Vikas Yadav Patade

Salt and drought tolerance of sugarcane under iso-osmotic salt and water stress: growth, osmolytes accumulation, and antioxidant defense

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) plants were treated with salt-NaCl or polyethylene glycol-PEG 8000 solutions (−0.7 MPa) for 15 days. Both the salt and PEG treatments significantly reduced leaf width, number of green leaves, and chlorophyll stability index. Osmotic adjustment (OA) indicated that both the stresses led to significant accumulation of osmolytes and sugars. Salt stressed plants appeared to use salt as an osmoticum while the PEG stressed plants showed an accumulation of sugars. Oxidative damage to membranes was not severe in plants subjected to salt or PEG stress. The salt stressed plants showed an increase in the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), while PEG stress led to an increase in SOD but not APX activity as compared to the control. Thus, results indicate that the iso-osmotic salt or PEG stress led to differential responses in plants especially with respect to growth, OA, and antioxidant enzyme activities.

Radiation induced in vitro mutagenesis for sugarcane improvement

Induced mutations have contributed significantly to plant improvement and more than 2500 mutant varieties have been developed through mutation breeding. Many examples related to different vegetatively propagated species show that the mutation induction can be empowered by in vitro techniques. Sugarcane is one of the most important industrial crops, used as major source of sugar and recently to produce ethanol, an important renewable biofuel source. At BARC, our group has been engaged in employing in vitro culture techniques in the improvement of sugarcane. Different in vitro culture systems have been developed and radiation induced mutagenesis has been undertaken using embryogenic callus cultures of cv. CoC-671 followed by the in vitro selection and field evaluation of the selected lines. Results on radiation-induced mutagenesis combined with in vitro selection have been reviewed. Also the novel approaches of overcoming intrasomatic competition during in vitro mutagenesis and use of using ion beam irradiation have been discussed.

Gamma Irradiation of Embryogenic Callus Cultures and In vitro Selection for Salt Tolerance in Sugarcane (Saccharum officinarum L.)

Abstract Radiation induced mutagenesis followed by in vitro selection was employed for salt tolerance in popular Indian sugarcane ( Saccharum officinarum L.) cv . CoC-671. Embryogenic calli were gamma irradiated and exposed to different levels of NaCl (42.8, 85.6, 128.3, 171.1, 213.9, 256.7, 299.5, or 342.2 mM). The relative growth rate (RGR) decreased progressively with increasing salt stress and was the least with a salt stress of 256.7 mM (0.25 ± 0.009), almost 10 fold lesser than the control. The RGR was significantly lower in 85.6 mM and higher salt stressed calli than the control. The survival percent also decreased, with an increase in NaCl concentration. In case of 10 and 20 Gy irradiated calli, regeneration was observed up to 85.6 mM NaCl selection, medium, whereas, higher treatments (128.3 mM and beyond) exhibited browning initially. However, in the subsequent subcultures, regeneration was obtained in the case of 10 and 20 Gy irradiated calli on 128.3 and 171.1 mM NaCl selections. Higher dose of gamma irradiation (40 Gy) also showed regeneration, but only with 85.6 mM NaCl selection. The unirradiated calli regenerated the highest number of plantlets followed by 10 and 20 Gy irradiated calli on salt selection. A total of 147 plantlets were selected from different salt levels. The salt selected plants are being tested for their field performance.

Investigation of arsenic accumulation and tolerance potential of Sesuvium portulacastrum (L.) L.

Sesuvium portulacastrum (L.) L., a facultative halophyte, is considered a suitable candidate for the phytoremediation of metals. An investigation of As accumulation and tolerance was conducted in Sesuvium plants upon exposure to As(V) (100-1000 μM) for 30 d. Plants demonstrated a good growth even after prolonged exposure (30 d) to high As(V) concentrations (1000 μM) and a significant As accumulation (155 μg g⁻¹ dry weight) with a bioaccumulation factor of more than ten at each concentration. The results of shoot and root dry weight, malondialdehyde accumulation, photosynthetic pigments, and total soluble proteins demonstrated that plants did not experience significant toxicity even at 1000 μM As(V) after 30 d. However, metabolites (total non-protein thiols and cysteine) and enzymes (serine acetyltransferase, cysteine synthase and γ-glutamylcysteine synthetase) of thiol metabolism, in general, remained either unaffected or showed slight decline. Hence, plants tolerated high As(V) concentrations without an involvement of thiol metabolism as a major component. Taken together, the results indicate that plants are potential As accumulator and may find application in the re-vegetation of As contaminated sites.

An in vitro radiation induced mutagenesis-selection system for salinity tolerance in sugarcane

In vitro radiation induced mutagenesis-selection was employed for salt tolerance in popular Indian sugarcane cv. Co 86032. Radiosensitivity characterized in terms of growth rate and cell viability indicated 20 Gy as LD50 for embryogenic sugarcane callus cultures. Response of embryogenic calli exposed to different NaCl concentrations (85.6, 128.3, 171.1, 213.9 or 256.7 mM) indicated significant progressive decrease in cell viability and increase in membrane damage rate with increasing salt concentration. Based on these results, 20 Gy irradiated calli were stressed with 85.6 or 171.1 mM NaCl for selection for salt tolerance. Both irradiated or non-irradiated calli exhibited reduced growth rate compared to their respective controls, however, the extent of growth rate reduction was more in un-irradiated stressed calli. Further, in order to study the effect of auxin on salt selection, the calli were cultured on NaCl selection medium supplemented with or without auxin (2, 4-D). The growth rate was higher when NaCl media was supplemented with the auxin than that of without auxin. The calli exposed to 171.1 mM NaCl accumulated more proline than the control and 85.6 mM NaCl stressed irradiated or nonirradiated calli. Proline accumulation in the calli exposed to 85.6 mM NaCl without 2, 4-D was significantly higher than that of the calli stressed at the same salt concentration with 2, 4-D. The irradiated calli stressed with 85.6 mM NaCl medium with or without 2, 4-D accumulated more proline than the nonirradiated calli exposed to the respective salt treatments. Thus, the results suggest in vitro mutagenesis-selection as a powerful tool for enhanced tolerance to abiotic stresses, which may improve the competitiveness of the popular sugarcane cultivars and their commercial cultivation in saline areas.

Morphological and molecular diversity analysis among the Indian clones of Sesuvium portulacastrum L.

Sesuvium portulacastrum L. (seapurslane) is a halophyte used as pioneer species in sand dune fixation and stabilization of saline soil. Studies on the morphological and molecular diversity were carried out for the 14 clones of Sesuvium collected from the different coastal regions of India. Significant differences were observed for morphological traits viz., length, width, diameter and area of leaf, internodal distance and stem diameter for different clones when compared with the clone from Gujarat state (GJ1). A UPGMA dendrogram for morphological traits based on the Pearson’s similarity coefficient clustered the clones into three groups considering 80% polymorphism as criteria. Molecular diversity among the clones was studied using Randomly Amplified Polymorphic DNA (RAPD), Internal Transcribed Spacer (ITS) and markers specific to Ac homologous region. Of the total 749 RAPD loci amplified with 70 random primers, 294 were polymorphic with 39.25% diversity. A phylogenetic tree constructed with UPGMA and SHAN, grouped the clones into three major clades based on RAPD data. The molecular diversity studied with ITS and markers specific to Ac homologous region revealed 37.50% and 66.66% polymorphism and clustered the clones into three and four clades, respectively. The genetic diversity analysis revealed wide variations among the S. portulacastrum clones, reflecting a high level of diversity within the species which might be due to anthropogenic impact and geographic environmental conditions. Further, the various clones from the different eco-geographic coastal localities might have originated from native places of wild abundance. To the best of our knowledge, this is the first attempt to evaluate both morphological and genetic diversity among the Sesuvium clones collected from the distant habitats of the coastal regions of the India.

