Pesticides and Sunflower Breeding

Abstract

The amount and quality of yields depend on the successful protection of crops from diseases, pests, weeds, and abiotic factors. The sunflower is a plant in which most diseases and pests are overcome genetically. The chemical method is also used in the production of sunflower, but it is important to say that there are still no genetically modified (GM) sunflowers on the market. By applying the classical breeding methods, new sunflower elite inbreed B lines that are resistant to two classes of herbicides (imidazolinones and sulfonylureas) were received. The aim of this study is to summarize the knowledge of pesticides and their use, as well as the breeding methods and resistance to herbicides in the sunflower.

1. Introduction

The amount and quality of yields depend on the successful protection of crops from diseases, pests, weeds, and abiotic factors. The first attempts to do so were in ancient times when diseased plants and plant pests were manually removed. Later on, additional measures were taken, such as the application of different crop rotation schemes. The first chemicals in agriculture were used only two centuries ago (for fungicides). Nowadays, there are attempts to apply biological methods of plant protection, for example, attracting wild birds.

Nonetheless, the most widely used method is still the chemical one, which is based on the application of certain chemicals—pesticides (from Latin, pestis—infection, and cedo—to kill), which affect the living cells of the adversary and result in either their death or the inhibition of their development. Its main tasks are:

1. to protect plants from diseases and pests;

2. to destroy pests and weeds;

3. to treat the diseased plants if possible.

The chemical ingredient that determines the biological action of different types of pesticides is commonly referred to as the active substance. When applying the chemical method, it is assumed that this substance will show its action only when it comes into contact with the pathogens (bacteria and fungi), harmful insects, Acari, nematodes, rodents, slugs, harmful birds, weeds, or whatever else is being combated. For this purpose, the used detergent is applied either directly on the adversary, on its food, or on the surface on which it moves (soil, water basins, and air). However, the implementation of the pesticides often happens when plants are the most vulnerable and, thus, can be fatally damaged. Anyhow, this overlap is close to unavoidable.

Advantages: The chemical method is popular in agricultural practice because it allows to effectively protect even the most threatened crops with minimal human labor costs. Moreover, it makes it possible to quickly cover large areas using modern machines such as ground-based field sprayers, aerosol generators, and agricultural aircraft. These reduce the cost of production and contribute to the improvement of quality while meeting the growing demands of the market. All in all, they guarantee multiple return on investments.

Disadvantages: When applied alone, the chemical method never leads to the complete elimination of the danger of a given adversary. Instead, a system of methods for plant protection must be applied—often a combination of different pesticides. On top of that, the improper use of chemicals creates conditions for the pollution of not only the treated agricultural products, but also their immediate surroundings—neighboring crops and water bodies, the soil, and the environment. These lead to the poisoning of wildlife (bees, beetles, ants, fish, etc.). The favorable ratio between beneficial and harmful insects is also violated and that leads to the massive multiplication of the harmful ones as well as the appearance of new pest species that previously were of no concern. In the case of weeds, at the expense of destroyed species, others that are not affected by the same chemical agent appear and take over. In the worst-case scenario, resistant populations of the adversaries are obtained, so measures to prevent and overcome this phenomenon must be taken.

Some of the requirements of a pesticide are as follows:

• its physical and chemical properties should not deteriorate when stored (up to 2 years after its production);

• during the preparation of the working solution not to hydrolyze and not to form secondary metabolic compounds that are phytotoxic;

• to not cause deterioration of biochemical composition and nutritional qualities of the resulting plant products.

Additionally, how well a detergent does its job depends on a number of factors, such as the type of chemical, the adversary being controlled, the used machines, the environmental conditions, and so on. Additionally, for the successful application of the chemical method of plant protection, it is necessary to know:

• the basic composition and physical properties of the applied detergent;

• the doses and concentrations at which the best results are obtained;

• the rules to be followed during their application in practice;

• the mechanism of their action on pests and on the protected plants;

• the extent to which these agents are harmful to natural biocenoses and agrophytocenoses and measures to prevent pollution;

• the impact of the given pesticide on humans.

The grouping of pesticides:

• according to the pest against which they are applied:

  • Fungicides: they are used to prevent and combat plant diseases (Fungicides against fungi; Bactericides against bacteria; Viricides against viruses and mycoplasmas).

  • Zoocides: they are used to control harmful plant pests (Insecticides against insects; Acaricides against herbivorous Acari; Nematicides against nematodes; Rodenticides against rodents; Lymacides against slugs; Avicides against birds).

  • Herbicides: they are used to control weeds in crops.

• according to their mode of action: systemic, penetrating, contact, gastric and gas;

• according to the origin of the active substance, which they contain: of mineral origin, of plant origin, and synthetic;

• according to the method of their application—powders (for dusting), soluble powders (stabilizers are added to them to keep the suspension stable), granular, liquids (solutions and emulsions), and gaseous detergents (ex., Fumigants);

• according to the time of their application—for spraying in winter, for application during the vegetation period, etc.;

• according to the mechanism of their action on pathogenic organisms—protective, lethal, and curative;

• according to the place of their application.

Powder pesticides (for dusting with agricultural aircraft): It is easy and simple to use them as no prior preparation is required, no water is used, and no working solutions are prepared. At the same time, however, they greatly pollute the work area as they are easily blown away. That also makes it so they can be applied only during windless hours (especially in dry weather). Furthermore, the chemicals are poorly retained on the plants and easy to wash away, which leads to a rapid reduction in their effectiveness and more pollution.

Soluble powders: Spraying is a widely used method of applying these. Well-trained workers, the presence of special containers and clothing, and increased inspection of sprayers for corrosion of metal parts are all a must because the working solution has to be prepared with great care. However, a smaller amount of detergent per unit area is used, which covers the plants well due to its improved retention, especially if wetting agents and adhesives are added to the solution. In addition, of great importance for the good absorption and distribution of the applied chemicals are the surface of the leaf blade (presence of waxy coating and hairiness), the angle of the leaves on the stem, their shape and size, as well as their immobility after the passing of the sprayer. As a rule, plants wet from rain or dew are never sprayed since, when the drops of the working solution fall on the wet plant parts, they are repelled, quickly diluted with the available water, and tend to flow down in large drops from the leaves.

Granular pesticides: The advantages of using them are that their application overlaps with sowing, that they allow for reduced contact of the worker with the detergent, and the possibility of using more toxic substances while limiting environmental pollution by the targeted application of the chemicals in, for example, rows and nests.

Aerosol mist covers large areas and penetrates well into the crowns of perennial species and of crops with a merged surface, but cannot be used even in the slightest wind, because it is easily carried outside the treated areas, which causes environmental pollution. Owing to this disadvantage, aerosols are used mainly in greenhouses and empty warehouses.

Fumigants are highly toxic gases, which quickly cover entire warehouses and penetrate everywhere. This requires fumigation to be carried out only by well-trained workers with appropriate work clothes and gas masks in compliance with all rules for safe work with chemicals.

The distinction between pesticides is not strict, as a number of detergents from one group can be applied against two or more groups of adversaries. For example, dinitro-ortho-cresol detergents have been used as insecticides for winter spraying, have both good fungicidal and acaricidal effects, have been used as contact herbicides in cereals and legumes, and have been good nematicides when imported into the soil. A number of fungicides (caratan, morastan, acrex, acricide, etc.) used to control powdery mildew were also good acaricides. Zinc phosphide has been used to control harmful rodents, but it has also been a successful remedy against some harmful insects (mole cricket, woodlice, etc.) and against soil-dwelling nematodes.

In any case, chemical products reduce agricultural harvest. Biotechnology could improve this by helping in developing insect-resistant genetically modified (GM) or herbicide-resistant GM crops. Although sustainable management calls for complete knowledge of the biology of the target adversary and its relationship with other components of the agroecosystem, the areas sown with GM crops increase every year. There are currently 32 GM crops worldwide, eight of which are grown in the European Union. Up-to-date data on the global area of transgenic crops and the resulting desired effect (new trait, enzyme, gene or factor) can be found at https://www.isaaa.org/gmapprovaldatabase/default.asp.

So, what is better: organic food, pesticides treatment, or GM crops?

Organic food is safer than regular food but is trickier to produce. It is important to have a proper rotation sequence with all crops, exact dates of planting and harvesting, and tillage practices. Besides, production can be high risk due to potential losses from diseases, insects, birds, and weeds. Biological (beneficial insects, pathogens, and host resistance) or mechanical (temperature, weather events, and trapping) control may be performed.

Pesticides treatment: In the strip-till and no-till technology, with the reduction of the soil treatments, the soil surface remains covered by the residues of the previous crops, and weed control can be difficult. In place of cultivation, a farmer can suppress weeds by managing a cover crop, mowing, crimping, or herbicide application. However, these may result in an increase in total farm expenses or even worse, environmental pollution, and oversaturation of the soil with detergents that adversely affect the development of the next crop.

GM crops, in general, need fewer field operations, such as tillage, which allows more residues to remain in the ground, sequestering more CO₂ in the soil and reducing greenhouse gas emissions. Owing to the likelihood of genetically modified organisms (GMOs) causing problems in humans and animals when consumed, spatial insulation is done to avoid unwanted cross-pollination, i.e., separation of fields with GM crops from confectionery cultures.

It is difficult to find a balance between all these, but using technologies for growing crops, with timely pest control, with the least possible chemical treatment and the best varieties selected for a given microclimate, high yields can be obtained. In fact, using both pesticides and biotech crops is the most sustainable option.

To further examine the topic, special attention will be given to pesticides used on sunflowers and on sunflower breeding itself.

The chemical method is often used in sunflower production (Figure 1), but it is important to say that there is still no GM sunflower on the market. All of the herbicide-tolerant traits of the sunflower were grown through traditional plant growing and not biotech means. While genetic engineers aim to produce GMO versions of many food crops, they probably will not succeed in manipulating the sunflower’s genes any time soon for two reasons. First, it is difficult to genetically change the sunflower. Second, the sunflower has many wild species to which transgenes can switch, and if that occurs, the result will be mass multiplication of the infected wild forms and pollution of the environment. Consequently, pesticide treatment and sunflower resistance to the used chemicals is crucial.

Figure 1.

A plant infested by Downy mildew.

2. Resistance to herbicides in the sunflower

Over the last decade, increasing amounts of sunflower fields have been treated with pesticides. For example, herbicides have been extensively used for weed control in North America since 1973. That is done in attempts to combat the growing weed problem caused by the weeds’ competition for moisture, nutrients, and depending on species for light and space as well. One result of the mentioned competition is substantial yield losses in sunflower production ranging from 20 to 70%. Herbicide use is an effective solution when planting sunflowers in a no-till or minimum-till cropping system. In fact, the chemical method may be beneficial if ground cover is needed to prevent soil erosion from wind and water. Nonetheless, that is not always the case. One of the registered preplant herbicide glyphosate, used for nonselective annual perennial grass and broadleaf weed control, is still the subject of much debate, as several studies report its negative effects on the environment [1]. Anyhow, new methods for testing the outcomes of the environmental exposure to glyphosate in sunflower production are being proposed, so the given results may end up being different [2].

However, the only way chemical treatment is possible is if the grown sunflower species are tolerant to the used pesticides. Thus, herbicide-resistant crops are becoming increasingly common in agricultural production. Berville et al. treated seeds of F₁ hybrids with gamma rays (100 Gy, 200 Gy, 300 Gy, and 400 Gy) and 0.2% ethyl methyl sulfonate and obtained mutants tolerant to bifenox and glyphosate [3]. Furthermore, the knowledge of sunflower genetics and breeding has been greatly expanded since the time that Škorić defined his hybrid model [4, 5, 6].

Resistance to herbicides from the class of imidazolinones (IMI) and of sulfonylureas (SU) is becoming one of the most important sunflower traits. Its benefits can be observed in Spain where imidazolinone resistance (transferred by sunflower breeding) has resulted in a broad spectrum of weed control (over 40 broadleaf and 20 grass weed species) and is highly effective in the control of the parasite broomrape (Orobanche cumana Wallr.). This tolerance has potential to be applied in all regions of the world for controlling several broadleaf weeds and even may control the broomrape in areas of the world where this parasitic weed attacks sunflower [7]. Be that as it may, the broomrape produces an extremely large number of seeds, and it is likely that if this control measure is widely used, isolation with herbicide resistance will become an issue. Previously, sulfonylurea (SU) and imidazolinone (IMI) herbicides were widely used to control wide sunflowers in the fields of corn, soybean, and other crop rotations that later developed herbicide resistance [1].

According to Sala et al., there are two primary mechanisms of herbicide tolerance (HT) in sunflower [8]:

1. tolerance caused by mutations in target sites of the herbicide (target-site tolerance);

2. tolerance caused by mutations in nontarget sites (nontarget-site tolerance).

Target-site tolerance involves a reduced sensitivity of target specific enzymes or proteins, and thus, this type of tolerance is mostly monogenic—as IMI and SU resistance [9]. Nontarget tolerance, on the other hand, involves several mechanisms, such as reduced uptake or translocation of the herbicide, increased rate of herbicide detoxification, decreased rate of herbicide activation, or sequestration of the herbicide away from the target site into the vacuole or the apoplast [10]. Target- and nontarget-site mechanisms can also be implemented together, such as in one of the current technologies of weed control, Imisun sunflowers [11, 12].

2.1 Development of IMI-resistant sunflower

Imidazolinone (Imazethapyr, Persuit) resistance in wild population of annual sunflower (Helianthus annuus L.) was first identified in Kansas in 1996, in a soybean field treated with the herbicide for 7 consecutive years [13].

The USDA-ARS (North Dakota State University (NDSU), Fargo, ND, USA) research group transferred this resistance into cultivated sunflower genotypes and released the public populations oil maintainer IMISUN-1 (Reg. no. GS-18, PI 607927), oil restorer IMISUN-2 (Reg. no. GS-19, PI 607928), confection maintainer IMISUN-3 (Reg. no. GS-20, PI 607929), and confection restorer IMISUN-4 (Reg. no. GS-21, PI 607930) [14]. Similar programs with the aim to incorporate IMI resistance from the wild H. annuus from Kansas into elite lines and developed IMI-resistant hybrids were run by Alonso in Spain [7], by Malidža et al. in Novi Sad, Serbia [15], and by several private companies in Argentina [16].

Notable results were achieved by Sala et al. [17], who obtained mutants resistant to imidazolinones by inducing mutations with a solution of ethyl methanesulfonate. The authors identified an IMI-resistant single partially dominant nuclear gene that they coded CHLA-PLUS and proved at the molecular level (with simple-sequence repeat (SSR) marker for the AHASL1 gene) that while it is different from Imr₁, both of them are allelic variants of the locus AHASL1.

It has been shown experimentally that the gene CHLA-PLUS has a higher degree of IMI resistance than the gene Imr₁ Imr₂. Breeding centers wishing to use the CHLA-PLUS gene for breeding purposes have to sign a contract with the company BASF. At the same time, BASF provides a protocol for screening for resistance at the molecular level (Clearfield®Protocol SF30). This established trademark production system for sunflower provides growers with a new technology, which ensures broad-spectrum post-emergent grass and broadleaf weed control combined with high-performing sunflower hybrids from leading seed companies or public institutions.

However, in recent years, probably due to overdose or incomplete absorption of the herbicide by the plants and accumulation in the soil, there has been a problem with the next year’s wheat crop (crop rotation). Because of that, IMI-resistant wheat breeding selection programs have been launched.

2.2 Development of resistant to sulfonylurea (tribenuron-methyl)

With sunflower breeding for IMI resistance, work has been started on the development of hybrids resistant to herbicides from the tribenuron-methyl group of sulfonylureas. Two resistance sources have been discovered.

The first one was derived from SU-resistant wild Helianthus annuus plants collected from the same area in Kansas where IMI resistance was found in 2002. The USDA-ARS (NDSU) research group incorporated this genetic resistance into cultivated sunflower and released public lines maintainer SURES-1 (Reg. no. GS-28, PI 633749) and restorer SURES-2 (Reg. no. GS-29, PI 633750) [18].

The second SU resistance was detected by DuPont within an artificial mutagenesis project conducted in the early 1990s. This material was reselected, purified, and tested by Pioneer/DuPont during 1998–2000. Several mutation events were evaluated, and selectivity to the sunflower mutation event SU7 was confirmed for a narrow range of SU herbicides. Pioneer/DuPont and the Institute of Field and Vegetable Crops (IFVC), Novi Sad, Serbia, were first to place SU-resistant hybrids on the market. First observations from commercial production indicated that although it was the case of a single dominant gene, it was necessary for both parents to possess the SU gene. When resistance is incorporated in only one parental line (Rf), a problem of how to produce 100% tolerant hybrid seeds arose and farmers often had susceptible plants in commercial crops [19].

2.3 Enzymes and genes

Imidazolinone-tolerant plants with altered acetohydroxyacid synthase (AHAS) genes and enzymes have been discovered in many species. IMI and SU herbicides are the specific inhibitors of acetohydroxyacid synthase (AHAS, EC 2.2.1.6). Species differ in herbicide susceptibility and can develop resistance to different classes of AHAS inhibitors. With few exceptions, resistances to AHAS-inhibiting herbicides, in otherwise susceptible species, are caused by point mutations in genes encoding AHAS that reduce the sensitivity of the enzyme to herbicide inhibition.

Acetolactate synthase (ALS), also called acetohydroxyacid synthase (AHAS), is the first enzyme in the biosynthesis of three vital amino acids in plants: valine, leucine, and isoleucine. Four different classes of herbicides inhibit ALS, thus causing the herbicidal effect. The most common are imidazolinones (IMI) and sulfonylureas (SU). They have been widely used since their introduction in the early 1980s, and now, they constitute one of the major weed control mode-of-action classes for many crops. Resistant (tolerant) plants rapidly metabolize the herbicide in herbicidally inactive form. Sensitivity is likewise due to the lack of metabolic detoxification. Advantages of ALS-inhibiting herbicides are as follows: very low application rate, broad spectrum of weed control (broadleaf and grassy weed species), broad range of crop selectivity, etc.

Their disadvantages may be the following:

1. Their extensive use and genetic mutability of the trait have led to the development of resistance in many species.

2. More than one gene may be involved in resistance, and they may not be totally dominant. In these cases, both parental lines have to be resistant in order to reach a commercially accepted resistance of the hybrid (more costly breeding process).

3. Because of cross resistance, careful herbicide management is required to ensure their long-time usefulness. Cross resistance refers to plant resistance to multiple herbicides that have the same mechanism of action [19].

IMI resistance in sunflower is controlled by two genes with a semidominant type of gene action—a major gene having a semidominant type of gene action (Imr1), and a second gene (Imr2) with a modifier effect when the major gene is present [16]. Malidža et al. reported that resistance to imidazolinone herbicides was controlled by a single gene with partial dominance [15]. These different findings regarding the mode of inheritance of IMI resistance could perhaps be explained by mutations being present at multiple loci in the original population of wild Helianthus annuus.

On the other hand, Jocić et al. reported that the sunflower resistance to tribenuron-methyl is controlled by a single dominant gene [20].

The three loci LG 2 (AHAS3), LG 6 (AHAS2), and LG 9 (AHAS1), flanked by mapped SSR markers, were shown on the public sunflower map of simply inherited traits [21] (Table 1). Eleven more sunflower AHAS ESTs were found. When the DNA sequences of various amplicons were aligned, three paralogous AHAS genes were discovered and named AHAS1, AHAS2, and AHAS3. Single-nucleotide polymorphism (SNP) markers were developed for AHAS1 and AHAS3, and single-strand conformation polymorphism (SSCP) markers were created for a six-base-pair INDEL in AHAS2 and a G/A SNP in AHAS3. In addition, an SSR marker was developed for AHAS1 based on the poly-Thr repeat in the putative transit peptide of AHAS1 [22].

