Acta Biológica Colombiana (Jul 2004)
Isolation and cultive of protoplast in passion fruit
Abstract
In this research we adjust the protoplasts culture and regeneration conditions, necessary to advance into somatic hybrids obtention. Isolation of Passiflora edulis var. flavicarpa protoplasts from in vitro seedlings cotyledons and leaves was carried out. These tissues were plasmolysed in a CPW13M solution, then exposed to enzymatic solutions. The better yields were 6,48 x 106 and 4,60 x 106 protoplasts/500 mg of tissue, reached with the mixture Cellulase R-10 1% and Pectolyase Y-23 0,05% from leaves, and the enzymatic solution Cellulase 2% and Macerozyme 0,4% from cotyledons tissue, respectively. The best densities for protoplasts culture were 5 x 104 protoplasts/ml obtained from cotyledons and 1,5 x 105 protoplast/ml from leaves, employing agarose droplets system, bathing by KM8p liquid media with glucose 100g/l and cefotaxime 300μg/ml. When first cell divisions occurred, the osmotic concentration was reduced by removing the liquid media and adding equal volume of fresh mix media KM8p:KM8 in a ratio of 3:1. Every seven days, this action was repited using mix media in ratios of 2:1, 1:1, and 1:3, until colonies and callus formation was achieved. Then, the call were transferred into MS media with BAP 2 mg/l and IBA 1 mg/l for plant regeneration on light conditions. After six weeks shoot differentiation was promoved, finally those shoots were subcultured to free regulators media for rooting.
