Dr.Harsh kumar sir
Dr. Kundan sir
ANTHER: A part of stamen
POLLEN: A fertilising powder
discharged from flowers anther.
ANTHER AND MICROSPORE(POLLEN)
CULTURE: It is the process of formation of
haploid plants from microspores (pollen)
cultured individually or anthers.
Anther culture for production of
haploids reported in about 250 species.
Solanaceae, cruciferae, gramineae are most common.
Anther/pollen culture is referred as
ANDROGENESIS : occurs when
pollen/microspore shift from a gametophytic to
sporophytic pathway of embryo formation.
shift occur prior to premitotic & postmitotic :
either vegetative or generative divide to
In experiments using Datura innoxia,
induction frequencies of almost 100% and
a yield of more than one thousand
plantlets or calluses have occurred under
optimal conditions from one anther.
Success can be determined within 24
hours as cells begin to divide.
figure : Anther culture and
haploid plants regeneration.
(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) 80day-old regenerated haploid plant from
anther culture (left-hand side).
1964, 1966 Datura innoxia (Guha and
Maheshwari), the Indian scientists.
1967 ( Bourgin and Nitsch ) : 1st haploid
plants from isolated anthers of Nicotiana.
during last decade : anther culture of rice,
wheat , maize, brassica, pepper and crop sp.
The microspores divide by an equal
division and two identical daughter cells
Vegetative and generative cells are not
distinctly formed in the pathway.
Example: Datura innoxia
The division of uninucleate microspores is
unusual , resulting in the formation of
vegetative and generative cell.
The sporophyte arises through further
division in the vegetative cell and
generative cell does not divide.
Examples: Nicotiana tabacum , Hordeum
vulgare , Triticum aestivum , Triticale.
The uninucleate microspore undergoes a
normal division but pollen embryos are formed
from generative cell alone.
The vegetative cell does not divide.
Examples: Hyoscyamus niger
Both generative and vegetative cell divide
further to the development of sporophyte.
Examples: Datura metal, Atropa
belladona, Datura innoxia(occasionally).
In Brassica napus , 1st division is
symmetric and the pollen embryos
develop the vegetative cell.
Pathways to pollen
1) Genotype of donor plant : determine the
frequency of pollen plant production.
_ eg. In Hordeum each genotype differs with
respect to androgenic response in anther
_ high responsive anthers should
2) Anther wall factor : act as conditioning factors
promote culture growth.
_ report: glutamine alone or in combination
serine and myoinositol could replace the
3) Stage of pollen : stage of pollen varies with
_ before/after 1st pollen mitosis- Datura,
4) Physiological status of donor plant :
a) grown under best environmental
conditions with good anthers.
b) flowers obtained at the beginning
of flowering season are highly
5) Pretreatment of anthers : appropriate
treatment required for good success of
haploid production(depend on donor plant
# Temperature influence:
a) induction of androgenesis is
stored at low temperature prior to
culture.e.g. maize , rye.
b) pretreatment of anthers at higher
response is not known but interference
with starch accumulation in pollen and
degradation of tapetum cellular
matrix,etc.may be involved.
Cold Treatment (3 to 5 C) Enhances
Symmetric Division of Microspores or
Division of Vegetative Nuclei
3 to 5 C
3 to 5 C
Cold Pretreatment of Anthers Enhances the
Cold treatment imposed prior to the first pollen
mitosis increases the frequency of symmetric
divisions of the microspore leading to embryo
Heat treatment(320 c for 8
hrs ), gamma- rays,
Stress by mannitol, calcium
Glutamine and sugar
(transfer to high glucose
Cold treatment( 4 - 100 c for 3-
6) Effect of light : pretreatment of anthers at
elevated temperatures( 3 0 c) stimulate
androgenesis in some Brassica and
7) Culture medium :
- it vary with the genotype and age of the anther.
- culture maintained on an auxin medium for
longer period develop a friable callus.
- a compound related to auxin namely 2,3,5triiodobenzoic acid(TIBA) gives +ve result at low
- incorporation of activated charcoal/2chloroethyl-phosphate stimulates androgenesis
DIFFERENT TYPES RELATED CULTURE
Simple agar plates containing
Complete nutrient medium
Non- solanaceae plants
Medium fortified with growth
adjuvents (yeast extract, casein
hydrosylate, coconut milk)
N6 and Yu-Pei
Potato – 2 medium(potato-1 +
six low salt concentration)
1) Selection of explants(eg. Flower bud)
2) preparation of explant
3) disinfection of bud
4) selected buds are pretreated
5) surface sterlization
7) transfer to culture room
8) transplanted to small pots in
less time consuming.
Requires skill to remove anthers without
Not much successful in case of cereal
Risk of chimera and callus formation from
1. Often fail to grow in-vitro.
2. Tissue or callus comprises a chimera of
diploid, tetraploid and haploid cells.
3. Formation of albinos especially with cereals
and effect the loss of plants due to albinism.
4. It is not economically viable for haploid
5. Callus in a medium supplemented with growth
regulators is usually detrimental for haploid