In vitro mutagenesis induction
Significant differences were detected among treatments, for all the morphological characters evaluated in the shoots, except for the diameter of the stem. The highest values for number and length of shoots were obtained with the dose of 0.5-Gy, while with the 1-Gy dose there was a slight increase in the number of shoots, although these were significantly smaller, than those from the control treatment. A higher number of leaves was observed in the shoots irradiated with 3-Gy (Table 1).
On the other hand, the dose of 9-Gy, had a negative effect, both in the number of shoots, as in its length and number of leaves. Shoots subjected to 11-Gy also had significantly smaller leaves than those of the control, while shoots irradiated with 13-Gy showed no significant difference with the control. The 15-Gy dose produced fewer shoots, which made them larger than those of the control. Although no significant differences were observed in the number of shoots, derived from the 19-Gy dose, it was found that they were significantly smaller and had fewer leaves, and therefore their development was slower (Fig. 1).
It was interesting to note, that most of the doses used, did not affect the survival of the irradiated shoots. However, survival of shots subjected to 5-Gy was 50%, unlike those subjected to 7-Gy and 19-Gy which only had a mortality of 25%; therefore, the median lethal dose (50%LD) could not be adequately defined in this study. On the other hand, the shoots submitted to the doses of 9, 15, 17 and 19Gy, lost their capacity of multiplication, for what, it was not possible to achieve the generation of new shoots, from their nodal segments.
In vitroselection for water deficit
Morphological response of vitroplants submited to PEG
The irradiated shoots were rooted and cultured in vitro, in 10 and 15% PEG. In all of them, it was observed a decrease in the morphological variables evaluated, in comparison with what was observed in the treatment without PEG. As the PEG concentration increased, the differences with the control treatment (without PEG), were more evident. A better morphological response to drought, caused by PEG (Table 2), was observed in vitroplants generated from irradiated shoots.
It was found, that vitroplants from irradiated 3 and 13-Gy shoots, grown on non-PEG media, had the highest fresh weight values, compared to those from non-irradiated or 7-Gy irradiated shoots. Vitroplants derived from irradiated 3-Gy shoots, were characterized by increased stem diameter thickness, however, no difference was observed with those derived from non-irradiated and 7-Gy irradiated shoots. However, no differences were observed among those from non-irradiated and 7-Gy irradiated shoots. In the same way, the vitroplants of shoots submitted to 13-Gy, showed longer roots than those of the control treatment.
The vitroplants from shoots irradiated at 3-Gy and grown at 10% PEG, showed a larger stem diameter, compared to those from 7 and 13-Gy, but without differences with those from the control treatment. These also had the highest fresh weight, compared to those derived from 7-Gy, but without differences with those from the control and 13-Gy treatments. The vitroplants from 13-Gy had the highest number of leaves, differing significantly from those from the control and 3-Gy treatments. The root length of the vitroplants derived from 13-Gy was significantly greater than those derived from 7-Gy, but without differences from those derived from the non-irradiated and 3-Gy treatments.
It was interesting to observe, the existence of significant differences in 7 of the 9 morphological variables evaluated, in the cultivated vitroplants in 15% of PEG. The vitroplants, from three radiation treatments evaluated, were characterized by having longer leaves, than those derived from the control treatment. The 3-Gy vitroplants showed a fresh weight and a stem diameter significantly larger, than those of the control, while those coming from 13 and 3-Gy showed wider leaves and longer roots, compared to the control treatment. The 13-Gy vitroplants had a greater number of leaves, which were thicker, than those observed in the control treatment (Table 2).
Biochemical response of vitroplants submited to PEG
No correlation was observed between proline concentration and the concentration of PEG added to the culture medium (Figs. 2a, 2b and 2c). However, significant differences were detected among vitroplants with different radiation doses. In 0% of PEG 13-Gy vitroplants had a lower proline concentration (0.45 ± 0.19) (H (3, N = 8) = 4,631, p = 0.0022). Not significant differences were found in vitroplants grown at 10% PEG (H (3, N = 8) = 1.89, p = 0.59). In 15% PEG the control treatment (0-Gy) had the highest concentration of proline (3.57 ± 0.18) in comparison with 7 and 13 Gy (0.99 ± 0.21 and 1.2 ± 0.13 respectively) but without significant differences with 3-Gy (2. 5 ± 0.43), (H (3, N = 8) = 20.08479, p = 0.0002.
A correlation was found in the accumulation of GB osmolyte. GB increased with increasing PEG concentration (Fig.2). The treatment whit 0% of PEG had a low concentration of GB however 3 and 13Gy showed a higher concentration of GB (0.58 ± 0.03, and 0.62 ± 0.08 μmol of betaine glycine / dry weight, respectively) differing of 7Gy (0.38 ± 0.01 and 0.39 ± 0.05 μmol of betaine glycine / dry weight) (H (3, N = 8) = 23.37, p = 0.000).
The control vitroplants (0-Gy) growed in 10% PEG, had a GB concentration of 4.06 ± 1.78 μmol betaine glycine / dry weight, which differed from the content shown by the 7 and 13-Gy vitroplants (2, 67 ± 0.21 and 2.52 ± 1.28 μmol betaine glycine / dry weight respectively); no difference was detected, however, with those of 3-Gy (3.09 ± 0.46 μmol betaine glycine / dry weight), (H (3, N = 8) = 9.96, p = 0.012).
The treatment with 15 % of PEG showed that 13-Gy vitroplants had the highest concentrations of GB (21.46 ± 4.55 μmol glycine betaine / dry weight), differing significantly from other treatments (H (3, N = 8) = 23.21, p = 0.000). 3-Gy vitroplants accumulate 12.32 ± 3.65 μmol glycine betaine / dry weight; 0-Gy vitroplants showed a GB content of 9. 21 ± 2.27 μmol of glycine betaine / dry weight, and 7Gy vitroplants had the lowest amount of GB (5.93 ± 0.20 μmol of glycine betaine / dry weight) These results show, that 13-Gy vitroplants produced a 133% increase in GB concentration, in relation to what was detected in the control vitroplants (Fig 2).