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Artigo 01 - Crop Biotechnology and The Future of Food
Artigo 01 - Crop Biotechnology and The Future of Food
https://doi.org/10.1038/s43016-020-0072-3
The global population continues to rise, as does the likelihood of reduced yields of major food crops due to the changing
climate, thus making the development of genetically improved, stress-resilient crops a research priority. The convergence
of low-cost genome sequencing with improved computational power and high-throughput molecular phenotyping technolo-
gies has accelerated the identification of genes underlying important agronomic traits relevant to food production and quality.
Here, we discuss the evolution of plant improvement, and how researchers leverage genomic analyses and revolutionary
new plant breeding technologies like site-directed nucleases to enhance food crop traits through agricultural biotechnology.
Deployment of these products from the laboratory to the field remains hindered by biological and regulatory bottlenecks that
require further development.
T
he production of food via agriculture began approximately for characteristics including upright vegetative structure, uniform
10,000 years ago, and its history is punctuated by strides in flowering, seed retention on the plant for easier harvest, and reduc-
progress both technological and biological. During the rela- tions in seed dormancy and toxic chemicals in edible tissues13. This
tively recent Green Revolution of the 1960s, international research suite of traits, termed the ‘domestication syndrome’, typically ren-
investment in agricultural improvement of the cereal grains wheat, ders domesticated crops poorly adapted to growth outside of human
rice and maize resulted in new high-yielding varieties that saw cultivation. Through iterative selective breeding, specific versions of
widespread cultivation and gave greater food security in many parts genes (alleles) underlying these traits and their surrounding DNA
of the world1. The subsequent rise in molecular genetic tools has regions became uniformly present across the domesticated breed-
ushered in the era of genomic breeding, wherein molecular breed- ing population14. (Box 1 provides a glossary of key terms.) Examples
ing and genetic engineering have gained prominence2. include the teosinte branched 1 allele that is a major contributor to
By 2050, the global population is predicted to reach 9.7 billion. modern-day maize plant architecture15 and the Bitter fruit allele
Meeting this higher food demand, if consumption practices and responsible for reducing cucumber fruit bitterness16. Continued
food waste do not change, requires estimated food production selection of these domesticated species further optimized agro-
increases of 25 to 100 per cent3,4. At the same time, crop yield is stag- nomic traits, and geographic dispersal established locally adapted
nating in many parts of the world5, and climate change threatens the landrace cultivars17. For some species, further breeding led to glob-
worldwide agricultural system6,7 with yields and nutritional content ally adapted elite cultivars produced by seed companies and research
predicted to decline for major crops8–10. Additionally, crop patho- institutions. While substantial research has been invested in pro-
gen and insect pest ranges are shifting into new territory towards ducing new varieties of select crops (for example, maize, wheat and
the global poles11. These challenges to sustained food security will rice), so-called orphan crops—often staple foods like cassava, millet
require multiple solutions encompassing social, technological and and sweet potato for people in developing areas of the world—have
economic change. One part of the solution is intrinsic improvement received less international genomic breeding focus18.
of cultivated crops2. Genetic diversity is crucial for continued crop improvement.
Increasing genomic and phenotypic information is becoming Many crops possess reduced genetic diversity relative to their
available as technology improves and costs decrease. New plant wild ancestors, though generally this effect is more prevalent in
breeding techniques reduce the time necessary to enhance agro- annual plants (for example, soybean and wheat) than in long-lived
nomic traits relative to conventional breeding. These techniques perennial species that extensively outcross and are often clonally
have potential to deliver improvements such as greater abiotic and propagated19. Domesticated species may harbour elevated numbers
biotic stress tolerance to minimize yield losses, and the improve- of deleterious mutations (see ref. 20 and references within). Novel
ment of food nutrition and quality. Underutilized and regionally genetic diversity can be introduced via induced mutagenesis using
important crops, often adapted to grow on marginal lands, can be irradiation (for example, X-rays and gamma rays) or chemical
further improved and grown more widely to diversify the global treatment, causing DNA breakages, deletions and/or base changes
diet. We discuss how the application of new biomolecular and that result in new alleles; a subset will have effects on agronomic
mechanistic tools provides greater understanding of the genetics and quality traits21. This process creates random genome-wide
and physiology underlying crop plant performance, and we address mutations, necessitating a multigenerational screening and selec-
how those new tools are being applied to innovations in food tion pipeline to identify high-performing mutant individuals. The
production and quality. global FAO/IAEA Mutant Variety Database (http://mvd.iaea.org)
documents over 3,300 mutant varieties representing over 230 plant
Evolution of plant breeding in the computational era species that have been officially released since 1950. Examples
Humans have manipulated plant genomes for millennia, long before include mutant alleles for the semi-dwarfing trait key in Green
the modern understanding of DNA underlying heritable genetics12. Revolution varieties of wheat, maize and rice22, and seedless
Early domestication of wild annual species occurred via selection mandarin citrus (PAU Kinnow-1)23. Landrace varieties and related
Department of Plant Pathology, University of California, Davis, CA, USA. 2The UC Davis Genome Center, University of California, Davis, CA, USA.
