about anther culture

1. By,
Pillai Aswathy viswanath
PG 2 Botany
St. Thomas college
kozhencherry

2.  The dominant life form of higher plants
is the free-living sporophyte.
 The sporophyte is the resultant of
fertilization of male and female gametes
and contains a set of chromosomes from
each parent which genomic constitution
is 2n.

3.  Cells of the gametophytes carry half
the sporophytic set off of
chromosomes [n]
 In diploid plants, which contain two
sets [2n] of chromosomes
 Haploid plants are defined as
sporophytes having only a single set of
chromosomes (n;gametophytic number
of chromosome).

4.  The ability to produce haploid plants is
a tremendous benefit in genetic, plant
breeding, plant physiology and
embryology studies.
 Study of genetic recombination in
higher plants.
 Haploids are use for mutation study
 Heritability studies are simplified, due
to haploid plant having only one set of
chromosome hence recessive mutation
are easily identified.

5.  Several strategies and methods have
been worked for the production of
haploid plants.
Two methods:
Androgenic methods
Gynogenic methods

6.  Androgenic Methods:
haploid production of plants
through anther or microspore culture
has been referred to as androgenesis
 Gynogenic methods:
haploid production of plants
from ovary or ovule culture has been
referred to as gynogenesis

7.  It is the formation of sporophyte from
the male gametophyte on artificial medium
 It is most commonly found in family
solanaceae and poaceae
 In androgenesis immature pollen grains are
induced to follow the sporophytic mode of
development by various physical and
chemical stimuli.

8.  There are two methods for in vitro
production of androgenic haploids
 They are :
Anther culture
isolated Pollen [microspore]
culture.

9. Anther Culture

10.  The male reproductive part
of a flower is called the
stamen.
 It is composed of a long tube
called a filament and has a
pollen-producing structure on
the end. This oval-shaped
structure is called
the anther.
 it produces the male
gametophyte known as pollen.
 Pollen: A fertilizing powder

11.  Anther culture is the process
of using anthers to culture
haploid plantlets.
 The technique was discovered
in 1964 by Guha and
Maheshwari.
 This technique can be used in
over 200 species, including
tomato, rice, tobacco,
barley,datura ,brassica ,etc.

12.  The success of androgenesis
dependent on the variety used, the
growth condition of the plants, and the
quality of the donor material.
 The natural flowering conditions are
normally the best environment for
donor plants to produce anthers to be
used in successful regeneration
experiments.

13.  Age of the donor plants should be noted
 Usually anther from the flower buds will
give better response during
androgenesis

14.  Once the donor material containing the
microspores has been selected , it
requires specific pretreatment
conditions
 The pretreatment can be applied at
different levels of explants , such as
intact flowers (e.g. for barley complete
spikes), isolated anthers, or isolated
microspores.

15.  With regard to different explants, the
type, levels, and duration of
pretreatments are different, and the
regeneration efficiencies vary as well.
 It is widely supposed that
pretreatment plays a key role for
anther callus induction.
 The main pretreatments applied to
anther culture are cold treatment, hot
treatment , and so on.

16.  Cold treatment: In general, cold
treatment between 3-6 degree C for 3-
15 days gives good response.
 As a result of cold treatment , weak or
non-viable anther and microspores are
killed also it will retards aging of the
anther wall
 It will showed pronounced activity in
multiplication of embryonic cells.

17.  Hot treatment: Floral buds or the entire
plant in some species when subjected to
30 degree C for 24h or 40 degree for
1h stimulates embryogenesis.
 Also cause dissolution of microtubules
and dislodging of the spindle which
causes abnormal division of the
microspore nucleus

18.  Before culture, surface
sterilization of flower buds was
carried out in the Laminar Air
Flow Cabinet.
 Young flower buds with immature
anthers are surface sterilized
and rinsed with sterile water
 The calyx from the flower buds
will be removed by flamed
forceps

19.  The corolla is slit open and stamens are
removed and placed on the sterile petri
dish
 Each anther is gently separated from
the filament
 Care should be taken to avoid injury to
anthers since it may induce callus
formation from anther walls.
 The intact uninjured anthers are
inoculated horizontally on nutrient media

20. Media:
 The medium requirement may vary with
the species
 For most of the species, the commonly
used media for anther culture include MS
(Murashige and Skoog, 1962) medium.
 The constituents of the basal medium and
combinations of growth regulators are
also an important factor in eliciting
successful androgenesis

21.  Anther culture media is often solidified
using agar.
 Agar may contain compounds inhibitory to
the androgenic process in some species
 The use of liquid medium has been
advocated by some researchers as a way
to avoid the potentially inhibitory
substances in gelling agents.
 Anthers may be placed on the surface of
the medium
 Alternatively, microspores may be
isolated and cultured directly in liquid
medium.

