Transformation Of Sugarcane Research Articles

Genetic Transformation of Sugarcane, Current Status and Future Prospects.

Sugarcane (Saccharum spp.) is a tropical and sub-tropical, vegetative-propagated crop that contributes to approximately 80% of the sugar and 40% of the world’s biofuel production. Modern sugarcane cultivars are highly polyploid and aneuploid hybrids with extremely large genomes (>10 Gigabases), that have originated from artificial crosses between the two species, Saccharum officinarum and S. spontaneum. The genetic complexity and low fertility of sugarcane under natural growing conditions make traditional breeding improvement extremely laborious, costly and time-consuming. This, together with its vegetative propagation, which allows for stable transfer and multiplication of transgenes, make sugarcane a good candidate for crop improvement through genetic engineering. Genetic transformation has the potential to improve economically important properties in sugarcane as well as diversify sugarcane beyond traditional applications, such as sucrose production. Traits such as herbicide, disease and insect resistance, improved tolerance to cold, salt and drought and accumulation of sugar and biomass have been some of the areas of interest as far as the application of transgenic sugarcane is concerned. Although there have been much interest in developing transgenic sugarcane there are only three officially approved varieties for commercialization, all of them expressing insect-resistance and recently released in Brazil. Since the early 1990’s, different genetic transformation systems have been successfully developed in sugarcane, including electroporation, Agrobacterium tumefaciens and biobalistics. However, genetic transformation of sugarcane is a very laborious process, which relies heavily on intensive and sophisticated tissue culture and plant generation procedures that must be optimized for each new genotype to be transformed. Therefore, it remains a great technical challenge to develop an efficient transformation protocol for any sugarcane variety that has not been previously transformed. Additionally, once a transgenic event is obtained, molecular studies required for a commercial release by regulatory authorities, which include transgene insertion site, number of transgenes and gene expression levels, are all hindered by the genomic complexity and the lack of a complete sequenced reference genome for this crop. The objective of this review is to summarize current techniques and state of the art in sugarcane transformation and provide information on existing and future sugarcane improvement by genetic engineering.

Modern Biotechnologies: Innovative and Sustainable Approaches for the Improvement of Sugarcane Tolerance to Environmental Stresses

Sugarcane (Saccharum spp.) is one of the most important industrial cash crops, contributing to the world sugar industry and biofuel production. It has been cultivated and improved from prehistoric times through natural selection and conventional breeding and, more recently, using the modern tools of genetic engineering and biotechnology. However, the heterogenicity, complex poly-aneuploid genome and susceptibility of sugarcane to different biotic and abiotic stresses represent impediments that require us to pay greater attention to the improvement of the sugarcane crop. Compared to traditional breeding, recent advances in breeding technologies (molecular marker-assisted breeding, sugarcane transformation, genome-editing and multiple omics technologies) can potentially improve sugarcane, especially against environmental stressors. This article will focus on efficient modern breeding technologies, which provide crucial clues for the engineering of sugarcane cultivars resistant to environmental stresses.

Construction of a recombinant vector containing SAI gene using Gateway-based RNAi system: a preliminary study in the development of high sucrose sugarcane

Recombinant plasmid construction that produces hairpin molecules for gene knockdown is not an easy method. Gateway cloning system is a technology to transfer DNA fragments utilizing clonase and specific sites. This study aimed to construct RNAi expression vector containing soluble acid invertase (SAI) gene using Gateway system to produce high-sugar content sugarcane. PCR was conducted to generate specific SAI fragment using cDNA template from two sugarcane varieties: Bululawang and PSJK922. Of the two specific primer pairs designed, only one pair amplified the SAI 161 bp gene fragment. Amplification occurred in both cDNA samples. TA (Thymine-Adenine pair) cloning was performed with pCR8/GW/TOPO plasmid and a clonase reaction was carried out to insert the specific fragments into the pHELLSGATE 8 hairpin plasmid. The results showed that two of the four colonies analyzed by associated restriction enzymes had plasmid pHELLSGATE8::hpSAI that contained a hairpin component of the SAI fragment. One attempt to construct the hairpin RNA using Gateway system in this study indicates the easiness of the system when compared to traditional cloning. The DNA sequence in the hairpin portion of this plasmid will be analysed to confirm SAI gene fragment, and it will be followed by transformation of sugarcane and SAI characterization.

RNAi-Based Gene Silencing in Sugarcane for Production of Biofuel.

