Anther culture:- the in vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for the purpose of generating haploid plantlets. Culturing anthers for the purpose of obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and grain legumes.

1. In vitro production of haploid plants
 Haploid : possesses gametophytic number of
chromosomes.
 Haploids are potential material to develop
homozygous lines and for the study of mutations.
 Spontaneous frequency of haploid : 0.001 to 0.1 %
through apomixis and parthenogenesis.
 1964, Guha and Maheshwari reported direct
development of plantlets and embryos from
microspores of Datura innoxia by anther culture.
 Haploids : monoploids (from diploid) or polyhaploids
(from polyploids). (x , n, 2n)
 Androgenesis : haploids from anthers
 Gynogenesis : haploids from ovule and ovary.

2. ANTHER CULTURE
Anther culture:- the in vitro culturing of anthers containing
microspores or immature pollen grains on a nutrient medium for
the purpose of generating haploid plantlets.
Culturing anthers for the purpose of obtaining Double Haploid is
not easy with many field crop species, particularly with the
cereals, cotton, and grain legumes.
Invitromethod
2

3. 4
Collect Anther
Anther Inoculation
Proliferating
anthers
Callus
Formation
Haploid plantlet
Colchicine treatment
Transplant
PROCEDURE FOR ANTHER CULTURE
DH Plant
Invitromethod
Embryo development

4. Anther culture
 Young flower buds with immature anthers in which the
microspores are confined within the anther sac are surface
sterilized and rinsed with sterile water.
 The calyx of the flower bud is removed by flamed forceps.
 Corolla is slit open and stamens are removed and placed in
sterile petriplate.
 One of the anthers is crushed to know the stage of pollen
development.
 Anther is gently separated from filament and intact,
uninjured anthers are inoculated horizontally on nutrient
media.
 To dissect the minute flowers like Brassica, stereo
microscope can be utilized.
 10-20 anthers are plated on one petriplate.

5.  In responsive anther, the wall tissue turn brown and
within
3-8 weeks they burst open due to the pressure
exerted by the growing pollen callus or pollen plants.
 After attaining the height of 3-5 cm, the individual
plantlets (embryogenesis) or shoots emerging from
callus (organogenesis) are separated and transferred
to suitable medium which supports further growth.
 Anther culture is very quick as compared to
microspore culture and it also influence development
of microspores acting as a conditioning factor.

6. Microspore culture
 Anthers are collected from sterilized flower buds in small beaker
containing basal media.
 Microspores are squeezed out by pressing anthers against the side
of the beaker with glass rod.
 Anther debris are removed by nylon sieve (40 to 100μ).
 The filtrate is centrifuged (150 g for 5 min.), supernatant with debris
is discarded and pellet of pollen is resuspended in fresh media and
several washes are given.
 Final suspension is pipetted into small petri dish with solid or liquid
media.
 Each dish is sealed with parafilm to avoid dehydration and
inoculated.
 Nurse culture or feeder layer : microspores are cultured on
anther by keeping semi permeable filter paper between anther

7. Advantages of microspore culture
over anther culture
 Uncontrolled effect of anther wall (possibility of
diploid plants and secretion of growth inhibiting
substances) and other associated tissue are
eliminated but anther wall has a stimulatory effect.
 Microspores are ideal for mutagenic studies
because, they can be evenly exposed to
chemicals or physical mutagen.
 Higher yields of plantlets per anther can be
obtained.

