Introduction

Planting forests is an effective way to further the greening of our planet while also contributing to many related goals, such as ecological sustainability or applying biomass in the industrial sector (FAO 2015; UNDP 2016). Following their successful application in crop plants, genetically modified breeding techniques have been applied to woody plants to meet certain specific demands, such as overcoming the barrier of extreme conditions with high/low temperature, salinity or drought, or supplying high lignin/cellulose components to biomass (FAO 2010; Häggman et al. 2013; Osakabe et al. 2011). This has led to much work in this area, and to the approval of three transgenic tree events for commercialization—namely, two events of Populus in China and one event of Eucalyptus in Brazil (ISAAA 2019; USDA 2019). Before approvals were authorized, these genetically engineered trees containing the transgenic events underwent national regulatory review and risk assessments aligned with international standards (Biosafety-Clearing-House 2015; CBD-COP-MOP8 2016; ISAAA 2019), just as for transgenic crops developed over the last 20 years (ISAAA 2016, 2017; “National Academies of Sciences, Engineering, and Medicine” 2016; Parisi et al. 2016).

The Government of Japan signed the Cartagena Protocol on Biosafety on November 21, 2003, and the Cartagena Act, the domestic law to ensure the implementation of the Cartagena Protocol, entered into force on February 19, 2004 (Watanabe et al. 2004; Government of Japan 2003). Prior to the enactment of the Cartagena Act, regulations regarding the biosafety of genetically modified organisms had individual guidelines for each ministry and agency depending on the purposes and context—i.e., guidelines for research and industry, guidelines for environmental and laboratory safety—but all these separate guidelines were unified by the Cartagena Act (Watanabe et al. 2004). The Cartagena Act regulates organisms harboring extracellularly processed nucleic acids (living modified organisms; LMOs) and their uses, and aims to prevent their use from affecting biodiversity (Government of Japan 2003). The Cartagena Act divides the uses of LMOs into roughly two types: applications without measures to prevent dispersion into the environment (“Type 1 Use”), and applications with measures to prevent dispersion into the environment (“Type 2 Use”) (Government of Japan 2003). All field trials of LM plants correspond to Type 1 Use regardless of the scales and purpose, and require risk assessments under the Cartagena Act and associated regulations (MoE and MAFF 2007). Because the protection goals of the environmental risk assessments are to conserve Japan’s diversity of native organisms, the assessments seek to evaluate risk from three perspectives: the competitive impact, the crossing ability, and the production of harmful substances (MoE and MAFF 2007). The first perspective concerns the competitive impact of transgenic plants on surrounding plants (native species)—namely, whether they have greater invasiveness or weediness potential (MoE and MAFF 2007). The second is the crossing ability between transgenic plants and conventional plants, which could allow vertical gene flow into the next hybrid generation, potentially replacing the wild relatives (MoE and MAFF 2007). The third perspective seeks to address the risk of novel or enhanced toxin production, since the dying plant bodies or roots systems of transgenic plants may release various chemical components that potentially represent a greater risk of harmful effects to other species (MoE and MAFF 2007), i.e., plant species or soil microbes. In the case of requesting permission for Type 1 Use of LM plants in Japan, the applicant is requested to submit assessment data on the existence of native organisms affected by the LM plant, and level of impact if the affected organisms are existing, from each of the three perspectives (MoE and MAFF 2007). The Ministry of Agriculture, Forestry and Fisheries indicates some specific methods for these assessments (MoE and MAFF 2007). In addition, when submitting the assessment data for Type 1 Use in Japan, any data obtained in a specific netted room or isolated field in Japan are usually required, although there are some exceptions (MoE and MAFF 2007).

