Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63–19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division–elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin–cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.
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