1.3.4. Fruit and Vegetable Quality Improvement

The primary goal in fruit and vegetable breeding is to improve quality and prolong shelf life after harvest. Quality refers to both external and internal factors. External quality refers to fruit size, colour, and texture, which can be discerned by the naked eye. Internal quality must be measured using equipment, and includes the levels of nutrients such as sugars, vitamins, and bioactive compounds including lycopene, anthocyanins, and malate. For example, in tomato, the ovary locule number, which determines 50% of the genetic

variation in fruit size, is determined by multiple QTLs [59]. Researchers at Cold Spring Harbor Laboratory designed eight sgRNAs and used CRISPR-Cas9 to edit the promoter region of the tomato CLAVATA-WUSCHEL (CLV-WUS) stem cell gene *CLV3* to obtain fruits that are larger and more numerous than wild-type fruits [60]; editing of fruit-size determining QTLs, such as the QTLs for locule number (lc) and fasciated number (fas), generated germplasm resources with an increased number of locules [61].

Fruit and vegetable colour and texture are important traits for consumers. For example, European and American consumers prefer red tomatoes, whereas Asian consumers prefer pink tomatoes [62,63]. CRISPR-Cas was used to modify phytoene synthase 1 (*PSY1*), MYB transcription factor 12 (*MYB12*), and anthocyanin 2 (*ANT2*) to obtain yellow, pink, and purple tomatoes, respectively [64–66]. The carotenoid isomerase gene of Chinese kale (*BoaCRTISO*) is responsible for catalysis, then conversion of lycopene precursors to lycopene. When *BoaCRTISO* was targeted and edited, the colour of mutants changed from green to yellow [67]. The primary goal of improving the intrinsic quality of fruits and vegetables is to improve their nutrient and bioactive compound content. Carbohydrates and vitamins are essential nutrients. Many genes are involved in the synthesis and metabolism of sucrose and carotenoids. One of the carotenoids, provitamin A, can be absorbed by the human body and converted into vitamin A. For example, CRISPR-Cas was used to knock out *MPK20* (mitogen-activated protein kinase 20), blocking the transcription and protein products of multiple genes in the sucrose metabolism pathway [68]. Biofortification, the biotechnological improvement of the absorption, transport, and metabolism of minerals by plants, increases the levels of micronutrients that are beneficial to human health; long-term consumption of these micronutrients can effectively prevent cardiovascular disease and cancer [69].

Anthocyanins [70], malate [71], γ-aminobutyric acid (GABA) [72], and lycopene [73] are bioactive compounds. Adjusting key metabolic-pathway-related genes via CRISPR-Cas9 can enrich these nutrients in fruits. For example, in tomatoes, butylamine content was increased 19-fold through editing multiple genes in the GABA synthesis pathway, and malate content was improved by regulating aluminium-activated malate transporter (*ALMT9*) [72].

CRISPR-Cas9 can also be used to reduce the content of substances in vegetables that are not conducive to human health, by targeting mutations that inactivate genes in biosynthetic pathways. In potato tubers, for example, excessive content of steroidal glycoalkaloids (SGAs), such as α-solanine and α-chaconine, affects their taste and makes them less safe for human consumption, hence low content is an indicator of high quality. CRISPR-Cas9 was used to delete *St16DOX* (steroid 16α-hydroxylase) in the potato SGA biosynthetic pathway, resulting in SGA-free potato lines [74].

Prolonged shelf-life is an important breeding goal in fruit and vegetable production. CRISPR was used to knock out ripening inhibitor (*RIN*) or DNA demethylase (DNA demethylase 2, *DML2*) to slow fruit ripening, thereby prolonging their shelf life. However, regulating these two genes in fruit alters peel colour and reduces flavour and nutritional value, severely reducing the fruit's palatability and sensory qualities [75,76]. In tomatoes, inhibiting the expression of the pectate lyase (*PL*) and alcobaca (*ALC*) genes effectively extended shelf life, without affecting the sensory qualities or nutritional value [77,78].
