**2. The lignification process and RR soybeans**

The term lignin is used to designate a group of substances with similar chemical units. According to Panobianco (1997), the chemical structure of lignin is very complex and still not very well defined. Butler & Bailey (1973), cited by Silva (1981), refer to lignin as a polymer, 3-methoxy-phenyl-propanol and 3-5-dimethoxy-phenyl-propanol, bonded in varied proportions and in random sequence, leading to a great variety of products, which makes exact definition difficult. According to Esau (1976), lignin consists of an organic substance or mixture of organic substances with high carbon content, but different from carbohydrates, and which is found associated with cellulose on the walls of numerous cells. The term lignin is used to designate a group of substances with similar chemical units reported as polymers derived from "p-coumaryl", "conyferyl" e "sinapyl" alcohols (Lewis &

Junior et al., 1999; Kuiper et al., 2001; Edmisten et al., 2006; Nodari & Destro, 2006), indicating overproduction of this substance of up to 20% more in RR cultivars. Such variation may occur not only in the vegetative parts of plants, but also in reproductive parts,

The term lignin is used to designate a group of substances with similar chemical units indicated as polymers derived from "p-coumaryl", "conyferyl" e "sinapyl" alcohols (Lewis & Yamamoto, 1990). Impermeable to water, lignin is also very resistant to pressure and not very elastic and it is the most abundant plant polymer after cellulose, being found in greater quantity in the cell wall, around 60% to 90% (Egg-Mendonça, 2001), and its deposition

According to the authors, overproduction of lignin observed in the RR soybean plant in the US, and more recently in Brazil, is leading to deep stem fissures, with a significant number of plants in the field presenting bent or broken stems, and this effect possibly arises in the

Although the exact cause of the lignin behavior in this mechanism is still unknown, the hypothesis of overproduction of lignin in RR soybean plants is based on the fact of the precursors of the lignin molecule being formed in the same metabolic pathway, the pathway of shikimic acid, inhibited by the glyphosate herbicide. The inhibition of EPSPS enzymes by glyphosate present in this pathway leads to a deficiency in the production of amino acids and consequent death of the plants. That way, the sequence CP4 EPSPS, introduced in the genome of commercial soybean cultivars responsible for production of the protein CP4 enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme that participates in the biosynthesis of aromatic amino acids in plants and microorganisms, may be presenting the

Nevertheless, research in this area is still quite limited and the few results published do not compare conventional cultivars with their respective RR genetically modified versions, but refer to comparison between diverse genotypes and therefore do not isolate the effect of the inserted transgene. In this context, it is relevant to discuss the results of more recent research dealing with this issue in this chapter, principally looking at comparisons between

For that reason, in this chapter we will discuss results of research dealing with the physiological quality and the lignin content in RR and conventional soybean seeds submitted to different harvest times and spraying with glyphosate herbicide, produced in

The term lignin is used to designate a group of substances with similar chemical units. According to Panobianco (1997), the chemical structure of lignin is very complex and still not very well defined. Butler & Bailey (1973), cited by Silva (1981), refer to lignin as a polymer, 3-methoxy-phenyl-propanol and 3-5-dimethoxy-phenyl-propanol, bonded in varied proportions and in random sequence, leading to a great variety of products, which makes exact definition difficult. According to Esau (1976), lignin consists of an organic substance or mixture of organic substances with high carbon content, but different from carbohydrates, and which is found associated with cellulose on the walls of numerous cells. The term lignin is used to designate a group of substances with similar chemical units reported as polymers derived from "p-coumaryl", "conyferyl" e "sinapyl" alcohols (Lewis &

conventional materials and their RR versions, which are essentially derivatives.

two different time periods and submitted to direct imbibition in water.

**2. The lignification process and RR soybeans** 

such as pods and seeds.

occurs during the formation of the cell wall.

presence of water deficit and high temperatures.

pleiotropic effect, thus modifying the lignin content in the plant.

Yamamoto, 1990). Impermeable to water, lignin is also very resistant to pressure and not very elastic and it is the most abundant plant polymer after cellulose, being found in greater quantity in the cell wall, around 60% to 90% (Egg-Mendonça, 2001), and its deposition occurs during the formation of the cell wall.

The growth and development of the cell wall may be divided into two phases: growth of the primary wall, a phase in which the cell increases in size, and growth of the secondary wall, a phase in which deposition of lignin polymers occurs to the extent that the cell wall becomes progressively thicker as of the internal edge of the primary wall, in the direction of the center of the cell. The inclusion of lignin on the cell wall originates in the middle lamella, going in the direction of the interior of the secondary wall. According to Jung & Alen (1995), the effect of this lignin deposition pattern makes the middle lamella/primary cell wall region more intensely lignified.

This lignin deposition is important not only to lend rigidity and resistance to plant tissue, such as stem and leaves, but especially for the seed coat of soybean seeds, it has been correlated with resistance to mechanical damage (Alvarez, 1994; Panobianco, 1999), providing mechanical resistance to the tissue and protection against infestations by microorganisms to the cell wall (Rijo & Vasconcelos, 1983, cited by Tavares et al., 1987).

## **2.1 Lignification and the soybean seed coat**

The seed coat is one of the main factors which determine germination capacity, vigor and longevity of seeds. It has a protective function during imbibition, avoiding cell rupture and loss of intracellular substances (Duke & Kakefuda, 1981), and also protects the embryonic axis (Carvalho & Nakagawa, 2000). It is derived from the integuments of the ovule where the primine gives rise to the testa and the secundine gives rise to the tegmen.

By means of a cross section of the testa of a soybean seed, three layers may be distinguished, the epidermis, the hypodermis and the inner parenchyma (Swanson et al., 1985). This last layer, composed of the spongy parenchyma, is present in the entire testa of the seed, except for the hilar region. It has from 6 to 8 cell layers, tangential to the surface of the testa, formed by thin walls and absent protoplasm, with the outermost part of this parenchyma being formed by large, elongated cells, while the innermost part by smaller and significantly branched cells (Esau, 1977).

The intermediate layer of the testa, the hypodermis, is formed of cells in hourglass form, or pillar cells, or even osteosclereid cells. It consists of a uniform cell layer through the entire testa, except for the hilar region. The cell wall of its sclerenchyma cells is not uniform, with the presence of large intercellular spaces (Corner, 1951).

The epidermis, outside of the testa, remains uniseriate and gives rise to the palisade layer, characteristic of leguminosae seeds. This layer consists of macrosclereids (Malpighi cells) with wall of unequal thickness, having a cuticle present over their outermost wall. It cells are elongated and arranged perpendicular to the surface of the testa, with thick cell walls (Esau, 1976).

In soybean seeds, the thickness of the four testa layers altogether, including the cuticle, starting from the surface, may vary from 70 to 100 micrometers, there being variation among cultivars. Nevertheless, this characteristic is a constant with each cultivar and is controlled genetically (Caviness & Simpson, 1974). The presence or lack of pores and their quantity, shape and size on the surface of the testa is also controlled genetically. The pores seem to be related to water absorption, such that in hard seeds they are either absent or they exist in small quantity (Calero, 1981).

Morphological characteristics associated with the thickness and structure of the seed coat has also been related to the quality of soybean seeds. With the aid of a Scanning Electron Microscopy (SEM), it is possible to obtain a direct image of the atoms on the surface of a material, formed by secondary electrons and emitted from the surface of the irradiated specimen by the beam of primary electrons or by those scattered, which, in spite of generating poorer quality images, may indicate differences in the elementary composition of the sample. Designed basically for surface examination of samples, SEM allows the observation of internal surfaces if fractured and exposed, using principally secondary electrons (Alves, 2006).

Silva (2003), by means of scanning micrography of transversal sections of the testa of soybean seeds of the cultivars M-Soy 8400 and M-Soy 8411 observed three visible cell layers: palisade cell layers, an hourglass cell layer, and spongy parenchyma cells. The author evaluated the behavior of these cell layers that compose the testa of soybean seeds when they were exposed to five periods of accelerated aging (0, 24, 48, 72 and 96 hours) at 42º C and approximately 100% relative air humidity. For the cultivars evaluated, reduction in the thickness of the testa of the soybean seed was verified, which suggests collapse of the cells that compose such layers, which may be related to reduction of germination potential.

