The efficiency of in vitro regeneration in litchi (Litchi chinensis Sonn.) is highly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins. This study evaluated the effects of phenylacetic acid (PAA) and its derivatives—4-chlorophenylacetic acid (CPA) and 4-iodophenylacetic acid (IPA)—on callus proliferation, somatic embryogenesis, and plantlet regeneration in ‘Feizixiao’ litchi, as well as to establish an efficient regeneration protocol. The inclusion of CPA or IPA in callus proliferation medium significantly enhanced the proliferation rate, with 20 mg·L⁻¹ CPA being the most effective. The highest number of somatic embryos per gram of fresh embryonic callus weight (gFW⁻¹) (1131 embryos·gFW⁻¹) was observed with 40 mg·L⁻¹ PAA in proliferation medium. The addition of 10 mg·L⁻¹ IPA to the proliferation medium yielded the highest plantlet regeneration rate (50 plantlets·gFW⁻¹). Supplementing the somatic embryo induction medium with 5 mg·L⁻¹ PAA resulted in 460 somatic embryos·gFW⁻¹ and 86 regenerated plantlets·gFW⁻¹. These findings indicate that PAA and its derivatives are effective PGRs for the in vitro regeneration of litchi, providing a valuable protocol for the propagation of elite cultivars.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a vital exogenous auxin for the induction and proliferation of litchi embryogenic callus. At present, its molecular regulation mechanism remains unclear. In this study, transcriptome sequencing samples were selected based on different cell growth phenotypes observed in ‘Feizixiao’ litchi embryogenic callus cultured in liquid medium with or without 2,4-D. By integrating transcriptome profiling with weighted gene co-expression network analysis (WGCNA), we identified key genes and signaling pathways dynamically responsive to 2,4-D concentration changes. We identified 558 commonly differentially expressed genes (DEGs), of which 117 were up-regulated and 387 were down-regulated; functional enrichment analysis revealed significant enrichment in the “plant hormone signal transduction” and “phenylpropanoid biosynthesis” pathways. In the former pathway, genes such as AUX28, GH3.17, GH3.6, and ARR5 were up-regulated; in the latter, by comparison, β-glucosidase 47 and Peroxidase 61 exhibited increased expression levels induced by 2,4-D. Furthermore, among these DEGs, 57 transcription factors belonged to 24 families. Notably, VRN1, FEZ, and DOF5.4 were significantly and rapidly induced by 2,4-D. WGCNA results demonstrated a significant positive correlation between the yellow module and 2,4-D treatment. Small heat shock protein (sHSP) genes constituted the core hub genes in the yellow module. Through Venn analysis of DEGs and key modules, 38 cross-genes were identified, of which non-specific lipid-transfer protein-like genes (nsLTP) were found to be specifically up-regulated without 2,4-D. The transcription factors and genes identified work in synergy to ensure the formation and sustained proliferation of embryogenic callus by precisely regulating the dynamic balance of auxin and cytokinin within cells and maintaining the stability of cell structure. Our findings provide a crucial theoretical foundation for understanding the molecular mechanism of 2,4-D in regulating litchi embryogenic callus proliferation.

The efficiency of in vitro regeneration in litchi (Litchi chinensis Sonn.) is highly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins. Although the effects of various growth regulators on the in vitro regeneration have been studied, more types of auxins need to be investigated for their influence on somatic embryogenesis and plantlet regeneration of litchi. In this study, the effects of phenylacetic acid (PAA) and its derivatives—4-chlorophenylacetic acid (CPA) and 4-iodophenylacetic acid (IPA)—were assessed in relation to callus proliferation, somatic embryogenesis (SE), and plantlet regeneration in ‘Feizixiao’ litchi. The inclusion of CPA or IPA in callus proliferation medium significantly enhanced the proliferation rate, with 20 mg·L⁻¹ CPA the most effective treatment. Embryonic calli following CPA and IPA treatments exhibited a deeper yellow color, smaller cell clusters, and thicker cytoplasm compared to the control and PAA-treated calli. The highest number of somatic embryos per gram of fresh embryonic callus weight (gFW⁻¹) (1131 embryos·gFW⁻¹) was observed when 40 mg·L⁻¹ PAA was added to the proliferation medium. The addition of 10 mg·L⁻¹ IPA to the proliferation medium yielded the highest plantlet regeneration rate (50 plantlets·gFW⁻¹). The optimal supplementation of the somatic embryo induction medium was 5 mg·L⁻¹ PAA, which resulted in 460 somatic embryos·gFW⁻¹ and 86 regenerated plantlets·gFW⁻¹. These findings indicate that PAA and its derivatives are effective PGRs in the in vitro regeneration of litchi, and provide a valuable protocol for the propagation of elite cultivars.