Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R₁ progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R₁ progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.     

Use of genomic and genic SSR markers for assessing genetic diversity and population structure in Indian and African finger millet (Eleusine coracana (L.) Gaertn.) germplasm

Genetic diversity and population structure were analyzed in 67 diverse finger millet accessions of African and Indian origin. A total of 69 alleles were generated with a mean of 4.0 alleles per locus and with an average gene diversity of 0.471. Molecular diversity parameters showed higher values in African accessions. Nineteen rare and nine unique alleles were observed and the identified accessions can be a potential source for further improvement of finger millet. Clustering of south Indian accessions with African lowland types and north/highland Indian accessions with that of African highlands was also observed. Structure analysis revealed the distinctness of Ugandan accessions compared to other African accessions and five major sub-populations useful for parental selection, conservation, and utilization of finger millet.     

Genetic variability and relationships among thirty genotypes of finger millet (Eleusine coracana L. Gaertn.) using RAPD markers

Ragi or finger millet (Eleusine coracana L.) is an important crop used for food, forage, and industrial products. It is distributed in tropical and temperate regions of the world. The germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or varieties identification has relied on morphological characters like growth habit, leaf architecture or floral morphology. Investigation through RAPD (random amplified polymorphic DNA) markers was undertaken for identification and determination of the genetic variation among thirty genotypes of ragi of the family Poaceae. Thirteen selected decamer primers were used for genetic analysis. A total of 124 distinct DNA fragments ranging from 300-3000 bp was amplified by using selected random RAPD marker. The genetic similarity was evaluated on the basis of the presence or absence of bands. Cluster analysis was made by the similarity coefficient. It indicated that the 30 genotypes of ragi form two major clusters, first, a major cluster having only one genotype, i. e. Dibyasinha and a second major cluster having twenty-nine genotypes. The second major cluster again subdivides into two minor clusters. A first minor cluster has only three varieties, i. e. Neelachal, OEB-56 and Chilika. The genotypes Neelachal and OEB-56 exhibit a 86% similarity with each other and 80% similarity with Chilika. A second minor cluster has 26 genotypes and is divided into two sub-minor clusters. The first sub-minor cluster has only one genotype (VL-322). The second sub-minor cluster again subdivides into two groups. One group has one genotype and the second group again is divided into two sub-groups, one with 13 genotypes and the other with 11 genotypes. The highest similarity coefficient was detected in a genotype collected from southern India and the least from northern India. The genotypes of finger millet collected from diverse agroclimatic regions of India constitute a wide genetic base. This is helpful in breeding programs and a major input into conservation biology of cereal crop.     

Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantsk _i andl ₅₀ for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol     

Somaclonal variability as a source for creation of new varieties of finger millet Eleusine coracana (L.) Gaertn

Data on selection and characterizing of finger millet Eleusine coracana (L.) Gaertn. somaclonal variants regenerated from callus culture are presented and described. Among the tested genetically stable lines the somaclonal variant SE-7 was the most interesting due to its acquired important agricultural traits, such as a higher seed and biomass yield, rapid seed germination at lower temperatures, shortening of the main plant development stages. Data analysis shows that somaclonal variability can be a source to obtain initial material for further selection of new finger millet varieties.     

Protein content and amino acid spectrum of finger millet [Eleusine coracana (L.) Gaertn.] as influenced by nitrogen and sulphur fertilizers

Summary The effect of N and S fertilizers on yield, protein content and amino acid composition of the tropical cereal finger millet was studied in field and sand culture experiments. In the field the grain yield measured from 2400 to 4100 kg/ha, and the seed crude protein from 7 to 13% with rates of N up to 150 kg/ha. Seed crude protein was high in methionine and cystine, but contained less sulphur amino acids when the seed was high in protein.Supplies in excess failed to increase the lower methionine and cystine contents of the crude protein.Sulphur deficiency drastically reduced the synthesis of sulphur amino acids, concomitantly raising the aspartic acid/asparagine content of the leaves up to 45 per cent of their total N content. An S:N weight ratio of 1:10 in the seeds and 1:15 in the leaves indicates adequate supply of sulphur. The importance of finger millet for human nutrition is considered in relation to increasing substitution with other crops, use of nitrogen fertilizers, and the actual sulphur status of soils in East Africa.Sponsored by the Norwegian Agency for International Development.Sponsored by the Norwegian Agency for International Development.     