Menezes et al. (2009) evaluating the thickness and structure of the soybean seed coat (Figures 1 and 2) and the association of these characteristics with the physiological quality of the seeds, concluded that traits used for evaluation of physiological quality may be correlated with the lignin content of the seed coat. Nevertheless, according to the author, it was not possible to establish a relationship between the physiological quality of the soybean seeds and the anatomical aspects of the seed coat evaluated by SEM, emphasizing the need for refining the methodologies available for this purpose due to the difficulties of establishing the work area of common structures on the seeds, and of having observed that cell structures vary in different positions on the seed coat, which makes comparison of these structures among seeds of different genotypes difficult. In spite of that, in a general way, it was possible to observe that the lignin thickness on the palisade cell layers was greater when compared to the hourglass cell layers.

Fig. 1. Scanning micrography of the testa of the cultivar CD 201; A: palisade cell layer; B: hourglass cell layer and C: spongy parenchyma. Source: Menezes et al. (2009).

Morphological characteristics associated with the thickness and structure of the seed coat has also been related to the quality of soybean seeds. With the aid of a Scanning Electron Microscopy (SEM), it is possible to obtain a direct image of the atoms on the surface of a material, formed by secondary electrons and emitted from the surface of the irradiated specimen by the beam of primary electrons or by those scattered, which, in spite of generating poorer quality images, may indicate differences in the elementary composition of the sample. Designed basically for surface examination of samples, SEM allows the observation of internal surfaces if fractured and exposed, using principally secondary

Silva (2003), by means of scanning micrography of transversal sections of the testa of soybean seeds of the cultivars M-Soy 8400 and M-Soy 8411 observed three visible cell layers: palisade cell layers, an hourglass cell layer, and spongy parenchyma cells. The author evaluated the behavior of these cell layers that compose the testa of soybean seeds when they were exposed to five periods of accelerated aging (0, 24, 48, 72 and 96 hours) at 42º C and approximately 100% relative air humidity. For the cultivars evaluated, reduction in the thickness of the testa of the soybean seed was verified, which suggests collapse of the cells that compose such layers, which may be related to reduction of germination potential. Menezes et al. (2009) evaluating the thickness and structure of the soybean seed coat (Figures 1 and 2) and the association of these characteristics with the physiological quality of the seeds, concluded that traits used for evaluation of physiological quality may be correlated with the lignin content of the seed coat. Nevertheless, according to the author, it was not possible to establish a relationship between the physiological quality of the soybean seeds and the anatomical aspects of the seed coat evaluated by SEM, emphasizing the need for refining the methodologies available for this purpose due to the difficulties of establishing the work area of common structures on the seeds, and of having observed that cell structures vary in different positions on the seed coat, which makes comparison of these structures among seeds of different genotypes difficult. In spite of that, in a general way, it was possible to observe that the lignin thickness on the palisade cell layers was greater

Fig. 1. Scanning micrography of the testa of the cultivar CD 201; A: palisade cell layer; B:

hourglass cell layer and C: spongy parenchyma. Source: Menezes et al. (2009).

electrons (Alves, 2006).

when compared to the hourglass cell layers.

As is common in leguminosae, there is a particularly impermeable region on the walls of the upper part of the macrosclereids, which reflects light more intensely than the rest of the wall (Esau, 1965). What is called the conspicuous light line is visible in many wild soybean species, but is less prominent in cropped species (Alexandrova & Alexandrova, 1935, cited by Carlson & Lersten, 1987). This palisade layer drew the interest of researchers through the fact of its structure, and in certain hard seeds of leguminosae, being the cause of the high degree of impermeability of the seed coat, consequently affecting germination capacity (Esau, 1976).

Hard or impermeable seeds, according to Woodstock (1988), may be the result of compacted organization of cellulose microfibriles on the cell wall. This, for its part, may be impregnated with waterproof substances, such as lignin, waxes, suberins or tannin. They are abundantly composed of cellulose and hemicellulose polysaccharides, and of phenylpropanoid polymers such as lignin (McDougall et al., 1996).

In accordance with McDougall et al. (1996), the impermeability of the seed coat provided by lignin, exercises a significant effect on the speed and capacity of water absorption through it, thus interfering in the quantity of leached materials released to the outside during the imbibition phase of the seed germination process. Crocker (1948) already mentioned the need for better understanding of this mechanism since it was considered to be the best example of efficiency against water penetration and should therefore be better utilized by breeders in adjusting this characteristic to their needs. As general characteristics of soybean cultivars with a less permeable seed coat, one may cite better conservation potential, lower levels of infection by pathogens, greater vigor and viability, as well as resistance to reabsorption of moisture after maturation (Panobianco, 1999).

