Keywords

14.1 Introduction

Sugarcane is a tall perennial tropical grass that produces unbranched stems of 2–4 m or taller and around 5 cm in diameter. It is cultivated to produce sugar (sucrose) which is extracted from the solid stems or stalks. Sucrose is synthesized in the leaves, exported, and accumulated in the stem. The stem is differentiated into joints comprising a node and an internode where sucrose content gradually increases from young immature to mature internode. The length and diameter of the internode are affected by environmental factors such as water supply, nutrition, and temperature (Verma et al. 2020a). The condition favorable to harvest sucrose is dependent on the ripening state that normally takes place during the cooler or drier times of the year. Under the best ripening condition, a tonne of sugar can be produced from 7 to 12 tonnes of cane. After sugarcane harvesting, it is normal to regrow sugarcane once or several times, and this cultivation method is known as ratooning.

Sugarcane, a C4 plant, is more efficient to use light, water, and nitrogen availability compared to C3 plants (Kellogg 2013). Under full sunlight, C4 plants continue to assimilate CO2 into carbohydrates, which increase as the available light increases. During the daytime, the stomata are slightly closed to minimize transpiration without any effect on carbon assimilation. The C4 plants also produce more biomass and have a higher photosynthetic rate per unit of water input and nitrogen. The operation of primary carboxylation of phosphoenolpyruvate (PEPC), which is located in mesophyll cell and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present in bundle sheath cells, makes the CO2 assimilation more efficient with low rate of photorespiration. Assimilation of CO2 produces various forms of organic carbon that will produce sucrose as a mobile carbon compound and is distributed to other tissues for carbon and energy source. The key enzyme for the synthesis of sucrose is sucrose-phosphate synthase (SPS), which catalyzes the production of sucrose-6-phosphate, which is converted to sucrose by the action of sucrose-phosphate phosphatase (Huber and Huber 1996). The SPS activity has been reported to play an important role in sucrose accumulation and biomass production in plants, including sugarcane (Anur et al. 2020). Molecular analysis revealed that SPS activity is structurally regulated by phosphorylation concerning changes in light intensity and water availability. The regulation of PEPC and SPS by environmental conditions such as water supply might express the important role of the enzymes in determining the growth and productivity of sugarcane under water deficit conditions.

Trends in climate change over the past few decades have induced biotic and abiotic stress on plants that have an impact on many agricultural productions, including sugarcane. Plants show a variety of physiological changes under climate change ranging from enhanced abiotic stress to accelerated pathogen infection. Climate change is assumed to cause an increase of temperature, flooding, and drought stress. However, many reports have been focused on the effect of drought stress on plant productivity (Verma et al. 2020a, 2021a, b). The physiological study revealed that plants possess the nature of resilience to survive under a limited water environment. Water stress induces a wide range of changes in gene expression and biochemical alteration to adjust plant growth under the stress condition (Verma et al. 2020b, c, 2021c). Molecular identification classified two groups of drought-inducible genes, the genes for protein abiotic stress tolerance and regulatory proteins such as transcription factors (Shinozaki and Yamaguchi-Shinozaki 2007). In addition, it is well reported that the accumulation of sugar, proline, and betaines helps plants adapt to drought stress (Chen and Murata 2002). These small molecule metabolites perform an essential function to protect cells from damage due to water stress. Glycine betaine (GB) is a non-toxic compatible solute that protects plants under water deficit and osmotic stress (Sakamoto and Murata 2002). Understanding of basic mechanism underlying drought tolerance will be beneficial to anticipate the impact of climate variability and develop genetic engineering for sugarcane (Verma et al. 2019).

Plant pathology has long been considered to study the environmental influence on plant diseases. Temperature change may favor the development of different pathogens such as bacterial diseases and the incidence of vector-borne diseases. In sugarcane, growth and productivity are affected by several diseases such as insects, fungal, bacterial, and viral infections. Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the most destructive viruses for sugarcane which reduces the yield up to 45% (Putra et al. 2014). The SCMV infection inhibits the development of stem diameter and length of internode from the early growth to the harvest period. This virus has been reported as a dominant pathogen that infects sugarcane in several countries, including Indonesia (Addy et al. 2017). Therefore, several methods have been developed to solve the problems of SCMV infection, such as viral elimination using in vitro meristematic culture, antivirus, and hot water treatments (Dewanti et al. 2016). However, the methods are not found to provide complete protection to sugarcane against viral infection in the field. Molecular study revealed that the SCMV genome contains genes encoding for ten functional proteins, including coat protein (CP) (Zhu et al. 2014). The gene encoding for CP is the most widely used component to induce resistance against viruses using genetic engineering in plants, including sugarcane.

Genetic improvement of sugarcane has been performed through conventional breeding programs, including intercrossing between the hybrids to increase sucrose production, induce stress tolerance, and gain diversity of alternative products. Although the breeding programs were successfully implemented, it is a laborious task and takes around 12 years or more. Modern commercial varieties have also been developed through interspecific hybridization between Saccharum species and allied genera of Miscanthus and Erianthus species. However, the conventional breeding for sugarcane resulted in polyploid and aneuploid with chromosome number of 2n = 80–120 that leads to meiotic instability, production of aneuploid gametes, and production of sterile seeds. Biotechnological tools are required to solve critical problems related to sugarcane improvement for sustainable agriculture.

Progress on molecular techniques and genetic transformation is needed to create new sugarcane cultivars using a biotechnological approach. Biotic and abiotic stresses alter sugarcane metabolism impacting its growth and productivity. To survive, plants exhibit several biochemical and molecular mechanisms which make them withstand or secure stress. Understanding biochemical and physiological mechanisms in response to biotic and abiotic stress is a major challenge for the developing biotechnology for sugarcane. The objective of this chapter is to improve sugarcane quality and quantum under environmental stress using biotechnological approaches.

14.2 Critical Points of Agrobacterium-Mediated Transformation in Sugarcane

14.2.1 Sugarcane Micropropagation

An efficient sugarcane tissue culture protocol is a valuable tool for sugarcane research activities, such as large-scale in vitro propagation and cultivar improvement. Conventional vegetative propagation is prone to several diseases, including gumming, Fiji, and other diseases. Therefore, establishing sugarcane tissue culture plays an essential role in producing disease-free plant material and reducing the seed production time. Notably, the sugarcane tissue culture has paved the way in improving sugarcane cultivars via sugarcane genetic transformation. Bower and Birch (1992) developed the first genetic transformation method in sugarcane using tissue culture. This method has been applied for engineering agronomic traits in various sugarcane cultivars (Bower and Birch 1992).

Plant cells have the capacity of totipotency, the ability of cells to regenerate into complete plants containing roots, stems, and leaves. This totipotency capacity can be triggered from meristematic tissue by growth regulators or hormone supplementation in tissue culture media to induce somatic embryogenesis callus and then regenerated to plantlets. In sugarcane, somatic embryogenic callus is derived from meristematic leaf roll tissue grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4 D) (Lee 1987). Then, the embryogenic sugarcane callus could easily regenerate to plantlets on hormone-free MS medium (Widuri et al. 2016). This simple micropropagation technique has been considerably applied for providing large-scale sugarcane seed demand. However, somaclonal variation may also arise from somatic embryogenesis in sugarcane that causes the development of variant phenotypes in sugarcane. Interestingly, the phenotypic variation caused by somatic embryogenesis generally reverts to its parental phenotype in sugarcane. The occurrence of somaclonal variations in somatic embryogenesis has been used to obtain new sugarcane varieties that are resistant to biotic or abiotic stress. In addition, somatic embryogenesis has played an essential role in the genetic transformation system to improve sugarcane cultivars.

