Preservation of Features of Anatomical Polymorphism of Deschampsia antarctica É. Desv. (Poaceae) During In Vitro Clonal Reproduction

We studied the anatomical features of the leaf structure of Deschampsia antarctica É. Desv. (Poaceae) obtained from seeds collected from different habitats of the maritime Antarctic. These plants have been maintained in culture in vitro for more than 7 years. The plants include stable chromosome forms comprising diploids (2 n = 26), a diploid plant with B-chromosomes (2 n = 26 + 0–3B), a triploid with rearranged chromosome morphology, and myxoploids with different ratios of diploid and nondiploid cells in the root meristem. The D. antarctica plants that were studied generally had a similar anatomical structure in culture in vitro and in nature. At the same time, plants of different cultivated genotypes also displayed their own leaf structure features. In particular, qualitative features included asymmetric four-ribs and five-rib leaves instead of typical three-rib leaves for some genotypes, some individuals characterized by the presence of unicellular nonglandular pointed trichomes on an adaxial side, differences in vascular bundle sheath, and other features. No clear dependence of the anatomical structural features on the chromosomal status of the studied genotypes was evident. At the same time, differences were evident in traits that included the number of stomata, thickness of the outer cell walls of the epidermis, bundles sheath state, number of leaf ribs, and the persistent presence of trichomes under prolonged in vitro cloning. The findings indicate that D. antarctica plants collected from different locations of the maritime Antarctic for long-term cultivation in vitro under standard cultivation conditions retain the anatomical characteristics of the wild plants. The polymorphism of in vitro-cultivated plants is not related to the polymorphism of their karyotype, but is apparently due to the heterogeneity of the original plants and possibly the epigenetic fixation of a number of anatomical features produced in the natural microhabitats of D. antarctica populations.

INTRODUCTION OF KARELIAN BIRCH TO THE CULTURE IN VITRO

At present, the biological diversity of tree species is drying up. One of the main reasons for extinction is the destructive anthropogenic impact. According to the latest data, it became known that the Karelian birch was included in the Red Book of the Republic of Karelia as an endangered and diminishing species. The in vitro clonal micropropagation technology can help to quickly restore the population of Karelian birch. And also the technology under consideration will help to massively produce seedlings and seedlings of Karelian birch for both decorative and silvicultural purposes.

Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

Effect of sowing dates of donor plants and concentration of 2,4-D on the purity of productive anther formation of common barley (Hordeum vulgare L.) in anther culture in vitro.

Creating dihaploid lines of agricultural plants is a labour-intensive but essential step in variety production in modern plant breeding. This stage allows significantly accelerate the process of creating new varieties of common barley and other crops. Barley digaploids are produced mainly by anther culture and microspore culture. The authors preferred anther culture in vitro. In the present study, the influence of climatic factors in the cultivation of donor plants on the yield of productive anthers at different sowing dates was established. The authors also identified the more stable culti- vars with a high anther production regardless of sowing date (Signal, Laureate and Eifel). Varieties showed the highest number of embryo-like structures formation at the first and third sowing dates (Zu Suren, Zu Zaza); and sorts with a high rate of productive anther formation at the second sowing date (Acha, Exploer) were identified. Different concentrations of 2,4-D in N6 medium on the frequency of embryogenesis and yield of productive anthers were studied. As a result of this study, the authors found that different concentrations of 2,4-D (1 mg/l and two mg/l) had no significant effect on the for- mation frequency of productive anthers in all the varieties studied. When the embryogenesis capacity of the cultivars was reviewed, all the samples were found to be positive in anther culture. However, the array Zu Suren had a significantly lower effective anthers yield than the samples Signal and Acha. As a result of correlation analysis, the authors found a close relationship between the length of the ear tube of donor plants and the frequency of formation of productive anthers (r = -0.69). A close relationship with the development of optimal microspore phase for the induction of androgenesis in anthers extracted from the ear tube with an average length of 6 cm was determined. This information can significantly speed up the selection of donor plants, but it is recommended to confirm the stage of microspore development microscopically for each new cultivar used.

