William F. Campbell

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.

Analysis of the spaceflight effects on growth and development of Super Dwarf wheat grown on the Space Station Mir.

The hypothesis being tested is that Super Dwarf wheat, Triticum aestivum L., plants in the Svet Greenhouse onboard the Russian Space Station Mir will complete a life cycle in spaceflight, providing that the environmental conditions necessary for adequate growth on Earth are supplied. Twenty six seeds of wheat were planted in each of 2 rows of 2 root compartments for a total of 104 seeds in Svet. Germination rate at 7 d was 56 and 73% on Mir and 75 and 90% in ground-based controls. Plants were grown throughout the whole cycle of ontogenesis (123 d) with samples gathered at different times to validate the morphological and reproductive stages of the plants. Young plants showed vigorous early seedling growth, with large biomass production, including the formation of 280 floral spikes. Upon return to Earth, comparative analyses showed that the number of tillers and flowers per spikelet were 63.2% and 40% greater, respectively, in Mir-grown plants than in the controls. By contrast, the stem length (52.4%), spike mass (49.2%) and length (23.1%), awn length (75.7%), number of spikelets per spike (42.8%) and number of seeds per spike (100% sterile) from Mir-grown plants were substantially less than the controls. Distribution of moisture and roots throughout the substrate was very good. All florets on Mir-grown spikes ceased development at the same stage of ontogeny. Lack of caryopses formation was attributed to male sterility occurring at different stages of staminal development. Anthers failed to dehisce and pollen grains were smaller and shriveled compared to the controls, suggesting a chronic stress had occurred in the Svet growth chamber. Recent ground-based studies indicated that ethylene, which was measured at 0.3 to 1.8 mg kg-1 in the Mir, almost certainly could have induced male sterility in the wheat plants grown on the Mir.

Comparative floral development of Mir-grown and ethylene-treated, earth-grown Super Dwarf wheat

To study plant growth in microgravity, we grew Super Dwarf wheat (Triticum aestivum L.) in the Svet growth chamber onboard the orbiting Russian space station, Mir, and in identical ground control units at the Institute of BioMedical Problems in Moscow, Russia. Seedling emergence was 56% and 73% in the two root-module compartments on Mir and 75% and 90% on earth. Growth was vigorous (produced ca. 1 kg dry mass), and individual plants produced 5 to 8 tillers on Mir compared with 3 to 5 on earth-grown controls. Upon harvest in space and return to earth, however, all inflorescences of the flight-grown plants were sterile. To ascertain if Super Dwarf wheat responded to the 1.1 to 1.7 micromoles mol-1 atmospheric levels of ethylene measured on the Mir prior to and during flowering, plants on earth were exposed to 0, 1, 3, 10, and 20 micromoles mol-1 of ethylene gas and 1200 micromoles mol-1 CO2 from 7 d after emergence to maturity. As in our Mir wheat, plant height, awn length, and the flag leaf were significantly shorter in the ethylene-exposed plants than in controls; inflorescences also exhibited 100% sterility. Scanning-electron-microscopic (SEM) examination of florets from Mir-grown and ethylene-treated, earth-grown plants showed that development ceased prior to anthesis, and the anthers did not dehisce. Laser scanning confocal microscopic (LSCM) examination of pollen grains from Mir and ethylene-treated plants on earth exhibited zero, one, and occasionally two, but rarely three nuclei; pollen produced in the absence of ethylene was always trinucleate, the normal condition. The scarcity of trinucleate pollen, abrupt cessation of floret development prior to anthesis, and excess tillering in wheat plants on Mir and in ethylene-containing atmospheres on earth build a strong case for the ethylene on Mir as the agent for the induced male sterility and other symptoms, rather than microgravity.

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.

Plant growth during the greenhouse II experiment on the Mir orbital station

We carried out three experiments with Super Dwarf wheat in the Bulgarian/Russian growth chamber Svet (0.1 m2 growing area) on the Space Station Mir. This paper mostly describes the first of these NASA-supported trials, began on Aug. 13, 1995. Plants were sampled five times and harvested on Nov. 9 after 90 days. Equipment failures led to low irradiance (3, then 4 of 6 lamp sets failed), instances of high temperatures (ca. 37 degrees C), and sometimes excessive substrate moisture. Although plants grew for the 90 d, no wheat heads were produced. Considering the low light levels, plants were surprisingly green, but of course biomass production was low. Plants were highly disoriented (low light, mirror walls?). Fixed and dried samples and the root module were returned on the U.S. Shuttle Atlantis on Nov. 20, 1995. Samples of the substrate, a nutrient-charged zeolite called Balkanine, were taken from the root module, carefully examined for roots, weighed, dried, and reweighed. The Svet control unit and the light bank were shipped to Moscow. An experiment validation test (EVT) of plant growth and experimental procedures, carried out in Moscow, was highly successful. Equipment built in Utah to measure CO2, H2O vapor, irradiance, air and leaf (IR) temperature, O2, pressure, and substrate moisture worked well in the EVT and in space. After this manuscript was first prepared, plants were grown in Mir with a new light bank and controller for 123 d in late 1996 and 39 days in 1996/1997. Plants grew exceptionally well with higher biomass production than in any previous space experiment, but the ca. 280 wheat heads that were produced in 1996 contained no seeds. Ethylene in the cabin atmosphere was responsible.

Ground-based studies with Super-Dwarf wheat in preparation for space flight

Several experiments were carried out to test responses of a Super-Dwarf cultivar of wheat (Triticum aestivum L.) to various environmental parameters that were anticipated to be present in our attempts to grow the wheat in a small growth chamber on the Russian Space Station, Mir, or that proved to be present in a 1995 trial space experiment. Under low photosynthetic photon flux (40-400 micromoles m-2 s-1 PPF), development (e.g. anthesis) was retarded, but heads (often sterile) always formed, even if light was so low that plants died before the heads could mature. Longer photoperiods promoted flowering, but night interruptions combined with short days did not provoke a long-day response as occurs with true long-day plants. The long-day effect could prove to be a summation of photosynthetic products. Heat stress (40 degrees C for 1-24 h) did not influence flowering but killed plants that were 13-16-day-old (no effect on younger plants). Concentrations of iodine or silver-fluoride disinfectants present in the water used for plants on Mir (1.0-4.0 mg L-1) did not affect plant growth although higher concentrations (8.0-1.6 mg L-1) were inhibitory. GA3 or indoleacetic acid applied every other day at concentrations from 1.0 x 10(-6) mg L-1 to 3.162 x 10(-4) mg L-1 did not change the height of Super-Dwarf wheat, suggesting that this cultivar is not a gibberellin mutant.

Morphological Analyses of Spring Wheat (CIMMYT cv. PCYT-10) Somaclones

The objectives of this study were to induce callus from single immature wheat embryos, produce multiple seedlings from the induced callus, and analyse the somaclonal regenerants for potential grain production in a space garden. Immature wheat, Triticum aestivum L. (cv. PCYT-10), embryos were excised 10 to 12 days post-anthesis and cultured on modified Murashige & Skoogs inorganic salts. Embryos cultured on medium containing kinetin (6-furfurylaminopurine) at 0.5 mgl−1 plus 2 or 3 mgl−1 dicamba (1-methoxy-3,6-dichlorobenzoic acid) or 0.2 mgl−1 2,4-dichlorophenoxyacetic acid produced calli from which 24, 35 and 39% of the explant tissue exhibited regenerants, respectively. The size of flag leaves, plant heights, tillers per plant, spike lengths, awn lengths, and seeds per spike were significantly different in regenerants of two-selfed recurrent generations (SC1, SC2) than in parental controls. However, there were no significant differences in spikelets per spike between the SC2 and parental controls. Desirable characteristics that were obtained included longer spikes, more seeds per spike, supernumerary spikelets, and larger flag leaves, variants that should be useful in wheat improvement programs.

Factors Affecting Somatic Embryogenesis in Wheat

The successful application of plant biotechnologies for the improvement of wheat (Triticum aestivum L.) will require reliable procedures for regenerating plants from tissues, single cells, and protoplasts. The optimization of such procedures would be facilitated by an understanding of factors that promote the normal development of somatic embryos. Aberrant somatic embryos were first recognized in wheat tissue cultures by Ahloowalia (1982), Ozias-Akins and Vasil (1982, 1983a), Maddock et al. (1983) and Magnusson and Bornman (1985). Recently, somatic embryogenesis and other aspects of wheat tissue culture were reviewed (Maddock 1985; Bajaj and Gosal 1986). This review focuses on more recent findings and presents concepts amenable to further experimentation.

Studies on flower initiation of super-dwarf wheat under stress conditions simulating those on the space station, Mir

Super-Dwarf wheat plants were grown in growth chambers under 12 treatments with three photoperiods (18 h, 21 h, 24 h) and four carbon dioxide (CO2) levels (360, 1,200, 3,000 and 7,000 micromoles mol-1). Carbon dioxide concentrations affected flower initiation rates of Super-Dwarf wheat. The optimum CO2 level for flower initiation and development was 1,200 micromoles mol-1. Super-optimum CO2 levels delayed flower initiation, but did not decrease final flower bud number per head. Longer photoperiods not only accelerated flower initiation rates, but also decreased deleterious effects of super-optimum CO2. Flower bud size and head length at the same developmental stage were larger under longer photoperiods, but final flower bud number was not affected by photoperiod.