Commercial Sugarcane Hybrids Research Articles

Overexpression of EaALDH7, an aldehyde dehydrogenase gene from Erianthus arundinaceus enhances salinity tolerance in transgenic sugarcane (Saccharum spp. Hybrid)

Aldehyde Dehydrogenases (ALDH), a group of enzymes, are associated with the detoxification of aldehydes, produced in plants during abiotic stress conditions. Salinity remains a pivotal abiotic challenge that poses a significant threat to cultivation and yield of sugarcane. In this study, an Aldehyde dehydrogenase gene (EaALDH7) from Erianthus arundinaceus was overexpressed in the commercial sugarcane hybrid cultivar Co 86032. The transgenic lines were evaluated at different NaCl concentrations ranging from 0 mM to 200 mM for various morpho-physiological and biochemical parameters. The control plants, subjected to salinity stress condition, exhibited morphological changes in protoxylem, metaxylem, pericycle and pith whereas the transgenic events were on par with plants under regular irrigation. The overexpressing (OE) lines showed less cell membrane injury and improved photosynthetic rate, transpiration rate, and stomatal conductance than the untransformed control plants under stress conditions. Elevated proline content, higher activity of enzymatic antioxidants such as sodium dismutase (SOD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) and low level of malondialdehyde MDA and hydrogen peroxide (H2O2) in the transgenic lines. The analysis of EaALDH7 expression revealed a significant upregulation in the transgenic lines compared to that of the untransformed control during salt stress conditions. The current study highlights the potentials of EaALDH7 gene in producing salinity-tolerant sugarcane cultivars.

Long read transcriptome sequencing of a sugarcane hybrid and its progenitors, Saccharum officinarum and S. spontaneum.

Commercial sugarcane hybrids are derivatives from Saccharum officinarum and Saccharum spontaneum hybrids containing the full complement of S. officinarum and a few S. spontaneum chromosomes and recombinants with favorable agronomic characters from both the species. The combination of the two sub-genomes in varying proportions in addition to the recombinants presents a challenge in the study of gene expression and regulation in the hybrid. We now report the transcriptome analysis of the two progenitor species and a modern commercial sugarcane hybrid through long read sequencing technology. Transcripts were profiled in the two progenitor species S. officinarum (Black Cheribon), and S. spontaneum (Coimbatore accession) and a recent high yielding, high sugar variety Co 11015. The composition and contribution of the progenitors to a hybrid with respect to sugar, biomass, and disease resistance were established. Sugar related transcripts originated from S. officinarum while several stress and senescence related transcripts were from S. spontaneum in the hybrid. The hybrid had a higher number of transcripts related to sugar transporters, invertases, transcription factors, trehalose, UDP sugars, and cellulose than the two progenitor species. Both S. officinarum and the hybrid had an abundance of novel genes like sugar phosphate translocator, while S. spontaneum had just one. In general, the hybrid shared a larger number of transcripts with S. officinarum than with S. spontaneum, reflecting the genomic contribution, while the progenitors shared very few transcripts between them. The common isoforms among the three genotypes and unique isoforms specific to each genotype indicate that there is a high scope for improvement of the modern hybrids by utilizing novel gene isoforms from the progenitor species.

Comparative transcriptome profiling to unravel the key molecular signalling pathways and drought adaptive plasticity in shoot borne root system of sugarcane

