Rex L. Smith

Nitrogen Fixation in Grasses Inoculated with Spirillum lipoferum.

Field-grown pearl millet (Pennisetum americanum) and guinea grass (Panicum maximum), lightly fertilized and inoculated with Spirillum lipoferum, produced significantly higher yields of dry matter than did uninoculated controls. Up to 42 and 39 kilograms of nitrogen per hectare were replaced by inoculation for pearl millet and guinea grass, respectively. The data demonstrate that nitrogen fixation by these grass-Spirillum systems is efficient and is achieved at a reasonable energy cost to the plant.

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Abstract Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N 2 -fixing ability was demonstrated by C 2 H 2 reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum ). These N 2 -fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N 2 -fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N 2 -fixing bacteria associated with grass roots.

Transformation of bahiagrass (Paspalum notatum Flugge)

Abstract. Bahiagrass (Paspalum notatum Flugge), a forage species widely used in the southeastern United States, and from Central Mexico to Argentina, was targeted for improvement through genetic engineering. Embryogenic callus, initiated from germinating seedlings, was bombarded with a vector containing the bar selectable marker/reporter gene that confers resistance to phosphinothricin (glufosinate) herbicide (trade names Liberty, Ignite and Finale). Thirty-two transgenic plants were recovered. These plants were identified by the polymerase chain reaction (PCR) and verified by Southern analysis. Transgenic plants with bar, as well as non-transgenic plants without bar, regenerated from bombarded callus and selected with glufosinate, developed strong and stable resistance to glufosinate during selection. This unusual resistance in non-transgenic plants has persisted for over a year and is passed on to new tillers. The development of resistance in non-transgenic cells reduced the herbicide selection efficiency and made it necessary to identify transgenic plants by PCR where the 32 transgenic plants were recovered from 674 glufosinate-resistant plants, giving a very low selection efficiency.

Mitochondrial DNA rearrangements in Pennisetum associated with reversion from cytoplasmic male sterility to fertility

Endonuclease restriction fragment patterns of Pennisetum americanum L. mitochondrial DNAs (mtDNAs) from a cytoplasmic male-sterile (CMS-A1), fertile revertants and a normal fertile cytoplasm were variable, while chloroplast DNA from those lines lacked variation. Comparisons between mtDNAs of CMS-A1 (parental) and fertile revertant lines revealed the presence of a unique 4.7 kbp PstI fragment in the sterile line that was not detected in any of the revertant lines. A 9.7 kbp PstI fragment was found in all of the revertants, but not in the CMS-A1. Neither of those fragments was found in the normal cytoplasm mtDNA. Hybridization studies revealed two sets of multiple homologies: 1) the 4.7 kbp fragment had homology with a 10.9 kbp and a 13.6 kbp fragment; and 2) the 9.7 kbp fragment was homologous with the 13.6 kbp fragment. The presence of those two repeated mitochondrial sequences on the altered fragments suggests that they may be involved in the recombinational associated events with reversion from CMS to fertility in P. americanum.

Development and application of RFLP and rapd dna markers in genetic improvement of Pennisetum for biomass and forage production

Abstract The objective of this research was to use new molecular techniques to improve efficiency of traditional breeding programs of Pennisetum for biomass and forage production. Restriction fragment length polymorphic (RFLP) and random amplified polymorphic DNA (RAPD) genetic markers were developed to characterize the Pennisetum purpureum genome and those markers were used to facilitate genetic study and breeding improvement. Standard RFLP and RAPD methodologies were adapted and/or modified to work well with Pennisetum and the DNA marker system was developed. This marker system was used to “fingerprint” the P. purpureum plant introduction collection, to determine the genetic diversity in that collection, to measure heterozygosity of selected lines and to measure hybridization/self fertilization rates using different crossing methods. Linkage relationships were studied between the DNA markers and quantitative trait loci (QTL), especially those loci associated with biomass/forage productivity, conversion to methane and rumen digestibility. Linkage analyses revealed 64 markers were linked to QTL of 26 plant traits. Those QTL-linked markers form the basis for genetic study of important biomasstforage productivity and quality traits. While the research was conducted with biomass for energy objectives, it is equally applicable to forage production and quality.

