A study was made of changes in fast neutron effectiveness during the hydration and germination of tomato seeds. The main findings and conclusions are the following,Section 3.6
Samples of unirradiated seeds and their constituent parts (seedcoat+endosperm and embryo) were taken at short intervals up to 72 hours after sowing, and samples of roots, hypocotyledons and cotyledons from 72 to 144 hours after sowing. Their water content, dry weight and elementary composition (C, H, N, O and Ash) were determined.
At 27°C, the water content reaches a 'plateau' after ca 20 hours. Further uptake depends upon the rupture of the seedcoat as a result of internal forces developed by metabolic processes. This post-germination water uptake is exponential until the 'saturation' phase, but it commences much earlier in the root than in the hypocotyledon and only much later in the cotyledons. This sequence corresponds with the time of liberation from the seedcoat.
Appreciable changes in the dry weight of the embryo and the endosperm commence ca 8 hours after 50% germination. These comprise the resorption by the embryo of 75% of the dry matter of the endosperm (completed ca 60 hours after 50% germination) and de novo
synthesis. At 104 hours after 50% germination, the former process has contributed 2/3 and the latter 1/3 to the total dry weight increase of the seedling. The increase in dry matter occurs first in the cotyledons (resorption of endosperm) and commences only ca 24 hours later in the other parts of the embryo. Approximately 56 hours after 50% germination, the dry weight of the cotyledons reaches a maximum after which it decreases; at this time the dry weight of the other organs increases rapidly.
The elementary composition of the dry matter is static until germination (apart from an early increase in N, by KNO 3
uptake from the medium). Subsequently, the C and H contents decrease markedly in all parts of the young seedling, while the N, O and Ash contents increase. These changes commence very soon after germination in the roots and ca 20 hours later in the hypocotyledons. Changes in the cotyledons evidently commence at the beginning of endosperm resorption, but become considerable only at 56 hours or more after 50% germination.Section 3.7
The rad doses relative to those in water (D H2O
) were 0.841 in dry embryos, 0.886 (105% relative to dry embryos) 24. hours after sowing, 0.916 (109%) 48 hours after sowing, i.e. 8 hours after 50% germination, and 0-977 (116%) 104 hours after 50% germination. The amounts of energy absorbed per embryo per krad D H2O
increased from 118 erg per dry embryo (100%) to 172 erg (146%) at 24 hours after sowing and 250 erg (212%) shortly after germination; until germination, the percentage values also represent the relative amounts of energy per 'average' embryo cell.Section 4.3
Four irradiation experiments were performed. The shapes of the dose/response relationships were studied for various M 1
and M 2
Germination delay increases less than proportionally to the dose. The induced delays are probably less with 24 hours prehydration than with irradiation of dry seeds. These and other facts, which are discussed, indicate that non-genetic disturbances are mainly involved, including possible damage to intracellular membranes. Stimulation of germination was noted in one experiment only; possible reasons for the inconsistency of this phenomenon are given. Irradiation increases the rate of seed ageing; this is attributed to complex genetic-cytoplasmio interactions which are discussed.
Under optimal conditions of culturing, the dose/response relationships for all growth characters are slightly S-shaped, without evidence of a true threshold; the relationships for some characters are almost linear over most of the dose range. Approximately straight regressions are usually obtained on normal probability paper. An apparent discrepancy in the dose/response curves relating to the growth of those organs already differentiated in the embryo was resolved by showing that these curves result from at least two growth components, differing in radiation sensitivity. One of these depends solely upon the elongation of existing cells. The 'residual length' attained by these organs at a lethal dose should be subtracted from the lengths actually measured, in order to obtain net growth. Failure to do this may lead to large errors in the estimation of radiation sensitivity; in this respect, the methods employed in the IAEA-sponsored international programme of biological monitoring of neutron sources with barley seeds should be reconsidered.
Sigmoidal dose/response curves with a threshold are obtained for all quantal characters and are amenable to probit analysis. Similar curves are obtained for quantitative discrete characters, but the underlying distributions are different and probit analysis is not appropriate.
Sub-optimal culturing conditions may cause considerable exaggeration of curvature and/or increased thresholds in the dose/response relationships. Such circumstances also lead to higher radiation tolerance estimates. In extreme cases, and for certain characters. increases over the control may occur and these should not be confused with 'stimulation' effects. Such increases were never observed under optimal conditions of nutrient supply and spacing.
The characters leaf number below the lot inflorescence and days to flowering show linear or upwardly curved Increases with dose. In so far as flowering delays are not fully accounted for by the increases in leaf number (at the higher doses), they can be attributed to delays at the earliest stages of seedling development rather than to an increased plastochron at later stages.
The dose/response relationships pertaining to both weight (or number) of seeds/M 1
fruit and the various categories of aberrant M 2
individuals (non-germinating seeds. sublethals and mutant seedlings) are apparently linear, except for a tapering off at sublethal doses in the case of weight of seed/M 1
fruit. These results are discussed.Section 4.4
Radiation sensitivity estimates were obtained by means of ED 50
's or regressions on dose. The ED 50
's for different characters show a close connection to the degree of curvature and/or the presence and width of a shoulder in their dose/response relationships. Cumulative root- and cotyledon growth are more affected by radiation than are cumulative growth of either the lot or 2nd leaf. all recorded after 25 clays. Whilst such data are of descriptive interest# they do not permit conclusions on relative sensitivities at the meristem level, let alone at the cellular level. Even extrapolation to other stages of growth is hazardous, because it is shown that the effect of a given dose may depend greatly upon the growth stage considered.
