Advancements and prospects of CRISPR/Cas9 technologies for abiotic and biotic stresses in sugar beet

Sugar beet is a crop with high sucrose content, known for sugar production and recently being considered as an emerging raw material for bioethanol production. This crop is also utilized as cattle feed, mainly when animal green fodder is scarce. Bioethanol and hydrogen gas production from this crop is an essential source of clean energy. Environmental stresses (abiotic/biotic) severely affect the productivity of this crop. Over the past few decades, the molecular mechanisms of biotic and abiotic stress responses in sugar beet have been investigated using next-generation sequencing, gene editing/silencing, and over-expression approaches. This information can be efficiently utilized through CRISPR/Cas 9 technology to mitigate the effects of abiotic and biotic stresses in sugar beet cultivation. This review highlights the potential use of CRISPR/Cas 9 technology for abiotic and biotic stress management in sugar beet. Beet genes known to be involved in response to alkaline, cold, and heavy metal stresses can be precisely modified via CRISPR/Cas 9 technology for enhancing sugar beet’s resilience to abiotic stresses with minimal off-target effects. Similarly, CRISPR/Cas 9 technology can help generate insect-resistant sugar beet varieties by targeting susceptibility-related genes, whereas incorporating Cry1Ab and Cry1C genes may provide defense against lepidopteron insects. Overall, CRISPR/Cas 9 technology may help enhance sugar beet’s adaptability to challenging environments, ensuring sustainable, high-yield production.


Introduction
Sugar beet (Beta vulgaris L.) is cultivated in temperate regions of the world, however, its cultivation has spread to the tropical and subtropical zones of India (Mall et al., 2022a;Misra et al., 2022a).Sugar beet is well known for its sugar production in many countries of the world covering a significant area under cultivation (Table 1) and contributing to around 30% of the world's sugar requirement (Zicari et al., 2019).The root of sugar beet is an important source of natural sucrose as a sweetening agent (Mall et al., 2022b) and has diverse industrial applications (Misra et al., 2018).Sugar beet production faces major threats from biotic and abiotic stresses (Mulet, 2022).For instance, common viral (beet necrotic yellow vein virus, BNYVV (Ramchandran et al., 2021), bacterial (like Pseudomonas aptata), and fungal diseases (like Cercospora (Misra et al., 2022c); Alternaria (Misra et al., 2020a), as well as nematodes (Meloidogyne incognita), and insects (like Aphis fabae (Mulet, 2022), Spodoptera litura (Santeshwari et al., 2020;Baitha et al., 2022)) hampers the production and productivity of the crop.Salinity, drought, heat (high temperatures), and cold are some of the abiotic stresses that severely impact sugar beet production all over the world (Misra et al., 2020b) (Table 2).Besides, other abiotic stresses like ozone build-up, flooding, nutritional deficiency, and heavy metal poisoning of the soil can also be challenging (Shabbir et al., 2022).In order for sugar beet plants to produce their optimum amount of sugar, enough moisture availability on a daily basis is necessary so as to allow for efficient transpiration and photosynthesis processes (Ober and Rajabi, 2010;Barratt et al., 2023).However, such ideal circumstances under natural environmental conditions have not been observed frequently owing to scanty rainfall or limited irrigating options.Despite the concerted efforts in developing tolerant varieties of sugar beet through conventional breeding and genome editing for improving the sugar and ethanol yield (Pattanayak et al., 2023), the scope still exists in the field of development of sugar beet varieties tolerance/resistance to abiotic and/or biotic stresses.
Molecular biology has witnessed a massive transformation due to the emergence and development of the CRISPR/Cas system as a biotechnological tool.The CRISPR/Cas 9 system is reportedly an efficient technology (Ahmad et al., 2021).The microbial adaptive immune system, CRISPR may target any genomic region by using a synthetic short guide RNA (sgRNA) (Jinek et al., 2012).Its strength comes in its capacity to effectively and precisely cause double-strand breaks in DNA at any location in the genome.CRISPR mediated genome editing can be used to change practically any sequence to expose its role in the genome (Asmamaw and Zawdie, 2021).On the basis of the genes encoding the effector modules CRISPR-Cas systems can be classified where different cas proteins have unique characteristics and functional roles (Chaudhuri et al., 2022) (Table 3).CRISPR/Cas 9 mediated genome editing requires a protospacer adjacent motif (PAM) for Cas nuclease for initiation of the cutting process.The PAM is located 3-4 nucleotides downstream from the specific site where cleavage needs to be done (Gleditzsch et al., 2019).The advances in genome editing techniques, particularly CRISPR/Cas 9 system, will benefit the cultivation of sugar beet by developing varieties resistant to abiotic and biotic stress conditions.This review discusses the current understanding of the mechanism of CRISPR/Cas 9 technology and its application in the improvement of sugar beet cultivars against abiotic/biotic stresses.
2 Site-directed nucleases (SDNs) and comparison of zinc finger nucleases, transcription activator like effector nucleases with CRISPR/Cas 9 technology Targeted genome engineering has been emerged as an alternative to traditional plant breeding approaches, aiming to achieve a variety of crop improvement goals and sustainable food production (Misra et al., 2018).Genetic engineering (GE) techniques have been deployed to enhance the quality attributes of sugar beet (e.g., shelf life) (Monteiro et al., 2018) and improve its tolerance to biotic and abiotic stresses (Wan et al., 2021).In this context, the application of site-directed nucleases (SDNs) has evolved as a suitable GE technique for introducing desirable characteristics into plants (Aglawe et al., 2018).Zinc finger nucleases (ZFNs), transcription activator like

