There are numerous challenges associated with the large-scale production of walnut In vitro-plantlets. It is imperative to develop new environmental control systems for its In vitro propagation. Additionally, there is a lack of knowledge regarding the impacts of lighting systems on the morpho-physiological traits and biomass accumulation in walnut tissue-cultured explants. In this study, walnut nodal shoots were subjected to eight different light spectra, including white, blue, red, green, far-red, blue-red combination, red-far-red combination, and a fluorescent lamp serving as a control, over a period of 28 days. The results indicated that combined spectra treatments, such as blue-red and red-far-red, led to improved biomass accumulation (total fresh and dry weight) compared to other monochromatic light spectral treatments. Furthermore, Light-emitting diode (LED) treatments had a discernible impact on the morpho-physiological traits of walnut In vitro-explants. Specifically, white light spectra enhanced Specific leaf area (SLA), while the green light spectra increased leaf water content (LWC) when compared to other light treatments. Additionally, the application of far-red light elevated leaf mass area (LMA) and water content per unit leaf area (LWCA). The findings of this study demonstrate that the quality, morphological, and growth characteristics of In vitro explants of walnut can be enhanced by utilizing specific light spectra. Manuscript profile