Silage maize variety testing systems usually evaluate new varieties within properly defined maturity groups; within a maturity group, all varieties are harvested on a single harvest date. In a small country like Belgium where growing conditions are similar across the maize growing areas, all tested silage maize varieties, comprising different maturity groups, are harvested on a single date. Under this testing system, only few varieties are harvested at the physiological stage where they theoretically show their optimal performance. In the current research, eight silage maize varieties were monitored from 25 to 40% whole-crop dry matter (DM) content at three locations in Belgium during the period 2007–2009. The optimal harvest date was calculated as the date where whole-crop DM yield, ear DM yield and organic matter digestibility were maximal. The variety rank at the optimal harvest date was compared with the variety rank at any studied single harvest date. Harvest dates where the variety rank was not statistically different to the rank at the optimal harvest date were pooled in a ‘harvest window’. Based on the current data, the harvest window comprised a flexible harvest period of about 14 days. It was therefore concluded that applying a single harvest date is scientifically justified for the ranking of silage maize varieties in Belgium, when the whole-crop DM content is between 28·1 and 40·6% with a maximum difference of 7·2% between all compared varieties.

In agricultural production systems, nitrogen (N) losses to the environment can occur through nitrous oxide (N2O) emissions and nitrate (NO3−) leaching. The objectives of the present study were to evaluate: (1) if urine excreted by non-lactating dairy cows pulse-dosed with dicyandiamide (DCD) and applied to lysimeters reduced N2O-N emissions and NO3−-N leaching on two soil types; and (2) if urine + DCD would increase herbage production over winter. Lysimeters were used to measure N2O emissions and NO3-N leaching. The soils used were a free-draining acid brown earth of sandy loam to loam texture (termed free-draining) and a poorly drained silt loam gley (termed poorly drained). Grass plots were established on the free-draining soil to measure herbage production. The N loading rate of the urine + DCD was 508 kg N/ha and the urine without DCD (urine only) was 451 kg N/ha. Total NO3−-N leaching losses from the free-draining and poorly draining soils were reduced from 100 and 81 kg NO3−-N/ha on the urine-only treatment, respectively, to 9 and 11·6 kg NO3−-N/ha on the urine + DCD treatment, respectively. Total N2O-N emissions from the free-draining and poorly drained soils were reduced significantly from 13·6 and 12·1 kg N2O-N/ha on the urine-only treatment, respectively, to 2·23 and 5·24 kg N2O-N/ha on the urine + DCD treatment, respectively. Applying urine with DCD to pastures inhibited the nitrification process for up to 56 days after treatment application. In the current experiment, there was no significant effect on spring herbage production when urine + DCD was applied to grass plots. Therefore, feeding DCD to dairy cows to apply DCD directly in urine patches was shown to be an effective mitigation strategy to reduce NO3−-N leaching and N2O-N emissions but did not appear to increase spring herbage production.

The spatial distribution of soil organic carbon (SOC) can be affected by environmental factors such as land use change, type of vegetation, soil redistribution processes and soil management practices. Because data are scarce in mountain agroecosystems, improving knowledge on the relationships between land use, soil redistribution processes and SOC fractions is of interest, especially in rapidly changing Mediterranean landscapes. Typically, SOC is divided into two distinct carbon fractions: the active and decomposable fraction (ACF) with rapid turnover rates, which acts as a short-term carbon reservoir, and the stable carbon fraction (SCF) with lower turnover rates that acts as a long-term reservoir. In the present study SOC, ACF and SCF contents were measured by the dry combustion method and converted to inventories expressed as mass per unit surface area (kg/m2). The SOC distribution patterns were related to land use and soil redistribution processes in soil profiles along a representative mountain agroecosystem toposequence in northeast Spain. The soil depth profiles were identified as stable, eroded and depositional sites using fallout 137caesium (Cs). Significantly higher amounts of SOC were found in forest soils (36 ± 20·2 g/kg) compared to abandoned (21 ± 14·3 g/kg) and cultivated arable land (11 ± 6·3 g/kg), suggesting that cultivation decreases SOC content. In addition, stable soil profiles had significantly higher SOC content (42 ± 24·3 g/kg) than at depositional and eroded profiles (18 ± 14·5 and 17 ± 13·1 g/kg, respectively). A positive and statistically significant relationship between SOC and 137Cs inventories suggested that both are moved and associated with similar soil redistribution processes.

To cope with phosphorus (P) deficiency, plants adapt root morphology to enhance inorganic P (Pi) acquisition from soil by allocating more biomass to roots, but whether the responses can be modified across gradients of P supply is not fully understood. The present study examined changes in root-length density (RLD), root-hair density (RHD) and root-hair length (RHL) of wheat (Triticum aestivum L.) in two contrasting soils, the Rough and Barnfield soils. Wheat plants were grown for 3 weeks in thin-plate rhizotrons in two soils with additions of 0, 10, 25, 50, 100 and 200 mg P/kg soil. Contrary to published literature, as P additions increased it was observed that a concomitant increase in RHL (250 to 1054 µm in the Rough soil and 303–1075 µm in the Barnfield soil) and RHD (57 to 122/mm in the Rough soil and 56–120/mm in the Barnfield soil), while RLD generally decreased (2480–1130 cm/cm3 in the Rough soil and 1716–865 cm/cm3 in the Barnfield soil). The levels of added P that resulted in critical P concentrations in the soils enabling maximum shoot biomass production were 50 mg/kg P in the Rough soil and 100 mg/kg P in the Barnfield soil, and these additions influenced root morphological changes. Under severe P deficiency, P supply increased RHL and RHD, but RLD was decreased. Improvement in lateral root and root-hair responses in wheat at extreme P deficiency may be a worthy target for breeding more sustainable genotypes for future agroecosystems.

Eight winter oilseed rape and two spring oilseed rape field experiments were performed in the UK in harvest years 2009–12. Each experiment consisted of at least one hybrid and one open-pollinated variety grown at five seed rates from 10 or 20 seeds/m2 to 160 or 200 seeds/m2. Linear plus exponential curves were used to describe the yield response to seed rate and to calculate economically optimal seed rates. Plant counts were then used to derive optimal plant population densities. These ranged from <10 to 39 plants/m2 for six winter oilseed rape experiments between 73 and >155 plants/m2 in two winter oilseed rape experiments with severe spring droughts, and from 47 to 65 plants/m2 for spring oilseed rape. Optimal plant population densities were lower for hybrid than for open-pollinated varieties, due to a combination of the higher cost of hybrid seed and, for some experimental sites, hybrid varieties compensating better for low plant populations. Across all sites, sowing winter oilseed rape at 30 seeds/m2 rather than common commercial rates of 70 seeds/m2 for hybrids and 100 seeds/m2 for open-pollinated varieties would have increased average gross margin by £29/ha. Sowing spring oilseed rape at 70 seeds/m2 rather than commonly used rates of 120 or 150 seeds/m2 would have increased average gross margin by £64/ha.

Effects of soil tillage systems and nitrogen (N) fertilizer management on spring wheat yield components, grain yield and N-use efficiency (NUE) were evaluated in contrasting weather of 2013 and 2014 on a clay soil at the Royal Agricultural University's Harnhill Manor Farm, Cirencester, UK. Three tillage systems – conventional plough tillage (CT), high intensity non-inversion tillage (HINiT) and low intensity non-inversion tillage (LINiT) for seedbed preparation – were compared at four rates of N fertilizer (0, 70, 140 and 210 kg N/ha). Responses to the effects of the management practices were strongly influenced by weather conditions and varied across seasons. Grain yields were similar between LINiT and CT in 2013, while CT produced higher yields in 2014. Nitrogen fertilization effects also varied across the years with no significant effects observed on grain yield in 2013, while in 2014 applications up to 140 kg N/ha increased yield. Grain protein ranged from 10·1 to 14·5% and increased with N rate in both years. Nitrogen-use efficiency ranged from 12·6 to 49·1 kg grain per kg N fertilizer and decreased as N fertilization rate increased in both years. There was no tillage effect on NUE in 2013, while in 2014 NUE under CT was similar to LINiT and higher than HINiT. The effect of tillage and N fertilization on soil moisture and soil mineral N (SMN) fluctuated across years. In 2013, LINiT showed significantly higher soil moisture than CT, while soil moisture did not differ between tillage systems in 2014. Conventional tillage had significantly higher SMN at harvest time in 2014, while no significant differences on SMN were observed between tillage systems in 2013. These results indicate that LINiT can be used to produce similar spring wheat yield to CT on this particular soil type, if a dry cropping season is expected. Crop response to N fertilization is limited when soil residual N is higher, while in conditions of lower residual SMN, a higher N supply is needed to increase yield and improve grain protein content.

