John G. Carman

EMBRYOGENIC CELLS IN PLANT TISSUE CULTURES: OCCURRENCE AND BEHAVIOR

SummaryPlants develop from meristems where cells proliferate and are partitioned into layers that eventually differentiate to form the various tissues and organs of the plant. A phenomenon unique to certain plant tissues is the ability to inducede nova a range of developmental patterns, including embryogenesis. The temporal and spacial distribution of these developmental competencies suggests that regulatory proteins, rather than a lack of signals or signal receptors, shield specific developmental genes from signals that otherwise would confuse development. Studies involving embryogenic cells demonstrate that they are not strongly shielded from developmental signals, thus they are not determined. Furthermore, the normal development of zygotic and somatic embryos is readily perturbed by abnormal physicochemical environments. This suggests that embryogeny remains developmentally plastic until differentiation is largely completed. The ability to induce somatic embryogenesis from specific tissues by specific signals is providing opportunities to further the molecular characterization of the menagerie of genetic regulation involved in development.

Improved somatic embryogenesis in wheat by partial simulation of the in-ovulo oxygen, growth-regulator and desiccation environments

The effects of O2, growth-regulators and desiccation on callus growth and somatic embryo (embryoid) development were investigated in cultures of immature embryos of two lines of Triticum aestivum L. Callus and embryoid formation were induced on media that contained N6-furfurylamin-opurine (kinetin) and either 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-o-anisic acid, either with or without abscisic acid (ABA). Cultures containing differentiated embryoids were then exposed to high concentrations of both ABA and indole-3-acetic acid, after which samples were desiccated to approx. 10% tissue moisture. Incubating cultures in 3.2 mmol·l-1 O2 (approx. 9%, low-O2) increased embryoid formation sixfold in one wheat line and nearly threefold in another. In the former line low-O2 caused the formation of mostly embryogenic callus. Low-O2 also decreased precocious germination of immature embryos, decreased callus growth, and improved development and viability of the resultant embryoids. Including 1.9 μmol·l-1 ABA in the callus-induction medium reduced germination of immature embryos and reduced the incidence of embryoids with visible abnormalities. Despite the improved morphology, significantly fewer of the embryoids produced on ABA-containing medium germinated. Desiccation significantly enhanced germination of these embryoids as well as those produced on ABA-free medium.

Induction of embryogenic Triticum aestivum L. calli. I. Quantification of genotype and culture medium effects

Somatic embryo (embryoid) formation from immature-embryo-derived calli was quantified in replicated experiments involving 10Triticum aestivum L. genotypes. Several published media formulations, which had previously been optimized for wheat tissue culture, were tested for each genotype. Embryos from each plant were randomly assigned to each medium. Percentage precocious germination of immature embryos and mean percentage scutellar callus per explant were recorded. Embryoids per callus were determined by microscopic examination at 28 and 56 days. There were highly significant differences among genotypes, media, and individual plants from which explants were taken. A medium based on double the Murashige and Skoog (MS) inorganic salt concentration was significantly better than other media. Inclusion of all MS vitamins appeared essential for optimal response. Two genotypes were tested in a second experiment where both 3,6-dichloro-o-anisic acid (9.05 μM) and 6-furfurylaminopurine (0.46 μM) were substituted for 2,4-dichlorophenoxyacetic acid (4.52 μM) in either double or normal MS medium. This substitution significantly increased embryoid formation at 28 days. Additions of either 6-furfurylaminopurine or coconut water increased precocious germination of both embryo explants and embryoids.