Sugarcane root system comprises of superficial sett roots as well as deeply-penetrating shoot borne roots (SBR) with latter being the permanent root system. In sugarcane, the healthy SBR contributes to a better crop yield and it also helps to produce multiple ratoon crops after the harvest. There is a dearth of in-depth knowledge on SBR system architecture and its functional role in modern day commercial hybrids. A comprehensive phenotypic, anatomical and whole transcriptome profiling, conducted between the commercial sugarcane hybrids and a wild germplasm Erianthus, found a developmental delay in both initiation and establishment of the SBR in commercial hybrid compared to Erianthus. The SBR system in Erianthus proved to be an extensive drought-adaptive root system architecture that significantly contributes to drought tolerance. On the other hand, SBRs in the commercial hybrids showed an irreversible collapse and damage of the root cells under drought stress. The outcomes from the comparative analysis of the transcriptome data showed a significant upregulation of the genes that regulate important stress signalling pathways viz., sugar, calcium, hormone signalling and phenylpropanoid biosynthesis in the SBRs of Erianthus. It was found that through these key signalling pathways, Erianthus SBRs triggered the downstream signalling cascade to impart physiological responses like osmoprotection, modification of the cell walls, detoxification of reactive oxygen species, expression of drought responsive transcription factors, maintenance of cell stability and lateral root development. The current study forms a basis for further exploration of the Shoot Borne Root system as a valuable breeding target to develop drought tolerant sugarcane genotypes.

Diurnal metabolism of energy-cane and sugarcane: A metabolomic and non-structural carbohydrate analysis

Energy-cane is a cross breed between commercial sugarcane and S. spontaneum, a hybrid that has higher fiber content, low sucrose and greater productivity compared to commercial sugarcane hybrids. It has been recently shown that diurnal and seasonal changes impact several physiological characteristics of sugarcane. However, little is known about how the diurnal cycle affects the production of carbohydrates and metabolites in energy-cane. In the present study, we investigate the diurnal cycle of adult sugarcane and energy-cane by analyzing the carbohydrates and metabolites profiles of leaves and internodes. Our results show fluctuations in the soluble sugars content (sucrose, glucose and fructose) and the metabolites profile throughout of day, which indicates different growth strategies and carbon use among these cultivars. In addition, a high concentration of starch was observed in the mature internodes of energy-cane. Some biological pathways related to biomass production, such as the phenylpropanoid pathway, were more active in energy-cane compared to sugarcane. Our results reveal that sugarcane and energy-cane metabolism are different throughout the day, a better understanding of the metabolism of these cultivars opens the possibility of creating more efficient plants through breeding.

DdRADseq-mediated detection of genetic variants in sugarcane.

The article presents an optimization of the key parameters for the identification of SNPs in sugarcane using a GBS protocol based on two Illumina NextSeq and NovaSeq platforms. Sugarcane (Saccharum sp.), a world-wide known feedstock for sugar production, bioethanol, and energy, has an extremely complex genome, being highly polyploid and aneuploid. A double-digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-fibre biotype for the detection of single nucleotide polymorphisms (SNPs). In this work we tested two Illumina sequencing platforms, read size (70 vs. 150bp), different sequencing coverage per individual (medium and high coverage), and single-reads versus paired-end reads. We also explored different variant calling strategies (with and without reference genome) and filtering schemes [combining two minor allele frequencies (MAFs) with three depth of coverage thresholds]. For the discovery of a large number of novel SNPs in sugarcane, we recommend longer size and paired-end reads, medium sequencing coverage per individual and Illumina platform NovaSeq6000 for a cost-effective approach, and filter parameters of lower MAF and higher depth coverages thresholds. Although the de novo analysis retrieved more SNPs, the reference-based method allows downstream characterization of variants. For the two best performing matrices, the number of SNPs per chromosome correlated positively with chromosome length, demonstrating the presence of variants throughout the genome. Multivariate comparisons, with both matrices, showed closer relationships among commercial hybrids than with the high-fibre biotype. Functional analysis of the SNPs demonstrated that more than half of them landed within regulatory regions, whereas the other half affected coding, intergenic and intronic regions. Allelic distances values were lower than 0.07 when analysing two replicated genotypes, confirming the protocol robustness.