1
Center on Food Security and the Environment, Stanford University, Stanford, CA, USA. ✉e-mail: [email protected]
3
Agrobacterium tumefaciens. A naturally occurring plant patho- Nanomaterials. A broad class of chemically and structurally diverse
genic bacterium that delivers DNA into host cells as part of its synthetic materials with at least one dimension between 1 and 100
infection process. Modified lab strains deliver DNA of interest nanometres, such as gold particles or carbon nanofilaments, which
into susceptible plant cells, where it is randomly integrated into can be used as delivery systems for biological molecules into plant
the host genome. This property was exploited to develop mutant and animal cells. Nanomaterials can be designed for cell and
populations in model systems for functional gene analysis, which organelle specificity, making them applicable to the agricultural
has provided the basis of much fundamental plant research over and biomedical fields.
the past decades.
Omics. The generic term for study of large-scale data of a
Allele. Alternative forms of a gene at a chromosomal location that biological class, such as the total complement of genes or chemical
arise through mutation. Different alleles may vary by small DNA metabolites present in an organism. Detection technologies
changes, encode multiple forms of a protein or possess structural vary by type of molecular component being assessed, may
changes that change when and how a gene is active. The diversity quantitate total or relative abundance, or provide other specific
of alleles for a trait in a population is often greater than those information.
present in any single individual; breeding is the directed accumu
lation of desired alleles from a population into an elite variety. Orphan crops. Also termed underutilized crops, underinvested
crops, or crops for the future. Orphan crops encompass all food
Clonal propagation. Asexual propagation that retains specific types: cereals, root and tubers, legumes, and vegetables. Although
combinations of alleles present in the parent plant. This process they provide nutrition to large numbers of people, these crops
can be used for rapid generation of additional crop plants like have historically received little attention by the international
those that reproduce infrequently or are sterile (for example, research community, and thus genomic data and molecular tools
bananas), or in grafting to fuse plants with desired traits to a remain limited.
genetically dissimilar rootstock.
Particle bombardment. Also termed biolistics or gene gun,
Epigenetics. Heritable chemical markers on DNA that influences this delivery system uses helium under high pressure to propel
how genes are read, causing activation or repression of gene microparticles bonded with DNA, RNA and/or protein into cells.
function. These markers are enzymatically added or removed, and These particles break through cell walls and penetrate to the
typically vary based on cell identity and response to external cues. interior, where the biomolecules dissociate and perform biological
functions.
Genomic selection. An accelerated breeding approach that
utilizes models trained on genomic and phenotypic data to Selective breeding. The reiterative process of assessing genetically
computationally predict breeding values for candidate lines based heterogeneous organisms for desired traits and combining
on their genotype, to improve varieties. these advantageous characteristics to improve cultivars. Genetic
diversity can originate from multiple sources, including DNA
Haploid/diploid. The number of complete genome copies an recombination, sexual outcrossing or induced mutations.
organism possesses is termed ploidy. Gamete cells like pollen
and ovules typically are haploid, containing just one copy of each Site-directed nuclease. A protein enzyme that cleaves DNA at
chromosome, one genome copy. Adult animals and many plant locations specified by chemical interactions between cellular
species are typically a fusion of two gametes and possess one pair of DNA and protein molecules (for example, zinc-finger nuclease
each chromosome, termed diploid. Additional copies of genomes or TALEN) or RNA (for example, CRISPR-Cas) sequences.
can arise through whole genome duplication or hybridization. For Canonical nucleases break both strands of double-stranded DNA,
example, bread wheat is hexaploid (six genomic copies), while while modified enzyme variants can cleave only one strand, or
strawberries are octoploid. perform other biochemical functions.
