Perceptions of plant breeding methods–from ‘phenotypic selection’ to ‘genetic modification’ and ‘new breeding technologies’

Figures & data

Table 1. Artificially-made interspecific hybrids used in agriculture and horticulture.

Interspecific hybrid	Trait enhancement	Reference
Chestnut (Castanea)	Blight resistant Chinese (C. mollissima) and Japanese (C. crenata) species used in a backcrossing programme with American chestnuts (C. dentata) to produce blight resistant timber chestnuts; Other crosses provide Phytopthora spp. (ink disease) in Europe	Pereira-Lorenzo et al. 2016
Papaya	Enhanced papaya ringspot virus-type P (PRSV-P) resistance	Drew et al. 1998
Tomato (Kumato –hybrid SX 387)	Colour ranging from a green to reddish brown or purple, varying in flavour from almost no flavour to sweeter than typical tomatoes due to a higher fructose content	United States Patent 2009
Triticale	Combine the superior agronomic performance and end-use qualities of wheat with the stress tolerance (both biotic and abiotic) and adaptability of rye	Randhawa et al. 2015; Mergoum et al. 2019
Raphanobrassica	Combines the superior biomass yield, quality, and palatability of kale, with the increased drought, insect, and disease tolerance of radish	Chakwizira et al. 2021; Dumbleton et al. 2022

Table 2. Comparison of benefits and challenges of New Breeding Technologies compared with transgenesis

Comparator	Transgenesis	New Breeding Technologies	Reference
Precision of mutation	Low; Relatively random and untargeted	High; CRISPR Cas can modify one or a few target sites with precision; some Cas effector proteins are more accurate than others	Ma et al. 2015; Lee et al. 2019b
Gene modification without introducing foreign DNA	Not possible	Possible through gene disruption or deletion (SDN1)	Veillet et al. 2019
Versatility, flexibility, adaptability	Moderate in requiring a gene construct to allow expression of the introduced gene	High–can be facilitated through gene disruption or deletion, but can also include gene modification or gene introduction	Pandey et al. 2019; Wheatley and Yang 2021
Efficiency and time to delivery	Moderate	Relatively fast	Zhang et al. 2014; Wolter et al. 2019
Inclusion of genetic diversity from wild relatives	Poor and time consuming	Effective option	Wolter et al. 2019
Agrobacterium and/or biolistics mediated gene insertion	Agrobacterium and biolistics	Agrobacterium and biolistics	Kuluev et al. 2019; Basso et al. 2020

Table 3. Examples of cultivars or germplasm developed using different methods of genetic engineering and genetic modification–those in bold have been submitted for de-regulation or commercialised. Refer also to Penna and Jain 2023) for a summary of successful examples of genome editing in fruit crops.

