Figure 1.
Evaluation of allelopathy through sandwich method. (a): 6-well micro-plate; (b): leaves placed between agar layers. (Source: Fujii et al., 2004).
Tomato cv. Moneymaker was modified by the insertion of a miraculin gene, which can modify a sour taste into a sweet taste. Environmental safety assessment for this special transgenic crop is an important step in assessing how safe this tomato is before it is released into the environment. Evaluation of invasiveness, allelopathy and unintended effects is highly essential for environmental safety assessment. The evaluation of invasiveness was carried out by growing a mixture of transgenic and non-transgenic tomatoes with ratios of 0:100 and 100:0 (sole-cultivation) and 25:75, 50:50 and 75:25 (mix-cultivation). Wet and dry biomasses of three-week-old tomato plants were measured. Soil microbes were evaluated by determining microbial populations (culturable) and estimating soil respiration. Microbial populations were determined through total plate count, while soil respiration was estimated using the titration method to calculate the levels of carbon dioxide released during the incubation. It was found that the aggressiveness of the miraculin transgenic tomato was equal to that of its counterpart. There were also no significant differences in microbial populations and soil respiration of miraculin transgenic tomato compared with those of wild type. In addition, miraculin transgenic tomato did not produce allelopathy that interfered with surrounding crops. It is concluded that transgenic tomato is equal to its counterpart in invasiveness, with no effect to soil microbes and no potential allelopathy found.
Citation: Nono Carsono, Fadlilah Aida Rahmani, Rangga Jiwa Wibawa, Santika Sari, Anas, Ryo Ohsawa, Ayako Shimono, Hiroshi Ezura. Invasiveness, allelopathic potential and unintended effects of miraculin transgenic tomato to soil microbes[J]. AIMS Agriculture and Food, 2022, 7(4): 872-882. doi: 10.3934/agrfood.2022053
| [1] | Nono Carsono, Faza A. Maulana, Iqbal F. Elfakhriano, Ade Ismail, Noladhi Wicaksana, Santika Sari, Hiroshi Ezura . The comparative analysis of agronomic, compositional, and physiological traits of miraculin transgenic tomato in the confined field trial. AIMS Agriculture and Food, 2023, 8(1): 187-197. doi: 10.3934/agrfood.2023010 |
| [2] | Soukaina Ouansafi, Fahde Abdelilah, Mostafa Kabine, Hind Maaghloud, Fatima Bellali, Karima El Bouqdaoui . The effects of soil proprieties on the yield and the growth of tomato plants and fruits irrigated by treated wastewater. AIMS Agriculture and Food, 2019, 4(4): 921-938. doi: 10.3934/agrfood.2019.4.921 |
| [3] | Simon Wambui Mburu, Gilbert Koskey, Ezekiel Mugendi Njeru, John M. Maingi . Revitalization of bacterial endophytes and rhizobacteria for nutrients bioavailability in degraded soils to promote crop production. AIMS Agriculture and Food, 2021, 6(2): 496-524. doi: 10.3934/agrfood.2021029 |
| [4] | Mohammad Zahirul Islam, Young-Tack Lee, Mahmuda Akter Mele, In-Lee Choi, Ho-Min Kang . Effect of fruit size on fruit quality, shelf life and microbial activity in cherry tomatoes. AIMS Agriculture and Food, 2019, 4(2): 340-348. doi: 10.3934/agrfood.2019.2.340 |
| [5] | Muhd Arif Shaffiq Sahrir, Nornasuha Yusoff, Kamalrul Azlan Azizan . Allelopathy activity under laboratory, greenhouse and field conditions: A review. AIMS Agriculture and Food, 2023, 8(1): 78-104. doi: 10.3934/agrfood.2023004 |
| [6] | Md. Shaha Nur Kabir, Kamal Rasool, Wang-Hee Lee, Seong-In Cho, Sun-Ok Chung . Influence of delayed cooling on the quality of tomatoes (Solanum lycopersicum L.) stored in a controlled chamber. AIMS Agriculture and Food, 2020, 5(2): 272-285. doi: 10.3934/agrfood.2020.2.272 |
| [7] | Hayriye Fatma Kibar, Ferhan K. Sabir . Chitosan coating for extending postharvest quality of tomatoes (Lycopersicon esculentum Mill.) maintained at different storage temperatures. AIMS Agriculture and Food, 2018, 3(2): 97-108. doi: 10.3934/agrfood.2018.2.97 |
| [8] | Janice Liang, Travis Reynolds, Alemayehu Wassie, Cathy Collins, Atalel Wubalem . Effects of exotic Eucalyptus spp. plantations on soil properties in and around sacred natural sites in the northern Ethiopian Highlands. AIMS Agriculture and Food, 2016, 1(2): 175-193. doi: 10.3934/agrfood.2016.2.175 |
| [9] | S. Ehsan Zohoori, Amir Mohamadi-Nejad, Reza Moghaddasi . Effective ways to the global competitiveness of food industry companies. AIMS Agriculture and Food, 2018, 3(3): 345-357. doi: 10.3934/agrfood.2018.3.345 |
| [10] | Cátia Morgado, José M. Pestana, Manuela M. Guerra, Carlos Brandão . Microbiological quality in convenient ready-to-eat vegetables during shelf life. AIMS Agriculture and Food, 2018, 3(4): 372-383. doi: 10.3934/agrfood.2018.4.372 |
Tomato cv. Moneymaker was modified by the insertion of a miraculin gene, which can modify a sour taste into a sweet taste. Environmental safety assessment for this special transgenic crop is an important step in assessing how safe this tomato is before it is released into the environment. Evaluation of invasiveness, allelopathy and unintended effects is highly essential for environmental safety assessment. The evaluation of invasiveness was carried out by growing a mixture of transgenic and non-transgenic tomatoes with ratios of 0:100 and 100:0 (sole-cultivation) and 25:75, 50:50 and 75:25 (mix-cultivation). Wet and dry biomasses of three-week-old tomato plants were measured. Soil microbes were evaluated by determining microbial populations (culturable) and estimating soil respiration. Microbial populations were determined through total plate count, while soil respiration was estimated using the titration method to calculate the levels of carbon dioxide released during the incubation. It was found that the aggressiveness of the miraculin transgenic tomato was equal to that of its counterpart. There were also no significant differences in microbial populations and soil respiration of miraculin transgenic tomato compared with those of wild type. In addition, miraculin transgenic tomato did not produce allelopathy that interfered with surrounding crops. It is concluded that transgenic tomato is equal to its counterpart in invasiveness, with no effect to soil microbes and no potential allelopathy found.
