Carlos Eduardo Assis da Silva¹, Lucas Rodrigues Xavier¹, Roberta Pena Da Paschoa¹, Kaliane Zaira Camacho Maximiano Cruz¹, Caio Cezar Guedes Corrêa¹, Daniel Dastan Rezabala Pacheco¹, Karina Vieira da Silva¹, Claudete Santa-Catarina¹, Vanildo Silveira¹

Somatic embryogenesis has emerged as a promising biotechnological strategy, enabling large-scale regeneration of genetically uniform plants and playing a key role in genetic transformation and gene edition protocols in plants. The ethylene signaling inhibitor, silver nitrate (AgNO₃), has shown positive effects on somatic embryogenesis in different species, promoting an increase in somatic embryo differentiation. However, the underlying molecular mechanisms and hormonal interactions of AgNO₃ remains elusive. Therefore, the present study aimed to evaluate the physiological, hormonal, and proteomic effects of AgNO₃ on sugarcane somatic embryo maturation, elucidating molecular mechanisms associated with the acquisition of embryogenic competence and identifying targets with potential biotechnological applications. In this study, embryogenic calli were induced in MS (Murashige and Skoog) medium supplemented with 10 µM 2,4-D (2,4-dichlorophenoxyacetic acid). After the third callus subculture, a pre-treatment of maturation with AgNO₃ was applied at concentrations of 0 (control), 0.5, and 5 µM. After 21 days, the calli were transferred to a hormone-free maturation medium, kept in the dark for 7 days, and then maintained under a 16h photoperiod to evaluate the effects of AgNO₃ on embryogenic competence. Somatic embryo number, fresh and dry mass were evaluated after 42 days of callus maturation treatment. The 0.5 µM AgNO₃ treatment resulted in a significant increase in the number of embryos (65.5) and dry mass (0.02158 g), compared to the control (40.1 embryos and 0.01732 g). In contrast, the 5 µM concentration drastically reduced the number of embryos (15.6) and showed signs of oxidation. To determine the effects of silver nitrate on the proteomic profile, callus samples were collected after pre-treatment with the different concentrations and subjected to bottom-up proteomic analysis. As a result, 2258 proteins were identified, of which 448 were considered differentially regulated in at least one treatment comparisons. Proteomic analysis revealed that the 0.5 µM treatment led to the accumulation of proteins related to meristem maintenance (SCL32, NMT1), cell cycle regulation (PCNA2), and cell wall biosynthesis (RHM2). In contrast, the 5 µM treatment resulted in the accumulation of proteins related to oxidative stress (PRXIIE, APX1) and auxin conjugation (GH3.1), along with a reduction in hormonal signaling proteins (CAND1). To study how silver nitrate modulates the hormonal profile of embryogenic cells, samples of the callus after pre-treatment were used for targeted metabolomic analysis and relative quantification of the hormones IAA, ABA, and ACC. Hormonal quantification indicated that the 0.5 µM treatment significantly increased IAA and ABA levels. Previous work has shown that high ABA levels promote storage reserve accumulation, while increased IAA may contribute to cell differentiation and embryo polarity, which may be associated with the increase in the number of somatic embryos after pre-treatment with silver nitrate. In conclusion, AgNO₃ at 0.5 µM enhances somatic embryogenesis through increased ABA and IAA levels and regulation of key proteins. However, higher concentrations cause phytotoxic effects and hormonal imbalance, impairing embryo development. These findings highlight potential molecular targets for future biotechnological applications in plant transformation and genetic engineering.

Agradecimentos: Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Programa de Pós-graduação em Biotecnologia Vegetal (PGBV), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) E Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ)