Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The target audience(s) of this project generating low-lignin high-qualityalfalfa varieties are key players in the $19 billion U.S. forage market and the $120 billion U.S. meat & dairy industries. Commercialization of Astalake Bio's technology offers an increase in yields while fine-tuning lignin deposition in cell walls, thereby ameliorating alfalfa feed quality and growers' revenue. Currently, there are two genetically engineered low-lignin alfalfa products on the market: HarvXtra® that was developed by Forage Genetics International (FGI) and "IQ Alfalfa (IQA™)" that was developed by Calyxt, Inc in collaboration with Alfalfa Partners® at Seed Co.FGI used a RNAi method and Calyxt used the TALEN genome-editing method to silence key lignin genes COMT and/or CCoAOMT to reduce lignin in alfalfa biomass. Astalake Bio'slow-lignin technology applied to alfalfa could potentially outperform the abovementioned HarvXtra® and IQA™ varieties due to competitive advantages: Higher yielding and biomass quality (high digestibility) combined with novel anti-methanogenic properties (mitigation of cattle lethal bloat and GHG emissions). Our approach is ultimately compatible with the genetic introgression to other elite germplasms via crossbreeding and can also be stacked with other beneficial traits (for example, herbicide tolerance: Roundup Ready®) via licensing, partnerships, and collaborations. If the demonstrated Phase I project outcomes will be verified at greenhouse/field environments in future research phases, the verifiedbiotechnology platform could be applied to other perennial forage/pasture species that currently cover 121.1 million acres in the United States (6% of the U.S. surface area). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We generated the initial low-lignin genetic events introduced in an alfalfa elite variety. The molecular, metabolic, and phenotypic characterizations fromselected best eventsgrown in laboratory settings revealed the targeted increase in biomass yield accompanied by reduced cell wall lignin content and ruminant enteric methane emission with in vitro measurements. These outcomes potentially contribute to improving alfalfa farming profitability, feed digestibility, and livestock performance for efficient meat and milk production. Plant biotech researchers have been generating a series of low-lignin alfalfa lines with transgene-free genome editing (i.e., CRISPR and TALEN) or RNAi approaches that reduce expression of lignin biosynthetic genes. One disadvantage of these approaches is the requirement for genome sequence information in targeted plant varieties, which is not always available. The Astalake Bio's low-lignin technology applied to alfalfa could potentially outperform the abovementioned engineeredlines due to its dominant genetic characteristics that maintain the traits for future crossbreeding.

Publications