The transgenic approach is a promising technique to support food security and improve the quality human life. This technology has high potency to improve production, nutrients, and crop resistance to biotic and abiotic stresses [1]. In 2016, genetically modified crops were planted by farmers in 185.1 million hectares across 26 countries, with 54% of biotech crops in developing countries and 46% in industrial countries [2]. In the United States, as a biotech mega-country, 72.9 million hectares have been growing with commercialized transgenic crops, such as maize (35.05 million hectares), soybean (31.84 million hectares), cotton (3.70 million hectares), alfalfa (1.23 million hectares), canola (0.62 million hectares), sugar beet (0.47 million hectares), papaya (1000 hectares), squash (1000 hectares) and potatoes (2500 hectares) [2].
Sugaya et al. [3] and Sun et al. [4,5] successfully produced transgenic strawberry, lettuce and tomato expressing miraculin gene under the control of a CaMV-35S promoter. The gene is derived from miracle fruit (Richadella dulcifica) from West Africa [6]. This transgenic tomato has a potential as an alternative low-calorie food. Miraculin can change a sour taste on the tongue into a sweet taste [7]. Miraculin is not sweet by itself, but if it is inserted into a plant with a sour taste, or we eat miraculin before the sour fruit, the fruit will taste sweet [8]. For example, before enjoying a lemon, if one eats fruits with the glycoprotein miraculin, then the tongue will taste sweetness. When the miraculin is on the tongue, the sweetness level can be over one hour when we feel an acidic solution [9]. The production of miraculin from the original plant (miracle fruit) is limited because it produces fruit when more than four years old [10]. This study suggests to mass production of miraculin because the tomato is convenient to cultivate in tropical countries, such as Indonesia.
The use of transgenic crops can be positive in many aspects, but it also raises concerns about the environment, human health and biodiversity, including miraculin transgenic tomato. Measures to anticipate transgenic crops and their commercialization have been regulated in the laws that apply in each country. The Organization for Economic Cooperation and Development (OECD) in 1993 [11] explained that to assess the biosafety risks of transgenic crops, one can use the concept of substantial equivalence, where the transgenic product is substantially equivalent to the product of non-transgenic origin except in the nature that is engineered [12,13,14]. Some components of the environmental risk assessment in the Cartagena Protocol, the European Union and countries that grow transgenic crops, such as Argentina, Australia, Canada, China and the USA, are invasiveness, weediness and the impacts of transgenic crops on the soil environment, agricultural cultivation systems and nontarget organisms [15]. The goal of the risk assessment is to identify, characterize and evaluate the safety of cultivation of transgenic plants with respect to health and the environment [16]. Examining the effect of miraculin transgenic tomato plants on microbial soil is important because a quarter of the Earth's total biomass is estimated to be soil microorganisms, especially a group of microflorae such as bacteria, fungi and actinomycetes, which are very dominant populations in the soil. Moreover, the microflora of the soil is a good indicator to determine changes in an ecosystem [17].
Biosafety assessment is one of the requirements that must be met in terms of the release of genetically modified (GM) or transgenic crop varieties. Biosafety assessment testing of miraculin transgenic tomato includes substantial equivalence assessment through invasiveness (environmental safety/biodiversity), allergenicity and toxicity testing (food safety), and it has a very important role for the utilization and release of GM plants, both for human health and the environment. All tests should be conducted with the aim of determining the potential environmental safety and food safety of the genetically engineered crops [18].
Invasiveness testing was done by figuring the biomass value, and this test is very important to determining the safety of miraculin transgenic tomato for the environment or biodiversity. Miraculin tomato also must be assessed for unintended effects in terms of miraculin transgenic tomato that would possibly affect soil microorganisms. Unintended effect testing on non-pathogenic bacteria is beneficial, and it should be conducted to determine the impact of the miraculin tomato on the environment around the planting site [19]. Kusano et al. [10] stated that multi-platform analysis is needed to assess the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin.
