Aplicación de algunas técnicas de estadística multivariada al estudio de la conservación in vitro de germoplasma de Dioscorea alata L.

Rational use of multivariate statistics, modeling and biological knowledge can help the researcher to design a tested crucial experiment and to reach consistent conclusions. This work based on this statement and on a previous report of in vitro conservation of D. alata. It aims to apply some multivariate statistical techniques to determine the main factors which contribute to differentiate and to group the treatments. The most suitable factors for the preservation of genetic resources of this species were also defined. The D. alata conservation process was evaluated by principal component analysis at 9 months. Percentage of survival, phenolization, explants with leaves, rooting, sprouting and leaf senescence, stem length, root number and number of novo nodes per explants were the analyzed variables. Results showed that the survival rate variables, percentage of explants with leaves, stem length, number of novo nodes and number of roots express much of the observed variation in the processes of growth and development. On the other hand, deterioration and aging of in vitro nodal segments buds is better explained by the percentage of phenolization variables and leaf senescence. The best treatments for in vitro conservation of D. alata based on variables with higher contribution in the multivariate statistical analysis, corresponded to the culture medium D - 571 with the 1.5% mannitol, 0.1 or 1 mg.l-1 BA and 2 g.l-1 of activated charcoal.Keywords: activated charcoal, benzylaminopurine, cluster analysis, principal components, mannitol, yamEl uso racional de la estadística multivariada, el modelamiento y el conocimiento biológico pueden ayudar al investigador a diseñar con criterio un experimento crucial y llegar a conclusiones consistentes. Teniendo en cuenta lo anterior y el antecedente de un informe de conservación in vitro de D. alata, este trabajo tuvo como objetivo aplicar algunas técnicas de estadística multivariada para determinar las variables de mayor contribución a la diferenciación y agrupamiento de los tratamientos así como, definir el más adecuado para la preservación de los recursos genéticos de esta especie. Se evaluó mediante el análisis de componentes principales el proceso de conservación de D. alata a los 9 meses a través del porcentaje de: supervivencia, fenolización, explantes con hojas, enraizamiento, brotación y senescencia foliar; la longitud del vástago, el número de raíces y el número de nudos de novo por explante. Los resultados demostraron que las variables porcentaje de supervivencia, porcentaje de explantes con hojas, longitud del vástago, número de nudos de novo y número de raíces permiten explicar gran parte de la variación observada en los procesos de crecimiento y desarrollo. Por otra parte, el deterioro y envejecimiento de yemas de segmentos nodales conservados in vitro se explica en mayor medida por las variables porcentaje de fenolización y de senescencia foliar. Los mejores tratamientos para la conservación in vitro de D. alata L. sobre la base de las variables de mayor contribución en el análisis estadístico multivariado, correspondieron al medio de cultivo D - 571 con 1.5% de manitol; 0.1 ó 1 mg.l-1 de BA, y 2 g.l-1 de carbón activado.Palabras clave: bencilaminopurina, carbón activado, componentes principales, conglomerados jerárquicos y ligamientos completos, manitol, ñam

<i> In vitro </i> establishment and multiplication of <i>Citrus aurantifolia</i> Christm. Swing. var. `Mexicana' from seeds

Citrus fruits are crops with high economic and medicinal value. The creole lime (Citrus aurantifolia Christm. Swing) var. `Mexican' is widely consumed in Cuba but the incidence of pests and diseases has affected its production. The aim of this work was to achieve its establishment and in vitro multiplication. Seeds from mature fruits were disinfected with NaOCl (0.5, 1.0, 1.5%) and different immersion times (5, 10 and 20 minutes). At 7 days the number of explants contaminated with microorganisms, with necrosis and alive was quantified. Subsequently the effect of growth regulators on the establishment stage (6-BAP, GA3 and IBA) and in multiplication stage (6-BAP, GA3) was determined. The results indicated that the sodium hypochlorite was effective in disinfection of seeds and in its survival. With 1% NaOCl for 20 min the best results were obtained. It was shown that for seed germination adding growth regulators is not required but for multiplication the inclusion of 1.0 mg l-1 GA3 and 2.0 mg l-1 6-BAP yielded higher number of shoots and coefficient multiplication. The established protocol could be used in future studies of plant breeding. Key words: micropropagation, nodal cutting, plant growth regulator

Conservación in vitro de Dioscorea alata L. clon caraqueño (Dioscoreaceae)