Hybrid vigour. The improved growth and yield displayed by specific Transgenic. Plants that have integrated genetic material artificially
hybrid offspring relative to either parent line. This performance is introduced from a foreign source are classified as transgenic. This
due to a unique combination of alleles created by crossbreeding transgene DNA is commonly from related plant species, bacteria
specific genetically distinct inbred parents and becomes variable in or synthetic origin, and includes regulatory sequences that can
subsequent generations due to further segregation of these alleles. specify cell- or tissue-specific gene expression.
wild populations also serve as sources to introduce diversity, such Precise genetic-marker-assisted breeding was used to introduce
as desired stress-adaptive traits, using genetic markers strongly the Sub1A-1 gene into several farmer-preferred rice cultivars
linked to or underlying the desired phenotype to guide breeding throughout South and Southeast Asia29, while selecting against
selection24,25. For example, the landrace rice cultivar Flood Resistant introduction of undesired genes of the donor parent, accruing
(FR) 13A possesses the ability to survive in complete water sub- benefits to generationally disadvantaged farmers30.
mergence for multiple weeks, but lacks the desired high yield and
high-quality grain of other commercial cultivars26. Severe flooding Using omics technology to augment traditional breeding
and prolonged, complete submergence of rice plants results in crop programmes. Recent technological advances have increased the
and yield losses for most commercial varieties. Crossbreeding gen- ubiquity of molecular ‘-omics’ studies in plant science, a term
erated an FR13A-derived rice population, and subsequent genetic denoting the total complement of a biological unit like genes
mapping identified the gene underlying its submergence tolerance (genome) or proteins (proteome). With modern high-throughput
as Sub1A-1, a gene absent from intolerant varieties26–28 (Fig. 1b). genome sequencing technology more accessible at lowering costs
d e
WT mult 8
Fig. 1 | Examples of genetically altered agricultural species. a, Inflorescence with fruits from maize progenitor teosinte (left) and primitive domesticated
maize (right). b, Submergence tolerance is conferred by the presence of the Sub1a-1 gene (right) compared with control rice (left) after two weeks of
submergence, followed by two weeks of recovery30. c,d, Eggplant variety Uttara showing injury from eggplant fruit and shoot borer (c) and lack of injury in
Bt eggplant variety131 (d). e, Wild tomato S. pimpinellifolium flower WT morphology (left) and after Cas9 targeted mutagenesis of the MULTIFLORA locus
(right) during de novo domestication113. Scale bars, 2 cm. Figure reproduced with permission from John Doebley (a); ref. 30, Laboratory of Pamela Ronald
(b); ref. 131, Cold Spring Harbor Laboratory Press (c,d); ref. 113, Springer Nature America, Inc (e).
(US$1,000–10,000s), the depth of crop variety and breadth of Computational correlative association studies synthesize the
species genomic data is quickly expanding. For example, collabora- information contained in these agronomic, proteomic, transcrip-
tive sequencing efforts have produced whole genome sequences for tomic and/or metabolomic datasets to find genes with large and
3,000 rice varieties from 89 countries31, and the African Orphan small effects in crop phenotype to guide manipulation of complex
Crops Consortium aims to sequence 101 genomes of impor- traits42 (Fig. 2). Recent research for tomato flavour improvement
tant native crops, including finger millet (Eleusine coracana) and exemplifies this application. Flavour is a complex combination of
Bambara groundnut (Vigna subterranea)32. To capture the diver- the taste of numerous sugars, acids and bitter chemical metabolites
sity of specific gene families within a large group, genomic DNA and the smell of volatile aromatic compounds, governed by numer-
samples can be preferentially enriched prior to sequencing. This ous genetic determinants that make it a difficult breeding goal43.