GRAPHICAL ABSTRACT
Frontiers in Genetics frontiersin.org(TAL) effector nucleases (TALENs), and Clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR/Cas) technology are instances of SDN methods.SDNs enable precise changes at predetermined places in a genome, avoiding any unintended random mutagenesis.They offer unparalleled control over targeted genome alterations, proving to be more cost-effective and efficient than conventional plant breeding and genetic engineering methods.SDNs have the potential to aid in crop improvement and enhance food security in many sugar beet producing countries (Table 4).SDNs are categorised into SDN1, SDN2, and SDN3 based on the outcomes of genomic alterations and double strand break repair (Ghouri et al., 2023).SDN1 does not require template and causes gene disruptions via InDels (small insertions or deletions of bases).SDN2 uses a homologous template to repair or modify the gene at one or more locations.SDN3 requires the use of a whole gene as a template and results in gene substitution or foreign DNA insertion (Chen and Gao, 2020).
The CRISPR/Cas system is a convenient replacement for ZFNs and TALENs in generating targeted genomic alterations (Ahmad et al., 2021).Both ZFNs and TALENs are utilized to mutate genomes at specific loci (Boti et al., 2023).However, these systems require two distinct DNA binding proteins flanking the region of interest, each having a C-terminal FokI nuclease module.Custom proteins are necessary for targeting DNA sequences.The process of designing and constructing custom proteins in both these technologies (Zinc finger motifs in ZFNs, while DNA binding domains obtained from TALE in TALENs, are required) is laborious and time-consuming.In CRISPR-mediated genome editing, PAM is located 3-4 nucleotides downstream from the specific site where cleavage will occur (Gleditzsch et al., 2019).Therefore, accessibility to the protospacer adjacent motif (PAM) site, as the main determining factor for altering any functioning sequence, is easier (Akram et al., 2023).CRISPR technology depends on RNA-guided sequences, which can be designed effortlessly (Bajpai et al., 2023).
The target specificity in ZFNs and TALENs is more challenging as zinc fingers or TALEs identify short DNA sequences and require the combination of multiple modules to target the desired region, which may result in off-targets (Jyoti et al., 2023).The probability of off-targets is very low in CRISPR because it utilizes guide RNA to target the desired site, which can be easily programmed.Furthermore, the application of custom protein engineering in ZFNs and TALENs causes lesser adaptation and more laborious efforts (Kalaitzandonakes et al., 2022) while altering in sgRNA is easier for targeting the desired gene in CRISPR technology.The use of sgRNA in CRISPR technology is also beneficial in the delivery stage, making it a simple process compared to ZFNs and TALENs (Pankaj and Kumar, 2023).Due to the complexity involved in ZFNs and TALENs, including design and construction, target specificity, flexibility, and delivery), these methods are not preferred compared to CRISPR (Bhatia et al., 2023).Sugar beet (Beta vulgaris L.) production is drastically affected by biotic and abiotic factors, which reduce the rate of photosynthesis, expansion of the canopy, development of the root system, and consequently, the accumulation of sucrose content in the plant (Misra et al., 2022a).Abiotic stresses, including temperature fluctuations, water scarcity, salinity, metal toxicity, and UV radiation, are harmful to the sugar beet crop, and severely affect its yield across the world (Yu et al., 2020).These stressors greatly restrict the distribution of sugar beet crops, affect their developmental processes, and decrease sugar beet productivity (Ober and Rajabi, 2010).Improved understanding of multiple molecular mechanisms, like pathway signalling, activation of transcription factors, transcript modification (post-transcriptional modification), translation of processed transcript, and protein modifications after the translation process, underlying stress responses of sugar beet crops at multiple levels, would be helpful in increasing the sugar beet production and sustainability through the application of CRISPR/Cas 9 (Yu et al., 2020;Misra et al., 2022a).Abiotic stress tolerance is a complex trait that is mediated by multiple genes.The components of metabolic, regulatory, and signalling networks in plants interact and crosstalk extensively under abiotic stress conditions (Garg et al., 2014;Mickelbart et al., 2015).Several abiotic stress resistance-conferring genes have been identified in plants (Table 5) and introgressed into related crops through the application of biotechnological tools (Razzaq et al., 2021).
Gene activation or repression occurs by targeting transcriptional activator or repressor complexes to particular sites within the gene promoter region with catalytically inactivated Cas endonuclease.For this purpose, CRISPR-based approaches like CRISPRa and CRISPRi could be beneficial in characterizing genes for abiotic stress tolerance (Osakabe et al., 2016).CRISPR activation, abbreviated as CRISPRa, is a CRISPR variation in which a catalytically dead (d) Cas 9 is coupled with a transcriptional effector to control target gene expression.When the guide RNA and the effector arm reach the genomic location, the dCas9 is unable to produce a cut, and the effector instead triggers downstream gene expression.CRISPRa technique is used to increase gene expression by targeting the promotor region upstream of the transcription start site (TSS), whereas the CRISPR interference (CRISPRi) approach suppresses gene expression by targeting the promotor region downstream of the TSS.The application of CRISPRa/i requires precise identification of the TSS location (Davis et al., 2018;Thomas et al., 2019).CRISPRi is a technology that uses dCas9's programmable binding capacity to inhibit gene expression by preventing or interfering with RNA polymerase binding, transcription factor binding, and transcriptional elongation.sgRNA specific to a gene sequence's upstream regulatory region (e.g., promoter) or transcription initiation site could direct dCas9 to bind and inhibit transcription initiation or elongation, effectively silencing gene expression.To inhibit target gene expression, dCas9 is coupled with the transcription repression domain of the Kruppel associated box (KRAB).dCas9 alone or in combination with KRAB is an effective tool for knocking off one or more genes (Piatek et al., 2015).CRISPRi and CRISPRa regulate gene expression by increasing or suppressing RNA polymerase, respectively.Thus, CRISPRi and CRISPRa are promising approaches for exploring and regulating stress-regulatory genes, as well as developing abiotic stress tolerant varieties.CRISPRi/a has been utilized successfully in plants to modify expression by a factor of 1000 (La Russa and Qi, 2015).CRISPRi and CRISPRa technologies can also be used in sugar beet under abiotic stress conditions.Although there is a lack of information available on this aspect, there are prospects to be worked on.The application of these technologies can aid in regulating gene expression in response to abiotic stress conditions (McCarty et al., 2020).These could be correlated with gene expression in sugar beet under drought, salinity, or temperature stress to provide tolerance.Altering these gene expressions could produce resilient sugar beet varieties with respect to abiotic stress conditions.Inhibiting or activating the targeted stress-responsive genes in sugar beet under abiotic stress conditions will help in understanding the regulatory mechanism.This, in turn, will assist in developing sugar beet varieties tolerant to adverse environmental conditions.Furthermore, Cas 12a could also be explored for targeting the specific genes contributing to abiotic stress pathways, like drought, salinity, temperature, etc.By altering the genes related to specific abiotic stress conditions in sugar beet, the plant's potential to adapt and survive under adverse conditions could be increased.For instance, Chen X. et al. (2018) reported the application of Cas12a in targeting drought-responsive genes (positive regulation of gene) in Arabidopsis.Altering these targeted drought-responsive genes through Cas 12a improved the plant's drought tolerance potential.Such plants exhibited better water retention capacity, reduction in wilting, and higher survivability under drought conditions.
The development and evolution of sugar beet varieties tolerant to abiotic stress induced by climate change and global warming are imperative (Yolcu et al., 2021).Therefore, CRISPR/Cas 9 technology is necessary to create highly resistant sugar beet varieties against biotic and abiotic stresses.The CRISPR/Cas 9 technology involves specific steps for developing novel sugar beet varieties for different abiotic stress tolerances (Figure 1).

