In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

 This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots. Received: 1 April 1999 / Revision received: 8 July 1999 / Accepted: 17 August 1999     

Genotype identification and assessment of genetic relationships in pearl millet [Pennisetum glaucum (L.) R. Br] using microsatellites and RAPDs

 The potential of DNA markers such as microsatellites, minisatellites and RAPDs was investigated in pearl millet [Pennisetum glaucum (L.) R. Br] with respect to their abundance and variability. Southern analysis, using 22 different di-, tri-, tetra- and penta-oligonucleotide probes and five minisatellite probes, identified (GATA)₄ as the most useful probe for the detection of multiple polymorphic fragments among pearl millet cultivars and landraces from India. The clustering patterns of pearl millet cultivars and landraces based on (GATA)₄ and RAPD (randomly amplified polymorphic DNA) markers differed. The landraces, representing eight states in India, could not be grouped based on their geographical distribution with the DNA markers. RAPD analysis revealed a high degree of genetic diversity among the cultivars and landraces employed in this study. The probability of an identical match by chance for any two genotypes using (GATA)₄ and RAPDs was 3.02×10^-20 for cultivars and 5.2×10^-9 for landraces. The microsatellite (GATA)₄ and RAPDs provide useful tools for genotype identification and for the assessment of genetic relationships in pearl millet. Received: 19 October 1997 / Accepted: 9 December 1997     

Long-term storage of Pennisetum glaucum (L.) R. Br. pollen

Summary Pearl millet [Pennisetum glaucum (L.) R. Br.] pollen has been successfully stored for 2,615 and 2,911 days at -18° and -73 °C, respectively, and continues to be viable. Viability of pollen stored at -73 °C appears to be little affected either by pollen storage moisture contents below 7.2% or by storage in glass vial or zip-lock plastic bag containers. Pollen moisture content appears to be more critical for maintaining viability at -18°C than at -73°C. Glass vials appear to be more desirable for longer term (>3 years) storage at -18°C.     

Genetic variation in grain-filling ability in dwarf pearl millet [Pennisetum glaucum (L.) R. Br.] restorer lines

The d2 dwarfing gene in pearl millet [Pennisetum glaucum (L.) R. Br.] carries a yield penalty due to an associated reduction in individual grain mass. This reduction, however, varies with genetic background, indicating that it may be possible to select against poor grain filling in d2 dwarfbackgrounds, given an effective measure of grain filling. This study was conducted to assess genetic variability forgrain-filling ability (in contrast to simply grain size),and its relationship to grain yield,indwarf pearl milletrestorer (R) lines. The grain-filling ability (GFA) of an individual R line was defined as the least squares estimate of its effect on individual grain mass in the analysis of variance, following a linear covariance adjustment for grain number. The study was based on 93dwarf hybrids involving31 d2 dwarfR-lines, evaluated over 3 years. Half of the variation in individual grain mass in the 93 hybrids was related to variation in grain number. Covariance adjustment in individual grain mass for grain number resulted in highly significant differences among hybrids and R lines in GFA. The R-line combining ability for GFA accounted for 26% of the variation in the R-line combining ability for yield, compared to 46% for the combining ability for grain number, and just 8% for the combining ability of individual grain mass. The combining ability for GFA was independent of the combining ability for various pre-flowering effects, including grain number, but was related to the combining ability for individual grain mass and harvest index. Improvement in individual grain mass achieved through selection for GFA should translate directly into yield improvement, whereasimprovement by direct selectionfor individual grain mass is less-likely to do so. Received: 9 April 2000 / Accepted: 16 May 2000     

Host plant resistance to grain mould in germplasm accessions of pearl millet (Pennisetum glaucum [L.] R. Br.)

Abstract

The paucity of information on the moulds in Indian pearl millet (Pennisetum glaucum) led to the studies that were conducted at ICRISAT, India to evaluate (a) 447 germplasm accessions of 32 countries for mould reaction in rainy season, (b) threshed grain mould rating (TGMS) and mycoflora on grains of each accession, and (c) mould scores in field and in vitro. Post physiological maturity evaluation showed that 16% of the accessions secured a mould rating of 2. In TGMS, 18% were mould free and 57% secured a rating of 2 on a 1 – 9 scale. Assessment of twenty representative accessions in vitro against individual and mixed conidial suspensions (1 × 10⁽⁶⁾ conidia ml^(?1)) of Fusarium moniliforme, F. pallidoroseum and Curvularia pennisetti indicated significant correlation (r = 0.97) between the overall field and in vitro scores of mixed spores inoculations. The mycoflora for TGMS in blotter test revealed that Fusarium moniliforme, F. pallidoroseum, Curvularia pennisetti, Helminthosporium spp., Alternaria spp. and Colletotrichum spp. to be the major fungi affecting pearl millet grain. It is advisable to harvest panicles at the physiological maturity stage to obtain better quality grains. A strong negative correlation between TGMS and % GS (r = 0.4601) and positive correlation between TGMS and % UGS (r = 0.4654) indicated that, the lesser the threshed grain mould rating higher the % seed germination.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Mitochondrial DNA-RFLP Analysis Distinguishes New CMS Sources in Pearl Millet (Pennisetum glaucum (L.) R. Br.)

Restriction fragment length polymorphism (RFLP) of mitochondrial (mt) DNA provides a rapid and effective method to assess heterogeneity among male sterile cytoplasms. Six isonuclear A-lines (81 A₁, with Tift 23A₁, cytoplasm, ICMA 88001 (= 81A_v) with Violaceum cytoplasm, 81A (=81A₄) with monodli = violaceum cytoplasm, Pb 310A₂ and Pb 311A₂ with A₂ cytoplasm from L 66A, and Pb 406A₃ with A₃ cytoplasm from L 67A), nine cytoplasmic male-sterility sources from Large-Seeded Genepool (LSGP 6, LSGP 14, LSGP 17, LSGP 22, LSGP 28, LSGP 36, LSGP 43, LSGP 55 and LSGP 66) and two each from Early Genepool (EGP 33 and EGP 15) and Population Varieties (PV 1 and PV 2) were characterized for variation in their mitochondrial genomes following Southern blot hybridizations using homologous (pearl millet 13.6 kb, 10.9 kb, 9.7 kb and 4.7 kb clones) and heterologous (maize atp6 and coxl clones) mitochondrial DNA (mtDNA) probes. Following cluster analysis based on similarity indices for the RFLP banding patterns observed, we identified seven cytoplasmic groups within LSGP. Two (LSGP 43 and LSGP 66) of these were quite distinct from each other as well as from other cytoplasms. This clearly indicates that besides serving as a source of diversity for agronomic and adaptation traits, broad-based gene pools can also provide diverse sources of cytoplasmic male sterility. These new CMS sources were also compared with standard CMS systems and cytoplasm-specific restriction fragments were identified.     

Genetic architecture of purple pigmentation and tagging of some loci to SSR markers in pearl millet, Pennisetum glaucum (L.) R. Br

This report describes the construction of integrated genetic maps in pearl millet involving certain purple phenotype and simple sequence repeat (SSR) markers. These maps provide a direct means of implementing DNA marker-assisted selection and of facilitating "map-based cloning" for engineering novel traits. The purple pigmentation of leaf sheath, midrib and leaf margin was inherited together 'en bloc' under the control of a single dominant locus (the 'midrib complex') and was inseparably associated with the locus governing the purple coloration of the internode. The purple panicle was caused by a single dominant locus. Each of the three characters (purple lamina, purple stigma and purple seed) was governed by two complementary loci. One of the two loci governing purple seed was associated with the SSR locus Xpsmp2090 in linkage group 1, with a linkage value of 22 cM, while the other locus was associated with the SSR locus Xpsmp2270 in linkage group 6, with a linkage value of 23 cM. The locus for purple pigmentation of the midrib complex was either responsible for pigmentation of the panicle in a pleiotropic manner or was linked to it very closely and associated with the SSR locus Xpsmp2086 in linkage group 4, with a suggestive linkage value of 21 cM. A dominant allele at this locus seems to be a prerequisite for the development of purple pigmentation in the lamina, stigma and seed. These findings suggest that the locus for pigmentation of the midrib complex might regulate the basic steps in anthocyanin pigment development by acting as a structural gene while other loci regulate the formation of color in specific plant parts.     

Quantitative trait loci associated with constitutive traits control water use in pearl millet [Pennisetum glaucum (L.) R. Br.]

There is substantial genetic variation for drought adaption in pearl millet in terms of traits controlling plant water use. It is important to understand genomic regions responsible for these traits. Here, F₇ recombinant inbred lines were used to identify quantitative trait loci (QTL) and allelic interactions for traits affecting plant water use, and their relevance is discussed for crop productivity in water‐limited environments. Four QTL contributed to increased transpiration rate under high vapour pressure deficit (VPD) conditions, all with alleles from drought‐sensitive parent ICMB 841. Of these four QTL, a major QTL (35.7%) was mapped on linkage group (LG) 6. The alleles for 863B at this QTL decreased transpiration rate and this QTL co‐mapped to a previously detected LG 6 QTL, with alleles from 863B for grain weight and panicle harvest index across severe terminal drought stress environments. This provided additional support for a link between water saving from a lower transpiration rate under high VPD and drought tolerance. 863B alleles in this same genomic region also increased shoot weight, leaf area and total transpiration under well‐watered conditions. One unexpected outcome was reduced transpiration under high VPD (15%) from the interaction of two alleles for high VPD transpiration (LG 6 (B), 40.7) and specific leaf mass and biomass (LG 7 (A), 35.3), (A, allele from ICMB 841, B, allele from 863B, marker position). The LG 6 QTL appears to combine alleles for growth potential, beneficial for non‐stress conditions, and for saving water under high evaporative demand, beneficial under stressful conditions. Mapping QTL for water‐use traits, and assessing their interactions offers considerable potential for improving pearl millet adaptation to specific stress conditions through physiology‐informed marker‐assisted selection.     

Heat Shock Proteins of Sorghum [Sorghum bicolor (L.) Moench] and Pearl Millet [Pennisetum glaucum (L.) R.Br.] Cultivars with Differing Heat Tolerance at Seedling Establishment Stage

The production of heat shock proteins was compared in sorghumand pearl millet genotypes differing in seedling establishmentcharacteristics under heat stress. Two major heat shock proteins(hsps) of apparent mol. wt. 65 kD and 62 kD were seen in allthe genotypes of sorghum tested when the incubation temperatureof the 40 h seedlings was altered from 35 ?C to 45 ?C for 2h. Under identical conditions, pearl millet genotypes showedmore hsps and the apparent mol. wt. of these ranged from 30–70kD. The hsp bands were more prominent in whole seedlings androots as compared to plumules. Differences in the productionof hsps were seen in sorghum and pearl millet genotypes withcontrasting heat tolerance at seedling establishment stage butthe significance of these needs to be studied further. Key words: Heat shock proteins, sorghum, genotypic differences     

Assessment of Genetic Diversity in Napier Grass (Pennisetum purpureum Schum.) using Microsatellite,Single-Nucleotide Polymorphism and Insertion-Deletion Markers from Pearl Millet (Pennisetum glaucum [L.] R. Br.)

Plant Molecular Biology Reporter - Napier grass (Pennisetum purpureum Schum.) is a well-established perennial fodder crop of African origin which recently has also drawn attention for its potential...     

Histology of,and physical factors affecting,transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombardment

Transient GUS (-glucuronidase) expression was visualized in whole and sectioned embryos of Pennisetum glaucum (L.) R. Br. (pearl millet) after microprojectile bombardment with pMON 8678 DNA. Strongest GUS expression occurred in cells located in the center of GUS positive spots with decreasing intensity in surrounding cells. GUS positive cells could be seen up to 12 cell layers beneath the epidermis. Needle-like crystals of the GUS assay product were found throughout the cytoplasm of GUS positive cells. The number of GUS positive spots was correlated to the microprojectile spread pattern on the medium surface. Shorter bombardment distances (6.6 and 9.8 cm) and the standard accelerator speed gave the best results for transient expression but also caused maximum tissue damage. The speed and distance, however, had little influence on the ability of bombarded embryos to form compact callus. The developmental stage of the bombarded immature embryos was the determining factor in the formation of compact callus, from which plants were regenerated.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Characterisation of downy mildew resistant and susceptible pearl millet (Pennisetum glaucum (L.) R.Br.) genotypes using isozyme,protein, randomly amplified polymorphic DNA and inter-simple sequence repeat markers