RNA interference (RNAi) is an innate cellular mechanism triggered by a double-stranded RNA (dsRNA) molecule causing selective inhibition of gene expression. Here, we demonstrated the RNAi technology for gene silencing in sugarcane for biofuel production. This chapter describes an efficient model system that established to target the caffeic acid O-methyltransferase (COMT) gene and the RNAi construct is designed and delivered through Agrobacterium mediated stable sugarcane transformation. Also, the approach for an analysis of resulting putative transgenic plants for a targeted RNAi mediated gene silencing is described.

Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy.

We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.

High-throughput in vitro culture system targeting genetic transformation in sugarcane

Conventional recombination breeding suffers setbacks for genetic improvement of sugarcane owing to its narrow gene pool, large complex genome, rare flowering, low fertility, long breeding cycle, and complex environmental interactions. However, production of transgenic plants can be a better alternative to improve quality traits and resistance to biotic and abiotic stresses. The authors report a ready in hand efficient in vitro culture protocol for genetic transformation in a popular sugarcane cv. Sabita (CoOR 03151) of Odisha. 2, 4-D at 3.0 mg/l resulted in highest callus induction frequency (87.8%) with white, friable, and nodular embryogenic calli suitable for plantlet regeneration. 2 mg/l BAP resulted in moderately higher number of shoots/responsive callus and higher percentage of plant survival. However, addition of TDZ (0.05 m/l) with 2 mg/l BAP in R medium (modified MS) may be considered optimum for regeneration of profuse healthy plantlets. A combination of PBZ (0.05 mg/l) with BAP (2 mg/l) and TDZ (0.05 mg/l) revealed profuse multiple shoots (25 microshoots/responsive callus) and higher percentage of survival during follow-up plant establishment than above hormonal supplementation. Among various hormone recipes in MS medium with 3.0 mg/l NAA resulted in excellent rhizogenesis response (88.0%) with more or less normal rooting within 2 weeks. The above efficient and highly reproducible in vitro culture system can be amenable for genetic transformation in this crop.

Spectrophotometric Estimation of Total Phenolic Content and Antioxidant Capacity of Molasses and Vinasses Generated from the Sugarcane Industry

To identify a possible use for some agroindustrial wastes generated from the sugarcane industry, we evaluated the antioxidant capacity of B and C molasses and vinasses from the sugar and bioethanol production processes. Molasses and vinasses were characterized by physicochemical methods. Subsequently, the samples were diluted in distilled water at five concentrations to obtain aqueous extracts. Total phenolic content (TPC) of the samples was determined using a spectrophotometric method and was expressed in mg equivalents of gallic acid. The antioxidant capacity of each sample was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl radical) and ABTS (2,2-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) methods, as well as the ferric-reducing power in FRAP (Ferric-reducing Antioxidant Power) assay. We found that, both for TPC and antioxidant capacity, the vinasses showed significantly higher values than the B and C molasses. These results showed a strong correlation between TPC and antioxidant capacity and revealed a remarkable increase in TPC and total antioxidant agents present in the extracts throughout the sugarcane transformation process. These findings allowed identifying vinasses as the by-product with the best antioxidant properties. Our work constitutes a first step in the study of molasses and vinasses as a promising antioxidant agent and as a novel resource to test in proliferative trials in cellular systems in vitro.Graphical

Prospect of Genetic Transformation in Sugarcane-A Review

Conventional breeding suffers setbacks for genetic improvement of sugarcane owing to its large complex genome, rare flowering, low fertility, long breeding cycle and complex environmental interactions. However, production of transgenic plants can be a better alternative to improve quality traits and resistance to biotic and abiotic stresses. In this pursuit, the authors presented a detailed review of current strategies of genetic transformation in sugarcane using a number of important candidate genes.

PGVG: a new Gateway-compatible vector for transformation of sugarcane and other monocot crops.

The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the β-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.

Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Scope of Genetic Transformation in Sugarcane-A Review

Sugarcane is a cash crop of national importance. Its complex genome, narrow gene pool, long breeding cycle, rare flowering and complex environmental interactions hinders progress in genetic improvement. But, the crop serves as an excellent material for in vitro culture. Therefore, genetic transformation can be a better alternative to incorporate resistance to diseases and abiotic stresses, and genetic improvement of quality traits. In this pursuit, the authors presented a detailed review of the status of in vitro culture and current strategies of genetic transformation in sugarcane using a number of important candidate genes.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

An Efficient Protocol for Agrobacterium-mediated Transformation of Sugarcane by Optimizing of Duration of Co-cultivation and Age of Callus

An Efficient Protocol for Agrobacterium-mediated Transformation of Sugarcane by Optimizing of Duration of Co-cultivation and Age of Callus

Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane

Sugarcane (Saccharum spp. hybrids) accounts for 80% of the table sugar produced worldwide and is also a prime feedstock for biofuel production. However, very few studies are available for directly comparing Agrobacterium tumefaciens-mediated transfer of T-DNA (AMT) and biolistic transfer of minimal expression cassettes (BLT MC) regarding transgene complexity and expression stability. In this study, the transformation efficiency, transgene integration pattern, expression level, and expression stability were compared in the commercially important sugarcane cultivar CP88-1762. A total of 312 transgenic lines derived from AMT and 250 lines derived from BLT MC were identified by PCR from genomic DNA using nptII-specific primers. Lines were analyzed with both qPCR (TaqMan®) and NPTII ELISA to determine the nptII transgene copy number and expression level. The results of Southern blot analysis on selected lines were highly correlated to the qPCR results. There were no significant differences between the two transformation systems for transformation efficiency, frequency of single copy integration, or level and stability of transgene expression when carried out with the same expression cassette, tissue culture, and selection procedure in 12 independent experiments. These findings suggested that both BLT MC and AMT provide suitable platforms for generation of elite sugarcane events.

Effective selection and regeneration of transgenic sugarcane plants using positive selection system

We compared the transformation frequency of a positive selection system, which employs an Escherichia coli-derived pmi (phosphomannose isomerase) gene as the selectable marker and mannose as the selective agent, to commonly used negative selection systems of bar (phosphinothricin acetyltransferase) /phosphinothricin (PPT), npt II (neomycin phosphotransferase) /G418 (geneticin), and hpt (hygromycin phosphotransferase) /Hygromycin. Transgenic sugarcane plants were obtained following biolistic transformation of callus with four different selectable marker genes. Under different selective agents, transformed callus was selectively proliferated on MS medium containing 13.57 μM of 2,4-D, regenerated on MS medium with 4.44 μM of BA, 9.29 μM of KT, and 0.89 μM of NAA, and rooted on half of MS medium with 13.38 μM of NAA and 0.44 μM of 6-BA. The results demonstrated that the transformation frequency of 4.2% scored in the pmi/mannose positive system was significantly higher than those in the negative selection systems (0.47% for bar/PPT, 1.38% for npt II/G418, and 0.63% for hpt/Hygromycin, respectively). Transgenic sugarcane plants using the pmi/mannose system were confirmed by polymerase chain reaction (PCR) analysis and chlorophenol red (CPR) assay. The CPR assay was found to efficiently and qualitatively detect the transgenic sugarcane from the nontransformed plants because medium color changes in the CPR assay were highly responsive to pmi gene expression and enzyme activity. Thus, a pmi/mannose positive selection system for sugarcane transformation was developed to obviate the use of negative selection systems based on herbicides and antibiotics.

Sugarcane (Saccharum spp. hybrids).

Genetic transformation of sugarcane has a tremendous potential to complement traditional breeding in crop improvement and will likely transform sugarcane into a bio-factory for value-added products. We describe here Agrobacterium tumefaciens-mediated transformation of sugarcane. Embryogenic callus induced from immature leaf whorls was used as target for transformation with the hypervirulent Agrobacterium strain AGL1 carrying a constitutive nptII expression cassette in vector pPZP200. Selection with 30 mg/L geneticin during the callus phase and 30 mg/L paromomycin during regeneration of shoots and roots effectively suppressed the development of non-transgenic plants. This protocol was successful with a commercially important sugarcane cultivar, CP-88-1762, at a transformation efficiency of two independent transgenic plants per g of callus.

<b>Plant regeneration and histological study of the somatic embryogenesis of sugarcane (<i>Saccharum</i> spp.) cultivars RB855156 and RB72454</b> - doi: 10.4025/actasciagron.v36i1.16342

The development of an efficient regeneration system is crucial for the genetic transformation of sugarcane. The aims of this study were to regenerate plants from somatic embryos and analyze the origin and division pattern of cells during the different embryonic developmental stages of the sugarcane cultivars RB855156 and RB72454. For both cultivars, the best results for embryogenic callus induction were obtained with explant cultures on culture medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by callus culture on solid medium with 4.5 µM 2,4-D. A higher rate of shoot induction was observed in RB855156 with the addition of 8.9 and 17.8 µM benzylaminopurine (BAP); this higher rate was also observed in RB72454 with the addition of 17.8 µM BAP. Murashige and Skoog (1962) (MS) medium containing 2.5 and 5.0 µM indolebutyric acid (IBA) for RB855156 and RB72454, respectively, was suitable for rooting. The plantlets were successfully acclimatized and the plant survival rate was 100% for both cultivars. Histological analysis revealed that the shoots in both cultivars originated from somatic embryogenesis

Advances in Agrobacterium-Mediated Sugarcane Transformation and Stable Transgene Expression

The sugarcane plant, with complex genetic characteristics and a lengthy breeding process, is challenging for genetic improvement through traditional breeding. Genetic engineering offers an opportunity for trait-specific sugarcane variety improvement. Transformation and transgene expression are key technologies to enable the application of biotechnology to sugarcane improvement, and technical challenges have been reported in both of these areas. An efficient Agrobacterium-mediated transformation system for multiple diverse sugarcane varieties has been developed to an industrial scale. The transgenic events generated have demonstrated high level transgene expression over multiple seasons in the field. Syngenta is now applying this technology on a larger scale in sugarcane improvement research.

Herbicide-tolerant sugarcane (Saccharum officinarum L.) plants: an unconventional method of weed removal

Unnecessary weed growth in sugarcane fields forces plants to compete for nutrients and sunlight for survival, which most often leads to significant yield losses. As chemical herbicides cannot differentiate between crop plants and weeds, the development of herbicide-tolerant crops is anticipated. Four sugarcane varieties, CPF-234, CPF-213, HSF-240, and CPF-246, were used to develop glyphosate herbicide tolerance. A glyphosate-tolerant gene of 1368 bp cloned directionally under the 35S promoter with the \Beta-glucuronidase (GUS) reporter gene was used as the transgene. Through the biolistic transformation of sugarcane, calli of all cultivars were transformed with glyphosate-tolerant gene constructs. Efficient regeneration conditions were optimized on 1.5-2.5 mg/L kinetin, 1.5-2.5 mg/L 6-benzylaminopurine (BAP), and 1 mg/L gibberellic acid (GA_{3}). The transformants exhibited the best shoot regeneration on a medium containing 2 mg/L kinetin, 2 mg/L BAP, 2 mg/L GA_3, and 1 mg/L indole-3-acetic acid. Based on initial screenings through GUS assay, the transformation efficiency was 22%, 32%, 17%, and 13% for cultivars 246, 234, 213, and 240, respectively. In transformed sugarcane plants, the transgene of 1368 bp was amplified. ELISA with gene-specific coated monoclonal IgG confirmed the transgene protein expression. It was revealed that all acclimatized transgenic sugarcane plants survived the glyphosate spray application of 900 mL/0.404 ha, except for the control nontransformed plants. However, at spray application of 1100 mL/0.404 ha, transgenic plants having the transgene protein OD of 0.2 to 1.0 did not survive, while those that had a transgene protein OD range of between 1.0 and 2.0 did. In addition, weeds growing alongside transgenic sugarcane plants turned brown and subsequently died at glyphosate spray applications of both 900 and 1100 mL/0.404 ha.

Genetic Improvement of Sugarcane for Drought and Salinity Stress Tolerance Using Arabidopsis Vacuolar Pyrophosphatase (AVP1) Gene

Sugarcane plant is a glycophyte, hence its growth and sucrose contents are severely affected by drought and salinity stresses. Bioengineering approaches offer a plausible and rapid solution to mitigate these losses. Therefore for genetic improvement of sugarcane against these stresses, the present study was conceived to transform Arabidopsis Vacuolar Pyrophosphatase (AVP1) gene--confers tolerance against drought and salinity--into sugarcane through Agrobacterium. For this purpose, highly regenerable apical buds of sugarcane variety CP77-400 were used as explants. EHA105 strain of Agrobacterium harboring pGreen0029 vector containing AVP1 gene driven under 35SCaMV promoter was employed for transformation. The key factors studied include application of acetosyringone, cefotaxime, kanamycin, and co-cultivation period for successful transformation. Maximum regeneration frequency of 77.5 % was achieved on MS media containing 1 mg/l BAP, 1 mg/l Kn, 1 mg/l GA₃, 0.25 mg/l NAA, 50 μM acetosyringone, 500 mg/l cefotaxime, and 150 mg/l kanamycin on 3 days of co-cultivation. The results revealed that apical buds are distinctive viable tissues for sugarcane transformation and regeneration to produce a large number of CP77-400 transgenic plants in shorter period of time without intervening mosaics and chimeras. The AVP1 transcripts expression in transgenic lines at various levels was detected by RT-PCR. Longer and profuse root system was observed in transgenic plants in comparison with control plants. Concomitantly, only transgenic plants were able to withstand higher NaCl salt stress as well as scarcity of water thus, showing tolerance against salinity and drought stresses.