Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Experimental analysis of a nitrogen removal process simulation of wastewater land treatment under three different wheat planting densities

Nitrogen contaminant transport, transformation and uptake simulation experiments were conducted in green house under three different planting density of winter wheat. They were Group A, planting density of 0.0208 plants/cm 2, Group B, 0.1042 plants /cm 2, and Group C, 0.1415 plants/cm 2. The capacity and ratio of nitrogen removal were different on three kinds of conditions of wastewater land treatment. From analysis of wastewater treatment capacity, wastewater concentration and irrigation intensity for Group C were suitable and nitrogen quantity added was 2 times of that for Group B, 2.6 times for Group A while nitrogen residue was only 7.06%. Hence, wastewater irrigation and treatment design with purpose of waste water treatment should select the design with maximum capacity, optimal removal ratio and least residue in soil, which was closely related to crop planting density, crop growth status and also background nitrogen quantity in soil.

Effect of Industrial Sludge Application on Soil Nitrogen and Wheat Plant Response

Wazirpur industrial area of Delhi generates a huge quantity of sludge per day, which is highly acidic in nature (pH 2.7 to 4.4) and contains macronutrients, micronutrients as well as toxic metals. A pot-culture experiment was conducted by taking the two soils (JNU and Chhattarpur) amended with sludge (0%, 10%, 20%, 30%), pretreated with lime (0%, 0.5% and 1%). Two wheat seedlings were planted per pot containing 3 kg sludge amended or control soil and the experiment was carried out till harvesting (four months) in a glass house. Lime treatments enhanced the N content in wheat plant in almost all cases. Sludge and lime treatments enhanced dry weight in wheat plants grown in Chhattarpur soil and dry weight increased with time. Maximum growth was observed in 0.5 lime treated and 20% sludge amended soils. But we have to take an account about any kind of metal toxicity before disposal of this waste to land.

WPA1 encodes a vWA domain protein that regulates wheat plant architecture

Plant height,spike,leaf,stem and grain morphologies are key components of plant architecture and related to wheat yield.A wheat(Triticum aestivum L.)mutant,wpa1,displaying temperaturedependent pleiotropic developmental anomalies,was isolated.The WPA1 gene,encoding a von Willebrand factor type A(vWA)domain protein,was located on chromosome arm 7DS and isolated by map-based cloning.The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing.Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants.The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield.

The Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter Wheat

Although plastic-covered ridge and furrow planting（RF） has been reported to produce substantial increases in the grain weight of winter wheat,the underlying mechanism is not yet understood.The present study used two cultivars,Xinong 538 and Zhoumai 18,and RF and traditional flatten planting（TF,control） with the objective of investigating the effect of RF on wheat grain filling and the possible relationship of hormonal changes in the wheat grains under RF to grain filling.The results indicated that RF significantly increased the grain weight,although the effects on grain filling were different： RF significantly increased the grain-filling rate and grain weight of inferior grains,whereas RF had no significant effect on grainfilling rate and grain weight of superior grains.The final grain weight of inferior grains under RF was 39.1 and 50.7 mg for Xinong 538 and Zhoumai 18,respectively,3.6 and 3.4 mg higher than the values under TF.However,the final grain weight of superior grains under RF was only 0.6 and 0.8 mg higher than under TF for Xinong 538 and Zhoumai 18,respectively.RF significantly decreased the ethylene and gibberellic acid content in the inferior grains and increased the indole-3-acetic acid,abscisic acid and zeatin ＋ zeatin riboside content in the inferior grains;however,no significant difference between RF and TF was observed for the hormonal content in the superior grains.Based on these results,we concluded that RF significantly modulated hormonal changes in the inferior grains and,thus,affected the grain filling and grain weight of the inferior grains;in contrast,RF had no significant effect on grain filling,grain weight and hormonal changes in the superior wheat grains.

