Pollen-mediated gene flow from transgenic cotton is constrained by physical isolation measures

The public concern about pollen-mediated gene flow (PGF) from genetically modified (GM) crops to non-GM crops heats up in recent years over China. In the current study, we conducted greenhouse and field experiments to measure PGF with various physical isolation measures, including 90, 80, 60 and 40 holes/cm2 separation nets and Sorghum bicolor, Zea mays and Lycopersicon esculentum separation crops between GM cotton and non-GM line (Shiyuan321) by seed DNA test during 2013 to 2015, and pollen grain dyeing was also conducted to assess the pollen flow in greenhouse during 2013. Our results revealed that (1) PGF varied depending on the physical isolation measures. PGF was the lowest with 90 holes/cm2 separation net and S. bicolor separation crop, and the highest with 40 holes/cm2 separation net and no isolation measure. (2) Similar to PGF results, 90 holes/cm2 separation net and S. bicolor separation crop could minimize the pollen dispersal. (3) PGF declined exponentially with increasing distance between GM cotton and Shiyuan321. Because of the production mode of farm household (limited cultivated area) in China, our study is particularly important, which is not only benefit for constraining PGF, but also has potential application value in practical production and the scientific researches.


Results
Greenhouse and field observation. The agronomic behavior of GM cotton and Shiyuan321 was similar between the greenhouse and field trials, and flowering was synchronous between two types of cottons throughout the flowering period, thus ensuring ample opportunity for pollen transfer. In greenhouses, wind velocities (most distance points: 0.5-0.6 m/s) were stable ( Figure S1). In field, no pesticide was used to control pests, and Apolygus lucorum, Aphis gossypii, Apis mellifera, Pieris rapae and Harmonia axyridis were observed in both years.

Evaluation of pollen dispersal with different physical isolation measures. Pollen grain dyeing
was conducted in a greenhouse during 2013 to assess the pollen flow with various physical isolation measures. As shown in Table 1, dyed pollens with various separation nets and crops were observed most at the distance of 3.2 and 4.0 m, respectively. The 90 holes/cm 2 separation net and S. bicolor separation crop could minimize the pollen dispersal, whereas the constraints of pollen dispersal by 40 holes/cm 2 separation net and L. esculentum separation crop were the weakest, and dyed pollens were observed at all distance points with no isolation measure (CK).
Hybridization was detected during the 2 yr of the study, and varied by the physical isolation measure and distance ( Table 2). More specifically, the average PGF of all sampled distances during the 2 yr were 3.69%, 4.40%, 6.31% and 8.81% with 90, 80, 60 and 40 holes/cm 2 separation nets, respectively (F 3,164 = 4.534, P = 0.004), 4.69%, 8.85%, 13.13% and 19.17% with S. bicolor, Z. mays, L. esculentum separation crops and no isolation measure (CK), respectively (F 3,188 = 10.766, P < 0.001). Net isolation (5.80%) was more effective to constrain PGF than crop isolation (8.89%) (t = 2.710, df = 226.845, P = 0.007). As shown in Table 2  Assessment of pollen-mediated gene flow in field experiment. A total of 5,040 seeds (60 seeds/ sample × 3 physical isolation measures × 7 distances × 2 directions × 2 yr) were sampled to assess PGF by DNA analyses in field experiment. Similar to greenhouse experiment, hybridization was varied by the physical isolation measure and distance in field experiment. More specifically, the average PGF of all sampled distances during the 2 yr were 2.86%, 3.99% and 13.51% with 90 holes/cm 2 separation net, S. bicolor separation crop and no isolation measure (CK), respectively (F 2,249 = 51.017, P < 0.001), however PGF with net isolation and crop isolation shown no significant difference (t = 1.342, df = 166, P = 0.182)( Table 3). As shown in Table 3 and Fig. 2, PGF also declined with increasing distance, ranging from 29.17% at 4.8 m to 6.67% at 25.6 m in 2014 and from 22.50% at 4.0 m to 5.83% at 19.2 and 25.6 m in 2015 with no isolation measure (CK), whereas PGF with 90 holes/cm 2 separation net and S. bicolor separation crop were relatively low among different distance points. Line (y = lnx) was the best-fit regression curve in Fig. 2 (CK R 2 = 0.8560; S. bicolor R 2 = 0.8748; 90 holes/cm 2 R 2 = 0.8832).

