The AFIN Board of Directors has decided to run a series of science-related webinars for the benefit of producers and other interested parties. 

This episode features a discussion with Mohammed Musthafa Mukthar to Enhancement of Total Shoot Lipid Content of Alfalfa and Sainfoin Using Chemical Mutagenesis and CRISPR-Cas9-Mediated Genome Editing

Mohammed Mukthar is a Research Assistant at the Forage Breeding and Agronomy Lab at the Lethbridge Research and Development Centre (Agriculture and Agri-Food Canada), led by Dr. Hari Poudel. He recently completed his Master’s degree at the University of Alberta, where he
defended his thesis on improving lipid content in legume forages using chemical mutagenesis and CRISPR-Cas9-mediated genome editing.
During his graduate research, Mohammed gained expertise in conventional and molecular breeding techniques, as well as transcriptomics. He has presented award-winning poster and oral presentations at national conferences and co-authored a review paper.
Currently, in his role as a Research Assistant, he continues to work on the lipid improvement project while also contributing to other forage-related programs in the lab.

Outside of research, Mohammed enjoys walking, reading, and writing blogs.

Enhancement of Total Shoot Lipid Content of Alfalfa and Sainfoin Using Chemical Mutagenesis and CRISPR-Cas9-Mediated Genome Editing

Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, Lethbridge.

Lack of statutory lipids in the vegetative tissues of forages leads to increased methane emissions primarily due to the reactions of methanogens during the fermentation process. While this issue can be overcome through lipid supplementation in ruminants; diet, it is often costly
and sometimes impractical. Alfalfa is a notable legume forage crop in terms of ruminant ration and export value. Sainfoin, on the other hand, is gaining considerable interest because of the bloat-free status. However, available cultivars of both crops contain ~1-3% lipid on dry matter basis. As such, the first objective of the study is to generate the first generation of chemically mutagenized legume forages with increased TSLC. Around 500 plants of each alfalfa (cv. AC Blue J) and sainfoin (cv. AAC Mountainview) were established in greenhouse after 0.5-1% of ethyl methanesulfonate (EMS) treatment. This M 1 population was analyzed for TSLC using near-infrared spectroscopy (NIRS) and validated using Gas Chromatography-Mass Spectrophotometry (GC-MS). Approximately 16 -18 high and low-lipid genotypes were selected
for morphology study, and two genotypes from each category were used for RNA sequencing to identify genes related to lipid biosynthesis. The second objective of the study is to enhance TSLC in alfalfa by downregulating PEROXISOMAL ABC TRANSPORTER 1 (PXA1) and SUGAR DEPENDENT 1 (SDP1) genes using CRISPR/Cas9 mediated genome editing tool.
Gene editing frequency droplet digital PCR (GEF-ddPCR) assays and Sanger sequencing were used to confirm the existence of mutations at the target sites. Eight unique PXA1-edited and 11 unique SDP1-edited genotypes have been identified with GEF ranging from 25% to 75%. Lipid content analysis revealed that some edited genotypes of PXA1 and SDP1 exhibited significant
disruptions in lipid accumulation and fatty acid composition as indicated by the increase in either leaf or stem lipid content or both. However, there were no consistent morphological variations in either the PXA1 or SDP1 mutants, with the exception of poor morphological traits observed in SDP1-gRNA3 mutants and consistent delayed flowering in all SDP1 mutants. The application of these breeding methods to develop new germplasm with high TSLC may provide sustainable feed to the ruminants and contribute to the reduction of greenhouse gas emissions.