Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.)

Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×10⁶ protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - PCV packed cell volume - MES morpholinoethanesulfonic acid     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

Comparison of media for their aptitude in wheat anther culture

Different media were evaluated with anthers of five spring wheat (Triticum aestivum L.) genotypes for their ability to produce embryos and green plants in anther culture. Our first experiment showed that the addition of a combination of 19 amino acids significantly increased the number of embryos and green plants obtained. The mean number of green plants per 100 anthers for the two genotypes in this experiment, HY320 and B723, went from 28.2 without amino acids in the medium, to 46.7 with addition of amino acids. Our second experiment with the genotypes HY320, Wim and Laval-19 showed that liquid medium with Ficoll is more efficient for anther culture (9.9 green plants/100 anthers) than solid (0 green plants), gelationous media (2.5 green plants/100 anthers) or liquid medium with Membrane Rafts (0 green plants; Hoechst Celanese Corp.). Our third experiment revealed that the effect of replacement of sucrose by maltose varied with the genotype of the donor plant. Maltose partially inhibited the androgenesis of three responsive genotypes, HY320, Wim and Reliance (40.3 green plants/100 anthers instead of 43.9 with sucrose), while maltose significantly increased the androgenesis of the recalcitrant genotype Laval-19 (10.8 green plants/100 anthers instead of 5.4 with sucrose). An amino acid x maltose interaction was also observed. Amino acids without maltose increased androgenesis, but the addition of maltose to the amino acid-enriched medium eliminated this positive effect of the amino acids.     

Molecular cytological evidence for gradual telomere synthesis at the broken chromosome ends in wheat

Telomere formation of the normal and broken chromosomes of common wheat,Triticum aestivum, was investigated byin situ hybridization using the biotin-labeled probe of telomere repetitive sequences (pAtT4) ofArabidopsis thaliana with subsequent amplification by an antibody. After double and triple amplification, prominent signals appeared at all the telomeric regions of the normal chromosomes. Prominent signals also emerged at the broken ends of the telocentric and deletion chromosomes that had passed through more than one generation since the appearance. However, broken ends that had passed through only the stages of gametogenesis, fertilization, embryogenesis and root development did not show complete signals such as found in normal telomeres. These findings indicate that a certain time or stage is required for synthesis of the telomeric repetitive sequences with a complete length. Nevertheless, because the broken ends without complete telomere sequences were also healed, restoration of the normal complement of telomere sequences is not necessary for healing of broken ends.     

An improved medium for in vitro grain development from detached wheat spikelets

Wheat spikelets detached from the spike at anthesis were cultured on solidified media and successfully produced mature grains. These grains resembled normal grains and contained well-developed, embryos. Lower concentrations of glutamine favored dry weight increase in developing grains. Such grains were indistinguishable from grains from greenhouse-grown plants in germination on moist blotting sheets. The technique of individual spikelet culture can be used to study physiology and development of wheat grains and kernels and to study host-pathogen interactions in wheat floret diseases such as Karnal bunt.     

Detecting specific repeated sequences in large,complex genomes by using representative difference analysis and double-probe verification

We present a modification of the representative difference analysis (RDA) technique used to target AT-rich repeated sequences, such as transposable elements, with a double-probe verification system. RDA is a subtractive/amplification PCR-based technology used to identify specific sequences that are different between 2 related genomes.Vsp I restriction enzyme was used to target AT-rich sequences. RDA products were cloned with a high efficiency. Double-probe verification is based on reverse dot-blot of cloned RDA products and uses a positive and a negative probe. We tested thisVsp I-modified RDA on different combinations of bread wheat (Triticum aestivum) and relatives.Triticeae members have large, complex genomes with various ploidy levels. RDA experiments were performed with single or bulked DNA. Reverse dot-blot double-probe verification detected specific repeated sequences quickly and efficiently. Together, the 2 systems provide a powerful tool for obtaining specific transposable elements and repeated sequences that are different between related genomes, regardless of genome size and ploidy.     

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.     

Recent advances in wheat transformation

Summary Since the first report of wheat transformation in the early 1990s, genetic engineering of wheat has evolved rapidly. Several laboratories worldwide have reported the production of fertile transgenic wheat plants using a variety of methods. While there are several innovative and promising approaches for wheat transformation using different explants as targets for transformation, different methods of transformation, and different selection schemes, the most common approach to wheat transformation is the bombardment of tissue derived from immature embryos followed by selection based on resistance to the bar gene. Even with all these successful reports, hurdles still exist for this recalcitrant crop. Of these hurdles, low transformation rates, tools for transgene expression, and transgene silencing in subsequent generations are probably the most critical. This review will provide an overview of wheat transformation in the past decade, addressing both positive and negative factors that effect transformation while highlighting the successes of the past and prospects for the future.     

Ear culture as a technique to overcome hybrid necrosis in wheat

Hybrid necrosis in wheat is a problem for gene transfer in wheat breeding. Hybrid necrosis occurs due to dominant complementary interaction of two genes, Ne1 and Ne2. A cross between wheat (Triticum aestivum L.) varieties C306 (drought tolerant, Ne1 carrier) and WL711(high yielding, Ne2 carrier) produced necrotic F1 hybrids, which died before ear emergence and produced no seeds. To overcome the problem of hybrid necrosis, ears enclosed in the leaf sheath were taken and cultured to maturity in liquid medium containing 5% sucrose and 0.04% glutamine. The necrotic hybrids produced only a few seeds per ear compared to parents, but individual grain weight was similar in the hybrid and the parents. The F1 ear culture study has been repeated for three years and F2 seeds obtained. In 1996–97, the cultured ears of F1 hybrids produced 62 seeds, of which only 52 showed germination and were grown under normal field conditions. Out of the 52 seeds, 50% were non-necrotic and showed segregation for various physiological traits. The results reveal that hybrids ears had the potential to form viable seeds. Culturing of wheat ears before ear emergence and production of viable F2 seeds from necrotic hybrids is a simple and efficient method for overcoming the problem of hybrid necrosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular mapping of a quantitative trait locus for aluminum tolerance in wheat cultivar Atlas 66

Genetic improvement of aluminum (Al) tolerance is one of the cost-effective solutions to improve wheat (Triticum aestivum) productivity in acidic soils. The objectives of the present study were to identify quantitative trait loci (QTL) for Al-tolerance and associated PCR-based markers for marker-assisted breeding utilizing cultivar Atlas 66. A population of recombinant inbred lines (RILs) from the cross Atlas 66/Century was screened for Al-tolerance by measuring root-growth rate during Al treatment in hydroponics and root response to hematoxylin stain of Al treatment. After 797 pairs of SSR primers were screened for polymorphisms between the parents, 131 pairs were selected for bulk segregant analysis (BSA). A QTL analysis based on SSR markers revealed one QTL on the distal region of chromosome arm 4DL where a malate transporter gene was mapped. This major QTL accounted for nearly 50% of the phenotypic variation for Al-tolerance. The SSR markers Xgdm125 and Xwmc331 were the flanking markers for the QTL and have the potential to be used for high-throughput, marker-assisted selection in wheat-breeding programs.