Trait	Gene/locus	Linkage group (LG)	Population/s, line/s [23]
Herbicide resistance	AHAS3	LG 2	RHA280 × RHA801,; NMS373 × ANN1811,; (HA425 × HA89) × HA89,; IMISUN-2 × ZENB9
	AHAS2	LG 6
	AHAS1	LG 9

Table 1.

The mapped loci in relation to the SSR markers.

In the study of Kolkman et al., DNA polymorphisms were not found between herbicide-susceptible and herbicide-resistant inbred lines in the AHAS2 and AHAS3 coding sequences, but two mutations in the sunflower AHAS1 gene were identified, i.e., an Ala205Val mutation and a Pro197Leu mutation, conferring resistance to AHAS-inhibiting herbicides (mutation codons in the acetohydroxyacid synthase gene AHAS1 that confers resistance to sulfonylurea (SU) and imidazolonone (IMI) herbicides). Pro197 and Ala205 are conserved amino acids in AHAS enzymes in numerous species [9]. The mutation of Pro197 is one of the most common mutations found in plants resistant to AHAS-inhibiting herbicides but mutations of Ala205 in inhibitor-resistant plants had, thus far, only been reported in cocklebur, Arabidopsis, and sunflower [23].

There exist three genes encoding for catalytic subunits of the AHAS enzyme: AHASL1, AHASL2, and AHASL3 (Figure 2). Known mutations for herbicide tolerance so far described were located in AHASL1. Formally described alleles of this gene, the site of the aminoacidic substitution controlling tolerance in each case (following Arabidopsis thaliana nomenclature), and the herbicide tolerance trait developed from each allele/mutant are provided.

Figure 2.

Genetics of AHAS-inhibitor herbicide tolerance in sunflower [8].

3. Sunflower breeding for tolerance to herbicides: Our results

In the past decade, our company also achieved significant results in sunflower breeding for resistance to some herbicides from the imidazolinones and sulfonylureas classes.

3.1 Genetic sources and geneplasm

Two wild species (Figure 3) were used—Helianthus annuus (two different accessions from ND and MN, USA) and Helianthus argophyllus, sources of herbicide resistance [24]. H. petiolaris, which was also used, showed partial tolerance, too.

Figure 3.

A. H. argophyllus. B. H. petiolaris. C. H. annuus (w.f.).

According to Olson et al. [25], several populations of wild sunflower (H. annuus and H. petiolaris) from the USA and Canada have been screened for resistance to imazethapyr and imazamox herbicides. Eight percent of 50 wild sunflower populations had some resistance to imazamox and 57% had some resistance to tribenuron in the central USA. In additional, according to Miller and Seiler, in Canada, 52% out of 23 wild H. annuus populations had some resistance to tribenuron [26].

Several lines have been used as a source of herbicide resistance genes from cultivated sunflower: oilseed maintainer HA425 (Reg. no. GP-254, PI 617098) and restorer RHA 426 (Reg. no. GP-255, PI 617099) [27] and SURES-1. The resistance of every plant from the lines was identified in advance by treatment with herbicides.

A variety of lines obtained by hybridization and mutagenesis for herbicide resistance were also studied [28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41]. Different plant responses to treatment with a number of herbicides have been reported, but the main point is that with increasing doses, plant breakage at the base of the stem is observed. Imidazolinone-tolerant materials were susceptible to sulfonylurea herbicide. Some of the lines were also included in the subsequent breeding program since they have suitable parental base line geneplasm (B and R) for hybrid’s registration.

3.2 New herbicide-resistant lines were obtained by using the breeding method

Herbicide resistance from wild species and resistant lines was transferred to cultivated sunflower by hybridization (interspecific and intergeneric). Self-pollination and yearly treating of the selected material were carried out. A high percentage of resistant plants was obtained from different crosses.

When treated with Pulsar 40 + Stomp 330 ЕК in 2009, 17 plants were killed and 4 were slightly affected out of 21 plants in total from hybrid material of the cross L. 1607 × Matricaria sp. For three of them, a normal seed set was obtained [24]. Since then, a new elite inbreed line (Figure 4) has been develop, and it can be deployed in the making of herbicide resistant hybrids.

Figure 4.

