One of the most important problems concerning the role of the male gametophyte in the evolution of higher plants is the genetic relationship between the haploid and diploid phases: are the genes expressed in the gametophytic phase the same as those controlling sporophytic functions, or does a specialized set of genetic factors exist? As far as characters determined by single genes are concerned, observation of segregation in a gametophytic population deriving from a single heterozygous plant provides a means of revealing haplo-diploid expression. This can be made by means of specific staining of pollen grains, provided that variants and suitable staining techniques exist. A more general method is provided by the analysis of the electrophoretic pattern of dimeric (or multimeric) enzymes. If the enzyme is of sporophytic origin, pollen extracts from plants heterozygous for electrophoretic mobility display the same banding pattern as sporophytic tissues (two homodimers and one heterodimer), whereas in the case of haploid transcription the pollen extracts reveal only the two parental homodimeric bands. However, it cannot be ruled out that causes other than haploid transcription may be responsible for preventing the activity of the heterodimeric enzyme, while this can be discarded if the heterodimeric form is found in pollen with heterozygous gene duplication.

This pollen type can be obtained for most of the maize genome using appropriate B-A translocations. Because of the B centromere nondisjunction at the second microspore division, a heterozygous B-A translocation, having an AA^BB^A genotype, produces pollen in which one of the two sperm cells has two B^A chromosomes, and the other none. In the progeny of a cross for which TB-A plants A(F)A^BB^A (F) are used as male parent and normal A(S)A(S) plants as female parent, seeds with hyperploid embryo, heterozygous for electrophoretic mobility, can be selected.

Hyperploid embryos are selected by means of genetic markers, chromosome dosage effects and/or root tip chromosome counting. At flowering, the resulting plants produce pollen that is in part partially diploid and heterozygous for electrophoretic mobility for enzymes coded by genes localized on the B-A translocated chromosome arm.

This method has been tested by analyzing electrophoretic variants of ADH-I, and has been used to study the gametophytic expression of GOT-1. For both the enzymes, sporophytic extracts of inbred lines revealed one band, while three bands were found in the hybrid; pollen extracts were identical with sporophytic extracts of the inbreds, while hybrid genotype gave rise to only two bands. Pollen from TB-A hyperploid plants revealed three bands, thus indicating that, when both alleles are present in the pollen grain the active heterodimeric enzyme is formed.

The method can also be used to map genes with gametophytic expression, specifying dimeric (or multimeric) enzymes and particularly to study genes with gametophytic expression.

M. Sari-Gorla, C. Frova, M. Corbella and E. Ottaviano

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