Saffron Protoplast Method Enables Single-Cell RNA Sequencing

Published in BMC Biotechnology in July 2026, the study addresses a significant technical barrier in plant molecular biology: the efficient removal of rigid cell walls to obtain high-quality cells suitable for advanced genomic analyses. By optimizing enzymatic digestion conditions for apical buds, petals, leaves, and roots, the team established a method that facilitates both single-cell RNA sequencing and transient gene expression assays in a species previously lacking a robust genetic transformation system.

The primary challenge in preparing plant protoplasts lies in the variability of cell wall composition across different tissues and developmental stages. The research team, led by Xiaoyuan Xi and Liqin Li, aimed to overcome this by systematically examining the effects of D-mannitol concentration, enzyme combinations, and digestion duration on protoplast yield and viability. Saffron plants were cultivated in the experimental fields of Huzhou City, Zhejiang Province, with specific attention to the timing of tissue collection to ensure optimal cell quality for downstream applications.

The optimal enzymatic solution contained 0.6 M D-mannitol, 1.5% cellulase, and 0.5% macerozyme, with a digestion time of three hours at 28 degrees Celsius. Under these conditions, the team achieved a protoplast yield of approximately 8.81 × 10⁶ per gram of fresh weight, with 95% of the cells exhibiting intact membranes and high viability as assessed by fluorescein diacetate and trypan blue staining.

Root protoplast isolation required a more complex enzymatic mixture due to the recalcitrant nature of the cell walls in fibrous roots. The optimal conditions included 0.7 M D-mannitol, 2.0% cellulase, 0.5% macerozyme, and 0.5% pectinase, with a four-hour digestion period. This protocol yielded 5.69 × 10⁵ protoplasts per gram of fresh weight. However, the viability of root-derived protoplasts was markedly lower than that of other tissues, with only 61% of cells remaining healthy and energetic, reflecting the inherent difficulties of isolating cells from mature, differentiated monocot tissues.

Petal protoplasts were isolated from light purple petals, which offered a balance between moisture content and pigment levels that did not interfere with molecular assays. The optimal digestion involved 0.7 M D-mannitol, 1.5% cellulase, and 0.5% macerozyme for 3.5 hours, resulting in the highest yield among all tissues at 1.36 × 10⁷ protoplasts per gram of fresh weight. The team noted that white petals were too prone to rupture, while dark purple petals contained high levels of anthocyanin that could compromise sequencing data.

For mesophyll protoplasts, the researchers identified the second segment of mature saffron leaves as the ideal sampling location, as other segments exhibited either excessive shrinkage or rupture. The optimal conditions mirrored those for petals, using 0.7 M D-mannitol, 1.5% cellulase, and 0.5% macerozyme, but with a four-hour digestion time. This protocol yielded 7.84 × 10⁵ protoplasts per gram of fresh weight with 96% viability. The team also found that tearing leaves into two pieces rather than cutting them into small segments produced a higher yield and increased the vigor of the isolated cells.

To validate the utility of the isolated protoplasts for single-cell RNA sequencing, the team constructed two high-quality libraries using apical bud protoplasts from non-flowering and flowering stages. Using the 10×Genomics Chromium platform, they captured 11,502 and 15,068 cells, respectively, with median unique molecular identifiers per cell of 2,594 and 3,869. The analysis identified 13 distinct cell clusters in the saffron apical bud, with specific marker genes successfully identified for clusters predominantly distributed in flowering buds. This demonstrates that the protoplasts retain sufficient transcriptomic integrity to reveal developmental trajectories and cellular diversity.

The study also established a transient gene expression system in saffron mesophyll protoplasts using a heat shock combined with PEG-mediated transformation method. The team transfected protoplasts with a GFP-expressing plasmid and observed strong, ubiquitous signals in the nucleus, cell membrane, and cytoplasm. The maximum transfection efficiency reached more than 20%, which the authors note is sufficient for protein subcellular localization assays but may require further optimization for more complex functional studies. This system provides a rapid tool for gene function identification without the need for a stable genetic transformation system.

The researchers emphasized that the yield and quality of protoplasts are highly dependent on developmental stage and sampling location. For instance, apical buds at the flowering transition stage yielded higher quality cells than those at later stages of flower organ differentiation, which produced more debris. The variation in results across tissues is attributed to intrinsic differences in cell wall composition, such as the presence of secondary wall thickening in roots, which reduces enzymatic accessibility. These findings underscore the necessity of tissue-specific optimization in plant protoplast research.

Despite the success of the protocol, the authors acknowledged several limitations, including the scalability of the methods for high-throughput applications and the long-term maintenance of protoplast cultures. The efficiency of plant regeneration from these protoplasts has yet to be fully optimized, which remains a critical hurdle for germplasm innovation. The team suggested that future studies should focus on multi-dimensional optimization of culture media and the development of strategies to sustain mitotic activity over extended periods.

The study contributes to the broader field of plant biotechnology by providing a detailed, reproducible method for saffron protoplast isolation. By overcoming the technical challenges associated with diverse saffron organs, the researchers have enabled a platform for mechanistic insights into organogenesis, growth, and development. The high-quality single-cell libraries and transient expression results indicate that this method can accelerate research on the molecular mechanisms governing saffron biology, potentially leading to the identification of key genes influencing medicinal efficacy and petal color.

