GMOs global landscape

Transgenesis has been rapidly implemented in world agriculture in terms of cultivated area. According to the latest reports from the International Service for the Acquisition of Agri-biotech Applications (ISAAA), issued in 2018 and 2019, GM crops have accumulated a total cultivated area of 2.53 billion hectares in 23 years of implementation of this technology (Fig. 1) [6, 8]. In 2019, the last reported year, an area of 190.4 million hectares was cultivated with GMOs in a total of 29 countries, with the Americas being the continent with the largest cultivated area in the world (Table 1).

Fig. 1

Cultivated area with GM crops worldwide. Plotted from data published in the ISAAA briefs of Global Status of Commercialized Biotech/GM Crops in 2018 and 2019 [6, 8]

Table 1 Countries with the largest GM crops cultivated area in 2019. The GM crops that have the largest cultivated area in each country are mentioned.

The most widely cultivated GM crops are soybean, maize, cotton, and canola, with an area of 188.6 million hectares, which corresponds to 99% of the area cultivated with GMOs worldwide (Fig. 2). About 90% of the area cultivated with GMOs is found in 5 countries (United States, Brazil, Argentina, Canada, and India) (Table 1). Most of the commercially available GM crops have been developed using transgenesis based on recombinant DNA technology, mainly to confer traits such as insect resistance, herbicide tolerance, and tolerance to abiotic stress (> 99% of total commercial traits)[8] (Table 2) or other non-frequent traits related to improved food fortification such as provitamin A biosynthesis in “golden rice” and “golden banana”, or increased starch content in EH92-527–1 potato [9,10,11,12,13]. These transgenic crops have been mainly used for food, livestock and poultry feed, and as ingredients for processed food such as protein extracts, oils and sugar; or for other industries such as ethanol (biofuel) or natural fibre production [14, 15].

Fig. 2

Adapted from the ISAAA brief of Global Status of Commercialized Biotech/GM Crops in 2019.

Cultivated area with GM crops reported for 2019. Adoption rate is shown as the percentage of cultivated area with GM crop compared to the total cultivated area for that crop, being GMO or not. [8]. *Other crops: Sugar beet, potato, apple, squash, papaya and eggplant.

Table 2 Worldwide GM crops cultivated area by traitThe era beyond transgenesis: genome editing toolsNew breeding techniques

Along with the process of transgenesis, in the last decade new technologies have been developed that allow editing the genome, or modify its expression, of the target organism in a precise, fast, and relatively cheaper way than other techniques, minted under the acronym "NBT" ("new breeding techniques") (Fig. 3). The genome editing process is based on the use of nucleases able to generate double-strand breaks (DSBs) in specific sequences when guided by proteins or RNA [16]. These breaks are then repaired by the cellular endogenous DNA repair machinery via non-homologous end joining (NHEJ), allowing targeted modifications, such as insertions or deletions, potentially knocking out targeted genes. Moreover, DSBs can also be repaired by homology-directed repair (HDR) using endogenous or delivered template DNA sequence, leading to gene replacement or insertion of sequences of different sizes, from one to many hundreds of nucleotides [17].

Fig. 3

New breeding techniques used for GM crops development. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the bacterial system of clustered regularly interspaced short palindromic repeats (CRISPR), employing Fok1 or Cas9 nucleases, are used to target DNA sequences to promote downstream modifications. ZFN, TALEN and Cas9 induce double-strand breaks that are corrected by NHEJ or HDR, modifying the target sequence with deletions or different size insertions. Modified Cas9, such as catalytically null (“dead” Cas9 or dCas9) is used coupled to transcriptional repressor or activators to regulate gene expression. Other forms of modified Cas9, such as coupled to reverse transcriptase (RT) or deaminases, are used to modify target sequence with specific template primers (prime editing) or switch specific bases (base editing).

The extension, location and downstream effects of these editions will determine the phenotype of the new variety, with novel traits that are, in principle, independent of exogenous gene constructs, thus differentiating them from transgenesis. Nonetheless, the delivery methods used to insert genome editor expression cassettes or ribonucleoprotein complexes represent an obstacle to obtain commercial varieties free of exogenous DNA. This is noteworthy not only for regulatory concerns but also for the acceptance of the final products by consumers [18, 19].

