Pineapple exhibits crassulacean acid metabolism (CAM) and has been used as a model to study the CAM pathway. It is a monocotyledonous triploid fruit that belongs to the family Bromeliaceae. Pineapple ( Ananas comosus L.) is the second most economically important fruit crop in the world after banana, and the improvement of its qualities are aimed at increasing yield, taste and resistance to environmental changes. Conclusionīased on our findings, the presently described method is an efficient and effective strategy for pineapple protoplast isolation and transformation it is convenient and time saving and provides a greater platform for transformation studies. Further findings showed that the system is also suitable for protein–protein interaction studies.
Additionally, colocalization analysis of two pineapple Mg 2+ transporter genes in pineapple protoplasts was consistent with the results in a tobacco transient expression system, confirming that the protoplast isolation method can be used to study subcellular localization. The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Compared to previously reported protocols, our protoplast isolation method is markedly faster (saving 4.5 h), requires only a small quantity of tissue sample (1 g of leaves) and has high yield (6.5 × 10 5).
A cocktail of 1.5% (W/V) Cellulase R-10 and 0.5% (W/V) Macerozyme R-10 resulted in 51% viable protoplasts with 3 h digestion. To address the above limitations, we have developed an efficient system for protoplast isolation and subcellular localization of desired proteins using pineapple plants derived from tissue culture. This has limited the full exploration of the genetic repository of pineapple as well as the study of its genes, protein localization and protein interactions. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. An efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species.
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