Isolation and regeneration of protoplasts from leaf explants of Rhyncholaelia digbyana

Abstract

A protocol for the isolation and regeneration of protoplasts from leaf explant of Rhyncholaelia digbyana is presented. The protoplasts were isolated using hemicellulase enzymes at 1.5, 2.25, and 3% (w/v), pectinase at 0.5 and 0.75% (w/v) and cellulase at 1 and 2% (w/v). Protoplast counting was carried out with a Neubauer camera and an optical microscope at 40X, and viability was determined with Evans blue dye at 0.025% (w/v). The protoplasts were cultivated following the standard plate method, using the K&M media with 0.06 M of saccharose, 2.3% of Gelrite and plant growth regulators. It produced a yield of 386250±1875 protoplasts/g of tissue using an enzymatic combination of 1.5% (w/v) of hemicellulase, 0.5% (w/v) of pectinase and 1% (w/v) of cellulase with an incubation time of 4 h. The colonies were observed after two months of culture and the highest number of colonies (1.66±0.50) was obtained when the protoplasts were cultured in Kao medium with 4.53 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.912 µM of Zeatin and 3.10 µM of 6-Benzylaminopurine (BA). The nuclear DNA content estimated by flow cytometry for R. digbyana was 27.39±3.8 pg of DNA equivalent to 26.79×10 9 pb and the number of mitotic chromosomes counted was 2n=40.

Aislamiento enzimático de protoplastos a partir del mesófilo de vitroplantas de Gynerium sagittatum Aubl. Beauv. cv. “Criolla”

“…Una vez finalizado cada periodo de incubación del material vegetal en las soluciones enzimáticas, se filtraron con la ayuda de mayas de nylon de 60 µm de diámetros (Mota et al, 2018), luego se centrifugaron las muestras a 2.500 rpm por 5 minutos en una microcentrifuga de mesa con control de temperatura (Micro CL 2112). El pellet obtenido fue lavado dos veces con una solución compuesta de: Manitol 0.4 M, CaCl 2 0.5 mM, KH 2 PO 4 0.7 mM (Parihar et al, 2015) y pH ajustado a 5.5 con NaOH 1N.…”

“…Por otra parte, las interacciones entre los factores celulasa y tiempo de incubación mostraron que el mayor número de protoplastos que se puede obtener es 72.073 protoplastos/mL, con la utilización de 2.0% de celulasa por 9 horas de incubación Figura 3B. Estos resultados muestran que a medida que trascurre el tiempo de incubación y a una concentración de 2.0% de celulasa hay probablemente una mayor eficiencia de la enzima sobre el sustrato, aunque periodos extensos del material vegetal en la solución enzimática puede acarrear daños sobre los protoplastos (Mota et al, 2018).…”