Mutagenesis and Identification of Sugarcane Mutants Using Survival on Polyethylene Glycol and Leaf Damage under Managed Water Stress

Drought causes severe damage to sugarcane, reducing its product yield. Given Thailand’s weather conditions and topography, a breeding program to develop new sugarcane genotypes with a high tolerance for water stress is important to the sugarcane industry. This study created new water stress tolerant sugarcane genotypes using ethyl methanesulfonate (EMS) mutagenesis in the sugarcane cultivar Khon Kaen 3. Using 16 mM of EMS for 4 h induced callus mutagenesis (survival rate, 57.5%). The survival rates of calli treated with 10 mM of EMS for 2 and 4 h in selective media with 15% PEG were higher than that of non-EMS-treated calli. The selected calli survived and grew on selective media with 20% PEG, while non-EMS-treated calli did not grow. The mutant plantlets developed from EMS-treated calli on selective media with 20% PEG for 4 weeks had varying survival rates: 72.25% (10 mM of EMS for 2 h), 75.85% (10 mM of EMS for 4 h), and 60.61% (16 mM of EMS for 4 h). Both healthy mutant sugarcane plants (2,086) and non-mutant plants (234) were cultured on the media with 20% PEG for 16 weeks. Of these, 462 mutant sugarcane plantlets survived and developed on the media, but all the non-mutant sugarcane plantlets died during the selection process. Mutagenesis induced using treatment 4 produced the highest frequency of mutant sugarcane plantlets with water-stress tolerance (45.5%). In total, 136 selected mutant sugarcane plants were transplanted to a greenhouse for evaluation under managed water stress. Fourteen mutant sugarcane plants stayed green after the third cycle of water stress, but the KK3 sugarcane cultivar showed damage on 50% of the leaves. Thus, EMS mutagenesis and evaluation using in vitro and greenhouse methods were successful in developing new sugarcane clones with high water-stress tolerance, which is important for sugarcane breeding programs.

Drought Sensitivity of Sugarcane Cultivars Shapes Rhizosphere Bacterial Community Patterns in Response to Water Stress

Rhizosphere bacteria, the main functional microorganisms inhabiting the roots of terrestrial plants, play important roles in regulating plant growth and environmental stress resistance. However, limited information is available regarding changes occurring within the structure of the root microbial community and the response mechanisms of host plants that improve adaptability to drought stress. In this study, we conducted an experiment on two sugarcane varieties with different drought tolerance levels under drought and control treatments and analyzed the rhizosphere bacterial communities using 16S rRNA high-throughput sequencing. Correlation analysis results clarified the influence of various factors on the rhizosphere bacterial community structure. Drought stress reduced the diversity of the bacterial community in the rhizosphere of sugarcane. Interestingly, the bacterial community of the drought-sensitive sugarcane cultivar GT39 changed more than that of the drought-tolerant cultivar ZZ9. In addition, ZZ9 had a high abundance of drought-resistant bacteria in the rhizosphere under optimal soil water conditions, whereas GT39 accumulated a large number of drought-resistant bacteria only under drought stress. GT39 mainly relied on Actinobacteria in its response to drought stress, and the abundance of this phylum was positively correlated with soil acid phosphatase and protease levels. In contrast, ZZ9 mainly relied on Bacilli in its response to drought stress, and the abundance of this class was positively correlated with only soil acid phosphatase levels. In conclusion, drought stress can significantly reduce the bacterial diversity and increase the abundance of drought-resistant bacteria in the sugarcane rhizosphere. The high abundance of drought-resistant bacteria in the rhizosphere of drought-tolerant cultivars under non-drought conditions is an important factor contributing to the high drought adaptability of these cultivars. Moreover, the core drought-resistant bacteria of the sugarcane rhizosphere and root exudates jointly affect the resistance of sugarcane to drought.