Morphologic and allozymic variation between long-term grazed and non-grazed populations of the bunchgrass Schizachyrium scoparium var. frequens

SummaryPlant populations of Schizachyrium scoparium var. frequens with a history of long-term grazing by domestic herbivores were characterized by shorter and narrower leaf blades and tillers of lower weight than plants from populations with a history of no grazing. Following four biweekly defoliation events plants from the grazed populations additionally displayed lower specific leaf weights, lower amounts of biomass removed per tiller upon defoliation and a greater number of new and regrowing tillers. Production values per plant remained similar between the two populations because of a greater number of tillers per plant in the grazed population.A large amount of allozymic polymorphism was observed in both the grazed and non-grazed populations. Results of the electrophoretic analyses indicated that a minimum of 66% of the plants sampled represented separate genotypes. No distinction could be made between grazed and non-grazed populations in terms of allozymic partitioning. The morphological variation observed between these two populations may represent a shift in the relative dominance of a series of genotypes variously adapted to herbivory.

Taxonomy and Biogeography of Apomixis in Angiosperms and Associated Biodiversity Characteristics

Apomixis in angiosperms is asexual reproduction from seed. Its importance to angiospermous evolution and biodiversity has been difficult to assess mainly because of insufficient taxonomic documentation. Thus, we assembled literature reporting apomixis occurrences among angiosperms and transferred the information to an internet database (http://www.apomixis.uni-goettingen.de). We then searched for correlations between apomixis occurrences and well-established measures of taxonomic diversity and biogeography. Apomixis was found to be taxonomically widespread with no clear tendency to specific groups and to occur with sexuality at all taxonomic levels. Adventitious embryony was the most frequent form (148 genera) followed by apospory (110) and diplospory (68). All three forms are phylogenetically scattered, but this scattering is strongly associated with measures of biodiversity. Across apomictic-containing orders and families, numbers of apomict-containing genera were positively correlated with total numbers of genera. In general, apomict-containing orders, families, and subfamilies of Asteraceae, Poaceae, and Orchidaceae were larger, i.e., they possessed more families or genera, than non-apomict-containing orders, families or subfamilies. Furthermore, many apomict-containing genera were found to be highly cosmopolitan. In this respect, 62% occupy multiple geographic zones. Numbers of genera containing sporophytic or gametophytic apomicts decreased from the tropics to the arctic, a trend that parallels general biodiversity. While angiosperms appear to be predisposed to shift from sex to apomixis, there is also evidence of reversions to sexuality. Such reversions may result from genetic or epigenetic destabilization events accompanying hybridization, polyploidy, or other cytogenetic alterations. Because of increased within-plant genetic and genomic heterogeneity, range expansions and diversifications at the species and genus levels may occur more rapidly upon reversion to sexuality. The significantly-enriched representations of apomicts among highly diverse and geographically-extensive taxa, from genera to orders, support this conclusion.

Induction of embryogenic Triticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on induction of embryogenic calli from immature embryos of a responsiveTriticum aestivum L. genotype (PCYT 10). Effects were quatified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 μM 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 μM or 4.65 μM significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 μM signficantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mgl-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D inT. aestivum callus cultures.

Canopy photosynthesis and transpiration in micro-gravity: Gas exchange measurements aboard Mir

The SVET Greenhouse on-board the Orbital Station Mir was used to measure canopy photosynthesis and transpiration rates for the first time in space. During the Greenhouse IIB experiment on Mir (June-January 1997), carbon and water vapor fluxes from two wheat (cv. Superdwarf) canopies were measured using the US developed Gas Exchange Measurement System (GEMS). Gas analyzers capable of resolving CO2 concentration differences of 5 micromoles mol-1 against a background of 0.9% CO2, are necessary to measure photosynthetic and respiratory rates on Mir. The ability of the GEMS gas analyzers to measure these CO2 concentration differences was determined during extensive ground calibrations. Similarly, the sensitivity of the analyzers to water vapor was sufficient to accurately measure canopy evapotranspiration. Evapotranspiration, which accounted for over 90% of the water added to the root zone, was estimated using gas exchange and used to estimate substrate moisture content. This paper presents canopy photosynthesis and transpiration data during the peak vegetative phase of development in microgravity.

