Presentation

On
Anther culture

Submitted to Submitted by
…………………… Neha Gupta
…………………… M.Sc.3rd semester
…………………… of Biotechnology
 Introduction of anther culture
 Haploid production
 Pathways of development
 Culture medium
 Growth regulators
 Stage of pollen development
 Culture environment
 Pretreatments
 Other factors
 Pollen culture
 Application of its
 Reference
 Haploid plants may be obtained from pollen grains by
placing anthers or isolated pollen grains on a suitable culture
medium; this constitutes anther and pollen culture.
 The anthers may be taken form plants grown in the field or in
pots, but ideally these plants should be grown under
controlled temperature, light and humidity; the optimum
condition may diller form species to species.
 Flower buds of the appropriate developmental stage one
collected, surface sterilized and their anthers one excised and
placed horizontally on culture medium. .
 Care should be taken to avoid injury to anthers since it may
induce callus formation from anther walls.
 In many plant species, somatic embryos from the pollen grains of
cultured anthers are directly produced eg. In Datura , Atropa,
Brassica compestris, B. napus, several Nicotiana sp. etc.In such
cases the plants obtained from germination of embryos one
generally haploid but some polyploids one also produced.
 But in many other species like rice (O.Sativa) barley
(H.Vulgare), wheat, tomato triticale etc.Pollen grains produce
callus from which plantlets may be regenerated under suitable
culture conditions.
 In these cases, the ploidy level of plants varies considerably more
than in those where embryos are produced.
 Of these following are examples of important crop species :
 Potato (s. tuberosum ), barley, wheat (Triticum Sp.), rice,
Brassica campestris, triticale, many other members of solanaceae
and some vegetables.
 The significance of haplods in genetics and plant breeding has
been realized for a long time.
 Spontaneous production of haploids usually occurs through the
process of parthenogenesis (embryo development from an
unfertilized egg).
 the development of numerous pollen plantlets in anther cultures
of Datura innoxia first repotted by two Indian scientists –Guha
and Maheshwari was a major break, through in haploid
breeding of higher plants.
 The technique of haploid production through anther culture has
been extended successfully to numerous plant species,
including many economically important plants, such as cereals
and vegetable, oil and tree crops.
 The early divisions in responding pollen grains may occur in one of the
following four ways.
 The uninucleate pollen grains may divide symmetrically to yield two
equal daughter cells both of which undergo further divisons. Ex- Datura
innoxia (pathway-I)
 In some other cases eg. – N.tabacum, Datura metel, barley, wheat,
triticale chillies etc, the uninucleate pollen divides unequally. the
generative cells degenerate immediately or after undergoing one or two
divisions.
 The callus fembryo originates due to successive divisons of the
vegetative cell. (Pathway II)
 But in few species eg.- Hyoscyamus niger , the pollen embryos originate
the generative cell alone, the vegetative cell either doesn’t divide or
divides only to a limited extent forming a suspensor like structure
(Pathway III).
 Finally in some species ex. Datura innoxia the
uninucleate pollen grain divide unequally, producing
generative and vegetative cells, but both these cells
divide repeatedly to contribute to the developing and
vegetative cells, but both these cells divide repeatedly
to contribute to the development embryo /callus.
(Pathway-IV)
 In Brassica napus, the first division is symmetrical and
the pollen embryos exclusive the vegetative cell; this
may be regarded as (Pathway V)
 Medium requirements may vary with the species, the genotype
the age of donar platns and anthers and conditions under which
the donar plants are grown.
 For example – pollen grains of Datura and tobacco produce
embryos on the agar medium containing only 2-4% sucrose,
while elaborate media eg:- N6 Potato- 2 media had to be
formulated for cereals.
 Sucrose is essential for anther cultures the connection may
range from 3% for barley to 6% for wheat and potato, but 2-3%
sucrose is most commonly used.
  
 A complete tissue culture medium is required : MS,
LS(Linsmaer and Skoog)and Some other.
 In solanaceous plants, pollen embryogenesis doesn’t
require any growth regulator but low levels of, auxins,
cytokinins and even GA3 appear beneficial; 0.1 mg/L
IAA gave the best results.
 In Hyoscyamus niger an auxin ex. 2mg/L 2, 4-D
enhanced the frequency of responding anthers but had no
effect on the number of embryogenic pollen grains.
 In contrast, cytokinins reduced the number of pollen
grains producing embryos most likely by interfering with
cell division in induced pollen grains.
 In species where callus is formed ex.- Cereals, auxins and
cytokinins are almost invariably used either in
combination or in sequence, but the role played by them is
not known.
 It seems that different GRs may be required for best results
with different plant species.
 The presence of an auxin may determine the mode of
subsequent development of androgenic cell masses. Wheat
anthers cultured on a medium having 2-4-D produce
callus, while those kept on a coconut milk supplemented
medium give rise to embryos.
 
 The optimum stage of pollen varies with species. for many
species, induding Datura ,tobacco etc. the optimum stage
is just before or just after the pollen mitosis, while the
early binucleate stage is most suitable for species like
Atropa belladona and Nicotiana sylvestris, and is
absolutely essential for Nicotiana knightiana.
 It may be pointed out that pollen development doesn’t
proceed uniformly within the same anther so that a single
anther may contain pollen grains at early binucleate and
midbinucleate pollen grains.
 Anther cultures are generally maintained in alternating periods
of light at 280C and darkness at 220C but the optimum
conditions vary with the species.
 The walls of responsive anthers turn brown and after 3-8 weeks
they burst open due to developing callus or embryos.
 In tobacco optimum temp. is around 250C. pollen embryos
aren’t formed in D. Innoxia anthers cultured at 200C or below.
 Clearly temperature optima varies with species.
 In some species ex- grape, potato, Datura etc. exposure of
anthers to light during their first 24 hr of culture enhances the
frequency of haploid callus or responding anthers.
 Exposure of excised / flower buds to a low temperature for some time ex-
at 3-50C or 2 days or at 7-80C for 12 days for tobacco, prior to removal of
anthers for culture may markedly enhance the recovery of haploid plants.
 In contrast the best pretreatment for cereals like wheat, rice, barley etc.
seems to be 3-28 days at 4-100C this increases the frequency of green
plants.
 The pretreatment temp and duration may be considerably affected by plant
species, genotype and stage of anther development.
 The mechanism of cold pretreatment response is not known but
interference with starch accumulation in pollen and degradation of
tapetum cellular matrix etc. may be involved.
 In addition pretreatments like centrifugation irradiation with x-rays and
gamma rays and reduced atmospheric pressure are reported to promote
androgenesis.
 Isolated pollen grains when cultured invivo give rise to haploid embryos
or callus this approach is called pollen culture.
 Pollen may be isolated either by squeezing or float culturing the anthers.
 In float culture excised anthers are floated on a shallow liquid medium in
a petridish. The anthers dehisce in a few days releasing their pollen
grains into medium. These anthers continue to shed pollen so that their
serial subculture yields pollen samples in different stages of
androgenesis.
 Initially isolated pollen grains were cultured either in hanging drops or
on a filter paper raft placed on cultured anthers.
 Pollen culture offers certain advantages over anther culture due to
elimination of anther wall ex :-
 Studies on differentiation and development are easier and more precise.
 No callus formation can occur from wall tissues.
 Products from different pollen grains ordinarily don’t get mixed up.
 Homozygous lines of the cross pollinating species and
hybrids are highly desirable to increase the efficiency of
selection and production of homozygous plants.
 On the other hand, homozygous plants can be obtained in a
single generation by diploidization of the haploids.
Haploids are also extremely useful for detecting recessive
mutants which may not express themselves in the
heterozygous diploid background and therefore can be
easily lost.
 Some of the applications of this haploid production are
discussed :-
 The most imp. Application of androgenic haploids is
in the production of stable homozygous dihaploids in
a single generation equivalent to the Fx generation of
pedigree breeding and considerably shortening the
breeding cycle.
 Haploid breeding involving anther culture has been
most successfully used in china to produce several
agronomically superior varieties of wheat, rice,
maize, and pepper many of which are under large
scale cultivation
 Besides yielding haploids in vitro androgenesis
provides a unique opportunity to screen the
gametophytic variation, caused by recombination
and segregation during meiosis at the sporophytic
level.
 Detection and isolation of recessive mutants in
the haploid state and rapid obtainment of the
mutated gene in a homozygous diploid state is a
special merit of haploidy in higher plants.
 Application of mutagenic treatment at the
microspore stage, which is a single called
structure has the added advantage of obtaining
solid mutants.
 Agrobcterium is a superior vehicle for
transformation of dicots but its use with
monocots is very limited.
 Therefore alternative methods such as micro
injection electroporation etc. are being tried.
 Anther problem with somatic protoplasts or cell
transformation in most monocots and some dicots
is the poor regenerability of plants after DNA
insertion.
 Biotechnology Expanding Horizons –
B.D.SINGH
 Plant Tissue Culture :

 Theory and Practice , a Revised Edition

 S.S. Bhojwani and M.K.Razdan

 Internet (Google)