In this study, an environmental safety assessment for transgenic miraculin tomato was conducted for invasiveness in biosafety containment (BC) and confined field trials (CFT), unintended effects on soil microbes (limited to culturable microbial populations) and potential allelopathy. Until now, there has been no study conducted for this very special tomato in a tropical country like Indonesia. Our purpose was to ensure that transgenic miraculin tomato is safe.
The experiments were conducted in biosafety containment, ICABIOGRAD, Bogor, and a confined field trial at the Experimental Station of the Faculty of Agriculture, Universitas Padjadjaran, Jatinangor Campus. Evaluation for invasiveness was performed according to a method developed for canola [20]. The experiment was arranged in a randomized block design with five treatments (two treatments of monoculture and three treatments of mixture). Various compositions of non-transgenic and transgenic tomatoes were tested, i.e., 0:100 and 100:0 (sole-culture) and 25:75, 50:50 and 75:25 (mix-culture). Wet and dry biomasses of three-week-old tomato plants after planting were measured using relative yield [21]. For drying, tomato plants were stored in a dryer at 55 ℃ for six days.
Relative yield was calculated as follows [20]:
| ra=Xab╱Xaa, | (1) |
| rb=Xba╱Xbb, | (2) |
where ra = relative yield of non-transgenic tomato, rb = relative yield of transgenic tomato, Xab = biomass of non-transgenic (mix-cultivation), Xba = biomass of transgenic (mix-cultivation), Xaa = biomass of non-transgenic (sole-cultivation), Xbb = biomass of transgenic (sole-cultivation).
| Relativeyieldtotal(RYT)=ra+rb, | (3) |
| Aggressiveness(A)=(ra−rb)/RYT, | (4) |
where A = 0 = equal competitiveness, A = (+) = high competitiveness, A = (−) = low competitiveness.
The soil respiration and microflora population experiments were conducted at the Laboratory of Biology and Biotechnology of Soil Science Department, Faculty of Agriculture, Universitas Padjadjaran. The experiment was arranged in a randomized block design with two treatments and repeated four times. They were arranged in zig-zag formation with 70 cm × 50 cm per plant. Both soil samples were evaluated at the times before planting, maximum vegetative period and after harvesting.
Soil respiration was estimated using the titration method to calculate the levels of carbon dioxide released during the incubation of the growing medium. About 100 grams of growing medium and 10 ml KOH solution were incubated in stoppered glass for 7 days. After that, the KOH solution was titrated by HCl 1N solution. Finally, the content of CO2 from respiration activity was estimated. Meanwhile, microbial populations were determined through total plate count method with Nutrient Agar (NA) medium for Bacteria, Potato Dextrose Agar medium for fungi and Dextrose Nitrate Agar medium for Actinomycetes. Incubation was performed at 25 ℃ for 2 days for bacteria, 5 days for fungi and 10 days for actinomycetes. Data for soil respiration and microflora population in the rhizospheres of transgenic tomato plants and non-transgenic cv. Moneymaker plants were then compared using Student's t-test at a 95% confidence level.
The evaluation of potential allelopathy was done with a sandwich method developed by Fujii et al. [22]. Transgenic and non-transgenic leaves and Ageratum conyzoides leaves were put in the same Petri dish. These leaves were placed between layers of agar on 6-well micro-plates, and lettuce seeds were used as seed tests on the surface of the agar. Data for allelopathy were measured by calculating seed germination and the seedling growth of lettuce. Analysis of variance (ANOVA) was performed for these data, followed by Tukey's test.
The results of the invasiveness evaluation in biosafety containment can be seen in Table 1 and Table 2, while the results for the confined field trial can be seen in Table 3 and Table 4. Based on Table 1 and Table 2, transgenic tomato cv. Moneymaker showed aggressiveness values of −0.17 in mixture and 0 in monoculture. Table 3 and Table 4 show aggressiveness values of −0.006 in mixture and 0 in monoculture. Meanwhile, wet weight of tomato plants in biosafety containment and confined field trial showed biomass values of −0.10 (Table 1) and −0.012 (Table 3), respectively. In dry weight, biomass was −0.19, and aggressiveness was −0.32 in mixture and 0 (zero) in monoculture (Table 2) in biosafety containment. Meanwhile, in the confined field trial, biomass was −0.015, and aggressiveness was −0.057 in mixture and 0 (zero) in monoculture (Table 4).
