Biotechnological Interventions for the Improvement of Sugarcane Crop and Sugar Production Biotechnological Interventions for the Improvement of Sugarcane Crop and Sugar Production

Sugarcane, not only fulfills 70% of world sugar needs but is also a prime potential source of bioethanol. It is majorly grown in tropical and subtropical regions. Researchers have improved this grass to great extent and have developed energy cane with ability to accumulate up to 18% sucrose in its Culm. Improvement of this crop is impeded by its complex genome, low fertility, long production cycle and susceptibility to various biotic and abiotic stresses. Biotechnological interventions hold great promise to address these impediments paving way to get improved sugarcane crop. Further, being vegetatively propagated in most of the agroecological regions, it has become more attractive plant to work with. This chapter highlights, how advanced knowledge of omics (genomics, tran - scriptomics, proteomics and metabolomics) can be employed to improve sugarcane crop. In addition, potential role of in vitro techniques and transgenic technology has also been discussed for developing improved sugarcane clones with enhanced sugar recovery.


Introduction
Sugarcane is a major contributor to world sugar and more than 70% of global saccharine needs are fulfilled by this sweet grass. It has been cultivated since pre-historic times as a sugar source. Further, it has great potential to be used for the production of bioethanol and has been grown in many countries as an energy crop. Brazil is the top most consumer of sugarcane biofuel followed by USA and fulfill 50% of their energy needs through biofuel. They run more than 5.0 million automobiles on hydrous ethanol at an ethanol content of 95.5% [1]. These facts direct us to strive for the improvement of sugarcane crop so that global energy needs may be fulfilled sustainably. Various promising varieties have been developed so far but they are posed to certain drastic stresses including biotic as well as abiotic stresses. Similarly, efforts have been made to improve sugar recovery. Since, crop productivity and quality can only be improved by employing innovative technologies. Plant tissue culture and genetic engineering has great potential to resolve problems faced by this crop [2]. Transgenic technology can do a lot to address all the aforementioned yield limiting constraints as any of the alien genes may be introduced into the plant through genetic transformation methodologies. Different methods of transformation i.e. biolistic [3,4] Agrobacterium [5] and electroporation [6] have been employed to engineer valuable agronomic traits like resistance against weedicides [7], viruses [8] and insects [3]. Efforts have been made to engineer metabolic pathways for improved sugar content [9] and for the production of biopolymers and bioplastics. Omics approaches have contributed a lot to understand and explore sugarcane genome to develop improved clones. Milestones in structural and functional genomics are also convincing. Different types of markers have been developed to speed up molecular breeding through early identification of superior genotypes [10]. Thus, biotechnological interventions have great potential to promote sugarcane not only as future energy crop but also a factory house for the production of therapeutics and industrial compounds.
These interventions have been discussed here to focus critical areas of research that can attract researchers for the improvement of this crop. Understanding molecular mechanisms involved in metabolic pathways and sucrose accumulation will prove a real milestone in developing future energy crop. Similarly, importance of in vitro techniques have been highlighted that how advancements in tissue culture techniques are important for germplasm conservation, development of somaclonal variants and genetic transformation. Likewise, potential of transgenic technology has been discussed to develop insect resistant, disease resistant and herbicide tolerant plants. Omics, a real potential area of future research, has been overviewed to highlight the role of genomics, transcriptomics, proteomics and metabolomics in sugarcane crop improvement and in developing energy cane (Figure 1).

Tissue culture based approaches for sugarcane crop improvement
Since the pioneer work on callus induction at Hawaiian Sugar Planters' Association Experiment Station and the method developed by Nickell [11] for root production, sugarcane tissue culture appeared as a valuable tool for diverse research activities. Shortly after this, Heinz and Mee [12] published the first report on sugarcane regeneration. These in vitro techniques had huge impact on basic research and also on the research of commercial interest which includes maintenance of elite germplasm, production of agronomically superior somaclones, micropropagation of elite clones, healthy planting material and screening for abiotic/biotic stress tolerance.

Somatic embryogenesis
Somatic embryogenesis may be divided into two phases: induction and expression. During the induction phase, embryogenic competence is acquired by differentiated somatic cells whereas during expression phase, embryogenic cells differentiate into somatic embryos. Komamine et al. [13] evaluated that embryogenic cells did not require any exogenous stimuli in the form of growth hormones or vitamins at induction state. Rather, competent cells require exogenous stimuli at transitional state in very minute quantities. Somatic embryogenesis may either be direct or indirect. Direct somatic embryogenesis involves development of embryo directly on the surface of explant tissues i.e. stem segments, leaf segments, young inflorescence, zygotic embryo, protoplasts and microspores. Indirect somatic embryogenesis involves an intermediary step of callus induction followed by embryogenesis. Different factors have been found to play key role in the acquisition of embryogenic competence. Exogenous growth regulators promote embryogenic competence by affecting cell polarity, pH gradients and by modifying ionic balance all around the cells. Intracellular pH is very crucial for embryogenic competence acquisition. Another critical aspect is the physical isolation of a cell from others. Embryogenic competence acquisition by somatic cells is regulated by the expression of certain genes which involve either upregulation or down regulation of certain functional genes. In addition, physiological, morphological and metabolic variations are also very important for the acquisition of embryogenic competence. Thus, somatic embryogenesis is a great milestone in sugarcane biotechnology [14]. Originally this method was developed as a substitute of meristem culture and regeneration but now it has become an important component of genetic engineering as well. It has well been exploited for the preservation of mutants and transformed material till their approval or field establishment. Various high yielding, early maturing, high sucrose content and smut resistant varieties with good ratooning ability have also been developed through somatic embryogenesis.

