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.
Keywords: Antioxidant defense proteins; Dead cell analysis; In vitro stress; Long-term cultures.