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 312.13 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 191.93 | 86.13 | 0.61 | 0.33 | 0.94 | −0.33 |
| 50NT:50TR | 134.13 | 159.63 | 0.43 | 0.60 | 1.03 | −0.17 |
| 25NT:75TR | 98.38 | 244.50 | 0.32 | 0.92 | 1.24 | −0.01 |
| 0NT:100TR | 0.00 | 264.88 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.10 | |||||
| Aggressiveness | Mix-cultivation | −0.17 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 15.50 | 00.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 6.50 | 07.10 | 0.42 | 0.51 | 0.93 | −0.76 |
| 50NT:50TR | 9.68 | 10.83 | 0.63 | 0.78 | 1.40 | −0.11 |
| 25NT:75TR | 5.60 | 15.70 | 0.36 | 1.13 | 1.49 | −0.10 |
| 0NT:100TR | 0.00 | 13.93 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.19 | |||||
| Aggressiveness | Mix-cultivation | −0.32 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 65.73 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 59.45 | 21.94 | 0.91 | 0.34 | 1.25 | 0.00 |
| 50NT:50TR | 13.41 | 14.68 | 0.63 | 0.20 | 0.23 | 0.43 |
| 25NT:75TR | 22.52 | 57.64 | 0.34 | 0.90 | 1.25 | −0.00 |
| 0NT:100TR | 0.00 | 63.85 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.01 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 7.63 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 7.06 | 3.20 | 0.93 | 0.43 | 1.36 | −0.01 |
| 50NT:50TR | 2.59 | 2.83 | 0.34 | 0.38 | 0.72 | −0.06 |
| 25NT:75TR | 3.21 | 6.97 | 0.42 | 0.94 | 1.36 | −0.01 |
| 0NT:100TR | 0.00 | 7.42 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.02 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
The invasiveness values were obtained from tomato plants grown in biosafety containment (BC) and the confined field trials (CFT). Both biomass and aggressiveness for wet and dry weight showed values that were all below or close to zero, indicating that transgenic tomato plants do not have the potential to become invasive, or cultivation of transgenic tomato is safe even if they grow in the mixture. Results on the invasiveness of this transgenic miraculin tomato were quite the same as those reported by Sugaya et al. [3] for transgenic miraculin strawberry.
The effect of transgenic tomato plants on soil microbial populations (culturable microbial populations) was examined through the observation of bacteria, fungi and actinomycetes, as well as soil respiration, and compared with the control, tomato cv. Moneymaker. It has been known that microflorae such as bacteria, fungi and actinomycetes are very common in the soil. Measurement of soil respiration is one of the important indexes in determining the total microbial activity in the soil [17]. The observations of populations of bacteria, fungi and actinomycetes, soil pH and soil respiration can be seen in Table 5.
| Soil sample from rhizosphere | Before planting | Maximum vegetative period | After harvesting |
| The population of bacteria (× 109 CFU/gram soil) | |||
| Transgenic tomato plant | 6.62 a | 7.80 a | 4.09 a |
| Non-transgenic tomato plant | 7.38 a | 7.84 a | 4.15 a |
| Population of fungi (× 104 CFU/gram soil) | |||
| Transgenic tomato plant | 4.79 a | 7.05 a | 3.88 a |
| Non-transgenic tomato plant | 5.79 a | 6.60 a | 3.61 a |
| The population of actinomycetes (× 108 CFU/gram soil) | |||
| Transgenic tomato plant | 1.39 a | 1.86 a | 1.10 a |
| Non-transgenic tomato plant | 1.41 a | 2.18 a | 1.08 a |
| pH | |||
| Transgenic tomato plant | 6.94 | 7.07 | 7.18 |
| Non-transgenic tomato plant | 7.03 | 7.10 | 7.19 |
| Soil respiration (mg CO2-C/kg/day) | |||
| Transgenic tomato plant | 6.43 a | 15.00 a | 9.00 a |
| Non-transgenic tomato plant | 6.86 a | 14.57 a | 8.57 a |
| Note: Values with the same letter in the same column are not significantly different according to Student's t-test. | |||
DownLoad:
CSV
The highest populations of microbes in the rhizospheres of tomato plants, either transgenic or non-transgenic, at the relevant time of observation were those of bacteria, actinomycetes and fungi (Table 5). The high populations of bacteria and actinomycetes were observed presumably because the pH of the growing medium was suitable for their growth. The planting medium used in the experiment has a neutral pH (Table 5).
Bacteria generally grow well at pH 7, with actinomycetes at pH values between 6.5 and 8.0 [17]. Meanwhile, the populations of fungi that we observed were smaller than those of actinomycetes and bacteria because the fungi are more suited to living in soil that is acidic [17]. Table 5 tells us about the microbial population's dynamics, which are closely related to the differences in root exudates produced by the roots for plant growth [23]. These exudates act as a source of nutrients and a carbon source for microbial growth [24]. Increased populations of bacteria, fungi actinomycetes occurred at the maximum vegetative period, and then the populations decreased during harvesting time. This condition might be due to the production of root exudates at the highest level and richness in organic acids and proteins [24]. Root exudate production declines with the aging process of plant roots [23]. Decreased production of root exudates also means reduced availability of food in the rhizosphere, causing the populations of bacteria, fungi and actinomycetes to experience famine and death.
In this study, observations of the microbial populations were positively in line with soil respiration. Increasing the microbial population also increases the activity of soil respiration. The observations of soil respiration can be seen in Table 5. The observations of bacteria, fungi and actinomycetes populations and the measurements of soil respiration in rhizospheres of transgenic tomato plants showed no significant differences compared with non-transgenic tomato plants according to Student's t-test at the level of 95%. This condition indicates that the miraculin gene that was inserted into the tomato plants has no effect on the culturable microbial populations and soil respiration, although miraculin protein can be secreted by the roots of transgenic tomato plants, as reported by Hirai et al. [25].
