Application of d cryo-plate technique for the cryopreservation of in vitro-grown shoot tips of Tarenaya spinosa (Cleomaceae).

BACKGROUND: Tarenaya spinosa is a medicinal species used for treating respiratory and inflammatory diseases. Various biotechnological studies have been developed for in vitro establishment of plants and long-term conservation of this species. OBJECTIVE: This study aimed to establish a new cryopreservation protocol using the D cryo-plate technique for in vitro-grown shoot tips of T. spinosa. MATERIALS AND METHODS: Different steps of the cryopreservation procedure were evaluated in this work: preculture; sucrose concentration of calcium alginate gel; concentration and time of exposure to osmoprotective loading solution; time of exposure to silica gel; and regrowth on recovery medium. RESULTS: The optimal procedure included preculture with increasing sucrose concentration (from 0.25 to 0.50 M), encapsulation and dehydration over silica-gel for 60 min. Increasing sucrose concentration in the loading solution or exposure duration had a negative effect on recovery of cryopreserved shoot tips. However, the association of calcium alginate gel enriched with 0.6 M sucrose with post-rewarming culture with BAP for 2 weeks resulted in the most efficient cryopreservation protocol (76% survival and 70% shoot recovery). CONCLUSION: The plants developed after cryopreservation maintained their in vitro multiplication capacity and demonstrated the efficiency of long-term conservation by D cryo-plate technique for T. spinosa. Doi.org/10.54680/fr23610110512.

Cryopreservation of In Vitro-Grown Shoot Tips of the Medicinal Species Cleome Spinosa (Cleomaceae) Applying Vitrification-Based Techniques.

BACKGROUND: Few cryopreservation studies have been reported with the genus Cleome. Due to the use of C. spinosa in traditional medicine and its valuable pharmacological potential, the long-term conservation of the species will allow the safe maintenance of its germplasm. OBJECTIVE: This study compares two vitrification-based techniques on the cryopreservation of shoot tips of C. spinosa. MATERIALS AND METHODS: The effect of sucrose preculture and different vitrification solutions was evaluated using vitrification and V Cryo-plate techniques. The supplementation of recovery medium with BAP was also assessed. RESULTS: The V Cryo-plate proved to be the most efficient technique. Treatment of shoot tips with PVS2 at 0°C resulted in a higher regeneration response after cryopreservation when compared to treatment with PVS2 and PVS3 at 25°C. The highest survival (83.3%) and recovery (76.6%) were achieved for shoot tips exposed to PVS2 for 90 min at 0°C and recovered on MS medium supplemented with 0.5 mg L-1 BAP for 2 weeks. CONCLUSION: Plants regenerated from cryopreserved shoot tips maintained their in vitro multiplication capacity and showed a normal phenotypic aspect, demonstrating the efficiency of the cryopreservation protocol.

CRYOPRESERVATION OF ADVENTITIOUS ROOTS OF Cleome rosea Vahl (CLEOMACEAE) USING A VITRIFICATION TECHNIQUE AND ASSESSMENT OF GENETIC STABILITY.

BACKGROUND: Cleome rosea, a Brazilian native species, has medicinal potential. Previously a cryopreservation protocol for in vitro roots using the vitrification solution PVS2 has been developed. However, the genetic stability of the cryopreserved material is yet to be assessed. OBJECTIVES: To evaluate the effects of loading and vitrification solutions (PVS2 and PVS3) on post-cryopreservation recovery of C. rosea roots, and to assess their genetic stability using Random Amplified Polymorphic DNA (RAPD) markers. MATERIALS AND METHODS: Root segments were pretreated with increasing concentrations of sucrose (0.2 to 0.4 M), followed by osmoprotection with loading solution and treatment with one of the vitrification solutions tested. RESULTS: The highest recoveries using PVS2 and PVS3 were obtained when root segments were exposed to these solutions for 15 min, reaching 77% and 100% respectively. The RAPD band profiles were monomorphic with most of the primers used. This molecular analysis revealed high genetic similarity (similarity coefficients among 0.98 and 1.00) between the cryopreserved roots and their mother plants. CONCLUSION: Roots from in vitro-propagated plants of C. rosea, were successfully cryopreserved using the vitrification technique. No major variations were observed on the genetic stability of cryopreserved roots, validating the use of this protocol as an efficient long-term conservation option for this species.

