Effectiveness of selection under different mating systems for the improvement of protein content in wheat (Triticum aestivum L. em Thell.)

Summary The effectiveness of selection for the improvement of protein content under random intermating (recurrent selection) and selfing series (pedigree selection) was evaluated in a cross of winter and spring wheats, Atlas 66 × HD 1977. Selection of 10 per cent high protein families resulted in an increase of 3.25 per cent and 4.30 per cent of the mean of checks through pedigree and recurrent selection, respectively. The mean protein differences in both methods were not significant. The increase in protein content was accompanied by a decrease in the grains per spike, grain yield and 1000-kernel weight, and the decline was relatively higher in recurrently selected than pedigreed population. Since the pedigree method is simple, less time consuming, economically cheaper, has favourable shifts in association and better correlated responses, it was decided to follow a few cycles of pedigree selection in early segregating generations, after which one or two cycles of recurrent selection in the elite lines could be introduced to increase genetic variation and concentrate favourable genes for grain yield.From a dissertation submitted by the senior author in partial fulfilment of the requirements for the Ph.D. degree. Work supported by financial assistance in the form of a Senior Research Fellowship received by the senior author from the I.C.A.R., New Delhi.     

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 characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

Epistatic,additive and dominance variation in a triple test cross of bread wheat (Triticum aestivum L.)

Summary Triple test cross progenies resulting from the crossing of three testers (Kloka, UP 368 and an f₁ intermediate between them) and 24 varieties of bread wheat have been studied for plant height (cm), peduncle length (cm), ear length (cm), number of spikelets per spike and harvest index (ratio between economic and total yield). Epistasis was not significant for any of the characters studied. The testers were inadequate for plant height and for peduncle length although the testers varied considerably for these traits. Additive variance played a significant role in the inheritance of all the characters except number of spikelets per spike. The dominance variance was important for plant height, ear length and harvest index. The degree of dominance was in the over-dominance range for plant height. Complete dominance was operative for ear length, number of spikelets per spike and harvest index whereas for peduncle length only partial dominance was observed. The possibility of the isolation of the recombinants with high harvest index has been stressed.     

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     

Mapping QTLs for nitrogen uptake in relation to the early growth of wheat (Triticum aestivum L.)

The objective of this study was to map QTLs for N uptake (NUP) in wheat, and to investigate factors influencing NUP. Two independent field trials with low N (LN) and high N (HN) treatments were conducted in the growing seasons of 2002–2003 (trial 1) and 2003–2004 (trial 2) to measure NUP per plant (N accumulated in the aerial part at maturity stage) of a doubled haploid (DH) population consisting of 120 DH lines derived from winter wheat varieties Hanxuan 10 and Lumai 14. A hydroponic culture with all nutrients supplied sufficiently was conducted to investigate shoot dry weight (SDW), root dry weight (RDW), tiller number (TN) and NUP (total plant N uptake) per plant of this mapping population at seedling stage. SDW, RDW, TN and NUP investigated in the hydroponic culture were significantly and positively correlated with each other, and with NUP under both LN and HN conditions in the field trials. Nine and eight QTLs for NUP were detected under LN and HN conditions in the field trials, respectively. Four to five QTLs for SDW, RDW, TN and NUP were detected in the hydroponic culture. One SDW QTL, three RDW QTLs, two TN QTLs detected in the hydroponic culture were linked with QTLs for NUP under LN or HN condition in the field trials. The positive correlation and genetic linkage for the traits between the field trials and the hydroponic culture demonstrated that greater seedling vigor of root and shoot is an important factor influencing N uptake in wheat. Diaoguo An and Junying Su: These authors contributed equally to this work. Section Editor: H.J. Kronzucker     

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.     

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.     

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     

Peroxidase activities in relation to plant height and grain weight in bread wheat (Triticum aestivum L.)

