Training managment training 14-18 feb,2021

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Doubled Haploid Production
Doubled Haploid Production Techniques
of Horticultural Crops

2. Bangladesh Profile
# North-eastern part of South Asia.
# Land =143999 km2
# Population=160 million.
#Temp. 50c- 280c in winter ,
220c - 400c in summer.
# The total cultivated area of
horticultural crops is about 0.69 million
hectares which is about 5% of the total
cropped area

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4. Accelerated Mutation Breeding in horticultural
Crops

5. Plant Tissue Culture?
Leaf explants
Immature
cotyledon 30 days after
initiation
Weeks after
initiation
A technique of growing in vitro all plant parts whether a single cell ,
a tissue or an organ on synthetic medium under controlledand
aseptic conditions
Totipotency theory states that cells are autonomic and in principle
are capable of regenerating to give a complete plant
Cotyledon
4

6. In Vitro Mutagenesis in
Plant Breeding Program
Entire plant (seeds)
Pollen grain
Buds
Plant tissue explants ( callus,
buds, roots etc.)
Ultraviolet radiation (UV)
Ionizing radiation (X-rays,
gamma-rays, alpha and beta
particles)

7. Types of In Vitro Culture
Culture of Intact Plants (Seed and Seedling Culture)
Embryo Culture (immature embryo culture)
Organ Culture
1. Shoot tip culture
2. Root culture
3. Leaf culture
4. Meristem
5. Anther and pollen culture
6.Callus Culture
7.Cell Suspension Culture
8.ProtoplastCulture

8. General techniquesof plant tissue culture
Aseptic Technique
Sterilization of explant
Sterilization of media
Preparation of culture media
Incubation of culture
Sub culturing
Plant regeneration
Acclimatization

9. Applications of Tissue Culture
Micropropagation
Germplasm preservation for endangered plants
Somaclonal variation
Doubled haploid production
Protoplast fusion
Secondary metabolites production
Genetic engineering

10. Doubled Haploid Production
Haploid ; An individuals with the gametic chromosome
number in its somatic cells
Doubled Haploid: An individual with the doubled
chromosome number of the haploid
Anther Culture
Microspore culture
Ovary culture

11. i)Acceleration inbred line development
ii)Develop immediate homozygosis, shorten the time to
cultivar release
iii)Provide greater efficiency of selection in plant breeding
iv)Improve the precision of genetic and mapping studies
v)Accelerate gene pyramiding
vi)Improve efficacy and efficiency in screening for
resistance
Advantages of
Doubled Haploid Techniques

12. Doubled Haploid: Practical Issues
Widely used methods:
•
•
•
•
•
Anther Culture
Microspore culture
Ovule culture
Irradiated pollens
Wide hybridization (cereals)

13. CUCUMBER CROSSING PROTOCOL
USING IRRADIATED POLLEN: Practical Issues

14. Plants for crossing can be grown in the glasshouses.
Greenhouse grown plants
are generally easier to work
with and crossing success is
often better.

15. Pots are labeled and arranged
on glasshouse benches.
The label for each pot may
contain information of plant.

16. Tools used for crossing.

17. Examine the male flower early in the
morning. Locate a newly opened male
flower. The male flower appears atop
a slender green stem which contains
pollen on the stamens inside the
petal.
The anthers are
pale yellow.
Locate a female flower. The female
blossom has a tiny swollen ovary at its
base that looks like miniature cucumber
at the base of the petal. This is actually
the ovary on the plant where the fruit
forms.
Female
bloom.
Female
bloom.
Female
flower.

18. The female flower has been labeled and paper
bagging for avoiding cross pollination
Female
flower
bagging
Female
flower
bagging
and
tagging

19. When the female flower bloom, and stigma becomes feathery and
protrudes then the female flower would be ready to pollinate

20. Choose of male flower which will be gamma irradiated. The anther should be pale yellow.
Before irradiation pollen viability test in microscope. Keeping of male flowers in petri dishes and
wrapping with paraphin to avoid pollen contamination and dryness. Irradiation of pollen with
gamma irradiator (60Co)
Anther should be pale yellow
Pollen Viability test
Finally ready to irradiation Male flower Gamma irradiation
Examine male flower

21. After irradiation remove all
the petals to expose the
stamen.
Do not shake the
blossom vigorously
since this knocks off the
irradiated pollen grains.
Touch the stamens to the stigma
inside the female flowers and
again bagging for 2-3 days.
Stamens with
irradiated pollen
grains for
pollination
1
2
1 3
3
4

22. Successful fruit setting by pollination with 50, 100 & 200 Gy irradiated pollen
Cucumber blossom is fertilized. When the female flower dries up, the fruit grows and develops.
Shasalocal 50Gy Shasa-6 100Gy
Shasa-6 200Gy
Shasa-6 50Gy Shasalocal 100Gy
Shasa-1
100Gy
Shasa-6 50Gy
Shasa-6 control