The transgenic plant can become an undesirable plant in subsequent cultivation. However, being a weed is the result of the actions of many genes. Most crop varieties have been sufficiently domesticated to be unable to survive in the unmanaged field. It is unlikely that a single gene transfer would make a wild plant [26]. Some genes controlling traits inserted by gene transfer may result in a weedy plant that improves fitness. Therefore, it can be destructive to native species. Weeds of various crops compete with cultivated plants for growth factors. In some plants, such as Brassica napus, Brassica rapa and Helianthus annuus, that have some characteristics of weeds, their transgenic and novel traits can make the plants themselves weedy and invasive [27].
In the experiment of potential allelopathy with a sandwich method, there were no significant differences among the control, transgenic and non-transgenic treatments. In contrast, the treatment of Ageratum conyzoides was significantly different from the other treatments in terms of seed germination and the seedling growth of lettuce (Table 6).
| Treatment | Leaf weight | Germination percentage (%) | Root and shoot length (mm) |
| Control (lettuce) | 0 mg | 73.33 a | 7.58 a |
| Non-transgenic | 10 mg | 66.67 a | 5.64 a |
| 50 mg | 66.67 a | 5.30 a | |
| Transgenic | 10 mg | 66.67 a | 4.75 a |
| 50 mg | 53.33 a | 5.22 a | |
| Ageratum conyzoides | 10 mg | 33.33 b | 2.83 b |
| Note: Values with the same letter in the same column are not significantly different according to Tukey's test. | |||
DownLoad:
CSV
Kuiper et al. [28] explained that if a novel food or its component is found substantially equivalent to an existing food or its component, it can be treated in the same manner concerning safety. Substantial equivalence aims to measure whether the transgenic crop is safe, as compared with its counterpart. The transgenic product and its counterpart must have the same environmental conditions to avoid genotypic and phenotypic differences not related to the genetic transformation process [29].
It is presumed that this evaluation result has no allelopathic effects that endanger other crops, as seen from the lettuce germination, which can occur appropriately. The sandwich method principle is to wipe chemical compounds from leaves placed between two layers of agar media on a 6-well micro-plate, with the seed test placed on the surface of the agar layer [22]. Usually, lettuce is a seed test because it is susceptible to the effects of allelopathy [30]. This finding indicates that transgenic miraculin did not produce allelopathic compounds that interfere with surrounding crops.
Environmental safety assessment of miraculin in a limited facility and a confined field trial has been performed for invasiveness, soil microbes and allelopathic effects. Invasiveness testing showed that both biomass and aggressiveness in biosafety containment and confined field trial for wet weight and dry weight of transgenic miraculin tomato with non-transgenic miraculin tomato cultivar Moneymaker were negative and near zero. There were also no significant differences in microflora population and soil respiration of transgenic miraculin tomatoes compared with those of wild type. No allelopathic effects were found, as proved by a sandwich method. In conclusion, the results indicate that transgenic miraculin tomato cv. Moneymaker is substantially equivalent to the non-transgenic tomato except for the presence of the miraculin gene.
We are indebted to the Directorate General of Higher Education (DGHE) which partially funded the research through the Japan Society for the Promotion of Science–Directorate General of Higher Education (JSPS-DGHE) Bilateral Joint Research Projects.
The authors declare no conflict of interest.
| [1] |
Sharma KK, Sharma HC, Seetharama N, et al. (2002) Development and deployment of transgenic plants: Biosafety considerations. Vitro Cell Dev Biol-Plant 38: 106–115. https://doi.org/10.1079/IVP2001268 doi: 10.1079/IVP2001268
|
| [2] | James C (2016) Global Status of Commercialized Biotech/GM Crops: 2016, ISAAA. Available from: https://www.isaaa.org/resources/publications/briefs/52/. |
| [3] |
Sugaya T, Yano M, Sun HJ, et al. (2008) Transgenic strawberry expressing the taste-modifying protein miraculin. Plant Biotechnol 25: 329–333. https://doi.org/10.5511/plantbiotechnology.25.329 doi: 10.5511/plantbiotechnology.25.329
|
| [4] |
