Different Treatments for Sugarcane Juice Preservation.

This investigation aimed to optimize the time, pH, pressure, and temperature of sugarcane juice pasteurization and to develop a "ready to serve" bottled sugarcane juice with a high preservation efficiency. Fresh sugarcane juice was extracted from sugarcane genotype Co 89003, and beverage samples were collected using three different treatments: sulphitation of juice with the addition of potassium metabisulphite (KMS-25, 50, 100, and 150 ppm), acidification of juice (addition of citric acid, to reduce the pH of the juice to 4.8, 4.5, and 4.25), and steam treatment of the canes (5 min, 10, and 15 min at 7 psi). In all treatments, the juice was pasteurized in glass bottles @ 65 °C for 25 min and stored at low temperature (5 °C) in pre-sterilized glass bottles. Juice properties such as the ˚Brix, total sugar, pH, and total phenolic content decreased with storage, whereas the microbial count, titrable acidity, and reducing sugar content significantly increased during storage. The addition of KMS, citric acid, and the steam treatment reduced the browning of juice and maintained the color of juice during storage, by inhibiting the polyphenol oxidase enzyme activity, from 0.571 unit/mL to 0.1 unit/mL. Among the selected treatments, sugarcane juice with KMS (100 and 150 ppm) and steam treatment of the canes for 5 and 10 min at 7 psi showed the minimum changes in physico-chemical properties, sensory qualities, and restricted microbial growth. Thesulphitation treatment with pasteurization proved best for increasing the shelf life of sugarcane juice upto 90 days with refrigeration. Similarly, the steam-subjected cane juice (10 and 15 min at 7 psi) could be effectively preserved for upto 30 days with refrigeration, without any preservative.

Combining genomic selection with genome-wide association analysis identified a large-effect QTL and improved selection for red rot resistance in sugarcane.

Red rot caused by the fungus Colletotrichum falcatum is the main disease limiting sugarcane productivity in several countries including the major producer India. The genetic basis for red rot resistance is unclear. We studied a panel of 305 sugarcane clones from the Australian breeding program for disease response phenotype and genotype using an Affymetrix® Axiom® array, to better understand the genetic basis of red rot resistance. SNP markers highly significantly associated with red rot response (≤ 10-8) were identified. Markers with largest effect were located in a single 14.6 Mb genomic region of sorghum (the closest diploid relative of sugarcane with a sequenced genome) suggesting the presence of a major-effect QTL. By genomic selection, the estimated selection accuracy was ~0.42 for red rot resistance. This was increased to ~0.5 with the addition of 29 highly significant SNPs as fixed effects. Analysis of genes nearby the markers linked to the QTL revealed many biotic stress responsive genes within this QTL, with the most significant SNP co-locating with a cluster of four chitinase A genes. The SNP markers identified here could be used to predict red rot resistance with high accuracy at any stage in the sugarcane breeding program.

Whole genome analysis and functional characterization of a novel Bacillus thuringiensis (Bt 62) isolate against sugarcane white grub Holotrichia serrata (F).

In this study, we report the whole genome assembly of Bt 62, a novel isolate harbouring cry8 holotype gene identified by us earlier. Sequencing was carried out using a combination of Illumina NextSeq 500 and Oxford Nanopore sequencing Technologies (ONT). The final assembled genome was 6.13 Mb comprising a circular chromosome and four plasmids. The bioassay studies against Holotrichia serrata (F.) (Coleoptera: Scarabaeidae), a polyphagous pest infesting sugarcane and other crops, indicated significant toxicity to first instar grubs over untreated larvae achieving a highest mean mortality of 91.11% for various doses tested. In vitro proteolytic assay and histopathological studies of the midgut of infected white grubs revealed proteolytic processing of the protoxin and extensive degeneration of larval midgut epithelial cells. The results demonstrate that this novel isolate could be used as a biopesticide or its crystal toxin genes could be expressed in sugarcane and other crops for resistance against H. serrata.

Genomic Selection in Sugarcane: Current Status and Future Prospects.

