Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T₂–T₄ generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T₂, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T₃ and T₄ plants, the genetic construct was present in 2–4 copies. In T₃, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5–7 %) was selected.

The use of genome editing technologies opens wide opportunities for the targeted mutagenesis in important agricultural crops. In the context of global warming, sorghum, an important drought- and heat-tolerant crop is of particular importance. However, compared to other cereals, sorghum grain has a lower nutritional value, due to the resistance of its storage proteins (kafirins) to proteolytic digestion. A decrease in the synthesis of kafirins as a result of mutations or the expression of the RNAi genetic constructs modifies the ultrastructure of protein bodies and improves their digestibility by proteases. To obtain mutants with improved protein digestibility, we created four binary vectors for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding α- and γ-kafirin, respectively. Each of these vectors contained the cas9 endonuclease gene and a guide RNA targeted the nucleotide sequences encoding the kafirin signal polypeptides. By means of agrobacterial transformation, the created vectors were introduced into the genome of the grain sorghum cv. Avans. 14 transgenic plants were regenerated. Sequencing of 5 regenerants obtained using a vector for the k1C5 mutagenesis revealed 3 plants with mutations. The offspring of these mutants had a higher digestibility of grain proteins in vitro (86–92%) compared to the initial cv. Avans (63–67%). Notably, the T1 plants lacked the cas9 gene and the bar marker gene, which indicates the production of mutants with the edited k1C5 gene sequence, which lack the genetic construct that induced this mutation. Two mutants with mutations in the gKAF1 sequence were obtained. Thus, using the CRISPR/Cas technology, we have obtained mutants with improved digestibility of kafirins, which can be used in practical sorghum breeding.