Fig. 2. Comparson of the thickness (μm) of the palisade cell and hourglass cell layers obtained by SEM from the cultivar CD 206. A: palisade cell layer; B: hourglass cell layer and C: spongy parenchyma. Source: Menezes et al. (2009).

Tavares et al. (1987), studying structural characteristics of the seed coat of seeds of soybean lines, concluded that the total fiber content is not connected with impermeability; however, in regard to the type of fiber, an accentuated increase in the lignin values was observed in the lines with impermeable seed coats (4.69% to 7.70%), differentiated from the values 1.80% to 3.18% found in lines with permeable seed coats. According to Brauns & Brauns (1960), cited by Tavares et al. (1987), the hydrophobic trait of lignin affects the hydrophilic bonds of the middle lamella and the removal of lignin interferes in the biological resistance of hydration in around 10.5% to 17% of the original tissue.

The occurrence of hard seeds in leguminosae has been attributed to both genetic and environmental factors (Donnelly, 1970). The percentage of hard seed exhibits considerable variability depending on the species or cultivar, the degree of maturity, the maturation conditions and the storage time. Thus, low air humidity during maturation results in a considerable increase in seed hardness (Baciu-Miclaus, 1970; Martins, 1989).

In soybeans, differences in the lignin content of the seed coat has been observed by various authors (Tavares et al., 1987; Carbonell et al., 1992; Alvarez, 1994; Carbonell & Krzyzanowski, 1995; Panobianco, 1999; Menezes et al., 2009; Gris et al., 2010;), and, in addition, differences have been reported in regard to the lignin content in the plant between genetically modified RR and conventional cultivars.

#### **2.2 Lignin biosynthesis and RR soybeans**

The advent of genetically modified soybeans, tolerant to the Roundup Ready© herbicide (RR), revolutionized the world soybean market. With the introduction of the CP4 EPSPS sequence in the genome of commercial soybean cultivars, which confers tolerance to the active ingredient glyphosate, the protein CP4 enolpyruvylshikimate-3-phosphate-synthase (EPSPS) is produced, an enzyme that participates in the biosynthesis of aromatic amino acids in plants and microorganisms. In the case of conventional cultivars, the inhibition of these enzymes by glyphosate, present in the shikimic acid pathway, leads to a deficiency in production of essential amino acids and consequent death of the plants, which does not occur in RR cultivars.

A great deal of speculation has been generated in relation to the lignin contents in the plant between genetically modified RR cultivars and conventional cultivars (Coghlan, 1999; Gertz Junior et al., 1999; Kuiper et al., 2001; Edmisten et al., 2006; Nodari & Destro, 2006).

In the late 1990s, some farmers in Georgia complained about the poor performance of their RR soybeans in years with a spring with drought and heat conditions. Scientists then carried out a comparative laboratory study of genetically modified and conventional soybeans (Gertz Junior et al. 1999). They found that the genetically modified plants were shorter, had a lower fresh weight, had less chlorophyll content, and, at high soil temperature of 40 ºC to 50ºC, suffered from stem splitting. According to Coghlan (1999), the elevated levels of lignin deposited in the stem of soybean plants would be leading to this splitting due to the stiffening of the plants under high temperatures (45oC), a problem also detected in genetically modified RR soybean crops in the USA, and which was to have led to considerable losses through falling of plants in hotter years (Nodari & Destro, 2006) as a consequence of overproduction of lignin in RR cultivars (Kuiper et al., 2001).

According to these authors, under stress conditions, losses in RR soybeans can arrive at 40% in comparison with conventional soybeans, brought about by greater production of lignin, up to 20% greater (Coghlan, 1999; Gertz et al., 1999). Nodari & Destro (2006), in a study undertaken in nine soybean crops in the state of Rio Grande do Sul (Brazil), observed that in the presence of drought and high temperatures, the RR soybean crops suffered more losses than conventional soybeans. The authors observed a large number of plants with deep stem

in regard to the type of fiber, an accentuated increase in the lignin values was observed in the lines with impermeable seed coats (4.69% to 7.70%), differentiated from the values 1.80% to 3.18% found in lines with permeable seed coats. According to Brauns & Brauns (1960), cited by Tavares et al. (1987), the hydrophobic trait of lignin affects the hydrophilic bonds of the middle lamella and the removal of lignin interferes in the biological resistance of

