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Finger millet [Eleusine coracana (L.) Gaertn] is an excellent food and forage crop of arid and semiarid areas in Africa and Asia. It is well adapted to drought, heat, high salinity, poor soil fertility and low pH with an efficient C4 carbon fixation mechanism for high yield potential. To normalize the target gene expression data, the identification of suitable reference genes is essential. Ten candidate reference genes were selected and their expression stability was analyzed in various samples treated with different abiotic stress conditions. Five different statistical algorithms: geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to determine the stability of these genes. Our results revealed GAPDH, EEF1a, ACT and CYC as highly stable reference genes and PP2A and eIF4A as least stable reference genes across all the samples and suggesting that these genes could be used for accurate transcript normalization under abiotic stress. To the best of our knowledge, this is the first report on identification of suitable reference genes for accurate transcript normalization using qRT-PCR in finger millet under abiotic stress.
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Metal load is an abiotic stress that becomes stronger by continual industrial production, wastage, and long-range transport of contaminants. It deteriorates the conditions of agricultural soil that leads to lower growth of cereals as well as decreasing nutritional value of harvested grains. Cadmium (Cd) entry by food chain also affects the health of population. The present study is focused on finding out the superior cereal variety under increasing Cd regime. The plants were grown in increasing Cd levels (0-1000 µM) in the medium and were investigated on 15th day of the exposure. Various parameters like antioxidative enzymes and osmoprotectant levels were studied in both roots and shoots. Cd accumulation in plant organs was determined by atomic absorption spectrophotometry (AAS). Analysis of stress tolerance mechanisms through reactive oxygen species (ROS) scavenging and better partitioning of Cd in roots indicated kodo millet to be more stress tolerant than wheat.
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Cádmio/toxicidade , Paspalum/efeitos dos fármacos , Poluentes do Solo/toxicidade , Triticum/efeitos dos fármacos , Biodegradação Ambiental , Paspalum/genética , Paspalum/crescimento & desenvolvimento , Especificidade da Espécie , Estresse Fisiológico , Triticum/genética , Triticum/crescimento & desenvolvimentoRESUMO
Stevia rebaudiana (Bert.) Bertoni is an important medicinal plant used as noncaloric commercial sweetener. Plants regenerated with higher levels of copper sulphate in the medium exhibited enhanced activity of peroxidase and polyphenoloxidase (PPO) enzymes. Transmission electron microscopy (TEM) revealed increase in size and number of electron dense inclusions in the chloroplasts of plants regenerated at optimised level of copper sulphate (0.5 microM) in the medium. There was decrease in chlorogenic acid (CGA) content. Chl-a-fluorescence transient pattern (OJIP) showed that the photosynthesis process was more efficient at 0.5 microM CuSO4 in the medium.
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Cloroplastos/efeitos dos fármacos , Cloroplastos/ultraestrutura , Sulfato de Cobre/farmacologia , Fotossíntese/efeitos dos fármacos , Stevia/efeitos dos fármacos , Cloroplastos/enzimologia , Cloroplastos/metabolismo , Stevia/enzimologia , Stevia/fisiologiaRESUMO
The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l(-1)) + ascorbic acid (100 mg l(-1)) + citric acid (10 mg l(-1))] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 µM) and α-naphthalene acetic acid (NAA, 2.6 µM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 µM) + phloroglucinol (PG, 3.9 µM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 µM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.
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In the present work, the Nickel oxide (rGO-NiO), Silver (rGO-Ag), Copper oxide (rGO-CuO) doped Graphene Oxide are reported for catalytic reactions. A comparative study for catalytic activities of these materials are performed with nitroaromatic compound 4-nitroaniline and the results are statistically studied by using univariate analysis of variance and Post Hoc Test through Statistical Package for Social Sciences and it is observed that CuO doped Graphene material is showing better catalytic activity in minimum time. So, further research has been focused on the catalytic acitivity of rGO-CuO only and it is found that it is efficient in reducing other nitro compounds also such as Picric acid and Nitrobenzene. Dye degradation of Methylene blue is also performed using CuO decorated Graphene material and significant changes were observed using UV spectroscopy. The characterization of rGO-CuO is done with Fourier-transform Infrared Spectroscopy, Powder X-ray Diffraction, Thermogravimetric Analysis, Scanning Electron Microscope and Transmission Electron Microscopy.
