In 2009, big challenges facing the agricultural sector in the twenty-first century were presented to the world. Human population growth, increased life expectancy, loss of biodiversity, climate change and accelerated land degradation are the main factors contributing to rethink agriculture system production. In that scenery, modern biotechnology has set a stage for the advancement of agricultural practices and it is clearly an important ally to apply a broad array of technologies and innovative systems where they are most needed, such as enhancing crop productivity, increasing yields, and ultimately ensuring food security. One of the biggest challenges is related to technify production systems, but with no doubt, developing genetic improvement toward getting an efficient and sustainable agriculture, generating new seed qualities (new traits), such as, among others, to upset fatty acids content in oilseed crops have been growing up significantly due to industry interest. In this study, a review about the main advances in genetic improvement of some oilseed crops, starting with omics to understand metabolic routes and to find out key genes in seed oil production, and also, getting in use of modern biotechnology to alter the production of fatty acids, and to face biotic challenges in oilseed crops is presented.
- seed oil
- oilseed crops
- fatty acids
- genetic improvement
- genetic engineering
- modern biotechnology
Over the last decades, the adoption of oilseed crops has been growing up significantly due to industry interest in the composition of their seed oils, which are made up of a wide range of fatty acids with six predominant types: 16 or 18 carbon palmitic, stearic, oleic, linoleic and linolenic acids, and 12 carbon lauric acid, as well as other unusual fatty acids produced by wild plant species include those with chain lengths between 8 and 24 carbons . Due to their structure and composition, those oils are used as food/industrial feed  and as a range of product applications such as surfactants, soap, detergents, lubricants, solvents, paints, inks, chemical feedstocks and cosmetics . In this study, a review about the main advances in genetic improvement of oilseed crops, starting with omics to understand metabolic routes and to find out key genes in seed oil production, and also, getting in use of modern biotechnology including genetic engineering and new breeding techniques (NBTs), a modern-breeding tool that has allowed the functional study of genes with potential application for breeding in agriculture, focusing on oilseed crop genetic improvement with high precision and less uncertainty (avoiding whole genomes crossing), and of course, in less time is presented; those scientific efforts where it was sought to upset fatty acids production or biotic tolerance will also be presented.
2. Oilseed crops
Seed oil is mainly obtained from plants recognized as oilseed crops, among them are: soybean (
Seed oils applications depend on physical and chemical properties of their fatty acids composition . Those oils are mainly composed of five major fatty acids, including the saturated palmitic (C16:0) and stearic (C18:0) acids, the monounsaturated oleic acid (C18:1), and the polyunsaturated LA (C18:2) and ALA (C18:3) . A large variety of other less common but not less important fatty acids can be found in different species and used for various industrial applications. These fatty acids vary in the number of carbons in the chain (from 8 to 24), the number of double bonds, and the presence of epoxy, hydroxyl and other functional groups .
Despite the industry's significant interest in oil crops, it is reasonable to mention that agriculture has a challenging future. The Food and Agriculture Organization of the United Nations (FAO) in 2009 presented the big challenges facing the agricultural sector in the world for near future . Human population growth, increased life expectancy, loss of biodiversity, climate change and accelerated land degradation are main factors contributing to rethink agriculture system production. Thus, there is a need to technify agricultural production systems, but without doubt, developing genetic improvement toward getting an efficient and sustainable agriculture, generating new seed qualities (new traits), such as, among others, high content of PUFAs in oilseed crops, it will be an aiming. Biotechnology will be fundamental to overcome these challenges. Genetic engineering techniques may play an important role by elevating the content of individual fatty acids or drastically changing the oil quality by the introduction of a new fatty acid, thus increasing raw materials available for oleochemistry. In this perspective, it has came growing research efforts of scientist around the world seeking to expand the knowledge barrier on oilseed crops. Figure 2 shows continuous growth in the number of scientific publications in this field, records subtracted from literature databases: Scopus, Web of Science, and ScienceDirect.
3. Understanding metabolic routes in oilseed crops
Oilseed plants represent an important renewable source of fatty acids because they accumulate them in the form of triacylglycerol (TAG) as major storage components in seeds . In plants, the reactions for de novo fatty acid synthesis begin in plastids  and then exported to the cytoplasm following two inter-related metabolic pathways: an acyl-CoA-dependent pathway and an acyl-CoA-independent pathway .
