Obtaining Protoplasts from Leaf Tissue Plantlets of Rubus Glaucus Benth (Blackberry) to Develop Proembryos

Abstract

Blackberry is a plant native from the highlands of South America, which grows in the Ecuadorian Andes. This plant has been cultivated for centuries in Ecuador and has been used as food and medicine. Its fruit is widely consumed due to its high content of minerals and vitamins, especially vitamin C. For a large-scale production, this study was aimed to establish alternatives and appropriate protocols for obtaining proembryos of Rubus glaucus using callus and protoplasts from leaf tissues. The following hormones were used in different concentrations: Auxins (2,4-D and NAA), Cytokinins (TDZ and Zeatine) and Giberillin GA3 and a basic Murashige and Skoog (MS) media as culture medium. Several assays were performed to determine the most suitable concentration of phytohormons for embryogenic callus formation and for stablishing proembryos in cell suspensions. Statistical analysis was performed with the help of Analysis of Variance (ANOVA) and Duncan test. The best treatment (p < 0.0001) was reached with 0.5 ppm of 2, 4-D auxin in Callogenesis phase, this concentration was sufficient to form vegetative callus over a period of four weeks with the leaf explant covered totally. Regarding to the increase of vegetative growth of callus, the cytokinin Zeatin at 0.5 ppm enabled an average increase of 0.5 cm per growing callus and finally 0.5 ppm of GA3 enabled an average growth rate of 141 667 cells/mL in the protoplast suspension.