Discussion
Efficient pollen dispersal leading to the fertilization, is the major problem when considering the risk of transgene escape in particular for GM crops that can hybridize with non-GM lines or related wild species 1,34 . When the desired gene gets introgressed with other species by recombination, particularly weedy relatives and feral and volunteer crop plants, there is a potential risk for new and more invasive weeds to arise [35][36][37] , and domestication traits can also make weeds less invasive 38 . Regulation approaches of GM crops in Europe are mainly based on the process of making GM crops, whereas those of Canada are based on the GM product. The regulation of GM crops in USA focuses primarily on the characteristics of the product, and a useful oversight of the regulatory process is maintained. China implements a relative pragmatic approach to GM crop regulations, which are basically based on the products and depended on the economic interest of a given application 3 . Thus, preventing gene flow is important for risk management of releasing GM crops, and the physical isolation measure may be a good choice in practical production to constrain the pollen dispersal [24][25][26][27] . In the present study, we determined the pollen dispersal with various physical isolation measures and measured PGF between the GM cotton and non-GM cotton in greenhouse and field experiments during two years using the molecular technique, which was confirmed to be more reliable to determine PGF than biological assays because of the weaker resistance of hybrids for some assays 21 . Previous studies on PGF were mainly conducted in fields 14,39-41 , whereas our study was conducted in greenhouse and field, which provided a more complementary assessment. Zhang et al. 8 found that PGF from GM cotton decreased exponentially as dispersal distance increased, ranging from 10.13% at 1 m to 0.04% at 50 m for the tfd A gene, 8.16% at 1 m to 0.08% at 20 m for the Bt gene. Our early study 21 found that PGF with Shiyuan321 went from 30% to <5% within 1.6 m from the test plot, and a low level from 8.33% to <1% PGF continued to occur sporadically out to a distance of 25.6 m. Van Deynze et al. 33 found PGF was independent of direction from the source plot and declined exponentially with increasing distance from 7.65% at 0.3 m to <1% beyond 9 m with A. melifera. In the present study, we collected a total of 12,240 cotton seeds during the 2-yr study and then extracted DNA from seeds directly to determine PGF. Similar to other previous studies 14,26,42 , we found PGF with any isolation measure in greenhouse and field tended to decline significantly with increasing distance because of the decreased proportion of GM pollens over the distance.
The heights of S. bicolor, Z. mays and L. esculentum were nearly 3.2, 2.2 and 1.5 m respectively at the pollination of cottons. Cotton pollen diameter is 120-150 μm, and the hole sizes of four kind separation nets were bigger than the size of cotton pollen. Our results revealed that 90 holes/cm 2 separation net and S. bicolor separation crop could constrain the pollen dispersal effectively, whereas dyed pollens were observed at all distance points with no isolation measure (CK). The constraint of pollen dispersal was benefit for reducing the environment risk of transgene escape. Similar to pollen grain dyeing results, we found that PGF in greenhouse and field was constrained by various physical isolation measures, with 90 holes/cm 2 separation net and S. bicolor separation crop having lower PGF rates than other isolation measures. Meanwhile net isolation was more effective to constrain PGF than crop isolation no matter in greenhouse and field. The height of separation crop might be the key to constrain PGF. In general, the 20 m cotton buffer or 50 m bare ground is accepted to provide sufficient containment of GM cotton in small scale trials in Australia, compared to sometimes up to 800 m for foundation seed production with additional pollen buffer rows surrounding the crop in USA 18 . Zhang et al. 8 confirmed that the farthest distance of pollen dispersal from cotton was 50 m, and recommended a 60 m buffer zone to limit pollen dispersal from small-scale field tests. However, isolation distance requirement may vary under different environmental conditions, especially wind and pollinator activity 14,19,21,33 . In the present study, we could still detect the gene flow occurring at 25.6 m both with 90 holes/cm 2 separation net and S. bicolor separation crop in field, and the further isolation distance with S. bicolor buffer or double separation nets may be needed to constrain PGF in agricultural production and scientific researches. The application of nets may be also benefit for controlling many insect plagues, whereas the disadvantage of this method is the high expense, Figure 2. Regression analysis for PGF with different physical isolation measures in field experiment as a function of the distance. The determination coefficient (R 2 ) between PGF and distance was obtained using SigmaPlot 10.0 software. Regression analysis was based on the data of the 2 yr study. and the price of nets increases with the number of holes/cm 2 . The application of long isolation distance to constrain PGF is unrealistic in China because of the limited cultivated area, and agricultural ministries of each level should explore subsidy mechanics to promote the application of plant buffer or other physical isolation measures in practical production.
We observed that PGF occurred at a higher frequency in greenhouse than field during our 2-yr study, which might be related to the automatic wind in greenhouse. PGF or cross-fertilization in field might be more complex than those in greenhouse, which was related with changeable natural condition 19,21,33,43,44 . Meanwhile, the planting scale of GM crops and the genetic relationship between GM crops and adjacent crops would also influence PGF 21,45 . Thus, we should consider the local natural condition and agroecological environment before setting the proper isolation measure to constrain PGF. The development of transgenic technology in China has already drawn heated public concerns about the possible risk of GM food in recent years, and central government has a long way to extend consuming transgenic crops. The physical isolation measure will play a more important role in future agricultural production.