CHARACTERIZATION AND IN SILICO ANALYSES OF RTCS GENE FROM SUGARCANE ENCODING LOB PROTEIN FAMILY OF TRANSCRIPTION FACTORS: A KEY REGULATOR OF SHOOT-BORNE ROOT INITIATION

RTCS (Rootless concerning Crown and Seminal roots) is a member of the plant-specific LATERAL ORGAN BOUNDARIES (LOB) domain (LBD) protein family of transcription factors. The RTCS gene has been identified as a key regulator of shoot borne root initiation in maize. In this study an attempt has been made to characterize RTCS from a wild related genus of sugarcane Erianthus arundinaceus and compared to the RTCS gene isolated from commercial sugarcane hybrid genotypes. The candidate gene has been successfully isolated from two genotypes of E. arundinaceus (SES 288 and IND 04- 1335: EaRTCS) and two commercial sugarcane hybrid genotypes (Co 775 and Co 86032: ShRTCS). The isolated RTCS full length gene showed significant variation between EaRTCS and ShRTCS at the DNA and protein level. The RTCS amino acid sequence of all the four genotypes indicated the presence of the characteristic structural features of LBD proteins such as a C-motif, LOB domain sequence and leucine zipper-like dimerization domain. Prediction and analysis of secondary structure of RTCS protein in SES 288 and IND 04-1335 showed the presence of 65% alpha helix, 1% β strand and 38% disorder, while sugarcane commercial genotypes showed 58 % alpha helix, 1% β strand and 47% disorder. Both the sequences of EaRTCS and ShRTCS was found to be closely related to Zea mays RTCS.

Irrigation Water Use Efficiency and Water Productivity of Commercial Sugarcane Hybrids under Water-Limited Conditions

HighlightsSugarcane hybrids with improved IWUE have greater scope in sugarcane agriculture as irrigation water is getting scarce.Among sugarcane hybrids, Co 8371 registered high mean water productivity of 4.18 kg m-3, followed by Co 85019 (3.92 kg m-3), while in I2, six hybrids had significantly higher water productivity (Co 85019, Co 0212, Co 86249, Co 10026, Co 0218 and Co V92102) above 4 kg m-3.Deficit irrigation scheduling (irrigation at recommended interval, with 50% crop evapotranspiration replacement) appears to be far more useful than reducing frequency as well as quantity of irrigation water alone. Hybrid mean water productivity was 3.2, 2.7, and 2.1 kg m-3 in I0, I1, and I2, respectively.ABSTRACT. The escalating deficit rainfall scenario in India indicates that drought is a recurrent phenomenon associated with tropical sugarcane farming, and the availability of irrigation water for sugarcane cultivation will be much less in coming years. To meet the challenge of limited and costly water supply, tropical sugarcane growers will have to find ways of increasing the efficiency of irrigation to maintain high cane yields. More efficient irrigation systems, accurate irrigation scheduling, and the right choice of sugarcane hybrids are potential means of increasing irrigation water use efficiency (IWUE), water productivity (WP), and global water security. With the objective of optimizing irrigation water use, a field experiment evaluating the physiological efficiency of commercial sugarcane hybrids for WP in a sandy clay soil under water-limited conditions was conducted during 2016-2017 at the ICAR-Sugarcane Breeding Institute in Coimbatore, India. The replicated field experiment was laid out in split-plot design with three irrigation levels as the main plot and 33 sugarcane hybrids as subplots. The prevailing climatic conditions during the experiment represented a tropical wet and dry climate, with the wet season lasting from October to December due to the northeast monsoon. The results showed that full irrigation at recommended intervals with 100% crop evapotranspiration (ET) replacement (I0) produced significantly higher cane yield than deficit irrigation at recommended intervals with 50% crop ET replacement (I1) and skipping alternate irrigations with 50% crop ET replacement (I2). The deficit irrigation treatments (I1 and I2) had declines in cane yield of 41.2% and 56.4%, respectively. IWUE was similar in I0 and I1, while I2 had reduced IWUE by 23%. WP was significantly influenced by irrigation level; reduction in irrigation water reduced WP by 17.5% and 36.3% in I1 and I2 compared to I0. Among sugarcane hybrids, Co 85019, Co 13006, Co 10026, Co 99004, CoLk 8102, Co 86249, Co 8371, Co 94008, and Co 95020 yielded higher than the genotypic mean under both deficit irrigation treatments, suggesting their usefulness in deficit irrigation strategies. Sugarcane hybrids with high WP can play a pivotal role in sustaining sugarcane productivity and can reduce the large volumes of irrigation water consumed in water-scarce tropical India. Thus, considering water security, the implications of the results are of paramount importance in promoting the coordinated development and management of water, land, and related resources to maximize economic benefits and social welfare in an equitable manner without compromising the sustainability of vital ecosystems at local as well as national levels. Keywords: Cane yield, Global water security, Sugarcane, Water-limited condition.

