Direct induced androgenesis in culture in vitro in sugar beet (Beta vulgaris L.)

Abstract

Purpose. To develop the method of direct induced androgenesis of sugar beet in culture in vitro . Methods. Biotechnological, cytological, breeding, statistical. Results. Specific for sugar beet components of the method of direct induced androgenesis in culture in vitro was developed, in particular, the phase of development of microspores, optimal for initiation of androgenesis, the temperature mode of explants pretreatment, conditions of anthers cultivation were determined. According to the results of cytological analysis of microspores and sugar beet pollen, it was determined that the single-nucleus stage of the vacuolized microspores is optimal for inoculation of anthers on the nutrient medium, and pre-treatment of explants using low-temperature stress (4–8 ° С) for 3–15 days is a necessary factor that initiates the transition of microspores from gametophytic to sporophytic pathway of development. The composition of nutrient media, different in content of macroelements, amino acids, vitamins and growth regulators, for the cultivation of anthers, the initiation of processes of direct androgenesis and the formation of embryos have been developed. The modified Murazig-Skoog medium – 0.5 doses of macroelements with the addition of vitamins: B1 – 10.0 mg/l; B6 – 1.0 mg/l; PP – 1.0 mg/l; C – 1.0 mg/l and amino acids: glutamine – 250.0–500.0 mg/l, asparagine – 1.0–10.0 mg/l, arginine – 2.0–10.0 mg/l, tyrosine – 1.0–10.0 mg/l, hydroxyproline – 2.0–4.0 mg/liter was used as a basis. According to the results of research, three most effective nutrient media with different content of growth regulators have been determined: polystimulin A-6 – 1.0–3.0 mg/l for the active substance + 6-BAP – 0.3–0.8 mg/liter; 2.4-D – 1.0–2.5 mg/l + 6-BAP – 0.3–0.8 mg/l + ABK – 0.3–1.0 mg/l or 6-BAP – 0.1–0.6 mg/l. Cultivation of sugar beet anthers on the developed nutrient media allowed to obtain 0.15–1.32% of various types of androgen embryos and microclones of androgenic origin. Conclusions . The method of direct induced androgenesis of sugar beet is developed: the optimal stage of development of microspores for inoculation of anthers, the mode of temperature pre-treatment of explants, the composition of nutrient media for the initiation of direct in vitro androgens and the obtaining of various types of androgenic embryoids have been determined. The results of this work are important for the creation of haploids and homozygous double-haploid lines, which will accelerate the breeding process for obtaining new varieties and hybrids of sugar beet.