Improving Abiotic Stress Tolerance in Alfalfa

Project Title

Improving Abiotic Stress Tolerance in Alfalfa Through the Simultaneous Down-Regulation and/or Genome Editing-Mediated Knockout of Multiple Genes


Stacy Singer Ph.D.

Stacy Singer Ph.D. (Agriculture Agri-Food Canada Lacombe) and Surya Acharya Ph.D. (Agriculture Agri-Food Canada Lethbridge)

Status Project Code
Completed March, 2023 FRG.06.17


Alfalfa is susceptible to numerous stresses including drought, waterlogging, salinity, frost and/or winterkill. Sometimes stress tolerance improves when certain genes are under-expressed. Transgenic approaches to down-regulate particular genes have worked better than conventional breeding in some cases. However, transgenics also face public acceptance and regulatory hurdles. Genome editing (“fixing” genes that are already there, rather than adding new genes from a different species) may help avoid GMO concerns by down-regulating genes that are naturally present rather than by introducing new genes from a different species. 


  • To assess the efficacy of previously discovered genes in enhancing alfalfa performance under abiotic stress conditions when downregulated via RNA interference (RNAi). Researchers will attempt to make further improvements in abiotic stress tolerance, and, if technology permits, utilize CRISPR-Cas9 as an alternative approach for gene knockout. 

What They DID

To achieve this, we first selected 5 genes that had been shown previously to act as negative regulators of stress tolerance in other plant species and found these same genes in alfalfa. We then turned down their expression in alfalfa using RNA interference (RNAi) and tested the resulting plants to see if they were better able to withstand various types of environmental stresses in the greenhouse (drought, salt, and waterlogging). In instances where plants were found to be more tolerant to a particular stress, we carried out many physiological, biochemical, and transcriptomic assessments with plants grown under both normal and stressed growth conditions to pinpoint mechanisms and genes that contributed to enhanced stress tolerance. Finally, we successfully developed gene editing technology in alfalfa, and generated new germplasm with edits in our chosen target genes for the downstream production of potentially drought- and waterlogging-resilient plants that lack any foreign DNA.  

What They Learned

We identified 5 genes (MsCBF2, MsACB3, MsFAO3, MsTAC1, and MsHB2) in alfalfa that we thought could potentially function as negative regulators of environmental stress tolerance based on previous research in other plant species. We found that our TAC1-RNAi and HB2-RNAi plants exhibited improvements in their resilience to water-deficit and waterlogging. In the case of the TAC1-RNAi alfalfa plants, this meant that they began wilting at substantially lower soil moisture contents, grew better after drought treatment, and survived extreme drought to a much higher extent than wild-type plants. Our results suggested this improvement occurred, at least partly, due to a decrease in leaf water loss, higher baseline antioxidant capacity and improved response in terms of antioxidant enzyme activity under drought. These plants also had equivalent or higher biomass production under normal growing conditions and increased root volumes.  

The HB2-RNAi plants were better able to maintain their green coloration during waterlogging treatment, grew better after periods of waterlogging, and survived long-term waterlogging to a greater extent than wild-type plants. Our findings indicated that improvements in antioxidant enzyme responses under waterlogging stress, and as a result, less oxidative damage to the plants, played a role in their enhanced tolerance. These plants also had equivalent biomass and longer roots than wild-type plants under regular growth conditions. Interestingly, when we turned down the expression of both MsTAC1 and MsHB2 genes in alfalfa simultaneously, we found that the resulting plants were only more tolerant to drought, which suggests that certain drought and waterlogging resilience mechanisms may interfere with one another. Finally, we successfully used CRISPR/Cas9 technology to generate alfalfa genotypes with edits in MsTAC1 and MsHB2 genes, and in the case of MsTAC1, all four gene copies were edited in the first generation. 

What It Means

Identifying physiological and biochemical mechanisms behind drought and waterlogging tolerance, as well as the discovery of numerous genes involved in these processes, will improve how we breed drought and flood tolerant alfalfa, as well as other forage crops. The generation of edited alfalfa plants will also allow us to provide a new source of transgene-free germplasm that can be assessed in the field for improved productivity under challenging environmental conditions.