**2. Decreased content of kafirins**

The primary effect of the functioning of genetic constructs for RNA silencing of kafirin genes is a decreased level of transcripts of these genes. Such an effect was shown for the pPTN915 genetic construct, designed to suppress the expression of

#### **Figure 1.**

*SDS-PAGE of kafirins from kernels of transgenic plants from the T1 generation of the RNAi mutant, cv. Avans, isolated under reducing conditions (with the addition of 2-mercaptoethanol). 1 – Original non-transgenic cv. Avans; 2–7 – Individual plants from the T1 family: 2–6 – Plants with a floury endosperm, containing ubi1-intron; 7 – Plant with a vitreous endosperm, not containing ubi1-intron; M – Molecular mass markers. Kafirins were extracted according to [20]. The arrow marks* α*-kafirin; the dotted arrow marks* γ*-kafirin.*

**147**

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

the γ-kafirin gene [21]. Many studies using SDS-PAGE have also clearly demonstrated a decrease in the content of monomers and polymers of kafirins [20, 22, 23, 32]. In our experiments, SDS-PAGE of proteins extracted from kernels of transgenic plants of Zh10 in non-reducing conditions (without the addition of 2-mercaptoethanol, which breaks the S-S bonds and, thereby, destroys the polymers of the kafirins), showed a decreased content of γ-kafirin monomer (28 kDa), as well as 47 and 66 kDa oligomers, which are supposed to arise as a result of γ-kafirin polymerization [33]. SDS-PAGE of kafirins extracted under reducing conditions from the kernels of transgenic plants of the Avans cultivar (T1 generation) carrying the same genetic construct revealed also a noticeable decrease in the content of γ- and

The main goal of experiments on silencing of kafirin genes is to improve seed storage protein digestibility. Herewith, depending on the structure of the genetic construct, suppression of certain subclasses of kafirins, and the cultivars used in

Subsequently, new transgenic plants were obtained in the sorghum public line P898012 using other genetic constructs ABS042 and ABS044, created during the ABS (Africa Biofortified Sorghum) project [22]. In these plants, an improvement in the digestibility of flour subjected to the cooking procedure was recorded: from 28% in the control to 39% (for the ABS042 construct for silencing γ- and δ-kafirins), and up to 59% (for the ABS044 construct for silencing α-, γ- and

Analysis of ultrastructure of protein bodied showed that in transgenic lines with α-kafirin silencing protein bodies were irregular in shape and had invaginations similar to P721Q mutant [34, 35]. In transgenic lines with γ-kafirin silencing, a diameter of protein bodies was reduced in comparison with original non-transgenic line [36]. In addition, in one of the studied lines, 42–1, protein bodies were highly irregular in shape, with deep invaginations present at the periphery, while in the line 42–2, the protein bodies had small peripheral indentations that gave the bound-

In the experiments of T. Kumar et al. [20] the genetic constructs pPTN915 and pPTN1017 designed for the induction of silencing γ- or α-kafirin, respectively, were also introduced into the genome of the Tx430 line through agrobacterial transformation. *In vitro* digestibility of proteins extracted from the flour of transgenic kernels with silencing of γ-kafirin, subjected to cooking procedure, did not differ from the non-transgenic control, while the silencing of α-kafirin by pPTN1017

Transgenic plants of cv. Zh10 obtained in our experiments carrying the genetic

improved the *in vitro* protein digestibility of flour subjected to cooking.

construct pNRKAF for silencing *γ-KAFIRIN-1* gene, also had a significantly

For example, transgenic plants of cv. Tx430 carrying the ABS166 genetic construct containing inverted repeats of several kafirin genes (α, γ, δ) separated by the intron sequence of the alcohol dehydrogenase gene (*ADH1*) and controlled by the 19-kDa α-zein promoter from maize were characterized by improved *in vitro* protein digestibility. Pepsin treatment of the raw flour and flour that underwent the cooking procedure resulted in 78% and 61% digestibility, respectively, while in the non-transgenic control these indicators varied within 40–50% and 34–40%, respectively [34, 35]. The genetic construct for the silencing of δ- and γ-kafirins (ABS149) also improved the digestibility of raw flour, but did not affect the

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

**3. Improvement of** *in vitro* **protein digestibility**

experiments, the level of digestibility varied significantly.

α-kafirins (**Figure 1**).

digestibility of the cooked flour.

ary region a cracked appearance.

δ-kafirins).

#### *RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

the γ-kafirin gene [21]. Many studies using SDS-PAGE have also clearly demonstrated a decrease in the content of monomers and polymers of kafirins [20, 22, 23, 32]. In our experiments, SDS-PAGE of proteins extracted from kernels of transgenic plants of Zh10 in non-reducing conditions (without the addition of 2-mercaptoethanol, which breaks the S-S bonds and, thereby, destroys the polymers of the kafirins), showed a decreased content of γ-kafirin monomer (28 kDa), as well as 47 and 66 kDa oligomers, which are supposed to arise as a result of γ-kafirin polymerization [33]. SDS-PAGE of kafirins extracted under reducing conditions from the kernels of transgenic plants of the Avans cultivar (T1 generation) carrying the same genetic construct revealed also a noticeable decrease in the content of γ- and α-kafirins (**Figure 1**).
