Peggy Ozias-Akins

Plant regeneration from cultured immature embryos and inflorescences ofTriticum aestivum L. (wheat): Evidence for somatic embryogenesis

SummaryTissue cultures ofTriticum aestivum L. (wheat) initiated from young inflorescences and immature embryos possessed the potential for regeneration of whole plants. Both a friable and a compact type of callus were produced on Murashige and Skoogs medium with 2 mg/l 2,4-dichlorophenoxyacetic acid. The friable callus contained meristematic centers in which the peripheral cells ceased dividing, elongated, and could be easily separated. Roots were frequently formed in this type of callus. The compact, yellowish, and nodular callus arose from the epithelial and sub-epithelial cells of the embryo scutellum, and the rachis and glumes of the young inflorescence. Such callus had a smooth surface and characteristic chlorophyllous areas. Plants were regenerated only from the compact callus. The first sign of differentiation in the compact callus was the formation of a cleft or notch on the smooth surface, followed by the appearance of trichomes and the direct development of leafy structures which were not associated initially with any shoot meristems. Multiple shoots subsequently arose at the bases of the leafy structures, which are considered modifications of the scutellum, a definitive part of the cereal embryo. Accordingly, we suggest that while typical bipolar embryos are generally not formed, plant regeneration nevertheless takes place through embryogenesis and the precocious germination of the embryoids. Plants regenerated from immature embryo and inflorescence cultures were grown to maturity in soil, and were shown to have the normal chromosome number of 2n=6x=42.

Improved efficiency and normalization of somatic embryogenesis inTriticum aestivum (wheat)

SummaryTissue cultures derived from the scutellum of immature embryos ofTriticum aestivum L. (wheat) gave rise to somatic embryos with a well-defined scutellum and coleoptile as well as one or more shoot primordia and a root primordium. The normal somatic embryos were formed from compact, white callus tissue which was not observed until 4 or more weeks after culture initiation. The highest frequency of white embryogenic tissue formation and the most normal embryoids were obtained in the dark on Murashige and Skoogs medium with twice the concentration of inorganic salts plus sucrose (2%), inositol (100–200 mg/l), casein hydrolysate (100–200 mg/l), glutamine (500 mg/l), and 2,4-dichlorophenoxyacetic acid (1–2 mg/l).

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

SummaryProtoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

SummaryMitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.

Callus induction and growth from the mature embryo ofTriticum aestivum (Wheat)

SummaryCell proliferation from the mature embryo of wheat occurs on a defined medium in the presence of 2,4-D. Unorganized growth is observed when the 2,4-D concentration is equal to or greater than 2 mg/l, but increasing levels of 2,4-D inhibit cell division. Cell divisions begin after 4 days in culture from parenchyma cells within and near the procambial tissues of the embryo axis. By day 8 continuous meristematic zones are formed in association with vascular tissues, and DNA synthesis and cell divisions are distributed throughout these zones. No morphological evidence exists to show that these zones consist of proliferating root primordia, which are formed only after the level of 2,4-D falls below some critical concentration. When the concentration of 2,4-D is lowered, the meristematic zones first become dissected and then give rise to many root meristems.

Preferential amplification of mitochondrial DNA fragments in somatic hybrids of the Gramineae

SummarySomatic hybrid cell lines of Pennisetum americanum + Panicum maximum, and of Pennisetum americanum + Saccharum officinarum display unique mitochondrial DNA (mtDNA) restriction patterns suggestive of mitochondrial fusion and recombination. Apparent recombinant fragments of the hybrids were recovered, cloned, and hybridized to parental and somatic hybrid mtDNAs. In each somatic hybrid, “novel” fragments were found to be present at low copy number in one or both of the parental mtDNAs, and amplified 15–30 times in the hybrids. In pearl millet-sugarcane somatic hybrid cells, the amplification does not appear to involve enhanced recombination. The presumably amplified restriction fragment of the pearl millet-Guinea grass somatic hybrids is a junction fragment of a repeat, present in low copy number in both parents, and in high copy number in the hybrids. Thus protoplast and probable mitochondrial fusion results in a marked shift in the direction of mtDNA recombination events. We conclude that amplification of parental mtDNA fragments is a common event in somatic hybrid cells of these Gramineae.

Induction of Nitrogenase Activity in Azospirillum brasilense by Conditioned Medium from Cell Suspension Cultures of Pennisetum americanum (Pearl Millet) and Panicum maximum (Guinea Grass)

Summary Nitrogenase activity ( = acetylene reduction activity) was observed in Azospirillum brasilense cultures grown on conditioned medium obtained from embryogenic pearl millet ( Pennisetum americanum ) and Guinea grass ( Panicum maximum ) cell suspension cultures, but no bacterial growth or activity was observed on unconditioned medium. A peak of maximum activity was obtained with medium harvested mid-way through a 13 d culture period. Non-embryogenic suspension cultures of pearl millet and Guinea grass supported little or no bacterial growth or nitrogenase activity.