## 3 Results and Discussion
### Selection of LMO Monitoring Sites and LM Cotton Survey  
As shown in Figure 1, the MOE monitored and removed unintentionally released LMO plants in certain areas of the country from 2009 to 2013. However, there is a need to broaden the scope to protect all natural ecosystems in South Korea. Thus, nationwide monitoring sites for LM cotton were selected and surveyed in six administrative districts of South Korea from 2014 to 2018 (Figure 2A,B). Based on the monitoring results of the previous year, the number of survey sites was expanded from 2014 to 2018 and natural environments along LMO transport routes were investigated (Figure 2B). A total of 3921 survey sites were monitored and many suspicious samples were found in various natural environments (Figure 2). Over a five-year period, 451 suspicious samples were collected: 71 in 2014, 58 in 2015, 43 in 2016, 167 in 2017, and 112 in 2018 (Figure 2D), suggesting that the guidance manual provided to investigators played an important role in facilitating the identification of released LMOs at survey sites. Interestingly, the number of suspicious samples collected decreased from 2014 to 2016 (Figure 2D). However, the number of suspicious samples dramatically increased in 2017 and 2018 due to a massive spillage of LM cotton. In particular, unapproved LM cotton was accidentally cultivated at an exhibition site in 2017, which likely increased the number of suspicious cotton samples collected that year. Figure 1: Scheme of the new LMO monitoring strategy. A comparison of the old (2009–2013) and new (2014-2018) LMO monitoring strategies. Recognition of Introduced LMO Proteins Using the Immunochemical Strip Kit Test during LM Cotton Survey  
Transgenic crops expressing functional LMO proteins are being developed to reduce production costs and may improve product safety. However, the introduced proteins in LMOs may pose a variety of risk assessment and management challenges in LMO-released environments. Therefore, rapid recognition of LMO traits in collected samples during a survey may improve the efficiency of conservation efforts for the natural ecosystem. In the current study, introduced LMO proteins, such as Cry1A, Cry1F, Cry2A, Vip3A, EPSPS, PAT/bar, DMO, and AAD-12, were detected quickly in collected samples using immunochemical strip kits at the environmental survey sites. Over a five-year period, 255 and 196 cotton plants were identified as positive (LMO) and negative (non-LMO), respectively (Figure 3A). Assessing LMO traits with strip kits enabled the fast identification of unintentionally released LM cotton in South Korea as herbicide-resistant, insect-resistant, or both (Figure 3B). The percentages of LMO traits in the presence of stacked traits (more than one gene) for insect resistance, herbicide resistance, or both were 24.3%, 3.5%, and 72.2%, respectively (Figure 3B). Figure 2: Monitoring sites for LM cotton and numbers of suspicious samples collected from 2014 to 2018. (**A**) Locations of LMO monitoring survey sites (blue spots) and (**B**) the number of monitoring sites by year. (**C**) The spread of suspicious cotton samples in South Korea (green spots) and (**D**) the number of collected suspicious samples by year. Event-specific PCR analysis was performed using the endogenous reference gene for cotton and event-specific introduced genes to identify the event name, manufacturer, and type of trait. As shown in Table 2, 13 event types were detected in 255 samples, and a variety of LM cotton events were identified each year: 43 in 2014, 40 in 2015, 29 in 2016, 61 in 2017, and 82 in 2018. Nucleotide sequencing analysis was used to investigate event information in the LM cotton samples, revealing that the number of LMO-positive populations and trait types matched exactly with the data in Table 2 and Figure 3. LM cotton was identified nationwide, and its abundance dramatically increased in 2018, which could be due to the expansion of survey areas for unapproved LMO cultivation following a number of unintentional releases of LM cotton at an exhibition site in 2017.