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/L 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.

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA₃) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9).

Relevance One of the basic directions of the cabbage crop breeding is the creation of F₁ hybrids with a complex of economically valuable traits. This process is difficult and time-consuming as to get pure lines must be within 6-12 years hold inbreeding. Herewith not every line gives the desired heterotic effect that also requires additional verification. Methods Biotechnological method culture of isolated microspores in vitro, which allows in the first generation to receive a line with 100% homozygosity, was used to speed up the breeding process. Combination ability were performed in complete diallel cross on the basic morphological signs. Results Culture medium for cultivation of isolated microspores in vitro was optimized for each genotype of cabbage for the best embryoids regeneration. Maximum amount of embryoids was received on medium with pH 6.2 using ampicillin 100 mg/l and zeatin 1 mg/l: 466.7 ± 153.2 PCs/100 buds. A new source material for breeding – doubled haploid lines of cabbage was received. Lines – the best parents for F₁ hybrids with high yield, compact rosette of leaves, with optimum inside and short outside cabbage stump was created. Studies have shown that optimization of breeding process in case of creation of pure lines of cabbage in 3 years with microspore culture requires to reduce the breeding process in 2 times.

RelevanceBiotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular.MethodsAccessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species.ResultsAs a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter.