Chinmay Pradhan

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

Abstract A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoogs medium containing either N6-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil.

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb.

SummaryA complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l−1 (10.8 μM) was added in conjunction with 0.5 mg l−1 (2.2 μM) N6-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l−1 Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l−1 (13.3 μM) BA and 0.5 mg l−1 (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l−1 each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.

Efficient plant regeneration from cell suspension-derived callus of East Indian rosewood (Dalbergia latifolia Roxb.)

Abstract A procedure is outlined for the establishment of a proliferating cell suspension culture of East Indian rosewood (Dalbergia latifolia Roxb.) and efficient plant regeneration from callus derived from such cultures. Callus was induced from hypocotyl segments derived from 1-week-old axenic seedlings on Murashige and Skoog (1962) medium (MS) containing 10.8 μM naphthaleneacetic acid (NAA) and 2.2 μM benzyladenine (BA). Calli were increased by subculturing on MS supplemented with same growth regulators and 10% coconut water (CW). Friable calli were used to initiate cell suspension cultures. Optimum cell proliferation occurred in MS containing 10.8 μM NAA, 2.2 μM BA and 10% CW, using an initial inoculum cell density of 2%. Cell clumps composed of 20–25 cells harvested from suspension cultures at the exponential growth phase readily formed callus within 3 weeks following plating on the semi-solid MS as above. High-frequency shoot-bud differentiation was induced in these calli on MS containing 2.7 μM NAA and 13.3 μM BA. The regeneration frequency declined at higher BA concentrations. The organogenic potential of the cell suspensions was influenced by the age of the culture. Regenerated shoots were rooted on half-strength MS containing 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. The plantlets were acclimatized and established in soil.

Rapid In Vitro Propagation of East Indian Rosewood (Dalbergia latifolia Roxb.) through High Frequency Shoot Proliferation from Cotyledonary Nodes

An efficient protocol is described for large scale in vitro propagation of east Indian rosewood (Dalbergia latifolia Roxb.) using cotyledonary nodes derived from axenic seedlings. Of the four different cytokinins tested, BA was most effective in inducing multiple shoot buds in the explants. High frequency shoot proliferation (93%) coupled with maximum number of shoot formation (10-12 shoots/explant) was recorded on Murashige and Skoog’s medium supplemented with 2.0 mg/l BA. The frequency of shoot proliferation declined at higher levels of cytokinins. Shoot culture was multiplied by subculturing the original cotyledonary node on a fresh shoot multiplication medium each time aHer excising the newly formed shoots. Shoots obtained from each passage were multiplied further as nodal explants and each node produced 3-4 shoots. In two months from a single cotyledonary node, about 90 shoots were obtained. Rooting was induced in 72% of the regenerated shoots on half-strength MS containing IAA, IBA and IPA each at 1.0 mg/l. Rooted plantlets were hardened off and eventually established in soil.

Antimicrobial effect of silver zinc oxide (Ag-ZnO) nanocomposite particles

The antimicrobial effects of silver nanocomposite particles (Ag-ZnO NC) on microorganisms, the antimicrobial mechanism and applications in medical devices are not yet clear. Stable Ag-ZnO NC were prepared and their morphological sizes and shapes were characterized by scanning electron microscopy. The effect of Ag-ZnO NC was tested on Bacillus thuringiensis, Escherichia coli and Pseudomonas aeruginosa in antibacterial tests including growth kinetics, antimicrobial susceptibility (disc diffusion) and minimal inhibitory concentration (MIC). Different concentrations of nanocomposites (i.e. 10, 20, 50,100, and 200 μg) showed concentration-dependant efficacy on all three tested microorganisms. E. coli was fairly sensitive in 200 μg of NC, forming a ∼15 mm inhibition zone; followed by B. thuringiensis, having ∼9 mm of inhibition zone, while P. aeruginosa, a pathogenic bacterium, showed negligible inhibition zone with Ag-ZnO NC. Growth of E. coli under Ag-ZnO NC treatment was significantly delayed with an extended lag phase of 2 hrs and 30 mins. Scanning electron microscopy confirmed the bacteriostatic effect of Ag-ZnO NC, which was manifested in cell division arrest with significant cell elongations compared to the control. The free radical generation effect of Ag-ZnO NC was tested against all these organisms. The results suggest that Ag-ZnO NC can be used effectively against microbial growth. This may be of use in diverse medical devices for antimicrobial control and can be a proper substitute for antibiotics in curing human health.

Submergence Stress: Responses and adaptations in crop plants

Submergence stress frequently encountered in crop plants is a widespread limiting factor for crop production throughout the world especially in irrigated and high-rainfall environments which results in huge economic losses. This chapter covers various features of submergence stress with special reference to crop plants, viz. causes of submergence and biophysical and biochemical alterations in crops, and various defence mechanisms adopted by crop plants. A brief discussion on different types of naturally or artificially developed tolerance mechanisms are presented here.

Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees

An economic and efficient procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica W. using nodal explants obtained from about 20-year-old mature trees growing in the field, belonging to the family of Apocynaceae. Shoot development was maximum (90%) on Murashige and Skoog’s (MS) basal medium supplemented with α-naphthalene acetic acid (NAA, 2.0 mg/L), indole-3-acetic acid (IAA, 1.0 mg/L), and KIN (1.0 mg/L) with (10.06 ± 0.24) mean number of shoots per explants and the maximum shoot length was found to be 4.01 ± 0.37. The role of auxins were instrumental as rooting of the differentiated shoots was best in MS medium with combination of indole-3-butyric acid (IBA, 1.5 mg/L) and IAA (1.5 mg/L) with 13.14 ± 0.08 mean number of roots per shoots and the mean root length was found to be 5.61 ± 0.03. Regenerated plantlets were successfully acclimated in the green house and after a hardening period of 4 weeks, 90% transplantation success was achieved under the natural condition. The established in vitro propagated plants were identical and uniform on the basis of the morphology and growth characteristics to the donor plants used in the study.

