Sangeeta Srivastava

The water-deficit stress- and red-rot-related genes in sugarcane

Sugarcane is an important international commodity as a valuable agricultural crop especially in developing countries. Sequencing was carried out to generate >35,000 expressed sequence tags (ESTs) from healthy as well as red-rot-infected tissue of Indian subtropical variety of sugarcane. Subsequent clustering with existing sugarcane ESTs in public databases identified 4,087 clusters, including 85 clusters that preferentially express upon Colletotrichum falcatum (red-rot) infection, which were previously unreported. Real-time reverse transcription–PCR profiling of selected EST clusters identified several sugarcane clusters that show differential expression in response to biotic and abiotic stress conditions. Twenty-five stress-related clusters showed >2-fold relative expression during water-deficit stress in sugarcane. Similarly, EST clusters could be identified, which exhibit association with red-rot disease when assessed in red-rot-susceptible and red-rot-resistant varieties of sugarcane. Such EST clusters are good candidates for in-depth analysis to elucidate stress-responsive pathways in sugarcane and facilitate genetic manipulation to tailor this crop for tolerance to various stresses.

Inter simple sequence repeat profile as a genetic marker system in sugarcane

Molecular profiling for varietal differentiation assumes an important role in the light of UPOV initiatives and PVP act. Inter simple sequence repeats (ISSR), a PCR based genetic marker system circumvents the requirement for flanking sequence information, and thus has found wide applicability in a variety of plants. The potential of ISSR markers for molecular profiling was assessed in sugarcane using a panel of 42 varieties of subtropical India. ISSR amplification was achieved using di-, tri-and tetraoligonucleotide repeats. All the nucleotide motifs were amenable to amplification. The ISSR results suggested that the frequency and clustering of specific simple sequence repeats was variable and motif-specific, however, there was not much difference in the di-, tri-or tetra-nucleotide ISSR profiles. The ISSR amplification pattern was used to assess genetic similarity of these sugarcane genotypes by Dice similarity coefficients, which ranged from 0.68 to 0.97 with a mean similarity value of 0.878. The ISSR amplification proved to be a valuable method for determining genetic variability among sugarcane varieties and for identification of the cultivars.

Assessment of genetic purity of micropropagated plants of sugarcane by isozyme and RAPD analysis

Shoot tip expiants of nine sugarcane varieties(Saccharum species hybrid.), CoLk 8102, CoLk 8001, CoS 767, CoLk 9606, CoLk 9617, CoS 95255, BO 91, CoJ 64 and Co 1148, were established on a solid MS medium containing 0.8% agar, 3% sucrose and 0.05mg/l of IAA, BAP and kinetin. Shoot regeneration has been achieved in liquid MS medium containing 0.5mg/l each of IAA, BAP and kinetin. Multiple shoots were obtained on liquid MS medium containing 0.01mg/l IAA, 0.5mg/l each of 6- benzyl aminopurine (BAP), kinetin and rooting occurred in 1/2 strength liquid MS medium with 5.0 mg/l NAA and 0.01 mg/l each of BAP and kinetin. To detect the genetic purity of in vitro raised plantlets, expression of iso-peroxidases in donor plants andin vitro raised plantlets was compared through native PAGE. Different genotypes showed marked variation regarding number, position and intensity of bands. Micropropagated plantlets showed identical pattern of peroxidases to their parental clones. The genetic stability of micro propagated plantlets was also tested by RAPD analysis. The banding pattern of PCR amplified products from micro propagated plantlets was monomorphic based on RAPD profile in all the varieties. RAPD analysis and peroxidase isozyme pattern confirmed the genetic purity of sugarcane plantlets derivedin vitro.