Method	Germplasm	Trait	Status	Reference
Cisgenesis	Apple (Malus domestica)	Increased scab resistance	Demonstrates proof of concept	Vanblaere et al. 2011
	Barley	Improved grain phytase activity	Demonstrates proof of concept	Holme et al. 2012, 2020
	Durum wheat (Triticum durum)	Improved baking quality	Demonstrates proof of concept	Gadaleta et al. 2008
	Grape (Vitis vinifera)	Improved fungal disease resistance	Demonstrates proof of concept	Dhekney et al. 2011
	Potato	Expression of resistance genes for late blight (Phytophthora iInfestans) resistance	Demonstrates proof of concept	Haverkort et al. 2009; Jo et al. 2014
		Resistance to Potato virus Y	Demonstrates proof of concept	Miroshnichenko et al. 2020
Intragenesis	Potato	Gene silencing to levels of amylopectin	Demonstrates proof of concept	de Vetten et al. 2003
		Prevention of black spot bruising	Demonstrates proof of concept	Rommens et al. 2004
		Limited acrylamide production in French fries	Demonstrates proof of concept	Chawla et al. 2012
		Low acrylamide, resistance to bruising and discolouration	Commercially available	Waltz 2015; ISAAA database 2023
	Tomato	Resistance to tomato spotted wilt virus and caulifower mosaic virus	Has been de-regulated in USA	Kumar et al. 2020
Transgenesis	32 crop and flower species	525 different transgenic events providing 2063 GM foods and 1461 GM feeds; underpinned by 824 regulatory approvals	Commercially available	Reviewed by Caradus 2022
Oligonucleotide-directed mutagenesis	Canola	Mutations in canola AHAS homologues, to develop Cibus’ herbicide-tolerant SU Canola^TM	Commercially available	Gocal 2015
	Maize	Single-point mutation of genes encoding acetohydroxyacid synthase, at a position known to confer imidazolinone (herbicide) resistance.	Demonstrates proof of concept	Zhu et al. 2000
		Altered epicuticular wax composition	Demonstrates proof of concept	Char et al. 2015
	Rice	Chimeric RNA/DNA oligonucleotides targeting endogenous acetolactate synthase gene conferring resistance to some herbicides	Demonstrates proof of concept	Okuzaki and Toriyama 2004
Zinc finger	Maize	Insertional disruption of one target locus results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds	Demonstrates proof of concept	Shukla et al. 2009
TALENs	Potato	Reduced acrylamide formation	Demonstrates proof of concept	Clasen et al. 2016
		Reduced toxic steroidal glycoalkaloids formation	Demonstrates proof of concept	Sawai et al. 2014
	Rice	Improved rice bacterial blight resistance	Demonstrates proof of concept	Li et al. 2012
		Increase the synthesis of 2-acetyl-1-pyrroline which is a major fragrance compound	Demonstrates proof of concept	Shan et al. 2015
	Soybean	Oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%; increased shelf life and oil stability	Submitted for de-regulation	Haun et al. 2014; Kumar et al. 2020
		High oleic acid (increased shelf life and trans-fat free)	Submitted for de-regulation	Waltz 2019; Kumar et al. 2020
	Sugarcane (Saccharum spp. hybrids)	Reduce lignin content to improve the conversion of lignocellulosic biomass into ethanol	Demonstrates proof of concept	Jung and Altpeter 2016; Kannan et al. 2018
	Wheat	Mutation of all three mildew resistance locus homeologs confers heritable broad-spectrum resistance to powdery mildew	Submitted for de-regulation	Wang et al. 2014; Kumar et al. 2020
CRISPR CasX (i.e. Cas9 and Cas12 and its variants)	Apple	Increase resistance to fire blight disease (Erwinia amylovora)	Demonstrates proof of concept	Malnoy et al. 2016
	Banana	Knockout the integrated the virus sequences of the integrated endogenous banana streak virus (eBSV) in the B genome of Plantain to allow breeding and dissemination of hybrids	Demonstrates proof of concept	Tripathi et al. 2019
	Cacao (Theobroma cacao)	increased resistance to the cacao pathogen Phytophthora tropicalis	Demonstrates proof of concept	Fister et al. 2018
	Canola (Brassica napus)	Improved fatty acid composition	Demonstrates proof of concept	Huang et al. 2020
		Single base editing of gene producing Acetolactate synthase to confer tribenuron-methyl herbicide resistance	Demonstrates proof of concept	Wu et al. 2020
	Cassava (Manihot esculenta)	Reduced cassava brown streak disease	Demonstrates proof of concept	Gomez et al. 2019
	Citrus (Citrus sinensis)	Improvement of citrus canker resistance through targeted modification of the susceptibility gene	Demonstrates proof of concept	Peng et al. 2017
	Cotton (Gossypium hirsutum)	Resistance to Verticillium wilt	Demonstrates proof of concept	Li et al. 2017
	Cucumber (Cucumis sativus)	Improved virus resistance	Demonstrates proof of concept	Chandrasekaran et al. 2016
	False flax (Camelina)	Increased oil content	Submitted for de-regulation	Waltz 2018
	Grape	Reduced tartaric acid synthesis	Demonstrates proof of concept	Ren et al. 2016
		Increased resistance to powdery mildew (Erysiphe necator)	Demonstrates proof of concept	Malnoy et al. 2016.
	Green bristlegrass (Setaria viridis)	Delayed flowering	Submitted for de-regulation	Waltz 2018; Kumar et al. 2020
	Maize	Decreased sensitivity to ethylene and concomitant improvement in yield under drought	Demonstrates proof of concept	Shi et al. 2017
		Reduced ligule development, imposed male sterility, and improved herbicide acetolactate synthase resistance	Demonstrates proof of concept	Svitashev et al. 2015
		Waxy corn with starch composed exclusively of amylopectin	Submitted for de-regulation	Waltz 2018
	Potato	Altered starch quality	Demonstrates proof of concept	Andersson et al. 2017
		Chlorsulfuron herbicide resistance	Demonstrates proof of concept	Veillet et al. 2019
	Rice	Potential for improved salinity tolerance	Demonstrates proof of concept	Duan et al. 2016; Farhat et al. 2019
		Improved resistance to rice blast	Demonstrates proof of concept	Wang et al. 2016
		Increased amylose content and resistant starch content to 25% and 9.8%, respectively; altering fine structure and nutritional properties of starch	Demonstrates proof of concept	Sun et al. 2017
		Regulation of stomatal density and patterning	Demonstrates proof of concept	Yin et al. 2017
	Sorghum	Reduced kafirin levels and improved protein quality and digestibility	Demonstrates proof of concept, but not in all germplasm (Massel et al. 2023)	Li et al. 2018
	Soybean	improved herbicide acetolactate synthase resistance	Demonstrates proof of concept	Li et al. 2015
		Early flowering under long day conditions	Demonstrates proof of concept	Han et al. 2019
		Drought and salt tolerance	Submitted for de-regulation	Curtin et al. 2018; Waltz 2018; Kumar et al. 2020
		Increased Oleic oil content	Approved for sale in USA	Calyxt 2019
		Low presence of raffinose and stachyose sugars	Approved in Colombia	GDM Seeds 2022
	Tomato	High amounts of γ-aminobutyric acid (GABA)	Commercialised	Waltz 2022
		Increased vitamin D3 expression, to provide a plant based source of Vitamin D3	Demonstrates proof of concept	Li et al. 2022
		Altering morphology of wild-type tomato with high antioxidant content	Demonstrates proof of concept	Zsögön et al. 2018
		Increased resistance to pathogens, Pseudomonas syringae, Phytophthora capsici and Xanthomonas spp.	Demonstrates proof of concept	De Thomazella et al. 2021
		Chlorsulfuron herbicide resistance	Demonstrates proof of concept	Veillet et al. 2019
	Wheat (SDN1)	Low gluten	Held up by European GM regulations	Sánchez-León et al. 2018; Jouanin et al. 2019, 2020
RNA-directed DNA methylation (Epigenesis)	Maize	High amylopectin starch content	Submitted for de-regulation	Waltz 2016; Kumar et al. 2020
	Petunia	Induced cytosine methylation that affected changes in flower colouration	Demonstrates proof of concept	Kanazawa et al. 2011
	Soybean	Increase in variance for pods per plant, seed weight and maturity time	Demonstrates proof of concept	Raju et al. 2018
	Tomato	Induced cytosine methylation that affected rate of fruit ripening	Demonstrates proof of concept	Kanazawa et al. 2011
Grafting on GM rootstock	Cherry (Prunus avium)	RNA interference (RNAi) vector used for developing viral resistance Prunus necrotic ringspot virus in rootstocks	At proof of concept stage	Song et al. 2013
Synthetic biology	Cereals	Nitrogen fixation symbiosis	Proposed opportunity concept	Rogers and Oldroyd 2014
	Crop plants	Production of health producing phytochemicals, e.g. carotenoids/isoprenoids	Proposed opportunity concept	Fraser et al. 2009