In the dependent pathway, commonly known as the Kennedy pathway, the priming and elongation of nascent acyl chains requires acetyl- and malonyl-CoA, respectively, as direct precursors up to eighteen carbons in length . In this pathway, the glycerol-3-phosphate acyltransferase (G3PAT) is the first enzyme that catalyzes the transfer of a fatty acid to glycerol-3-phosphate (G3P) to form lysophosphatidic acid (LPA). Then, the LPA is acylated by the lysophosphatidic acid acyltransferase (LPAAT) to yield phosphatidic acid (PA). Next, PA is dephosphorylated by the phosphatidic acid phosphatase (PAP) to form diacylglycerol (DAG) and finally, a diacylglycerol acyltransferase (DGAT) catalyzes the acylation of DAG to the production of TAG . In the acyl-CoA-independent pathway, an alternative enzyme is used for the final acylation reaction, termed phospholipid:diacylglycerol acyltransferase (PDAT). PDAT directly transfers an acyl group from phosphatidylcholine (PC) to DAG, producing TAG .
Desaturation steps for fatty acids are catalyzed by plastidial stearoyl-acyl carrier protein (ACP) desaturases. After termination, free fatty acids are activated to CoA esters, exported from the plastid, and assembled into glycerolipids at the endoplasmic reticulum (ER) . In addition, further modifications (desaturation, hydroxylation, elongation, etc.) occur in the ER while acyl chains are esterified to glycerolipids or CoA . The low polarity of TAG is believed to result in the accumulation of this lipid between bilayer leaflets leading to the budding of storage organelles termed oil bodies . The accumulation of hydroxy fatty acids depends on many factors, including the performance of the desaturases and efficient channeling of hydroxy fatty acids into storage triacylglycerols . Fatty acid dehydrogenase (FAD) catalyzes the desaturation reaction, leading to the formation of unsaturated FA. Interestingly, studies have revealed that some desaturase enzymes (such as the FAD2 and FAD3 genes) could be regulated at the transcriptional level or at the post-translation level in response to low-temperature induction in model plants [17, 18]. Other important enzyme is FAH12 which belongs to a large family of fatty acid modification enzymes that are related to the
High-quality RNAseq data have allowed the identification and an accurate quantification of expression of transcription factors and key genes related with lipid metabolic pathways in soybean ,
Gathering the RNAseq information and advances in plant transformation technology is possible now engineering plants for the production of oilseed fatty acids. In a remarkable research, Arabidopsis plant was modified introducing a fatty acid hydroxylase from castor plant, it leading to produce some ricinoleic acid and an unusual fatty acid in the seed . Reactions in triacylglycerol biosynthesis have also been manipulated to increase seed oil content. Studies have suggested that the level of DGAT activity during seed development may have a substantial effect on the flow of carbon into seed oil. Thus, overexpression of cDNAs encoding either
However, in the last decade, the scientists have realized that the manipulation of single genes only contribute with limited value to change the metabolic pathways. Nowadays, there are strategies focused on more complex approaches involving simultaneous overexpression or suppression of multiple genes to achieve optimal metabolic flux . Understanding a metabolic network would facilitate the production of natural products and the synthesis of novel molecules in a predictable and useful manner . For this reason, the metabolic engineering in oilseed plants has attracted industrial and academic researchers in the last decade.
4. Modern biotechnology for genetic improvement in oilseed crops
The Convention on Biological Diversity (CBD) has defined biotechnology as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use” . In fact, biotechnology includes several agricultural as well as food manufacturing tools and techniques. However, when a biotechnology development uses new deoxyribonucleic acid (DNA) techniques, molecular biology, and reproductive technological applications ranging from gene transfer to DNA typing to cloning of plants and animals, it has been considerable modern biotechnology . The potential of modern biotechnology is widely known, as it makes the use of recombinant DNA technology to generate modified microorganisms, plants and animals to make them more suitable for several potential applications: improved crops, production of new antibiotics and hormones, xenotransplantation, gene therapy, bioremediation, and genetic editing, one of the most recent techniques.
