ESTs (Expressed sequence tags) and number of nucleotide sequences corresponding to
Abstract
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, transcriptomics, 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.
Keywords
- sugarcane
- omics
- transgenics
- crop improvement
- biofuel
1. 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]
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
2. 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
2.1. 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.
2.2. 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
2.3. Micropropagation to produce sanitated plants
Systemic buildup of infections particularly diseases (viral, bacterial and fungal) affect plant vigor and health. Unavailability of an efficient
2.4. Germplasm conservation
Another important application of
3. 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.
3.1. 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
3.2. 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].
3.3. 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 “
No. of ESTs | Nucleotide sequences | |
---|---|---|
20,701 | 7066 | |
341 | 234 | |
284,482 | 2267 | |
Mixed cultivar of |
73,778 | 10 |
SP80-3280 cultivar of |
135,534 | 54 |
CoS 767 cultivar of |
25,382 | – |
Q117 cultivar of |
9141 | 54 |
SP70-1143 cultivar of |
24,313 | 8 |
Co 1148 cultivar of |
1069 | 2 |
CP72-2086 cultivar of |
7993 | 4 |
Co 740 cultivar of |
310 | 25 |
CoC 671 cultivar of |
315 | 67 |
NCo376 cultivar of |
535 | 11 |
H50-7209 cultivar of |
27 | 3 |
F134 cultivar of |
4 | – |
Co 62175 cultivar of |
206 | 1 |
Co 86032 cultivar of |
30 | 101 |
Unknown cultivar of |
3904 | 339 |
285,216 | 13,382 |
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
3.4. 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].
3.5. 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].
4. 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
4.1. Developing genetically modified varieties with improved biotic stress tolerance
4.1.1. Herbicide resistance
Herbicide resistance is one of the major traits in transgenic plants. It is so desirable that more than 70% of the transgenic crops growing worldwide are herbicide resistant. Various herbicide resistance genes i.e.
4.1.2. 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. Annual loss of $10–$20 million were estimated to sugar industry at Lower Rio Grande Valley of Texas only by
Considerable advancements have been made to develop transgenic sugarcane having resistance against lepidopteran borers such as
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
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 (
4.2. 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
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
4.3. 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
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.
5. 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.
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 (
6. 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.
References
- 1.
Wheals AE, Basso LC, Alves DM, Amorim HV. Fuel ethanol after 25 years. Trends in Biotechnology. 1999; 17 :482-487 - 2.
Sundar AR, Ashwin NMR, Barnabas EL, Malathi P, Viswananthan R. Disease resistance in sugarcane – An overview. Scientia Agraria Paranaensis. 2015; 14 :200-212 - 3.
Khan MS, Ali S, Iqbal J. Developmental and photosynthetic regulation of Bacillus thuringiensis δ-endotoxin reveals that engineered sugarcane conferring resistance to ‘dead heart’ contains no toxins in cane juice. Molecular Biology Reports. 2011;38 :2359-2369 - 4.
Mustafa G. Development of plastid transformation in sugarcane [PhD Diss.]. Pakistan: Quaid-I-Azam Univ.; 2011 - 5.
Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B, Selvaraj N, Vasudevan A, Kasthurirengan S. Agrobacterium -mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Reports. 2004;23 :134-143 - 6.
Arencibia AD, Carmona ER, Cornide MT, Castiglione S, O’Relly J, Chinea A, Oramas P, Sala F. Somaclonal variation in insect-resistant transgenic sugarcane ( Saccharum hybrid) plants produced by cell electroporation. Transgenic Research. 1999;8 :349-360 - 7.
Leibbrandt NB, Snyman SJ. Stability of gene expression and agronomic performance of a transgenic herbicide-resistant sugarcane line in South Africa. Crop Science. 2003; 43 :671-677 - 8.
Gilbert RA, Gallo-Meagher M, Comstock JC, Miler JD, Jain M, Abouzid A. Agronomic evaluation of sugarcane lines transformed for resistance to sugarcane mosaic virus strain E. Crop Science. 2005; 45 :2060-2067 - 9.
Vickers JE, Grof CPL, Bonnett GD, Jackson PA, Morgan TE. Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs a performance of sugarcane clones in the field. Australian Journal of Agricultural Research. 2005; 56 :57-68 - 10.
Butterfield MK, Barnes JM, Heinze BS, Rutherford RS, Huckett BI. RFLP markers for resistance to eldana and smut from an unstructured sugarcane population. In: Proceeding International Society of Sugarcane Technology Molecular Biology. Workshop, Montpellier, France. Vol. 4; 2003. p. 14 - 11.