In this study, we report an efficient method for conservation in vitro of Dioscorea alata L. clone caraqueño during 9 and 12 months based on the culture medium modification (MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1) with different manitol (0; 0,5 and 1,5%) and BAP (0; 0,1 mg.L-1) levels. At 9 and 12 months of in vitro conservation, the following evaluations were determined: survival (%), leaf senility (%), shoot length, buds and microtuber explant count. Plant regeneration (%), shoot length, leaf number and bud explant count were determined at 8 weeks of the nodal cutting regeneration (from vitroplants conserved for 9 and 12 months) in the culture medium (MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1). .Shoot length, leaf number and bud explant count were evaluated at 5 weeks during the conventional micropropagation. The cultivation variants formed by media MS to 75% + vitamins MS + sucrose 30 g.L-1 + activated charcoal 2 g.L-1 and MS to 75% + vitamins MS + sucrose 30 g. l-1 + activated charcoal 2 g.L-1 + BAP 0, 1 mg.L-1 allowed an effective way the conservation of in vitro plants from nodal cutting of clone caraqueño yam during 9 and 12 months with high survival percentages, a significant number of microtubers, smallest leaf senility percentages and 100% regeneration in whole plants with a normal growth in micropropagation conditions.El trabajo tuvo como objetivo establecer, para Dioscorea alata L. clon caraqueño, un método eficiente para la conservación in vitro de durante 9 y 12 meses basado en la modificación del medio de cultivo (MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1) con distintos niveles de manitol y BAP. Los tratamientos consistieron en la adición en el medio de cultivo de manitol (0; 0,5 y 1,5%) y BAP (0; 0,1 mg.L-1). A los 9 y 12 meses de conservación in vitro se realizaron las siguientes evaluaciones: supervivencia (%), senescencia foliar (%), numero de nudos, longitud del vástago, número de microtubérculos. A las 8 semanas de la regeneración de los segmentos nodales (procedentes de vitroplantas conservadas a 9 y 12 meses) en el medio de cultivo (MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1) se determinó la regeneración de plantas completas (%), número de nudos, número de hojas y longitud del vástago. A las 5 semanas durante la micropropagación convencional se determinó el número de nudos, número de hojas y la longitud del vástago. Las variantes de cultivo formadas por el medio MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1 y el MS al 75% + vitaminas MS + sacarosa 30 g.L-1 + carbón activado 2 g.L-1 + BAP 0,1 mg.L-1 permitió de manera efectiva la conservación de vitroplantas a partir de segmentos uninodales de D. alata clon caraqueño durante 9 y 12 meses con altos porcentajes de supervivencia, un número significativo de microtubérculos, los menores porcentajes de senescencia foliar y 100% de regeneración en plantas completas con un crecimiento normal en condiciones de micropropagación

Safety and immunogenicity of the FINLAY-FR-1A vaccine in COVID-19 convalescent participants: an open-label phase 2a and double-blind, randomised, placebo-controlled, phase 2b, seamless, clinical trial

BACKGROUND: A phase 1, clinical trial to evaluate FINLAY-FR-1A vaccine in COVID-19 convalescent individuals was completed. Here, we report results of the phase 2, clinical trial. METHODS: We studied 450 convalescent participants with a history of asymptomatic, mild, or moderate COVID-19 at the National Institute of Hematology and Immunology and the National Centre for Sexual Education in Havana, Cuba. The study included adults aged 19-78 years who had recovered from COVID-19 and had had a negative PCR test at least 2 months before the initiation of the study. Phase 2 was done sequentially in two stages. The first stage to assess safety comprised an open, non-controlled phase 2a study in participants aged 60-78 years who received a single dose of the FINLAY-FR-1A vaccine (50 μg of recombinant dimeric receptor binding domain [RBD]). The second stage comprised the placebo-controlled, double-blind, phase 2b trial in participants aged 19-78 years, where participants were randomly assigned (4:1) into two groups: an experimental group vaccinated with a single dose of the FINLAY-FR-1A vaccine, and a control (placebo) group injected with vaccine excipient. The primary outcomes were safety, evaluated 28 days after vaccination by the occurrence of serious adverse events in all participants, and successful immune response, assessed by neutralising antibody ELISA, and defined as half-maximal surrogate virus neutralisation titres of 250 or more. Secondary endpoints included vaccine immunogenicity assessed by ELISA anti-RBD and live-virus neutralisation test. All randomly assigned participants were included in the safety analysis (safety population), and immunogenicity was evaluated in participants without study interruptions (per-protocol population). The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000366-En and WHO-ICTRP and is complete. FINDINGS: From April 9, 2021, to April 17, 2021, 663 COVID-19 convalescent participants were enrolled in the study; 213 participants did not meet the selection criteria and 450 volunteers were recruited. 20 participants aged 60-78 years were included in the open, single-group, phase 2a study and 430 participants were randomly assigned to the experimental (n=344) or control groups (n=86) in the phase 2b study of participants aged 19-78 years. 19 (95%) of 20 phase 2a volunteers achieved a successful immune response after vaccination. No vaccine-associated serious adverse events were reported in the whole study population. Minor adverse events were found, the most common being pain at the injection site (105 [29%] of 364 in the intervention group; 13 [15%] of 86 in the placebo group). A successful immune response was found in 289 (81%) of 358 participants 28 days after vaccination. The vaccine elicited a greater than 31-times increase in anti-RBD-IgG antibodies compared with prevaccination rates, and the seroconversion rate was 302 (84%) of 358 on day 28 after vaccination; the geometric mean titres of live-virus neutralisation test increased from 15·4 (95% CI 10·3-23·2) to 400·3 (272·4-588·1) and high response was found against alpha, beta, and delta variants of concern. INTERPRETATION: A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 strengthened the pre-existing natural immunity, with excellent safety profile. FUNDING: Cuba's Ministry of Science, Technology, and Environment