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Genetically modified sugarcane for bioenergy generation
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Genetically modified sugarcane for bioenergy generation Paulo Arruda1,2 Sugarcane breeding has significantly progressed over the past 30 years, but attempts to further increase crop yield have been limited due to the complexity of the sugarcane genome. An alternative to boost the crop yield is the introduction of genes encoding desirable traits in the elite sugarcane cultivars. Genetically modified sugarcane with increased yield and pest and disease resistance has already proven its value not only by the increased sugar content but also for the improvement of the crop performance. However, transgene stability is still a challenge since transgene silencing seems to occur in a large proportion of genetically modified sugarcane plants. In addition, regulatory issues associated with the crop propagation model will also be a challenge to the commercial approval of genetically modified sugarcane. Addresses 1 Centro de Biologia Molecular e Engenharia Gene´tica, Universidade Estadual de Campinas (UNICAMP), 13083-875 Campinas, SP, Brazil 2 Departamento de Gene´tica e Evoluc¸a˜o, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 13083-862 Campinas, SP, Brazil Corresponding author: Arruda, Paulo ([email protected])
Current Opinion in Biotechnology 2012, 23:315–322 This review comes from a themed issue on Energy biotechnology Edited by James C Liao and Joachim Messing Available online 15th November 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.10.012
Introduction Sugarcane, along with other major seed and foraging crops, belongs to the grass family Poaceae, a common ancestor of which gave rise to triticeae, rice, and the members of the Andropogoneae tribe about 80 million years ago (MYA) [1]. Rice diverged from the Andropogoneae approximately 60 MYA, while maize diverged from sorghum 11 MYA and sugarcane diverged from sorghum 8–9 MYA [2,3]. All of the sugarcane cultivars currently grown around the world resulted from crosses between Saccharum officinarum, a domesticated sugarproducing species with a basic chromosome number of 10 and chromosome constitution of 2n = 80, and Saccharum spontaneum, a wild species with a basic chromosome number of 8 and a chromosome constitution of 2n = 40–128 (Figure 1) [4,5]. The interspecific hybrid of these two species was backcrossed to S. officinarum www.sciencedirect.com
few times to generate a sugarcane germplasm that incorporates the high sugar content trait of S. officinarum in addition to disease and stress resistance traits of S. spontaneum [6]. However, as a result of a chromosomal stress produced by the interspecific cross, the genomic constitution of the hybrid became even more complex than its parent species, with a chromosome constitution varying between 2n = 100–130. The hybrid preserves intact 15– 25% of the chromosomes inherited from S. spontaneum and 60–70% inherited from S. officinarum. Around 5–10% of recombinants between the homeologous chromosomes of both species are also formed in the hybrid [4]. The sugarcane breeding process involves crossing superior varieties and selection among the F1 progenies individuals with favorable allelic combinations. But the chromosomal architecture of the sugarcane hybrid makes each cross a unique, unpredictable event due to the random sorting of chromosomes from both species and the formation of recombinants that affect the distribution of favorable and unfavorable alleles [7]. This architecture, combined with the multiplicity of alleles at each locus [8,9], makes the breeding process immensely complicated. In general, hundreds of thousands of F1 seedlings are screened for disease resistance, adaptability to distinct local environments, sucrose yield, agronomic manageability, and good milling characteristics [6]. The first round of screening for disease resistance is performed at nurseries, where the seedlings are sprayed with fungi or bacterial inoculums [6]. Resistant seedlings are then planted in distinct locations to evaluate the other desirable characteristics. This evaluation is performed for 3–5 cuttings (ratoons) to assure trait stability. After 7–10 years of evaluation, one or two clones, out of hundreds of thousands of the initial F1 seedlings, are released as commercial varieties [6]. With the recent interest in feedstock for second-generation cellulosic ethanol, the energy cane concept reemerged as an important topic of some breeding programs [6]. Instead of accumulating high levels of soluble sugar in the stalks, energy cane is bred to produce plants with high fiber content and high biomass. These traits can be obtained, for example, by backcrossing elite sugarcane varieties to S. spontaneum (Figure 1) [6]. Genetic modification of sugarcane could be a powerful tool to incorporate pest-resistant and disease-resistant traits into elite commercial sugarcane varieties and to increase their agronomic performance and sugar yield. Additionally, genetic modification could be used to increase the overall biomass of energy cane and to reduce Current Opinion in Biotechnology 2012, 23:315–322
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Figure 1
Hybrid Sugarcane
X
S. officinarum
Diesel
Juice
Sugar
Bagasse
1st Generation Ethanol
S. Spontaneum m Straw
Hybrid Energy Cane
Bioelectricity
Cellulosic Ethanol
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Schematic representation of sugarcane and energy cane production and their use in the bioenergy industry. Modern sugarcane cultivars derived from crosses between S. officinarum (a high sugar species) and S. spontaneum (a stress-resistant wild relative). The high sugar varieties were produced by backcrossing the interspecific hybrid to S. officinarum. Recently, high fiber, high biomass cultivars have been bred by backcrossing elite sugarcane varieties to S. spontaneum to produce a hybrid energy cane. The carbon fixed by the sugarcane plant is allocated as follows: 1/3 in the leaves, 1/3 in the juice and 1/3 in the bagasse. The straw primarily represents the leaves that are left in the crop field. The juice is used to produce sugar, ethanol, and feedstock for diesel production by the synthetic biology industry. The bagasse is currently used to produce bioelectricity, but bagasse and straw could also be used as feedstock for cellulosic ethanol. The energy cane enters a pipeline to produce cellulosic ethanol and bioelectricity.
the production cost of second-generation cellulosic ethanol by overexpressing in these plants the cellulolytic enzymes needed to convert cellulose into fermentable sugars. This review highlights the advances in the production of genetically modified sugarcane to incorporate traits associated with agronomic performance and bioenergy generation. Current status and limitations of producing genetically modified sugarcane are discussed.
Modern sugarcane varieties, energy cane and its processing Modern sugarcane varieties have been bred for increased sugar content in the stalk. Soluble sugars, mainly sucrose, makes up 1/3 of the photosynthetically fixed carbon and are stored in acidic vacuoles that occupy over 80% of parenchyma cells volume in the mature stalk [10]. These vacuoles are highly metabolically active, as they take up sucrose against a high osmotic gradient created by the accumulation of soluble sugars. The other 2/3 of the fixed carbon of sugarcane plants is stored as complex carbohydrates, mainly cellulose and hemicellulose, which compose the cell walls of leaves and stalks (Figure 1). Current Opinion in Biotechnology 2012, 23:315–322
The accumulation of high levels of sucrose in the stalk parenchyma cells involves complex regulatory mechanisms. Sucrose is synthesized in the leaf mesophyll cells and is then transported through the phloem to the developing culm. Part of this sucrose is used for cellulose synthesis and part is taken up by the stalk parenchyma cells and is stored in acidic vacuoles [11]. In mature sugarcane stalks, sucrose accumulates in these vacuoles at a concentration of 600–700 mM [12]. Accumulation of such high levels of sucrose involves adaptive mechanisms to protect the parenchyma cells from osmotic and water stress. Expression profile analysis suggests that the pathway involved in this process is distinct from the drought stress pathway commonly used by plants to protect from water restriction [13]. Manipulation of carbon partitioning between sugar and fiber is essential in the breeding programs, and it is a difficult task to overcome the negative correlation between sugar and fiber production [6]. In modern industrial plantations, the sugarcane stalks are harvested, while the leaves are left in the field to help restore the soil fertility. At sugarcane mills, the stalks are crushed, and the resulting juice is used for single step www.sciencedirect.com
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sucrose crystallization. The sugar remaining in the molasses is fermented to produce fuel ethanol (Figure 1) [14]. The bagasse powder that results from stalk crushing is used to produce bioelectricity, but it could also be a valuable feedstock source for cellulosic ethanol production [15]. The sugarcane juice can also be used as a feedstock for fuel production in the synthetic biology biotech industry (Figure 1) [14].
