Freedom to operate study

Farmers from the United States and Argentina began to plant GM soybean around 1996 after it started to be marketed under the Roundup Ready trademark in the USA. GM soybean occupies around 95.9 million ha in the world (almost 50% of the GM crops worldwide). In 2018, the GM soybean area planted in the USA was 34.08 million ha. Brazil occupied the second position in the world with a production of ~34.86 million ha. GM soybean cultivation has expanded throughout Argentina with 18 million ha in 2018. Monsanto (now Bayer) was not capable of achieving patent protection for GM soybean with tolerance for glyphosate (Roundup Ready soybean seed) in Argentina. The company had requested the IPRs it held in Europe, but the Supreme Court of Argentina rejected that request based on the national patent law that regulates the patent duration of 20 years. Monsanto has registered some GM soybean events related to glyphosate tolerance in Argentina, (e.g., MON87701 x MON89788 and MON89788), perhaps using plant breeder rights (^{Brookes & Barfoot, 2018}; ^{Fries et al., 2019}; ^{ISAAA, n.d.}).

An FTO study that considers the importance of generating GM soybean with marketing possibilities, before the laboratory activities and at the start of the biotechnological process, should be developed to avoid, as far as possible, the violation of third-party rights. Initially, a list of elements (materials and protocols) to be used in the development of a GM line must be in place. Then, a specific country search must be carried out based on the requested patents/current patents, including plant breeder's rights or "plant patents" (regarding the plant varieties) for each element. Data can be recovered from the United States patent office database (USPTO) (https://www.uspto.gov/patents/search), the European patent office database (ESPACENET) (https://worldwide.espacenet.com/), the Lens suite (https://www.lens.org/lens/), PATENTSCOPE from the World Intellectual Property Organization (https://patentscope.wipo.int/search/es/search.jsf), and Google Patents (https://patents.google.com/). In each of these websites, a regional patent database needs to be requested during the planning stage. Regional patent databases should be studied. Also, biological specialized databases can be reviewed, such as Nucleotide from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/nucleotide/), or Phytozome (https://phytozome-next.jgi.doe.gov/), to determine the origin of the selective biological information (^{Wyse & Luria, 2021}). Data about patents related to glyphosate tolerant soybean should be obtained during the period from 1996 to the present day (^{Jefferson, Graff, et al., 2015}; ^{Goforth, 2017}). Therefore, there is a growing need for competent lawyers specialized on GM intellectual property at this stage.

Patent data should focus especially on claims and year of application without forgetting the importance of the overall description of the invention. An FTO analysis involves the search of patents and their claims because they allow an understanding of the set of approaches that explain the developmental process of a GM crop: cloning vectors, DNA sequences, Agrobacterium strains, methods, transfer material, or confidential agreements (^{Chi-Ham et al., 2012}; ^{Miralpeix et al., 2014}; ^{Zanga et al., 2015}). At the same time, an FTO analysis provides a parallel strategy to develop the same product in a structured way with a focus on commercialization without requirements for additional payments from licenses (^{Sommer, 2012}). A GM soybean project based on FTO requires legal assistance from a lawyer, at least in its first instance of design. One of the first things that must be identified to achieve the goal of a GM soybean event is the soybean variety. Some countries have patent systems that prevent patent protection of certain products, such as germplasm for research and breeding (^{Correa, 2014}). Likewise, if native genetic material is relevant in a biotechno-logical project, a contractual instrument for the appropriate access and utilization of biological and genetic resources respectful of traditional knowledge may be necessary (^{Deplazes-Zemp, 2019}; ^{Heinrich & Hesketh, 2019}). A large number of countries have achieved significant progress in research and development on new soybean varieties in the light of local conditions (Tab. 2) (^{Cober et al., 2009}). Just in Africa, there are 13 countries developing local soybean varieties (Benin, Cameroon, Ethiopia, Ghana, Kenya, Mali, Mozambique, Nigeria, Rwanda, Sudan, Uganda, Zambia, and Zimbabwe) (^{Santos, 2019}). There is a program that involves the development of a genetic improvement of soybean for the tropical Colombian environment since 1960 (Tab. 2) (S. Caicedo, personal communication, 12th May 2017). Therefore, it is best to have a local soybean variety adapted to the environmental conditions of the region of interest, if possible.

TABLE 2 Standard soybean genotype collections. Transgenic processes can be influenced by virulence of Agrobacterium strains, plant species or plant varieties with predisposition to being infected (genetic background), and the design of expression cassettes. 

*Soybean genotypes adapted to various thermal zones (≥24°C, humidity ~80%), with productivity of ~3500 kg ha^-1, a vegetative period of -100 d, and highly sensitive to the photoperiod.

