Production of androgenic plants through Pollen embryogenesis in anther cultures ofBrassica carinata A. Braun

Pollen embryogenesis occurred in anther cultures of two genotypes ofBrassica carinata A. Braun. Pretreatment of anthers at 35°C for 3 or 6 days was essential for the induction of androgenesis on growth regulator-free culture medium. A combination of sucrose and glucose was better than sucrose alone. None of the pollen embryos germinated normally. Full plants were raised through adventitious bud differentiation from their hypocotyl.     

Hormone and sugar effects on rice sucrose transporter <Emphasis Type="Italic">OsSUT1</Emphasis> expression in germinating embryos

The rice (Oryza sativa L.) OsSUT1 gene encodes a sucrose transporter protein. OsSUT1 was suggested to contribute to phloem loading of sucrose. OsSUT1 expression is highly induced in embryos after seeds were imbibed in water and peaked at 2 days after imbibition, but mRNA levels decline gradually afterwards. In this study, we demonstrated that phytohormones and sugars regulate OsSUT1 expression. Antagonism of abscisic acid and gibberellic acid appeared to play an important role in regulating OsSUT1 expression during embryo germination. In addition, our data showed a glucose and sucrose effect on OsSUT1 expression that represented a bi-phase process. Initially, glucose and sucrose functioned as negative regulators of OsSUT1 expression in germinating embryos after a 1-day treatment; however, when the treatment duration was extended to 5 days, OsSUT1 expression was significantly enhanced. Therefore, we hypothesized that the glucose and sucrose effect might occur in combination with other side effects, such as changes in hormone content or catabolism. Based on the effects that sugar analogs have on OsSUT1 expression, we suggest that the signal transduction for regulating glucose-responsive OsSUT1 expression in embryos occurs via a hexokinase-mediated pathway.     

Enhancement of ABA responsiveness in wheat embryos by high temperature*†

Abstract. Mature wheat (Triticum aestivum L.) grain often possesses high-temperature dormancy which restricts the grain from germinating at warm temperatures (25–30°C). Isolated embryos from such grain exhibited little high-temperature dormancy when germinated in water. Dormancy was restored by the application of abscisic acid (ABA) to the embryos. The ability of ABA to block germination in isolated embryos was enhanced significantly by elevating the germination temperature. ABA was 100 times more effective in reducing embryonic germination at 30°C than at 15°C. These temperature effects on embryonic response to ABA are a useful system for studying the mechanism of ABA action in seed dormancy.     

Improvement of English walnut somatic embryo germination and conversion by desiccation treatments and plantlet development by lower medium salts

Summary Well-developed somatic embryos were selected from a repetivively somatic embryo line derived from embryonic axes of immature zygotic embryos of English walnut ‘No. 120’ (Juglans regia L.) for germination and conversion studies. In germinating dishes, somatic embryos germinated into only shoots, only roots, or both shoots and roots. Without any pretreatment, 28% somatic embryos germinated, while those treated with 2.5–5.0 mg 1⁻¹ (7.2–14.4 μmol) gibberellic acid (GA₃) germinated at 25–28% and those receiving a cold treatment of 2–3 mo. at 3–4°C germinated at 30–43%. However, only 4–19% of the germinating embryos showed both shoots and roots. Treated with desiccation, either with CaCl₂·6H₂O or Ca(NO₃)₂·4H₂O at 20°C in the dark for 3 d, somatic embryos germinated at 85–91%, 57–69% of which had both shoots and roots. Treatment with 2 mo. cold storage in combination with desiccation using Ca(NO₃)₂·4H₂O resulted in 92% of somatic embryos germinating, 70% of which showed both shoots and roots. No significant differences were observed between solid and liquid germination media. After transferring the germinating embryos to plantlet development media, 52–63% of those with both shoots and roots developed into plantlets while 11% with only shoots or 9% with only roots converted into plantlets. Plantlet development was improved by using lower medium salts and sucrose concentrations. The addition of activated charcoal enhanced root development, particularly root branching. Of 131 plants transplanted, 91 plants were acclimatized to a greenhouse.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Effect of plant growth conditions, plating density, and genotype on the anther culture response of soft white spring wheat hybrids

