Influence of copper and zinc sulfates on in vitro propagation efficiency of orchids (Dendrobium phalaenopsis): A step towards optimizing clonal propagation protocols

The Orchidaceae family, particularly Dendrobium orchids, plays a significant role in the global floriculture industry, yet developing efficient clonal propagation protocols is challenging. This study investigates the effects of copper sulfate (CuSO4) and zinc sulfate (ZnSO4) supplementation on the in vitro propagation of Dendrobium phalaenopsis orchids. Various concentrations of CuSO4 (2.0, 4.0, 6.0, and 8.0 mg/L) and ZnSO4 (4.0, 8.0, and 12.0 mg/L) were tested individually and in combination using Murashige and Skoog’s (MS) media, supplemented with different auxins and a constant cytokinin level. Dendrobium seeds (0.01 g per culture bottle) served as the starting material, with germination recorded by the first appearance of protocorms. Media with 6.0 mg/L CuSO4 resulted in the fastest protocorm emergence (31.56 days), while the combination of 4.0 mg/L CuSO4 and 12.0 mg/L ZnSO4 proved most effective overall, yielding a shorter germination time of 39 days, the highest shoot production (14 shoots), and the tallest shoots (12.56 cm) one-month post-germination. This combination also achieved the highest shoot multiplication rate (27.33) and enhanced chlorophyll content (2.41 mg/g) three months after transfer to multiplication media. Notably, during root formation, this treatment accelerated root initiation (31.11 days) and improved root vigor. The carryover effects of CuSO4 and ZnSO4 from in vitro to in vivo conditions were evident, with a 92.22% survival rate and no morphological differences from seed-grown plants. These findings highlight the synergistic benefits of CuSO4 and ZnSO4 in establishing an efficient method for orchid propagation with improved success rates in both in vitro and in vivo environments.