Summary The relationship of peroxidase activity with plant height and grain weight has been studied in seven different varieties of bread wheat belonging to diverse genotypes, and their F₁ crosses. The association between plant height and peroxidase activity was highly significant and negative. Based on the similarity index values of peroxidase isoenzymes, the seven wheat genotypes could be classified into two groups: the first group consisting of triple and quadruple dwarf varieties and the other of tall, single and double dwarf. A negative correlation between peroxidase activity and grain weight was also observed. However, the results of this study indicate a possibility of developing a dwarf plant type with low peroxidase activity and well filled grains.     

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     

Identification of quantitative trait locus of zinc and phosphorus density in wheat (Triticum aestivum L.) grain

Zinc (Zn) is an essential micronutrient for human beings. However, Zn malnutrition has become a major problem throughout the world. Wheat is the most important food crop in the world, therefore, developing Zn-enriched wheat varieties provides an effective approach to reduce Zn malnutrition in human beings. The aim of this study was to understand the genetic control of grain Zn density in wheat and hence, to provide genetic basis for breeding wheat with high grain Zn density using molecular approach. A doubled haploid (DH) population developed from a cross between winter wheat varieties Hanxuan10 and Lumai 14 was used to identify quantitative trait loci (QTLs) for Zn concentration and content in wheat grains. In addition, phosphorus (P) concentration and content in wheat grain were also investigated to examine possible interactions between these two nutrients. The wheat grains used in this study were harvested from the plants grown under normal condition in a field trial. We found the grain Zn concentrations of the DH population varied from 25.9 to 50.5 mg/kg and the Zn content varied from 0.90 to 2.21 μg/seed. The grain P concentrations of the DH population varied from 0.258 to 0.429 mg/kg, and the P contents varied from 0.083 to 0.186 mg/seed. A significant positive correlation was observed between Zn and P density in this experiment. The results showed that both grain Zn and P densities were controlled by polygenes. Four and seven QTLs for Zn concentration and Zn content were detected, respectively. All the four QTLs for Zn concentration co-located with the QTLs for Zn content, suggesting a possibility to improve both grain Zn concentration and content simultaneously. Four and six QTLs for P concentration and P content were detected, respectively. The two QTLs for grain Zn concentration on chromosomes 4A and 4D co-located with the QTLs for P concentration. The four QTLs for grain Zn content on chromosome 2D, 3A and 4A co-located with the QTLs for P contents, reflecting the positive correlations between the grain Zn and P density, and providing possibility of improving grain micro- and macronutrient density simultaneously in wheat. In order to improve human health, the effect of P–Zn relation in grain on the Zn bioavailability should also be considered in the future work.     

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.     

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.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

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.     

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.     

Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase

The Aspergillus niger phytase-encoding gene (phyA) has been constitutively expressed in wheat. Transgenic wheat lines were generated by microprojectile bombardment of immature embryos, using the bar-Bialaphos selection system. The bar and the phyA gene expression were controlled by the maize ubiquitin-1 promoter. To ensure secretion and glycosylation of the microbial phytase, an expression cassette was designed (Ubi-SP-Phy) where an -amylase signal peptide sequence was inserted between the promoter and the phytase coding region. A similar cassette was constructed without the signal peptide sequence (Ubi-Phy). Five lines of fertile wheat transformed with the Ubi-SP-Phy were generated and two lines with the Ubi-Phy construct. The inheritance of the phyA gene was monitored through three generations. Western blotting of leaf and seed derived protein revealed the presence of an immunoreacting polypeptide of the size expected for the Aspergillus phytase. Up to 25 days after pollination, the heterologous phytase was exclusively present in the pericarp-seed coat-aleurone fraction. Thereafter, it accumulated in the endosperm in amounts exceeding that found in the seed coat and aleurone. The phyA mRNA and derived protein could at no stage be detected in the embryo. The Ubi-SP-Phy transgenic seeds exhibited up to 4-fold increase of phytase activity while up to 56% increase was found in Ubi-Phy plants. It is concluded that a functional Aspergillus phytase can be produced in significant amounts in wheat grains. This may be of relevance for improving the phytate-phosphorus digestibility when wheat grains are used for non-ruminant animal feed.