Table 4. Review of some country differences in regulation of genetic engineering techniques and in particular New Breeding Technologies (NBTs) (also refer to Atanassova and Keiper 2018; Menz et al. 2020; and Turnbull et al. 2021; Jones et al. 2022).

Country or region	Regulatory system	Regulation of NBTs	Reference
Argentina	Process based but with updated legislation to provide clarity on the regulatory status of products of plant breeding innovations; regulatory framework explicitly addresses products obtained from NBTs	Uses a case-by-case determination of whether a ‘new combination of genetic material’ was generated with an absence of events (transgenes) that may have been used transiently during the development process. Crops modified using SDN-1, SDN-2 and ODM would generally not be regulated	Whelan and Lema 2015; Lassoued et al. 2020; OGTR 2021
Australia	Process based, but reviewed in 2016; however GM derived food can be used if shown to be as safe as non-GM food (must be labelled)	NBTs that do not involve the application of nucleic acid repair template (e.g. SDN-1) are excluded	MPI 2021; OGTR 2021; FAO 2022; FSANZ 2022
Canada	Regulates products based on the expression of a novel trait, rather than the method used in its development	Uses ‘novelty’ as a trigger to assess the environmental safety of plants with novel traits or novel feed prior to product release	CFIA 2016; Lassoued et al. 2020
European Union	Both process- and product- focused elements, but predominantly interpreted as a strict process-focused system–reviewed in 2016 but remains unchanged	All NBTs regulated as genetically modified	Lassoued et al. 2020; OGTR 2021
New Zealand	Processed based–not reviewed in 25 years; however GM derived food can be used if shown to be as safe as non-GM food (must be labelled)	All NBTs regulated as genetically modified as a result of a High Court ruling	Fritsche et al. 2018; Royal Society Te Apārangi 2019; FSANZ 2021, 2022; MPI 2021; Caradus 2022; FAO 2022
USA	US implemented a Coordinated Framework for the Regulation of Biotechnology to assess and regulate the products of biotechnology. Regulation of specific risks (i.e. product characteristics) rather than the application of default regulation to all products of biotechnology	Does not regulate plants with deletions, single-nucleotide substitutions, insertions from compatible plant relatives or null segregants, unless plant pest species are involved. Therefore the majority of SDN-1 and ODM outcomes and some SDN-2 and −3 outcomes in plants are outside USDA’s regulatory scope	FDA 2020; Lassoued et al. 2020; OGTR 2021

Table 5. Percentage of traits sought using New Breeding Technologies by researchers (Modrzejewski et al. 2019) and crop breeding companies (Jorasch 2020).

Trait category	Survey
	Researchers and web pages	Crop breeding companies
Agronomic value	36	26
Food and feed quality	29	18
Biotic stress tolerance	16	23
Abiotic stress tolerance	5	15
Herbicide tolerance	8	5
Industrial applications	6	9
Other*	–	5

* Other includes –flavour, shelf-life, digestibility, ornamental value (flower colour), and post-harvest quality.

Table 6. Comparison of using chemical or radiation mutagenesis with CRISPR site-directed nucleases gene editing (derived from Lema 2021).

Comparator	Technique
	Ionising radiation or chemical mutagenesis	CRISPR site-directed nucleases
Control over gene sequences targeted	None	Tight. Breakage directed to specific sequences of interest
Knowledge on the trait-associated mutation	None–regulators not required to ask	Known from the start, part of the breeding strategy and the regulatory analysis
Unintended consequences–off target mutations	Significant –regulators not required to ask	Unlikely but regulators will ask
Possibility of finding off-target mutations	Limited –regulators not required to ask	Easy, and can be provided to reduce regulatory burden
Versatility, flexibility, and adaptability	Random and most often resulting in gene disruption	High –can be facilitated through gene disruption or deletion, but can also include gene modification or gene introduction

Pereira-Lorenzo S, Costa R, Anagnostakis S, Serdar U, Yamamoto T, Saito T, Ramos-Cabrer AM, Ling Q, Barreneche T, Robin C, Botta R. 2016. Interspecific hybridization of chestnut. Polyploidy and Hybridization for Crop Improvement. 15:377–408.

 Google Scholar

Drew RA, Magdalita PM, O'Brien CM. 1998. Development of Carica interspecific hybrids. Acta Horticulturae. 2(461):285–292. doi:10.17660/ActaHortic.1998.461.31.

