Simple protoplast isolation system for gene expression and protein interaction studies in pineapple ( Ananas comosus L.)
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Plant Methods Open Access
RESEARCH
Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.) S. V. G. N. Priyadarshani1† , Bingyan Hu1†, Weimin Li1, Hina Ali1,2 , Haifeng Jia1, Lihua Zhao1,2, Simon Peter Ojolo1 , Syed Muhammad Azam1,2 , Junjie Xiong1,2 , Maokai Yan1,2, Zia ur Rahman1,2, Qingsong Wu3* and Yuan Qin1*
Abstract 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. Keywords: Protoplast, BAP, NAA, Transfection, Pineapple
*Correspondence: [email protected]; [email protected] † S. V. G. N. Priyadarshani and Bingyan Hu contributed equally to this work 1 State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Key Lab of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, College of Crop Sciences, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China 3 South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, Guangdong Province, China Full list of author information is available at the en
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