The possibility of freeze drying of plant material and its grinding in a shaking bead mill to determine the activity of nitrate reductase (NR) was studied. The effectiveness of applying this approach to high throughput mass screening of crops was shown. The assay was carried out on seedlings of common wheat (Triticum aestivum) of the following cultivars: Altigo, Vassa, Grom, Doka, Soberbash, Starshina, Fisht and spring wheat Novosibirskaya 67. The crops were grown during 4-5 weeks on substrate without nitrogen and on substrate supplemented with 50 millimol / l (mM) of potassium nitrate. Nitrate reductase in plants retained its activity after lyophilization and grinding of dried leaves in a mill. The proposed protocol for NR activity assay is suitable for plant lysates with an NR activity suffi cient to form nitrite in the range of 5–120 micromoles / l (μM) in 800 μl of reaction mix (for instance, freeze-dried sample originated from 100 mg of wheat seedling leaves). Centrifugation of a plant lysate at 20,000 g almost did not change NR activity compared to 12,000 g that is achievable for most lab centrifuges. Lysates from fresh leaves contained signifi cantly more total protein than lysates from lyophilized leaves (with an equal amount of starting wet material). The difference in the nitrate-reducing activity in lysates from fresh and lyophilized leaves was not as high as the difference in protein concentration. Thus, the activity of NR calculated per g of total protein was higher in lyophilized leaves than in fresh leaves. The activity of NR was signifi cantly induced by nitrate for all cultivars. The basal and nitrate-induced NR activity varied widely between the cultivars, and the induction ranged from 2.5 fold for Novosibirskaya 67 variety and 2.7 fold for Vassa to 5.4 for Altigo and 5.7 fold for Grom.
The breeding improvement of triticale is tightly associated with the introgression of dwarfing genes, in particular, gibberellin (GA)-insensitive Ddw1 from rye. Despite the increase in harvest index and resistance to lodging, this gene adversely affects grain weight and size. Growth regulation factor (GRF) genes are plant-specific transcription factors that play an important role in plant growth, including GA-induced stem elongation. This study presents the results of a two-year field experiment to assess the effect of alleles of the TaGRF3-2A gene in interaction with DDW1 on economically valuable traits of spring triticale plants grown in the Non-Chernozem zone. Our results show that, depending on the allelic state, the TaGRF3-2A gene in semi-dwarf spring triticale plants influences the thousand grain weight and the grain weight of the main spike in spring triticale, which makes it possible to use it to compensate for the negative effects of the dwarfing allele Ddw1. The identified allelic variants of the TaGRF3-2A gene can be included in marker-assisted breeding for triticale to improve traits.
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