EARLY SORTING FOR HERBICIDE RESISTANCE IN DOUBLED HAPLOIDS OF RAPESEED
Authors:   Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC), VNIISSOK, Moscow, Russia, Kozar Elena V. , Domblides Elena A. ,   Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC), VNIISSOK, Moscow, Russia
Published: February 2025

Rapeseed is an herbaceous plant of the cabbage family (Brassicaceae), it is an allotetraploid (2n=38) with two genomes A and C, and it originated from an interspecific cross between Brassica campestris L., the donor of genome A, and Brassica oleracea L., the donor of genome C. Genome A has ten chromosomes and genome C has nine chromosomes (Tan et al., 2005). Rapeseed is an important oilseed crop worldwide and its production is increasing year by year.

One of the most important parameters determining the profitability of rapeseed cultivation is its yield. The yield of this crop is affected by many facts, including weed infestation of fields by weeds that compete with rapeseed for sunlight, water, soil nutrients, and physical space in the field, which is a serious problem and limits the yield of this crop, reducing it by 23-64% compared to controls without weeds (Bijanzadeh et al., 2010; Guo et al., 2020). On the other hand, the presence of weeds worsens the quality of raw material, because the presence of weed seeds among rapeseed reduces the quality of oil and complicates its processing. Development of herbicide-resistant rapeseed varieties and hybrids is a priority area of breeding as one of the most effective tools for weed control (Tan et al., 2005). Herbicides to which resistant forms of rapeseed have been developed can be divided into six groups: glufosinate, glyphosate, bromoxynil, imidazolino-nones, triazinines and sulfonylureas (Goncharov and Gorlova, 2018). Rapeseed resistant to imidazolinones, triazinines and sulfonylureas has been bred by conventional plant breeding, which is attractive for countries where GMO cultivation is prohibited. The imidazolinone group of herbicides is attractive not only because resistance has been developed without genome editing, but also because of the low toxicity of this group of herbicides to mammals and low application rates. Imidazolinone herbicides were developed in the 1980s, resistant varieties and hybrids to imidazolinones are commercialized under the brand name of the Clearfield® production system (Goncharov and Gorlova, 2018), with the abbreviation “CL” in the names of varieties and hybrids. Imidazolinones belong to one of five chemical families of herbicide inhibiting AHAS. Acetolactate synthase (ALS or AHAS) is a key enzyme for the biosynthesis of branched-chain amino acids. Five AHAS loci have been reported in oilseed rape. Three loci, AHAS2, AHAS3 and AHAS4 originate from the A genome, while two loci, AHAS1 and AHAS5 originate from the C genome (Rutledge et al., 1991). Of these, the AHAS1 and AHAS3 genes are constitutively expressed and encode the major AHAS activities required for B. napus growth and development. Mutations in these genes determine the resistance of oilseed rape to imidazolinones (BnAHAS1R (Ser653Asp), BnAHAS3R(Trp574Leu) (Kozar and Domblides, 2024). The presence of even one mutation already provides some degree of resistance of rapeseed to imidazolinones, but for industrial production it is important that the resistance is very high, so that when varying the herbicide treatment there is full confidence that rapeseed will not be damaged. In addition, forms with both mutations at the same time are preferred for commercial use because they have a synergistic effect on resistance, and it has been shown that these mutations are maximally effective in the homozygous state of the alleles. In view of these aspects, breeding for resistance to imidazolinones without the use of DH-technologies is difficult.

Since markers have been developed for the imidazolinone resistance trait in rapeseed, one obvious technique for early selection of rapeseed for resistance is to use them. However, trait genetics is usually much more complex and in addition to the major determinant genes, there are many non-target genes that also influence the resistance trait. Because of this, marker-assisted selection has significant limitations, as it is not possible to develop markers for all possible variants of genes that provide a particular trait. Therefore, when using marker-assisted selection, it is possible that a number of resistant genotypes will not be identified, and it will not be possible to determine the degree of resistance of genotypes in comparison with each other at early stages of plant ontogenesis. On the other hand, in marker-assisted breeding there is always the difficulty that the developed markers are not always well reproducible in other laboratories.

All the problems described above can be solved by working with solid selective media under in vitro conditions. By using nutrient media with different addition of herbicides in the nutrient medium, all resistant forms will be identified, and by using several concentrations in one experiment on resistance assessment, it is possible to determine the degree of resistance of genotypes under in vitro conditions in comparison with each other. In our work, we used MS medium (Murashige and Skoog, 1962) with the addition of the following concentrations of imazomox: 2.5 mg L-1, 7.5 mg L-1, 25 mg L-1 and 37.5 mg L-1. Microshoots of doubled haploids propagated microclonally so that there were several microshoots per nutrient medium variant were used in the experiment. The experiment was conducted in the presence of susceptibility and resistance controls to imidazolinone group herbicides, and resistance was evaluated two weeks after transplanting plants to imozamox media. The results of the experiment showed that at a concentration of 2.5 mg L-1, the susceptibility control died, while at a concentration of 7.5 mg L-1 imazamox in MS solid nutrient medium, the resistance control survived, but the first signs of herbicide damage were visible. Medium supplemented with 25 mg L-1 imazamox allowed selection of plants with greater resistance than the resistant control. At a concentration of 37.5 mg L-1 plants did not survive. Thus, for the selection of resistant rape genotypes we recommend the medium with the addition of 7.5 mg L-1 imazamox. The system of selection of plants for resistance using selective media is very simple in technical execution and gives a great advantage in assessing the degree of resistance of each genotype at early stages, and each genotype due to parallel cultivation on media without the addition of herbicides, if necessary, is always saved for further experiments or breeding work.