Efeito de diferentes meios nutritivos e fitorreguladores visando à otimização da calogênese de Annona mucosa (Jacq. ) / Effect of different culture media and plant growth regulators for the optimization of callus induction of Annona mucosa (Jacq. )

Annona mucosa é uma árvore frutífera da família Annonaceae, produtora de importantes metabólitos secundários de interesse medicinal, como lignanas, acetogeninas e alcaloides. A cultura in vitro de calos representa um importante recurso para a produção contínua de metabólitos, viabilizando a conservação da biodiversidade química e a obtenção controlada de material para estudos biológicos e fitoquímicos. O objetivo deste trabalho foi otimizar a produção de calos friáveis de A. mucosa, avaliando o efeito de diferentes meios nutritivos e fitorreguladores. Segmentos de folha e de hipocótilo de plântulas germinadas in vivo foram utilizados como explantes e inoculados nos meios de cultura MS, WPM e B5 suplementados com picloram (2 - 20µM) isolado ou combinado com as citocininas BAP, KIN ou TDZ (0,2 - 1µM). As culturas foram mantidas a 26±2ºC, no escuro, com subcultivos mensais. A produção de calos foi avaliada por aferição do peso dos calos, após 90 dias. Em todos os tratamentos na presença da auxina picloram, o cultivo de hipocótilos resultou em maior porcentagem de formação de calos, sobretudo no meio de cultura WPM. A associação com TDZ produziu massa calogênica friável altamente proliferativa e ausente de oxidação, alcançando valores superiores àqueles obtidos em trabalhos prévios com a espécie. Os resultados viabilizam o uso do material em suspensões celulares e posterior caracterização fitoquímica para a exploração da produção in vitro de metabólitos da espécie.

The Annona mucosa is a fruit tree of the Annonaceae family that produces a range of secondary metabolites of medicinal interest, such as lignans, acetogenins and alkaloids. The callus culture represents a renewable source of valuable medicinal compounds and controlled supply of material for biological and phytochemical studies. Therefore, this study was carried out to investigate the effects of three nutrient media, different concentrations of picloram and cytokinin types, in order to optimize the biomass yield and friability of calluses of A. mucosa. Leaf and hypocotyl segments from seedlings produced from in vivo seed germination were used as explants, which were inoculated in MS, WPM and B5 culture media supplemented with picloram (2-20µM) only or in addition to the cytokinins BAP, KIN or TDZ (0,2 - 1µM ). Cultures were maintained at 26±2ºC in the dark, with monthly subcultures. After 90 days, biomass production was evaluated. In all treatments, hypocotyl explants provided the highest percentage of callus formation, particularly in WPM. The association with TDZ produced highly proliferative friable callus, with no oxidation, reaching higher values than the previous works with this species. The results enable the use of the calluses produced in cell suspensions and the subsequent phytochemical characterization, in order to explore the in vitro production of metabolites of the species.

Susceptibility of Giardia lamblia to Hovenia dulcis extracts.

Giardia lamblia is the causative agent of giardiasis, a common parasitic infection of the human and animal digestive tract. Although several drugs have been available to treat this infection, they present unpleasant side effects or cytotoxicity. In order to find a more natural treatment for the disease, we analyzed the effects of the methanolic extract and three fractions obtained from Hovenia dulcis Thunb. (Rhamnaceae) leaves on G. lamblia. Comparing all fractions, dichloromethane was more efficient in reducing Giardia growth. The exposition of G. lamblia to this fraction lead to degenerations in the surface, modifications in the cell shape and alterations in the localization of nuclei. Besides that, the adhesion of G. lamblia was also altered. Experiments revealed that the obtained fraction did not present cytotoxic effects in mammalian cells. In summary, dichloromethane fraction has strong antigiardial effects and could become an important new substance for the treatment of giardiasis.