Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.)
S. V. G. N. Priyadarshani,
Bingyan Hu,
Weimin Li,
Hina Ali,
Haifeng Jia,
Lihua Zhao,
Simon Peter Ojolo,
Syed Muhammad Azam,
Junjie Xiong,
Maokai Yan,
Zia ur Rahman,
Qingsong Wu,
Yuan Qin
Affiliations
S. V. G. N. Priyadarshani
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
Bingyan Hu
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
Weimin Li
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
Hina Ali
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
Haifeng Jia
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
Lihua Zhao
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
Simon Peter Ojolo
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
Syed Muhammad Azam
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
Junjie Xiong
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
Maokai Yan
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
Zia ur Rahman
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
Qingsong Wu
South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences
Yuan Qin
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
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.