An in situ study of growth of Lemongrass Cymbopogon flexuosus (Nees ex Steud.) W. Watson on varying concentration of Chromium (Cr +6 ) on soil and its bioaccumulation: Perspectives on phytoremediation potential and phytostabilisation of chromium toxicity

Chromium (Cr) contamination in soil is a growing concern in sustainable agricultural production and food safety. Remediation of Cr from contaminated soils is a challenging task which may not only help in sustaining agriculture but also in minimizing adverse environmental impacts. Pot culture experiments were performed with the application of varied concentration of Cr+6 to assess the Chromium accumulation potential of Lemongrass and to study the impact of toxic concentration of Cr+6 on morphological, physiological and biochemical parameters of the plant. The results showed an increasing accumulation trend of Chromium with increasing Chromium concentrations in both root and shoot of 60 days old Lemongrass plants, while the protein and chlorophyll contents decreased. Similarly, accumulation of Cr increased the levels of proline and antioxidant enzymes indicating the enhanced damage control activity. The potentiality of the plant with the capacity to accumulate and stabilize Cr compound in Cr contaminated soil by phytoremediation process has been explored in the present investigation.

Genotypic variations of ten Indian cultivars of Colocasia esculenta var. antiquorom Schott. evident by chromosomal and RAPD markers

Detailed karyotype, genome size and RAPD marker analysis were employed to assess genetic diversity in Taro (Colocasia esculenta var antiquorom Schott.). Karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosome with variations of ploidy from 2n = 2x = 28 (cv. Mothan, cv. Muktakeshi, cv. Sree Kiran, cv. Sree Pallavi, cv. Sunajhili) to 3n = 3x = 42 (cv. Banky, cv. DP-25, cv. Duradin, cv. H-3, cv. Telia) in the genome. Highly significant variations in the genomic length, volume and total form (TF) % were noted at variety level. Total genomic chromosome length varied from 46.96μm in cv. Sree Kiran to 100.49μm in cv. Duradin. Total genomic chromosome volume varied from 18.22μm3 in cv. Sunajhili to 38.22μm3 in cv. Duradin. Total form percentage was varied from 24.94% in cv. Sree Kiran to 39.04% in cv. H-3 confirming near metacentric to metacentric chromosomes in the karyotype. Significant variations in the 4C DNA content noted among the cultivars that ranged from 7.24 pg in cv. Sree Kiran to 18.24 pg in cv. Duradin; accordingly, genome size varied from ~7095 to 17875 Mbp. High genome size in all the triplod varieties with 3x = 42 chromosomes could be due to the presence of extra set of chromosomes in the genome or high amount of repetitive DNA. The variation in the genome size at the variety level might be attributed to loss or addition of highly repetitive sequences in the genome. Amplification of genomic DNA in 10 genotypes using Operon primers yielded 230 amplified DNA fragments, ranging in size from 200 to 2500bp out of which 79 bands were polymorphic. A total of 8 unique RAPD bands were observed among 10 taro genotypes that revealed primer wise polymorphism ranged from 16.66 to 47.36% with an average polymorphic percentage of 34.34%. Whereas, among the cultivars the polymorphic percentage varied from 3.70% between cv. DR-25 & cv. Duradin and cv. Telia & cv. H-3 to 41.94% between cv. Mothan & cv. Muktakeshi. Genetic similarity based on Jaccard’s coefficient varied from 0.54 to 0.96, indicating wide genetic variability among the varieties based on RAPD markers. Similarity measures and cluster analysis generally reflected the expected trends in relationships of diploid and triplod taro varieties. Dendrogram obtained from the genetic distances among the varieties could be useful for breeders to choose the diverse parents for breeding programme aimed at varietal improvement.

Chromium translocation, concentration and its phytotoxic impacts in in vivo grown seedlings of Sesbania sesban L. Merrill.

The present in vivo pot culture study showed hexavalent chromium (Cr+6) induced phytotoxic impacts and its translocation potential in 21 days old sesban (Sesbania sesban L. Merrill.) seedlings. Cr+6 showed significant growth retardation in 21 days old sesban (Sesbania sesban L. Merrill.) seedlings. Germination of seeds at 10,000 mg L-1 of Cr+6 exhibit 80% inhibition in germination. Seedling survival was 67% after 7 days of seedling exposure to 300 mg kg-1 of Cr+6. Shoot phytotoxicity was enhanced from 6% to 31% with elevated supply of Cr+6 from 10 mg kg-1 to 300 mg kg-1. Elevated supply of Cr+6 exhibited increasing and decreasing trends in % phytotoxicity and seedling tolerance index, respectively. Elevated supply of chromium showed decreased chlorophyll and catalase activities. Peroxidase activities in roots and leaves were significantly higher at increased supply of Cr+6. Cr bioconcentration in roots was nearly 10 times more than stems whereas leaves showed nearly double accumulation than stems. Tissue specific chromium bioaccumulation showed 53 and 12 times more in roots and shoots respectively at 300 mg kg-1 Cr+6 than control. The present study reveals potential of sesban for effective Cr translocation from roots to shoots as evident from their translocation factor and Total Accumulation Rate values.