Sugarcane is a C4 and agro-industry-based crop with a high potential for biomass production. It serves as raw material for the production of sugar, ethanol, and electricity. Modern sugarcane varieties are derived from the interspecific and intergeneric hybridization between Saccharum officinarum, Saccharum spontaneum, and other wild relatives. Sugarcane breeding programmes are broadly categorized into germplasm collection and characterization, pre-breeding and genetic base-broadening, and varietal development programmes. The varietal identification through the classic breeding programme requires a minimum of 12-14 years. The precise phenotyping in sugarcane is extremely tedious due to the high propensity of lodging and suckering owing to the influence of environmental factors and crop management practices. This kind of phenotyping requires data from both plant crop and ratoon experiments conducted over locations and seasons. In this review, we explored the feasibility of genomic selection schemes for various breeding programmes in sugarcane. The genetic diversity analysis using genome-wide markers helps in the formation of core set germplasm representing the total genomic diversity present in the Saccharum gene bank. The genome-wide association studies and genomic prediction in the Saccharum gene bank are helpful to identify the complete genomic resources for cane yield, commercial cane sugar, tolerances to biotic and abiotic stresses, and other agronomic traits. The implementation of genomic selection in pre-breeding, genetic base-broadening programmes assist in precise introgression of specific genes and recurrent selection schemes enhance the higher frequency of favorable alleles in the population with a considerable reduction in breeding cycles and population size. The integration of environmental covariates and genomic prediction in multi-environment trials assists in the prediction of varietal performance for different agro-climatic zones. This review also directed its focus on enhancing the genetic gain over time, cost, and resource allocation at various stages of breeding programmes.

Delineation of genotype × environment interaction for identification of stable genotypes for tillering phase drought stress tolerance in sugarcane.

Sugarcane is a trans-seasonal long-duration crop and tillering phase (60-150 days) is the most sensitive phase for moisture stress, causing significant reduction in biomass accumulation. The study focussed to assess the Genotype × Environment Interaction (GEI) for tillering phase moisture stress and to identify the stable genotypes in sugarcane. The study dealt with 14 drought tolerant genotypes and two standards (Co 86032 and CoM 0265) which were evaluated in two plant and one ratoon trials at four locations in Maharashtra, India. The moisture stress was imposed for 60 days from 90 to 150 days after planting and corresponded to tillering phase by withholding the irrigation. The AMMI ANOVA showed significant GEI for cane and CCS yield accounting 18.33 and 19.45 percent of variability respectively. Drought and genotype main effects were highly significant accounting 49.08 and 32.59 percent variability for cane yield and, 52.45 and 28.10 percent variability for CCS yield respectively. The first two interactive principal component (IPCA) biplots of AMMI showed diverse nature of all four environments and the Discriminative vs Mean biplots of Genotype + genotype × environment interaction (GGE) model showed that 'Pune' as the highly discriminating environment. The genotype ranking biplots of GGE showed that Co 85019 was the most stable genotype followed by Co 98017. Similar results were also observed in Yield vs IPCA1 biplot of AMMI, which revealed Co 85019 and Co 98017 as high yielding stable varieties. Yield related environmental maximum (YREM) showed thirteen and nine percent loss due to crossover interactions in Co 85019 for cane yield and CCS yield respectively. The multi-environment BLUP and genotype stability index (GSI) has reaffirmed that Co 85019 as a drought proof and stable genotype with high yield under tillering phase drought stress. The results suggested using Co 85019 for cultivation in drought prone regions and the usefulness of the methodology for identifying more such sugarcane varieties for the benefit of resource poor famers in drought affected regions.

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress.

The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes in the presence of glutathione. Recent studies established the presence of unique DJ-1/PfpI domain containing protein named glyoxalase III (Gly III) in prokaryotes, involved in the detoxification of MG into D-lactic acid through a single step process. In the present study, eleven transgenic sugarcane events overexpressing EaGly III were assessed for salinity stress (100 mM and 200 mM NaCl) tolerance. Lipid peroxidation as well as cell membrane injury remained very minimal in all the transgenic events indicating reduced oxidative damage. Transgenic events exhibited significantly higher plant water status, gas exchange parameters, chlorophyll, carotenoid, and proline content, total soluble sugars, SOD and POD activity compared to wild type (WT) under salinity stress. Histological studies by taking the cross section showed a highly stable root system in transgenic events upon exposure to salinity stress. Results of the present study indicate that transgenic sugarcane events overexpressing EaGly III performed well and exhibited improved salinity stress tolerance.

Biomass potential of novel interspecific and intergeneric hybrids of Saccharum grown in sub-tropical climates.

Sugarcane-derived biomass is a promising source of renewable energy to meet the growing demands for biofuel. Currently, modern sugarcane cultivars are unable to provide enough biomass due to their narrow genetic base and susceptibility to abiotic and biotic stresses. We have evaluated total of 23 hybrids derived from diverse genetic backgrounds of different Saccharum spp. and allied genera, one inbred and compared with commercial checks. Intergeneric hybrids (IGHs) KGS 99-100 and GU 04-432, produced significantly higher biomass (43.37 t ha-1 and 35.24 t ha-1, respectively) than commercial sugarcane have genes derived from Erianthus arundinaceus. Interspecific hybrids (ISHs) GU 07-3704 and 99-489, also produced significantly higher amounts of biomass (37.24 t ha-1 and 33.25 t ha-1, respectively) than commercial checks have genes from S. officinarum and S. spontaneum backgrounds. ISHs recorded significantly higher biomass yield, number of stalks and total dry matter percentage whereas, IGH group recorded significantly higher fibre percent. Furthermore, the clones resistant to red rot and sugarcane borers were identified. The estimated energy value for seven hybrid clones was found to be very high. Cluster analysis of genetic traits revealed two major clusters in traits improving biomass. Our study has revealed that the genetic diversity present in these hybrids could be used for improving biomass production and tolerance to abiotic and biotic stresses in cultivated sugarcanes.

Current breeding and genomic approaches to enhance the cane and sugar productivity under abiotic stress conditions.

Sugarcane (Saccharum spp.) crop is vulnerable to many abiotic stresses such as drought, salinity, waterlogging, cold and high temperature due to climate change. Over the past few decades new breeding and genomic approaches have been used to enhance the genotypic performance under abiotic stress conditions. In sugarcane, introgression of genes from wild species and allied genera for abiotic stress tolerance traits plays a significant role in the development of several stress-tolerant varieties. Moreover, the genomics and transcriptomics approaches have helped to elucidate the key genes/TFs and pathways involved in abiotic stress tolerance in sugarcane. Several novel miRNAs families /proteins or regulatory elements that are responsible for drought, salinity, and cold tolerance have been identified through high-throughput sequencing. The existing sugarcane monoploid genome sequence information opens new gateways and opportunities for researchers to improve the desired traits through efficient genome editing tools, such as the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system. TALEN mediated mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane significantly reduces the lignin content in the cell wall which is amenable for biofuel production from lignocellulosic biomass. In this review, we focus on current breeding with genomic approaches and their substantial role in enhancing cane production under the abiotic stress conditions, which is expected to provide new insights to plant breeders and biotechnologists to modify their strategy in developing stress-tolerant sugarcane varieties, which can highlight the future demand of cane, bio-energy, and viability of sugar industries.

Ectopic expression of DJ-1/PfpI domain containing Erianthus arundinaceus Glyoxalase III (EaGly III) enhances drought tolerance in sugarcane.

KEY MESSAGE: Sugarcane transgenic overexpressing EaGly III from Erianthus arundinaceus showed enhanced water deficit stress tolerance. Methylglyoxal (MG), an α-ketoaldehyde formed from either glycolysis or TCA cycle, is capable of causing total cellular damage via the generation of reactive oxygen species (ROS), advanced glycation end products (AGEs) and nucleic acid degradation. Glyoxalase pathway is a ubiquitous pathway known for detoxification of MG, involving key enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Recently, a novel and an additional enzyme in glyoxalase pathway, viz., glyoxalase III (Gly III), has been discovered which possesses DJ-1/PfpI domain recognized for detoxifying MG in a single step process without requirement of any coenzyme. In the present study, a Gly III gene isolated from Erianthus arundinaceus, a wild relative of sugarcane, overexpressed in commercially cultivated sugarcane hybrid Co 86032 was assessed for drought tolerance. Morphometric observations revealed that transgenic sugarcane overexpressing EaGly III acquired drought tolerance trait. Oxidative damage caused by triggering generation of ROS has been determined to be low in transgenic plants as compared to wild type (WT). Transgenics resulted in higher relative water content, chlorophyll content, gas exchange parameters, photosynthetic efficiency, proline content and soluble sugars upon water deficit stress. In addition, higher and stable level of superoxide dismutase and peroxidase activities were observed along with minimal lipid peroxidation during drought stress signifying the tolerance mechanism exhibited by transgenic events. There was no significant structural change observed in the root anatomy of transgenic plants. Altogether, EaGly III gene could be considered as a potential candidate for conferring water deficit stress tolerance for sugarcane and other agricultural crops.

Isolation, characterization and expression analysis of stress responsive plant nuclear transcriptional factor subunit (NF-YB2) from commercial Saccharum hybrid and wild relative Erianthus arundinaceus.

