Table 2.
Transmission and segregation of CRISPR/Cas9-mediated target mutagenesis and transgenes from T0 to T1 generation.
| T0 line | T0 plants |
Segregation of mutations and T-DNA in T1 progenies |
||||||
|---|---|---|---|---|---|---|---|---|
| Genotype | Zygosity | Cas9/gRNA/ hptII | Number of T1 plants tested | Targeted mutations | Expected segregation ratio | P-value | Cas9/gRNA/hptII | |
| sbeI-c6 | i1a/i1b | Bi-allelic | + | 26 | 6 i1a, 12 i1a/i1b, 8 i1b | 1:2:1 | 0.97 | 24+ : 2- |
| sbeI-c18 | i1 | Ho | + | 15 | 15 i1 | N.A | N.A | 15+ : 0- |
| sbeI-c28 | i1/i64 | Bi-allelic | + | 34 | 17 i1, 11 i1/i64, 6 i64 | 1:2:1 | 0.64 | 26+ : 8- |
| sbeI-c29 | d1 | Ho | + | 15 | 15 d1 | N.A | N.A | 15+ : 0- |
| sbeI-c31 | d1/d8 | Bi-allelic | + | 37 | 9 d1, 17 d1/d8, 11 d8 | 1:2:1 | 0.98 | 28+ : 9- |
| sbeI-c40 | i1/d6 | Bi-allelic | - | 35 | 10 i1, 17 i1/d6, 8 d6 | 1:2:1 | 0.99 | 0+ : 35- |
| sbeIIb-c1 | i1 | Ho | + | 22 | 22 i1 | N.A | N.A | 17+ : 5- |
| sbeIIb-c3 | d5/i248 | Ho | + | 38 | 38 (d5/i248) | N.A | N.A | 17+ : 21- |
| sbeIIb-c4 | i1 | Ho | + | 17 | 17 i1 | N.A | N.A | 17+ : 0- |
| sbeIIb-c7 | i1a/i1b | Bi-allelic | + | 36 | 9 i1a, 20 i1a/i1b, 7 i1b | 1:2:1 | 0.96 | 36+ : 0- |
| sbeIIb-c9 | i1 | Ho | + | 15 | 15 i1 | N.A | N.A | 15+ : 0- |
| sbeIIb-c15 | i1a/i1b | Bi-allelic | + | 19 | 5 i1a, 8i1a /i1b, 6 i1b | 1:2:1 | 0.95 | 18+ : 1- |
T0 plants can be bi-allelic, homozygote (Ho) and heterozygote (He). In d#, the # refers to the number of bp deleted from the target site; i#a or i#b, # refers to the number of bp inserted at target sites with different nucleotide or different positions (a or b). According to the χ2 test, P > 0.5, means in very good agreement with expected Mendelian segregation ratio; N.A, not applicable. For transgene analysis, ‘+’ represents that Cas9/gRNA/hptII was detected and ‘-’ shows Cas9/gRNA/hptII not detected. Some of the T0 lines such as sbe-c1 had only one T-DNA insertion as demonstrated by around 1/3 of Cas9/gRNA/hptII-free plants in T1 progenies, whereas sbeI-c6 T0 plant probably had two T-DNA insertions because only about 1/12 of T1 plants were Cas9/gRNA/hptII free. The failures to identify Cas9/gRNA/hptII-free plants at T1 generation for some lines were probably due to either multiple insertions or tight linkage to the mutations.