Genetic engineering crops based on recombinant DNA technology were first introduced for commercial production in 1990s. This technology uses the identification, isolation and manipulation followed by the introduction of desired gene(s) from one organism (for example, a plant or bacteria) to another, thus giving rise to a transgenic or genetically modified organism. This technique has been fast replacing plant breeding so as to incorporate characteristics that are impossible to achieve by breeding. Biotechnology has the potential to help overcome many of the short-comings of the species being promoted, especially where exogenous genes are needed because there are characters that are difficult to produce by traditional breeding, or where characters tissue-specific or temporal expression or suppression of endogenous genes would be valuable . For oilseed crops, modern biotechnology should allow the production of plants with specific fatty acids content.
In the following paragraphs, main advances in plant genetic improvement using modern biotechnology, focused on oilseed crops, those scientific efforts in soybean (
4.1. Soybean (
Due to its importance as a crop, genetic transformation techniques have been used extensively to improve the cropʼs valuable traits. Herbicide-tolerant (Roundup Ready) Soybean (
In terms of genetic transformation methods, many reports describing soybean transformation by particle bombardment using meristems as the target tissue have been published. The Biolistics particle delivery system for soybean transformation was evaluated in two different regeneration systems: from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long-term proliferative suspension culture of the cultivar Fayette . A method for high-frequency recovery of transgenic soybean by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques was made by . A targeting method to insert genes with biolistics to predefined soybean genome sites using the yeast FLP-FRT recombination system was made by . A double-barreled gene
Genetic engineering approaches have been applied to enrich the content of soybean oil for a particular fatty acid or class of fatty acids. One of those examples was made by . These authors developed transgenic soybean seeds by down-regulating the expression of
On the other hand, regarding to biotic factors,  developed transgenic soybean to improve resistance against SMV. HC-Pro coding sequences were introduced within a RNAi inducing hairpin construct and
4.2. Sunflower (
Sunflower is one of the most important oilseed crops cultivated on a global level. Its seeds have always been ground and pounded into flour for making bread, cracked and eaten as snacks, mixed with vegetables, and extracted for oil. The seeds are also a source of purple dye and have medicinal uses . Sunflower seeds are composed by 20% protein and 50% fat. In this crop oil, up to 90% of its fatty acids are unsaturated, namely oleic (C18:1, 16–19%) and linoleic (C18:2, 68–72%) acids. The remaining 10% of its fatty acids are palmitic (C16:0, 6%), stearic (C18:0, 5%), and minor quantities of myristic (C14:0), myristoleic (C14:1), palmitoleic (C16:1), arachidic (C20:0), behenic (C22:0) .
Several scientific efforts have been made to develop genetic improvement methods in sunflower, using modern biotechnology.
Perhaps one of the earliest works in sunflower was developed by , which introduced plasmid into isolated sunflower protoplast. Another effort was made by  who used microprojectile bombardment of half-shoot apices followed by co-culture with
One of the most important aspects of any transformation protocol is an efficient selection of transgenic plants. On the way to develop a procedure to minimize the number of transgenic escapes,  germinated sunflower seeds for 24 h on half-strength MS-based medium, before cutting the seeds to give two half embryos, each with one cotyledon. Once that, cotyledon explants were inoculated with
Leaving aside developments made around transformation methods, and focusing on advances toward genetic improvement with some functional characteristics, some efforts to improve oil production in sunflower have been made recently. Dağüstü et al.  introduced the
Some research interests have been around decreasing levels of palmitic and stearic acid of sunflower, due to their contribution on increasing the plasma cholesterol level in humans, associated with heart disease. Škorić et al.  induced mutations via seed treatment with γ-rays, X-rays, and mutagenic chemicals such as ethyl methanesulfonate (EMS) and dimethyl sulfate (DMS) to generate sunflower genotypes with high levels of C 18:2, C 18:1, C 18:0, C 16:1 and C 16:0.
4.3. Canola (
A group of researchers developed transgenic canola seeds with significantly increasing of oil content . Those authors showed that seed-specific overexpression of BnLEC1 and BnL1L genes (from canola), placed under the control of the truncated canola storage protein 2S-1 promoter, which is also known as the
In the same way to improve canola oil production, Qi et al.  isolated the RNA-binding motifs No2 (RRM2) of the flowering control locus A (FCA) protein (FCA-RRM2) from variety No. 1 “Nannongyou” of Canola, and then, it was introduced in cotyledon nodes using
Aimed at not good perception of erucic acid (cis-13-docosenoic acid) in the canola oil triglycerides, because of presumptive effects on growth retardation and pathogenic changes to internal organs when fed at high concentrations to laboratory animals, a research was made to decrease erucic acid level in Canola plants. Shi et al.  reported the development of canola transgenic with change in fatty acids compositions, using
4.4. Palm (
Cultivation of the oil palm (
It is reported that currently palm oil accounts for about 20% of world oils and fats production. It was forecast that, with the increase in world population, the demand for palm oil would grow faster than the rise in supply, so that, the supply of palm oil would also need to be increased to meet the above demand. It was therefore crucial to increase the yield of palm oil, improve its oil quality, and produce novel products via genetic engineering, as it could be achieved faster this way than by conventional breeding ways.