Transgenic Wheat Plants Obtained With Pollen-Tube Pathway Method

β-glucuronidase (GUS) gene (carried by pBI121 plasmid) has been introduced into hexaploid wheat c. v. Xiaoshan No. 3 with the pollen-tube pathway method. Five transgenic plants are selected from 106 T0 plants. All of these plants are identified by the Southern hybridization, and the GUS activity can be detected by using fluorogenic assay and histochemical stain.

Collision detection of virtual plant based on bounding volume hierarchy: A case study on virtual wheat

Visualization of simulated crop growth and development is of significant interest to crop research and production. This study aims to address the phenomenon of organs cross-drawing by developing a method of collision detection for improving vivid 3D visualizations of virtual wheat crops. First, the triangular data of leaves are generated with the tessellation of non-uniform rational B-splines surfaces. Second, the bounding volumes（BVs） and bounding volume hierarchies（BVHs） of leaves are constructed based on the leaf morphological characteristics and the collision detection of two leaves are performed using the Separating Axis Theorem. Third, the detecting effect of the above method is compared with the methods of traditional BVHs, Axis-Aligned Bounding Box（AABB） tree, and Oriented Bounding Box（OBB） tree. Finally, the BVs of other organs（ear, stem, and leaf sheath） in virtual wheat plant are constructed based on their geometric morphology, and the collision detections are conducted at the organ, individual and population scales. The results indicate that the collision detection method developed in this study can accurately detect collisions between organs, especially at the plant canopy level with high collision frequency. This collision detection-based virtual crop visualization method could reduce the phenomenon of organs cross-drawing effectively and enhance the reality of visualizations.

Effect of Plant Density on Wild Oat Competition with Competitive and Non-Competitive Wheat Cultivars

Wild oat （Avena ludoviciana） is one of the worst weeds in wheat fields. The effect of wheat density on wild oat competition with more or less competitive wheat cultivars （Triticum aestivum） was investigated at the Experimental Farm of Plant Pests and Diseases Research Institute, Karaj, Iran. The experiment was established as a factorial combination of wheat varieties Roshan as non-competitive and Niknejad as competitive; three wheat densities （recommended density, recommended＋25%, and recommended＋50%, corresponding respectively with 300, 375, and 450 plants m-2 for Roshan and 400, 500, and 600 plants m-2 for Niknejad） and four wild oat densities （0, 25, 50, and 75 plants m-2） were selected for this experiment. Hyperbolic equations were used to describe relationship between yield and weed density. Increase in wheat density reduced wild oat biomass. Maximum wild oat biomass was achieved at the highest density of the wild oat together with the lowest wheat density. The results showed that higher densities of wheat are able to suppress wild oat dry matter production. Inter-specific competition in Niknejad was 1.7 times more than that in Roshan. Maximum yields of Niknejad and Roshan in the presence of wild oat were obtained at recommended density＋25% and recommended density, respectively. Increase in wheat density leads to a decrease in wheat yield due to an increase in intra-specific competition. Increase in wild oat density results in the reduction of wheat yield through decrease in fertile tiller per plant and spike m-2.

Pathogenesis-related protein genes involved in race-specific allstage resistance and non-race specific high-temperature adultplant resistance to Puccinia striiformis f. sp. tritici in wheat