Elite line from cross H. annuus × Matricaria sp.

The introduction of genes for resistance to IMI from wild species or from resistant genotype into elite B or R lines is done by backcrossing accompanied with continual resistance screening and elimination of sensitive and yellow flash plants.

During the first 2 weeks, the phenotypical distinction of plants is observed: resistant, intermediate with less yellow flash, dead plants (susceptible) (Figure 5) and intermediate with severe “yellow flash” (Figure 6).

Figure 5.

(A) Susceptible plants and (B) resistant.

Figure 6.

Effect of imidazolonone herbicide treatments on the produced crop: 15 days after treatment the effect of “yellow flash” phenomenon is observed on some plants (the less resistant to completely susceptible plants).

Heterozygous plants are less tolerant than homozygous ones. Therefore, different herbicide concentrations are needed for screening and selecting phenotypically the genotypes, without injuring the tolerant or killing the heterozygous plants.

This is clearly shown in field conditions (Figure 7). However, there arises a new issue: stress. Thus, plants develop at different rates during the vegetation (growing) season.

Figure 7.

Breeding for tolerance to herbicides by hybridization. (A) Screening for resistance totribenuron-methyl (SU-res) and imidazolonone (IMI-res) herbicide in field trial conditions—susceptible (dead) and resistant (normally developing) plants. (B) One resistant from many plants and (C) plants at different rates of vegetation.

Now, we have 30 more inbreed lines and 150 forms in different generation (from hybridization and mutagenesis), resistant to herbicides (Pulsar or Express). Some of them have specifically morphological traits: mutation as white pollen color, fasciation, wrinkled leaf, zig-zag stem, and other (Figure 8). Many of the lines were with good combining ability, increased 1000-seed weight, high seed oil content, early maturity, and resistance to some diseases. Some of the lines could directly be used as parent forms of sunflowers for human food.

Figure 8.

Mutagenesis. 1. Zig-zag stem. 2. White pollen color. 3. Fasciation of stem, inflorescence, and leaves. 4. Wrinkled leaves. 5. Yellow spotting. 6. Altered shape of inflorescence with densely spaced disk flowers. 7. Different colors of the ray flower and center of the head. 8. Funnel-shaped ray flower.

Except through hybridization, genetic variability in cultivated sunflower can be increased by mutations. Induced mutations are caused by humans, by treating plants with various physical or chemical agents. Mutagens create a wide range of heritable changes in sunflower. Mutations are most frequently observed in morphological traits, oil quality, resistance to herbicides, resistance to low or high temperatures, and other traits. In fact, some of the new traits can be used as morphological markers.

There were some deformations of sunflowers (Figure 9) after treatment with herbicides. These defects often are a result of the impact of the stress factor and the inability of the plant to adapt to it. Some of the extreme cases are of fertile disk flowers (f) in the sterile inflorescence (st) or vice versa, but more often plants simply do not develop normally or cannot produce a next generation.

Figure 9.

Deformations of sunflower after treatment with herbicides. 1. Branching (A), hardening (B), elongation (C) and breaking at the base (D) of the stem. 2. Deformation of the inflorescence. 3. Fertile disk flowers (f) in the sterile inflorescence (st) or vice versa.

3.3 A new line with resistance to herbicides Pulsar and Express was developed

The aim of the first experiment was to establish genetic variability (geneplasm) and sources (wild species and cultivated sunflower forms and lines) of herbicide resistance. The field trial was separated in three parts: the first part for treatment with herbicide Pulsar 40 (120 mL/dka)—P, the second part used as a control (not treated)—K, and the third part for treatment with Express (5 g/dka)—SU (Figure 10). The materials were sprayed during the optimum time as to test them for resistance to IMI herbicides is the stage of three to five pairs of permanent leaves. The dose of Pulsar was found to effectively control several weeds and have a highly effective control of parasite broomrape (O. cumana), but it was not so capable of controlling Convolvulus arvensis properly.

Figure 10.

Р group, K group, and SU group.

