Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Associate Pro-Vice Chancellor and Senior Lecturer, Crop Production Technology, Royal Agricultural University, Cirencester Gloucestershire, GL7 6JS, United Kingdom.
Gene. 2021 Mar 30;774:145424. doi: 10.1016/j.gene.2021.145424. Epub 2021 Jan 9.
The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20 mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4 mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310 µg Gal/g FW) and bulb (980-1150 µg Gal/g FW) than the culture derived samples; callus (1.0-7.0 µg Gal/g FW) and bulblets (12-215 µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpOOMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.
卡尔顿水仙含有石蒜科生物碱，如加兰他敏、石蒜碱、石蒜伦碱、水仙碱、那可丁等，具有治疗早中期阿尔茨海默病、癌症、肿瘤等药物特性。利用植物体外系统进行生物碱生物合成已被视为药物发现的一种工具，相关途径开始被理解，但仍远未完成。因此，本研究强调观察在田间生长的鳞茎和发育中的细胞培养系统中，参与石蒜科生物碱生物合成途径的假定基因的相对表达。在含有生长调节剂的 MS 培养基上，从卡尔顿双胞胎鳞片外植体中开发组织培养。高生长素（20mg/L NAA）的 MS 培养基最适合愈伤组织生长和维持，而低生长素（4mg/L NAA）和 MS 基本培养基的培养基则产生最大数量的小鳞茎。田间组织显示出更高的加兰他敏含量；即基盘（1050-1310μg Gal/g FW）和鳞茎（980-1150μg Gal/g FW）高于培养物衍生样品；愈伤组织（1.0-7.0μg Gal/g FW）和小鳞茎（12-215μg Gal/g FW）的鲜重（FW）基础。研究中样品的 GC-MS 色谱图也显示存在其他重要生物碱，即石蒜碱、石蒜伦碱、石蒜宁、海曼胺、刺桐碱、石蒜碱和塔齐汀。从体外愈伤组织、小鳞茎和田间生长的鳞茎、基盘中提取的 RNA 用于 PCR 检测假定基因的相对表达；P450、PAL、TYDC 和 NpOOMT 均以肌动蛋白为内参。所选 P450s 和 TYDC 的转录本在体外和体内组织中均有表达。PAL 在愈伤组织中的表达高于田间样本。NpN4OMT 的表达在田间样本中明显高于体外组织。因此，体外组织可能是从植物中可重复、方便地提取生物碱的良好来源。
