Role of double haploids in vegetable crop improvement

Commercial
acceptance
Uniformity
Elite inbred
lines
Homozygousity
2

Limiting factors……….????
3

BASAVARAJ
Ph.D in vegetable Science
Role of double haploids in vegetable crop improvement
UNIVERSITY OF HORTICULTURAL SCIENCES,
BAGALKOT

Topic division
 Introduction
 Historical Background
 Methods of Double Haploids production
 Identification of haploids
 Double Haploids in crop improvement
 ConcludingThoughts
6

Haploids - defined as the sporophyte plants that contain a gametic
chromosome number (n).
(A) MONOPLOID - Haploids derived from diploid e.g. Cucumber n = 7(1x)
(B) POLYHAPLOID - Haploids derived from polyploids
i) Autopolyhaploids
e.g. Potato 2n= 4x=48
ii) Allopolyhaploids
e.g. Sweet potato 2n = 6x = 90
Introduction
7

Timeline of haploid development
•Blakeslee et al., (1922) - Datura stramonium
•Guha and Maheswari (1964) - Anther culture technique for the
production of haploids in the laboratory
•Niizeki and Oono (1968) - Production of rice haploids
•Tobacco (Nitsch and Nitsch, 1969), rice, maize, brassica, barley (Kasha
and Kao, 1970)
•First In vitro induced haploid by gynogenesis - San Noeum (1976) in
barley
8

 Doubled haploid methodologies have now been applied to over 250
species (Misra et al., 2014)
 Commercial varieties developed through DH protocols for many crops -
290 varieties have already released (Ferrie and Caswell, 2011)
9

A plant or line obtained by doubling
the chromosome number of a haploid plant or individual.
• Homozygous for all loci
• New variety in self pollinated crops or as parental inbred line
for hybrid production in cross pollinated crops.
(1) Induction of haploid and
(2) Doubling of chromosome number of the haploid individual.
10

In DH METHOD - Probability of getting the desirable genotype is (1/2)n
DIPLOID METHOD – Probability of getting the desirable genotype is
(1/4)n
Aa X Aa
AA : Aa : aa
(1 : 2 : 1)
Aa
AA : aa
(1 : 1)
A a
DOUBLE HAPLOID METHOD DIPLOID METHOD
‘n’ loci are segregating:-
11

Why Haploids
Development of homozygous lines.
Generation of exclusive male plants.
Induction of mutations.
Production of disease and insect resistance plants.
Production of salt tolerance plants.
Cytogenetical research.
12

Advantages of using Haploids
 Rapid technique – homozygous plants can be achieved in one
generation (inbred lines).
 DHs can be represent as new variety (self-pollinated crops) or as
parental inbred line for the production of hybrids (cross-
pollinated crops).
 Selection tool for the elimination of genotypes expressing strong
inbreeding depression in cross-pollinated crops.
 DH lines also are valuable tools in cytogenetical research.
13

Methods of haploid production
In vitro methods
1) Androgenesis
a. Anther culture
b. Pollen/ Microspore culture
2) Gynogenesis
a. Ovary slice culture
b. Ovule culture
In situ methods
1) Distant (wide) hybridization
2) Parthenogenesis
14

Androgenesis
Androgenesis is the process of induction and regeneration of
haploids and double haploids originating from male gametic cells.
Anther culture or isolated microspores undergo embryogenesis/
organogenesis directly.
potato, pepper and asparagus
15

1.ANDROGENESIS
In
vitro
method
• Process of embryo
development from the male
gametophytes i.e
microspores or anthers.
common guidelines of the
anther culture method
16

Fig 2. Microspore culture of cabbage: (A) first divisions of microspores in
nutrient medium, (B) regenerated embryos, (C) embryos at desiccation
treatment needed for regrowth, (D) Selfing of DH lines
17

(Palmer et al., 2005)
Gynogenesis
Unfertilized isolated ovules, ovaries of flower buds develop
embryos from cells of the embryo sac.
In vitro induction of maternal haploids is gynogenesis, is
another pathway to the production of haploid embryos
exclusively from a female gametophyte.
onion, sugar beet, cucumber, squash, gerbera, sunflower, wheat,
barley etc.
18