22.  In androgenesis most of the species
required complete nutrient medium
[mineral salts, vitamins and sucrose]
with growth regulators.
 For many species (2–3%) sucrose is
added to the media it will induced the
pollens for androgenesis

23.  Several reports are available in which
either one or more hormone has been
found necessary for an androgenic
response.
 Addition of activated charcoal to agar
medium is advocated, since charcoal is
thought to absorb inhibitory compounds
present in trace elements in the culture
medium

24.  For a few species, such as tobacco, it
is not necessary to add plant growth
regulators to the anther culture media.
 Most species, however, require a low
concentration of some form of auxin in
the media.
 Cytokinin is sometimes used in
combination with auxin, is necessary
for pollen embryogenesis and pollen
callusing

25.  The addition of other substances in the
medium such as glutamine, casein,
proline, biotin, inositol, coconut water,
silver nitrate and polyvinylpyrrolidone
 The addition of glutamine and
glutathione to the culture medium also
enhances the embryogenic response.

26.  After inoculation haploid plants develop
from anther culture either directly or
indirectly through a callus phase.
Direct androgenesis
Indirect androgenesis

27. Direct androgenesis:
 It is also called pollen derived
embryogenesis
 Here pollen grains directly acts as a
zygote and passes through various
embryogenic stages similar to zygotic
embryogenesis.
 When the pollen grains has reached
globular stage of embryo, the wall of the
pollen is broken and embryo is released

28.  The released
embryo develop
cotyledons, which
ultimately give rise
to plantlets
 Eg: Datura ,
Brassica campestris

29. Indirect androgenesis:-
 In indirect androgenesis
the pollen grains, instead
of normal
embryogenesis , divide
erratically to develop
callus
 Callus tissue which is
finally redifferentiates
and forms haploid
plantlets
 Eg: rice , wheat, tomato

31.  Depending on the composition of the
medium, development pollen may leads
to the formation of embryoids or a
mass of parenchymatous callus
 Based on the few initial divisions in the
pollen grains or responds of pollen
grains,4 pathways have been identified
in in vitro androgenesis

32.  The uninucleate pollen grains may divide
symmetrically to yield two equal
daughter cells, both of which undergo
further divisions it will contribute to
sporophyte development
 Vegetative and generative cells are not
distinctly formed in this pathway.
 Example: Datura innoxia

33.  In this case ,the uninucleate pollen
divides unequally which will result in the
formation of Vegetative and generative
cells
 The sporophyte or plantlet arises
through further division in the
vegetative cell while the generative cell
does not divide.
 Examples: Nicotiana tabacum ,
Hordeum vulgare , Triticum aestivum

35.  The uninucleate pollen undergoes
a normal division but pollen
embryos are predominantly formed
from generative cell alone. the
generative cell either does not
divide at all or does so only to
limited extent
 The vegetative cell does not
divide.
 Examples: Hyoscyamus niger

36.  In some species, the uninucleate pollen
grains divide unequally, producing
generative and vegetative cell but both
these cells divide repeatedly to
contribute to the development of
sporophyte
 Examples: Datura metal

38.  Of the above early pattern of
divisions , the responsive pollen grains
ultimately become multicellular and
burst open to release cellular mass
having an irregular shape
 This tissue gradually becomes globular
embryo and undergoes normal
embryonic differentiation

39.  Four to five weeks after inoculation of
anthers, the calli attained convenient
size.
 Then they were removed aseptically
from the petridish on a sterilized glass
plate inside the laminar airflow cabinet
and were placed again on freshly
prepared sterilized medium containing
appropriate hormonal supplements for
plant regeneration from the callus.

40.  Sub culture was done in the MS media
 The sub cultured calli continued to
proliferate and differentiated into
shoots.
 After shoot initiation, more light
intensity was used for shoot elongation.
 The plants with well developed shoots
and roots are then transferred to pots
 By this method ,the pollen grain give
rise to haploid plant

42.  The plants may originate not only from the
pollen grains , but also from various parts
of the anther
 During anther culture there is always the
possibility that somatic cells of the anther
that are diploid will also respond to the
culture condition and so produce unwanted
diploid calli or plantlets.