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

In this study, full-length (1282-1330bp) 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 756-762bp, 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

BackgroundGlyoxalase 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).ResultsIn 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.ConclusionsThis 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.

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.

Nuclear and cytoplasmic contributions from Erianthus arundinaceus (Retz.) Jeswiet in a sugarcane hybrid clone confirmed through genomic in situ hybridization and cytoplasmic DNA polymorphism

A hybrid between Erianthus arundinaceus (Retz.) Jeswiet and Saccharum spontaneum L. which are wild related species of sugarcane (Saccharum L., Family Poaceae), was repeatedly crossed as female parent with sugarcane commercial varieties to develop near commercial sugarcane clones. The cytoplasm type of the hybrid derivatives were confirmed to be of E. arundinaceus through the mitochondrial and chloroplast DNA polymorphism of nad 4/3-4 intron segment and psbC–trnS segment, respectively. The E. arundinaceus × S. spontaneum hybrid with somatic chromosome number 2n = 62 was confirmed to have 30 chromosomes from E. arundinaceus through genomic in situ hybridization (GISH). The (E. arundinaceus × S. spontaneum) × sugarcane hybrid (2n = 118) had 24 chromosomes from E. arundinaceus whereas its next generation hybrid with sugarcane (2n = 108) had only 12 Erianthus chromosomes. The commercial sugarcane hybrid Co 15015, which is the third generation hybrid with 2n = 106 was confirmed to have two E. arundinaceus chromosomes through GISH. It is the first report of sugarcane with both alien cytoplasm and chromosome contributions from E. arundinaceus.

Transcriptome Analysis and Functional Genomics of Sugarcane

Sugarcane is a genetically complex polyploid with increasing economic importance as a feedstock for biofuel production. The characterization of sugarcane genes and their association with biological traits such as sugar accumulation, biomass yield and stress tolerance has so far primarily relied on studies of the sugarcane transcriptome. Associations of gene expression with biological traits have been based on alterations in the timing and intensity of gene expression with various treatments and developmental stages, and in some instances, by genotypic correlations using segregating populations or genotypes contrasting for traits. The transcriptome of sugarcane is complex and includes transcripts of homo(eo)logues reflecting the highly polyploid genome of commercial sugarcane hybrids derived recently from two Saccharum species. The impact of this genomic complexity on transcription will be greatly informed by data emerging from the International Sugarcane Genome Sequencing Project. Increasingly, expressed gene sequences are exploited as genetic markers for traits in genome mapping and association studies. Reverse genetics approaches are possible in sugarcane but only very few sugarcane genes have had their specific functions examined in transgenic sugarcane plants. An integration of genome, transcriptome and metabolome data is emerging that will inform the molecular breeding of this important sugar and energy crop.

Genetic diversity in sugarcane hybrids (Saccharum spp complex) grown in tropical India based on STMS markers

