**1. Introduction**

Development of plant varieties and hybrids that possess the necessary traits and properties is the main goal of plant breeding. With the accumulation of knowledge in the field of genetics,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

physiology and molecular biology of plants, the ability of breeders and geneticists to create valuable varieties and hybrids has significantly expanded. Development of genetic engineer‐ ing approaches have allowed creating a significant number of cultivars and lines, resistant to biotic and abiotic stresses, with improved quality of final products, increased photosynthetic rate and nutrient‐use efficiency [1].

Creation of transgenic plants with the changed composition of proteins and improved nutri‐ tional value is one of the most promising areas of genetic engineering. These investigations are particularly relevant for cereals being the main source of food and feed protein. It is known that humans receive from cereals up to 50% of proteins (or up to 70% in developing countries) and up to 65% of calories, in which the storage proteins account for up to 80% of the total protein content in the mature seed [2]. To solve this problem, various genetic engineering technologies had been developed. These technologies allow the introduction of new genes and thereby modulate the synthesis of new proteins with higher nutritional value or in a highly specific way downregulate genes that control the synthesis of proteins with a low nutritional value or reducing the digestibility or assimilation of other proteins [3–5]. Genetic engineering techniques are quite promising for enrichment of cereal grain with essential amino acids, i.e., lysine, tryptophan and methionine [6]. To date, transgenic lines with a modified composition of seed storage proteins, with increased lysine content and with improved baking properties have already been obtained in all most important species of cereals—maize, rice and wheat [7–9].

These studies are extremely important for sorghum—a unique drought‐tolerant cereal crop having special importance for sustainable grain production in the arid regions. Today, sor‐ ghum is one of the five most widely cultivated cereal crops, and with the increase of climate aridity, observed in many regions of the globe, demand for sorghum will increasingly grow. However, the majority of sorghum cultivars and hybrids have relatively poor nutritive value in comparison with other cereals [10, 11]. One of the reasons of relatively low nutritive value of sorghum grain is resistance of its seed storage proteins (kafirins) to protease digestion [12]. The causes of the poor sorghum protein digestibility were studied extensively [10, 13, 14]. Among the factors that cause or may affect this phenomenon, there are chemical structures of kafirin molecules, some of which (α‐ and β‐kafirins) are abundant with sulfur‐containing amino acids capable to form S–S bonds, resistant to protease digestion; interactions of kafirins with non‐kafirin proteins and non‐protein components such as polyphenols and polysaccha‐ rides; spatial organization of different kafirins in the protein bodies of endosperm cells; endo‐ sperm structure (vitreous or floury).

It is generally accepted that the peripheral disposition of γ‐kafirin in protein bodies reduces digestibility of α‐kafirin—the major sorghum seed storage protein located central position in protein bodies and comprising up to 80% of total endosperm kafirins [13, 14]. This hypothesis is supported by studies of protein bodies of the mutant with improved protein digestibility. In this mutant, protein bodies shape has been changed from spherical to invaginate; the γ‐kafirin was located at the bottom of invaginations where it should not interfere with the digestion of the α‐kafirin [15]. Recent study also showed that a sorghum mutant with high digestibility of kafirins has a point mutation in the signal sequence of the α‐kafirin gene, which appar‐ ently disrupts its deposition in protein bodies [16]. One of the main characteristic features of kafirin proteins is their ability to form oligomers or polymers of high molecular weight. These oligomers comprise α‐ and γ‐kafirins that are linked together by disulfide (S–S) bonds [17, 18]. They are resistant to protease digestion and occur more in the vitreous endosperm fraction [14, 19].

physiology and molecular biology of plants, the ability of breeders and geneticists to create valuable varieties and hybrids has significantly expanded. Development of genetic engineer‐ ing approaches have allowed creating a significant number of cultivars and lines, resistant to biotic and abiotic stresses, with improved quality of final products, increased photosynthetic

