Temperature, humidity, ethylene levels, and the storage oxygen-to-carbon dioxide ratio must be controlled to slow down maturation and senescence in order to maintain produce shelf-life and quality. Low temperatures are used to reduce respiration, thereby extending shelf-life, but also have the added benefit of suppressing water loss, shrinkage, and fungal growth, which can occur due to physical injury and physiological disorders. Modifying the atmosphere to change the carbon dioxide-to-oxygen ratio and relative humidity using modified atmosphere packaging or large-scale storage of produce in controlled atmosphere rooms can extend the post harvest life of commodities . Ethylene biosynthesis and emission underpin post harvest quality and shelf-life in climacteric fruit and vegetables. Ethylene accelerates ripening, but also senescence; therefore, ethylene must be managed to optimize shelf-life. This is underscored by the number of ethylene inhibitors, absorbers, and blockers on the market .The goal of post harvest management is therefore to control this progression to senescence, i.e., to pause the ripening process for shipping and storage, and then to restart it with a minimal loss of quality. However, the processes that control the ripening-to-senescence transition dictate quality, creating a dilemma, whereby altering ripening biology via refrigeration, chemicals, or other means to lengthen shelf-life,blueberries in containers often unavoidably disrupts ripening outcomes and reduces quality.
This leads to consumer rejection and post harvest waste. The alternative—to maximize consumer preference by harvesting produce close to peak maturity stage, and with no chemical or physical treatment, will invariably increase post harvest losses due to the shortened shelf-life, and increased susceptibility to bruising and pathogenic infection .There is great excitement at the innovation gene editing and the associated technologies potentially bring for improving crop quality, especially for species and traits that have been relatively understudied, such as post harvest traits of horticultural crops. Manipulation of plant genomes in a precise manner has been achieved at a spellbinding pace since the era of genome editing. The current gene-editing tool of choice is CRISPR–Cas9. The researcher is able to generate mutations in narrowly defined regions of the genome, and it has been successfully applied to induce valuable traits in many crop species. Further, CRISPR can bypass other burdens like sterility, self-incompatibility, high heterozygosity, low frequency of recovering desired alleles and traits and long life cycles, which extend or halt entirely conventional breeding efforts. CRISPR is a prokaryotic system that protects organisms from viral infection. This naturally occurring mechanism in bacteria has been co-opted by scientists to remove unwanted nucleotides or to insert new or altered ones to promote traits seen as desirable in an organism of interest. For CRISPR editing, a synthetic guide RNA is designed to an identified protospacer adjacent motif in the sequence of interest, and this, along with the Cas protein sequence, is inserted into a cell where they are processed using the cell’s gene expression apparatus.
The Cas protein synthesized in the plant produces a double stranded break at the bases identified by the gRNA. Repair of the DSB in DNA is usually not faithful to the original sequence, and thus, non-synonymous mutations may be introduced into the genome. The precise changes in nucleotide sequences are difficult to predict, but indels of varying sizes and single nucleotide polymorphisms are most common, providing diverse genetic variants. DSB repairs occur naturally in almost all plant tissues, so this is not an inherently foreign process. Although genomic mutations generated by CRISPR mediated random repair mechanisms are easily achieved, the ability to specifically express the Cas protein in a controlled spatial-temporal manner, and in conjunction with other enzymes, is often desirable for basic and applied plant research. Precise site-directed editing can be used for single-base substitution of a gene of interest, which has been achieved in cereals, as well as horticultural crops such as tomato and potato. In addition, tissue-specific knockouts using a CRISPR technique, called CRISPRTSKO, can generate somatic mutations in cells, tissues, and organs by using specific promoters. Similarly, another gene-editing system uses an inducible chimeric transcription factor , to control the expression of Cas protein in planta. Apart from knock-out/in of gene coding regions, transcriptional modulation of gene expression can be achieved by CRISPR targeting of gene regulatory elements. New alleles generated by CRISPR/Cas in promoters and enhancers where transcription factors bind to direct gene expression, can lead to fine-tuned expression. Similarly, variants in upstream open reading frame sequences could enhance post-transcriptional modulation of gene expression, influencing phenotype. The expression of a gene may also be varied by changing its DNA methylation status. In tomato, orange, and bell pepper, DNA methylation regulates ripening by controlling ripening-related TFs or genes.