Somaclonal variations
Somaclonal variation have been employed to improve cane-quality, sugar recovery, yield, drought tolerance and disease resistance. To increase the incidence of genetic variation and to get positive modifications in the target plant genome, physical (ion beams, gamma rays) and chemical (sodium azide, sodium nitrite and ethylmethane sulfonate (EMS) mutagens have been tested [15]. Introduction of selection pressure at cellular level has been successful to isolate mutants with desired characters. For fungal pathogen resistance inoculation with fungal pathotoxins or culture filtrates has been very effective. Somaclonal variants of sugarcane were developed against eye spot disease by Larkin and Scowcroft. Mutagenesis has been used by various researchers to isolate embryogenic cells and plants tolerant against red rot [16].
Similarly, for abiotic stress tolerance polyethylene glycol, mannitol and sodium chloride has been used for the selection of plants against drought/salinity tolerance [17]. Various studies were conducted to evaluate the level of variability and transmission of variations into the next generation by vegetative propagation. These studies verified the occurrence of considerable variations in in-vitro derived plants. However, extensive field experiments showed that tissue culture derived phenotypic variations were often temporary as most of the variants relapsed to the parental phenotype in the first ratoon crop [18]. Few other studies also supported somaclonal variations but to little extent. Chowdhury and Vasil [19] were not able to recognize any considerable variation in the DNA of plants regenerated from cell suspension, protoplasts and callus cultures. Taylor et al. [20] performed random amplified polymorphic DNA (RAPD) analyses of plants regenerated from embryogenic culture. Only a few polymorphisms were observed. Anyhow, there are different opinions about the somaclonal variations and their transmission to next generation. Most of the researchers are of the view that these variations depend upon the genetic makeup and experimental conditions under which plants are screened and selected.

Micropropagation to produce sanitated plants
Systemic buildup of infections particularly diseases (viral, bacterial and fungal) affect plant vigor and health. Unavailability of an efficient in vitro mass multiplication system in sugarcane is a major constraint in the provision of disease free elite germplasm [21]. Sugarcane has long breeding cycle and it requires 10-15 years to complete selection cycle. Fuzz multiplication rate of newly released sugarcane varieties is always slow 1:6-1:8 [22] and diseases accumulate in the seed sets during multiplication. Unavailability of disease free planting material is a major limitation in the improvement of sugarcane crop. Normally, sugarcane reproduces vegetatively but seed propagation is also there under particular climatic conditions [23]. Nodal cuttings are being used for the propagation of commercial sugarcane. In this method of multiplication, meristematic or non-meristematic tissues are used as explant. Sugarcane plants have been regenerated directly from apical and axillary meristems and also from immature leaf tissues [24].
In vitro propagation of sugarcane through meristematic tissues responded better as compared with other types of plant tissues. Therefore, significant efforts have been made to explore meristematic tissues for mass multiplication. In the beginning of twenty-first century, some reports highlighted direct regeneration of sugarcane genotypes through thin layer culture of cells from immature leaf or inflorescence [24]. They reported to lessen the time span required for in vitro propagation. Significant efforts have been made to establish protocols for direct or indirect sugarcane regeneration. Almost each part of the sugarcane plant has been exploited for callus induction but only inflorescence and immature leaves [25] responded better to morphogenic callus. Callus based regeneration gained significance with the prediction that in-vitro induced mutations can play some key role in sugarcane improvement [26]. In vitro induced variability is beneficial for the development of new varieties but it becomes undesirable when true-to-type plants are required. Only fewer examples have been quoted to highlight useful variations in callus-derived plants. Meristem culture was successfully used to eradicate chlorotic streak disease, sugarcane mosaic virus [27], white leaf disease and ratoon stunting disease. Combination of meristem culture and heat treatment have proved very effective to eradicate pathogens of Fiji disease [28], SCMV [27] and downy mildew. It is an effective method to eliminate most of the bacterial and fungal diseases and is commonly used to eradicate diseases of unknown etiology as well. Disease free planting material of sugarcane obtained from apices culture is now routinely used for the international exchange of this crop. Researchers have explored that plants regenerated from thin cell layer culture can be used to produce disease free sugarcane plants from the infected ones with Leifsonia xyli, SCMV and FDV. In vitro culture techniques are used in Brazil and USA to produce healthy planting material for commercial applications. Cryotherapy has also appeared as a proficient method to eliminate phytoplasma from the crop plants and has also been used for long term storage of germplasm or production of disease free plants [29].

Germplasm conservation
Another important application of in vitro techniques that attracted researchers is germplasm conservation [30]. In-vitro storage of sugarcane germplasm had been established at the Centre de Cooperation Internationale en Recherche Agronomique pour le Development (CIRAD) in France, Sugarcane Breeding Institute in India, and BSES Limited (formerly the Bureau of Sugar Experiment Stations) in Australia. More than 200 hybrid clones of Saccharum spp. were preserved at 18°C for 12 months and no phenotypic modifications were observed in the recovered plants. However, with the advancements in tissue culture techniques, in vitro preservation became more valuable for the preservation of genetic resources especially of sugarcane [31]. The minimal medium used in in vitro preservation has been used successfully during short and medium term preservation, especially for meristems and shoot apices. Decline in explants metabolic activity is usually achieved by changing physical environment or composition of the media used [32]. The commonly used approaches are: lowering of incubation temperature and use of osmotically active compounds such as sorbitol, mannitol and sucrose. Moreover, growth inhibitors like abscisic acid (ABA) is also frequently used. Various factors i.e. vitamins, salts, osmotic stress and others have been explored by different researchers. Survival rate varied in all these experiments but nobody exploited genetic or cytological studies to assess the genetic stability of in vitro plants. For diploid species, Simple Sequence Repeat (SSR), Amplified Fragment Length Polymorphism (AFLP) and Inter-Simple Sequence Repeat (ISSR) have been used successfully to assess genetic stability of in-vitro plants [33]. But, for plants like sugarcane which has complex polyploid genome, these tools are inappropriate as interpretation of the results become tricky [34]. Using microscopic techniques for sugarcane is also difficult because of small size and large number of chromosomes and also due to the presence of various cytotypes [35]. In this context flow cytometry has got attraction as it ensures estimation of relative amounts of plants nuclear DNA quickly and precisely [36]. Cytometry is able to discriminate between plants derived different culture techniques and has extensively been used, in many economically important species such as Gossypium hirsutum, Vitis vinifera, Passiflora spp., Elaeis guineensis, Musa acuminata and Prunus cerasus [37]. Flow cytometric analysis of shoots was performed after every 6 months of storage. As a consequence, a discrete behavior of tested varieties was observed during storage and on average approximately 80% cultures were able to recover. From these findings it is concluded sugarcane genotypes can be maintained in minimal growth condition for extensive periods but may lead to genetic variations.

Omics approaches to improve sugarcane crop
The word "Omics" has become a broader term and it is impossible to cover it just in one topic. Omics approaches have explored understandings of complex interactions between genes, proteins and metabolites. These integrated approaches heavily rely on analytical methods, bioinformatics, computational analysis and many other disciplines of biology. Using genomics, proteomics, transcriptomics and metabolomics approaches, the consistency and predictability in plant breeding and transgenic technology has been improved. It has helped to produce high quality and stress resilient crops with enhanced nutritional value in less time and lower input usage. Omics has provided insights into the molecular mechanisms involved in insect resistance and tolerance to herbicides, cold, salinity and drought stresses [38]. To interpret the omics (genomics, transcriptomics, proteomics and metabolomics) approaches in sugarcane for higher yield, higher sucrose contents, biotic and abiotic stress tolerance, one should have knowledge about the genome structure, physiology and functional veracity of sugarcane with other related crops.

Genomics
Sugarcane has a large genome size of 7440 Mb (mega base pairs) having 2n = 100-300. The genome is supposed to be evolved as a result of a complex hybridization event. It is considered that Saccharum officinarum is octoploid. The monoploid genome size of Saccharum officinarum is 930 Mb and that of Saccharum spontaneum is 750 Mb, twice of the size of rice genome (~390 Mb).
Geneticists are trying to interpret the associations of complex sugarcane genome with other similar crop plants. The level of genome varies from diploid to decaploid among the Poaceae species [39]. The conservation and origin of gene function is suggested by gene order which is maintained by synteny of the genome [40]. The TE (transposable elements) intervening between coding genes strongly support the extension of genome in grasses. Transposons and retrotransposons are two categories of transposable elements. In plants, the most abundant retroelements are LTR (long terminal repeats) retrotransposons. Transposase proteins are involved in insertion-deletion mechanisms. Active sites of the transcription control the movement of retrotransposons, which reinsert them into the genome after each propagation cycle to increase copy number. Recent studies show that there exists a gene remodeling mechanism which results in the generation of new genes. As a result of gene remodeling, gene expression is altered by new regulatory networks [41]. The study of transposable elements in wheat and barley [42] provided close relationship of transposable elements with genome structure. The transposable elements in sugarcane can be activated and evaluated by functional transcriptomic approaches. The major limitation in sugarcane genetic improvement is its genome size. To sequence the genome of an organism Bacterial Artificial Chromosomes (BAC) are used. BAC (Bacterial Artificial Chromosomes) library was constructed with HindIII partial digestion for sugarcane cultivar 'R570' having more than 100,000 clones with 130 mega base pairs (Mb) insert size [43]. For map-based cloning of sugarcane, BAC resources will be highly esteemed and physical map of sorghum (http://www.genome.clemson.edu/tools/contig_viewer/index. html) will be used as complementary tool.

Transcriptomics
Transcriptomic approaches have emerged as an effective tool for functional characterization of unknown genes. In combination with proteomics and metabolomics, these approaches are very useful for the development of improved sugarcane clones. It reduces the complexity of data and targets. Only active genes in the cell or tissues are considered at the time of sampling. By employing transcriptomic approaches, one can easily compare similar type of tissues at different developmental stages in different organisms growing in different conditions [44].

ESTs
Due to large size and complexity of genome, the whole genome sequence of sugarcane was not available. The genome size of its modern cultivar is considered to be more than 10 GB. From 12th July, 2017, NCBI database has 83,138 GSSs (genome survey sequences), 285,216 ESTs (expressed sequence tags) and 13,382 nucleotide sequences including 491 sequences of mRNA under the search of "Saccharum". There are three main groups of ESTs including a large group resulting from a modern variety of sugarcane and two small groups from Saccharum officinarum and Saccharum arundinaceum ( Table 1) Many projects have been executed for sequencing sugarcane ESTs (expressed sequence tags) in Brazil (http://sucest.lad.ic.unicamp.br/en), South Africa and Australia [46]. Until now more than 0.3 million (300,000) ESTs have been generated. A database holding 0.238 million (238,000) ESTs (constructed from diverse organs and tissues) from 37 libraries was erected by SUCEST (the Brazilian ONSA consortium's sugarcane EST project). More than 43 thousand clusters (that may signify distinctive transcripts) were assembled by cluster analysis of the SUCEST. A BLAST search showed that almost 50% of these expressed sequence tag clones had no resemblance with known proteins. In Genbank, almost 40% of the clones represent full length protein sequences. The genes involved in diverse metabolic processes have successfully been recognized by analysis of SUCEST database. These analyses reveal that assemblage of ESTs is highly illustrative and indicate tagging of thousands of sugarcane genes [47]. ESTs represent gene encoding sequences, natural antisense transcripts, transacting siRNA precursors, miRNA and most commonly noncoding RNA. The information provided by EST dataset is an important starting point to know about the genome of an organism. It can also help to determine genes of agronomic importance (tolerance of biotic and abiotic stresses, sugar content and mineral nutrition). EST availability makes possible the analyses of gene expression on a large scale. Numerous studies have been conducted for in-silico analysis of transcript enrichment using different cDNA libraries [48].