Expressed sequence tag-simple sequence repeat (EST-SSR) markers were used to analyse genetic diversity among three Lens species. The SSR loci amplified successfully in wild species, with 94·82% transferability in Lens culinaris subsp. orientalis, 95·4% in Lens nigricans, 98·81% in L. culinaris subsp. odemensis, 94·82% in L. culinaris subsp. tomentosus and 96·55% in Lens ervoides. Ninety-nine alleles (average 3·41 alleles/locus) were detected by 29 SSR markers. Based on the unweighted pair group method with arithmetic mean cluster analysis, all the genotypes were grouped into three clusters at a similarity level of 0·30. The diversity analysis indicated no species-specific clustering of the wild and cultivated species. Wild species L. nigricans and L. culinaris subsp. odemensis, L. culinaris subsp. orientalis and L. ervoides were grouped in Cluster I, whereas the Mediterranean land races of L. culinaris subsp. culinaris and L. culinaris subsp. tomentosus formed a separate group in Cluster II A. Cluster II B comprised L. ervoides, L. culinaris subsp. orientalis and L. culinaris subsp. culinaris. Clusters II C, II D and II F included cultivated Indian lentil genotypes. Cluster II E comprised Indian and Mediterranean germplasm lines. Cluster II F included three early maturing germplasm lines, whereas Cluster III included only two germplasm lines. The functional annotation of SSR-containing unigenes revealed that a majority of genes were involved in an important transport-related function or were a component of metabolic pathways. A high level of polymorphism of EST-SSRs and their transferability to related wild species indicated that these markers could be used for molecular screening, map construction, comparative genomic studies and marker-assisted selection.

A field study was carried out to examine the response of microbial communities of a clay loam soil to long-term (30 years) effects of residue return and fertilization. The experimental design was a split-plot arrangement of treatments, consisting of three residue treatments (crop residues returned at rates of 0, 2500 and 5000 kg/ha) in combination with eight fertilization treatments (control, no fertilizer; N, mineral nitrogen (N) fertilizer; P, mineral phosphorus (P) fertilizer; K, mineral potassium (K) fertilizer; NP, mineral NP fertilizer; NK, mineral NK fertilizer; PK, mineral PK fertilizer; and NPK, mineral NPK fertilizer). Soil microbial communities were characterized by phospholipid fatty acid analysis. Results indicated that the more crop residues were returned, the lower ratio of fungi to bacteria was observed. However, soil microbial biomass was only found to be significantly higher in plots with residues returned at a rate of 5000 kg/ha but not 2500 kg/ha. This suggested there was a threshold for microbial biomass to increase under residue return for the clay loam soil studied. The fertilization effect on soil microbial biomass gradually decreased with increases in the amount of crop residues returned. A significant composition change was observed under N fertilization. Structural equation modelling indicated that soil microbial communities were influenced directly by residue return and indirectly by residue-induced change in ratio of carbon to N and fertilization-induced change in soil pH.

Trifoliate orange (Poncirus trifoliata (L.) Raf.) is a very useful taxon for the citrus industry since this rootstock is immune to the Citrus Tristeza virus and confers cold tolerance. Numerous trifoliate orange varieties exist but little is known regarding their behavioural variability when subjected to abiotic constraints. The diversity of 74 P. trifoliata accessions maintained in the INRA-CIRAD Citrus Germplasm Collection was investigated using simple sequence repeat markers. Two major genetic groups were clearly identified as a few homonyms, intergroup or intra-group hybrids and doubled-chromosome tetraploid forms. The Group 1 phenotype was characterized by larger flowers and leaves and smaller seeds than Group 2. Tetraploid accessions showed larger leaves and heavier seeds than all other diploid accessions, regardless of genetic classification. Eight genotypes belonging to both genetic groups, as well as two hybrids between the two groups, were selected to investigate their water deficit tolerance. Stress was applied by withdrawing irrigation for 4 weeks. Physiological parameters such as leaf stomatal conductance, quantum yield of photosystem II electron transport, soil water potential, leaf osmotic potential and transpiration rate were estimated. Some varieties, such as Rubidoux 0101033, were clearly more tolerant to water deficit than others, such as Pomeroy 0101040 and Pomeroy 0110081. Interestingly, accessions that had the highest soil water potential and were the least affected by stress belonged to genetic Group 2. Conversely, trifoliate oranges of genetic Group 1 were the least tolerant.

