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In one example of the application of ZFNs to crop breeding, the endogenous maize gene ZmIPK1 was disrupted by insertion of PAT gene cassettes, and this resulted in herbicide tolerance and alteration of the inositol phosphate profile of developing maize seeds [ 14 ]. As a proven technology, ZFN-mediated targeted transgene integration was also used for trait stacking in maize, that is for assembling a number of useful traits together to create an even greater potential for crop improvement [ 15 ].

Later, Cantos et al. Nevertheless, the design of ZFNs remains a complicated and technically challenging process, and one that often has low efficacy. 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 , Nicotiana , Brachypodium , barley, potato, tomato, sugarcane, flax, rapeseed, soybean, rice, maize, and wheat reviewed in [ 12 , 13 ].

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 [ 18 ]. By knocking out the maize GL2 gene, Char et al.

Paolo Ranalli (Author of Improvement of Crop Plants for Industrial End Uses)

In sugarcane, cell wall composition and saccharification efficiency have been improved by TALEN-mediated mutagenesis [ 21 , 22 ]. TALENs can be used to modify the nutritional profiles of crops: soybeans with high oleic acid and low linoleic acid contents were generated by disrupting fatty acid desaturase FAD genes, thus improving the shelf life and heat stability of soybean oil [ 23 , 24 ].

In potato tubers, the accumulation of reducing sugars during cold storage influences the quality of the product, and knocking out the vacuolar invertase VInv gene resulted in tubers that had undetectable levels of problematic reducing sugars [ 25 ]. In addition, the production of haploid plants that inherit chromosomes from only one parent can greatly accelerate plant breeding.

Crop improvement by TALEN-mediated gene insertion is well exemplified in the tomato, where incorporating TALENs and donor DNA into geminivirus replicons significantly increased their copy number and hence the efficiency of homologous recombination [ 29 ]; a strong promoter was inserted upstream of the gene controlling anthocyanin biosynthesis, and purple tomatoes with high anthocyanin levels were obtained [ 29 ]. To date, many crops such as rice, 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 reviewed in [ 37 , 38 ].

The most frequent application has been in the production of null alleles, or gene knockouts, predominantly achieved by the introduction of small indels that result in frame-shift mutations or by introducing premature stop codons Fig. Yield is a major concern in crop breeding. Li et al. Grain Weight 2 GW2 is a key gene in cereal crops, which when disrupted increases grain weight and protein content in wheat [ 41 ].

In rice, Sun et al. In the absence of GBSS expression in the endosperm, amylose was not synthesized, and this created a high amylopectin waxy maize with improved digestibility and the potential for bio-industrial applications [ 44 ]. The release of commercial hybrids with this trait is planned for The same gene has also been targeted in the potato by researchers at the Swedish Agricultural University to produce waxy potatoes, with improved cultivars aimed predominantly at the industrial starch market to be released in the next few years [ 45 ]. The technology also has been used to improve resistance to biotic stresses.

Zhang et al. The resulting plants were resistant to powdery mildew and did not show mildew-induced cell death [ 46 ]. Furthermore, powdery mildew-resistant tomatoes were generated by editing SlMLO1 [ 49 ], and bacterial speck-resistant tomatoes were created by disrupting SlJAZ2 [ 50 ]. Citrus canker is a severe disease that is responsible for significant economic losses worldwide, and CsLOB1 is a susceptibility gene for citrus canker. By modifying the CsLOB1 promoter, canker symptoms were alleviated in Duncan grapefruits [ 51 ] and Wanjincheng oranges had enhanced resistance to citrus canker [ 52 ].

In the cucumber, when the eIF4E eukaryotic translation initiation factor 4E gene was disrupted, broad virus resistance was generated [ 54 ]; the plants were shown to be immune to an Ipomovirus Cucumber Vein Yellowing Virus and were resistant to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W [ 54 ]. Polyphenol oxidase PPO is an enzyme that causes browning in many fruits and vegetables.

By knocking out this gene, Waltz and coworkers [ 55 ] developed a non-browning mushroom. In maize, when the thermosensitive genic male-sterile 5 gene TMS5 was knocked out, thermosensitive male-sterile maize was generated [ 58 ].

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Genome-editing techniques can also accelerate the domestication of crops. Recently, a promoter swap and dual amino-acid substitutions were achieved at the EPSPS locus in cassava, generating glyphosate tolerance [ 69 ]. Genome-editing technology already shows great potential in agriculture, but it is still limited by the low efficiency of HR, off-target effects, restrictive protospacer adjacent motif PAM sequences, and other issues. Fortunately, novel innovations are continually being added to the genome-editing toolkit to address these limitations.