Proteomics
Proteomics is the large-scale study of proteome (whole protein contents) and diverse properties of proteins. Through proteomics approaches, we can determine the structural and functional details of biological systems under different conditions. Proteomics has been a major field of functional genomics after the completion of many genome sequencing projects. It has also helped to understand the mode of actions, resistance mechanisms and bio-degradation of pesticides. However, in sugarcane, proteome study is a little bit complicated as no standard protein extraction protocol is available [49]. As compared to other monocots, sugarcane proteomics have not gained momentum yet. Finding protein extraction techniques is a stepping stone in shaping up sugarcane proteomics. Earlier, isoenzyme pattern was used as a tool in sugarcane varietal identification and taxonomy. Isoenzyme pattern was analyzed on 1D gradient polyacrylamide gels based on molecular weight differences [50]. Saccharum species (S. sinense, S. edule, S. robustum, S. spontaneum and S. officinarum) were discriminated from other related genus Eriochrysis, Imperata, Narenga, Eriochrysis, Miscanthus and Erianthus by isozyme pattern of acid phosphatases, leucine aminopeptidases and esterases. O'Farrell [51] introduced 2DE which increased interest in sugarcane proteomics. 2DE technique was then used to study sugarcane roots [52], stalks, leaves [53], meristematic cells and suspension cells [54]. Changes in 2DE protein pattern was observed under different stress conditions. Sample preparation is a crucial step and is necessary for reproducible results. Sugarcane tissues are rigid, fibrous in nature, have sucrose, phenolic compounds and other metabolites in its stalk. Protein extraction protocols have been optimized for the extraction of protein from leaves, meristem and cell suspension cultures but extraction of protein from stalk is still a challenge [55]. Since sugarcane stalk is the core site for sucrose metabolism and host-pathogen interaction but no reproducible protocol is available for the isolation of proteins from stalk tissues.

Metabolomics
Metabolomics is the study of metabolites within the cells, tissues or organism. Proteomics is the study of gene product produced whereas metabolomics explores whether gene products are metabolically active within an organism or not. It also includes role of metabolites in various cellular processes. Hence, metabolites are direct indicators of the performance of a plant under particular biotic or abiotic stresses [56]. Nutritional quality and plant health can be improved by monitoring the changes in metabolite profiling. So, the retrieved informations can effectively be used to develop improved crop varieties as well. Variations in metabolite pattern can also assist to distinguish the mode of action of pesticide which provides critical information for the discovery of new pesticides. Metabolomics may be employed to determine differences and similarities between parents and offsprings on the basis of metabolite composition. Mass spectrometry (MS) and NMR (nuclear magnetic resonance) techniques are used for metabolic profiling, to monitor the metabolic regulations and to analyze the impact of herbicides, pesticides, high temperature, intense light, humidity, soil type, salinity, fertilizers and pests on metabolite composition. One of the fundamental reasons for unavailability of data on sugarcane metabolites is the complexity of sugarcane genome and metabolomes.
Most of the research has been focused on differential gene expression. Second constraint is the limited availability of technology due to its sensitivity and labor intensity. Recently, metabolome (whole metabolites in a specific tissue) has been used as a tool for understanding metabolic regulations. This work was accomplished by some advanced technologies where multiple metabolites were determined in a particular tissue within an hour simultaneously. GCMS (gas chromatography-mass spectrometry) is a vastly used technique that separates the metabolites of different types and identify them on the basis of mass spectral matching and retention time. Identification and extraction methods were optimized for thirty sugarcane metabolites. Hence, metabolome studies are of pivotal importance to understand interaction between the genes and their resultant proteins which can be used to understand mechanisms of sucrose accumulation in sugarcane [57].

Transgenic approaches for crop improvement
Transgenic technology is the only technology through which alien genes may be introduced across the species. Sugarcane has well be explored to engineer for certain valuable agronomic traits and for enhanced sucrose contents. Most of the transformation events reported in sugarcane are through biolistic, anyhow Agrobacterium and electroporation have also been used. Success of engineered lines depend upon the integration and stable expression of introduced gene/s. Recalcitrancy, low transformation efficiency, transgene inactivation and difficult backcrossing are major bottlenecks in sugarcane transformation. Though limited reports are available for the field plantation of transgenic sugarcane yet a large number of research groups are involved in engineering sugarcane genome [58].

