A new isolation device for shortening gene flow distance in small-scale transgenic maize breeding
The GM maize material used was the GM insect-resistant maize variety (line) GIF, and the maize was a yellow grain strain provided by the Lai Jinsheng Teacher Laboratory of China Agricultural University. The conventional maize variety Meiyu 11 with white kernels was selected as the pollen receptor of GM maize. The inheritance of the seed (kernel) color can be considered to be a single gene, with one pair of alleles (yellow vs. white). The yellow allele is dominant, and the white allele is recessive. The experimental site was sown at the base of the agricultural GM environmental safety assessment of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Wujitangxia Village, Maihao Town, Wenchang City, Hainan Province (110° 45′ 44″ E, 19° 32′ 14″ N). Transgenic insect-resistant maize was sown three times, once every other week, so that the pollination period of GM maize overlapped with the silking period of the non-GM maize. Artificial on-demand sowing with three seeds per hole and a 4–5 cm sowing depth was adopted.
Field experiments were carried out during two seasons in 2016–2017 and 2017–2018. In the first planting season of 2016–2017, the farthest investigated distance of flow frequency was 60 m (Fig. 1A, Table 1). According to the results from the first investigation, the frequency of gene flow in the eight directions beyond 30 m was very low, almost zero (Table 1). Thus, in 2017–2018, the farthest investigated distance of flow frequency was adjusted to 30 m. In the second planting season, the total area was approximately 14,000 m2 (Fig. 1B, Table 2). As in Hainan off-season reproduction regions the work of breeding research institutes is particularly intensive, it is generally difficult to meet conventional isolation conditions. At the same time, this area also provided a reference for regions around the world that need close isolation. Therefore, we added bagging measures in the treatment areas during the maize tassel pollination period in the second planting season in order to further reduce the flow frequency.
Figure 1
Design of the experimental area. (A) In the period of 2016–2017, the design of the experimental area included one control area (A) and one isolation area (B). The dimensions of control area A and isolation area B in the figure are the same. (B) In the period from 2017 to 2018, the design of the experimental area included one control area (D) and three isolation areas (A, B and C). The solid line represents the isolation area, and the dashed line represents the control area without isolation devices. A1–A8 and B1–B8 in (A) and A1–A8, B1–B8, C1–C8 and D1–D8 in (B) represent eight directions of NE, N, NW, W, SW, S, SE and E, respectively. The dimensions of control area D and isolation areas A, B and C in the figure are the same. The blue numbers represent the size of the experimental areas. The green arrows represent the main wind direction during flowering.
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In the first year of the experiment, control and treatment areas were set up. The area of the control region was 10,000 m2 (100 m × 100 m). A 100 m2 (10 m × 10 m) plot was designated in the center for GM insect-resistant maize, and non-GM maize was planted around this central area. The treatment area with isolation measures was 10,000 m2 (100 m × 100 m). A 100 m2 (10 m × 10 m) plot was designated in the center for GM insect-resistant maize, and non-GM maize was planted around this area. Colored steel plates were used as an isolation measure. The isolation height was 4 m.
A colored steel plate was the isolation material used in these experiments (Fig. 2). Colored steel plates and steel plates are two different materials. At present, there are many colors of colored steel plates. As for which color was used in our isolation experiments, there was no strict requirement, only a desire to match with the surrounding environment. Colored steel plates have the advantages of having both an organic polymer and a steel plate, and many organic polymers have good colorability, formability, corrosion resistance, decoration and high-strength. This combines with the workability of a steel plate, which can be easily finished by stamping, cutting, bending, deep drawing, and other processing to form virtually any shape. This makes the products made of colored steel plates have excellent practicability, decoration, processing and durability.
Figure 2
Isolation device for natural ecological risk control of GM maize. (A) Schematic of the isolation device; (B) partial diagram of the isolation device; (C) sectional view of figure (B); (D) structural detail diagram of the square card; 1: rectangular steel frame, 1.1: steel frame wall, 1.1a: horizontal steel rod, 1.1b: vertical steel rod, 2: inclined support rod, 3: colored steel plate, 4: door for entry and exit, 5: hot-dip galvanized steel frame. 6: structure of the square card, 6.1: screw.
