X ray-and fast neutron-induced mutations in Arabidopsis thaliana and the effect of dithiothreitol upon the mutant spectrum

The genetic effects of X-ray and fast neutron seed-irradiation of Arabidopsis thaliana (L.) Heynh., and the influence of a pre-irradiation treatment with the radio-protector dithiothreitol (DTT), are the main subjects of this thesis.

Chapters I and II deal with the effects of radiation - with or without a pre-irradiation treatment - on M 1 -plant development,and on ovule sterility, embryonic lethality and frequency of chlorophyll deficient embryos in the M 1 -siliques. These investigations revealed that:- with increasing radiation doses, no further increase in chlorophyll mutant frequency is found at relatively high doses, for Arabidopsis progenies of the "main" inflorescence;- this observed "saturation" in chlorophyll mutant frequency is, at least partly, due to scoring of progenies from initial cells - forming the sub-epidermal cell layer - which have heterogeneous radiation-sensitivities;- this heterogeneity, which was indicated after some of the X-ray as well after some of the fast neutron treatments, is most likely caused by replacement of the original 'initial cells by less sensitive cells;- fast neutron-irradiation of pre-soaked (3 hours) Arabidopsis seeds is approximately seven times more effective than X-irradiation with respect to the induction of M 1 -ovule sterility and M 2 -embryonic lethality;- dithiothreitol (DTT) provides considerable protection against X- and fast neutron-irradiation, using M 1 -ovule sterility, and, to a lesser extent, the number of embryonic lethals and chlorophyll mutants as parameters. This, as compared to irradiation without DTT, leads to more mutants at a given level of M 1 -ovule sterility.To compare the genetic effects of different mutagens, a standardized mutagen dose with reference to a specific genetic effect is necessary. The applied doses of X-rays and fast neutrons as reported in chapter II - the X-ray doses were seven times the applied fast neutron doses - induced similar levels of ovule sterility and embryonic lethality in Arabidopsis. Therefore, these doses were applied in the analysis of the X-ray and fast neutron induced mutant spectra. In chapter III the results of a study on the effects of (1) radiation type, (2) radiation dose and (3) a DTT pre-irradiation treatment, on the spectra of X-ray- and fastneutron-induced morphological mutants are reported. These investigations demonstrated that;- the X-ray- and fast neutron-induced spectra of different types of viable mutants vary significantly;- throughout the studied dose range some mutant types occur significantly more frequently with fast neutrons than with X-rays, i.e. mutants with loosely packed leaves with long petioles and the eceriferum mutants;- mutants with closely packed leaves with short petioles were more frequently induced with X-rays. Other mutants, e.g. the vital chlorophyll deficient mutants, were induced with equal frequencies by X-rays and fast neutrons in the studied range of radiation doses;- certain mutant types are relatively less frequent after irradiation in the presence of DTT. Since DTT preferentially protects against radiation-induced single and double strand-breaks, this observation suggests that these mutants more often than other mutants, originate from strand-break damage. It is striking that these mutant types were more frequently induced with fast neutrons than with X-rays.

In order to compare different mutagenic treatments, the mutation frequency should be expressed per cell. For the transformation of the mutant frequency scores, expressed as the number of mutant plants among the total number of M 2 -plants (or embryos in the case of Müller's embryo test), a mutant frequency of twenty per cent in the offspring of a heterozygous parent and an equal viability of mutant and non-mutant plants have been assumed for all treatments in chapters I, II and III.In chapter IV, the segregation frequency of mutants induced by various X-ray and fast neutron doses, with and withoutDTT pre-treatment is studied.From this investigation the following conclusions can be drawn:- the average mutant segregation frequencies of X-ray-and fast neutron-induced mutants are equal;- a DTT pre-irradiation treatment does not influence the segregation frequency of radiation-induced mutants;- there is no consistent change in the mutant segregation frequency with increasing radiation dose;- progenies from single flowers in the top of the M 1 -main inflorescence originate, in general, from genetically homogeneous tissue;- the average mutant segregation frequency is 21.5 per cent. Since no difference in viability of mutant and non-mutant plants is observed, the mutant deficit is due to reduced transmission of the mutant gene through (probably the male) gametophytes;- the observations lead to the conclusion that the mutant segregation frequency in the offspring of a heterozygous parent can be estimated from the M 2 -segregation frequencies, provided that progenies from a single flower from the top of the M 1 -main inflorescence are tested. The obtained value can then be used directly for the calculation of the mutant frequency per cell.Arabidopsis mutants with a reduced or absent wax coating, i.e. eceriferum mutants, are relatively more frequently inducedwith fast neutrons than with X-rays (chapters III and V). The phenotypic analyses - by macroscopic and scanning-electronmicroscopic techniques, reported in chapter V - of the wax coating on stems and siliques of the wild type and of 30 fast neutron -, 11 X-ray-, and 10 EMS-Induced and of 1 spontaneous mutant, indicate that:- in eceriferum mutants with visually reduced wax coating, the quantity of rodlet-shaped wax extrusions is less than in thewild type;- in eceriferum mutants, with visually absent wax coating, the wax extrusions are affected in specific ways. Compared to the wild type, the rodlet-shaped crystals are absent or rare; the plate-like wax structures are absent or reduced, and often there is, in contrast to the wild type, a thick organised wax layer present on the siliques and stems.Genetic analysis of the eceriferum mutants, reported in chapter V, revealed that the mutants were conditioned by many different loci (minimum 14, maximum 44). The ultrastructural research of the eceriferum mutants with absent wax coating demonstrated that, at the level of resolution of the scanning-electron microscope, we can distinguish between differential effects of different loci. However, the number of mutants per locus was still too low to pronounce upon X-ray or fast neutron specificity for individual loci.

In chapter VI, various methods to select lines, mutated for a specific characteristic and with an otherwise undisturbed geneticbackground, are discussed. A mutation breeding programme is proposed. It is based on the determination of M l -heterozygous plants by means of single seed progeny testing. Subsequently, the desired mutant line is selected in the progeny of these M 1 - plants of which M 2 spare seed is available. As compared to M 2 -bulk testing, the mutation frequency can be increased considerably with the proposed method. Besides, the number of independent mutants is higher compared to M 1 -progeny testing (without spare seed) and M 2 -bulk testing. The mutant frequency and the number of spare seed determine to what extent.