Organelle antioxidants improve microspore embryogenesis in wheat and triticale

Low frequency of green plant production and albinism limits the use of isolated microspore culture (IMC) in cereal breeding programs. The present study was conducted in triticale and bread wheat IMC to increase the production of green plants and minimize albinism. NPB-99?+?10% Ficoll induction medium was supplemented with mitochondrial or plastid antioxidants, in a completely random design, to evaluate their contribution to successful microspore embryogenesis and green plant production. Each group of antioxidants was tested independently: first in triticale and then validated in various spring wheat genotypes. While the response differed by wheat genotype, induction medium supplemented with proline (10 mM) yielded a greater number of embryos/embryo-like structures and green plants in both triticale and wheat. No differences were found with respect to albinism in triticale or wheat except for the cv. Sadash. Among plastid antioxidants tested, glutathione (2 μM) proved to be the best antioxidant to increase embryo and green plant production. Salicylic acid also helped to reduce the number of albino plants in triticale and the wheat genotype SWS366. Overall, induction medium supplemented with proline or glutathione enhanced microspore embryogenesis in both triticale and wheat and increased the number of green plants in the recalcitrant genotypes.     

Genotype and age effect on in vitro embryo rescue of bread wheat x hexaploid triticale hybrids

The in vitro embryo rescue response of hybrid embryos of different ages and sizes of 16 bread wheat x hexaploid triticale crosses was studied. Response was dependent on the parental wheat genotype. However, the triticale genotype did not seem to affect the response of in vitro embryo rescue. Optimum age of the embryo for better recovery of complete plantlets coincided with onset of the drying of hybrid caryopses in the field. Embryo size was positively correlated with embryo germination and recovery of plantlets and negatively correlated with callus development. The best embryo germination (55.55%) and plantlet recovery (50%) were recorded in the hybrid HD 2380 wheat x JNIT 173 triticale.     

Hybrid necrosis in triticale and the expression of necrosis genes in allopolyploids

Summary The occurrence in triticale of four different genes causing hybrid necrosis is described: Ne1 and Ne2 in the B genome of wheat and Ner1 and Ner2 in the rye genome. Hybrid necrosis develops due to dominant complementary interaction of two genes. This interaction in triticale, however, may take place not only between genes belonging to the same genome but also between genes of different genomes. In triticale, these genes can cause hybrid necrosis in four different combinations. The inheritance of the phenomenon in triticale is, therefore, more complicated than it is in wheat or rye. To avoid hybrid necrosis in triticale, attention should be paid that no necrosis genes are introduced into the primary triticale stocks from the wheat and rye parents. The expression of necrosis genes is influenced by the level of ploidy. Any additional genome — A, B, D, or R — may exert a suppressing effect on the expression of necrosis genes. Therefore, when identifying genotypes of triticale with regard to their necrosis genes, the level of ploidy has to be accounted for. Moreover, the present results illustrate that gene expression in polyploids is not only determined by interactions with other single genes but that it may also be modified by the total genotype of the respective individual.     

Ventilatory mechanisms and the effect of hypoxia and temperature on the embryonic lesser spotted dogfish

Small, intermediate and large-sized embryos of the dogfish Scyliorhinus canicula utilize different ventilatory methods; small and intermediate embryos rely on body movement alone to stir either the jelly or sea water in the capsule, large embryos use conventional pharyngeal pumping to pump water through the case. The effects of environmental changes in O₂ tension (0.5–100% air saturation) and temperature (6–18°C) upon ventilatory mechanisms in the developing embryo in situ were studied using non-invasive ultrasonography. All three embryo classes increased ventilation rate with rising temperature: for small embryos, y=2.02x+3.295 ( P <0.01); for intermediate embryos, y=3.51x+0.395 ( P <0.01); and for large embryos, y=3.81x+9.39 ( P <0.01); where y=ventilatory frequency (tail beats min⁻¹ or pump cycles min⁻¹) and x=temperature (°C). Q ₁₀ (6–16°C)=5.0, 2.45, and 2.08 for small, intermediate and large embryos, respectively; corresponding Q ₁₀ (8–18°C) values were 2.09, 2.62, and 2.02. It is suggested that the extreme response of small embryos to 6°C is related to a different state of development in either chemoreceptors or muscle blocks. There was no significant change in ventilatory frequency induced by chronic (2 h) hypoxia. Dogfish embryos are oxyconformers at 8°C but oxyregulators at higher temperatures. Water flow through an eggcase occupied by a large embryo was studied also. Water enters the open eggcase of a large embryo, drawn in by the buccal/opercular pump of the respiring embryo, via holes at the posterior end of the eggcase. Expired water exits holes at the anterior end of the eggcase. The mean residence time for water in the case is 50 s at 8°C, giving a transit velocity of 1.36 mm s⁻¹.     

