Co‐expression of GR79 EPSPS and GAT yields herbicide‐resistant cotton with low glyphosate residues

Glyphosate‐resistant (GR) crops have been adopted on a massive scale by North and South American farmers. Currently, about 80% of the 120 million hectares of the global genetically modified (GM) crops are GR crop varieties. However, the adoption of GR plants in China has not occurred at the same pace, owing to several factors including, among other things, labour markets and the residual effects of glyphosate in transgenic plants. Here, we report the co‐expression of codon‐optimized forms of GR79 EPSPS and N‐acetyltransferase (GAT) genes in cotton. We found five times more resistance to glyphosate with 10‐fold reduction in glyphosate residues in two pGR79 EPSPS‐pGAT co‐expression cotton lines, GGCO2 and GGCO5. The GGCO2 line was used in a hybridization programme to develop new GR cottons. Field trials at five locations during three growing seasons showed that pGR79‐pGAT transgenic cotton lines have the same agronomic performance as conventional varieties, but were USD 390‐495 cheaper to produce per hectare because of the high cost of conventional weed management practices. Our strategy to pyramid these genes clearly worked and thus offers attractive promise for the engineering and breeding of highly resistant low‐glyphosate‐residue cotton varieties.     

Growth and Reproduction of Glyphosate-Resistant and Susceptible Populations of Kochia scoparia

Evolution of glyphosate-resistant kochia is a threat to no-till wheat-fallow and glyphosate-resistant (GR) cropping systems of the US Great Plains. The EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene amplification confers glyphosate resistance in the tested Kochia scoparia (L.) Schrad populations from Montana. Experiments were conducted in spring to fall 2014 (run 1) and summer 2014 to spring 2015 (run 2) to investigate the growth and reproductive traits of the GR vs. glyphosate-susceptible (SUS) populations of K. scoparia and to determine the relationship of EPSPS gene amplification with the level of glyphosate resistance. GR K. scoparia inbred lines (CHES01 and JOP01) exhibited 2 to 14 relative copies of the EPSPS gene compared with the SUS inbred line with only one copy. In the absence of glyphosate, no differences in growth and reproductive parameters were evident between the tested GR and SUS inbred lines, across an intraspecific competition gradient (1 to 170 plants m^-2). GR K. scoparia plants with 2 to 4 copies of the EPSPS gene survived the field-use rate (870 g ha^-1) of glyphosate, but failed to survive the 4,350 g ha^-1 rate of glyphosate (five-times the field-use rate). In contrast, GR plants with 5 to 14 EPSPS gene copies survived the 4,350 g ha^-1 of glyphosate. The results from this research indicate that GR K. scoparia with 5 or more EPSPS gene copies will most likely persist in field populations, irrespective of glyphosate selection pressure.     

Efficient particle bombardment-mediated transformation of Cuban soybean (INCASoy-36) using glyphosate as a selective agent

Soybean is highly affected by weeds in tropical countries, causing significant losses in yields. Transgenic herbicide resistant soybeans have been produced in a limited number of varieties and parental lines. This study was conducted to obtain glyphosate herbicide resistant transgenic soybean plants through particle bombardment of embryonic axes in a Cuban variety. Shoot regeneration in 25 mg/L of glyphosate occurred within a short period and plantlets developed roots in a medium without selection pressure, which favored the in vitro growth of plants at a transformation frequency of 3.1–6.0?%. Expression and integration of the cp4epsps gene was confirmed in the progeny by an immune-detection assay, PCR and Southern blot. All greenhouse evaluated transgenic soybean lines (T₁) displayed tolerance to 1.25 Kg/ha of glyphosate. Growth and seed development of transformed plants was similar to untransformed plants. The regeneration procedure using embryonic axes combined with the efficient selection of shoots in glyphosate enabled the production of transgenic plants of this Cuban genotype, showing high tolerance to the herbicide, good efficiency and reproducibility.     

Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri

The inheritance of glyphosate resistance in two Amaranthus palmeri populations (R1 and R2) was examined in reciprocal crosses (RC) and second reciprocal crosses (2RC) between glyphosate-resistant (R) and -susceptible (S) parents of this dioecious species. R populations and Female-R × Male-S crosses contain higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy numbers than the S population. EPSPS expression, EPSPS enzyme activity, EPSPS protein quantity, and level of resistance to glyphosate correlated positively with genomic EPSPS relative copy number. Transfer of resistance was more influenced by the female than the male parent in spite of the fact that the multiple copies of EPSPS are amplified in the nuclear genome. This led us to hypothesize that this perplexing pattern of inheritance may result from apomictic seed production in A. palmeri. We confirmed that reproductively isolated R and S female plants produced seeds, indicating that A. palmeri can produce seeds both sexually and apomictically (facultative apomixis). This apomictic trait accounts for the low copy number inheritance in the Female-S × Male-R offsprings. Apomixis may also enhance the stability of the glyphosate resistance trait in the R populations in the absence of reproductive partners.     

