University of Milan


University of Bologna

Gametophytic selection for Chlorsulfuron tolerance

--M. Sari Gorla, E. Ottaviano, E. Frascaroli and P. Landi

The purpose of this research was to prove the possibility of obtaining in the sporophytic generation a response to selection, applied at the gametophytic level, for specific resistance factors. Gametophytic selection can be particularly useful to select very rare mutants or allelic combinations, which is the case of resistance to herbicide molecules: owing to the peculiar characteristics of pollen (haploidy and large population size), the procedure can be much more effective than selection on the sporophyte, provided that the genes controlling resistance (or tolerance) are expressed both in the haploid and the diploid phase of the life cycle.

Chlorsulfuron was chosen on the basis of: i) its biological target (acetolactate synthase), which is expressed at the cellular level and so could be expressed in pollen too, and ii) its favourable characteristics as herbicide (low dosage, no toxicity for animals). However, it is degraded slowly in the soil, thus damaging susceptible crops sown after a resistant one. Maize is a susceptible plant species, but variability for tolerance has been described.

Selection was applied to pollen from heterozygous plants obtained from a tolerant (Va85) and a susceptible (B73) inbred, at different stages of the gametophytic phase: during microspore development or during pollen function. In the first experiment, tassels were detached about ten days before anthesis, put in liquid medium supplemented with the herbicide (20mg/l) for 24 hours and then grown in artificial conditions until flowering; the pollen produced was used to pollinate female plants of the sensitive genotype. In the second, the recipient plants, of the sensitive genotype, were treated by spraying their silks with a solution containing the chemical (200mg/l) after pollination with pollen from the F1 plants. Control plants were produced through the same procedures, except for the presence of the herbicide.

The response to selection was assayed on the resulting backcross sporophytic progeny. Plants from treated or non-treated pollen were grown in the presence of Chlorsulfuron (0.3ppb vol), and the two populations ("Control" and "Treatment") were compared with regard to two traits: seedling height and main root length (cm). The relative frequency distributions are reported in Figure 1a, which refers to the results of the first experiment (treatment during pollen development), and in Fig 1b (second experiment:

Figure 1. Frequency distribution of seedling height and root length of backcross populations obtained from treated (dotted) and non-treated (non-dotted) pollen. 1a: first experiment; 1b: second experiment.

treatment during pollen function); in each diagram the dotted area refers to the backcross progeny from treated pollen, the non-dotted to the backcross seedling from non-treated pollen; the arrows indicate the means. The results reveal a significant increase of the mean values for both characters in the treated populations when compared with the controls (F test); immature tassel treatment produced larger effects than silk treatment.

The response to selection, applied on pollen produced by single hybrid plants, in the resulting sporophytic progeny, demonstrates that genes controlling tolerance are expressed in pollen and are the same genes conferring tolerance to the plant. These data indicate the possibility of using male gametophytic selection for crop improvement: since the increase of tolerance has been obtained in only one generation of selection, it could prove to be a very good tool for increasing the efficiency of the traditional breeding methods.


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