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.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The flag leaf of wheat was examined for changes in quantity and activity of ribulose-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39), in the proteolytic degradation of RuBPCase and other native proteins, and in the ultrastructure of the leaf cells during grain development. Proteolytic degradation of RuBPCase at pH 4.8 increased until 8–10 d after anthesis, then declined, and increased again 16–18 d after anthesis. The second peak coincided with the onset of a preferential loss of immunologically recognizable RuBPCase. The specific activity and number of active sites per molecule of RuBPCase did not change during senescence. Examination of ultrastructure with the electron microscope showed little change in the appearance of the mitochondria as the flag leaf aged. Prominent cristae were still evident 35 d after anthesis. In contrast, the chloroplasts showed a progressive disruption of the thylakoid structure and an increasing number of osmiophilic glubules. The double membrane envelope surrounding the chloroplast appeared intact until at least 20 d after anthesis. The tonoplast also appeared intact up to 20 d. At later stages of senescence of the leaf the outer membrane of the chloroplast adjacent to the tonoplast appeared to break but the inner membrane of the envelope appeared intact until at least 35 d after anthesis.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase (EC. 4.1.1.39) I=Waters et al. 1980     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The activity of a range of endo- and exopeptidase enzymes have been measured in the glumes, flag leaf and stem during the period of grain development in wheat. The enzymes show a sequential pattern of appearance with activity peaks occurring at a number of intervals from anthesis until just prior to the cessation of grain growth. Of the enzymes studied only the haemoglobin- and casein-degrading activity and alanylglycine-dipeptidase activity increased during the period of rapid protein loss, while aminopeptidase, carboxypeptidase and leucyltyrosine dipeptidase reached maximum activity prior to this period.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.II. Peoples et al. (1980)     

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.     

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.     

Sucrose metabolism in developing endosperm of immature wheat (Triticum aestivum L.) grain

With a view to investigating the role of the enzyme pyrophosphate-fructose-6-phosphate-1-phosphotransferase (PFP) in sucrose breakdown in developing endosperm of wheat grain, the activity of PFP and related enzymes such as phosphofructokinase (PFK), fructose-6-bisphosphatase (FBPase), fructose-6-phosphate-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (F2, 6-P2ase) and the contents of the various intermediates of the pathway serving either the substrate or the effectors of these enzymes such as glu-6-P,glu-1-P,fru-6-P,fru-1,6-P2,DHAP,G3P, UDP-glucose, ADP-glucose, Pi,PPi and fru-2,6-P2 have been determined at 5 days intervals starting from day-5 after anthesis until day-40 after anthesis. These enzymes except PFK-2 had their peak activity at day-25 after anthesis. The activity of PFP was several fold higher than that of PFK at each stage of grain development. PFK-2 exhibited the lowest activity. The various intermediates again had their maximum concentration either at day-20 or day-25 after anthesis. Among hexose phosphates studied, glu-6-P was present in highest concentration at each stage of grain development. The level of Pi was much higher than those of PPi and fru-2,6-P2. Similarly, concentration of UDP-glucose was higher than that of ADP-glucose. Based on these results, it is proposed that the major role of the enzyme PFP in developing wheat grain is to provide PPi for sucrose breakdown via sucrose synthase.     

Inheritance of grain polyphenol oxidase (PPO) activity in multiple wheat (Triticum aestivum L.) genetic backgrounds

Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS markers, PPO18, PPO29 and STS01, were used to identify lines with putative alleles at the Ppo-A1 and Ppo-D1 loci conditioning low or high PPO activity. ANOVA showed significant genotypic effects on PPO activity (P?<?0.0001) in all populations. The generations and generation?×?genotype effects were not significant in any population. A putative third (null) genotype at Ppo-A1 (no PCR fragments for PPO18) was discovered in NW07OR1066 and NW07OR1070 derived populations, and these had the lowest mean PPO activities. Results demonstrated that both Ppo-A1 and Ppo-D1 loci affect the kernel PPO activity, but the Ppo-A1 has the major effect. In three populations, contrary results were observed to those predicted from previous work with Ppo-D1 alleles, suggesting the markers for Ppo-D1 allele might give erroneous results in some genetic backgrounds or lineages. Results suggest that selection for low or null alleles only at Ppo-A1 might allow development of low PPO wheat cultivars.     

Genetic diversity of reaction of common wheat (Triticum aestivum L.) cultivars to light intensity

The effect of low light intensity (LI) on the period from sprouting to earing was studied in 12 cultivars of the spring common wheat under controlled conditions. Differences between cultivars with respect to their responses to LI (RLIs) were found both for those that were photoperiod-sensitive and those that were almost photoperiod-neutral. Specifically, a prolonged photoperiod and a low LI differently increased the period from sprouting to earling in different cultivars. Genetic analysis of the RLI demonstrated, for the first time, that the weak response was incompletely dominant in F1. The results of genetic analysis agree with the hypothesis that the cultivars Pitic 62 and Novosibirskaya 22 differ in alleles of two loci controlling the RLI in wheat.     

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.)

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.     

Preliminary screening for water stress tolerance and genetic diversity in wheat (Triticum aestivum L.) cultivars from Sudan

The present study was carried out to screen 12 Sudanese wheat (Triticum aestivum L.) cultivars for their response to water stress at early germination stages and to characterize sources that could be used in breeding programs to develop wheat cultivars with better adaptation to water stress. The effect of osmotic stress on the early growing stages was evaluated, in vitro, using five concentrations of Polyethylene glycol. Genetic diversity was studied using 24 allele specific simple sequence repeats (SSR) markers associated with drought tolerance in wheat. The presence of the drought genes and their chromosomal location was also investigated by isolating and sequencing the dehydration responsive element binding protein (dreb1). Results of the in vitro screening among the cultivars showed significant differences in the root length, shoot length and root/shoot ratio. The 24 drought specific SSR markers used revealed 50 alleles, with an average of 2.0 alleles per locus. Of these, 60% were polymorphic with polymorphism information content (PIC) ranging from 0.16 to 0.89. A dendrogram based on the similarity values generated from the SSR data revealed three major clusters. Of the five specific primers for dreb1 genes, only primer P25F/PR produced amplification products with the expected fragment sizes. Sequencing and BLAST results of the cloned fragments excised from the gels showed 99% homology to the dreb1 gene on chromosome 3A.     

Tolerance to ion toxicities enhances wheat (Triticum aestivum L.) grain yield in waterlogged acidic soils

Aims

The high concentrations of Mn, Fe and Al in acid soils during waterlogging impair root and shoot growth more severely in intolerant than tolerant wheat genotypes. This study aims to establish whether this difference in vegetative growth and survival during waterlogging (1) is verifiable across a range of tolerant/intolerant genotypes and acid soils, and (2) results in improved recovery after cessation of waterlogging and enhanced grain yield.

Methods

Wheat genotypes contrasting in their tolerance to ion toxicities were grown in four acid soils until 63DAS and maturity, with a 42-day waterlogging treatment imposed at 21 DAS.

Results

The shoot Al, Mn and Fe concentrations increased by up to 5-, 3- and 9-fold respectively due to waterlogging in various soils. Compared to the intolerant lines, Al-, Mn- and Fe-tolerant genotypes maintained a relatively lower increase in shoot concentrations of Al (79 vs. 117%), Mn (90 vs. 101%) and Fe (171 vs. 252%) and demonstrated better waterlogging tolerance at the vegetative stage expressed in relative root (38% vs. 25%) and shoot (62% vs. 52%) growth. After cessation of waterlogging and the continued growth to maturity, tolerant genotypes maintained a relatively lower plant concentration of Al, Mn and Fe, but produced a higher above-ground biomass (74% vs. 56%) and most importantly demonstrated improved waterlogging tolerance (a relative grain yield of 78% vs. 54%) compared to intolerant genotypes. Maturity following waterlogging stress was delayed less in tolerant than intolerant genotypes (114 vs. 124%, respectively), which would reduce the potential yield loss where post-anthesis coincides with drought.

Conclusions

The results confirm the validity of a novel approach of enhancing waterlogging tolerance of wheat genotypes grown in acid soil via increased tolerance to ion toxicities.     

Assessment of grain yield indices in response to drought stress in wheat (Triticum aestivum L.)

Drought stress constricts crop production in the world. Increasing human population and predicted temperature increase owing to global warming will lead ruthless problems for agricultural production in near future. Hence, use of high yielding genotypes having drought tolerance and scrutinize of drought sensitive local cultivars for making them tolerant may be the proficient approaches to cope its detrimental outcomes. The current study was executed during 20015–2016 and 2016–2017 in field using randomized complete block design under factorial arrangements on 50 wheat genotypes for exploring their sensitivity and tolerance against drought. Some of the attributes of grain yield and drought tolerance indices were recorded. Grain yield showed negative correlations with tolerance index (TOL), drought index (DI) and stress susceptibility index (SSI) while positive correlation with mean productivity (MP) and geometric mean productivity (GMP) under drought condition. These findings depicted that tolerant genotypes could be chosen by high MP and GMP values and low SSI and TOL values. Based on the results, genotypes GA-02, Faisalabad-83, 9444, Sehar-06, Pirsabak-04 and Kohistan-97 were more tolerant and recognized as suitable for both normal and drought conditions. Genotypes of Chenab-00, Kohsar-95, Parwaz-94 and Kohenoor-83 confirmed more sensitive due to high grain yield loss under drought stress.     

The potential of genotypically diverse cultivar mixtures to moderate aphid populations in wheat (Triticum aestivum L.)

Plant species diversity has long been considered a primary driver of arthropod community structure; however, recent ecological research has demonstrated that plant genotypic diversity can also play a major role in influencing the composition of arthropod communities. Genotypic diversity has already been exploited in some agricultural systems to improve disease control and appears to hold promise for managing some insect species as well. To explore the potential for using genotypic diversity within a crop species to help manage insect pests, we used laboratory-based studies to investigate the influence of wheat (Triticum aestivum L.) genotypic diversity on aphid (Rhopalosiphum padi L.) population growth. Increasingly diverse mixtures of wheat genotypes supported lower aphid populations compared with monocultures and were equally productive as single variety plantings. In the absence of aphids, genotypic mixtures were more productive than monocultures. We also analyzed the volatile organic compounds emitted by non-infested genotypic mixtures to provide insight on a possible mechanism influencing aphid populations. Mixtures and monocultures of wheat emitted the same compounds, but mixtures emitted greater amounts of volatile compounds than monocultures. Our results suggest that genotypic mixtures can strongly influence the growth rate and size of aphid populations; therefore, cultivar mixtures appear to hold good potential to be an effective tool for managing insect pests in crop fields.     

Effects of foliar applied benzyladenine on grain yield and grain protein in wheat (Triticum aestivum L.)

The effects of foliar applications of nitrogen and benzyladenine (BA) on grain yield and grain protein of wheat grown under field conditions were studied over 2 years with 5 cultivars at 2 locations. Nitrogen (N) at 20 kg.ha^–1, and BA at 100 or 800 mg.l^–1 were applied alone or combined at pre and post-anthesis; applications of BA at 8 mg.l^–1 were also made on individual ears in order to study the effect on cell number. Weekly determinations of the chlorophyll content of the flag leaf were conducted after anthesis to study leaf senescence. At harvest, yield, yield components and grain protein percentage were determined. N and BA applications delayed chlorophyll loss in the flag leaf, but modified neither yield nor yield components. Foliarly applied BA increased grain protein in four of the five cultivars tested. It is concluded that delay of the senescence induced by BA might allow more energy to be available for N uptake by the crop leading to an increase in grain protein.Research supported by a CAFPTA grant 1656/86 and by CONICET, PID 30017700/85.CONICETComisión de Investigaciones Cientificas de la Provincia de Buenos AiresInstituto de Fisiologia Vegetal     

Identifying loci influencing grain number by microsatellite screening in bread wheat (Triticum aestivum L.)

Grain number (GN) is one of three major yield-related components in wheat. We used the Chinese wheat mini core collection to undertake a genome-wide association analysis of grain number using 531 SSR markers randomly located on all 21 chromosomes. Grain numbers of all accessions were measured in four trials, i.e. two environments in four growing seasons. Association analysis based on a mixed linear model (MLM) revealed that 27 SSR loci were significantly associated with mean GN (MGN) estimated by the best linear unbiased predictor (BLUP) method. These included numerous breeder favorable alleles with strong positive effects at 23 loci. Significant or extremely significant differences were detected on MGN between varieties conveying favored allele and varieties with other alleles. Moreover, statistical simulation showed that the favored alleles have additive genetic effects. Although modern varieties combined larger numbers of favored alleles, the numbers of favored alleles were not significantly different from those in landraces, especially those alleles contributing mostly to the phenotypic variation. These results indicate that there is still considerable genetic potential for use of markers for genome selection of GN for high yield in wheat.     

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.     

Contribution of the wheat (Triticum aestivum L.) flag leaf to grain yield in response to plant growth regulators

In West-Europe, intensive cereal management uses plant growth regulators (PGRs) especially for wheat. A green-house experiment compared the effects of two PGRs on flag leaf characteristics and yield of winter wheat. Chlormequat chloride + choline chloride (CCC) and chlormequat chloride + choline chloride + imazaquin (CCC+I) were applied to winter wheat at growth stage 5 (Feekes Large scale). CCC and CCC+I significantly increased flag leaf surface area at anthesis. Both treatments also enhanced chlorophyll content of the main stem flag leaf. The grain filling period was extended with PGR application by 2 days. CCC and CCC+I significantly increased net CO₂ assimilation rates during the flag leaf life. No effects of PGR spraying were observed on the pattern of ¹⁴C labelled assimilate distribution. Increased grain yield was due to the increase in average grain weight. The results indicate that PGR treatments increased flag leaf contribution to grain filling. The addition of imazaquin (I) to chlormequat (CCC) improved the effects of CCC.     

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.)

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997–2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01–3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype–phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.