**2. Obtaining of transgenic plants with genetic construct for RNA silencing of the γ‐kafirin gene**

To obtain transgenic plants with silencing of gamma‐kafirin gene, the binary silencing vector, pNRKAFSIL, has been designed. This vector contained a hairpin insert that consisted of an inverted repeat of the fragment of the γ‐kafirin gene and *ubi1* intron as the spacer between the arms of the inverted repeat (**Figure 1**). The 307‐bp fragment of the γ‐kafirin gene was

**Figure 1.** Map of the pNRKAFSIL vector containing hairpin insert consisted from inverted repeat of the fragment of the γ‐kafirin gene ("INVKAF" and "DIRKAF") and *ubi1* intron as the spacer between the arms of the inverted repeat (published with the permission of the publishing house "Nauka").

isolated by PCR from genomic DNA of sorghum. The sequence corresponded to bases 280– 588 of GeneBank accession number M73688 [29]. This construct was driven by the CaMV 35S‐promoter. The T‐DNA region of this vector contained selectable marker *bar* gene driven by *nos*‐promoter. The binary vector pNRKAFSIL was introduced in *Agrobacterium tumefaciens* GV3101.

To obtain transgenic plants with genetic construct for RNA silencing of the γ‐kafirin gene, cocultivation of immature embryos of sorghum cv. Zheltozernoe 10 (Zh10) (15–17 days after pollination) with cell suspension of the *A. tumefaciens* strain GV3101/pNRKAFSIL was performed.

Activation of *vir*‐genes was made according to the published protocol [30] with some modi‐ fications. *A. tumefaciens* strain GV3101/pNRKAFSIL vector was grown on an Agrobacterium (AB) minimal medium [31] with the antibiotics for 3 days at 28° C. After that a loop of the Agrobacterium cells were transferred into the flask with 20 ml of liquid yeast extract peptone (YEP) medium with the antibiotics and grown for 9 h under continuous shaking (220 rpm) at 28° C. Then, the cells were collected by centrifugation and suspended in a small volume (5–6 ml) of modified AB medium without phosphates with the addition of 200 μM acetosyrin‐ gone (Sigma‐Aldrich, USA) and were incubated for 18 h under gentle shaking (60–70 rpm) at 22–23° C. After incubation, the cells were collected by centrifugation and suspended in inocu‐ lating medium PHI‐I [32] with the addition of 200 μM acetosyringone to a final OD600=0.6. This suspension was used for inoculation of immature embryos.

Agrobacterial transformation was based on previously published protocols [20, 32] with some modifications. Immature embryos after pre‐cultivation for 3 days on the agar M11 medium [33] were placed onto sterile filter paper wetted with inoculating medium and were inoculated with an agrobacterial cell suspension in PHI‐I medium for 10 min at room temperature. The Agrobacterium inoculum was then removed, and the filter with embryos was transferred into another Petri dish on a dry filter and was wetted with cocultivation medium (M11 medium supplemented with 200 μM acetosyringone). The cocultivation step was performed for 3 days at 23 ± 1°C in the dark. After cocultivation, the embryos were transferred to the M11 medium with the addition of 200 mg/l timentin solidified with 2.5 g/l phytagel and were cultured at 27 ± 1°C in the dark for 7 days. Then, the embryos with developing embryogenic calli were subcultured to the fresh medium of the same composition with the addition of 2.5 mg/l glu‐ fosinate ammonium (GA) and were cultivated at 28°C in the dark for 3–4 weeks.

From two experiments on cocultivation of immature sorghum embryos of Zh10 with *A. tumefaciens* strain GV3101/pNRKAFSIL 35 embryogenic calli survived after selection on the medium with 2.5 mg/l GA (**Table 1**; **Figure 2A**). For plant regeneration, the herbicide‐tolerant calli were transferred onto regeneration medium (murashige and skoog (MS), 1.0 mg/l kinetin, 1.0 mg/l Indole‐3‐Acetic Acid (IAA)) and maintained at 25° C under a photoperiod of 16 h light and 8 h dark. Initiation of shoot development was observed in 13 calli transferred to the regeneration medium, but in the majority of the cultures, shoot development was arrested at early stages. Nevertheless, few regenerants were obtained (**Figure 2B**), one of which turned out to be PCR‐ positive in the experiment with primers to the *bar* gene (**Figure 3A**).


