An efficient hairy root system for genome editing of a β-ODAP pathway gene in Lathyrus sativus
Grass pea (Lathyrus sativus) is an ideal legume crop for resource-poor farmers, having resistance to various biotic and abiotic stresses. The seeds of this plant are rich in protein and are the only known dietary source of L-homoarginine. Moreover, it thrives with minimal inputs making it a promising crop in grain legume breeding programs with immense potential for food security. Despite these advantages, the global area under its cultivation has decreased because of the presence of an antinutrient compound, {beta}-N-oxalyl-L-,{beta}-diamino propionic acid ({beta}-ODAP), which results in neurolathyrism both in humans and animals. Multiple efforts in the past have resulted in the development of improved varieties with low ODAP. Still, due to variations in response to the environment, stable low-ODAP lines have not been developed for large-scale cultivation. In this paper, we report in planta characterization of Oxalyl-CoA Synthetase (OCS) involved in the oxalylating step leading to {beta}-ODAP production. We established a hairy root transformation system for Lathyrus and demonstrated the genome editing of LsOCS. Further, we show that oxalate accumulates in these hairy roots due to loss-of-function of the OCS gene. This is the first report of functional analysis of a Lathyrus gene in Lathyrus. The hairy root genome editing system we developed can be used as a quick system for functional studies of Lathyrus genes.