Introduction

Sugarcane (Saccharum officinarum L.) is an important cash crop. Sugarcane has immense potential for production of sugar which accounts for 70% of world’s sugar. Besides, it has enormous potential for biofuel (ethanol) production. In spite of all efforts, India is still a slow runner in productivity than most of sugarcane growing countries of the world which may be attributed to sensitivity to salt, drought and biotic stresses (Khaliq et al., 2005). Drought alone accounts about 17% potential yield loss. Being a typical glycophyte, its growth is severely affected under salt stress leading to significant reduction in yield potential (Suprasanna, 2010). Due to its high heterozygosity, crossing of the desired parents provides enough variability for an early clonal selection. However, conventional breeding has not resulted major stride in developing elite sugarcane varieties due to narrow gene pool, higher ploidy (2n=100-120), rare flowering, low fertility, large genome size, long breeding cycle and complex environmental interactions. Besides, the breeding work in sugarcane is time consuming and laborious. Compared to cisgenic breeding, molecular manipulation via in vitro culture may reduce time, labor and costs involved. In this context, production of transgenic sugarcane can be a better alternative which can pave a way forward to integrate desired gene(s) into its genome and the transgenic plant can be multiplied extensively using micro-propagation techniques. Such a technique avoids the problem of linkage drag normally associated with conventional breeding besides shortcutting the period of breeding in sugarcane.

1 Genome Complexity

The size of the sugarcane genome is about 10 Gb while its genome complexity is due to the mixture of euploid and aneuploid chromosome sets with homologous genes present in from 8 to 12 copies (Souza et al., 2011). The estimated monoploid genome size is about 750–930 Mb (the monoploid genome size of the two parental species, S. officinarum and S. spontaneum, are 930 Mb and 750 Mb, respectively), closest to sorghum genome (~730 Mb) and about twice the size of the rice genome (~380 Mb) (D’Hont and Glaszmann, 2001). A high gene-copy number, the integration of two chromosome sets from two different species, and a significant repeat content hinder the understanding of how the genome functions and obtaining a genuine assembled monoploid genome (Figueira et al., 2012).

2 Current Transgenic Strategies  

Sugarcane industry demands huge production of sugarcane. This can be achieved by providing a large number of planting materials to the sugarcane farmers that are robust and climate resilient. Conventional breeding takes 10 to 15 years for breeding. It involves random shuffling of genes without guaranteed transfer of a desired gene and the situation is complicated due to highly complex nature of the sugarcane genome. Genetic engineering can be a more directed alternative to conventional plant breeding (Pillay, 2013). During this process, specific gene(s) of interest is identified, isolated and introduced into target organism (Singh, 2012). Success of the transformation depends on transformation system that minimizes somaclonal variation, stable integration of the transgene into the genome of the target tissue, strong promoter driving high level expression of the transgene and selection of transformed cells (Rashid & Lateef, 2016). Plant regeneration system via direct embryogenesis (Snyman et al., 2006) may be suitably used for the purpose. 

The method chosen for sugarcane transformation is dependent on the type of tissue being modified. Transformation efficiency of seeds (Mayavan et al., 2013), apical meristems (Khan et al., 2013), axillary bud explants (Manickavasagam et al., 2004), young leaf whorl (Eldessoky et al., 2011), somatic embryogenic callus (de Alcantara et al., 2014), protoplasts (Arencibia et al., 1995) and cell aggregates of suspension culture (Efendi and Matsuoka, 2011) have been reported. Among these, embryogenic callus are the preferred explant for transformation owing to their high regeneration response (Taparia et al., 2012a; 2012b). The genetic transformation of sugarcane protoplasts and cell suspension has been carried out by using polyethylene glycol (Aftab and Iqbal, 2001) and electroporation (Rakoczy-Trojanowska, 2002). Microprojectile delivery system can produce large number of transiently expressing sugarcane suspension cultures (Rani, 2012). Bower and Birch, (1992) reported production of transgenic plants by micro-projectile bombardment of embryogenic callus using either gene gun or particle inflow gun. A pressurized helium gas is used to accelerate the gold particle coated with DNA for bombardment into the target tissue (Rashid and Lateef, 2016). Prior to bombardment, the target tissue is placed onto a conditioning medium containing osmoticum that will allow easy penetration of the DNA into the cells and their nuclei (Slater et al., 2008). However, Agrobacterium-mediated genetic transformation is a method of choice due to reduced transgene silencing (Kumar et al., 2014a; 2014b) and its simple methodology (Brumbley et al., 2008) and a high efficiency of transgene integration with few copies (Kumar et al., 2013). 