Ten soft white spring wheat (Triticum aestivum L.) F₁ hybrids were grown under three temperature regimes, and anthers were cultured at two plating densities to investigate the effect of plant growth conditions, plating density, and genotype on embryo induction and plant regeneration. Anthers from plants grown at high temperature (25 °/18 °C) or from plants transferred from low (15 °/12 °C) to high temperature generally produced more embryos and green shoots, with a lower frequency of albinos, than did anthers from plants grown at low temperature. However, plating densities of 10 versus 20 anthers per milliliter, had little effect on anther response. Four of the five hybrids with `Fielder' as the female parent produced more embryos and green shoots than did hybrids with `AC Reed' as the female parent. Received: 12 July 1996 / Revision received: 1 April 1997 / Accepted: 30 April 1997     

Environmental factors controlling seed germination and seedling recruitment of Stipa bungeana on the Loess Plateau of northwestern China

Germination studies are important for collecting information on field seedling recruitment, plant conservation and restoration. This study investigated the role of light, temperature, nitrogen, water stress and burial depth in controlling germination of Stipa bungeana seeds. S. bungeana seeds are photo-inhibited; light significantly decreased seed germination regardless of temperature and water conditions. Seeds germinated at 10–30° C, and the highest germination was 72 % and 88 % at 20° C in light and dark, respectively. Thermal model analysis showed that presence of light significantly increased average thermal requirement [θ _T (50)] from 105°Cd to 186°Cd at sub-optimal temperature, implying that light delays seed germination. Hydrotime model analysis showed that presence of light caused a shift in the median base water potential [Ψ _b(50)] from ?0.68 to ?0.26 MPa, which partly explains why light decreased both percentage and speed of germination, even at optimal conditions. As burial depth increased, seedling emergence initially increased and then decreased; the highest seedling emergence recruitment was 43 %, for seeds buried at a depth of 1 cm. Field observations showed that seedling emergence occurred primarily from July to September, and scarcely occurred from April to June. These results suggest that the light inhibitory effect is an adaptive mechanism that prevents S. bungeana seeds from germinating on the soil surface. To attain highest seedling establishment, seeds of S. bungeana should be sown at a soil depth of 1 cm prior to the rainy season, using seeds stored for 1 year.     

Chronic hypoxic incubation blunts thermally dependent cholinergic tone on the cardiovascular system in embryonic American alligator (Alligator mississippiensis)

Environmental conditions play a major role in shaping reptilian embryonic development, but studies addressing the impact of interactions between chronic and acute environmental stressors on embryonic systems are lacking. In the present study, we investigated thermal dependence of cholinergic and adrenergic cardiovascular tone in embryonic American alligators (Alligator mississippiensis) and assessed possible phenotypic plasticity in a chronic hypoxic incubation treatment. We compared changes in heart rate (f _H) and mean arterial blood pressure (P _M) for chronically hypoxic and normoxic-incubated embryos after cholinergic and adrenergic blockade following three different acute temperature treatments: (1) 30 °C (control incubation temperature), (2) acute, progressive decrease 30–24 °C then held at 24 °C, and (3) acute, progressive increase 30–36 °C then held at 36 °C. f _H progressively fell in response to decreasing temperature and rose in response to increasing temperature. P _M did not significantly change with decreasing temperature, but was lowered significantly with increasing acute temperature in the normoxic group at 90 % of development only. Propranolol administration (β adrenergic antagonist) produced a significant f _H decrease at 24, 30, and 36 °C that was similar at all temperatures for all groups. For normoxic-incubated embryos at 90 % of development, atropine administration (cholinergic antagonist) significantly increased f _H in both 24 and 36 °C treatments, but not in the 30 °C control treatment. This atropine response at 24 and 36 °C demonstrated acute thermally dependent cholinergic tone on f _H late in development for normoxic-incubated, but not chronically hypoxic-incubated embryos. Collectively, data indicated that cardiovascular control mechanisms in embryonic alligators may be activated by thermal extremes, and the maturation of control mechanisms was delayed by chronic hypoxia.     