Notes: EC = embryogenic cultures; GA = glufosinate ammonium.

1 PCR with primers to *bar* gene.

isolated by PCR from genomic DNA of sorghum. The sequence corresponded to bases 280– 588 of GeneBank accession number M73688 [29]. This construct was driven by the CaMV 35S‐promoter. The T‐DNA region of this vector contained selectable marker *bar* gene driven by *nos*‐promoter. The binary vector pNRKAFSIL was introduced in *Agrobacterium tumefaciens*

**Figure 1.** Map of the pNRKAFSIL vector containing hairpin insert consisted from inverted repeat of the fragment of the γ‐kafirin gene ("INVKAF" and "DIRKAF") and *ubi1* intron as the spacer between the arms of the inverted repeat

To obtain transgenic plants with genetic construct for RNA silencing of the γ‐kafirin gene, cocultivation of immature embryos of sorghum cv. Zheltozernoe 10 (Zh10) (15–17 days after pollination) with cell suspension of the *A. tumefaciens* strain GV3101/pNRKAFSIL was

Activation of *vir*‐genes was made according to the published protocol [30] with some modi‐ fications. *A. tumefaciens* strain GV3101/pNRKAFSIL vector was grown on an Agrobacterium

Agrobacterium cells were transferred into the flask with 20 ml of liquid yeast extract peptone (YEP) medium with the antibiotics and grown for 9 h under continuous shaking (220 rpm) at

C. After that a loop of the

(AB) minimal medium [31] with the antibiotics for 3 days at 28°

(published with the permission of the publishing house "Nauka").

GV3101.

94 Plant Engineering

performed.

2 Combined progeny from PCR‐positive plants from T1 generation.

**Table 1.** Selection of transgenic plants by cocultivation of immature sorghum embryos of Zheltozernoe 10 with the *A. tumefaciens* GV3101/pNRKAFSIL.

**Figure 2.** Embryogenic callus developing on M11 medium with 2.5 mg/l glufosinate ammonium (A) and regenerated plants (B) obtained in experiment on *Agrobacterium*‐mediated genetic transformation of immature sorghum embryos with *A. tumefaciens* strain GV3101/pNRKAFSIL.

Self‐pollinated progeny (T1 ) of this plant (#94) was tested for herbicide tolerance by germi‐ nation on a medium containing 2.5 mg/l of the selective agent (**Figure 4**). This concentration causes browning and death of sensitive non‐transgenic plants. Herbicide‐tolerant plants were found, and the sensitive plants predominated over tolerant ones (**Table 1**). Some of herbicide‐ tolerant plants that were tested with the primers to the *bar* gene were proved to be PCR‐positive (**Figure 3B**). In the progeny of PCR‐positive T1 plants (i.e., in the T2 generation) that were grown on the medium with 2.5 mg/l GA, the frequency of herbicide tolerant plants was significantly higher (**Table 1**) and some of these plants were also PCR‐positive (data not shown).

These data testify that the progeny of plant #94 inherited the transgenic construct. A low fre‐ quency of tolerant plants in the T1 generation might be explained by silencing of the *bar* gene driven by *nos*‐promotor because silencing of transgene is a common phenomenon in sorghum

**Figure 3.** PCR analysis of genomic DNA of plants from T<sup>0</sup> (A) and T1 (B) generations obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL with primers to *bar* gene. (A) 1—original non‐transgenic line, Zheltozernoe 10; 2—negative control without template DNA; 3—T<sup>0</sup> plant (#94); 4—pNRKAFSIL; M—100‐bp ladder. (B) 1–6—individual plants from T1 generation; 7—pNRKAFSIL; 8—negative control without template DNA; M—100‐bp ladder. Amplified fragment of the *bar* gene (444 bp) is marked by arrow (published with the permission of the publishing house "Nauka").

Development of Transgenic Sorghum Plants with Improved *In Vitro* Kafirin Digestibility http://dx.doi.org/10.5772/intechopen.69973 97

**Figure 4.** Segregation for tolerance to 2.5 mg/l glufosinate ammonium in the progeny of PCR‐positive plant #94 obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL. Note green tolerant plants and necrosis in sensitive plants (tolerant plants survived selection, have been transferred from the agar medium to tap water to improve their survival in soil).

genetic transformation [34]. In the T2 generation, segregation of GA‐tolerant vs. GA‐sensitive plants corresponds to a monogenic ratio 3:1 (χ<sup>2</sup> = 0.286; 0.50 < *P* < 0.75) (**Table 1**).

