Abstract
Abstract
Phalaenopsis hybrida
is one of the most commercially valuable ornamental orchids worldwide. Conventional micropropagation using pedicel axillary buds suffers from limited explant availability and low multiplication efficiency. Leaf-based in-vitro regeneration enables repeated harvesting of explants from donor plants, representing a promising approach for mass seedling production and gene-editing-assisted breeding. Nevertheless, existing regeneration protocols are hampered by three major limitations: severe explant browning, poor callus regeneration efficiency, and the lack of integrated culture media covering the complete workflow of callus induction, adventitious bud differentiation and rooting. In this study, sterile leaves of the widely cultivated cultivar ‘Big Chili’ were used as explants. We systematically evaluated the effects of thidiazuron (TDZ) concentration, combined supplementation of citric acid and α-naphthaleneacetic acid (NAA), as well as interactive effects between leaf age and explant inoculation orientation. Single-factor gradient experiments together with 2 × 2 two-way analysis of variance (ANOVA) were performed to select optimal culture media and standardized cultivation parameters for each developmental stage, and a complete leaf regeneration pipeline was established. The optimal medium for callus induction was 1/2 MS medium supplemented with 3mg·L⁻¹ TDZ, 150 mL·L⁻¹ coconut water, 0.1 g·L⁻¹ citric acid, 20 g·L⁻¹ sucrose and 5.5 g·L⁻¹ agar (pH 5.8). Two-way ANOVA revealed a significant positive interaction between leaf age and inoculation orientation. The highest callus induction rate was achieved when newly-expanded young leaves were cultured with the adaxial surface upwards under continuous darkness at 25°C for 90 days. The best medium for adventitious bud differentiation was 1/2 MS medium containing 1.0mg·L⁻¹ 6-benzylaminopurine (6-BA), 0.4mg·L⁻¹ NAA, 2.5mg·L⁻¹ adenine, 150 mL·L⁻¹ coconut water, 0.5 g·L⁻¹ activated carbon, 20 g·L⁻¹ sucrose and 5.5 g·L⁻¹ agar (pH 5.8), yielding an adventitious bud differentiation rate of 39.82%. For rooting and plantlet strengthening, the optimum medium was MS medium supplemented with 1.0mg·L⁻¹ 6-BA, 0.4mg·L⁻¹ NAA, 150 mL·L⁻¹ coconut water, 1.0 g·L⁻¹ activated carbon, 20 g·L⁻¹ sucrose and 5.5 g·L⁻¹ agar (pH 5.8), with a rooting rate of 50.81%. This work defines the critical threshold concentration of TDZ and the coordinated regulatory roles of leaf age and inoculation mode in governing leaf-explant dedifferentiation. Our findings advance the fundamental understanding of cell totipotency within Orchidaceae, and provide a standardised technical protocol for industrial-scale seedling production and genetic transformation of
P. hybrida
.