Sugarcane in vitro propagation is also achieved without callus intervening through direct regeneration and multiplication from apical meristem or axillary buds. Explants from axillary buds can minimize genetic changes and avoid 2,4 D in culture media, which can cause somaclonal variation. In vitro propagation using axillary buds of sugarcane minimizes the somaclonal variation event, so it is used routinely for in vitro propagation of sugarcane (Manickavasagam et al. 2004). However, sterilizing axillary buds from field-grown stalks requires a potent sterilant such as mercury chloride (HgCl2), which is generally avoided because of its toxicity. Alternatively, shoot apical meristem is applied for mass multiplication of sugarcane shoots. Several methods have been developed to improve in vitro sugarcane multiplication from shoot apical meristem in MS media. Temporary immersion of shoot apical meristem into MS media containing high concentration of BAP (benzylaminopurine) resulted in weak, tiny, and non-separable shoots (Biradar et al. 2009). In addition, organic nitrogen sources in MS media may play an essential role in the multiplication of sugarcane shoot apical meristem. Some amino acids such as asparagine, cysteine, casein, glutamine, and glycine are primarily used in culture media as organic nitrogen sources (Saad and Elshahed 2012). The addition of 100 ppm glutamine and 2 ppm glycine into MS media produced robust and healthy sugarcane plantlets (Sugiharto, unpublished data). Glutamine and glycine may stimulate the multiplication of shoot apical meristem, which is suitable for micropropagation and genetic transformation of sugarcane.

14.2.2 Agrobacterium-Mediated Transformation

Genetic transformation is a valuable technology based on inserting genes into the genome to improve plant traits such as yield, pathogen resistance, and stress tolerance. Initially, the genes were introduced into plant cells directly using polyethylene glycol (PEG) treatment, electroporation, or particle bombardment (Rathus and Birch 1992). These direct transformation methods were less efficient due to multi-copy gene integration, high cost, requiring sophisticated equipment, and skillful labor (Dai et al. 2001). Meanwhile, the transformation method using agrobacterium is a powerful tool to introduce genes of interest into the plant genome. This method has been widely used to introduce genes into numerous dicot crops, including canola, cotton, potatoes, soybeans, and tomatoes. During the initial years, monocot plants were considered recalcitrant to Agrobacterium transformation because of their narrow host range. However, in recent years, Agrobacterium transformation was successfully carried out even in monocotyledonous plants by improving plant regeneration techniques and manipulation of factors affecting transgene delivery and integration into the plant genome. For example, co-cultivation media supplementing with acetosyringone, a phenolic compound that activates the virulence gene of Agrobacterium, can increase the T-DNA transfer efficiency into rice callus (Xi et al. 2018), maize embryos (Ishida et al. 1996), and banana suckers (May et al. 1995). Agrobacterium-mediated transformation system was also carried out successfully in sugarcane using meristematic explants (Arencibia et al. 1998). This technique provides several advantages, including low copy number of gene integration, low cost, and technical simplicity. However, reproducible transformations using Agrobacterium are required for routine genetic manipulation of sugarcane. So, optimizing critical factors affecting this transformation system is necessary to have a reproducible method, low somaclonal variation, and high efficiency of transformants multiplication.

The embryonic callus was mainly used as an explant in the plant transformation system. However, using in vitro regenerated shoots derived from apical meristem or axillary buds as explant offers several advantages in sugarcane transformation. Agrobacterium-mediated transformation using axillary buds explant resulted in stable transgenic sugarcane with transformation efficiency of about 50% (Manickavasagam et al. 2004). Unfortunately, the preparation of axillary buds from field-grown sugarcane is a tedious process due to the high possibility of bacterial contaminants. Alternatively, apical meristems derived from in vitro shoots are also suitable for obtaining contaminant-free explants (Sugiharto et al. 2005). Micropropagation of shoot from apical meristem has been developed using the MS media supplemented with glutamine and glycine, which results in healthy and rapid shoot growth. The basal segment of the healthy grown shoot was excised traversal around 0.2–0.3 cm and then used as an explant for genetic transformation (Sugiharto 2018). This method produces transgenic sugarcane plants in 4 months with a 4–10% transformation efficiency (Apriasti et al. 2018). Thus, the basal segment of in vitro shoot can act as a suitable and effective explant for routine genetic transformation in sugarcane.

The genetic transformation in sugarcane is not as simple as the preparation of explant but requires fine-tuning of various parameters. Several undetermined factors, such as selection of promoter, a selectable marker, and Agrobacterium strain, should be adjusted to improve transformation efficiency. DNA regulatory elements called promoters control gene expression in particular strengths and patterns. The promoter affects transformation efficiency, and the choice influences transgenic production (Liu et al. 2003). Many plant DNA promoters are well-characterized and classified into constitutive, tissue-specific, cell type-specific, organelle-specific, and inducible promoters. The Cauliflower mosaic virus 35S (CaMV 35S) promoter is a constitutive promoter commonly used in the transformation of dicot and monocot plants, including sugarcane (Apriasti et al. 2018). However, current research shows that ubiquitin, an endogenous plant promoter, effectively directs the constitutive expression in sugarcane. The rice polyubiquitin (RUBQ) 2 promoter increases GUS gene expression 1.6-fold compared to Zea mays polyubiquitin (ZmUbi) 1 promoter in sugarcane callus (Liu et al. 2003). Comparison of the effectiveness of CaMV and ubiquitin promoter showed that the ubiquitin significantly induced a higher expression of the targeted gene in sugarcane (Widyaningrum et al. 2021). Therefore, the ubiquitin promoter is widely used to drive the expression of transgenes in the transformation of sugarcane and other monocotyledonous plants.

The selectable markers and selective agents are critical factors affecting the plant’s genetic transformation. The selective agent, such as antibiotic or herbicide, suppressed the growth of the non-transformed cell in the selective media. The selectable marker gene transforms into the plant cell, and the gene of interest facilitates the transformed cells to survive in the selective media. Selectable marker genes that are commonly used in plant genetic transformation are the kanamycin resistance gene (nptII), hygromycin resistance gene (hptII), and herbicide Basta/phosphinothricin resistance gene (bar). Determination of explant sensitivity and appropriate concentration of the selective agent in the media is critical for the success of the genetic transformation. Excessive concentrations of selective agents in the media not only kill non-transformed cells but also suppress the growth of transformed cells (Miki and McHugh 2004). Evaluation of kanamycin and hygromycin as selective agents in Gramineae showed that both antibiotics suppressed cell suspension culture of Triticum monococcum, Panicum maximum, and Saccharum officinarum (Hauptmann et al. 1988). The hygromycin showed more effectiveness than kanamycin as a selective agent in the genetic transformation of rice (Lin and Zhang 2005) and maize (Que et al. 2014). In addition, herbicide Basta has also been used as the selective agent in the genetic transformation of monocots, such as rice (Rathore et al. 1993) and oil palm (Parveez et al. 2007). The comparative studies of nptII, hptII, and bar effectivity in sugarcane genetic transformation are limited. However, the agrobacterium-mediated transformation of sugarcane generally uses the hptII gene as a selectable marker because a low concentration of hygromycin, 25 mg/L, in a selective media is sufficient to discriminate between transformant and non-transformant plants (Arencibia et al. 1998).

The Agrobacterium strain and its density during explant infection contribute to the efficiency of the plant’s genetic transformation. The LBA4404 strain is commonly used for the genetic transformation of monocot plants. While the GV3101 strain has the highest transformation efficiency than AGL1, EHA105, and MP90 strains in the dicot plant (Chetty et al. 2013). The agrobacterium density at OD600 = 0.5 during infection processes increases cotton’s transformation efficiency (Jin et al. 2005), and higher density also leads to bacterial overgrowth that is difficult to eliminate from the explant after co-cultivation. The use of GV3101 strain for the infection of in vitro shoot explant at OD600 = 0.5 is the best example for routine transformation of sugarcane.

