A Whole-Plant Screening Test to Select Freezing-Tolerant and Low-Dormant Genotypes.

Winter survival is a determinant factor for the persistence of perennials grown in northern climates. High winter survival cultivars, however, have lower yield due to their early transition into a dormant state in the fall. Here we describe a whole plant assay entirely performed indoor in growth chambers and walk-in freezers to identify low-dormant genotypes with superior freezing tolerance within populations of open pollinated species. Three successive freezing stresses are applied to a broad base of 3000 genotypes to progressively eliminate 97% of the population and to retain only the 3% best performing genotypes. This approach can be used to generate recurrently selected populations in different species.

Biochemical and molecular responses during overwintering of red clover populations recurrently selected for improved freezing tolerance.

Low freezing tolerance reduces the persistence of red clover under northern climate. The incidence of winter damages in perennial crops could increase in the future due to the adverse effects of the predicted warmer fall temperature on plant cold acclimation. To accelerate breeding progress, two cultivars of red clover Christie (C-TF0) and Endure (E-TF0) were exposed to a recurrent selection protocol for freezing tolerance performed indoor. New populations were obtained after five (C-TF5 and E-TF5), six (C-TF6 and E-TF6), and seven (C-TF7 and E-TF7) cycles of recurrent selection. These populations were overwintered under natural conditions and monitored for freezing tolerance and cold-induced molecular traits. Freezing tolerance was improved by up to 6 °C in recurrently selected populations when compared to initial cultivars confirming that further progress are achieved with advanced cycles of selection. Monthly analysis of biochemical changes shows that higher starch concentrations at the onset of the fall hardening period are contributing to the acquisition of superior freezing tolerance through its impact on sucrose accumulation. They also contribute to the vigor of spring regrowth by sustaining more pinitol and proline synthesis. Larger concentrations of these metabolites in populations with higher levels of freezing tolerance (TF7) hint at their involvement in winter survival of red clover. Among genes differentially expressed in response to both cold acclimation and recurrent selection, a concomitant cold induction of APPR9 and cold repression of 1-aminocyclopropane-carboxylate synthase suggests a link between the repression of a pathway regulated by ethylene and the improvement of freezing tolerance in red clover.

Effect of photoperiod prior to cold acclimation on freezing tolerance and carbohydrate metabolism in alfalfa (Medicago sativa L.).

Cold acclimation proceeds sequentially in response to decreases in photoperiod and temperature. This study aimed at assessing the impact of photoperiod prior to cold acclimation on freezing tolerance and related biochemical and molecular responses in two alfalfa cultivars. The fall dormant cultivar Evolution and semi-dormant cultivar 6010 were grown in growth chambers under different photoperiods (8, 10, 12, 14 or 16h) prior to cold acclimation. Freezing tolerance was evaluated as well as carbohydrate concentrations, levels of transcripts encoding enzymes of carbohydrate metabolism as well as a K-3dehydrin, before and after cold acclimation. The fall dormant cultivar Evolution had a better freezing tolerance than the semi-dormant cultivar 6010. The effect of photoperiod prior to cold acclimation on the level of freezing tolerance differed between the two cultivars: an 8h-photoperiod induced the highest level of freezing tolerance in Evolution and the lowest in 6010. In Evolution, the 8h-induced superior freezing tolerance was associated with higher concentration of raffinose-family oligosaccharides (RFO). The transcript levels of sucrose synthase (SuSy) decreased whereas those of sucrose phosphatase synthase (SPS) and galactinol synthase (GaS) increased in response to cold acclimation in both cultivars. Our results indicate that RFO metabolism could be involved in short photoperiod-induced freezing tolerance in dormant alfalfa cultivars.

Alleviation of Drought Stress and Metabolic Changes in Timothy (Phleum pratense L.) Colonized with Bacillus subtilis B26.

Drought is a major limiting factor of crop productivity worldwide and its incidence is predicted to increase under climate change. Drought adaptation of cool-season grasses is thus a major challenge to secure the agricultural productivity under current and future climate conditions. Endophytes are non-pathogenic plant-associated bacteria that can play an important role in conferring resistance and improving plant tolerance to drought. In this study, the effect of inoculation of the bacterial endophyte Bacillus subtilis strain B26 on growth, water status, photosynthetic activity and metabolism of timothy (Phleum pratense L.) subjected to drought stress was investigated under controlled conditions. Under both drought-stress and non-stressed conditions, strain B26 successfully colonized the internal tissues of timothy and had a positive impact on plant growth. Exposure of inoculated plant to a 8-week drought-stress led to significant increase in shoot and root biomass by 26.6 and 63.8%, and in photosynthesis and stomatal conductance by 55.2 and 214.9% respectively, compared to non-inoculated plants grown under similar conditions. There was a significant effect of the endophyte on plant metabolism; higher levels of several sugars, notably sucrose and fructans and an increase of key amino acids such as, asparagine, glutamic acid and glutamine were recorded in shoots and roots of colonized plants compared to non-colonized ones. The accumulation of the non-protein amino acid GABA in shoots of stressed plants and in roots of stressed and unstressed plants was increased in the presence of the endophyte. Taken together, our results indicate that B. subtilis B26 improves timothy growth under drought stress through the modification of osmolyte accumulation in roots and shoots. These results will contribute to the development of a microbial agent to improve the yield of grass species including forage crops and cereals exposed to environmental stresses.

Genomic Prediction of Biomass Yield in Two Selection Cycles of a Tetraploid Alfalfa Breeding Population.

Alfalfa (Medicago sativa L.) is a widely planted perennial forage legume grown throughout temperate and dry subtropical regions in the world. Long breeding cycles limit genetic improvement of alfalfa, particularly for complex traits such as biomass yield. Genomic selection (GS), based on predicted breeding values obtained using genome-wide molecular markers, could enhance breeding efficiency in terms of gain per unit time and cost. In this study, we genotyped tetraploid alfalfa plants that had previously been evaluated for yield during two cycles of phenotypic selection using genotyping-by-sequencing (GBS). We then developed prediction equations using yield data from three locations. Approximately 10,000 single nucleotide polymorphism (SNP) markers were used for GS modeling. The genomic prediction accuracy of total biomass yield ranged from 0.34 to 0.51 for the Cycle 0 population and from 0.21 to 0.66 for the Cycle 1 population, depending on the location. The GS model developed using Cycle 0 as the training population in predicting total biomass yield in Cycle 1 resulted in accuracies up to 0.40. Both genotype × environment interaction and the number of harvests and years used to generate yield phenotypes had effects on prediction accuracy across generations and locations, Based on our results, the selection efficiency per unit time for GS is higher than phenotypic selection, although accuracies will likely decline across multiple selection cycles. This study provided evidence that GS can accelerate genetic gain in alfalfa for biomass yield.