Interactions of the stripe rust pathogen （Puccinia striiformis f. sp responses. Among various genes involved in the plant-pathogen related （PR） protein genes determine different defense responses tritici） with wheat plants activate a w^ae range OT nost nteractions, the expressions of particular pathogenesis-Different types of resistance have been recognized and utilized for developing wheat cultivars for resistance to stripe rust. All-stage resistance can be detected in seedling stage and remains at high levels throughout the plant growth stages. This type of resistance is race-specific and not durable. In contrast, plants with only high-temperature adult-plant （HTAP） resistance are susceptible in seedling stage, but become resistant when plants grow older and the weather becomes warmer. HTAP resistance controlled by a single gene is partial, but usually non-race specific and durable. The objective of this study was to analyze the expression of PR protein genes involved in different types of wheat resistance to stripe rust. The expression levels of 8 PR protein genes （PR1, PRI.2, PR2, PR3, PR4, PR5, PR9 and PRIO） were quantitatively evaluated at 0, 1, 2, 7 and 14 days after inoculation in single resistance gene lines of wheat with all-stage resistance genes YrTrl, Yr76, YrSP and YrExp2 and lines carrying HTAP resistance genes Yr52, Yr59, Yr62 and Yr7B. Races PSTv-4 and PSTv-37 for compatible and incompatible interactions were used in evaluation of PR protein gene expression in wheat lines carrying all-stage resistance genes in the seedling- stage experiment while PSTv-37 was used in the HTAP experiment. Analysis of quantitative real-time polymerase chain reaction （qRT-PCR） revealed that all of the PR protein genes were involved in the different types of resistance controlled by different Yr genes. However, these genes were upregulated at different time points and at different levels during the infection process among the wheat lines with different Yr genes for either all-stage resistance or HTAP resistance. Some of the genes were also induced in compatible interactions, but the levels were almost always higher in the incompatible interaction than in the compatible interaction at the same time point for each Yr gene. These results indicate that both salicylic acid and jasmonate signaling pathways are involved in both race-specific all-stage resistance and non-race specific HTAP resistance. Although expressing at different stages of infection and at different levels, these PR protein genes work in concert for contribution to different types of resistance controlled by different Yr genes.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins （AGPs） and hydrogen peroxide （H202） have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 （KN 199） and Xinchun 9 （XC9）, together with low-frequency regeneration wheat line Chinese Spring （CS）, presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L-1 AGP or 0.005 to 0.01‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L-1 AGP application level in callus induction medium plant regeneration rates of 8.49,409.06 and 283.16% were achieved for Jimai 22 （JM22）, Jingdong 18 （JD18） and Yangmai 18 （YM18）, respectively; whereas at 100 mg L-1 AGP level, CS （105.44%）, Chuannong 16 （CN16） （80.60%） and Ningchun 4 （NC4） （62.87%） acted the best. Moreover CS （79.05%）, JM22 （7.55%）, CN16 （101.87%）, YM18 （365.56%）, Yangmai 20 （YM20） （10.48%）, and CB301 （187.40%） were more responsive to 0.005 %o of H2O2, and NC4 （35.37%） obtained the highest shoot regeneration rates at 0.01%o of H2O2. Overall, these two methods, inter-culture and AGP （or H2O2） application, can be further applied to wheat transgenic research.

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.

Study of the Adult-Plant Resistance at Different Growth Stages to Stripe Rust in Wheat

Components of resistance were investigated for five adult-plant resistant (APR) wheat (Triticum aestivum) cultivars at sixgrowth stages and two temperatures in the greenhouse, and disease progress in a field trial. Chinese cultivar Chuanyu 12displayed high to intermediate infection type (IT) in the greenhouse but was highly resistant in the field. Weebill showedan intermediate IT in the greenhouse and also in the field. Chpaio, Tukuru and Saar, known to carry combinations of Yr18and 2-3 additional minor genes, were highly resistant in both experiments. Greenhouse experiments indicated that thelower IT of APR cultivars initiated at tillering stage. Latent periods (LP) for APR cultivars were generally longer as thegrowth stage progressed. We conclude that APR to stripe rust can be best characterized in field trials although significantcorrelations are seen between field severity and IT and LP measured in the greenhouse.