Herbicide resistance
Herbicide resistance is one of the major traits in transgenic plants. It [59]. Gallo-Meagher and Irvine [60] reported bar gene transformation in sugarcane by biolistic gun. Resultant transformants showed tolerance against basta which authenticated effectiveness of bar gene not only for herbicide tolerance trait but also as a selectable marker gene, for the selection of putative transformants. Enriquez-Obregon et al. [61] reported transformation of uid1 and bar genes in sugarcane by Agrobacterium mediated transformation. They obtained GUS-positive and BASTA resistant calli. Similarly, BASTA resistant variety (Ja 60-5) of sugarcane has also been developed through Agrobacterium mediated transformation. Transformation frequencies reached up to 10-35% by employing different transformation protocols. The PAT (phosphinothricin acetyltransferase) and neo (neomycin phosphotransferase) genes were reported to be transformed in SP80-180 genotype of sugarcane by biolistic method [62]. The selected transformants were resistant against commercial formulation of ammonium glufosinate. Southern blot analysis was used to confirm the stable integration of neo and PAT genes. While western blot analysis and RT-qPCR were used to analyze the expression of these genes. Another report was given by Manickavasagam et al. [5]. They developed herbicide tolerant sugarcane plants by Agrobacterium mediated transformation. This was first report of Agrobacterium mediated transformation in which axillary buds from 6 months old plants were used as explant. Agrobacterium with binary vector pGA492 having β-glucuronidase, neomycin phosphotransferase II and bar genes in between the T-DNA regions was used for transformation. This study proved that phosphinothricin (5.0 mg/L) is more effective selective agent as compared with kanamycin and geneticin. Southern blot analysis was used to confirm the transformants. Leibbrandt and Snyman [7] reported the transformation of pat gene in NCo 310 genotype of sugarcane which confers resistance to the herbicide Basta. Stable transgene expression was evaluated in glasshouse and field conditions.

Insect resistance
Insect pests are one of the major yield limiting agents which cause serious losses to crop yield. Economically important insect pests of sugarcane can be categorized into borers, sap sucking pests, white grubs and termites. Sugarcane pests show extensive variation in species composition in different tropic and subtropic agro climatic regions. All around the world, sugarcane is facing problems of insect pests and diseases which are seriously affecting sugar production. No exact estimates are available for these cumulative losses caused by the insect pests and diseases. Anyhow, economic losses caused by certain pests has been estimated. observed on the expression of transgene [69]. Transgene expression was determined as 1-10 and 0.2-6.0 ng/mg of total soluble proteins in the leaves and stem respectively, which was seven times higher than reported by Arencibia et al. [64]. Plants also showed better resistance to sugarcane stem borer, when tested by in vivo and in vitro insect bioassay. Expression of the transgene was further increased with increase in GC content of cry1Ac gene and was determined as 2.2-50 ng/mg of total soluble proteins when GC content was increased to 54.8%. Hence expression of cry1Ac gene in sugarcane increased with increase in its GC content [65].
Proteinase inhibitors (PIs) derived from both the animals and plants had been introduced in sugarcane to confer resistance against borers. A soybean PI was mixed in the artificial diet of insects and it appeared to have detrimental effects on growth of D. saccharalis. Falco and Silva-Filho [66] developed transgenic sugarcane expressing Soybean Kunitz Trypsin Inhibitor (SKTI) and Soybean Bowman-Birk Inhibitor (SBBI) driven by maize ubi-1 promoter. Transgenic lines were evaluated against D. saccharalis by feeding excised leaf tissue and by infecting plants with neonate larvae in the green house. Transgenic lines with SBBI did not show significant change in the larvae mortality whereas it was slightly higher when fed on the leaves of SKTI expressing plants. Leaves of transgenic plants with SKTI or SBBI inhibited insect growth and metabolism, hence resulted in reduced insect weight. Dead hearts were also observed in almost all of the plants tested in green house. Without knowing the level of expression, it is very difficult to establish relationship between inhibitor content and dead heart to determine their role in borer resistance. Aportinin appeared to be more effective in inhibting gut proteinases of S. excerptalis as compared with those of C. infuscatellus and C. sacchariphagus indicus. In succeeding studies, aprotinin gene under maize ubi-1 promoter was introduced in sugarcane and transgene expression was determined as 0.16-0.50% of the total soluble leaf proteins. In vivo screening assays also revealed very low mortality of S. excerptalis larvae but weight of insects was reduced by 99.8% hence insect growth was affected to great extent.
Besides proteinase inhibitors and cry toxins, certain other molecules have also been explored for the effective control of insect pests in sugarcane. Plant derived insecticidal proteins (lectins) are more valuable for the control of insects as compared with bacterial insecticidal proteins. Wheat germ lectin, snowdrop lectin (Galanthus nivalis agglutinin, GNA) and avidin were used as dietary proteins in bioassays against larvae of sugarcane white grubs. Wheat germ lectins and snowdrop appeared insecticidal and growth inhibitor for Antitrogus parvulus larvae. Avidin also appeared to inhibit growth of A. consanguineus [67]. Such positive results encouraged researchers to exploit potential of these genes for the control of white grubs. Sétamou et al. [68] included extracts of snowdrop lectin expressing transgenic lines (0.89% in leaf sheath) in the artificial diet (0.47% lectin of total extractable proteins) and examined responses of D. saccharalis and E. loftini larvae. When artificial diet containing 0.50% of transgenic GNA was fed to the larvae of E. loftini, a considerable decrease in larvae survival, pupation percentage, adult emergence, pupal weight, longevity and fertility was observed. Transgenic sugarcane expressing lectin under phloem specific RSs-1 (rice sucrose synthase-1) or maize ubi-1 promoters were developed via Agrobacterium mediated transformation. Reduced survival rate, development, fertility and feeding was observed in the larvae of C. lanigera feeding on transgenic plants. Aphid population density was decreased up to 60-80%, and even up to 95% in some lines [69]. Maximum resistance was observed when phloem specific promoter (RSs-1) was used in transformation against sucking pests like wooly aphids. Field performance of transgenic lines is dependent on the expression level and stability of transgene. The most critical factors in this regard are: promoter strength, codon usage, gene silencing and site of integration. Rice polyubiquitin (RUBIQ2) promoter has proved as the strongest promoter for transgene expression in sugarcane [70]. Anyhow, maize ubi-1 promoter has also extensively been used in sugarcane for the optimal expression of transgenes. Though maize ubi-1 is a constitutive promoter but it does not give same level of expression in all plant parts, e.g. leaves, roots and stem. Keeping in view the tissue specificity and feeding habits of insect pests, it is necessary to use tissue specific or wound inducible promoters which will overexpress insecticidal proteins only in the target tissues and will prove more effective for the control of insect pests.