Genetic transformation and expression stability Tissue culture and plant regeneration, a prerequisite for genetic transformation, have been established in sugarcane for almost 40 years [16]. Embryogenic calli are produced from transverse sections, cut just above the apical meristem of leaf whorls, inoculated in Murashige and Skoog (MS) basal medium [17] supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D), casein hydrolysate, sugars, vitamins, and coconut water (Figure 2) [18,19]. Plates are incubated in the dark to induce nodular embryogenic calli that are subcultured every 2 weeks. The highly nodular embryogenic calli are the preferred tissue for genetic transformation using either particle bombardment or Agrobacterium-mediated transformation. Plant regeneration and rooting is induced by transferring the embryogenic calli to medium without 2,4D and incubating under a 16 h photoperiod regime
(b)
(c)
(d)
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Genetic modification of sugarcane through transformation of embryogenic calli. The base of the leaf whorls is sliced and plated in MS medium supplemented with sugar and phytohormones. (a) Nodular embryogenic calli formed after a few weeks of inoculation. (b) Transformed embryogenic tissues, as evidenced by the GFP marker. (c) Regeneration of plants in MS medium without hormones. (d) Selection of transformed plants in MS medium containing a selective agent, such as an herbicide or antibiotic. www.sciencedirect.com
Sugarcane transformation has been mostly done by particle bombardment of embryogenic calli [20]. When this technology was first established, the successful recovery of a high number of transformed events depended on the use of the few sugarcane genotypes that produced transformable embryogenic calli. Since then, tissue culture methods have been improved, allowing the production of transformable embryogenic calli from dozens of elite sugarcane varieties [18]. This is primarily due to better explant quality rather than to the culture medium modifications. Agrobacterium-mediated transformation of embryogenic calli was first achieved using embryogenic calli [21,22] following a protocol that includes a dissection step, an appropriate co-cultivation medium and a selection scheme that enhances the rate of Agrobacterium infection and transformation [21] (Figure 2). In the last few years, this protocol has been improved and routinely used by several groups [19,23–25]. Transformation efficiency has increased such that over 5 transformed plants per plate are successfully recovered when the selective gene is placed under the control of a strong constitutive promoter [19]. Transformation cassettes used for sugarcane transformation usually incorporate antibiotic resistance genes [26], herbicide resistance genes [27], or phosphomannose isomerase (PMI) [28]. PMI allows for the positive selection of transformed tissues growing in mannose-containing medium. Cassettes also incorporate reporter genes, such as glucuronidase (GUS) [29] or green fluorescent protein (GFP) [30]. In general, resistance and reporter genes are placed under the control of strong constitutive promoters, such as the promoter of the gene encoding the 35S coat protein of cauliflower mosaic virus (35SCaMV) [23] or the polyubiquitin (UBI) promoter from maize [31] or rice [29].
Figure 2
(a)
(Figure 2) [18]. Regenerated plants are then transferred to pots for acclimation and growth after which the stalks of the adult plant are used for plant propagation.
Genetically modified sugarcane produced by particle bombardment transformation has been shown to be highly unstable. Transformed plants usually present complex construct integration with high copy numbers of the inserted transgene; sometimes the transgene is inserted in tandem, while it can also be inserted throughout the plant genome. These modified plants usually show high activity in young transformed seedlings, but transgene expression is generally turned off in mature plants in the first, second, or third ratoons [32]. Expression instability has been attributed to gene silencing induced by high copy number integration. Because sugarcane possesses a high degree of ploidy, allele silencing has been suggested to be more frequent in this crop compared to diploid plants [32]. Silencing-mediated transgene instability seems to have a greater effect when the constructs incorporate promoters Current Opinion in Biotechnology 2012, 23:315–322
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Table 1 Genetically modified sugarcane with high sugar content and insect, virus and stress resistance. Trait
Promoter/target gene
Promoter/selective marker
Reference
Insect resistance Insect resistance Insect resistance Herbicide resistance Herbicide resistance Herbicide resistance SCYLV resistance SCYLV resistance SCYLV resistance SrMV resistance FDV resistance Drought resistance Drought resistance Increased sugar content Increased trehalulose Increased sugar content Increased sugar content Cellulase, endoglucanase
CaMV35S/CryIAb UBI/CryIAb UBI/CryIAc NA UBI/Bar CaMV 35S/Bar UBI/CP UBI/CP UBI/CP UBI/CP UBI/ORF1 AIPC/P5CS CaMV 35S/TS CaMV 35S: UBI/PFP UBI/TS Emu/SSFT UBI/SI UBI/CBHI, CBHII, EG
None CaMV 35S/Bar UBI/G418 CaMV35S/Bar UBI/Kam NA UBI/Kam UBI/Kam UBI/Kam UBI/Kam UBI/Kam UBI/Bar CaMV 35S/Kam UBI/Kam UBI/Kam Emu/Kam UBI/Kam UBI/Kam
[1] [4] [48] [15] [16] [35] [17] [18] [53] [25] [38] [39] [51] [21] [22] [24] [49] [23]
Abbreviations: SCYLV — sugarcane yellow leaf virus; SrMV — Sorgum mosaic virus; FDV — Fiji disease virus; CaMV35S — 35S cauliflower mosaic virus promoter; CryIAb — Bt endotoxin; Bar — phosphinotricin acetyl transferase; UBI — maize polyubiquitin promoter; CP — Cpat protein; ORF1 — open reading frame 1; AIPC — ABA-inducible promoter complex; P5CS — pyrroline-5-carboxylate synthetase; TS — trehalulose synthase; SSFT — sucrose–sucrose fructosil transferase; SI — sucrose isomerase; CBHI, CBHII — cellobiohydrolase I and II; EG — endoglucanase; Kam — kanamycin; NA — not applicable.
from sugarcane genes. To test if gene silencing is associated with sugarcane promoters coming out of non-functional alleles, the promoters of eight alleles of a unique gene were fused to the GUS reporter gene and introduced into transgenic plants by particle bombardment [33]. The transgenes were highly active in young seedlings, but none of these transgenes sustained expression in mature stalks, indicating highly efficient transgene silencing from these promoters. Gene silencing could also be caused by the particle bombardment transformation technique itself. Recently, genetically modified sugarcane expressing the Cry1Ab toxin gene from Bacillus thuringiensis driven by the maize UBI promoter was produced using either particle bombardment or Agrobacterium-mediated transformation [23]. Plants produced with Agrobacterium-mediated transformation showed higher frequency of single-site integration, increased stability of transgenes and higher expression level of the toxin, compared to plants produced by particle bombardment, which contained multiple integrations. Transgene instability in sugarcane may be higher than in other diploid crops, although this instability is enhanced by particle bombardment transformation. Further work is needed to explore different target gene/promoter combinations to fully answer the question of transgene instability in sugarcane.