The occurrence of hard seeds in leguminosae has been attributed to both genetic and environmental factors (Donnelly, 1970). The percentage of hard seed exhibits considerable variability depending on the species or cultivar, the degree of maturity, the maturation conditions and the storage time. Thus, low air humidity during maturation results in a

In soybeans, differences in the lignin content of the seed coat has been observed by various authors (Tavares et al., 1987; Carbonell et al., 1992; Alvarez, 1994; Carbonell & Krzyzanowski, 1995; Panobianco, 1999; Menezes et al., 2009; Gris et al., 2010;), and, in addition, differences have been reported in regard to the lignin content in the plant between

The advent of genetically modified soybeans, tolerant to the Roundup Ready© herbicide (RR), revolutionized the world soybean market. With the introduction of the CP4 EPSPS sequence in the genome of commercial soybean cultivars, which confers tolerance to the active ingredient glyphosate, the protein CP4 enolpyruvylshikimate-3-phosphate-synthase (EPSPS) is produced, an enzyme that participates in the biosynthesis of aromatic amino acids in plants and microorganisms. In the case of conventional cultivars, the inhibition of these enzymes by glyphosate, present in the shikimic acid pathway, leads to a deficiency in production of essential amino acids and consequent death of the plants, which does not

A great deal of speculation has been generated in relation to the lignin contents in the plant between genetically modified RR cultivars and conventional cultivars (Coghlan, 1999; Gertz

In the late 1990s, some farmers in Georgia complained about the poor performance of their RR soybeans in years with a spring with drought and heat conditions. Scientists then carried out a comparative laboratory study of genetically modified and conventional soybeans (Gertz Junior et al. 1999). They found that the genetically modified plants were shorter, had a lower fresh weight, had less chlorophyll content, and, at high soil temperature of 40 ºC to 50ºC, suffered from stem splitting. According to Coghlan (1999), the elevated levels of lignin deposited in the stem of soybean plants would be leading to this splitting due to the stiffening of the plants under high temperatures (45oC), a problem also detected in genetically modified RR soybean crops in the USA, and which was to have led to considerable losses through falling of plants in hotter years (Nodari & Destro, 2006) as a

According to these authors, under stress conditions, losses in RR soybeans can arrive at 40% in comparison with conventional soybeans, brought about by greater production of lignin, up to 20% greater (Coghlan, 1999; Gertz et al., 1999). Nodari & Destro (2006), in a study undertaken in nine soybean crops in the state of Rio Grande do Sul (Brazil), observed that in the presence of drought and high temperatures, the RR soybean crops suffered more losses than conventional soybeans. The authors observed a large number of plants with deep stem

Junior et al., 1999; Kuiper et al., 2001; Edmisten et al., 2006; Nodari & Destro, 2006).

consequence of overproduction of lignin in RR cultivars (Kuiper et al., 2001).

considerable increase in seed hardness (Baciu-Miclaus, 1970; Martins, 1989).

hydration in around 10.5% to 17% of the original tissue.

genetically modified RR and conventional cultivars.

**2.2 Lignin biosynthesis and RR soybeans** 

occur in RR cultivars.

splitting and a significant quantity of these plants had bent or broken stems, around 50% to 70% of the plants, according to the authors, possibly due to overproduction of lignin in the RR material (Figure 3).

Fig. 3. Plants of the "Maradona" variety with broken stem (left), split (middle) and intact stem (right). Source: Nodari & Destro (2006).

The plants are responsible for the production of secondary metabolites that perform innumerable functions, among which the terpenes, the phenolic compounds and the alkaloids are considered as the most important. The secondary compounds are biosynthesized through three basic metabolic pathways, the acetate-mevalonate, the acetatemalonate and the acetate-shikimate (Érsek & Kiraly, 1986), also denominated simply as mevalonic acid pathway, malonic acid pathway and shikimic acid pathway, respectively (Taiz & Zeiger, 1998).

In superior plants, the shikimic acid pathway occurs in plastids, there also being evidence that it is present in the cytosol (Hrazdina & Jensen, 1992). This important metabolic pathway begins with phosphoenolpyruvate (PEP), derived from glycolysis, and the erythrose 4-P coming from the monophosphate pentose pathway and the Calvin cycle, resulting in the biosynthesis of the phenylalanine amino acids, tyrosine and tryptophan (Salisbury & Ross, 1992) (Figure 4).

