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. 2025 Jul 2;25(1):825.
doi: 10.1186/s12870-025-06835-w.

The mechanism of PGR-free direct organogenesis in Lycium ruthenicum leaf explants revealed by RNA-Seq and phytohormone metabolome co-analysis

Lujia Li  1 , Yucheng Wang  1 , Wenhui Liu  1 , Yue Gao  1 , Jiawen Wang  1 , Huifang Lu  1 , Weiman Xu  1 , Qin-Mei Wang  2
Affiliations

The mechanism of PGR-free direct organogenesis in Lycium ruthenicum leaf explants revealed by RNA-Seq and phytohormone metabolome co-analysis

Lujia Li et al. BMC Plant Biol. .

Abstract

Background: Lycium ruthenicum is an economically important shrub known for its resistance to drought and saline-alkali conditions. This study identified a particular L. ruthenicum clone whose leaf explants can undergo direct organogenesis on plant growth regulator (PGR)-free media, and both rooting and shooting abilities of the leaf-tip explants were significantly greater than those of the leaf-middle explants. However, the underlying mechanisms remain unclear.

Results: RNA-Seq analysis revealed that the differentially expressed genes (DEGs) associated with rooting and shooting in both leaf-tip and leaf-middle explants were enriched in the Plant hormone signal transduction KEGG pathway. For the first time, we identified 16 and nine differentially accumulated metabolites (DAMs) linked to direct root and shoot organogenesis from leaf explants, respectively. The stronger direct rooting ability observed in leaf-tip explants was associated with (i) up-regulation of sucrose synthase (SUS), scarecrow-like 21 (SCL21), transport inhibitor response 1 (TIR1), and auxin response factor (ARF) genes; (ii) up-regulation of IAA, IAA-Leu-Me, and BAP; (iii) down-regulation of eight DAMs. Moreover, the enhanced direct shooting ability in leaf-tip explants was correlated with (i) 13 DAMs including upregulated JA and JA-ILE, (ii) up-regulated SUS and (iii) down-regulated jasmonate ZIM domain 2 (JAZ2) gene. Notably, JAZ2 and these 13 DAMs represent newly discovered factors associated with the stronger shooting ability of leaf explants. Additionally, this study conducted correlation analyses between DAMs and DEGs related to rooting and shooting in leaf explants, as well as the enhanced rooting and shooting capacities of leaf-tip explants.

Conclusions: This study identifies the key DEGs and DAMs correlated with the direct organogenesis of L. ruthenicum leaf explants. Building upon these findings, a mechanistic model for the stronger direct organogenesis observed in leaf-tip explants was established. These findings offer valuable theoretical guidance for optimizing direct organogenesis systems in plant leaves.

Keywords: Black wolfberry; Direct organogenesis; Leaf explant; Plant hormone; Transcriptome.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The in vitro plants of tissue culture clone 11 of Lycium ruthenicum from Key Laboratory of Forest Tree Genetics, Breeding and Cultivation of Liaoning Province (41° 49′ 25’’ N; 123° 34′ 10’’ E, 60 m a. s. l.) were used as research materials. This study complies with relevant institutional, national, and international guidelines and legislation. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
PGR-free direct organogenesis of the L. ruthenicum leaf-tip explants. a The leaf-tip explants rooting on the 15th day after inoculation (arrow indicates root); b The leaf-tip explants shooting at the base of the root (arrow indicates explant); c Surviving transplanted plants
Fig. 2
Fig. 2
The DEGs of various comparison groups of the L. ruthenicum leaf explant RNA-Seq. a Number of DEGs in different comparison groups; b The up- and down-regulated DEGs between the same types of leaf explants at adjacent periods; c The up- and down-regulated DEGs between the leaf-tip and leaf-middle explants at the same periods
Fig. 3
Fig. 3
L. ruthenicum DEGs and DAMs (RTE vs. RME) annotated to the Plant hormone signal transduction KEGG pathway. DEGs of red boxes were up-regulated, of green boxes were down-regulated in the RTE. Blue boxes suggested that there were both up- and down-regulated DEGs in the RTE. Red dot indicate the up-regulated DAM in the RTE
Fig. 4
Fig. 4
L. ruthenicum DEGs and DAMs (STE vs. SME) annotated to the Plant hormone signal transduction KEGG pathway. DEGs of red boxes were up-regulated, of green boxes were down-regulated in the RTE. Blue boxes suggested that there were both up- and down-regulated DEGs in the RTE. Red dots indicate the up-regulated DAMs in the STE
Fig. 5
Fig. 5
Expression levels of the 11 DEGs in L. ruthenicum revealed by qRT-PCR (a, c) and RNA-Seq (b, d)
Fig. 6
Fig. 6
Venn diagram of DAMs in different comparison groups of L. ruthenicum. a and b Venn diagram of DAMs between the same types of the leaf explants at adjacent periods; c Venn diagram of DAMs between the leaf-tip and leaf-middle explants at the same periods
Fig. 7
Fig. 7
A hypothesis model of stronger direct organogenesis (rooting and shooting) ability in the L. ruthenicum leaf-tip explants. An asterisk (*) or pound sign (#) denotes the first reported DAM/DEG associated with enhanced rooting or shooting capacity in leaf explants, respectively. Suc, Sucrose; UDP-Glc, UDP-glucose
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