Arabidopsis thaliana LEAFY COTYLEDON1 (LEC1) gene is shown to have numerous diverse functions in plant development, including the regulation of embryo morphogenesis and maturation, hypocotyl elongation, flowering transition, etc. However, the functions of LEC1 orthologs in different plant species have not been extensively studied. In this study, we obtained a line of Medicago truncatula, a model leguminous plant, carrying the loss-of-function mutation in the MtLEC1 (MtNF-YB10) gene, orthologous to LEC1, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas9) genome editing system. Edited plants with loss of MtNF-YB10 function did not demonstrate any severe abnormalities during their normal growth and gave viable seeds, but their capability for somatic embryogenesis in vitro was dramatically reduced. The T1 progeny of unedited plants with a Cas9-gRNA cassette insertion was also analyzed based on the suggestion that editing could occur during seed formation. However, no edited plants were found in the T1 generation. These results suggest divergent functions of LEC1 orthologs and make it possible to investigate potential specific MtNF-YB10 functions.

Genome modification of legumes, peas in particular, is accompanied by significant challenges. Establishing a reliable reporter system to identify tissue that expresses foreign DNA may help to optimize and develop transformation protocols for these species. The RUBY system, based on the synthesis of red betalain from tyrosine, offers a convenient solution for monitoring the efficiency of transgene introduction. To evaluate the effectiveness of RUBY application in pea tissue culture, we combined agrobacterial transformation with an in vitro cultivation system, inducing callus development. Transformed explants demonstrated RUBY pigmentation, but it disappeared during cultivation. We hypothesized that this issue is caused by tyrosine depletion. To check this suggestion, we tested whether tyrosine supplementation could maintain RUBY coloring. In the later stages, pigmentation still could not be preserved. However, our modified conditions increased the percent of colored shoot apex explants during the early cultivation stages. Thus, it is likely that some explants transformed with the RUBY cassette do not synthesize a sufficient amount of betalain due to the deficit of endogenous tyrosine. In this case, adding exogenous tyrosine would enhance betalain production and improve the detectability of tissues containing the RUBY cassette. These data can be used for the optimization of RUBY application conditions for peas and other species.

Legumes are important agricultural and food crops, however, some legume species have a low regenerative capacityin vitro, which complicates obtaining the genetically modified plants with improved properties and analysing gene function. To search for genes that stimulate somatic embryogenesis and to increase the regeneration frequency in legumesin vitro, we designed a screening vector system that will allow faster cloning of genes encoding potential regulators of morphogenesis by preserving restriction sites in the final vector. The construction of vectors is based on the Golden Gate [1] modular cloning method. Using type II restriction endonucleases, DNA fragments form sticky ends and are combined in a given order to form multigene constructs intended forAgrobacterium-mediated transformation. In order to identify efficient variants for gene expression, we used a number of promoters: CaMV 35S(long), CaMV 35S(double), nopaline synthase (nos), actin 2 (act2), and a number of terminators: 35S CaMV, nos, act2 forMtWOX9-1overexpression.Medicago truncatulaWOX9-1 (MtWOX9-1) is a WUSCHEL- related homeobox transcription factor for which a positive effect on the formation of somatic embryos in callus culture was previously shown [2]. Based on the analysis of embryogenic tissue, the optimal combination of promoters and terminators will be selected to assemble the vector for screening of morphogenetic regulators. The work is supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement 075-10-2021-093, Project PBB-RND-2243).

BACKGROUND: Genetic modification of pea remains challenging, which can likely be attributed to its low regeneration efficiency. To obtain transgenic pea plants, shoot regeneration followed by rooting is usually applied. Another regeneration pathway, somatic embryogenesis, is not used for pea genome modification due to very low frequency of this process. If a gene stimulating somatic embryogenesis was identified, it could be used as a morphogenic regulator to enable regeneration of pea plants from genetically modified callus cells. The search for such genes relies on the development of a cultivation system, allowing production of a significant amount of callus tissue in which a potential morphogenic regulator is ectopically expressed. AIM: The aim of study was to evaluate the possibility of obtaining pea callus tissue expressing foreign DNA with or without usage of selective agents, specifically, kanamycin and hygromycin B. METHODS: In this study, we combined agrobacterial transformation protocol with a method of callus induction from pea shoot apex explants. To evaluate the effectiveness of this transformation system with different selective agents, we used two different reporters: RUBY and DsRed. RESULTS: Our results demonstrate that transformation of pea shoot apices using the developed system yields a significant percentage of calli containing tissue regions which express foreign DNA. Addition of the antibiotics as selective agents doesn’t increase frequency of calli expressing introduced DNA. CONCLUSION: Results obtained in this study suggest that searching for regeneration stimulators is feasible in Pisum sativum without usage of selective agents.