Can gene editing reduce postharvest waste and loss of fruit, vegetables, and ornamentals?

Abstract

Postharvest waste and loss of horticultural crops exacerbates the agricultural problems facing humankind and will continue to do so in the next decade. Fruits and vegetables provide us with a vast spectrum of healthful nutrients, and along with ornamentals, enrich our lives with a wide array of pleasant sensory experiences. These commodities are, however, highly perishable. Approximately 33% of the produce that is harvested is never consumed since these products naturally have a short shelf-life, which leads to postharvest loss and waste. This loss, however, could be reduced by breeding new crops that retain desirable traits and accrue less damage over the course of long supply chains. New gene-editing tools promise the rapid and inexpensive production of new varieties of crops with enhanced traits more easily than was previously possible. Our aim in this review is to critically evaluate gene editing as a tool to modify the biological pathways that determine fruit, vegetable, and ornamental quality, especially after storage. We provide brief and accessible overviews of both the CRISPR–Cas9 method and the produce supply chain. Next, we survey the literature of the last 30 years, to catalog genes that control or regulate quality or senescence traits that are “ripe” for gene editing. Finally, we discuss barriers to implementing gene editing for postharvest, from the limitations of experimental methods to international policy. We conclude that in spite of the hurdles that remain, gene editing of produce and ornamentals will likely have a measurable impact on reducing postharvest loss and waste in the next 5–10 years.

References

[1] Wolter, F., Schindele, P. & Puchta, H. Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites. BMC Plant Biol. 19, 176 (2019).

[2] Pradhan, P., Fischer, G., van Velthuizen, H., Reusser, D. E. & Kropp, J. P. Closing yield gaps: how sustainable can we be? PLoS ONE 10, e0129487 (2015).

[3] Alfa, H. H. & Arroo, R. R. J. Over 3 decades of research on dietary flavonoid antioxidants and cancer prevention: What have we achieved? Phytochem Rev. 18, 989-1004 (2019).

[4] Fraga, C. G., Croft, K. D., Kennedy, D. O. & Tomas-Barberan, F. A. The effects of polyphenols and other bioactives on human health. Food Funct. 10, 514-528 (2019).

[5] Liskova, A. et al. Dietary phytochemicals targeting cancer stem cells. Molecules 24, 899 (2019).

[6] Saiwal, N., Dahiya, M. & Dureja, H. Nutraceutical insight into Vegetables and their Potential for Nutrition Mediated Healthcare. Curr. Nutr. Food Sci. 15, 441-453 (2019).

[7] FAO. Global Food Losses and Food Waste - Extent, Causes and Prevention. http://www.fao.org/docrep/014/mb060e/mb060e00.pdf (FAO, 2011).

[8] Porat, R., Lichter, A., Terry, L. A., Harker, R. & Buzby, J. Postharvest losses of fruit and vegetables during retail and in consumers’ homes: quantifications, causes, and means of prevention. Postharvest Biol. Tec. 139, 135-149 (2018).

[9] Kitinoja, L., Tokala, V. V. & Brondy, A. A review of global postharvest loss assessments in plant-based food crops: recent findings and measurement gaps. J. Postharvest Technol. 6, 1-15 (2018).

[10] Bahadur, K. C. K. et al. When too much isn’t enough: does current food production meet global nutritional needs? PLoS ONE 13, e0205683 (2018).

[11] HPLE. Food Losses and Waste in the Context of Sustainable Food Systems: A Report. http://www.fao.org/3/a-i3901e.pdf (2014).

[12] Kader, A. A. Increasing food availability by reducing postharvest losses of fresh produce. In Proc. 5th International Postharvest Symposium, Vols 1-3 2169-2175 (2005).

[13] NASEM (ed). in Reducing Impacts of Food Loss and Waste: Proceedings of a Workshop (Committee on Reducing Food Loss and Waste: A Workshop on Impacts; Science and Technology for Sustainability Program; Policy and Global Affairs. National Academies of Sciences, Engineering, and Medicine, 2019).

[14] ReFED. A Roadmap to Reduce U.S. Food Waste by 20 Percent (ReFED, 2016).

[15] Kader, A. A. in Postharvest Technology of Horticultural Crops Vol. Publication 3529 (ed Kader, A. A. ) 39-48 (University of California Agriculture and Natural, 2011).

[16] Hewett, E. W. Postharvest Innovation: Current Trends and Future Challenges in the Global Market. In Southeast Asia Symposium on Quality Management in Postharvest Systems and Asia Pacific Symposium on Postharvest Quality Management of Root and Tuber Crops> Vol. 989, 25-37 (2013).

[17] Hewett, E. W. Postharvest research for quality horticultural products. Middle East Hortic. Summit 1051, 63-70 (2014).

[18] Kader, A. A. & Saltveit, M. E. Postharvest Physiology and Pathology of Vegetables (eds Bartz, J. A. & Brecht, J. K.) (CRC Press, 2002).

[19] Kays, S. J. Quality maintenance of fresh produce. Acta Hortic. 875, 27-31 (2010).

[20] Saltveit, M. E. & Morris, L. L. Chilling Injury of Horticultural Crops (ed Wang, C. Y.) 3-15 (CRC Press, 1990).

[21] Bower, J. H., Blasi, W. V. & Mitcham, E. J. Effect of ethylene in the storage environment on quality of ‘Bartlett pears’. Postharvest Biol. Tec. 28, 371-379 (2003).

[22] El-Kereamy, A. et al. Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant. 119, 175-182 (2003).

[23] Gong, Y. P., Fan, X. T. & Mattheis, J. P. Responses of ‘Bing’ and ‘Rainier’ sweet cherries to ethylene and 1-methylcyclopropene. J. Am. Soc. Hortic. Sci. 127, 831-835 (2002).

[24] Friedman, H. in Plant Breeding Reviews (ed Goldman, I.) Ch 3, 61-94 (Wiley, 2020).

[25] Liu, Z. Y. & Jiang, W. B. Lignin deposition and effect of postharvest treatment on lignification of green asparagus (Asparagus officinalis L.). Plant Growth Regul. 48, 187-193 (2006).

[26] Ritenour, M. A., Ahrens, M. J. & Saltveit, M. E. Effects of temperature on ethylene-induced phenylalanine ammonia-lyase activity and russet spotting in harvested iceberg lettuce. J. Am. Soc. Hortic. Sci. 120, 84-87 (1995).