According to Resende et al. (2003), the enzymes that participate in the initial and intermediary steps of the lignin biosynthesis pathway are common to the phenylpropanoid pathway (Figure 5). The metabolism of the phenylpropanoids includes a complex series of biochemical pathways that provide the plants with thousands of combinations. Many of these, according to Boatright et al. (2004), are intermediate in the synthesis of structural substances of the cells, such as lignin, if formed from shikimic acid, which forms the basic units of the cinnamic and p-coumaric acids (Simões & Spitzer, 2004).

Source: Adapted from Taiz & Zeiger (1998).

Fig. 4. Schematic representation of the shikimic, malonic and mevalonic acid pathways. ¶

Fig. 5. Lignin biosynthesis pathway. Source: Baldoni (2010).

**Eritrose 4 P** Pentose Fosfato Glicólise

**Pentose phosphate Glycolysis**

**Malonic acid Terpenes**

A A Aromáticos A A Alifáticos **Terpenos**

Fig. 4. Schematic representation of the shikimic, malonic and mevalonic acid pathways. ¶

**Shikimic acid Krebs Cicle** Acetyl CoA **Mevalonic acid**

**Ác. Mevalô nico**

**P E P**

**Compostos Fenólicos**

**Phenolic compounds**

¶

CO2 Fotossíntese Carboidratos

Photosynthesis Carbohydrates

**Ác. shiq uímico** Ciclo Krebs Acetil CoA

**A A Aromatic A A Aliphatic Ác. Malônico**

**Produtos secundárioscom N**

Fig. 5. Lignin biosynthesis pathway. Source: Baldoni (2010).

**Secondary products with N**

Source: Adapted from Taiz & Zeiger (1998).

Erythrose 4P

Lignin synthesis involves various enzymes and knowledge of them is important in studies in which the quality of soybean seeds and the lignin content is related (Baldoni, 2010). The complexity of the lignin biosynthesis pathways is attributed to various multifunctional enzymes, which also correspond to different gene families (Xu et al., 2009).

A considerable quantity of genes is attributed as participant in lignin synthesis, such as genes which regulate the activity of the enzymes phenylalanine ammonia-lyase (PAL), Cinnamate 4-Hydroxylase (C4H), 4-cumarate-CoA ligase (4CL), 4 Hydroxycinnamate 3- Hydroxylase (C3H), 5-Adenosyl-Methionine: Caffeate/5-Hydroxy (OMT), Ferulate-5- Hydroxylase (F5H), Hydroxycinnamoyl COA Reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) (Boudet, 2000; Boudet, 2003; Darley et al., 2001).

Although the exact cause of lignin behavior under stress conditions in RR soybean cultivars is still unknown (Coghlan, 1999), possibly the alterations in the content of this biopolymer in the plant is due to the fact of the precursors of the lignin molecule being formed in the shikimic acid pathway, which is inhibited by the glysophate herbicide in conventional plants. The inhibition of EPSPS enzymes, present in this pathway by the glyphosate, lead to a deficiency in the production of amino acids and consequent death of the plants. That way, the CP4 EPSPS sequence introduced in the genome of the commercial soybean cultivars denominated RR, responsible for the production of the protein CP4 enolpyruvylshikimate-3 phosphate synthase (EPSPS), an enzyme that participates in the biosynthesis of aromatic amino acids in plants and microorganisms, may present the pleiotropic effect, thus modifying the lignin content in the plant.

In spite of all those studies suggesting the pleiotropic effect of the transgene under high stress conditions in laboratory tests in the USA, some authors suggest that it might not be detected until specific environmental conditions are observed, which usually does not occur in field conditions. In this sense, the quantification of lignin in the plant, and consequently in pods and the seed coat of soybeans, become necessary in field conditions, principally with a view toward comparisons between conventional materials and their RR versions, which are essentially derivatives, since the previous reports refer to diverse genotypes, thus not isolating the effect of the inserted transgene. It is worth highlighting that scientific studies that truly prove the pleiotropic effect of the RR transgene under any characteristics are rare in the literature, with most of them being based only on observations and not on scientific results.

Therefore, we will further discuss some results of research obtained in Brazil in which the relation lignin versus RR and conventional soybean cultivars under diverse aspects was evaluated, emphasizing contents of this polymer in the plant, pod and seed coat.
