*3.2. Exploiting Canola Meal Potential*

Despite the protein-rich content of canola meal (38%, by meal weight) and wide adoption of low glucosinolate (GSL) varieties, antinutritional compounds, typically, phenolics, sinapine, and phytate, are still challenging the use of by-products of canola oil production as animal feed [16,91]. The expansion of biofuel production from canola [89] has led to significantly additional volumes of canola meal as a by-product and promoted feed industry [16,92]. However, in the study of Skugor et al. [93], the replacement of rapeseed meal for soybean meal as 20% supplement of diet for growing finishing pigs in 3 months, produced elevated expression of major control factors of energy generation, reduced protein synthesis in muscle tissue, and greater expression of oxidative stress, which was caused by higher fiber content and GSL compounds and other secondary metabolites in rapeseed meal. Other studies on animal nutrition also had similar conclusions about antinutritional compounds in canola meal, including high fiber content, lignin, and GSLs [94,95].

Since the release of the first "double-low" canola cultivar with low levels of both erucic acid and glucosinolates (GSL), breeding efforts to further reduce GSL content in canola cultivars including *B. rapa*, *B. napus*, and *B. carinata* are still in progress [1]. While GSL inclusion in diet affects health of livestock e.g., damaging liver, kidney, and thyroid gland and impairing fertility [96], this secondary metabolite could be useful as a cancer prevention and plant protection agen<sup>t</sup> [97,98]. The importance of GSLs in plant defense has been demonstrated [99] and explains the increase in susceptibility to pests and diseases of the "00" (low seed erucic acid and GSL) rapeseed cultivars [100].

Over the at least 15 years, knowledge on metabolism of GSLs in Brassica crops and Arabidopsis, and the underlying genetic architecture has been achieved [97,98,100,101]. To date, there are at least 130 different classes of plant GSL, almost all from the Brassicales order, summarized by Blaževi´c et al. [102] and approximately 40 different GSLs synthesized in Arabidopsis [103]. Understanding the regulation of GSL biosynthesis through molecular genetic tools (SNP marker identification, QTL mapping) and gene technology (gene cloning, gene knock-out) enables controlling in planta GSL profiles and their targeted position for expression [100]. Using an associative Transcriptomics platform comprising 355,536 SNPs and a transcriptome reference, Kittipol et al. [100] found a region on chromosome A3 of *B. napus* had strong association with variation in root aromatic GSL and confirmed

the gene *Bna*.HAG3.A3 as a key regulator for the trait expression. Although studies of genetic factors controlling expression of GSL in vegetative parts and seeds of canola are being performed, they are clearly the keys to enhance pest resistance for canola varieties, and increase nutrition value and use of rapeseed meal. Future canola varieties with differential expression of GSLs in term of types, levels, and position in plant from which the obtained meal may increase in its intake for the feed industry and expand to other markets (as in cancer treatment, herbicide industry).

Among efforts to accommodate higher-value markets for canola meal, breeding towards enhancement of canola protein content is the most achievable and economical solution [16,104]. Canola protein extracted from the meal with a well-balanced amino acid profile could be a new protein source for humans, providing that allergen causing proteins, napin and cruciferin, are controlled along with antinutritional compounds residing in the seed hull [104]. Therefore, improvement targets for protein from canola meal should be protein content, protein types (napin, cruciferin), and reduction in fiber and GSL [104]. To recognize this, recently Canadian Protein Industries announced a major investment in breeding to improve protein and reduce fiber to diversify markets for food and feed applications of canola meal [105].

Major genetic factors controlling seed cellulose are being investigated in *B. napus* and the model plant *Arabidopsis* [106], providing fundamental knowledge about the genetic control of fiber and protein content of seed. Using RNA interference technology, the functions of the two genes encoding cellulose synthase subunits in *Arabidopsis thaliana*; AtCESA1 and AtCESA9, were elucidated by generating transgenic *A. thaliana* containing the hairpin RNA (hpRNAi) constructs which specifically spliced their complementary targets [106].

Yellow-seed rapeseed varieties of *B. napus*, *B. rapa*, and *B. juncea* have been shown to have lower fiber content than brown-seeded canola, thus, showed higher feed efficiency in an in vivo experiment on chickens than brown-seed canola [94]. The characteristic genetic background of yellow-seeded *B. napus* could be differentiated from the black-seeded ones by comparing the correlation between differentially expressed genes (DEGs) from the transcriptomic data and the observed traits in the two *B. napus* types, from which down-regulated DEGs in the yellow seeds were linked to reduction in flavonoid and lignin contents, while other DEGs linked to fatty acid biosynthesis and metabolism [107]. Post-translation regulation of seed coat traits could be an effective approach with less effect on cellulose biosynthesis in other plant parts [106]. There have been at least 2 examples of development of yellow seeded line; through directed mutation, an improved yellow-seeded rapeseed line was produced [108]. In addition, in 2007, a new *B. napus* line was introduced by Canadian scientists with low fiber, yellow canola seeds comprising high oleic, low linolenic oil [109]. However, yellow-seed canola is still ye<sup>t</sup> to be commercially successful.