Modified sugarcane for enhanced sugar accumulation Sugarcane stores fixed carbon as soluble sugars, mostly sucrose, in vacuoles of parenchyma cells of mature stalks Current Opinion in Biotechnology 2012, 23:315–322
[10]. However, transforming photosynthetically fixed carbon into stored sucrose is a complex process that involves osmotic regulation, sugar sensors, sucrose cleavage and feedback regulation of photosynthesis [11]. The acidic vacuoles occupy approximately 80% of the total cell volume and accumulate sucrose up to a concentration of 600–700 mM [12]. The limiting step in increasing the sugar yield in sugarcane is the high osmotic gradient between the vacuoles and symplast within the stalk parenchyma cells [11]. Attempts to overcome this osmotic limitation include the downregulation of the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) through constitutive overexpression of antisense RNA to the PFP-b gene (Table 1) [34]. These genetically modified plants present reduced PFP levels in young internodes and almost undetectable levels of PFP in mature internodes. However, while these plants presented a significant increase in the sucrose concentration of the immature internodes, the amount of sucrose in the mature internodes was unaffected. A detailed analysis of the PFP-modified plants showed alterations in sugar metabolites in immature internodes, with an eightfold increase in the hexose-phosphate:triose-phosphate ratio, a restriction of the triose phosphate-to-hexose phosphate cycle and an increase in sucrose synthesis [35]. However, in mature internodes, these effects were not significantly different between the modified plants and the controls. On the other hand, these modified plants presented significantly higher fiber content, indicating that the osmotically imposed limit resulting from sucrose over accumulation diverted the carbon flux from soluble sugars to fiber. www.sciencedirect.com
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An alternative approach in which the limiting step imposed by osmotic constraints was bypassed involved the production of genetically modified sugarcane that overexpressed in the vacuoles a bacterial sucrose isomerase (SI), an enzyme that converts sucrose into the isomer isomaltulose (IM) [36] (Table 1). These plants almost doubled the amount of sugar in their juice accumulating up to 500 mM of isomaltulose without affecting the accumulation of sucrose or the carbon partitioning between sugar and fiber. The accumulation of sugars in these genetically modified plants could result from the conversion of monosaccharides into disaccharides via the overexpressed SI in the vacuole to alleviate the osmotic stress. A cell line derived from these plants accumulates twofold more sucrose than control cell lines derived from the same sugarcane variety [37]. This increase is not accompanied by parallel accumulation of IM. The sugar metabolism of these cells showed reduced extracellular invertase, depletion of extracellular sucrose and sucrose synthase activity, and higher symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) [37]. The enzymatic activity of both sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) is faster in these modified cell lines as well. The authors postulate that a signaling mechanism involving T6P enhances sucrose synthesis in sugarcane [37]. Overexpression of a sucrose–sucrose fructosil transferase (SSFT) in sugarcane has also been used to boost sugar accumulation in the parenchyma cells vacuoles of mature stalk [38] (Table 1). SSFT uses sucrose as a substrate to produce linear beta-(2,6)-linked fructans, a polysaccharide that is not naturally produced in sugarcane. These modified plants accumulate twice the amount of sugars in mature stalks as compared to control plants. The overexpression of this enzyme in vacuoles establishes a sugar sink to a polymer that is less osmotically active, which therefore may alleviate the osmotic constraints of the vacuole. Not all genetically modified sugarcane lines transformed to produce alternative sugars in their vacuoles increase sugar overproduction. Sugarcane plants that overexpress a vacuole-targeted bacterial trehalulose synthase showed accumulation of up to 600 mM trehalulose in mature stalks, but almost all of the sucrose in the vacuoles was converted into the sucrose isomer [39]. Thus, it seems that alteration of sugar metabolism through the manipulation of key sucrose biosynthesis enzymes does not necessarily overcome the osmotic constraints of the mature vacuole. On the other hand, the overexpression of enzymes that produce disaccharides or sugar polymers can bypass the osmotic limit and lead to increased accumulation of soluble sugars in the mature sugarcane stalk. www.sciencedirect.com
Modified sugarcane for second-generation ethanol production Two-thirds of the photosynthetically fixed carbon in sugarcane is stored in the form of cellulose and hemicellulose (Figure 1). Sugarcane leaves are currently left in the field after harvesting, but millions of tons of the bagasse power are produced annually in the sugarcane mills [6]. Although this sugarcane bagasse is currently used for bioelectricity generation [14], it is also an excellent feedstock for second-generation ethanol production [40,41]. However, cellulosic ethanol is economically limited by the high cost of the enzymes necessary to convert cellulose and hemicellulose into fermentable sugars [42]. The overexpression of cellulolytic enzymes in genetically modified plants could help to reduce the cost of enzyme production. Additionally, delivery of the enzyme to sugarcane mills would be an easy task because the plant feedstock would already contain the enzyme. Technologies for expressing and storing enzymes in plants have significantly progressed over the last few years, as overproduction, storage, and stability issues could be addressed by targeting the foreign enzymes to specific cellular organelles [43]. Engineered enzymes with increased thermostability and improved hydrolytic efficiency has also been developed [42]. Fungal cellobiohydrolases (CBH I and CBH II) and bacterial endoglucanase (EG) have been overexpressed in genetically modified sugarcane leaves. Targeting CBHI and CBHII to vacuoles and EG to chloroplasts resulted in high enzymatic activity in mature leaves, demonstrating the feasibility of expressing cellulose hydrolytic enzymes in sugarcane plants [43]. This approach could be of considerable interest to sugar mills that have the technology to process sugarcane and energy cane. Cellulolytic enzymes could be produced throughout the entire energy cane plant and used for processing both its own biomass and the bagasse from sugarcane crushing.
Modified sugarcane for biotic and abiotic stress resistance One of the first genetically modified sugarcane plants was produced to constitutively overexpress the B. thuringiensis (Bt) CryIAb endotoxin [44]. Despite low expression levels, the modified plants presented significant larvicidal activity against the sugarcane borer Diatraea saccharalis. Recently, Cry1Ab genetically modified sugarcane was produced using particle bombardment and Agrobacterium-mediated transformation [23]. The transgene was also pyramided with the trypsin inhibitor apronitin. The Agrobacterium-transformed plants showed high levels of transgene expression and superior resistance to sugarcane borer [23]. In another example, sugarcane plants were transformed with a truncated codon optimized form of the Bt endotoxin gene cry1Ac by particle bombardment [45]. In field trials, high levels of cry1Ac expression correlated with superior resistance to heavy infestation by Current Opinion in Biotechnology 2012, 23:315–322
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sugarcane stem borer and in some cases, the modified plants were completely immune to insect attack [45]. Genetically modified sugarcane for biotic and abiotic stress resistance has also been created. Overexpression of virus coat proteins or downregulating virus by antisense mRNA has produced resistant plants to sugarcane mosaic virus (SCMV) [46,47,48,49], sugarcane yellow leaf virus (SCYLV) [50] and Fiji disease virus (FDV) [51]. In some cases, these modified plants have demonstrated high levels of virus resistance and trait stability in field trials [48]. Plants overexpressing the trehalose synthase gene from Grifola frondosa accumulated high levels of trehalose and presented increased drought tolerance compared with control plants [52]. Another drought-resistant strategy involved the overexpression of the gene encoding pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in proline synthesis. These modified plants had increased proline accumulation and increased biomass yields when plants were submitted to drought stress [53].
Constraints for the commercial development of modified sugarcane The potential of application of biotechnology tools to increase sugar yield and agronomic performance of sugarcane are promising as yield gains using conventional breeding may have reached a ceiling due to the enormous difficulty imposed by the complexity of the sugarcane genome. However, the mechanisms of transgene silencing in sugarcane must still be elucidated. In diploid crops, genetically modified events can be converted to homozygous lines soon after transformation and can then quickly be tested for stability in few generations. Candidate events can further be introgressed into elite lines by successive backcrossing steps. In sugarcane, this is not possible due to the high degree of ploidy. Therefore, the transgene inserted into one of the 10 sugarcane homologous/homeologous chromosomes will always stay in the heterozygous form. This could be a factor of instability depending on the locus of insertion. Therefore, large numbers of transformation events, perhaps exceeding the number currently used by the biotech industry to generate corn events (in the order of 1000 per target gene), may be necessary to find stable events that can be tested for 3–4 cuttings (6–8 years). Later the commercial events would have to maintain stability for the life of the variety (15–20 years). Alternatively, the candidate event could be incorporated into the breeding program as one of the parental strains during the initial crossing seasons. However, this strategy would face difficulties for approval by the Biosafety Commissions because selection schemes in sugarcane breeding require large field test areas and multiple different locations. The method of transformation also presents challenges. Particle bombardment produces high copy number integration of the transgene, along with an unknown number of fragmented DNA Current Opinion in Biotechnology 2012, 23:315–322
pieces, generated during the bombardment that could integrate into the genome. These fragmented DNA pieces are difficult to identify and cannot be eliminated by successive backcrosses or self-pollination. Agrobacterium-mediated transformation is more amenable than particle bombardment to regulatory approval because single copy integration occurs at higher rates and the probability of integration of fragmented DNA pieces into the host genome is much lower. Another issue refers to the regulatory approval of construct, rather than the transformed events. In general, tens of different varieties are used to cover the growing area of industrial plantations. Therefore, a candidate gene must be incorporated into each one of these different varieties by transformation rather than by backcrossing. Approval of events for each variety is time-consuming and economically impeditive. Therefore, a construct-based approval might be the only way to develop economically viable, comercial genetically modified sugarcane.
Concluding remarks Genetic modification has enormous potential for increasing the sugar yield and improving the agronomic performance of sugarcane through the incorporation of desirable traits in elite varieties. Genetically modified sugarcane plants that incorporate genes for insect resistance, virus resistance and drought resistance have already passed the proof of concept. Strategies to increase sugar content in mature sugarcane stalk have also been developed by overexpressing genes that encode enzymes that convert sucrose into other disaccharides or sugar polymers. Plants with these modifications show an increase in overall sugar yield. Conversely, sugarcane has also been genetically modified to overproduce cellulolytic enzymes for producing second-generation cellulosic ethanol. However, although research has provided some insight into the mechanisms of transgene silencing in sugarcane, there is still much to be understood about the production of long-term stable, genetically modified sugarcane. Agrobacterium-mediated transformation is already routinely used by several groups in academia and is extensively used in the biotech industry as well. This transformation technology progress could help solve the regulatory issues associated with transgene integration. Finally, the research community and biotech industry must join efforts with the Biosafety Commission to establish the best models for the commercial approval of genetically modified sugarcane as this technology would be key to maintain and possibly increase the role of sugarcane as a major crop for sustainable energy production.
Acknowledgements The author gratefully acknowledges support by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP). The author is a recipient of a CNPq productivity fellowship. www.sciencedirect.com
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14. Arruda P: Perspective of the Sugarcane Industry in Brazil. Trop Plant Biol 2011, 4:3-8. 15. Goldemberg J, Coelho ST, Guardabassi P: The sustainability of ethanol production from sugarcane. Energy Policy 2008, 36:2086-2097. 16. Lakshmanan P, Geijskes J, Aitken KS, Grof CLP, Bonnett GD, Smith GR: Sugarcane biotechnology: the challenges and opportunities. In Vitro Cell Dev Biol-Plant 2005, 41:345-363. 17. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962, 15:473-497. 18. Basnayake SWV, Moyle R, Birch RG: Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars. Plant Cell Rep 2011, 30:439-448. The authors describe an improved protocol for the generation of transformable embryogenic calli from a set of elite sugarcane cultivars. www.sciencedirect.com
31. Christensen AH, Quail PH: Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 1996, 5:213-218. 32. Birch RG, Bower RS, Elliott AR: Highly efficient, 5-sequencespecific transgene silencing in a complex polyploid. Trop Plant Biol 2010, 3:88-97. The authors describe a possible mechanism by which transgenes could be silenced in genetically modified sugarcane. 33. Mudge SR, Osabe K, Casu RE, Bonnett GD, Manners JM, Birch RG: Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species. Planta 2009, 229:549-558. 34. Groenewald JH, Botha FC: Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes. Transgenic Res 2008, 17:85-92. This paper describes the alterations in sugar metabolism caused by the downregulation of PFP and the metabolic constrains limiting increment of sucrose accumulation in the acidic vacuoles of parenchyma cells. Current Opinion in Biotechnology 2012, 23:315–322
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35. van der Merwe MJ, Groenewald JH, Stitt M, Kossmann J, Botha FC: Downregulation of pyrophosphate: D-fructose-6phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexosephosphate levels. Planta 2010, 231:595-608. 36. Wu L, Birch RG: Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotechnol J 2007, 5:109-117. The authors show that targeted expression of a bacterial sucrose isomerase to the vacuoles of sugarcane parenchyma cells leads to an overproduction of isomaltulose and a dramatic increase in the levels of soluble sugars. The transgene expression also increased photosynthesis, sucrose transport, and sink strength. 37. Wu L, Birch RG: Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line. Funct Plant Biol 2010, 37:1161-1174. 38. Hellwege E, Knuth K: Sugarcane plants with increase storage carbohydrates content. Patent WO2006/066969A2. 39. 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 Biotechnol J 2011, 9:32-37. 40. Seabra Joaquim EA, Macedo Isaias C: Comparative analysis for power generation and ethanol production from sugarcane residual biomass in Brazil. Energy Policy 2011, 39:421-428. 41. Somerville C, Youngs H, Taylor C, Davis SC, Long SP: Feedstocks for lignocellulosic biofuels. Science 2010, 329:790-792. 42. Sainz MB: Commercial cellulosic ethanol: the role of plantexpressed enzymes. In Vitro Cell Dev Biol-Plant 2009, 45:314-329. 43. 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 Biotechnol J 2011:1-13 doi: 10.1111/j.14677652.2011.00597.x. The authors show that the targeted expression of the cellobiohydrolases CBH I and CBH II in leaf vacuoles and bacterial endoglucanase in chloroplasts, as driven by a strong constitutive promoter, led to a significant accumulation of the enzymes in mature leaves. The authors demonstrate the utility of this approach as a potential technology for second-generation ethanol from sugarcane bagasse 44. Arencibia A, Vazquez RI, Prieto D, Tellez P, Carmona ER, Coego A, Hernandez L, De la Riva GA, Selman-Housein G: Transgenic
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sugarcane plants resistant to stem borer attack. Mol Breed 1997, 3:247-255. 45. Weng LX, Deng HH, Xu JL, Li Q, Zhang YO, 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 Res 2011, 20:759-772. 46. Butterfield MK, Irvine JE, Garza MV, Mirkov ET: Inheritance and segregation of virus and herbicide resistance transgenes in sugarcane. Theor Appl Genet 2002, 104:797-803. 47. Gilbert RA, Gallo-Meagher M, Comstock JC, Miller JD, Jain M, Abouzid A: Agronomic evaluation of sugarcane lines transformed for resistance to Sugarcane mosaic virus strain E. Crop Sci 2005, 45:2060-2067. 48. Gilbert RA, Glynn NC, Comstock C, Davis MJ: Agronomic performance and genetic characterization of sugarcane transformed for resistance to sugarcane yellow leaf virus. Field Crops Res 2009, 111:39-46. The authors present a detailed analysis of the field performance of genetically modified sugarcane for virus resistance. 49. Ingelbrecht IL, Irvine JE, Mirkov TE: Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homologydependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiol 1999, 119:1187-1197. 50. Zhu YJ, McCafferty H, Osterman G, Lim S, Agbayani R, Lehrer A, Schenck S, Komor E: Genetic transformation with untranslatable coat protein gene of sugarcane yellow leaf virus reduces virus titers in sugarcane. Transgenic Res 2011, 20:503-512. 51. McQualter RB, Dale JL, Harding RM, McMahon JA, Smith GR: Production and evaluation of transgenic sugarcane containing a Fiji disease virus (FDV) genome segment S9derived synthetic resistance gene. Aust J Agric Res 2004, 55:139-145. 52. 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.). J Integr Plant Biol 2006, 48:453-459. 53. Molinari HBC, Marura CJ, Daros E, de Camposa MKF, de Carvalhoa JFRP, Bespalhok Filho JC, Pereira LFP, Vieira LGE: Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiol Plant 2007, 130:218-229.
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Genetically modified sugarcane for bioenergy generation Paulo Arruda1,2 Sugarcane breeding has significantly progressed over the past 30 years, but attempts to further increase crop yield have been limited due to the complexity of the sugarcane genome. An alternative to boost the crop yield is the introduction of genes encoding desirable traits in the elite sugarcane cultivars. Genetically modified sugarcane with increased yield and pest and disease resistance has already proven its value not only by the increased sugar content but also for the improvement of the crop performance. However, transgene stability is still a challenge since transgene silencing seems to occur in a large proportion of genetically modified sugarcane plants. In addition, regulatory issues associated with the crop propagation model will also be a challenge to the commercial approval of genetically modified sugarcane. Addresses 1 Centro de Biologia Molecular e Engenharia Gene´tica, Universidade Estadual de Campinas (UNICAMP), 13083-875 Campinas, SP, Brazil 2 Departamento de Gene´tica e Evoluc¸a˜o, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 13083-862 Campinas, SP, Brazil Corresponding author: Arruda, Paulo ([email protected])
Current Opinion in Biotechnology 2012, 23:315–322 This review comes from a themed issue on Energy biotechnology Edited by James C Liao and Joachim Messing Available online 15th November 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.10.012
Introduction Sugarcane, along with other major seed and foraging crops, belongs to the grass family Poaceae, a common ancestor of which gave rise to triticeae, rice, and the members of the Andropogoneae tribe about 80 million years ago (MYA) [1]. Rice diverged from the Andropogoneae approximately 60 MYA, while maize diverged from sorghum 11 MYA and sugarcane diverged from sorghum 8–9 MYA [2,3]. All of the sugarcane cultivars currently grown around the world resulted from crosses between Saccharum officinarum, a domesticated sugarproducing species with a basic chromosome number of 10 and chromosome constitution of 2n = 80, and Saccharum spontaneum, a wild species with a basic chromosome number of 8 and a chromosome constitution of 2n = 40–128 (Figure 1) [4,5]. The interspecific hybrid of these two species was backcrossed to S. officinarum www.sciencedirect.com
few times to generate a sugarcane germplasm that incorporates the high sugar content trait of S. officinarum in addition to disease and stress resistance traits of S. spontaneum [6]. However, as a result of a chromosomal stress produced by the interspecific cross, the genomic constitution of the hybrid became even more complex than its parent species, with a chromosome constitution varying between 2n = 100–130. The hybrid preserves intact 15– 25% of the chromosomes inherited from S. spontaneum and 60–70% inherited from S. officinarum. Around 5–10% of recombinants between the homeologous chromosomes of both species are also formed in the hybrid [4]. The sugarcane breeding process involves crossing superior varieties and selection among the F1 progenies individuals with favorable allelic combinations. But the chromosomal architecture of the sugarcane hybrid makes each cross a unique, unpredictable event due to the random sorting of chromosomes from both species and the formation of recombinants that affect the distribution of favorable and unfavorable alleles [7]. This architecture, combined with the multiplicity of alleles at each locus [8,9], makes the breeding process immensely complicated. In general, hundreds of thousands of F1 seedlings are screened for disease resistance, adaptability to distinct local environments, sucrose yield, agronomic manageability, and good milling characteristics [6]. The first round of screening for disease resistance is performed at nurseries, where the seedlings are sprayed with fungi or bacterial inoculums [6]. Resistant seedlings are then planted in distinct locations to evaluate the other desirable characteristics. This evaluation is performed for 3–5 cuttings (ratoons) to assure trait stability. After 7–10 years of evaluation, one or two clones, out of hundreds of thousands of the initial F1 seedlings, are released as commercial varieties [6]. With the recent interest in feedstock for second-generation cellulosic ethanol, the energy cane concept reemerged as an important topic of some breeding programs [6]. Instead of accumulating high levels of soluble sugar in the stalks, energy cane is bred to produce plants with high fiber content and high biomass. These traits can be obtained, for example, by backcrossing elite sugarcane varieties to S. spontaneum (Figure 1) [6]. Genetic modification of sugarcane could be a powerful tool to incorporate pest-resistant and disease-resistant traits into elite commercial sugarcane varieties and to increase their agronomic performance and sugar yield. Additionally, genetic modification could be used to increase the overall biomass of energy cane and to reduce Current Opinion in Biotechnology 2012, 23:315–322
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Figure 1
Hybrid Sugarcane
X
S. officinarum
Diesel
Juice
Sugar
Bagasse
1st Generation Ethanol
S. Spontaneum m Straw
Hybrid Energy Cane
Bioelectricity
Cellulosic Ethanol
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Schematic representation of sugarcane and energy cane production and their use in the bioenergy industry. Modern sugarcane cultivars derived from crosses between S. officinarum (a high sugar species) and S. spontaneum (a stress-resistant wild relative). The high sugar varieties were produced by backcrossing the interspecific hybrid to S. officinarum. Recently, high fiber, high biomass cultivars have been bred by backcrossing elite sugarcane varieties to S. spontaneum to produce a hybrid energy cane. The carbon fixed by the sugarcane plant is allocated as follows: 1/3 in the leaves, 1/3 in the juice and 1/3 in the bagasse. The straw primarily represents the leaves that are left in the crop field. The juice is used to produce sugar, ethanol, and feedstock for diesel production by the synthetic biology industry. The bagasse is currently used to produce bioelectricity, but bagasse and straw could also be used as feedstock for cellulosic ethanol. The energy cane enters a pipeline to produce cellulosic ethanol and bioelectricity.
the production cost of second-generation cellulosic ethanol by overexpressing in these plants the cellulolytic enzymes needed to convert cellulose into fermentable sugars. This review highlights the advances in the production of genetically modified sugarcane to incorporate traits associated with agronomic performance and bioenergy generation. Current status and limitations of producing genetically modified sugarcane are discussed.
Modern sugarcane varieties, energy cane and its processing Modern sugarcane varieties have been bred for increased sugar content in the stalk. Soluble sugars, mainly sucrose, makes up 1/3 of the photosynthetically fixed carbon and are stored in acidic vacuoles that occupy over 80% of parenchyma cells volume in the mature stalk [10]. These vacuoles are highly metabolically active, as they take up sucrose against a high osmotic gradient created by the accumulation of soluble sugars. The other 2/3 of the fixed carbon of sugarcane plants is stored as complex carbohydrates, mainly cellulose and hemicellulose, which compose the cell walls of leaves and stalks (Figure 1). Current Opinion in Biotechnology 2012, 23:315–322
The accumulation of high levels of sucrose in the stalk parenchyma cells involves complex regulatory mechanisms. Sucrose is synthesized in the leaf mesophyll cells and is then transported through the phloem to the developing culm. Part of this sucrose is used for cellulose synthesis and part is taken up by the stalk parenchyma cells and is stored in acidic vacuoles [11]. In mature sugarcane stalks, sucrose accumulates in these vacuoles at a concentration of 600–700 mM [12]. Accumulation of such high levels of sucrose involves adaptive mechanisms to protect the parenchyma cells from osmotic and water stress. Expression profile analysis suggests that the pathway involved in this process is distinct from the drought stress pathway commonly used by plants to protect from water restriction [13]. Manipulation of carbon partitioning between sugar and fiber is essential in the breeding programs, and it is a difficult task to overcome the negative correlation between sugar and fiber production [6]. In modern industrial plantations, the sugarcane stalks are harvested, while the leaves are left in the field to help restore the soil fertility. At sugarcane mills, the stalks are crushed, and the resulting juice is used for single step www.sciencedirect.com
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sucrose crystallization. The sugar remaining in the molasses is fermented to produce fuel ethanol (Figure 1) [14]. The bagasse powder that results from stalk crushing is used to produce bioelectricity, but it could also be a valuable feedstock source for cellulosic ethanol production [15]. The sugarcane juice can also be used as a feedstock for fuel production in the synthetic biology biotech industry (Figure 1) [14].
Genetic transformation and expression stability Tissue culture and plant regeneration, a prerequisite for genetic transformation, have been established in sugarcane for almost 40 years [16]. Embryogenic calli are produced from transverse sections, cut just above the apical meristem of leaf whorls, inoculated in Murashige and Skoog (MS) basal medium [17] supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D), casein hydrolysate, sugars, vitamins, and coconut water (Figure 2) [18,19]. Plates are incubated in the dark to induce nodular embryogenic calli that are subcultured every 2 weeks. The highly nodular embryogenic calli are the preferred tissue for genetic transformation using either particle bombardment or Agrobacterium-mediated transformation. Plant regeneration and rooting is induced by transferring the embryogenic calli to medium without 2,4D and incubating under a 16 h photoperiod regime
(b)
(c)
(d)
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Genetic modification of sugarcane through transformation of embryogenic calli. The base of the leaf whorls is sliced and plated in MS medium supplemented with sugar and phytohormones. (a) Nodular embryogenic calli formed after a few weeks of inoculation. (b) Transformed embryogenic tissues, as evidenced by the GFP marker. (c) Regeneration of plants in MS medium without hormones. (d) Selection of transformed plants in MS medium containing a selective agent, such as an herbicide or antibiotic. www.sciencedirect.com
Sugarcane transformation has been mostly done by particle bombardment of embryogenic calli [20]. When this technology was first established, the successful recovery of a high number of transformed events depended on the use of the few sugarcane genotypes that produced transformable embryogenic calli. Since then, tissue culture methods have been improved, allowing the production of transformable embryogenic calli from dozens of elite sugarcane varieties [18]. This is primarily due to better explant quality rather than to the culture medium modifications. Agrobacterium-mediated transformation of embryogenic calli was first achieved using embryogenic calli [21,22] following a protocol that includes a dissection step, an appropriate co-cultivation medium and a selection scheme that enhances the rate of Agrobacterium infection and transformation [21] (Figure 2). In the last few years, this protocol has been improved and routinely used by several groups [19,23–25]. Transformation efficiency has increased such that over 5 transformed plants per plate are successfully recovered when the selective gene is placed under the control of a strong constitutive promoter [19]. Transformation cassettes used for sugarcane transformation usually incorporate antibiotic resistance genes [26], herbicide resistance genes [27], or phosphomannose isomerase (PMI) [28]. PMI allows for the positive selection of transformed tissues growing in mannose-containing medium. Cassettes also incorporate reporter genes, such as glucuronidase (GUS) [29] or green fluorescent protein (GFP) [30]. In general, resistance and reporter genes are placed under the control of strong constitutive promoters, such as the promoter of the gene encoding the 35S coat protein of cauliflower mosaic virus (35SCaMV) [23] or the polyubiquitin (UBI) promoter from maize [31] or rice [29].
Figure 2
(a)
(Figure 2) [18]. Regenerated plants are then transferred to pots for acclimation and growth after which the stalks of the adult plant are used for plant propagation.
Genetically modified sugarcane produced by particle bombardment transformation has been shown to be highly unstable. Transformed plants usually present complex construct integration with high copy numbers of the inserted transgene; sometimes the transgene is inserted in tandem, while it can also be inserted throughout the plant genome. These modified plants usually show high activity in young transformed seedlings, but transgene expression is generally turned off in mature plants in the first, second, or third ratoons [32]. Expression instability has been attributed to gene silencing induced by high copy number integration. Because sugarcane possesses a high degree of ploidy, allele silencing has been suggested to be more frequent in this crop compared to diploid plants [32]. Silencing-mediated transgene instability seems to have a greater effect when the constructs incorporate promoters Current Opinion in Biotechnology 2012, 23:315–322
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Table 1 Genetically modified sugarcane with high sugar content and insect, virus and stress resistance. Trait
Promoter/target gene
Promoter/selective marker
Reference
Insect resistance Insect resistance Insect resistance Herbicide resistance Herbicide resistance Herbicide resistance SCYLV resistance SCYLV resistance SCYLV resistance SrMV resistance FDV resistance Drought resistance Drought resistance Increased sugar content Increased trehalulose Increased sugar content Increased sugar content Cellulase, endoglucanase
CaMV35S/CryIAb UBI/CryIAb UBI/CryIAc NA UBI/Bar CaMV 35S/Bar UBI/CP UBI/CP UBI/CP UBI/CP UBI/ORF1 AIPC/P5CS CaMV 35S/TS CaMV 35S: UBI/PFP UBI/TS Emu/SSFT UBI/SI UBI/CBHI, CBHII, EG
None CaMV 35S/Bar UBI/G418 CaMV35S/Bar UBI/Kam NA UBI/Kam UBI/Kam UBI/Kam UBI/Kam UBI/Kam UBI/Bar CaMV 35S/Kam UBI/Kam UBI/Kam Emu/Kam UBI/Kam UBI/Kam
[1] [4] [48] [15] [16] [35] [17] [18] [53] [25] [38] [39] [51] [21] [22] [24] [49] [23]
Abbreviations: SCYLV — sugarcane yellow leaf virus; SrMV — Sorgum mosaic virus; FDV — Fiji disease virus; CaMV35S — 35S cauliflower mosaic virus promoter; CryIAb — Bt endotoxin; Bar — phosphinotricin acetyl transferase; UBI — maize polyubiquitin promoter; CP — Cpat protein; ORF1 — open reading frame 1; AIPC — ABA-inducible promoter complex; P5CS — pyrroline-5-carboxylate synthetase; TS — trehalulose synthase; SSFT — sucrose–sucrose fructosil transferase; SI — sucrose isomerase; CBHI, CBHII — cellobiohydrolase I and II; EG — endoglucanase; Kam — kanamycin; NA — not applicable.
from sugarcane genes. To test if gene silencing is associated with sugarcane promoters coming out of non-functional alleles, the promoters of eight alleles of a unique gene were fused to the GUS reporter gene and introduced into transgenic plants by particle bombardment [33]. The transgenes were highly active in young seedlings, but none of these transgenes sustained expression in mature stalks, indicating highly efficient transgene silencing from these promoters. Gene silencing could also be caused by the particle bombardment transformation technique itself. Recently, genetically modified sugarcane expressing the Cry1Ab toxin gene from Bacillus thuringiensis driven by the maize UBI promoter was produced using either particle bombardment or Agrobacterium-mediated transformation [23]. Plants produced with Agrobacterium-mediated transformation showed higher frequency of single-site integration, increased stability of transgenes and higher expression level of the toxin, compared to plants produced by particle bombardment, which contained multiple integrations. Transgene instability in sugarcane may be higher than in other diploid crops, although this instability is enhanced by particle bombardment transformation. Further work is needed to explore different target gene/promoter combinations to fully answer the question of transgene instability in sugarcane.
Modified sugarcane for enhanced sugar accumulation Sugarcane stores fixed carbon as soluble sugars, mostly sucrose, in vacuoles of parenchyma cells of mature stalks Current Opinion in Biotechnology 2012, 23:315–322
[10]. However, transforming photosynthetically fixed carbon into stored sucrose is a complex process that involves osmotic regulation, sugar sensors, sucrose cleavage and feedback regulation of photosynthesis [11]. The acidic vacuoles occupy approximately 80% of the total cell volume and accumulate sucrose up to a concentration of 600–700 mM [12]. The limiting step in increasing the sugar yield in sugarcane is the high osmotic gradient between the vacuoles and symplast within the stalk parenchyma cells [11]. Attempts to overcome this osmotic limitation include the downregulation of the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) through constitutive overexpression of antisense RNA to the PFP-b gene (Table 1) [34]. These genetically modified plants present reduced PFP levels in young internodes and almost undetectable levels of PFP in mature internodes. However, while these plants presented a significant increase in the sucrose concentration of the immature internodes, the amount of sucrose in the mature internodes was unaffected. A detailed analysis of the PFP-modified plants showed alterations in sugar metabolites in immature internodes, with an eightfold increase in the hexose-phosphate:triose-phosphate ratio, a restriction of the triose phosphate-to-hexose phosphate cycle and an increase in sucrose synthesis [35]. However, in mature internodes, these effects were not significantly different between the modified plants and the controls. On the other hand, these modified plants presented significantly higher fiber content, indicating that the osmotically imposed limit resulting from sucrose over accumulation diverted the carbon flux from soluble sugars to fiber. www.sciencedirect.com
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An alternative approach in which the limiting step imposed by osmotic constraints was bypassed involved the production of genetically modified sugarcane that overexpressed in the vacuoles a bacterial sucrose isomerase (SI), an enzyme that converts sucrose into the isomer isomaltulose (IM) [36] (Table 1). These plants almost doubled the amount of sugar in their juice accumulating up to 500 mM of isomaltulose without affecting the accumulation of sucrose or the carbon partitioning between sugar and fiber. The accumulation of sugars in these genetically modified plants could result from the conversion of monosaccharides into disaccharides via the overexpressed SI in the vacuole to alleviate the osmotic stress. A cell line derived from these plants accumulates twofold more sucrose than control cell lines derived from the same sugarcane variety [37]. This increase is not accompanied by parallel accumulation of IM. The sugar metabolism of these cells showed reduced extracellular invertase, depletion of extracellular sucrose and sucrose synthase activity, and higher symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) [37]. The enzymatic activity of both sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) is faster in these modified cell lines as well. The authors postulate that a signaling mechanism involving T6P enhances sucrose synthesis in sugarcane [37]. Overexpression of a sucrose–sucrose fructosil transferase (SSFT) in sugarcane has also been used to boost sugar accumulation in the parenchyma cells vacuoles of mature stalk [38] (Table 1). SSFT uses sucrose as a substrate to produce linear beta-(2,6)-linked fructans, a polysaccharide that is not naturally produced in sugarcane. These modified plants accumulate twice the amount of sugars in mature stalks as compared to control plants. The overexpression of this enzyme in vacuoles establishes a sugar sink to a polymer that is less osmotically active, which therefore may alleviate the osmotic constraints of the vacuole. Not all genetically modified sugarcane lines transformed to produce alternative sugars in their vacuoles increase sugar overproduction. Sugarcane plants that overexpress a vacuole-targeted bacterial trehalulose synthase showed accumulation of up to 600 mM trehalulose in mature stalks, but almost all of the sucrose in the vacuoles was converted into the sucrose isomer [39]. Thus, it seems that alteration of sugar metabolism through the manipulation of key sucrose biosynthesis enzymes does not necessarily overcome the osmotic constraints of the mature vacuole. On the other hand, the overexpression of enzymes that produce disaccharides or sugar polymers can bypass the osmotic limit and lead to increased accumulation of soluble sugars in the mature sugarcane stalk. www.sciencedirect.com
Modified sugarcane for second-generation ethanol production Two-thirds of the photosynthetically fixed carbon in sugarcane is stored in the form of cellulose and hemicellulose (Figure 1). Sugarcane leaves are currently left in the field after harvesting, but millions of tons of the bagasse power are produced annually in the sugarcane mills [6]. Although this sugarcane bagasse is currently used for bioelectricity generation [14], it is also an excellent feedstock for second-generation ethanol production [40,41]. However, cellulosic ethanol is economically limited by the high cost of the enzymes necessary to convert cellulose and hemicellulose into fermentable sugars [42]. The overexpression of cellulolytic enzymes in genetically modified plants could help to reduce the cost of enzyme production. Additionally, delivery of the enzyme to sugarcane mills would be an easy task because the plant feedstock would already contain the enzyme. Technologies for expressing and storing enzymes in plants have significantly progressed over the last few years, as overproduction, storage, and stability issues could be addressed by targeting the foreign enzymes to specific cellular organelles [43]. Engineered enzymes with increased thermostability and improved hydrolytic efficiency has also been developed [42]. Fungal cellobiohydrolases (CBH I and CBH II) and bacterial endoglucanase (EG) have been overexpressed in genetically modified sugarcane leaves. Targeting CBHI and CBHII to vacuoles and EG to chloroplasts resulted in high enzymatic activity in mature leaves, demonstrating the feasibility of expressing cellulose hydrolytic enzymes in sugarcane plants [43]. This approach could be of considerable interest to sugar mills that have the technology to process sugarcane and energy cane. Cellulolytic enzymes could be produced throughout the entire energy cane plant and used for processing both its own biomass and the bagasse from sugarcane crushing.
Modified sugarcane for biotic and abiotic stress resistance One of the first genetically modified sugarcane plants was produced to constitutively overexpress the B. thuringiensis (Bt) CryIAb endotoxin [44]. Despite low expression levels, the modified plants presented significant larvicidal activity against the sugarcane borer Diatraea saccharalis. Recently, Cry1Ab genetically modified sugarcane was produced using particle bombardment and Agrobacterium-mediated transformation [23]. The transgene was also pyramided with the trypsin inhibitor apronitin. The Agrobacterium-transformed plants showed high levels of transgene expression and superior resistance to sugarcane borer [23]. In another example, sugarcane plants were transformed with a truncated codon optimized form of the Bt endotoxin gene cry1Ac by particle bombardment [45]. In field trials, high levels of cry1Ac expression correlated with superior resistance to heavy infestation by Current Opinion in Biotechnology 2012, 23:315–322
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sugarcane stem borer and in some cases, the modified plants were completely immune to insect attack [45]. Genetically modified sugarcane for biotic and abiotic stress resistance has also been created. Overexpression of virus coat proteins or downregulating virus by antisense mRNA has produced resistant plants to sugarcane mosaic virus (SCMV) [46,47,48,49], sugarcane yellow leaf virus (SCYLV) [50] and Fiji disease virus (FDV) [51]. In some cases, these modified plants have demonstrated high levels of virus resistance and trait stability in field trials [48]. Plants overexpressing the trehalose synthase gene from Grifola frondosa accumulated high levels of trehalose and presented increased drought tolerance compared with control plants [52]. Another drought-resistant strategy involved the overexpression of the gene encoding pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in proline synthesis. These modified plants had increased proline accumulation and increased biomass yields when plants were submitted to drought stress [53].
Constraints for the commercial development of modified sugarcane The potential of application of biotechnology tools to increase sugar yield and agronomic performance of sugarcane are promising as yield gains using conventional breeding may have reached a ceiling due to the enormous difficulty imposed by the complexity of the sugarcane genome. However, the mechanisms of transgene silencing in sugarcane must still be elucidated. In diploid crops, genetically modified events can be converted to homozygous lines soon after transformation and can then quickly be tested for stability in few generations. Candidate events can further be introgressed into elite lines by successive backcrossing steps. In sugarcane, this is not possible due to the high degree of ploidy. Therefore, the transgene inserted into one of the 10 sugarcane homologous/homeologous chromosomes will always stay in the heterozygous form. This could be a factor of instability depending on the locus of insertion. Therefore, large numbers of transformation events, perhaps exceeding the number currently used by the biotech industry to generate corn events (in the order of 1000 per target gene), may be necessary to find stable events that can be tested for 3–4 cuttings (6–8 years). Later the commercial events would have to maintain stability for the life of the variety (15–20 years). Alternatively, the candidate event could be incorporated into the breeding program as one of the parental strains during the initial crossing seasons. However, this strategy would face difficulties for approval by the Biosafety Commissions because selection schemes in sugarcane breeding require large field test areas and multiple different locations. The method of transformation also presents challenges. Particle bombardment produces high copy number integration of the transgene, along with an unknown number of fragmented DNA Current Opinion in Biotechnology 2012, 23:315–322
pieces, generated during the bombardment that could integrate into the genome. These fragmented DNA pieces are difficult to identify and cannot be eliminated by successive backcrosses or self-pollination. Agrobacterium-mediated transformation is more amenable than particle bombardment to regulatory approval because single copy integration occurs at higher rates and the probability of integration of fragmented DNA pieces into the host genome is much lower. Another issue refers to the regulatory approval of construct, rather than the transformed events. In general, tens of different varieties are used to cover the growing area of industrial plantations. Therefore, a candidate gene must be incorporated into each one of these different varieties by transformation rather than by backcrossing. Approval of events for each variety is time-consuming and economically impeditive. Therefore, a construct-based approval might be the only way to develop economically viable, comercial genetically modified sugarcane.
Concluding remarks Genetic modification has enormous potential for increasing the sugar yield and improving the agronomic performance of sugarcane through the incorporation of desirable traits in elite varieties. Genetically modified sugarcane plants that incorporate genes for insect resistance, virus resistance and drought resistance have already passed the proof of concept. Strategies to increase sugar content in mature sugarcane stalk have also been developed by overexpressing genes that encode enzymes that convert sucrose into other disaccharides or sugar polymers. Plants with these modifications show an increase in overall sugar yield. Conversely, sugarcane has also been genetically modified to overproduce cellulolytic enzymes for producing second-generation cellulosic ethanol. However, although research has provided some insight into the mechanisms of transgene silencing in sugarcane, there is still much to be understood about the production of long-term stable, genetically modified sugarcane. Agrobacterium-mediated transformation is already routinely used by several groups in academia and is extensively used in the biotech industry as well. This transformation technology progress could help solve the regulatory issues associated with transgene integration. Finally, the research community and biotech industry must join efforts with the Biosafety Commission to establish the best models for the commercial approval of genetically modified sugarcane as this technology would be key to maintain and possibly increase the role of sugarcane as a major crop for sustainable energy production.
Acknowledgements The author gratefully acknowledges support by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP). The author is a recipient of a CNPq productivity fellowship. www.sciencedirect.com
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