Peroxidase-antiperoxidase labeling of Azospirillum brasilense in field-grown pearl millet

Abstract Bacterial cells of Azospirillum brasilense (Tarrand) from their natural habitat were labeled with peroxidase-antiperoxidase (PAP), identified, and observed using the transmission electron microscope. Pure cultures of A. brasilense, axenically inoculated pearl millet root samples, and field-grown inoculated pearl millet root samples were embedded in Luffs araldite. Thin sections were treated using the immunological PAP method. Identification was possible because of the heavy outlining of the cells with a dense deposit of osmium. Pleomorphic forms of A. brasilense were observed in axenic pearl millet root cultures. Encapsulated forms were larger than vibrioid forms, and both types reacted with antiserum against the bacterial strain.

Developmental changes in abundance of the VSPβ protein following nuclear transformation of maize with the Soybean vspβ cDNA

BackgroundDeveloping monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter.ResultsFrom 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill.ConclusionDevelopmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.

The influence of shading on associative N2 fixation

SummaryThe effect of reduced solar radiation on associative N2-fixation and plant parameters was studied in three field experiments (1978–80). ‘Gahi-3’ pearl millet (Pennisetum americanum (L.) K. Monch.) field plots were shaded with saran shade cloth that reduced solar radiation by 50% and 75%. Acetylene reduction activity (ARA) was reduced by shading in one of the three experiments. The two non-responding experiments were conducted on a wall-drained, low-activity site (ARA means ranging 17–68 n moles ethylene core−1 h−1), the responding experiment was conducted on a poorly drained, high-ARA site.Shading affected the plants drastically, reducing fresh weight and dry matter yields up to 46% (50% shade) and 57% (75% shade). Shading also reduced dry matter percentage from 19.6 (no shade) to 15.3 (75% shade) and increased nitrogen content from 0.6% (no shade) to 1.53% (75% shading). However, shading did not affect protein yield. Inoculation withAzospirillum brasilense had no measurable effect on yield or acetylene reduction in the first two experiments.In the third experiment, shading reduced mean ARA of inoculated plots over 100% but had no significant effect on control plots. Inoculation significantly increased ARA in the nonshaded plots but not in shaded plots. Acetylene reduction activity was high, with means ranging between 208 and 465 n moles ethylene evolved core−1 h−1. Soil moisture and millet growth stage also affected acetylene reduction activity.

USE OF QUANTITATIVE SEROLOGY IN PREDICTING THE GENOTYPIC RELATIONSHIPS OF OAT CULTIVARS

SummaryQuantitative serological techniques were used to estimate the genotype relationships of six oat (Avena sativa L.) cultivars to a seventh. Reciprocal comparisons were made in which leaf, pollen, whole grain, and scutellum antigens were used. The reciprocal comparisons did not estimate genotypic relationships in the same order, but produced reciprocal results, i.e., when one method estimated lower relationships the other estimated higher, often above 100%. The reciprocal results were attributed to antigens common to each cultivar and these antigens masked and confounded the qualitative differences needed to estimate genotypic relationships. The different responses of common and qualitative antigens in the reciprocal comparisons made possible an analysis method that removed much of the masking effect of common antigen differences. In these tests, whole grain and embryo mean serological relationships agree well with best genetic estimates of genotypic relationships.

Acetylene Reduction Activity and Non-Structural Carbohydrate Content of Hemarthria Altissima CV. Bigalta, After Defoliation

Cut vs. uncut plants of Hemarthria altissima cv. Bigalta were studied to ascertain if the defoliated plants had significantly lower acetylene reduction activity (ARA) than unclipped plants. (Fig. a). Non-structural carbohydrate determinations were made of dried root samples. Roots sectioned with a freezing microtome were observed to contain starch in the cortical cells in the center of the root. ARA per gram of root, was significantly lower in the cut plants (p=. 02), and percent carbohydrate content was also significantly lower in the cut plants 17 days after clipping (p=. 001). A summary of percent carbohydrates found in the stems, crowns and roots of cut vs. uncut Hemarthria altissima is also shown. (Fig. b). Observations of root and shoot development indicated that cut plants had increased shoot growth, but initiated fewer new roots than the uncut plants. The ARA results are similar to, but at lower rates than reported with legume seedlings (Cralle, Heichel 1981) (Vance et al. 1979) and in field pastures (Halliday, Pate 1976).