Selection of Flower
Extraction of ovary
Surface
sterilization
Slicing of ovary
Development of
haploid embryo
Transferred to
regeneration medium
Cultured on
rooting medium
DH Plant
In
vitro
method
Ovules transferred
to basal medium
Transferred to
Induction medium
Colchicine treatment
2. GYNOGENESIS
19

Fig 1. Production of onion haploid plants with in vitro gynogenesis. (A) In
vitro culture of un-pollinated flower buds on B5 medium ((B) germination of
haploid embryos after 60 -180 days in culture; (C) elongation of haploid plantlets
and (D) acclimatization of haploid plants in the greenhouse.
20

In situ Method
Wide Hybridization (inter-specific ybridization)
1: DHs
• Solanum tuberosum L. x S. phureja (wild )
ssp. tuberosum (2n=4x =48 ) (2n=2x=24)
(female) ( male)
(Maine, 2003 and Rokka, 2009 )
21

Abelmoschus esculantus X A. tuberculatus
Solanum incanum X S. melongena
Raphanus sativus X Brassica oleraceae
22
(Maine, 2003 and Rokka, 2009 )

2. Parthenogenesis
 In this method the egg cell develops into an embryo
without fertilization by the sperm nucleus
 Pollination with irradiated pollen
In
vivo
method
23

Direct Method:
 Cytological technique – counting the chromosome number in root tip
cells
 Flow cytometry,
Indirect Method: These include
 stomatal size,
 chloroplast number of the guard cells and
 morphological observations.
Identification of Haploids
24

1. Morphological observation
Morphological variability of regenerants by visual observation
and colour. Haploids are smaller and exhibit a lower plant
vigour compared to donar plants.
2. Chloroplast number in stomatal guard cells
25

3. Flow cytometry and Chromosomal count
Determination of ploidy level of regenerated
plants of Popular by flow cytometry (a-c) and
chromosome counting (d-f)
(Ying Li., et. al., 2013)
Diploid
Triploid
Haploid
o Simple option for large
scale ploidy determination.
o Enables detection of the
mixoploids regenerants and
determination of their
proportion.
26

 Haploid plant may grow up to a flowering stage, but viable
gametes can not be formed due to lack of one set homologous
chromosomes. Consequently there is no seed formation.
 Double haploid contain two set of chromosome which are
exactly identical.
 Haploid can be diplodized (by duplication of chromosomes) to
produced homozygous plants.
 There are mainly two approaches for diploidization
1. Colchicines treatment .
2. Endomitosis.
(Satyanarayana 2008)
Diploidization of haploid plant
27

Factors Affecting Haploid Induction
Donor plant genotype
Physiological condition of donor plants (i.e. low temp
and high illumination)
Developmental stage of gametes, microspores and
ovules
Culture medium composition
Physical factors during tissue culture (light,
temperature)
28

Objective
Compare the homozygous, uniform, true inbred lines with popular
commercial hybrids of Heading broccoli.
30

USVL048 derived from fertile and heterozygous hybrid Marathon
USVL131
31
(Farnam, 2013, USA)

Fig. 1. Fall field plots of USVL131 (left) and USVL048 (right) grown at Charleston, SC, and showing
heads at a mature, market-sized stage.
USVL131 USVL048
32
(Farnam, 2013, USA)

Variety DTH
(days)
Dome
(rating)
Bead
size
(mm)
Head
quality
(rating)
Head
mass
(g)
Stem
diam
(cm)
USVL048 91 1.8 1.33 4.0 236 33.7
USVL131 84 1.9 1.49 4.6 218 32.2
Liberty 79 2.0 1.30 3.8 246 37.8
Patron 77 2.1 1.31 4.2 243 37.0
Captain 72 2.1 1.63 4.5 173 31.3
Gypsy 75 2.1 1.53 4.2 272 36.5
Marathon 86 1.8 1.27 4.0 223 35.2
Legacy 86 2.0 1.23 3.7 272 37.9
LSD0.05 5 NS 0.07 0.4 30 4.1
Table 1. Trait means for days from transplant to harvest (DTH), degree of dome (Dome) rating,
bead size, head quality rating, head mass, and stem diameter for the inbred (Farnam, 2013, USA)
33
Dome rating on a 1 to 5 scale with 1 = very high dome, 3 = moderate dome, and 5 = flat.
Quality rating on a 1 to 9 scale with 1 = exceptional, 3 = excellent, 5 = good, marketable 7 =
poor, nonmarketable 9 = not recognizable as broccoli, nonmarketable