The raw reads generated in this study have been submitted to the NCBI BioProject database, ensuring transparency and reproducibility for the scientific community. The availability of these data, along with the detailed protocol, provides a valuable resource for researchers working with saffron and other monocot species with similar cell wall characteristics. The work represents a significant step forward in the molecular biology of saffron, a crop of considerable economic and medicinal importance.

In conclusion, the establishment of tissue-specific protoplast isolation methods for saffron offers a versatile tool for both basic research and applied biotechnology. The ability to perform single-cell RNA sequencing and transient gene expression in this species opens new avenues for understanding its complex regulatory networks.

The study was conducted as a methodological optimization and validation effort, with all experiments performed in triplicate to ensure statistical robustness. Data were analyzed using one-way analysis of variance, with differences considered significant at P < 0.05, P < 0.01, or P < 0.001. This rigorous statistical framework supports the conclusion that the observed variations in protoplast yield and viability across different enzymatic conditions and tissue types are not due to random variation but reflect genuine biological and technical differences in the isolation process.

Protoplasts, which are plant cells with their rigid cell walls removed, are essential for various molecular biology applications, including tissue culture, single-cell RNA sequencing, and transient gene expression analyses. Their totipotency, rapid growth, and synchrony make them valuable tools for botanists. However, the presence of cell walls in plants, which vary in thickness and composition depending on species, tissues, developmental stages, and environmental conditions, poses a significant challenge to obtaining high-quality protoplasts. This variability necessitates the development of tissue-specific isolation methods to ensure successful generation of high-yield and high-quality protoplasts.

The saffron plant, Crocus sativus, has a life cycle divided into reproductive growth from June to November and vegetative growth from December to May. The specific timing of tissue collection is critical, as the study utilized apical buds at the flowering transition stage, roots 0.5–2 cm from the tip, light purple petals, and the second segment of leaves 10–15 cm in length. This precise selection of developmental stages and sampling locations was crucial for achieving optimal protoplast quality.

The enzymatic digestion process involved the use of cellulase R-10 and macerozyme R-10, with pectinase added for root isolation. The enzymatic solutions were prepared with varying concentrations of D-mannitol, ranging from 0.4 to 0.7 M, and enzyme concentrations of 0.5% to 2.0% for cellulase and 0.5% to 1.0% for macerozyme. The solutions were warmed at 55°C for 10 minutes and cooled to room temperature before the addition of CaCl2 and bovine serum albumin. This careful preparation of the enzymatic mixture was essential for effective cell wall degradation without compromising cell viability.

The purification of protoplasts differed based on the tissue type. Mesophyll and petal protoplasts were purified by filtration through cell sieves, centrifugation, and natural sedimentation, while apical bud and root protoplasts required sucrose gradient centrifugation. This differential purification approach was necessary to remove impurities and ensure the integrity of the isolated cells. The yield was evaluated as the number of protoplasts released per gram of fresh weight, and cell viability was assessed using fluorescein diacetate staining, with viable protoplasts exhibiting green fluorescence.

The construction of single-cell RNA sequencing libraries involved loading cell suspensions into the 10×Genomics Chromium single-cell microfluidics device to generate single-cell gel beads-in-emulsion. The libraries were generated using the Single Cell 3’ Library & Gel Bead Kit V3, with barcoded, reverse-transcribed full-length cDNAs amplified by PCR and ligated to sequencing adapters. The paired-end libraries were sequenced using the Illumina NovaSeq 6000 platform, and qualitative analysis was performed using an Agilent 4150 Bioanalyzer. This high-throughput sequencing approach allowed for the detailed analysis of gene expression at the single-cell level, providing insights into the cellular diversity and developmental trajectories of saffron apical buds.

The transient gene expression system was established using a heat shock combined with PEG-mediated transformation method. The plasmid pCAMBIA1302_35S::GFP was used to visualize the subcellular localization of GFP in mesophyll protoplasts. The transformation process involved incubating the protoplasts with PEG-CaCl2 solution at 42°C for 10 minutes, followed by resuspension in WI solution and incubation at 18°C in the dark for 12–24 hours. This method achieved a transfection efficiency of more than 20%, demonstrating the functionality of the transient expression system for rapid gene characterization in saffron.

The study highlights the importance of optimizing protoplast isolation methods for specific tissues and developmental stages. The varying yields and viabilities observed across different tissues underscore the need for tailored approaches in plant molecular biology. By establishing these efficient methods, the researchers have provided a valuable resource for future studies aimed at understanding the molecular mechanisms governing saffron development and improving germplasm through genetic engineering.

Subject of Research: Tissue-specific protoplast isolation and single-cell RNA sequencing in saffron

Article Title: An efficient method for tissue-specific protoplast isolation suitable for single-cell RNA sequencing and transient gene expression analysis in saffron (Crocus sativus L.)

Article References: Xi, X., Song, J., Feng, M., Qian, X., Sun, H., & Li, L. (2026). An efficient method for tissue-specific protoplast isolation suitable for single-cell RNA sequencing and transient gene expression analysis in saffron (Crocus sativus L.). BMC Biotechnology, 26(1), Article 87. https://doi.org/10.1186/s12896-026-01187-1

Image Credits: AI Generated

DOI: 10.1186/s12896-026-01187-1

Keywords: saffron, protoplast isolation, single-cell RNA sequencing, transient gene expression, plant cell biology, Crocus sativus, enzymatic digestion, molecular biology, germplasm innovation, BMC Biotechnology, efficient, method