Effect of tiller pre-treatments on microspore vitality and symmetric divisions
Based on microspores response, the rye lines were ranked into three categories: recalcitrant, moderate and responsive. When plotted against microspore viability, it can be seen that the moderate and responsive lines have significantly higher viability than the recalcitrant lines (Fig. 1b, c, S1). Microspores isolated from control tillers of the recalcitrant lines (lines 1, 2, 6, 9, 11, 12, 13, 15) had 3.5- and 3.9-fold lower vitality in comparison with responsive and moderate lines (lines 3, 4, 5, 7, 8, 10, 14), respectively (S1, S2).
Although microspore viability was significantly influenced by the genotype of donor plants (Table 2; Fig. 1b, c, S1, S2), tiller pre-treatments (Control, GSH, MAN, GSH/MAN, MAN/GSH) had a significant effect on the mean microspore vitality and the frequency of symmetric divisions leading to androgenic structure (AS) formation under in vitro conditions in all categories (Fig. 1, S3, S4). Glutathione had an effect on each genotype, but not to the same degree, or in the same way. Pre-treatments with glutathione alone (GSH) or in combination with mannitol (GSH/MAN and MAN/GSH) enhanced microspore viability up to the level of ca. 50% viable microspores (S2) whereas MAN and its combination with GSH (MAN/GSH) increased the frequency of dividing microspores up to 12–14% for the moderate and responsive categories (S4). GSH/MAN and MAN/GSH tillers treatment usually increased (ca. 1.20-fold) microspore survivability after the isolation procedure and during the first days of in vitro culture (Fig. 1b). Combination of GSH and MAN improved microspore vitality in comparison with MAN on its own. Too high stress intensity (MAN) had a negative effect on microspore viability (S2) but simultaneously stimulated embryogenesis initiation (S4).
In the recalcitrant lines, microspore viability and frequency of symmetric divisions was significantly influenced by the tillers treatments (Table 3). Pre-treatments with glutathione alone (GSH) or in combination with mannitol (GSH/MAN and MAN/GSH) stimulated microspore viability 1.8-, 2.3- and 2.6-fold for GSH, GSH/MAN and MAN/GSH, respectively (Fig. 1b). The order of treatments was crucial for the resistant lines. MAN/GSH was the only treatment able to increase microspore viability up to ca. 50% viable cells (S2). Moreover, mannitol alone (MAN) or in combination with glutathione was the most effective in triggering symmetrical divisions. As a result, the frequency of embryogenic divisions was 4.6–5.3-fold higher (up to the level of ca. 20%) in comparison with the control (Fig. 1b, S4).
Androgenesis induction efficiency in isolated microspore cultures
Androgenic responsiveness of microspore suspensions varied significantly among the 15 studied rye lines (S5), which were arranged by the level of susceptibility to embryo formation within three categories (responsive, moderate and recalcitrant) after 8 weeks of culture. The androgenic capacity was significantly influenced by the genotype of donor plants, tiller pre-treatment, type of medium and interactions between Line × Medium, Treatment × Medium and Line × Treatment × Medium (Table 4; Fig. 2a).
The studied rye lines presented a wide spectrum of responses regardless of medium type—the mean AS production per spike ranged from 0 to 4.6 AS per 105 mcs. The majority (11 lines) responded to the applied treatments and formed AS (eight lines under control, five under GSH, two under MAN and five under MAN/GSH; S5). The most effective among the tested treatments was MAN/GSH, where mean AS formation was almost twofold or 15-fold higher in comparison with control and MAN, respectively (Fig. 2c). 190-2 induction medium was significantly better than W-14 and yielded 2.5-fold higher effectiveness of AS formation (Fig. 2d). Embryogenesis efficiency on the tested media was genotype-specific and related to different responses to the type of medium—e.g. for lines 14 and 8, the number of developed embryos increased 2.78-fold or 43.90-fold on 190-2 and W-14 medium, respectively (S5). Although such an impressive effect was observed for W-14 medium, the highest number of embryos was obtained on 190-2 medium (16.38 AS per 105 mcs per spike; S5). Furthermore, on the 190-2 medium, under control, GSH and MAN/GSH pre-treatments, all genotype categories produced AS (with the highest effectiveness obtained at 26 °C for the responsive lines; S6). Therefore, from the applied temperature regimes, culture incubation at 26 °C was chosen as optimal (Fig. 2e).
Microscopic observations (conducted on 0, 7, 14, 28 and 42d of in vitro culture) of microspore suspensions obtained from control spikes revealed that AS (at least to the stage of multicellular structures released from the exine) were derived only for responsive and moderately susceptible genotypes. However, properly developed AS around 5 mm in diameter were obtained only in the case of responsive lines. For moderately responsive genotypes, AS development was inhibited already after 4 weeks of culture at the stage of few-celled structure (Fig. 2a).