DNA sequence organization in finger millet (Eleusine coracana)

Approximately 39 to 49% of the genome of finger millet consists of repetitive DNA sequences which intersperse with 18% of single copy DNA sequences of 1900 nucleotide pairs. Agarose gel filtration and electrophoresis experiments have yielded the sizes of interspersed repeated sequences as 4000–4200 nucleotide pairs and 150–200 nucleotide pairs. Approximately 20% of the repeated DNA sequences (4000–4200 nucleotide pairs) are involved in long range interspersion pattern, while 60% of the repeated DNA sequences (150–200 nucleotide pairs) are involved in short period interspersion pattern. Based on the data available in literature and the results described here on DNA sequence organization in plants, it is proposed that plants with haploid DNA content of more than 2.5 pg exhibit mostly the short period interspersion pattern, while those with haploid DNA content of less than 2.5 pg show diverse patterns of genome organization. NCL Communication No.: 2708     

Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.     

Molecular cloning,characterization and three-dimensional structure prediction of Lipoxygenase from Finger millet [Eleusine coracana (L.) Gaertn.] germinating seedlings

Lipoxygenases (LOXs) are functionally diverse class of non-heme iron containing dioxygenases. They involve in multiple physiological processes in plants and animals. In current study a full length cDNA of LOX has been cloned from finger millet germinating seedlings using PCR and RACE PCR methods. The ORF (2661 bp) encodes 887 amino acid residues, with an approximate molecular weight of 100.8 kDa and an isoelectric point of 5.90. The sequence exhibited significant similarity to already known LOXs. It comprises of a putative PLAT domain and an iron-binding catalytic domain. Sequence analysis demonstrated that this LOX is most probably a type 9-LOX, and shared highest identity (85 %) with the Setaria italica 9-LOX. TargetP predicted EcLOX as a cytoplasmic protein. It has an optimum pH of about 6.4 and increased lipid peroxidation levels were observed on day 5 of germination. Semi-quantitative RT-PCR analysis showed that EcLOX gene expression was preferentially higher on day 4 after germination, and in growing leaf and root tissues. Its expression could be induced by Methyl Jasmonate (MeJA), but not by Salicylic acid (SA). The 3-dimensional structure of the EcLOX was modeled using in silico approach and Molecular dynamics (MD) simulations were performed. Further, to understand the protein active site, docking studies were performed with substrates and inhibitors. In summary, EcLOX protein was characterized using various molecular, biochemical and in silico methods which improves our understanding of these fascinating enzymes.     

Obtaining the transgenic lines of finger millet Eleusine coracana (L.). with dinitroaniline resistance

The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant α-tubulin gene (TUAml), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 μM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgenic nature of plants. The analysis of seed of T₁ of transgenic lines confirmed heterozygous character of inheritance of trifluralin resistance.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.     

Microbial population and biochemical changes in fermenting finger millet (Eleusine coracana)

Natural fermentation of finger millet (Eleusine coracana) was carried out for 48 h. Microbiological and chemical analysis was performed throughout the fermentation process. The fermentation was heterolactic dominated by lactic acid bacteria accompanied by the production of lactic and acetic acids with decrease in pH and increase in titratable acidity. The microbial population increased until 18 to 24 h accompanied by a rapid decrease in total and reducing sugars. The microflora stabilized between 24 and 48 h, during which time the total and -amylase activities increased with accumulation of sugars. Total free amino acids also increased. Yeast counts were low and moulds and coliforms were absent. Repeated fermentations showed consistency in the qualitative and quantitative changes in microflora. Five predominant types of bacteria, strains belonging to Leuconostoc, Pediococcus and Lactobacillus were identified. Of these only one type, Pediococcus, dominated (>80%) in the latter half of fermentation.     

Genomic in situ hybridization identifies genome donor of finger millet (Eleusine coracana)

Eleusine coracana, commonly called finger millet, is an important cereal of semi-arid regions, cultivated in parts of Africa and India for its grain. It is reported to be an allotetraploid with a chromosome number 2n = 4x = 36, and diploid species E. indica, with chromosome number 2n = 2x = 18, is considered to be one of its genome donors. In situ hybridization of the E. coracana genome with the genomic DNA of various diploid species of the genus confirmed that E. indica is one of the genome donors to E. coracana and that E. floccifolia is another genome contributor to this allotetraploid species. In situ hybridization also showed a close genomic relationship between 4 diploid species, E. indica, E. floccifolia, E. tristachya and E. intermedia, and also between these and tetraploid species E. coracana. The common genomic in situ hybridization (GISH) signals of the genomic DNA of E. indica and E. tristachya on 15–18 chromosomes of E. coracana clearly indicated that these 2 species have a close genomic similarity. GISH on 25–27 chromosomes of E. coracana withthe genomic DNA of E. intermedia and cross in situ hybridization signals on the chromosomes of E. coracana with genomic DNA of E. intermedia and E. indica or E. intermedia and E. floccifolia has showed that E. intermedia may be an intermediate species of E. indica and E. floccifolia. Received: 15 May 2000 / Accepted: 4 September 2000     