However, genome-wide association studies have shown that single-base changes are usually responsible for variations in elite traits in crop plants [ 71 ]; hence, efficient techniques for producing precise point mutations in crops are needed urgently. The base-editing technologies employ Cas9 nickase nCas9 or dead Cas9 dCas9 fused to an enzyme with base conversion activity. Cytidine-deaminase-mediated base editing CBE has been used in rice, Arabidopsis , wheat, maize, and tomato reviewed in [ 75 , 76 ]. Recently, this technology has been used in watermelon and wheat to create herbicide-resistant plants [ 77 , 78 ].

Fortunately, Gaudelli and colleagues [ 74 ], using several rounds of directed evolution and protein engineering, were able to develop an efficient ABE. In rice, Yan et al. Hua et al. An ABE has also been used with rapeseed protoplasts and in Arabidopsis , and the desired phenotypic alterations and germline transmission were observed in Arabidopsis [ 81 ]. In addition to generating point mutations, CBE can also be used to produce nonsense mutations that disrupt genes of interest and knockout their gene functions [ 83 ]. All-in-all, base-editing tools have given genome editing a new dimension, broadening its potential applications by means of nucleotide-specific modifications at specific genomic sites.

Conventional genome editing involves the delivery and integration into the host genome of DNA cassettes encoding editing components. Integration occurs at random, and therefore can generate undesirable genetic changes. Even if the DNA cassettes are degraded, the resulting fragments may be integrated and could produce undesirable effects [ 84 ]. Prolonged expression of genome-editing tools increases off-target effects in plants since nucleases are abundant in these organisms [ 19 , 26 , 85 ].

Moreover, the introduction of foreign DNA into plant genomes raises regulatory concerns in relation to GM organisms [ 86 ]. Therefore, DNA-free genome editing is a groundbreaking technology, producing genetically edited crops with a reduced risk of undesirable off-target mutations, and meeting current and future agriculture demands from both a scientific and regulatory standpoint.

DNA-free genome editing has been accomplished using both protoplast-mediated transformation and particle bombardment. Similarly, Malnoy et al. Unfortunately, efficient, regenerable protoplast systems are not available for a number of agriculturally important higher crop species, and therefore there has been a search for other DNA-free genome editing methods. Particle bombardment-mediated DNA-free genome-editing technology has been developed in wheat and maize [ 89 , 90 , 91 ]. Recently, a combination of base editing and DNA-free genome editing has been described in wheat [ 78 ], with an average frequency of C-to-T conversion of 1.

This development should greatly facilitate both the application of base editing to plant breeding and the commercialization of edited plants. Therefore, Cas9 variants were needed to overcome this limitation. Cpf1 recognizes T-rich PAMs and generates cohesive ends with four or five nucleotide overhangs rather than blunt-end breaks, which complements the characteristics of Cas9 to a large extent Fig. Recently, Cpf1 from Francisella novicida FnCpf1 was used for targeted mutagenesis in tobacco and rice [ 93 ], and the Cpf1 ortholog from a Lachnospiraceae bacterium LbCpf1 generated targeted mutations in rice [ 94 , 95 ].

A variant AsCpf1 Cpf1 ortholog from Acidaminococcus sp. BV3L6 demonstrated high genome-editing efficiencies in human cells [ 96 ], but was less efficient in rice [ 97 ] and in soybean and rice protoplasts [ 98 , 99 ]. When tested for their ability to induce targeted gene insertions via HR, the FnCpf1 and LbCpf1 nucleases generated precise gene insertions at a target site in rice, at a higher frequency than most other genome-editing nucleases [ ]. LbCpf1 has also been used for targeted gene replacement in rice [ ]. In plants, cellular processes are often regulated by complex genetic networks, and the manipulation of agronomic traits depends on the precise engineering of complex metabolic pathways, which requires the concerted expression of multiple genes.

Re-orienting crop improvement for the changing climatic conditions of the 21st century

Therefore, molecular tools with the capability to manipulate multiple genes simultaneously are of great value in both basic research and practical applications. One of the advantages of CRISPR systems over other genome-editing methods is their potential for multiplexing, the simultaneous editing of multiple target sites [ 31 ]. Xie et al. Taking advantage of this characteristic, Wang et al. Multiple sgRNAs can also be used to target a single gene to improve rates of editing in crops that have low transformation or editing efficiencies. Now that the complete genomes of many crops have been sequenced, the challenge of the post-genomic era is to analyze the functions of all crop genes systematically, as most of the genes sequenced to date have unknown functions and may control important agronomic traits.

