Alfalfa (Medicago sativa L.) is the most commonly grown forage legume in North America due to its excellent agronomic qualities. However, its lack of persistence under severe winter conditions reduced its utilisation in the eastern provinces of Canada. Cold tolerance has been shown to be the most important factor in winter survival. Selection for cold tolerance is difficult due to the complexity of field evaluation. A method of selection performed under environmentally-controlled conditions has been used for the identification of genotypes having superior cold tolerance.
Briefly, 1500 plants (cv Apica) were cold hardened for 2 weeks at 2°C and for 2 additional weeks at -2°C before they were subjected to three successive freezing stresses. The freezing temperatures were -10°C for the first and the second stress and -12°C for the third. Plants were selected on the basis of their cold tolerance and the vigour of their regrowth, and intercrossed to produce a first cycle population potentially more cold tolerant (CT1). Similarly a second cycle of selection was performed using CT1 to produce a CT2 population. The original population (Apica) and the two populations derived from our selection (CT1 and CT2) were evaluated for their cold tolerance (LT50), cryoprotective sugar accumulation and for the expression of two genes (msaCIA and msaCIC) regulated by cold temperature (Castonguay et al., 1995; Castonguay et al., 1997).
The results of this study indicated that alfalfa cold tolerance (LT50) and regrowth after exposure to cold temperatures could be improve by a selection based on successive freezing stresses. Cold tolerance progressively increased at each cycle of selection from -16.9oC for Apica to -17.4oC for CT1 and -19.1oC for CT2. A repercussion of this selection was the significant increase of the cryoprotective sugars, stachyose and raffinose, at the crown level. The CT1 and CT2 populations selected for superior cold tolerance accumulated significantly higher levels of those two sugars in February, comparatively to the original population (stachyose : 12.45, 14.21 and 16.94 mg g-1 DW, raffinose : 15.34, 18.70 and 21.73 mg g-1 DW for Apica, CT1 and CT2 respectively). The selection did not modify the expression of the msaCIA and msaCIC genes. Our results indicated that the use of successive freezing stresses is an efficient approach for the development of alfalfa populations with superior cold tolerance.
Castonguay, Y., P. Nadeau, P. Lechasseur, and L. Chouinard. 1995. Differential accumulation of carbohydrates in alfalfa cultivars of contrasting winterhardiness. Crop Sci. 35 : 509-516.
Castonguay, Y., P. Nadeau, S. Laberge, and L.-P. Vézina. 1997. Changes in gene expression in six alfalfa winter hardening conditions. Crop Sci. 37 : 332-342.