8. METHODS OF CHROMOSOME DOUBLING (Diploidization)
1. Endomitosis
2. Endoreduplication
3. C-mitosis
4. Nuclear fusion
ENDOMITOSIS is described as chromosome multiplication
and separation but failure of spindle leads to one
restitution nucleus with chromosome number doubled.
It has also been called ‘Nuclear Restitution’.
ENDOREDUPLICATION is phenomenon of DNA or
Chromosome doubling without Cytokinesis. 9

9. C-MITOSIS- endomitosis under the influence
of colchicine
NUCLEAR FUSION- it occurs when two or more
nuclei divide synchronously and develop a common
spindle. Thus, two or more nuclei could result with
doubled, polyploid or aneuploid chromosome number.
OTHER CHROMOSOME DOUBLING AGENTS
1. Acenanaphthene 2. Chloramphenicol
3. Nitrous oxide 4. Parafluorophenyl alanine
5. 8- hydroxyquinone 20

10. COLCHICINE
1820- alkaloid isolated by French
chemists P.S. Pelletier and J. Caventon
It is a toxic natural alkaloid and secondary
metabolite, extracted from plants of the
genus Colchicum (autumn crocus,
Colchicum autumnale, also known as
"meadow saffron").
Extracted from seeds and corms of
Colchicum.
Increase fertile plant and reduce albinism
in anther culture. 22

11. Solution of colchicine which interferes with
cell division, but DNA is doubled
N-​[(7S)-1,​2,​3,​10-tetramethoxy-9-oxo-5,​6,​7,​9-
tetrahydrobenzo​[a]​heptalen-7-yl] acetamide
Systematic (IUPAC) name – C22H25NO6
Colchicine inhibits microtubule polymerization
by binding to tubulin. Availability of tubulin is
essential to mitosis, and therefore colchicine
effectively functions as a "mitotic poison" or
“spindle poison”.
CHEMISTRY OF COLCHICINE
HOW COLCHICINE WORK ????
23

12. In DH METHOD - probability of getting the desirable genotype is (1/2)n
DIPLOID METHOD – probability of getting the desirable genotype is (1/4)n
GENETICS OF DH POPULATION
27
DOUBLE HAPLOID METHOD DIPLOID METHOD
If ‘n’ loci are segregating:-

13. Gynogenic haploids
 Haploids from ovary and ovule
 Promising in some of the species where anther
culture is unsatisfactory. e.g. Gerbera.
 San Noeum (1976) : first successful effort for
gynogenic haploid in barley.

14. Applications of ovary culture
 Useful to study embryo development, fruit
development, different aspects of fruit physiology
including respiration, maturation and disease.
 Process of parthenogenesis can be studied by
culture of unpollinated ovaries.
 To study the role of floral organs, e.g. calyx is
necessary for proper fruit development in many
species.
 Pre-requisite for in vitro fertilization (test tube
pollination) to overcome self-incompatibility (pre-
fertilization barrier).
 Induction of polyembryony.
 To study fruit development in apomictic species (role

15. Applications (Ovule culture)
 To obtain viable interspecific hybrids by culturing
fertilized ovule.
 Production gynogenic haploids.
 Regeneration of parasitic plant in absence of host.
e.g. striga and orabanke.
 Ovule culture of orchid plant. (naturally the seeds
germinate in association with fungus, if the
fungus is not present, many seeds are lost.)
 Induction of polyembryony (from nucellus).
 Virus eradication

16. Significance and uses of haploids
1. Development of homozygous lines
2. Hybrid development from 100 % homozygous lines.
3. Induction of mutation
4. Induction of genetic variability (polyploids, aneuploids)
5. Cytogenetic research (pairing pattern studies to know
evolutionary history)
6. Release of the variety : Wilt resistant tobacco, tall and high
sugar sugarcane
7. Hybrid sorting in haploid breeding : Chances of getting
superior gamete is more than obtaining superior segregant in
F2 population.
8. Disease resistance : Barley- Mosaic resistance, Blast
resistant rice varieties.
9. Insect resistance : Brown plant hopper resistant rice
10. Salt tolerance : Salt tolerant rice varieity (Hua Bain 124-4)

17. Problems
1. High level management and expertise.
2. Appearance of diploids and tetraploids, lack of selective
cell division.
3. Callus formation is detrimental in androgenesis.
4. High incidence of albinism in case of androgenesis.
5. During derivation of haploid, selection can not be
applied.
6. Polyploids generally outgrows haploids
7. Doubling does not always result in production of
homozygotes.