Self‐pollinated progeny (T1

96 Plant Engineering

with *A. tumefaciens* strain GV3101/pNRKAFSIL.

(**Figure 3B**). In the progeny of PCR‐positive T1

**Figure 3.** PCR analysis of genomic DNA of plants from T<sup>0</sup>

2—negative control without template DNA; 3—T<sup>0</sup>

plants from T1

quency of tolerant plants in the T1

) of this plant (#94) was tested for herbicide tolerance by germi‐

generation might be explained by silencing of the *bar* gene

generation) that were grown

(B) generations obtained by genetic transformation

plant (#94); 4—pNRKAFSIL; M—100‐bp ladder. (B) 1–6—individual

plants (i.e., in the T2

nation on a medium containing 2.5 mg/l of the selective agent (**Figure 4**). This concentration causes browning and death of sensitive non‐transgenic plants. Herbicide‐tolerant plants were found, and the sensitive plants predominated over tolerant ones (**Table 1**). Some of herbicide‐ tolerant plants that were tested with the primers to the *bar* gene were proved to be PCR‐positive

**Figure 2.** Embryogenic callus developing on M11 medium with 2.5 mg/l glufosinate ammonium (A) and regenerated plants (B) obtained in experiment on *Agrobacterium*‐mediated genetic transformation of immature sorghum embryos

on the medium with 2.5 mg/l GA, the frequency of herbicide tolerant plants was significantly

These data testify that the progeny of plant #94 inherited the transgenic construct. A low fre‐

driven by *nos*‐promotor because silencing of transgene is a common phenomenon in sorghum

(A) and T1

generation; 7—pNRKAFSIL; 8—negative control without template DNA; M—100‐bp ladder. Amplified

with *A. tumefaciens* GV3101/pNRKAFSIL with primers to *bar* gene. (A) 1—original non‐transgenic line, Zheltozernoe 10;

fragment of the *bar* gene (444 bp) is marked by arrow (published with the permission of the publishing house "Nauka").

higher (**Table 1**) and some of these plants were also PCR‐positive (data not shown).

The inheritance of T‐DNA in subsequent generations, including T<sup>4</sup> , was confirmed by PCR analysis using primers to the marker gene *bar*, with each of the three T2 families studied con‐ tained PCR‐positive plants (**Figure 5A**).

To verify the presence of the genetic construct for RNA silencing of the γ‐kafirin gene in the transgenic plants, we performed a PCR analysis of a number of plants from T3 and T4 genera‐ tions for the presence of ubiquitin intron. In the studied plants, amplification of a fragment of this gene was observed, which confirmed the presence of a genetic construction for γ‐kafirin silencing in the genome of the obtained transgenic plants (**Figure 5B**).

**Figure 5.** PCR analysis of genomic DNA of sorghum plants from T<sup>3</sup> and T4 generations obtained by genetic transformation with *A. tumefaciens* GV3101/pNRKAFSIL with primers to *bar* gene (A) and *Ubi*‐intron (B). (A) 1—Т<sup>3</sup> 94‐2‐04‐1; 2—Т<sup>3</sup> 94‐2‐04‐3; 3—Т<sup>4</sup> 94‐2‐11‐2‐4; 4—Т<sup>3</sup> 94‐3‐04‐3; 5—Т<sup>4</sup> 94‐3‐08‐2‐1; 6—Т<sup>4</sup> 94‐3‐08‐2‐3; 7—Т<sup>4</sup> 94‐2‐11‐2‐1; 8—pNRKAFSIL; M—100‐bp ladder and 9—negative control (without DNA template). Amplified fragment of the *bar* gene (444 bp) is marked by arrow. (B) 1—Zh10, original non‐transgenic line; 2—Т<sup>3</sup> 94‐2‐04‐1; 3—Т<sup>4</sup> 94‐2‐11‐2‐1; 4—Т<sup>0</sup> Ogonek; 5—Т<sup>3</sup> 94‐2‐04‐2; 6—Т<sup>4</sup> 94‐3‐08‐3‐3; 7—pNRKAFSIL; M—100‐bp ladder and 8—negative control (without DNA template). Amplified fragment of *Ubi*‐intron (584 bp) is marked by arrow.