23. Not successful fruit setting by pollination with 300 & 400 Gy irradiated pollen
Successful fruit setting by pollination with 200 Gy irradiated pollen
Shasa-6 200Gy
Shasalocal 200Gy

24. NP 400 300 200 100 50 cont. NP 400 300 200 100 50 cont.
Gamma irradiation (Gy) Gamma irradiation (Gy)
Shasa-6 Shasalocal
NP 400 300 200 100 50 cont. Seed developed in irradiated fruit
Gamma irradiation (Gy)
Shasa-1

25. FDA
Pollen viability using fluorescence staining
i) Bright fluorescence with FDA indicates viability ii) No fluorescence with FDA
indicates dead pollen
iii) With DAPI staining the three nuclei of mature pollen can be seen,
iv) non-staining (empty) indicates non-viable pollen
DAPI

26. Dose treatments of 50-200 Gy may be used as mutagenic treatments. Pollen is
viable and can be used in crossing to introduce mutations via fertilisation.
Dose treatments of 300-400 Gy and greater kill pollen and these may be used
in pollinations to induce haploid embryo development.
Effects of irradiation on pollen viability
Cucumber genotypes
Shasa-1 Shasalocal Shasa-6
Dose(G
y)
Pollen viability (%) Pollen viability (%) Pollen viability (%)
FDA DAP I FDA DAP I FDA DAP I
0 52.68 a 71.58 a 48.90 a 63.12 a 61.28 a 83.42 a
50 51.50 a 69.86 a 45.52 a 56.59 a 60.21 a 82.11 a
100 44.61 b 61.62 b 36.51 b 43.46 b 42.68 b 62.19 b
200 8.52 c 22.68 c 24.51 c 35.19 b 36.52 c 53.98 c
300 0d 0d 0d 0c 0c 0c
400 0d 0d 0d 0c 0c 0c

27. Doses
(Gy)
Shasa-1 Shasalocal Shasa-6
Fruit
set/6
female
flowers
(no.)
Fruit
setting
(%)
Fruit
setting/6
female
flowers
(no.)
Fruit setting
(%)
Fruit
setting/6
female
flowers
(no.)
Fruit
setting
(%)
0 5 83 5 83 6 100
50 5 83 5 83 6 100
100 5 83 4 66 5 83
200 4 66 3 50 5 83
300 0 0 0 0 0 0
400 0 0 0 0 0 0
Table 1. Mean number of fruit set by pollination with irradiated pollen

28. Anther culture for haploid production using gamma irradiation
Four tomato genotypes : Phili, BINA-2671, BINA-2853 and Roton
Irradiation dose : 0, 75, 150, 300Gy
Flowers collection: Flowers were collected for anther culture which the plant
produced 2-6mm length of bud at morning.
Culture media : Murashige and Skoog Basal Medium

29. Plate 6. Preparation of MS media(1) , anther platting (2-6) callus formation of
anther(3-4)
1 2
5
4
4
3

30. Irradiated plant
Dose (Gy)
Name of Tomato
genotypes
Number of total
anther in per
Petri-dish
Total number of
response anther
for callus
formation
% of response
anther for callus
formation
0 Phili 34a 26a 76b
BINA-2671 32a 28a 87a
BINA-2853 29a 22ab 75b
Roton 23b 16c 69c
75 Phili 36a 17c 70c
BINA-2671 30a 21b 75b
BINA-2853 25b 18c 75b
Roton 28ab 15c 66d
150 Phili 23b 28a 77b
BINA-2671 30a 23b 76b
BINA-2853 24b 20b 80ab
Roton 20c 20b 71bc
300 Phili 15d 10d 73bc
BINA-2671 20c 15c 75b
BINA-2853 16d 10d 62d
Roton 13cd 8d 61d
The common letter did not probability differ at 5% level of significance as per DMRT

31. Effect of pollen irradiation on pollen viability and fruit set in tomato
# Pollen was irradiated at 0 (control), 50, 100, 200, 300 and 400 Gy of
gamma ray (source activity was 150 gray/min)

32. 1 2 3
4
5
6
7
8
1. Emasculation of flower
2. Bud length 2-6 mm young flower
selected
3. Separation of male part
4.Bagging of female part with net bag
5. Collected flowers for gamma
irradiation
6. Gamma irradiation of anther with
pollen
7. Pollination with irradiated pollen
8. Bagging again to avoid contamination

33. Fruit setting at 50 Gy Fruit setting at 100 Gy
Fruit setting at 200Gy
Fruit setting at 0 Gy
Not Fruit setting at 400 Gy
Not Fruit setting at 300 Gy

34. # Pollination with irradiated pollen at 50 t0 200 Gy gave fruit sets ranging
between 75 to 85 % which was comparable to pollination with control pollen.
# Pollination with 300 and 400Gy irradiated pollen did not give any fruit set in
the four tomato genotypes studied indicating that high dose treatments may
have lethal effects on tomato pollen

35. Tomato