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An efficient and reproducible protocol has been developed for in vitro propagation of Pithecellobium dulce (Roxb.) Benth (a multipurpose leguminous tree) from field grown nodal segments (axillary bud). Shoot bud induction occurred from nodal explants of 15-years-old tree on Murashige and Skoog (MS) basal medium supplemented with 4.4 µM 6-benzyladenine (BA) and multiplication was achieved on MS medium supplemented with 4.4 µM BA + 0.73 µM phenylacetic acid (PAA) i.e. up to 7 shoot buds in the period of 5-6 weeks. Addition of adenine sulphate (AdS) to this medium further enhanced the number of shoot buds up to 10. Proliferating shoot cultures were established by repeatedly subculturing primary culture on fresh medium (MS + 4.4 µM BA + 0.73 µM PAA) after every 25 days. In vitro rooting was achieved on MS medium supplemented with 2.46 µM Indole-3-butyric acid (IBA) + 41.63 µM activated charcoal (AC). The micropropagated shoots with well developed roots were acclimatized in green house in pots containing sand, soil and manure (1:1:1). Genetic stability of micropropagated clones was evaluated using Random amplified polymorphic DNA (RAPD) and Inter simple sequence repeat (ISSR) markers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic uniformity of micropropagated plants. This is the first report of an efficient protocol for regeneration of P. dulce through organogenesis, which can be used for further genetic transformation and pharmaceutical purposes.
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BACKGROUND: Capsicum or chilli is an important crop in India which exhibits immense structural and genetic variations reflecting their intra- and inter-specific relationships. The aim of this study was to establish relationships amongst 54 Capsicum accessions through analysis of genetic and population structure using ISSR markers. RESULTS: Out of 19, successful DNA amplifications were shown by 7 ISSR primers and a total of 80 bands were identified ranging between 8 and 14 with an average of 11.43 bands/primer. A significant degree of polymorphic information content (PIC), discriminating power (DP), resolving power (RP), effective multiplex ratio (EMR), and marker index (MI) were identified as 0.39, 0.70, 6.40, 5.88, and 2.30, respectively, using ISSR markers in chillies. The cross-transferability ranged from 8.0 to 72.15% with an average of 52.63% among chillies. Amongst genetic information, grand mean values were 0.264, 0.180, 0.376, 0.296, and 0.180, which correspond to Shannon's information index (I), expected heterozygosity (He), Nei's gene diversity, total diversity among species (Ht), diversity within species (Hs), respectively. Further, the coefficients of gene differentiation (Gst) and gene flow (Nm) were 0.393 and 0.773, representing higher genetic variation among the population which was confirmed by analysis of molecular variance (AMOVA). CONCLUSION: ISSR markers represented a potent system for the estimation of relationships or variation studies and generated information useful for planning crop management and improvement strategies in chilli breeding.
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Enantioselective synthesis through photocatalysis is one of the highly preferred approaches towards preparation of optically active compounds. This review elaborates and critically analyzes the different strategies of photocatalytic enantioselective reactions through H-bonding, transition metal catalysis, phase-transfer catalysis (PTC), chiral Lewis acid catalysis, N-heterocyclic carbene catalysis, and amine catalysis, and also explores ion pairs. In addition, it explains the different catalysis modes with multifunctional approaches for enantioselective photocatalytic reactions.
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Aminas , Ácidos de Lewis , Catálise , EstereoisomerismoRESUMO
Heavy metal contamination of water bodies has been a cause of grave concern around the globe. Analysis of various industrial effluents has revealed a perilous level of Cr (VI) and Ni (II). Pseudomonas aeruginosa is an extracellular polymeric substances (EPSs) producing bacterium. EPS has a great potential in the sequestration of heavy metal ions. In the present study efforts have been made to understand the effect of time, pH, and temperature on production of EPS by P. aeruginosa (MTCC 1688). The extracted EPS has been applied for removal of Ni (II) and Cr (VI) ions from aqueous system. The results revealed that highest EPS yield (26 mg/50 mL) can be obtained after 96 h of incubation at pH 6 and 32 °C temperature in 50 mL of culture. Treatment of 10 mg/L Cr (VI) and Ni (II) with 30 mg/L EPS resulted in the removal of 26% and 9% of Cr (VI) and Ni (II), respectively. Fourier-transform infrared spectral analysis revealed the involvement of -OH, -NH, C-O, diketone, and ester functional groups of EPS in the attachment of Cr (VI) ion while involvement of amide and -C[bond, double bond]O groups in Ni (II) binding with EPS. Scaling-up the production of EPS using bioreactor may further help in developing an efficient process for treatment of water polluted with Cr and Ni.
