Antibiotics are widely applied for plant cultivation in vitro to eliminate bacterial contamination. However, they can have both positive and negative effects on the cells of cultivated plants, and these effects largely depend on the type antibiotic used and its concentration. The objective of the present study was to estimate the effect of β-lactam antibiotics ampicillin (Amp) and cefotaxime (Cef) on microspore embryogenesis induction in vitro in the Brassica species. The performed experiments confirmed cefotaxime inhibits microspores in B. napus and B. oleracea, even in concentrations as low as 50 mg/L. The highest embryo yield was obtained for B. napus in the NLN-13 medium with added ampicillin in concentrations of 50–100 mg/L as an antimicrobial agent. This embryo yield was significantly higher than that obtained in a medium without supplemented antibiotics and two times higher than in the medium with added cefotaxime. Analogous results were obtained for B. oleracea and B. rapa.

Red cabbage belongs to the economically important group of vegetable crops of the Brassicaceae family. A unique feature of this vegetable crop that distinguishes it from other members of the family is its unique biochemical composition characterized by high anthocyanin content, which gives it antioxidant properties. The production mainly uses F1 hybrids, which require constant parental lines, requiring 6–7 generations of inbreeding. Culture of isolated microspores in vitro is currently one of the promising methods for the accelerated production of pure lines with 100% homozygosity. The aim of this study is to investigate the factors and select optimal parameters for successful induction of red cabbage embryogenesis in isolated microspore culture in vitro and subsequent regeneration of DH plants. As a result of research, for the first time, it was possible to carry out the full cycle of obtaining DH plants of red cabbage from the induction of embryogenesis to their inclusion in the breeding process. The size of buds containing predominantly microspores at the late vacuolated stage and pollen at the early bi-cellular stage has to be selected individually for each genotype, because the embryoid yield will be determined by the interaction of these two factors. In the six samples studied, the maximum embryoid yield was obtained from buds 4.1–4.4 mm and 4.5–5.0 mm long, depending on the genotype. Cultivation of microspores was carried out on liquid NLN culture medium with 13% sucrose. The maximum number of embryoids (173.5 ± 7.5 pcs./Petri dish) was obtained on culture medium with pH 5.8 and heat shock at 32 °C for 48 h. Successful embryoid development and plant regeneration by direct germination from shoot apical meristem were achieved on MS culture medium with 2% sucrose and 0.7% agar, supplemented with 6-benzylaminopurine at a concentration of 1 mg/L. Analysis of the obtained regenerated plants, which successfully passed the stage of adaptation to ex vitro conditions by flow cytometry, showed that most of them were doubled haploids (up to 90.9%). A low number of seeds produced by self-fertilization in DH plants was observed.

Haploid technologies are used to create homozygous lines for accelerated breeding. We aimed to optimize the technology for using the isolated microspore culture in vitro to obtain doubled haploids of the carrot (Daucus carota L.). We studied two carrot varieties with different responsiveness to embryogenesis, Altajskaya lakomka and Breeding line 17. Carrot microspores were isolated from buds and cultivated in liquid nutrient media supplemented with an antibiotic and activated carbon in vitro. They were exposed to different thermal treatments. The experiment showed the benefits of combining cold pre-treatment of buds (5°C for 1 day) with heat shock of isolated microspores in vitro (32°C for 2 days). The induction of embryogenesis on the NLN-13 medium was twice as high as on the MSm-13 medium. The use of 1% activated carbon in 0.5% agarose increased the yield of embryoids by more than 1.5 times. 100 mg/dm3 of ampicillin was found to be the most efficient concentration. After 30 days of cultivation under optimized conditions, the yield was 161.3 and 44.0 embryoids per Petri dish for the cultivar Altajskaya lakomka and Breeding line 17, respectively. The induction of carrot embryogenesis is determined by the type and duration of thermal stress, the composition of the nutrient medium, the use of activated carbon as a sorbent, the addition of β-lactam antibiotics, and the type of explant exposed to thermal treatment. Our technology enabled us to obtain homozygous doubled haploid lines of carrots during a year, and these lines were included in the breeding process to create F1 hybrids.