Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Two distinct P element subfamilies in the genome of Drosophila bifasciata

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5 end and 14 by at the 3 end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity.     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N₂ fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled ¹⁵N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for ¹⁵N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an ¹⁵N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for ¹⁵N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with ¹⁵N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with ¹⁵N. These two studies conclusively show that within a short period of time, N is transferred from both the N₂-fixing legume to the associated non-legume and also from the non-legume to the N₂-fixing legume. The occurrence of a bi-directional N transfer between N₂-fixing and non-N₂-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Optimization of transient gene expression in pollen of Norway spruce (Picea abies) by particle acceleration

In order to optimize transient gene expression in Norway spruce pollen after DNA delivery with particle bombardment, effects of different conditions during homhardmenl were analysed using β-glucuroniduse (GUS) driven by the rice Act I promoter and Inciferase (LUS) driven by the tomato !at 52 promoter as reporter genes. Transient gene expression was significantly increased hy using two bombardments. Also the distance from the stopping plate to the sample was critical to gam maximum gene expression. There was no significant difference between gold and tungsten particles, and the number of positively stained pollen increased with increasing DNA concentration, from 5 to 40 pg DNA added in the DNA/tungsten solution The DNA delivery to Norway spruce pollen was most efficient at a chamber pressure above 70 kPa.     

Transfer of nitrogen from damaged roots of white clover (Trifolium repens L.) to closely associated roots of intact perennial ryegrass (Lolium perenne L)

An experiment is described in which the magnitude of N transferred from damaged white clover roots to perennial ryegrass was determined, using ¹⁵N labelling of the grass plant. There was no effect on the growth and N-fixation of the clover plants after removing part of the root system. The ¹⁵N data suggested that N had been acquired by all grass plants, even in plants grown alone with no further N supplied after labelling. However, after quantifying the mobile and stored N pools of the grass plants it was evident that significant transfer of N from clover to grass only took place from damaged clover roots. Dilution of the atom% ¹⁵N in the roots of the grass plants grown alone, and in association with undamaged clover roots, was explained by remobilisation of N within the plant.