Optimization and Presentation of a Protocol for Protoplast Isolation, Regeneration and Gene Transfer in Potato

The unique characteristics of plant protoplasts have made them a powerful tool for researchers to study the genetic modification of plant cells. The most important prerequisite for the use of protoplasts is the ability to extract them easily and in large quantities, and to cultivate and regenerate them for cell colony formation and plant production. The most common method of extracting protoplasts from plant tissues is the enzymatic method. Concentration and composition of enzymes and isolation and culture steps are key factors in protoplast extraction. The aim of this study was to investigate the effect of these factors on protoplasts of in vitro potato (Solanum tuberosum L.) and to develop an efficient and suitable method for their extraction, transfection, callus formation and regeneration.

For protoplast isolation from commercial and common Agria cultivar of potato, different treatments of Cellulase and Macerozyme enzymes concentration, incubation time in enzyme solution and isolation steps on leaf tissue, stem and aerial part of in vitro plantlets were investigated. The effects of different hormone concentration on culture and regeneration of protoplasts were studied. After protoplast isolation, the efficiency of transfection by PEG 4000 using pBin61-GFP plasmid was evaluated.

The best results were obtained from leaf explants and the highest number of protoplasts (2×106 protoplasts per ml) were extracted from leaf mesophilic tissues after 16 hours' incubation in enzyme solution (1% Cellulase and 0.2% Macerozyme). The transfection efficiency of protoplasts by PEG with 40% concentration and duration of 30 minutes was 50%. The MS medium containing 5 mg/l NAA and 0.1 mg/l BAP was selected as the best culture medium for callus induction. In this hormonal treatment, 100% of callus induction from protoplasts was obtained after about one month. The best medium for plant regeneration and shooting from callus was the combination of 2 mg/l Zeatin with 0.1 mg/l GA3 and 0.01 mg/l NAA with 86% efficiency.

Based on the results, the described protocol for protoplast extraction and gene transfer in this study provides necessary steps for genetic engineering, transformation and gene expression in the potato plant.