Carbohydrates, minerals and free amino acids in Triticum aestivum L. kernels during early embryony

Summary Culture procedures for zygotic and somatic embryos can be improved by simulating the nutrient and hormone levels observed in ovules during zygotic embryogenesis. To meet this objective, we identified potentially important nutrients in wheat Triticum aestivum L. kernel fractions that contained ovule fluids, which were collected by centrifugation. Malate dehydrogenase assays suggested that these samples contained apoplastic and cytoplasmic fluids. Kernel fractions were analyzed for minerals, free amino acids, and water soluble carbohydrates. On a mole percentage basis, K, P, Mg, and S accounted for 85 % of the minerals in the ovule solutions while Ser, Ala, NO 3 − , NH 4 + , Gly, Glx and Pro accounted for 81 % of the free amino acids, NO 3 − , and NH 4 + fraction. Sucrose, fructose, and glucose in these solutions ranged from 30 to 160 mmol kg−1 fresh mass (FM) in sucrose equivalents. These solutions also contained high concentrations of short and longer-chain fructans. Loosely-joined endosperm cells, collected through centrifugation, comprised about 10% of kernel FM at 6d past anthesis (DPA), but less than 1% of kernel FM by 18 DPA. Concentrations of maltose, sucrose, neokestose and 1-kestose in the pelleted endosperm increased to high levels by 18 DPA, which is evidence that stored fructans and amylopectins in the endosperm are hydrolyzed and used as nutrients by the growing embryo. The liquid fraction of younger kernels may closely approximate ovule fluid, and embryo culture procedures have been improved by incorporating the nutrient analysis information obtained therefrom.

Thein ovulo environment and its relevance to cloning wheat via somatic embryogenesis

SummaryInduction of somatic embryogenesis refers to processes that initiate embryo-forming cellular activity. Competence of somatic tissues for embryo formation is measured by the number of somatic embryos produced following induction. Recent findings indicate that competence of wheat explants can be enhanced by treatments that alter hormone levels during differentiation of explantsin vivo. Wheat spikes have been detached at anthesis and cultured with culms in detached-spike-culture medium. The addition of abscisic acid, indole-3-acetic acid or zeatin to the medium depresses embryogenic competence of immature-embryo explants (excised and cultured 11–13 days after anthesis) while deleting hormones from the medium enhances competence. Differentiation of somatic embryos is improved by exposing embryogenic cultures to conditions that simulate nutritional, hormonal and gas-phase conditions of ovules. In particular the lowering of oxygen availability to callus cultures promotes growth of embryogenic callus and suppresses growth of nonembryogenic callus. Finally procedures that simulate seed desiccation significantly increase germination percentages of somatic embryos of wheat.

Hormones in wheat kernels during embryony

Summary Abscisic acid (ABA), indole-3-acetic acid (IAA) and six cytokinins in wheat (Triticum aestivum L.) grain components were extracted, from 0 to 25 d post anthesis (DPA), and quantified by noncompetitive indirect ELISA. At 3 DPA, which coincides with early embryo and endosperm cell divisions, whole kernel hormone levels in µmol kg –1 dry mass (DM) were: zeatin (Z) 30, IAA 4, zeatin riboside 1.8, ABA 1.0, other cytokinins < 0.6. Endosperm Z content declined from about 13 µmol kg –1 DM at 6 DPA to 0.1µmol kg –1 DM by 9 DPA and remained low thereafter. Generally by 9 DPA, embryo differentiation is nearly complete, and the endosperm mitotic index has decreased to near zero. At 13 DPA, Z content in embryos was 0.9 µmol kg –1 DM, and then decreased to 0.3 µmol kg –1 DM by 25 DPA. Endosperm IAA content remained low through 9 DPA (about 6 µmol kg –1 DM) and then increased sharply to about 38 µmol kg –1 DM by 19 and 25 DPA. In contrast, embryo IAA content decreased from about 34 µmol kg –1 DM at 13 DPA to about 15 µmol kg –1 DM at 19 and 25 DPA. Endosperm ABA content fluctuated between 1.0 and 4.5 µmol kg –1 DM between 6 and 25 DPA while embryo ABA content fluctuated between 3.4 and 4.8 µmol kg –1 DM between 13 and 25 DPA. These hormone changes may be