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HAPLOID CULTURE

Contents:
• Introduction to haploid culture
• History
• Types of haploid culture:
in vivo and in vitro
• Androgenesis:
A. Anther culture- protocol, advantages,
disadvantages
B. Pollen culture- protocol, advantages
 Culture media for androgenesis
 Process involved in androgenesis
• Gynogenesis:
 Method
 Advantages and disadvantages of ovule culture
• Diploidisation
• Applications of haploid culture
• Limitations/ problems associated with
haploid culture
• References
INTRODUCTION:
• Haploids: sporophytes with gametophytic
chromosome number
• Have been produced in a variety of plant
species
• In nature: produced by parthenogenesis
• Can arise: in-vivo
in vitro
HISTORY:
• 1921: A.D. Bergner- discovered haploid plants
in Datura stramonium
• 1951: Tulecke cultured pollen grains of Ginkgo
biloba (Gymnosperm)
• 1964: Guha and Maheshwari- haploid embryos
from Datura innoxia
• 1967: Bourgin and Nitsch- haploid plants from
Nicotiana
TYPES OF HAPLOID CULTURES:

• IN VIVO:
(a) Spontaneous ocurance in low
frequency
(b) Ovule androgenesis
(c) Chemical treatment
(d) Temperature shocks
(e) Irradiation effects
• IN VITRO METHODS:
(a) Androgenesis: production of haploid
plants from:
1.anthers
2.pollens (microspores)
(b) Ovule culture (Gynogenesis): production
of haploid plants from unfertilized egg cell
ANDROGENESIS
ANDROGENESIS:
A. ANTHER CULTURE:
• Process of using anthers to culture haploid plantlets.
• Protocol-
1.Flower buds collected from the plants
2.Buds taken in a sterile petri dish
3. Chilling for 12 days (7-80 celsius)
4. Surface sterilization using 0.01% HgCl2
5. Rinsing 3-4 times using double distilled water
6. Buds carefully teased and anthers are removed
7.Anthers dissected out from each bud
8.One anther from each group is removed and squashed in
acetocarmine
9.Filaments cut from anthers to avoid callusing from cut
ends in vitro
10.Suitable anthers placed on culture medium,
incubated at 25o C in darkness
11.Young embryos emerge from cultured anthers
in about 2 weeks
12.Cultures shifted to light (300 lux): 2 weeks
13.Complete plantlets: observed after 4-5 weeks
Anther culture
Advantages of anther culture:
• Fairly simple technique
• High induction frequency (a large
proportion of the anthers used in culture
respond)
• Haploids can be produced in large
numbers very quickly.
Disadvantages of anther culture:

• When working with some species, the majority of


plants produced are non-haploid
• In cereals, very few green plants are obtained;
Mostly: albinos or green-albino chimeras
obtained
• Tedious to remove the anthers without causing
damage
• sometimes a particular orientation is necessary
to achieve a desired response
B. POLLEN/ MICROSPORE CULTURE:
• In vitro culture of male gametophytic cells i.e. microspores

• Protocol:
1. Collection of anthers from sterilized flower buds
2. Squeezing of anthers by pressing them against the sides of
beaker with a glass rod
3. Removal of anther tissue by filteration through a nylon sieve
(having a pore diameter slightly wider than the diameter of
pollen)
4. Centrifugation of pollen suspension: 150g, 5 mins
5. Supernatent discarded & pellet of pollen resuspended in
fresh media
6. Microspores obtained are mixed with an appropriate culture
media
7. Final suspension pipetted into small
petridishes
8. Sealing of dishes with paraffin
9. Responsive microspores form embryos or calli
10. Development into plants by transferring to a
suitable media
Pollen culture:
Advantages of pollen culture

• Uncontrolled effects of anther wall are


eliminated
• The sequence of androgenesis can be
observed
• Ideal for mutagenic studies
• High yields obtained
Culture media for androgenesis:
• Requirements vary with:
1. species
2. genotype
3. age of donor plants and anthers
• Can be simple: 2-4% sucrose
Eg. Nicotiana tabacum
• Complete media required for Solanaceous
species
Eg. N6, Potato 2 media
• Adjuvants: yeast extract, casein
hydrosylate, vitamins etc- for non
Solanaceous species
• Growth media:
1. Not required for Solanaceous species
2. Auxins, CK, GA3 can be added in low
levels
3. Gelling agents: in case of Barley
4. Activated charcoal
5. Amino acids like glutamine, proline etc.
Process involved in Androgenesis:

• Pollen grains

Bigger Smaller
Stain deeply Stain lightly
Starch grains +nt Starch grains –nt
S- GRAINS
• S grains respond during anther culture
• Pathways involved: early divisions in pollen
grains may occur in one of the following ways:
(a) Pathway-1: Eg. Datura innoxia
Pollen unequal division Generative cell (GC):
degenerates
Vegetative cell (VC):
forms callus/ embryo
(b) Pathway-2: Eg. N. tabacum
GC
Pollen symmetrical div. equal divide to form
callus/ embryo
VC
(c) Pathway-3: Eg. Hyoscyamus niger
VC does not divide
Pollen unequal div.
GC divides forms callus
or embryo
(d) Pathway-4: Eg. Some species of Datura
innoxia
VC
Pollen unequal div. divides forms
callus/embryo
GC
• Process: occurs in 2 ways-
(a) DIRECT EMBRYOGENESIS:
origination of embryos from microspores
without callusing
(b) INDIRECT EMBRYOGENESIS:
microspores undergo proliferation to form
callus
• (a)Anther at the
onset of the culture.
(b) Anther after 6
days in culture.
• (c, d) Embryos
emerging from the
anthers after 30
days in culture,
showing roots (c)
and shoots (d).
• (e–g) Plantlets with
cotyledons (e) and
with leaves
• (f, g) subcultured in
growing medium.
• (h) 80-day-old
regenerated haploid
plant from anther
culture (left-hand
side) and a diploid
control of the same
age (right-hand
side).
GYNOGENESIS:
GYNOGENESIS
• In vitro triggering of megaspores or female
gametophytes to sporophytic development.
• First done by San Noeum (1976) in Barley.
• Zhu and Whu (1979) cultured unpollinated
ovaries of Nicotiana tabacum.
• Alternative method for production of haploids
where anther culture has unsatisfactory results.
• Successfully applied in wheat, rice, maize,
tobacco, Gerbera etc.
• Rate of success varies with species.
• Can be cultured in pollinated or unpollinated stage.
• Conditions:
1. Optimum stage: mature embryo sac
stage
2. Media: Nitsch’s or White’s media, MS
media, Miller’s basal media
3. Growth regulators: MCPA (2-methyl-4-chloro
phenoxy acetic acid)
4. Cold treatment of flower buds: 40 C
5. Light and temperature of incubation
Method of ovule culture:

flower buds excised 24-48 hours before anthesis

calyx, corolla, stamens removed

surface sterilization of ovaries

tip of distal part of pedicel cut off

ovary implanted with cut end, inserted in medium


Advantages & disadvantages of
ovule culture:
• Advantages:
1. Has given positive results in F.
Compositae, Chenopodiaceae etc.
where androgenic response is poor
2. Only approach to produce haploids in
male sterile species.
• Disadvantages:
1. So far successful in less than two dozen species.
2. Frequency of responding ovaries is quite low
Diploidisation:
• Haploid plants grow normally under in vitro
conditions up to flowering stage
• Viable gametes are not formed due to absence
of absence of one set of chromosomes.
• So diploidisation is necessary.
• Done by:
1.Colchicine treatment: 0.5% colchicine solution
or colchicine lanolin paste.
2. Endomitosis
Application of haploids:
• Development of pure homozygous lines.
• Hybrid development.
• Transfer of desired alien genes: disease and insect
resistance, salt tolerance.
• Production of transgenic plants.
• Cytogenetic research.
• Generation of exclusively male plants.
• In genome mapping.
• Induction of mutation for modification.
Problems associated or
limitations of haploid culture:
• Failure of anthers to grow in vitro often.
• Developing callus: comprises of n, 2n, 3n, 4n cells.
• Selective cell division (of haploid microspores only
and not of unwanted tissue): often impossible.
• Formation of albinos especially in cereals
• Not economically viable.
• Polyploids outgrow haploids.
• Doubling of haploids is time consuming.
References:
• Books:
1.Introduction to plant tissue culture:
second edition- M.K. Razdan
(Oxford & IBH Publishing Co. Pvt. Ltd.)
2. Introduction to Plant Biotechnology:
H.S. Chawla
3. Biotechnology: P.K. Gupta
(Rastogi Publications)
4. Biotechnology: B. D. Singh
(Kalyani Publishers)
• Internet references:
1. www.plantuoguelph.ca
2.http://www.joensuu.fi/
3. www.apsnet.org
4. www.molecular-plant-biotechnology.info/
5. www.nature.com/nature/journal/
etc
THANK
YOU..!!