[27] Chalutz, E., Devay, J. E. & Maxie, E. C. Ethylene-induced isocoumarin formation in carrot root tissue. Plant Physiol. 44, 235 (1969).

[28] Albornoz, K., Cantwell, M. I., Zhang, L. & Beckles, D. M. Integrative analysis of postharvest chilling injury in cherry tomato fruit reveals contrapuntal spatio-temporal responses to ripening and cold stress. Sci. Rep. 9, 2795 (2019).

[29] Jasin, M. Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet. 12, 224-228 (1996).

[30] Carroll, D. Genome engineering with zinc-finger nucleases. Genetics 188, 773-782 (2011).

[31] Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278 (2014).

[32] Satheesh, V., Zhang, H., Wang, X. T. & Lei, M. G. Precise editing of plant genomes - prospects and challenges. Semin. Cell Dev. Biol. 96, 115-123 (2019).

[33] Pandey, P. K. et al. Versatile and multifaceted CRISPR/Cas gene editing tool for plant research. Semin. Cell Dev. Biol. 96, 107-114 (2019).

[34] Enciso-Rodriguez, F. et al. Overcoming self-incompatibility in diploid potato using CRISPR-Cas9. Front. Plant Sci. 10, 376 (2019).

[35] Nadakuduti, S. S., Buell, C. R., Voytas, D. F., Starker, C. G. & Douches, D. S. Genome editing for crop improvement - applications in clonally propagated polyploids with a focus on potato (Solanum tuberosum L.). Front. Plant Sci. 9, 1607 (2018).

[36] Sharma, S., Kaur, R. & Singh, A. Recent advances in CRISPR/Cas mediated genome editing for crop improvement. Plant Biotechnol. Rep. 11, 193-207 (2017).

[37] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).

[38] Chen, K. L., Wang, Y. P., Zhang, R., Zhang, H. W. & Gao, C. X. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667-697 (2019).

[39] Metje-Sprink, J., Menz, J., Modrzejewski, D. & Sprink, T. DNA-free genome editing: past, present and future. Front. Plant Sci. 9, 1957 (2019).

[40] Puchta, H. The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J. Exp. Bot. 56, 1-14 (2005).

[41] Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells (vol 19, pg 770, 2018). Nat. Rev. Genet. 19, 801- 801 (2018).

[42] Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441 (2017).

[43] Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438 (2017).

[44] Veillet, F. et al. The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato. Plant Cell Rep. 38, 1065-1080 (2019).

[45] Veillet, F. et al. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor. Int. J. Mol. Sci. 20, 402 (2019).

[46] Decaestecker, W. et al. CRISPR-TSKO: a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis. Plant Cell 31, 2868-2887 (2019).

[47] Brand, L. et al. A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol. 141, 1194-1204 (2006).

[48] Wang, X. et al. An inducible genome editing system for plants. Nat. Plants 6, 766-772 (2020).

[49] Zhang, H. W. et al. Genome editing of upstream open reading frames enables translational control in plants. Nat. Biotechnol. 36, 894 (2018).

[50] Li, T. D. et al. Domestication of wild tomato is accelerated by genome editing. Nat. Biotechnol. 36, 1160 (2018).

[51] Zsogon, A. et al. De novo domestication of wild tomato using genome editing. Nat. Biotechnol. 36, 1211 (2018).

[52] Cheng, J. F. et al. Downregulation of RdDM during strawberry fruit ripening. Genome Biol. 19, 212 (2018).

[53] Gao, Y. et al. Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants. Hortic. Res. 6, 39 (2019).

[54] Huang, H. et al. Global increase in DNA methylation during orange fruit development and ripening. Proc. Natl Acad. Sci. USA 116, 1430-1436 (2019).

[55] Papikian, A., Liu, W. L., Gallego-Bartolome, J . & Jacobsen, S. E. Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems. Nat. Commun. 10, 729 (2019).

[56] Wang, W. et al. Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. Crispr J. 1, 65-74 (2018).

[57] Schaeffer, S. M. & Nakata, P. A. CRISPR/Cas9-mediated genome editing and gene replacement in plants: transitioning from lab to field. Plant Sci. 240, 130-142 (2015).

[58] Eshed, Y. & Lippman, Z. B. Revolutions in agriculture chart a course for targeted breeding of old and new crops. Science 366, 705 (2019).

[59] Hamant, O. Plant scientists can’t ignore Jevons paradox anymore. Nat. Plants 6, 720-722 (2020).

[60] Haynes-Maslow, L., Parsons, S. E., Wheeler, S. B. & Leone, L. A. A qualitative study of perceived barriers to fruit and vegetable consumption among low-income populations, North Carolina, 2011. Prev Chronic Dis. 10, E34 (2013).

[61] Lucan, S. C., Barg, F. K. & Long, J. A. Promoters and barriers to fruit, vegetable, and fast-food consumption among urban, low-income african americans-a qualitative approach. Am. J. Public Health 100, 631-635 (2010).

[62] Miller, V. et al. Availability, affordability, and consumption of fruits and vegetables in 18 countries across income levels: findings from the Prospective Urban Rural Epidemiology (PURE) study. Lancet Glob. Health 4, E695-E703 (2016).

[63] Mook, K., Laraia, B. A., Oddo, V. M. & Jones-Smith, J. C. Food security status and barriers to fruit and vegetable consumption in two economically deprived communities of Oakland, California, 2013-2014. Prev. Chronic Dis. 13, 150402 (2016).

[64] Tallant, A., Rettig, M. & Tennyson, S. Barriers and facilitators for fruit and vegetable consumption among adults in rural counties. Fam. Consum. Sci. Res. J. 47, 87-100 (2018).

[65] Possingham, J. V. Fruit and vegetable quality in 21st century-the influence of Japan. J. Jpn Soc. Hortic. Sci. 67, 1250-1254 (1998).

[66] Florkowski, W. J. et al. in Postharvest Handling 3rd edn (edsFlorkowski, W. J., Shewfelt, R. L., Brueckner, B. & Prussia, S. E.) 147-166 (Academic Press, 2014).

[67] Kader, A. A. Flavor quality of fruits and vegetables. J. Sci. Food Agr. 88, 1863-1868 (2008).

[68] Florkowski, W., Shewfelt, R. & Prussia, S. E. in Postharvest Handling- A Systems Approach (eds Florkowski, W. J., Shewfelt, R. L. & Prussia, S. E. ) 592 (Academic Press, 2014).

