1.3.5. Application of CRISPR-Cas9 to Crop Domestication

The domestication of wild species into commercial cultivated species requires changes in numerous crop traits, including seed setting, size, consistency of maturation, flowering, photoperiod sensitivity, and the nutritional value of the fruit [79]. Plant domestication mostly affects the genes controlling plant morphology, plant growth habit, floral induction, fruit size and number, dispersal, and architecture, as well as the nutritional composition. To achieve the ideotype, alleles controlling favourable nutritional attributes and stress resilience from wild relatives are introduced into cultivated species via traditional domestication technology, but this process is very time-consuming in bringing about changes

to many loci. With its ability to precisely manipulate the genome, CRISPR-Cas9 can substantially accelerate de novo domestication.

The tomato is a model crop for artificial domestication using CRISPR-Cas9. In tomato plants, the joint is a weak region of the stem which allows the fruit to drop from the plant, making the fruit prone to fall after ripening, thus improving seed dispersal. Many years of artificial domestication based on harvesting habits has generated cultivars with jointless fruit stems, in which the fruit do not fall after maturation [79,80]. Roldan et al. [81] used CRISPR-Cas to mutate *MBP21* (MADS-box protein 21), obtaining a new jointless germplasm resource.

Parthenocarpy (fertilisation-independent seedless fruit development) is an important agronomic fruit and vegetable trait and can help ensure stable yield in fluctuating environments. It satisfies consumer preferences for seedless over seeded fruits and provides savings in energy consumption when separating the seeds for industrial production. In tomatoes, *SlAGL6* (SlAGAMOUS-like 6) is essential for parthenocarpy during high temperature stress. *SlAGL6*-mutant plants grow normally and have the same fruit weight and morphology as wild-type plants. Therefore, this gene is an important resource for creating new parthenocarpic germplasms. Homozygous or biallelic mutant plants obtained by modifying *SlAGL6* produced parthenocarpic fruits and fruits with a maximum of 10 seeds, respectively [82]. CRISPR-Cas has also been used to knock out *SlARF7* (auxin response factor 7) and *SlIAA9* (indole-3-acetic acid inducible 9) to obtain seedless tomatoes. Seedless tomatoes are obtained from the T0 generation of the biallelic and homozygous *SlIAA9*-mutant Micro-Tom cultivar and the commercial Ailsa Craig cultivar [83,84].

Plant yield depends primarily on the number of flowers, which in turn is determined by inflorescence structure. *BOP* (blade-on-petiole) is homologous to genes associated with leaf complexity and silique dehiscence in tomato and *Arabidopsis*. Knocking out *BOP* via CRISPR-Cas9 altered inflorescence morphology. CRISPR-Bop1/2/3 triple mutants flower faster and have simpler inflorescence structure than wild-type plants [85]. The site-directed editing of six key genes that determine yield in wild tomatoes (*Solanum pimpinellifolium*) has resulted in morphological changes in aspects such as size, fruit number, and nutritional composition [86]. In domesticated wild tomatoes, genes associated with morphology, number of flowers, fruit yield, and vitamin C synthesis have been improved by editing their coding sequences, cis-regulatory sequences, and upstream open reading frames [81]. APETALA2a (*AP2a*), NON-RIPENING (*NOR*), and FRUITFULL (*FUL1*/*TDR4* and *FUL2*/*MBP7*) have been modified to accelerate tomato maturation, producing plants that mature earlier in natural environments [87].

Crop sensitivity to photoperiod restricts their planting areas and regulating the photoperiod-associated genes can accelerate domestication. The disruption of self-pruning 5G (*SP5G*) generated a rapid surge in flowering that leads to an early fruit harvest [88].

Dwarf-crop breeding is an important direction in domestication research, as dwarf plants are resistant to lodging under high wind conditions. Compared with normal plants, it is more convenient to pick fruits from dwarfed plants [89]. Dwarf plants transport nutrients more readily over the shorter distances from the roots to the leaves [89]. The application of CRISPR-Cas9 technology in the genomes of several commercially important fruit and vegetables has been achieved as outlined in Table 1.


**Table 1.** List of target genes and traits modified via CRISPR-Cas9 technology in fruit and vegetable

 crops.


**Table 1.** *Cont.*