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When maize was harvested after ripening, the investigated directions of control plots were NE, N, NW, W, SW, S, SE and E, labeled with A1–A8, respectively, and those of the isolation plots were labeled with B1–B8, respectively. The location of GM insect-resistant maize from 1 m, 3 m, 5 m, 10 m, 15 m, 20 m, 30 m, 40 m, 50 m and 60 m was investigated along these eight directions. The farthest investigation distances for NE, NW, SW and SE were 60 m, and other directions were 40 m. Ten maize plants were harvested randomly at each point (the first ear). Plants were marked in the order of P1, P2, P3, … P10, dried and stored for further testing. The total number of kernels harvested per corn ear was recorded.
In the second year of the experiment, one control and three treatments were set up. The control plot and the three treatment areas with isolation measures covered an area of 3500 m2 (50 m × 70 m). A 100 m2 (10 m × 10 m) plot was designated in the center of the plot to plant GM maize, and non-GM maize was planted around this central area. Colored steel plates were used as an isolation measure. Bagging of tassels of transgenic maize plants was performed during the pollination period. No bagging was conducted in the control area.
When the maize was harvested after ripening, the investigated directions of control plots were NE, N, NW, W, SW, S, SE and E, labeled D1, D2, D3, D4, D5, D6, D7 and D8, respectively. Isolation area A was marked A1, A2, A3, A4, A5, A6, A7 and A8 along the same eight directions. Isolation areas B and C were marked with B1, B2, B3, B4, B5, B6, B7 and B8, and C1, C2, C3, C4, C5, C6, C7 and C8, respectively. The location of GM insect-resistant maize from 1 m, 3 m, 5 m, 10 m, 15 m, 20 m and 30 m was investigated along these eight directions. The farthest investigation distances for NE, NW, SW and SE were 30 m, and the farthest investigation distances for N, W, S and E were 20 m. Ten maize plants were harvested randomly at each point (the first ear). Plants were marked in the order of P1, P2, P3, … P10, dried and stored for further testing. The total number of kernels harvested per corn ear was recorded.
The endosperm was identified by dominant and recessive traits. According to the number of endosperm traits of GM insect-resistant maize harvested at different directions and distances from GM insect-resistant maize, the pollen transmission distance and outcrossing rate of GM insect-resistant maize were then determined. This method can only be applied to dominant endosperm traits such as yellow or non-waxy grains.
The outcrossing rate was calculated according to formula (1):
$$ P = frac{N}{T} times 100, $$
(1)
where P is the outcrossing rate percentage (%), N is the number of corn kernels containing exogenous genes (the number of the yellow seeds) per ear of corn in units of granules, and T is the total grains (the number of the yellow seeds and white seeds) per ear in units of granules. The outcrossing rates of exogenous genes in different directions and distances were determined, and then the pollen flow distance was determined.
As descriptive statistics, the arithmetic mean as well the standard deviation of outcrossing rates were calculated. The outcrossing rate at each point (1 m, 3 m, 5 m, … 60 m) in the experiment was the mean of the outcrossing rate (P1, P2, P3, … P10) of 10 corn plants at that point.
Details of the isolation device for gene flow risk control of GM maize
The isolation device for gene flow risk control of GM maize, as shown in Fig. 2, comprises a rectangular steel frame (1). The rectangular steel frame 1 was composed of four steel frame walls (1.1), each of which was composed of multiple horizontal steel poles (1.1a) and vertical steel poles (1.1b). Each vertical steel pole was fixed 20–30 cm deep in the soil, and the angle between the inclined support pole (2) and the vertical steel pole was 30°–45°. The vertical steel pole of the four steel frame walls intersected the horizontal steel pole of the top. There were eight inclined supporting poles at the intersection of the vertical steel pole at the four corners of the rectangular steel frame and the horizontal steel pole at the top of the rectangular steel frame, and one inclined supporting pole was fixed through the square card structure (6). The four-sided steel frame wall of the rectangular steel frame was equipped with a colored steel plate (3), and one side of the isolation device was provided with an entry and exit (4). Horizontal steel bars at the top of the rectangular steel frame were provided with a hot-dip galvanized steel frame (5). The hot-dip supporting steel frame was a quadrilateral, and the four corners of the hot-dip supporting steel frame were fixed in the middle of the horizontal steel pole through the hoop. The clamp structure (6) included a side opening and a hollow rectangular frame. The top of the inclined support rod was obliquely inserted into the square clamp structure and fixed on the vertical steel rod through a screw (6.1). The dimensions of the steel rod and the inclined supporting rod were 6000 mm in length, 40 mm in diameter and 2 mm in thickness, and the colored steel plate was 0.425 mm in thickness. The size of the device and the number of inclined supporting rods were determined according to the actual situation in the field. More