Methods
Plant materials. The inbred line Shiyuan321 (pollen receptor) and GM cotton (pollen donor) were obtained from the Cotton Research Institute, Chinese Academy of Agricultural Sciences, and were purified by consecutive two-generations of self-crosses, to ensure that each line was homozygous. The GM cotton was obtained by transferring the insect resistance gene Cry I Ac and the glyphosate resistance gene CP4 EPSPS from Agrobacterium tumefaciens into the embryonic calli of Shiyuan321 via microprojectile bombardment. The hemizygous progeny can be easily identified by PCR analyses because of the high resistance gene expression 23,46-48 . Greenhouse layout and procedures. To evaluate the impacts of physical isolation measures on PGF, the greenhouse trials (L60 m × W8 m × H4 m) were carried out in the Shangzhuang Experimental Station of China Agricultural University (40°14′N, 116°19′E), Beijing, China during 2013 and 2014. Greenhouse provided a relatively closed environment, which was not benefit for pollen dispersal. Thus, several electric fans (2 m height) were installed every 4.5 m, turned on (from 6:00 to 18:00) to promote pollen dispersal, and wind speeds were monitored at 8:00, 12:00 and 16:00 (as 3 replications) using an anemograph (ZRQF-F303, Beijing Detector Instrument Co., Beijing, China) 19 . The agronomic behavior of GM cotton and Shiyuan321 was similar to a standard cultivar in the field. Plants were monitored at least twice weekly for florescence emergence and anthesis from early July through late August (24-29 °C, full-bloom stage for all plants) to ensure the flowering synchrony between two types of cottons 49 . Treatment rows were spaced 0.8 m apart and plants within rows were spaced 0.5 m apart. Fig. 3  PGF evaluation. For each distance point, 60 seeds [20 seeds from each height of flowers (lower, middle, and upper)] were screened for hybrid confirmation by polymerase chain reaction (PCR) amplification of the fragment sequence of Cry I Ac. DNA was extracted using the cetyltrimethyl ammonium bromide (CTAB) protocol 51 with the addition of 20 g/L polyvinylpyrrolidone (PVP) in the extraction buffer. A 1 μL sample of DNA was used to perform spectroscopic quantitation using a NanoDrop 2000 spectrophotometer (Thermo Fisher, USA). Then, 80 ng DNA samples were used to amplify the target gene with primers of 5′-GAAGGATTGAGCAATCTCTAC-3′ and 5′-CAATCAGCCTAGTAAGGTCGT-3′ 19 . The PCR reaction was typically cycled 30 times at 94 °C for 60 s, 56 °C for 60 s, and 72 °C for 90 s 19 . Routine screening for PCR assay was performed by agarose gel electrophoresis (10 g agarose/L) with GM cotton as positive control and Shiyuan321 as negative control. Statistical analysis. All statistical analyses were conducted using the SPSS 16.0 software (IBM, Armonk, NY). All descriptive statistics were given as the mean values and standard errors of the mean (mean ± SE). The data were analyzed using Tukey HSD test and intendent t test at the P = 0.05 level of significance. Regression analyses (a potential dispersal model: y = lnx) were performed with SigmaPlot 10.0 software (Systat Software Inc., San Jose), and the determination coefficient (R 2 ) between PGF and distance was obtained from SigmaPlot 10.0 software.