Our experience in the FTO analysis of GM soybean shows that patent application protects the following: i) DNA promoters or termination regions (CaMV35S, Gmubi, Nos) including their use in vectors for genetic transformation of mono- and dicotyledonous plants; ii) epsps gene and its variants and possible uses for genetic transformation of plants; iii) transit peptide (e.g., Petunia hybrid); iv) vectors (pCAMBIA vector); v) transformation and regeneration methods, and vi) plant varieties through a special protection known as "breeder's right" (1978 Act and 1991 Act on Common Provisions for the Protection of the Rights of Breeders of Plant Varieties - International Union for the Protection of New Varieties of Plants - UPOV) (^{Brenner, 1998}; ^{Dattée, 2009}). According to international guidelines, a natural gene sequence cannot be patented; however, in some countries, patenting a gene sequence isolated from the environment is allowed if it is cloned in a vector (^{Jefferson, Köllhofer, et al., 2015}). The pCAMBIA series is a set of protected plant molecular biology vectors with complete access for research and used for developing new varieties of plants (^{Jefferson, 2008}).

A difficulty in this respect is that several methods are covered by patents that sometimes are not taken into consideration for the development of GM soybean for glyphosate tolerance. On some occasions, it is said that these techniques are being used by the public sector without giving further details. The following section describes our specific methods used when processing GM soybean for glyphosate tolerance and some frequently related patents.

Laboratory procedures and IPR

This section provides a technical guide framework relevant to the design and development of soybean transformation for glyphosate tolerance, based on our experience and perception of IPRs trends. According to ^{Nottenburg and Roa Rodríguez (2008)}, those elements that have been patented are transformation vectors, vector genes, transgenes, vector design, methods for making recombinant Agrobacterium with an engineered vector, recombinant Agrobacterium incorporating engineered vectors, improved Agrobacterium strains for transformation, methods of preparing plant tissue for transformation, methods of transforming specific plants, and transformed plants and plant cells.

First, the available soybean seeds must be germinated and regenerated. Soybean seeds must be surface sterilized for 16 h using chlorine gas (4.1 ml of 10 N HCl, 100 ml 5% NaClO). Sterilized seeds are germinated in 0.7% agar medium, pH 5.8, for 5 d (27°C, 16/8 h photoperiod). Then, seed regeneration is monitored using a culture medium containing 1X Gamborg vitamins, 1X B5 salts, 30 g L^-1 sucrose, 1.67 mg L^-1 BAP, 3 mM MES, and 0.7% agar at pH 5.7. Regeneration containers are incubated for a 16/8 h photoperiod at 27°C. Five days later, the number of germinating seedlings must be determined. Regeneration is continuously monitored for four weeks. Regeneration rates are calculated based on the number of explants with shoots and the number of shoots by explant. Some patents cover similar but non-identical protocols enabling seed regeneration (e.g., EP1517991A4 (application June 22, 2002, and withdrawn status) and US5824877A (application July 22, 1988, and expired status)).

The next step is to design an expression cassette that must be introduced into a transformation vector. We prefer pCAMBIA vectors whose principal characteristics involve high copy numbers, 35S promoter, kanamycin or hygromycin B as selection markers, and GUS as screening marker. Also, pCAMBIA vectors have an FTO with availability for academic research without charges, and licenses for profit companies (for more details please visit https://cambia.org/). Ideally, an expression cassette should be designed in such a way that all the elements are stable with the help of molecular biology tools (PCR, enzyme digestion, cloning and ligation, etc.). This expression cassette can also be designed with an aggregation of genetic elements on a modularized principle through bioinformatics for chemical synthesis. When the expression cassette is obtained from a specialized company, it is important for the agreement to have an unrestricted right of utilization, and to avoid clauses as "only for research". The expression cassette includes a promoter, a sequence of chloroplast transit peptide (e.g., CTP from petunia), a CP4-epsps gene (from Agrobacterium sp. strain CP4), and a terminator sequence. Expression of transgenes is a random process caused by the codon usage or RNA regulation. According to the plant codon usage associated with transcriptional regulation and translational efficiency, mRNA stability, splicing at the mRNA level, use of special promoters, removal of polyadenylation signals and cryptic splice sites, and elimination of any potential RNA secondary structure adjacent to the translational start codon, the use of modified prokaryotic genes has allowed reaching levels of expression up to 100 times greater than transgenes without modifications (^{Jackson et al., 2014}). A search of patent databases shows that the patents covering the CP4-epsps gene expired in 2014.

