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

Λεπτομέρειες βιβλιογραφικής εγγραφής
Τίτλος:	An efficient method for tissue-specific protoplast isolation suitable for single-cell RNA sequencing and transient gene expression analysis in saffron (Crocus sativus L.).
Συγγραφείς:	Xi X; TCM (Traditional Chinese Medicine) Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory drugs, Huzhou Central Hospital, Fifth School of Clinical Medicine of Zhejiang, Chinese Medical University, 1558 Sanhuan North Road, Huzhou, Zhejiang, 313000, China.; Affiliated Huzhou Hospital, Zhejiang University School of Medicine, Huzhou, Zhejiang, China.; Huzhou Central Hospital, Affiliated Central Hospital of Huzhou University, Huzhou, Zhejiang, China., Song J; TCM (Traditional Chinese Medicine) Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory drugs, Huzhou Central Hospital, Fifth School of Clinical Medicine of Zhejiang, Chinese Medical University, 1558 Sanhuan North Road, Huzhou, Zhejiang, 313000, China., Feng M; School of Pharmacy, Hangzhou Normal University, Hangzhou, Zhejiang, China., Qian X; TCM (Traditional Chinese Medicine) Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory drugs, Huzhou Central Hospital, Fifth School of Clinical Medicine of Zhejiang, Chinese Medical University, 1558 Sanhuan North Road, Huzhou, Zhejiang, 313000, China., Sun H; TCM (Traditional Chinese Medicine) Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory drugs, Huzhou Central Hospital, Fifth School of Clinical Medicine of Zhejiang, Chinese Medical University, 1558 Sanhuan North Road, Huzhou, Zhejiang, 313000, China., Li L; TCM (Traditional Chinese Medicine) Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory drugs, Huzhou Central Hospital, Fifth School of Clinical Medicine of Zhejiang, Chinese Medical University, 1558 Sanhuan North Road, Huzhou, Zhejiang, 313000, China. liliqin@hzhospital.com.; Affiliated Huzhou Hospital, Zhejiang University School of Medicine, Huzhou, Zhejiang, China. liliqin@hzhospital.com.; Huzhou Central Hospital, Affiliated Central Hospital of Huzhou University, Huzhou, Zhejiang, China. liliqin@hzhospital.com.
Πηγή:	BMC biotechnology [BMC Biotechnol] 2026 Jul 08; Vol. 26 (1). Date of Electronic Publication: 2026 Jul 08.
Τύπος έκδοσης:	Journal Article
Γλώσσα:	English
Στοιχεία περιοδικού:	Publisher: BioMed Central Country of Publication: England NLM ID: 101088663 Publication Model: Electronic Cited Medium: Internet ISSN: 1472-6750 (Electronic) Linking ISSN: 14726750 NLM ISO Abbreviation: BMC Biotechnol Subsets: MEDLINE
Imprint Name(s):	Original Publication: [London] : BioMed Central, 2001-
Ιατρικοί όροι (MeSH):	Protoplasts/metabolism , Protoplasts/cytology , Crocus/genetics , Crocus/cytology , Crocus/metabolism , Sequence Analysis, RNA/methods, Single-Cell Analysis/methods ; Single-Cell Gene Expression Analysis
Περίληψη:	Background: Protoplasts have been widely utilized in tissue culture, single-cell RNA sequencing, and transient gene expression analyses. However, the primary challenge in their preparation lies in the efficient removal of rigid cell walls to obtain high-quality plant cells. The aim of this study is to establish an effective method for protoplast isolation from saffron (Crocus sativus L.), thereby accelerating advances in molecular biology research in this species. Result: Tissue-specific protoplasts were successfully isolated from apical buds, petals, leaves, and roots of saffron, with yields of approximately 8.81 × 10⁶, 1.36 × 10⁷, 7.84 × 10⁵, and 5.69 × 10⁵/g FW, and viabilities of ~ 95%, ~ 94%, ~ 96%, and ~ 61%, respectively. The optimal enzymatic digestion conditions varied by tissue type: apical buds (0.6 M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28 °C, 3 h); roots (0.7 M D-mannitol, 2.0% cellulase, 0.5% macerozyme, 0.5% pectinase, 28 °C, 4 h); petals (0.7 M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28 °C, 3.5 h); and mesophyll (0.7 M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28 °C, 4 h). The quality of the protoplasts was affected by developmental stages and sampling locations. Furthermore, two high-quality single-cell libraries were constructed using protoplasts from non-flowering and flowering apical buds, respectively. We successfully visualized the subcellular localization of GFP in the mesophyll protoplasts. Conclusion: This study presents the first detailed report on protoplast isolation, evaluation, and application in saffron. By overcoming the technical challenges of tissue-specific protoplast isolation from diverse organs, we established a highly efficient method that enables both the development of novel germplasm and mechanistic insights into saffron organogenesis, growth, and development. (© 2026. The Author(s).)
Competing Interests:	Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
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Grant Information:	82304668 National Natural Science Foundation of China; 32570418 National Natural Science Foundation of China; 2026ZF85 Zhejiang Provincial Traditional Chinese Medicine Science and Technology Program
Contributed Indexing:	Keywords: Efficient; Saffron; Single-cell RNA sequencing; Tissue-specific protoplast isolation; Transient gene expression
Molecular Sequence:	BioProject PRJNA1457925; PRJNA1457928
Entry Date(s):	Date Created: 20260709 Date Completed: 20260711 Latest Revision: 20260813
Update Code:	20260814
PubMed Central ID:	PMC13352814
DOI:	10.1186/s12896-026-01187-1
PMID:	42421006
Βάση Δεδομένων:	MEDLINE

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ISSN:	1472-6750
DOI:	10.1186/s12896-026-01187-1