method has been used to define genetic variation in disease resis- Indeed, historic selection for a gene underlying pale green, evenly
tance gene repertoires in Solanaceae and Triticeae (RenSeq33,34), and ripening tomato fruits also had a deleterious effect on total sugar
gluten gene families in bread wheat (GlutEnSeq35). content44; breeding for increased fruit size or pink colouration also
Robotics and other technologies are speeding acquisition of altered its chemical profile45. Multiple studies paired quantification
data on macrophysiology of crop plants36, while an assortment of of a select number of tomato metabolites (~20–80) with genetic
high-throughput detection technologies can be employed to pro- markers in wild and cultivated tomato fruits to identify genes
duce physiological data on RNA, protein, metabolites and other underlying specific potential flavour determinants46–48. Consumer
molecular metrics shaped by the plant’s genomics and its environ- flavour preference surveys were paired with analysis of 398 mod-
ment. Network-based analyses using a broad array of transcrip- ern, heirloom and wild accessions to identify which compounds
tomic data can identify major regulatory hubs underlying adaptive and genetic markers were correlated with positive taste preference49.
responses to environmental stress (for example, refs. 37–39). Plant Work by Zhu et al. expanded the metabolite panel to quantify 980
proteomic analysis may detect context-dependent protein abun- distinct fruit chemical metabolites in 610 wild and commercial
dance and the presence of transient chemical modifications that red-fruited tomato accessions and paired that information with
alter their biological function40. Recently, a proteomic survey of 13 genetic sequence and gene expression network analysis, describing
species across the green plant lineage has been reported, identifying how domestication and breeding have altered tomato metabolites45.
known and novel conserved multi-protein complexes and protein Most recently, the first meta-analysis of previous association studies
interactions; genes encoding proteins in these complexes likely play in tomato46,47,49 compiled data from 775 tomato accessions and iden-
a role in important agronomic traits41. tified 305 genes tied to sugars, acids and flavour-related volatiles50.
Transcriptomics
Metabolomics
O
N N
O N N
Fig. 2 | Omics data can be used to inform marker-assisted breeding, genomic selection and site-directed nuclease targets. Plant population diversity
can be characterized for agronomic traits through field phenotyping and using omics technologies that capture genome sequence (genomics) and
RNA (transcriptomics), protein (proteomics) and metabolite abundance (metabolomics) under a number of conditions. Computational algorithms and
correlation analysis of these different data sets can uncover linkages between genes and macro and molecular phenotypes and identify novel genes with
potential agronomic value. These data can be used to generate markers for use in marker-assisted selection breeding, to produce predictive models for
use in genomic selection or guide introgression of genes from wild sources without the use of biotechnology (blue text). Use of omics data and phenotypic
analysis can also identify gene candidates for manipulation via site-directed nucleases (including targeted mutagenesis and targeted gene insertion)
and inform conventional introduction of genes or multi-gene clusters for creation of stable transgenic lines using biotechnology tools (green text).
Credit: Scott Camazine / Alamy Stock Photo (protein structure)
The genetic locations identified in these studies represent potential non-homologous end joining, in which broken DNA strands are
targets for manipulation, either through conventional breeding or reattached, often creating small deletions or insertions of DNA
biotechnological approaches. bases on either end of the break, and homology directed repair,
Additionally, data from genome sequencing, gene association in which the gap is bridged using a template that shares sequence
studies and phenotypic data can be used to generate and train with the DNA region surrounding the break54. These repair pro-
machine-learning predictive models for use in genomic selec- cesses can be exploited to enable the introduction of several genetic
tion plant breeding (reviewed in ref. 51). This accelerated process changes of different classifications, including: mutation of gene
uses a trained computational model to select lines based on their function through random error-prone DNA repair via end joining
genetic markers without repeated phenotyping during breeding (classified as site-directed nuclease application 1 (SDN1), see Fig. 3);
cycles. Such models can be repeatedly improved with data from allele replacement through repair mechanisms using supplied
high-throughput phenotyping technologies in both research and short DNA fragments as a template (SDN2); or targeted insertion
field settings, utilizing stress-phenotyping via imaging sensors to of DNA at the cut site using supplied long template DNA (SDN3).
detect disease, drought and nutrient deficiency, among others52. In non-reproductive plant cells, non-homologous end joining
repair occurs at high efficiency, while homology directed repair
Gene-editing tools enable customization of germplasm occurs with low efficiency55. Introduced DNA breakages can also
The discovery and application of targetable site-directed nuclease stimulate targeted recombination between homologous chromo-
(SDN) enzymes ushered in a new era of plant mutagenesis breeding, somes. Cas9-induced cuts were used to trigger recombination
giving researchers powerful tools for precision manipulation of crop in tomato somatic cells to alter fruit colour56, suggesting the poten-
genomes (broadly termed ‘gene editing’) to leverage the wealth of tial application of the technology to break inheritance linkages
genomic data. Among these, RNA-guided CRISPR-Cas technology between physically close genes or combine favourable alleles onto
has become a dominant tool since 2013, when gene editing capac- one chromosome.
ity was demonstrated in plant cells. The gene editing toolkit has Improvement of cultivar performance and product quality can
bloomed in the short period that has followed, with Cas enzyme be achieved via deactivation of genes with undesirable effects, or
variants discovered or developed that enable A/T to G/C DNA modification of regulatory regions governing when and how genes
base-pair swapping, direct gene repression or activation, directed are expressed57. Targeted mutagenesis without inserting foreign
DNA methylation and RNA targeting. For specific details of current DNA at the target site (SDN1) accounts for roughly 90 per cent of
CRISPR technology, Cas variants and gene-editing methods, read- SDN usage thus far in published agricultural research58 due to its
ers are referred to a recent review53. relative ease and can be viewed as an evolution of mutation breed-
ing previously discussed. Cas cutting specificity is determined by
Application of site-directed nucleases to molecular breeding an associated guide RNA molecule that can bind to the target DNA
of food crops. Site-directed nucleases, also called sequence- sequence. Supplying the enzyme with multiple guide RNAs allows
specific nucleases, create a targeted breakage in DNA. Two distinct for simultaneous changes at multiple locations across chromosomes
mechanisms within the plant cell exist to repair this damage: within a single cell59 or allows for large deletions of DNA60 when
compound lycopene, responsible for the fruit’s red colour, and the closer to future use as a winter cover crop and source of oilseed
health-promoting compound γ-aminobutyric acid were highly for food114.
enriched90–92 by manipulating biosynthesis genes to favour accumu- As costs further decline and genomic data and resources accrue,
lation of these compounds. In lettuce, removal of regulatory DNA gene editing can facilitate rapid improvement and greater adoption
regions enhanced production of vitamin C in the leaf threefold93. of orphan crops and landrace cultivars115. Lemmon and co-workers
In sorghum, deletion of multiple α-kafirin gene regions encoding generated whole genome sequence, gene expression data, and gene
seed storage proteins enhanced protein digestibility and increased editing protocols for the groundcherry Physalis grisea with the goal
overall essential amino acid lysine and total protein content of the of transforming this fruit into a mass-produced berry crop among
grain94. Soybean seed oil genes were altered to increase fourfold the the rank of strawberry and raspberry. Weedy, sprawling ground-
percentage of the polyunsaturated fat oleic acid95, and oleic acid lev- cherry plant growth was compacted, and fruit size and number
els could also be modulated by different mutation combinations of increased116,117. Genome editing protocols have also proved suc-
redundant genes in Camelina sativa96. cessful in cassava, an orphan crop grown for uses in both food and
Gene editing has also been applied to traits that prolong the post- manufacturing. The cassava tuber starch profile was altered through
harvest shelf life in certain food crops, potentially reducing food modification of amylose synthesis genes, conferring desirable cook-
waste along the supply chain97. Transgenic silencing of polyphenol ing and food processing properties118. Careful elimination of genes
oxidase genes reduces potato tuber browning after processing98, responsible for toxic compound formation could also enhance
and this trait was subsequently commercialized after application to nutritional quality and processing in cassava119 and grass pea
transgenic non-browning Arctic apples99. Recently, targeted muta- (Lathyrus sativus)120. As genomic information and biotech methods
genesis was used to create varieties of non-browning, transgene-free on wild and orphan species continues to develop, editing of known
white button mushrooms100, potatoes101 and romaine lettuce102,103. domestication genes111,121 will further expand crop diversity and
Development of ripe tomatoes that remain firm longer has been a food options.
long-sought goal briefly realized in the Flavr Savr tomato, in which
transgenic gene silencing reduced activity of cell-wall modify- Insertion of transgenes and synthetic gene clusters
ing enzyme that contributes to fruit softening104. Recent manipu- The process of plant transformation, in which genetic material is
lation of a different gene has yielded firmer ripe fruits105 with introduced and integrated into the heritable plant genome, has been
greater resistance to fungal rot106 that retain flavour-contributing a fundamental tool in the development of cultivated genetically
metabolite profiles107. engineered food, fibre and biofuel crops. Trait design can introduce
single or multiple genes to affect a phenotype, or introduce DNA
Creating multi-generational hybrid vigour. Agriculture of the sequences that cause silencing of endogenous genes. Inclusion of
past century has widely exploited the genetic phenomenon of regulatory DNA elements can produce selective effects that are diffi-
hybrid vigour, or heterosis, to produce high-performing, genetically cult to achieve via targeted mutagenesis. For example, seed-specific
uniform F1 hybrid crops that possess variable yield in subsequent gene silencing of widely expressed gossypol synthesis genes in cot-
generations due to genes segregating during sexual reproduction. ton was used to engineer seed with minimal levels of the toxic com-
Mutagenesis of three genes creates the genotype Mitosis instead of pound, making them fit for human consumption while preserving
Meiosis (MiMe) in diploid rice, which results in clonal diploid gam- gossypol content in vegetative parts as a necessary chemical deter-
etes instead of haploid gametes due to the doubling of the genome rent against pathogens and insect pests122. Transgenic products have
without two rounds of segregation and cell division108. When historically received higher scrutiny from government regulatory
MiMe is paired with the pollen mutation matrilineal that prevents bodies, vastly increasing costs and time to develop and commercial-
transfer of pollen-derived DNA to the embryo, self-fertilization in ize transgenics compared with conventional varieties123,124. In 2018,
F1 hybrids produced clonal seeds that retain the F1 diploid geno- 26 countries cultivated 191.7 million hectares of genetically engi-
type109. Another study in rice achieved a similar result by combining neered crops, with the United States, Brazil, Argentina, Canada and
the MiMe genotype with transgenic cell-specific expression of the India collectively representing 91 per cent of the global transgenic
embryogenic gene Baby Boom 1, which stimulates embryo develop- crop area125.
ment without fertilization110. However, this application remains in One of the most prevalent engineered traits is insect resistance
the early phases and further optimization is necessary before com- conferred by genes originating from the soil bacterium Bacillus
mercial adoption, as clonal seed production was reported to occur thuringiensis (Bt). The Bt trait has been applied to many crops,
at low rates ranging from 5 to 29 per cent. including maize, soybean, cotton and eggplant126, and the cumula-
tive use of Bt maize, soybean and cotton crops has resulted in 37 per
Increase the diversity of cultivated species through rapid domes- cent less global pesticide use127 and potential insect pest-suppressive
tication. Another promising approach to expand the food palate benefits to nearby non-Bt crops128. In India, Bt cotton pesticide
is through de novo domestication, in which domestication application was reduced by 50 per cent and, accordingly, reduced
alleles are introduced to wild species possessing desired stress acute pesticide poisonings were seen in cotton growers129. The Bt
tolerance or adaptation to marginal lands, compressing thousands trait in maize has also led to increased consumer safety through
of years of human mutagenesis and selective breeding to a few reduced grain contamination with mycotoxins produced by fungal
plant generations111. This strategy has been demonstrated in the infections that can follow insect damage130. In Bangladesh, where
wild tomato relative Solanum pimpinellifolium, as simultane- introduction of four varieties of Bt eggplant (Fig. 1c,d) in 2014
ous editing via CRISPR of four112 and six113 known domestication marked the first genetically engineered food crop released in a
genes was sufficient to remodel shoot and flower morphology developing country and first Bt vegetable, net returns for farmers
(Fig. 1e) and increase the size and number of fruits to be similar increased sixfold in part due to a 61% reduction in pesticide costs131.
to cultivated tomato Solanum lycopersicum within a single genera- The breadth of phenotypic change possible with transgenesis
tion. De novo domesticated fruits contained 500 per cent higher is far greater than can be achieved through gene editing alone in
levels of lycopene than a commercial tomato variety113 and retained part due to the wide genetic diversity that can be utilized, includ-
disease resistance and salinity tolerance traits of the wild parent112. ing across species with innate reproductive barriers (for example,
Similarly, recent stacking of mutations modifying flowering time, ref. 132). Assembling a repertoire of disease-resistance genes from
seed retention, removal of undesired chemicals and enrichment of numerous accessions or species, as well as rationally designed novel
oleic acids in seeds brings the weed pennycress (Thlaspi arvense L.) resistance genes133, could provide durable, broad-based resistance
Use of site-directed nucleases for targeted gene insertion Bypassing tissue culture methods for producing modified
or replacement. Commonly used methods of Agrobacterium plants. The use of tissue culture to generate germplasm can yield
tumefaciens-mediated and DNA-coated particle bombardment unintended genomic changes affecting fitness or yield of the end
transformation introduce transgenic sequences randomly in the product, necessitating additional screening of individual lines.
crop genome with potential for endogenous gene disruption or This is a long-known phenomenon named somaclonal variation160.
misexpression of neighbouring genes through trans- or cis-gene Whole genome sequencing revealed an increase in DNA deletions
regulation. Another potential drawback of random DNA inser- and variations in cultured tissues of rice161, cotton162 and potato163.
tion is that, when multiple transgenes are used, these transgenes Epigenetic marks widespread in the genome can be lost or retained,
will likely integrate into different chromosomes and each will with these changes persisting for multiple generations after regen-
segregate independently, complicating downstream breeding. To eration from tissue culture164–166. In some cases, these plants have
address these potential shortcomings, researchers are investigating altered gene expression profiles that resemble their source tissue167
the use of SDNs to precisely introduce DNA elements into the plant and display aberrant interactions with beneficial and pathogenic
genome at targeted DNA breakages via the plant’s DNA homology microorganisms. While sufficient screening and field testing can
directed repair pathway. Multiple introduced genes can be incor- identify properly functioning individuals, the development of new
porated at a single genomic safe harbour, a defined chromosomal methods bypassing or limiting tissue culture is desirable151.
region with minimal positional effects to maximize efficacy of the Newly developed nanomaterials enable passive delivery of bio-
transgene without perturbing necessary cell functions144. Ainley molecules such as DNA, RNA and proteins into cells for, among
et al. developed a DNA ‘landing pad’ that when integrated into the other applications, integration into, or gene editing of, host
genome allows sequential stacking of modular transgenes through genomes. Physical and chemical properties of these materials such
defined sequence-specific nuclease cut sites. Its use in maize dem- as size and electric charge can be tailored to allow uptake across
onstrated co-segregation of inserted traits in the subsequent gen- plant cell walls and membranes of both plant and animal cells (see
eration145. Despite these advances, site-specific integration of DNA ref. 168 and references within), with application as simple as a foliar
via SDNs remains challenging in plants, as homology-directed spray. Carbon nanomaterials have been demonstrated to passively
repair is not always active during the plant cell life cycle, and it is traverse the plant cell wall to deliver DNA into leaf cells of arugula,
difficult to deliver sufficient target DNA near the DNA breakage wheat, cotton and the tobacco relative Nicotiana benthamiana169,
for high-efficiency insertion146. Efforts to address this include bias- and selectively into arugula, spinach and tobacco leaf cell chloro-
ing the cell’s repair machinery to favour homology-directed repair plasts170. Expression of the delivered genes was short lived (less than
(for example, ref. 147) and employing plant viral replication machin- 10 days) as the DNA did not integrate into the host genome169; such
ery to increase the amount of donor DNA, the latter of which a system could be useful for CRISPR gene-editing in which integra-
has enhanced targeted insertion efficiency in tobacco148, tomato149 tion is not necessary for function.
and wheat150. Gametic tissues in crop plants that propagate via sexual repro-
duction are being targeted to bypass tissue culture regeneration.
New methods to expand plant transformation technology Zhao et al. used a magnetic field to deliver transgene-carrying
Generating transformed plants is often a major bottleneck for crop nanoparticles through apertures where the cell wall is thin and
improvement151. Biological characteristics, such as recalcitrance more permeable in cotton, pepper and pumpkin pollen, produc-
to foreign DNA uptake and genomic integration or capacity for ing viable transgenic pollen171. Manual fertilization of flowers
regeneration into whole plants after transformation, limit genetic using this pollen produced seeds with stable, heritable transgene
engineering to amenable plant cultivars. The rigid plant cell wall integration. Transgenic pollen can also serve as the delivery vector
presents a specific challenge to plant transformation protocols for gene-editing protein/RNA complexes. Using methods deve
relative to those of mammalian cells, which lack such a barrier. loped previously172,173, a specific type of maize pollen carrying the
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Received: 22 October 2019; Accepted: 1 April 2020; 33. Jupe, F. et al. Resistance gene enrichment sequencing (RenSeq) enables
Published online: 19 May 2020 reannotation of the NB-LRR gene family from sequenced plant genomes