Variation in response of accessions of minor millets,Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and Eragrostis tef (Zucc.) Trotter (tef) to salinity in early seedling growth

The response to increasing NaCl concentration of seedlings of 25 accessions of Ethiopian land races of each of Pennisetum americanum (L.) Leeke (pearl millet) and Eleusine coracana (L.) Gaertn (finger millet), and 15 accessions of Eragrostis tef (Zucc.) Trotter (tef), was examined after two week's growth in NaCl solution culture. Although increasing NaCl concentration significantly reduced seedling root lengths, there was considerable variation within, and between accessions within each species.Analysis based upon a non-linear least square inversion method, using root length data, revealed significant differences in accessions of P. americanum and E. tef on the basis of the estimated salinity threshold, C _t , the NaCl concentrations at which root length begins to decrease. C _t did not differ significantly between E. coracana accessions. Estimates of C₅₀ and C₀, mininum concentrations causing a 50% decrease in root length, and zero root growth respectively, revealed differences between and within accessions for all three species. Overall, finger millet was more tolerant than tef, which was more tolerant than pearl millet. There is clear evidence that differences in tolerance are genetically based from broad sense heritability estimates.     

Determination of calcium responsiveness towards exogenous application in two genotypes of Eleusine coracana L. differing in their grain calcium content

In order to investigate the influence of exogenous application of calcium (Ca) on its accumulation in finger millet, two genotypes (GPHCPB-1 and GPHCPB-45), which posses low and high grain Ca contents, respectively, were subjected to regular fertigation of varying levels of Ca in Hoagland’s nutrient medium. The responsiveness of both the genotypes towards increasing exogenous application of Ca (0.1, 5.0, 10 and 20) was determined in terms of changes in tissue Ca levels, agro-morpho-physio-biochemical parameters. Sharp increase of Ca content in root, stem and spike was observed up to excess level of Ca (10 mM) in GPHCPB-1 while in case of GPHCPB-45 an increase in Ca content was observed only up to sufficient level of Ca (5 mM) and above that its accumulation remained constant or declined in both the genotypes. In case of leaf the level of Ca increased linearly at all concentrations of supplied Ca in both the genotypes. Both the genotypes behave differentially as GPHCPB-45 genotype accumulated more Ca and was also superior in root length, root dry matter accumulation, plant height and relative water content at Ca deficient condition (0.1 mM) as compared to GPHCPB-1 genotype. The continuous rise in stem diameter, biomass, seed yield, chlorophyll content, SPAD value, seed oxalic acid and phytic acid content were recorded in both the genotypes up to excess or toxic levels of supplied Ca. On the basis of present study it was concluded that Ca accumulation in plant is determined by both genetic (genotype dependent) as well as environmental factors (availability of Ca in rhizosphere).     

Solute accumulation, solute potential, germinability and seedling vigour of seeds of finger millet (Eleusine coracana Gaertn.) raised under rain-fed conditions and under irrigation

Abstract The nature of the solutes accumulated in the grains of ten finger millet genotypes grown under rain-fed conditions with moisture stress during panicle and grain development were determined. Although grain yield decreased under stress, the solute potential (SP) of seeds from stressed plants was significantly lower (more negative), probably due to the increase in sucrose content. There were no significant changes in the content of total amino acids, free proline, reducing sugars or potassium. The contribution of sucrose to total solute potential was approximately 32% in seeds obtained from stressed plants as compared with only 15% in seeds from irrigated treatment. The germinability and seedling vigour of the seeds from stressed plants was significantly higher under simulated stress with polyethylene glycol (PEG), but was similar to that of seeds of non-stressed plants under normal conditions.     

Three alpha-amylases from malted finger millet (Ragi,Eleusine coracana,Indaf-15)--purification and partial characterization

Three alpha-amylases (E.C. 3.2.1.1) were purified to apparent homogeneity from 72 h finger millet malt by three step purification via fractional acetone precipitation, DEAE-Sephacel ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 6.5, 2.9, 9.6% and fold purification of 26, 17 and 31, respectively. alpha-Nature of these amylases was identified by their ability to rapidly reduce the viscosity of starch solution and also in liberating oligosaccharides of higher D.P. and were accordingly designated as amylases alpha-1((b)), alpha-2 and alpha-3, respectively. These amylases, having a molecular weight of 45+/-2 kDa were found to be monomeric. The pH and temperature optima of these alpha-amylases were found to be in the range of 5.0-5.5 and 45-50 degrees C, respectively. K(m) values of these amylases for various cereal starches varied between 0.59 and 1.43%. Carbodiimide (50 mM) and metal ions such as Al(3+), Fe(2+), and Hg(2+) (5 mM) have completely inhibited these enzymes at 45 degrees C. Amino acid analysis of these enzymes indicated high amounts of glycine which is an unusual feature of these enzymes.