Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.     

Apomixis in <Emphasis Type="Italic">Eulaliopsis binata</Emphasis>: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.     

An efficient screen for reproductive pathways using mature seeds of monocots and dicots

Seed samples of 32 species (obligate and facultative sexuals and apomicts of monocots and dicots) were investigated by flow cytometry to reveal the pathway of reproduction. Ten different pathways of seed formation could be reconstructed considering whether the female and/or male gametes were reduced or unreduced, the embryos arose via the zygotic or parthenogenetic route and the endosperm via the pseudogamous or autonomous route. The screen is suited to select sporophytic or gametophytic mutants in sexual species, to identify pure sexual or obligate apomictic genotypes from facultative apomictic species, and to analyze the inheritance of the individual reproductive processes. Corresponding unique results are presented for Arabidopsis, Arabis, Hypericum and Poa. The screen of mature seeds by flow cytometry yielded more information about the reproductive behavior of individual plants than any other available test, and is very useful both in basic research and plant breeding.     

Regulation of organogenesis and somatic embryogenesis by auxin in melon,Cucumis melo L.

Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA 3indoleacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog     

Influence of auxin on the establishment of bilateral symmetry in monocots

To study the influence of auxin on the shift from radial to bilateral symmetry during monocot embryogenesis, the fate of young wheat (Triticum aestivum L.) zygotic embryos has been manipulated in vitro by adding auxins, an auxin transport inhibitor and an auxin antagonist to the culture medium. The two synthetic auxins used, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), induced identical phenotypes. In the most severe cases, the shift from radial to bilateral symmetry was blocked resulting in continuous uniform radial growth. The natural auxin indole-3-acetic acid (IAA) induced the same phenotype. The effect of 2,4,5-T and 2,4D depended on their concentrations and on the developmental stage of the isolated embryos. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin transport inhibitor, the overall embryo symmetry was abnormal. The relative position of the shoot apical meristem in comparison with the scutellum was anomalous. The quality of shoot apical meristem and the scutellum differentiation was altered compared with normal developed embryos. No root meristem was differentiated. The effect of TIBA depends on its concentration and on the developmental stage of the isolated embryos. By contrast, 2-(pchlorophenoxy)-2-methylpropionic acid (PCIB) which is described as an auxin antagonist, has no visible direct effect on the embryonic symmetry. These observations indicate that auxin influences the change from radial symmetry to embryonic polarity during monocot embryogenesis. A model of auxin action during early wheat embryo development is proposed.     

Comparative cyto-embryological investigations of sexual and apomictic dandelions (Taraxacum) and their apomictic hybrids

. In the autonomous apomictic Taraxacum officinale (common dandelion), parthenogenetic egg cells develop into embryos and central cells into endosperm without prior fertilisation. Unreduced (2n) megaspores are formed via meiotic diplospory, a nonreductional type of meiosis. In this paper, we describe the normal developmental pathways of sexual and apomictic reproduction and compare these with the development observed in the apomictic hybrids. In sexual diploids, a standard type of megasporogenesis and embryo sac development is synchronised between florets in individual capitula. In contrast, we observed that megasporogenesis and gametogenesis proceeded asynchronously between florets within a single capitulum of natural triploid apomicts. In addition, autonomous endosperm and embryo development initiated independently within individual florets. Parthenogenetic initiation of embryo development in outdoor apomicts was found to be temperature-dependent. Egg cells produced in natural apomicts were not fertilised after pollination with haploid pollen grains although pollen tubes were observed to grow into their embryo sacs. Both reductional and diplosporous megasporogenesis were observed in individual inflorescences of triploid apomictic hybrids. Embryo and endosperm development initiated independently in natural and hybrid apomicts.     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

The beneficial role of different auxins and polyamines at successive stages of somatic embryo formation and development of Panax ginseng in vitro