 Google Scholar

United States Patent. 2009. Tomato plants. Patent No.: US 7,612.261 B2; [accessed 25 February 2023]. https://patentimages.storage.googleapis.com/17/d5/0d/dcba5fd7f54abf/US7612261.pdf.

 Google Scholar

Randhawa HS, Bona L, Graf RJ. 2015. Triticale breeding—progress and prospect. In: Eudes F, editor. Triticale. Cham: Springer; p. 15–32. doi:10.1007/978-3-319-22551-7_2

 Google Scholar

Mergoum M, Sapkota S, ElDoliefy AEA, Naraghi SM, Pirseyedi S, Alamri MS, AbuHammad W. 2019. Triticale (x Triticosecale Wittmack) breeding. In: Al-Khayri J, Jain S, Johnson D, editors. Advances in plant breeding strategies: cereals. Cham: Springer; p. 405–451. doi:10.1007/978-3-030-23108-8_11

 Google Scholar

Chakwizira E, Maley S, Dumbleton A. 2021. Water use efficiency of raphanobrassica and forage rape. Agronomy Journal of New Zealand. 51:35–46.

 Google Scholar

Dumbleton A, Foley F, Westwood C, Box G. 2022. The development of Pallaton raphanobrassica for New Zealand farming systems. Journal of New Zealand Grasslands. 83:107–114. doi:10.33584/jnzg.2021.83.3505.

 Google Scholar

Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, et al. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant. 8:1274–1284. doi:10.1016/j.molp.2015.04.007.

 PubMed Web of Science ®Google Scholar

Lee MA, Howard-Andrews V, Chester M. 2019b. Resistance of multiple diploid and tetraploid perennial ryegrass (lolium perenne L.) varieties to three projected drought scenarios for the UK in 2080. Agronomy. 9: Article 159. doi:10.3390/agronomy9030159.

 Web of Science ®Google Scholar

Veillet F, Perrot L, Chauvin L, Kermarrec M-P, Guyon-Debast A, Chauvin J-E, Nogué F, Mazier M. 2019. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor. International Journal of Molecular Sciences. 20: Article 402. doi:10.3390/ijms20020402.

 Web of Science ®Google Scholar

Pandey PK, Quilichini TD, Vaid N, Gao P, Xiang D, Datla R. 2019. Versatile and multifaceted CRISPR/Cas gene editing tool for plant research. Seminars in Cell & Developmental Biology. 96:107–114. Academic Press. doi:10.1016/j.semcdb.2019.04.012

 PubMed Web of Science ®Google Scholar

Wheatley MS, Yang Y. 2021. Versatile applications of the CRISPR/Cas toolkit in plant pathology and disease management. Phytopathology. 111:1080–1090. doi:10.1094/PHYTO-08-20-0322-IA.

 PubMed Web of Science ®Google Scholar

Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu J-K. 2014. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal. 12:797–807. doi:10.1111/pbi.12200.

 PubMed Web of Science ®Google Scholar

Wolter F, Schindele P, Puchta H. 2019. Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites. BMC Plant Biology. 19: Article 176. doi:10.1186/s12870-019-1775-1.

 Web of Science ®Google Scholar

Kuluev BR, Gumerova GR, Mikhaylova EV, Gerashchenkov GA, Rozhnova NA, Vershinina ZR, Khyazev AV, Matniyazov RT, Baymiev A, Baymiev A, Chemeris AV. 2019. Delivery of CRISPR/Cas components into higher plant cells for genome editing. Russian Journal of Plant Physiology. 66:694–706. doi:10.1134/S102144371905011X.

 Web of Science ®Google Scholar

Basso MF, Arraes FB, Grossi-de-Sa M, Moreira VJ, Alves-Ferreira M, Grossi-de-Sa MF. 2020. Insights into genetic and molecular elements for transgenic crop development. Frontiers in Plant Science. 11:Article 509. doi:10.3389/fpls.2020.00509.

 PubMed Web of Science ®Google Scholar

Penna S, Jain SM. 2023. Fruit crop improvement with genome editing, in vitro and transgenic approaches. Horticulturae. 9: Article 58. doi:10.3390/horticulturae9010058.

 Web of Science ®Google Scholar

Vanblaere T, Szankowski I, Schaart J, Schouten H, Flachowsky H, Broggini GAL, Gessler C. 2011. The development of a cisgenic apple plant. Journal of Biotechnology. 154:304–311.

 PubMed Web of Science ®Google Scholar

Holme IB, Dionisio G, Brinch-Pedersen H, Wendt T, Madsen CK, Vincze E, Holm PB. 2012. Cisgenic barley with improved phytase activity. Plant Biotechnology Journal. 10:237–247.

 PubMed Web of Science ®Google Scholar

Holme IB, Madsen CK, Wendt T, Brinch-Pedersen H. 2020. Horizontal stacking of PAPhy_a cisgenes in barley is a potent strategy for increasing mature grain phytase activity. Frontiers in Plant Science. 11:Article 592139. doi:10.3389/fpls.2020.592139.

 PubMed Web of Science ®Google Scholar

Gadaleta A, Giancaspro A, Blechl AE, Blanco A. 2008. A transgenic durum wheat line that is free of marker genes and expresses 1DY10. Journal of Cereal Science. 48:439–445. doi:10.1016/j.jcs.2007.11.005.

 Web of Science ®Google Scholar

Dhekney SA, Li ZT, Gray DJ. 2011. Grapevines engineered to express cisgenic vitis vinifera thaumatin-like protein exhibit fungal disease resistance. Vitro Cellular & Developmental Biology - Plant. 47:458–466. doi:10.1007/s11627-011-9358-3.