The Selection of Transgenic Recipients from New Elite Wheat Cultivars and Study on Its Plant Regeneration System

In the protocol of wheat transformation, to use elite wheat cultivars as exogenous gene recipients can speed up the process of commercial field applications of transgenic wheat. However, it is necessary to screen wheat cultivars with good tissue culture response （TCR） continuously from plenty of elite wheat cultivars released for wheat transformation, and it is also important to find a plant regeneration system that is suitable for these cultivars. So, the TCR of mature and immature embryos of six wheat cultivars Chuannong 11 （CN11）, Chuannong12 （CN12）, Chuannong17 （CN17）, Chuannong18 （CN18）, Chuannong19 （CN19）, and Chuannong21 （CN21）, which possess superior agronomic traits, were investigated by using a good TCR wheat cultivar Bobwhite as control. The results indicated that only the immature and mature embryos of CN12, CN17, and CN18 exhibited good TCR compared with Bobwhite. No significant differences were observed between embryos of Bobwhite and of the three cultivars in TCR. Mature embryo-derived calli of CN12 were used as explants for transformation by particle bombardment of SAMDC gene. Seven transformants were obtained and the efficiency was 2.3%. This research supplies three new elite recipient cultivars for wheat transformation. The wheat plant regeneration system used in this research is different from those successful ones reported previously and it could be a reference for other wheat genotypes. Furthermore, Bobwhite and the three wheat cultivars were proved to be 1RS/1BL translocation, by methods of A-PAGE, C- banding, and genomic in situ hybridization （GISH）. These results imply that probably there is some relationship between 1RS/1BL translocation and TCR of wheat embryos. So this research gives us a hint that we should pay more attention to the 1RS/1BL translocations when we screen the wheat cultivars with good TCR and also that the mechanism of the effect of 1RS/ 1BL translocation on TCR is worthy of being investigated.

Planting Date, Seeding Rate, and Cultivar Impact Agronomic Traits and Semolina of Durum Wheat

Durum wheat (Triticum durum Desf.) is a market class of wheat subject to price discounts in the marketplace if quality standards are not met. This study was conducted in order to determine how certain agronomic practices might impact durum wheat quality. The effects of planting date (PD), cultivar, and seeding rate on agronomic and semolina quality traits were investigated in field trials conducted near Hettinger and Minot, ND in 2014 and 2015. The interaction of PD and cultivar was significant for many of the traits evaluated. There was a significant PD X cultivar interaction or PD and cultivar effect for yield in all environments. Planting date X cultivar interacted for test weight at all environments. In general, a delay in PD resulted in a significant decrease in yield and test weight for all cultivars. However, Carpio yielded more than other cultivars in high yielding environments while the yield and test weight of Joppa was more adversely affected by delays in PD. Seeding rate did not have a consistent effect on any agronomic or quality trait. Protein content, kernel yellow pigment content, falling number (FN), and vitreous kernels were more dependent on cultivar, regardless of PD and environment. Semolina extraction, gluten index (GI), and wet gluten (WG) values tended to decrease with a delay in PD. These data continue to support cultivar selection as a critical component for obtaining high-yielding, high-quality durum wheat. However, PD and environment can impact certain agronomic and end-use traits, regardless of cultivar grown.

A New Method to Determine Central Wavelength and Optimal Bandwidth for Predicting Plant Nitrogen Uptake in Winter Wheat

Plant nitrogen （N） uptake is a good indicator of crop N status. In this study, a new method was designed to determine the central wavelength, optimal bandwidth and vegetation indices for predicting plant N uptake （g N m-2） in winter wheat （Triticum aestivum L.）. The data were collected from the ground-based hyperspectral reflectance measurements in eight field experiments on winter wheat of different years, eco-sites, varieties, N rates, sowing dates, and densities. The plant N uptake index （PNUI） based on NDVI of 807 nm combined with 736 nm was selected as the optimal vegetation index, and a linear model was developed with R2 of 0.870 and RMSE of 1.546 g N m-2 for calibration, and R2 of 0.834, RMSE of 1.316 g N m-2, slope of 0.934, and intercept of 0.001 for validation. Then, the effect of the bandwidth of central wavelengths on model performance was determined based on the interaction between central wavelength and bandwidth expansion. The results indicated that the optimal bandwidth varies with the changes of the central wavelength and with the interaction between the two bands in one vegetation index. These findings are important for prediction and diagnosis of plant N uptake more precise and accurate in crop management.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.