According to , palm fruits have two storage tissues, mesocarp and kernel, that can be the target for accumulating genetically modified products. The substrates and intermediates implied in the production of storage oil or protein in these tissues may be channeled to alter the levels of existing products or to produce novel value-added products without deleterious effects on the plants. During oil palm fruit development, at level of period of oil accumulation as well as fatty acid composition, the mesocarp and kernel tissues show differences. A study reported by  showed the regulation of the gene expression during period of oil synthesis in both tissues. The expression profile of the mesocarp-specific gene in different oil palm tissues, as well as at different developmental stages of the mesocarp and at the cellular level indicated a strong correlation with that of a fatty acid biosynthetic gene, stearoyl-ACP desaturase. Using promoter-reporter constructs, assays and transformations, these authors demonstrated that this promoter is conducive to the development of genetic improvement research modern biotechnology, since specific genes can be located there generating high degree of expression.
Different efforts have been made to get biotechnological palm crops which can generate metabolites of interest. Genetic engineering in oil palm is relatively recent as it was initiated at Malaysian Palm Oil Board (MPOB) in the late 1980s . Related to transformation protocols, particle bombardment and
Seeking to improve oleate oil palm production, some studies had some strategies where transformation vectors were constructed: (1) antisense palmitoyl-ACP thioesterase gene driven by CaMV35S promote, (2) antisense palmitoyl-ACP thioesterase and sense KAS II genes driven by mesocarp-specific promoter, (3) antisense palmitoyl-ACP thioesterase, sense KAS II and sense stearoyl-ACP desaturase genes driven by a mesocarp-specific promoter and, (4) antisense palmitoyl-ACP thioesterase, antisense oleoyl-CoA desaturase, sense KASII and sense stearoyl-ACP desaturase genes driven by a mesocarp-specific promoter, and then, those constructs were bombarded into oil palm embryogenic cultures. Molecular and biological assays were made and some plantlets were transferred to soil in the biosafety screenhouse . It is important to refer that KAS II is one of the main enzymes contributing toward high palmitic acid.
Stearate oil palm is an another key oil with great industrial interest such as cocoa butter substitute and personal care products such as lotions, shaving cream, and rubbing oils. Biochemical studies have demonstrated that oil palm contains an active stearoyl-ACP desaturase, and therefore down-regulating the activity of stearoyl-ACP desaturase could reduce the conversion of stearoyl-ACP into oleoyl-ACP . Three transformation vectors were constructed carrying an antisense stearoyl-ACP desaturase gene driven by ubiquitin, CaMV35S, and mesocarp-specific promoters . These authors reported that those constructs were bombarded into oil palm embryogenic cultures and a few lines of resistant polyembryogenic cultures and then full plant regeneration were obtained. The transgenic plant’s stearic acid content increased as a result of concomitant reduction of oleic acid levels.
Regarding to biotic factors,  introduced a synthetic
4.5. Castor bean (
Castor or castor bean (
Introduction of foreign genetic material defining specific agronomically important traits into castor through genetic transformation techniques have been attempted. Transgenic research in castor bean (
Castor semilooper (
In the same way, toward obtaining biotechnological strategies for the control of castor semilooper,  reported the use of
On the other hand, two approaches were used to develop castor cultivars with reduced levels of toxin.  reported on “knocking out” the genes responsible for ricin production as well as genes responsible to produce ricinine and CB-1A. Then, conventional sexual hybridization was used to develop F6 lines of castor that have a 75–70% reduction in ricin and
4.6. Cotton (
Chapman et al.  reported the development of transgenic cotton plants with increased seed oleic acid levels. Using an Agrobacterium-mediated system transformation, these authors introduced a binary vector previously designed to suppress expression of the endogenous cottonseed enzyme fatty acid desaturase 2 (Fad2) by subcloning a mutant allele from a rapeseed fad2 gene. It is known that FAD2 enzyme, in the endoplasmic reticulum of plant cells, catalyzes conversion of oleic acid to linoleic acid so that, decreasing this enzyme activity would be an increase of oleic acid content in cottonseed oil. At the end of the research, these authors’ increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in primary transformants and 47% of oleic acid content in their progeny, which represent an increasing of three times comparing with standard cottonseed oil.
Due to consumption of the saturated fatty acid, overall cholesterol levels increases, more specifically low-density lipoprotein (LDL) which is considered “bad cholesterol,” and it is well known worldwide that its consumption increases risk of cardiovascular disease ; a group of researchers started a study to improve the quality of cottonseed oil.  used RNAi technology to regulate fatty acid metabolism of cottonseed inhibiting GhFAD2-1 and GhFATB gene expression levels, simultaneously. These genes encoding the microsomal oleate desaturase and palmitoyl-acyl carrier protein thioesterase, respectively, play significant roles in regulating the proportions of saturated and polyunsaturated fatty acids in cottonseed lipids. Using this technology, they decreased palmitic acid and linoleic acid content and increased oleic acid content, but unfortunately, they got an adverse effect on seed germination and seed vigor. In spite of achieving an adequate balance in the content of fatty acids, thinking in human consumption of cottonseeds oil, it is necessary to explore others effective regulating strategies to improve the quality of cottonseed oil.
On the other hand, recently, Wang et al.  reported a genome-wide analysis in several
4.7. Peanuts (
Research about transgenic peanut crops has been undertaken for the development of fungi resistant. This crop is susceptible to many types of pathogens including those caused by fungi. Chenaulr et al.  reported the development of transgenic peanuts which were introduced two hydrolase genes, a glucanase from alfalfa (
4.8. Olive (
Olive oil production and consumption are increasing in importance around the world. Spain is the largest producer with an average 1 million tons per year, followed by Italy and Greece with 560 and 350 thousand tons, respectively . This crop contributes significantly not only to the global economy but also to food security in terms of its nutritional value. It is well known that olive and olive oil play an important role in prevention of coronary heart disease and certain cancers, due to their high levels of monosaturated fatty acids and phenolic compounds .
Olive is characterized by a long history of cultivation, as it was one of the first tree species to be domesticated, and by wide diversity. A very large number (over 1600) of cultivated varieties characterize this species. This diploid specie (2n = 46) has a small genome (2200 Mb) and it is predominantly allogamous in nature [94, 95]. During a long period of time, local and old cultivars have been evaluated by different genetics, morphological, and agronomics approaches. Recently, the huge genetic variability of this specie has been evaluated using molecular markers, including SSRs, particularly advantageous because olive is a clonally propagated, perennial, slow growing, highly heterozygous cultivated species with a very large uncharacterized genome .
As any other extensive crop, the olive has urgent challenges; they are summarized into six big areas such as: (i) olive growing; (ii) processing, byproduct, and environmental issues; (iii) virgin olive oil sensory quality; (iv) purity, authentication, and traceability; (v) health and nutrition; and (vi) consumers. Moreover, the olive varieties renewal have been hampered by the extreme longevity of olive trees, the long period of juvenility of their offspring, and the diffidence of the public to accept genotypes obtained with advanced biotechnological approaches. Modern biotechnological techniques are suitable for olive improvement because they both allow direct correction of main defects, combining with existing known superior cultivars, and can also support traditional breeding using the great genetic variability present in the species, to guide crossing of genotypes chosen among the olive populations of different sites . Modern biotechnology along with traditional and
However, some aspects of the olive biotechnology remain challenging; for example, the olive propagation is still a laborious practice. As regards traditional propagation, rooting of cuttings and grafting stem segments onto rootstocks are possible. The regeneration of whole plants from ovules, on the other hand, is used only occasionally. Micropropagation of olive is not easy mainly due to explant oxidation, difficulties in explant disinfection, and labor-oriented establishment of
As it was seen throughout the review, last three decades, for these oilseed crops: soybean (
This book chapter was supported by “Component No.1 - Biotechnology”, project code 4600000480 financed by Colombian general system of royalties (SGR, Spanish acronym), in partnership with Secretaría de Agricultura y Desarrollo Rural of Departamento de Antioquia, Universidad Pontificia Bolivariana, Universidad Nacional de Colombia and Universidad EAFIT.