Sun HJ, Cui ML, Ma B, et al. (2006) Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580: 620–626. https://doi.org/10.1016/j.febslet.2005.12.080 doi: 10.1016/j.febslet.2005.12.080
|
| [5] |
Sun HJ, Kataoka H, Yano M, et al. (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotechnol J 5: 768–777. https://doi.org/10.1111/j.1467-7652.2007.00283.x doi: 10.1111/j.1467-7652.2007.00283.x
|
| [6] |
Al-Bachchu MA, Jin SB, Park JW, et al. (2011) Functional expression of miraculin, a taste modifying protein, in Transgenic Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.). J Korean Soc Appl Biol Chem 54: 24–29. https://doi.org/10.3839/jksabc.2011.003 doi: 10.3839/jksabc.2011.003
|
| [7] |
Kurihara K, Beidler LM (1968) Taste-modifying protein from miracle fruit. Science 161: 1241–1243. https://doi.org/10.1126/science.161.3847.1241 doi: 10.1126/science.161.3847.1241
|
| [8] |
Theerasilp S, Kurihara Y (1998) Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 263: 11536–11539. https://doi.org/10.1016/S0021-9258(18)37991-2 doi: 10.1016/S0021-9258(18)37991-2
|
| [9] |
Endo C, Hirata A, Takami A, et al. (2018) Effect of miraculin on sweet and sour tastes evoked by mixed acid solutions. Food Nutr Sci 6: 757–764. https://doi.org/10.4236/fns.2015.69078 doi: 10.1002/fsn3.617
|
| [10] |
Duhita N, Hiwasa-Tanase K, Yoshida S, et al. (2011) A simple method for purifying undenatured miraculin from transgenic tomato fruits. Plant Biotechnol 28: 281–286. https://doi.org/10.5511/plantbiotechnology.11.0207a doi: 10.5511/plantbiotechnology.11.0207a
|
| [11] | OECD (1993) Safety Evaluation of Foods Derived by Modern Biotechnology: Concepts and Principles. Available from: https://www.oecd.org/science/biotrack/41036698.pdf. |
| [12] | Tabei Y (2003) Risk Assessment of GM Crops and the Challenge of Building Public Acceptance in Japan. Available from: https://www.fftc.org.tw/en/publications/main/624. |
| [13] |
Bergmans H (2006) Basic framework for risk assessment for transgenic plants developed by the OECD: 20 years after the OECD "Blue Book". Environ Biosafety Res 5: 213–218. https://doi.org/10.1051/ebr:2007010 doi: 10.1051/ebr:2007010
|
| [14] |
Ramessar K, Peremarti A, Gómez-Galera S, et al. (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: A case of the science not supporting the politics. Transgenic Res 16: 261–280. https://doi.org/10.1007/s11248-007-9083-1 doi: 10.1007/s11248-007-9083-1
|
| [15] | Mackenzie R, Burhenne-Guilmin F, La Viña AGM, et al. (2003) An explanatory guide to the cartagena protocol on biosafety, Cambridge: IUCN. |
| [16] | Ministry of Environment, Forest and Climate Change, Government of India (2016) Environmental Risk Assessment of Genetically Engineered Plants: A Guide for Stakeholders. Available from: https://ibkp.dbtindia.gov.in/DBT_Content_Test/CMS/Guidelines/20181115134854330_Guidelines%20for%20the%20Environmental%20Risk%20Assessment%20of%20Genetically%20Engineered%20Plants,%202016.pdf. |
| [17] | Rao NSS (1994) Soil microorganisms and plant growth, 2 Eds., Jakarta: UI Press. |
| [18] | Bahagiawati, Herman M (2008) Peraturan Perundang-undangantentang Keamanan Produk Bioteknologi dan Status Perakitan Tanaman Produk Bioteknologidi Indonesia. Available from: http://www.litbang.pertanian.go.id/info-aktual/854/file/Bagian-1.pdf. |
| [19] | Herman M (2010) Fourteen years of development of the biosafety and food safety regulations of genetic engineering products and their implementation in Indonesia. J AgroBiogen 6: 113–125. |
| [20] |
Kusano M, Redestig H, Hirai T, et al. (2011) Covering chemical diversity of genetically-modified tomatoes using metabolomics for objective substantial equivalence assessment. PLoS ONE 6: e1698. https://doi.org/10.1371/journal.pone.0016989 doi: 10.1371/journal.pone.0016989
|
| [21] | Canadian Food Inspection Agency, The Biology of Glycine max (L.) Merr. (Soybean), 1996. |
| [22] |
Olubode OO, Ogunsakin TA, Salau AW, et al. (2015) Influence of intercrop population and applied poultry manure rates on component crops productivity responses in a snake tomato/celosia cropping system. Am J Plant Sci 6: 1040–1057. https://doi.org/10.4236/ajps.2015.67109 doi: 10.4236/ajps.2015.67109
|
| [23] |
Fujii Y, Shibuya T, Nakatani K, et al. (2004) Assessment method for allelopathic effect from leaf litter leachates. Weed Biol Manag 4: 19–23. https://doi.org/10.1111/j.1445-6664.2003.00113.x doi: 10.1111/j.1445-6664.2003.00113.x
|
| [24] |
Walker TS, Harsh PB, Erich G, et al. (2003) Root exudation and rhizosphere biology. Plant Physiol 132: 44–51. https://doi.org/10.1104/pp.102.019661 doi: 10.1104/pp.102.019661