Binding a methylation modifying protein to a CRISPR complex with a deactivated Cas9 may be a feasible approach to edit regions targeted for de/methylation in ripening-related genes, thus controlling shelf-life. CRISPR-Cas also enables modulation of traits in species that are difficult to obtain through traditional breeding. Approximately 70% of angiosperms are polyploid, which increases the effort needed for introducing new alleles by crossing and selection. Transmission of Cas activity in the progeny of CRISPR-expressing lines holds promise for transgenerational gene-editing in polyploid plants. This method was shown to introduce newly mutated alleles, not only in F1 but also in F2 and F3 plants. De novo domestication, a new idea in crop improvement, has been demonstrated in multiple species of the wild Solanum genus by CRISPR targeting. Novel alleles of selected “domestication genes” are generated in wild species, land races, or non-commercial genotypes to speed-up their transformation to elite varieties suitable for cultivation and post harvest practices of modern agriculture. In conclusion, various CRISPR techniques and approaches can be used to introduce nuanced changes in the expression of single or multiple genes, however, it also has real value as a tool to dissect the network of biological pathways responsible for ripening, senescence, and quality. It is expected to help identify hitherto unknown genes, that when altered, can promote favorable post harvest phenotypes. These desirable phenotypes are discussed in “Produce post harvest attributes that would minimize PLW” section.Consumers have shown that fruit and vegetables with desirable appearance, texture, taste, and flavors will have higher salability. The criteria for a favorable appearance include produce of the right color and color uniformity, correct shape and dimensions,planting blueberries in pots and often a glossy surface area free from defects. Identifying and manipulating the genes determining these pathways could improve quality. Consumers also have specific notions of what “unacceptable” produce is, and this has consequences for the generation of post harvest waste. This may vary culturally and according to socio-economic status, but general trends are identifiable. Produce with characteristics reminiscent of rotten, infested, or unripe material will be rejected. This is widely accepted as an evolutionary strategy to avoid poisoning or illness from contaminated food, as well as a learned response based on a previous negative experience. Therefore, lesions or aromas due to age or bruising are associated with “bad” fruit and vegetables and will be rejected not only as “low quality” but as potentially dangerous, despite the produce being largelyintact and actually safe. While quality of flavor is widely believed to be a strong predictor of repeat purchase, visible appearance has a strong role in initial selection or rejection at the point of purchase, and later discarding in the home. These negative traits all interact with the consumer priorities mentioned above and contribute to post harvest waste.Although our knowledge of basic fruit and produce biology is incomplete, there has been extensive work that points to the action of individual genes which, when altered in expression, may deliver useful phenotypes. Manipulating these biological processes by gene editing is a promising new avenue for reducing PLW. Many traits, however, are determined by networks of genes, and although distinct, some networks overlap so that changes in one may have unintended consequences in another. A major challenge is to understand the complicated regulation of these pathways in order to fine-tune them in a beneficial way. Gene editing has the potential to clarify the role of individual constituents in conjunction with the production of novel varieties.As mentioned in “The challenge of the post harvest supply web” section, ethylene is a master regulator of ripening; in climacteric fruit, ethylene production must be managed to optimize shelf-life , but genetic solutions may be more effective.
In climacteric fruit, ethylene synthesis, regulation, and perception lead to the transcription of ripening-regulated genes that determine quality attributes desired by consumers. When ACO and ACS expression is genetically suppressed or silenced in a range of species, e.g., petunia, tomato, melon, papaya, and kiwifruit, ethylene production is decreased and shelf-life is extended due to slowed ripening processes. In tomato, the regulation of ethylene biosynthesis is mediated by a complex network centered around the master regulatory proteins: CNR, RIN, and NOR, which are required for normal ripening. The recent use of CRISPR to induce targeted deletions or substitutions in CNR and NOR, and in other transcription factors , AP2a, FUL1, and FUL2 revealed multiple and redundant levels of regulation in the ripening pathway. Using CRISPR to create fruit varying sequentially in one or more of these transcription factors may improve our understanding of the molecular regulation of ethylene response in horticultural crops. This knowledge would allow us to control ethylene production so that ripening proceeds at the rate and with the timing that is optimal for supply chain dynamics while maintaining quality. This would directly mitigate PLW.Global demand for fresh-cut ornamentals has increased in the past years, with an estimated value of $16B in 2015. The top producers, the Netherlands, Ecuador, Columbia, and Kenya, export floral products long distances, primarily to Europe, North America, and East Asia. However, ornamental crops are highly perishable and up to 50% of the farm value may be lost along the cold-chain, and each extra day in transit leads to a 15% loss of value. Further, after consumer purchase, ornamental shelf-life, i.e., vaselife, is typically only 10–12 days, so rapid transport along a cold-chain is essential. Ethylene has a critical role in accelerating flower senescence in some species, and targeting components of the ethylene signal transduction pathway has been successful in extending vase-life in carnation and petunia. Gene editing was also used to mutate ACO1 in petunia thereby increasing flower longevity. In species that are not ethylene-responsive, vaselife could also be extended by inhibiting general senescence proteins.The triterpenoids and waxes coating the harvested parts of horticultural crops may have a bigger influence on quality and shelf-life than previously recognized. The plant cuticle is the first layer of defense against water loss and pathogen infestation. The cuticle is also responsible for multiple traits involved in fruit quality and shelf-life, such as surface brightness, the characteristic “bloom” of grapes, blueberries and plums, and potentially modulating texture changes. Fruit cuticle composition actively changes depending on the environment and organ developmental stage, which affects its protective function during fleshy fruit ripening. The interaction between the bio-mechanical properties of the fruit cuticle and cell wall polysaccharides affects the development of surface cracks in cherries, apples, and tomato. These aesthetically undesirable traits for consumers can also reduce produce shelf-life. Identifying genes key to cuticle compound biosynthesis could improve fruit response to environmental stresses during post harvest storage and reduce pathogen susceptibility.The breakdown of the cell wall during fruit ripening is a crucial process in the development of fruit sensorial quality. Softening the fruit is essential for increasing its appeal to animals and humans for consumption, and thus seed dispersal. Ripening and senescence, together with fungal attack, accelerate the rate of CW degradation, leading to rotting. Rotting and ripening are discussed separately, even though they overlap biologically in relation to CW softening and fruit shelf-life. The modern, worldwide food supply chain often necessitates that the breakdown of the cell walls, either by ripening, senescent processes or by fungal rot, be halted or slowed. CW softening processes are catalyzed by multiple enzymes that respond to developmental and environmental cues and occur over a variety of timelines, depending on the organism and tissue in question. CW degradation is orchestrated by polygalacturonase , pectin methylesterase , pectate lyase , and β-galactosidase. PG, PME, PL, and β-Gal vary in their biotechnological potential to control firmness/fruit softening .