Short-term salt and PEG stresses regulate expression of MicroRNA, miR159 in sugarcane leaves

MicroRNAs (miRNAs) are involved in the regulation of stress-responsive gene expression and stress adaptation. Transcript expression of mature miR159 was studied in sugarcane leaves stressed for long-term (15 days) NaCl (150 mM) or iso-osmotic PEG 8000 (20% w/v) and for short period (up to 24 h) with NaCl (200 mM) or PEG (20% w/v). The results revealed no significant changes in transcript levels of the miRNA in response to the long-term stress however, short-term salt or PEG stress led to significant up-regulation over the control. Progressive increase in transcripts of the miRNA was observed under short-term PEG stress with exposure period. Using computational tools, various members of the MYB transcription factor family were predicted as the potential targets of miR159. The transcript expression of the MYB-related gene indicated up-regulation at 1 h of salt stress with concomitant slight downregulation of the miRNA. In addition, under short-term PEG stress, the transcript levels of MYB and miR159 were the opposite of each other, suggesting MYB as a potential target of miR159. To our knowledge, this is the first preliminary evidence on the involvement of miR159 in abiotic stress responses in sugarcane.

Exogenous Application of Proline Alleviates Salt Induced Oxidative Stress More Efficiently than Glycine Betaine in Sugarcane Cultured Cells

Antioxidant defense system provides protection against oxidative damage caused by abiotic stresses including salinity. Ameliorative effects of l-proline, l-glutamine, glycine betaine (GB) on growth, proline accumulation and antioxidant enzyme activities were studied using cultured cells of sugarcane against salt (NaCl) stress. NaCl stress reduced growth rate significantly over the control however, proline or glutamine supplementation resulted in growth revival. Proline supplementation to media with or without salt increased accumulation of free proline significantly than the controls and other (proline, GB and glutamine) treatments. Salt stress led to increase in superoxide dismutase (SOD) and glutathione reductase activity whereas guaiacol peroxidase (GPX), catalase and ascorbate peroxidase activities were significantly suppressed. Proline supplementation to the salt medium improved the GPX activity over the salt media supplemented with glutamine or glycine betaine. The activity ratio between SOD and H2O2 scavenging enzyme activities, which is considered as a working hypothesis for biochemical marker for salt tolerance, was lower in salt medium supplemented with proline. Thus, the higher growth rate and the lower activity ratio suggest maximum salt stress ameliorative potential of proline in sugarcane cultured cells.

Calcium Signaling and Its Significance in Alleviating Salt Stress in Plants

Environmental stresses such as salinity, temperature, drought and heavy metals negatively impact the agricultural productivity. Of these, salinity stands as a major problem mainly in the developing countries. Calcium is an essential nutrient that regulates the plant growth and development and it has evolved as a ubiquitous secondary messenger in mediating complex responses towards various developmental and environmental cues. Thus, understanding the calcium signaling and consequent calcium-dependent events is essential to improve plant productivity under extreme environment. The first step in calcium signaling is the induction of [Ca2+]cyt-transient/signatures which is defined as the repetitive oscillations or spiking of [Ca2+]cyt level. This in turn activates a set of calcium binding proteins including Ca2+ sensors/decoders, protein kinases and transcription factors. The interplay between Ca2+ signatures and these proteins together contributes towards the stimulus specificity. Various efforts have been made to manipulate calcium signaling events either by exogenous calcium supplementation or by genetic modification of calcium signaling related genes in many plant species and considerable progress has been made in managing the plant responses toward salt stress. Additionally, these studies also help in understanding the effect of salt stress on the process of calcium signaling. The present review deals with the basic steps of calcium signaling process and its possible modulations that can lead to the enhanced salt tolerance in plants.

Profiling of culture-induced variation in sugarcane plants regenerated via direct and indirect somatic embryogenesis by using transposon-insertion polymorphism

Transposable elements constitute a large fraction of plant genomes, and changes due to TE mobilization have provided powerful genetic and molecular tools. Somaclonal variation is a common phenomena caused either by various extrinsic and intrinsic factors related to in vitro culture or transposon activity has been detected under cell culture or tissue culture milieu. In this study, variability among direct somatic embryogenesis (DSEM) and indirect somatic embryogenesis (ISEM) regenerants of sugarcane cv. CoC-671 was studied using Ac transposon insertion polymorphism. A total of 254 amplification products were obtained ranging from 0.5 Kb to 2 Kb. The DNA profile revealed genetic polymorphism among the ISEM regenerants compared to DSEM regenerants. There was n uniform insertion pattern of the Ac like transposons among the DSEM regenerants, whereas it was variable in case of ISEM regenerants. The results on the insertion polymorphism of Ac homologous regions among the ISEM regenerants indicate that the transposition occurred during in vitro culture and that this marker system could be useful in profiling of genetic variation.