Developing genetically modified varieties with improved abiotic stress tolerance
Abiotic stresses may alter physiological status of a plant either directly or indirectly by disturbing its metabolism, growth and development. Among abiotic stresses salinity, drought and low temperature are the fundamental factors that significantly influence plant performance. To combat these stresses plant triggers a cascade of physiological and biochemical reactions. Commonly sugars and other osmolytes accumulate in response to abiotic stresses (drought, salinity and low temperature). Water is an essential component for life but its scarcity is increasing day by day throughout the world. Currently almost 65% of fresh water is being used for irrigation, indicating that survival would not be possible without developing drought tolerant varieties in the near future. Sugarcane is a high delta crop and requires heavy irrigation but is now expanding in the regions where water availability is limited, so only drought tolerant varieties can be grown with success [71].
Molecular studies have explored that any of the plant growing in stress conditions strives to withstand those conditions by activating certain stress responsive genes/proteins. They include antioxidant enzymes, late embryogenesis abundant proteins, Arabidopsis HARDY genes [72], various transcription factors and certain protease inhibitors. Transcriptomic analyses of sugarcane under drought stress has lead to the identification of stress related genes. These genes should be figured out that how critical they are for stress tolerance. Molecular Systems Biology can be used to characterize regulatory networks using model plant. A novel sugarcane gene Scdr1 (sugarcane drought responsive 1) was overexpressed in tobacco. Its overexpression resulted in increased tolerance against salinity, drought and oxidative stress as was evident by increased photosynthesis, water content, germination rate, biomass, chlorophyll content and reduced accumulation of ROS. Physiological parameters were also less affected as compared to wild type plants. The relationship between anatomical structure and drought tolerance have also been investigated. In the roots of sugarcane, number of vessels per unit area, more veins, widened vesicles in bulliform cells, thick cuticle and less stomata per unit area have close association with drought tolerance [73]. Drought tolerant varieties have better growth of mechanical tissues around the vascular bundle and their thick-walled cells have higher degree of lignification. Various genes that encode transcription factors bZIP, DREB, MYB [74] and RD26 have also been evaluated to enhance stress tolerance in plants. Transcription factors of MYB superfamily which are in abundant, also play crucial role in growth and development under stress conditions [75]. Expression profiling of sugarcane was performed under stress conditions to identify abiotic-stress-inducible genes. Wild type Q117 sugarcane plants were exposed to salinity, drought and cold stresses. Variations in the expression level of four genes encoding for galactinol synthase (GolS), late embryogenesis abundant protein 3 (LEA3), early response to dehydration protein 4 (ERD4) and pyrroline-5-carboxylase synthetase (P5CS) were evaluated by real-time PCR. P5CS and GolS were strongly induced under salt stress whereas, LEA3 and ERD4 were induced under cold and drought stress respectively. Overexpression of CBF4 (C-repeat binding factor 4) gene from Arabidopsis thaliana in Q117 led to a considerable increase in the expression of P5CS and ERD4 as compared with wild-type plants under normal conditions. Anyhow expression of LEA3 and GolS did not appear to be affected in transgenic plants. These results suggested presence of active abiotic stress-inducible genes in sugarcane and that expression of Arabidopsis CBF4 gene in sugarcane can activate stress tolerance genes under normal conditions [76].
Plants use complex mechanisms to adapt ionic/osmotic stresses and accumulate compatible solutes to cope with these stress conditions. Salt stress has drastic effects on photosynthetic activity which affects crop production, product quality and of course sucrose accumulation in cane stalk. These stresses disturb homeostasis at cellular level and even at plant level. It is very critical for the plant to maintain low level of toxic ions in the cell but under salt stress, Na + and Cl − ions accumulate in the cytoplasm due to their inability to pump them out. The level of Na + ion is regulated by specific transporters i.e. plasma membrane Na + /H + antiporter SOS1. When AVP1 (Arabidopsis Vacuolar Pyrophosphatase) gene was overexpressed in sugarcane by Kumar et al. [77], increased tolerance against drought and salinity was observed in the transgenic plants. Profused and longer roots were observed in transgenic plants as compared to control. Concurrently, survival of transgenic plants under drought and salt stress indicated their increased level of tolerance against drought and salinity. Constitutive expression of AVP1 gene improved plant growth under different abiotic stresses. Many explanations have been anticipated including better vacuolar ion sequestration, enhanced auxin transport, increased heterotrophic growth, and more sucrose transport from source to sink tissues. Mutant plants which lack functional AVP1 gene and transgenic plants for AVP1 were used to evaluate its role. It becomes clear that AVP1 is a protein with multiple functions. Systems Biology can be of great help for the complete understanding of these complex biological networks [78].

