Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.)

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.

Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

“…However, it is difficult to isolate protoplasts with high yield and viability from mature leaf tissues of many plants. Hence, young leaves of in vitro grown plantlets of Phalaenopsis [26], Dendrobium [27], Tanacetum [28], grape (Vitis vinifera) [29], pepper (Capsicum annuum L.) [30] and pineapple [31] have been used. Nevertheless, callus induction is time consuming and impractical for protoplast isolation.…”

A simple method to co-purify genomic DNA, RNA, and proteins for functional studies

“…One advantage of our method is that it is compatible with samples in small quantities as starting materials, such as protoplasts. Protoplast assays have been widely used in plant biology to rapidly and efficiently test: 1) the cellular localization of proteins and RNAs (Priyadarshani et al, 2018, Rolland, 2018, 2) plant responses to biotic stresses at transcriptional and post-transcriptional levels (Asai et al, 2002, He et al, 2007, 3) protein-protein interactions (Schweiger andSchwenkert, 2014, Priyadarshani et al, 2018), 4) gene functions (Hamel et al, 2011, Li et al, 2013, 5) viral replications (Qi andDing, 2002, Owen et al, 2016), etc. Due to the limit of sample quantity, current studies are often limited to microscopic analysis or only analyze one type of biological components (DNA, RNA, or protein) in protoplasts, undermining the value of this transient transgenic approach.…”

“…We are interested in testing whether this method is efficient for samples in small quantity, such as protoplasts. Protoplast system allows rapid analysis of protein subcellular localization (Priyadarshani et al, 2018, Rolland, 2018, gene functions (Hamel et al, 2011, Li et al, 2013, plant responses to stresses (Asai et al, 2002, He et al, 2007, viral replication processes (Qi andDing, 2002, Owen et al, 2016), etc. However, previous studies were restrained to microscopic analysis or to only analyze RNA or proteins due to the limited amount of materials.…”

Correction to: Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.)

“…In the original version of this article [ 1 ], the spelling of the tenth author name, Moakai Yan, was incorrect. The corrected name is given in this correction article.…”