Nickell LG. Tissue and cell culture of sugarcane: Another research tool. Hawaii Plant Records. 1964; 57 :223-229 - 12.
Heinz DJ, Mee G. Plant differentiation from callus tissue of Saccharum species. Crop Science. 1969;9 :346-348 - 13.
Komamine A, Kawahara R, Matsumoto M, Sunabori S, Toya T, Fujiwara A, Tsukahara M, Smith J, Ito M, Fukuda H, Nomura K. Mechanisms of somatic embryogenesis in cell cultures: Physiology, biochemistry, and molecular biology. In Vitro Cellular & Developmental Biology-Plant. 1992; 28 :11-14 - 14.
Lakshmanan P, Geijskes RJ, Wang L, Elliott A, Grof CP, Berding N, Smith GR. Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane ( Saccharum spp. interspecific hybrids) leaf culture. Plant Cell Reports. 2006;25 :1007-1015 - 15.
Koch AC, Ramgareeb S, Snyman SJ, Watt MP, Rutherford RS. An in vitro induced mutagenesis protocol for the production of sugarcane tolerant to imidazolinone herbicides. Proceedings of the International Society for Sugar Cane Technologists. 2010;27 :1-5 - 16.
Sengar AS, Thind KS, Kumar B, Pallavi M, Gosal SS. In vitro selection at cellular level for red rot resistance in sugarcane (Saccharum sp .). Plant Growth Regulation. 2009;58 :201-209 - 17.
Suprasanna P, Rupali C, Desai NS, Bapat VA. Partial desiccation augments plant regeneration from irradiated embryogenic cultures of sugarcane. Plant Cell, Tissue and Organ Culture. 2008; 92 :101-105 - 18.
Irvine JE, Benda GTA, Legendre BL, Machado GR. The frequency of marker changes in sugarcane plants regenerated from callus culture. II Evidence for vegetative and genetic transmission, epigenic effects and chimeral disruption. Plant Cell, Tissue and Organ Culture. 1991; 26 :115-125 - 19.
Chowdhury MKU, Vasil I. Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars ( Saccharum spp.). Theoretical and Applied Genetics. 1993;86 :181-188 - 20.
Taylor PWJ, Geijskes JR, Ko HL, Fraser TA, Henry RJ, Birch RG. Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture. TAG Theoretical and Applied Genetics. 1995; 90 :1169-1173 - 21.
Kaur R, Kapoor M. Plant regeneration through somatic embryogenesis in sugarcane. Sugar Tech. 2016; 18 :93-99 - 22.
Jalaja NC, Neelamathi D, Sreenivasan TV. Micropropagation for Quality Seed Production in Sugarcane in Asia and the Pacific. Food and Agriculture Organization of the United Nations. India; 2008 - 23.
Bakker H. Sugar Cane Cultivation and Management. New York: Kluwer Academic/Plenum Publishers; 1999 - 24.
Geijskes RJ, Wang LF, Lakshmanan P, McKeon MG, Berding N, Swain RS, Elliott AR, Grof CPL, Jackson J, Smith GR. Smartsette seedlings: Tissue culture seed plants for the Australian sugar industry. Proceedings of the Australian Society of Sugarcane Technologists, PK Editorial Services. 2003; 25 :6-6 - 25.
Ali S, Iqbal J, Khan MS. Genotype independent in vitro regeneration system in elite varieties of sugarcane. Pakistan Journal of Botany. 2010; 42 :3783-3790 - 26.
Mustafa G, Khan MS. Differential role of indolebutyric acid in sugarcane root development. Sugar Tech. 2016; 18 :55-60 - 27.
Kristini A. The use of tissue culture to eliminate some important diseases in sugarcane [M.Sc. thesis]. Australia: University of Queensland; 2004 - 28.
Wagih ME, Gordon GH, Ryan CC, Adkins SW. Development of an axillary bud culture technique for Fiji disease virus elimination in sugarcane. Australian Journal of Botany. 1995; 43 :135-143 - 29.
Wang QC, Valkonen JPT. Elimination of viruses and phytoplasma by cryotherapy of in vitro-grown shoot tips: Analysis of all cases. Advances in Horticultural Science. 2007; 21 :265-269 - 30.
Bajaj YP. Cryopreservation of plant cell, tissue, and organ culture for the conservation of germplasm and biodiversity. In: Cryopreservation of Plant Germplasm I. Springer Berlin Heidelberg. Germany; 1995. p. 3-28 - 31.
