**10. Genome editing in peanut**

The CRISPR/Cas9 system is based on the prokaryotic type II CRISPR system, which was derived from a gene editing mechanism in bacteria. It's a relatively new technique that allows researchers to change the DNA of an organism for the sake of research. Breeders can use this technique to add, remove, or modify genetic materials at a precise point in the genome. In comparison to ZFNs and TALENs, the CRISPR/Cas9 system stands out for its ease of use, efficiency, and low cost, as well as its capacity to target multiple genes [77]. Gene-editing technology has a lot of potential for improving peanut oleic acid. The first gene editing in the model plants *Arabidopsis thaliana* and *Nicotiana benthamina* using CRISPR/Cas9 was reported in 2013 [71, 78]. Since then, it has been widely used in many plant species for gene function research, and its current widespread use in crop breeding shows promise for future breeding programmes. The limited specificity of sgRNA in CRISPR/Cas9 may result in off-target DNA sequences. An unanticipated or undesirable mutation will occur in the organism's genome as a result of this consequence. Despite the fact that cas9 nickase was developed to decrease the off-target effect, improvement is still required [79]. The use of gene editing techniques makes the creation of double-strand breaks in chromosomes much faster than using conventional breeding techniques. Double stranded breaks (DSBs) can be utilized to deliver targeted disease resistance and genome alterations to improve agronomic parameters such as yield and nutritional content by harnessing the natural cellular DNA repair process [80, 81]. To characterize the functions of peanut AhNFR1 and AhNFR5 genes in the nodulation symbiosis, researchers used hairy root-mediated CRISPR knockout. The findings not only confirmed that using CRISPR/Cas9 in combination with a hairy root transformation system is a quick way to characterize gene functions in roots, but they also improved our understanding of the role of the NFR genes in peanut nodulation [82].

### **11.** *Carthamus tinctorius*

Safflower (*C. tinctorius*) is a versatile crop that can be grown in the tropics and subtropics in semi-arid climates [83]. Safflower seed cakes provide a high protein source, feed for animals and birds, and traditional medicine. Safflower oil is rich in oleic as well as linoleic acid [84]. In addition to these traditional applications, safflower is increasingly being used to synthesize transgenic goods, including pharmaceutical ginseng, human insulin, and apolipoprotein [85]. Safflower has evolved into a platform for industrial food production due to its low outcrossing rate and weediness, distinctive appearance from other oilseed crops, and excellent agronomic characteristics, such as the taproot architecture that allows it to access subsoil water reserves [86]. It has been commercially successful to genetically modify safflower, but there is no detailed description of how to generate and analyze transgenic T1 plants in the public domain. The lack of reliable regeneration of transgenic T1 progeny in safflower has enormous implications for this economically-important plant's capacity to be used as a high yielding industrial crop. Safflower is undoubtedly a challenging crop to genetically engineer, and there is substantial literature that describes limitations of tissue culture techniques for safflower [87, 88].

#### **12. Crop improvement of safflower**

Although safflowers produce some of the healthiest oils for human consumption, their agronomical features of drought resistance and arid region adaptation prevent them from becoming a major crop. The lack of oil and yield is due to its low oil content and susceptibility to diseases and insect pests as compared to other oilseed crops like canola and cotton. Plant breeding has produced a range of cultivars that have different fatty acid profile oils, quantities, and quality, with the primary use being edible and industrial oils, along with a minor use as bird seed. This comprises specialized oils with high -linoleic acid (gamma-linoleic acid, GLA) and higher tocopherol content, which are thought to offer health benefits. Safflower oil offers potential in the biofuel industry as well as foundation for pharmaceutical manufacture in GM safflower seed [85, 89–91]. Current Australian varieties contain up to 42% oil whereas in United States have developed cultivars with oil content levels ranging from 45 to 55% [92]. In India, the most prevalent breeding approach for safflower cultivar production is choice from indigenous varieties, and multiple germplasm lines with required qualities have been created. Through selection and/ or hybridization with local lines, this material can then be used for breeding in other countries. Safflower cultivars were produced in the twentieth century in the United States, Canada, and Argentina, using material imported from India, Russia, and Turkey [93]. The most complicated variables in safflower are seed yield and oil content, and selection for these traits is impeded by substantial genetic-environmental interactions. For the production of hybrid safflower plants, dominant and recessive genetic male sterility (GMS), cytoplasmic male sterility (CMS), and

temperature sensitive genetic male sterility (TGMS) systems have been established. In India, GMS safflower lines (including spiny and non-spiny flowered lines) with a 20–25% increase in seed and oil output are available. In India, CMS and TGMS lines are also commercially accessible. Despite the development of hybrid safflower production technologies and the testing of hybrids, practical production of hybrid safflower is still a long way off [94, 95].
