Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants.

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.

Triploid Hybrid Vigor in Above-Ground Growth and Methane Fermentation Efficiency of Energy Willow.

Hybrid vigor and polyploidy are genetic events widely utilized to increase the productivity of crops. Given that bioenergy usage needs to be expanded, we investigated triploid hybrid vigor in terms of the biology of biomass-related willow traits and their relevance to the control of biomethane production. To produce triploid hybrid genotypes, we crossed two female diploid Swedish cultivars (Inger, Tordis) with two male autotetraploid willow (Salix viminalis) variants (PP-E7, PP-E15). Field studies at two locations and in two successive years recorded considerable midparent heterosis (MPH%) in early shoot length that ranged between 11.14 and 68.85% and in the growth rate between 34.12 and 97.18%. The three triploid hybrids (THs) developed larger leaves than their parental cultivars, and the MPH% for their CO2 assimilation rate varied between 0.84 and 25.30%. The impact of hybrid vigor on the concentrations of plant hormones in these TH genotypes reflected essentially different hormonal statuses that depended preferentially on maternal parents. Hybrid vigor was evinced by an elevated concentration of jasmonic acid in shoot meristems of all the three THs (MPH:29.73; 67.08; 91.91%). Heterosis in auxin-type hormones, such as indole-3-acetic acid (MPH:207.49%), phenylacetic acid (MPH:223.51%), and salicylic acid (MPH:27.72%) and benzoic acid (MPH:85.75%), was detectable in the shoots of TH21/2 plants. These hormones also accumulated in their maternal Inger plants. Heterosis in cytokinin-type hormones characterized the shoots of TH3/12 and TH17/17 genotypes having Tordis as their maternal parent. Unexpectedly, we detected abscisic acid as a positive factor in the growth of TH17/17 plants with negative MPH percentages in stomatal conductance and a lower CO2 assimilation rate. During anaerobic digestion, wood raw materials from the triploid willow hybrids that provided positive MPH% in biomethane yield (6.38 and 27.87%) showed negative MPH in their acid detergent lignin contents (from -8.01 to -14.36%). Altogether, these insights into controlling factors of above-ground growth parameters of willow genotypes support the utilization of triploid hybrid vigor in willow breeding to expand the cultivation of short rotation energy trees for renewable energy production.

RING-Type E3 Ubiqitin Ligase Barley Genes (HvYrg1-2) Control Characteristics of Both Vegetative Organs and Seeds as Yield Components.

Previously, studies on RING-type E3 ubiquitin ligases in cereals were preferentially focused on GW2 genes primarily controlling seed parameters in rice and wheat. Here we report cloning two HvYrg genes from barley that share significant homology with rice GW2 gene. In antisense genotypes efficiency of gene silencing varied between genes and transgenic lines: ASHvYrg1: 30-50% and ASHvYrg2: 20-27%. Reduced activity of both genes altered shoot system with increasing number of side shoots. Changes in leaf width, weight, or plant weight and height reached significant levels in some transgenic lines. Lowering expression of the two barley HvYrg genes caused opposite responses in spike development. Plants with ASHvYrg1 gene construct showed earlier heading time and prolonged grain-filling period, while plants from ASHvYrg2 genotype flowered in delay. Digital imaging of root development revealed that down-regulation of HvYrg1 gene variant stimulated root growth, while ASHvYrg2 plants developed reduced root system. Comparison of seed parameters indicated an increase in thousand grain weight accompanied with longer and wider seed morphology. In summary we conclude that in contrast to inhibition of GW2 genes in rice and wheat plants, down-regulation of the barely HvYrg genes caused substantial changes in vegetative organs in addition to alteration of seed parameters.

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis.

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.