Enhanced induction of microspore embryogenesis after <Emphasis Type="Italic">n</Emphasis>-butanol treatment in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) anther culture

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.     

Genetic variability of the low-molecular-weight glutenin subunits in spelt wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> ssp.<Emphasis Type="Italic"> spelta</Emphasis> L. em Thell.)

The low-molecular-weight glutenin subunit composition of a collection of 403 accessions of spelt wheat (Triticum aestivum ssp. spelta L. em. Thell) was analyzed by SDS-PAGE. Extensive variation was found, including 46 different patterns for zone B and 16 for zone C. Patterns within zone B exhibited from two to six bands and patterns in zone C had between four and six bands in SDS-PAGE gels. A higher number of bands was observed when urea was added to the gels. Zone B exhibited between six and 11 bands, and we identified 14 new patterns in this zone. For zone C, up to ten new patterns that comprised between five and nine bands were detected. For both zones, 86 patterns were found. The variability detected in this material is greater than that detected in other hulled wheats.Communicated by H.F. Linskens     

HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) progenitors

The allelic compositions of high- and low-molecular-weight subunits of glutenins (HMW-GS and LMW-GS) among European spelt (Triticum spelta L.) and related hexaploid and tetraploid Triticum species were investigated by one- and two-dimensional polyacrylamide-gel electrophoresis (PAGE) and capillary electrophoresis (CE). A total of seven novel glutenin alleles (designated A1a*, B1d*, B1g*, B1f*, B1j*, D1a* at Glu-1 and A3h at the Glu-3 loci, respectively) in European spelt wheat were detected by SDS-PAGE, which were confirmed further by employing A-PAGE and CE methods. Particularly, two HMW-GS alleles, Glu-B1d* coding the subunits 6.1 and 22.1, and Glu-B1f* coding the subunits 13 and 22*, were found to occur in European spelt with frequencies of 32.34% and 5.11%, respectively. These two alleles were present in cultivated emmer (Triticum dicoccum), but they were not observed in bread wheat (Triticum aestivum L.). The allele Glu-B1g* coding for 13* and 19* subunits found in spelt wheat was also detected in club wheat (Triticum compactum L.). Additionally, two alleles coding for LMW-GS, Glu-A3h and Glu-B3d, occurred with high frequencies in spelt, club and cultivated emmer wheat, whereas these were not found or present with very low frequencies in bread wheat. Our results strongly support the secondary origin hypothesis, namely European spelt wheat originated from hybridization between cultivated emmer and club wheat. This is also confirmed experimentally by the artificial synthesis of spelt through crossing between old European emmer wheat, T. dicoccum and club wheat, T. compactum.Communicated by H.F. Linskens     

Genetic control of gliadin components in wheat <Emphasis Type="Italic">Triticum spelta</Emphasis> L.

The componental composition of electrophoretic spectra of gliadin in Triticum spelta L. was studied. By analogy with common wheat T. aestivum L., it was established that genes controlling gliadin components in spelt are also located in short arms of chromosomes of homeological groups 1 and 6. Analysis of gliadin spectra in F₂ grains from the crosses k-20539 × Ershovskaya 32 and k-20558 × Ershovskaya 32 revealed linkage of some components and their grouping into blocks (alleles) of coinherited gliadin components. Alleles of gliadin-coding loci identical to alleles of common wheat and new alleles earlier unknown for wheat populations have been identified.     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Induction,regeneration, and biolistic sensitivities of different genotypes of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The induction, regeneration, and biolistic sensitivities of different genotypes of common wheat (Triticum aestivum L.) have been determined in order to develop an efficient system for transformation of Russian cultivars of spring wheat. Short-term (two days) cold treatment (4°C) has been demonstrated to distinctly increase the frequency of morphogenetic callus induction. The optimal phytohormonal composition of the nutrient medium ensuring an in vitro regeneration rate of the common wheat cultivar Lada as high as 90% has been determined. The optimal temporal parameters of genetic transformation of wheat plants (10–14 days of culturing after initiation of a morphogenetic callus) have been determined for two transformation methods: biolistic without precipitated DNA and transformation with the plasmid psGFP-BAR. Analysis of the transient expression of the gfp gene has confirmed that 14 days of culturing is the optimal duration.     

Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.     

Isolated microspore culture overperforms anther culture for green plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Barley isolated microspore culture (IMC) was compared to anther culture (AC) for its efficiency in green plant (GP) regeneration. With six cultivars investigated, IMC resulted in significantly more GPs (3.6–287 per 100 anthers) than AC (0–29.6), which was on average 9.3-fold more efficient (113.7 vs 12.2). GPs were produced via IMC from all the genotypes tested, whereas no green shoot was generated by AC in two of the cultivars. In spite of genotype dependency for regeneration rates, the average GP percentage of IMC was just slightly higher than that of AC. Effects of microspore developmental stages and medium-sterilization methods on IMC were examined with the aim of optimizing culture conditions. We found that the optimum stage for cold pretreatment of spikes was different from that for mannitol starvation of anthers. Significant variations in microspore embryogenesis and regeneration were observed among five stages tested. Optimal stages for the two pretreatments were accordingly determined. Percentages of viable microspores were strongly influenced by protocols of medium-aseptisation. Although filter-sterilized media yielded two-time higher frequencies of living microspores and significantly more GPs than autoclaved ones, the filtration protocol was rather labor-intensive and time-consuming. Therefore a new procedure by combining filtering with autoclaving was subsequently developed as it was more effective than autoclaving and more convenient than filter-sterilization. The method described here could be useful for large-scale preparation of culture media.     

Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

Overexpression of <Emphasis Type="Italic">AtHDG11</Emphasis> enhanced drought tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Drought is one of the major abiotic stresses restricting the yield of wheat (Triticum aestivum L.). Breeding wheat varieties with drought tolerance is an effective and durable way to fight against drought. Here we reported introduction of AtHDG11 into wheat via Agrobacterium-mediated transformation and analyzed the morphological and physiological characteristics of T2 generation transgenic lines under drought stress. With drought treatment for 30 days, transgenic plants showed significantly improved drought tolerance. Compared with controls, the transgenic lines displayed lower stomatal density, lower water loss rate, more proline accumulation and increased activities of catalase and superoxide dismutase. Without irrigation after booting stage, the photosynthetic parameters, such as net photosynthesis rate, water use efficiency and efficiency of excitation energy, were increased in transgenic lines, while transpiration rate was decreased. Moreover, the kernel yield of transgenic lines was also improved under drought condition. Taken together, our data demonstrate that AtHDG11 has great potential in genetic improvement of drought tolerance of wheat.     

Genetic marking of glume color in <Emphasis Type="Italic">Triticum spelta</Emphasis> L. var. <Emphasis Type="Italic">caeruleum</Emphasis> using gliadins

Using gliadins as genetic markers, Triticum spelta L. var. caeruleum accessions were analyzed to identify genetic control of the dark color of glumes. The research material was F₂ and BC₁ plants from crosses between spelt accessions and white-glumed common wheat varieties. The segregation for glume color fitted the monogenic control of the trait. The electrophoretic analysis of gliadins in grains from the hybrid plants has shown that the Gli-Alj* allele in the T. spelta var. caeruleum accessions is linked to the allele for the dark (black) color of glumes at the Rg-A1 locus.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

A search for high-molecular-weight subunits of glutenin from <Emphasis Type="Italic">Triticum timopheevi</Emphasis> Zhuk. in the lines of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. <Emphasis Type="Italic">Triticum timopheevi</Emphasis> Zhuk.)

The study is a continuation of investigation of prolamins in brown rust-resistant introgressive lines of common wheat, produced with participation of Triticum timopheeevi Zhuk. [1]. Two wheat lines with a substitution of the Glu-1 loci of T. timopheevi were identified. Line 684 had high-molecular-weight glutenin subunits encoded by 1Ax, as well as by 1Ay gene, which was silent in commercial lines. It was demonstrated that line 684 could serve as a source of the Glu-A _t ¹ locus. Line 186 carried the Glu-B1/Glu-G1 substitution. Comparative analysis of storage proteins from the introgression lines of common wheat Triticum aestivum L. with those from parental forms demonstrated polymorphism among the lines, resulted from natural varietal polymorphism, and introgression of the Glu-3 and Gli-1 loci from the genome of T. timopheevi.     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.     

The parameters of grain filling and yield components in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L. var. <Emphasis Type="Italic">durum</Emphasis>)

Final grain dry weight, a component of yield in wheat, is dependent on the duration and the rate of grain filling. The purpose of the study was to compare the grain filling patterns between common wheat, (Triticum aestivum L.), and durum wheat, (Triticum turgidum L. var. durum), and investigate relationships among grain filling parameters, yield components and the yield itself. The most important variables in differentiating among grain filling curves were final grain dry weight (W) for common wheat genotypes and grain filling rate (R) for durum wheat genotypes; however, in all cases the sets of variables important in differentiating among grain filling curves were extended to either two or all three parameters. Furthermore, in one out of three environmental conditions and for both groups of genotypes, the most important parameter in the set was grain filling duration (T). It indicates significant impact of environmental conditions on dry matter accumulation and the mutual effect of grain filling duration and its rate on the final grain dry weight. The medium early anthesis date could be associated with further grain weight and yield improvements in wheat. Grain filling of earlier genotypes occurs in more temperate environments, which provides enough time for gradual grain fill and avoids the extremes of temperature and the stress of dry conditions.     

Comparative analysis of the differential regeneration response of various genotypes of <Emphasis Type="Italic">Triticum aestivum</Emphasis>, <Emphasis Type="Italic">Triticum durum</Emphasis> and <Emphasis Type="Italic">Triticum dicoccum</Emphasis>

An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant) different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall 80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks. This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits in the three species of wheat.     

Genetic mapping of stem rust resistance gene <Emphasis Type="Italic">Sr13</Emphasis> in tetraploid wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis> L.)

Wheat stem rust caused by Puccinia graminis f. sp. tritici, can cause significant yield losses. To combat the disease, breeders have deployed resistance genes both individually and in combinations to increase resistance durability. A new race, TTKSK (Ug99), identified in Uganda in 1999 is virulent on most of the resistance genes currently deployed, and is rapidly spreading to other regions of the world. It is therefore important to identify, map, and deploy resistance genes that are still effective against TTKSK. One of these resistance genes, Sr13, was previously assigned to the long arm of chromosome 6A, but its precise map location was not known. In this study, the genome location of Sr13 was determined in four tetraploid wheat (T. turgidum ssp. durum) mapping populations involving the TTKSK resistant varieties Kronos, Kofa, Medora and Sceptre. Our results showed that resistance was linked to common molecular markers in all four populations, suggesting that these durum lines carry the same resistance gene. Based on its chromosome location and infection types against different races of stem rust, this gene is postulated to be Sr13. Sr13 was mapped within a 1.2–2.8 cM interval (depending on the mapping population) between EST markers CD926040 and BE471213, which corresponds to a 285-kb region in rice chromosome 2, and a 3.1-Mb region in Brachypodium chromosome 3. These maps will be the foundation for developing high-density maps, identifying diagnostic markers, and positional cloning of Sr13.     

Physical mapping of <Emphasis Type="Italic">puroindoline b</Emphasis>-<Emphasis Type="Italic">2</Emphasis> genes and molecular characterization of a novel variant in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L.)

The puroindoline genes (Pina and Pinb) are the functional components of the common or bread wheat (Triticum aestivum L.) grain hardness locus that are responsible for kernel texture. In this study, four puroindoline b-2 variants were physically mapped using nulli-tetrosomic lines of bread wheat cultivar Chinese Spring and substitution lines of durum wheat (Triticum turgidum L.) cultivar Langdon. Results indicated that Pinb-2v1 was on 7D of Chinese Spring, Pinb-2v2 on 7B of Chinese Spring, Pinb-2v3 on 7B of Chinese Spring and Langdon, and Pinb-2v4 on 7A of Chinese Spring and Langdon. A new puroindoline b-2 variant, designated Pinb-2v5, was identified at the puroindoline b-2 locus of durum wheat cultivar Langdon, with a difference of only five single nucelotide polymorphisms compared with Pinb-2v4. Sequencing results indicated that, in comparison with the Pinb-2v3 sequence (AM99733 and GQ496618 with one base-pair modification of G to T at 6th position, designated Pinb-2v3a) in bread wheat cultivar Witchta, the coding region of Pinb-2v3 in 12 durum wheat cultivars had a single nucleotide change from T to C at the 311th position, resulting in a corresponding amino acid change from valine to alanine at the 104th position. This new allele was designated Pinb-2v3b. The study of puroindoline b-2 gene polymorphism in CIMMYT and Italian durum wheat germplasm and discovery of a novel puroindoline b-2 variant could provide useful information for further understanding the molecular and genetic basis of kernel hardness and illustrating gene duplication events in wheat.     

A <Emphasis Type="Italic">Pseudo-Response Regulator</Emphasis> is misexpressed in the photoperiod insensitive <Emphasis Type="Italic">Ppd-D1a</Emphasis> mutant of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the “green revolution” converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a ‘Chinese Spring’ wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Beales and A. Turner contributed equally to the work.