Location: Forage and Range ResearchTitle: Physiological and molecular characterization of lucerne (Medicago sativa L.) germplasm with improved seedling freezing tolerance
|ANOWER, M - South Dakota State University|
|FENNELL, ANNE - South Dakota State University|
|BOE, ARVID - South Dakota State University|
|WU, YAJUN - South Dakota State University|
Submitted to: Crop and Pasture Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2016
Publication Date: 6/28/2016
Citation: Anower, M.R., Fennell, A., Boe, A., Mott, I.W., Peel, M., Wu, Y. 2016. Physiological and molecular characterization of lucerne (Medicago sativa L.) germplasm with improved seedling freezing tolerance. Crop and Pasture Science. 67:655-665.
Interpretive Summary: Freezing temperatures early in the growing season can cause severe damage and death of young seedlings, a problem of particular concern in northern growing climates of the Great Plains region of the United States. Identifying plants with natural freezing tolerance is an important first step in developing germplasm sources that can be used to improve crop freezing tolerance. We screened 14 germplasm sources of alfalfa to determine their relative tolerance to freezing temperatures. Two of those collections, RS (Rivers Side) and FR (Foster Ranch) were collected from the Grand River National Grasslands in South Dakota, an environment with naturally harsh growing conditions. Both RS and FR had greater freezing tolerance than the other 12 collections tested, including freezing tolerant varieties Apica (AP) and CUF-101 (CUF). Electrolyte leakage closely correlated with plant survival rates after freezing. LT-50 is the temperature at which 50% of electrolytes leak out of the cells due to freezing damage. The LT-50 for RS, FR, AP, and CUF was -18, -9.6, -8.5, and -5 degrees C, respectively. Analysis of accumulation of soluble sugars, a protection to freezing, was not correlated with freezing tolerance in these collections. Quantification of gene expression levels of transcription factors MsCBF1, MsCBF2 and CAS15B, known to facilitate freezing tolerance, showed that RS, FR, and AP all had rapid increase in expression of CBF genes. However, CUF only showed a moderate increase in CAS15B expression, suggesting that CBF expression may be a more reliable marker than CAS15 for freezing tolerance in these alfalfas. These findings suggest that multiple mechanisms likely contribute to the complex trait of freezing tolerance, and that RS and FR are nautral sources of freezing tolerance in alfalfa that can be used in breeding programs to increase cold tolerance of alfalfa varieties.
Technical Abstract: We conducted greenhouse experiments to compare 14 alfalfa germplasms for their survival following freezing. Among these germplasms are collections adapted to the Grand River National Grasslands in South Dakota. Our hypothesis was that these collections developed tolerance to survive the frigid growth conditions that are common to the upper Midwestern U.S. Two of those collections, RS (River side) and FR (Foster ranch) showed greater freezing tolerance than the other 12 germplasms tested based on their consistent survival rates with or without cold acclimation. In multiple freezing studies, RS and FR had average survival rates of 74% and 69%, respectively, in contrast to the commercial cultivar Apica (AP) and CUF-101 (CUF) (55% and 19%), respectively. The LT50 of leaf electrolyte leakage was closely correlated with survival rates. Leaf LT50 improved 2-3 times after 3 days of cold acclimation based on leaf electrolyte leakage analysis, reaching -18, -9.6, -8.5, and -5 degrees C for RS, FR, AP, and CUF, respectively. Comparison of the total soluble sugars and relative water content in shoots before and after cold acclimation showed that they were not correlated with the freezing tolerance and could not be used to explain the responses of RS and FR during cold acclimation. Transcript analysis of cold responsive MsCBF1, MsCBF2, and CAS15B genes showed that RS, FR, AP, and CUF exhibited distinct patterns of cold induction. While RS, FR and AP, showed a rapid increase in expression level of one or two of these genes compared to CUF, CUF only showed a moderate induction in MsCBF2 and CAS15B transcripts suggesting that expression of these genes may be a useful molecular market for identifying freezing tolerance in alfalfa. Furthermore, these findings support the hypothesis that freezing tolerance in alfalfa is a complex trait and a combination of different mechanisms may greatly improve its freezing tolerance.