Once the designed expression cassette is synthetized according to the instructions given in the preceding paragraph, it must be introduced into A. tumefaciens. The recombinant strain can be maintained on a Luria-Bertani (LB) medium (50 mg L^-1 kanamycin). A hypervirulent strain of A. tumefaciens, ATCC53213, carrying a pMON546 vector with a petunia epsps gene has been described by ^{Kishore and Shah (1988)} and ^{Shah et al. (1986)} in the patent US4971908A (application April 22, 1988, and expired status) and the patent US4940835A (application July 7,1986, and expired status), respectively. Agrobacterium strains must be characterized using PCR: i) Ach5FtsZ primers (F: 5'-GAACTTACAGGCGGGCTGGGT-3', R: 5'-CGC-CGTCTTCAGGGCACTTTCA-3', product: 369 pb) are specific to A. tumefaciens LBA4404; ii) C58GlyA primers (F: 5'-CCACCACCACGACGCACAAGTCT-3', R: 5'- TGC- CGAGACGGACACCCGAC-3', product: 423 pb) are useful for the detection of C58C1, EHA101, EHA105, and GV3101 strains; iii) pTiBo542 primers (5'- CCCGCTGAGAAT- GACGCCAA-3', R: 5'- CCTGCGACACATCGTTGCT- GA-3', product: 766 pb) are specific to EHA101 and EHA105 (differentiated from C58C1, LBA4404 and GV3101 strains), and iv) nptI primers (F: 5'- CTGCGATTCCGACTCGTC-CA -3', R: 5'- CGGGCAATCAGGTGCGACA-3', product: 572 pb) are special for EHA101 only (^{Deeba et al., 2014}). In our experience, A. tumefaciens strains EHA101 and EHA105 are useful for soybean transformation, probably because of their virulence. We tested another bacterium, Sinorhizobium meliloti (see https://cambia.org/), to see if we could recreate the soybean infection; however, the results were not encouraging. Agrobacterium cultures (OD650 = 0.6-1.0 at 28°C, 250 rpm) must be used for infection of explants (^{Guo et al., 2020}). A bacterial pellet obtained by culture spinning (8000 rpm, 4 min) must be resuspended in cocultivation media (1X Gamborg vitamins, 0.1X B5 salts, 1.67 mg L^-1 BAP, 0.25 mg L^-1 GA₃, 3% sucrose, 20 mM MES, 200 μM acetosyringone, pH 5.7).

Cotyledons must be excised at the junction between the hypocotyl and the half-way point of the cotyledon, five mm below the cotyledonary node, and a cut is performed to divide the cotyledonary explants. The plumule is eliminated and two small incisions are made on the cotyledonary node. Explants are infected with recombinant A. tumefaciens (containing a designed expression cassette as described above) in coculture broth (30 min), and then inoculated on coculture solid medium (1X Gamborg vitamins, 0.1X B5 salts, 1.67 mg L^-1 BAP, 0.25 mg L^-1 GA₃, 3% sucrose, 3.9 g L^-1 MES, 200 acetosyringone, 0.7% agar, and pH 5.7) with the adaxial side down. The co-cultivation plates are incubated in the dark for 3 d at 28°C. The explants are then rinsed in sterile water twice. After the last rinse (using 350 mg L^-1 cefotaxime), explants are transferred into shoot induction medium (1X Gamborg vitamins, 1X B5 salts, 30 g L^-1 sucrose, 1.67 mg L^-1 BAP, 3 mM MES, 0.7% agar, pH 5.7 containing 100 mg L^-1 timentin, and 350 mg L^-1 cefotaxime) for two weeks. The shoot induction medium must be refreshed for an additional period of two weeks (^{Paz et al., 2004}; ^{Song et al., 2013}).