Gene knockout is a frequently used and effective strategy for identifying gene functions; hence, large-scale mutant libraries at the whole-genome level are of great value for functional genomics and for crop improvement. Genome-wide mutant libraries in rice have been constructed by two teams. Lu et al. Meng et al.

These two groups selected rice for genome-wide targeted mutagenesis mainly because of its relatively small genome, rich genomic resources, and highly efficient transformation system. As techniques evolve, the construction of mutant libraries in other valued crop species should not be too long delayed. Besides gene knockouts and knockins, genome editing tools can also be used to regulate gene expression. Gene regulation mainly involves the repression and activation of genes and is often achieved by fusing transcriptional repressors or activators to the DNA-binding domains of genome-editing constructs such as zinc finger protein ZFP , TALE, or dCas9 , thereby targeting the regulatory regions of endogenous genes [ ].

Mutants in which KASII was activated displayed the desirable agronomic trait of decreased levels of palmitic acid and total saturated fatty acid [ ]. Furthermore, both AsCpf1 and LbCpf1 have been used to repress transcription in Arabidopsis, thus underlining the great promise of Cpf1 for modulating plant transcriptomes [ 99 ]. Rodriguez-Leal et al.

Introduction to Crop Improvement Methods [Year-2]

In this way, they could systematically assess the association of cis-regulatory regions with phenotypic traits, which should be helpful in enhancing tomato breeding. Targeting the uORF of LsGGP2 generated a mutant lettuce with improved tolerance to oxidative stress and increased ascorbate content [ ]. This strategy provides a generalizable, efficient method for manipulating the translation of mRNAs, which can be applied to dissect biological mechanisms and improve crops.

Unlike applications that aim primarily to alter DNA sequences, the effects of genome editing on gene regulation act at the transcript level, and could be used to reveal the function of many non-canonical RNAs that are related to crop improvement. As most non-coding transcripts are nuclear and lack open reading frames, genome editing that modulates transcription directly is optimally suited to interrogating the function of such RNAs. Over the past several decades, traditional breeding that depends on access to plant populations with sufficient variability has made great contributions to agriculture.

However, this variability is mainly derived from spontaneous mutations or from mutations that are induced by chemical mutagens or physical irradiation. Such mutations are usually rare and occur at random. Moreover, many types of variation might not arise in elite varieties, and consequently time-consuming, laborious breeding programs are needed to introduce desirable alleles into elite crops.


By contrast, genome editing as an advanced molecular biology technique can produce precisely targeted modifications in any crop [ 4 , 5 ]. Given the availability of a variety of genome-editing tools with different applications Fig. Once appropriate genome-editing tools have been selected, the target sequences are designed and introduced into the most suitable vectors, and the appropriate genetic cargo DNA, RNA, or RNPs for delivery is selected Fig. After the genetic cargo has entered the target plant cells, the target sequences will be modified, and edited calli will be regenerated and will ultimately give rise to edited plants Fig.

It may well be that protoplast-based systems are not readily available, or even possible, in a species of choice. Furthermore, regeneration by tissue culture may be difficult or limited to a few model genotypes. In these cases, it may be beneficial to design methodologies that do not require regeneration, such as the use of pollen, or the use of immature embryos that can be coaxed to germinate in vitro. With the progress already made in the development of genome-editing tools and the development of new breakthroughs, genome editing promises to play a key role in speeding up crop breeding and in meeting the ever-increasing global demand for food.

Moreover, the exigencies of climate change call for great flexibility and innovation in crop resilience and production systems. In addition, we must take into account government regulations and consumer acceptance around the use of these new breeding technologies. Following publication of the original article [1], the authors reported the following two errors. Solutions for a cultivated planet.

Global food demand and the sustainable intensification of agriculture. Genetically engineered crops: from idea to product. Annu Rev Plant Biol. Chen K, Gao C. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep. Gao C. Genome editing in crops: from bench to field. Natl Sci Rev. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet. Voytas DF, Gao C. Breeding of triticale with improved yield and lodging resistance for irrigated environments is achievable and can be pursued with confidence in breeding programs. Plant sciences, sustainable farming systems and food quality.

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Abstract Research into winter cereal breeding in Australia has focused primarily on studying the effects of rainfed environments.