3.1 Transgenic Plant Regeneration
Agrobacterium-mediated method of callus infection obtained 40 seedlings after a series of regeneration processes (e.g.co-culture, screening, differentiation, rooting, etc.). The seedlings were transplanted and the herbicide screening of 3-leaf stage seedlings obtained 15 herbicide-resistant plants. The obtained resistant plants were transplanted into the soil and cultivated in the greenhouse for molecular detection.
3.2 PCR detection results of maize transgenic plants
Genomic DNA was extracted from the leaves of herbicide-tolerant plants. The non-transformed plant was used as a negative control and the pCAMBIA3300-cry1Iem-Bar plasmid was used as a positive control. The Cry1Iem and Bar genes were amplified by PCR (Fig. 2). Ten positive plants were obtained. The obvious Cry1Iem gene and Bar gene bands.
3.3 Transgenic Plants Southern Hybridization Test
Southern hybridization was carried out on the plants tested positive by PCR, and the test results are shown in Fig. 3. A total of four Southern-positive plants were obtained, The plants ere single-copy bands. The size of each hybridization band was significantly different, and negative control found no obvious hybridization bands. The results of Southern hybridization indicated that the Cry1Iem gene has been integrated randomly into the genome of maize in different parts.
3.4 Transgenic Plants RT-PCR Detection
In order to further study the transcript of exogenous gene in maize, total RNA was extracted from the seedling of Southern hybrid plants and transcribed into cDNA as a template. The Cry1Iem gene was amplified by PCR using the primers Cry1Iemr-F and Cry1Iemr-R. The internal standard gene (primer: Actin) was simultaneously amplified. The amplification products were detected by 1% agarose gel electrophoresis respectively. The result is shown in Fig. 4. Four transgenic positive plants can amplify two bands of Cry1Iem and Actin genes, while non-transgenic plants only Actin strip without Cry1Iem band. The results of RT-PCR indicated that the Cry1Iem gene has been successfully transcribed in transgenic plants.
3.5 Transgenic Plants Pnsect Resistance Identification
The results of selective and non-selective antifeeding experiments of Bt maize against Ostrinia furnacalis at the end of 2nd instar were consistent (Fig. 5). Bt maize had good antifeeding activity against Ostrinia furnacalis within 8, 16 and 24 hours after the experiment (antifeeding rate was over 50%), but the antifeeding effect of Bt maize gradually lost with time, and the antifeeding rate was almost zero at 48 hours. The results showed that the susceptibility of corn borer to Bt corn decreased with the increase of its age and the time of feeding on Bt corn. In addition, at the same time, the selective antifeeding rate was higher than the non-selective antifeeding rate, which indicated that the larvae of Ostrinia furnacalis ate less Bt maize leaves under the condition of selectivity, while the larvae of Ostrinia furnacalis ate more Bt maize leaves under the condition of non-selectivity, so that the non-selective antifeeding rate was lower.
3.6 Identification of insect resistance in transgenic plants
Experiments on the inoculation of transgenic and non-transgenic plants showed that the T2 transgenic plants positive for PCR had strong resistance to Asian corn borer, and the plants grew well after two weeks of infestation (Fig. 6-A). The transgenic control group was affected by severe insect pest (Fig. 6-B). There were a large number of large wormholes in the heart and outer leaves, which were highly sensitive to corn borer, and the leaf level was greater than 7 grades. The leaf damage of the plant was investigated according to the plant's leaf damage status, and the average leaf damage level of the corn borer was determined. The average damage value of each plant was calculated in three repeated experiments. The plant resistance was evaluated according to the 9-level resistance evaluation standard, and the average value of the three repeated experiments was evaluated. The resistance level of this strain. Statistical analysis was carried out on the resistance of each strain see Tab. 2. The insect resistance of different transformation events was different. The strains L1, L4 and L5 reached high resistance level, and the plants were almost not eaten by Asian corn borer. There were only a few needle-like wormholes, which were significantly different from the control; the strains L2 and L7 showed insect resistance, and the heart leaves had individual wormholes, which were significantly different from the control; the strains showed L3 and L6. For the worms, the heart leaf and the outer leaves have a large number of large wormholes, and there is no significant difference compared with the control. However, a small number of plants in each insect-resistant strain showed a certain degree of susceptibility, again indicating the genetic separation of the offspring. These susceptibility and high-sensitivity events may be due to insufficient expression of Cry1Iem protein or environmental factors, resulting in the loss of insect resistance.
Table 2
The resistances to Asian corn borers of transgenic seeding
Line No.
|
Origin
|
Leaf-feeding level
|
Resistant grade
|
L1
|
T1-1-1
|
1.30±0.43a
|
(HR)
|
L2
|
T1-2-2
|
3.02±0.17a
|
(R)
|
L3
|
T1-2-3
|
7.02±2.17c
|
(S)
|
L4
|
T1-2-4
|
1.22±0.26a
|
(HR)
|
L5
|
T1-3-1
|
1.46±0.36a
|
(HR)
|
L6
|
T1-4-2
|
6.21±0.19c
|
(S)
|
L7
|
T1-5-1
|
3.12±0.31a
|
(R)
|
CK
|
H99
|
7.62±0.42
|
(S)
|
Note: The data in table are the means ± standard deviations of leaf-feeding levels from every line, a, b, c represents a significant difference from the non-transgenic inbred line respectively as:at a p<0.01 , b 0.01<p<0.05 and c 0.05<p, same as the following.
3.7 Identification of insect resistance in transgenic plants during silking
According to the larvae experiment of the transgenic plants in the T2 generation of maize transgenic plants, most of the PCR-positive plants were resistant to Asian corn borer, and the ears were not affected by corn borer, while the corresponding H99 inbred lines were not. In the transgenic corn plants, the ears are severely damaged by corn borer.
According to the average of the degree of damage to the ear of each line, the resistance of the lines was evaluated. The results are shown in Tab. 3. The resistance of the transgenic lines to the corn borer was significantly different at the earing stage. It is the strongest and reaches the high level of resistance. Compared with the control, the difference is extremely significant. L2, L4 and L5 have better resistance and reach the level of insect resistance. Compared with the control, the difference is extremely significant; the resistance of the strain L7 is relatively weak. The level of medium resistance was reached, and the difference was extremely significant compared with the control; while L3 and L6 had no obvious insect resistance compared with the control, and showed the level of insect resistance. Compared with the identification results of seedling resistance, the insect resistance levels of each strain decreased, but the relative levels of insect resistance of each strain were basically the same. The analysis may be due to the decrease of the expression level of Cry1Iem gene in the ear stage. The insect-resistant effect decreased, or the objective damage situation referenced in the evaluation criteria for seedling and ear-breaking insect resistance was inconsistent.
Table 3
Survey result in the field under artificial infestation with Ostrinia furnacalis at silking
Line No.
|
Origin
|
Mean damage rating scales
|
Resistant grade
|
L1
|
T1-1-1
|
1.00±0.21a
|
(HR)
|
L2
|
T1-2-2
|
3.22±0.31a
|
(R)
|
L3
|
T1-2-3
|
7.67±0.42c
|
(S)
|
L4
|
T1-2-4
|
2.21±0.23a
|
(R)
|
L5
|
T1-3-1
|
2.01±0.69a
|
(R)
|
L6
|
T1-4-2
|
6.02±0.45c
|
(S)
|
L7
|
T1-5-1
|
4.55±0.35a
|
(MR)
|
CK
|
H99
|
6.75±0.44
|
(S)
|
Notes:The data in table are the means±standard deviations of ear damagerating scales from threerepeated experiments.