 Web of Science ®Google Scholar

Haverkort AJ, Struik PC, Visser RGF, Jacobsen E. 2009. Applied biotechnology to combat late blight in potato caused by phytophthora infestans. Potato Research. 52:249–264.

 Web of Science ®Google Scholar

Jo KR, Kim CJ, Kim SJ, Kim TY, Bergervoet M, Jongsma MA, Visser RG, Jacobsen E, Vossen JH. 2014. Development of late blight resistant potatoes by cisgene stacking. BMC Biotechnology. 14: Article 50. doi:10.1186/1472-6750-14-50.

 Web of Science ®Google Scholar

Miroshnichenko D, Timerbaev V, Okuneva A, Klementyeva A, Sidorova T, Pushin A, Dolgov S. 2020. Enhancement of resistance to PVY in intragenic marker-free potato plants by RNAi-mediated silencing of eIF4E translation initiation factors. Plant Cell, Tissue and Organ Culture (PCTOC). 140:691–705. doi:10.1007/s11240-019-01746-9.

 Web of Science ®Google Scholar

de Vetten N, Wolters A, Raemakers K, van der Meer I, ter Stege R, Heeres E, Heeres P, Visser R. 2003. A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nature Biotechnology. 21:439–442.

 PubMed Web of Science ®Google Scholar

Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang L, Perry R, Swords K. 2004. Crop improvement through modification of the plant’s own genome. Plant Physiology. 135:421–431.

 PubMed Web of Science ®Google Scholar

Chawla R, Shakya R, Rommens CM. 2012. Tuber-specific silencing of asparagine synthetase-1 reduces the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape and yield. Plant Biotechnology Journal. 10:913–924. doi:10.1111/j.1467-7652.2012.00720.x.

 PubMed Web of Science ®Google Scholar

Waltz E. 2015. USDA approves next-generation GM potato. Nature Biotechnology. 33:12–13. doi:10.1038/nbt0115-12.

 PubMed Web of Science ®Google Scholar

ISAAA database. 2023. GM Approval Database. https://www.isaaa.org/gmapprovaldatabase/.

 Google Scholar

Kumar K, Gambhir G, Dass A, Tripathi AK, Singh A, Jha AK, Yadava P, Choudhary M, Rakshit S. 2020. Genetically modified crops: current status and future prospects. Planta. 251: Article 91. doi:10.1007/s00425-020-03372-8.

 Web of Science ®Google Scholar

Caradus JR. 2022. Ergot alkaloids in New Zealand pastures and their impact. New Zealand Journal of Agricultural Research. 65:1–41. doi:10.1080/00288233.2020.1785514

 Web of Science ®Google Scholar

Gocal G. 2015. Non-transgenic trait development in crop plants using oligo-directed mutagenesis: cibus’ rapid trait development system. In: Eaglesham A, Hardy RW, editors. NABC report 26. New DNA-editing approaches: methods, applications and policy for agriculture. Ithaca (NY): North American Agricultural Biotechnology Council; p. 97–105.

 Google Scholar

Zhu T, Mettenburg K, Peterson DJ, Tagliani L, Baszczynski CL. 2000. Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Nature Biotechnology. 18:555–558. doi:10.1038/75435.

 PubMed Web of Science ®Google Scholar

Char SN, Unger-Wallace E, Frame B, Briggs SA, Main M, Spalding MH, Vollbrecht E, Wang K, Yang B. 2015. Heritable site-specific mutagenesis using TALENs in maize. Plant Biotechnology Journal. 13:1002–1010. doi:10.1111/pbi.12344.

 PubMed Web of Science ®Google Scholar

Okuzaki A, Toriyama K. 2004. Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice. Plant Cell Reports. 22:509–512. doi:10.1007/s00299-003-0698-2.

 PubMed Web of Science ®Google Scholar

Shukla V, Doyon Y, Miller J, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, et al. 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature. 459:437–441. doi:10.1038/nature07992.

 PubMed Web of Science ®Google Scholar

Clasen BM, Stoddard TJ, Luo S, Demorest ZL, Li J, Cedrone F, Tibebu R, Davison S, Ray EE, Daulhac A, Coffman A. 2016. Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnology Journal. 14:169–176. doi:10.1111/pbi.12370.

 PubMed Web of Science ®Google Scholar

Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T, Takebayashi Y, Kojima M, Sakakibara H, Aoki T, et al. 2014. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato. The Plant Cell. 26:3763–3774. doi:10.1105/tpc.114.130096.

 PubMed Web of Science ®Google Scholar

Li T, Liu B, Spalding M, Weeks DP, Yang B. 2012. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nature Biotechnology. 30:390–392. doi:10.1038/nbt.2199.

 PubMed Web of Science ®Google Scholar

Shan Q, Zhang Y, Chen K, Zhang K, Gao C. 2015. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnology Journal. 13:791–800. doi:10.1111/pbi.12312.

 PubMed Web of Science ®Google Scholar

Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F. 2014. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnology Journal. 12:934–940. doi:10.1111/pbi.12201.

 PubMed Web of Science ®Google Scholar

Waltz E. 2019. Appetite grows for biotech foods with health benefits. Nature Biotechnology. 37:573–575. doi:10.1038/d41587-019-00012-9.