Developing genetically modified varieties with improved sugar recovery
Sugarcane has capacity to store more than 25% sucrose of its fresh weight, so a great potential is there to increase sugar recovery. Advancements in biotechnological tools has helped to understand metabolic pathways involved in sucrose accumulation in sugarcane. Enzymes and control points involved in sucrose metabolic pathway, photosynthetic efficiency, degree of phloem loading/unloading, rate of sucrose assimilation and carbon partitioning within the stem and vacuoles are the key targets which needs to be explored for increasing sucrose accumulation. Expression analysis of genes in relation to sucrose accumulation can be of great help to understand role of various genes involved in sucrose metabolism. Until now many studies have been reported on the genes involved in sucrose metabolism directly or indirectly. SPS (sucrose phosphate synthase), SPP (sucrose phosphate phosphatase), SuSy (sucrose synthase), HK (hexokinase), FK (fructokinase) VAI (vacuolar acid invertase), NAI (neutral acid invertase), CWI (cell wall invertase), SAI (soluble acid invertase), PFK (ATP dependent phosphofructokinase), PFP (pyrophosphate dependent phosphofructokinase), UDPase (UDP glucose pyrophosphorylase), ADP-G-PP-(ADP-Glucose pyrophosphorylase) and sucrose transporters (SUT1 and SUT4) are the key enzymes involved in sucrose metabolic pathway [79]. Engineering these enzymes through genetic transformation may lead to increased sucrose accumulation and of course sugar recovery (Figure 2).
Fructose 6-phosphate 1-phosphotransferase catalyzes the principal reaction of glycolysis i.e. the reversible conversion of pyrophosphate (PPi) and fructose 6-phosphate (Fru 6-P) into inorganic phosphate (Pi) and fructose 1,6-bisphosphate (Fru 1,6-P2). Pyrophosphate  dependent phosphofructokinase (PFP) is also partially responsible for being cycled between the hexose phosphate and triose phosphate pools. This cycling was downregulated by constitutive expression of untranslatable and antisense forms of PFP-b gene. Approximately 70% activity was decreased in young internodal tissues and no activity was observed in mature tissues. Hendrik and Botha [80] reported decrease in sugar yield as the result of decrease in the PFP activity. A significant increase in sucrose content (in more than 50% of the lines) was observed in the immature internodes, but even 30% downregulation of Pyrophosphate dependent phosphofructokinase (PFP) activity did not affect the mature internodes as compared to wild type. Mature internodes of most of the transgenic lines showed higher sucrose accumulation but was not significant. Hence Pyrophosphate dependent phosphofructokinase (PFP) activity in internodal tissues of sugarcane has a positive relation with respiration and is inversely related with sucrose content. In transgenic plants, no significant difference was observed in development and growth of plants both under greenhouse and field conditions. So PFP (pyrophosphate dependent phosphofructokinase) influences the sucrose accumulation ability of biosynthetically active and young culm of sugarcane. Equilibrium of glycolytic intermediates (stored sucrose) is restored when ATP dependent phosphofructokinase and the PFP activity is sufficient.
Sugarcane culm is an important experimental system to elucidate biochemical and molecular mechanisms involved in sucrose accumulation or carbon partitioning for the application of gene expression studies in this context [81]. Vacuolar targeted expression of sucrose isomerase gene doubled sucrose accumulation in the culm of greenhouse growing plants. Engineered sugarcane plants not only depicted enhanced sucrose transport but also photosynthesis and sink strengths were improved. These results highlighted importance of sugarcane as an energy crop as more carbon source would result in more biofuel production. Higher level of sucrose and accumulation of isomaltulose (a high value sugar) has also been reported in sugarcane [82]. An experimental study was conducted to explore biosynthesis of isomaltulose (IM) through engineering metabolic pathways. Sucrose (α-D-glucopyranosyl 1,2-D-fructofuranose) is converted into isomaltulose (α-D-glucopyranosyl-1,6-D-fructofuranose) by some bacteria. This sucrose is resistant to several microorganisms as is not metabolized by invertases. Easy digestion (likewise glucose and fructose) by humans is another significant advantage of this sweetener. Instead of salivary invertases, intestinal disaccharidase is involved in the digestion of isomaltulose, so its digestion is relatively slow. Anyhow, it is beneficial because it does not affect insulin and blood glucose levels. Owing to be acariogenic, non-hygroscopic, stable and slowly digestible sweetener, it has mounting market. Biosynthesis of isomaltulose (IM) involves a sucrose isomerase (SI) that does not require cofactor and substrate activation [83]. More isomaltulose (IM) is produced in sugarcane culmn when highly efficient sucrose isomerase (SI) is targeted to vacuole. Further, IM (isomaltulose) could be accumulated without any prominent decrease in sucrose content. Sucrose contents appeared to be doubled in selected transgenic lines but further studies would be required for commercial scale application of this trait i.e. patterns of developmental expression, compartmentation and enzyme stability resulting in high isomaltulose (IM) content. Hence, sucrose isomers can be produced in sugarcane by transgenic technology. Isomaltulose was produced either by expressing sucrose isomerase in the apoplast or in the vacuole. Apoplast-targeted expression did not show any significant increase in isomaltulose (IM) whereas, vacuole-targeted expression of transgene resulted in significant increase in the isomaltulose (IM) accumulation under greenhouse conditions [91]. Hamerli and Birch [84] reported the first field trial of transgenic sugarcane producing trehalulose (TH). Synthetic sweeteners, an alternative to sucrose are produced through fermentation or chemical reactions which are very expensive. Production of sweeteners in sugarcane through targeted expression of transgene in the mature stem can be an economical alternative. For targeted delivery of proteins into the plastids and vacuoles, transit peptides have already been established in transformants. Zhang et al. [85] worked out not only to develop abiotic stress tolerant sugarcane but also on engineering metabolic pathways for improved trehalose (a valuable sugar moiety) content. Directing sucrose accumulation to vacuole in spite of cytosol may prove an effective strategy for enhanced sucrose accumulation because vacuole occupies large space in the cell. Hence, biotechnological interventions can do a lot to improve sugar recovery in this sweet grass.

