Professor Indian Institute of Technology Kharagpur, Kharagpur-721302, India Kharagpur, West Bengal, India
Body of Abstract: Fatty acid profiles of the seed storage lipids determine the nutritional quality of edible fats and oils. Indian mustard (Brassica juncea; AABB; 2n=36) is an important oilseed crop of tropical and subtropical regions worldwide, including the Indian subcontinent. Mustard oil extracted from the seeds of B. juncea is a staple oil used in Indian cooking, naturally enriched with antioxidants, and is known for its distinct pungent aroma. However, the mustard oil inherently contains high level (40-55%) of nutritionally undesirable erucic acid (C22:1) and low level (10-15%) of nutritionally desirable oleic acid (C18:1), depending upon the cultivars. Previously, using RNA interference (RNAi) approach, we have down-regulated the cytoplasmic enzyme, fatty acid elongase (BjFAE) through seed-specific transgene expression in B. juncea. These transgenic lines exhibited ~80% reduction in unwanted erucic acid content along with the increased levels of desirable fatty acids in mustard oil. In order to develop “zero-erucic acid” containing mustard oil, CRISPR-Cas9 technology has been employed for targeted editing of BjFAE1 gene. Five different dual guide RNA constructs were separately introduced into two high yielding B. juncea cultivars, which naturally produce mustard oil with high erucic acid content ( >45%) to create 200-400 bp deletions or frameshift mutations in both the homoeologs of BjFAE1 gene. Fatty acid profile analysis of the total lipid extracted from the mature seeds of T1 knockout lines revealed that the erucic acid content is significantly reduced to < 20% when one of the BjFAE1 alleles is mutated, while the double mutants of both alleles result in nearly zero /non-detectable erucic acid. Furthermore, the levels of healthy fatty acids were increased in different degrees in the seed oil of all the mutants. These B. juncea knockout lines having zero/low-erucic acid trait are important resources for future breeding program to develop elite cultivars of Brassica spp.