Effect of kinetin and zeatin on androgenesis parameters in triticale (Xtriticosecale wittmack) anther culture in vitro

Haploid and doubled haploid techniques make it possible to obtain homozygous lines and reduce the time for selection. In triticale breeding, the main problem of the androgenesis method (anther culture and isolated microspore culture) used to obtain haploid plants, is low efficiency of green plants regeneration. In the present investigation, for the first time in the culture of isolated anthers, it was established that the positive effect of zeatin is due to the effectiveness of the induction stage of androgenesism with an increase in the number of embryogenic structures. The aim of this research was to evaluate the androgenetic potential of various triticale genotypes on an initial nutrient medium differing in the source of cytokinins (kinetin and zeatin). In this work, breeding samples of winter triticale of the selection of the Federal Agrarian Research Center of the South-East were used: No. 1 (Zimogor/winter soft wheat, L.39), No. 2 (DH-21/Kapriz), No. 3 (Vocaliz//MAG/Vodoley), No. 4 (MAG/Vodoley//TI-17) and No. 5 (Zubr/TI-17). Donor plants were grown on bogar in the field conditions (a scientific station, the Federal Agrarian Research Center of the South-East, Saratov, 2023). The cut shoots were placed in a refrigerator and kept at 4 °С for 14 days. The ears were sterilized with a commercial preparation Belizna (OOO VHA, Russia) for 8 minutes, followed by three rinsing with sterile distilled water. For the anther culture, a solid C-17 nutrient medium containing 2 mg/l 2,4-D («SERVA», Germany), 0.4 mg/l kinetin (SERVA, Germany) or 0.4 mg/l zeatin (SERVA, Germany) and 9 % sucrose (SERVA, Russia) was used as a base medium. Anthers were cultured in the dark for 5-6 weeks in the climate control unit KS-200 (ОАО Smolenskoye SKTB SPU, Russia) at 25-28 °С. Androgenetic structures larger than 2 mm in size were transferred to the MS nutrient medium for plant regeneration with 0.5 mg/l IAA, 0.2 mg/l kinetin and 3 % sucrose. The regeneration condition were a 16-hour photoperiod, lighting 10 thousand lx and a temperature of 25-28 °С. The plants were vernalized in vitro at 4 °С for 2 months, then transplanted in the nutrient soil (TERRAVITA, OOO NORD PULP, Russia) in separate cups and cultured in a vegetable box until the tillering stage. The effectiveness of androgenesis was assessed by the number of embryogenic anthers (EA/100 CA) and the number of androgenetic structures (AS/100 CA) per 100 cultivated anthers, the number of androgenetic structures per 100 embryogenic anthers (AS/100 EA), the total number of regenerants per 100 cultivated anthers and per 100 androgenetic structures (R/100 CA and R/100 AS), the number of green and albino plants per 100 cultivated anthers and per 100 androgenetic structures (GR/100 CA and AR/100 CA, GR/100 AS and AR/100 AS). The AC yield averaged 9.8 (variation 3.0-23.2) on a medium with kinetin and 15.2 (variation 4.8-31.0) on a medium with zeatin. In total, 2972 AS were formed from 21216 CA, of which 1118 were formed on a medium with kinetin and 1854 with zeatin. The androgenesis induction stage was on average 1.7 times higher for five genotypes when zeatin was used compared to kinetin. The average values of regeneration efficiency per 100 CA were 2.2 for kinetin (variation 0.5-6.7) and 3.2 (variation 0.5-8.3) for zeatin. However, the advantages of zeatin in the yield of regenerants from androgenetic structures the differentiation of which determines the success of regeneration, occurred only in two breeding samples. In three breeding samples, there was a statistically significant (p ≤ 0.05) decrease in the yield of regenerants from AC formed on a medium with zeatin. The number of green plants per 100 AC averaged 7.0 for kinetin and 4.7 for zeatin across genotypes. Thus, the improved efficiency of triticale anthers culture on the nutrient medium with zeatin in terms of androgenesis induction did not lead to an increase in the parameters of plant regeneration. The positive effect of zeatin on the production of triticale haploid plants in anther culture is due to the effectiveness of the induction stage, that is, a higher frequency of androgenetic structures formation, rather than their improved differentiation in comparison with kinetin.