Agrobacterium tumefaciens is a gram-negative bacteria and the strains e.g., AGL0, AGL1, EHA105 and LBA4404 carrying vectors like pAHC27, pEmuKN, pR11F (Pillay, 2013), pGreen0029 (Kumar et al., 2013), pBract 302 (Reis et al.2014), pMLH7133 (Efendi and Matsuoka 2011), Pu912 (McQualter and Dookun-Saumtally, 2007), pGFP35S (Rasul et al., 2014), pWBvec10a (Joyce et al., 2010), pKYLX80 (Gilbert et al., 2005) etc. are in vogue used for genetic transformation. Agrobacterium strain EHA105 harboring pGreen0029 vector was used for genetic transformation against drought and salinity tolerance in sugarcane.

The selection system and co-cultivation medium are the most important factors determining the success of Agrobacterium -mediated transformation and transgenic plant regeneration (Joyce et al., 2010). The most important and widely used selectable marker is npt II (neomycin phosphotransferase II) gene conferring resistance to kanamycin and geneticin (Joyce at al., 2010) that increases the efficiency of transgene integration and recovery of transgenic plants. Inhibitory effects of selective agent are tissue and species specific (Yu et al., 2003). Therefore, it is necessary to know the minimal inhibitory concentration of selective agent for different sugarcane cultivars before attempting genetic transformation.

3 Gene Promoters for Sugarcane Transformation

The regulation of gene expression involves DNA sequence upstream of the transcribed region and transcription factors that stabilizes RNA polymerases in these promoter regions to start transcription. Genomic synteny with other C4 plant species e.g., sorghum and maize can be used for map-based isolation of genes, identification of regulatory networks involving cis regulatory elements and promoters (Waclawovsky et al., 2010). A number of researchers used promoters e.g., Emu, Maize Adh 1, CaMV 35S, Maize ubiquitin promoter, TMV 35S, Rab17 for construction of gene cascade in sugarcane genetic transformation (Reis et al., 2014). AVP1 (Arabidopsis Vacuolar Pyrophosphatase-1) gene driven under 35S CaMV promoter was used for genetic transformation against drought and salinity tolerance in sugarcane (Kumar et al., 2014a).  However, compared to 35S CaMV (a constitutive promoter), the stress inducible rd29A promoter is in vogue useful for improving drought, cold and salt stress without any penalty on growth (Kasuga et al., 2004). Maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a cavendish banana streak badnavirus promoter were used for production bioplastic (PHB) production in transgenic sugarcane. Among these, cavendish banana streak badnavirus promoter was shown to be most potent for PHB production at seedling stage, but activity of this promoter reduced as the plants mature (Petrasovits et al., 2012). 

4 Transcriptional Factors for Sugarcane Transformation

A number of studies have been done to improve abiotic stress tolerance using transcriptional factors. Over-expressing CBFs/DREBs in transgenic Arabidopsis (Gilmour et al., 2000), canola (Jaglo et al., 2001), tomato (Hsieh et al., 2002) conferred freezing and drought (Haake et al., 2002) tolerance. Over-expression of transcription factors is in vogue done using stress regulated promoters. Ries et al. (2014) revealed induced over-expression of AtDREB2A CA in sugarcane to improve drought tolerance. Similarly, SodERF3 (a novel sugarcane ethylene responsive factor) gene of sugarcane enhanced salt and drought tolerance in tobacco plants (Trujillo et al., 2008). However, Oh et al., (2005) revealed constitutive over-expression of CBF3 and ABF3 in rice that led to the development of transgenic rice with elevated tolerance to drought and salinity without any growth inhibition or any phenotypic aberrations. Sugarcane transgenics expressing MYB transcription factors show improved glucose release with a significant decrease in acid-insoluble lignin (Poovaiah et al., 2016).

