2. PRODUCTION OF DOUBLE
HAPLOID PLANTS
Presented by:-
Desai Vruddhi K.
Reg. No- 04-AGRMA-01581-2017
Department of Genetics & Plant Breeding
C.P.College of Agriculture
Sardarkrushinagar, Dantiwada
3. INTRODUCTION
• A doubled haploid (DH) is a genotype formed
when haploid cells undergo chromosome
doubling. Artificial production of doubled
haploids is important in plant breeding .
• Haploid cells are produced from pollen or egg
cells or from other cells of the gametophyte, then
by induced or spontaneous chromosome doubling,
a doubled haploid cell is produced, which can be
grown into a doubled haploid plant.
4. HISTORY
• 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
• wide crossing Kasha and Kao, (1970) - Barley
Burk et al., (1979) - Tobacco Doubled haploid
methodologies have now been applied to over 250
species
5. Why we need double haploids(DH)
• Development of homozygous lines
• Fixation of heterosis
• Mutation studies and easy to induce mutation
• Production of biotic and abiotic stress resistant
plants
• Cytogenetical research
• Induction of genetic variability at haploid level
• Double haploids in genome mapping
• Evolutionary studies
6. What are uses of double haploids
• Development of purelines
• Development of cultivars
• Development of hybrids as parents
• Construction of genetic maps
• Gene tagging/location genes
• Identification of moleculars markers for trait
selection.
7. How to induce haploids
1. wide crossing
2. Irradiation and- chemical treatment,
3. Selection of twins, and
4. By anther and pollen culture.
8. Methods to Induce DHs
• 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
9. ANTHER CULTURE
1.collection of unopened flower buds
2.surface sterilized with tween 80 and mercuric chloride
3.Anthers excised from flower buds and kept seperately
4.Anthers in first meiotic division is selected by
acetocarmine test
5.Inoculated in the medium containing glutamine, L-serine
and inositol
6. Incubated the culture at 25˚C for 15 days. Here, anthers
grow in to embryoids.
7. embryoids transfer to rooting medium under 3000 lux
illumination after 4-5 weeks the embryoids became
plantlets.
8.After aclimatization, transfer to green house
10.
11. POLLEN/MICROSPORE CULTURE
1.Anther collected from flower buds and pollen grains are isolated
and inoculated in the nutrients medium with the concentration
of pollen 0.5ml.
2.Nitsh (1974) medium is used for pollen culture
3. Anthers are place on the medium. some times nurse culture may
used.
4. A paper disc is placed over anther or callus.
5 O.5ml pollen suspension is poured on the disc
6 Petridish covered with lid and incubated at 50˚C for 4 weeks.
Pollengrains grown in to individual clones with 60% efficacy.
7 Clones are transferred to callus induction medium to produce calli.
8 Calli transfered to shooting medium and then to rooting medium
to produce plantlets
12.
13.
14. Importance of pollen and anther
culture
• Utility of anther and pollen culture for basic
research cytogenetic studies.
• Study of genetic recombination in higher plants.
• Study of mode of differentiation from single cell
to whole organism.
• Study of factor controlling pollen embryogenesis
of higher plants
• Formation of double haploid that are homozygous
and fertile.
15. Ovary Slice Culture
Ovary slice culture technique involves culture of transverse
sections of unpollinated ovaries on culture media. The
following protocol was used to induce in vitro gynogenesis
in Tea (Hazarika and Chaturvedi 2012):
a. For Ovary slice culture in Tea, unopened and unpollinated
mature flower-buds (6-10 mm) size were collected early in
the morning. Some of the buds were fixed in FAA (5:5:90
v/v/v Formaldehyde: Acetic acid: 70% Ethanol), for 48 h,
and then stored in 70% alcohol. Later on, the appropriate
developmental stage of the embryo sac was determined by
histological analysis.
16. conti…
b. The flower buds were surface sterilized with
0.1% HgCl2 for 7 minutes, followed by rinsing
with sterile distilled water at least thrice.
c. Carefully dissected transverse sections of ovaries
were cultured on Murashige and Skoog's media
supplemented with varying concentrations of
Auxins and Cytokinins.
d. Six ovary slices containing unpollinated ovules
were cultured in 60 mm X 15 mm pre sterilized
disposable Petriplates containing 10 ml MS
medium.
e. The sealed Petriplates were subjected to various
regimes of temperature and light treatments.
17. Ovule culture
• The unfertilized ovary is surface sterilized and the ovules
were taken and placed into culture. Excision of ovule,
followed by culture on specific media may be either
extremely easy to accomplish, as in case of large-seeded
species in which only a single ovule is present, or time-
consuming and intricate, in small-seeded polyovulate
species. Two types of ovule support systems have been
developed. The filter paper support system involves
culturing of the ovules on top of filter paper placed over
liquid medium, whereas the vermiculite support technique
demands placing the ovules on a sterile vermiculite/liquid
media mixture (vermiculite support) with the micropylar
side down. Unpollinated ovule culture has been used for
haploid production in sugar beets and onions.