Androgenesis of Red Cabbage in Isolated Microspore Culture In Vitro

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.

Obtaining doubled haploids of Cucurbita pepo L.

Doubled haploids have been widely used worldwide in breeding programs and fundamental research as valuable homozygous material for about 100 years. The species Cucurbita pepo L. are represented by a huge variety of forms, include highly productive vegetable crops and have a wide distribution in the world. Despite the great economic importance, the creation of effective protocols to ensure stable production of doubled haploids in this species remains an urgent task. DH plants are of interest not only because of the acceleration of the breeding process, but also because of the realization of the huge potential of gametoclonal variability inherent in this highly polymorphic species. In this review, we analyzed the main technologies used for obtaining doubled haploids in vegetable crops of C. pepo: parthenogenesis in situ stimulated by treated/irradiated pollen, gynogenesis in vitro (unpollinated ovule culture in vitro) and androgenesis in vitro (anther/microspore culture in vitro). An analysis is presented of the research carried out from the beginning of the discovery of haploid plants to the current advances and evaluation of the prospects in the field of DH plant production. The main critical factors influencing the efficiency of each technology and its individual steps are considered. The developed technology of doubled haploids obtaining using non-pollinated ovary culture in vitro is presented. This technology allows to obtain up to 55 embryoids per one cultivated ovary (28 embryoids/ 100 cultivated ovules) To introduce haploid technologies into the breeding process it is necessary to evaluate the obtained plants for ploidy level. The use of direct counting of chromosomes in apical cells may present a certain difficulty in this species due to their large number (2n=40) and their small size. Depending on the level of laboratory equipment, ploidy determination using flow cytometry of cell nuclei and counting the number of chloroplasts in stomatal guard cells in the epidermis of the abaxial side of the leaf may be more convenient methods. The prospects for the use of molecular markers for assessment for homozygosity in DH technologies used, including C. pepo, are discussed in the review.

Embryo culture in vitro in the experimental evaluation of drought resistance in cereals (review)

Drought is an unfavorable combination of meteorological conditions when plants experience a long-term water deficiency both in the air and soil. This is one of the most common abiotic stressors, which leads not only to significant crop losses but also rises threat to food security. Researchers are actively developing ways to breed drought-tolerant cultivars of economically valuable crops, especially cereals – the main food resource. One of the promising areas of biotechnological evaluation of the resistance of existing and newly created cereal genotypes to drought for breeding purposes is the use of culture in vitro. In this case, embryos at the particular stage of development are used as explants (so-called embryo culture in vitro). The review aims to analyze the literature and own data on the production of cereal regenerants in embryo culture in vitro under selective experimental conditions of imitation of physiological drought. It has been shown that in vitro cultivation of immature embryos at a critical stage of relative autonomy is especially promising. This kind of embryo does not depend on the physiological factors of the maternal organism and can autonomously give rise to the fully developed plant under adequate conditions in vitro and later ex vitro. This allows the biotechnologist to obtain regenerants directly, excluding an additional time-consuming stage of the formation of morphogenic calli in vitro. As follows, the time required for expensive experiments is also reduced. Data on the identification of the critical stage of the relative autonomy of the cereal embryogenesis are presented. Criterion (proposed by the authors) for identifying this stage by the ability of the embryos to complete embryogenesis and form the seedlings on a hormone-free medium in vitro and give rise to the full developed regenerants ex vitro has been analyzed. Furthermore, the analysis of the laboratory germination of the obtained caryopses was carried out. It was discovered that in spring soft wheat, for example, such stage, corresponding to the formation of all organs in the embryo, occurs 15 days after pollination. The issues of using relatively autonomous embryos in the biotechnological assessment of the genotype drought resistance under selective conditions in vitro are considered.