Various strategies have been developed globally to conserve germplasm by propagating plants. One important technique is in vitro propagation and preservation through tissue culture. In many investigated plants, the long in vitro conservation is plagued with several limitations like genetic variations, developmental errors in cells or tissues due to induced stress. This provoked us to conduct a study of Catharanthus roseus culture maintained for over fourteen long years and a newly established 8-month-old culture. The present study investigated and compared the two tissue types differing by their age. The biomass accumulation, the biochemical differences of the two, dead cell analysis with aging via confocal microscopy, and liquid chromatography-mass spectroscopy (LC-MS)-based proteomic differences were studied in old and newly established Catharanthus culture. The proteomic study reveals more than 120 upregulated or high abundance proteins in old culture as compared to newly established Catharanthus. The identified upregulated proteins are stress protein 69, heat shock proteins (HSP), isocitrate dehydrogenase, pyruvate dehydrogenase, and others. These proteins had an association with antioxidant activities, related to stress, and a few are linked to respiration. Our study reveals the presence of a robust antioxidant defense mechanism, i.e., 51.94%, 78.8%, and 61% higher SOD, APX, and CAT activities in older cultures (O) as compared to newly established tissues (N), which perhaps act against stress and may play a key role in ameliorating negative impacts of long-term in vitro conditions. The inherent strong antioxidant defense system in old cultures added resilience and enabled the culture to revive growth quickly (within 1–2 days) following transfer to new medium as compared to new culture (7–10 days). The biomass accumulation was more (37.08 %) in old tissues as compared to new culture. The 2C DNA or genome size of C. roseus especially the 14-year-old culture–derived regenerated plant was measured by flow cytometry. The 2C DNA size of this Catharanthus (old culture) plant is 1.516 pg, which is very similar to new culture–derived plants’ and field-grown plants’ genome size. No anomaly in genome size was noted in plants of old culture, as opposed to common perception.

全球已经制定了各种策略来通过繁殖植物来保护种质。一项重要的技术是通过组织培养进行体外繁殖和保存。在许多研究的植物中，长期的体外保存受到一些限制的困扰，例如遗传变异、诱导应激导致的细胞或组织发育错误。这激发了我们对长春花进行研究14 年以上的文化和新建立的 8 个月大的文化。本研究调查并比较了两种不同年龄的组织类型。在旧的和新建立的长春花培养物中研究了生物量积累、两者的生化差异、通过共聚焦显微镜分析老化的死细胞以及基于液相色谱-质谱 (LC-MS) 的蛋白质组学差异。蛋白质组学研究显示，与新建立的长春花相比，旧培养物中有 120 多种上调或高丰度的蛋白质. 鉴定出的上调蛋白是应激蛋白 69、热休克蛋白 (HSP)、异柠檬酸脱氢酶、丙酮酸脱氢酶等。这些蛋白质与抗氧化活性有关，与压力有关，还有一些与呼吸有关。我们的研究揭示了强大的抗氧化防御机制的存在，即与新建立的组织 (N) 相比，旧培养物 (O) 中的 SOD、APX 和 CAT 活性分别高 51.94%、78.8% 和 61%，这可能会起作用对抗压力，并可能在减轻长期体外条件的负面影响方面发挥关键作用。与新培养物（7-10 天）相比，旧培养物中固有的强大抗氧化防御系统增加了弹性，并使培养物在转移到新培养基后（1-2 天）能够快速恢复生长。生物量积累更多（37. 08 %) 在旧组织中与新培养物相比。2C DNA 或基因组大小C. roseus尤其是 14 岁的培养再生植物通过流式细胞术进行了测量。这种长春花（旧培养）植物的 2C DNA 大小为 1.516 pg，与新培养植物和田间种植植物的基因组大小非常相似。与普遍看法相反，在古老文化的植物中没有发现基因组大小异常。