Genetic Engineering for Enhancing Sugarcane Tolerance to Biotic and Abiotic Stresses

Abstract

Sugarcane, a vital cash crop, contributes significantly to the world’s sugar supply and raw materials for biofuel production, playing a significant role in the global sugar industry. However, sustainable productivity is severely hampered by biotic and abiotic stressors. Genetic engineering has been used to transfer useful genes into sugarcane plants to improve desirable traits and has emerged as a basic and applied research method to maintain growth and productivity under different adverse environmental conditions. However, the use of transgenic approaches remains contentious and requires rigorous experimental methods to address biosafety challenges. Clustered regularly interspaced short palindromic repeat (CRISPR) mediated genome editing technology is growing rapidly and may revolutionize sugarcane production. This review aims to explore innovative genetic engineering techniques and their successful application in developing sugarcane cultivars with enhanced resistance to biotic and abiotic stresses to produce superior sugarcane cultivars.

1. Introduction

Sugarcane (Saccharum spp. hybrids) is a major tropical and sub-tropical crop cultivated in over 121 countries across 27 million hectares of land worldwide. It contributes about 80% of the world’s total sugar production and is the most efficient feedstock for bio-ethanol and diesel, accounting for 40% of the world’s biofuel production [1]. In addition, sugarcane is the source of valuable products such as paper, acetic acid, plywood, fibers, bio-fertilizer, animal feed, board, and industrial enzymes [2]. Conventional breeding methods for sugarcane aim to develop new hybrid varieties with higher yields and increased sugar contents [3]. However, the high genetic complexity of sugarcane hybrids, and a variable number of chromosomes (ranging from 53 to 143 chromosomes with an estimated genome size of 10 Gb pose significant challenges to the breeders [4]. At the same time, conventional breeding is costly, labor-intensive, and time-consuming, taking up to 10–15 years to release a new elite variety [5]. To acquire more of the intricate nature of the sugarcane genome, numerous initiatives have led to a variety of genome sequencing projects, encompassing parental species and hybrid genotypes [6,7,8,9,10,11,12,13]. More recently, the Chinese Academy of Sciences has initiated a pilot program in collaboration with the National Key Laboratory project, specifically targeting tropical crop breeding. Such scientific collaboration is anticipated to yield significant outcomes and contribute positively to the field of sugarcane genetic improvement in the future.

On the other hand, sugarcane cultivation is adversely affected by biotic and abiotic stresses, including weeds, diseases, insects and pests, drought, salinity, low temperatures, heavy metals, and low soil fertility (Figure 1). More importantly, genetic engineering and genome editing have emerged as powerful tools to address these challenges and increase yield productivity without losses caused by these stresses. Sugarcane is a vegetatively propagative crop and breeding aspects make it an excellent candidate for crop improvement through genetic engineering, to produce green energy. Transgenic technology allows the precise transfer of one or more stacks of genes from unrelated plants or different organisms. Since the 1990s, different genetic transformation techniques have been successfully developed in sugarcane, including electroporation, Agrobacterium tumefaciens, the biolistic bombardment method, and so on [14,15]. In the following three decades, Agrobacterium-mediated transformation and other new methods have become routine exercises in many research laboratories worldwide. Moreover, genome editing can edit, insert, or replace specific sequences within the genome. A number of genes promoting resistance to biotic and abiotic stresses have been introduced into sugarcane for genetic improvement. Therefore, this review summarizes the advances in genetic improvement of sugarcane and its potential to increase yield productivity, especially under biotic and abiotic stresses.

2. Biotic Stress

2.1. The Enhancement of Herbicide Tolerance

Due to a sessile nature, plants must confront various living organisms in the environment (Figure 2). Weed infestation is a serious problem that negatively impacts the yield of sugarcane, resulting in significant economic losses [16,17]. Introducing genes that confer herbicide tolerance into sugarcane can aid in the breeding of transgenic sugarcane tolerant to herbicides, reducing labor intensity and costs while ensuring a higher yield of sugarcane. Traditional breeding for herbicide-tolerant traits is lagging due to the lack of herbicide-tolerant gene pools in wild-sugarcane relative species [18]. An alternative strategy should be adopted for developing crops that are tolerant to broad-spectrum herbicides [19]. The advances in transgenic sugarcane that have been engineered for herbicide tolerance are summarized in

Table 1. Genetic modifications for herbicide-tolerant traits in sugarcane.

Variety	Trait Method	Explant	Promoter	Gene	Reference
SP80–180	Particle bombardment	Axillary bud	Ubi-1	bar	[20]
ROC22	Agrobacterium mediated	Embryogenic calli	Ubi-1	EPSPS, Cry1Ab	[21]
CPF-234, CPF-213, HSF-240 & CPF-246	Particle bombardment	Embryogenic calli	CaMV35S	Glyphosate	[22]
TUC 03-12	Particle bombardment	Embryogenic calli	Rice Actin	EPSPS	[23]
CPF-246	Agrobacterium mediated	Embryogenic calli	Ubi-1	Cry1Ac, Cry2 & GT	[24]
ROC22	Agrobacterium mediated	Embryogenic calli	CaMV35S	bar	[25]
ROC22	Agrobacterium mediated	Embryogenic calli	Ubi-1	bar	[26]
RA87-3	Particle bombardment	Embryogenic calli	Rice Actin	EPSPS	[27]
NCo310	Particle bombardment	Meristem		Pat	[28]
Ja60-15	Agrobacterium mediated	Embryogenic calli	Rice-Ubi	bar	[29]
Co92061 and Co671	Agrobacterium mediated	Somatic embryos	CaMV35S	bar	[30]

.

The integration of the bar gene into the genome of sugarcane using the biolistic transformation method resulted in the production of herbicide-tolerant transgenic sugarcane [20]. Using Agrobacterium-mediated transformation to introduce the herbicide-tolerant genes bar and CP4-EPSPS into the main sugarcane varieties, resulted in transgenic sugarcane exhibiting excellent herbicide tolerance [19,31]. By incorporating the genome of embryonic calli with a glyphosate-tolerant (GT) gene, the first study published the genetic transformation of four sugarcane genotypes through the bombardment of embryonic calli with the GT gene. These results showed that 88% of the transgenic plants survived on the first application of glyphosate, whereas all the non-transformed plants died. In the second application, increasing the dose of glyphosate resulted in the accumulation of GT gene-encoded products that survived [22]. However, transgenic glyphosate-tolerant sugarcane was developed for commercial release through the genetic transformation of cultivar RA87-3. The objective was to obtain glyphosate-tolerant transgenic events in two recently released cultivars, TUC 95-10 and TUC 03-12, by introducing EPSPS and nptII genes using the microprojectile method. A transgenic event tolerant to the glyphosate herbicide was successfully obtained through the biolistic transformation using the TUC 03-12 genotype as the starting material. The resulting genotype showed greater than 99% similarity with the parental genotype [23,32].