• Low regeneration frequency can be attributed to poor gynogenic
efficiency and population structure.
• Parents with good gynogenic potential can be utilized for
introgression breeding in onion where a reduction in the
breeding cycle is beneficial.
• (Allium cepa var aggregatum) having the highest gynogenic
potential of 55.2%
34
(Anandan et al., 2014, Pune)
Gynogenic potential in onion

Objective: Characterizing Indian genotypes for gynogenic potential
35

Fig. 2. Gynogenic regenerant emerging
from ovary cultures
Fig. 3. Normal plantlet regenerated
through gynogenesis.
36

Table 2. Gynogenic potential among Indian short day onion varieties
Genotypes No. of
explants
No. of regene-
rants
Gynogenesis
(%)
Agri Found Rose 891 17 1.9
AgriFound Dark Red 200 4 2.0
Arka Kalyan 189 6 3.2
B780 295 10 3.3
Bhima Kiran 2300 65 2.8
Bhima Red 2000 38 1.9
Bhima Shakti 1250 26 2.1
Bhima Shubhra 441 20 4.5
Bhima Shweta 3800 34 0.9
Bhima Super 1000 35 3.5
N-2-4-1 500 15 3.0
Pusa White Round 286 4 1.3
RLKM-1 165 5 3.1
W514 165 3 1.8
37

Objective:
 To determine antioxidant properties
of 25 DH lines of white cabbage in
comparison with two standard
cultivars.
AUK, Poland Leja et al. (2012) 38

Materials and methods
• 25 DH lines of white cabbage by androgenesis
• Two standard cultivars Lennox F1 and Attraction F1
• Freshly harvested cabbage heads were either analysed immediately or
stored for 4 months in commercially cold chambers at 1-20 C and 80-85%
RH.
• Determine:
Total phenols = Photometric method with Folins reagent
Ascorbic acid = Iodate-titration
Radical scavenging activity (RSA) = % of DPPH free radical
neutralized for 30 min.
39

Table 3 :The antioxidant properties (ascorbic acid content) in DH lines of white
cabbage.
Pedigree Ascorbic acid content (mg 100 g-1f.w. )
Freshly harvested cabbage Long term stored cabbage
Lennox F1 35.61 48.88
Attraction F1 38.72 45.32
Parent 1 38.44 35.96
Parent 2 21.12 28.80
DH lines
4005 29.48 46.20
4011 51.48 45.32
4015 38.30 39.17
4013 42.94 48.88
4016 34.82 33.44
4019 41.10 35.64
4027 19.65 35.20
4031 40.76 42.12 40
Leja et al. (2012)

Table 4: The antioxidant properties ( Total Phenols) in DH
lines of white cabbage.
Pedigree Total phenols (mg 100g-1f.w. )
Lennox F1 48.87 71.99
Parent 1 48.65 65.62
Parent 2 49.50 62.56
DH lines
4007 59.13 54.95
4008 55.98 45.35
4011 62.09 53.15
4013 52.65 76.08
4015 59.68 87.66
4017 60.64 79.82
4019 43.15 57.60
4026 54.36 80.63
4027 66.03 68.74
Leja et al. (2006) 41
Leja et al. (2012)

Table 5: The antioxidant properties(RSA) in DH lines of
white cabbage.
Pedigree Radical scavenging activity (%)
Lennox F1 6.41 15.14
Parent 1 5.19 13.79
Parent 2 3.20 5.23
DH lines
4005 4.26 2.49
4008 6.72 3.57
4015 6.41 17.40
4017 10.33 13.53
4021 2.76 12.75
4024 9.46 12.70
4029 9.66 11.73
4031 10.75 11.64 42
Leja et al. (2012)

• Most promising lines seem to be 4017.
• Among the lines of good antioxidant properties,
particularly after long term storage, special attention
should be paid to DH lines 4013, 4014 an 4024 of
high RSA accompanied with high phenol content.
43