Nine wheat genotypes, bred for the high-input agronomical conditions of Henan Province (China), were tested under the high-yielding Mediterranean conditions of Spain. Two cultivars widely grown in the zone were included as controls. Crop growth and leaf chlorophyll (Chl) content, leaf stomatal conductance (gs) and canopy temperature (CT) were measured during the crop cycle and stable carbon (C), oxygen (O) and nitrogen (N) isotope compositions (δ13C, δ18O and δ15N) were analysed on different plant parts. The lower yield of the Chinese genotypes compared with the controls was due to fewer grains/unit area, associated with lower tillering and a plant height clearly below the optimal range. Moreover, Chinese wheat exhibited a lower spike fertility index than the controls, and this was associated with a less compact spike structure. The physiological characteristics that were related to better performance under high-yielding Mediterranean conditions consisted of a higher green aerial biomass, particularly during the reproductive stage, together with more favourable water conditions (higher gs and lower CT and δ13C), the capacity to take up water during grain fill (higher δ18O) and a more efficient uptake (lower δ15N) and utilization (lower leaf N and Chl content) of N fertilizer. It is concluded that Chinese genotypes exhibited a low acclimation capacity to the moderate stress typical of the high-yielding Mediterranean conditions.

To support tropical maize (Zea mays L.) breeding efforts, the current work aimed to assess harvest index (HI) in modern hybrids and determine the effect of different planting densities on grain yield and HI under well-fertilized (HN) and nitrogen (N) deficient conditions. Harvest index and grain yield of 34 hybrids on average reached 0·42 and 7·06 t/ha (five environments), indicating a large potential for improvement in HI relative to temperate hybrids. Ear weight (r = 0·88), HI (r = 0·78) and shoot dry weight (r = 0·68) were strongly associated with grain yield. In the second experiment, seven hybrids were evaluated at planting densities of 5, 7, 9 and 11 plants/m2 under HN (six environments) and N deficient (LN) conditions (four environments) to assess the effect of planting density on grain yield and HI. Grain yield increased by 40·4 and 21·8% under HN and LN conditions when planting density was increased relative to the lowest planting density. Harvest index increased from 0·42 at 5 plants/m2 to 0·45 at 11 plants/m2 under HN conditions and decreased from 0·44 at 5 plants/m2 to 0·42 at 9 plants/m2 under LN conditions. Harvest index was maximized at planting densities of 8·33 plants/m2 and 5·30 plants/m2 under HN and LN conditions, respectively, while grain yield was maximized at 9·93 plants/m2 and 7·89/m2. Optimal planting density maximizing both HI and grain yield were higher than planting densities currently used in tropical germplasm. It can be concluded that productivity in tropical maize could be increased both under intensive (+40·4%) and lower-input management (+21·8%) by increasing planting densities above those currently used in smallholder agriculture in Latin America and Sub-Saharan Africa, in environments targeted by the International Maize and Wheat Improvement Center.

Following the surface application of granulated urea to grassland, high ammonia (NH3) losses of up to 30% have been reported. The addition of a urease inhibitor (UI) to urea granules could be a way to abate these losses. Field experiments were conducted at two intensive grassland sites in 2007 and 2008 to evaluate the potential of the new UI N-(2-nitrophenyl) phosphoric triamide (2-NPT; concentrations of 0·75, 1·0 and 1·5 g N/kg) to reduce NH3 emissions resulting from the application of granulated urea. Ammonia losses were continuously measured on plots fertilized with urea, urea + 2-NPT, calcium ammonium nitrate and a control (0N). The measurements were made with a dynamic chamber system. All measurement periods were started after a period of precipitation with a following rainless period being forecasted. Results over measurement periods of 10 days following fertilization are presented. Ammonia losses following the application of granulated urea varied between 4·6 and 11·8 kg N/ha, corresponding to 4·2 up to 14·0% of the applied nitrogen. The addition of 2-NPT to urea granules at three concentrations significantly reduced NH3 losses by 69–100%. Comparable losses of NH3 were observed for urea containing the UI 2-NPT as well as calcium ammonium nitrate, and were not significantly different from the control treatment. No relationships between losses, meteorological factors and soil moisture were observed. The addition of the UI 2-NPT to urea granules applied on grassland effectively reduced NH3 losses.

The direct response of a divergent selection programme for total cell wall ester-linked diferulate concentration in maize pith stalk tissues and its indirect effect on cell wall degradability and corn borer resistance have been previously evaluated. Since increased total diferulate concentration is expected to improve crop performance in response to corn borers, the objective of the present research was to evaluate the indirect response of the divergent selection for diferulates on agronomic traits under corn borer infestation. For this purpose, five maize populations with contrasting total diferulate concentrations were evaluated four environments for performance under protected and infested conditions. Measured traits were: days to anthesis, days to silking, plant height, stalk lodging, grain moisture at harvest and grain yield. High diferulate populations showed a significant reduction in anthesis (precocity), and were 11 cm taller than the starting population, while low diferulate populations were 9 cm shorter, and showed nearly 1 t/ha lower grain yield than the original and high diferulate populations. The analysis showed that cycles of selection were positively correlated with flowering, plant height and grain yield. The infestations with borers produced >1 t/ha of reduction in grain yield; although the higher diferulate populations showed a better performance under infestation than the low diferulate populations. This positive effect on the grain yield by increasing diferulate content can be considered an extra in order to breed for resistance to corn borers.

The present study explored the optimal long-term seed renewal strategy for malting barley for use either of farm-saved seed (FSS) or commercially certified seed (CCS). The dynamic optimization problem was solved by a dynamic programming routine with known parameters for one-period returns and transition equations. The model parameters represented Finnish farm-level returns and costs, as well as genetic improvements in barley yield potential. The results suggested that there is a farm-level economic incentive not to use CCS every year, despite the higher yield potential of CCS compared with FSS. The simulations highlighted the reasons behind the observed yield gap between genetic improvements in yield potential and attained yields at the farm scale.

High temperature stress at critical growth stages is a major risk factor for wheat in many wheat growing areas globally. Developing weather indices relating to yield reductions in wheat is an urgent requirement for weather-index-based crop insurance. The objectives of the present study were to: (i) identify critical phenological stage(s) for heat stress, (ii) quantify the impact of heat stress at critical growth stage(s) and (iii) work out thresholds of temperature for obtaining above average, average and below average yield of wheat. For achieving these objectives, 11 years’ experimental data for three cultivars (HD-2285, K-8804 and K-9107) under three sowing dates at the Kanpur Centre located in the Indo-Gangetic Plains of Uttar Pradesh, India were used. Among the eight phenological stages, the milk stage (growth stage 73) was identified as most sensitive for high maximum and minimum temperatures to adversely affect yield. The rate of yield reduction with unit increase in maximum and minimum temperatures (°C) was found to be highest in K-8804 and lowest in HD-2285. The optimum ranges of maximum temperature during anthesis, milk, dough and maturity stages are 19·7–21·9, 24·2–26·5, 26·1–28·8 and 29·5–30·8 °C, respectively and those for minimum temperature are 4·3–6·2, 8·3–9·7, 11·5–12·4 and 13·0–15·1 °C, respectively. The thresholds of temperature during critical stages and quantification of heat stress on yield will be of use in devising weather-index-based crop insurance products in wheat and also for breeding temperature-stress-resistant genotypes. This method of devising weather indices in the present study can be used in other crops and regions of the world as an adaptation strategy for climate change.

Relationships between milk fatty acids (MFA) and methane (CH4) emissions from dairy cattle were explored. Data from a total of 12 studies including 39 treatments were gathered in the database. Methane was expressed as daily emissions (g/d), relative to dry matter intake (g/kg), milk production (g/kg milk) and body weight (g/kg). The univariate correlations between MFA and CH4 were based on absolute means and on relative changes of each treatment compared with its corresponding control. Saturated fatty acids, odd- and branched-chain FA and long-chain poly-unsaturated FA were positively related to CH4, while cis- and trans-isomers of mono-unsaturated FA were negatively related to CH4. However, most of the coefficients of determination (R2) of these univariate regressions ranged from 0·2 to 0·7, indicating that individual MFA only explain a limited part of the variation in CH4. Significant relationships between MFA and CH4 varied depending on the unit in which emissions were expressed. Similarly, some MFA seemed more suited to predict relative changes in CH4 emissions rather than absolute amounts. The present findings contribute to the exploration of the potential of MFA as biomarkers for CH4 emissions from dairy cattle, although differences between studies in the detail of MFA analysis and hence the number of MFA reported in the current study, complicates this kind of literature survey.