Eco-friendly management of red rot disease of sugarcane with Trichoderma strains

Eighty strains of Trichoderma harzianum and T. viride occurring in sugarcane rhizosphere soil of sub-tropical zone were isolated on Trichoderma specific medium and tried screened in vitro against Colletotrichum falcatum. Highly potent ones (Th 37 & Th 38) were tried to manage red rot disease under field conditions. Healthy setts were treated by applying Trichoderma mixed culture (TMC) of T. harzianum strains grown in sterilized maize bran (2 kg) and subsequently mixed with sterilized press mud or FYM (20 kg) / ha. The treatment was also compared with spore suspension containing 106 conidia / ml and metabolites (2.5% culture filtrate). Combinations were also tried for improving efficiency. The trials were repeated thrice in CoLk 7701, a highly susceptible variety / genotype. Disease management was ascertained by inoculation challenged with potent Colletotrichum falcatum pathotype (Cf 09). Infection of C. falcatum was protected in 45–55% plants. There was no infection in such plants as against 97–100% infection in check. Red rot infection was also considerably suppressed in 20–25% plants. Where grade of infection was reduced to 2–4 in 0–9 scale. Testing was made at an appropriate temperature (300 C) and high relative humidity (90%) during July–August. The protection offered might be due to direct parasitic action of T. harzianum on C. falcatum and also systemic resistance induced in sugarcane. Application of T. harzianum is useful for management of red rot and cultivation of moderately resistant varieties for several years. It is also useful for reducing the economical losses in susceptible varieties. The yield was also enhanced by 15–20 t / ha due to improved germination and shoot biomass. Application of Trichoderma biopesticide is eco-friendly, economical and efficient for improving soil health also.

Trichoderma induced improvement in growth, yield and quality of sugarcane

Trichoderma harzianum andT. viride were significantly effective in improving germination (6-14%), tiller population (21-78%), millable canes (5-30%), yield (6-38%) and CCS t/ha (30-34%) over the control in plant cane of CoS 94257. Metabolites of both the species @ 2.5% were found to be more efficient and significantly better than spore suspension (106 conidia/ml) and TMC containingTrichoderma @ 2 kg/ha in 20 kg sterilized farm yard manure (FYM). The metabolites improved tiller count (53-78%), millable canes (27-30%) and yield (34-38%). The yield boost up by the metabolites was up to 81.9 t/ha withT. viride and 79.8 t/ha withT. harzianum over 59.3 t/ha of control. The differences in yield due to both the species were significant.T. viride alone was tested for improving the yield of ratoon. The emergence of clumps was enhanced with spore suspension, TMC and metabolites ofT.viride. The metabolites were, significantly superior for increasing the number of clumps (43.2%) than the other treatments (1.7-20.1%) and control.T. viride metabolites were also better for producing more tillers (75%) and millable canes (40%). The improvement in yield ranged from 53.16 t/ha to 76.31 t/ha with metabolites and 72.13 t/ha with double doze of TMC, half applied at clump emerging stage and half at tiller stage. CCS t/ha was also enhanced in ratoon crop by 40% with metabolites and 36% with double doze of TMC. Application ofT. harzianum andT.viride was found to be economical, non hazardous and useful for soil health. The benefit cost ratio suggests that by expending*Rs. 1000/ ha onTrichoderma in sugarcane a benefit of Rs. 11500/ in plant cane/ha and Rs. 16500/ in ratoon /ha may be obtained.

Development and utilisation of conserved-intron scanning marker in sugarcane

Genetic dissection of economic traits in sugarcane requires sufficiently informative molecular markers that are currently lacking in this highly valued crop. Through comparative analysis of publicly available expressed-sequence data of sugarcane, sorghum and barley, and the whole rice genome-sequence survey, novel functional markers based on conserved-intron scanning primers (CISP) were developed and evaluated in different accessions across various taxonomic ranks of sugarcane. Polymorphism was moderate (55.2%), whereas 94.7% of the markers developed amplified fragments in selected genotypes. Mean polymorphism information content value was 0.582 (range 0.320–0.715), which was comparable to that with genic microsatellite markers (0.52) but lower than that with EST-SSR (0.73). Genetic-similarity coefficient ranged from 0.39 to 0.95, indicating variable levels of divergence depending on the taxonomic rank assessed. Cluster analysis revealed that the genotypes grouped in accordance with the taxonomical classification of sugarcane, with a relatively good support from a Mantel’s test (r = 0.847) and a moderate bootstrap value (65–89%). The CISP markers reported in the present study have potential utility for genetic-diversity analysis and application in sugarcane-breeding programs.