Creation of transgenic plants with the changed composition of proteins and improved nutri‐ tional value is one of the most promising areas of genetic engineering. These investigations are particularly relevant for cereals being the main source of food and feed protein. It is known that humans receive from cereals up to 50% of proteins (or up to 70% in developing countries) and up to 65% of calories, in which the storage proteins account for up to 80% of the total protein content in the mature seed [2]. To solve this problem, various genetic engineering technologies had been developed. These technologies allow the introduction of new genes and thereby modulate the synthesis of new proteins with higher nutritional value or in a highly specific way downregulate genes that control the synthesis of proteins with a low nutritional value or reducing the digestibility or assimilation of other proteins [3–5]. Genetic engineering techniques are quite promising for enrichment of cereal grain with essential amino acids, i.e., lysine, tryptophan and methionine [6]. To date, transgenic lines with a modified composition of seed storage proteins, with increased lysine content and with improved baking properties have already been obtained in all most important species of

These studies are extremely important for sorghum—a unique drought‐tolerant cereal crop having special importance for sustainable grain production in the arid regions. Today, sor‐ ghum is one of the five most widely cultivated cereal crops, and with the increase of climate aridity, observed in many regions of the globe, demand for sorghum will increasingly grow. However, the majority of sorghum cultivars and hybrids have relatively poor nutritive value in comparison with other cereals [10, 11]. One of the reasons of relatively low nutritive value of sorghum grain is resistance of its seed storage proteins (kafirins) to protease digestion [12]. The causes of the poor sorghum protein digestibility were studied extensively [10, 13, 14]. Among the factors that cause or may affect this phenomenon, there are chemical structures of kafirin molecules, some of which (α‐ and β‐kafirins) are abundant with sulfur‐containing amino acids capable to form S–S bonds, resistant to protease digestion; interactions of kafirins with non‐kafirin proteins and non‐protein components such as polyphenols and polysaccha‐ rides; spatial organization of different kafirins in the protein bodies of endosperm cells; endo‐

It is generally accepted that the peripheral disposition of γ‐kafirin in protein bodies reduces digestibility of α‐kafirin—the major sorghum seed storage protein located central position in protein bodies and comprising up to 80% of total endosperm kafirins [13, 14]. This hypothesis is supported by studies of protein bodies of the mutant with improved protein digestibility. In this mutant, protein bodies shape has been changed from spherical to invaginate; the γ‐kafirin was located at the bottom of invaginations where it should not interfere with the digestion of the α‐kafirin [15]. Recent study also showed that a sorghum mutant with high digestibility

rate and nutrient‐use efficiency [1].

92 Plant Engineering

cereals—maize, rice and wheat [7–9].

sperm structure (vitreous or floury).

Improving of sorghum genetic transformation technology [20, 21] makes it possible to solve this problem by using RNA interference (RNAi) that allows targeted downregulation of indi‐ vidual genes. In recent years, RNAi technology has become widely used for changing the composition of the storage proteins and starch in different cereal species [3–5].

In maize, with using of genetic constructs harboring inverted repeats of genes of α‐zeins (19 and 22 kDa), transgenic lines with suppressed synthesis of these proteins were obtained [22, 23]. It was found that repression of the synthesis of zeins possessing a relatively low nutri‐ tional value leads to accumulation of other proteins with a higher nutritional value. Maize plants with gene silencing of α‐zeins were characterized by doubled content of essential amino acids tryptophan and lysine in the kernels. These experiments showed that gene silenc‐ ing of 22 kDa α‐zein resulted in the formation of the floury endosperm. Such a modification in the type of endosperm was associated with abnormalities in the formation of the structure of protein bodies, namely the violation of deposition of 19 kDa α‐zein into the center of a protein body, or a modification of its interaction with β‐ and γ‐zeins [22].

In sorghum, transgenic lines with genetic constructs capable of RNAi silencing of different kafirin classes were obtained [24–27]. Transgenic plants harboring these constructs were char‐ acterized by improved *in vitro* protein digestibility (IVPD) that was accompanied by opaque floury endosperm. Unfortunately, the floury endosperm reduces the practical value of these lines, because the reduction of the vitreous layer increases the fragility of kernels and increases the susceptibility to fungal infection.

In our experiments, we obtained transgenic sorghum plants with genetic construct for silenc‐ ing of the gamma‐kafirin gene [28]. These plants retained sectors of vitreous endosperm in their kernels and were characterized by high level of *in vitro* kafirins digestibility. In this chap‐ ter, we review these experiments and present new data, confirming inheritance of the genetic construct and its effect on endosperm protein spectrum and endosperm texture.
