**7. Conclusions**

*Grain and Seed Proteins Functionality*

In addition to instability at the epigenetic level, we have found the genetic instability of introduced construct for RNAi-silencing. In this regard, analysis of the progeny of the RNAi mutant #1–1 (cv. Avans), carrying a construct for silencing γ-*KAFIRIN-1*, is indicative. Of the 4 studied T1 plants grown in the experimental field plot, all plants were transgenic, because carried the *nos*-promoter driving the expression of the marker gene *bar*, located in T-DNA of pNRKAF, along with a genetic construct for the γ-*KAFIRIN-1* gene silencing (**Figure 7A**). At the same time, one of these plants (#3) lacked the *ubi1*-intron, which is a part of the genetic construct for silencing (**Figure 7B**). All kernels developed in the panicle of plant #3 had the vitreous type of endosperm, characteristic to the original cultivar (**Figure 4A**), while in the panicles of other plants, in which the *ubi1-*intron was present, the kernels had a floury type of endosperm (**Figure 4B**), characteristic for transgenic

*202 bp fragment (B). Amplified gene-specific fragments are marked with arrows [50].*

*PCR analysis of transgenic sorghum plants (T4 generation) carrying a genetic construct pNRKAF [23] with primers to the ubi1-intron (A) and nos-promoter (B). 1 (A, B) – Original non-transgenic line Zh10; 2–-14 (A), 2-12 (B) – DNA of individual transgenic plants from the T4 families; 15 (A), 14 (B) – A. tumefaciens GV3101/pNRKAF (positive control); 16 (A), 14 (B) – DNA markers; 15 (A) – Negative control (no DNA). The ubi1-intron specific primers amplified the 267 bp fragment (A). The nos-specific primers amplified the* 

In addition, in Zh10 transgenic plants from the T4 families with high digestibility of kafirins, probable elimination of the *nos*-promoter, which controls the expression of the marker gene *bar* in the pNRKAF genetic construct [23] was found [49]. **Figure 8** clearly shows that in the plants from the T4 families, amplification of the *ubi1-*intron fragment was observed, while amplification of the *nos-*promoter located in the construct in front of the marker gene *bar* was absent. Thus, these plants probably turned out to be functionally marker-free transgenic plants. This fact is of significant interest, since the presence of marker genes in the genetic constructs

hinders the practical use of transgenic lines in practical plant breeding.

**154**

**Figure 8.**

plants with γ-kafirin silencing.

The research findings presented in this chapter provide strong evidence that RNA interference can be used for the improvement of the nutritional value of grain sorghum. RNAi mutants are characterized by significantly improved digestibility of kafirins and higher content of essential amino acids, in particular lysine. In some cases, these mutants retain vitreous endosperm that is highly important for grain hardiness and in ensuring the resistance of kernels to fungal diseases.

Nevertheless, in most cases the kernels with suppressed synthesis of γ- or α-kafirins have floury endosperm that strongly reduces their use in sorghum breeding. Such a correlation between the traits of high digestibility of kafirins and the floury type of endosperm, which was originally observed in the P721Q mutant and lines created on its basis is a serious problem (see review [8]). In maize, the correlation between the floury endosperm and the increased lysine content was disrupted using modifier genes that enhanced the accumulation of γ-zein [42, 51, 52]. However, in sorghum, an increase in the synthesis of γ-kafirin may decrease the level of kafirin digestibility due to a high content of sulfurcontaining amino acids, which contribute to the polymerization of kafirins. Possibly, one of the ways to solve this problem may be down-regulation of genes that encode protease inhibitors, which can also affect the level of digestion of kafirins by exogenous proteases. In this case, the resulting lines would have a hard endosperm in combination with a high digestibility of kafirins.

### **Acknowledgements**

The work was funded in part by the Russian Foundation for Basic Research, grant 19-016-00117.

#### **Author details**

Lev A. Elkonin\*, Valery M. Panin, Odissey A. Kenzhegulov and Saule Kh. Sarsenova Federal Agricultural Research Centre of South-East, Saratov, Russia

\*Address all correspondence to: lelkonin@gmail.com

© 2021 The Author(s). Licensee IntechOpen. 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.
