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Aislamiento enzimático de protoplastos a partir del mesófilo de vitroplantas de Gynerium sagittatum Aubl. Beauv. cv. “Criolla”
Enzymatic isolation of protoplasts from the mesophilus of vitroplants Gynerium sagittatum Aubl. Beauv. cv. “Criolla”
DOI:
https://doi.org/10.15446/rev.colomb.biote.v25n1.104551Keywords:
caña flecha, celulasa, macerozima, tiempo (es)Arrow cane, cellulase, macerozyme, time (en)
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Gynerium sagittatum es una gramínea ampliamente utilizada en la costa Caribe colombiana como fuente de fibra natural para la elaboración de artesanías, particularmente por la comunidad Zenú. En la presente investigación se evaluó el efecto de diferentes concentraciones de enzimas: celulasa y macerozima a diferentes tiempos de incubación y sus interacciones en el aislamiento de protoplastos. Los protoplastos se obtuvieron del mesófilo foliar de vitroplantas de G. sagittatum expuesto a combinaciones enzimáticas de celulasa (1.5 y 2.0%), con macerozima (0.3, 0.6 y 0.9%), durante 3, 6 y 9 horas de incubación, para un total de 18 tratamientos con 5 réplicas cada uno. Los mayores números de protoplastos aislados correspondieron a T18 (2.0% celulasa, 0.9% macerozima), T12 (2.0% de celulasa, 0.3% macerozima), T3 (1.5% de celulasa, 0.3% de macerozima) y T6 (1.5% de celulasa, 0.6% de macerozima) por 9 horas de incubación cada uno, con valores de 88.625, 83.000, 75.000 y 53.375 protoplastos/mL respectivamente. El tiempo de incubación fue significativo en el aislamiento de los protoplastos (p<0.05). Las predicciones entre factores mostraron que una interacción de 2.0% de celulasa y 0.9% de macerozima permite obtener 44.302 protoplastos/mL, mientras que las interaciciones tiempo de incubación-celulasa y tiempo de incubación-macerozima mostraron que es posible obtener 72.073 y 71.212 protoplastos/mL con 2.0% de celulasa y 0.9% macerozima por 9 horas de incubación cada una respectivamente. Los resultados indican que la aplicación de estas enzimas permite obtener cantidades considerables de protoplastos de G. sagittatum a partir de explantes cultivados in vitro.
Gynerium sagittatum is a graminaceous plant widely used in the Caribbean coast of Colombia as a natural fiber source for the elaboration of handicrafts, particularly by the Zenú community. In the present investigation, the effect of different concentrations of cellulase and macerozyme enzymes at different incubation times and their interaction in the isolation of protoplasts was evaluated. Protoplasts were obtained from leaf mesophyll of G. sagittatum vitroplants exposed to enzymatic combinations of cellulase (1.5 and 2.0%), with macerozyme (0.3, 0.6 and 0.9%), for 3, 6 and 9 hours of incubation, for a total of 18 treatments with 5 replicates each. The highest numbers of isolated protoplasts corresponded to T18 (2.0% cellulase, 0.9% macerozyme), T12 (2.0% cellulase, 0.3% macerozyme), T3 (1.5% cellulase, 0.3% macerozyme) and T6 (1.5% cellulase, 0.6% macerozyme); at 9 hours incubation. The protoplast number for these treatments were: 88.625, 83.000, 75.000 and 53.375 protoplasts/mL respectively. Incubation time was significant in the isolation of protoplasts (p<0.05). The predictions between the factors showed that with an interaction of 2.0% cellulase and 0.9% macerozyme it is possible to obtain 44.302 protoplasts/mL, likewise, the incubation time-cellulase and incubation time-macerozyme interactions showed that it is possible to obtain 72.073 and 71.212 protoplasts/mL with 2.0% cellulase and 0.9% macerozyme for 9 hours of incubation respectively. The results indicate that the use of these enzymes and time, allows the isolation of of protoplasts from G. sagittatum in vitro plants.
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