Root proteome alterations in sugarcane promoted by the regrowth cycle in commercial production

The decrease in agricultural productivity in successive cutting of sugarcane plants is associated with several extrinsic and intrinsic factors. However, no studies have focused on the physiological potential of sett roots in successive cuts in sugarcane culture. There have been no proteomic studies on sugarcane sett roots at different stages of cutting. In this study, the UPLC-ESI-TOF-MS system and bioinformatics tools were used to identify proteins of sett roots in the first and fifth cuts of sugarcane cultivar RB966928 in the sprouting stage. Differences in the proteome of sett roots of RB966928 in the first and fifth cuts detected in this study supports the hypothesis that the proteome of sett roots may change after successive cuts in sugarcane culture. A reduction in the number of proteins was observed in the roots of the fifth cut, whereas 34% of proteins, identified exclusively in the first cut, were absent in the fifth cut. Proteome analysis of sett roots in the first and fifth cuts showed that the changes after successive cuts were quantitative (number of proteins) and mainly qualitative. In this study, the detailed list of proteins identified in the first cut but absent in the fifth cut is relevant. The findings of this study may aid further research that employ biotic or abiotic elicitors to induce gene expression of essential proteins absent in sett roots of the fifth cut, and thus increasing the agricultural productivity and longevity of cane fields

Transcriptomic Analysis of Changes in Gene Expression During Flowering Induction in Sugarcane Under Controlled Photoperiodic Conditions

Flowering is of utmost relevance for the agricultural productivity of the sugarcane bioeconomy, but data and knowledge of the genetic mechanisms underlying its photoperiodic induction are still scarce. An understanding of the molecular mechanisms that regulate the transition from vegetative to reproductive growth in sugarcane could provide better control of flowering for breeding. This study aimed to investigate the transcriptome of +1 mature leaves of a sugarcane cultivar subjected to florally inductive and non-inductive photoperiodic treatments to identify gene expression patterns and molecular regulatory modules. We identified 7,083 differentially expressed (DE) genes, of which 5,623 showed significant identity to other plant genes. Functional group analysis showed differential regulation of important metabolic pathways involved in plant development, such as plant hormones (i.e., cytokinin, gibberellin, and abscisic acid), light reactions, and photorespiration. Gene ontology enrichment analysis revealed evidence of upregulated processes and functions related to the response to abiotic stress, photoprotection, photosynthesis, light harvesting, and pigment biosynthesis, whereas important categories related to growth and vegetative development of plants, such as plant organ morphogenesis, shoot system development, macromolecule metabolic process, and lignin biosynthesis, were downregulated. Also, out of 76 sugarcane transcripts considered putative orthologs to flowering genes from other plants (such as Arabidopsis thaliana, Oryza sativa, and Sorghum bicolor), 21 transcripts were DE. Nine DE genes related to flowering and response to photoperiod were analyzed either at mature or spindle leaves at two development stages corresponding to the early stage of induction and inflorescence primordia formation. Finally, we report a set of flowering-induced long non-coding RNAs and describe their level of conservation to other crops, many of which showed expression patterns correlated against those in the functionally grouped gene network.

Hydraulic conductivity and diffusivity of an Oxisol cultivated with sugarcane fertigated with nitrogen and potassium

This study had the objective to evaluate the effect of irrigation and fertigation (NK) in the hydraulic conductivity and diffusivity of an Oxisol cultivated with sugarcane. The experimental design comprised randomized blocks in a 5 × 2 factorial scheme, with four replications. Treatments consisted of five levels of water replacement (100, 75, 50, 25 and 0%), with and without fertirrigation (NK). The planting of sugarcane, cultivar RB85-5453, was performed in a double row (W-shaped), 8 m long, with 1.80 m spacing between the double rows, the distance between the crops in the double row was 0.40 m, with a total area of 52,8 m2 in each paddock. For treatments with water, replacement (WR) a drip tube was placed in the ground at a depth of 0.20 m among the furrows of the double row. The drip tube (DRIPNET PC 16150) comprised a thin wall, 1.0 bar pressure, nominal discharge 1.0 L h-1, and 0.50 m spacing between drippers. Nitrogen was applied by fertirrigation at a dose of 100 Kg ha-1, at 30-day intervals, with 10 applications throughout the development of the sugarcane culture. Potassium fertilization was done partially, in 30% of the furrows, and the remaining part was treated with the irrigation water. Nitrogen and potassium were spread only in the treatment with 0% water replacement. Was evaluated hydraulic conductivity and diffusivity versus logarithmic pressure head, at a depth of 10 cm, using RETC software. The hydraulic diffusivity for water replacement of 25 and 50% with fertigation was 160.3 and 14.9 cm2 days-1 for the lower values of the logarithm of the pressure head.

