The Influencing Factors of In Vitro Regeneration and Bulblet Enlargement of Two Ploidy Lilium longiflorum

Lilium longiflorum is a diploid lily species valued for its tolerance to humid–hot environments and pleasant fragrance. However, its poor cold hardiness and low bulb-forming capacity limit its cultivation. To overcome these deficiencies, autotetraploids were previously generated in our laboratory via somatic doubling. In order to expand the reproductive efficiency of the two, this study optimized in vitro regeneration and bulblet enlargement protocols. We analyzed the effects of various plant growth regulators and sucrose concentrations, alongside the expression of genes related to carbohydrate metabolism and hormone signaling. Results revealed divergent regenerative pathways: diploids favored direct organogenesis (optimal medium: MS + 30 g/L sucrose + 0.5 mg/L 6-BA + 0.2 mg/L NAA + 1.0 mg/L glyphosate), whereas tetraploids thrived via a TDZ-induced callus pathway (1/2 MS + 30 g/L sucrose + 1.0 mg/L NAA + 0.2 mg/L TDZ). During bulblet enlargement, diploids were predominantly regulated by IBA and prone to proliferation (optimal enlargement medium: MS + 60 g/L sucrose + 2.0 mg/L IBA), while tetraploids were sucrose-sensitive and prioritized single-bulb hypertrophy (MS + 60 g/L sucrose + 0.5 mg/L IBA + 0.1 mg/L 6-BA + 0.1 mg/L CPPU). qRT-PCR indicated that LlAGPS1, LlGBSSI, LlSWEET15, LlMYC2, and LlSAUR32 were highly expressed in tetraploids during rapid enlargement (24–36 d), suggesting a role in bulb hypertrophy, whereas upregulated LlSUS4 and LlCWIN3 in diploids correlated with proliferation. The study provides a practical technical reference for the industrialized propagation of high-quality L.longiflorum bulbs and provide a theoretical foundation for understanding ploidy-dependent development in Lilium.