Alkaline stress tolerance
Aside from ionic toxicity and osmotic stress, high pH in the alkaline soils disrupts cell pH stability, destroys cell membrane integrity, and reduces root vitality and photosynthetic activity (Fang et al., 2021).Under the condition of high saline stress, beet varieties that are cultivated along with wild types (naturally occurring/non-domesticated beet varieties) exhibited high antioxidant enzyme activities (Wang et al., 2017;Li B. et al., 2021).Wu et al. (2019) reported that with increasing sodium bicarbonate concentrations, Na + concentrations were enhanced significantly in shoots and roots of sugar beet plants under alkaline conditions while a steady level of potassium ion concentrations was observed.Maintenance of K + and Na + homeostasis could be a key strategy for sugar beets adjusting to alkaline stress.Several genes (like WRKY, NAC, MYB, etc.) have been reported to be involved in conferring abiotic stress tolerance in plants (Chinnusamy et al., 2006;Hennig, 2012;Khadiza et al., 2017).Wu et al. (2019) reported 58 WRKY genes, and among them, 9 genes were found to be responsible for alkaline stress responses (~15 mM-100 mM NaCHO 3 ) in both shoot and root parts.It evidently proved the increased expression of the BvWRKY10 gene (in the terminal and lateral shoots) and BvWRKY16 gene (in roots) under alkaline stress.WRKY proteins have been known to be associated with the response of plants to biotic and abiotic stress conditions (Jiang et al., 2017).The quantitative alterations and tissue-specific expressions of different BvWRKY genes have clearly shown its implications for alkaline stress tolerance in sugar beet.Research to alter the expression of BvWRKY genes using CRISPR/Cas 9 technology is required for the development of stress-resistant varieties.The BvWRKY family genes are specific and play crucial roles in wider aspects of sugar beet development processes like germination, root development, photosynthesis, etc., including response to alkaline stress conditions (Wu et al., 2019).Therefore, these genes can be engineered through CRISPR/Cas 9 technology to develop alkaline stress-resistant varieties in sugar beet (Table 6).Furthermore, long noncoding RNAs (lncRNAs) in response to alkaline stress had also been identified and characterized in sugar beet leaves.Besides, the interactions of candidate genes and miRNAs with the lncRNAs under stress conditions have also been reported in sugar beet (Zou et al., 2020).The use of CRISPR/Cas 9 technology on these identified/characterized lncRNAs will be helpful in evaluating their function in sugar beet and its expression can be modified to provide alkali stress tolerance in sugar beet.Additionally, genes belonging to the bHLH (basic helix-loop-helix) family in sugar beet involved in salt stress tolerance have also been well known.Wang Y. et al. (2021) reported the BvbHLH93 gene as a salt-responsive gene that has been shown to confer tolerance under salt stress conditions in sugar beet.By increasing antioxidant activity and decreasing ROS generation, the BvbHLH93 gene modulates salt stress tolerance in sugar beets.Furthermore, the BvbHLH93 gene's ability to reduce RbohD and RbohF gene expression through modulating polyamine metabolism warrants additional investigation in sugar beet.CRISPR/Cas 9 technology will help in understanding the role and expression of these genes in salt stress conditions.This will aid in knowing the regulation of this gene in conferring tolerance to sugar beet for salt stress conditions.

Cold stress tolerance
Chilling temperature affects sugar beet cultivation, production, and economic yield (Moliterni et al., 2015;Bhattarcharya, 2022).• Provide resistance Cold stress conditions have a serious impact on the sugar beets at various developmental stages, including early germination, sugar metabolism, growth, and bolting in the roots (Hoffmann and Kluge-Severin, 2011).The seedling stage of sugar beet is particularly prone to low temperature stresses.Cold stress during this stage leads to severe degeneration and growth retardation in the root system, consequently decreasing its sugar content (Moliterni et al., 2015;Jalilian et al., 2017).Porcel et al. (2018) revealed that overexpression of BvCOLD1 gene in sugar beet exhibits cold tolerance potential along with other abiotic stress tolerance and overcoming boron deficiency.Keller et al. (2021) found that improved freezing-tolerant sugar beet genotypes accumulated more raffinose in the pith portion, which is vulnerable tissue to freeze damage.This finding demonstrated that raffinose and its precursors protect sugar beets from freezing damage.Recognizing the importance of raffinose in providing cold tolerance to sugar beet plants, Kito et al. (2018) isolated and characterized two genes, BvRS1 and BvRS2, from the sugar beet plant.These genes code for the expression of raffinose synthase, which is a crucial enzyme during raffinose biosynthesis.An increase in transcription levels of BvRS1 and BvRS2 genes was observed in response to chilling stress in both leaves and roots (Kito et al., 2018).Furthermore, the production and build-up of oligosaccharides belonging to the raffinose family are particularly important during cold hardiness.Galactinol synthase (GolS) is considered a key regulator of the synthesis of such oligosaccharides and their accumulation (Vinson et al., 2020).
The expression levels of GOLS2 and GOLS3 genes, responsible for galactinol synthase, as well as BvRS2 and BvRS5, were high during chilling temperatures (Keller et al., 2021).Interestingly, the product of the BvRS5 gene product and raffinose content increased exceptionally during freezing temperature in the taproots of tolerant sugar beet varieties, GT2 and GT3.In comparison with other sugar beet germplasm, GT2 exhibited high expression of GOLS and RS genes and raffinose content in roots, indicating chilling resistance in GT2 (Keller et al., 2021;Yolcu et al., 2021).Membrane proteins also frequently recognize cold stress conditions in sugar beet.These proteins activate a Ca 2+ signal in the cytosol.Ca 2+ -binding proteins may act as a bridge between the Ca 2+ signal and several downstream transcription factors (Iqbal et al., 2022).It was reported that B. vulgaris Integral Membrane Protein gene resembles AtERDL6, which was previously reported for cold tolerance (Qi et al., 1995).
Freezing conditions may be responsible for the increased transcription rate of BvIMP gene and vacuolar carbohydrates trafficking in sugar beet leaves, crucial for chilling stress response and germination of seed (Yu et al., 2020;Reyer et al., 2021).
Utilizing CRISPR/Cas technology, scientists have successfully enhanced cold stress tolerance in plants by editing genes allied with cold stress and raffinose synthesis.In the case of Vitis vinifera, the VaDof17d gene plays a critical role in the cold-responsive pathway and the production of raffinose family oligosaccharides.This is evidenced by the enhanced expression of galactinol synthase (GolS) and raffinose synthase genes.Mutating the Dof17d-ED gene through CRISPR/Cas 9 technology resulted in reduced cold tolerance and reduced levels of raffinose family oligosaccharides during cold stress (Wang et al., 2021).Therefore, genes including BvIMP, BvRS1, and BvRS2 (Table 6) can be engineered in sugar beet through CRISPR/ Cas 9 technology to develop site-specific mutants.These mutants could be instrumental in enhancing cold tolerance in sugar beet crops by boosting their expression.

Heat stress tolerance
Increased temperatures and water scarcity tend to drastically affect the water content in plants where excess transpiration decreases the rate of water intake and causes permanent wilting in the plant (Kumar et al., 2004).Sugar beet is greatly affected by altered climatic conditions and disturbances in weather (Abou-Elwafa et al., 2020).High temperatures hamper major metabolic activities such as germination of seed, seed viability and its vigor, etc., leading to a threat to the survival of crop plants (Stevanato et al., 2019).Critical physiochemical processes, including photosynthesis and photosystem (PSII) activity also greatly affected due to blockage in the electron transport chain under high temperature stress (Murakami et al., 2000;Moore et al., 2021).The recent expansion of sugar beet cultivation in tropic and sub-tropical regions has drawn attention to farming it during the summer season (Abou-Elwafa et al., 2020).
Recognizing the importance of sugar beet cultivation across the world during the summer season, Ismail et al. (2020) explored the role of the transcription factor BvHSF gene, which showed higher expression levels under heat stress.The sugar beet crop showed enhanced expression levels in response to water scarcity, heat stress, and drought stress.Heat shock transcription factors (HSFs) are pivotal transcription factors in plants, critical for their response to various abiotic stresses such as heat, cold, salt, and drought (Fan et al., 2021).These HSF family members act by binding to the reverse repeat region of heat shock elements (HSEs), facilitating the transcription of heat shock proteins (HSPs) and assisting in the plant's stress adaptation mechanisms (Guo et al., 2016;Jacob et al., 2017).Wu et al. (2022) demonstrated that CRISPR/Cas 9 knockout mutants, specifically targeting single copy MpHSF genes (Mphsfa1V and Mphsfb1 mutants), resulted in different indel editing sites, showcasing enhanced thermotolerance in plants.Therefore, BvHSF genes can be engineered in sugar beet through CRISPR/ Cas 9 technology to enhance their expression in sugar beet crops for increased tolerance towards heat resistance by creating in-dels (Table 6).

Drought stress tolerance
Drought stress also negatively impacts sugar beet root growth and development during the early phases of growth.Furthermore, the introduction of drought stress later in the growing season reduces leaf area and the number of leaves, ultimately resulting in lowered photosynthetic efficiency (Abou-Elwafa et al., 2020).
Generally, an increase in the expression of multiple droughtresponsive genes and transcription factors enhances the plant's ability to tolerate drought conditions (Fang and Xiong, 2015;Kumar et al., 2020;Santosh Kumar et al., 2020).Conversely, upregulation of drought-sensitive genes in plants heightens their vulnerability to drought stress due to imbalances in hormonal levels, reduced antioxidant activities, and heightened production of reactive oxygen species (ROS).CRISPR/Cas 9 based genome editing provides a promising avenue for enhancing drought tolerance in plants.This technique involves targeting negative regulators or drought-sensitive genes, allowing scientists to modify specific genetic elements and create crops that are more resilient to water scarcity.By precisely altering these genes, droughtresistant varieties can potentially be developed, ensuring sustainable agriculture in the face of changing environmental conditions.WRKY transcription factors are pivotal regulators of plant growth, development, and responses to both biotic and abiotic stresses.Among these factors, WRKY3 and WRKY4 genes in plants play a significant role in orchestrating the defense mechanisms against drought stress (Li B. et al., 2021).For instance, genetic manipulation of the OsWRKY5 transcription factor has revealed significant insights into drought tolerance in plants.OsWRKY5, a key regulator, was found to hinder the plant's ability to withstand drought.During the seedling and heading phases, OsWRKY5 was primarily expressed in growing leaves, and its expression decreased under drought stress conditions.Researchers conducted experiments using genome-edited loss-offunction alleles, oswrky5-2 and oswrky5-3, to enhance drought tolerance.These edited alleles resulted in increased drought resistance, as evidenced by improved plant growth even under water scarcity (Lim et al., 2022).Conversely, when OsWRKY5 was overexpressed in the activation-tagged line oswrky5-D, plants exhibited greater susceptibility to drought stress.Overexpression of OsWRKY5 led to heightened sensitivity to abscisic acid (ABA), a plant hormone involved in stress response, and encouraged ABAdependent stomatal closure.By editing the OsWRKY5 genome, researchers successfully enhanced the plant's ability to produce grains even under drought stress conditions.This breakthrough offers valuable insights into improving crop resilience against water shortage, a critical factor in agricultural sustainability.Another example is for obtaining drought tolerance through CRISPR technology is enhancing the expression of AREB1, a specific transcription factor.In contrast, plants with a knocked-out AREB1 gene exhibit increased sensitivity to drought stress (Singh et al., 2016).Roca Paixão et al. ( 2019) demonstrated enhanced drought stress tolerance through the utilization of CRISPR/ dCas9 fusion with a Histone Acetyl Transferase (AtHAT) gene in Arabidopsis.These genes and transcription factors could also be targeted in sugar beet crops for attaining drought stress tolerance.