Martins ERF, Dahmer N, Sforça DA, Silva CBC, Bundock P, Henry RJ, Souza GM, van Sluys MA, Landell MGA, Carneiro MS, Vincentz MAG, Pinto LR, Vencovsky R, Souza AP. SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids. Scientific Reports. 2013; 3 :3399 - 32.
Scherwinski-Pereira JE, Costa FHS. Conservação in vitro de recursos genéticos de plantas: estratégias, princípios e aplicações. In: Barrueto Cid LP, editor. Cultivo in vitro de plantas. Brasília: Embrapa Informação Tecnológica; 2010. p. 177-234 - 33.
Singh SR, Dalal S, Singh R, Dhawan AK, Kalia RK. Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers. In Vitro Cellular and Developmental Biology-Plant. 2013;49 :572-583 - 34.
Park JW, Solis-Gracia N, Trevino C, da Silva JA. Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex. Molecular Breeding. 2012;30 :987-999 - 35.
Suganya A, Nithiyanantham R, Premachandran MN, Balasundaram N, Govindaraj P, Selvi A, Nair NV. Cytological studies in hybrids between a sugarcane cultivar and three cytotypes of wild Saccharum spontaneum L. Journal of Sugarcane Research. 2012;2 :34-40 - 36.
Smulders MJM, de Klerk GJ. Epigenetics in plant tissue culture. Plant Growth Regulation. 2011; 63 :137-146 - 37.
Vujović T, Cerović R, Ružić D. Ploidy level stability of adventitious shoots of sour cherry ‘Cacanski Rubin’ and Gisela 5 cherry rootstock. Plant Cell, Tissue and Organ Culture. 2012; 111 :323-333 - 38.
Van Emon JM. The omics revolution in agricultural research. Journal of Agricultural and Food Chemistry. 2015; 64 :36-44 - 39.
Paterson AH, Bowers JE, Bruggmann R, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M. The Sorghum bicolor genome and the diversification of grasses. Nature. 2009;457 :551-548 - 40.
Manetti ME, Rossi M, Cruz GMQ, Saccaro NL, Nakabashi M, Altebarmakian V, Rodier-Goud M, Domingues D, D’Hont A, Van Sluys MA. Mutator system derivatives isolated from sugarcane genome sequence. Tropical Plant Biology. 2012; 5 :233-243 - 41.
Feschotte C. Transposable elements and the evolution of regulatory networks. Nature Review Genetics. 2008; 9 :397-405 - 42.
Shirasu K, Schulman AH, Lahaye T, Schulze-Lefert P. A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Research. 2000; 10 :908-915 - 43.
Tomkins JP, Yu Y, Miller-Smith H, Frisch DA, Woo SS, Wing RA. A bacterial artificial chromosome library for sugarcane. TAG Theoretical and Applied Genetics. 1999; 99 :419-424 - 44.
Casu R, Hotta CT, Souza GM, Henry RJ, Kole C. Functional genomics: Transcriptomics of sugarcane-current status and future prospects. In: Genetics, Genomics and Breeding of Sugarcane. Science Publishers. Brazil; 2010. p. 167-191 - 45.
Souza GM, Berges H, Bocs S, Casu R, D’Hont A, Ferreira JE, Henry R, Ming R, Potier B, Van Sluys MA, Vincentz M. The sugarcane genome challenge: Strategies for sequencing a highly complex genome. Tropical Plant Biology. 2011; 4 :145-156 - 46.
Casu RO, Dimmock CH, Thomas ME, Bower NE, Knight DE, Grof CH, McIntyre L, Jackson P, Jordan D, Whan V, Drenth J. Genetic and expression profiling in sugarcane. Proceedings of the International Society of Sugarcane Technologists. 2001; 24 :626-627 - 47.
Grivet L, Arruda P. Sugarcane genomics: Depicting the complex genome of an important tropical crop. Current Opinion in Plant Biology. 2002; 5 :122-127 - 48.
Simpson AJG, Perez JF. ONSA, the Sao Paulo virtual genomics institute. Nature Biotechnology. 1998; 16 :795-796 - 49.
Barnabas L, Ramadass A, Amalraj RS, Palaniyandi M, Rasappa V. Sugarcane proteomics: An update on current status, challenges, and future prospects. Proteomics. 2015; 15 :1658-1670 - 50.
Heinz DJ. Isozyme prints for variety identification. Sugarcane Breed Newsletter. 1969; 24 :8 - 51.
O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry. 1975; 250 :4007-4021 - 52.
