Enzymatic Antioxidant System Activation Assures the Viability of Guadua chacoensis (Bambusoideae, Poaceae) Embryogenic Cultures during Cryopreservation.

This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs' ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.

Comparative proteomic analysis and antioxidant enzyme activity provide new insights into the embryogenic competence of Guadua chacoensis (Bambusoideae, Poaceae).

Somatic embryogenesis (SE) involves modifications of cellular, biochemical, genetic, and epigenetic patterns. Our work investigated proteins as markers of embryogenic response and characterized the redox state of embryogenic cultures (EC) of Guadua chacoensis. We identified a total of 855 proteins; 129 were up- and 136 down-accumulated in EC as compared with non-embryogenic culture (NEC). Additionally, 37 and 22 proteins were identified as unique in EC and NEC, respectively. Heat-shock proteins as unique proteins and increased activity in Superoxide Dismutase and Guaiacol Peroxidase in EC suggest that the embryogenic response requires activation of the stress response mechanism. Ribosomal, translational, and glycolytic proteins in EC seem to be associated with protein synthesis and energy sources for embryo development, respectively. Accumulation of cell wall-related proteins, such as Arabinogalactan and Polygalacturonase inhibitors, and signaling transduction proteins, including Chitinase, Phospholipase, and Guanine nucleotide-binding proteins in EC seems to be associated with embryogenic response. Enhancement of H2O2 content in EC compared to NEC suggests a possible role as a secondary messenger in SE. Altogether, the present study identified marker proteins of embryogenic response in G. chacoensis and revealed the activation of ROS scavenging enzymes to assure cell redox homeostasis and SE responses. SIGNIFICANCE: Somatic embryogenesis is a promising technique for the propagation and conservation of bamboo species; however, this route has been the least understood and studied until now. This study corresponds to the first work approaching proteomics complemented with biochemical analyses in the somatic embryogenesis of bamboo, bringing robust and precise information that can improve our understanding of this complex morphogenetic route.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL-1 of L-glutamine, 1,050 mgL-1 of L-asparagine, 350 mgL-1 of L-arginine, and 35 mgL-1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.