All applied combinations of spike treatments improved the process of androgenic embryo formation. Glutathione alone (GSH) or in combination with mannitol (GSH/MAN, MAN/GSH) was the most effective. Globular AS of responsive lines were formed after 28 days of culture in all variants of tiller pre-treatments. However, well-developed AS (bigger than 5 mm in diameter) were obtained only after three treatments: control, GSH and MAN/GSH. MAN in combination with GSH (MAN/GSH) accelerated embryo formation as multicellular globular structures were formed after just 14 days of in vitro culture in comparison with control, GSH, MAN and GSH/MAN (Fig. 2a).
Interestingly, in the case of recalcitrant lines, combined MAN and GSH treatment (in both variants) was the only one where AS were formed. The positive effect of GSH/MAN and MAN/GSH on globular embryo formation was observable after four weeks of culture. The effect persisted till the 42nd day of culture, but only when MAN was applied for the first 14 days of the treatment. In these cultures, good-quality AS at the coleoptilar stage, able to germinate, were formed (Fig. 2a). However, green haploid/DHs regeneration was not observed.
Androgenesis efficiency in anther cultures
Androgenic responsiveness of the studied rye lines estimated by anther culture method varied significantly (at P ≤ 0.05) in respect of androgenic structure induction (AS per spike), total plant regeneration (RTotal per spike and RTotal 100 AS−1) and green plant regeneration ability (RGreen per spike and RGreen 100 AS−1).
The capacity for androgenesis induction (AS per spike) among the 15 studied rye lines was influenced by the genotype of donor plants, type of medium and interactions between Line × Treatment (Table 5; Fig. 3a–c, S7).
The positive effect of exogenously applied GSH on its effectiveness was observed only at the induction phase and depended on the genotype (Fig. 3b, S7, S8) and on the type of induction medium—e.g. for line 8, mean AS per spike increased 2.08-fold on W-14 medium when tillers were GSH-treated (S8).
The final androgenesis induction effectiveness averaged for the 15 lines of rye presented a wide spectrum of responses—with the mean number of AS per spike ranging from 0 to 21.3 (Fig. 3c) and the mean total regeneration ability from 4 to 30 RTotal per spike. Although the type of tiller pre-treatment and the induction medium did not significantly influence the regeneration, interactions between Line × Medium, Line × Treatment and Line × Medium × Treatment significantly influenced four regeneration parameters: RTotal per spike, RTotal 100 AS−1, RGreen per spike and RGreen 100 AS−1 (Table 5).
The average androgenesis induction efficiency for the most responsive lines (7, 8, 12, 14) was almost threefold higher in comparison to the moderately responsive lines (1, 4, 10, 11) and 14-fold higher in comparison to the recalcitrant ones (2, 3, 5, 6, 9, 13, 15). The mean parameter AS per spike for those groups was 11, 3 and 0.7, respectively. For all three groups, the most important was the effect of the interaction between Line × Treatment.
Plant regeneration was achieved for four rye lines (4, 11, 12, 14) with the effectiveness ranging from 0.3 to 3.2 RGreen per spike and from 0.3 to 6.0 RTotal per spike (S7). In total, 12 green plants were obtained, with the highest number of regenerants obtained for line 11 (Fig. 3d; S7). In the case of this line, MAN was the only treatment which resulted in 5.60 RGreen per spike regenerated from AS induced on 190-2 medium (S7). The majority (55%) of regenerants of line 11 (three plants), line 12 (one plant) and line 14 (three plants) had diploid (2n) number of chromosomes, which characterizes double haploids (DHs). The remaining 45% of green regenerants represented haploids (three plants obtained from lines no. 11, 12 and 14) and tetraploids (18%, two plants obtained from line 11). Exemplary cytograms and phenotypes of regenerants are shown in Fig. 3e.
Androgenesis efficiency: anther cultures vs microspore cultures
Androgenic responsiveness varied significantly among the 15 studied rye lines (S5–S8) in both types of cultures: anther cultures (AC) and microspore suspensions (MC). The genotype of donor plants, type of medium and interactions between Line × Treatment × Medium significantly influenced the androgenic capacity in AC and MC.
In general, higher mean effectiveness of androgenesis induction was obtained in anther cultures in all categories of genotypes with different responsiveness to that process. Low temperature (control) and GSH were the most effective for both AC and MC (Table 6). The observed tendency to increase the final effectiveness in MC of recalcitrant lines (2.4-fold on W-14 medium; S7) resulted from GSH effect on microspores viability (ca. twofold; S7), whereas MAN seemed to stimulate regeneration ability (RTotal per spike and RGreen per spike) in recalcitrant lines (S7, S8).