India Education

aayush_005 · #1 (**permalink**) 09-14-2008, 10:43 AM

The term haploid refers to those plants which
possess a gametophytic number of chromosomes
(single set) in their sporophytes (2n).
First report of formation of haploids in Datura innoxia
was given by Guha and Maheshwari, at Delhi university,
in 1964.
Subsequently, Bourgin and Nitsch in 1967 ‐‐‐‐ reported
haploid plants from isolated anthers of Nicotiana.

Methods of haploid production
1. Androgenesis
i. Anther culture
ii. Pollen culture/ Microspore
Culture
2.Gynogenesis
3. Bulbosum Technique (Genome
elimination by distant hybridization)

Anther culture : Anther culture is the technique by which the
developing anthers are cultured on a nutrient medium where
the microspores within the cultured anthers develop into
callus or embryoids that give rise to haploid plantlets through
organogenesis or embryogenesis
Pollen Culture: is the technique by which the pollen grains
(uninucleate stage ) are excised from the intact anthers are
cultured on the nutrient medium where the microspores give
rise to haploid plantlets by organogenesis or embryogenesis
Gynogenesis: Inductionof haploids from unpollinated ovaries
or ovules
Bulbosum technique: Distant hybridization

Androgenesis
􀂉 From the male gametophyte of an
angiosperm plant, i.e. microspore (immature
pollen)
􀂉 The underlying principle is to stop the
development of pollen cell to direct
development in a plant (no gamete phases)
􀂉 Anther techniques are simple, quick and
efficient:
􀂉 immature anthers sterilized
􀂉 acetocarmine test for pollen
development
􀂉 solid media, pollen callus → shoots

Pathways of microspore division
Pathway I : Microspore divide equally into 2 identical daughter
cells which contribute to the sporophyte development. eg.,
Datura
Pathway II : Division of uninucleate microspores is unequal
resulting in theformation of VC and GC. Divisions in the VC
give rise to sporophyte , eg., Nicotiana
Pathway III: reverse of pathway III , sporophyte results from
the division in GC eg., Hyoscyamus niger
Pathway IV: Division of microspore is assymetrical as in
pathway II. Both the GC and VC take part equally

Microspore culture
􀂉 Haploid plants can also be produced through culture
of male gametophytic cells i.e. microspores or immature
pollen→ embryo (directly) or via callus
Advantages:
􀂉 uncontrolled effects of the anther wall and other
associated tissue are eliminated
􀂉 the sequence of androgenesis can be observed starting
from a single cell
􀂉 microspores are ideal for mutagenic and transformation
studies
􀂉 high yield of plants per anther can be obtained

Success of androgenesis
Anther culture ability is genetically
controlled:
􀂉 testing various cultivars in an simple medium
Physiological status of the donor plant
􀂉 no pesticides
􀂉 optimal environmental conditions: light,
photoperiod,
􀂉 temperature, nutrition, CO2 concentration
􀂉 outdoor plants more responsive that greenhouse
material
􀂉 young plants better than old
The composition of culture media: MS (1962,
White (1932) and Nitschand Nitsch(1969), 2-4%
sucrose and iron source

􀂉 Success of androgenesis
􀂉 Stage of pollen: anthers with microspores
ranging from tetrad to the binucleate stage are
responsive
􀂉 optimum stage of pollen for each species
􀂉 Development of the pollen can be stopped by
taking the pollen away from its normal
environment in the living plant and placing in
other specific conditions
􀂉 this induction is enhanced by giving certain
treatments: cold pretreatment low temp of 3‐5⁰ C for
48‐72 h or even more has been found beneficial, hot
treatment, chemical treatment (ethrel)

Process of androgenesis
􀂉 embryogenesis within 2 weeks
􀂉 Microspores undergo divisions → 40‐50‐celled
proembryo → embryo development simulating
normal zygotic embryo formation
􀂉 The plantlets originating from the callus generally
exhibit various levels of ploidy
􀂉 endomitosis and/or fusion of various nuclei
􀂉 ploidy status must be analysed for obtaining
homozygous lines: counting of plastids in stomata,
chromosome number, number of nucleoli, flow
cytometric analysis

Diploidization
􀂉 haploids can be diploidized to produce
homozygous plants:
􀂉 colchicine treatment (spindle inhibitor→chromosome
duplication)
􀂉 endomitosis: haploid p cells are unstable in culture and
have a tendency to undergo endomitosis (=
chromosome duplication without nuclear division), can
be enhanced in callus by auxin cytokinin medium
􀂉 Homozygous diploid callus or embryoids may form by
the spontaneous nuclear fusion of two similar nuclei of
the cultured pollen afer first division.

Gynogenic haploids (gyno = female)
􀂉 Megaspores or female gametophytes of angiosperms
can be triggered in vitro gg to sporophytic development
􀂉 Culture of unpollinated ovaries and ovules represents an
alternative for the production of haploid plants in
species for which anther culture has failed (e.g. albino plants)
􀂉 Not used as much as androgenic method
􀂉 problems in dissection of unfertilized ovules/ovaries
􀂉 promising for gymnosperms
􀂉 In addition to unpollinated ovaries, pollinated can be
also used in some cases
􀂉 Mainly employed in gymnosperms, like, Ephedra, Zamia and
some cycads

Gynogenic haploids
􀂉 Flower buds excised 24‐48 h prior anthesis
(unpollinated)
􀂉 The origin of gynogenic haploids differs in
species
􀂉 eggs/antipodal cells/ synergids
􀂉 Genotype of the donor plant is the most
important factor since each genotype shows
a different response
􀂉 Production of male sterile plants

Significances and uses of haploids
1. Development of pure homozygous lines: reduction in time
from 6‐8 years to a few months or a year
2. Hybrid development
3. Induction of mutations:
􀂉 majority of induced mutations are recessive and
therefore not expressed in the diploid cells
􀂉 resistance to antibiotics, herbicides, toxins, UV light,
gamma radiation, extreme temperatures
􀂉 Induction of genetic variability
􀂉 various ploidy levels and mutants
5. Generation of exclusively male plants, for example, in
Asparagus officinalis male plants have a higher production
and yield, ″super male plants″ with YY

6. Cytogenetic studies
􀂉 haploids can be used to produce aneuploids (= the loss or
gain of from the normal euploid number): monosomics in
wheat, trisomics in potato and nullisomics in tobacco
7.Significance in the early release of varieties: wheat, rice,
tobacco
8. Disease resistance
9. Insect resistance
10. Salt tolerance
11. Doubled haploids in genome mapping
􀂉 for molecular screening studies a much smaller sample
of doubled haploids is required for desirable
recombinants
􀂉 the identification of markers is much more secure

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