[69] Beckles, D. M. Factors affecting the postharvest soluble solids and sugar content of tomato (Solanum lycopersicum L.) fruit. Postharvest Biol. Tec. 63, 129-140 (2012).

[70] Barrett, D. M., Beaulieu, J. C. & Shewfelt, R. Color, flavor, texture, and nutritional quality of fresh-cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing. Crit. Rev. Food Sci. 50, 369-389 (2010).

[71] Mogren, L. et al. The hurdle approach-a holistic concept for controlling food safety risks associated with pathogenic bacterial contamination of leafy green vegetables. A review. Front. Microbiol. 9, 1965 (2018).

[72] Breslin, PaulA. S. An evolutionary perspective on food and human taste. Curr. Biol. 23, R409-R418 (2013).

[73] Diehl, D. C. et al. Exploring produce industry attitudes: relationships between postharvest handling, fruit flavor, and consumer purchasing. Horttechnology 23, 642-650 (2013).

[74] Hebrok, M. & Boks, C. Household food waste: drivers and potential intervention points for design. An extensive review. J. Clean. Prod. 151, 380-392 (2017).

[75] Jaeger, S. R. et al. Consumers’ visual attention to fruit defects and disorders: a case study with apple images. Postharvest Biol. Tec. 116, 36-44 (2016).

[76] Hamilton, A. J., Lycett, G. W. & Grierson, D. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, https://doi.org/10.1038/346284a0 (1990).

[77] López-Gómez, R. et al. Ripening in papaya fruit is altered by ACC oxidase cosuppression. Transgenic Res 18, 89-97 (2009).

[78] Nagata, M. et al. Modification of tomato fruit ripening by transformation with sense or antisense chimeric 1-aminocyclopropane-1-carboxylate synthase genes. Acta Hortic. 394, 213-218 (1995).

[79] Oeller, P. W., Min-Wong, L., Taylor, L. P., Pike, D. A. & Theologis, A. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254, https://doi.org/10.1126/science.1925603 (1991).

[80] Xiong, A.-S. et al. Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Rep. 23, 639-646 (2005).

[81] Xue, C. et al. Genome wide identification and functional characterization of strawberry pectin methylesterases related to fruit softening. BMC Plant Biol. 20, 13 (2020).

[82] Atkinson, R. G. et al. Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line. J. Exp. Bot. 62, 3821-3835 (2011).

[83] Ayub, R. et al. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat. Biotechnol. 14, 862-866 (1996).

[84] Lu, P. T. et al. Genome encode analyses reveal the basis of convergent evolution of fleshy fruit ripening. Nat. Plants 4, 784-791 (2018).

[85] Yu, Q.-h et al. CRISPR/Cas9-induced targeted mutagenesis and gene replacement to generate long-shelf life tomato lines. Sci. Rep. 7, 11874 (2017).

[86] Wang, R. F. et al. Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis. Sci. Rep. 9, 1696 (2019).

[87] Ito, Y., Nishizawa-Yokoi, A., Endo, M., Mikami, M. & Toki, S. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruitripening. Biochem. Biophys. Res. Commun. 467, 76-82 (2015).

[88] MarketWatch. Floriculture Market 2019 Global Industry Size, Share, Forecasts Analysis, Company Profiles, Competitive Landscape and Key Regions 2024. Available at 360 Research Reports. https://www.marketwatch.com/press-release/floriculture-market-2019-global-industry-size-share-forecasts-analysis-company-profiles-competitive-landscape-and-key-regions-2024-available-at-360-research-reports-2019-09-26 (2019).

[89] Xia, Y., Deng, X., Zhou, P., Shima, K. & Teixeira da Silva, J. A. in Floriculture, Ornamental and Plant Biotechnology - Advances and Topical Issues Vol. 1-4 (ed Teixeira da Silva, J. A. ) (Global Science Books, Ltd., 2006).

[90] Pranuthi, P., Suseela, T., Swami, D. V., Salomi Suneetha, D. R. & Sudha Vani, V. Effect of packing and storage conditions on physiological loss in weight, diameter of the flower, electrolyte leakage in extending the vase life of Carnation cv. Kiro. Int. J. Curr. Microbiol. Appl. Sci. 7, 1278-1287 (2018).

[91] Mamias, S. The Floriculture Supply Chain: Characteristics And Prospects (Union Fleurs-International Flower Trade Association, The Netherlands, 2018).

[92] Fredenburgh, F. The 4,000 mile flower delivery. http://www.bbc.com/future/bespoke/made-on-earth/the-new-roots-of-the-flower-trade/ (2019).

[93] Savin, K. W. et al. Antisense Acc oxidase RNA delays carnation petal senescence. Hortscience 30, 970-972 (1995).

[94] Woodson, W. R., Park, K. Y., Drory, A., Larsen, P. B. & Wang, H. Expression of ethylene biosynthetic-pathway transcripts in senescing carnation flowers. Plant Physiol. 99, 526-532 (1992).

[95] Wu, M. J., Vandoorn, W. G. & Reid, M. S. Variation in the senescence of carnation (Dianthus-Caryophyllus L) Cultivars .1. Comparison of flower life, respiration and ethylene biosynthesis. Sci. Hortic. 48, 99-107 (1991).

[96] Chang, X. X. et al. A petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence. PLoS ONE 9, e88320 (2014).

[97] Tan, Y. Y. et al. PhGRL2 protein, interacting with PhACO1, is involved in flower senescence in the petunia. Mol. Plant. 7, 1384-1387 (2014).

[98] Wang, H. et al. Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor. PLoS ONE 8, e65800 (2013).

[99] Xu, J. et al. CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity. Plant Biotechnol. J. 18, 287-297 (2020).

[100] Reid, M. S. & Jiang, C.-Z. in Horticultural Reviews Vol. 40 (ed. Janick, J.) 1-44 (John Wiley & Sons, Inc., 2012).

[101] Lara, I., Belge, B. & Goulao, L. F. The fruit cuticle as a modulator of postharvest quality. Postharvest Biol. Technol. 87, 103-112 (2014).

[102] Lara, I., Heredia, A. & Dominguez, E. Shelf life potential and the fruit cuticle: the unexpected player. Front. Plant Sci. 10, 770 (2019).

