Improved conservation of callus and rhizome microcuttings of Podophyllum hexandrum germplasm using the slow growth storage approach

Abstract

Abstract Podophyllum hexandrum Royle (syn. Podophyllum emodi Wall.), commonly known as Himalayan mayapple, is an endangered medicinal plant recognized as the primary natural source of podophyllotoxin, a potent compound with anticancer and antiviral properties. In this study, we developed an optimized protocol for the long-term preservation of P. hexandrum germplasm using a slow growth storage (SGS) technique, successfully preserving the viability and genetic stability of both callus and rhizome cuttings. In vitro cultured callus and rhizome microcuttings of P. hexandrum were conserved using the slow growth storage (SGS) technique in Murashige and Skoog (MS) medium under cold conditions (5 °C), supplemented with different concentrations of sucrose, mannitol, and sorbitol in combination with calcium pantothenate and spermidine, to induce slow growth and maintain tissue viability. It was observed that sorbitol (5.5%) combined with spermidine (2 mg L−1), calcium pantothenate (3 mg L−1), and 6-benzylaminopurine (BA) (1.5 mg L−1) showed better efficacy than the mannitol (6.5%) combination in preserving and regenerating callus and rhizome microcuttings. In contrast, the combination with sucrose (6.5%) was the least effective. This study developed an effective in vitro protocol for conserving P. hexandrum, an endangered medicinal plant, through slow growth storage. A medium containing sorbitol, mannitol, spermidine (2 mg L−1), and calcium pantothenate (2 mg L−1) enhanced tissue viability, stress tolerance, and long-term survival of callus and rhizome explants while maintaining genetic stability during cold storage. These findings suggest that this protocol provides a reliable approach for the ex-situ conservation of P. hexandrum, ensuring the availability of genetically stable plant material for future research and medicinal use. This is the first report on the germplasm conservation of callus and rhizome microcuttings of P. hexandrum grown in Pakistan using the slow growth technique.