Abstract
BackgroundAn efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. 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.ResultsTo 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. 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. 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 × 105). The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Additionally, colocalization analysis of two pineapple Mg2+ 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. Further findings showed that the system is also suitable for protein–protein interaction studies.ConclusionBased 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.
Highlights
An efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species
BAP is the main synthetic cytokinin used in pineapple tissue culture, and from the hormone combinations tested, we found that the best medium for the induction of callus and buds was as follows: full strength Murashige and Skoog (MS) medium supplemented with 3% sucrose, 4 mg/l BAP and 0.2 mg/l 1-naphthaleneacetic acid (NAA) and 3 g/l phytagel
The hormone combination of the above medium led to a significant difference (F = 10.33 and P = 0.001) in callus and bud induction compared to the other hormone combinations (Fig. 1e)
Summary
An efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. Quick and effective transformation systems are still lacking for many plant species including pineapple. 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. Pineapple is important as a model and crop plant, its self-incompatibility and long lifespan continue to pose challenges for breeding programs [6]. Agrobacterium-mediated transformation in pineapple is time consuming and technically challenging, with transformation efficiency reported to be as low as 0.12–2.69% [7] As a result, this bottleneck in Agrobacterium-mediated pineapple transformation hinders crop improvement achieved through molecular breeding methods. An easy and efficient transformation method is urgently needed to overcome these limitations and to facilitate the functional characterization of genes, the localization and interaction of proteins and transgenic studies in pineapple
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