Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

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.     

Subcellular localization of mineral deposits in the roots of wheat (Triticum aestivum L.)

Summary Mineral distribution in the roots of wheat (Triticum aestivum L. cv. Wheaton) was investigated using X-ray microanalysis of bulk frozen hydrated roots in SEM and of freeze substituted sections in TEM. Results obtained using the two methods agreed reasonably well. A total often elements were detected: Na, Mg, Si, P, S, Cl, K, Ca, Mn, and Fe. Of these Si, P, Ca, and Mn were incorporated into biomineralized structures. Silica was deposited in the endodermal walls in the older parts of the root. Silicon was also detected in the large central metaxylem lumina in the basal zone of the root, and in the smaller peripheral metaxylem and the immediately contiguous pericycle and outer parenchyma cells bridging the small metaxylem vessels to the endodermal layer. In the basal zone of the root some of the inner cortical cells contained intracellular electron opaque deposits. These were associated with the cell walls, had non-opaque inclusions and microanalysis revealed that they consisted of calcium, phosphorus and manganese.Abbreviations A apical zone of root - M midzone of root - B basal zone of root - SEM scanning electron microscope - TEM transmission electron microscope     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

RELP markers linked to two Hessian fly-resistance genes in wheat (Triticum aestivum L.) from Triticum tauschii (coss.) Schmal

Summary Restriction fragment length polymorphism (RFLP) markers linked to genes controlling Hessian fly resistance from Triticum tauschii (Coss.) Schmal. were identified for two wheat (Triticum aestivum L.) germ plasm lines KS89WGRC3 (C3) and KS89WGRC6 (C6). Forty-six clones with loci on chromosomes of homoeologous group 3 and 28 clones on those of group 6 were surveyed for polymorphisms. Eleven and 12 clones detected T. tauschii loci in the two lines, respectively. Analysis of F₂ progenies indicated that the Hessian fly resistance gene H23 identified in C3 is linked to XksuH4 (6.9 cM) and XksuG48 (A) (15.6 cM), located on 6D. The resistance gene H24 in C6 is linked to XcnlBCD451 (5.9 cM), XcnlCD0482 (5.9 cM) and XksuG48 (B) (12.9 cM), located on 3DL.Paper No. 810 of the Cornell Plant Breeding Series     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

The chromosomal location of factors determining the presence of phenolic compounds in wheat (Triticum aestivum L.)

Summary Using thin-layer chromatography and nulli-tetrasomic and ditellosomic series of Triticum aestivum L. cv. Chinese Spring, it has been possible to relate the phenolic compounds found in adult plant leaves and 12 day-old seedling leaves with the chromosomes or chromosome arms 1 B, 2 BL, 3 BL, 5 A, 6 AL, 7 B and 7 DS.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Modelling potassium uptake by wheat (Triticum aestivum) crops

Spring wheat was grown in the field under deficient and sufficient levels of soil K and with high and low supplies of fertiliser nitrogen. Measurements were made of K uptake, soil nutrient supply parameters, root growth and, in solution culture, root influx parameters. Mechanistic models predicted uptake reasonably well under K-deficient conditions, but over-predicted uptake, by as much as 4 times, under K-sufficient conditions. The over-prediction was apparently due to poor characterisation of plant demand.     

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21–30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv Chris and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv Chris, with the F1 of Chris × Sinton displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.Abbreviations PAA Phenylacetic acid - MMS modified MS medium - MS Murashige and Skoog's medium 1962 - FHG Hunter's FHG medium 1988     

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.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Distribution of magnesium in wheat (Triticum aestivum L.) in relation to supply

The aims of this study were to describe the distribution of magnesium (Mg) and its retranslocation within wheat, in order to develop diagnostic procedures for Mg deficiency. Plants were grown in solution culture with both constant supply (0, 5, 10, 20, 40, 80, 160 MMg) and discontinued supply (40 M and 160 M decreased to nil).Magnesium was depleted from old leaves when Mg supply to the roots was halted. However, initial deficiency symptoms occurred on young leaves under constant but inadequate supply, contrasting with previous reports. Magnesium concentrations were also lower in young leaves compared to old leaves. Symptoms of yellowing and necrosis occurred if the leaf tissue contained <1194 gg^–1, irrespective of leaf age. The minimum Mg concentration in whole shoots associated with maximum shoot weight was 932 gg^–1; for the youngest emerged blade (YEB) it was 861 gg^–1. Symptoms were apparent on the young leaf before a reduction in shoot weight was measurable. The concentration of Mg in the YEB and whole shoot were better related to solution Mg concentration than was the Mg concentration in the old leaf.     

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.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.