[103] Benichou, M. et al. in Postharvest Biology and Technology of Temperate Fruits (eds Mir, S. A., Shah, M. A. & Mir, M. M.) 77-100 (Spring International Publishing, 2018).

[104] Petit, J., Bres, C., Mauxion, J. P., Bakan, B. & Rothan, C. Breeding for cuticle-associated traits in crop species: traits, targets, and strategies. J. Exp. Bot. 68, 5369-5387 (2017).

[105] Rid, M., Markheiser, A., Hoffmann, C. & Gross, J. Waxy bloom on grape berry surface is one important factor for oviposition of European grapevine moths. J. Pest Sci. 91, 1225-1239 (2018).

[106] Loypimai, P., Paewboonsom, S., Damerow, L. & Blanke, M. M. The wax bloom on blueberry: application of luster sensor technology to assess glossiness and the effect of polishing as a fruit quality parameter. J. Appl Bot. Food Qual. 90, 154-158 (2017).

[107] Storey, R. & Price, W. E. Microstructure of the skin of d’Agen plums. Sci. Hortic. 81, 279-286 (1999).

[108] Trivedi, P. et al. Developmental and environmental regulation of cuticular wax biosynthesis in fleshy fruits. Front. Plant Sci 10, 431 (2019).

[109] Bruggenwirth, M. & Knoche, M. Cell wall swelling, fracture mode, and the mechanical properties of cherry fruit skins are closely related. Planta 245, 765-777 (2017).

[110] Knoche, M. & Grimm, E. Surface moisture induces microcracks in the cuticle of ‘Golden Delicious’ apple. Hortscience 43, 1929-1931 (2008).

[111] Dominguez, E. et al. Tomato fruit continues growing while ripening, affecting cuticle properties and cracking. Physiol. Plant. 146, 473-486 (2012).

[112] Giovannoni, J . Molecular biology of fruit maturation and ripening. Annu Rev. Plant Phys. 52, 725-749 (2001).

[113] Cantu, D. et al. Ripening-regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR but not RIN or ethylene. Plant Physiol. 150, 1434-1449 (2009).

[114] Payasi, A., Mishra, N. N., Chaves, A. L. S. & Singh, R. Biochemistry of fruit softening: an overview. Physiol. Mol. Biol. Plants 15, 103-113 (2009).

[115] Posé, S. et al. Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits. J. Exp. Bot. 64, 3803-3815 (2013).

[116] Brummell, D. A. & Harpster, M. H. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311-340 (2001).

[117] Jiménez-Bermudez, S. et al. Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiol. 128, 751-759 (2002).

[118] Santiago-Domenech, N. et al. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening. J. Exp. Bot. 59, 2769-2779 (2008).

[119] Yang, L. et al. Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould. Plant Biotechnol. J. 15, 1544-1555 (2017).

[120] Tieman, D. M., Harriman, R. W., Ramamohan, G. & Handa, A. K. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell. 4, 667-679 (1992).

[121] Wang, S. Y., Zhou, Q., Zhou, X., Zhang, F. & Ji, S. J. Ethylene plays an important role in the softening and sucrose metabolism of blueberries postharvest. Food Chem. 310, 125965 (2020).

[122] Wen, B., Strom, A., Tasker, A., West, G. & Tucker, G. A. Effect of silencing the two major tomato fruit pectin methylesterase isoforms on cell wall pectin metabolism. Plant Biol. 15, 1025-1032 (2013).

[123] Smith, D. L., Abbott, J. A. & Gross, K. C. Down-regulation of tomato beta-galactosidase 4 results in decreased fruit softening. Plant Physiol. 129, 1755-1762 (2002).

[124] Moctezuma, E., Smith, D. L. & Gross, K. C. Antisense suppression of a beta-galactosidase gene (TBG6) in tomato increases fruit cracking. J. Exp. Bot. 54, 2025-2033 (2003).

[125] Carey, A. T. et al. Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits. J. Exp. Bot. 52, 663-668 (2001).

[126] Uluisik, S. et al. Genetic improvement of tomato by targeted control of fruit softening. Nat. Biotechnol. 34, 950 (2016).

[127] Smith, C. J. S. et al. Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol. Biol. 14, 369-379 (1990).

[128] Wormit, A. & Usadel, B. The multifaceted role of pectin methylesterase inhibitors (PMEIs). Int. J. Mol. Sci. 19, 2878 (2018).

[129] Encinas-Basurto, D. et al. Alterations in volatile metabolites profile of fresh tomatoes in response to Alternaria alternata (Fr.) Keissl. 1912 infection. Chil. J. Agr. Res. 77, 194-201 (2017).

[130] Peris, J. E., Rodriguez, A., Pena, L. & Fedriani, J. M. Fungal infestation boosts fruit aroma and fruit removal by mammals and birds. Sci. Rep. 7, 5646 (2017).

[131] Rodrigo, J. E. P., Laffitte, J. M. F. & García, L. P. Los mamíferos frugívoros prefieren frutos de cítricos infectados por Penicillium:¿ se equivocaba Janzen? Rev. Ecosistemas 24, 5-13 (2015).

[132] Rodriguez, A., Alquezar, B. & Pena, L. Fruit aromas in mature fleshy fruits as signals of readiness for predation and seed dispersal. N. Phytol. 197, 36-48 (2013).

[133] Vilanova, L. et al. Differential contribution of the two major polygalacturonases from Penicillium digitatum to virulence towards citrus fruit. Int J. Food Microbiol. 282, 16-23 (2018).

[134] Silva, C. J. et al. Tomato fruit susceptibility to fungal disease can be uncoupled from ripening by suppressing susceptibility factors. Preprint at https://www.biorxiv.org/content/10.1101/2020.06.03.132829v1.full (2020).

[135] Porat, R., McCollum, T. G., Vinokur, V. & Droby, S. Effects of various elicitors on the transcription of a beta-1,3-endoglucanase gene in citrus fruit. J. Phytopathol. 150, 70-75 (2002).

[136] Distefano, G. et al. Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection. Transgenic Res. 17, 873-879 (2008).

[137] Muccilli, V. et al. Substantial equivalence of a transgenic lemon fruit showing postharvest fungal pathogens resistance. J. Agr. Food Chem. 68, 3806-3816 (2020).

[138] Ballester, A. R. et al. Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance. Mol. Plant Pathol. 12, 879-897 (2011).

