T. D. Nikam

In vitro culture of Safflower L. cv. Bhima : initiation, growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate.

Antioxidant enzyme activities and protein profiling under salt stress in indica rice genotypes differing in salt tolerance

The effect of NaCl stress (0–300 mM) was investigated in terms of antioxidant enzymes activity and their isozymatic pattern and SDS-PAGE proteins banding pattern in three rice cultivars (cvs), Panvel-3 (tolerant), Kalarata (moderately tolerant) and Karjat-3 (sensitive). Interestingly, superoxide dismutase activity in roots and glutathione reductase activity in both shoots and roots were decreased significantly under high salinity levels in Karjat-3 and Kalarata, while, a sharp increase was observed in Panvel-3. Catalase and peroxidase activities were increased with salt stress of all cvs, with highest magnitude in Panvel-3. There was an induction of two new POX isoforms in Panvel-3 and Kalarata in stressed plants. Four SOD isoforms were observed in all the genotypes, irrespective of non/saline conditions. Total 33 proteins bands ranging from 17–154.5 kDa were either expressed de novo or up/down-accumulated due to NaCl stress. In Panvel-3, three new bands, one of 32 kDa in shoots while two of 37 and 116.7 kDa appeared in roots under salt stress. A new band of 37 kDa was also observed in roots of Kalarata under salinity stress. Thus, salinity tolerance nature of Panvel-3 may be correlated with higher enzyme activities and expression of some new polypeptides under salt stress.

SODIUM CHLORIDEINDUCED CHANGES IN MINERAL NUTRIENTS AND PROLINE ACCUMULATION IN INDICA RICE CULTIVARS DIFFERING IN SALT TOLERANCE

ABSTRACT The effect of increasing sodium chloride (NaCl; 0 to 300 mM) stress was investigated on plant growth, mineral nutrients, and proline accumulation in two indica rice cultivars differing in salt tolerance. The shoots and roots of ‘Karjat-3’ (salt sensitive cv.) showed greater reduction in fresh weight, dry weight, and water content under increasing salinity stress when compared to ‘Panvel-3’ (salt tolerant cv.). The magnitude of chloride (Cl) accumulation with increasing salinity varied between cultivars, with lesser accumulation in ‘Panvel-3’, whereas, calcium (Ca) content was more in ‘Panvel-3’ than ‘Karjat-3’ at all salinity levels. Increase in shoot-sodium (Na) did not show notable variation between cultivars under salinity stress; however, Na accumulation was notably lower in roots of ‘Panvel-3’ than ‘Karjat-3’. Under increasing salinity stress, ‘Panvel-3’ maintained significantly lower sodium/potassium, sodium/calcium and sodium/nitrogen ratios than ‘Karjat-3’, both in shoots and roots, with more differential response in roots. Regardless of treatment, proline concentration was considerably higher in ‘Panvel-3’ than in ‘Karjat-3’. The salt tolerance of cv. ‘Panvel-3’ was positively correlated with better growth, higher proline accumulation and enhanced uptakes of beneficial minerals such as potassium (K) and Ca.

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.

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.

Sesuvium portulacastrum (L.) L. a promising halophyte: cultivation, utilization and distribution in India

Seapurslane, Sesuvium portulacastrum (L.) L. (Aizoaceae) is a pioneer, psammophytic facultative halophyte naturally growing in the subtropical, mediterranean, coastal and warmer zones of the world. The plant is utilized as a vegetable by the local peoples as well as forage for domestic animals in the coastal area. Environmentally, it is utilized for the bioreclamation of saline soil in the arid and semiarid regions and an alternative culture to problematic soils. The Sesuvium clones collected from the coastal regions of India lead to conclusions with respect to its geographical distribution and prospective utilization in the bioremediation of coastal and saline areas, as an emerging source of secondary metabolite-phytoecdysteroids in sericulture industry and cultivation on large scale for food as well as forage, of this underutilized, neglected wild and also cultivated plant species.

High frequency shoot regeneration in Agave sisalana

Callus cultures of Agave sisalana were initiated from rhizome, and stem explants on MS, SH, Gamborg and Whites medium supplemented with different concentrations of BA, kinetin, NAA, IAA and 2,4-D either in combination or singly. Optimum numbers of shoots were obtained from stem and rhizome explants either directly or from callus. The capacity for the shoot regeneration remained constant in the callus for more than 32 months. Regenerated shoots rooted readily within 21-35 days on fine sand with half the strength of inorganic salts of MS medium. 100% of the rooted plants successfully adapted to field conditions and grown in the soil. Regenerated plants were morphologically similar to the field grown mother plants.

Plant Salt Stress: Adaptive Responses, Tolerance Mechanism and Bioengineering for Salt Tolerance

Salinity is an important abiotic environmental stress factor threatening agricultural productivity throughout the world. The detrimental effects of salinity stress are observed at cellular, organ and whole plant level at osmotic phase (early/short-term response) and ionic phase (late/long-term response). High salinity exerts its negative impact on major plant processes such as disrupting the osmotic and ionic equilibrium, protein synthesis, photosynthesis, energy, and lipid metabolism. To adapt and tolerate salt stress, plants have evolved physiological and biochemical mechanisms orchestrated by multiple biochemical pathways of ion homeostasis, osmolytes synthesis, ROS scavenging, and hormonal balance. At the molecular level, such adaptation involves activation of cascade(s) of gene modulations and synthesis of defense metabolites. In recent years, several candidate genes have been identified and employed to facilitate genetic engineering efforts to improve salt tolerance in crop plants. However, there is a further need of improvement for successful release of salt tolerant cultivars at the field level. In this article we present the physiological, biochemical and molecular signatures of plant responses to salinity, and outline their use in genetic engineering to improve salt stress tolerance.

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.

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoogs basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.