The herbicide chlorsulfuron is commonly applied to cereals and may persist in alkaline soil long enough to damage subsequent sensitive crops. Sewage water, a potential source of the heavy metal cadmium (Cd), is used to irrigate agricultural soils in many developing countries. The current work evaluated the effects of the residual herbicide chlorsulfuron and Cd on maize seedlings, with particular attention to the mechanism of their action on plant growth. Maize seeds were planted in soil that had been sprayed with chlorsulfuron and Cd, after which residues in both seedlings and the soil were measured. The chlorsulfuron dose was correlated with the amount of residue found in seedlings but not in the soil. In all, 39 metabolites were identified in seedlings using the Automated Mass spectral Deconvolution and Identification System software program and the retention index method. The combination of chlorsulfuron and Cd significantly reduced multiple metabolites in the shikimate pathway, malic acid and citric acid production in the tricarboxylic acid cycle, and lactic acid, glucose, aspartic acid, asparagine and 3-glycerophosphoric acid production. In addition, chlorsulfuron caused an increase in multiple amino acids, including tyrosine, methionine and asparagine, and a marked decrease in caffeic and cinnamic acids (the secondary metabolites derived from the shikimate pathway and galactose). Finally, chlorsulfuron and Cd stress markedly increased shikimate acid, decreased 3-glycerophosphoric acid and caused negative correlations between the amounts of phenylalanine and tyrosine and those of quinic- and cinnamic-acid. In conclusion, chlorsulfuron and Cd did not have a synergistic effect on maize seedlings; rather, the combination of these pollutants had an antagonistic effect on some amino acids.

Identifying and applying the optimum fertilizer nitrogen (N) rate is a permanent challenge for farmers. Prediction of fertilizer N requirement, based on crop chlorophyll measurements (CMs), relies on a strong relationship between fertilizer N supply and leaf chlorophyll concentration at a given crop growth stage. A methodological approach is described, aiming to develop an algorithm that uses CM inputs to derive the economically optimum fertilizer N rate for top-dressing, without using a reference plot for data normalization. The method was tested on maize (Zea mays L. cvar Jabali) at experimental and farmer sites in the central (‘Bajío’) region of Mexico over 3 years (2010–12). Increasing fertilizer N supply at planting significantly influenced chlorophyll concentration at the seventh unfolded maize leaf stage (GS 17 on the Zadoks scale). Maize grain yields increased with increasing total fertilizer N supply and fitted quadratic models, which allowed economically optimum fertilizer N rates (Nopt) to be calculated. The Nopt ranged from 160 to 300 kg N/ha and corresponding grain yields ranged from 7·7 to 14 t/ha. Grouped data analysis (sites–years) confirmed a highly significant relationship between the Nopt and the chlorophyll concentration at GS 17, which could be described by a linear model: Nopt = 513·3–0·58 × CM. This model predicted the top-dressing Nopt within a fertilizer N management regime adapted to local maize cropping systems and led to similar grain yields across test sites compared with the same parameters calculated based on grain yield response trials. The current approach is variety-specific, so development of so-called correction factors accounting for variety-related differences in chlorophyll concentration is described. The results demonstrated the feasibility of the proposed algorithms to support decision-making on the optimum fertilizer N rate to apply in maize production systems with one top-dressing application.

Genetically modified (GM) plants expressing Bt toxin provide protection against lepidopteran pests. The only GM crop in Pakistan is Bt cotton, which was illegally imported and adopted rapidly by cotton producers. Farmers gained access to the seed of many unapproved Bt genotypes before the matter was picked up and formal approval granted by the relevant governmental agencies. The present study was conducted to evaluate the samples of Bt cotton, collected from farmers and seed dealer, for transgene integration and expression. Seeds of 52 cotton genotypes, labelled as Bt, were collected from various farmers and seed dealers. An immunoblot strip test was carried out, which showed that only 0·86 of the samples collected were synthesizing Cry1Ac toxin. According to multiplexed polymerase chain reaction (PCR) results, 0·86 of the genotypes tested were positive for the Mon531 event (an ‘event’ is a specific genetic modification in a specific species) and 0·14 were negative for any transgene. Transcript analysis of transgenes in positive genotypes by real-time Rt-PCR confirmed the synthesis of mRNA in all genotypes but with significant variation. The concentration of Bt toxin revealed by enzyme linked immunosorbent assay (ELISA) showed that only 0·02 genotypes had the reported optimum level. The real-time PCR and ELISA results further confirmed the attenuation of transgene expression at transcriptional and translational level by various internal and external factors. The same type of event was found in all genotypes, with significant variation in toxin level, revealing the impact of genetic background on transgene expression. The findings support the recommendation to improve the existing quality criteria for transgenic cotton variety approval and certification in Pakistan, with the inclusion of toxin concentration in the list of parameters to be considered.