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India has a diverse range of agro-ecological conditions which support the cultivation of different rice varieties differing in the adaptation which is so important for sustainable development of rice crop. Specific ecotypes of rice adapted to diverse conditions have divergence in their morphology, physiology, biochemistry, molecular function, agronomy, and stress response. In the present study, 12 different rice varieties viz., PB-1, PB-1509, Pusa-RH-10, CSR-30, HKR-47, PR-126, Govind, Sharbati, ADT-37, ADT-39, ADT-45, White Ponni, were selected for the study of intrinsic biochemical behaviour and these varieties belong to different Agro-ecological zones and basmati or non-basmati rice varieties. Amongst intrinsic biochemicals activity, the differential response of radical scavenging, superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POX) activities, were observed in the selected rice varieties at 14 days old seedling stage, developed under controlled growth conditions. Comparatively, North India region rice varieties displayed an enhanced intrinsic biochemical response than south India region rice varieties. Similarly, basmati rice varieties showed increased biochemical response compared to non-basmati rice varieties. Thus, the differential biochemical responses (radical scavenging, SOD, CAT, and POX activities) observed creates a significant difference between rice varieties and provides valuable information about rice ecotype-biochemical interaction for sustainable adaptive value under different ecological conditions.
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Extraction of biosurfactants from plants is advantageous than from microbes. The properties and robustness of biosurfactant derived from the mesocarp of Balanites aegyptiaca have been reported. However, the dark brown property of biosurfactant and lack of knowledge of its biocompatibility limits its scope. In the present work, the decolorization protocol for this biosurfactant was optimized using hydrogen peroxide. The hemolytic potential and biocompatibility based on cell toxicity and proliferation were also investigated. This study is the first report on the decolorization and toxicity assay of this biosurfactant. For decolorization of biosurfactant, 34 full factorial design was used, and the data were subjected to ANOVA. Results indicate that 1.5% of hydrogen peroxide can decolorize the biosurfactant most efficiently at 40 °C in 70 min at pH 7. Mitochondrial reductase (MTT) and reactive oxygen species (ROS) assays on M5S mouse skin fibroblast cells revealed that decolorized biosurfactant up to 50 µg/mL for 6 h had no significant toxic effect. Hemolysis assay showed ~ 2.5% hemolysis of human RBCs, indicating the nontoxic effect of this biosurfactant. The present work established a decolorization protocol making the biosurfactant chromatically acceptable. Biocompatibility assays confirm its safer use as observed by experiments on M5S skin fibroblast cells under in vitro conditions.
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Balanites/química , Materiais Biocompatíveis/química , Tensoativos/química , Animais , Materiais Biocompatíveis/farmacologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Teste de Materiais/métodos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Tensoativos/farmacologiaRESUMO
Metallic nanoparticles are traditionally synthesized by wet chemical techniques, where the chemicals used are often toxic and flammable. In the present study, the spore crystal mixture of Bacillus thuringiensis was used for the synthesis of silver nanoparticles. Nanoparticles were characterized using UV-Vis absorption spectroscopy, XRD and TEM. X-ray diffraction and TEM analysis showed the average particle size of 15 nm and mixed (cubic and hexagonal) structure. This is for the first time that any bacterial spore crystal mixture was used for the synthesis of nanoparticles. Further, these biologically synthesized nanoparticles were found to be highly toxic against different multi drug resistant human pathogenic bacteria.
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Bacillus thuringiensis/química , Nanopartículas Metálicas/química , Prata/química , Esporos Bacterianos/química , Cristalização , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Espectrofotometria , Difração de Raios XRESUMO
An efficient protocol for physico-chemical characterization of gum exudates collected from the drumstick tree (Moringa oleifera Lam.) has been reported in the present study. Extraction of gum metabolites was done using a series of water, alcohol, acid, and alkali solvent systems. The gum was sparingly soluble in water at room temperature and formed a colloidal solution. Solubility of the gum gradually increased in the solvent gradient (80% ethanol, deionized water, 0.05 M HCl, and 0.05 M NaOH) at 90 °C. Further, electron microscopy revealed that the acetyl group is essential in maintaining the structural integrity, and deacetylation of gum resulted in formation of a mesh of scattered and fibrous particles. Treatment of gum with deionized water resulted in development of a hydrocolloidal matrix with a pore size of 0.5 µm, which upon deacetylation was reduced up to 0.2 µm. The polymer was amorphous in nature and showed maximum thermal stability in ethanol. Gas chromatography-mass spectrometry of the gum polymer revealed that carbohydrate derivatives constituted its major part (>75%). Maximum carbohydrate concentration was obtained in the ethanol soluble fraction, along with fatty acids (10%) and secondary metabolites (9%). The results provided very first confirmation of the hydrocolloidal properties and thermostability of the gum exudates obtained from the drumstick tree, which can further be used to develop an eco-friendly and nontoxic bioligand.