Comparative performance of bread wheat and hexaploid triticale cytoplasms

Summary Thirteen wheat-like advanced-generation triticale x wheat derivatives, having tetraploid wheat cytoplasm from triticale, were reciprocally crossed with three improved bread wheats, and the resulting F₁s were evaluated for determining the comparative performance of the bread wheat and triticale cytoplasms for different traits. Significant reciprocal differences in the mean performance were observed for days to heading, days to maturity, spikes/plant, flag-leaf area, peduncle length, plant height, spike length, grains/spike, 1,000-grain weight, grain yield and grain protein content, and most of them were in favour of hexaploid wheat cytoplasm. However, this superiority of the hexaploid cytoplasm was not universal for a particular trait, implying that the differences in the performance of the evaluated reciprocal crosses depended not solely on the cytoplasmic background, but also on the interplay of the specific genotype with the cytoplasm.     

Oviposition preferences of the Hessian fly and their consequences for the survival and reproductive potential of offspring

1. The relationship between oviposition preference and offspring performance was investigated experimentally for the Hessian fly Mayetiola destructor (Say) using 10 grass genotypes that represented five different genera and six different species. Oviposition preferences were quantified in a choice test using arrays of 200–400 plants. Offspring performance was estimated by recording survival during three phases of the insect–plant association and by measuring wing length, a correlate of adult reproductive potential. Density effects were examined for all offspring variables, and were taken into account when offspring performance was compared across the 10 grasses. 2. Egg counts from the oviposition choice test revealed a consistent ranking of plants: 18ITSN triticale > Otane bread wheat = Caldwell bread wheat = Stacy bread wheat > 3424 bread wheat = PND durum wheat = Fleet barley = Valetta barley = Matua brome grass > Awapuni oat. 3. Survival and adult wing length varied significantly on the 10 plant types. Survival was ranked: Otane bread wheat > 3424 bread wheat = Fleet barley = Matua brome grass > Caldwell bread wheat = Valetta barley > 18ITSN triticale = PND durum wheat = Stacy bread wheat > Awapuni oat. Adult male and female wing lengths were greatest on Otane, the bread wheat that also provided the best survival. 4. For Hessian flies on each of the 10 plant types, data on survival, wing length, and wing length–fecundity relationships were combined into a single fitness measure. When these fitness measures were compared with egg counts, no overall pattern emerged. On seven of the 10 plant types, there was a positive linear relationship between egg counts and offspring fitness. On the other three plant types, egg counts were high while fitness was low. Possible reasons why Hessian fly females lay eggs on plants that are poor hosts for their offspring are discussed.     

An evaluation of gene expression during somatic embryogenesis of two temperature-sensitive carrot variants unable to complete embryo development

The synthesis of putative stage-specific polypeptides during somatic embryogenesis of the carrot ( Daucus carota L. cv. Danvers) was investigated in the temperature-sensitive variants OB-2 and OB-3. These variants undergo normal embryo development to produce mature plantlets at the permissive temperature (24°C), but are arrested at the oblong stage to form elongated embryos without cotyledons at the restrictive temperature (33°C). Using two-dimensional polyacrylamide gel electrophoresis of in vivo labelled polypeptides, the patterns of stage-specific polypeptides in both lines were compared in: (1) oblong embryos grown at continuous 24°C vs oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of identical morphology but different developmental fates); and (2) heartshaped embryos grown at constant 24°C vs enlarged oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of the same age but different morphologies). The 22 putative stage-specific, polypeptides observed in this study fall into four classes: (1) line-specific, (2) age-specific, (3) unsynchronized, and (4) synchronized polypeptides. Only the last class, which consists of 4 polypeptides, exhibits synthesis patterns which are consistent with the polypeptides being causally involved in somatic embryo development. It is concluded that stage-specific behavior as assayed by PAGE analyses of simple 'present or absent' comparisons is insufficient to identify most of the polypeptides that may be relevant for somatic embryogenesis.     

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T₀ lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T₁ seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.     