Heavy metal stress tolerance
Exposure of plants to toxic heavy metals causes different metabolic and physiochemical changes that depend on the concentration of these metals in soil, plant species, varieties, and abiotic conditions (Jamla et al., 2021;Thakur et al., 2022).Toxic metals like Pb damage the vacuolar membrane of sugar beet roots (Trela et al., 2012;Beata et al., 2022).Pb is one of the most toxic metals for plant cells, and it has a negative effect on the growth of plants, photosynthesis, respiration, and electron transport chain (Sharma and Dubey, 2005).Cd stress in B. vulgaris caused retarded growth, chlorosis, and increased root/plant ratio along with a decline in the rate of respiration in root-tips and photosynthesis (Greger and ögren, 1991;Larbi et al., 2002;Liu et al., 2022).Haque et al. (2021) observed that higher levels of Cd in sugar beet plants cause growth retardation due to an insufficient amount of Fe, resulting in decreased photosynthetic activity, and oxidative stress occurring in cells.Cd-treated plants display sensitivity to oxidative stress, leading to an increase in levels of O 2 − and H 2 O 2 in roots and shoots.Additionally, Haque et al. ( 2021) reported the antioxidant defense mechanism in sugar beet under a higher concentration of toxic metal and observed that Cd stress enhances the activity of catalase (CAT) enzyme in the shoots, whereas the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) do not increase either in roots or in shoots.Genes involved in heavy metal stress tolerance in sugar beets have been explored and proven to be important.There are two MTP genes, BmMTP10 and BmMTP11, reported for metal-resistant proteins from wild species of sugar beet (B.maritima).The detoxification process of Ni was controlled by genes from wild sugar beet (B.maritima) named as toxic nickel concentration (NIC), i.e., NIC3, NIC6, and NIC8 (Bozdag et al., 2014;Yolcu et al., 2021).It is estimated that all these genes are required for protection against Ni toxicity.In a similar study, under Cd toxicity, sugar beet roots showed higher expression of putative BvHMA3 and BvNRAMP3 genes, suggesting that these genes are involved in the Cd uptake process (Haque et al., 2021).
Heavy metal-associated proteins (HMPs) and natural resistance-associated macrophage proteins (Nramp) are vital for heavy metal transport and detoxification within plant cells (Singh et al., 2016;Li W. et al., 2020).In rice, essential transporter genes such as OsLCT1 and OsNramp5 have been identified as key players in the absorption of Cd by the roots (Chang et al., 2020).Through CRISPR/Cas 9 enabled gene-expression manipulation, significant strides have been made in reducing the levels of Cd and Pb in rice grains.Specifically, the knockout of OsNRAMP1 using CRISPR/ Cas9 technology, as demonstrated in studies by Chu et al. (2022) and Wang F. Z. et al. (2017), has led to a substantial decrease in Cd and Pb content.Therefore, BvHMA3 and BvNRAMP3 genes can also be engineered in sugar beet through CRISPR/Cas 9 technology to develop heavy metal resistant varieties in sugar beet by creating site-specific mutagenesis.
4 Application of CRISPR/Cas 9 technology for improving biotic stress resistance

Pathogen resistance mechanism
The CRISPR/Cas 9 adaptive immune system for viral, bacterial, fungal resistance operates in three steps (Figure 2), regardless of the various shapes: 1) adaptation, 2) expression and maturity, and 3) interference.Protospacers, unique short DNA snippets from the invasive pathogen, are recognized by the Cas protein and inserted into CRISPR repeats as new spacers during the adaptation process.The host can then establish immunological memory and be equipped to recognize the same invasive infections in the future concerning this new spacer, which also serves as a genetic record (Paul et al., 2021).
For some CRISPR/Cas 9 systems to acquire the protospacer, the target DNA must have a short PAM (of 3-5 nucleotides) (Bolotin et al., 2005;Deveau et al., 2008;Shah et al., 2013).The CRISPR array is translated into a precursor-CRISPR RNA (pre-RNA), which is then processed to produce short mature CRISPR RNA (crRNA) through endo-nucleolytic cleavage and contains the sequences of the invading pathogen that have been learned (Carte et al., 2008;Haurwitz et al., 2010).At its 5′ end, each crRNA has a single spacer (a brief RNA segment that complements the DNA sequence of the foreign genetic material), and at its 3' end, it has a CRISPR repeat sequence.
An active Cas-crRNA effector complex is formed when the mature crRNA combines with one or more Cas effector proteins.This complex searches for and attacks the cell's foreign nucleic acids during the interference phase.Using the specific PAM sequence either upstream or downstream of the protospacer, and relying on Watson-Crick base pairing, the crRNA component of the complex serves as a guide to identify the target DNA (Semenova et al., 2011;Jiang et al., 2013;Westra et al., 2013;Fineran et al., 2014;Zetsche et al., 2015).Once the target DNA is successfully recognized, the Cas nuclease cleaves and digests it.Different classes of the CRISPR immune system, based on various effector Cas proteins and PAM recognition sequences, have emerged in recent years.
Invading foreign DNA fragments of virus particles are recognized and eliminated by the CRISPR/Cas 9 system, allowing it to identify and remove DNA or RNA sequences that facilitate continued invasion (Barakate and Stephens, 2016).CRISPR/Cas 9 technology modifies the plant's inherent defense mechanism by detecting and removing harmful genes hidden within plant viruses.It can also be utilized to develop agricultural cultivars that are more resistant to specific plant viruses.This approach has fundamentally transformed virus resistance research because of its ability to use sequence-specific nucleases (Hsu et al., 2014).