SDS and Native page protein profile for identification and characterization of elite sugarcane genotypes

The genetic markers provided by protein and enzyme polymorphism in plant population have been used to assess the degree of genetic variability and species relationship. Electrophoretic protein analysis holds potential utility in breeding programmes of sugarcane through helping in the unequivocal characterization of varieties used. Nine sugarcane cultivars were characterized by analyzing protein polymorphism in leaf tissues through SDS and Native PAGE. The electrophoregram showed a total of twenty bands of SDS proteins with molecular weight from 10 to 200 KDa and twenty four bands of native proteins having molecular weight of 10 to 708 KDa with difference in number of bands, bandwidth and intensity among different genotypes of sugarcane. The cultivars could be differentiated on the basis of SDS and Native protein polymorphism of total soluble leaf proteins profile which showed genetic homology as well as divergence among the different genotypes. The protein gel electrophoresis approach could thus be defined as a valuable means for distinguishing accessions of sugarcane through detecting their polymorphism.

Effect of methyl jasmonate on sugarcane seedlings

Sugarcane seedlings derived from single bud setts of CoLk 8102 were exposed to methyl jasmonate (1,2.5 and 5 ppm). Enzyme activities of catalase, peroxidase, Superoxide dismutase, nitrate reductase, isoenzyme analysis of Superoxide dismutase, proline and soluble protein content were determined after one month. The activities of catalase, Superoxide dismutase, nitrate reductase were lower at 1 ppm compared to control but at 5 ppm these were comparable to the control. The activity of peroxidase was higher than control under all MeJA treatments. The treatment with 2.5 and 5 ppm MeJA increased the number of isoenzymes of Superoxide dismutase in a dose dependent manner while 1 ppm MeJA treated ones had lesser number of isoenzymes as compared to control. Proline content was lower at 1 ppm but increased at 5 ppm. The reduction in enzymes activities at 1 ppm MeJA and increase at 5 ppm MeJA suggest methyl jasmonate regulated expression of these enzymes in sugarcane. These results suggest existence of the methyl jasmonate signalling pathway in sugarcane.

Ribosomal DNA Sequence Based Characterization of Trichoderma Isolates Antagonistic to Colletotrichum falcatum Causing Red Rot Disease of Sugarcane

Trichoderma bioagent isolates cultured from the sugarcane rhizosphere of subtropical India, and established efficient against Colletotrichum falcatum Went (red rot pathogen) in sugarcane are described based on phylogenetic analysis of nucleotide sequences. DNA sequences of the isolates included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1-5.8S- ITS2). The 5.8S region was found conserved and much of the sequence variability was due to indels or transition/transversion mutations in the ITS1 and 2 regions. Both the isolates belonged to the T. harzianum clade. These isolates are distinct yet have close genetic similarity with several established strains/isolates of T. harzianum reported worldwide. Correct identification of these bioagents would augment the effective utilization of these fungi of immense agricultural importance in managing the red rot disease of sugarcane.

Genetic divergence among interspecific hybrids of sugarcane

Fourteen Interspecific Hybrids of Sugarcane (ISH lines) along with two commercial genotypes were evaluated for yield and quality attributes to find out the genetic divergence among the genotypes and the factors governing it, and to isolate genotype of divergent and distinct characteristics. The ISH lines were grouped into three clusters containing 6, 5 and 5 genotypes each. Intracluster D2 values ranged from 1.923–2.540 and intercluster from 3.707–4.986. Maximum divergence was present between cluster I and II and minimum between II and III indicating their close relationship. The contribution of cane yield, purity, % juice and CCS% at 12 months was maximum towards divergence.