Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments

Abstract

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the ‘mustard bomb’ reaction in reducing pungency upon the mastication of fresh tissue—a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Glucoraphanin Accumulation via Glucoraphanin Synthesis Promotion during Broccoli Germination

“…Thus, based on its structure, glucoraphanin can be considered an aliphatic glucosinolate [ 7 ]. Research has shown that glucoraphanin can be degraded to sulforaphane by myrosinase (MYR; e.g., TGG1 and TGG2) after the breakdown of broccoli and can further impact human health [ 8 , 9 , 10 ]. For example, the regular consumption of broccoli may provide some protection against COVID-19 as glucoraphanin can be degraded to sulforaphane to act against viral replication in vivo [ 11 ].…”

Identifying sustainability assessment parameters for genetically engineered agrifoods

“…First generation of biotechnologies that relied on transgenic modification (i.e., transfer of genetic material from one organism to another) in food and agricultural commodities have been on the market since 1996, largely in commodity crops like soybeans, corn, and cotton (International Service for the Acquisition of Agri‐biotech Applications [ISAAA], 2019). Broader varieties of GE animal‐based foods and fresh produce have entered the US market, such as the faster‐growing salmon that relies on transgenic modification (Food and Drug Administration [FDA], 2022a) and RNA interference derived nonbrowning apples (FDA, 2015) and gene‐edited mustard (Karlson et al, 2022). At the same time, there have been several recent recommendations that emerging technologies in food and agriculture systems should be evaluated according to a broad range of environmental, economic, and societal considerations in order to ensure sustainability (Kuiken et al, 2021; White House, 2022; Wickson et al, 2017).…”

“…Broader varieties of GE animal-based foods and fresh produce have entered the US market, such as the faster-growing salmon that relies on transgenic modification (Food and Drug Administration [FDA], 2022a) and RNA interference derived nonbrowning apples (FDA, 2015) and gene-edited mustard (Karlson et al, 2022). At the same time, there have been several recent recommendations that emerging technologies in food and agriculture systems should be evaluated according to a broad range of environmental, economic, and societal considerations in order to ensure sustainability (Kuiken et al, 2021;White House, 2022;Wickson et al, 2017).…”

Mutagenesis in the Age of Next-Generation-Sequencing and Genome Editing

“…Hudson demonstrated that induced mutation by N-methyl nitrosourea (NMU) is an effective approach to improve protein and oil content in soybean [15]. Karlson et al described an approach to develop nutritious fresh leaf greens from a tetraploid Brassica juncea, or brown mustard [16]. Leaf pungency due to a reaction of the myrosinase enzyme with its glucosinolate substrates is a major factor preventing the use of mustard leaves as nutritious leafy greens.…”

“…Leaf pungency due to a reaction of the myrosinase enzyme with its glucosinolate substrates is a major factor preventing the use of mustard leaves as nutritious leafy greens. By knocking out the type-I myrosinase multigene family in a tetraploid Brassica juncea with CRISPR-Cas12a, Karlson et al developed a leafy green mustard plant that has a stable reduction in leaf pungency in human sensory and biochemical analyses [16]. Despite the power of isolating mutants of essential genes through conventional mutagenesis, the process can be challenging.…”