Agrobacterium Mediated Transformation Optimizations for Sugarcane (Saccharum Officinarum L.) Cultivar SPF-234 with Direct Organogenesis

Sugarcane (Saccharum officinarum L.) is the most important food and energy crop worldwide. In the present study, an efficient Agrobacterium mediated transformation and regeneration system for sugarcane cultivar SPF-234 was established. Agrobacterium tumefaciens strains EHA101and LBA4404 using vector pIG121 Hm, having GUS, HPTII and NPTII genes were used. Polymerase chain reaction (PCR) and histochemical assays confirmed the GUS gene expression. A 620 bp fragment from GUS positive plants was amplified. The GUS expressing putative transformants were 35% of the total plants formed under 30 minute immersion time and 72 hr of incubation period. The co-cultivation media having 60 µM acetosyringone produced 66% GUS expressing plants for LBA4404 and 58% for EHA101. The maximum average number of directly produced shoot (59.5%) from leaf explant was in M6 media having 1.00 mg/l 6-Benzylaminopurine (BAP) and 2.5 mg/l Naphthaleneacetic acid (NAA). A significant decrease (17%) was observed when auxin (NAA) concentration was increased to 4.0 mg/l. The best response of shoot elongation was observed in SE4 media having equal concentration (2.00 mg/l) of both kinetin and BAP. Increased concentrations of kinetin significantly decreased shoot elongation of the subject cultivar. Agrobacterium strain LBA4404 performed better for genetic transformation of the said sugarcane cultivar.This quick and less expensive transformation and direct regeneration system could be exploited for sugarcane on commercial scale in general, and for this elite cultivar in particular.

Comparative profiling of drought induced root metabolic responses in sugarcane wild relative Erianthus arundinaceus (IND 04-1335) and a commercial variety Co 99004

Aim: To study the metabolic changes in roots of a drought tolerant wild relative of sugarcane Erianthus arundinaceus clone (IND 04-1335) and a commercial sugarcane cultivar Co 99004. Methodology: Setts of Erianthus arundinaceus (IND 04-1335) and a commercial variety Co 99004 were planted in medium size pots in replication. After 45 days of planting, drought stress was imposed by withholding irrigation. The corresponding control pots were maintained under continuous irrigation. On 26th day of drought, stress morphological and physiological traits such as leaf drying, canopy temperature, leaf relative water content and chlorophyll fluorescence were recorded. Root samples were subjected to metabolomic analysis using GC-MS. Results: After 26 days of drought exposure, IND 04-1335 were found to be tolerant without any drought induced morphological symptoms. The comparative metabolite profiling identified a total of 143 metabolites in the control and drought exposed roots. Hierarchical cluster analysis showed that roots of IND04-1335 control and Co 99004 stress had the most similar metabolite profiles, while the profile of IND04-1335 stressed root was distinctive.The metabolomic profile of IND04-1335 under drought stress showed an increased accumulation of sugars (melezitose, trehalose), sugar alcohols (mannitol), amino acid (proline) and carotenoids (rhodopin, carotene) as compared to Co 99004. Interpretation: Differentially accumulated metabolites in IND04-1335 under drought stress may play an important role as osmoregulants, antioxidants and chelating agents thereby imparting better tolerance mechanism to this genotype.

Selectivity index of indaziflam to sugarcane cv. IACSP95-5000 in two soil textures

Selectivity index is a way of assessing the discrimination of herbicide to a given crop by observing its effects on the crop and the weeds. The aim was to obtain the selectivity index of indaziflam herbicide to sugarcane cultivar IACSP95-5000 as a function of five weed species in two soils textures. The experiment was carried out in a greenhouse at Piracicaba, São Paulo, Brazil. The treatments consisted of indaziflam doses (0; 12.5; 25; 50; 100; 200; 400; 800 and 1,600 g of the active ingredient (ai) ha-1), applied in pre-emergence of the sugarcane and of the weeds Urochloa decumbens, Urochloa plantaginea, Digitaria horizontalis, Panicum maximum and Rottboellia cochinchinensis. In sandy loam soil, a 100% control for all weeds was provided at 25 g ai ha-1. In clay soil, for D. horizontalis the 90% reduction in total dry mass (ED90) was obtained at 25 g ai ha-1, for R. cochinchinensis at 193 g ai ha-1, for U. plantaginea at 152 g ai ha-1, for P. maximum at 124 g ai ha-1, and for U. decumbens at 94 g ai ha-1. Indaziflam was selective to IACSP95-5000 in both soils, with 10% of reduction in dry mass (ED10) at 137 g ai ha-1 for soil with a sandy loam texture and 353 g ai ha-1 for clay soil. The selectivity index was higher than 1 for all weeds in clay soil. It was not possible to obtain the selectivity index for sandy loam soil due to species susceptibility to the herbicide.

Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.