CRISPR tools for disease diagnosis in sugar beet
The efficient implementation of control measures or management strategies heavily relies on the correct and timely identification of diseases and causative organisms.Consequently, disease diagnosis is critical and serves as the starting point for disease management.In this regard, Cas proteins play a significant role in managing diseases at the initial stages in plants.Cas proteins are attractive candidates for repurposing nucleic acid detection due to their programmability and extreme selectivity in binding and cleaving nucleic acids.Advances in understanding various Cas proteins have paved the way for the development of ultrasensitive, mobile, and cost-effective nucleic acid-based pointof-care (POC) testing equipment.Cas 9 proteins have been utilized to create robust and reliable nucleic acid detection technologies, but the recent discovery of Cas 13a and Cas 12a, with guaranteed cleavage activity, has revolutionized the field of nucleic acid detection (Gootenberg et al., 2017;Chen J. S. et al., 2018;Gootenberg et al., 2018;Azhar et al., 2021;Jiao et al., 2021).
A new molecular diagnostic approach known as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) technology (Chen J. S. et al., 2018), based on the CRISPR-Cas 12a system had been utilized for detecting BNYVV in sugar beet roots (Ramachandran et al., 2021).In this diagnostic method, Cas 12a cleaves any surrounding single-stranded DNA without regard for its target.This trait is known as collateral activity, and it has been exploited to develop DETECTR.In this approach, a complementary guide RNA first directs Cas12a to a target dsDNA (Figure 3).When Cas 12a binds to the correct target, it cleaves ssDNA reporter molecules coupled with a quencher and a fluorophore.A fluorescence quencher (FQ)-labeled reporter was employed to monitor the transcleavage activity induced by the Cas12a-gRNA complex binding to the guide-complementary target DNA.In the presence of target DNA, the Cas 12a-gRNA complex's trans-cleavage activity is triggered, leading to the cleavage of surrounding FQ-labeled ssDNA reporters (Chen J. S. et al., 2018).The fluorescent signal generated by the separation of the quencher and the fluorophore detects the indiscriminate cleavage (Kocak and Gersbach, 2018).DETECTR exhibits enhanced sensitivity when combined with RPA preamplification.This diagnostic technique requires the amplification of viral fragments from template DNA under isothermal conditions.The one-step reverse transcriptase recombinase polymerase amplification (RPA) method was employed in sugar beet.The precise sensitivity of this method is its standout feature, as it can detect a single molecule of viral particle within a microliter of the sample (Kocak and Gersbach, 2018).
Another common CRISPR-based technology applied in sugar beet for viral resistance is nucleic acid sequence-based amplification CRISPR cleavage (NASBA).This technology helped the plants achieve total viral resistance.This technique involves targeting viral DNA using a guide RNA (gRNA) and cutting the viral DNA with the Cas 9 enzyme.The trans-activating crRNA (tracrRNA) base pairs with the repeat sequence in the crRNA to form a unique dual RNA hybrid structure guide that directs Cas9 to cleave the target DNA.A chimeric sgRNA combines crRNA and tracrRNA into a single RNA transcript.The two nuclease domains (RuvC and HNH) present in Cas 9 cut the target and non-target DNA strands, respectively.A short trinucleotide PAM is also required for the initial target sequence identification; without it, the target sequence cannot be recognized.Successful identification results in a double-strand upstream of the 3′-NGG PAM (Li J. et al., 2022).This method is based on Cas 9 selective cleavage of target DNA and the toehold switch principle.This approach can distinguish between genotypes as it can identify a single base difference based on the presence or absence of a PAM sequence.This method utilizes sequence-based amplification, PAMdependent target detection, Cas 9 cleavage, and a toehold sensor (Pardee et al., 2016).Yildirim et al. (2019) utilized CRISPR/Cas 9 technology to confer various resistances in sugar beet against two curly top viruses (Beet curly top viruses (BCTV) and beet curly top Iranian viruses (BCTIV)).These viruses belong to two separate genera within the Geminiviridae family, specifically curtovirus and becurtovirus families, respectively.The gRNA/cas-9 endonuclease system was transiently overexpressed to check BCTV and BCTIV in sugar beet plants.Sugar beet plants overexpressing the gRNA/Cas 9 constructs exhibited decreased viral DNA accumulation, and this accumulation was also observed to be delayed compared to plants without the overexpression of gRNA/Cas 9 constructs.The CRISPR/ Cas 9 system used in providing resistance to sugar beet plants against viral diseases, specifically targets the dsDNA of a geminivirus with gRNA.This approach helps restrict virus reproduction by disrupting critical replication genes (Khatodia et al., 2017).Ji et al. (2015) demonstrated the application of CRISPR technology against geminivirus in plants.Efficient antivirus sgRNAs can be identified to recognize specific sites in the viral genome.This approach has high potential for developing multiple resistances to all Geminiviruses.The viral resistance mechanism in sugar beet through the CRISPR/Cas 9 system is illustrated in the figure below (Figure 4).The advent of CRISPR/Cas 9 has opened up numerous prospects for developing superior crop varieties through genome editing with great precision and accuracy.CRISPR techniques usher in a new era of breeding systems in which plant immunity is enhanced by disrupting the compatible connection between infections and hosts (Karmakar et al., 2022).CRISPR-based available tools like SHERLOCK, FLASH, and LEOPARD wherein Cas 12 and Cas 13 proteins have been utilized, will be helpful in the identification, diagnosis and management of diseases in sugar beet.

Beet cyst nematode (Heterodera schachtii schmidt) resistance
The host range of the beet cyst nematode (BCN) is wide and includes numerous species from abundant plant families, including Chenopodiaceae and Brassicaceae.In sugar beet cultivation (Beta vulgaris L.), Heterodera schachtii is a serious pest.It is known that cultivated Beta species lack the genes that provide resistance to nematodes.Beta procumbens, a wild species, and its allied species (B.webbiana and B. patellaris) are the sources of resistance genes (Cai et al., 1997).BCN is resistant to the Hs1 pro−1 locus.The native Hs1 pro−1 gene encodes a 282-amino acid protein with incomplete leucine-rich repeats and a potential membrane-spanning region.This protein is produced in sugar beet roots.The expression of the matching complementary DNA gave resistance to BCN infection in susceptible sugar beet (Cai et al., 1997).The Hs1 pro−1 (Table 7) promoter promotes nematode feeding site-specific GUS expression in both sugar beet and Arabidopsis, indicating a shared mechanism for regulating Hs1 pro−1 expression in these two species (Thurau et al., 2003).Kumar et al. (2021) identified the resistance gene, Hs4 (about 230 kb-sized area).CRISPR-Cas-mediated deletion (CRISPRi) and overexpression (CRISPRa) in susceptible sugar beet roots were used to characterize a candidate gene.The gene encodes a rhomboid-like protease predicted to be bound to the endoplasmic reticulum.CRISPR Cas mutagenesis in the resistant sugar beet cultivar, NEMATA, deleted the ORF1.The roots of the knockout clones were extremely susceptible as huge numbers of J4 females and cysts packed with eggs observed in these roots.The expression of ORF in sugar beet roots determined nematode resistance/susceptibility. Beet roots expressing ORF 1 were resistant to nematodes while low ORF expression led to moderate susceptibility.Roots that did not express were susceptible to the nematode.Thus, CRISPRa can be a potential tool for the overexpressing targeted genes, which can confer tolerance to abiotic and biotic stress conditions (Horlbeck et al., 2016;Rai et al., 2019).

