Sunita Dalal

Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.

In vitro screening and evaluation of some Indian medicinal plants for their potential to inhibit Jack bean and bacterial ureases causing urinary infections

Abstract Context: Bacterial ureases play an important role in pathogenesis of urinary infections. Selection of plants was done on the basis of their uses by the local people for the treatment of various bacterial and urinary infections. Objective: Our investigation screens and evaluates 15 Indian medicinal plants for their possible urease inhibitory activity as well as their ability to inhibit bacteria causing urinary infections. Materials and methods: Plant extracts in three different solvents (methanol, aqueous, and cow urine) were screened for their effect on Jack-bean urease using the phenol–hypochlorite method. Subsequently, seven bacterial strains were screened for their ability to release urease and further antimicrobial-linked urease inhibition activity and minimum inhibitory concentration of the tested extracts were evaluated by the agar well diffusion and microdilution method, respectively. Results: Five plants out of 15 crude extracts revealed good urease inhibitory activity (≥20% at 1 mg/ml conc.) and IC50 values for these extracts ranged from 2.77 to 0.70 mg/ml. Further testing of these extracts on urease-producing bacterial strains (Staphylococcus aureus NCDC 109, S. aureus MTCC 3160, Proteus vulgaris MTCC 426, Klebsiella pneumoniae MTCC 4030, and Pseudomonas aeruginosa MTCC 7453) showed good anti-urease potency with an MIC ranging from 500 to 7.3 µg/ml. Discussion and conclusion: The results of screening as well as susceptibility assay clearly revealed a strong urease inhibitory effect of Acacia nilotica L. (Fabaceae), Emblica officinalis Gaertn. (Phyllanthaceae), Psidium guajava L. (Myrtaceae), Rosa indica L. (Rosaceae), and Terminalia chebula Retz. (Combretaceae). Our findings may help to explain the beneficial effect of these plants against infections associated with the urease enzyme.

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF βC1 in satellite DNA β which are predicted to function as silencing suppressors. In the present study suppressor function of ORF βC1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing βC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with βC1 gene from ToLCBB. However, in the case of 35S-βC1 CLCuMB and 35S-βC1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which βC1 interfere in RNA-silencing pathway is different.

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.

Grain filling duration and temperature pattern influence on the performance of wheat genotypes under late planting

Terminal heat referred to as increase in temperature during grain filling, is one of the important stress factors for wheat production and is responsible for decline in wheat production in many environments worldwide. In order to meet the challenges of high temperature ahead of global warming, concerted efforts are needed to evaluate wheat genotypes for heat tolerance and develop genotypes suitable for such stressed environments. Twenty-seven advanced wheat genotypes developed for stress and normal environments by different research centres were evaluated during 2009–10 and 2010–11 under timely sown (normal) and late sown (heat stress) environments. Analysis of variance revealed that the genotypes differed significantly in grain filling duration (GFD), grain growth rate (GGR) and thousand-grain weight (TGW). Out of 27 genotypes, 16 were found to be tolerant for thousand-grain weight under late planting (heat stress) during 2009-10 but only 12 were tolerant during 2010–11. Many of the genotypes registered more reduction in thousand-grain weight during 2010–11 as compared to 2009–10; the temperatures during 2009–10 were higher. The differences in grain filling duration under two conditions during both seasons as well as difference in temperatures during first half of grain filling explain the reduction pattern in the genotypes. GFD had significant negative correlation with temperatures during post heading period and the difference in GFD under two environments had positive correlation with these temperatures. The reduction in GFD had regression of 33.3% on reduction in GGR and reduction in GGR had regression of 41.6% on reduction in TGW genotypes AKW 1071, DBW 17, HS 277, K 7903, K 9107, NW 1014 and RAJ 3765 had less sensitivity to stress environments during both years.

Isolation and identification of promoter elements of Cotton leaf curl Multan betasatellite associated with Tomato leaf curl viruses

Betasatellite DNAs associated with Old World monopartite begomoviruses encode a single gene (βC1) which mediate the vital functions required for viral pathogenicity. A region of upstream translation start site of βC1 of Cotton leaf curl Multan betasatellite (CLCuMB) associated with Tomato leaf curl New Delhi virus was tested for its promoter activity. Transcript mapping studies revealed that the transcript had a very short leader sequence of 12 nt and 3′ end was 9 nt downstream of stop codon at 186 nt coordinate position. Transient expression studies in Nicotiana benthamiana using the β-glucuronidase reporter gene driven by a βC1 promoter of CLCuMB identified a 169 nt region (between 720 and 869 nt coordinate) upstream of βC1 which is important for βC1 transcription. Histochemical GUS staining of selected solanaceous hosts indicated that βC1 promoter was active in leaves of N. benthamiana and Nicotiana tabacum and leaves and fruit of tomato. Temporal regulation studies on viral transcript using semi-quantitative PCR showed that the βC1 is a late phase gene and is expressed only 48 h post-inoculation.

A Cross Sectional Study on Prevalence of Antibiotic Resistance and Role of Efflux Pumps in Fluoroquinolone Resistance by using Efflux Pump Inhibitors in Isolated Cultures from Poultry, Dairy Farms and MTCC Strains from Reservoirs

Aims: Emergence of antibiotic resistance in bacterial strains has always remained a crucial concern. Mutations in antibiotic target sites, over expression of efflux pump are the major modes of development of bacterial antibiotic resistances. The present study was conducted to determine antibiotic resistance and role of efflux pumps in fluoroquinolone resistance by using efflux pump

In vitro and in silico Approach to Evaluate the Urease and Collagenase Inhibitory Activity of Embilica officinalis Gaertn Fruit

Aim: The key virulent factors of bacteria are enzymes. Urease and collagenase enzyme play a vital role in pathogenesis of wide array of bacterial strains and cause numerous diseases. So the aim of present study was to find out the potent drug candidate from

Effect of Wedelolactone on CCl4 Induced Liver Damage

The hepatoprotective activity of ethanolic extract and wedelolactone isolated from EcliptaalbaL. (Family: Asteraceae) was studied against CCl induced, acute hepatotoxicity in rats. 4 Hepatoprotective activity was studied by estimating biochemical analysis of blood viz. serum enzyme activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), protein and bilirubin. The treatment with wedelolactone showed a dosedependent reduction of CCl induced toxicity, indicating the compound could preserve the normal 4 functional status of the liver.