Abstract

In vitro regeneration methods that efficiently produce entire plants from single cells are crucial for clonal propagation and genetic engineering. Shoots can be directly induced from mature organs like leaves, bypassing callogenesis in some dicotyledonous plants, like tobacco. Tissue culture exposes plant tissues to specific nutrients, light, and hormones in sterile, in vitro conditions to produce genetically identical plants rapidly. Nonetheless, the technique encounters various bottlenecks, notably hyperhydricity or vitrification, a morphological and physiological disorder that involves excessive hydration and abnormal shoot development, causing a glassy, water-soaked appearance due to excessive water uptake. To counter this, we employed RMOP medium with varying phytagel concentrations (1.3g/L, 2.6g/L, 3.9g/L and 5.2g/L) and added different levels of sugar-alcohols e.g., mannitol and sorbitol (250mg/L, 500mg/L, and 750mg/L). Optimal vitrification reduction occurred at 3.9g/L phytagel, though 5.2g/L reduced vitrification but with stunted growth. In alternative media, 2.6g phytagel with 250mg/L mannitol displayed reduced vitrification with robust growth. In conclusion, 3.9g phytagel fosters plant growth, yet its expense raises concern.

Keywords: Tissue culture, Hyperhydricity, Phytagel, Sorbitol, Manitol