[139] Gonzalez-Candelas, L., Alamar, S., Sanchez-Torres, P., Zacarias, L. & Marcos, J. F. A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection. BMC Plant Biol. 10, 194 (2010).

[140] Chen, J. Y., Shen, Y. T., Chen, C. Y. & Wan, C. P. Inhibition of key citrus postharvest fungal strains by plant extracts in vitro and in vivo: a review. Plants 8, 26 (2019).

[141] Sanzani, S. M., Schena, L. & Ippolito, A. Effectiveness of phenolic compounds against citrus green mould. Molecules 19, 12500-12508 (2014).

[142] Khoo, H. E., Azlan, A., Kong, K. & Ismail, A. Phytochemicals and medicinal properties of indigenous tropical fruits with potential for commercial development. Evid. Based Compl. Alt. 2016, 7591951 (2016).

[143] Zhang, Y. et al. Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold. Curr. Biol. 23, 1094-1100 (2013).

[144] Zhang, Y. et al. Different reactive oxygen species scavenging properties of flavonoids determine their abilities to extend the shelf life of tomato. Plant Physiol. 169, 1568-1583 (2015).

[145] Bassolino, L. et al. Accumulation of anthocyanins in tomato skin extends shelf life. N. Phytol. 200, 650-655 (2013).

[146] Mes, P. J., Boches, P., Myers, J. R. & Durst, R. Characterization of tomatoes expressing anthocyanin in the fruit. J. Am. Soc. Hortic. Sci. 133, 262-269 (2008).

[147] Nookaraju, A. et al. Molecular approaches for enhancing sweetness in fruits and vegetables. Sci. Hortic. 127, 1-15 (2010).

[148] Beckles, D. M., Hong, N., Stamova, L. & Luengwilai, K. Biochemical factors contributing to tomato fruit sugar content: a review. Fruits 67, 49-64 (2012).

[149] Beauvoit, B. et al. Putting primary metabolism into perspective to obtain better fruits. Ann. Bot. 122, 1-21 (2018).

[150] Fridman, E., Carrari, F., Liu, Y. S., Fernie, A. R. & Zamir, D. Zooming in on a quantitative trait for tomato yield using interspecific introgressions. Science 305, 1786-1789 (2004).

[151] Petreikov, M. et al. Carbohydrate balance and accumulation during development of near-isogenic tomato lines differing in the AGPase-L1 allele. J. Am. Soc. Hortic. Sci. 134, 134-140 (2009).

[152] Centeno, D. C. et al. Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening. Plant Cell. 23, 162-184 (2011).

[153] Powell, A. L. T. et al. Uniform ripening encodes a golden 2-like transcription factor regulating tomato fruit chloroplast development. Science 336, 1711-1715 (2012).

[154] Sagar, M. et al. SlARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development. Plant Physiol. 161, 1362-1374 (2013).

[155] Sagor, G. H. M. et al. A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene. Plant Biotechnol. J. 14, 1116-1126 (2016).

[156] Shammai, A. et al. Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit. Plant J. 96, 343-357 (2018).

[157] Yuan, Y. et al. (2018). SlARF10, an auxin response factor, is involved in chlorophyll and sugaraccumulation during tomato fruit development. J. Exp. Bot. 69, 5507-5518 (2018).

[158] Cheng, J. T. et al. Overexpression of the tonoplast sugar transporter CmTST2 in melon fruit increases sugar accumulation. J. Exp. Bot. 69, 511-523 (2018).

[159] Smith, A. M., Denyer, K. & Martin, C. R. What controls the amount and structure of starch in storage organs. Plant Physiol. 107, 673-677 (1995).

[160] Andersson, M. et al. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep. 36, 117-128 (2017).

[161] Beckles, D. M. & Thitisaksakul, M. in Encyclopedia of Biotechnology in Agriculture and Food (eds Heldman, D., Hoover, D. & Wheeler, M.) 8 (CRC Press, Boca Raton, 2014).

[162] Wang, H. X. et al. CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (Ipomoea Batatas) for the improvement of starch quality. Int. J. Mol. Sci. 20, 4702 (2019).

[163] Li, H. T., Gidley, M. J. & Dhital, S. High-amylose starches to bridge the “fiber gap”: development, structure, and nutritional functionality. Compr. Rev. Food Sci. Food Saf. 18, 362-379 (2019).

[164] Jobling, S. Improving starch for food and industrial applications. Curr. Opin. Plant Biol. 7, 210-218 (2004).

[165] Hofvander, P., Andersson, M., Larsson, C. T. & Larsson, H. Field performance and starch characteristics of high-amylose potatoes obtained by antisense gene targeting of two branching enzymes. Plant Biotechnol. J. 2, 311-320 (2004).

[166] Jobling, S. A., Westcott, R. J., Tayal, A., Jeffcoat, R. & Schwall, G. P. Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes. Nat. Biotechnol. 20, 295-299 (2002).

[167] Schwall, G. P. et al. Production of very-high-amylose potato starch by inhibition of SBE A and B. Nat. Biotechnol. 18, 551-554 (2000).

[168] Visser, R. G. F. et al. Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol. Gen. Genet. 225, 289-296 (1991).

[169] Dong, S. Y. & Beckles, D. M. Dynamic changes in the starch-sugar interconversion within plant source and sink tissues promote a better abiotic stress response. J. Plant Physiol. 234, 80-93 (2019).

[170] Luengwilai, K. & Beckles, D. M. Starch granules in tomato fruit show a complex pattern of degradation. J. Agric. Food Chem. 57, 8480-8487 (2009).

[171] McKibbin, R. S. et al. Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1. Plant Biotechnol. J. 4, 409-418 (2006).

[172] Regierer, B. et al. Starch content and yield increase as a result of altering adenylate pools in transgenic plants. Nat. Biotechnol. 20, 1256-1260 (2002).

[173] Burton, W. G. The sugar balance in some British potato varieties during storage. II. The effects of tuber age, previous storage temperature, and intermittent refrigeration upon low-temperature sweetening. Eur. Potato J. 12, 81-95 (1969).

[174] Amir, J., Kahn, V. & Unterman, M. Respiration, Atp level, and sugar accumulation in potato-tubers during storage at 4-degrees. Phytochemistry 16, 1495-1498 (1977).

[175] Mottram, D. S., Wedzicha, B. L. & Dodson, A. T. Acrylamide is formed in the Maillard reaction. Nature 419, 448-449 (2002).

