Background An 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. Results 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. 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. Conclusion Based 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.

中文翻译：

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用于菠萝（Ananas comosus L.）基因表达和蛋白质相互作用研究的简单原生质体分离系统。

背景有效的转化方案是研究和利用任何植物物种遗传潜力的首要条件。快速转化系统对于基因功能分析以及蛋白质及其体内相互作用的研究也至关重要。然而，目前包括菠萝在内的许多植物物种仍然缺乏快速有效的转化系统。这限制了对菠萝遗传库的全面探索以及对其基因、蛋白质定位和蛋白质相互作用的研究。结果为了解决上述限制，我们开发了一种有效的系统，使用来自组织培养的菠萝植物进行所需蛋白质的原生质体分离和亚细胞定位。1.5% (W/V) 纤维素酶 R-10 和 0.5% (W/V) Macerozyme R-10 的混合物经过 3 小时的消化可产生 51% 的存活原生质体。与之前报道的方案相比，我们的原生质体分离方法明显更快（节省 4.5 小时），仅需要少量组织样本（1 g 叶子），并且产量高（6.5 × 105）。使用具有不同细胞器标记的原生质体中的细胞器定位来验证分离的原生质体的质量。此外，菠萝原生质体中两个菠萝Mg2+转运蛋白基因的共定位分析与烟草瞬时表达系统的结果一致，证实原生质体分离方法可用于研究亚细胞定位。进一步的研究结果表明该系统也适用于蛋白质-蛋白质相互作用研究。结论根据我们的研究结果，目前描述的方法是菠萝原生质体分离和转化的一种高效且有效的策略；方便、省时，为转化研究提供了更大的平台。