Agricultural engineering

Agricultural engineering combines various disciplines of mechanical, civil, electrical, food science, environmental, software, and chemical engineering to improve the efficiency of farms and agribusinesses while ensuring sustainability of natural and renewable resources. This includes designing and building agricultural infrastructure, such as dams or water reservoirs; engineering solutions for pollution control; and developing new forms of biofuels and non-food resources, like algae and agricultural waste. This includes using genetic technologies for developing foods to increase agricultural yield.

Gene knockout, or loss-of-function (LoF) alleles, are an important source of genetic variation. This is a genome-editing tool that can enable the precise and targeted modifications of an organism's genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. Discoveries in functional molecular genomics have led to an increasing appreciation for their importance in crop evolution.

Part of the interest in LoF mutations is the type and number of mutations that can break a gene's molecular function could be greater than the mutations that lead to gain-of-function changes in protein activity or expression. Such mutations can include frame-shifts, premature stop codons, large insertions and deletions, splice site disruptions, and amino acid substitutions. The number of mutations that can cause LoF is also large because many positions along the length of a gene are vulnerable to disruptive mutations. This can have implications for crop evolution as it can influence the variants.

There are many reasons targeted knockout mutations are becoming a particularly compelling direction for modern breeding efforts. Studies of the genetic basis of past crop evolution have revealed that LoF mutations can provide a significant contribution to crop improvement. They are also often easy to identify from sequence data and are easy to annotate from whole genome sequence data in search of candidates for molecular breeding. As well, genome editing technologies, especially CRISPR/Cas based gene editing, are good at generating targeted knockout mutations. In contrast, the editing efficiency of gain-of-function alleles is much lower and faces additional levels of complexity and challenges.

The evolutionary history of crops demonstrates that breaking the right genes can have a positive impact on numerous traits, including yield. Knowledge of the potential for LoF alleles to increase crop yields motivates current efforts to knockout target genes as a breeding tool. In rice for example, CRIPSR/Cas was used to create knockout alleles in landraces mimicking natural LoF alleles in sd1 already known to be responsible for semi-dwarf rice varieties. Likewise, DEP1 and GN1A are two genes associated with rice yield, and loss of these genes has resulted in an increased number of grains per panicle and an increase in gain yield. And unlike traditional breeding, which is generally limited to crosses between species to produce fertile offspring, targeted knockout mutations can extend beyond species boundaries. This means discoveries made in crop relatives can be translated into downstream molecular breeding.

Loss-of-function can be effective at conferring resistance to certain kinds of biotic stress. Many plant pests and pathogens interact with plant molecular pathways, and crops can benefit from the knockout of these genes. The expectation is consistent with discoveries of natural mechanisms of disease resistance and broader surveys of natural genetic diversity that have found LoF to be common in gene annotations as antagonistic biotic interactions. For example, the parasitic plant Striga hermonthica uses the product of the gene LGS1, which is a molecule secreted by sorghum roots into the soil, to find host plants. And it has been discovered that sorghum varieties lacking functional alleles of this gene have evolved greater resistance. It is also important to note that not all beneficial knockout mutations are universally beneficial. For example, there may be a knockout mutation that increases resistance to one pathogen but decreases resistance to others.

One of the uses for targeted knockout to accelerate crop breeding is for improved nutritional traits such as increased bioavailability of macro- and micro-nutrients and the decrease of undesirable toxins. The logic is that LoF can be used in the evolution of such traits is that desirable phenotypes may be achieved by knocking out genes responsible for the production, uptake, or tissue specific accumulation of toxic, anti-nutritive, or undesirable elements or molecules. Toxic heavy metals, such as cadmium, can accumulate in many crops, posing a threat to communities relying on food grown in contaminated soil. But, a breeding program based on knockouts that can translate discoveries between crop species, NRAMP5 and other members of this gene family have emerged as promising target for engineering low-cadmium varieties of crops such as cacao.