Currently, the three most widely used NBT are zinc finger domain-coupled nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the bacterial system of clustered regularly interspaced short palindromic repeats (CRISPR) coupled to the nucleases Cas9, Cas12, or Cpf1, among others [16, 17]. ZFN and TALEN systems are based on nucleases, such as Fok1, coupled to tandem zinc finger protein domains or TALE protein repeats, respectively, recognizing specific DNA motifs by protein-DNA interaction. Once this protein-guided DNA interaction occurs, Fok1 nucleases dimerize and perform their enzymatic activity on the double-stranded DNA [16] (Fig. 3). For example, ZFN gene editing has been used on commercially relevant crops to modify endogenous genes involved in different phenotypes such as development in tomato [20], starch metabolism in rice [21], sexual fertility in apple and fig [22], RNA silencing genes in soybean [23] or confer resistance to imidazolinone herbicides in wheat [24]. Potentially commercial traits of interest have been developed using TALEN. For instance, to modify sugar metabolism and improve herbicide tolerance in potato [25, 26], increase oleic acid content in peanut and soybean oil [27, 28], or reduce lignin content and improve saccharification in sugarcane [29, 30]. Early flowering is another trait of interest, allowing to face seasonal and logistic hurdles, and wild cabbage is an example of these research efforts performed by TALEN [31].

CRISPR systems

On the other hand, the CRISPR system is based on RNA-guided DNA pairing, taking advantage of the recently growing knowledge of bacterial CRISPR/Cas complex. This RNA–DNA interaction allows sequence specificity design in a more efficient, versatile, and cheaper way than ZFN and TALEN systems [16,17,18, 32]. Furthermore, the use of tailored Cas complexes has expanded the toolkit for genome editing by incorporating base-switching enzymes, transcription regulators, or adding translational modifications [18]. To date, several variations have been made to the CRISPR/Cas system in order to obtain different modifications on the DNA sequences or regulate gene expression (Fig. 3). Nuclease-inactivated Cas9 (dCas9 for “catalytically dead Cas9”) allows Cas9 targeting DNA sequences guided by a guide RNA (gRNA) without producing strand breaks [33]. The coupling of dCas9 with transcriptional repressors or activators constitutes an adaptable tool to modify gene expression (34). Moreover, fusion of Cas9 with deaminases has been shown to be useful for C-T and A-G base replacement (known as base editors) [33, 35].

In addition to precise base editing, the potential of modified CRISPR/Cas9 systems has been taken into a broader perspective with “prime-editing” tools. In this approach, a reverse transcriptase (RT) is fused to Cas9, and the gRNA is concatenated with template RNA for RT activity. This allows targeting the sequence of interest and editing multiple bases at once without template DNA [36]. In addition to targeting gene expression, CRISPR/Cas systems can also be used to modulate translation by editing upstream open reading frames (uORFs). These uORFs can regulate translation of the primary ORF, as demonstrated in lettuce modified in the LsGGp2 uORF, enhancing vitamin C biosynthesis [37].

CRISPR systems have been experimentally used on most commercial GM crops such as maize, soybean and cotton, along with several other crops, e.g., apple, carrot, orange, raps, lettuce, grapes, pear, strawberry, cucumbers, wheat, rice, and tomato, in addition to ornamental flowers such as Dendrobium officinale (orchid), Ipomoea nil (morning glory), Petunia hybrid (petunia) and Torenia fournieri (wishbone flower) [16, 18, 32, 38,39,40]. The traits obtained by CRISPR also cover a wide range of biotechnological interests such as sugar, fatty acid or pigment metabolism, herbicide tolerance, pathogen resistance, and development modifications, among others.

Regulatory frameworks

Despite their differences, countries’ regulatory frameworks can be broadly classified as process-oriented or product-oriented [14, 41]. The first determines criteria and regulations according to the methods used to generate new plant varieties, while the second applies criteria based on the new characteristics of a biotechnological event and makes comparisons with their conventional counterparts, both applying a case-by-case evaluation system. Noteworthy, these product- or process-oriented regulatory guides commonly share elements with each other, making legal frameworks difficult to categorize [41, 42]. Parallel to the “regulatory style” of each country, the Cartagena Protocol, signed by more than 140 countries, constitutes an instance of international law with binding legal principles for the countries that have ratified it [7].

Most of the current regulations worldwide have been created to address transgenic-derived crops, which have the largest participation on markets. However, frameworks have been updated in several countries, mainly developed countries, to take genome-edited crops into account. Here we address the current status of regulatory frameworks for GM crops around the globe, in which, for the purpose of this review, we have categorized the main regulatory focus of each country as product- or process- oriented (although it is not a legal classification in any of these countries) (Table 3).

Table 3 GMO regulatory frameworks around the wordAmericasLatin America

In Latin America there are different types of regulation, from very permissive to moratorium. The lack of consensus is a characteristic of the region, where countries with similar regulations do not coordinate or share information on seeding requests. The “biotechnological mega-countries” (a term coined by ISAAA for countries growing more than 50.000 Ha of GM crops) in the region, being Argentina, Brazil, Bolivia, Colombia, Mexico, Paraguay and Uruguay, have regulations that allow the cultivation and/or trade of GMO, rendering them as important players in the global market. Other Latin America countries with planted GMOs such Costa Rica and Honduras, have similar regulations compared to the biotechnological mega-countries of the region. Chile has a unique regulatory framework where GMO crops can be planted for seed production and export, and research purposes but not for domestic food or feed uses [43, 44]. Despite the regulation similarities within these countries, their definitions and lists of approved events are not synchronized, which delays the application of events throughout the region and weakens regional trade [14].