Seed dormancy-breaking and germination requirements ofDrosera anglica,an insectivorous species of the Northern Hemisphere

Seeds of Drosera anglica collected in Sweden were dormant at maturity in late summer, and dormancy break occurred during cold stratification. Stratified seeds required light for germination, but light had to be given after temperatures were high enough to be favorable for germination. Seeds stratified in darkness at 5/1 °C and incubated in light at 12/12 h daily temperature regimes of 15/6, 20/10 and 25/15 °C germinated slower and to a significantly lower percentage at each temperature regime than those stratified in light and incubated in light. Length of the stratification period required before seeds would germinate to high percentages depended on (1) whether seeds were in light or in darkness during stratification and during the subsequent incubation period, and (2) the temperature regime during incubation. Seeds collected in 1999 germinated to 4, 24 and 92 % in light at 15/6, 20/10 and 25/15 °C, respectively, after 2 weeks of stratification in light. Seeds stratified in light for 18 weeks and incubated in light at 15/6, 20/10 and 25/15 °C germinated to 87, 95 and 100 %, respectively, while those stratified in darkness for 18 weeks and incubated in light germinated to 6, 82 and 91 %, respectively. Seeds collected from the same site in 1998 and 1999, stratified in light at 5/1 °C and incubated in light at 15/6 °C germinated to 22 and 87 %, respectively, indicating year-to-year variation in degree of dormancy. As dormancy break occurred, the minimum temperature for germination decreased. Thus, seed dormancy is broken in nature by cold stratification during winter, and by spring, seeds are capable of germinating at low habitat temperatures, if they are exposed to light.     

The size and germinability of Scots pine pollen in different temperatures in vitro

The effect of genotype, the origin of genotype, and germination temperature on Scots pine pollen grain size, hydration rate, germinability, and tube growth was studied in vitro. The mean sizes of dry and germinated pollen grains varied among pollen genotypes in different ways, thus the hydration rate varied among genotypes. Pollen from Scots pine that originates in northern Finland hydrated more than pollen from a population in southern Finland. Germination temperature had no effect on the hydration rate. Germinability and tube growth rate of northern genotypes were higher at 20 °C than at 15 °C. Differences among southern genotypes were not significant. At 15 °C, the germinability and pollen tube growth rate of northern genotypes were lower than southern genotypes. At 20 °C, the differences were not significant. It appears that germination and growth of pollen from northern populations are enhanced at higher temperatures whereas pollen from southern populations is unaffected.     

Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light

We investigated sugar uptake and transport in rice (Oryza sativa) embryo during grain germination. Endogenous sugar levels, accumulation of starch granules, and gene expression of a rice sucrose transporter (OsSUT1) were examined using rice embryos germinated with or without exogenous sugar supply. Starch granules remarkably accumulated in the cells around vascular bundles as a consequence of the sugar taken up by the embryos, indicating that the taken-up sugars are transiently converted into starch. In situ detection for OsSUT1 mRNA indicated its localization in the phloem companion cells. Furthermore, northern-blot and in situ hybridization analyses showed that OsSUT1 expression is not detectable in embryos subjected to sugar starvation conditions, whereas its expression is enhanced by an increased endogenous sugar level. Overall results indicate that the expression of companion cell-specific sucrose transporter, OsSUT1 is regulated by the endogenous sugar status as well as light exposure.     

