In vitro propagation of a Saccharum officinarum (L.) and Sclerostachya fusca (Roxb.) A. Camus hybrid

Summary Callus induction and plant differentiation were obtained in an intergeneric hybrid of Saccharum officinarum and Sclerostachya fusca. The sub clones showed morphological variation. Chromosome numerical variation was not observed but structural aberrations were noticed in some sub clones. The study indicates the use of tissue culture technique for inducing intergeneric gene transfer in Saccharum hybrids.     

Direct plant regeneration from cultured young leaf segments of sugarcane

Young leaf segments (1.0–1.5 cm) excised from spindle explants of three commercial sugarcane varieties viz. Co J 64, Co J 83 and Co J 86 were cultured on different media compositions based on Murashige and Skoog (1962) salts. Cultured explants exhibited swelling followed by direct shoot regeneration on media containing naphthaleneacetic acid, in all the three varieties. Highest frequency 83.12% shoot regeneration occurred on Murashige and Skoog medium supplemented with naphthaleneacetic acid (5.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹) in variety Co J 83. Medium devoid of naphthaleneacetic acid and supplemented with only kinetin did not induce direct shoot regeneration in any of the varieties thus tried. Subsequently profuse rooting of shoots was observed on the same medium and complete plantlets were recovered within 6 weeks. The plantlets were hardened and transferred to soil. Tissue culture derived field-grown plants were normal and exhibited faster growth and better tillering. This developed single step method of direct plant regeneration can be used for rapid mass cloning and genetic transformation of sugarcane.     

Molecular Identification and Phylogenetic Analysis of Sugarcane Yellow Leaf Phytoplasma (SCYLP) in Egypt

Samples of sugarcane leaves were collected from different commercial fields and breeding stations in Egypt. Aetiology of sugarcane phytoplasma disease was investigated using nested PCR. Phytoplasma‐specific primers (P1/P7 and R16F2n/R16R2) were used to amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism analyses revealed that the tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group. Phylogenetic analyses of 60 screened accessions of 16S ribosomal RNA gene sequences of Candidatus phytoplasmas comprising those collected from Egypt (this study) and those extracted from GenBank showed that they split into two distinct clusters. All the phytoplasmas form a stable phylogenetic subcluster, as judged by branch length and bootstrap values of 100% in the 16S group cluster. Results of phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. Conversely, based on the analysis of the 16S‐23S region, examined isolates segregated into four different clusters suggesting a notable heterogeneity between them. These results are the first record of the presence of phytoplasma in association with sugarcane yellow leaf in Egypt.     

Charge balance and acidity regulation during growth of sugar-cane cell suspensions

Abstract. An analysis of nutrient uptake by batch cultures of sugar-cane cells was performed to gain information about the ionic balance during uptake of charged metabolites. Whereas younger cultures (up to 1 week old) have to compensate excess cation influx with proton efflux, older cultures show balanced cation–anion uptake.
Younger cells produce a small amount of carboxylic acids to furnish protons for charge compensation at the cytoplasmic membrane. Older cells synthesize organic acids more abundantly to generate protons necessary for the proton demand of nitrate and sulphate assimilation. Despite these assimilation reactions only a small percentage of carbon, which is taken up mainly as hexose, needs to be oxidized to carboxylic acids for that purpose. In contrast, younger cultures preferentially use the amino acids of the medium instead of assimilating nitrate. The use of amino acids as a nitrogen source does not require a significant part of metabolism for biochemical pH-stability, whereas an efficient proton circulation on the cytoplasmic membrane seems to be of major importance.
A balance study of the main metabolized elements, carbon, nitrogen and sulphur was performed to get a quantitative impression of the fate of these nutrients during growth of cell cultures.     

Three founding ancestral genomes involved in the origin of sugarcane

Background and AimsModern sugarcane cultivars (Saccharum spp.) are high polyploids, aneuploids (2n = ~12x = ~120) derived from interspecific hybridizations between the domesticated sweet species Saccharum officinarum and the wild species S. spontaneum.MethodsTo analyse the architecture and origin of such a complex genome, we analysed the sequences of all 12 hom(oe)ologous haplotypes (BAC clones) from two distinct genomic regions of a typical modern cultivar, as well as the corresponding sequence in Miscanthus sinense and Sorghum bicolor, and monitored their distribution among representatives of the Saccharum genus.Key ResultsThe diversity observed among haplotypes suggested the existence of three founding genomes (A, B, C) in modern cultivars, which diverged between 0.8 and 1.3 Mya. Two genomes (A, B) were contributed by S. officinarum; these were also found in its wild presumed ancestor S. robustum, and one genome (C) was contributed by S. spontaneum. These results suggest that S. officinarum and S. robustum are derived from interspecific hybridization between two unknown ancestors (A and B genomes). The A genome contributed most haplotypes (nine or ten) while the B and C genomes contributed one or two haplotypes in the regions analysed of this typical modern cultivar. Interspecific hybridizations likely involved accessions or gametes with distinct ploidy levels and/or were followed by a series of backcrosses with the A genome. The three founding genomes were found in all S. barberi, S. sinense and modern cultivars analysed. None of the analysed accessions contained only the A genome or the B genome, suggesting that representatives of these founding genomes remain to be discovered.ConclusionsThis evolutionary model, which combines interspecificity and high polyploidy, can explain the variable chromosome pairing affinity observed in Saccharum. It represents a major revision of the understanding of Saccharum diversity.     