Therefore, at the XXXIV Extraordinary Meeting of the Southern Agricultural Council (CAS), held in 2017, the Ministers of Agriculture of Brazil, Chile, Paraguay and Uruguay, and the Secretary of Livestock, Agriculture and Fisheries of Argentina, declared it necessary to promote activities of regional cooperation and exchange of information for the approval of GMOs and to train "experts in new technologies" (related to NBT). The common characteristic in the regional regulations relies in evaluating food biosafety and field release (environmental and biodiversity) in a case-by-case and product-oriented manner, according to the institutions mandated for such purposes in each country [14].

On the prohibitive side of Latin American region, Ecuador, Peru, and Venezuela do not allow the commercial cultivation of GMOs. Ecuador, whose 2008 constitution defines the country as "free of transgenic crops and seeds", has made its position more flexible and allows the use of GM seeds for research purposes, through the Organic Law of Agrobiodiversity, Seeds and Promotion of the Sustainable Agriculture, decreed in 2017. In the case of Venezuela, the Venezuelan Seed Law, decreed in 2015, prohibits all GM crops in its territory.

In Peru, a transition towards a prohibitive policy has been observed. In 1999, through Law 27104, the regulation of GMOs was established, managing and controlling their confined use and release; in addition to regulating its introduction, commercialization, research, transportation, and storage, among others. It even decreed a law for the labelling of foods made with ingredients that contain GMOs (Law 29888). However, through the enactment of Law 29811, in 2011, a moratorium on the entry and production of GMOs in Peruvian territory was established for ten years, emphasizing the need to assess risks, protect biodiversity, and generate a new regulatory framework. This moratorium excludes GMOs cultivated for research purposes and in January 2021 the Peruvian Congress enacted an extension of the moratorium for fifteen more years from the end of the first ten-year period (Law 31111).

USA and Canada

The United States of America (USA) and Canada share a common regulatory style, considering the new GM plant varieties as conventional based on case-by-case biosafety analysis. This permissive style has allowed these countries to use their previous legislation to adapt it to the evaluation of GMOs [14]. Following this line, the USA, despite being the main producer of GM crops in the world, does not have federal legislation as a general framework to regulate GMOs. Depending on whether the purpose of the GM product is for human, animal and/or environmental use, its authorization and regulation fall under the standards of the Food and Drug Administration (FDA); the Animal and Plant Health Inspection Service (APHIS); or the Department of Agriculture (USDA) and/or the USA Environmental Protection Agency (EPA), respectively.

The case of Canada is unique in the world, considering a new term in its regulatory framework: “plant with novel traits” (PNT). In the Canadian regulatory framework, a new plant variety is considered a PNT if it meets certain differentiating criteria with its conventional counterpart, regardless of the methodology used to generate it, be it transgenesis, conventional breeding or NBT. Therefore, a new plant variety can be considered a PNT in Canada while being considered a GMO for the rest of the world. Despite the broad definition criteria, Canada has a biosafety evaluation system focused on toxicity, allergenicity, impact on field release and even impacts on organisms other than the PNT, through the Canadian Food Inspection Agency (CFIA) [11].

Europe

The regulations of the European Union (EU) have been classified as restrictive since they determine high biosafety standards for human and animal consumption, environmental impact and consumer interests, as stated in the first article of Regulation 1829/2003 EU. This standard gives much of the responsibility on the applicant to demonstrate the safety of the GM product and to monitor its cultivation or use as food. In addition, the EU regulation provides a framework for citizen participation by making public the Authority's opinions regarding new requests. Citizens can send their comments to the evaluation committee within a period of thirty days, through article 6 number 7 of the aforementioned Regulation. Such have been the levels of control in the EU in terms of applications to cultivate GMOs that in more than two decades only two biotechnological events have been approved for cultivation and in the last years only one is cultivated in Spain and Portugal (insect-resistant corn, MON810) [8].

Despite this, the EU is one of the main importers of GMOs for human consumption, being mainly soybeans and its derivatives (90–95% GMOs of total imports), maize (20–25% GMOs of total imports) and canola (25% GMOs of total imports) [8]. In addition to seeding restrictions, it has regulations on traceability and labelling of GMOs. The general standard of the EU regulatory style is based on the definition of process-oriented GMOs, defining a GMO in article 2 of Directive 2001/18/EC, as “if the method of genetic modification is carried out in such a way that does not occur by natural crossing and/or recombination”. This definition does not take into account the type of modification, be it gene insertion, regulatory sequences, specific nucleotide changes, etc. Therefore, it does not discriminate the type of methodology used to generate a GMO. On the other hand, this definition also includes conventional plant breeding, on which cases it has an allowing “historical” criterion.