Temperature-induced Changes in the Synthesis of Unsaturated Fatty Acids by Acanthamoeba castellanii

ABSTRACT. Major fatty acid components of Acanthamoeba castellanii lipids extracted after growth at 30°C include myristate, palmitate, stearate and the polyunsaturates linoleate, eicosadienoate, eicosatrienoate and arachidonate, with oleate as the sole major monounsaturated fatty acid. By comparison, growth at 15°C gave increased linoleate, eicosatrienoate and arachidonate, but decreased oleate and palmitate. When the growth temperature was shifted downwards from 30°C to 15°C, increased lipid unsaturation occurred over a period of 24 h; thus decreases of oleate and eicosadienoate were accompanied by increases in linoleate, eicosatrienoate, arachidonate and eicosapentaenoate. An upwards shift from 15°C to 30°C gave negligible alterations in fatty acid composition over a similar period. At 15°C organisms rapidly use [1-¹⁴C] acetate for de novo fatty acid synthesis; stearate is converted via oleate to further desaturation and chain elongation products. Similar short term experiments at 30°C indicate only de novo synthesis and Δ9-desaturation; synthesis of polyunsaturates was a much slower process. Rapid incorporation of [1-¹⁴C] oleate at 30°C was not accompanied by metabolic conversion over two hours, whereas at 15°C n-6 desaturation to linoleate was observed. Temperature shift of organisms from 15°C to 30°C in the presence of [1-¹⁴C] acetate revealed that over half of the fatty acids in newly-synthesised lipids were saturated, but the proportions of unsaturated fatty acids increased with time until the total polyenoate components reached 17% after 22 h. A shift of temperature in the reverse direction gave a corresponding figure of 60% for polyunsaturated fatty acids. These results emphasize the importance of n-6 desaturation in the low temperature adaptation of Acanthamoeba castellanii .     

Efficient production of haploid wheat (Triticum aestivum) through crosses between Japanese wheat and maize (Zea mays)

Summary Four Japanese wheat varieties, three crossable and one non-crossable with Hordeum bulbosum, were pollinated with maize pollen of 5 genotypes. By the application of 2,4-dichlorophenoxyacetic acid after pollination, embryos kept developing on wheat plants until 14 days after pollination. The frequency of embryo formation was significantly different among the maize genotypes, varying from 18.0% to 31.9%, but not among the wheat varieties. By bagging spikes with flag leaves the frequency of embryo formation was increased by about 7%. Ten- to twelve-day-old embryos gave higher frequencies of plant formation (83.6%) than 14-day-old embryos(50.0%). All 6 regenerated plants investigated cytologically were found to be haploid. Twelve of the 14 colchicine-treated plants produced florets setting seeds. The overall efficiency of our procedure is considered to be higher than that reported by Laurie and Bennett (1988).     

Differential rRNA genes expression in bread wheat and its inheritance

The expression of the ribosomal RNA (rRNA) genes from rye, located within the nucleolus organizer regions (NORs), is repressed by cytosine methylation in wheat x rye hybrids and in triticale, as consequence of nucleolar dominance. Our previous study revealed that bread wheat cultivars with a maximum number of four Ag-NORs presented high level of rDNA cytosine methylation when compared to others with a maximum of six Ag-NORs. In order to evaluate the inheritance of the Ag-NORs number and NOR methylation patterns, we produced F₁ hybrids between bread wheat cultivars with four Ag-NORs and bread wheat cultivars with six Ag-NORs (in the direct and reciprocal senses). The F₂ progenies of these F₁ hybrids were also evaluated for the NOR number and methylation patterns. Parent bread wheat cultivars with a maximum of four Ag-NORs after treated with 5-azacytidine evidenced a maximum of six Ag-NORs per metaphase cell and a maximum of six nucleoli per interphase nucleus, confirming that the expression of the rRNA genes in bread wheat is related to cytosine methylation. Most of the F₁ hybrids showed a maximum number of four or six Ag-NORs, similarly to that of the female parent suggesting a non-mendelian inheritance, while other hybrids presented four or six Ag-NORs in both senses of the cross. The F₁ NOR methylation patterns showed some fragments common to their parents but also novel fragments suggesting genomic and/or chromosome rearrangements after hybridization. Despite the different NOR patterns among the parents, an invariable NOR pattern was found among the F₁ plants suggesting a tendency to stability, which was also transmitted to the F₂. The F₂ progenies showed plants with a maximum of four, five and/or six Ag-NORs. The ratio of plants with four, five and/or six Ag-NORs per F₂ progeny was variable and did not follow any specific mendelian proportion. These results allowed us to suggest that the inheritance of the number of Ag-NORs by the F₁ and F₂ plants did not follow any mendelian inheritance and were not correlated to NOR methylation patterns in contrast to what was verified for their parents.     

普通小麦与粗山羊草正,反交的育性研究

普通小麦与粗山羊草杂交需借助幼胚培养方可获得杂种,取授粉１２～１６天的幼胚进行拯拯救,成苗率较高,效果较好。而不同杂交方式对共杂种成胚率、成苗率及思胚拯救率影响很大。以普通小麦为母本,粗山羊草为父杂交时,１５个杂交组合平均杂种成胚率,成苗率及幼胚拯率分别为５．３５％和５８．７３％,而在１２个反交组合中,其平均成胚率,成苗率及幼胚拯救率分别为９８．８８％、１７．１０％和２４．４４％。由此可见,普通小     

rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat x rye hybrids

Ribosomal RNA genes originating from one parent are often suppressed in interspecific hybrids. We show that treatments during germination with the cytosine analogue 5-azacytidine stably reactivate the expression of the suppressed rRNA genes of rye origin in the wheat x rye amphiploid, triticale, by preventing methylation of sites in the rye rDNA. When 5-azacytidine is applied to embryos of triticale and wheat x rye F₁ hybrids nine, or more, days after fertilization, rye rRNA gene expression is stably reactivated in the resulting seedling. Earlier treatments have no effect on rye rRNA gene expression, indicating that undermethylation of DNA early in embryo development is reversible. After 9 days, the methylation status of rRNA genes in maintained throughout development. Since the change in expression follows a methylation change at particular restriction-enzyme sites, the data establish a clear correlation between gene activity and methylation in plants.     

Opposite effects of temperature on breaking of dormancy and on induction of frost resistance in apple embryos

Apples ( Pyrus mains L., ev. Golden Delicious) were placed at 0, 12 and 35°C. At different intervals, seeds were taken from fruits, and the ability of isolated embryos to germinate was determined. In parallel, the survival of similar embryos frozen for 30 min at -7°C was estimated. Storage of apples at 0°C led to complete removal of embryo dormancy within ca 10 weeks. At 12°C, dormancy was only partly eliminated, and at 35°C breaking of dormancy did not occur. The onset of secondary embryo dormancy was thus evident as the result of 35 and 12°C treatments. On the other hand, the storage of apples at 35°C was the most effective treatment for induction of frost resistance in the embryos. At 12°C the same level of frost resistance was reached much slower. Even a prolonged storage of apples at 0°C did not allow frost survival of all embryos. The above relations were confirmed in experiments with transfer of fruits from one temperature of storage to another. The temperature of fruit storage affected the dormancy removal in embryos and induction of resistance to rapid freezing in opposite directions.     

Gibberellin status and responsiveness in shoots of tall and dwarf genotypes of diploid rye (Secale cereale)

The recessive dwarfing alleles of rye ( Secale cereale L.), ct1 and ct2 , caused a 35–55% reduction in the length of leaf 2 compared with corresponding tall lines grown at both 10°C and 20°C. The dwarf lines were 45–50% as responsive to applied GA₃ as the tall lines at 20°C but the absolute GA-responsiveness of the dwarfs was greater at 10°C than at 20°C. There was no significant difference in the contents of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₃ and GA₈ in the leaf extension zone of tall and dwarf seedlings grown at 20°C. It was concluded that the mechanism whereby GA homeostasis is maintained is functional in both tall and dwarf lines despite marked differences in leaf extension rate. The recessive rye mutations may cause loss of function late in the GA-cell elongation pathway or, alternatively, indirectly affect GA-responsiveness in vegetative tissues. The genetic and physiological evidence indicates that ct1 and ct2 are unrelated to the GA-insensitive Rht genes in hexaploid bread wheat.     

Molecular characterization and functional analysis of elite genes in wheat and its related species

The tribe Triticeae includes major cereal crops (bread wheat, durum wheat, triticale, barley and rye), as well as abundant forage and lawn grasses. Wheat and its wild related species possess numerous favourable genes for yield improvement, grain quality enhancement, biotic and abiotic stress resistance, and constitute a giant gene pool for wheat improvement. In recent years, significant progress on molecular characterization and functional analysis of elite genes in wheat and its related species have been achieved. In this paper, we review the cloned functional genes correlated with grain quality, biotic and abiotic stress resistance, photosystem and nutrition utilization in wheat and its related species.     

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos

Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

     

Microspore culture of small grain cereals

Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis.In wheat, the effect of basic media (W₁₄, A2, CHB₃, P4-m) was compared and among them the W₁₄, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB₃ and MS_m media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB₃ media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.