Plant nuclear factor (NF-Y) is a transcription activating factor, consisting of three subunits, and plays a key regulatory role in many stress-responsive mechanisms including drought and salinity stresses. NF-Ys function both as complex and individual subunits. Considering the importance of sugarcane as a commercial crop with high socio-economic importance and the crop being affected mostly by water deficit stress and salinity stress causing significant yield loss, nuclear transcriptional factor NF-YB2 was focused in this study. Plant nuclear factor subunit B2 from Erianthus arundinaceus (EaNF-YB2), a wild relative of sugarcane which is known for its drought and salinity stress tolerance, and commercial Saccharum hybrid Co 86032 (ShNF-YB2) was isolated and characterized. Both EaNF-YB2 and ShNF-YB2 genes are 543 bp long that encodes for a polypeptide of 180 amino acid residues. Comparison of EaNF-YB2 and ShNF-YB2 gene sequences revealed nucleotide substitutions at nine positions corresponding to three synonymous and six nonsynonymous amino acid substitutions that resulted in variations in physiochemical properties. However, multiple sequence alignment (MSA) of NF-YB2 proteins showed conservation of functionally important amino acid residues. In silico analysis revealed NF-YB2 to be a hydrophilic and intracellular protein, and EaNF-YB2 is thermally more stable than that of ShNF-YB2. Phylogenetic analysis suggested the lower rate of evolution of NF-YB2. Subcellular localization in sugarcane callus revealed NF-YB2 localization at nucleus that further evidenced it to be a transcription activation factor. Comparative RT-qPCR experiments showed a significantly higher level of NF-YB2 expression in E. arundinaceus when compared to that in the commercial Saccharum hybrid Co 86032 under drought and salinity stresses. Hence, EaNF-YB2 could be an ideal candidate gene, and its overexpression in sugarcane through genetic engineering approach might enhance tolerance to drought and salinity stresses.

Isolation and characterization of water-deficit stress-responsive α-expansin 1 (EXPA1) gene from Saccharum complex.

In this study, full-length (1282-1330 bp) α-expansin 1 (EXPA1) gene from three different accessions belonging to Saccharum complex (Saccharum officinarum-SoEXPA1, Erianthus arundinaceus-EaEXPA1, and Saccharum spp. hybrid-ShEXPA1) was isolated using RAGE technique and characterized. The intronic and coding regions of isolated expansin genes ranged between 526-568 and 756-762 bp, respectively. An open reading frame encoding a polypeptide of 252 amino acids was obtained from S. officinarum and commercial sugarcane hybrid, whereas 254 amino acids were obtained in E. arundinaceus, a wild relative of Saccharum. Bioinformatics analysis of deduced protein revealed the presence of specific signature sequences and conserved amino acid residues crucial for the functioning of the protein. The predicted physicochemical characterization showed that the protein is stable in nature with instability index (II) value less than 40 and also clearly shown the dominance of random coil in the protein structure. Phylogenetic analysis revealed high conservation of EXPA1 among Saccharum complex and related crop species, Sorghum bicolor and Zea mays. The docking study of EXPA1 protein showed the interaction with xylose, which is present in xyloglucan of plant cell wall, elucidated the role of the expansin proteins in plant cell wall modification. This was further supported by the subcellular localization experiment in which it is clearly seen that the expansin protein localizes in the cell wall. Relative expression analysis of EXPA1 gene in Saccharum complex during drought stress showed high expression of the EaEXPA1 in comparison with SoEXPA1 and ShEXPA1 indicating possible role of EaEXPA1 in increased water-deficit stress tolerance in E. arundinaceus. These results suggest the potential use of EXPA1 for increasing the water-deficient stress tolerance levels in crop plants.

Comparative analysis of glyoxalase pathway genes in Erianthus arundinaceus and commercial sugarcane hybrid under salinity and drought conditions.

