Table 1 Assessing T-DNA excision and targeted editing detection in grapevine and apple transgenic lines.

From: Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Vector

Genotype

plant ID

Copy Number CN n = nptII; c = Cas9

CN after induction A = induced plant 6 h. 42 °C 3x; B = induced bud 3 h. 42 °C 3x; C = induced bud 3 h. 42 °C 5x

Cas9 induction

Targeted editing percentage

Vitis vinifera

Vector 1

Chardonnay

GT89.3

0.71 ± 0.22 (n)

n.i.

Constitutive

50%

Thompson seedless

GT90.1

0.92 ± 0.06 (n)

A 0.98 ± 0.09 (n)

Constitutive

0%

Vector 2

Microvine

GT92.2

1.81 ± 0.09 (n)

n.i.

Constitutive

n.a.

GT103.1

0.97 ± 0.17(n)

B1 0.83; B2 0.84; C 0.87

Constitutive

B1 4.8% + 5.6%; B2 5% + 5.3%; C 3% + 3%

GT103.2

0.85 ± 0.04 (n)

C 0.86

Constitutive

C 16.6% + 14%

Vector 3

Sugraone

GT109.3

0.85 (c)

A 0.75 ± 0.13

Inducible

B 1.4%

Vector 4

Sugraone

GT110.4

1.2 ± 0.4 (c )

A1 0.78; A2 0.83; A3 0.67; C1 0.96; C2 0.92

Inducible

A1 0%; A2 0%; A3 0%; C1 0%; C2 0%

GT110.5

0.8 ± 0.25 (c )

A1 0.85; A2 0.73; C1 0.82; C2 0.71

Inducible

A1 12%; A2 29%; C1 6%; C2 6.6%

GT110.6

0.79 ± 0.13 (c )

A1 0.93; A2 0.79; C1 0.88; C2 0.92

Inducible

A1 5%; A2 3.3%; C1 9%; C2 4%

GT110.8

0.82 (c )

A 0.85

Inducible

A 0%

GT110.11

1.14 (c )

n.i.

Inducible

n.a.

GT110.15

1.34 (c )

n.i.

Inducible

n.a.

GT110.18

0.78 (c )

n.i.

Inducible

n.a.

GT110.20

1.24 (c )

n.i.

Inducible

n.a.

Malus x domestica

Vector 5

Gala

V1-4

1.1 (n)

n.i.

Constitutive

0%

V1-10

0.9 ± 0.1 (n)

A1 0.61; A2 0.15; A3 0.2; A4 1.14; A5 0.48; A6 0.07; A7 0.13; A8 0.26; A9 0.3; A10 0.3

Constitutive

100%

V1-14

0.6 (n)

n.i.

Constitutive

50%

Golden Delicious

V2-3

1.0 ± 0.1(n)

A1 0.13; A2 0.25; A3 0.62; A4 0.74; A5 0.32; A6 0.67; A7 0.1; A8 0.28; A9 0.11; A10 0.63

Constitutive

100%

V4-5

0.6 (n)

n.i.

Constitutive

0%

V4-27

0.8 (n)

n.i.

Constitutive

100%

V4-34

1.6 ± 0.3 (n)

A1 1.35; A2 1.48; A3 1.98; A4 1.77; A5 1.4; A6 1.7; A7 1.63; A8 1.64; A9 1.76; A10 0.0

Constitutive

100%

Vector 6

Gala

V6-2

1.10 ± 0.12 (n)

n.i.

Constitutive

100%

V6-10

0.95 ± 0.13 (n)

n.i.

Constitutive

100%

  1. Using the binary vectors illustrated in Fig. 1, fourteen grapevine and nine apple transgenic lines, from different genotypes, were produced and included in the present study. The presence of T-DNA in the genome of not-induced and heat-shock induced (via the strategies A, B, C) plants was evaluated by quantifying the nptII (n) or Cas9 (c) copy number (CN) using a Taqman Real time-PCR method described in the Materials and Methods Section. CRISPR/Cas9 on-target editing was detected on the predicted target site by NGS as reported in the Materials and Methods Section. Not induced (n.i.); not applicable (n.a.). Data related to vector 5 are the one reported in Pompili et al.12.
Back to article page