Biochemical responses of glyphosate resistant and susceptible soybean plants exposed to glyphosate

Glyphosate is a wide spectrum, non-selective, post-emergence herbicide. It acts on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products, leading to plant death. Transgenic glyphosate-resistant (GR) soybean [Glycine max (L.)] expressing an glyphosate-insensitive EPSPS enzyme has provided new opportunities for weed control in soybean production. The effect of glyphosate application on chlorophyll level, lipid peroxidation, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GOPX) and superoxide dismutase (SOD) activities, soluble amino acid levels and protein profile, in leaves and roots, was examined in two conventional (non-GR) and two transgenic (GR) soybean. Glyphosate treatment had no significant impact on lipid peroxidation, whilst the chlorophyll content decreased in only one non-GR cultivar. However, there was a significant increase in the levels of soluble amino acid in roots and leaves, more so in non-GR than in GR soybean cultivars. Root CAT activity increased in non-GR cultivars and was not altered in GR cultivars. In leaves, CAT activity was inhibited in one non-GR and one GR cultivar. GOPX activity increased in one GR cultivar and in both non-GR cultivars. Root APX activity increased in one GR cultivar. The soluble protein profiles as assessed by 1-D gel electrophoresis of selected non-GR and GR soybean lines were unaffected by glyphosate treatment. Neither was formation of new isoenzymes of SOD and CAT observed when these lines were treated by glyphosate. The slight oxidative stress generated by glyphosate has no relevance to plant mortality. The potential antioxidant action of soluble amino acids may be responsible for the lack of lipid peroxidation observed. CAT activity in the roots and soluble amino acids in the leaves can be used as indicators of glyphosate resistance.     

No fitness cost of glyphosate resistance endowed by massive <Emphasis Type="Italic">EPSPS</Emphasis> gene amplification in <Emphasis Type="Italic">Amaranthus palmeri</Emphasis>

Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.     

大豆微核心种质对草甘膦的耐受性鉴定

以萌发初期大豆弯曲的子叶节为靶点,利用草甘膦(Roundup)点施鉴定法,分析了221份大豆微核心种质的草甘膦耐受性及其与抗草甘膦转CP4-EPSPS基因大豆AG5601的差异,结果表明,草甘膦对不同大豆种质的抑制程度与点施的草甘膦浓度呈正相关,Roundup稀释浓度为1/1000、1/10000时,草甘膦对大豆生长几乎无影响;Roundup稀释浓度为1/10时,草甘膦显著抑制大豆生长,导致植株死亡;Roundup稀释浓度为1/100时,不同大豆种质对草甘膦耐受性差异显著,并鉴定出对草甘膦具有较好耐受性的种质10份。虽然大豆微核心种质对草甘膦的耐受程度远远低于AG5601,但不同大豆种质对草甘膦耐受性存在显著差异,这为利用转基因和杂交转育技术培育抗草甘膦转基因大豆的受体或轮回亲本的选择提供了理论依据。     

Glyphosate affects lignin content and amino acid production in glyphosate-resistant soybean

Farmers report that some glyphosate-resistant soybean varieties are visually injured by glyphosate. Glyphosate is the main herbicide that directly affects the synthesis of secondary compounds. In this work, we evaluated the effect of increasing rates of glyphosate on lignin and amino acid content, photosynthetic parameters and dry biomass in the early maturity group cultivar BRS 242 GR soybean. Plants were grown in half-strength complete nutrient solution and subjected to various rates of glyphosate either as a single or in sequential applications. All parameters evaluated were affected by increasing glyphosate rates. The effects were more pronounced as glyphosate rates increased, and were more intense with a single total application than sequential applications at lower rates.     

Glyphosate-induced impairment of plant growth and micronutrient status in glyphosate-resistant soybean (Glycine max L.)