The production of viable plantlets via somatic embryogenesis in Panax ginseng requires different culture media corresponding to successive developmental stages. The effects of several auxins and polyamines have been tested at various steps. Multiplication of the embryogenic root-derived callus has been optimized on half-strength MS medium supplemented with 3-(benzo[b]selenyl)acetic acid (BSAA, a synthetic auxin) and kinetin; exogenously applied polyamines were deleterious at this stage, causing browning of the callus, diminished capacity of embryo initiation, and an increased tendency to hyperhydricity. BSAA again appeared to be the most favourable auxin at the initiation stage, but here its action was reinforced by the presence of polyamines, spermidine being the most efficient. Among the auxin needed at the next step, i.e., for the regeneration of embryos, the two seleniated auxins BSAA and seleniated 2,4-dichlorophenoxyacetic acid (2,4-D-Se) were the more efficient. For the harmonious development of plantlets, i.e., the simultaneous outgrowth of shoots and roots, the polyamines were favourable, with a greater efficiency for spermine.     

Novel inter-series hybrids in Solanum, section Petota

Sexual hybrids between distantly related Solanum species can undergo endosperm failure, a post-zygotic barrier in inter-species hybridizations. This barrier is explained by the endosperm balance number (EBN) hypothesis, which states that parents must have corresponding EBNs for viable seed formation. Tests for inter-crossability were made involving the Mexican species Solanum pinnatisectum Dunal. (series Pinnatisecta, A^piA^pi, 1EBN), autotetraploids of this species, Solanum verrucosum Schlechtd. (series Tuberosa, AA, 2EBN), haploids (2x, 2EBN) of the South American S. tuberosum L. (series Tuberosa, A₁A₁A₂A₂, 4EBN), and F₂ haploid-species hybrids with South American species (AA, 2EBN) S. berthaultii Hawkes, S. sparsipilum (Bitter.) Juz. and Bukasov and S. chacoense Bitter. The development of hybrid endosperms was investigated for these combinations by confocal microscopy with regard to cell-division timing and tissue collapse. Novel sexual diploid (AA^pi) and triploid (AA^piA^pi) inter-series hybrids were generated from S. verrucosum × S. pinnatisectum crosses by using post-pollination applications of auxin. F₁ embryos were rescued in vitro. The hybrid status of recovered plants was verified by microsatellite marker analysis, and the ploidy was determined by chromosome counting. The application of phytohormones in inter-ploidy S. pinnatisectum × S. tuberosum crosses, however, did not delay endosperm collapse, and embryos were not formed. Other diploid, 1EBN species tested in remote hybridizations with Group Tuberosum were S. cardiophyllum Lindl., S. trifidum Correll, and S. tarnii Hawkes and Hjert., series Pinnatisecta, and S. bulbocastanum Dunal., series Bulbocastana. Based on the analysis of post-zygotic reproductive barriers among isolated species of section Petota, we propose strategies to overcome such incompatibilities.     

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.352 larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (B_III hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin::#-glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.     

Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Expression of the apomictic potential and selection for apomixis in sorghum line AS-1a

Expression of elements of apomixis was studied for ten seasons in sorghum line AS-1a and its backcross hybrids in the 9E and A3 sterile cytoplasms. Cytoembryological analysis revealed aposporous embryo sacks (apo-ESs), their initial cells, and, rare, parthenogeneic proembryos in ovules of line AS-1a and its BC₂ and BC₃ hybrids on the 9E cytoplasm. The A3 sterile cytoplasm suppressed the development of parthenogenetic proembryos, but did not affect the apo-ES formation. The frequency of apomictic elements increased in seasons with high daily temperatures and total precipitation deficiency in the period of ovule and megagametophyte development (r = −0.805, p <.01). Selection was used to isolate the families where the frequency of ovules with apo-ESs was 28% and the frequency of parthenogenetic proembryos was 14%. Emasculated panicles of line AS-1a were pollinated with pollen of line Volzhskoe-4v, which carried the Rs marker dominant gene, responsible for the anthocyan color of coleoptyles and leaves in seedlings. Plants of the maternal type were found in the progenies of these crosses at a frequency of 1.4–28.1%. The genetic structure of the endosperm in grains with maternal-type seedlings was inferred from the electrophoretic patterns of storage proteins (kafirins). The kafirin spectra of grains producing maternal-type seedlings was similar to the spectrum of line AS-1a and differed from the spectra of grains producing hybrid seedlings, indicating that the endosperm developed autonomously when apomictic grains formed in line AS-1a. The results showed that lines with facultative apomixis can be constructed in functionally diploid plants.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS_3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MS_WH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.     