 PubMed Web of Science ®Google Scholar

Jung JH, Altpeter F. 2016. TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol. Plant Molecular Biology. 92:131–142. doi:10.1007/s11103-016-0499-y.

 PubMed Web of Science ®Google Scholar

Kannan B, Jung JH, Moxley GW, Lee S-M, Altpeter F. 2018. TALEN-mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield. Plant Biotechnology Journal. 16:856–866. doi:10.1111/pbi.12833.

 PubMed Web of Science ®Google Scholar

Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J-L. 2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology. 32:947–951. doi:10.1038/nbt.2969.

 PubMed Web of Science ®Google Scholar

Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala Kanchiswamy C. 2016. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Frontiers in Plant Science. 7: Article 1904. doi:10.3389/fpls.2016.01904.

 Web of Science ®Google Scholar

Tripathi JN, Ntui VO, Ron M, Muiruri SK, Britt A, Tripathi L. 2019. CRISPR/cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding. Communications Biology. 2: Article 46. doi:10.1038/s42003-019-0288-7.

 Web of Science ®Google Scholar

Fister AS, Landherr L, Maximova SN, Guiltinan MJ. 2018. Transient expression of CRISPR/Cas9 machinery targeting TcNPR3 enhances defense response in theobroma cacao. Frontiers in Plant Science. 9:Article 268. doi:10.3389/fpls.2018.00268.

 PubMed Web of Science ®Google Scholar

Huang H, Cui T, Zhang L, Yang Q, Yang Y, Xie K, Fan C, Zhou Y. 2020. Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in brassica napus. Theoretical and Applied Genetics. 133:2401–2411. doi:10.1007/s00122-020-03607-y.

 PubMed Web of Science ®Google Scholar

Wu J, Chen C, Xian G, Liu D, Lin L, Yin S, Sun Q, Fang Y, Zhang H, Wang Y. 2020. Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing. Plant Biotechnology Journal. 18:1857–1859. doi:10.1111/pbi.13368.

 PubMed Web of Science ®Google Scholar

Gomez MA, Lin DZ, Moll T, Chauhan RD, Hayden L, Renninger K, Beyene G, Taylor NJ, Carrington J, Staskawicz B, Bart R. 2019. Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence. Plant Biotechnology Journal. 17:421–434. doi:10.1111/pbi.12987.

 PubMed Web of Science ®Google Scholar

Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X. 2017. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnology Journal. 15:1509–1519. doi:10.1111/pbi.12733.

 PubMed Web of Science ®Google Scholar

Li C, Unver T, Zhang B. 2017. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton (gossypium hirsutum L.). Scientific Reports. 7: Article 43902. doi:10.1038/srep43902.

 Web of Science ®Google Scholar

Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A. 2016. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular Plant Pathology. 17:1140–1153. doi:10.1111/mpp.12375.

 PubMed Web of Science ®Google Scholar

Waltz E. 2018. With a free pass, CRISPR-edited plants reach market in record time. Nature Biotechnology. 36:6–7. doi:10.1038/nbt0118-6b.

 PubMed Web of Science ®Google Scholar

Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z. 2016. CRISPR/Cas9-mediated efficient targeted mutagenesis in chardonnay (Vitis vinifera L.). Scientific Reports. 6: Article 32289. doi:10.1038/srep32289.

 Web of Science ®Google Scholar

Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, Hakimi SM, Mo H, Habben JE. 2017. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal. 15:207–216. doi:10.1111/pbi.12603.

 PubMed Web of Science ®Google Scholar

Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM. 2015. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiology. 169:931–945. doi:10.1104/pp.15.00793.

 PubMed Web of Science ®Google Scholar

Andersson M, Turesson H, Nicolia A, Fält A-S, Samuelsson M, Hofvander P. 2017. Efficient targeted multiallelic mutagenesis in tetraploid potato (solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Reports. 36:117–128. doi:10.1007/s00299-016-2062-3.

 PubMed Web of Science ®Google Scholar

Duan YB, Li J, Qin RY, Xu RF, Li H, Yang YC, Ma H, Li L, Wei PC, Yang JB. 2016. Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis. Plant Molecular Biology. 90:49–62. doi:10.1007/s11103-015-0393-z.

 PubMed Web of Science ®Google Scholar

Farhat S, Jain N, Singh N, Sreevathsa R, Das PK, Rai R, Yadav S, Kumar P, Sarkar A, Jain A. 2019. CRISPR-Cas9 directed genome engineering for enhancing salt stress tolerance in rice. Seminars in Cell & Developmental Biology. 96:91–99. doi:10.1016/j.semcdb.2019.05.003.

 PubMed Web of Science ®Google Scholar

Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y-G, Zhao K. 2016. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One. 11: Article e0154027. doi:10.1371/journal.pone.0154027.

 Web of Science ®Google Scholar

Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, Du W, Du J, Francis F, Zhao Y, Xia L. 2017. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant Science. 8: Article 298. doi:10.3389/fpls.2017.00298.

 Web of Science ®Google Scholar

Yin X, Biswal AK, Dionora J, Perdigon KM, Balahadia CP, Mazumdar S, Chater C, Lin HC, Coe RA, Kretzschmar T, Gray JE. 2017. CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Reports. 36:745–757. doi:10.1007/s00299-017-2118-z.

 PubMed Web of Science ®Google Scholar

Massel K, Hintzsche J, Restall J, Kerr ED, Schulz BL, Godwin ID. 2023. CRISPR-knockout of β-kafirin in sorghum does not recapitulate the grain quality of natural mutants. Planta. 257:8. doi:10.1007/s00425-022-04038-3.