18. Factors influencing anther culture
1) Physiological status of donor plant
2) Effect of temperature
3) Culture medium
4) Anther wall factor
5) Stages of microspore
6) Stages of embryo sac
7) Genotype of donor plants
8) Effect of female flower position
19. 1) Genotype of donor plants:- The genotype of the
donor plant plays a significant role in determining
the frequency of pollen production.
Example :- Horedum of each genotype differs with
respect to androgenic response in anther culture.
2) Anther wall factor:- The anther wall provide the
nourishment in the development of isolated
pollen of a number of species.
There are reports that glutamine alone or in
combination with serine and myo inositol could
replace the anther wall factor for isolated
cultures.
20. 3) Culture medium:- For anther culture, medium
Requirements vary with genotype and the age of the
anther as well as condition under which donor plants
are
grown.
• Incorporation of activated charcoal into the
medium has stimulated the induction of
androgenesis.
• The iron in the medium plays a very important
role for the induction of haploids .
• Potato extracts, coconut milk and growth
regulators like auxin and cytokinin are used for
anther and pollen culture.
21. 4) Stages of microspores:-
• Anther are most productive when cultured at
the uninucleate microspore stage.
• Example, barley, wheat, rice etc
• anther of some species give the best response
if pollen is cultured at first mitosis or later
stage
• Example:- datura, tobacco
22. 5) Effect of temperature:- Temperature enhance
the induction frequency of microspore
androgensis.
• The low temperature treatment to anther or
flower bud enhance the haploid formation.
• The low temperature effects the number of
factors such as dissolution of microtubules
lowering of absicisic acid maintenance of
higher ratio of viable pollen capable of
embryognesis.
23. 6) Physiological status of donor plant:-
• Physiological status of donor plant such as water
stress nitrogen requirement and age of donor
plant highly affect the pollen embryogenesis.
• Plants starved of nitrogen may give more
responsive anthers compared to those that are
well fed with nitrogenous fertilizers.
24. Advantages of DHs
• The ability to produce homozygous lines after a single
round recombination saves a lot of time for the plant
breeders.
• Studies conclude that random DH’s are comparable to
the selected lines in pedigree inbreeding.
• The other advantages include development of large
number of homozygous lines, efficient genetic analysis
and development of markers for useful traits in much
less time. More specific benefits include the possibility
of seed propagation as an alternative to vegetative
multiplication in ornamentals, and in species such as
trees in which long life cycles and inbreeding
depression preclude traditional breeding methods,
doubled haploidy provides new alternatives.
25. Disadvantages of DHs
• The main disadvantage with the DH population is that
selection cannot be imposed on the population. But in
conventional breeding selection can be practised for
several generations: thereby desirable characters can be
improved in the population.
• In haploids produced from anther culture, it is observed
that some plants are aneuploids and some are mixed
haploid-diploid types. 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. Hence one has to take several factors into
consideration before deploying doubled haploidy in
breeding programmes.
26. Case study
Recent advances and application of doubled
haploids in wheat breeding
• W. Tadesse*, M. Inagaki, S. Tawkaz, M. Baum and M.
van Ginkel
abstract:
• Genetic improvement to develop varieties with high
yield potential and resistance/tolerance to abiotic and
biotic stresses with acceptable end use qualities is the
most viable and environment friendly option to
increase wheat yield in a sustainable fashion. In vitro
haploid production followed by chromosome doubling
greatly enhances the production of complete
homozygous wheat lines in a single generation and
increases the precision and efficiency of selection
process in wheat breeding.
27. Conti…
• It also enables the detection of linkage and gene
interactions, estimate genetic variance and the
number of genes for quantitative characteristics,
produce genetic translocations, substitutions and
chromosome addition lines, and facilitate genetic
transformation and mutation studies. Wheat
cultivars developed from doubled haploids using
anther-culture and maize induction systems have
been released for cultivation in both developed
and developing countries. In this review, the
origin and production of haploids, techniques in
anther-culture and their application in wheat
breeding are summarized.
28. The procedure of anther-culture in wheat is
illustrated in Figure
• a = pre-treatment of the donor plants at 4°C; b = anthers in liquid induction
medium; c = developing of embryos in liquid induction medium; d=embryo
converting to green on solid regeneration medium; e= green plants in
regeneration medium; f = haploid plants at acclimatization stage; g =
haploid plants under colchicine treatment (0.2%); h = doubled haploid lines
in the field showing uniformity within lines (Tawkaz, 2011).