[176] Gokmen, V. & Palazoglu, T. K. Acrylamide formation in foods during thermal processing with a focus on frying. Food Bioprocess Technol. 1, 35-42 (2008).

[177] Morrell, S. & Aprees, T. Control of the hexose content of potato-tubers. Phytochemistry 25, 1073-1076 (1986).

[178] Lorberth, R., Ritte, G., Willmitzer, L. & Kossmann, J. Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat. Biotechnol. 16, 473-477 (1998).

[179] Cochrane, M. P., Duffus, C. M., Allison, M. J. & Mackay, G. R. Amylolytic activity in stored potato-tubers .2. The effect of low-temperature storage on the activities of alpha-amylase and beta-amylase and alpha-glucosidase in potato-tubers. Potato Res. 34, 333-341 (1991).

[180] Claassen, P. A. M., Budde, M. A. W. & Vancalker, M. H. Increase in phosphorylase-activity during cold-induced sugar accumulation in potato-tubers. Potato Res. 36, 205-217 (1993).

[181] Zrenner, R., Schuler, K. & Sonnewald, U. Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers. Planta 198, 246-252 (1996).

[182] Yu, X. Y. et al. Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development. J. Exp. Bot. 59, 2969-2977 (2008).

[183] Cheng, M., Lowe, B. A., Spencer, T. M., Ye, X. D. & Armstrong, C. L. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. Vitr. Cell Dev. 40, 31-45 (2004).

[184] Bhaskar, P. B. et al. Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato. Plant Physiol. 154, 939-948 (2010).

[185] Richael, C. et al. POTATO CULTIVAR W8. USA patent (2015).

[186] Klein, D., Gkisakis, V., Krumbein, A., Livieratos, I. & Kopke, U. Old and endangered tomato cultivars under organic greenhouse production: effect of harvest time on flavour profile and consumer acceptance. Int J. Food Sci. Technol. 45, 2250-2257 (2010).

[187] Causse, M., Buret, M., Robini, K. & Verschave, P. Inheritance of nutritional and sensory quality traits in fresh market tomato and relation to consumer preferences. J. Food Sci. 68, 2342-2350 (2003).

[188] Causse, M. et al. Consumer preferences for fresh tomato at the european scale: a common segmentation on taste and firmness. J. Food Sci. 75, S531-S541 (2010).

[189] Sinesio, F. et al. Sensory quality of fresh french and dutch market tomatoes: a preference mapping study with italian consumers. J. Food Sci. 75, S55-S67 (2010).

[190] Zhang, B. et al. Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation. Proc. Natl Acad. Sci. USA 113, 12580-12585 (2016).

[191] Klee, H. J. & Tieman, D. M. The genetics of fruit flavour preferences. Nat. Rev. Genet. 19, 347-356 (2018).

[192] Ferrão, L. F. V. et al. Genome-wide association of volatiles reveals candidate loci for blueberry flavor. N. Phytol. 226, 1725-1737 (2020).

[193] Tieman, D. et al. The chemical interactions underlying tomato flavor preferences. Curr. Biol. 22, 1035-1039 (2012).

[194] Zhao, J. T. et al. Meta-analysis of genome-wide association studies provides insights into genetic control of tomato flavor. Nat. Commun. 10, 1534 (2019).

[195] Mathieu, S. et al. Flavour compounds in tomato fruits: identification of loci and potential pathways affecting volatile composition. J. Exp. Bot. 60, 325-337 (2009).

[196] Galili, G., Galili, S., Lewinsohn, E. & Tadmor, Y. Genetic, molecular, and genomic approaches to improve the value of plant foods and feeds. Crit. Rev. Plant Sci. 21, 167-204 (2002).

[197] He, Q. & Luo, Y. Enzymatic browning and its control in fresh-cut produce Stewart Postharvest Rev. 6, 1-7 (2007).

[198] González, M. N. et al. Reduced enzymatic browning in potato tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein complexes delivery of the CRISPR/Cas9 system. Front Plant Sci. 10, 1649- 1649 (2020).

[199] Armstrong, J. & Lane, W. D. Genetically modified reduced-browning fruit producing plant and produce fruit thereof and method of obtaining such. USA patent (2009).

[200] Brooks, J. Consumer feedback: The world is ready for Arctic® apples. Availabe online: https://www.okspecialtyfruits.com/consumer-feedback-theworld-is-ready-for-arctic-apples/ (accessed on May 30th), (Okanagan Specialty Fruits Inc. Summerland, British Columbia, 2012).

[201] Waltz, E. Gene-edited CRISPR mushroom escapes US regulation. Nature 532, 293 (2016).

[202] Beckles, D. M. & Thitisaksakul, M. How environmental stress affects starch composition and functionality in cereal endosperm. Starch 66, 58-71 (2014).

[203] Brandi, M. T., Cox, C. E. & Teplitski, M. Salmonella interactions with plants and their associated microbiota. Phytopathology 103, 316-325 (2013).

[204] Machado-Moreira, B., Richards, K., Brennan, F., Abram, F. & Burgess, C. M. Microbial contamination of fresh produce: what, where, and how? Compr. Rev. Food Sci. Food Saf. 18, 1727-1750 (2019).

[205] Haymaker, J. et al. Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S. Environ. Res. 172, 630-636 (2019).

[206] Olaimat, A. N. & Holley, R. A. Factors influencing the microbial safety of fresh produce: a review. Food Microbiol. 32, 1-19 (2012).

[207] Teplitski, M., Barak, J. D. & Schneider, K. R. Human enteric pathogens in produce: un-answered ecological questions with direct implications for food safety. Curr. Opin. Biotechnol. 20, 166-171 (2009).

[208] Montano, J. et al. Salmonella enterica serovar typhimurium 14028s genomic regions required for colonization of lettuce leaves. Front. Microbiol. 11, 6 (2020).

[209] Jacob, C. & Melotto, M. Human pathogen colonization of lettuce dependent upon plant genotype and defense response activation. Front. Plant Sci. 10, 1769 (2020).

[210] Melotto, M. et al. Breeding crops for enhanced food safety. Front. Plant Sci. 11, https://doi.org/10.3389/fpls.2020.00428 (2020).

