DH lines of winter triticale (× Triticosecale Wittm.) used in the study originated from a cross between the German breeding line ‘Saka3006’ (SaKa Pflanzenzucht GbR, Germany) and the Polish cultivar ‘Modus’ (Plant Breeding Strzelce Ltd.). The ‘Saka3006’ × ‘Modus’ mapping population composed of 90 DH lines was produced at Hohenheim University (Stuttgart, Germany) and kindly provided by Dr Eva Bauer. Seven DH lines studied in the first experiment were recognized earlier with the use of anther culture method as highly differentiated in respect of their embryogenic potential (Żur et al. 2014a, b, 2015; Krzewska et al. 2015). Five of them (DH18, DH19, DH28, DH47 and DH119) were used in a more detailed study as the object of biochemical analyses.
Plant growth conditions
Germinating kernels were placed in perlite pre-soaked with Hoagland solution (HS) and vernalized for 7 weeks at 4 °C and 8 h/16 h (day/night) photoperiod. Vernalized seedlings were planted into pots containing a mixture of soil, de-acidified substrate peat and sand (2/2/1; v/v/v), and grown in a greenhouse at 25 °C and 16 h/8 h photoperiod.
Pre-treatment of tillers used for microspore embryogenesis induction
Tillers from donor plants were collected when the majority of microspores were at mid- to late uni-nucleate stage of development, placed in HS medium and stored for 3 weeks at 4 °C in the dark. Simultaneously with low temperature pre-treatment, a part of tillers was also treated with 0.3 mM reduced glutathione (GSH) supplemented to HS medium. The concentration of GSH used in the study was chosen based on the results of a preliminary experiment in which concentrations in the range between 0.3 and 1 mM were tested (data not shown).
In the first experiment, cut tillers were exposed to: (1) low temperature (4 °C) for 21 days (LT), (2) LT and GSH, with GSH supplemented to HS medium at the start of LT pre-treatment (LT + GSH) or (3) LT and GSH, with GSH supplemented to HS medium on the last 3–8 days before microspore isolation (LT + 3–8d GSH).
In the second experiment, the above-mentioned tiller treatments with LT and LT + GSH were again applied to five DH lines of triticale. Additionally, the combination of LT and modified, short GSH pre-treatment, supplemented to HS medium precisely on the last 4 days before microspore isolation (LT + 4dGSH) was used for two DH lines of triticale (DH19, DH47).
The scheme of the experiment is presented in Online Resource 1.
Microspore isolation and culture
The spikes were sprayed with 96% ethanol, surface sterilized in a 20% solution of commercial bleach (‘Domestos’) for 15 min and rinsed 4–5 times with sterile deionised water. Then, the spikes were cut into ca. 2 cm segments and blended in 0.3 M mannitol with the use of Waring blender (Fisher Scientific Inc.). The resulting slurry was filtered through a 74 µm metal sieve (200 mesh; CD-1, Sigma-Aldrich) and pelleted (100×g, 5 min). After removing the supernatant, the microspores were resuspended in 0.3 M mannitol and gently layered onto a 21% maltose solution for density gradient centrifugation (80×g, 5 min). Viable microspores settled at the interface between mannitol and maltose were collected, washed in 0.3 M mannitol and pelleted again (100×g, 5 min). Then, the supernatant was removed and the pelleted microspores were resuspended in 1 ml modified 190-2 medium (Zhuang and Xu 1983, modified according to; Pauk et al. 2000). The total number of collected microspores was estimated with a Neubauer counting chamber and then the suspension was sampled for in vitro culture and biochemical analyses. Microspore suspension with the final density of approximately 70 × 103 microspores per ml (mcs/ml) was transferred to 15 × 60 mm petri dishes and co-cultured with immature ovaries (10 per 1 ml of suspension) which have been dissected simultaneously with microspore isolation. The cultures were incubated in the dark at 26 °C.
Starting from the sixth week of culture, embryo-like structures (ELS) of 1 mm in size were transferred onto 0.6% agar solidified regeneration medium 190-2 (Zhuang and Xu 1983) supplemented with 0.5 mg/l kinetin, 0.5 mg/l NAA and 30 g/l sucrose, pH 6.0. The cultures were kept at 26 °C, with 16 h/8 h (day/night) photoperiod in a dim light [80–100 µmol (hν) m−2 s−1 (PAR)].
The measurements of isolated microspore yield, viability, and the frequency of embryogenesis initiation as well as the visualization of further stages of embryogenic development
Usually 5–6 spikes were used for one isolation procedure. The number of isolated microspores was estimated with the use of a Neubauer counting chamber. The number of isolated microspores received per one spike of a donor plant was considered as ‘isolated microspore yield’ (Y).