However, sugarcane borers and weeds threaten production, quality, and yield. Genetic modification using stack genes technology (combining two or more genes) was performed by introducing modified cane borer-tolerant (CEMB-Cry1Ac) and glyphosate-tolerant (CEMB-GT) genes into sugarcane. Transgenic plants showed efficient tolerance to cane borers and tolerance to glyphosate, indicating sustainable tolerance in sugarcane [24]. A transgenic sugarcane cultivar (ROC22) was regenerated using herbicide screening, containing the bar gene to obtain sense and antisense strigolactones biosynthesis genes (ScD27.2). All transgenic lines tested positive for herbicide tolerance and contained the target gene. Furthermore, PCR detection and 1% Basta (Glufosinate) application on leaves revealed bar in all lines [25]. A recent study has presented an efficient transformation system mediated by Agrobacterium, which utilizes the herbicide-tolerant bar gene as the selectable marker. The transgenic lines were confirmed through PCR and exhibited tolerance to herbicides. Moreover, the protocol demonstrated several advantages, including a higher yield of transgenic lines from calli, improved selection, and increased transformation efficiency [26]; these results showed that a dependable tool now offers a standard procedure for cultivating robust transgenic sugarcane plants.

Additionally, gene editing technology has provided new strategies for breeding herbicide-tolerant sugarcane varieties. CRISPR/Cas9-mediated multi-allelic gene mutation techniques mutate the acetolactate synthase gene (ALS) to develop broad-spectrum herbicide-tolerant sugarcane plants [33]. The development of environmentally friendly herbicide-tolerant sugarcane varieties through genetic engineering and gene editing technology will help to reduce the use of harmful herbicides and make sugarcane cultivation more environmentally friendly and sustainable in the future.

2.2. Enhancing Disease Tolerance in Sugarcane

The defense mechanisms of sugarcane are triggered in response to biotic stressors, including different pathogens, such as fungi, bacteria, viruses, and insects [34,35,36,37,38]. These biotic stressors result in serious diseases in sugarcane (Figure 2), such as red rot (Colletotrichum falcatum), smut (Sporisorium scitamineum), pineapple disease (Ceratocystis paradoxa), red stripe (Acidovorax avenae), leaf scald (Xanthomonas albileneans), grassy shoot disease (Phytoplasma), and mosaic virus (ScMV) and yellow leaf virus (ScYLV) infection, as well as sugarcane stem borer (Diatraea saccharalis), African sugarcane stalk borer (Eldana saccharina), sugarcane weevil (Sphenophorus levis), and various pests such as borers, pyrilla, thrips, grasshoppers, sucking pests, and cane grubs (Melanaphis sacchari) [16,39,40]. Sugarcane crops are susceptible to over 100 different types of pathogens [41]. Breeding programs play a crucial role in genotype screening for disease resistance. However, sugarcane breeders face significant challenges simultaneously introducing resistance to all pathogens [42]. Therefore, biotechnological strategies have been developed to produce commercial clones with superior agronomic traits; these genetic modifications are summarized in

Table 2. List of genetic engineering traits for disease tolerance in sugarcane.

Target Disease	Variety	Trait Method	Explant	Promoter	Gene	Reference
SCMV	SPF-234 & NSG-311	Particle bombardment	Calli	Ubi	CP	[1]
SrMV	CP65-357 & CP72-1210	Particle bombardment	Calli	Ubi-1	CP	[43]
FDV	Q124	Particle bombardment	Calli	Ubi	Segment 9 of ORF 1	[44]
SCYLV	H62-4671	Particle bombardment	Cell cultures	Ubi	CP	[45]
SCMV	Bululawang	Agrobacterium mediated	Shoots	CaMV35S	CP	[46]
SrMV	ROC22	Agrobacterium mediated	Leaf	CaMV35S	CP	[47]
SCMV	CP 84-1198 and CP 80-1827	Particle bombardment	Calli	Ubi	CP	[48]
SCYLV	CP 92-1666	Particle bombardment	Calli	Ubi	CP	[49]
SCMV	Bululawang	PDR & RNAi	Lateral buds		CP	[50]
SCSMV	ROC22	Agrobacterium mediated	Calli	Ubi 1	Pac1	[51]
Red rot	CoJ 83	Agrobacterium mediated	Axillary bud	CaMV35S	β-1,3-glucanase	[52]
Red rot	S2006SP-93	Particle bombardment	Calli	Ubi	Chitinase class-II	[53]
Colletotrichum	SPF-234	Particle bombardment	Calli	Ubi	SWO, SWT	[54]
Smut	KRFo93-1	Particle bombardment	Calli	CaMV35S	BSR1	[55]
Leaf scald	Q63 & Q87	Particle bombardment	Calli	Ubi	Albicidin detoxifying	[56]

.

The most prevalent viral diseases affecting sugarcane crops include sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and yellow leaf syndrome caused by Sugarcane yellow leaf virus (SCYLV) [41,57,58]. To address this, the preliminary virus capsid protein (CP) was utilized to produce transgenic plants that are immune to viruses. These plants produce a viral protein that co-suppresses and bypasses several stages of the viral life cycle and reduces disease manifestation. The use of untranslated SrMV strain CP enabled the cultivation of transgenic sugarcane plants. These plants display complete susceptibility to fully tolerant phenotypes [43]. In another study, transgenic lines with enhanced tolerance to Fiji disease virus (FDV) were obtained using particle bombardment. The transgene encodes a translatable version of FDV segment 9 of 1 open reading frame (ORF) of the virus genome. The tolerance of the transgenic plants was evaluated using a glasshouse experiment. However, the phenotypes of transgenic plants were not entirely consistent with resistance mechanisms based on post-transcriptional gene silencing (PTGS) [44].

Transgenic sugarcane against SCYLV was developed through the biolistic bombardment of cell cultures with an untranslatable CP gene. The resistance level exhibited by some transgenic plants was comparable to that of the completely resistant cultivar. These results were based on the virus titer and disease symptom development [45]. A strong innate defensive response against viral infections has been demonstrated using RNA interference (RNAi). In the host defense system, viruses produce suppressors of the host RNA interference (RNAi) pathway. A potential approach to studying viral pathogenesis and managing mosaic disease in sugarcane may involve identifying the microRNAs (miRNAs) encoded by the sugarcane streak mosaic virus (SCSMV) and further requires understanding the host genes that are related to miRNA targets [59]. Moreover, the study examined the resistance of transformed plants to the SCMV, using both the full 927 base pairs (bp) of the CP-SCMV gene sequence and truncated sequences of 702 bp. These results demonstrated that plants with the complete gene sequence exhibited a more protective strategy against the virus than those with truncated sequences [46]. Furthermore, the expression of short hairpin RNAs (shRNAs) confers resistance against SCMV infection [1,47]. These findings validated that RNAi technology can confer resistance against SCMV and the ubiquitin promoter is effective in the development of transgenic sugarcane lines [60].

Moreover, a study was conducted to evaluate the agronomic performance and viral resistance of transgenic lines transformed with SCYLV resistance, using antisense expression of a portion of the viral CP gene. The parental genotype outperformed the transgenic lines in sugarcane yield. However, the transgenic lines displayed lower SCYLV infection (0–5%) than the parental variety (98%). These variations in yield could be attributed to somaclonal variation during in vitro regeneration of transgenic plants. These findings suggest that transgenic sugarcane exhibits variable agronomic performance and disease resistance [48,49]. These results warrant further investigation to develop more efficient and reliable transgenic sugarcane cultivars. The use of transgenic sugarcane lines expressing the SCMV-CP gene led to a higher yield than the wild-type, with all evaluated lines producing significantly more cane and sucrose per hectare than the parental variety. In addition, transgenic lines exhibited a lower incidence of SCMV than the parental variety. These findings indicated that the introduction of transgenic sugarcane expressing SCMV-CP could be a viable solution for enhancing agronomic traits and viral disease resistance [61]. Taken together, these studies demonstrated that transgenic plants undergo minor hitherto discernible changes in morphology, physiology, and phytopathology despite low levels of genomic changes. Therefore, it is essential to assess somaclonal variation within transgenic populations to ensure the proper management and evaluation of transgenic sugarcane for field trials [62,63].