 Web of Science ®Google Scholar

Li A, Jia S, Yobi A, Ge Z, Sato SJ, Zhang C, Angelovici R, Clemente TE, Holding DR. 2018. Editing of an alpha-kafirin gene family increases, digestibility and protein quality in sorghum. Plant Physiology. 177:1425–1438. doi:10.1104/pp.18.00200.

 PubMed Web of Science ®Google Scholar

Li Z, Liu Z-B, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT, Falco CE, Cigan SC, M A. 2015. Cas9-guide RNA directed genome editing in soybean. Plant Physiology. 169:960–970. doi:10.1104/pp.15.00783.

 PubMed Web of Science ®Google Scholar

Han J, Guo B, Guo Y, Zhang B, Wang X, Qiu LJ. 2019. Creation of early flowering germplasm of soybean by CRISPR/Cas9 technology. Frontiers in Plant Science. 10:Article 1446. doi:10.3389/fpls.2019.01446.

 PubMed Web of Science ®Google Scholar

Curtin SJ, Xiong Y, Michno JM, Campbell BW, Stec AO, Čermák T, Starker C, Voytas DF, Eamens AL, Stupar RM. 2018. CRISPR/cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of glycine max and medicago truncatula. Plant Biotechnology Journal. 16:1125–1137. doi:10.1111/pbi.12857.

 PubMed Web of Science ®Google Scholar

Calyxt. 2019. First commercial sale of Calyxt high oleic soybean oil on the U.S. market. Press Release; [accessed 19 January 2023]. https://calyxt.com/wp-content/uploads/2019/02/20190226_PR-Calyno-Commercialization.pdf.

 Google Scholar

GDM Seeds. 2022. The Colombian government approves soy edited by GDM with low presence of sugars; [accessed 19 January 2023]. https://www.gdmseeds.com/wp-content/uploads/2023/01/ING-Soja-Colombia.pdf.

 Google Scholar

Waltz E. 2022. GABA-enriched tomato is first CRISPR-edited food to enter market. Nature Biotechnology. 40:9–11. doi:https://doi.org/10.1038/d41587-021-00026-2.

 PubMed Web of Science ®Google Scholar

Li J, Scarano A, Mora Gonzalez N, D’Orso F, Yue Y, Nemeth K, Saalbach G, Hill L, Oliveira Martins C, Moran R, et al. 2022. Biofortified tomatoes provide a new route to vitamin D sufficiency. Nature Plants. 8:611–616. doi:10.1038/s41477-022-01154-6.

 PubMed Web of Science ®Google Scholar

Zsögön A, Čermák T, Naves E, Notini MM, Edel KH, Weinl S, Freschi L, Voytas DF, Kudla J, Peres LEP. 2018. De novo domestication of wild tomato using genome editing. Nature Biotechnology. 36:1211–1216. doi:10.1038/nbt.4272.

 Web of Science ®Google Scholar

De Thomazella DPT, Seong K, Mackelprang R, Dahlbeck D, Geng Y, Gill US, Qia T, Pham J, Giuseppe P, Lee CT, et al. 2021. Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. Proceedings of the National Academy of Sciences. 118: Article e2026152118. doi:10.1073/pnas.2026152118.

 Web of Science ®Google Scholar

Sánchez-León S, Gil-Humanes J, Ozuna CV, Giménez MJ, Sousa C, Voytas DF, Barro F. 2018. Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnology Journal. 16:902–910. doi:10.1111/pbi.12837.

 PubMed Web of Science ®Google Scholar

Jouanin A, Schaart JG, Boyd LA, Cockram J, Leigh FJ, Bates R, Wallington EJ, Visser RG, Smulders MJ. 2019. Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families. BMC Plant Biology. 19:Article 333. doi:10.1186/s12870-019-1889-5.

 PubMed Web of Science ®Google Scholar

Jouanin A, Gilissen LJ, Schaart JG, Leigh FJ, Cockram J, Wallington EJ, Boyd LA, van den Broeck HC, van der Meer IM, America AH, Visser RG. 2020. CRISPR/cas9 gene editing of gluten in wheat to reduce gluten content and exposure—reviewing methods to screen for coeliac safety. Frontiers in Nutrition. 7:Article 51. doi:10.3389/fnut.2020.00051.

 Web of Science ®Google Scholar

Waltz E. 2016. CRISPR-edited crops free to enter market, skip regulation. Nature Biotechnology. 34: Article 582. doi:10.1038/nbt0616-582.

 Web of Science ®Google Scholar

Kanazawa A, Inaba JI, Shimura H, Otagaki S, Tsukahara S, Matsuzawa A, Bm K, Goto K, Masuta C. 2011. Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants. The Plant Journal. 65:156–168. doi:10.1111/j.1365-313X.2010.04401.x.

 PubMed Web of Science ®Google Scholar

Raju SKK, Shao M-R, Sanchez R, Xu Y-Z, Sandhu A, Graef G, Mackenzie S. 2018. An epigenetic breeding system in soybean for increased yield and stability. Plant Biotechnology Journal. 16:1836–1847. doi:10.1111/pbi.12919.

 PubMed Web of Science ®Google Scholar

Song G-q, Sink KC, Walworth AE, Cook MA, Allison RF, Lang GA. 2013. Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing. Plant Biotechnology Journal. 11:702–708. doi:10.1111/pbi.12060.

