# A New Approach for Environmental Risk Assessments of Living Modified Organisms in South Korea
## 3 Results and Discussion
### Risk Assessment Using an Agroinfiltrated Plant  
To verify the applicability of the infiltrated plant as a replacement for the LM crop, a bioassay for _S. litura_ was performed using an artificial diet and an agroinfiltrated plant. The Vip3A protein concentration used in the diet-overlay bioassay was determined using the methods of [PERSON] et al. and [PERSON] et al. (2017); [PERSON] et al. (2017). A measurement endpoint is generally demonstrated as a significant increase in lethal toxin concentration, killing 50% of the insects (LC\({}_{50}\)) [PERSON] et al. (2017). The response criteria used to assess mortality included severe growth inhibition and death. Therefore, the time in days until 50% lethality (LT\({}_{50}\)) is necessary to perform a risk assessment of the new approach. First, the LT\({}_{50}\) was investigated by insect bioassays using an artificial diet and an agroinfiltrated plant (Table 1). As shown in Table 1, we confirmed that the LT\({}_{50}\) value of transient agroinfiltrated plants was more efficient than the artificial diet. In addition, the LT\({}_{50}\) value of agroinfiltrated plants was lower than that of LM crops or LM proteins [PERSON] et al. (2017); [PERSON] et al. (2017). This result suggests that agroinfiltrated plants can be used as an effective material for LMO risk assessment using insect bioassays. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Feed Type** & **Number of _S. litura_** & \
\begin{tabular}{c} **LT\({}_{50}\)\(\pm\) SE** \\ **(Day)** \\ \end{tabular} &
\begin{tabular}{c} **LT\({}_{70}\)\(\pm\) SE** \\ **(Day)** \\ \end{tabular} \\ \hline Artificial diet & & & \\ (+Vip3 Aa) & 100 & 5.8 \(\pm\) 0.20 & 10.2 \(\pm\) 0.41 \\ Agroinfiltrated leaf & 100 & 4.7 \(\pm\) 0.45 & 9.2 \(\pm\) 3.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The survival time of _S. litura_ larvae using the insect bioassay. Figure 4: The stability of Vip3 Aa in the recombinant protein-treated artificial diet and infiltrated plant leaves. The stability of the Vip3 Aa protein in the recombinant Vip3 Aa-treated artificial diet (100 ng/mg) and third-day plant leaves after Vip3 Aa-agroinfiltration. Each sample was collected per 3 h, and the protein expression level was quantified by immunoblot analysis with anti-Vip3 Aa Ab. Protein intensity was measured using the ImageJ (version 1.53n) program. The red arrow indicates the last indication time point of Vip3 Aa protein, and inset data shows the western blot assay data until 24 h. All values represent the mean \(\pm\) standard deviation (SD) of triplicate experiments. We performed a bioassay for _S. litura_ using an artificial diet and agroinfiltrated plant (Figure 4). The duration of the bioassay can drastically affect the final toxicity outcome of the LM protein [41]. Therefore, we fed the protein-treated artificial diet and the agroinfiltrated leaves to the first instar larvae of _S. litura_ (n = 10, duplicate) for 12 d (refer to Table 1) (approximately twice that of the LT50 value). The recombinant protein was added to the artificial diet every 24 h, and the leaves on the third day after infiltration were replaced every 48 h (Figure 5). In the insect bioassay using the recombinant Vip3 Aa-treated artificial diet, dead individuals were detected from the fifth day (LC, 40 ng/mg) and the third day (HC, 100 ng/mg) after feeding (Figure 5a,c). The total number of deaths in the low- and high-concentration groups was 6 and 70, respectively. Bioassays against _S. litura_ larvae showed that both the 100 ng/mg Vip3 Aa-treated artificial diet and agroinfiltrated leaves had a similar toxicity after 12 d (Figure 5c,d). However, in the agroinfiltration leaf-feeding experiment, dead individuals were found on the first day after feeding and 72 of 100 larvae were overlaid with Vip3 Aa-expressed plant leaves (Figure 5b,d). Based on these results,  
Figure 5: In insecticidal activity for _S. litura_ using the recombinant Vip3 Aa-treated-artificial diet and the Vip3 Aa-agorin infiltrated plant leaf.