Objective
To screen cucumber cultivars resistance to CMV, and to establish
double haploid plant lines by ovule culture
44

• Screening of cucumber accessions for CMV resistance
• The cotyledon stage were inoculated with the virulent
CMV I (HC-53)
• DAS-ELISA
• Double haploid production
• Screening of CMV resistant double haploid lines
Fig. 5. Double haploid at the stage that was used for
CMV I resistant screening 45

Results
Countries Origin Reaction Total
Resistant Susceptible
America 5 3 8
India 2 0 2
Japan 1 3 4
Pakistan 0 1 1
Malaysia 0 1 1
Nederland 0 1 1
Philippines 1 0 1
China 6 0 6
Thailand 29 47 76
Total 44 56 100
Table 6. Reaction of cucumber lines to mechanical inoculation with CMV
ELISA reading: R ≤ 0.23 MR ≥ 0.24 ≤0.45 S ≥ 0.46; Healthy plant = 0.21
46
(Plapung et al., 2014, Thailand)

Double haploid production
Fig. 6. Embryogenesis and regeneration of plantlets derived from ovule culture
A-B =sprouting embryo, C=shoot-like organogenesis, D= regenerated plantlets.
47

Screening of CMV resistant DH
Accessions Code DH lines Level of resistance
Resistant
(R)
Moderately
Resistant(MR)
Susceptible
(S)
CSL 0006 86 1 1 - -
CSL 0011 91 5 1 4 -
CSL 0021 101 1 - - 1
CSL 0052 132 1 - 1 -
CSL 0094 174 1 - 1
TOTAL 9 2 5 2
Table 8. Reactions of DH line cucumbers resistance to CMV
Resistance screening based on the ELISA reading: R ≥ 0.23; MR ≥ 0.24 ≤ 0.45; S ≥
0.46 (2x of Negative control); Healthy plant = 0.21
48

• Double haploid cucumber production and screening for
resistance to this virus can accelerate breeding programs
through the use of homozygous double haploid lines (DHL)
• The different levels of CMV resistance observed in the DH
cucumbers.
49

• To study invitro response of cultivated anthers
of different Bulgarian pepper lines, varities and
hybrids
• To determine the suitable media for direct
embryogenesis.
Bulgaria Irikova et al. (2011)
50

Donor plants from eight lines : 145, 146, 603, 668, 1312, 1241, 1924,
1957,
Seven varieties : Maritsa, Kurtovska kapya, Albena, Zlaten medal,
Stryama, Hebar, Buketen and
Four hybrids : 50 (1647 × S) F1; 53 (1647 × 1312) F1; 73 (1647 × 668)
F1; 91 (1647 × V) F1 of sweet pepper (Capsicum annuum L.)
Two induction media : C and Cm with two duration of anther
culture 12 and 40 days
Two regeneration media : R and Rm
The frequency of callusogenesis anthers,
indirect organogenesis, direct embryogenesis
and obtained plant regenerants noticed
51

Composition of induction and regeneration media
52

Table 9. Frequency of callusogenesis, embryogenesis and regenerants in anther culture
0f different pepper genotypes (%)

 Important to determine both the induction and
regeneration medium as well as the duration of
cultivation of anthers.
Genotype not liable to induction of androgenesis
probably have an embryogenic potential, but they need
longer time.
54

• To study productive parameters
of monogerm and multigerm sugar
beet DH lines
A.I. Bulgaria Kikindonov et al. (2016)
55

• Monogerm and multigerm double haploids lines derived from
gynogenesis of some diploid lines like 19, 760, 316.
• During diploidization of haploid explants 4 tetraploids arose among
them two were included in this study.
• Same invitro techniques were used for creation of dihaploids in
monogerm lines
The tested productive parameters are root yield (t/ha), sugar
content (%) and white sugar yield (t/ha)
56