Eucalyptus spp. are currently the most important trees used for forestry plantation. This genus is native to Australia, Papua New Guinea, Indonesia and the island of Mindanao in the Philippines (Eldridge et al. 1994; Nishimura 1987). Eucalyptus camaldulensis, one of the economically important species of Eucalyptus, has been widely cultivated all over the world (Boland et al. 2006; CAB-International 2000; Doran and Brophy 1990; Nishimura 1987). In our previously reported work, three novel salt-tolerant transgenic E. camaldulensis lines overexpressing the RNA-Binding-Protein (McRBP) gene from M. crystallinum, driven by a constitutive MC8 promoter were developed and evaluated in a semi-confined screen house (Tran et al. 2019). They showed clear tolerance to both acute salinity stress (400 mM NaCl within 6 weeks) and chronic salinity stress (70 mM NaCl within 24 weeks), but there was no difference in their phenotype under non-stress conditions (Tran et al. 2019). From these results, we concluded that these lines of transgenic McRBP-E. camaldulensis lines could be the candidates for practical use in plantations or afforestation in highly saline soil (Tran et al. 2019). However, these results come from the experiments conducted in the screen house and not been in an outdoor environment. In addition, before releasing the genetic engineering trees into the environment, the ERA based on the experimental field trial studies (Government of Japan 2003; MoE and MAFF 2007). In this current work, we assessed the potential effects on biodiversity of an experimental confined field trial of these three transgenic McRBP-E. camaldulensis lines in comparison with those of independent non-transgenic E. camaldulensis clonal lines in a manner complying with the Japanese biosafety regulatory flamework.

Materials and methods

Plant materials and cultivation conditions

This study was designed to examine the potential effects on biodiversity of three transgenic McRBP-E. camaldulensis lines, i.e., 2–5–4, 2–5–6 and 2–5–7 (Tran et al. 2019). Transgenic E. camaldulensis lines were generated by shoots induced from seedlings derived from industrially produced bulk seeds. E. camaldulensis seedlings were derived from different seeds from the cross-pollinated population. Thus, the transgenic E. camaldulensis lines differ not only with respect to transgenic events but also in terms of the host genetic background. All the transgenic lines used in this work were T0 generation and vegetative propagated progenies. Because it is difficult to prepare a near-isogenic non-transgenic E. camaldulensis line from transgenic lines, three independent non-transgenic clonal lines, i.e., cam2, cam6 and CML2, derived from plantation forest trees were used as comparators (Tran et al. 2019).

All plant materials were cultivated in 15-cm diameter pots for over 6 months in a semi-confined screen house at the University of Tsukuba, Tsukuba, Ibaraki prefecture, Japan under the stipulations of Japan’s Ministry of Education, Culture, Sports, Science and Technology biosafety regulatory framework for Type 2 Use (Ministerial Ordinance No. 1, 2004), which were described in detail previously (Tran et al. 2018b, 2019; Yu et al. 2009). The semi-confined screen house was a special netted house with all windows (roof and side windows) covered by a mesh screen to inhibit pollen dispersal and the entry of insects, a front chamber in the entrance to prevent exposure of the plants to the outside environment, and two parallel channels to store drainage. In addition, the temperature was controlled by opening and closing the windows and by a gas heater (Tran et al. 2018b, 2019; Yu et al. 2009).

Assessment of the allelopathic impact of plant bodies on peripheral plants by sandwich assay

To estimate the potential risks posed by dying plant bodies on the peripheral plant population, we determined the allelopathic activity of leachates released from dried leaves by the sandwich assay as described in previous reports (Fujii et al. 2003; Tran et al. 2018a). The leaves were collected from three plants each of the transgenic and non-transgenic lines cultivated for more than 6 months in the screen house and dried at 60 °C for 24 h. Two doses of dried leaf tissue (10 mg and 50 mg) were placed in the middle of two 5-mL low-melting-point agar layers in a 6-well plate (34 mm in diameter and 16 mm in depth). In this study, we chose Lettuce (Lactuca sativa var. capitata) as the indicator plant. Lettuce was often chosen as the indicator species for evaluating allelopathic activities because of its properties as simultaneous and rapid germination, reliable germination and homogeneity, high sensitivity to bioactive substances and easiness of purchase with low cost (Fujii et al. 2003). This species was generally used to assess the allelopathic impacts of many plant species (Fujii et al. 2003; Itani et al. 2013; Mardani et al. 2015; Morikawa et al. 2012) as well as to evaluate bioassays used in the ERA of transgenic plants (Kikuchi et al. 2009; Ko et al. 2019; Oguchi et al. 2014; Tran et al. 2018a; Yu et al. 2013a, b). Five lettuce seeds (cv. Gokuwase Cisco; Takii Seed, Kyoto, Japan) were sowed into each well. After incubating at 25 °C for 3 days in the dark, the radicle and hypocotyl lengths were measured. For each dose of dry leaf tissue, nine wells were used for each evaluation. Six wells were used for blank agar media (not containing dry leaf tissue).