With dry seed irradiation. the effects of fast neutrons (in % per krad) on seeds/M 1
fruit are much more severe than on vegetative growth and development, and are moreover ca 3 times the % increases in 'the total of all aberrant M 2
categories. Among the latter, the increases in the % non-germinating seeds with dose are generally higher than those of the % visibly mutated seedlings.
The relative proportions of the various effects changed considerably with prehydration; the changes were examined by means of Dose Reduction Factors (see 4.6 below).Section 4.5
The radiation responses of 2 cultivars, 'Moneymaker' (101) and 'Glorie' (GL) were compared. Seeds of GL were ca 1.14 times as sensitive as those of ML The 100-seed weight ratio GL/MM was 1.00/1-14 and the 100-embryo weight ratio 1,00/1-15 (difference highly significant). The size of the shoot apex probably differed to the same extent and the difference in sensitivity is attributed mainly to this factor. The percentage root meristem cells in G 1
was virtually the same in both cultivars. The average amount of DNA per nucleus was 5% lower in GL than in MM (difference insignificant). seed prehydration caused in GL, compared to MM, a more pronounced sensitization with regard to both M 1
seed set reduction and the production of non-germinating M 2
seeds, and a lesser sensitization in respect of the induction of recessive 'visible' mutations.Section 4.6
The Dose Reduction Factors (DRF) associated with the various stages of prehydration/germination were calculated for each character. The DRF's relating to the seedling characters increase much more rapidly with increasing prehydration time than those for late somatic characters and M 1
fertility. Furthermore, in the M 2
, the proportions of non-germinating seeds, sublethals and mutant seedlings are shifted in the direction of non-germinating seeds.
Only a small proportion of the increases in neutron effectiveness (DRF-1.0) can be explained by increased rad doses. The amount of energy absorbed per prehydrated embryo relative to that per dry embryo, which can be considered fairly representative of the relative energy absorption per (meristem) cell prior to germination, accounted for only 13-30% of the increase in neutron effectiveness as judged by M 1
fertility and the total of all aberrant M 2
categories. Consequently, most of the effectiveness enhancement with prehydration, and also the 'spectral' shift among the aberrant M 2
categories, must be ascribed to other factors: (i) intracellular differences in water uptake and swelling (possibly more rapid hydration of the nucleus), (ii) increases in the mobility of intracellular structures and of molecules, ions and radicals, and corresponding increases in metabolic activity, (iii) progression through the successive stages of the cell cycle, and increases in cell number and (iv) resorption of endosperm sub stances. These various factors are discussed.
The more pronounced prehydration sensitization with regard to the early, compared to the late M 1
characters is attributed to (i) the structure of the embryonic shoot apex., which allows an increasing amount of replacement of damaged cells towards the centre of the me ristematic region, (ii) possibly a more rapid sensitization, during germination, of the peripheral cells of the shoot apex than of the more centrally located cells, owing to the more rapid onset and high er rates of division of the former, and (iii) a decreasing impact of damage to the early organs as development proceeds.
Differences in the degree of sensitization between experiments are tentatively ascribed to several endogenous and external variables, including dose rate. It is concluded that a good reproducibility is attainable if all conditions are standardized,Section 4.7
An analysis of simple within-treatment correlation between various quantitative somatic characters has shown that r-values tend to decrease
(1) with advancing stage of development at the time of recording
(2) with increasing genetic and non-genetic radiation damage, especial when the characters studied concern organs derived from different initials
(3) with increasing interval between the dates of recording
(4) with increasing time lapse after the moment when one of the cha racters has reached its ultimate value at the date of recording, assuming that the value of the other character is still increas ing
(5) at early stages of growth, especially in the case of organs of which the initial growth is determined by cell elongation inde pendently of cell division, or of which the moment of growth ini tiation is very variable
(6) under sub-optimal conditions of culturing
(7) by a change in culturing conditions, especially
if this affects one character more than the other.
Radiation may increase the degree of within-treatment correlation if the effects of radiation on one character exert a direct influence on the second character.
Both the somatic M 1
characters and the weight (or number) of seeds/M 1
fruit are virtually uncorrelated with the presence of recessive mutations detectable in M 2
With a view to interpreting differences in radiation sensitivity it is necessary to devote increasing attention to the histological constitution of the shoot apices of irradiated objects, and to cellular and intracellular parameters, rather than aiming at more precise estimates of the atomic composition at the tissue level. Whereas all sensitivity differences may ultimately be explained in terms of biophysics and microdosimetry, it is unlikely that major advances in this field will come from studies on multicellular organisms.Section 5.2
Cumulative root- and cotyledon growth are suitable characters for an early evaluation of the mean effectiveness of neutron treatments and allow the repetition of mutagenic treatments, though only with a given material and under comparable circumstances. Under optimal and standardized conditions, tomato seeds could probably be used, in addition to barley seeds, for the biological monitoring of neutron irradiation facilities.
The virtual absence of within-treatment correlation between M 1
and M 2
characters allows the elimination of all badly growing and partially sterile M 1 plants without a noticeable reduction in the frequency of recessive mutations. This is of value in mutation breeding by means of micro-mutations. However, when chromosomal rearrangements are de sired, the reverse procedure, i.e. the selection of partially sterile individuals, is indicated.Section 5.3
A good reproducibility of neutron effectiveness estimates is possible with regard to the seedling characters, M 1
fertility, and the sum of all aberrant M 2
categories (non-germinating seeds,, sublethals and mutant seedlings) but probably not with regard to these M 2
categories analysed separately.Section 5.4
Dry seeds are the most efficient for the induction of recessive mutations. Germinating seeds are probably the most efficient for the production of drastic chromosomal rearrangements.