Table 6: Root yield, sugar content and white sugar yield of dihaploid
sugar beet lines.
Dihaploid line
Root Yield % to the
initial line
Sugar content % to
the initial line
White sugar yield %
to the initial line
Monogerm dihaploid lines
DH 19-27 103.1 101.5 105.2
DH 19-66 112.5 101.5 113.1
Initial line 19 37.32 14.80% 4.39 t/ha
DH 54-4 105.7 97.9 101.9
DH 150-4 104.2 104.8 110.7
Initial line 760 42.83 t/ha 13.69% 4.80 t/ha
Multigerm dihaploid lines
DH 52 102.7 100.9 106.6
TH 48 107.9 105.4 113.6
DH 58 109.0 98.2 107.9
TH 55 117.9 99.4 116.3
Initial line 316 44.81 t/ha 15.76% 5.79t/ha
Kikindonov et al. (2016)
A.I. Bulgaria
57

Hybrid Root Yield % to
the standard
Sugar content % to
the standard
White sugar yield
% to the standard
DH 19-27×DH 52 111.7 95.6 104.6
DH 19-66×DH 52 111.5 95.5 103.7
DH 54-4×DH 52 113.7 96.8 109.2
DH 150-4×DH 52 111.8 100.4 112.0
DH 19-27×DH 58 112.7 97.4 109.1
DH 19-66×DH 58 113.2 100.1 113.7
DH 54-4×DH 58 111.4 99.1 110.9
DH 150-4×DH 58 109.5 102.1 111.2
Standard group 49.21 t/ha 16.08% 6.55 t/ha
Table 7 : Root yield, sugar content and white sugar yield of diploid hybrids of
the dihaploid sugar beet lines
Kikindonov et al. (2016)
A.I. Bulgaria 58

Table 8: Root yield, sugar content and white sugar yield of triploid
hybrids of dihaploid sugar beet lines.
Hybrids Root yield % to the
standard
Sugar content % to
the standard
White sugar yield %
to the standard
DH 19-27 x TH 48 102.3 103.0 107.3
DH 19-66 x TH 48 105.1 104.3 109.3
DH 54-4 x TH 48 109.5 102.3 111.8
DH 150-4 x TH 48 109.8 101.6 111.5
DH19-27 x TH 55 104.9 97.8 112.3
DH 19-66 x TH 55 115.4 104.5 110.6
DH 54-4 x TH 55 111.7 100.4 112.2
DH 150-4 x TH 55 110.2 102.7 113.3
standard 50.22 t/ha 15.28 6.23 t/ha
A. I. Bulgaria Kikindonov et al. (2016) 59

• Best performing monogerm line DH 19-66 and DH
150-4 and the multigerm pollinators DH 58 and TH
55
• Highest white sugar yield by crosses of monogerm
line DH 150-4 and multigerm pollinator DH 58 and
TH 55.
60

DH Varieties Developed vegetables
Crop Genotype Method Country Breeder(s)
Asparagus Guelph Millenium Female x DH
super male
Canada Dave Wolyn
Asparagus Ringo, Golia, Argo,
Eros
DH female x DH
super male
Italy Falavigna et al.,
1999
Eggplant Petra, Seven, Cristal,
Milar , Seneagal
F1 from DH
parent(s)
Spain Ramon Dolcet-
Sanjuan
Pepper Carisma, Tajo, Pekin,
Olmo, Alcor, Aneto,
Beret, Collado
F1 from DH
parent(s)
Spain Ramon Dolcet-
Sanjuan
61
Achievements
Meenakshi et al., 2017

62
Frequency of haploid occurrence is low
selection cannot be imposed on the population.
Success of DH method is genotype dependent
Success is unpredictable and can consume valuable resources
The over-usage of doubled haploidy may reduce genetic variation in
breeding germplasm
Health and legal concerns related to handling the doubling chemical
agent
Disadvantages of DH breeding technique

Conclusion
Haploids speed up the breeding efforts and allows recycling of
germplasm and fixing of the elite inbreeds for advanced hybrid testing
Due to the shortened breeding cycle from 6-7 generations down to 1-
3, there is a substantial cost savings and increased capacity to fast
track breeding objectives
63

Role of double haploids in vegetable crop improvement

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Role of double haploids in vegetable crop improvement

Similar to Role of double haploids in vegetable crop improvement (20)

More from Basavaraj Panjagal

More from Basavaraj Panjagal (13)

Recently uploaded

Recently uploaded (20)

Role of double haploids in vegetable crop improvement