Microspore viability (V) was determined on the isolation day by fluorochromatic reaction to fluorescein diacetate (FDA; 0.01%; λEx = 465 nm, λEm = 515 nm, green fluorescence; Heslop-Harrison and Heslop-Harrison 1970). The samples were examined under Nikon Eclipse-E600 equipped with a differential interference contrast (DIC) system. Images were collected with Nikon DS-Ri1 digital camera and processed with NIS-Elements AR 3.0 Imaging Analysis, Microsoft PowerPoint and Corel PhotoPaint 10.0.
The frequency of embryogenic microspores was assessed on the isolation day with an inverted light microscope (NIKON TS-100/100F) by calculating the percentage of star-like structures (SLS) and microspores undergoing the first symmetrical division, as typical hallmarks of microspore embryogenesis initiation (Touraev et al. 1997a; Indrianto et al. 2001; Dubas et al. 2010). Next phases of embryogenic development were examined microscopically in petri dishes containing microspore suspensions after 7, 14 and 21 days of in vitro culture.
Total antioxidant activity estimated by DPPH method
Isolated microspores (samples of ca. 70 mg fresh weight) were immediately frozen in liquid nitrogen and then stored until the analyses in a deep freezer at − 60 °C. The samples were freeze dried and ground with ball mill MM400 (Retsch, Haan, Germany) in Eppendorf vials, to which 1 ml of 50% ethanol was then added, and shaken for two hours at room temperature. The extracts were centrifuged for 20 min in a centrifuge at 18,000×g (MPW-350R, Warsaw, Poland) and the supernatant was used for the measurements. Total antioxidant activity (free radical-scavenging activity) in the tissues was measured by DPPH method according to Brand-Williams et al. (1995) with some modifications adapting the protocol to 96-well microtitre plates and to the measurement of absorbance with a microtitre plate reader (Płażek et al. 2011). A solution of 0.5 mM of stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, SIGMA) in methanol was used. Absorbance was determined after 30 min of the reaction at 37 °C at 515 nm using reader Model 680 (Bio-Rad Laboratories, Hercules, CA, USA). The results are expressed as µmoles of Trolox (SIGMA) equivalents. For each pre-treatment, at least three independent measurements were made.
Measurement of reduced (GSH) and oxidized (GSSG) glutathione
The collected microspores were homogenized in ice-cold 6% meta-phosphoric acid and centrifuged for 20 min at 12,000×g, at 4 °C.
Total glutathione was determined by the method of Knörzer et al. (1996). Samples (100 µl supernatant) were neutralized with 25 µl 50 mM potassium phosphate buffer (pH 7.5) and 25 µl triethanolamine. The assay mix contained 20 µl neutralized plant extract and up to 900 µl potassium phosphate buffer (pH 7.5) containing 1 mM dithio-bis-nitrobenzoic acid, 0.2 mM NADPH, and 1 unit glutathione reductase (GR). The absorbance was recorded at 412 nm for 3 min The activity of GR enzyme is proportional with glutathione concentrations. Control reaction was carried out without plant extract. Glutathione concentration was calculated from the calibration curve prepared with GSH solutions of known concentrations.
Oxidized glutathione (GSSG) was determined by the same method (Knörzer et al. 1996) with the exception that reduced glutathione (GSH) was blocked with 2-vinylpyridine in the supernatant. 8 µl of 2-vinylpyridine were added to the neutralized extract, mixed and incubated for 1 h at room temperature. Then, 50 µl of neutralized supernatant was added to the assay mixture. Reduced GSH was calculated from the difference between total and GSSG concentrations. For each pre-treatment, at least three independent measurements were made.
The biochemical analyses were conducted on microspores isolated from (1) LT pre-treated tillers after 21 days at 4 °C in HS and (2) LT + GSH pre-treated tillers after 21 days at 4 °C in HS supplemented with 0.3 mM GSH. For two DH lines (DH19 and DH47), the effect of 4-day GSH pre-treatment (LT + 4dGSH) was also analysed. Additionally, freshly cut, non-treated tillers (NT) were used as control.
All data after testing for normal distribution were examined by two-way analysis of variance (ANOVA), after which post hoc comparison was conducted using Duncan’s multiple range test (p ≤ 0.05). Nonparametric Spearman’s rank-order correlation coefficients (R) were used to visualize interrelationships among the studied traits. All statistical analyses were performed using STATISTICA version 12 (Stat Soft Inc.) package.