Pacheco CM, Pestana-Calsa MC, Gozzo FC, Nogueira RJMC, Menossi MTC. Differentially delayed root proteome responses to salt stress in sugar cane varieties. Journal of Proteome Research. 2013; 12 :5681-5695 - 53.
Zhou G, Yang LT, Li YR, Zou CL, Huang LP, Qiu LH, Huang X, Srivastava MK. Proteomic analysis of osmotic stress-responsive proteins in sugarcane leaves. Plant Molecular Biology Reporter. 2012; 30 :349-359 - 54.
Ramagopal S, Carr JB. Sugarcane proteins and messenger RNAs regulated by salt in suspension cells. Plant Cell Environment. 1991; 14 :47-56 - 55.
Que Y, Su Y, Guo J, Wu Q, Xu L. A global view of transcriptome dynamics during Sporisorium scitamineum challenge in sugarcane by RNA-seq. PLoS One. 2014;9 :e0106476 - 56.
Aliferis K, Chrysayi-Tokousbalides M. Metabolomics in pesticide research and development: Review and future perspectives. Metabolomics. 2011; 7 :35-53 - 57.
Bosch S, Rohwer JM, Botha FC. The sugarcane metabolome. Proceedings of the South African Sugar Technologists Association. 2003; 7 :129-133 - 58.
Basnayake SW, Morgan TC, Wu L, Birch RG. Field performance of transgenic sugarcane expressing isomaltulose synthase. Plant Biotechnology Journal. 2012; 10 :217-225 - 59.
Joyce P, Hermann S, O’Connell A, Dinh Q, Shumbe L, Lakshmanan P. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods. Plant Biotechnology Journal. 2014;12 :411-424 - 60.
Gallo-Meagher M, Irvine JM. Herbicide resistant transgenic sugarcane plants containing the bar gene. Crop Science. 1996; 36 :1367-1374 - 61.
Enriquez-Obregon GA, Vazquez-Padron RI, Pieto-Samsonov DL, De la Riva GA, Selman-Housein G. Herbicide-resistant sugarcane ( Saccharum officinarum L.) plants byAgrobacterium -mediated transformation. Planta. 1998;206 :20-27 - 62.
Falco MC, Neto AT, Ulian EC. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell Reports. 2000; 19 :1188-1194 - 63.
Mukunthan N, Srikanth J, Singaravelu B, Asokan S, Kurup NK, Goud YS. Assessment of woolly aphid impact on growth, yield and quality parameters of sugarcane. Sugar Tech. 2008; 10 :143-149 - 64.
Arencibia A, Vázquez RI, Prieto D, Téllez P, Carmona ER, Coego A, Hernández L, Gustavo A, Selman-Housein G. Transgenic sugarcane plants resistant to stem borer attack. Molecular Breeding. 1997; 3 :247-255 - 65.
Weng LX, Deng HH, Xu JL, Li Q, Zhang YQ, Jiang ZD, Li QW, Chen JW, Zhang LH.Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials. Transgenic Research. 2011; 20 :759-772 - 66.
Falco MC, Silva-Filho MC. Expression of soybean proteinase inhibitors in transgenic sugarcane plants: Effects on natural defense against Diatraea saccharalis . Plant Physiology and Biochemistry. 2003;41 :761-766 - 67.
Allsopp PG, McGhie TK. Snowdrop lectin and wheatgerm lectins as antimetabolites for the control of sugarcane whitegrubs. Entomologia Experimentalis et Applicata. 1996; 80 :409-414 - 68.
Sétamou M, Bernal JS, Legaspi JC, Mirkov TE, Legaspi BC. Evaluation of lectin-expressing transgenic sugarcane against stalkborers ( Lepidoptera: Pyralidae ): Effects on life history parameters. Journal of Economic Entomology. 2002;95 :469-477 - 69.
Zhangsun DT, Luo SL, Chen RK, Tang KX. Improved Agrobacterium -mediated genetic transformation of GNA transgenic sugarcane. Biologia. 2007;62 :386-393 - 70.
Zhang LH, Weng LX, Jiang ZD. Sugarcane. In: Pua EC, Davey MR, editors. Biotechnology in Agriculture and Forestry Transgenic Crops. Springer. Germany; 2007 - 71.
Somerville C, Youngs H, Taylor C, Davis SC, Long SP. Feedstocks for lignocellulosic biofuels. Science. 2010; 329 :790-792 - 72.
Aydin S, Buyuk İ, Aras ES. Expression of SOD gene and evaluating its role in stress tolerance in NaCl and PEG stressed Lycopersicum esculentum . Turkish Journal of Botany. 2014;38 :89-98 - 73.
Malik KB. Some anatomical characteristics of sugarcane varieties in relation to drough resistance. Agriculture Research. 1986; 11 :43-49 - 74.
Ahuja I, de Vos RC, Bones AM, Hall RD. Plant molecular stress responses face climate change. Trends in Plant Sciences. 2010; 15 :664-674 - 75.
Zhang L, Guangyao Z, Jizeng J, Xu L, Kong X. Molecular characterization of 60 isolated wheat MYB genes and analysis of their expression during abiotic stress. Journal of Experimental Biology. 2012; 63 :203-214 - 76.
McQualter RB, Dookun-Saumtally A. Expression profiling of abiotic-stress inducible genes in sugarcane. In: XXVI Congress, International Society of Sugar Cane Technologists; 2007. pp. 878-888 - 77.
Kumar T, Khan MR, Abbas Z, Ali GM. Genetic improvement of sugarcane for drought and salinity stress tolerance using Arabidopsis vacuolar pyrophosphatase (AVP1) gene. Molecular Biotechnology. 2014; 56 :199-209 - 78.
Schilling RK, Tester M, Marschner P, Plett DC, Roy SJ. AVP1: One protein, many roles. Trends in Plant Science. 2017; 22 :154-162 - 79.
Chandra A, Verma PK, Islam MN, Grisham MP, Jain R, Sharma A, Roopendra K, Singh K, Singh P, Verma I, Solomon S. Expression analysis of genes associated with sucrose accumulation in sugarcane ( Saccharum spp. hybrids) varieties differing in content and time of peak sucrose storage. Plant Biology. 2015;17 :608-617 - 80.
Hendrik JG, Botha FC. Down-regulation of pyrophosphate: Fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes. Transgenic Research. 2008; 17 :85-92 - 81.
Moore PH. Temporal and spatial regulation of sucrose accumulation in the sugarcane stem. Functional Plant Biology. 1995; 22 :661-679 - 82.
Wu L, Birch RG. Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotechnology Journal. 2007; 5 :109-117 - 83.
Wu L, Birch RG. Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene. Applied and Environmental Microbiology. 2005;71 :1581-1590 - 84.
Hamerli D, Birch RG. Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane. Plant Biotechnology Journal. 2011 Jan 1; 9 (1):32-37 - 85.
Zhang SZ, Yang BP, Feng CL, Chen RK, Luo JP, Cai WW, Liu FH. Expression of the Grifola frondosa trehalose synthase gene and improvement of drought-tolerance in sugarcane (Saccharum officinarum L.). Journal of Integrative Plant Biology. 2006;48 :453-459 - 86.
Mustafa G, Sarwar Khan M. Prospecting the utility of antibiotics as lethal selection agents for chloroplast transformation in sugarcane. International Journal of Agriculture & Biology. 2012; 14 :307-310 - 87.
Botha FC. Energy yield and cost in a sugarcane biomass system. Proceedings of the Australian Society of Sugar Cane Technologists. 2009; 31 :1-10 - 88.
Matsuoka S, Ferro J, Arruda P. The Brazilian experience of sugarcane ethanol industry. In Vitro Cellular and Developmental Biology-Plant. 2009; 45 :372-381 - 89.
Ming R, Moore PH, Wu KK, D’Hont A, Tew TL, Mirkov TE, da Silva J, Schnell RJ, Brumbley SM, Lakshmanan P, Comstock JC, Glaszmann JC, Paterson AH. Sugarcane improvement through breeding and biotechnology. Plant Breeding Reviews. 2006; 27 :17-118 - 90.
Arruda P. Perspective of the sugarcane industry in Brazil. Tropical Plant Biology. 2011; 4 :3-8 - 91.
Sainz MB. Commercial cellulosic ethanol: The role of plant expressed enzymes. In Vitro Cellular and Developmental Biology-Plant. 2009; 45 :314-329 - 92.
Harrison MD, Geijskes J, Coleman HD, Shand K, Kinkema M, Palupe A, Hassall R, Sainz M,Lloyd R, Miles S, Dale JL. Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane. Plant Biotechnology Journal. 2011; 9 :1-13 - 93.
Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJJ, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T. The path forward for biofuels and biomaterials. Science. 2006; 311 :484-489 - 94.
Jaffe G. Withering on the Vine: Will Agricultural Biotech’s Promises Bear Fruit? Washington, DC: Center for Science in the Public Interest; 2005 - 95.
Anonymous. US DOE. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda. DOE/SC-0095. Washington, DC: Office of Science and Office of Energy Efficiency and Renewable Energy, U.S. Department of Energy; 2006