5 Status of Transgenic Studies in Sugarcane

In recent years, development of transgenics is increasing rapidly in sugarcane. Until now, massive research efforts have been focused on sugarcane with the aim of establishing an efficient and reliable sugarcane transformation system to improve various traits of interest in sugarcane. Transformation in sugarcane was carried out to incorporate reporter genes (Hansom et al., 1999) and to produce plants resistance to herbicides (Falco et al., 2000), viruses (Gilbert et al., 2005), bacteria (Zhang et al., 1999), fungi (Enriquez et al., 2001) and insect pests (Braga et al., 2003). Sugarcane has been also genetically modified for sugar yield, improved cellulosic ethanol production from sugarcane biomass and quality traits (Vickers et al., 2005), pharmaceuticals (Wang et al., 2005), novel sugars with potential benefits to consumer (OGTR, 2004). Besides, genetic modification underlying altered plant architecture, plant growth, enhanced nitrogen use efficiency, enhanced water use efficiency, drought and salinity tolerance (Reis et al. 2014) have been attempted in sugarcane.

5.1 Enhanced Sugar Content

Sugarcane accounts for 80% of sucrose produced worldwide, with the remaining 20% coming from sugarbeet. Plants generate ATP (adinosine tri-phosphate) and NADP (nicotinamide adenine dinucleotide) by photosynthesis to fix absorbed carbon dioxide (CO2) into triose phosphates (e.g., glyceraldehyde-3-phosphate) via Calvin Cycle. Triose phosphates in leaf chloroplasts are transported into the cytosol and converted into sucrose (hexose) by SPS (sucrose phosphate synthase) and SuSy (sucrose synthase). Identification and isolation of candidate genes involved in biosynthesis and follow-up transport of sucrose would immensely useful for planning successful recovery of transgenic plants with high sucrose accumulation. The sugar in leaf mesophyll cells is targeted for phloem loading. For this, sugar is transported into the apoplast via sucrose transporters (ShSUT1) on plasmamembrane (apoplastic transport) (Rae et al., 2005) or to other cells through plasmodesmata (symplastic transport). Apoplastic invertases play a significant role in recycling of sugar by breaking hexose to triose form. Thus, it can be envisaged that molecular manipulation underlying decreasing cytosolic invertase and PFP (pyrophosphate dependent phosphofructokinase) activity, as well as increased sucrose transport and subsequent storage in the cell vacuole can increase sucrose levels within the sugarcane culm. 

Sucrose content is a highly desirable trait in sugarcane. Sugarcane is commercially grown for high sucrose content (about 20%) in matured stem internodes. A number of studies are now available for transgenic manipulation of sugar content using candidate genes related to sugar biosynthesis and transport to sink (stem internode). Conradie (2011) attempted to transfer the tonoplast-bound AtV-PPase gene linked to the maize ubiquitin promoter, in sugarcane callus. The transgenic callus did not reveal significant accumulation of sucrose in the vacuole but changed in transgenic plants. 

Papini-Terzi et al. (2009) have identified genes differentially expressed in internodes using cDNA microarrays. There is a strong coincidence between drought responsive and sucrose biosynthesis genes and but a limited overlap with ABA signaling. Several protein kinases and transcription factors are likely to be regulators of sucrose accumulation. Besides, aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Transgenic research can elucidate the role of these genes (Papini-Terzi et al. 2009). It has been shown that sugar crystallized from GM sugarcane plants does not contain DNA or proteins of the introduced transgene (s). This paves the way for commercialization of GM sugarcane and derived products (Joyce et al., 2013).

5.2 Reduced Lignin Content and Increased Ethanol Production

It has been demonstrated that a strong negative correlation does exist between lignin deposition in cell wall and sugar released by in planta enzymatic hydrolysis (Chen and Dixon, 2007). As many as ten enzymes are associated with biosynthesis of lignin (Jung et al., 2012a). Lignin content can be reduced by down regulation of any of these genes using transgenic approaches. Down regulation of cinnamoyl-CoA reductase (CCR) in poplar resulted in up to 50% reduction in lignin content, resulting in a concomitant increase in cellulose content (Leple et al., 2007). Similarly, RNAi suppression of a sugarcane COMT (caffeic acid 3-O-methyltransferase) gene reduced lignin content by up to 14%, resulting in an increase of 29% in saccharification yield (Jung et al. 2012b). Besides, RNAi-mediated down regulation of the 4CL (4-coumarate: CoA ligase) gene led to moderate reduction in lignin (4 to 5%), but increased the saccharification efficiency by 19-20% without compromising plant performance (Altpeter, 2012). In contrast, overexpression of PvMYB4 in switchgrass reduced lignin content by 40~70%.

Lignin, after cellulose accounts for 30% of the organic carbon in the biosphere. It is considered as an important evolutionary adaptation of plants during their transition from the aquatic environment to land. Down-regulation of lignin biosynthesis pathway enzymes and/or in planta expression of cell wall degrading enzymes will increase the efficiency of bio-ethanol production. Now, microbial genes encoding cellulose-degrading enzymes have been precisely engineered into sugarcane to allow a more efficient conversion of cellulose to ethanol (Harrison et al., 2011).

5.3 Fungal and Bacterial Disease Resistance

Over 100 fungi and ten bacteria are pests of sugarcane in different parts of the world (Singh and Waraitch, 1981). Among these, red rot and leaf scald are of special mention. Red-rot is a major fungal disease, causing severe loss (18~31%) in yield and quality of the sugarcane. This is also called ‘cancer’ of sugarcane caused by Colletotrichum falcatum. The fungus is highly variable which causes frequent break down of resistant mechanism in tolerant varieties. Genotypes with higher activity of polyphenol oxidase (PPO), Phenylalanine Ammonia lyase (PAL) and Tyrosine Ammonia lyase (TAL) was shown to be linked with red rot resistance (Srivastava and Solomon, 1990; Madan et al., 1991). The Sugarcane Breeding Institute (SBI) at Coimbatore used anti-fungal genes drawn from other plant sources such as alfalfa plant for glucanase and paddy plant for chitenase and Dahlia flowers for anti-microbial peptides for developing the transgenic sugarcane varieties (Annonymous, 2006). Differential display (DD)-RT-PCR followed by silver staining made it possible to detect resistant gene chitenase using resistant gene analogue (RGA) primers (Viswanathan et al. 2009). COC-671 is a high sucrose yielding transgenic sugarcane variety has been screened for red rot resistance. 

Leaf scald is a widespread and devastating bacterial disease prevalent in monocotyledonous plants in the Poaceae family, including Saccharum spp. and other grasses. It is caused by a gram-negative bacteria Xanthomonas albilineans that produces a plastid degrading toxin ‘albicidin’ with antibiotic property. Transgenic sugarcane plants that express a bacterial albicidin detoxifying gene (albD- encoding an esterase enzyme with albicidin hydrolase activity) from Pantoea dispersa provides biocontrol against leaf scald disease (Zhang and Birch, 1997; Zhang et al., 1999). Plants with albicidin detoxification capacity equivalent to 1–10 ng of AlbD enzyme per mg of leaf protein did not develop white chlorotic lines in inoculated emerging leaves, whereas all untransformed control plants developed severe symptoms.

5.4 Virus Resistance

Transgenic technique for virus resistance was mostly targeted to sugarcane mosaic virus (SCMV) (Gilbert et al., 2005), yellow leaf virus (Gilbert et al., 2009). The SCMV (10 kb single stranded RNA genome) is a sub-group of Potyviruses. It can significantly reduce stalk yield and sucrose content (Gao et al., 2011). The virus damages chloroplasts and blocks photosynthesis (Chauhan et al., 2015). The transgenic approach involves inserting a gene derived from the virus itself (Gilbert et al., 2009). A suitable promoter bound- nucleotide sequence encoding coat protein or an anti-sense RNA molecule complementary to the plus strand viral RNA or a hairpin dsRNA molecule for the coat protein gene can serve the purpose. However, the coat protein gene (B2, B36, B38, B48, and B51) mediated protection (CPMP) is an effective strategy to improve virus resistance. Smith (1997) produced a range of transgenic plants containing the SCMV coat protein gene for protection against the virus infection. Over-expressing viral CPs in the transgenic plants interferes with virion assembly as well as viral movement within the plant (Mehta et al., 2013). The transgenic line with CP gene B48 using microprojectile bombardment was highly resistant to SCMV with less than 3% incidence of infection (Yao et al., 2017). In maize, a typical h-type thioredoxin gene (ZmTrxh) within a major quantitative trait locus (Scmv1) imparts strong resistance to SCMV at the early infection stage (Liu et al., 2017). Weng et al., (2010) used biolistic transformation with the capsid gene of the yellow leaf virus (YLV). Zhu et al. (2011) revealed extreme reduction of YLV virus titers in at least six out of nine transgenic lines of cv.H62-467 using untranslatable coat protein gene. 

Improving plant antiviral resistance by gene silencing has proven to be effective in several plant-virus biosystems. RNA silencing can be suitably used for defense against invasive nucleic acids such as viruses and transposable elements (Voinnet, 2005). Transgenic sugarcane resistant to sorghum mosaic virus based on coat protein gene silencing by RNA Interference has been attempted (Guo et al., 2015). The technique was also found suitable to suppress lignin biosynthesis in sugarcane (Osabe et al., 2009; Jung et al., 2012a). Besides, the RNAi-mediated virus resistance has been reported in soybean (Kim et al., 2013), tobacco (Niu et al., 2011), potato (Ntui et al., 2013), barley (Wang et al., 2000), tomato (Lin et al., 2011), maize (Zhang et al., 2011) and rice (Shimizu et al., 2012).

Since RNA-mediated resistance is sequence-specific, viruses not closely similar to the transgene sequence can circumvent attempts at silencing. Furthermore, they can cause reversal of silencing, as revealed by recovered expression of the silenced marker genes in transgenic plants following virus infection (Voinnet et al., 1999). The virus counter-defense against silencing is reported to be mediated by viral RNA silencing suppressor proteins (reviewed in Carrington et al., 2001). RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation (Guo et al., 2015). Besides, the CP gene of SCMV-E strains has been successfully transferred into S. officinarum for protection against SCMV (Yao et al., 2015).

It is now well established that transgenes in plants can suppress expression of homologous endogenous genes. Viral RNAs homology-dependent gene silencing can occur either at the level of transcription or posttranscriptional process due to specific degradation of the homologous RNA molecules (Mueller et al., 1999). Ingelbrecht et al. (1999) transformed sugarcane with an untranslatable form of the sorghum mosaic potyvirus strain SCH CP gene using particle gun bombardment. Transgenic sugarcane plants infected with sorghum mosaic potyvirus strain SCH; had shown fully susceptible to completely resistant phenotype, and a recovery phenotype was also observed. These results led to the hypothesis that gene silencing is a natural plant defense mechanism. On the contrary, recovery of fully susceptible plants in a few virus resistant transgenic lines could be due to the fact that viruses have evolved a mechanism to counter it (Anandalakshmi et al., 1998).

5.5 Insect Pests Resistance

Sugarcane is attacked by a wide range of insects including stem borers, sucking pests and canegrubs causing an estimated loss of around 10%. Progress towards insect resistance in this crop is indeed limited due to genomic complexity and lack of resistance source in the sugarcane germplasm. However, a number of transgenic sugarcane lines have been developed with genes encoding Cry proteins (δ-endotoxins) (Weng et al., 2006), proteinase inhibitors (Nutt et al., 2001), snow drop lectins (Zhangsun et al., 2007), ribosome inactivating proteins, secondary plant metabolites for resistance to borers, sucking insects or grubs (Khan, 2001; Srikanth et al., 2010; Bates et al., 2005). Setamou et al. (2002) had shown reduction in longevity of adult female rice borer (Eoreuma loftini) and sugarcane borer (Diatraea saccharalis) in transgenic sugarcane using snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene. Similarly, transgenic sugarcane harboring cry1Ac gene exhibited enhanced resistance against sugarcane borer in addition to high sugarcane set yield (Gao et al., 2016). Arencibia et al. (1997) developed stem borer resistant sugarcane by introduction of a gene from a soil bacterium. 

5.6 Marker Gene Transfer

Several studies showed efficient transformation with marker gene in sugarcane (Taparia et al., 2012a; 2012b) using callus as explant. Khan et al. (2013) was able to transfer GUS, HPTII (hygromycin resistant) and NPTII (kanamycin resistant) genes to shoot tip cultures of sugarcane variety HSF-240 by using Agrobacterium tumefaciens strain EHA101 with vector pIG121 Hm. Transformation in sugarcane was also carried out to incorporate reporter genes (Hansom et al., 1999) and notable among these is the green fluorescent reporter gene transferred to sugarcane.

5.7 Plastid Transformation

Only a few researchers tried plastid transformation though it has several advantages. Chloroplasts are maternally inherited; hence there is no danger of transgene transfer through pollen to related weeds. Multi-gene transfer can be conveniently carried out in chloroplasts which is rather difficult with nuclear genome. Each single plant cell contains enormous number of plastid genomes/plastid. Therefore, high level of transgene expression is inevitable and also there is less chance of transgene silencing. Higher yields of transgene product have been realized in dicot species as cotton, soybean, lettuce, carrot, tomato and tobacco (Wang et al., 2009). 

The most successful method for inserting foreign genes into chloroplasts is particle gun bombardment that allows site-specific integration. However, PEG-mediated plastid transformation was successfully carried out by Kofer et al. (1998). In this method, the gene cascade is flanked by plastid DNA sequence to promote transfer of the transgene in to plastid genome by homologous recombination. The aadA gene conferring resistance to spectinomycin and streptomycin is in vogue used in plastid transformation in higher plants. A few reports are available for chloroplast genetic transformation in rice and wheat (Lee et al., 2006; Cui et al., 2011). Christopher (2007) carried out a project on sugarcane chloroplast transformation for production of human vaccines against Rotavirus.

5.8 Biodegradable Plastics

Considering high biomass production in sugarcane, the development of transgenic sugarcane for biodegradable polymers e.g., polyhydroxybutyrate (PHB) is a potential area of research (Petrasovits et al., 2007). Polyhydroxybutyrate (PHB) production was shown to be improved in transgenic tobacco due to enhanced translation efficiency of bacterial PHB biosynthetic genes e.g., acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) (Matsumoto et al., 2011). In sugarcane, the maize chlorophyll A/B -binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in the crop (Petrasovits et al., 2012).

5.9 Herbicide Resistance

A number of bacterial genes are now available for detoxification of herbicides that are in vogue used for weed control (Enríquez-Obregón et al., 1998). In addition to reduce production cost by way of weed control, the herbicide resistance genes (e.g., bar and pat) are also potentially used as selective markers for screening of putative transformants (Manickavasagam et al., 2004) in transformation experiments. Nasir et al. (2014) was successful to transfer 1368bp glyphosate tolerance gene under the control of CaMV-35S promoter with the β-glucuronidase (GUS) reporter gene to four varieties of sugarcane (CPF-234, CPF-213, HSF-240, and CPF-246) with transformation efficiency as high as 32% using biolistic transformation. Transgenic sugarcane plants resistant to the commercial herbicide BASTA was produced by Agrobacterium tumefaciens-mediated delivery of.bar gene encoding phosphinothricine (PPT) acetyltransferase (Enriquez-Obregon et al., 1998). Similarly, Gallo-Meagher and Irvine (1996) have obtained herbicide resistant transgenic sugarcane plants of the commercial cultivar NCo 310 using particle bombardment of bar gene with somatic embryogenic calli.

5.10 Drought and Salinity Tolerance

Drought resistance being a complex trait, and keeping in view the complex ploidy status of sugarcane genome, genetic modification of the crop for drought tolerance remains to be a challenging task. Identification of differentially expressed candidate genes with regulatory function to water deprivation; can pave the way for production of drought tolerant transformed plants (Kirch et al., 2005). In 2011, a transgenic sugarcane variety containing the transgene DREB2A has been released in Brazil which offers tolerance against heat, drought and salinity.

Increase in drought tolerance was correlated with proline accumulation in transgenic sugarcane (Molinari et al., 2007). The drought stress induced gene Δ1-pyrroline-5-carboxylate synthetase (P5CS) meant for proline production; was also shown to confer tolerance to salt stress in transgenic sugarcane (Guerzoni et al., 2015). A drought tolerant transgenic sugarcane variety harboring a bacterial gene for betaine production; has been released for commercial cultivation in Indonesia. Zhang et al., (2006) reported the expression of Grifola frondosa TSase gene for improving drought tolerance in sugarcane. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter. Compared to non-transgenic plants, the transgenic plants can accumulate high levels of trehalose, up to 8.805–12.863 mg/g fresh weight. These transgenic plants had increased tolerance to drought and have increased yields under drought conditions. Similarly, Wang et al., (2005) developed the transgenic sugarcane plants harboring G. frondosa synthase gene confirmed by PCR and Southern analysis. They reported that some transgenic plants showed multiple phenotypic alterations and some plants demonstrated improved tolerance to osmotic stress. In another study, Over-expression of heterologous P5CS gene under stress inducible promoter (AIPC) was also reported to enhance drought tolerance in sugarcane (Molinari et al., 2007). They suggested that stress inducible proline accumulation in the transgenic sugarcane plants under water deficit stress acts as a component of antioxidant defense system rather than as osmotic adjustment mediator. The Arabidopsis CBF4 gene transferred to sugarcane under the control of the maize ubiquitin promoter and the nos terminator was reported to improve drought tolerance (McQualter and Dookun-Saumtally, 2007).  Besides, genetic transformation of sugarcane for drought tolerance has been attempted using Arabidopsis Vacuolar Pyrophosphatase (AVP1) gene (Kumar et al., 2014a; 2014b) and Arabidopsis bax inhibitor-1 gene (Ramiro et al., 2016). 

5.11 Quality Traits and Other Products of Commercial Importance

Genetic modification underlying production of naturally occurring compounds for use in bioplastics, antibiotic resistance, altered juice colour (Manickavasagam, 2004; Mitchell, 2011) have been attempted in sugarcane. Further, genetic engineering of sugarcane varieties that can produce high-value compounds such as pharmaceutically important proteins, functional foods, nutraceuticals, biopolymers, precursors, enzymes and biopigments are paving ways to launch sugarcane as a biofactory in coming years (Suprasanna, 2010).

Polyphenol oxidase (PPO-a copper protein) catalyzes the oxidation of phenolics to quinones and thus causes oxidative browning of juice. Salicylhydroxamic acid (SHAM) completely inhibited the PPO activity. Besides, treatment with Cycloheximide (CHX) or upon heating markedly reduced the browning of juice colour. Isolation of the gene encoding PPO paves the way for use of genetic manipulation to down regulate the PPO gene in the existing commercial cultivars. Overexpression of polyphenol oxidase in transgenic sugarcane results in darker juice and raw sugar (Vickers et al., 2013). Besides, overexpression of polyphenol oxidase in transgenic tomato resulted insect resistance (Thipyapong et al., 2007).

6 Epilogue

A number of transgenic techniques have been used for transfer of useful genes from diversified genetic background. However, Agrobacterium-mediated genetic transformation proved to have several advantages over direct gene transfer techniques in sugarcane. Increased sugar yield and biomass production coupled with biotic and abiotic resistance is always the top most priority in sugarcane transgenic research. In sugarcane, the potential benefits realized by transgenic research far outweigh the risks.  The present review reveals that development of genetically modified climate resilient genotypes with improved quality features and resistance to dreadful diseases seems to be a near reality in sugarcane.

Acknowledgement

I sincerely acknowledge and thank all researchers for their valuable contributions included in this pursuit.

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