ИСТИНА

Buckwheat (Fagopyrum esculentum Moench) is a pseudo-cereal crop that belongs to the Polygonaceae family. Due to its high nutritional value and bioactive compounds, it is widely used in the food and pharmaceutical industries. Gene editing offers opportunities to introduce desirable traits and remove undesired ones to generate new, improved buckwheat varieties faster than classical breeding. One of the approaches to obtain genetically modified buckwheat is through transformation and gene editing, followed by regeneration of transgenic plants through hairy root culture. Two self-compatible buckwheat varieties KK8 и Shinano-SC were used in this study. Agrobacterium rhizogenes strains A4 and R1000 were tested for induction of hairy roots and transfer of T-DNA. The binary vectors used in this work included GFP and BAR expression cassettes for selection of transformed plants, as well as CRISPR/Cas9 expression cassettes for knockout of DFR and LFY genes. All vectors and expression cassettes were created using the modules of the MoClo Toolkit and CRISRP Plants kits (Addgene) cloning system. Both A. rhizogenes strains efficiently induced hairy root growth in buckwheat explants, but only hairy roots induced by R1000 strain had green fluorescence, i.e., successfully transferred the T-DNA from the binary vectors into the plant genome. Callus cultures from hairy roots of F. esculentum can be obtained with a different set of hormones, but we found the classical composition of MS3 medium with 2 mg/L 2,4-Dichlorophenoxyacetic acid and 2 mg/L Kinetin for callus induction to be optimal. For regeneration from callus culture, the MS3 medium composition including 6-Benzylaminopurine (2 mg/L), Kinetin (0.2 mg/L), and Indole-3-acetic acid (0.2 mg/L) was optimal. It is also noteworthy that the fluorescence of the GFP marker gradually disappeared as the regenerants grew, even though the morphogenic callus from which they were derived was fully fluorescent and the regenerated plants still contained T-DNA based on PCR test. Regenerated plants of the KK8 и Shinano-SC varieties were successfully acclimated to non-sterile soil conditions.