 PubMed Web of Science ®Google Scholar

Rogers C, Oldroyd GED. 2014. Synthetic biology approaches to engineering the nitrogen symbiosis in cereals. Journal of Experimental Botany. 65:1939–1946. doi:10.1093/jxb/eru098.

 PubMed Web of Science ®Google Scholar

Fraser PD, Enfissi EMA, Bramley PM. 2009. Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Archives of Biochemistry and Biophysics. 483:196–204. doi:10.1016/j.abb.2008.10.009.

 PubMed Web of Science ®Google Scholar

Atanassova A, Keiper F. 2018. Plant breeding innovation: a global regulatory perspective. Cereal Chemistry. 95:8–16. doi:10.1002/cche.10021.

 Web of Science ®Google Scholar

Menz J, Modrzejewski D, Hartung F, Wilhelm R, Sprink T. 2020. Genome edited crops touch the market: a view on the global development and regulatory environment. Frontiers in Plant Science. 11: Article 586027. doi:10.3389/fpls.2020.586027.

 Web of Science ®Google Scholar

Turnbull C, Lillemo M, Hvoslef-Eide TA. 2021. Global regulation of genetically modified crops amid the gene edited crop boom – A review. Frontiers in Plant Science. 12: Article 630396. doi:10.3389/fpls.2021.630396.

 Web of Science ®Google Scholar

Jones MGK, Fosu-Nyarko J, Iqbal S, Adeel M, Romero-Aldemita R, Arujanan M, Kasai M, Wei X, Prasetya B, Nugroho S, et al. 2022. Enabling trade in gene-edited produce in Asia and australasia: the developing regulatory landscape and future perspectives. Plants. 11. doi:10.3390/plants11192538

 Web of Science ®Google Scholar

Whelan AI, Lema MA. 2015. Regulatory framework for gene editing and other new breeding techniques (NBTs) in Argentina. GM Crops & Food. 6:253–265. doi:10.1080/21645698.2015.1114698.

 PubMed Web of Science ®Google Scholar

Lassoued R, Macall DM, Smyth SJ, Phillips PW, Hesseln H. 2020. How should we regulate products of new breeding techniques? Opinion of surveyed experts in plant biotechnology. Biotechnology Reports. 26: Article e00460. doi:10.1016/j.btre.2020.e00460.

 Google Scholar

OGTR. 2021. Office of the Gene Technology Regulator. Technical review of the Gene Technology Regulations 2001 Decision Regulation Impact Statement; [accessed 28 December 2022]. https://www.ogtr.gov.au/sites/default/files/files/2021-07/decision_ris.pdf.

 Google Scholar

MPI. 2021. Ministry for Primary Industries. Labelling requirements for genetically modified food; [accessed 28 December 2022]. https://www.mpi.govt.nz/food-business/labelling-composition-food-drinks/specific-product-labelling/labelling-requirements-for-genetically-modified-food/.

 Google Scholar

FAO. 2022. Gene editing and agrifood systems. Food and Agriculture Organization of the United Nations, Rome. doi:10.4060/cc3579en

 Google Scholar

FSANZ. 2022. Food Standards Australia New Zealand. GM Foods in Australia and New Zealand; [accessed 28 December 2022]. https://www.foodstandards.gov.au/consumer/gmfood/Documents/GM%20Foods%20in%20Australia%20and%20New%20Zealand.pdf.

 Google Scholar

CFIA. 2016. Canadian Food Inspection Agency. Regulating agricultural biotechnology in Canada: an overview; [accessed 28 December 2022]. https://inspection.canada.ca/plant-varieties/plants-with-novel-traits/general-public/regulating-agricultural-biotechnology/eng/1338187581090/1338188593891.

 Google Scholar

Fritsche S, Poovaiah C, MacRae E, Thorlby G. 2018. A New Zealand perspective on the application and regulation of gene editing. Frontiers in Plant Science. 9:Article 1323. https://www.frontiersin.org/articles/10.3389fpls.2018.01323.

 PubMed Web of Science ®Google Scholar

Royal Society Te Apārangi. 2019. Gene editing: scenarios in the primary industries; [accessed 12 January 2023]. https://www.royalsociety.org.nz/assets/Uploads/Gene-Editing-Scenarios-Primary-industries-DIGITAL.pdf.

 Google Scholar

FSANZ. 2021. Food Standards Australia New Zealand. Genetically modified foods; [accessed 28 December 2022]. https://www.foodstandards.govt.nz/consumer/gmfood/Pages/default.aspx.

 Google Scholar

FDA. 2020. US Food and Drug Administration. Food from new plant varieties; [accessed 28 December 2022]. https://www.fda.gov/food/food-ingredients-packaging/food-new-plant-varieties.

 Google Scholar

Modrzejewski D, Hartung F, Sprink T, Krause D, Kohl C, Wilhelm R. 2019. What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects: a systematic map. Environmental Evidence. 8: Article 27. doi:10.1186/s13750-019-0171-5.

 Web of Science ®Google Scholar

Jorasch P. 2020. Potential, challenges, and threats for the application of new breeding techniques by the private plant breeding sector in the EU. Frontiers in Plant Science. 11:Article 582011. doi:10.3389/fpls.2020.582011.

 PubMed Web of Science ®Google Scholar

Lema M. 2021. Breaking barriers with breeding: a primer on new breeding innovations for food security. ISAAA Brief. 56:23–31.

 Google Scholar