[211] Albornoz, K. et al. Investigating postharvest chilling injury in tomato (Solanum lycopersicum L.) fruit using magnetic resonance imaging and 5-azacytidine, a hypomethylation agent. Acta Hortic. 1278, https://doi.org/10.17660/ActaHortic.12020.11278.17635 (2020).

[212] Biswas, P., East, A. R., Hewett, E. W. & Heyes, J. A. in Horticultural Reviews Vol. 44 (ed Jules Janick) (John Wiley & Sons, 2017).

[213] Liu, Y. D. et al. SlGRAS4 mediates a novel regulatory pathway promoting chilling tolerance in tomato. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13328 (2020).

[214] Ferguson, I., Volz, R. & Woolf, A. Preharvest factors affecting physiological disorders of fruit. Postharvest Biol. Tec. 15, 255-262 (1999).

[215] de Freitas, S. T., Jiang, C. Z. & Mitcham, E. J. Mechanisms involved in calcium deficiency development in tomato fruit in response to gibberellins. J. Plant Growth Regul. 31, 221-234 (2012).

[216] Kader, A. A. in Postharvest Techology of Horticulture Crops (ed Kader, A. A. ) 279-285 (University of California ANR Natural Resources, 2002).

[217] Kader, A. A. in Postharvest Technology of Horticultural Crops (ed Kader, A. A. ) 39-48 (University of California, Agriculture and Natural Resources, 2002).

[218] Thompson, A. K. Fruit and Vegetable Storage: Hybobaric, Hyperbaric and Controlled Atmosphere. https://doi.org/10.1007/978-3-319-23591-2 (Springer, 2016).

[219] Pedreschi, R. & Lurie, S. Advances and current challenges in understanding postharvest abiotic stresses in perishables. Postharvest Biol. Technol. 107, 77-89 (2015).

[220] Altpeter, F. et al. Advancing crop transformation in the era of genome editing. Plant Cell 28, 1510-1520 (2016).

[221] Cohen, S. N., Chang, A. C. Y., Boyer, H. W. & Helling, R. B. Construction of biologically functional bacterial plasmids in-vitro. Proc. Natl Acad. Sci. USA 70, 3240-3244 (1973).

[222] Song, G. Q., Prieto, H. & Orbovic, V. Agrobacterium-mediated transformation of tree fruit crops: methods, progress, and challenges. Front. Plant Sci. 10, 26 (2019).

[223] Sanford, J. C. Biolistic Plant Transformation. Physiol. Plant. 79, 206-209 (1990).

[224] Fitch, M. M. M. in Biotechnology of Fruit and Nut Crops (ed Litz, R. E.) 173-208 (CABI Publishing, Cambridge, MA, 2005).

[225] Oliveira, M. & Fraser, L. in Biotechnology of Fruit and Nut Crops (ed Litz, R. E.) 2-28 (CABI Publishing, Cambridge, MA, 2005).

[226] Fernandez-Pinan, S. et al. Agrobacterium tumefaciens and Agrobacterium rhizogenes-mediated transformation of potato and the promoter activity of a suberin gene by GUS staining. J. Vis. Exp. https://doi.org/10.3791/59119 (2019).

[227] Doyle, C. et al. A simple method for spray-on gene editing in planta. Preprint at https://doi.org/10.1101/805036 (2019).

[228] Maher, M. F. et al. Plant gene editing through de novo induction of meristems. Nat. Biotechnol. https://doi.org/10.1038/s41587-019-0337-2 (2019).

[229] Debernardi, J. M. et al. A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nat. Biotechnol. 38, 1274-1279 (2020).

[230] Kong, J. et al. Overexpression of the transcription factor GROWTH-REGULATING FACTOR5 improves transformation of dicot and monocot species. Front. Plant Sci. 11, 1389 (2020).

[231] Aurand, R. et al. Anatomical and biochemical trait network underlying genetic variations in tomato fruit texture. Euphytica 187, 99-116 (2012).

[232] Nunez-Lillo, G. et al. High-density genetic map and QTL analysis of soluble solid content, maturity date, and mealiness in peach using genotyping by sequencing. Sci. Hortic. 257, 108734 (2019).

[233] Hayes, R. J., Galeano, C. H., Luo, Y. G., Antonise, R. & Simko, I. Inheritance of decay of fresh-cut lettuce in a recombinant inbred line population from ‘Salinas 88’ x ‘La Brillante’. J. Am. Soc. Hortic. Sci. 139, 388-398 (2014).

[234] Ogundiwin, E. A., Peace, C. P. & Gradziel, T. M. Molecular genetic dissection of chilling injury in peach fruit. Acta Hortic. 78, 633-638 (2007).

[235] Soltys-Kalina, D. et al. Novel candidate genes AuxRP and Hsp90 influence the chip color of potato tubers. Mol. Breed. 35, 224 (2015).

[236] da Costa, J. H. P., Rodriguez, G. R., Pratta, G. R., Picardi, L. A. & Zorzoli, R. QTL detection for fruit shelf life and quality traits across segregating populations of tomato. Sci. Hortic. 156, 47-53 (2013).

[237] Howard, N. P. et al. Two QTL characterized for soft scald and soggy breakdown in apple (Malus x domestica) through pedigree-based analysis of a large population of interconnected families. Tree Genet. Genomes 14, 2 (2018).

[238] Ding, Y. D. et al. Network analysis of postharvest senescence process in citrus fruits revealed by transcriptomic and metabolomic profiling. Plant Physiol. 168, 357-U642 (2015).

[239] Tang, N., Deng, W., Hu, N., Chen, N. & Li, Z. G. Metabolite and transcriptomic analysis reveals metabolic and regulatory features associated with Powell orange pulp deterioration during room temperature and cold storage. Postharvest Biol. Technol. 112, 75-86 (2016).

[240] Metje-Sprink, J., Sprink, T. & Hartung, F. Genome-edited plants in the field. Curr. Opin. Biotechnol. 61, 1-6 (2020).

[241] Mika, A. Flux and Uncertainty in the CRISPR Patent Landscape (The Scientist, 2017).

[242] Terry, M. UC-Berkely renews U.S. patent dispute with the broad institute over CRISPR. https://www.biospace.com/article/crispr-patent-battle-isn-t-quite-over-yet/ (2019).

[243] Rizk, C. CRISPR patent fight turns ugly as UC accuses broad researchers of lying about claims. https://www.genomeweb.com/business-news/crispr-patent-fight-turns-ugly-uc-accuses-broad-researchers-lying-about-claims (2019).