43.  Sometimes the development of
microspores inside the anther may be
interrupted due to growth inhibiting
substances leaking out of the anther
wall in contact with nutrient medium.
 Anthers often fail to grow in vitro or
the initial growth is followed by abortion
of the embryos
 Isolated microspore or pollen grains

44.  In 1953,Tulecke was able to obtain the
callus from isolated pollen cultures of
gymnosperms
 This raised the possibility of obtaining
haploids plants from isolated
microspores or pollen culture

45. Isolated Pollen [Microspore]
Culture

46.  For microspore culture , anther are collected
from sterilized flower buds and kept in a small
beaker containing basal media
 The microspore are then squeezed out of the
anthers by pressing them against the side of
beaker with a glass rod
 Anther tissue debris is removed by filtering
the suspension through a nylon sieve

47.  This pollen suspension is then centrifuged
 The supernatant containing fine debris is
discarded and the pellet of pollen is
resuspended in fresh media and washed at
least twice
 the final suspension is then pipetted in to
small petri dishes

48.  To ensure good aeration, the layer of
liquid in the dish should be as thin as
possible
 Each dish is then sealed with parafilm
to avoid dehydration and is incubated at
28 degree C in darkness for the first
14 days of culture

49.  After 14 days ,the culture are
transferred to suitable media
 Their the microspores forms embryos or
calluses which may later differentiate to
form whole haploid plants

51. Genotype Of Donor Plant
 The genotype of the donor plant plays a
major role in determining the success or
failure of an Androgenesis.
 Some genotypes of a given species may
show androgenesis, while some others may
not.
 The genetics of androgenic response has
been analysed in several crops like wheat,

52.  The natural flowering conditions (light
intensity, day-length, temperature, humidity,
etc.) are normally the best environment for
donor plants to produce anthers to be used in
successful regeneration experiments.
 Any infection or stress to the donor plants will
lead to less success or complete failure for
induction of androgenesis and further
regeneration.

53.  Age of the donor plants affect the
embryogenic potential of the cultured anther
 Usually anther from the flower buds give
better response during androgenesis
 Anther derived from the older inflorescences
show decline in the frequency of haploid
production due to reduced pollen viability

54. Anther wall factor
 Sometimes the development of microspores
inside the anther may be interrupted due
to growth inhibiting substances leaking
out of the anther wall in contact with
nutrient medium.
 During anther culture there is always the
possibility that somatic cells of the anther
that are diploid will also respond to the
culture condition and so produce unwanted

55. Culture medium
 The culture medium also play a vital role ,
since the requirements will vary with the
genotype and age of the anther as well as the
condition under the donor plant are grown
 The medium should contain the correct amount
and proportion of inorganic nutrients

56. Growth regulators
 In cereals , auxin , particularly 2-4 D
promotes the induction of pollen callus
 Kinetin or cytokinins are essential for
induction of pollen embryos in solanaceae
 The concentration of sucrose plays an
important role in induction of pollen
haploid plants

57.  Activated charcoal is added to the culture
medium
It helps in 2 ways:-
 The removal of inhibitors from the agar
medium
 The adsorption of 5-hydroxymethylfurfural,a
product of sucrose dehydration during
autoclaving , assumed to be an inhibitor of
growth in anther culture

58. Physical factors
 Temperature and light are two physical
factors which plays an important role in
the culture of anthers
 Frequency of haploid formation and
growth of plantlets are generally better
in light
 A temperature in the range of 23-28
C is usually suitable for divisions in

59.  But it has been observed that chilling
of anthers before inoculation ,
increases the number of pollen
embryoids
 On the other hand , high –
temperature treatment of anther
culture of brassica napus before
transferring them to normal
temperature resulted in more embryoid
production

60. Other factors
 Certain organic supplements added to
culture medium often enhance the
growth of anther culture
Some of these include:
 The hydrolyzed products of proteins
such as casein (found in milk),nucleic
acids

61.  Coconut milk obtained from tender
coconuts is often added to tissue
culture media
 It contains a complex mixture of nucleic
acids , sugar , growth hormones and
some vitamins.
 In addition, amino acids like glutamine,
proline, serine, etc. enhance the
frequency of responsive anthers

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biotechnology,in vitro principles , techniques and
application ,(2011),MJP publishers
 Bajaj, Y.P.S. 1983. In vitroproduction of haploids.
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Techniques for Propagation and Breeding . Ed. D.A.
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