The 2020 and 2021 placement of the crops is laid out in the same way, but the treatment doses are increased—165 mL/dka for Pulsar and 6.2 g/dka for Express (Figure 11). This increase is in order to clear the heterozygous plants faster, i.e., to leave only those fully resistant (homozygous) to herbicides.

Figure 11.

Р group, K group, and SU group in experiment, 2020.

The drought during (Figure 12) this period was an additional stress factor besides the increased concentration of the chemicals. But, despite everything, we still have new elite B lines.

Figure 12.

Drought impact.

Herbicide resistance (IMI and SU) was successfully transferred from the different sources into elite R and B lines. Two years ago, we, Mihsan breeding group, reported a new form of sunflower with a new type of combined resistance to herbicides [42] (Figure 13). The study continued, and now, we have a line in homozygous state that is suitable for molecular analysis, which is the only thing that can show exactly what has happened.

Figure 13.

Mutant line with new morphological traits, 2015. A and B: Mutant plant. C: Leaf. D: Inflorescence and leaf.

The initial cross was between our mutant line and one American line, IMI type. The aim was to transfer IMI resistance into the morphologically specific line. The first treatment was done on the F₂ generation. Seeds from IMI resistant F₂ plants were divided into three groups. Every one of two groups was treated separately with herbicide Pulsar (Р group) or Express (SU group). The received result was a very high percentage (60–95%) of alive plants in both the groups. After self-pollination, in F₄ hybrid generation, four numbers (all from seeds of only one plant from the previous year) from the SU group and five numbers from the Р group were with 100% alive plants. The original F₂ plants are the initial parents of two of these numbers. The mentioned two numbers from the Р group were used as donors of pollen for hybridization of seven not-resistant to herbicides B lines after emasculation. The aim was to understand how this new trait transmits (the mode of inheritance). F₁ and F₂ plants were treated with Pulsar. A different percentage (20–80%) of alive plants was received in every one of crosses. F₂ plants were received from alive isolated F₁ plants. The results were varying—numbers with all dead plants, numbers with all alive plants, and numbers with different percentage (7–92%) alive plants.

In 2020 and 2021, the treatment doses were increased for both detergents. As a result, in 2021, four numbers with plants in the sixth hybrid generation were homozygous for trait resistance to both herbicides. These were already new elite B lines. Out of the treated 11 lines, in one, all the numbers were dead in both the groups of treatment. In this same sixth generation, and with increasing treatment doses, a different and very interesting result was obtained, namely, in three lines, all numbers showed complete resistance to herbicide Express, but when treated with Pulsar, some of the plants were affected. It is also interesting to note that 95% of the affected plants die after the Pulsar treatment, and up to 2% of the plants produce only a few seeds. On the other hand, the plants affected by Express branch and deform their inflorescence, but about 80–90% of them survive. In a small proportion of these plants, however, no seed set is obtained after isolation and self-pollination. Three numbers of F4 plants from crosses between not-resistant B lines and plants with this new trait—resistance to both herbicides—show complete resistance to both herbicides.

4. Conclusion

In conclusion, it can be said that sunflower breeders use various methods in order to get new genetic variability (by interspecific and intergeneric hybridization and mutagenesis) in new elite lines that after having their combining abilities examined are later used for making new hybrids. The choice of methods is closely connected to the breeding goals set in advance, as well as the available staff, equipment, inheritance of the most significant agronomic traits, available genetic resources, and other factors. Molecular marker-assisted selection (MAS) can also be used to check herbicide tolerance.

The chemical industry is constantly creating new pesticide agents that are highly selective, slightly poisonous to humans, decompose quickly, and do not contaminate crop production. In this way, many of the disadvantages of the chemical method are avoided, and the possibility of widespread application is improved. Despite the increased use of biofertilizers and various biological control methods (entomopathogenic bacteria and fungi, predatory insects, parasitic insects, etc.), the creation of resistant varieties (both to the pests themselves and to the used chemicals), and many more, which make it possible to reduce the use of pesticides, the question arises: Which is the best and safest option for us, humans? Everyone has to answer for themselves, finding the balance between all the options, while trying to preserve the environment.

Acknowledgments

I would like to thank Mihsan Ltd. for giving me the opportunity to present these results and photographs.