Production of Haploids

Haploid Culture
•Haploid plant (n) = recessive mutations displayed n+n=
double haploid

•Occur spontaneously in inter-specific cross or induced by

irradiating pollen prior to pollination. Extremely poor efficiency.

•1964 Guha and Maheshwari cultured Datura innoxia anthers

and found that large portion of culture contains haploid cells.

.
The haploid embryos must be
germinated in vitro.
 Anther/Pollen culture
• Method to produce
haploid plants
• Spontaneous occurrence
in low frequency
• Induction by physical
and/or chemical
treatment
• Chromosome elimination
following interspecific
hybridization
 Androgenesis
• History
– 1964, 1966 Datura innoxia (Guha and
Maheshwari)
– 1967 Nicotiana tabacum (Nitsch)
• Critical factor - change in developmental
pattern from mature pollen to
embryogenesis.
Factors influencing androgenesis
– Genotype of donor plants
– Anther wall factors
– Culture medium and culture density
– Stage of microspore or pollen development
– Effect of temperature and/or light
– Physiological status of donor plant
Ovary Culture for Production of
Haploids
 Ovary Culture : Introduction
• Ovary or ovule is yet another method for
production of haploid plants.
• They are called gynogenic haploids.
• First developed by Noem (1976) in Hordeum
vulgare.
• Later haploids of rice, wheat, maize and
sunflower, sugar beet, tobacco are developed.
 Ovary Culture : Introduction
• Gynogenic haploids are produced/ attempted
only when androgenic haploids are failure,
because the method is very sensitive.
• Appropriate excised flower buds are excised 1
or 2 days prior to ANTHESIS.
• After removal of other floral parts, ovaries are
surface sterilized.
 Ovary Culture : Introduction
• The ovaries are now detached from pedicel
and are inoculated with it.
• If liquid medium is used, the ovaries are
floated over the medium with the help of
filter paper with pedicel inserted through it.
• commonly used media are
• MS, White, N6, Nitsch + growth factors.
 Ovary Culture : Introduction
• This technique is mainly useful in male sterile
lines.
• There are some difficulties associated with in
• Dissection of unfertilized ovary/ ovules is
rather difficult.
• Only one ovary/ flower as opposed to
anthers/ flower
• Very slow growth and response.
 Production of Homozygous Lines
• Haploids plants can be produced and grown
but not propagated, as there is no seed set.
• Reason is lack of viable gametes.
• It is attempted to diplodize the haploid plants.
 Colchicine treatment
• Colchicine is the most effective agent
• It is a strong inhibitor of spindle fibers and
enforce C-mitosis.
• C- mitosis can lead to chromosome duplication
• Colchicine application is done by different
ways
• Apply colchicine paste to the axils of
leaves
The haploid plants can be treated with
colchicine to obtain doubled haploids.
 Colchicine treatment
• Decapitate the main axis
• The axilary leaf bunches turn oout to be
dipliods
• Or seedling treatment—wash –transfer
• Cotton swab method for axilary
buds/main axis.
• Endomitosis in invitro cultures
 Colchicine treatment
• Chemical treatment : all spindle fibre
inhibiting chemicals.
• All these techniques develop/ produce
homozygous lines
 ONION OVARY CULTURE

ovaries

ovaries

haploid
DH tetraploid
CUCUMBER OVARY CULTURE

embryo
s

OVULES

embryo sac
 ZUCCHINI OVARY CULTURE

embryos development of embryos

developed from
ovules
 Uses of haploids
• Completely homozygous plants
• Inbred lines
• Mutation studies
• Breeding (equal ploidy levels)
• Mapping