Beet necrotic yellow vein virus resistance
The multipartite genome of Beet necrotic yellow vein virus (BNYVV) consistis of five positive-stranded RNAs, providing an enticing framework for the production of several foreign proteins (Jiang et al., 2019).Additionally, NbPDS guide RNAs were delivered through BNYVV-based vectors to transgenic plants expressing Cas9 for genome editing.This delivery resulted in a photobleached phenotype in systemically infected leaves.The BNYVV-based vectors will facilitate the expression and production of multiple proteins in sugar beet and related crop plants.gRNA can be delivered using BNYVV-based vectors for CRISPR/Cas 9 plant genome editing (Jiang et al., 2019).
In a breeding line developed by the Holly Sugar Company in the USA, partial resistance to BNYVV was found to be caused by a single dominant gene (Rz1) (Lewellen et al., 1987;Scholten et al., 1996).Accessions WB42 (Rz2) and WB41 (Rz3) of Beta vulgaris subsp.maritima from Denmark have also been identified as having BNYVV resistance (Lewellen et al., 1987;Gidner et al., 2005).Compared to the Rz1 gene, the dominant Rz2 gene seems to confer a higher level of resistance (Paul et al., 1993).
Detection of BNYVV is extremely sensitive and specific to the infected roots, as measured by the reporter signal.Ramachandran et al. ( 2021) established isothermal RT-RPA and CRISPR-based  (Continued on following page) Frontiers in Genetics frontiersin.org14 Misra et al. 10.3389/fgene.2023.1235855virus diagnostic techniques for detecting BNYVV in sugar beet roots with rhizomanial disease.Using this CRISPR-based technique, BNYVV in sugar beet roots baited for rhizomania can be identified, generating a readily identifiable fluorescence signal as compared to healthy reference root samples.The BNYVV RNA-1 sequence was chosen as the target since it is one of the least diverged components of BNYVV.The CRISPR-Cas 12a-based BNYVV detection approach has paved the way for a sensitive, focused, and high-throughput detection platform for the assessment of rhizomania (Ramachandran et al., 2021).
The creation and validation of such CRISPR-based BNYVV diagnostic approaches for sugar beet roots offer advantages in terms of sensitivity and resilience in isothermal circumstances.Hence, it would be a helpful tool for the companies involved in evaluating viruses to drive disease control strategies (Ramachandran et al., 2021).Furthermore, the technology established for virus identification in underground root tissue can be applied to create a CRISPR-based detection platform for viruses and soil-borne disease-causing pathogens in other crops as well.

Beet curly top virus resistance
Beet curly top virus is a single stranded DNA virus that belongs to the genus Curtovirus and has been known to cause outbreaks in sugar beet as a disease.This virus has a strong impact on sugar beet yield, reducing it to 30% and above.The beet leaf hopper (Circulifer tenellus) is the vector of this disease (Strausbaugh et al., 2012).Yildirim et al. (2019) utilized 20 gRNAs which targeted viral DNAs of this virus, and the plant was transformed with Cas 9 enzyme including vector (pKIR1.1)to provide antiviral resistance.Viral movement is inhibited by the gRNA/Cas 9 construct.Overexpressing the gRNA/cas-9 constructs in sugar beet plants resulted in a delayed and diminished accumulation of both viral DNA.In this manner, sugar beet was harnessed to build full viral resistance in the species.
Yildrim et al. ( 2022) used a genome-based characterization technology of beet curly top Iranian virus in sugar beets and isolated and identified the Turkish variants of this virus along with its pathogenicity.Ebrahimi et al. (2022) explained the presence and pathogenicity of beet curly top Iranian virus in sugar beet.This helps in the management of this viral disease.Furthermore (Yildrim et al., 2023), reported that first-time broad-spectrum resistance against Becurtovirus using CRISPR technology where four gRNAs, involved in beet curly top Iranian virus, were cloned into vector consisting of Cas 9 and later agroinoculated into the virus infected leaves of sugar beet.Briddon et al. (1989) demonstrated that the genes for the capsid protein (CP), C4, and C2 are essential for viral pathogenesis and the emergence of disease symptoms in plants.The CP gene mutation caused the virus to lose its infectiousness and ability to propagate.Targeting these genes through the CRISPR/Cas 9 tool in sugar beet will help in the management of this viral disease.
The importance of miRNAs in sugar beet curly top virus resistance (BCTV) has been shown.Majumdar et al. (2022) determined that differentially expressed (DE) miRNAs are, in certain cases, only present in the R lines.Future functional evaluation of these potential miRNAs, either by overexpression in germplasm that is BCTV sensitive or by employing them as molecular markers to scan various sugar beet genomes, will aid in establishing BCTV resistance.Furthermore, Eini et al. (2022) demonstrated the development of very effective geminiviral replicons (GVR) from BCTV with a broad host range for recombinant gene expression in plants.

Cercospora leaf spot resistance
In response to infection by the leaf-spot fungus Cercospora beticola, two isoforms of acid chitinase (SE1 and SE2) were found in the leaves of sugar beet.However, only one of the isoforms (SE2) had exochitinase Frontiers in Genetics frontiersin.orgactivity and could successfully hydrolyze chito-oligosaccharides (Nielsen et al., 1993).In resistant vs. susceptible cultivars, the sugar beet SE2 gene (B.vulgaris SE2) is expressed at a substantially higher level following Cercospora infection (Nielsen et al., 1993).The leaves of sugar beet infected by C. beticola also contained two isoforms of another acid chitinase (SP1 and SP2), which are similar to SE1 and SE2.Infected sugar beet plant leaves had very high levels of BvSP2 (B.vulgaris SP2) gene expression, however, BvSP2 protein accumulation was only found in the vicinity of the infection sites (Nielsen et al., 1994).Sugar beet leaves responded vigorously when exposed to a cell-wall protein solution made from the non-pathogenic oomycete Pythium oligandrum isolate.This response involved a substantial increase in BvSE2 gene expression, which peaked at 4 h following the vaccination (Takenaka and Tamagake, 2009).However, since oomycetes lack chitin, BvSE2 may be produced as part of a coordinated response to other proteins involved in the disease (Collinge et al., 1993).

Insect-pest resistance in sugar beet
Generally, resistance in insect-pest populations occur due to mutations in genes, which facilitates the encoding for receptor molecules and disrupt the interaction between the insect and the toxin.In rice, CRISPR/Cas9-dependent knockout of CYP71A1 mutant gene enables it encode a functional tryptamine 5hydroxylase (Lu et al., 2018).Tryptamine 5-hydroxylase is responsible for transforming tryptamine to serotonin and increases plant resistance against plant hoppers.A similar approach could be applied to the sugar beet plants to develop resistance against leaf chewing insects like H. armigera and S. litura.CRISPR/Cas 9mediated mutagenesis of GmUGT led to the development of transgenic plant (Zhang et al., 2022).The Arabidopsis ugt72b1 mutant exhibited aggravated cell wall lignification and an increase in flavonoid content (Lin et al., 2016).Both cell wall lignification and flavonoids contribute to resistance against leaf-chewing insects.Cell wall lignification serves as the first physical barrier against leaf-chewing insects (War et al., 2012).Hence, the enhanced resistance against leafchewing insects due to GmUGT mutations could be attributed to aggravated cell wall lignification and altered flavonoids (Zhang et al., 2022).Sugar beet plants transformed with the cry1Ab gene exhibited powerful defense against lepidopteron insects (Jafari et al., 2009).Sedighi et al. (2011) also reported a similar success story against an Egyptian leafworm (Spodoptera littoralis) infestation.Sugar beet plants have been transformed with Cry1Ab and Cry1C which exhibit resistance against the cabbage armyworm (Spodoptera frugiperda) (Kimoto and Shimamoto, 2001;Kimoto and Shimamoto, 2002) and on lepidopteron with Cry1C and Cry2A as well (Lytvyn et al., 2014).Regev et al. (1996) described that the Cry1C protein controls larvae of Spodoptera spp.
One of the most efficient methods for pest control is utilizing the toxic qualities of Cry protein's present in the Gram-positive pathogenic bacterium Bacillus thuringiensis.Different families of Cry proteins (or Bt proteins) exhibit highly selective toxicity against members of specific insect orders (Lytvyn et al., 2014).The order Lepidoptera exhibits a toxic response against proteins encoded by Cry1 and Cry9 genes (Table 8).Diptera and Lepidoptera show toxicity against proteins encoded by Cry2 genes.Coleopteran insects exhibit a toxic response against proteins encoded by Cry3 genes.Diptera also show a toxic response against the protein encoded by the Cry2 gene (Yuan et al., 2007).

Conclusion and future prospects
Global laboratories are increasingly turning to CRISPR/Cas 9 editing as their instrument of choice for determining how genes work and how they might be used in other contexts.This technology is being utilized in various crop development efforts to reduce biotic and abiotic stressors.The great precision, efficacy, efficiency, cost-effectiveness, and time efficiency of editing procedures have led to their development as useful tools.The advancement in molecular tools like CRISPR/Cas 9 has opened up new approaches for genome editing in sugar beet.This technology can be used for the generation of resistant/tolerant sugar beet breeding lines/germplasm to withstand abiotic/biotic stress.In sugar beet, specific genes can be silenced or knocked out to change their functionality.The plant may benefit and adapt to the abiotic stress environment.Tolerance to such circumstances may be linked to adjustments in their physiological and biochemical mechanisms.The subsequent breeding cycles produces sugar beet cultivars that are more resilient to such challenges due to the adoption of carefully chosen tolerant breeding lines.Under drought stress conditions, this improvement becomes apparent in the plant, highlighting the plant's enhanced water usage efficiency.This can also be tested for sugar beet.Furthermore, investigations into the use of CRISPR/Cas 9 to create novel quantitative features/traits with gain-of-function mutations through replacements in sugar beet could be seen.CRISPR/Cas 9 technology promises to make a significant contribution to understanding the gene regulatory networks underlying abiotic stress response/adaptation and crop improvement initiatives to create stress-tolerant plants.
Additionally, CRISPR/Cas 9 technology applications hold great potential for addressing the challenges faced in sugar beet crops during biotic stress.Biotic stresses, resulting from pathogen and pest occurrences, significantly affect sugar beet production and yield.Traditional breeding methods have certain limitations in achieving rapid and precise genetic modification.CRISPR/Cas 9 technology has emerged as a promising solution with revolutionary approach to improve sugar beet tolerance to biotic stresses.Despite success of CRISPR/Cas 9 technology in controlling biotic stress, particularly diseases in economically significant crops, its use in insect management has not been fully utilized.Modest success has been achieved despite the intellectual exercise in creating techniques for insect pest resistance in both insects and plants.In contrast to other stresses, the main drawback has been the scarcity of target genes.Therefore, it is crucial for scientists to focus on finding sources of resistance that might serve as a foundation for insect control.To achieve this, it is necessary to evaluate the available germplasm, including wild relatives of certain crops, for pest response and to identify stressresponsive genes using multi-omics techniques.Targeted mutations turning susceptible plants into those that can control their respective pests are not far off in space or time, with such studies already in vogue.These factors, along with regulatory restrictions on gene-edited crops, may help the technique succeed in advancing not only science but also societal acceptance.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Frontiers in Genetics frontiersin.org27 Misra et al. 10.3389/fgene.2023.1235855

FIGURE 1
FIGURE 1 CRISPR/Cas 9 mechanism for abiotic stress tolerance in sugar beet.

FIGURE 2
FIGURE 2CRISPR/Cas 9 system mechanism for pathogen (Bacterial/Fungal/Viral) resistance in the plants.Adaptive, processing, and interference stages are three steps involved in this system.

FIGURE 3
FIGURE 3Development of viral resistance in sugar beet through DETECTR technology.The Cas12a-gRNA complex identifies target DNA that was amplified by recombinase polymerase amplification (RPA).When the target is recognised, it fragments the surrounding FQ-labeled ssDNA reporters, allowing the fluorescence to be recovered.

TABLE 3
Different types or classes of Cas proteins, emphasizing their unique characteristics and functional roles.

TABLE 4
Legislation of different sugar beet producing countries for genome editing crops.

TABLE 5
Identified genes in other crops for providing abiotic stress tolerance through CRISPR/Cas technology.

TABLE 7 (
Continued) CRISPR/Cas 9 technology application in major sugar beet diseases for pathogen (bacterial/fungal/viral) resistance.Genes that can impart tolerance to sugar beet crop against insect pests (larval stage) via CRISPR/Cas 9 technology.

TABLE 8 (
Continued) Genes that can impart tolerance to sugar beet crop against insect pests (larval stage) via CRISPR/Cas 9 technology.

TABLE 8 (
Continued) Genes that can impart tolerance to sugar beet crop against insect pests (larval stage) via CRISPR/Cas 9 technology.(b)Some genomes successfully edited in plants by the CRISPR/Cas tool kits against insects