14.3 DNA Recombinant Technology

14.3.1 Cloning Gene

Recombinant DNA technology is defined as combining DNA fragments from different sources or inserting foreign DNA into the genome to obtain valuable characters or products from a living organism. These technologies include gene isolation, cloning, genetic transformation, and gene insertion into the genome of living organisms. Recombinant DNA technology has developed since the discovery of DNA polymerase, reverse transcriptase, DNA ligase, and restriction endonucleases enzymes that can copy, cut, and ligate DNA fragments. DNA polymerase and reverse transcriptase provide the possibility to copy DNA from the genome or messenger RNA, respectively. DNA ligase acts as glue for joining two adjacent DNA fragments via a phosphodiester bond. More than 600 commercially available restriction endonucleases serve as scissors that are able to cut DNA at a particular site (Roberts et al. 2007). Cutting and re-ligation using restriction endonucleases and DNA ligase facilitate the transfer of one DNA fragment to another. The isolated DNA fragment can then be inserted into a plasmid, a circular DNA molecule distinct from the bacterial chromosome. The plasmid can replicate independently during cell division, thereby enabling the amplification of the inserted DNA fragment.

Using these techniques, several genes from sugarcane have been cloned and characterized. The gene encoding sucrose-phosphate synthases (SPS), SoSPSl, and SoSPS2 were isolated from the cDNA of sugarcane leaves. SoSPSl is expressed predominantly in leaves, whereas SoSPS2 is expressed in both leaves and roots (Sugiharto et al. 1997). The drought-inducible gene SoDip22, which is expressed in bundle sheath cells, was also isolated from the cDNA of sugarcane leaves (Sugiharto et al. 2002). In addition, the genes encoding for sucrose transporter protein (Novita et al. 2007) and coat protein of SCMV (Apriasti et al. 2018) have also been successfully isolated from sugarcane.

14.3.2 Gene Overexpression

Gene overexpression is defined as an attempt to increase the transcript level of a coding gene using a promoter or other regulatory element. This technique intends to achieve higher levels of RNA transcription and protein expression. Several promoters have been known as constitutive promoters, such as CaMV35S, ZmUbi1, OsAct1, OsTubA1, and OsUbq1. Some promoters have been used to generate transgenic sugarcane with high sucrose content, virus tolerance, cold tolerance, and drought tolerance. For example, the CaMV35S promoter drives SoSPS1 expression to increase sucrose content in sugarcane (Anur et al. 2020), while ZmUbi1 controlled the expression of RNAi constructs to generate virus-resistant sugarcane (Widyaningrum et al. 2021). In addition, the ZmUbi1 promoter was also utilized to drive Alpha (α)–tubulin (TUA) and ATP citrate lyase (ACL) to develop cold and drought tolerance sugarcane (Chen et al. 2021; Zhu et al. 2021).

Sanford and Johnson (1985) described a concept that inserting a gene from a virus into the host genome would confer resistance to the host against the virus, which was then known as pathogen-derived resistance (PDR). For example, the viral coat protein expressed in plants provides resistance to the virus by inhibiting the virion disassembly in the early infection event (Baulcombe 1996). This mechanism is supported by experiments that plants expressing the coat protein show resistance to virion inoculation but are sensitive to virus inoculation in the form of RNA, indicating that coat protein inhibits early infection events (Powell-Abel et al. 1986; Hemenway et al. 1988). Virion disassembly is required to allow viral genome replication and RNA expression in the host cell to produce organelles of new viruses. Reassembly of virus organelle into virion is essential for the long-distance movement to allow virus entry into vascular tissue (Saito et al. 1990). So, the inhibition of virion disassembly by coat protein in the initial infection event prevents virus replication and long-distance movement (Fig. 14.1a).

Fig. 14.1
figure 1

Model of PDR, RNA silencing, and CRISPR/Cas strategy for inducing virus resistance in the plant. (a) PDR strategy achieved by expressing viral coat protein or inactive movement protein in the transgenic plant. Coat proteins inhibit virion disassembly in the initial infection, while inactive movement protein (MP) inhibits cell to cell RNA virus movement through plasmodesmata (PD). (b) RNA silencing is triggered by hairpin RNA, artificial pre-miRNA, or the activity of RDR on sense/antisense RNA. The dsRNA is processed to small interference RNA (siRNA). The siRNA incorporates into AGO protein effectors, which provide sequence specificity to cleave homolog RNA target. The activity of RDR enables amplification and production of secondary siRNA corresponding to regions outside of the primary siRNA target (transitivity). (c) Three variants of Cas protein were used in the strategy to target plant viruses. The spCas9 was utilized for targeting DNA virus while FnCas9 and Cas13a were employed for targeting RNA virus. All Cas variants required specific sgRNA, which provide sequence specificity to the virus genome

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Plasmodesmata, channels connecting cytoplasm between adjacent cells, mediate the spreading of viruses from one cell to another. The movement of viruses between adjacent cells is facilitated by movement proteins (MPs) encoded by the viral genome. The MPs complex polymer binds to virus RNA to facilitate movement along microtubules toward plasmodesmata (Carrington et al. 1996). Then, MPs modify the plasmodesmata component to increase its size exclusion limits (SEL), facilitating the movement of either naked RNA or virion to cross plasmodesmata channels (Lazarowitz and Beachy 1999). Unlike CP (coat proteins), plants expressing a functional MP are more susceptible to tobacco mosaic virus (TMV) infection, whereas overexpression of inactive MPs (lacking movement function) confers resistance to the TMV virus (Lapidot et al. 1993; Cooper et al. 1995). The inactive MPs and wild-type MPs possibly compete for the binding site at the plasmodesmata component, resulting in inhibition of virus dispersal (Baulcombe 1996). Interestingly, inactive MPs confer resistance to various virus groups (Cooper et al. 1995). It seems that inactive MPs complex can recognize genome RNA from several viruses and prevent cell to cell movement (Fig. 14.1a).

14.3.3 RNA Interference

Gene silencing is a conserved mechanism in the eukaryotic organism that employs small interference RNA (siRNA) and protein effectors to suppress homolog gene expression at the transcriptional or post-transcriptional levels. Gene silencing was initiated by forming double-strand RNA (dsRNA) and subsequently processed to small interference RNA (siRNA). One of the two strands of siRNA incorporates into protein effectors to form RNA-induced silencing complex (RISC) and provide sequence specificity to cleave homolog RNA target in post-transcriptional gene silencing (PTGS) or mediate chromatin methylation in transcriptional gene silencing (TGS). PTGS is later known as RNA interference (RNAi). Although TGS and PTGS are mechanistically related and share molecular machinery, in this chapter, the discussion is focused on PTGS/RNAi in relation to virus resistance traits in sugarcane.

The dsRNA is naturally found in replication intermediates or highly structured genomic RNA of the virus. RNA virus replication was mediated by viral RNA-dependent RNA polymerase (RdRP), resulting in perfectly paired dsRNAs known as replication intermediates. On the other hand, the RNA genome of the virus arranges in highly base-paired structure with several imperfect dsRNA and hairpin loop structures. DICER processes replication intermediates and imperfect dsRNA to 21 and 22 primary siRNAs (Molnár et al. 2005). The formation of primary siRNA by DICER is the initiation phase of the RNA silencing mechanism to deal with viruses.

The DICER protein comprises three functional domains lying from the N- to C-terminus: RNA helicase, PAZ (Piwi/Argonaut/Zwille), RNAse III a b, and dsRNA-binding domain. The plant genome generally encodes four different DICER-LIKE (DCL) proteins that produce a distinct length of siRNAs (Song and Rossi 2017). DCL1 produces variable size microRNA (miRNA), a small RNA encoded in the genome (Bartel 2004). DCL2, DCL3, and DCL4 process dsRNA to 22, 24, and 21-nucleotide (nt) siRNA, respectively (Nagano et al. 2014; Benoit 2020; Wu et al. 2020). The 21-nt and 22-nt siRNA guide RNA degradation in PTGS, while 24 nt siRNA mediate chromatin methylation in TGS (Tan et al. 2020). The DCL2 and DCL4 have a redundant function in processing viral-derived RNA and play an essential role in systemic antiviral silencing in the plant (Qin et al. 2017; Chen et al. 2018).

The siRNA assembles into Argonaute (AGO) protein and provides specificity to Argonaute (AGO) protein effectors to cleave homolog RNA. The AGO protein comprises two domains, the PAZ domain for binding single-stranded nucleic acid and PIWI-domain containing RNAse-H-like fold (Hutvagner and Simard 2008). Seven AGO are critical players of gene silencing and viral defense, i.e., AGO1, AGO2, AGO5, AGO7, and AGO10 play a role in targeting RNA degradation in PTGS; AGO4 and AGO6 mediate chromatin methylation in TGS (Carbonell and Carrington 2015). The AGO1 is a significant player in plant defense mechanisms against invading viruses, indicated by its upregulation in response to the viral attack (Várallyay et al. 2010). However, the activity of AGO1 has interfered with the protein suppressors encoded by the virus by inhibiting its transcription level or cleavage activity (Xiuren Zhang et al. 2006; Csorba et al. 2010; Várallyay et al. 2010). When the AGO1 is inactivated, the plant activates the second layer of defense mechanism against invading virus by expressing AGO2 (Harvey et al. 2011).

The amplification of virus siRNA is required to ensure the efficiency of RNA silencing against virus attacks. RNA-dependent RNA polymerase (RDR) mediates the formation of secondary siRNA from cleaved RNA of the virus. RDR synthesizes dsRNA from RNA lacking a 5′ triphosphate cap or poly-A tail and then processed into 21 or 22 secondary siRNA by DICER (Luo and Chen 2007; Willmann et al. 2011). The mRNA lacking poly-A tail or 5′ triphosphate cap is converted to dsRNA by RDR through a primer-independent or dependent approach, respectively (Curaba and Chen 2008). The activity of RDR enables the production of secondary siRNA corresponding to regions outside the primary siRNA target (Fig. 14.1b) (Moissiard et al. 2007). Amplification and transitivity of siRNA determine the strength and persistence of antiviral defense against the virus (Baulcombe 2007). Three homologous plant RDR genes, RDR1, RDR2, and RDR6, are required in the biogenesis of secondary siRNA from RNA viruses. For example, the biogenesis of secondary siRNA from the Tobacco rattle virus (TRV) required the combined activity of the three RDR genes (Livia et al. 2008).

Several techniques had been reported to generate dsRNA, such as co-suppression, sense-antisense construct, and hpRNA (Fig. 14.1b) (Waterhouse et al. 1998). The dsRNA using co-suppression or sense-antisense construct usually results in low silencing efficiency (Stoutjesdijk et al. 2002). A more effective approach to produce dsRNA is to clone both sense and antisense sequences, separated by an intron, under the control of a promoter to generate self-complementary RNA (Wesley et al. 2001). The approach was initially known as hpRNA, but later it was known as RNAi construct. This technology was applied to confer resistance against several families of RNA viruses in soybean, tomato, and tobacco (Andika et al. 2005; Hu et al. 2011; Zhang et al. 2011; Ammara et al. 2015). The RNAi was also reported to mediate effective resistance against SCMV and sorghum mosaic virus (SrMV) in sugarcane. The trait introduced by RNAi was inherited in plant progeny, indicating that the RNAi construct is stable in the offspring of the transgenic plant (Chuang and Meyerowitz 2000).

14.3.4 Genome Editing

Genome editing provides flexibility and effectivity to manipulate plant genomes for diverse purposes such as gene study, increased productivity, conferring resistance to biotic or abiotic stress, and improving plant quality. Genome editing is a genetic engineering technique that employs engineered nuclease to generate double-strand breaks (DSB) at the specific location, which are then repaired by the cell’s internal mechanisms through non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in a mutation or insertion. Four genome editing systems have recently been developed to manipulate plant genomes, such as ZFN, TALEN, and the CRISPR/Cas9 system. The CRISPR/Cas9 system has been widely used because of its simplicity, robustness, and cost-effectiveness.

The CRISPR/Cas9 is an adaptive immune system against invading viruses or foreign genetic elements in prokaryotes. In this immune system, the bacteria acquire a short sequence from viruses known as a spacer and integrate it between two sequences repeat of the CRISPR array in the genome, allowing them to remember and develop immunity against viruses. Bacteria transcribe the spacer-repeat array into a long precursor CRISPR RNA (pre-crRNA) and subsequently process it to a small mature crRNA guide. Repeat sequence in the crRNA forms base pairs with an additional small non-coding RNA known as trans-activating crRNA (tracrRNA) to form dual-RNA structure. The tracrRNA is encoded by trans-activating the CRISPR RNA gene located upstream of the CAS operon in the CRISPR locus. The Cas9 nuclease protein recruits a dual crRNA-tracrRNA structure to identify the complementary target sequences in viral DNA. The Cas9-crRNA-tracrRNA complex recognizes a short DNA motif termed Protospacer Adjacent Motif (PAM), where Cas9 binds and unwinds the dsRNA to facilitate duplex formation between spacer of crRNA and DNA target sequence (Jiang and Doudna 2017; Hille et al. 2018). PAM is a short-conserved sequence adjacent to the crRNA target site, recognized specifically by the Cas9 protein. The commonly used Streptococcus pyogenes Cas9 (spCas9) protein recognizes PAM motif 5′-NGG-3′, where N can be any nucleotide of DNA (Sternberg et al. 2014). The PAM motif appears only in adjacent target sequences but not in crRNA, facilitating discrimination of self and non-self DNA, thereby preventing the immune system from attacking the host (autoimmunity) (Sashital et al. 2012; Rath et al. 2015). This immune system confers resistance to the bacterial population and is inherited vertically to their progeny (Marraffini 2015).

The CRISPR/Cas9 immune system is adopted as a genetic engineering tool to manipulate the sequence of a eukaryotic genome. The CRISPR/Cas9 system comprises two components, i.e., single guide RNA (sgRNA) and CRISPR-associated 9 (Cas9) protein. The sgRNA is a single RNA transcript formed by a combination of crRNA and tracrRNA separated by a linker loop, mimicking the dual-RNA structure required by Cas9 to direct site-specific DNA cleavage. By modifying the 20 bp spacer sequence at 5′ end of gRNA, it is possible to target any sequence in the genome (Martin et al. 2012). Cas9 gene is constitutively expressed in plant cells driven byCaMV35S or ZmUbi promoter for modifying the plant genome. Nuclear localization signals (NLS) are fused to Cas9 protein to direct its expression to the nucleus. The sgRNA is expressed under U6 or U3 promoters to facilitate transcription, which starts with nucleotides G for U6 or A for U3 promoters. For targeting plant genomes, the sgRNA spacer follows a consensus sequence G(N)19–22 or A(N)19–22, where the first G or A may or may not pair with the target sequence (Belhaj et al. 2013). Recently, CRISPR/Cas9 was utilized to generate sugarcane herbicide resistance by generating DSB in the Acetolactate synthase (ALS) gene and introduced ALS sequence containing amino acid substitutions W574L and S653I via HDR mechanism (Oz et al. 2021).

The causal agent of disease that causes a significant loss in sugarcane production is mostly mediated by RNA viruses. Since spCas9 is targeting DNA, a variant that targets RNA is required to engineer resistance against viral RNA. Two Cas9 variants, Francisella novicida Cas9 (FnCas9) and Leptotrichia shahii Cas13a are components of prokaryotic adaptive immunity against RNA viruses. The FnCas9 recognizes PAM 5′-NGG-3′ at its target RNA locus, whereas Cas13a does not require PAM, making it more flexible than FnCas9 (Sampson et al. 2013; Abudayyeh et al. 2017). Both FnCas9 and Cas13a provide the possibility to develop plants resistance to RNA viruses (Fig. 14.1c). An example from the monocot plant, the Cas13a and sgRNA targeting southern rice black-streaked dwarf virus (SRBSDV) or rice stripe mosaic virus (RSMV) expressed in rice showed mild symptoms and less virus accumulation (Zhang et al. 2019). A similar approach using FnCas9 or Cas13a might be applied to combat RNA virus attacks in sugarcane.

14.4 Biotechnology to Increase Sucrose Production

Sucrose is a dominant mobile sugar that has a crucial role in plant growth and development, various types of gene expressions, and sugar signaling pathway (Gifford et al. 1984). However, the function of sucrose in microorganisms still remains unclear. Recent biochemical and molecular studies reported that sucrose synthesis in prokaryotic cells provides new insight into sugar metabolism in terms of its origin (Salerno and Curatti 2003). Sucrose is the common form of sugar that is generated from photosynthesis products. Further, sucrose is exported from source leaves to carbon-importing sink tissues for allocation of carbon resources. There is evidence that sucrose not only provides the fuel for plant growth but also has an important influence on the expression of genes that are involved in signaling function and cell differentiation (Lunn and MacRae 2003). Sucrose accumulates in the stem as the primary storage reserve in sugarcane. Thus, it is suggested that the activity of sucrose biosynthesis enzymes influences sucrose loading into the phloem and sink (Zhu et al. 1997; Castleden et al. 2004).

Based on the phylogenetic origin, the enzymes involved in sucrose metabolism have been characterized as sucrose biosynthesis-related proteins (SBRPs). The group of enzymes that are classified under SBRP comprised of sucrose-phosphate synthase (SPS; EC 2.4.1.14), sucrose synthase (SuS; EC 2.4.1.13), and sucrose-phosphate phosphatase (SPP; EC 3.1.3.24). SPS is responsible for yielding sucrose with inorganic phosphate (Pi), whereas SPP catalyzes an irreversible pathway for producing free sucrose. Subsequently, SuS catalyzes a reversible reaction in which sucrose is hydrolyzed into fructose and uridine diphosphate glucose (UDP-G). In addition, invertase (INV; EC 3.2.1.26) is involved in their reversible cleavage of sucrose (Cumino et al. 2002; Salerno and Curatti 2003). Both SuS and INV are assigned a role in breaking down sucrose under most physiological conditions in plant cells (Fig. 14.2).

Fig. 14.2
figure 2

Model of sucrose metabolism pathway in plant cell

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It is well known that SPS is a key enzyme in the sucrose synthesis pathway. SPS catalyzes the reaction of S6P (sucrose-6-phosphate) formation from the substrate UDP-G and fructose-6-phosphate (F6P) (Leloir and Cardini 1955; Amir and Preiss 1982). SPS plays a physiological role by modulating photosynthetic carbon flux into sucrose. The activity of plant SPS is under complex regulation involving allosteric effectors, glucose-6-phosphate (G6P), and Pi. Plant SPS is activated by G6P in a concentration-dependent manner up to 5 mM. An increase of G6P concentration is correlated with increased sucrose formation and decreasing concentration of the cytosolic Pi (Doehlert and Huber 1983; Huber and Huber 1996; Sawitri et al. 2016) (Fig. 14.2).

The active form of SPS occurred as a result of the dephosphorylated enzyme. It has been previously postulated that phosphorylation at certain serine residue modulates SPS activity in response to dark-light transition. Thus, the accumulation of G6P might be associated with light conditions. In order to determine the residues responsible for phosphorylation, alteration of a serine residue at position 162, which corresponds to residue S158 in spinach, has been attempted in sugarcane SPS. Substitution of S158 to alanine in spinach SPS showed consistency with dephosphorylated form and is not regulated by light modulation (Lunn et al. 1999; Toroser et al. 1999). However, there was no insight into the phosphorylation state of sugarcane SPS except that loss of S162 in the N-terminal domain deletion mutant has no significant effect on SPS activity (Sawitri et al. 2016).

The gene encoding SPSs have been successfully cloned from various C3 and C4 plants, such as Arabidopsis (Park et al. 2008) and maize (Worrell et al. 1991), respectively. In addition, the response of photosynthetic SPS is more sensitive to G6P rather than non-photosynthetic SPS. In some plants, including sugarcane, SPSs have different isomeric forms with different deduced amino acid sequences. The comparison between sugarcane SPS and SPSs from other species showed that SoSPS1 has the highest homology of about 95% identical to maize SPS and less but significant homology to spinach SPS (56%), sugar beet SPS (56%), and potato SPS (55%). Sugarcane SoSPS2 has significant homology to maize (50%), spinach (58%), sugar beet (57%), and potato (56%). The corresponding sequences revealed 49% identity between SoSPS1 and SoSPS2 (Sugiharto et al. 1997). Consequently, SoSPS1 provides a potential application to be engineered since it has been considered as a representative enzyme for photosynthetic carbon allocation with the regulatory function (Sawitri et al. 2016).

The protein stability and abundance of SoSPS1 in plant cells are relatively poor (Huber and Huber 1996). Therefore, constructing a recombinant protein expression system offers new prospects to enhance its protein production level for enzyme characterization and biotechnology application. Several studies reported the expression of plant and cyanobacteria SPS genes (Worrell et al. 1991; Sonnewald et al. 1993; Lunn et al. 1999; Chen et al. 2007) in Escherichia coli but resulting recombinant enzymes did not show a clear property of enzyme regulation. Previous reports revealed that deletion of the N-terminal domain tends to increase the specific activity by tenfold as compared to full-length plant SPS. Although N-terminal deletion in SPS is not allosterically regulated by G6P, the application of these mutants will be one of the strategies to increase the sucrose accumulation in sugarcane.

Many studies demonstrate to elucidate the role of SPS in controlling carbon partitioning in plants. Overexpression of SPS showed an increase of photosynthetic rate and sucrose: starch ratio in leaves of transgenic tomato (Worrell et al. 1991; Galtier et al. 1993) and Arabidopsis (Signora et al. 1998). Whereas, overexpression of SPS also contributes to enhancing sucrose accumulation in tomato fruit (Nguyen-Quoc et al. 1999), while overexpression of SPS in cotton resulted in improved fiber quality (Haigler et al. 2007). The effect of overexpressed SPS in plant growth and biomass has also been investigated in transgenic Arabidopsis, poplar, and tobacco (Park et al. 2008; Maloney et al. 2015). These reports revealed that overexpression of SPSs affected not only increased sucrose accumulation in leaves but also played a pivotal role in starch metabolism and carbon partitioning in sink tissue.

It will also be interesting to determine the regulatory mechanism of SoSPS1 involved in carbon partitioning. Carbon partitioning is a critical process in distributing chemical energy converted by the plant through photosynthesis. In sugarcane, overexpression of the SoSPS1 gene revealed that SPS accumulation and its activity increased, followed by increased sucrose accumulation and improved growth traits, such as increased plant biomass in transgenic sugarcane. The elevated sucrose levels showed that SPS is not only modulating sucrose synthesis but also concomitant with degrading INV activity in the leaves (Anur et al. 2020). It suggested that INV controls the sucrose levels so as not to exceed the level of photosynthesis gene suppression; therefore, INV plays an important role in maintaining the balance between the sucrose signaling pathway and metabolism.

Although high sucrose content is accumulated in the sugarcane stalk, the sucrose translocation and accumulation mechanism remains unclear. Synthesis of sucrose is predominantly reported in leaves and translocated to the sink tissues through several types of sugar transporters, such as sucrose transporter (SUT) and SWEET proteins (Wang et al. 2013). Several studies showed that the overexpression of a sucrose transporter gene increased sucrose unloading and sink strength (Rosche et al. 2002; Cheng et al. 2018). Manipulation of the SUT and SWEET genes was reported to increase the SPS activity and sucrose unloading in the sink tissue (Lin et al. 2014). Multiple target genes are considered for genetic engineering to increase sucrose accumulation in sugarcane. Therefore, the engineering of sugar transporters in cooperation with increased SPS activity generates a new alternative for enhancing sucrose accumulation and improving crop yield, including sugarcane.

14.5 Biotechnology of Water Stress Tolerance

14.5.1 Biochemical and Molecular Aspects of Water Stress Responses

Water deficit or drought stress is one of the most important environmental factors limiting sugarcane growth and productivity. The drought stress significantly affects sugar production, which is determined by Brix, Pol, and reducing sugar (Begum et al. 2012). On the other hand, gradual water deficit during sugarcane maturation reduces growth but increases sucrose accumulation in the stem (Inman-Bamber and Smith 2005). A new perspective to the sugarcane production system has been reported that sugarcane previously exposed to drought stress will perform better under water stress on the next cultivation (Marcos et al. 2018). However, these controversial issues lead to studies on the effect of drought stress in sugarcane at biochemical and molecular levels.

Sugarcane is a C4 plant and is considered to have a higher water use efficiency. The operation of the C4 cycle with PEPC in mesophyll cell and Rubisco in bundle sheath cell generate suppression of photorespiration. PEPC is believed to have high affinity for CO2 assimilation from the atmosphere and allows high-rate carbon assimilation when stomata are slightly closed (Lopes et al. 2011). During midday, with a high temperature and light intensity, the C4 plants leaves are slightly rolling to reduce transpiration. The PEPC is a primary enzyme for carbon assimilation, and that activity is affected by water stress (Ghannoum 2009).

Measurement of the carbon assimilating enzyme activity showed that sucrose content and shoot dry weight fluctuated according to the SPS activity in Saccharum species (Sugiharto 2005). Furthermore, observation of sugarcane grown in the field revealed that the SPS activity, as well as sucrose contents, was higher in dry land than in wet land. The biochemical analysis found that halting the process of watering resulted in increased SPS activity and sucrose content in sugarcane leaves (Sugiharto 2018). The activity of SPS is enhanced by water stress due to covalent modification of the enzyme that is caused by protein phosphorylation of serine residue at position 424 (Huber and Huber 1996). In addition, identification of drought-response genes showed that water deficit is associated with changed gene expression associated with sucrose accumulation in sugarcane (Iskandar et al. 2011). These results suggested that sucrose may act as an osmoregulator and helped the sugarcane getting adapt to water deficit conditions.

Water deficit induces gene expression for the protein responsible for the drought stress tolerance in plants. The molecular study revealed that a drought-inducible protein named SoDip22 was identified in the water stress-tolerant sugarcane phenotype (Sugiharto et al. 2002). The amino acid sequence of SoDip22 exhibited similarity to ABA, stress, and ripening-inducible protein from various plant species. However, further study on the function of the protein on water stress response has never been conducted.

It is well established that drought stress regulates several genes expression, including the transcription factors (TFs) in plants. The TFs are the proteins that play the vital molecular switches of gene expression and regulate plant development in responses to various types of stress. The key TFs regulating drought-responsive gene transcription have been identified in plants such as MYB, MYC, DREB/CBF, ABF/AREB, NAC, and WRKY (Osakabe et al. 2014). For example, CBF/DREB1 and DREB2 from rice have been identified, and their overexpression improved drought tolerance in rice (Shinozaki and Yamaguchi-Shinozaki 2007). The overexpression of GmDREB1 from soybean consistently improved the yield performance of transgenic wheat when grown under limited water conditions in the field (Zhou et al. 2020). Recently, the TFs of R2R3-MYB have been identified and play a positive role in responding to drought-induced senescence in sugarcane (Guo et al. 2017). Therefore, the potential use of TFs families such as WRKY, NAC, MYB is an important clue for the engineering of stress-tolerant sugarcane (Javed et al. 2020). However, the research on TFs as a target to genetically engineer drought tolerance sugarcane is still meager. Most recently, it was reported that overexpression of AtBBX29, a member of B-box proteins, increased drought tolerance and delayed senescence under the water deficit condition in transgenic sugarcane (Mbambalala et al. 2021).

Measurement of enzymes activity responsible for scavenger and detoxification of reactive oxygen species (ROS) during drought stress showed that superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) are higher in drought stress-tolerant plants as compared to sensitive sugarcane cultivars (Cia et al. 2012; dos Santos and Silva 2015). The activities of ROS scavenging enzymes were suggested as a marker of drought stress tolerance in sugarcane. Genetic engineering of ROS enzymes has been conducted to increase drought tolerance in plants but has not been developed in sugarcane.

14.5.2 Genetic Engineering to Enhance Glycine Betaine Biosynthesis

Plants have various strategies to survive under water deficit by inducing the accumulation of small molecules referred to as compatible solutes or osmoprotectants (Rhodes and Hanson 1993). Glycine betaine (GB) (N,N,N-trimethyl glycine) is one of the most studied osmoprotectants which helps plants to acclimate to drought conditions (Chen and Murata 2002). The GB stabilizes the structure of macro molecules and helps in the proper functioning of the cell membrane (Sakamoto and Murata 2002). The accumulation of GB has been reported in some species such as Amaranthus, sorghum, sugar beet under drought stress conditions, and that accumulation contributes to the acclimation of the plant cell to water stress (Bohnert et al. 1995). The addition of exogenous GB at 10 mM has been reported to increase the growth and yield of maize under salt-stress conditions (Yang and Lu 2005). However, the economic analysis and other disadvantages of the exogenous application should be well considered. Although the detailed function of GB has not been established well, the genetic engineering to enhance GB synthesis in sugarcane that naturally does not accumulate GB is discussed in this section.

GB is synthesized from two-step reactions, conversion of choline to betaine aldehyde and the betaine aldehyde to GB that is catalyzed by choline dehydrogenase (CDH) and betaine aldehyde dehydrogenase (BADH) in the microorganism and mammalian cells. In plants, the conversion of choline to betaine aldehyde is catalyzed by choline monooxygenase (CMO) and then converted into GB by BADH. In addition, a single-step reaction for the conversion of choline into GB by choline oxygenase (COD) was found in Arthrobacter globiformis and Arthrobacter pascens. Interestingly, the microbial CDH was found capable to catalyze two-step reactions, the oxidation of choline into BADH, and further conversion to GB (Cánovas et al. 2000). This result has been confirmed using purified CDH from Halomonas elongata that showed a similar substrate specificity to both choline and BADH (Gadda and McAllister-Wilkins 2003).

The genes involved in the pathway of GB biosynthesis have been cloned from various organisms. The genes for CDH and BADH referred to as betA and betB were isolated from Escherichia coli (Andresen et al. 1988) and bacteria Halomonas elongata (Gadda and McAllister-Wilkins 2003). The COD gene that catalyzes a single-step reaction for GB synthesis was also successfully cloned from bacteria Arthrobacter pascens and Arthrobacter globiformis (Rozwadowski et al. 1991). In higher plants, genes for CMO and BADH have been isolated from GB accumulator species such as spinach, sugar beet, and amaranth (Rathinasabapathi 2000). The genes encoding for the pathway of GB synthesis from microorganisms have become a major target to develop environmental stress-tolerant plants. Genetic engineering of GB accumulation using the genes from microorganisms has been reported in tomato (Solanum lycopersicum), potato (Solanum tuberosum), rice (Oryza sativa), and maize (Zea mays) (Quan et al. 2004).

The genetic engineering of GB biosynthesis has been conducted mainly with the gene for a single-step reaction of GB synthesis in plants. Overexpression of gene for COD targeted in chloroplast accumulated low level of GB in Arabidopsis (Hayashi et al. 1998) and rice (Sakamoto and Murata 1998). In addition, constitutive expression of bacterial COD in naturally lacking GB plant species also reported lower levels of GB. The low GB level in transgenic plants was caused by a low level of choline substrate in the site targeted GB synthesis. A substantial increase in GB content was obtained when the transgenic plants were supplied with choline or phosphocholine (Huang et al. 2000).

Introducing betA encoding for CDH from E.coli showed increased activity of CDH and resulted in salt tolerance in tobacco (Lilius et al. 1996) and maize (Saneoka et al. 1995) plants. The increased CDH activity elevated GB content which is correlated with the degree of salt tolerance. These results indicated that the GB plays a key role in osmotic adjustment. Furthermore, the overexpression of betA resulted in drought-tolerant maize, and the grain yield was significantly higher than the wild-type control after water-deficit conditions (Quan et al. 2004). A similar result reported that overexpression of the betA gene elevated the GB content and created drought tolerance in transgenic cotton (Lv et al. 2007). It was established that CDH from microorganisms has the capacity to catalyze the oxidation of choline to betaine aldehyde and further converted into GB (Cánovas et al. 2000). These results indicated that overexpression of the betA gene from microorganisms enhanced GB level and salt-drought tolerances and improved plant growth and productivity.

Genetic engineering to enhance GB synthesis and drought tolerance has been developed using the betA gene in sugarcane. The betA isolated from Rhizobium meliloti was constructed into the binary vector by Ajinomoto Co. Inc. Japan (Australian Patent Office No. 737600) and introduced into Agrobacterium tumefaciens LBA4404 for sugarcane transformation. The Agrobacterium-mediated transformation was performed using embryogenic explant callus in the Laboratory of Biotechnology PT. Perkebunan Nusantara XI in collaboration with Ajinomoto company and University of Jember. After selection using appropriate antibiotics, the selected sugarcane transformant was acclimatized in the greenhouse for further analysis.

PCR and Southern hybridization analysis showed stable betA gene integration in the transgenic sugarcane leaves genome. The GB content was elevated in the leaves of transgenic lines ranging from 182 to 880 ppm after drought treatment but not detected in the wild-type or non-transgenic sugarcane parental. The transgenic lines showed prolonged wilting symptoms compared to the wild-type sugarcane after drought stress. Interestingly, the root morphology was longer and deeper distributed in the soil media, showing the character of drought-tolerant sugarcane (Smith et al. 2005). Moreover, the elevation of GB content also enhanced salt tolerance in the transgenic sugarcane lines. These results indicated that overexpressing the betA gene enhances GB content and helps the sugarcane to get adjusted to drought and salt stress.

Evaluation of the growth and productivity of the transgenic sugarcane lines were conducted in the confine limited field trial under the supervision of the Indonesian Genetically Modified Product Biosafety Commission. The stem internode of transgenic lines was grown normally and not affected by drought stress, but the internode of non-transgenic shortened due to growth retardation. Total cane yield significantly increased in the transgenic lines compared to the non-transgenic counterpart. The sugar production in the transgenic lines is 10–30% higher than wild-type parental sugarcane in non-irrigated dry land (Waltz 2014). The transgenic sugarcane has been completed with biosafety certifications by the Indonesian Biosafety Commission, released by the Indonesian Ministry of Agriculture (No 4571/Kpts/SR.120/8/2013), and cultivated by the sugarcane company.

14.6 Biotechnology of Virus Resistance

14.6.1 Viruses and Sugarcane Mosaic Disease

Mosaic diseases are a major constrain causing significant losses in sugarcane yield and have become a severe problem for sugarcane plantations. The disease reduces total leaf chlorophyll content and photosynthetic capacity, affecting sucrose accumulation and ultimately causing yield losses in sugarcane (Irvine 1971). The causal agents of mosaic disease in sugarcane are three viruses: SCMV, SrMV, and SCSMV.

SCMV and SrMV are classified into genus Potyvirus in the Potyviridae family based on serological tests and the host range of viruses (Hall et al. 1998; Gibbs and Ohshima 2010). SCMV and SrMV are naturally transmitted by aphids from plant to plant in a non-persistent manner (Gadhave et al. 2020). Potyvirus genome is positive single-strand RNA (+ ssRNA) attributed with genome-linked protein at 5′ terminal and poly-A tail at 3′ terminal (Gell et al. 2014). This RNA genome encoded a single large polyprotein, which is cleaved by self-encoded protease into ten individual mature proteins, i.e., protein 1 (P1), helper component proteinase (HC-pro), protein 3 (P3), 6 K protein 1 (6 K1), cylindrical inclusion protein (CI), 6 K protein 2 (6 K2), viral protein genome-linked (VPg), nuclear inclusion a (NIa) protein, nuclear inclusion b (NIb) protein, and coat protein (CP) (Revers et al. 2007). To distinguish between SCMV and SrMV, Yang and Mirkov (2007) developed RT-PCR coupled RFLP method. Sequence alignment confirmed the gaps and nucleotide differences between SCMV and SrMV at the 3′-terminal of the genome that spanned the Nib, CP, and 3′-untranslated regions. Two sets of specific primers were designed using the gaps and nucleotide differences and then used in the RT-PCR coupled RFLP method to distinguish between SCMV and SrMV (Yang and Mirkov 2007).

SCMV and SrMV are major pathogens causing a severe threat to sugarcane plantations globally. SCMV could reduce sugarcane yield up to 45% in India for susceptible varieties. Mosaic diseases caused by SCMV are reported with an incidence of up to 78% in East Java and Indonesia (Addy et al. 2017). SCMV infection cases, including new strains or genome variation, are still reported from many countries, indicating that the virus is a severe problem in the sugarcane-based industry (Wu et al. 2012). While SrMV is the most common pathogen associated with sugarcane mosaic disease compared to SCMV in China. It is also reported that SrMV is a causal agent for mosaic disease in Louisiana, with incidences ranging from 0 to 10% (Rice et al. 2019). High incidence of coinfection of SCMV and SrMV was reported from China, in which coinfection resulted in heavy mosaic symptoms. In contrast, a single virus infection showed symptomatic or asymptomatic conditions indicating that coinfection is more virulent than a single infection (Xu et al. 2008). The incidence of coinfection of SCMV and SrMV is also common in Tucumán, Argentina (Perera et al. 2008). SCMV and SrMV are common pathogens for sugarcane and can also infect sorghum, maize, and Columbus grass (Sorghum almum) (Fan et al. 2003; Xu et al. 2010; Mollov et al. 2016; Klein and Smith 2020).

SCSMV was previously known as sugarcane mosaic virus-strain F (SCMV-F) and classified into genus Potyvirus in the Potyviridae family. The virus was identified from quarantined sugarcane exhibiting mosaic symptoms imported from Pakistan. The SCMV-F is transmitted from plant to plant in a mechanical mode rather than a vector-transmitted fashion (Damayanti and Putra 2010; Putra et al. 2015). Phylogenetic study shows that protein encoded by 3′ terminal sequence of the SCMV-F is highly similar to Wheat streak mosaic virus (WSMV) and Brome streak mosaic virus (BSMV). To reflect this similarity, the SCMV-F was renamed to Sugarcane streak mosaic virus (Hall et al. 1998). However, the serological test revealed no cross-reaction between SCSMV and members of Potyvirus (SCMV, SrMV) and Rymovirus (WSMV, BSMV). The genomic structure of SCSMV is identical to the member of the Potyviridae family, including Ipomovirus, Potyvirus, Rymovirus, and Tritimovirus (Xu et al. 2010). However, the sequence similarity of SCSMV and potyviral-related genera was comparatively low, indicating that SCSMV does not belong to Potyvirus and should be classified into a new genus in the family Potyviridae (Rabenstein et al. 2002; Viswanathan et al. 2008a). International Committee on Taxonomy of Viruses (ICVT) has designated Poacevirus as the new genus name for SCSMV, Triticum mosaic virus, and Caladenia virus A (Wylie et al. 2017). The identification of SCSMV from an unknown field sample or germplasm is carried out using an RT-PCR-based method using a specific primer amplified CP region at the 3′ end of the virus genome (Hema et al. 2003).

Mosaic diseases caused by SCSMV infection are mostly reported from Asian countries such as India (Chatenet et al. 2003), China (Li et al. 2011; He et al. 2013), and Indonesia (Damayanti and Putra 2010), but recently, it was also identified in Côte d’Ivoire, Africa (Sorho et al. 2020). SCSMV was observed in 30% sugarcane fields across Java, Indonesia, and found to reduce sugar yield by about 20% in highly susceptible varieties (Putra et al. 2014, 2015). Sugarcane mosaic diseases caused by a single infection of SCMV rarely occur (Xu et al. 2008). SCSMV predominantly infects sugarcane in a coinfection manner with SCMV (Rao et al. 2006) and SrMV (Luo et al. 2016). Coinfection is a common incident that causes mosaic disease, so that method is required to identify several viruses simultaneously. An RT-PCR-based method was developed by designing two primer sets in the single tube RT-PCR reaction (Duplex RT-PCR) to identify 860 bp and 690 bp coat proteins corresponding to SCMV and SCSMV, respectively (Viswanathan et al. 2008b). Similarly, Feng et al. (2020) also developed multiplex RT-PCR methods to identify multiple viruses in a single tube reaction from sugarcane samples (Feng et al. 2020).

14.6.2 Strategy to Develop Virus-Resistant Plants

The mosaic disease is reported globally and has become a severe threat to sugarcane plantations. SCMV, SrMV, and ScSMV are the primary causative agent of mosaic disease, in which a single dominant virus infection or mixed infection occurs depending on time and place. SCMV infection is dominant in India, China, and Indonesia in the 1980s. In recent years, the mixed infection has been frequently observed in China (SCMV-SrMV and SrMV-SCSMV) (Xu et al. 2008; Luo et al. 2016), India (SCMV-SCSMV) (Rao et al. 2006), and Indonesia (SCMV-SCSMV) (Putra et al. 2015; Addy et al. 2017). SCMV is still the most severe and prevalent virus observed in sugarcane plantations worldwide. Aphids naturally spread SCMV and SRMV, so they are more easily transmitted than SCSMV, which are mechanically transmitted.

SCMV and SrMV are transmitted by aphids in a non-persistent manner, while SCSMV spreads from plant to plant in a mechanical manner. Controlling the dispersal of the virus using chemicals is impossible, and regulating aphids as vectors is impractical (Wu et al. 2012). Therefore, the cultivation of resistant varieties is the most effective way to control the mosaic disease (Gonçalves et al. 2012). Natural resistance traits to SCMV, SrMV, and SCSMV were exploited from sugarcane germplasm and may serve as basis of sugarcane breeding programs (Li et al. 2018a, b). However, introducing resistance traits to the elite sugarcane cultivar by conventional breeding is complicated due to its poor fertility and complex polyploid-aneuploid genome, resulting in an extended breeding period (Lakshmanan et al. 2005). Therefore, genetic engineering has become an essential tool to introduce virus resistance traits into elite sugarcane cultivars by utilizing molecular approaches, such as PDR, RNAi, and CRISPR/Cas9.

The first genetic engineering approach in sugarcane to introduce virus resistance traits is PDR. This approach uses a sequence from the pathogen’s genome and is introduced into the plant’s genome under the control of a specific promoter. The most widely used viral sequence in PDR is coat protein. Joyce et al. (1998) used CP sequence under the control of either Emu (synthetic promoter) or Ubi (ubiquitin promoter) and introduced it into sugarcane using particle bombardment. Only one line from the Emu transgenic line showed resistance to SCMV. In comparison, Ubi transgenic plants indicated ten lines of resistance, four lines with a mild symptom, and ten lines with the ability to recover from the SCMV infection in the challenge test. The data indicated that the CP sequence in the sugarcane genome could confer resistance to SCMV infection (Joyce et al. 1998). The promoter also plays an essential role in controlling CP expression to acquire resistance.

Apriasti et al. (2018) compared the efficiency of complete (927 bp) and N-terminal truncated (702 bp) sequence of CP gene to induce PDR against SCMV infection in sugarcane. Both sequences were introduced into sugarcane via Agrobacterium-mediated transformation. The complete and truncated CP genes were expressed at protein levels in the transgenic sugarcane. As a result, the complete sequence of CP generated a higher resistance to SCMV infection than its truncated version, indicating that complete coat protein is possibly required for effective blockage of viral disassembly and replication (Apriasti et al. 2018).

RNAi was more widely used than PDR to introduce virus resistance traits in sugarcane. The RNAi mechanism is initiated by the formation of siRNA, which plays a role in degrading viral RNA through a complex process known as PTGS. The siRNAs are processed from dsRNA, generated from hairpin repeat, sense, and antisense RNA. Ingelbrecht et al. (1999) have constructed an untranslatable sense CP gene from SrMV cassette driven by ubiquitin promoter. The cassette was transformed into sugarcane using particle gun bombardment to generate transgenic plants. In the SrMV infection test, plants with susceptible phenotype, recovery phenotype, and completely resistant phenotype were observed among transgenic plants (Table 14.1). The resistant plants show a high transcription rate of CP transgene, but its mRNA levels are low or undetectable (Ingelbrecht et al. 1999). Probably, the gene silencing machinery processed mRNA of CP transgene immediately into siRNA so that the mRNA was undetectable. The untranslatable form of sense CP can induce virus resistance in sugarcane through the PTGS mechanism.

Table 14.1 Biotechnology approach used for generating virus-resistant sugarcane
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Guo et al. (2015) constructed 423 bp CP gene from SrMV in hairpin structures driven by CaMV35S promoter in RNAi vector. The RNAi construct was delivered to sugarcane callus via agrobacterium-mediated transformation to generate a stable transgenic plant. The transgenic plant with the interference sequence shows a resistance rate of 87.5% in the artificial SrMV inoculation challenge (Guo et al. 2015). Aslam et al. (2018) engineered a stable short hairpin (shRNA) carrying siRNA driven by polyubiquitin promoter for targeting the CP gene of SCMV. The shRNA constructs were introduced into sugarcane callus via particle bombardment to generate a transgenic plant. Upon SCMV virus inoculation challenge, the transgenic sugarcane shows virus RNA reduction ranging from 10 to 90%, indicating that most transgenic sugarcane lines expressing shRNA were resistant to SCMV infection (Aslam et al. 2018).

Widyaningrum et al. (2021) compared the effectivity of CaMV35S and ZmUbi promoter in controlling the hairpin structure of CP (997 bp) to induce resistance against SCMV infection. Both the CaMV35S and ZmUbi promoters driving the CP hairpin structure were introduced to sugarcane by Agrobacterium-mediated transformation. In the SCMV infection test, hairpin CP driven by the CaMV promoter generated 57.69% resistant lines, whereas the ZmUbi promoter generated 82.35% resistant lines. The result indicated that the ZmUbi promoter is more effective than the CaMV35S promoter in driving CP RNAi expression to induce SCMV resistance in sugarcane (Widyaningrum et al. 2021).

Recently, Hidayati et al. (2021) performed a comparative study examining the efficacy of PDR and RNAi in generating sugarcane resistance against SCMV infection and found that RNAi is more effective than PDR (Hidayati et al. 2021). This finding implies that gene silencing-induced virus RNA degradation is more effective in combating virus attack than inhibiting virion disassembly by coat protein. Possibly, sugarcane carrying RNAi of coat protein accumulated a high level of siRNA inducing virus RNA degradation that operates by a mechanism similar to PTGS. In agreement with this hypothesis, the use of RNAi in downregulating endogenous genes is more efficacious than co-suppression because of its effectiveness in producing dsRNA and siRNA to trigger gene silencing (Stoutjesdijk et al. 2002).

14.7 Conclusion

A new sugarcane cultivar with essential traits such as drought tolerance, disease resistance, and high biomass yield has been developed employing novel strategies using biotechnological approach. To develop drought and stress-tolerant sugarcane, overexpression of the gene encoding bacterial betA for increasing betaine content helps sugarcane to acclimate to water deficit environment. In addition, pathogen-derived resistance (PDR) and RNA interference (RNAi) technologies have been applied to engineer sugarcane cultivars having resistance to mosaic virus. Along with improving sugarcane traits through abiotic stress tolerance and biotic stress resistance, improving the efficiency of carbon partitioning by genetic engineering of SPS can be utilized as an essential strategy for increasing yield and biomass allocation.