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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

ISOLATION AND PRELIMINARY CHARACTERIZATION OF A MAIZE LOW PHYTIC ACID MUTANT

PILU S.R*, NIELSEN E.**

* Dipartimento di Produzione Vegetale (Di.Pro.Ve.), Università degli Studi di Milano, Via Celoria 2, 20133 Milano

salvatore.pilu@unimi.it

** Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, via Ferrata 1, 27100 Pavia

nielsen@ipvgen.unipv.it

maize, mutagenesis, low phytic acid mutant, TLC

Phosphorous is an essential nutrient for all living organisms. However, its massive use in agriculture may have deleterious effect on the environment by leaching into streams, lakes and rivers. Furthermore, P mined from rock deposit s is not an unlimited resource, and may be depleted within 60-70 years. Therefore, the utilisation and excretion of P from agricultural husbandry is being attracting concern, and efforts should be directed to increase and improve the recycling of this mineral element.

Phytic acid (phytine), myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP₆), is the major storage compound of phosphorous in plants, accumulating predominantly in seeds (up to 4-5% of dry weight) and pollen. In cereal and in by products of oil seeds, which are the major ingredients used by the feed industry, up to 80% of P is present as phytate P, which has generally very little bio-availability for monogastric animals due to their lack of phytase activity. As a consequence, it is common to supplement the animals diet with inorganic phosphate or, in the last decade, with microbial phytase (Pointillart et al., 1987). The latter practice has been shown to increase the utilisation of feed P by animals, as well as to decrease the amount of the phytic P excreted with manure, causing eutrophication of surface water. Recently, Aspergillus phytase was directly accumulated in transgenic seeds, which can then be included in feeds for transgenic animals (Pen et al., 1993).

Mutational breeding offers a third opportunity: the isolation of cereal mutants accumulating less phytic P and more free phosphate in the seed (Raboy et al. 1990, Rasmussen et al. 1998), and we have chosen to apply this approach in maize.

Since normal mature maize seeds contain high phytate-phosphate and low free phosphate levels, a screening for high level of free phosphate in seed should provide a quick and inherently sensitive assay for lpa (low phytic acid) genotype. In order to identify lpa mutants, a population of EMS (ethyl methanesulfonate)-induced mutants was generated using the pollen-treatment method. Approximately 600 M₂ family were screened. The first screening was carried out by titrating free phosphate by the molybdate staining assay. Putative mutants were then challenged by a TLC (Thin layer chromatography) method allowing the simultaneous detection of free phosphate and phytate.

Most of the mutations isolated this way turned out to perturb germ or aleurone development. However, one monogenic recessive mutation (named lpa 3-1), even causing approximately an ten fold increase in the amount of free phosphate titratable in a -/- homozygous seed, did not affect normal germination or seedling growth. Moreover, also in +/- heterozygous seeds, free phosphate content was increased 2-3 fold compared to wild type.

Genetic analysis of this mutation, as well as its further biochemical characterisation aiming at identifying the biosynthetic step involved, are under way.