In the present investigation, downy mildew resistant and susceptible pearl millet genotypes were characterised using seed protein and isozymes at pre- [45 days after sowing (DAS)] and post-infection (57 DAS, i.e. 7 days after infection) stage, as well as molecular markers at seedling stage without infection. Native polyacrylamide gel electrophoreis (PAGE) isozyme banding pattern of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and esterase showed some inducible band(s) due to disease infection and differentiated resistant and susceptible genotypes. Total seed protein profiling revealed the presence of two unique protein of ～97 and ～100 kDa in resistant genotypes. Randomly amplified polymorphic DNA (RAPD) analysis did not show any specific marker for disease resistant and susceptible genotypes. However, inter-simple sequence repeat (ISSR) markers showed six markers in resistant genotypes viz., UBC-825 (900 bp), UBC-827 (900 bp), UBC-857 (1000 bp, 700 bp, 375 bp and 200 bp). Moreover, a single unique band UBC-857 (400 bp) was present in only susceptible genotypes. Overall pooled analysis of isozymes, protein profiling, RAPD and ISSR data showed two distinct clusters of resistant and susceptible genotypes. These results suggested that seed protein profiling and ISSR markers may be used for large scale screening of germplasm for disease reaction trait.     

Osmotic adjustment to water stress in pearl millet (Pennisetum americanum [L.] Leeke) under field conditions

Abstract. Osmotic adjustment, a mechanism whereby plants maintain positive turgor despite low water potential (ψ), was investigated in pearl millet ( Pennisetum americanum [L.] Leeke) in three types of field experiment at Hyderabad, India:

• (1)

  Osmotic adjustment during the growing season was evaluated by comparing solute potential (ψ_s) of leaves taken at midday from irrigated and droughted plots and allowed to rehydrate in the laboratory. The degree of seasonal adjustment was also estimated by comparing observed values of ψ_s in the field with those expected if ψ_s decreased solely in proportion to water loss. Both types of assessment indicated the maximum seasonal adjustment to be about 0.2 MPa. The cultivars BJ 104 and Serere 39 differed in their capacity to adjust osmotically over the season; Serere 39 was least able to osmoregulate.
• (2)

  Measurements of diurnal variations in ψ and ψ_s in BJ 104 revealed osmotic adjustment during the afternoon hours. At a given value of ψ, turgor (ψ_p) was about 0.1 MPa higher in irrigated, and over 0.2 MPa higher in droughted plants, in the afternoon, than in the morning.
• (3)

  Osmotic adjustment of different leaves within the canopy was investigated. Upper leaves had lower ψ than basal leaves. Differences in ψ were matched by gradients in ψ_s, so that turgor was similar for all leaf layers.

     

Toxic and repellent properties of Xylopia aethiopica (Dunal) A. Richard on Tribolium castaneum Herbst infesting stored millets,Pennisetum glaucum (L.) R. Br.

The toxicity and repellency of Xylopia aethiopica seed extract was investigated in the laboratory against Tribolium castaneum Herbst. Concentration and days after treatment (DAT) caused a significant increase in T. castaneum adult mortality with an interaction effect of both on mortality when filter paper was impregnated with X. aethiopica extract. At 0.2 ml/60 cm² extract, significant mortality was observed at three–seven DAT when compared with one DAT. At 0.4 ml/60cm², 100% mortality was recorded at the lowest exposure period of one DAT. When 0.2 ml extract was applied to 5 g millet seeds, mortality at five–seven DAT was significantly higher than mortality observed in the control. Although repellency was dose-dependent, the percentage of T. castaneum that were repelled from treated filter paper was not significant. At 0.4 ml/60 cm², Class II repellency (26.7%) was observed. The results suggest that X. aethiopica can only effectively control T. castaneum populations that have infested millet but do not prevent cross-infestation via repellency.     

Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T₁ progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T₁ plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.     

Phylogeny and origin of pearl millet (Pennisetum glaucum [L.] R. Br) as revealed by microsatellite loci

During the last 12,000 years, different cultures around the world have domesticated cereal crops. Several studies investigated the evolutionary history and domestication of cereals such as wheat in the Middle East, rice in Asia or maize in America. The domestication process in Africa has led to the emergence of important cereal crops like pearl millet in Sahelian Africa. In this study, we used 27 microsatellite loci to analyze 84 wild accessions and 355 cultivated accessions originating from the whole pearl millet distribution area in Africa and Asia. We found significantly higher diversity in the wild pearl millet group. The cultivated pearl millet sample possessed 81% of the alleles and 83% of the genetic diversity of the wild pearl millet sample. Using Bayesian approaches, we identified intermediate genotypes between the cultivated and wild groups. We then analyzed the phylogenetic relationship among accessions not showing introgression and found that a monophyletic origin of cultivated pearl millet in West Africa is the most likely scenario supported by our data set.