Variation in temperature and light but not salinity invokes antioxidant enzyme activities in germinating seeds of Salsola drummondii

Seeds with efficient antioxidant defence system show higher germination under stress conditions; however, such information is limited for the halophyte seeds. We therefore studied lipid peroxidation and antioxidant responses of a leaf-succulent halophyte Salsola drummondii during seed germination under different salinity levels (0, 200 and 800 mM NaCl), temperature (10/20, 20/30 and 25/35°C) and light regimes. Seeds absorbed water and germinated in less than 1 h in non-saline control while increases in salinity decreased the rate of water uptake as well as seed germination. Non-optimal temperatures (10/20 and 25/35°C) and complete dark condition reduced seed germination in comparison to those seeds germinated under optimal temperature (20/30°C) and 12-h photoperiod, respectively. Generally, higher lipid peroxidation and antioxidant enzyme activities were observed in seeds at non-optimal temperature and in those seeds germinated in dark. Decrease in reduced ascorbic acid content was found in highest salinity and temperature treatments, while reduced glutathione content did not change significantly with changes in salinity, temperature and light regimes. These results indicate variation in temperature and light but not salinity enhances antioxidant enzyme activities in germinating seeds of Salsola drummondii.     

The role of gibberellic acid (GA3) in the removal of dormancy inFraxinus excelsior L. seeds

The seeds ofFraxinus excelsior L. were stratified at 17-20 °C (warm stratification), at 4-6 °C (cold stratification) and at alternating temperature (warm — cold stratification). The seeds subjected to warm stratification only, remained dormant. The seeds stratified only at 4-6 °C germinated gradually during a long period of time. The seeds subjected to warm — cold stratification, however, germinated with great intensity within a relatively short period of time. GA₃ was shown to stimulate the growth of embryos markedly, and its effect on the germination of seeds depended on the temperature of stratification. GA₃ applied during the cold stratification accelerated the removal of dormancy by shortening the period of stratification and by influencing the germination of seeds. The results obtained indicate a similarity between the effect of temperature 17-20 °C during the warm stratification and that of gibberellic acid. Both those factors applied separately affect favourably after-ripening of the embryos and accelerate the germination of seeds.     

Effects of ascorbic acid,alpha-tocopherol,and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l^?1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l^?1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l^?1) and ascorbic acid (5 and 10 mg l^?1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l^?1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l^?1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l^?1 α-tocopherol. When 50 mg l^?1 α-tocopherol was combined with 5 or 10 mg l^?1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l^?1 of reduced glutathione and 5 mg l^?l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.     

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

Key message

We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified.

Abstract

Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.     

Biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a newly isolated cold-adapted sulfamethoxazole-degrading bacterium

Sulfamethoxazole is a common antibiotic that is frequently detected in wastewater and surface water. This study investigated the biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a cold-adapted bacterium. Strain HA-4, which uses sulfamethoxazole as its sole source of carbon and energy, was isolated at a low temperature (10 °C) and identified as P. psychrophila by physico-biochemical characterization and 16S rRNA gene sequence analysis. Strain HA-4 removed sulfamethoxazole at temperatures ranging from 5.0 °C to 30 °C, with the maximal removal rate at 10 °C. The maximal removal rate of sulfamethoxazole by strain HA-4 was 34.30 % after 192 h at 10 °C. The highest percentage of unsaturated fatty acid was determined to be 23.03 % at 10 °C, which adheres to the characteristic for cold-adapted psychrophiles and psychrotrophs. At low concentrations of sulfamethoxazole, the growth kinetics correlated well with the Haldane model. The single-substrate parameter values of sulfamethoxazole on cell growth were determined to be μ _max?=?0.01 h^?1, K _s?=?20.91 mg/l and K _i?=?170.60 mg/l. Additionally, the major intermediates from sulfamethoxazole biodegradation by strain HA-4, including aniline, 3-amino-5-methylisoxazole, 4-aminothiophenol and sulfanilamide, were identified by GC-MS and high-resolution mass spectrometry (HR-MS) analysis. The results demonstrate that strain HA-4 has the potential to degrade sulfamethoxazole at low temperatures.     

Pollen,ovules, and pollination in pea: Success,failure, and resilience in heat

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars (“CDC Golden” and “CDC Sage”) were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of “CDC Sage” being more stable compared with “CDC Golden.” Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in “CDC Golden,” but not in “CDC Sage.” After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity.