*3.5. Herbicide Tolerance*/*Resistance*

Weed invasion is ubiquitous and a persistent threat to crop yield, especially to major field crops including canola [153], for which chemical treatment is the most e ffective control and constitutes considerable expenditure, approximately 20–36% of total operating cost of canola production [154,155]. Use of herbicide tolerance (HT) crops in combination with a broad spectrum herbicide as glyphosate has been assumed as revolutionary in weed managemen<sup>t</sup> [156,157] as evidenced by their benefits to agronomy, economics, and the environment [158,159], providing flexibility in scheduling weed treatment and crop yield enhancement [160].

HT canola varieties have been obtained either through conventional breeding e.g., Clearfield ® and triazine-tolerant (TT) canola varieties, or genetic engineering e.g., InVigor ® (Bayer CropScience, Leverkusen, Germany) and Roundup Ready (RR, Monsanto) [158]. The first TT canola variety, developed through the incorporation of the TT trait from *B. campetris* and *B. rapa* by backcrossing [161], was quickly adopted in Canada and Australia in the 1990s. In 1995, the first imidazolinone-tolerant *B. napus* was registered, of which the trait was developed through mutagenesis by BASF, and the first transgenic canola variety tolerant to the herbicide glyphosate (Roundup) was developed by Monsanto [2]. Glyphosate tolerance in the initial RR cultivars was conferred by the expression of CP4 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from *Agrobacterium* sp. strain CP4, the enzyme involved in the shikimate pathway essential for the biosynthesis of aromatic amino acids [162]. This is the most common genetically modified (GM) trait in the current biotech crops, with over 80 transformation events being approved for commercial release [163]. Roundup Ready canola has gained approvals for commercial release in major canola producing countries including Canada, Japan, USA, and Australia within 1994 and 2003, where the obtained oil has been approved for food use in addition to European countries, Mexico, Philippines, and Singapore [164]. At least 34 transformation events have been approved in canola varieties to produce the crops with glyphosate HT alone or stacked with other GM traits [163]. Since their commercial release, HT canola varieties have dominated in canola growing countries [165]. However, the intensive cultivation of HT canola and overuse of herbicides, which are often broad-spectrum herbicides with wider application windows, has increased selection pressure on this trait in weed, which has resulted in HT weeds [157]. An increase of up to 15-fold in glyphosate herbicide use in cultivation compared to 1974 [166] was recorded, contributed by the rapid spread of RR crops globally [156,160]. Overreliance on a few HT crops and single active herbicidal ingredients has caused leaking of HT traits [160,167–169], with cases relating to all of the introduced HT systems being reported worldwide [170].

Recently, negative e ffects on human and animals from glyphosate exposure in the field and through the food chain have caused restrictions or bans on its applications in Austria, France, Germany, and Vietnam [156]. Therefore, other alternatives for glyphosate applications in agriculture, including weed management, are required for the development of sustainable agriculture with reduced reliance on one active ingredient as glyphosate [169,171]. Due to the lack of novel broad-spectrum herbicides, newly developed HT crops still rely on glyphosate and glufosinate, along with other common types of herbicides such as ALS inhibitors, synthetic auxins, HPPD inhibitors, and ACCase inhibitors [160]. To date, combination or stacking HT traits in one variety is the choice of seed companies, starting with the release of RT technology producing hybrids (Hyola ® 525RT, Bayer ® 3000TR) with glyphosate (RR) and triazine (TT) tolerance, and followed by "stacked" varieties with Triazine tolerance and Clearfield ® (TT+CL), which are tolerant to both triazine and imidazolinone (IMI) herbicides [172]. Strict control of the use of HT varieties and combining other weed managemen<sup>t</sup> practices is an e fficient way to manage herbicide tolerance of weeds.