Our experience implies a longer-term evaluation of tolerance by using lower concentrations of glyphosate of 0 mg L^-1 for shoot initiation 1 (regeneration 1), then a glyphosate concentration of 25 mg L^-1 for shoot initiation 2 (regeneration 2), and then a concentration of glyphosate reduced to 6 mg L^-1 (shoot elongation) and 0 mg L^-1 (rooting), respectively. Herbicide selection includes shoot induction without glyphosate (0 ^M) (the first two weeks) and 148 (subsequent two weeks). After four weeks in shoot induction medium, explants are transferred into a shoot elongation medium (1X MS salts, 1X Gamborg vitamins, 0.5 mg L^-1 GA3, 0.1 mg L^-1 IAA, 0.7 mg L^-1 BAP, 30 g L^-1 sucrose, 3 mM MES, 50 mg L^-1 asparagine, 50 mg L^-1 glutamine, 70 mg L^-1 vancomycin, 350 mg L^-1 cefotaxime, 35 glyphosate, 0.7% agar, and pH 5.7). A new culture medium must be prepared every two weeks for six weeks (16/8 h photope-riod, 26°C). Every shoot from the elongation step that has at least 2 cm is moved to the rooting medium (0.66X MS salts, 1X Gamborg vitamins, 3 mM MES, 30 g L^-1 sucrose, 0.7% agar, and pH 5.7). The whole process is summarized in Figure 1. Some alternative selection strategies have been described (^{Clemente et al., 2000}; Monsanto Technology, 2011; ^{Dow AgroSciences, 2014}) (Tab. 3). We have used a strategy for the transformation and selection of GM events that is consistent with the strategic FTO. Therefore, the reporter genes from pCAMBIA vectors are removed using restriction enzymes and, in this way, the CP4 epsps gene is both a desired trait and gene reporter avoiding unjustified additional IPRs. The use of low concentrations for glypho-sate selection in the regeneration media could increase in vitro escapes (non-germline transformation or chimerism in primary transformants). It is for this reason that a selection marker is important (e.g., antibiotic resistance or green fluorescent protein genes) (^{Miki & McHugh, 2004}). Selection markers have been at the center of controversy about biosafety concerns; therefore, new approaches to produce marker-free GM plants were developed for novel events (^{Darbani et al., 2007}; ^{Tuteja et al., 2012}). Since the glyphosate-tolerant GM soybean development has an FTO focus related to the CP4 epsps gene (as mentioned above), the transformation of a high number of explants is essential. The efficiency of soybean transformation can be explained by plant defense mechanisms such as mitogen-activated protein kinases, defense proteins, reactive oxygen species, or hormones (^{Imam et al., 2016}). Soybean transformation has a very narrow efficiency (glyphosate-tolerant events vs. infected explants) of less than ~6% using Agrobacterium (^{Hinchee et al., 1988}), probably since A. tumefaciens has low infective capacity on soybean tissues. Additionally, it is dependent on the soybean genotype and A. tumefaciens strain (^{Song et al., 2013}). These reasons are enough to strongly support the evaluation of a great set of explants, considering the additional loss of material as a result of contamination, seed quality, or genotype resistance.

FIGURE 1 Steps in the transformation of soybean by A. tumefaciens. 

Transformants that survive long term glyphosate-selection must be transferred to the soil and subjected to leaf painting with glyphosate at 2300 g acid equivalent (ae) h^-1 and the tolerance behavior could be scored one week after the treatment. ELISA and herbicide painting analysis must also be conducted in T0 plants (hemizygous dominant) (^{Passricha et al., 2016}), and the phenotypic response must be correlated with molecular analysis. With these new materials, it is possible to obtain a high number of homo-zygous individuals for the trait. Possible GM lines must be evaluated using PCR (transgene presence), ELISA (EPSPS protein), Southern blot (transgene copy number), and real time PCR (transgene expression). The rooted primary transformants must be individualized and propagated in soil for a period of 30 d. The plantlets could be screened with ~0.2% glyphosate (~300 per plant) to assess their survival rate 10 d after aspersion (^{Guo et al., 2020}).

The T-DNA and Vir proteins form a complex that controls the supply of a single T-DNA strand (T-strand) into the plant chromosomes. The assimilation of the T-strand is, probably, due to homology repair, or to non-homologous, or perhaps microhomology-mediated end joining that repairs DNA double-strand breaks. The host DNA structure, tissue expression, and soybean genotypes play a relevant role in transformation efficiency (^{Hintz et al., 1992}; ^{Cober et al., 2009}; ^{Jamsheed et al., 2013}) (Tab. 2). The presence of 5-25 bp of homology between RB/LB of the T-DNA and the plant genome, the frequent insertion of the T-DNA in promoter regions and gene rich regions, and the correspondence of T-DNA tag density and gene density between GC/AT content provide support for the hypothesis of microhomology-facilitated end joining mechanism, which explains why the T-DNA insertion occurs in several locations (introns, terminators, telomeres, or repetitive sequences) (Jamsheed et al., 2013; ^{Bourras et al., 2015}). In the present case, there are several patents that include new approaches to the molecular mechanisms used by A. tumefaciens during plant transformation: WO2007132193A1 (entitled: modified vird2 protein and its use in improved gene transfer), WO2004035731A2 (entitled: increasing host plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis vip1 gene), US20150267213A1 (entitled: strains of Agrobacterium modified to increase plant transformation frequency), WO2016125078A1 (entitled: Agrobacterium-mediated genome modification without T- DNA integration), WO2002052026A2 (describes methods to DNA integration through homologous recombination pathway), and US6800791B1 (an engineered A. tumefaciens strain to transfer proteins to plant cell). This is just a small window on the global patents currently taking place about Agrobacterium mechanisms, and several of these patents are still valid.

TABLE 3 Selection strategies in GM soybean for glyphosate tolerance.