In vitro anther and isolated microspore culture as tools in Pepper breeding

Concept of microspore culture

The aim of a microspore culture is to get as fast as possible a haploid plant, which has several advantages for plant breeding. Such as easier research in plant breeding, because dominant features don’t drown out recessive ones. (Pierik, 1997)
First step is to isolate from a heterozygous, diploid plant the anther with pollen which are right in the stage of pre-first-cell-division (optimal for most plants). Because there are different ways of pollen development a pre-treatment can influence the direction. Treatment with ice cold water per example leads preferably to symmetric cell division or the following divisions of the vegetative nucleus. (Pierik, 1997)
In the second step the isolated microspores are cultivated on an adequate nutrient medium and a mono(ha)ploid plant will arise which is steril and therefore not yet useful for plant breeding. (Pierik, 1997)
Therefore a third step with the aim to get a diploid plant is needed. In most cases the plant is treated with colchicine which allows the plant to double its set of chromosomes. The haploid plant becomes a diploid, homozygous plant, which is now very useful for plant breeding. (Pierik, 1997)

How to determine the ploidy level

Basically it’s chromosome counting which is best done in dividing cells (eg.root tip cells from young seedlings) during metaphase when the DNA is condensed to chromosomes. Staining the chromosomes makes them better visible and counting is easier. Since counting manually is tedious the method of flow cytometry (laser based counting) is a good option. For flow cytometry pre-treatments of the plant cells are required. Such as staining (increase visibility), squashing (separate nucleus from other cell components) and washing (if there are still compounds which interfere an unproblematic counting). (Ochatt et al., 2011)

The role of an ovary co-culture

As Lantos et al. (2009) describe ovary co-cultures in anther or microspore cultures increase the success. But it is little known about the reasons. They assume that proteins excreted by the ovary promote the development in anther or microspore cultures. For successful promotion it does not specially need an ovary co-culture of the same species. (Lantos et al., 2009)

Literature:

Pierik, R.L.M. (1997). In Vitro Culture of Higher Plants. Department of Horticulture, Netherlands

Lantos C., Gémes-Juhasz A., Somogyi G., Ötvös K., Vagi P., Mihaly R., Kristof Z., Somogyi N., Mihaly R. (2009). Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Springer Science+Business Media B.V. 2009

Ochatt S.J., Patat-Ochatt E.M., Moessner A. (2011). Ploidy level determination within the context of in vitro breeding. Springer Science+Business Media B.V. 2011