Simultaneous Cellulase Production, Saccharification and Detoxification Using Dilute Acid Hydrolysate of S. spontaneum with Trichoderma reesei NCIM 992 and Aspergillus niger

Bioethanol production from lignocellulosic materials has several limitations. One aspect is the high production cost of cellulases used for saccharification of substrate and inhibition of fermenting yeast due to inhibitors released in acid hydrolysis. In the present work we have made an attempt to achieve simultaneous cellulases production, saccharification and detoxification using dilute acid hydrolysate of Saccharum spontaneum with and without addition of nutrients, supplemented with acid hydrolyzed biomass prior to inoculation in one set and after 3 days of inoculation in another set. Organisms used were T. reesei NCIM 992, and Aspergillus niger isolated in our laboratory. Cellulase yield obtained was 0.8 IU/ml on fourth day with T. reesei. Sugars were found to increase from fourth to fifth day, when hydrolysate was supplemented with nutrients and acid hydrolyzed biomass followed by inoculation with T. reesei. Phenolics were also found to decrease by 67%.     

Characterisation of an ethyl methanesulfonate-derived drought-tolerant sugarcane mutant line

Sustainable sugarcane production in areas prone to frequent and severe drought can be achieved by creating resilient sugarcane varieties. In this study, a unique mutant line, M9.2, was generated from a drought susceptible commercial sugarcane cultivar, N19, through the exposure of callus cells to the ethyl methanesulfonate mutagen and subsequent in vitro osmotic selection on polyethylene glycol. The study aimed to characterise the M9.2 mutant, in comparison with the parental genotype, in terms of its physiological and biochemical performance and proteome profile when exposed to moderate (14 days without water) and severe (21 days without water) water deficit stress in glasshouse pot trials. In comparison to the parental counterparts, the mutant plants were able to sustain the quantum efficiency of photosystem II (Fv/Fm) throughout the stress. Under mild stress, the mutant plants displayed elevated stomatal conductance, high concentrations of proline, accumulated less H₂O₂ and phenotypically displayed limited wilting and no visible signs of leaf senescence. Under severe stress, the mutant plants accumulated less malondialdehyde and more antioxidant enzymes (superoxide dismutase and catalase) than the parental line. Differential protein expression was also observed according to two-dimensional difference gel electrophoresis patterns of proteins expressed in the M9.2 mutant versus the parental plants during moderate stress. Analysis revealed proteins related to photosynthesis (pyruvate orthophosphate dikinase, un-fragmented ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and chlorophyll a/b-binding protein 3) and carbohydrate metabolism (sucrose synthase) were up-regulated in the mutant. Differentially expressed proteins were further linked to energy metabolism, methylation homeostasis and DNA repair. This study characterises the new M9.2 mutant with beneficial drought-tolerant traits which have the potential to be exploited in future sugarcane breeding programmes.     

Molecular investigation of the genetic base of sugarcane cultivars

 Molecular diversity was analysed among 162 clones of sugarcane using DNA restriction fragment length polymorphism (RFLP). One hundred and nine of them were modern cultivars of interspecific origin; most of them were bred in Barbados or in Mauritius. Fifty three were from Saccharum officinarum species, which is the major source of genes in modern cultivars, prevailing over the part of the genome incorporated from the wild species Saccharum spontaneum. Twelve low-copy nuclear DNA probes scattered over the genome were used in combination with one or two restriction enzymes. A total of 399 fragments was identified, 386 of which were polymorphic. Each sugarcane clone displayed a high number of fragments per probe/enzyme combination, illustrating the polyploid constitution of the genome. Among the S. officinarum clones, those from New Guinea had the largest variability and encompassed that present among clones collected from the Indonesian Islands and those known to have been involved in the parentage of modern cultivars. This is in agreement with the hypothesis that New Guinea is the centre of origin of this species. The clones from New Caledonia formed a separate group and could correspond to S. officinarum clones modified through introgression with other members of the ‘Saccharum complex’. Despite the low number of S. officinarum clones used for breeding cultivars, more than 80% of the markers present in the whole S. officinarum sample were also found in modern cultivars due probably to a high heterozygosity related to polyploidy. Among the cultivars, the two main groups, originating from Barbados and Mauritius, were clearly separated. This appeared essentially due to S. spontaneum alleles present in Mauritian cultivars and absent in Barbadan ones, probably in relation to the regular use of early generation interspecific hybrids in the breeding program employed in Mauritius. Received: 9 November 1998 / Accepted: 19 November 1998     

Isolation from sugarcane cultures of variants resistant to antimetabolites

Variants resistant to antimetabolites are useful for investigating metabolic regulation and biochemical genetics in organisms. In this study, suspensions of mutagenized sugarcane ( Saccharum sp.) cells, originating from a stalk parenchyma explain of the Hawaiian variety 50–7209, were used to investigate the feasibility of isolating variants resistant to l -canavanine, glyphosate [N-(phosphonomethyl)glycine], ophiobolin A and orthovanadate. Rigorous retesting of clones which grew on selection media led to the identification of three cell lines, two of which were resistant to glyphosate and one to orthovanadate. No variants were isolated which showed a persistent resistance either to l -canavanine or ophiobolin A.
The results demonstrate that resistant variants do occur, or can be induced, in sugarcane cell suspensions and that they can be rescued and cultivated.