RNAi-Mutants of Sorghum bicolor (L.) Moench with Improved Digestibility of Seed Storage Proteins

Abstract

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.

Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene

“…It is noteworthy that among the F 1 hybrids with the CMS line A2 KVV-114, one plant that was PCR positive for the nos-promoter but PCR negative for the ubi1-intron was found. We previously found a similar case among T 1 plants (Elkonin et al, 2021). These facts indicate that in some cases there may be a disturbance of the integrity of the integrated T-DNA and the elimination of the genetic construct for silencing.…”

“…1-3, 190-1, 190-2, 190-4), as well as the progeny of plant No. 190-3, which was PCR negative for the ubi1-intron, but PCR positive for the nos-promoter (Elkonin et al, 2021).…”

“…The original mutant with high digestibility of grain proteins (Avans-1/18) was obtained in an experiment on Agrobacte rium-mediated genetic transformation of the new commercial cultivar Avans of grain sorghum (Sorghum bicolor (L.) Moench) using Agrobacterium tumefaciens strain GV3101, carrying the binary vector pNRKAFSIL (Elkonin et al, 2021). This vector contains a genetic construct consisting of a frag ment (309 bp) of the γ-kafirin gene (GenBank acc.…”

“…In one of the T 0 plants, all kernels had a floury endosperm type and were characterized by a high level of kafirins digestibility (up to 93 %), which was significantly higher than the level of digestibility in the cv. Avans (57 %) (Elkonin et al, 2021). T 1 plants inherited these traits, as well as resistance to ammonium glufosinate caused by the bar gene, which was also present in the T-DNA of the pNRKAFSIL vector (Borisenko et al, 2022).…”

Genetic manipulation of anti-nutritional factors in major crops for a sustainable diet in future

“…Considering the earlier observations for reducing the anti-nutrients, it can be observed that RNAi and gene editing are the two major techniques that are used in tissue-specific reduction in major crops ( Figure 3 ) ( Perera et al 2018 ; Elkonin et al 2021 ). Although the initial investigations have been conducted with reduced anti-nutrients, a standard protocol for strategic reduction of anti-nutrients is crucial in crops such as pulses needs to be reinforced in future.…”