This investigation demonstrated potential detrimental side effects of glyphosate on plant growth and micronutrient (Mn, Zn) status of a glyphosate-resistant (GR) soybean variety (Glycine max cv. Valiosa), which were found to be highly dependent on the selected growth conditions. In hydroponic experiments with sufficient Mn supply [0.5 μM], the GR cv. Valiosa produced similar plant biomass, root length and number of lateral roots in the control treatment without glyphosate as compared to its non-GR parental line cv. Conquista. However, this was associated with 50% lower Mn shoot concentrations in cv. Conquista, suggesting a higher Mn demand of the transgenic cv. Valiosa under the selected growth conditions. Glyphosate application significantly inhibited root biomass production, root elongation, and lateral root formation of the GR line, associated with a 50% reduction of Mn shoot concentrations. Interestingly, no comparable effects were detectable at low Mn supply [0.1 μM]. This may indicate Mn-dependent differences in the intracellular transformation of glyphosate to the toxic metabolite aminomethylphosphonic acid (AMPA) in the two isolines. In soil culture experiments conducted on a calcareous loess sub-soil of a Luvisol (pH 7.6) and a highly weathered Arenosol (pH 4.5), shoot biomass production and Zn leaf concentrations of the GR-variety were affected by glyphosate applications on the Arenosol but not on the calcareous Loess sub-soil. Analysis of micronutrient levels in high and low molecular weight (LMW) fractions (80% ethanol extracts) of young leaves revealed no indications for internal immobilization of micronutrients (Mn, Zn, Fe) by excessive complexation with glyphosate in the LMW phase.     

Real-time quantification of wild-type contaminants in glyphosate tolerant soybean

Background

Trait purity is a key factor for the successful utilization of biotech varieties and is currently assessed by analysis of individual seeds or plants. Here we propose a novel PCR-based approach to test trait purity that can be applied to bulk samples. To this aim the insertion site of a transgene is characterized and the corresponding sequence of the wild-type (wt) allele is used as diagnostic target for amplification. As a demonstration, we developed a real-time quantitative PCR method to test purity of glyphosate tolerant (Roundup Ready^®, RR) soybean.

Results

The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant. On the other hand, no amplification product was obtained from RR soybean varieties, indicating that the wt sequence is single copy and represents a suitable marker of conventional soybean presence. In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy.

Conclusion

In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy. The main advantages of the assay here described derive from its applicability to bulk samples, which would allow to increase the number of single seeds or plants forming the analytical sample, thus improving accuracy and throughput while containing costs. For these reasons this application of quantitative PCR could represent a useful tool in agricultural biotechnology.     

Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.     

马铃薯/大豆套作对3个大豆品种光合指标和产量的影响

以大豆品种中黄30(早熟)、冀豆17(中熟)和齐黄34(晚熟)单作为对照,在大田条件下,研究马铃薯/大豆套作模式中3个品种生育期叶面积指数的变化及干物质积累的特征,分析套作马铃薯收获前后对大豆光合指标、产量及其构成因素的影响.结果表明： 生育前期阴蔽导致套作大豆叶面积指数(LAI)降低,干物质积累变缓,营养生长期相对延长,不同品种套作大豆光合速率(P_n)、蒸腾速率(T_r)和气孔导度(g_s)均低于单作.生育后期套作大豆生长加快,尤其是马铃薯收获后晚熟品种增幅显著提高.此时,套作大豆受光条件得到较大改善,表现出较强的补偿效应,LAI、干物质积累、P_n和g_s相对于单作上升幅度加大,接近于单作大豆,但不同品种补偿能力不同.与单作相比,套作模式下不同大豆品种的有效荚数、单株粒数及每荚粒数均有所降低,其中早熟品种分别显著下降22.0%、36.0%、17.6%,中熟品种下降5.1%、13.1%、8.9%,晚熟品种下降5.7%、7.6%、2.1%.套作模式下,中、晚熟大豆品种的产量较早熟品种分别高92.4%和163.4%,总土地当量比(LER)分别达到1.81和1.84.表明中、晚熟大豆品种与马铃薯组合套作优势更强,有利于马铃薯收获后大豆的补偿生长,促进套作大豆产量提高,充分发挥了复合群体的产量优势.     

抗草甘膦转基因大豆对根际土壤细菌及根瘤菌的影响

转基因大豆是全球种植最广泛的转基因作物,抗除草剂是其最主要的转基因特性.微生物群落是土壤质量的监测指标之一,抗草甘膦转基因大豆及配施草甘膦是否影响大豆根际土壤细菌及根瘤菌群落尚不清楚.本研究基于大田试验,以非转基因亲本大豆‘中豆32’为对照(CK),分析转G10-epsps基因耐除草剂大豆SHZD32-01(GR)及配施草甘膦(GR+G)在大豆各生育时期对根际土壤细菌和根瘤菌的影响.结果表明: 与CK相比,GR、GR+G处理对苗期和成熟期根际土壤pH、总有机碳(TOC)、总氮(TN)、NH₄⁺-N含量等产生影响;GR处理显著增加结荚期根际土壤细菌群落丰度及多样性,GR+G处理显著增加结荚期根际土壤细菌群落多样性,但显著降低苗期和结荚期根际土壤细菌群落丰度;GR、GR+G处理改变了部分优势细菌类群的相对丰度,但不同生育期根际土壤优势细菌类群均为变形菌门、酸杆菌门、拟杆菌门、绿弯菌门、浮霉菌门和放线菌门;GR、GR+G处理改变了根瘤菌类群的相对丰度,但未影响慢生根瘤菌和中华根瘤菌两种主要大豆根瘤菌的相对丰度,且GR+G处理结荚期根际土壤根瘤菌相对丰度显著降低.环境因子分析显示,根际土壤放线菌和根瘤菌群落丰度主要受土壤pH影响.抗草甘膦转基因大豆或配施草甘膦显著影响结荚期根际土壤细菌和根瘤菌,但其影响随大豆的生长而消失.     

