Anther Culture

Background
The purpose of anther and pollen culture is the production of haploid plants by the induction of
embryogenesis from repeated divisions of microspores or immature pollen grains (Dodds and
Roberts, 1985). The chromosome complement of these haploids can be doubled by colchicine
treatment or other techniques to yield fertile homozygous diploids. The resultant diploids can be
used in plant breeding to improve crop plants (Sunderland and Cocking, 1978).

The haploid nature of embryoids should be determined by standard chromosome staining
procedures (acetocarmine or Feulgen reaction) prior to colchicine treatment. Similarly, the effect of
colchicine on chromosome doubling needs to be monitored by chromosome staining.
Materials
tobacco buds
sharp pointed forceps, surgical scissors
95% ethanol
petri dishes of culture medium (1/2 strength MS, 2% sucrose 0.8% agar, glutamine [800
mg/liter], serine [100 mg/liter])
Methods
1. Obtain two buds at the appropriate stage. This occurs in tobacco when the sepals and the petals
in the bud are the same length.
2. Holding the bud by the pedicel between the thumb and first finger, dip the entire bud in 95%
ethanol for 15 seconds
3. Remove bud and allow excess alcohol to drip off.
4. With a pair of sterile forceps, remove the outer layer of tissue, the sepals.
5. Next, remove the inner layer of tissue, the petals, exposing the anthers.
6. Open the petri dish containing the medium for the induction of haploids. Remove each anther
from the bud and drop it onto the medium. Do not damage the anther or include any filament
tissue.
7. Repeat for another bud.
8. When finished, seal the plates and place in incubator (25°C).
9. In 2–3 weeks examine for somatic embryo initiation. Embryoid-forming cells are characterized
by dense cytoplasmic contents, large starch grains and a relatively large nucleus. Embryoids
appear opaque among translucent cells. Embryoids also exhibit high dehydrogenase activity
and can be detected by tetrazolium staining (Dodds and Roberts, 1985).
Acknowledgements
Paul Bottino (Botany Department, University of Maryland, College Park) provided the
sterilization procedure for Experiment 2 and Methods for Experiments 4 and 5.

Literature Cited
Bottino, P. J. 1981. Methods in plant tissue culture. Kemtec Educational Corp., Kensington,
Maryland, 72 pages.
Butcher, D. N., and D. S. Ingram. 1976. Plant tissue culture. Arnold, London, 67 pages.
Dodds, J. H., and L. W. Roberts. 1985. Experiments in plant tissue culture. Second edition.
Cambridge University Press, New York, 232 pages.
Street, H. E. 1973. Plant tissue and cell culture. Blackwell Scientific Publications, Oxford, 320
pages.
Sunderland, N., and E. C. Cocking. 1978. Plant tissue culture in China—major change ahead?
Nature, 274:643-44.
Sunderland, N., and M. Roberts. 1977. New approach to pollen culture. Nature, 270:236-238.
Ting, I. P. 1982. Plant physiology. Addison-Wesley, Reading, Massachusetts, 642 pages.
Wetherell, D. F. 1982. Introduction to "in vitro" propagation. Avery Publishing Group Inc.,
Wayne, New Jersey, 16 pages.