Assessment of the allelopathic impact of roots on peripheral plants by succeeding crop assay

To evaluate the potential risk conferred by the allelopathic activity of roots onto the peripheral wild plant population, the succeeding crop assay (Atosaku test) was performed among the transgenic and non-transgenic plants (Asakawa et al. 1992). Approximately 100 g of soil without E. camaldulensis roots was collected from three cultivated pots for each of the transgenic lines and non-transgenic lines and was distributed into 6 plastic cell trays (4-cm square, 4-cm deep). Five lettuce seeds were sown into each soil cell, and were incubated at 25 °C under a dark condition. The growth of lettuce seedlings was measured after sowing 5 days (Asakawa et al. 1992; Tran et al. 2018a). Eighteen cells were used for each evaluation and six cells were used for blank new soil.

Assessment of potential impact on the population of soil microorganisms by the spread plate method

In order to estimate the potential impact of harmful substances secreted from the roots on the soil microorganism community, the numbers of culturable aerobic soil microorganisms were compared between transgenic McRBP-Eucalyptus and the non-transgenic plants. Approximately 60 g of soil without E. camaldulensis roots was collected from three cultivated pots for each of the transgenic and non-transgenic lines. Evaluation of the numbers of culturable aerobic soil microorganisms were performed according to our previous report (Ko et al. 2019; Oguchi et al. 2014; Tran et al. 2018a; Yu et al. 2013b). Two kinds of plate culture media were used: the oxytetracycline-glucose-yeast extract (OGYE) medium to assess soil fungi and the peptone-tryptone-yeast extract-glucose (PTYG) medium to evaluate soil actinomycetes and bacteria other than actinomycetes. 30 g of soil was dried at 80 °C for 24 h as the dry weight sample. The remaining 30 g soil was mixed with 270 mL of sterile 15 mM phosphate buffer (pH 7.0). The diluted soil suspension was spread on petri dishes (9 cm in diameter) containing OGYE/PTYG media and was incubated in the dark at room temperature. Fungal colonies were counted after 3 days of incubation, and actinomycete and bacterial colonies were counted after 7 days of incubation (Oguchi et al. 2014; Tran et al. 2018a).

Statistical analysis

The data were subjected to a statistical analysis using either one-way/two-way analysis of variance (ANOVA) or the split plot analysis of variance (Perry et al. 2009). Each ANOVA was performed using R ver. 3.6.0 software (2019-04-26) and/or Microsoft Excel 2016 MSO (16.0.9001.2102) (Microsoft, Redmond, WA). The Tukey–Kramer multiple comparisons test was used as necessary, with R.

Results

Transgenic RBP-E. camaldulensis and the Japanese biosafety regulation framework for transgenic plants in Type 2 Use

In this study, the host plant was E. camaldulensis Dehnh, commonly known as River red gum, which is an evergreen angiosperm tree belonging to the family Myrtaceae, genus Eucalyptus and section Exsertaria (OECD 2016). It is native to Australia, and distributed over a wide area with latitudes ranging from 2° 48′ S along the Mary River in the tropical Northern territory, to 38° 15′ S in the cool, temperate, southwestern Victoria region, and at altitudes ranging from 20 to 70 m, and in areas with rainfalls of 200–1100 mm annually (CAB-International 2018; Eldridge et al. 1994). Eucalyptus is an exotic genus in most regions in the northern hemisphere. This genus is a poor invader, because pollen dispersal and pollination rarely occur by wind; in most cases they are performed by specific birds that can only be found in Australia (Cremer 1977; Griffin 1980; OECD 2016; Ruthrof et al. 2003). Furthermore, seeds and seedlings of Eucalyptus have high mortality when exposed to inconvenient conditions (OECD 2016).