A comparative study aimed to evaluate the efficacy of two transgenic sugarcanes, generated through Pathogen-derived resistance (PDR) and RNAi methods using gene-encoding coat protein (CP) of SCMV, against SCMV through artificial viral inoculation. These results indicate that the RNAi approach targeting the CP gene proves more effective in producing resistance against SCMV infection in transgenic sugarcane compared to the PDR approach [50]. These results highlight promising strategies for developing SCMV-resistant sugarcane through genetic engineering. Breeding virus-resistant sugarcane varieties is the main goal of the sugarcane breeding program. Double-stranded RNA-specific ribonuclease (PAC1) encoded by the Pac1 gene from Schizosaccharomyces pombe was introduced into a virus-sensitive sugarcane cultivar by Agrobacterium-mediated transformation. These results showed that transgenic plants possess significantly milder symptoms and lower viral loads than the wild-type, providing a basic foundation for breeding virus-resistant sugarcane [51].

To combat fungal diseases such as brown rust in sugarcane, glucanase and chitinase genes have been expressed in transgenic sugarcane plants. Transgenic sugarcane resistant to red rot was developed by overexpressing the β-1,3-glucanase gene from Trichoderma spp. as the primary target of fungal attack. Microscopic examination of parenchyma cells in the stalks of these plants was filled with sucrose, which inhibited the growth of C. falcatum hyphae. Transgene overexpression after infection was upregulated and successfully transmitted to the second generation of clones [52]. Moreover, the potential of transgenic sugarcane lines expressing the barley chitinase class II gene against C. falcatum infection was assessed. Protein extracts from transgenic sugarcane plants inhibited the growth of C. falcatum mycelia, and transgenic lines demonstrated strong resistance against C. falcatum. Additionally, the mRNA expression of the transgene in C. falcatum inoculated transgenic sugarcane lines was outperformed by control parental plants [53]. Two genes, SUGARWIN1 (SWO) and SUGARWIN2 (SWT), were overexpressed in sugarcane to improve resistance against C. falcatum. Transgenic plants showed significant inhibition of the fungal pathogen infection and proliferation, indicating potential for developing crops with better fungal resistance [54]. Smut disease, caused by S. scitamineum, is considered a destructive disease of sugarcane. Rice receptor-like cytoplasmic kinase, known as broad-spectrum resistance 1 (BSR1) in sugarcane, confers resistance against smut under greenhouse conditions. BSR1 overexpressing line displayed normal growth and morphology [55], suggesting that BSR1 overexpression is effective in conferring broad-spectrum disease resistance in the different crops.

2.3. The Reinforcement of Insect and Pest Resistance

Insects and pests cause significant reduction in the yield and quality of sugarcane using different stem borers (Figure 2), such as Lepidoptera (D. saccharalis), root borers (Emmalocera depressella), top borers (Chilo terrenellus), pink borers (Sesamia inferens), and pink stem borers (Sesamia cretica) [64,65]. The extensive use of pesticides can harm other life on land such as beneficial insects, vertebrates, humans, and the environment. Pesticides may also pollute different soil zones before reaching deep water levels. Modern biotechnological approaches have made rapid progress in the genetic engineering of sugarcane plants to protect against pests by transferring genes derived from plants, pests, and bacteria [66,67,68] (

Table 3. List of transgenic sugarcane expressing foreign genes for enhanced insect and pest resistance.

Variety	Trait Method	Explant	Promoter	Candidate Gene	Target Pest	Reference
Ja60-5	Electroporation	Calli	CaMV35S	cry1Ab	D. saccharalis	[15]
TUC95-10/TUC03-12	Particle bombardment	Calli	pCab1	Bt	Diatraea saccharalis.	[18]
SP80-1842 & SP80-3280	Particle bombardment	Calli	Maize Ubi-1	skti, sbbi	D. saccharalis	[20]
ROC22	Agrobacterium mediated	Calli	Ubi-1	cry1Ab	D. saccharalis	[21]
YT79-177 & ROC16	Particle bombardment	Calli	Maize Ubi-1	cry1Ac	P. venosatus	[64]
CP65-357	Paint-sprayer delivery		Maize Ubi-1	Snowdrop lectin	Eoreuma loftini, D. saccharalis	[69]
ROC25	Agrobacterium mediated	Calli	Ubi-1	Amaranthus viridis & skti	D. saccharalis	[70]
CoC92061 & Co 86032	Particle bombardment	Calli	Maize Ubi-1	Aprotinin	S. excerptalis	[71]
CPF-246	Agrobacterium mediated	Calli	Maize Ubi-1	Vip3A	C. infuscatellus	[72]
GT54-9(C9)	Agrobacterium mediated	Leaves	CaMV 35S	cry1Ac	Sesamia cretica	[73]
SP 803280	Agrobacterium mediated	Calli	35S & FMV	cry1Ab, cry2Ab	D. saccharalis	[74]
Zhongzhe1 (ZZ1)	Particle bombardment	Direct embryo	Ubi	PPA	Ceratovacuna lanigera Zehntner	[75]
SP80-3280 & 1842	Particle bombardment	Calli	Maize PEPC	cry1Ab	D. saccharalis	[76]
YT79-177 & ROC16	Particle bombardment	Calli	Maize Ubi-1	Synthetic-cry1Ac	P. venosatus	[77]
FN81–745 & Badila	Agrobacterium mediated	Calli	RSs-1,Ubi-1	Gna	Ceratovacuna lanigera	[78]
Gui94-119	Particle bombardment	Calli	Ubi	cry1Ac	D. saccharalis	[79]
SP80-185	Plasmid transformation	Calli	Maize ubi-1	HIS Cane CPI -1	S. levis	[80]
CoC671	Agrobacterium mediated	Leaf roll	CaMV35S	cry1Aa3	C. infuscatellus, C. sacchariphagu & S. excerptalis	[81]
Co 86032 & CoJ 64	Particle bombardment	Calli	Maize Ubi-1	cry1Ab	C. infuscatellus	[82]
FN15	Particle bombardment	Calli	CaMV35S	cry1Ac	D. saccharalis	[83]
LK 92-11	Agrobacterium mediated	Calli	CaMV35S	cry1Ab	D. saccharalis	[84]
SP80-185	Particle bombardment	Calli	Maize ubi-1	CaneCPI-1	S. levis	[85]
FN15 & ROC22	Particle bombardment	Calli	CaMV35S	cry1Ac	D. saccharalis	[86]
ROC22	Particle bombardment	Calli	ST-LSI	cry2A	C. sacchariphagus, S. nivella, C. infuscatellus, A. schistaceana & S. inferens	[87]
Event CTC175-A	Agrobacterium mediated	Calli	PEPC	cry1Ab	D. saccharalis	[88]
Event CTC91087-6	Agrobacterium mediated	Calli	Maize ubi-1	Cry1ac	D. saccharalis	[89]
Bululawang	Agrobacterium mediated	Calli	RUBISCO	CryIAb-CryIAc	Scripophaga excerptalis	[90]

).