BACKGROUND: Glyoxalase pathway is a reactive carbonyl species (RCS) scavenging mechanism involved in the detoxification of methylglyoxal (MG), which is a reactive α-ketoaldehyde. In plants under abiotic stress, the cellular toxicity is reduced through glyoxalase pathway genes, i.e. Glyoxalase I (Gly I), Glyoxalase II (Gly II) and Glyoxalase III (Gly III). Salinity and water deficit stresses produce higher amounts of endogenous MG resulting in severe tissue damage. Thus, characterizing glyoxalase pathway genes that govern the MG metabolism should provide new insights on abiotic stress tolerance in Erianthus arundinaceus, a wild relative of sugarcane and commercial sugarcane hybrid (Co 86032). RESULTS: In this study, three glyoxalase genes (Glyoxalase I, II and III) from E. arundinaceus (a wild relative of sugarcane) and commercial sugarcane hybrid (Co 86032) were characterized. Comparative gene expression profiles (qRT-PCR) of Glyoxalase I, II and III under salinity and water deficit stress conditions revealed differential transcript expression with higher levels of Glyoxalase III in both the stress conditions. Significantly, E. arundinaceus had a higher expression level of glyoxalase genes compared to commercial sugarcane hybrid. On the other hand, gas exchange parameters like stomatal conductance and transpiration rate were declined to very low levels under both salt and drought induced stresses in commercial sugarcane hybrid when compared to E. arundinaceus. E. arundinaceus maintained better net photosynthetic rate compared to commercial sugarcane hybrid. The phylogenetic analysis of glyoxalase proteins showed its close evolutionary relationship with Sorghum bicolor and Zea mays. Glyoxalase I and II were predicted to possess 9 and 7 isoforms respectively whereas, Glyoxalase III couldn't be identified as it comes under uncharacterized protein identified in recent past. Chromosomal mapping is also carried out for glyoxalase pathway genes and its isoforms. Docking studies revealed the binding affinities of glyoxalase proteins in both E. arundinaceus and commercial sugarcane hybrid with their substrate molecules. CONCLUSIONS: This study emphasizes the role of Glyoxalase pathway genes in stress defensive mechanism which route to benefit in progressive plant adaptations and serves as potential candidates for development of salt and drought tolerant crops.

Differential gene expression profiling through transcriptome approach of Saccharum spontaneum L. under low temperature stress reveals genes potentially involved in cold acclimation.

Sugarcane (Saccharum sp.) is predominantly grown in both tropics and subtropics in India, and the subtropics alone contribute more than half of sugarcane production. Sugarcane active growth period in subtropics is restricted to 8-9 months mainly due to winter's low temperature stress prevailing during November to February every year. Being a commercial crop, tolerance to low temperature is important in sugarcane improvement programs. Development of cold tolerant sugarcane varieties require a deep knowledge on molecular mechanism naturally adapted by cold tolerant genotypes during low temperature stress. To understand gene regulation under low temperature stress, control and stressed (10 °C, 24 h) leaf samples of cold tolerant S. spontaneum IND 00-1037 collected from high altitude region in Arunachal Pradesh were used for transcriptome analysis using the Illumina NextSeq 500 platform with paired-end sequencing method. Raw reads of 5.1 GB (control) and 5.3 GB (stressed) obtained were assembled using trinity and annotated with UNIPROT, KEGG, GO, COG and SUCEST databases, and transcriptome was validated using qRT-PCR. The differential gene expression (DGE) analysis showed that 2583 genes were upregulated and 3302 genes were down-regulated upon low temperature stress. A total of 170 cold responsive transcriptional factors belonging to 30 families were differentially regulated. CBF6 (C-binding factor), a DNA binding transcriptional activation protein associated with cold acclimation and freezing tolerance was differentially upregulated. Many low temperature responsive genes involved in various metabolic pathways, viz. cold sensing through membrane fluidity, calcium and lipid signaling genes, MAP kinases, phytohormone signaling and biosynthetic genes, antioxidative enzymes, membrane and cellular stabilizing genes, genes involved in biosynthesis of polyunsaturated fatty acids, chaperones, LEA proteins, soluble sugars, osmoprotectants, lignin and pectin biosynthetic genes were also differentially upregulated. Potential cold responsive genes and transcriptional factors involved in cold tolerance mechanism in cold tolerant S. spontaneum IND 00-1037 were identified. Together, this study provides insights into the cold tolerance to low temperature stress in S. spontaneum, thus opening applications in the genetic improvement of cold stress tolerance in sugarcane.

De novo sequencing and transcriptome analysis of a low temperature tolerant Saccharum spontaneum clone IND 00-1037.

Saccharum spontaneum L., a wild relative of sugarcane, is known for its adaptability to environmental stresses, particularly cold stress. In the present study, an attempt was made for transcriptome profiling of the low temperature (10°C) tolerant S. spontaneum clone IND 00-1037 collected from high altitude regions of Arunachal Pradesh, North Eastern India. The Illumina Nextseq500 platform yielded a total of 47.63 and 48.18 million reads corresponding to 4.7 and 4.8 gigabase pairs (Gb) of processed reads for control and cold stressed (10°C for 24h) samples, respectively. These reads were de novo assembled into 214,611 unigenes with an average length of 801bp. Further, all unigenes were aligned to GO, KEGG and COG databases in order to identify novel genes and pathways responsive upon low temperature conditions. The differential gene expression analysis revealed that about 2583 genes were upregulated and 3302 genes were down regulated during the stress. This is perhaps the comprehensive transcriptome data of a low temperature tolerant clone of S. spontaneum. This study would aid in identifying novel genes and also in future genomic studies pertaining to sugarcane and its wild relatives.