Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.47

METHODOLOGIES FOR THE MOLECULAR CHARACTERISATION OF POLLEN MUTANT

M. VIANELLO, E. GATTI, M. VILLA, M. SARI GORLA

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano

pollen mutants, gaMS-1, maize, development

The purpose of this research is the study of the genetic control of pollen development and function in maize. The knowledge of the genetic and molecular components regulating these processes can allow the development of new strategies of genetic improvement of important crops. Furthermore this is an excellent model for important basic researches on developmental processes, including establishment of cell polarity, cell fate determination, cell differentiation and morphogenesis.

For these studies pollen mutants represent an important tool. We have isolated many mutants of pollen development, both pre-meiotic and post-meiotic. The latter are characterized by a 1:1 ratio of fertile and sterile pollen grains within a single anther. In particular, gaMS-1 produces mutant grains that at anthesis are still at a stage of maturation corresponding to that of a binucleate microspore.

However, the analysis of this type of mutants presents some drawbacks, and it is important to set up some specific strategies for its optimisation.

First of all, due to the sterility of the mutants grains, the mutation can be maintained only in heterozygous condition and it is not possible to obtain homozygous plants. Consequently, it is not possible to obtain a homogeneous pollen population, exclusively composed by mutant pollen grains. Thus, in order to analyze transcripts or proteins differentially expressed by the two types of grains it is necessary to compare wild type pollen with a mixture of normal and mutant grains derived by segregating plants. This method greatly reduces the power of resolution of the analysis, since only quantitative differences can be detected.

To overcome this problem, a method for the separation of the two pollen types produced by heterozygous plants has been developed to obtain suspensions containing up to 95% of each grain type, by using a gradient of Percoll, from 100% up to 40%.

A further limitation is the material availability: due to the above-mentioned problems, large amounts of it are normally very difficult to be obtained. Thus it is important to develop systems for the analysis of DNA of a limited number of grains. From the suspension of the pollen previously separated by Percoll, a manual removal of the about 5% of different grains of pollen was made, using a glass capillary. The DNA from the cleaned pollen suspension has been amplified directly, avoiding the extraction protocol. The pollen has been homogenised, and the obtained suspension submitted to a PCR reaction.

Fresh pollen, collected during flowering time, is maintained in 70% ethanol. The amplification of DNA from these fixed pollen grains has been performed following the above mentioned protocol.

A RNA isolation protocol (LiCl-method) has been set up, to be used on limited amount of pollen.