Africa

The African continent has been slowly adopting GM crops with different regulations between countries, similar to Latin America. Africa is home to some of the countries with the largest area planted with GM crops in the world (South Africa, Sudan, Nigeria, Eswatini and Ethiopia) (Table 1), in addition to Malawi and Kenya. With regard to the cultivated plant varieties, South Africa has cultivated maize, soybeans and cotton, while other countries cultivate mainly IR/Bt cotton [8], for a total of 2.9 million hectares of GM crops in 2019.

South Africa was the first African country to regulate GM crops through the Genetically Modified Organisms Act No. 15 of 1997, while other countries began to regulate this technology since the early 2000’s (Kenya and Malawi) or the last decade (Egypt, Ethiopia, Eswatini, Ghana, Nigeria, Sudan, Burkina Faso and Uganda). Furthermore, Egypt, Ghana and Uganda do not allow GM crops cultivation for commercial purposes [14]. Burkina Faso has been producing Bt cotton since 2008 but stopped its production in 2016 due to quality concerns. Its regulation allows cultivation of GM crops, but there is currently no commercial production [45]. Although Egypt was a pioneering African country in developing and planting GM maize in 2008, GM cultivation was banned four years later due to a lack of biosafety laws [46].

Asia and Oceania

Asia is the main source of GM cotton, with India being the country with the largest cultivated area (11.9 million hectares of Bt cotton in 2019) [8]. Despite the approval of Bt cotton in India in 2002, several other food and non-food GM crops are not allowed and have been planted illegally since then, such as virus-resistant papaya, Bt brinjal/eggplant and IR/HT cotton [47]. Like India, Pakistan and China are also ones of the main producers of Bt cotton [8]. Beyond the domestic and export production of GM crops, China has led the research and development of GMOs obtained by NBT, being the main source of published articles and patent applications in this regard [48, 49]. The Ministry of Agriculture and Rural Affairs is the institution responsible for new approvals and demands strict field and environmental assessments for new events, delaying the process from development to commercialization. This marks a difference with the USA and Canadian regulatory frameworks, that allow faster track for the application of new events [14].

Philippines is one of the key players in the market of GMOs in Southeast Asia, being a leading producer of GM maize in the region, and also an important commercial target for GM rice that harbours enzymes for the biosynthesis of the vitamin A precursor (golden rice), which is produced mainly in China [9]. Like Philippines, Indonesia also has a product-oriented regulation with the difference of a smaller production of GMO limited to sugarcane [8]. Similar to these cases, Vietnam and Bangladesh, in Asia mainland, also have a permissive regulatory style regarding GMOs but only one species is the main focus of production being maize and brinjal/eggplant, respectively [8].

In the Pacific region, New Zealand has a strict regulatory framework that takes Māori culture into consideration, prohibiting crops that may alter traditions, sites, flora, and fauna [50]. This has led to no GM crops being cultivated commercially in the country. Moreover, this regulatory framework also considers new plant varieties developed by NBT through the regulation of GMOs [41].

Japan and Australia allow the cultivation of GM crops but with different regulatory approaches. Japan leads in GM crops approvals behind the USA, but its strict confined field trials and environmental risk assessments have not allowed commercial production of GMOs for food or feed, but only for ornamental blue rose flower [51]. On the other hand, Australia has allowed commercial production of GM crops, being a major producer of cotton, canola, and safflower (ranked 13th in area cultivated with GMOs in 2019) (Table 1) [8]. Despite their different approaches to commercial cultivation of GMOs, Japan and Australia share common criteria for evaluating and defining new plant varieties developed by NBT, considering unguided repair of site-directed nuclease activity (SDN-1) organisms as non-GMO [52, 53].

Beyond GM cropsGM microorganisms

Agriculture has been the activity with the most extensive research, development, and application of GMOs. However, several other fields have been taking advantage of this technology. Closely related to crops, the use of yeast has been a historical tool for the production of bread and alcoholic beverages (such as wine and beer). Furthermore, due to the extensive knowledge of yeast genetics and cell biology, the biotechnological application of yeasts, as well as other fungal species, has rapidly evolved and spans various industries, such as biofuel production, medical applications, and alcoholic beverages itself. For example, genetic modification of yeast strains has been experimentally tested to modulate ethanol yields [54, 55].

Although the use of GM yeasts in industrial applications such as bioethanol and pharmaceutical production is not a problem (the commercialization of recombinant insulin is an example of this), the use of GM yeasts for food production has faced the same problems associated with GM plants, i.e., legal restrictions and consumer rejection, which lead to the limited commercial success that recombinant yeasts have had in the food industry [