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Being an economical and nutritional crop, Capsicum appeases people's peppery taste and is found to bewidely distributed all over the world having vast diversity. In the present study, genetic polymorphism, cross transferability (CT) and genetic diversity were examined among the 54 different accessions of Capsicum species including 49 of Capsicum annuum, three of C. baccatum and two of C. frutescens, using a set of 36 start codon targeted (SCoT) primers. Of the total, 35 SCoT markers showed successful amplification profile among chilli germplasms and an average primer polymorphism was reported as 81.52% which ranged from 50% (SCoT-6) to 100% (SCoT-11). A total of 365 amplicons were obtained with an average of 10.43 bands per primer and the length of the bands ranged from 150 bp to 1.2 kb. Further, polymorphic information content value of SCoT markers ranged from 0.42 (for SCoT-25) to 0.86 (SCoT-27) with an average of 0.78. The average value of CT of SCoT markers was 44.08% ranged from 14.25% to 57.26% among different chilli accessions. A dendrogram was constructed and established genetic relationship among 54 capsicum species, with the help of translation initiation codon polymorphisms or SCoT primer amplification. This study suggests the effectiveness of SCoT marker system for characterizing and assessing genetic diversity of Capsicum germplasm, which can be used for evolutionary studies and to identify agronomically important traits.
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Capsicum/genética , Códon de Iniciação , Cruzamentos Genéticos , Polimorfismo Genético , Amplificação de Genes , Marcadores Genéticos , Variação Genética , Genoma de Planta , Genômica/métodos , FilogeniaRESUMO
Plastic and polythene as hydrophobic materials become a grave concern due to their non-biodegradable nature, cumbersome recycling and waste management. Cuticular wax derived from Calotropis procera is explored as an eco-friendly and safe hydrophobic material. The effects of duration of exposure to solvent, solvent type, size and side of the leaf on cuticular wax yield have been studied. Leaf with the smallest area (10 cm2-25 cm2) was found to be the most suitable to isolate the wax. GC-MS analysis of the wax revealed that the wax consists of mainly esters, alkane and alkene. Mitochondrial reductase (MTT) and lactate dehydrogenase (LDH) assay have been carried out on M5S cell line at various concentrations and the results indicate that up to 1 µg/ml (acetone as solvent) and 3 µg/ml (chloroform as solvent) use of wax has no toxic effect. To evaluate the hydrophobic potential of the wax in developing hydrophobic paper water regains and contact angle has been measured. The gain in hydrophobicity of the paper is evident from the rise in contact angle (≥90Ë) of paper coated with wax. Scanning electron micrograph and FTIR spectra generated physical and chemical evidence of coating of wax on paper.
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Calotropis , Folhas de Planta/química , Ceras/química , Ceras/toxicidade , Alcanos/análise , Alcenos/análise , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ésteres/análise , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Papel , Solventes/químicaRESUMO
Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) shows high levels of variation in fruit color, fruit stripe pattern, seed coat color, and size. Thirty-eight accessions of C. colocynthis plants from different parts of semi-arid Rajasthan were collected and genetic diversity was assessed using random-amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 65 RAPD decamer primers, 50 primers produced 549 scorable bands of which 318 were polymorphic. Polymorphic banding patterns with the number of amplified fragments varied from 5 (OPA-08 and OPF-9) to 19 (OPT-20) in the molecular size range of 150-6000 bp. Percent polymorphism ranged from 22.2% (OPA-09) to 83.3% (OPE-12) with 55.14% polymorphism. Out of the 20 ISSR primers screened, 13 primers produced 166 amplification products, of which 99 were polymorphic. The number of bands amplified per primer varied between 9 (UBC-807, 802) and 16 (UBC-803, 812) with average band size between 250 and 4000 bp. Percent polymorphism ranged from 45.4% (UBC-815) to 73.3% (UBC-814) with 65.05% polymorphism. Dendrogram constructed on the basis of RAPD + ISSR polymorphism separated the accessions into four distinct clusters at 72% variation with Jaccard's similarity coefficient ranging from minimum 0.64 to 0.95. The matrices for RAPD and ISSR were also compared using Mantel's test and obtained correlation value (r = 0.7947). Discriminating power of RAPD and ISSR markers was assessed by calculating polymorphic information content, multiplex ratio, marker index, and resolving power. Approx. 50% RAPD and ISSR markers showed PIC value and heterozygosity (H) ≥ 0.50, indicating marker as informative. The primers that showed higher polymorphism had higher RP, MR, and MI values.
