotypes compared with of anther culture.
A large number of regenerated plantlets, which were obtained through
gynogenesis, consisted of haploids (57.8%) and diploids (34.6%).
Oryzalin is effective chemical for chromosomal doubling.
W14/W15 and FLS genes were used as genetic markers in order to confirm
whether the diploid plant obtained from unfertilized ovule culture is a DH or
not.
Inference

49. Conclusion
Doubled haploids (DH) technology is highly desirable can be
implemented in all environments.
Rapid production of homozygous lines.
Speed up the breeding by fixing of the elite inbreds.
Shortened breeding cycle and cost saving.
Elimination of unfavorable genes and enrichment of favorable
genes.
Improvement of germplasm lines.
Early release of commercial hybrids and resource saving.
Combination with other tools including precision phenotyping,
bioinformatics, genomic selection and modeling can improve the
efficiency of the breeding process.

50. Thank you!