Potential of biotechnology to promote sugarcane as a future energy crop
Plant biomass from grasses including sugarcane, can be used as a renewable source of energy by converting their cellulose, hemicellulose and lignin into bioethanol. Plant derived biofuels reduce dependence on fossil fuels and is of great importance in the countries where oil reserves are limited. Engineering plastid genome of sugarcane may prove a great milestone in this regard [86]. Biofuels produced from plant lignocellulosic biomass (second generation biomass) have advantage over first generation biomass in term of CO2 balance and net energy. Another advantage is that they have no competition for supplies with food industries. As a result, production of bioethanol from 2nd generation biomass is more economical. Sugarcane is one of the most economical source of bioethanol all over the world. Brazil is the leading country in this regard and 50% of the country energy needs are fulfilled by sugarcane ethanol [87]. National fuel alcohol program (ProAlcooL) was launched by Brazil. Major aim of this program was to replace usage of gasoline with bioethanol. In Brazil, 6.19 billion gallons (23.4 billion liters) of ethanol was produced from sugarcane whereas 15% of the total electricity was generated from sugarcane bagasse during 2014 [88]. The genetic foundation of current sugarcane breeding program started with interspecific hybrid varieties originated from early breeding activities in West Indies, India (e.g. Co 206, Co 207) and Indonesia (e.g. POJ 2878, POJ 2364). Sugar yield was increased up to 1-2% per annum by sugarcane programs and most of this increase is attributed to genetics. Conventional breeding in sugarcane has certain limits, as a result desired results could not be achieved. Saccharum spp. is genetically complex having 2n = 100-130 with intricate genomic makeup evolved through highly successful interspecific hybridization between Saccharum spontaneum and Saccharum officinarum, which have well been explored to develop commercial varieties. Ming et al. [89] summarized usage of conventional and molecular approaches for the genetic improvement of sugarcane making it world's most efficient crop in terms of conversion of solar energy into chemical energy.
In sugarcane, about two-third of the photosynthetically fixed carbon is stored in the form of cellulose and hemicellulose. Sugarcane mills produce millions of tons of bagasse annually in addition to the leaves which are left behind in the field. Sugarcane bagasse is an excellent 2nd generation source for production of ethanol and bioelectricity [90]. High cost of enzymes limit the conversion of hemicellulose and cellulose into cellulosic ethanol. Production cost of enzymes can be decreased by the overexpression of cellulolytic enzymes in GM (genetically modified) plants to meet the demand of sugarcane mills. Adoption of new technologies may help to overcome issues relevant to the stability, storage and overproduction of enzymes in plants. Sainz [91] reported that highly thermostable and hydrolytically efficient enzymes were produced by genetic engineering. Transgenic sugarcane plants overexpressing bacterial endoglucanase (EG) and fungal cellobiohydrolases (CBH I and CBH II) were developed. Targeting EG to chloroplasts and cellobiohydrolases to vacuoles resulted in elevated enzymatic activity in the mature plant leaves. This increased enzyme activity demonstrated that cellulose hydrolytic enzymes can be produced in sugarcane plants [92] and will boost up energy production from cane and its by-products including bagasse. In addition to the traditional agricultural products (food, feed and fiber), plants are emerging as a valuable source of energy, fuel, biomaterials and chemical precursors for the industry. Advancements in research are of pivotal importance to meet the increasing demand of quality raw materials [93]. Genetic engineering techniques are playing important role to achieve this goal and are envisioned to play leading role in the production and processing technology. For instance, input cost can be reduced by producing raw material in plants as plants have proved an effective platform for the production of industrially important compounds. GM microbes are commonly used at industrial scale for rapid conversion of raw materials into desired product. Conversely, a few GM crops have gained commercial status in spite of wide spread eagerness and renowned potential of genetic engineering for crop improvement [94]. Biotechnology occupies a central role in US Department of Energy (DOE) to develop crops with modified cell wall composition. The DOE has received encouraging appreciation because of their research on bioenergy crops and production of valuable processing enzymes by engineering metabolic and biochemical pathways [95]. To successfully attain national goal of bioeconomy, genetic engineering is appearing as a major contributor. A wide range of plants like corn, poplar, switchgrass, canola, sorghum, soybean (Saccharum L.) had been used to produce bioenergy but sugarcane is far better choice as is a perennial crop which does not require reseeding after each growing cycle. Hence, sugarcane is the most valuable crop for the production of bioethanol which can further be improved by employing biotechnological innovations.

Conclusions
Conventional research has contributed to great extent and has delivered its maximum. So, the only hope to get improved agricultural crops is the implication of advanced research. Adoption of biotechnological interventions have proved their worth and more than 18 million farmers in 26 countries are growing GM crops on an area of 185.1 million hectares (457.4 million acres) which is increasing each year. More than 90% of these crops are either insect resistant or herbicide tolerant resulting a massive decrease in the usage of chemical pesticides by 37%, increased crop yields by 22%, and increased farmer's profits by 68%. Though, biotechnological interventions have produced agronomically improved genotypes yet scientists are currently working to engineer sugarcane crop as a platform for large scale production of chemicals with industrial as well as therapeutic significance. Hence, Biotechnological interventions hold great promises to develop a better sugarcane crop with improved agronomic traits, sugar contents and biofuel production.