Giovanni Rodrigues Vianna

In Brazil, renewable biofuel is mainly produced from soybean [ Glycine max (L.) Merr.] oil. Typically, soybean‐derived oil contains approximately 25% of the monounsaturated oleic acid, 13% of the saturated fatty acid palmitic, and 55% of the polyunsaturated fatty acid (PUFA) linoleic acid. The high PUFA profile negatively affects biofuel performance, reducing oxidative stability, whereas saturated fatty acid content reduces viscosity and cold flow performance. Increasing the content of oleic acid at the expense of PUFA concentration is a desirable trait for biofuel quality. In an earlier study conducted by our group, using RNA interference (RNAi) technology, a cellular mechanism responsible for post‐transcriptional gene silencing, generated a high‐oleic, low‐palmitic acid soybean line, designated event 65‐25. Now, in this report, we show the stability of the RNAi line 65‐25 for 15 generations (10 years). Small interfering RNA (siRNA) originating from the RNAi construction was detected in the generation of the original 65‐25 event. Additionally, analyses of the expression of fatty acid biosynthesis genes, particularly the genes encoding for delta‐12 desaturase (FAD2), delta‐15 desaturase (FAD3), and the fatty acyl‐acyl carrier protein thioesterase B (FATB), were compared to the wild‐type (WT) soybean line, Conquista from Embrapa, with the high‐oleic lines. In the analyzed lines derived from 65‐25, FAD2 , FAD3 , and FATB genes were downregulated compared to WT plants. Oil profile analysis showed that the RNAi lines retained a more than twofold increase in oleic acid seed content relative to WT line. Our data confirm that the RNAi transgene remained stable for over 15 generations in soybean seeds. These RNAi lines consistently downregulate key genes in the fatty acid biosynthesis pathway— FAD2‐1 , FAD2‐2 , FAD3 , and FATB1 —leading to enhanced oleic acid content in mature seeds.

Genome editing technologies have revolutionized the production of microorganisms, plants, and animals with phenotypes of interest to agriculture. Editing previously sequenced genomes allows for the punctual, discreet, precise, and accurate alteration of DNA for genetic analysis, genotyping, and phenotyping, as well as the production of edited organisms for academic and industrial purposes, among many other objectives. In this context, genome editing technologies have been causing a revolution in Brazilian agriculture. Thanks to the publication of Normative Resolution No. 16 (in Portuguese Resolução Normativa No. 16-RN16) in 2018, Brazilian regulatory authorities have adapted to the new genetic manipulation technologies available to the scientific community. This review aims to describe the effects of updates to the regulatory framework for edited organisms in Brazil and to point out their impacts on research and development of emerging technologies in the Brazilian agricultural sector. The implementation of RN16 rationalized the regulatory aspects regarding the production, manipulation, exploration and commercial release of edited organisms and led to the faster, cheaper and safer obtaining of edited technologies, which are more productive and better adapted to different environmental conditions in Brazil.

The modification of plant genomes is an essential step towards the development of new crops to increase food production. Initially, genome modification relied on conventional plant breeding and the introduction of genetic traits using crossing techniques to generate new varieties. In the 1980s, trans and cis modification allowed the incorporation of specific traits into plant genomes, largely due to the development of DNA delivery systems such as Agrobacterium and biolistics. This technological breakthrough has boosted a second generation of genetically modified crops that have had a major impact on agriculture. However, the stochastic nature of the DNA delivery systems, with little control over where the genome modification occurred, required regulatory measures to ensure environmental, human, and animal safety, which has hindered the ability and speed to generate new and more adapted varieties. New technological advances have made it possible to increase the precision of genome modifications leading to a third generation of agricultural products. These technological advances rely on enzymes targeting specific genomic regions, making it possible to introduce mutations and new traits into the plant genome. Meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas9 are valuable tools that allow for specific genomic modifications. These tools have made it possible to develop new, safer varieties in a shorter timeframe. In this review, we explore the functional mechanisms of these new breeding tools, their advantages and drawbacks, and their potential to explore metabolic engineering and synthetic biology in plants.

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.

Currently, the market demands products committed to protecting human health and the environment, known as clean products. We developed a protocol using DNA fragments containing only the gene sequence of interest, to replace the circular vectors containing genes for antibiotic resistance and other undesirable sequences, for obtaining transgenic soybeans for microparticle bombardment. Vector pAC321 was digested with the restriction enzyme PvuII to produce the 6159 bp ahas fragment, which contains the mutated ahas gene from Arabidopsis thaliana (Brassicaceae), under the control of its own promoter and terminator. This gene confers resistance against imazapyr, a herbicidal molecule of the imidazolinone class, capable of systemically translocating and concentrating in the apical meristematic region of the plant, the same region used for the introduction of the transgenes. This fragment was used to generate 10 putative transgenic soybean lines.

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α′ subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg⁻¹ seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α′) subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9 g kg⁻¹) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.

A Fundação de Pesquisas Científicas de Ribeirão Preto (FUNPEC-RP) foi criada em 10 de março de 1983, é pessoa jurídica de direito privado, sem fins lucrativos com autonomia administrativa e financeira.Tem como principais objetivos apoiar e promover o desenvolvimento de ensino e pesquisa e a prestação de serviços à comunidade. A FUNPEC-RP tem também o importante papel na produção e disseminação de conhecimento.