|
| [25] |
Hirai T, Fukukawa G, Kakuta H, et al. (2010) Production of recombinant miraculin using transgenic tomatoes in a closed cultivation system. J Agri Food Chem 58: 6096–6101. https://doi.org/10.1021/jf100414v doi: 10.1021/jf100414v
|
| [26] | Koch M (2004) Manual on Biosafety Risk Assessment and Risk Management for Cameroon, Cameroon National Biosafety Committee. Available from: https://pdfkiwi.com/preview/manual-on-biosafety-risk-assessment-and-risk-management-for-6048570e81c02. |
| [27] |
Giraldo PA, Shinozuka H, Spangenberg GC, et al. (2019) Safety assessment of genetically modified feed: Is there any difference from food? Front Plant Sci 10: 1592. https://doi.org/10.3389/fpls.2019.01592 doi: 10.3389/fpls.2019.01592
|
| [28] |
Kuiper HA, Kleter GA, Noteborn HPJM, et al. (2002) Substantial equivalence—an appropriate paradigm for the safety assessment of genetically modified foods? Toxicology 181–182: 427–431. https://doi.org/10.1016/S0300-483X(02)00488-2 doi: 10.1016/S0300-483X(02)00488-2
|
| [29] |
Singh OV, Jain RK, Ghai S, et al. (2006) Genetically modified crops: Success, safety assessment, and public concern. Appl Microbiol Biotechnol 71: 598–607. https://doi.org/10.1007/s00253-006-0449-8 doi: 10.1007/s00253-006-0449-8
|
| [30] | Itani T, Nakahata Y, Kato-Naguchi H (2013) Allelopathic activity of some herb plant species. Int J Agri Biol 15: 1359–1362. |
| 1. | Nono Carsono, Faza A. Maulana, Iqbal F. Elfakhriano, Ade Ismail, Noladhi Wicaksana, Santika Sari, Hiroshi Ezura, The comparative analysis of agronomic, compositional, and physiological traits of miraculin transgenic tomato in the confined field trial, 2023, 8, 2471-2086, 187, 10.3934/agrfood.2023010 |
Figures(3) / Tables(6)
Nono Carsono, Fadlilah Aida Rahmani, Rangga Jiwa Wibawa, Santika Sari, Anas, Ryo Ohsawa, Ayako Shimono, Hiroshi Ezura. Invasiveness, allelopathic potential and unintended effects of miraculin transgenic tomato to soil microbes[J]. AIMS Agriculture and Food, 2022, 7(4): 872-882. doi: 10.3934/agrfood.2022053
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 312.13 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 191.93 | 86.13 | 0.61 | 0.33 | 0.94 | −0.33 |
| 50NT:50TR | 134.13 | 159.63 | 0.43 | 0.60 | 1.03 | −0.17 |
| 25NT:75TR | 98.38 | 244.50 | 0.32 | 0.92 | 1.24 | −0.01 |
| 0NT:100TR | 0.00 | 264.88 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.10 | |||||
| Aggressiveness | Mix-cultivation | −0.17 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 15.50 | 00.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 6.50 | 07.10 | 0.42 | 0.51 | 0.93 | −0.76 |
| 50NT:50TR | 9.68 | 10.83 | 0.63 | 0.78 | 1.40 | −0.11 |
| 25NT:75TR | 5.60 | 15.70 | 0.36 | 1.13 | 1.49 | −0.10 |
| 0NT:100TR | 0.00 | 13.93 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.19 | |||||
| Aggressiveness | Mix-cultivation | −0.32 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 65.73 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 59.45 | 21.94 | 0.91 | 0.34 | 1.25 | 0.00 |
| 50NT:50TR | 13.41 | 14.68 | 0.63 | 0.20 | 0.23 | 0.43 |
| 25NT:75TR | 22.52 | 57.64 | 0.34 | 0.90 | 1.25 | −0.00 |
| 0NT:100TR | 0.00 | 63.85 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.01 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 7.63 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 7.06 | 3.20 | 0.93 | 0.43 | 1.36 | −0.01 |
| 50NT:50TR | 2.59 | 2.83 | 0.34 | 0.38 | 0.72 | −0.06 |
| 25NT:75TR | 3.21 | 6.97 | 0.42 | 0.94 | 1.36 | −0.01 |
| 0NT:100TR | 0.00 | 7.42 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.02 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
DownLoad:
CSV
| Soil sample from rhizosphere | Before planting | Maximum vegetative period | After harvesting |
| The population of bacteria (× 109 CFU/gram soil) | |||
| Transgenic tomato plant | 6.62 a | 7.80 a | 4.09 a |
| Non-transgenic tomato plant | 7.38 a | 7.84 a | 4.15 a |
| Population of fungi (× 104 CFU/gram soil) | |||
| Transgenic tomato plant | 4.79 a | 7.05 a | 3.88 a |
| Non-transgenic tomato plant | 5.79 a | 6.60 a | 3.61 a |
| The population of actinomycetes (× 108 CFU/gram soil) | |||
| Transgenic tomato plant | 1.39 a | 1.86 a | 1.10 a |
| Non-transgenic tomato plant | 1.41 a | 2.18 a | 1.08 a |
| pH | |||
| Transgenic tomato plant | 6.94 | 7.07 | 7.18 |
| Non-transgenic tomato plant | 7.03 | 7.10 | 7.19 |
| Soil respiration (mg CO2-C/kg/day) | |||
| Transgenic tomato plant | 6.43 a | 15.00 a | 9.00 a |
