Supplementary file 1_Indirect regeneration of long-term callus cultures in gladiolus: a protocol for stable genetic fidelity.docx

Gladiolus is an important and economically valued ornamental plant grown worldwide. One of the major challenges in its micropropagation is maintaining genetic stability during indirect regeneration and long-term callus maintenance. The objective of this study was to develop an optimized indirect shoot regeneration protocol for three commercial Gladiolus cultivars with consistent genetic traits. Callus initiated from the basal part of extended mother corm sprout (EMCS) explants in MS medium supplemented with 2 mgL-1 2,4-D, 2 mgL-1 NAA and 1 mgL-1 BAP. The synthesis of phenolic compounds was effectively controlled by the addition of 150 mgL-1 ascorbic acid, 100 mgL-1 citric acid, and 500 mgL-1 activated charcoal. This medium led to an 80% decrease in the accumulation of phenolic compounds across all cultivars in comparison to the control. For shoot regeneration, calli which were maintained over the long term were transferred to MS medium supplemented with 2 mgL-1 BAP, 2 mgL-1 Kin and 0.25 mgL-1 NAA. This significantly enhanced shoot regeneration percentage (95.55%) and number (39.44 shoots per explant). Additionally, cormel formation was significantly enhanced (16.66 cormels per explant) at the base of regenerated plantlets using MS medium containing 9% sucrose and 2 mgL-1 indole-3-acetic acid, without any cormel formation in the control. Cormels were effectively acclimatized in the greenhouse with 100% survival rate. To demonstrate genetic stability, regenerated plantlets were evaluated by flow cytometry and Inter Simple Sequence Repeat (ISSR) markers, verifying their genetic identification with the mother plants. This study provides a reliable and scalable protocol for the commercial micropropagation of gladiolus, with promising applications in breeding programs that aim at transferring desirable traits such as disease resistance or specific floral features.

唐菖蒲（Gladiolus）是全球广泛栽培的重要经济观赏植物。其微繁殖的核心挑战之一，是在间接再生与长期愈伤组织维持过程中维持遗传稳定性。本研究旨在为三个商业唐菖蒲品种开发优化的间接芽再生方案，以获得性状一致的再生植株。以伸长母球芽外植体（extended mother corm sprout, EMCS）基部为外植体，在添加2 mg·L⁻¹ 2,4-二氯苯氧乙酸（2,4-D）、2 mg·L⁻¹ α-萘乙酸（NAA）及1 mg·L⁻¹ 6-苄氨基腺嘌呤（BAP）的MS培养基中诱导愈伤组织。通过添加150 mg·L⁻¹抗坏血酸、100 mg·L⁻¹柠檬酸与500 mg·L⁻¹活性炭，可有效控制酚类物质的合成；相较于对照组，该培养基可使所有供试品种的酚类物质积累量降低80%。将长期维持培养的愈伤组织转移至添加2 mg·L⁻¹ BAP、2 mg·L⁻¹激动素（Kin）及0.25 mg·L⁻¹ NAA的MS培养基中进行芽再生，可显著提升芽再生率（95.55%）与单外植体芽数（39.44个芽）。此外，使用添加9%蔗糖与2 mg·L⁻¹吲哚-3-乙酸（IAA）的MS培养基，可显著促进再生幼苗基部形成子球茎，单外植体可形成16.66个子球茎，而对照组未出现任何子球茎。所获子球茎可在温室中有效驯化，成活率达100%。为验证再生植株的遗传稳定性，本研究通过流式细胞术（flow cytometry）与简单序列重复区间扩增多态性（Inter Simple Sequence Repeat, ISSR）标记对再生幼苗进行评估，证实其与母株遗传一致性。本研究为唐菖蒲的商业化微繁殖提供了可靠且可规模化的技术方案，在旨在转移抗病性或特定花部性状等优良性状的育种项目中具有良好应用前景。