Somatic embryogenesis in Eucalyptus globulus

Somatic embryos were obtained from 1% of cotyledon pieces and hypocotyls of mature embryos of Eucalyptus globulus Labill. cultured on media containing a high concentration of picloram or IBA. 2,4-D and other synthetic auxins did not yield somatic embryos or embryogenic callus. Somatic embryos arose indirectly via callus, being visible after four months, and directly, where little callus or adventitious root initiation occurred. Somatic embryos, formed directly from explants, were visible within five weeks. Various structural abnormalities of somatic embryos were observed, especially after induction on media containing picloram. Only two out of fifteen somatic embryos showed hypocotyl and radical elongation, but plantlets did not develop further.     

Effects of Growth Factors FGF2 and IGF2 on the Development of Parthenogenetic Mouse Embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA × C57BK/6)FF₁. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18–21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 g/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). More than a half of FGF-2-treated parthenogenetic embryos developed to the stage of 40 and some of them, to the stage of 50 somites. The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryosin vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatmentin vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

Synthesis and activity of another seleniated auxin: 2,4-dichlorophenylselenoacetic acid

The synthesis of 2,4-dichlorophenylselenoacetic acid (2,4-D-Se) may be completed in three steps starting from 2,4-dichloroaniline. The selenium is inserted in the molecule by reaction of a diazonium salt with potassium selenocyanate. 2,4-D-Se has been tested as an auxin in several bioassays including the regeneration of somatic embryos, adventitious root formation and the associated temporary increase of endogenous auxins at the induction phase, and callus formation, and compared with the natural auxin indoleacetic acid (IAA), the classical synthetic auxin(s) naphthaleneacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D), and with the synthetic seleniated IAA, 3-(benzo[b]selenienyl) acetic acid, BSAA. These biological assays classified 2,4-D-Se together with BSAA among the most powerful synthetic auxins. The role of selenium is briefly discussed.     

The Mechanism of Low Temperature Dormancy in Mature Seeds of Syringa Species

The mechanism of seed dormancy at low temperatures (15-9°C) was investigated in the seeds of Syringa josikaea, S. reflexa and S. vulgaris. Low temperature dormancy in Syringa species was mainly imposed by endosperm embedding the radicle. Different degrees of embryo dormancy may occur in S. reflexa seeds. In most cases the low temperature dormancy was broken completely by removing the endosperm around the radicle. The endosperm did not seem to contain significant quantities of germination inhibitors, and the results indicate that it prevents germination mainly due to its mechanical resistance. The mechanical resistance of endosperm did not change during chilling or during induction of dormancy by high temperature incubation. The strength of the endosperm decreased rapidly in non-dormant seeds before visible germination. Similar changes were not observed in dormant seeds. Generally, the strength of the endosperm was lower in the non- (or less) dormant species S. josikaea and S. vulgaris than in the more dormant S. reflexa seeds. The growth potential of the embryos, measured as their ability to germinate in osmotic solutions (mannitol or polyethylen glycol 4000), was increased by chilling and by GA₃-treatment. The growth potential of untreated S. josikaea and S. vulgaris embryos was generally higher than that of S. reflexa embryos. Acid ethyl acetate fractions of methanol extracts from embryos of all three species contained substances with GA³-like activity in the lettuce hypocotyl test. The activity was found at Rf 0.9–1.0 on paper chromatograms run in distilled water. No significant changes in the activity were detected during chilling or prior to visible germination.