The capacity to impact nutritional composition of plants with LoF alleles may be a reflection of the pool and flux of nature of biosynthetic pathways. In oilseed crops, this principle has been applied to engineer fatty acid profiles that are healthier and have longer shelf lives. CRISPER-mediated knockouts of FAD2 genes reduced both linolenic and linoleic acid by over 70 percent in Camelina. And in cassava, knocking out genes CYPJ9D1 and CYPJ9D2, both enzymes that catalyze the synthesis of cyanogens, in bitter varieties have been used to reduce cyanide by 92 percent. And, for other crops, it is known that LoF alleles in GBSSI, an amylose synthase gene, are responsible for crop products with low amylose content which is a desirable culinary trait for some.

Many of the already targeted gene knockouts to improve crops have involved genes known to improve traits with well studied functions previously found in model systems. While serving as valuable case studies and proofs of concept demonstrating the efficacy of knockouts as a tool for breeding, the next generation of breeding by knockouts will require a much larger scale discovery of new target genes. And while the understanding of what genes and knockouts can achieve, the question remains what genes should be targeted.

The discovery of natural knockout alleles.

The ability to generate whole genome sequence data for large populations represents a quantum leap in the ability to uncover genes where LoF has contributed to trait evolution and identify candidate genes for targeted knockouts. Previous genotyping approaches involved identifying genomic regions associated with traits of interest in crop populations, such as genotyping by sequencing, reduce representation sequencing, and SNP arrays to provide a thorough summary of genetic diversity at genomic scales. In contrast to earlier forms of genetic data, whole genome sequencing data provides details of functional molecular diversity in the context of genomic elements such as protein coding genes.

The potential effect of every variant in the genome can be predicted with an array of tools. From whole genome sequence data, predicted alleles have been identified in crop species. One such discovery emerging from genome resequencing projects is the scale of LoF variation in segregating crops. For example, in rice, 198,609 premature stop codons, including single nucleotide variants, were found when 3010 genotypes were sequenced. And with deeper investigations, these and other LoF variants such as frameshift mutations can be used in function-based GWA where associations of functional allele states are compared to identifiable candidate loci.

The technologies for gene knockout or loss-of-function use typical sequence-specific nucleases that can be induced to recognize specific DNA sequences and to generate double-standard breaks. The plant's endogenous repair systems can fix double-stranded breaks by either non-homologous end joining, which can lead to the insertion or deletion of nucleotides thereby causing gene knockouts, or by homologous recombination, which can cause gene replacements and insertions. Many such mutations have been produced through the use of genome editing technologies in a variety of plants, which have been shown to be useful for crop improvement.

Zinc-finger nuclease (ZFN) are fusions of zinc-finger-based DNA recognition modules and the DNA-cleavage domain of the FokI restriction enzyme. Each individual zinc finger typically recognizes and binds to a nucleotide triple, and fingers are often assembled into groups to bind specific DNA sequences. This technique has been used to modify arabidopsis, nicotiana, maize, petunia, soybean, rapeseed, rice, apple, and fig. For example, ZFNs in crop breeding to disrupt the endogenous maize gene ZmIPK1 with the insertion of PAT gene cassettes. This disruption resulted in herbicide tolerance and alteration of the inositol phosphate profile of developing maize seeds.

As a proven technology, ZFN-mediated targeted transgene integration has been used for trait stacking in maize, for assembling a number of useful traits together to create an even potential for crop improvement. ZFNs have also been used to identify safe regions for gene integration in rice, and these identified sites should serve as reliable loci for further gene insertion and trait stacking. However, the design of ZFNs remains a complicated and challenging process, often with low efficacy.

Like ZFNs, transcription activator-like effector nucleases (TALENs) are fusions of transcriptional activator-like effector (TALE) repeats and the FokI restriction enzyme. However, each individual TALE repeat targets a single nucleotide, allowing for more flexible target design and increasing the number of potential target sites relative to those that can be targeted by ZFNs.

Genome editing by TALENs has been demonstrated in a wide variety of plants, including Arabidopsis, Nicotania, Brachypodium, barley, potato, tomato, sugarcane, flax, rapeseed, soybean, rice, maize, and wheat. The first application of TALEN-mediated genome editing in crop improvement was in rice, where the bacterial blight susceptibility gene OsSWEET14 was disrupted and the resulting mutant rice were found to be resistant to bacterial blight. Analogously, TALENs have been used in wheat to knockout three TaMLO homeologs in order to create powdery mildew-resistant wheat.

TALENs can be used to modify the nutritional profiles of crops: soybeans with high oleic acid and low linoleic acid contents generated by disrupting fatty acid desaturase genes, and improving the shelf life and heat stability of soybean oil. In potato tubers, the accumulation of reducing sugars during cold storage influences the quality of the product, and knocking out vacuolar invertase gene resulted in tubers that had undetectable levels of problematic reducing sugars. Flavor has also been produced through the use of TALEN technology in rice, to disrupt the betaine aldehyde dehydrogenase gene. The technology has a lot of potential for crop trait improvement; however, the construction of TALE repeats remains a challenge and the efficiency of gene targeting has been variable.

CRISPR/Cas9 systems, especially the type II CRISPR/SpCas9 system from Streptococcus pyogenes, have been developed as versatile genome-editing tools for a variety of applications. Compared to ZFNs and TALENs, the CRISPR/Cas system is simple, efficient, and low cost, and is able to target multiple genes. Because of this, it has been exploited in plants and has been shown to be an effective solution to problems in plant breeding. Crops such as rize, maize, wheat, soybean, barley, sorghum, potato, tomato, flax, rapeseed, Camelina, cotton, cucumber, lettuce, grapes, grapefruit, apple, oranges, and watermelon have been edited by this technique.

The mechanism of CRISPR/Cas9 mediated genome engineering in plants.

The most frequent application has been in the production of null alleles, or gene knockouts, achieved by the introduction of small indels that result in frame-shift mutations or by introducing premature stop codons. Yield is often a concern in crop breeding. In rice, the LAZY1 gene was knocked out by CRISPER/Cas9, and a tiller-spreading phenotype was generated which increased crop yield under certain circumstances. This was also used to mutate Gn1a, DEP1, and GS3 genes of the rice cultivar Zhongua11, producing mutants with enhanced grain number, dense erect panicles, and larger grain size, respectively.

The nutritional profiles of crops can also be improved by CRISPR/Cas9. As with TALEN-mediated knockout in soybean to improve the shelf life and heat stability, CRISPR/Cas9 has been used to target FAD2 to improve oleic acid content while decreasing polyunsaturated fatty acids in the emerging oil seed plant Camelina sativa. In rice, CRISPR/Cas9 has been used to generate targeted mutations in SBEIIb to lead to a higher production of long chains in amylopectin to improve the fine structure and nutritional properties of starch. Using CRISPR/Cas9, Corteva AgriScience knocked out the maize waxy gene Wx1, which encodes the granule-bound starch synthase (GBSS) gene responsible for making amylose. Without GBSS expression in the endosperm, amylose was not synthesized, and this created a high amylopectin maize with improved digestibility and the potential for bio-industrial applications.

The technology has also been used to improve resistance to biotic stresses. This has been done by using CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homeologs of EDR1. The resulting plants were resistant to powdery mildew and did not show mildew-induced cell death. And in rice, the mutagenesis of OsERF922 and OsSWEET13 obtained rice blast resistance and bacterial blight resistance. Further, powdery mildew-resistant tomatoes were generated by editing S1MLO1 and bacterial speck-resistant tomatoes created by disrupting S1JAZ2. Citrus canker, a sever disease which kills crops worldwide, and with a susceptibility gene - CsLOB1 - has been modified and the modification has alleviated canker symptoms in Duncan grapefruits and Wanjincheng oranges.

There is an open door to the adoption of crops modified by CRISPR/Cas9, including a 2016 FDA approval to a CRISPR-edited mushroom, and a 2017 approval for false flax and drought-tolerant soybean, both CRISPR-edited crops. With these approvals, research has been boosted with a focus on the application of CRISPR to fruit crops. However, the technology faces some sociopolitical challenges, including public acceptance and government regulation. While transgene-free organisms edited by CRISPR/Cas9 are not regulated in the United States, questions of whether to govern the use of the technology are being discussed in China and Japan. Although, with further advances in the technology and the establishment of an evaluation system, there is a more optimistic and inclusive attitude towards CRISPR-edited crops.

With genome-editing technologies showing great potential in agriculture, it is still limited by the low efficiency and off-target effects, restrictive protospacer adjacent motif sequences, among other issues. New innovations to these and other problems are continually being added to the genome-editing toolkits to address those limitations. These include:

• Base editing: genome-wide association studies have shown that single-based changes are usually responsible for variations in traits, and efficient techniques for producing precise point mutations in crops are required. CRISPR/Cas9-mediated base-editing technology offers a genome-editing approach to convert one DNA base into another, without the use of a DNA repair template.
• DNA-free genome editing systems: conventional genome editing involves the delivery and integration into the hose genome of DNA cassettes encoding editing components. Integration occurs at random, and can cause undesirable genetic changes. As well, prolonged expression of genome-editing tools increases off-target effects in plants since nucleases are abundant in these organisms. Therefore, DNA-free genome editing would be capable of producing genetically edited crops with a reduced risk of undesirable off-target mutation, and thereby meet current and future agricultural demands from both scientific and regulatory perspectives.
• CRISPR/Cpf1 system: the type II CRISPR/SpCas9 system is simple and efficient, but is limited to recognizing sequences upstream of the appropriate 5'-NGG-3' PAMs, and restricts potential target sites. The type V CRISPR/Cpf1 system has demonstrated potential in this area through the recognition of T-rich PAMs, and the capability for generating cohesive ends with four or five nucleotide overhands rather than blunt-end breaks. This has been used for targeted mutagenesis in tobacco and rice.

Genetically modified organisms (GMO), especially crops, are often produced with beneficial traits through the transfer of genes (transgenes) or gene elements of known function into crop varieties. Despite the promise GMO crops hold for global food security, their use is affected by health and environmental safety concerns. Government regulatory frameworks with the aim of safeguarding human and environmental safety have led to barriers to the widespread adoption of new GMO traits. As a result, the advantages of GMOs and genetically modified traits have been restricted to a small number of cultivated crops.

For thousands of years, humans have used modification methods, such as selective breeding and cross-breeding, to modulate and select plants and animals with more desirable traits. For example, early farmers developed cross-breeding methods to grow corn with a range of colors, sizes, and uses. While modern strawberries are a cross between a species native to North American and a species native to South America. Most foods consumed today were created through these traditional breeding methods, but these methods take time and it can be difficult to make specific changes. After scientists developed genetic engineering in the 1970s, they were able to make similar changes in a more specific way and in a shorter amount of time. A brief timeline of genetic modification in agriculture includes:

• 1866 - Gregor Mendel breeds two different types of peas and identifies the basic process of genetics
• 1922 - the first hybrid corn is produced and sold commercially
• 1940 - plant breeders learn to use radiation or chemicals to randomly change an organism's DNA
• 1973 - biochemists Herbert Boyer and Stanley Cohen develop genetic engineering by inserting DNA from one bacteria into another
• 1982 - FDA approves the first consumer GMO product developed through genetic engineering: human insulin to treat diabetes
• 1986 - the federal government establishes the Coordinated Framework for the Regulation of Biotechnology. This policy describes how the U.S. Food and Drug Administration (FDA), U.S. Environmental Protection Agency (EPA), and U.S. Department of Agriculture (USDA) work together to regulate the safety of GMOs
• 1992 - FDA policy states that foods from GMO plants must meet the same requirements, including the same safety standards, as foods derived from traditionally bred plants
• 1994 - the first GMO crop created through genetic engineering - a GMO tomato - becomes available for sale after studies evaluated by federal agencies proved it to be as safe as traditionally bred tomatoes
• 1990s - the first wave of GMO produce created through genetic engineering becomes available to consumers, including: summer squash, soybeans, cotton, corn, papayas, tomatoes, potatoes, and canola
• 2003 - the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United Nations develop international guidelines and standards to determine the safety of GMO foods
• 2005 - GMO alfalfa and sugar beets are available for sale in the United States
• 2015 - FDA approves application for first genetic modification in an animal for use as food, a genetically engineered salmon
• 2016 - Congress passes a law requiring labeling for some foods produced through genetic engineering and uses the term "bioengineered" which starts to appear on some foods
• 2017 - GMO apples are available for sale in the United States
• 2019 - FDA completes consultation on first food from a genome edited plant

Genetically engineered food has been subject to controversy since its consumer introduction in the 1990s. This included a 1999 publication that showed Bt toxin had negative effects on butterfly populations in laboratory tests, which led to objections to its use. But follow-up studies in agricultural settings confirmed the safety of the technology. As well, during the late 1990s and early 2000s, poor yields of genetically engineered cotton in India were associated with a presumed increase in farmer suicides. Later, it was found that suicide rates among farmers were unchanged and there were economic benefits for most of the farmers from the genetically engineered cotton.

This awareness led to a wider call for regulations of genetically modified foods, leading to labelling requirements for genetically modified food in around sixty-four countries. Many groups have argued that labelling genetically modified food is important for consumer choice and for monitoring unforeseen problems associated with the technology. Whereas the groups opposing labels claim a law would unnecessarily eliminate consumer demand for genetically engineered crops, causing increases in food prices and resource utilization.

Non-GMO refers to foods considered to have been unaltered or genetically modified, with the genetic makeup of the organism is not considered to have been altered. Although, with early cross-breeding and selective breeding having been argued to have been genetic manipulative processes, the definition is more often understood to refer to any crops being altered genetically in a laboratory using any artificial technology, with cross-breeding or selective breeding being considered natural technologies.

The push for Non-GMO food comes from concerns around what part of the crop is altered and what the effects could be on human consumption or the wider environment. For example, concern around what a modified organism capable of withstanding the application of herbicide would mean for the use of herbicide overall, suggesting there would be an increased use, which would be problematic to the wide environment. Or the manipulation of a crop to produce toxins with an insecticide effect and what the wider effect on insect populations and, in turn, human health would be of such a crop.

The Non-GMO Project, which advocates for non-GMO foods, lists other projects to develop traits—such as resistance to browning in apples—and for creating new organisms through synthetic biology. Despite the claims of biotechnology companies and researcher, these manipulated crops do not offer increased yield, drought tolerance, enhanced nutrition, or any other consumer benefit. The advocacy group continues to suggest that with the lack of long-term studies on the effects of GMO foods, and their upstream effect on the entire food chain, the impact on human safety is poorly understood.

In many cases, the National Academies of Sciences, among others, have responded to these concerns, and holds a stance that genetically modified organism (GMO) food is safe, along with the majority of the world's scientific community. Further, from a health perspective, GMO food has been found, through studies, to be no different nutritionally than non-GMO food, and some GMO foods have been found to be healthier—such as peanuts genetically engineered to reduce the levels of aflatoxin and gluten-free wheat. Further, with the maturation of existing technologies, and the promised precision of CRISPR/Cas systems, the problems of unwanted effects in genetic modifications are reduced.