Development of protoplast isolation method in some cultivars of Ficus carica as crucial stage for implementation of single-cell RNA-seq technology in plant investigation

I.V. Mitrofanova, O.V. Krivenko, O.N. Kuleshova, I.V. Bulavin, E.S. Chelebieva, E.A. Vodiasova, O.V. Mitrofanova
Common fig is one of the most important subtropical fruits. It is used in food, cosmetic, and soap industries as well as in medicine. The Nikita Botanical Gardens has a large Ficus carica collection which includes 267 cultivars and hybrid forms. For study of this collection we used different methods of propagation and conservation. The genomic investigation of some fig cultivars was started, including the development of methods based on the current single-cell sequencing technology. The main objective of our study was to develop an approach to protoplast isolation from the leaves of F. carica valuable cultivars with quality for future use in the microfluidic systems, which would allow for library preparation of single-cell RNA-seq analysis. Protoplast isolation from the fig leaf explants, which were taken from in vitro cultured microshoots of ‘Sabrutsiya Rozovaya’ and ‘Datte de Naples’ cultivars, have been carried out. The direct method of enzymatic protoplast isolation with significant number of viable protoplasts of fig cultivars ‘Sabrutsiya Rozovaya’ (2·105), ‘Datte de Naples’ (1.39·105) and ‘Violette’ (1.67·105) was developed for using in 10x Genomics’ Chromium microfluidic system. Primary transcriptomes of two fig cultivars were obtained. Comparison between gene expression profiles in leaves of in vitro plantlets and in vivo adapted plants was conducted by bioinformatic methods. Transcriptome annotation was carried out by using genome assembly of ‘Horaishi’ cultivar, taken from NCBI GenBank. Thus, new approaches to study of the transcriptome variation during protoplast isolation in ‘Sabrutsiya Rozovaya’ and ‘Datte de Naples’ cultivars were developed and realized. Firstly, the assessment of the possibility of leaves plant material using in 10x Genomics’ Chromium platform for single-cell RNA-seq analysis was given. Perspective of current single-cell sequencing technology for solving fundamental challenges of plant morphogenesis on different stages of their development will be discussed.
Mitrofanova, I.V., Krivenko, O.V., Kuleshova, O.N., Bulavin, I.V., Chelebieva, E.S., Vodiasova, E.A. and Mitrofanova, O.V. (2021). Development of protoplast isolation method in some cultivars of Ficus carica as crucial stage for implementation of single-cell RNA-seq technology in plant investigation. Acta Hortic. 1310, 41-48
DOI: 10.17660/ActaHortic.2021.1310.7
https://doi.org/10.17660/ActaHortic.2021.1310.7
common fig, in vitro and in vivo plants, enzymatic protoplast isolation, transcriptome, single-cell RNA-seq
English
1310_7
41-48

Acta Horticulturae