| Non-transgenic tomato plant | 6.86 a | 14.57 a | 8.57 a |
| Note: Values with the same letter in the same column are not significantly different according to Student's t-test. | |||
DownLoad:
CSV
| Treatment | Leaf weight | Germination percentage (%) | Root and shoot length (mm) |
| Control (lettuce) | 0 mg | 73.33 a | 7.58 a |
| Non-transgenic | 10 mg | 66.67 a | 5.64 a |
| 50 mg | 66.67 a | 5.30 a | |
| Transgenic | 10 mg | 66.67 a | 4.75 a |
| 50 mg | 53.33 a | 5.22 a | |
| Ageratum conyzoides | 10 mg | 33.33 b | 2.83 b |
| Note: Values with the same letter in the same column are not significantly different according to Tukey's test. | |||
DownLoad:
CSV
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 312.13 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 191.93 | 86.13 | 0.61 | 0.33 | 0.94 | −0.33 |
| 50NT:50TR | 134.13 | 159.63 | 0.43 | 0.60 | 1.03 | −0.17 |
| 25NT:75TR | 98.38 | 244.50 | 0.32 | 0.92 | 1.24 | −0.01 |
| 0NT:100TR | 0.00 | 264.88 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.10 | |||||
| Aggressiveness | Mix-cultivation | −0.17 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 15.50 | 00.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 6.50 | 07.10 | 0.42 | 0.51 | 0.93 | −0.76 |
| 50NT:50TR | 9.68 | 10.83 | 0.63 | 0.78 | 1.40 | −0.11 |
| 25NT:75TR | 5.60 | 15.70 | 0.36 | 1.13 | 1.49 | −0.10 |
| 0NT:100TR | 0.00 | 13.93 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.19 | |||||
| Aggressiveness | Mix-cultivation | −0.32 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
| Seed composition | Shoot wet weight of non-transgenic (g) | Shoot wet weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 65.73 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 59.45 | 21.94 | 0.91 | 0.34 | 1.25 | 0.00 |
| 50NT:50TR | 13.41 | 14.68 | 0.63 | 0.20 | 0.23 | 0.43 |
| 25NT:75TR | 22.52 | 57.64 | 0.34 | 0.90 | 1.25 | −0.00 |
| 0NT:100TR | 0.00 | 63.85 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.01 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
| Seed composition | Shoot dry weight of non-transgenic (g) | Shoot dry weight of transgenic (g) | Relative yield of non-transgenic | Relative yield of transgenic | Relative yield total | Aggress-iveness |
| 100NT:0TR | 7.63 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 |
| 75NT:25TR | 7.06 | 3.20 | 0.93 | 0.43 | 1.36 | −0.01 |
| 50NT:50TR | 2.59 | 2.83 | 0.34 | 0.38 | 0.72 | −0.06 |
| 25NT:75TR | 3.21 | 6.97 | 0.42 | 0.94 | 1.36 | −0.01 |
| 0NT:100TR | 0.00 | 7.42 | 0.00 | 1.00 | 1.00 | 0.00 |
| Biomass | −0.02 | |||||
| Aggressiveness | Mix-cultivation | −0.06 | ||||
| Sole-cultivation | 0.00 | |||||
| Notes: Ra = relative yield of non-transgenic tomato; Rb = relative yield of transgenic tomato; RYT = relative yield total; A = value of aggressiveness. Biomass = average of all aggressiveness values; Aggressiveness in mixture = average value of the aggressiveness of seed composition in mixture; Aggressiveness in monoculture = average value of the aggressiveness of seed composition in monoculture. | ||||||
| Soil sample from rhizosphere | Before planting | Maximum vegetative period | After harvesting |
| The population of bacteria (× 109 CFU/gram soil) | |||
| Transgenic tomato plant | 6.62 a | 7.80 a | 4.09 a |
| Non-transgenic tomato plant | 7.38 a | 7.84 a | 4.15 a |
| Population of fungi (× 104 CFU/gram soil) | |||
| Transgenic tomato plant | 4.79 a | 7.05 a | 3.88 a |
| Non-transgenic tomato plant | 5.79 a | 6.60 a | 3.61 a |
| The population of actinomycetes (× 108 CFU/gram soil) | |||
| Transgenic tomato plant | 1.39 a | 1.86 a | 1.10 a |
| Non-transgenic tomato plant | 1.41 a | 2.18 a | 1.08 a |
| pH | |||
| Transgenic tomato plant | 6.94 | 7.07 | 7.18 |
| Non-transgenic tomato plant | 7.03 | 7.10 | 7.19 |
| Soil respiration (mg CO2-C/kg/day) | |||
| Transgenic tomato plant | 6.43 a | 15.00 a | 9.00 a |
| Non-transgenic tomato plant | 6.86 a | 14.57 a | 8.57 a |
| Note: Values with the same letter in the same column are not significantly different according to Student's t-test. | |||
| Treatment | Leaf weight | Germination percentage (%) | Root and shoot length (mm) |
| Control (lettuce) | 0 mg | 73.33 a | 7.58 a |
| Non-transgenic | 10 mg | 66.67 a | 5.64 a |
| 50 mg | 66.67 a | 5.30 a | |
| Transgenic | 10 mg | 66.67 a | 4.75 a |
| 50 mg | 53.33 a | 5.22 a | |
| Ageratum conyzoides | 10 mg | 33.33 b | 2.83 b |
| Note: Values with the same letter in the same column are not significantly different according to Tukey's test. | |||