Alternatively, the genetic transformation of several insecticidal proteins, such as lectins and protease inhibitors (PIs), has been used in genetic transformation. Other genes included avac (Amaranthus viridis L. agglutinin), skti (soybean Kunitz trypsin inhibitor), sbbi (Bowman–Birk inhibitor), and gna (Galanthus nivalis agglutinin) [69,70]. Transgenic sugarcane plants expressing either potato proteinase inhibitor II or the snowdrop lectin gene showed increased antibiosis in the larvae of the canegrub Antitrogus consanguineus under glasshouse conditions. Canegrubs feeding on the transgenic line transformed with the potato gene showed 4.2% weight gain in canegrubs fed on control plants. Similarly, larvae feeding on the roots of transgenic lines transformed with the snowdrop gene showed a 20.6% weight gain of grubs feeding on wild-type plants. In contrast, larvae from the Greyback Cane Beetle (Dermolepida albohirtum) fed on transgenic plants expressing snowdrop lectin and proteinase inhibitor decreased in weight compared to larvae fed on non-transformed plants [18]. However, the growth of D. saccharalis larvae fed on transgenic sugarcane transformed with skti and sbbi from soybean was restricted compared to that of larvae from control plants [20]. In vivo bioassay studies of transgenic sugarcane transformed with a synthetic bovine pancreatic trypsin inhibitor (aprotinin) gene against the top borer (Scirpophaga excerptalis) indicated that larvae fed on transgenic plants exhibited a decrease in larval weight of up to 99.8% [71]. Transgenic sugarcane plants overexpressing sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) were evaluated for their potential resistance through feeding assays with larvae of sugarcane billbug (S. levis); significantly less damage by larval attack has been observed in transgenic plants than non-transgenic plants [66]. The trypsin inhibitory activity of PIs from Erianthus arundinaceus, a wild-sugarcane relative, was evaluated and showed significant differences among different plant tissues. The highest inhibitory activity was found in meristematic tissue, and PIs isolated from the apical meristem effectively inhibited the mid-gut proteinases of early shoot borer (C. infuscatellus) and internode borer (Chilo sacchariphagus indicus) [91,92]. These studies have demonstrated the possibility of identifying new and efficient PIs for producing sugarcane plants that are resistant to stem borers. Furthermore, transgenic sugarcane lines displaying the Vip3A protein exhibited resistance to the sugarcane stem borer (C. infuscatellus). These results imply that incorporating a single copy of the Vip3A gene into transgenic sugarcane lines confers resistance to borers [72]. Consequently, these lines could potentially be used to produce insect-resistant transgenic sugarcane and could also be combined with the Bacillus thuringiensis (Bt) toxin in gene pyramiding to enhance resistance.

Nevertheless, the most important insecticidal proteins produced by the gram-positive spore-forming bacterium B. thuringiensis (Bt), which produces proteins during the vegetative phase (Vip) and sporulation phase (Cry), are toxic to a wide range of insects [73,93]. The development of transgenic Bt plants that are resistant to stem borers in sugarcane cultivation has been achieved using specific toxins. Studies have identified various Bt genes, including cry1Ab, cry1Aa3, cry1Ac, s-cry1Ac, m-cry1Ac, cry2A, and vip3A, that have been deployed (Table 3). More importantly, Bt proteins have been used to control sugarcane borer in maize for the past decade. However, practical use in sugarcane requires the delivery of high doses of protein and harbors the slow evolution of insect resistance. Two Bacillus proteins, Cry1Ab and Cry2Ab, have been expressed in commercial sugarcane varieties, demonstrating efficacy against sugarcane borer in the field. A strategy for trait deployment of tropical crops has also been described [74]. Moreover, the study aimed to develop transgenic sugarcane lines with increased resistance to the sugarcane borer D. saccharalis. The Bt genes were incorporated into embryogenic sugarcane calli. The transgenic lines with higher levels of Bt transcripts were identified and tested for resistance against the pest [18].

To explore the utilization of the Pinellia pedatisecta agglutinin PPA in controlling aphids in sugarcane, the resistance of independent transgenic sugarcane lines was assessed. Alterations in stomatal patterns, antioxidant enzymes, chlorophyll content, sugar, and tannins were examined before and after insect infestation. These findings revealed that PPA enhanced sugarcane resistance to sugarcane woolly aphids and insect resistance [75]. Taken together, there is an urgent need to fully utilize the recently sequenced sugarcane genome to investigate the interactions between sugarcane and its pathogens, as well as pathogens that are closely related to those affecting sorghum and other grasses sharing similar genomes [6,7,8,9,10,12,13]. However, it is expected that the reference genome for Saccharum spp. hybrids is still underway because of its complexity and high polyploidy, which may serve as a tool for searching resistance genes for many common diseases in sugarcane, and functional disease resistance will be maintained when genes are moved across the species using transgenic technology.

3. The Enhancement of Tolerance to Abiotic Stress

Sugarcane cultivation is influenced by a variety of abiotic factors, such as drought, salinity, high temperature, nutrient deficiencies, and heavy metals, which significantly decrease the average yield (Figure 1). To cope with adverse environments, plants must adopt tolerance mechanisms and undergo various morphological, anatomical, physiological, and cellular changes in response to abiotic stresses [94]. The plant response to stress is a multifaceted process that involves various signaling pathways, post-translational modifications, secondary metabolite synthesis, nitrogen metabolism, and the activity of various proteins, including transcription factors, kinases, and transporters. The genes responsible for regulating biotic and abiotic stress responses may function cumulatively or redundantly, activating both common and distinct downstream targets [95]. Adapting to these stresses is also linked to metabolic adaptation, which results in the accumulation of various organic solutes like proline, sugars, betaines, and polyols. To protect from the harmful effects of reactive oxygen species (ROS) (Figure 3), plants utilize antioxidant enzymes such as ascorbate peroxidase APX, SOD, and GR, and non-enzymatic antioxidants like carotenoids, ascorbic acid, and flavonoids [96,97]. Researchers have identified different genes that contribute to abiotic stress tolerance (

Table 4. Genetic engineering of sugarcane for abiotic stress tolerance.

Variety	Trait Method	Type of Explant	Promoter	Gene	Gene Function	Stress	Reference
ROC22	Agrobacterium		CaMV35S	TERF1	Gene regulation	Drought	[2]
RB855156	Biolistic	Embryogenic calli	pRab17	DREB2A CA	Gene regulation	Drought	[100]
NCo310	Biolistic	Embryogenic calli	UBI	AtBBX29	Gene regulation	Drought	[101]
CP-77-400	Agrobacterium	Apical buds	CaMV35S	AVP1	Osmotic regulation	Drought	[102]
CSSG-668	Biolistic	Embryogenic calli	CaMV35S	AVP1	Osmotic regulation	Drought	[103]
RB835089	Biolistic	Embryogeniccalli	UBI	BI-1	PCD-regulation	Drought	[104]
RB855156	Biolistic	Nodal buds	ABA-AIPC	P5CS	Proline synthesis	Salinity	[105]
Guitang21	Agrobacterium	Embryogenic calli	UBI	SoP5CS	Proline synthesis	Drought	[106]
Co 86032	Biolistic		UBI	EaGly III	Reduce oxidative stress	Salinity	[107]
Co86032	Agrobacterium		UBI	HSP70	Cellular stability	Drought/ Salinity	[108]
RB855536	Biolistic	Embryogenic calli	AtCOR15a	ipt	Cytokinin	Cold	[109]
ROC22	Agrobacterium	Calli	UBI	SoTUA	α-tubulin synthesis	Cold	[110]
ROC10	Agrobacterium		CaMV35S	TSase	Biomolecules stabilization	Drought	[111]
Co86032	Agrobacterium/ biobalistic	Embryogenic calli	UBI	PDH45/DREB2	Nucleic acids metabolism	Drought/ Salinity	[112]

) in model/water-tolerant plants, unrelated plants, or completely different organisms, including those involved in the overexpression of transcription factors (TFs) and effector proteins, which play crucial roles in activating genes in response to abiotic stresses [98,99].

The COR/DREB (Cold-induced regulated-dehydration-responsive binding element) family of regulatory proteins is the first known set to play a role in the regulation of abiotic stress genes in plants [113]. Studies have shown that the overexpression of Arabidopsis AtDREB2A in transgenic sugarcane can enhance drought tolerance under greenhouse conditions. In these experiments, transgenic plants exposed to drought stress exhibited higher relative water content (RWC), carbon assimilation, sugar content, and bud sprouting, without affecting biomass [100]. B-box (BBX) proteins are essential for the control of plant growth and development. Transgenic sugarcane overexpressing the AtBBX29 gene exhibited improved rates of photosynthesis and higher levels of antioxidants and osmolytes under drought conditions [101]. In another study, the accumulation of osmolytes, such as proline, soluble sugars, and glycine betaine, was observed in sugarcane plants engineered to overexpress the tomato ethylene-responsive factor gene (TERF), and these transgenic plants displayed reduced ROS and malondialdehyde (MDA) content [2].

The Arabidopsis H⁺-pyrophosphatase type I gene (AVP1) is involved in the control of apoplastic pH and auxin transport, and overexpression of the AVP1 gene in sugarcane results in increased RWC and osmotic and turgor potential in the leaves, indicating that improved drought and salinity tolerance. In addition, transgenic sugarcane plants exhibited profuse rooting systems [102,103]. Proapoptotic BAX proteins are responsible for controlling programmed cell death (PCD). Overexpression of the BAX inhibitor gene (BI-1) from A. thaliana in sugarcane plants increases drought tolerance by reducing the induction of cell-death pathways [104]. The drought-responsive gene BRK1 from S. spontaneum was transformed into sugarcane, and BRK1 transgenic lines had improved physiological parameters. Interlocking marginal lobes in epidermal leaf cells were observed in all transgenic BRK1 lines during drought stress compared with the wild-type. These findings suggest that BRK1 plays a potential role in sugarcane’s response to drought stress, promoting leaf epidermal cell morphogenesis and actin polymerization [114].

Plants exposed to salinity stress acquire osmoprotectant molecules, such as proline, which serve not only as a source of nourishment but also as scavengers such as ROS, to maintain cellular activities. By overexpressing the pyrroline-5-carboxylase synthase gene (P5C5), salinity-tolerant sugarcane was obtained [105]. Additionally, improved tolerance to water deprivation was achieved through overexpression of a related gene (SoP5C5). P5C5 plays a crucial role in proline production. The ROS-scavenging glyoxalase pathway enzymes, including glyoxalase I (Gly I), glyoxalase II (Gly II), and glyoxalase III (Gly III), help transgenic sugarcane plants to eliminate harmful compounds, such as glyoxylate, under abiotic stress conditions [106]. Furthermore, under salt stress, transgenic sugarcane overexpressing the EaGlyIII gene displayed improved RWC, photosynthesis, osmolytes, and ROS-scavenging enzyme activities, resulting in increased biomass and enhancing germination rates [107]. These results indicate that the overexpression of EaGly III could be a promising approach for salt and drought tolerance in sugarcane [115]. However, the ScD27.2 gene was introduced into sugarcane to study its role in drought tolerance. The results showed that interfering with ScD27.2 expression decreased tolerance to drought stress, indicating its role in sugarcane growth and development [25].

Plant cells respond to stressful conditions by producing a family of stress proteins known as heat shock proteins (HSPs). In E. arundinaceus, a sweet cane, HSP70 overexpression has a strong protective effect when plants are exposed to water and salt stress. Transgenic plants displayed increased photosynthetic efficiency, RWC, stress-induced gene expression, and cell membrane thermostability [108]. Transgenic sugarcane with the drought-tolerant Ea-DREB2B gene from S. arundinaceum, a wild species of S. officinarum, can regulate response to drought stress. In addition, interactions between soil fungi and bacterial communities in the rhizoplane, rhizosphere, and bulk soil were assessed. The results revealed that the host plant genotype plays a crucial role in strengthening plant–fungi interactions and enhancing beneficial fungal function in the root-related area of transgenic sugarcane, allowing response to drought stress. Moreover, changes in the soil environment caused by transgenic sugarcane alter bacterial communities [116,117].

However, limited studies have been conducted on cold-tolerant transgenic sugarcane (Table 4). These plants overexpress the bacterial isopentenyl-transferase gene (ipt) using the cold-inducible promoter AtCOR15 derived from A. thaliana. Compared with non-transgenic parent plants exposed to low temperatures, transgenic sugarcane exhibited increased chlorophyll, reduced MDA content, and decreased electrolyte leakage [109]. Transgenic plants overexpressing the tubulin gene (TUA), cloned from cold-tolerant sugarcane varieties, enhanced the cold tolerance of cold-susceptible sugarcane varieties, exhibiting increased soluble protein and sugar content, enhanced peroxidase activity, and reduced MDA content compared to non-transgenic plants [110]. Further investigation into the effects of cold stress could have substantial implications for the sugarcane industry, as cold stress negatively affects sugarcane crop yields and quality.

4. Production of New Compounds and Renewable Energy Sources

Genetically modified sugarcane has achieved significant improvement under biotic and abiotic stress conditions. Plant cells, as opposed to animal cells, are cost-effective and safe methods for producing new recombinant (r) proteins. Sugarcane plants have well-developed storage tissue systems, which make them promising candidates to produce high-value compounds and pharmaceuticals. Recent advancements in bio-pharming have enabled the production of a wide range of essential products, such as high biomass production, rapid growth, and efficient carbon fixation, making sugarcane more attractive for novel compound applications [41,57,118]. The utilization of sugarcane for molecular farming has gained significant momentum owing to its minimal transgene dispersal and high amount of extractable juice with low protein content. The culm of sugarcane comprises 70% dry weight and is predominantly composed of parenchyma cells, a desirable target for r-protein production. Within plant cells, vacuoles constitute a large portion of the cell volume, contributing 60% of the cane weight [57,119]. Thus, targeting r-proteins in the vacuole can result in high-protein yields and relatively simple purification.

Initial attempts were made to express proteins in the cytoplasm and purify from the leaves, resulting in low yields due to the presence of numerous other proteins that may interfere with downstream processing [120,121]. However, targeting proteins to vacuoles in the stem parenchyma may lead to higher levels of recombinant proteins. Previous studies have shown that newly identified vacuolar-targeting determinants (VT) of 78-bp or 18-bp in combination with strong constitutive promoter (Port ubi882) have proven to be effective in the production of large quantities of r-proteins. However, the presence of vacuolar proteolytic enzymes and the acidic nature of lytic vacuoles must be considered when selecting recombinant candidate proteins. It is estimated that 4.8–7.2 kg of r-protein can be produced per acre (40,000 canes per acre) with a purity of 50% after affinity chromatography [122]. This yield is comparable to commercially available r-proteins. The abundance of extractable juice with low protein content and well-established juice processing technology has motivated researchers to focus on the culm of sugarcane. Thus, culms have drawn attention due to their significant biomass output and potential for the large-scale production of recombinant proteins. Sugarcane could be an effective platform to produce recombinant proteins, and further research could significantly impact the biotechnology sector. Taken together, targeting r-proteins to the lytic vacuoles of sugarcane shows promising results in the production of recombinant proteins. Sugarcane can extract a large volume of juice coupled with newly identified vacuolar-targeting determinants and strong constitutive promoter, making it an efficient platform for molecular farming.

Trehalose is a naturally occurring non-reducing disaccharide that plays an important role in regulating carbon metabolism and photosynthesis. To investigate the effects of trehalose synthesis on sucrose accumulation in sugarcane, overexpression of Escherichia. coli, otsA (trehalose-6-phosphate synthase TPS), otsB (trehalose-6-phosphate phosphatase; TPP), and silenced native TPS expression altered the effect of trehalose synthesis. These findings suggest that manipulating Tre6P/trehalose metabolism could be a potential strategy for modifying the sugar profile of sugarcane stems [123]. Sucrose phosphate synthase (SPS) is a vital enzyme that plays a key role in regulating sucrose content in sugarcane by controlling the synthesis of sucrose. Enhancing SPS activity may be a useful approach to increasing sugarcane yield [124]. The effects of overexpressing the SoSPS1 gene on sucrose accumulation and carbon partitioning in transgenic sugarcane were studied. Overexpression of SoSPS1 resulted in increased levels of sucrose and enhanced enzymatic activity in the leaves and stalks, leading to improved plant growth [125]. Transgenic sugarcane with enhanced sucrose yield was developed by the over-expression (SoSPS1) gene. Furthermore, the nutritional and mineral composition of transgenic lines and non-transgenic sugarcane were analyzed after 11 months in field conditions. Protein and potassium content was higher in stems of transgenic lines. However, wild-type and transgenic sugarcane were found to be substantially equivalent in terms of nutritional and mineral compositions [126]. The development of new sucrose isomers, trehalulose and isomaltulose, is facilitated by the insertion of sucrose isomerase genes. The transgene expression in the sugarcane culms exhibited significantly greater expression levels than in leaf tissues. The overall sugar estimation conducted in internodes of the transgenic sugarcane revealed increased sugar concentrations in mature sugarcane culms. The transgenic sugarcane lines demonstrated the highest sugar recovery of 14.9%, compared to 8.5% in the control lines [127]. However, consistent attempts to modify sugar metabolism to increase sugar yield remain challenging for molecular farming.

Another field of study is bioplastics such as polyhydroxyalkanoates (PHAs), which are biodegradable. Previous studies have shown that these polymers can be produced from sugarcane, including polyhydroxybutyrate (PHB) [128,129]. Utilization of sugarcane as a renewable bioenergy crop is a new emerging task. The selection of superior multipurpose cultivars for agro-industries is based on genetic and biotechnological strategies. To meet the demand for renewable energy sources, sugarcane has received numerous genes promoting resistance to biotic and abiotic stresses [41,57,118]. Nonetheless, considerable emphasis has been placed on improving the production of bioenergy and biofuel. Various approaches have been devised to modify lignin, facilitate saccharification, and enhance second-generation bioethanol [130,131]. Recently developed transgenic sugarcane, termed oilcane, has been genetically engineered to direct carbon flux towards biosynthesis and accumulation of energy-rich triacylglyceride (TAG) molecules in vegetative tissues [132]. This oilcane was employed to produce five high-value bioproducts: hydroxymethylfurfural (HMF), furfural, acetic acid, fermentable sugars, and vegetative lipids [132,133,134]. Genetic and biotechnological strategies have been employed to improve sugarcane productivity, including the development of transgenic sugarcane that directs carbon flux toward the biosynthesis and accumulation of energy-rich molecules. These advances in sugarcane biotechnology hold great potential for the development of future energy sources for biofuel production. Conversely, the success of these developments depends on the careful selection of techniques employed for sugarcane transformation, choice of promoters and marker genes, target tissue/explants, and tissue culture system (Table 1, Table 2, Table 3 and Table 4). Further research and development in sugarcane biotechnology will be crucial for meeting the increasing demand for renewable energy sources and sustainable agro-industries. However, little progress has been made in cultivating sugarcane for commercialization through genetic engineering in a few countries (

Table 5. List of Sugarcane genetically modified (GM) event names approved for food/feed and commercialization.

Developer	Event Name	Trait Method	Gene	Gene Source	Product	Function	Country/Year
Centro de Tecnologia Canavieira (CTC)	CTB141175/01-A	Microparticle bombardment	cry1Ab	B. thuringiensis subsp. kurstaki	Cry1Ab delta-endotoxin	lepidopteron insects	Brazil 2017, Canada & United States 2018
	CTC-92015-7	Agrobacterium mediated	cry1Ac	B. thuringiensis subsp. Kurstaki strain HD73	Cry1Ac delta-endotoxin	lepidopteron insects	Brazil 2022
			nptII	E.coli Tn5 transposon	neomycin phosphotransferase II	neomycin & kanamycin antibiotics	Brazil 2022
	CTC75064-3	Agrobacterium mediated	cry1Ac	B.thuringiensis subsp. Kurstaki strain HD73	Cry1Ac delta-endotoxin	lepidopteron insects	Brazil 2020, Canada 2022
			nptII	E.coli Tn5 transposon	neomycin phosphotransferase II	neomycin & kanamycin antibiotics	Brazil 2020, Canada 2022
	CTC91087-6		cry1Ac	B. thuringiensis subsp. Kurstaki strain HD73	Cry1Ac delta-endotoxin	lepidopteron insects	Brazil 2018 & United States 2020
	CTC93209	Agrobacterium mediated	cry1Ac	B. thuringiensis subsp. Kurstaki strain HD73	Cry1Ac delta-endotoxin	lepidopteron insects	Brazil 2019
	CTC95019-5	Agrobacterium mediated	cry1Ac	B. thuringiensis subsp. Kurstaki strain HD73	Cry1Ac delta-endotoxin	lepidopteron insects	Brazil 2021
			nptII	E. coli Tn5 transposon	neomycin phospho transferase II	neomycin & kanamycin antibiotics	Brazil 2021
PT Perkebunan Nusantara XI (Persero)	NXI-1T	Agrobacterium mediated	EcBetA	E. coli	choline dehydrogenase	Osmoprotectant, glycine betaine	Indonesia 2011
			nptII	E. coli Tn5 transposon	neomycin phospho transferase II	neomycin & kanamycin	Indonesia 2011
			aph4 hpt)	E. coli	hygromycin-B phosphotransferase	hygromycin B	Indonesia 2011
PT Perkebunan Nusantara XI (Persero)	NXI-4T	Agrobacterium mediated	RmBetA	Rhizobium meliloti	choline dehydrogenase	osmoprotectantglycine betaine)	Indonesia 2013
PT Perkebunan Nusantara XI (Persero)	NXI-6T	Agrobacterium mediated	RmBetA	Rhizobium meliloti	choline dehydrogenase	Osmoprotectant, glycine betaine	Indonesia 2013
Estación Experimental Agroindustrial Obispo Colombres (EEAOC)	TUC-873RH-7	Microparticle bombardment	cp4 epsps (aroA:CP4)	A. tumefaciens strain CP4	(EPSPS) enzyme	glyphosate herbicide	Argentina 2015
			nptII	E. coli Tn5 transposon	neomycin phosphotransferase II	neomycin & kanamycin antibiotics	Argentina 2015
Monsanto Company & Bayer Crop Science	MON87427 × MON95379 × MON87411	Conventional breeding-cross hybridization-transgenic donor(s)	cry1Da_7	B. thuringiensis	crystalline protein prototoxin Cry1Da_7	S. italica promoter and O. sativa gos2 terminator-Rice actin 15 gene-intron	Brazil 2021
			cry1B.868	B. thuringiensis	crystalline protein prototoxin Cry1B.868	S. italica promoter and O. sativa gos2 terminator-Rice actin 15 gene-intron	Brazil 2021
			cry3Bb1	B. thuringiensis subsp. kumamotoensis	Cry3Bb1 delta endotoxin	Tolerance to coleopteran insects & corn rootworm	Brazil 2021

Source 2024. Crop Biotech Update April. Available online at: https://www.isaaa.org/gmapprovaldatabase/crop/default.asp? CropID=27&Crop=Sugarcane (accessed on 24 April 2024).

).

5. Status and Concerns about GM Sugarcane

Since its inception, the debate over genetically modified organisms (GMOs) has continued, with a particular focus on the release of herbicide-tolerant sugarcane and its potential impact on the sugar export market. Several studies have been conducted on sugarcane using various genetic transformation techniques. For example, the safety of transgenic sugarcane with the AVP1 gene [103] was assessed through animal feeding and genotoxicity assays. Acute and subchronic toxicity studies were conducted in rats, and no significant differences were observed among the different treatments [135]. Moreover, genotoxicity assessment revealed that GM sugarcane was neither genotoxic nor cytotoxic to rats. These results suggest that GM sugarcane is non-toxic to experimental animals and is suitable for commercial release. A field study conducted on transgenic sugarcane expressing insect and glyphosate tolerance genes showed poor agronomic and industrial traits compared to non-transformed plants [21]. Some studies have reported successful field trials on disease-resistant transgenic sugarcane varieties. For instance, two cultivars resistant to SCMV showed large variations in yield and disease resistance in the field condition [48]. In another trial, transgenic sugarcane lines transformed for SCYLV resistance showed reduced performance compared to the parental genotype, despite their viral resistance [49]. Conversely, a positive result was observed in a field experiment conducted on transgenic sugarcane lines expressing the CP gene of SCMV, with greater yield and lower SCMV disease incidence at four different experiment locations in China across two successive growing seasons [61]. However, only a few officially approved varieties (ISAAA 2024: Table 5) have been released.

A transgenic trait expressing the herbicide-resistant sugarcane CP4-EPSPS gene was developed in Argentina. In Argentina, the commercial approval/deregulation of transgenic events requires continuous effort. Extensive research has been conducted on health and environmental issues to evaluate the transgenic event impact on agricultural systems and food safety [27,32,136]. Later, the Ministry of Agriculture, Livestock, and Fisheries denied grants for final approval and commercialization. This may concern the export of sugars produced from such transgenic crops. Nonetheless, this process has led to the development of new regulations for the cultivation of sugarcane. Argentina’s regulatory system for assessing transgenic crops has served as a model in many other countries. In 2013, a significant change was made in the requirements for evaluating vegetatively propagated and highly polyploid genome crops such as sugarcane. This change allowed for fast-track evaluation of new events containing gene constructs equal or similar to those that have already been approved. This resolution has greatly accelerated the approval of new sugarcane varieties with the same or similar gene constructs regardless of their position in the genome. The introgression of a transgene through backcrossing is impossible. Thus, two possible ways to produce transgenic sugarcane varieties have emerged: forward breeding and the direct transformation of elite varieties. Both methods are time-consuming and often result in outdated technology by the time the transgenic variety is ready for commercial release. This policy has allowed the production of new varieties with desirable traits in a timely and cost-effective manner and has been adopted by regulatory agencies in Canada, the United States, and Brazil (Table 5). However, regulatory authorities in various countries play crucial roles in monitoring and overseeing the research and development of GMOs to ensure that their introduction does not pose any threat to human health or the environment. Moreover, due to deregulation, the cultivation and use of transgenic varieties are no longer subject to regulatory restrictions. Therefore, it is necessary to establish stewardship programs to safeguard these varieties and neighboring crops. Implementing current strategies would not only facilitate the successful reintroduction of GM sugarcane but also establish standards for introducing new transgenic traits into sugarcane.

Moreover, the first drought-tolerant transgenic sugarcane expressing the bacterial choline dehydrogenase gene, the osmoregulator glycine betaine, plays a crucial role in plant defenses against cellular dehydration. Persero (PT Perkebunan Nusantara XI) was commercially released in Indonesia (ISAAA 2024; Table 5). Two genetically modified sugarcane varieties produced by Embrapa Agro-energy were approved by the Brazilian regulatory authority, the National Biosafety Technical Commission (CTNBio). Brazilian science has developed the first sugarcane variety with CRISPR/Cas9 altered, Cana Flex I and II, which showed improved cell-wall digestibility. Currently, plants with altered genomes are controlled as transgenic in certain nations but are not regarded as GM in others. Recently, Brazilian authorization has been encouraging as it will significantly reduce the expenses and work required for commercial releases. It is anticipated that more nations will soon assist in the deregulation of varieties created using transgene technology.

6. Genome Editing for Sugarcane Improvement

Sugarcane is characterized by a huge genome size (~10 GB), high degree of complexity, auto-allopolyploid, limited genetic diversity, and extensive recombination between the two subgenomes. In addition, it is highly heterozygous in nature, taking a long generation time to propagate vegetatively and preserve its genetic makeup. However, alternative strategies such as genetic transformation and the application of different omics technologies have been widely employed for the genetic improvement of sugarcane [137]. Nonetheless, sugarcane can be edited using the CRISPR/Cas9 system, even at high levels of ploidy. CRISPR/Cas9 is a highly effective and dependable tool for targeted mutagenesis (Figure 4). To date, four classes of nuclease-mediated genome editing methods/technologies have been developed; meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system [138,139].

Targeted mutagenesis using TALENs was first demonstrated in the sugarcane gene responsible for lignin biosynthesis, caffeic acid O-methyltransferase (COMT). The gene was identified, and TALEN-mediated mutations were introduced into the COMT gene to assess effects on agronomic performance, lignin content, cell wall composition, and saccharification efficiency. These results showed a 19.7% reduction in lignin and a 44% increase in saccharification of cell wall-bound sugars, without influencing biomass, disease resistance, and logging under field conditions. These modifications significantly increased the production of sugarcane-derived biofuels [130,131]. Furthermore, targeted co-mutagenesis of more than 100 alleles of the COMT gene improves saccharification and enhances bioethanol yield from biomass without affecting agronomic performance [138,140]. However, optimizing SgRNA expression cassettes, vectors, and delivery systems is necessary for co-editing multiple alleles. Although TALEN and ZFN-based genome engineering of crops have proven useful, their application for precise gene editing has been limited because of the design and complex interactions of zinc fingers and DNA [138,141]. Of note, sugarcane is a vegetative propagating crop once mutagenesis occurs, and can be stably transmitted to progeny.

The CRISPR/Cas9 system is a groundbreaking technology that emerged as the most crucial tool for crop improvement following genetic transformation [142,143,144]. Among three types of CRISPR/Cas9 mechanisms, Types I, II, and III were classified based on the presence of cas 3, cas 9, and cas 10 genes, respectively; Type II is preferred for genome editing. Using this technology, plants classified under the non-GMO category can be produced, which boosts public acceptance as the target genome does not contain any foreign DNA molecules [145]. Insect pests and diseases are the major causes of reduced sugarcane yield; also, ratoon crops should be considered. Red rot is a major disease that results in significant yield loss in sugarcane. For example, the antifungal diene compound (AFD) is a potent antifungal agent produced in sugarcane that helps to alter dormancy in Colletotrichum spp. CRISPR/Cas9 technology can be utilized to modify genes involved in AFD biosynthesis, providing sugarcane resistance to red rot caused by Colletotrichum spp. [146].

Additionally, smut disease caused by S. scitamineum can be managed by modifying the genes involved in β-1,3-glucanase biosynthesis using CRISPR/Cas9. Deletion of Sspep1 plays a crucial role in various biological activities of S. scitamineum, such as mating, defense mechanisms, and virulence, and is helpful in smut disease resistance [147]. Furthermore, deletion of the Ssubc2 gene encoding kinase regulator in S. scitamineum plays a potential role in the growth and proliferation of fungi in sugarcane, resulting in disruption of mating and a decrease in the virulence potential of the fungus [148,149]. Deletion of the SsRSS1, which regulates salicylic acid, can result in a reduction in fungal virulence. For instance, R genes play a crucial role in determining the capacity of plants to resist insect pests and diseases, while susceptible genes (S genes) make plants sensitive to biotic stress [150]. The adoption of CRISPR/Cas9-mediated genome editing in sugarcane offers several benefits including multiplex capacity, adaptability, and ease of design. This approach is considered more advantageous than the TALEN [151].

CRISPR/Cas9 technology has the potential to improve the expression of the non-specific lipid transfer (ScNsLTP) gene in sugarcane, thereby providing tolerance to drought and cold stress [152]. The transcripts of ScGluD2, a novel member of subfamily D beta-1,3-glucanase, partially increased after 12 h of salt stress and were significantly upregulated from 6 to 24 h under abscisic acid (ABA), H₂O₂, and CdCl₂. These results suggest that ABA may act as a signaling molecule that helps to regulate oxidative stress and plays a critical role in stress-induced ScGluD2 transcripts [153]. Interestingly, ScGluD2 is a stress-responsive gene in sugarcane expressed under defense response against smut, salt, and heavy metal stress [154]. Using CRISPR/Cas9 technology, ScGluD2 expression can be enhanced to provide tolerance against heavy metal and saline stress in sugarcane.

The use of specific co-editing techniques involving multiple alleles and CRISPR/Cas9 technology can facilitate the rapid development of herbicide tolerance in sugarcane. Analysis of 146 individually engineered plants from five separate experiments in sugarcane revealed targeted nucleotide substitutions, resulting in alterations in W574L and S653I in the acetolactate synthase (ALS) gene in 11 lines. In addition to single targeted amino acid substitutions, W574L and S653I were present in 25 and 18 lines, respectively. Co-editing of three ALS alleles that confer herbicide tolerance has been established [33], and such techniques can be used to convert inferior to superior alleles through precise substitution mutations.

The application of TALEN-based technology enabled precise editing of conserved locations on the gene, resulting in the development of COMT-engineered lines that exhibited a 19.7% reduction in lignin content and a 43.8% improvement in saccharification. The lignin composition of COMT mutants increases from 29 to 32% [130]. Considering this fact, and the complex and polyploid nature of the sugarcane genome, precise mutation using CRISPR/Cas9 technology could serve as a valuable tool for reducing lignin content. The utilization of a multigene expression/suppression strategy resulted in the hyper-accumulation of triacylglycerol (TAG) and total fatty acids (FA) in the leaves, as two-fold higher than observed in unmodified energy cane [155,156]. The strongest link between TAG accumulation, ZmDGAT1 expression, and SDP1 repression was also observed. Moreover, genetically modified sugarcane upregulates TAG biosynthesis genes and downregulates TAG catabolism genes to direct carbon flux toward oil synthesis and accumulation in the leaf and stem tissues. Additionally, under field conditions, constitutive expression of WRI 1, along with lipogenic factors DGAT1-2, OLE1, and PXA1, resulted in hyper-accumulation of TAG and reduced biomass yield [157,158].

7. Future Perspectives and Conclusions

Sugarcane is a valuable cash crop that not only provides sugar but also serves as a renewable energy source. The application of genetic engineering and genome editing tools can reduce losses caused by biotic and abiotic stresses. Several transgenic sugarcane traits outperformed under greenhouse and field conditions. However, the commercial release of transgenic sugarcane varieties lags behind that of other crops, such as soybean, corn, and cotton. Nonetheless, some countries, including Argentina, recently allowed the rapid release of second varieties of sugarcane, with genetic modifications similar to previously released varieties. This action is expected to encourage the development of new transgenic varieties in other countries, such as India, China, Thailand, and the United States. With the continuous increase in the world population, excellent transgenic sugarcane varieties such as those with insect resistance, disease resistance, and drought tolerance will attract increasing attention from various countries, thus promoting sucrose production to meet people’s demands. Recent marketing approvals for transgenic sugarcane in Indonesia and Brazil are expected to accelerate the development of new transgenic sugarcane varieties that can provide effective solutions to the challenges faced by crops in different agro-ecological conditions. These improved sugarcane varieties are most urgently needed in Yunnan province, China, because of its huge mountains and extremely complex ecological environment compared to elsewhere in the world. At the same time, advanced biotechnological methods can be used to produce r-proteins, biomolecules, and industrial and pharmaceutical products from the stem vacuoles of sugarcane. Sugarcane can also serve as a platform to produce edible vaccines and other useful products, making plant-based production cost-effective. The successful implementation of sugarcane-based vaccine production could significantly reduce global demand; with the potential to fuel the industry, stimulate economic growth, and reduce carbon emissions, sugarcane is poised to be a game changer.

The CRISPR-Cas9 gene editing method has shown promising results in rice and wheat. However, identifying appropriate sequences for modification and predicting the potential consequences on the genome is a challenging task in sugarcane. However, genome editing in sugarcane has been hindered by transgene silencing at pre–post transcriptional levels. To address this, efficient promoters can be used to regulate specific genome-editing tools, such as Cas9. Like other genome-editing nucleases, Cas9 may also have off-target effects, resulting in unwanted mutations. The gRNA–Cas9 complexes, in addition to cutting target DNA, can also cleave off-target DNA sequences. Overall, several challenges still need to be addressed. The use of different Cas9 variants and other CRISPR-associated nucleases has the potential to be a powerful tool for successful genome editing in sugarcane in the near future.

Furthermore, to effectively utilize CRISPR/Cas9 technology for defending biotic and abiotic stress in sugarcane, a comprehensive understanding of the genomic sequences is indispensable. Fortunately, recent advances in genome sequencing technology and the availability of the reference genome, along with other transcriptome resources, can significantly enhance sugarcane genetic improvement. The CRISPR-Cas9 technology can efficiently edit multiple copies of sugarcane genes after knowing haplotypes clearly, including those with a high number of homologs and homeologs (orthologous and paralogous). Optimizing genome editing reagents and their delivery can facilitate the co-editing of numerous alleles, thereby increasing editing efficiency. Recent achievements in targeting multiple copies of genes in sugarcane have demonstrated that genome editing is possible for polyploid crops. Thus, this opens new avenues for future research to develop new elite varieties with enhanced tolerance to biotic and abiotic stressors. The utilization of gene editing technology is expected to increase soon due to its potential to significantly reduce the time and costs associated with commercial deregulation.