[244] Akst, J. USPTO restarts CRISPR patent dispute between broad and UC. https://www.the-scientist.com/news-opinion/uspto-restarts-crispr-patent-dispute-between-broad-and-uc-66050 (2019).

[245] Elsevier. CRISPR. https://www.elsevier.com/research-intelligence/campaigns/crispr (2016).

[246] Kocak, D. D. et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat. Biotechnol. 37, 657 (2019).

[247] Tan, Y. Y. et al. Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. Proc. Natl Acad. Sci. USA 116, 20969-20976 (2019).

[248] Institute, B. For journalists: statement and background on the CRISPR patent process. https://www.broadinstitute.org/crispr/journalists-statement-and-background-crispr-patent-process (2017).

[249] Rozen, I. Removing a major CRISPR licensing roadblock in agriculture. The Broad Institute. https://www.broadinstitute.org/news/removing-major-crispr-licensing-roadblock-agriculture (2017).

[250] Ahuja, V. Regulation of emerging gene technologies in India. BMC Proc. 12, 14 (2018).

[251] Consortium, E. Regulating genome edited organisms as GMOs has negative consequences for agriculture, society and economy. https://www.mpg.de/13748566/position-paper-crispr.pdf (2019).

[252] Ledford, H. CRISPR conundrum: strict European court ruling leaves food-testing labs without a plan. Nature 572, https://doi.org/10.1038/d41586-019-02162-x (2019).

[253] Weigel, D., Bock, R. & Coupland, G. Scientists call for modernization of EU gene-editing legislation. https://www.mpg.de/13761643/scientists-call-for-modernization-of-the-european-genetic-engineering-law (2019).

[254] Stokstad, E. United States relaxes rules for biotech crops. (Science Magazine, Plants & Animals Science and Policy, 2020).

[255] Callaway, E. CRISPR plants now subject to tough GM laws in European Union. Nature 560, https://doi.org/10.1038/d41586-018-05814-6 (2018).

[256] European Parliament . 32001L0018 (Official Journal of the European Communities, 2001).

[257] Priefer, C., Jörissen, J. & Bräutigam, K.-R. Food waste prevention in Europe - A cause-driven approach to identify the most relevant leverage points for action. Resour. Conserv. Recycling 109, 155-165 (2016).

[258] De Laurentiis, V., Corrado, S. & Sala, S. Quantifying household waste of fresh fruit and vegetables in the EU. Waste Manag. 77, 238-251 (2018).

[259] Tuncel, A. et al. Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes. Plant Biotechnol. J. 17, 2259-2271 (2019).

[260] Wang, D. D. et al. Characterization of CRISPR mutants targeting genes modulating pectin degradation in ripening tomato. Plant Physiol. 179, 544-557 (2019).

[261] Behboodian, B., Mohd Ali, Z., Ismail, I. & Zainal, Z. Postharvest analysis of lowland transgenic tomato fruits harboring hpRNAi-ACO1construct. Sci. World J. 439870. https://doi.org/10.1100/2012/439870 (2012).

[262] Kramer, M., Sanders, R., Bolkan, H., Waters, C., Sheeny, R. E. & Hiatt, W. R. Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: processing, firmness and disease resistance. Postharvest Biol. Technol. 1, 241-255 (1992).

[263] Quesada, M. A. et al. Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening. Plant Physiol. 150, 1022-1032 (2009).

[264] Elitzur, T. et al. Banana MaMADS transcription factors are necessary for fruit ripening and molecular tools to promote shelf-life and food security. Plant Physiol. 171, 380-391 (2016).

[265] Yang, L. et al. Silencing of Sl PL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf‐life and reduced susceptibility to grey mould. Plant Biotechnol. J. 15, 1544-1555 (2017).

[266] Tieman, D. M., Harriman, R. W., Ramamohan, G. & Handa, A. K. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. The Plant Cell 4, 667-679 (1992).

[267] Ban, Q. et al. Functional characterization of persimmon β-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation. Plant Science 274, 109-120 (2018).

[268] Paniagua, C. et al. Antisense downregulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening. J. Exp. Bot. 67, 619-631 (2016).

[269] Fu, D. Q., Zhu, B. Z., Zhu, H. L., Jiang, W. B., & Luo, Y. B. Virus‐induced gene silencing in tomato fruit. Plant J. 43, 299-308 (2005).

[270] Huang, L. C. et al. Delayed flower senescence of Petunia hybrida plants transformed with antisense broccoli ACC synthase and ACC oxidase genes. Postharvest Biol. Technol. 46, 47-53 (2007).

[271] Rommens, C. M., Ye, J., Richael, C. & Swords, K. Improving potato storage and processing characteristics through all-native DNA transformation. J. Agric. Food Chem. 54, 9882-9887 (2006).

[272] Rommens, C. M., Yan, H., Swords, K., Richael, C., & Ye, J. (2008). Low‐acrylamide French fries and potato chips. Plant Biotechnol. J. 6, 843-853 (2008).

[273] van Rijswick, C. World Fruit Map 2018: Global Trade Still Fruitful. RaboResearch Food & Agribusiness (2018).

[274] Houben, M. & Van de Poel, B. 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO): the enzyme that makes the plant hormone ethylene. Front. Plant Sci. 10, 695 (2019).

[275] Yang, S. F. & Hoffman, N. E. Ethylene biosynthesis and its regulation in higher-plants. Annu Rev. Plant Phys. 35, 155-189 (1984).

[276] Martinez-Romero, D. et al. Tools to maintain postharvest fruit and vegetable quality through the inhibition of ethylene action: a review. Crit. Rev. Food Sci. 47, 543-560 (2007).

[277] Hyde, P. T., Guan, X., Abreu, V. & Setter, T. L. The anti-ethylene growth regulator silver thiosulfate (STS) increases flower production and longevity in cassava (Manihot esculenta Crantz). Plant Growth Regul. 90, 441-453 (2020).

[278] de Wild, H. P. J., Otma, E. C. & Peppelenbos, H. W. Carbon dioxide action on ethylene biosynthesis of preclimacteric and climacteric pear fruit. J. Exp. Bot. 54, 1537-1544 (2003).

[279] Watkins, C. B. The use of 1-methylcyclopropene (1-MCP) on fruits and vegetables. Biotechnol. Adv. 24, 389-409 (2006).