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Withania coagulans (Stocks) Dunal (Solanaceae), also known as 'Panir Bandh' is an important medicinal plant that is extensively used as a home remedy for several diseases in the Indian subcontinent. The plant possesses specific steroidal lactones known as withanolides which show high level of pharmaceutical activity against a broad spectrum of microorganisms. Natural propagation of the plant occurs through Seed but due to unisexual nature of the flowers; chances of Seed setting are very limited and the plant is on the verge of extinction because of overexploitation and reproductive failure. Plant tissue culture techniques offer opportunities for ex situ conservation and mass multiplication of endangered plant species through micropropagation and also enhancement of in vitro biosynthesis of bioactive compounds. In this chapter we present protocols for the mass multiplication of W. coagulans, assessment of clonal fidelity by RAPD, and estimation of bioactive compounds (withanolides) by thin layer chromatography (TLC) and reverse phase HPLC developed in our laboratory.
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Lactonas/análise , Brotos de Planta/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos/métodos , Withania/crescimento & desenvolvimento , Vitanolídeos/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Lactonas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Withania/genética , Withania/metabolismo , Vitanolídeos/metabolismoRESUMO
Extracellular Polymeric Substances (EPS) of microbial origin are complex biopolymers and vary greatly in their chemical composition. They have a great potential in chelation of metal ions. In this work, the effect of growth phase, temperature and pH on production of EPS by two bacteria Azotobacter beijreinckii and Bacillus subtilis have been studied. Extracted EPS was used to remove Cr(VI) from aqueous system. A. beijreinckii produced maximum EPS after 24h at pH 7 and temperature 30°C while B. subtilis produced maximum EPS after 96h at pH 7 and temperature 37°C. For an initial concentration of 10ppm, 26% and 48% Cr(VI) removal was recorded for EPS derived from A. beijreinckii and B. subtilis respectively. The presence of functional groups on EPS and their interaction with Cr(VI) was confirmed using Fourier-transform infrared (FTIR) spectra analysis. In both the bacteria, carboxyl and phosphate groups show involvement in metal binding.
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Azotobacter/metabolismo , Bacillus subtilis/metabolismo , Cromo/metabolismo , Biopolímeros/química , Cromo/química , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
Expressed sequence tags (ESTs) are important resource for gene discovery, gene expression and its regulation, molecular marker development, and comparative genomics. We procured 10000 ESTs and analyzed 267 EST-SSRs markers through computational approach. The average density was one SSR/10.45 kb or 6.4% frequency, wherein trinucleotide repeats (66.74%) were the most abundant followed by di- (26.10%), tetra- (4.67%), penta- (1.5%), and hexanucleotide (1.2%) repeats. Functional annotations were done and after-effect newly developed 63 EST-SSRs were used for cross transferability, genetic diversity, and bulk segregation analysis (BSA). Out of 63 EST-SSRs, 42 markers were identified owing to their expansion genetics across 20 different plants which amplified 519 alleles at 180 loci with an average of 2.88 alleles/locus and the polymorphic information content (PIC) ranged from 0.51 to 0.93 with an average of 0.83. The cross transferability ranged from 25% for wheat to 97.22% for Schlerostachya, with an average of 55.86%, and genetic relationships were established based on diversification among them. Moreover, 10 EST-SSRs were recognized as important markers between bulks of pooled DNA of sugarcane cultivars through BSA. This study highlights the employability of the markers in transferability, genetic diversity in grass species, and distinguished sugarcane bulks.
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A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2-2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds.