Interaction between nitrogen and photon flux density in birch seedlings at steady-state nutrition

Birch ( Betula pendula Roth.) was investigated under steady-state nutrition and growth at different incident photon flux densities (PFD) and different relative addition rates of nitrogen. PFD had a strong influence on the relative growth rate at optimum nutrition and on the nitrogen productivity (growth rate per unit of nitrogen) but little effect on the formal relationships between nitrogen and growth, i.e. PFD and nitrogen nutrition are orthogonal growth factors. At a given suboptimum nitrogen (the same distance from optimum), increased PFD increased the relative growth rate and, therefore, the relative uptake rate and the required relative addition rate in accordance with the theoretical equality between these three parameters at steady-state nutrition. Correspondingly, at a given suboptimum relative addition rate, increased PFD decreased nitrogen status (larger distance from optimum) at an unchanged relative growth rate. Nutrient uptake rate, dry matter content, and partitioning of biomass and nutrients are strongly influenced by nitrogen status. PFD influences these characteristics, but only to an extent corresponding to its effect on the nitrogen status. The influence of PDF on the relative growth rate at optimum and on nitrogen productivity is well described by hyperbolic relationships, similar to reported PFD/photosynthesis relationships. These expressions for plant growth as well as the productivities of leaf area and quantum appear to be valuable characteristics of plant responses to light and nutrition. Although the calculated PFD/growth relationships indicate saturation at high values of PFD, a more realistic estimate of PFD at which saturation occurs is about 30 mol m⁻² day⁻¹, where the highest relative growth rate and nitrogen productivity were experimentally determined. No significant effect was observed because of day length differences between the present and previous experiments.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

Evidence for a periodic excretion of nitrogen by roots of grass-legume associations

Diurnal variation in ion content of the solution bathing roots of two plants growing together in sand culture was analysed for three pairs of grass-legume species (Lolium multiflorum andTrifolium pratense; Zea mays andGlycine hispida; Avena sativa andVicia sativa) and their monospecific controls. Biomass and nitrogen content of plants were determined. Ion concentration (NO ₃ ⁻ , NO ₂ ⁻ , NH ₄ ⁺ , and K⁺) and pH of root solutions were measured for Lolium-Trifolium plant pairs and controls at 6 hours intervals over 36 h, starting at 8 am within a circadian cycle. Root solutions were regularly depleted in NO ₃ ⁻ by the grasses (Lolium-Lolium control) throughout the cycle. For associations involving the legume (Lolium-Trifolium and Trifolium-Trifolium), NO ₃ ⁻ depletion was followed by NO ₃ ⁻ enrichment at night, from late afternoon to early morning; the enrichment was more marked for the Lolium-Trifolium association. Solutions which did not contain NO ₂ ⁻ ions, were enriched by trace amounts of NH ₄ ⁺ ions, largely depleted in K⁺ and alkalanized for all associations throughout the cycle. Repeating the experiment with the three pairs of species at the vegetative phase of development confirmed the previous results: NO ₃ ⁻ enrichment during the night for associations with legumes. When the experiment was repeated with older plants which had almost completed their flowering stage, depletion only was observed and no NO ₃ ⁻ enrichment. These data suggest that NO ₃ ⁻ enrichment results from N excretion from active nodulated roots of the legume, accounting for the increase in both biomass and nitrogen content of the companion grass in grass-legume association. The quantitative importance and periodicity of nitrogen excretion as well as the origin of nitrate enrichment are discussed.     

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Use of tissue culture to bypass wheat hybrid necrosis

Summary Hybrid necrosis in wheat is a barrier to gene transfer in wheat breeding practice. It is based on two complementary genes, Ne1 and Ne2. Recovery mutants (Re1, Re2 and Re3) which can grow to maturity were recovered from immature embryo cultures of necrotic hybrids between T. aestivum and T. durum. Cytological observation demonstrated that Re1 had 34 chromosomes instead of 35. This indicated that one of the chromosomes carrying the Ne genes was lost. Genetic study suggested that for Re1, the lost chromosome was chromosome 5B of the durum parental line. Re mutants are male sterile but can be maintained through a young ear culture method. Re mutants could be successfully pollinated by either parental line and the BC1 progeny is partially fertile. Re mutants were repeatedly induced in about 1% of the regenerated plants from immature embryo culture. This technique provides a practical way to bypass hybrid necrosis.     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

A comparison of anther and microspore culture as a breeding tool in Brassica napus

Summary A direct comparison of microspore culture and anther culture was made in Brassica napus using F₁ crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 21 for the population obtained from the Regent x Golden crosses but 11 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.