Understanding the pattern of diversity among the commercial sugarcane hybrids is highly useful to sugarcane breeders in planning and broadening the genetic base. Genetic analysis of 22 cultivated sugarcane hybrids representing all agro-eco climatic regions of tropical India was carried out using ten sequence tagged microsatellite sites (STMS) primers. A total of 127 markers were amplified, of which 78.74% were polymorphic with an average of 10 polymorphic products per STMS primer. Jaccard’s similarity coefficient value estimated between closely related hybrids was 0.889 while the lowest coefficient value of 0.574 was detected with distantly related hybrids. The average genetic similarity among the hybrids was ~84.8%. These results indicated the existence of low level of genetic diversity among the commercial hybrids under cultivation. Unweighted pair group method with arithmetic averages (UPGMA) cluster analysis detected five major clusters. Cluster II consisted of four varieties which had Co 775 as one of the parents. Variety CoC 671 and its somaclone Co 94012 were grouped into cluster IIa. Varieties grouped in the cluster III had either CoC 671 or Co 775 in their genealogy indicating the influence of parental genome contribution to clustering. Varieties developed for east coast zone were grouped in the clusters IIb, IIIb, IIIc and IVe which indicated the influence of adaptation of varieties to particular agro-climatic condition. The study also identified 12 unique markers which can be useful in varietal identification and rouging in seed plot.

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

BackgroundMicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.ResultsIn this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species S. officinarum and S. spontaneum. Using sugarcane MIR827 as a query, we found a novel MIR827 precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as SsCBP1. SsCBP1 was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of SsCBP1 by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots.ConclusionsUsing publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.

Genetic transformation of the euploid Saccharum officinarum via direct and indirect embryogenesis

The genetic nature of foreign gene transfer and integration into commercial sugarcane hybrids with complex genetic backgrounds is unclear and unpredictable. A pure genetic background in genetic engineering approaches might lead to more predictable results within Saccharum officinarum than in a hybrid genome. S. officinarum plants (Badila and Black Cheribon genotypes) expressing a reporter gene was regenerated from microprojectile-bombarded leaf disks and callus. The concentration of auxin added to the basal media, both prior to bombardment and during regeneration, significantly affected callus induction and somatic embryogenesis. A ten-fold decrease in 2,4-D concentration resulted in somatic embryos forming directly from sugarcane leaf disks. Somatic embryos were produced via direct embryogenesis from 7 days old Black Cheribon leaf disks on 0.5 mg/l 2,4-D. A callus interface initiated on 3 mg/l 2,4-D from Badila leaf disks was preferred for indirect embryogenesis to produce the maximum number of transgenic Badila plants. These approaches lead to the successful establishment of genetic transformation systems for two S. officinarum genotypes that could be used in the future for gene function studies and breeding purposes.

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 ‘outgroup’ varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.

Plant regeneration from a cryopreserved embryogenic cell suspension of a commercial sugarcane hybrid (Saccharum sp.)

Efficient plant regeneration was obtained from a cryopreserved embryogenic cell suspension of sugarcane established from leaf derived callus. Pregrowing the cells for three days in MS basal medium supplemented with 0.33 M sorbitol was essential to the process. The cells were cooled at a rate of 0.5°C/min to -40°C and then stored in liquid nitrogen. Thawing was carried out rapidly in water at +40°C, and the cells were then plated without washing onto filter paper discs placed on a semi-solid regeneration medium (MS basal + 3% sucrose + 0.13 mg/1 2,4-D +0.25 mg/1 BAP + 0.25 mg/1 kinetin + 0.25 mg/1 zeatin). The filter paper discs, along with the cells, were transferred to the same, fresh medium after five hours. After 24 hours the cells were scraped off, placed on fresh semi-solid medium and incubated at 28°C in the dark for two weeks before transfer to light. A regeneration efficiency of 92% was obtained (regenerated plants, expressed as a percent of unfrozen control). Plants regenerated from cryopreserved cells, and grown to maturity in the greenhouse, were morphologically identical to regenerated control plants.

Colchicine‐Induced Polyploids from Cell Suspension Cultures of Sugarcane1

A high percentage of endoreduplicated chromosomes resulted when cell suspension cultures of sugarcane (Saccharum officinarum L.) were treated with colchicine. Cytological examination of 46 plants from H 57‐1627 callus differentiated from the colchicine‐treated cells showed 22 to be polyploids, 2 to be mixoploids, and 22 to be diploids. This is the first report of chromosome doubling in commercial sugarcane hybrids. The proposed method of treating cells in suspension with colchicine should prove beneficial to sugarcane breeders and other breeders interested in obtaining polyploid plants.