Bottom Cooling During Culture Initiation Increases Survival and Reduces Hyperhydricity in Micropropagated Cannabis Plants

Hyperhydricity is characterized by morphological abnormalities and reduced plant vigour. This study investigated the use of a bottom cooling system (creating an approximate 2 °C temperature differential) during culture initiation to evaluate the impact on hyperhydricity in cannabis micropropagation. Nodal explants from two clonal triploid cultivars known to exhibit hyperhydricity, Higher Education 1 (HED-1) and Higher Education 2 (HED-2), were surface sterilized and placed in culture tubes using standard methods. Treatments included bottom cooling, metal pads without bottom cooling, and standard shelving (controls—no pad). Various morphological and physiological traits were assessed, including a detached leave water loss assay, dry mass, chlorophyll content, and survival rate. Plants cultured with bottom cooling showed significantly higher survival rates, healthier appearance, and improved physiological parameters compared to controls. In contrast, many control explants were hyperhydric with translucent and brittle leaves. Quantitative data revealed significant improvements in fresh weight (54.84% for HED-1 and 51.42% for HED-2), dry weight (36% for HED-1 and 8% for HED-2), chlorophyll fluorescence ratios (7.24% for HED-1 and 9.18% for HED-2), chlorophyll content (18.38% for HED-1 and 20.67% for HED-2), and cuticle/stomate function (30% for HED-1 and 27.27% for HED-2) using bottom cooling. Moreover, our morphological observation showed that almost 85% of control plants were hyperhydric, whereas only 10% of the plants cultured with a bottom cooling system were hyperhydric. This study confirmed that bottom cooling helps reduce the rate and impacts of hyperhydricity in cannabis and significantly improves the survival and quality of in vitro plants.