In Japan, E. camaldulensis is an exotic species, and is currently not used for plantations, but only as an ornamental tree (Nishimura 1987). The invasion risk assessment of E. camaldulensis was performed in an isolated field in Tsukuba city, Ibaraki prefecture, Japan in 2006. The results illustrated that E. camaldulensis is less invasive than wild weeds (Kikuchi et al. 2006). Taken together, these characteristics indicate that E. camaldulensis trees are unlikely to compete with native species, which is a point of concern in the Japanese regulation framework (MoE and MAFF 2007). Moreover, in regard to the third issue, the productivity of harmful substances, allelopathic assessment experiments of several transgenic Eucalyptus events were performed in both a semi-confined screen house (Type 2 Use) and confined field (Type 1 Use) and revealed no adverse impacts, as reported in our previous studies (Kikuchi et al. 2006, 2009; Oguchi et al. 2014; Tran et al. 2018a; Yu et al. 2013a, b).

In this study, each transgenic E. camaldulensis line harbored one copy of the T-DNA construct (Tran et al. 2019). The stability of transgene integration in the host plant genome and the expression of transgenes in three RBP-E. camaldulensis lines were demonstrated in our previous report (Tran et al. 2019). This T-DNA construct includes two transgenes—McRBP and NPTII—that can encode functional proteins. McRBP is a novel RNA-Binding-Protein gene from M. crystallinum. This gene encodes a protein sequence of 306 amino acid residues that contains two RNA-recognition motifs (RRM_1; Pfam identifier PF00076) and that acts as an RNA chaperone in vivo (Tran et al. 2019). By means of its RNA chaperone activities, McRBP improves the abiotic stress tolerance of the three above-mentioned transgenic E. camaldulensis lines but with no enzymatic activity (Tran et al. 2019). Moreover, in other reports, it was confirmed by a database (Allergen Database for Food Safety by National Institute of Health Science, Japan) search that McRBP does not contain any suspected allergenic sequences (Nakamura et al. 2005, 2014). In the case of the selectable marker gene NPTII, risk assessments have already been conducted, and it has been internationally agreed that expression of NPTII causes no considerable risk to human health or the environment (EFSA 2007; Fuchs et al. 1993; OGTR 2017). On the other hand, the T-DNA construct includes other DNA fragments, which are functional sequences such as promoters (MC8 promoter and NOS promoter), terminators (HSP terminator and NOS terminator) and transcriptional enhancer (5′ untranslated region of AtADH gene; AtADH-5′UTR), but they do not encode any protein or peptide fragment.

Familiarity of the organisms serving as donors of the nucleic acids is also important as a biosafety evaluation point for LMOs. M. crystallinum is the donor organism of the McRBP gene (Nakamura et al. 2005, 2014), and Arabidopsis thaliana is the donor organism of two DNA fragments, the HSP terminator (Nagaya et al. 2010) and AtADH-5′UTR (Sugio et al. 2008). The donor organism of NPTII is Escherichia coli (Fuchs et al. 1993; Rothstein et al. 1981). Escherichia coli is a gram-negative bacterium, a harmless member of the normal microbiota of the human intestinal tract (Percival, and Williams 2014). Agrobacterium (R. radiobactor) is a gram-negative soil bacterium that is able to transfer genes in the T-DNA region to plant cells and cause diseases in plants (Nonaka et al. 2017), and it is the donor organism of the NOS promoter (An et al. 1990; Shaw et al. 1984), NOS terminator (Bevan et al. 1983; Depicker et al. 1982) and the right and left border of T-DNA (Barker et al. 1983). In addition, the milk vetch dwarf virus is dependent on plants, and it is the donor organism of the MC8 promoter (Shirasawa-Seo et al. 2005). As a result of the assessment based on the family-friendliness of the nucleic acid donors thus far, the unintended biosafety risks arising from the nucleic acid-donating organisms would be limited.

