Krstić-Milošević Dijana, Banjac Nevena, Janković Teodora, Vinterhalter Dragan, Vinterhalter Branka
Department of Plant Physiology, Institute for Biological Research "Siniša Stanković"-National Institute of the Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia.
Insitute for Medicinal Plants Research "Dr Josif Pančić", Tadeuša Košćuška 1, 11000 Belgrade, Serbia.
Plants (Basel). 2021 Aug 11;10(8):1651. doi: 10.3390/plants10081651.
In vitro shoot culture of the endangered medicinal plant was established from epicotyl explants cultured on MS basal medium with 0.2 mg L 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8--glucoside and bellidifolin-8--glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 μM increased the accumulation of mangiferin, demethylbellidifolin-8--glucoside, and bellidifolin-8--glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.
以在添加0.2 mg/L 6-苄基腺嘌呤（BA）的MS基本培养基上培养的上胚轴外植体建立了濒危药用植物的离体芽培养体系，并首次对其氧杂蒽酮含量进行了评估。获得了5个芽系，它们在增殖率、芽伸长和氧杂蒽酮谱方面没有显著差异。用5 mg/L吲哚-3-丁酸（IBA）处理2天，然后在无植物生长调节剂的1/2 MS液体培养基中培养60天的芽，生根率最高（33.3%）。高效液相色谱分析显示，与野生植物相比，离体培养芽中氧杂蒽酮——芒果苷、雏菊叶龙胆酮、去甲基雏菊叶龙胆酮、去甲基雏菊叶龙胆酮-8-β-葡萄糖苷和雏菊叶龙胆酮-8-β-葡萄糖苷的含量较低。蔗糖、山梨醇和非生物诱导剂水杨酸（SA）、茉莉酸（JA）和茉莉酸甲酯（MeJA）浓度的增加改变了芽的生长和氧杂蒽酮的产生。以233.6 mM的最高浓度施用蔗糖和山梨醇可提高干物质百分比，而100 μM的SA可使芽生长提高2倍。与对照芽相比，蔗糖浓度的增加使芽培养物中氧杂蒽酮的积累提高了2-3倍。100-300 μM的诱导剂几乎同等程度地增加了芒果苷、去甲基雏菊叶龙胆酮-8-β-葡萄糖苷和雏菊叶龙胆酮-8-β-葡萄糖苷的积累，而500 μM最高浓度的MeJA使苷元去甲基雏菊叶龙胆酮和雏菊叶龙胆酮的含量比对照提高了7倍。所得结果有助于该植物的保护，并为大规模芽繁殖和具有药理活性的氧杂蒽酮生产的进一步研究铺平了道路。
