Факторы, влияющие на получение DH-растений в культуре микроспор in vitro редиса европейского

In this protocol for obtaining doubled haploids plants (DH), we propose a new method for microspore isolation. This method is useful for genotypes of the Brassicaceae family with low responsiveness to DH technology. For such crops, it allows increasing the embryo yield several times and sometimes obtaining embryos for the first time. This method of microspore isolation reduces the mechanical impact on the bud tissue, which minimizes somatic cell destruction and reduces to get it into the preparation through the filter, thus increasing its purity. The new isolation method also increases the relative concentration of embryogenic microspores in the preparation. This is possible because the anther tissues are not destroyed during the isolation process. Therefore, the anther retains its structure and microspores of early and late stages are trapped by the anther tissue, thus the anther acts as a sieve. Late stages are trapped because of their larger size, while early stages are trapped because they are even more tightly bound to the anther tissue. Together, these factors increase the efficiency of the technology for DH production in vitro microspore culture. This protocol article provides a detailed experimental protocol to the method presented in the experimental article (E.V. Kozar, E.G. Kozar, E.A. Domblides. Effect of the Method of Microspore Isolation on the Efficiency of Isolated Microspore Culture In Vitro for Brassicaceae Family. Horticulturae. 2022. Vol. 8, No. 10. P. 864. DOI 10.3390/horticulturae8100864) but does not repeat all the results documenting the efficacy of the actual method.

Isolated microspore culture in vitro (IMC) is an advanced technique for producing doubled haploids. We developed a modified microspore isolation method for the Brassicaceae family, which exceeds the results obtained by a standard microspore isolation method. We found that the new method allows an increase in the percentage of microspores at the embryogenic stage of development in the culture. In the spring rapeseed ‘Ratnik’ culture the percentage of microspores increases from 66.7% to 73%, and in the European radish ‘RBK’ from 34% to 61.9%. Moreover, the new method of microspore isolation made it possible to expand the range of linear bud sizes (from 3.5–4.0 to 3.0–4.5 mm for spring rapeseed ‘Ratnik’) suitable for IMC technology. In addition, the new method of microspore isolation reduced the debris in the preparation of spring rapeseed ‘Ratnik’ and European radish ‘RBK’ by 2.4 and 15 times, respectively. The best results were shown on Sareptian mustard No. 72, where the yield of embryoids increased by 7.5 times. Remarkably, the new method of microspore isolation allowed us to obtain the first embryoids of red cabbage No. 428, whereas no embryoids were obtained using the standard method of microspore isolation. In summary, the new method of microspore isolation allows an increase in the efficiency of IMC technology for Brassicaceae family crops.

The European radish is one of the most unresponsive crops in the Brassicaceae family to embryogenesis in in vitro microspore culture. The aim of this work was to study the process of embryogenesis of European radish and its biological features. In this study, the embryogenesis of European radish is described in detail with illustrative data for the first time. For the first time for the entire family Brassicaceae, the following were found: microspores with intact exines with ordered-like divisions; microspores completely free of exines; and a new scheme of suspensors attachment to the apical parts of embryoids. The morphology of double and triple twin embryoids was described, and new patterns of their attachment to each other were discovered. Uneven maturation of European radish embryoids at all stages of embryogenesis was noted. The period of embryoid maturation to the globular stage of development corresponded, in terms of time, to the culture of B. napus, and into the cotyledonary stage of development, maturation was faster and amounted to 17–23 days. The rate of embryoid development with and without suspensors was the same.

Resistance of agricultural crops to herbicides is an important topic that concerns many researchers. One of the most popular groups of herbicides is the imidazolinone group. Resistant forms of crops such as wheat (Triticum aestivum L.), sunflower (Helianthus annuus L.), corn (Zea mays L.), rice (Oryza sativa L.) and oilseed rape (Brassica napus L.) have been developed to this group of herbicides. All crops resistant to this group of herbicides have the commercial name Clearfield^®. In this review, the information concerning oilseed rape resistance to the imidazolinone group of herbicides is collected and summarized; it will be useful for breeders and researchers engaged in this direction. This review touches upon the topic of mechanisms of oilseed rape resistance to imidazolinones. Mutation variants known to date, which provide resistance to this group of herbicides, are described, and known molecular markers of them are presented. Approaches to the selection of oilseed rape for resistance to the imidazolinone group of herbicides are mentioned. Various methods of utilizing imidazolinone resistance to improve the purity of hybrid seeds are also considered.