Allelopathic impact of E. camaldulensis bodies on peripheral plants

The Japanese biosafety regulation framework requires multiple assessments of allelopathic activities as an evaluation of the potential harmful impacts on native plants (MoE and MAFF 2007). The sandwich method is a bioassay method in which the allelopathic activities evaluated by monitoring the germination and growth of the plant on agar (MoE and MAFF 2007; Fujii et al. 2003; Tran et al. 2018a). In this work, three transgenic and three non-transgenic eucalyptus leaves were assayed by the sandwich method and the potential effect of substances released from dying plant bodies on the surrounding plants was evaluated by sandwich assay (Table 1, Fig. 1). The hypocotyl and radicle growth of lettuce seedlings at 10 mg and 50 mg leaf supplementation compared to that of the blank control are shown in Fig. 1. Table 1 shows the analysis of variance (ANOVA) table comparing the allelopathic activities among three transgenic and three non-transgenic lines of E. camaldulensis. Significant differences were observed between the two doses tested (10 mg and 50 mg; α < 0.001), but no significant differences in the hypocotyl and radicle growth of the monitor plant were detected among the 6 lines (α = 0.05) (Table 1). Tukey’s honestly significant difference (HSD) test revealed a remarkable difference between the two doses (10 mg and 50 mg), but no significant difference among the six lines (α = 0.05; Fig. 1). These results suggest that the allelopathic productivity of the transgenic E. camaldulensis leaves is not significantly different from that of the non-transgenic Eucalyptus.

Table 1 Analyses of variance of measurements of the sandwich assay
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Fig. 1
figure 1

Assessment of allelopathic activities by sandwich assay. Allelopathic activities of leaves of the McRBP-transgenic and non-transgenic control E. camaldulensis lines were evaluated by the growth of the recipient lettuce seedlings sowed on agar media containing 10 mg or 50 mg of dried E. camaldulensis leaves. The relative growth of hypocotyls and radicles compared to that on the blank agar media are shown in a, b, respectively. The letters on the upper edge of panels indicate significant differences by the Tukey-HSD test (α = 0.05). Error bars indicate standard error (n = 3)

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Allelopathic impact of adjacent soil on peripheral plants

In case of release the transgenic plants to the environment, the possibility that substances produced by transgenic plants and infiltrate roots and their residues affect the surrounding vegetation through soil should be considered. To assess the potential allelopathic activity of soil adjacent the transgenic plants and their roots, the succeeding crop test was conducted among RBP-E. camaldulensis and their conventional plants (Table 2, Fig. 2). The hypocotyl and radicle growths of recipient lettuce seedlings in cultivated soils are shown in Fig. 2. An ANOVA test indicated that there was no significant difference observed between RBP group and non-transgenic group (α = 0.05; Table 2). Among the six lines, a difference was found in hypocotyl length (α = 0.05; Table 2). However, this difference was not due to an effect of the transgene but due to error, and it was supported by the results from Tukey-HSD test (Table 2, Fig. 2). Consequently, no significant difference was observed in the potential effects on other plants of the productivity of harmful substances by Eucalyptus roots between the transgenic Eucalyptus and the non-transgenic Eucalyptus.

Table 2 Analyses of variance of measurements of the succeeding crop assay
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Fig. 2
figure 2

Assessment of allelopathic activities by succeeding crop assay. Allelopathic activities of the secretory component from McRBP-transgenic and non-transgenic control E. camaldulensis lines were evaluated by the growth of the recipient lettuce seedlings sowed on soils collected from pots cultivated with E. camaldulensis for more than 6 months. The relative growth of hypocotyls and radicles compared to that on the fresh soil is shown in a, b, respectively. The letters on the upper edge of panels indicate significant differences by the Tukey-HSD test (α = 0.05). Error bars indicate standard error (n = 3)

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Impact on the population of soil microorganisms on adjacent soil

In case of release the transgenic plants to the environment, the possibility that substances produced by transgenic plants and infiltrate roots and their residues affect the soil microbioal population through soil should also be considered. The microbial population on soil adjacent to transgenic and non-transgenic reference plants was monitored by the spread plate method (Table 3, Fig. 3). The colony forming units (CFUs) of culturable soil microorganisms are shown in Fig. 3. Tukey-HSD tests indicated that there were no significant differences in the numbers of actinomycetes, bacteria (except actinomycetes), or fungi among the six lines consisting of three transgenic and three non-transgenic lines (Fig. 3). On the other hand, the ANOVA test indicated that the bacterial populations in the soil of the transgenic pots were significantly higher than those in the soil of non-transgenic pots (Table 3). Although the difference was significant, it appeared to have no adverse effect on the population of soil microorganisms on adjacent soil. We therefore tentatively propose that transgenic RBP-E. camaldulensis does not negatively impact the soil microbial biodiversity compared to the non-transgenic E. camaldulensis. A future evaluation on the confined field trial would be expected to provide additional detailed information on the impact of these transgenic Eucalyptus lines on soil microorganisms.

Table 3 Analyses of variance of numbers of culturable soil microorganism
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Fig. 3
figure 3

Assessment of potential impact on soil microorganisms. The microorganisms were extracted from soils collected from pots cultivated with E. camaldulensis for more than 6 months. The diluted soil extracts were spread on an OGYE plate to test for bacteria and actinomycetes or a PTYG plate to test for fungi. The CFU of actinomycetes, bacteria (except actinomycetes), and fungi are shown in a–c, respectively. The letters on the upper edge of panels indicate significant differences by the Tukey-HSD test (α = 0.05). Error bars indicate standard error (n = 3)

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In our previous reports, ERA of transgenic Eucalyptus lines harboring codA/Mangrin genes were performed in a semi-confined screen house (Type 2 Use) (Kikuchi et al. 2009; Tran et al. 2018a; Yu et al. 2013a, b). Their effects on biodiversity were not significantly different from those of the non-transgenic Eucalyptus, so they were granted approval for Type 1 Use. An isolated field trial (Type 1 Use) for some of these transgenic Eucalyptus lines was carried out with ERA. The results indicated that transgenic Eucalyptus did not have an adverse impact on biodiversity (Oguchi et al. 2014). The results from this study are similar to our previous results on Type 2 Use, in as much as there were no adverse allelopathic effects on peripheral plants or microbial populations (Kikuchi et al. 2009; Tran et al. 2018a; Yu et al. 2013a, b).

Discussion

Although the protection goals of the environmental risk assessment of transgenic plants differ by nation and region, ERA is basically performed by comparing the possible impacts on biodiversity between transgenic plants and the appropriate comparators. In this study, according to the Japanese regulatory framework on prior to the experimental confined field trial stipulated by the Minister of Agriculture, Forestry and Fisheries (MAFF 2013), we have confirmed that there is no significant difference between transgenic E. camaldulensis lines harboring the McRBP gene and the non-transgenic E. camaldulensis lines in terms of the potential impacts of the productivity of harmful substances by E. camaldulensis on the peripheral plants and soil microorganisms. From the above results, the three transgenic McRBP-E. camaldulensis lines did not confer additional risk to the receiving environment in the comparison to non-transgenic E. camaldulensis. Therefore, we conclude that using a genetic transformation technique to create McRBP recombination in E. camaldulensis improved the salinity stress tolerance without adversely affecting the biodiversity. Although, the evaluation of the potential impact on biodiversity by the step-by-step field trial would be required for the practical use of McRBP-E. camaldulensis.