Embryogenic callus suspension culture and plant regeneration in litchi (Litchi chinensis Sonn.)

【Objective】Litchi (Litchi chinensis Sonn.) is an economically valuable fruit species in southern China. Breeding new variety is very important for meeting consumer needs and improving economic efficiency. However, the species' allogamous reproductive strategy drives extreme genomic heterozygosity, presenting formidable barriers to precision breeding and genetic manipulation. To address these constraints, cell suspension culture emerges as a transformative biotechnological platform, offering dual strategic capacities. It would generate homogeneous cell populations essential for protoplast isolation, somatic hybridization, and transgenic development; Moreover, it would enable industrialscale biosynthesis of high- value phytochemicals. This study systematically optimized critical parameters governing litchi anther-derived callus growth in suspension systems, including macro-/micronutrient formulations (inositol, 2,4-D, sucrose), organic growth modulators [lactalbumin hydrolysate (LH), coconut water (CW)], and inoculation density thresholds. The multifactorial optimization strategy aimed to establish a robust, scalable suspension culture protocol yielding high-quality embryogenic cultures, thereby creating foundational biomaterials for advanced applications in litchi protoplast technology and cross-species genetic engineering initiatives.【Methods】Anther-derived embryogenic callus of Feizixiao litchi was used as the initial material. Suspension cultures were established in liquid MS medium under dark conditions (25±2 ℃, 120 r·min-1 ). A series of experiments were conducted to optimize culture conditions: Inositol (0-0.2 g·L-1 ), 2,4-D (0.1-2 mg·L-1 ), sucrose (10-40 g·L-1 ), LH (0-0.4 g·L-1 ), CW (0-200 mL·L-1 ), and inoculum densities (10-40 g·L-1 ) were tested for their effects on cell proliferation, single- cell viability, and biomass. Suspension cultures were subculture every 4 days, filtered through a 0.85 mm sieve, and monitored for growth parameters [fresh weight (FW), dry weight (DW), packed cell volume (PCV), pH, conductivity). Embryogenic callus proliferation, somatic embryo differentiation, maturation, and plant regeneration were induced using specific solid media supplemented with growth regulators (NAA, ZT, ABA) and high sucrose concentrations.【Results】Single-factor trial results showed that the effects of inositol, 2,4-D, sucrose, CW and initial inoculum density on the callus morphology (single cell, little cell group and density) and proliferation rate (FW, DW) were significant (P＜0.05), while the effect of LH was not significant. The embryogenic suspension system of litchi was established. The optimal treatment regimen was as follows: Anther embryogenic callus of Feizixiao litchi was inoculated into liquid medium containing MS + inositol 0.15 g ·L-1 + 2,4-D 1 mg ·L-1 + sugar 20 g · L- 1 + CW 50 mL · L- 1 . The initial inoculum density was 1.5 g per 50 mL of culture medium. The suspension culture was maintained under dark conditions at (25±2) ℃ with 120 rpm orbital shaking. The subculturing was performed every 4 days for 3-4 cycles, followed by filtration through 0.85 mm mesh, after that the subculturing interval was extended to 7 days. After approximately 20 days of total cultivation, an optimal suspension cell line was obtained, characterized by predominantly round or nearround cell morphology, excellent dispersion and homogeneity, rapid cell division and growth, abundant and active cytoplasmic structures, and a clear, transparent suspension. The growth parameters of the litchi suspension culture (single- cell yield, biomass accumulation, and cell packed volume) followed a sigmoidal ("S"-shaped) growth curve. The culture progression was divided into three phases: lag phase (days 1-2): minimal single-cell dispersion, slow increases in FW, DW and packed cell volume (PCV), logarithmic growth phase (days 3-6): rapid cell division with significant increases in single-cell count, FW, DW, and cell density, and stationary phase (days 7-16): stabilized cell growth with gradual increases in FW, DW and PCV. Beyond this phase, the prolonged culture led to cell senescence and fragmentation, accompanied by sharp declines in viable cell count and survival rate, with minimal increases in FW, DW and PCV. The pH of the suspension initially showed a slight increase followed by continuous decline throughout the culture period. The electrical conductivity exhibited a consistent downward trend during the entire suspension culture process.【Conclusion】An optimized embryogenic suspension system was successfully established for Feizixiao litchi using MS medium supplemented with inositol 0.15 g · L-1 , 2,4-D 1 mg·L-1 , sucrose 20 g·L-1 , and CW 50 mL·L-1 . This system exhibited high uniformity, rapid proliferation, and stable embryogenic potential, enabling efficient somatic embryo production and plant regeneration. The findings could advance litchi biotechnology applications, including mutagenesis, protoplast isolation, and synthetic seed production, and offer a reference for recalcitrant woody species.