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.     

Impact of the ahas transgene and of herbicides associated with the soybean crop on soil microbial communities

Although Brazil has recently reached the position as the second largest producer of genetically modified soybean [Glycine max (L.) Merr.], there are few reports on the effects of transgenic crops and the associated use of specific herbicides on soil microbial communities, both under the edaphoclimatic conditions in Brazil, and in other producer regions in the southern hemisphere. The aim of this study was to evaluate the effects of transgenic soybean containing the ahas gene conferring resistance to herbicides of the imidazolinone group, and of the herbicides associated with transgenic soybeans on the soil microbial community. Twenty field experiments were carried out during three growing seasons (summer of 2006/2007, short-season of 2007 and summer of 2007/2008), in nine municipalities located in six Brazilian states and in the Federal District. The experiments were conducted using a completely randomized block design with four replicates and three treatments: (1) conventional (non-transgenic) soybean cultivar Conquista with conventional herbicides (bentazone + acifluorfen-sodium and other herbicides, depending on the level of infestation in each region); (2) near-isogenic transgenic Cultivance (CV127) containing the ahas gene, with conventional herbicides; (3) transgenic Cultivance with specific herbicide of the imidazolinone group (imazapyr). As the objective of the study was to verify impacts of the transgene and herbicides on the soil microbial community of the whole area and not only a punctual rhizospheric effects, samples were taken at the 0–10 cm layer prior to cropping and at R2 soybean growth stage, between plant rows. Quantitative (microbial biomass C and N, MB-C and MB-N) and qualitative (DGGE of the 16S rDNA region) parameters of soil microbial community were evaluated. No qualitative or quantitative differences were found that could be attributed to the transgene ahas. A comparison of Cultivance soybean with conventional and imidazolinone-group herbicides applications also failed to reveal differences that could be attributed to the specific use of imazapyr, even after three consecutive croppings at the same site. Finally, no differences were detected between conventional (Conquista and conventional herbicides) and transgenic soybean managements (Cultivance and imazapyr). However, marked differences were observed in MB-C and MB-N between the different sites and times of year and, for the 16S rDNA-DGGE profiles, between different sites. In conclusion, microbial community evaluations were found to be sensitive and viable for monitoring different technologies and agricultural management methods, but no differences could be attributed to the ahas transgene for three consecutive cropping seasons.     

Glyphosate affects chlorophyll,photosynthesis and water use of four Intacta RR2 soybean cultivars

The planting of RR2 Intacta soybeans by farmers has been expanding strongly. However, some visual injuries have been noted after glyphosate application. The aim of this study was to evaluate the influence of glyphosate application on chlorophyll, photosynthesis and water use of four Intacta RR2 soybean cultivars. The experiment was conducted in a greenhouse, in a randomized block design with a 3 × 4 factorial scheme, consisting of three glyphosate rates and four soybean cultivars. The glyphosate formula used was isopropylamine salt + potassium salt. The parameters analyzed were phytotoxicity at 7, 14, 21 and 28 days after application, and total chlorophyll index at 0, 3, 7, 14, 21, 28, 35, 42 and 49 days after application. Furthermore, 40 days after application, the net CO2 assimilation rate (A), transpiration rate (E), stomatal conductance (G), and internal CO₂ concentration (Ci) were evaluated as well. The water-use efficiency (WUE) and carboxylation efficiency were calculated. The data were submitted to analysis of variance and compared by the Tukey’s test (p ≤ 0.05), followed by regression analysis. The phytotoxicity influence could be seen until 21 days after application, in which Monsoy 6210 IPRO cultivar was the most injured. The increasing doses promoted a reduction of the chlorophyll level up to 35 days after application in Monsoy 6410 IPRO. The cultivars tested here showed similar chlorophyll index values. On the 3rd, 7th and 14th DAA (Fig. 5a–c), there was a significant linear decline in the chlorophyll index with rising glyphosate dose for all four cultivars. The chlorophyll index cultivars were not influenced by the doses on the 42nd and 49th DAA. There was no difference in water use and carboxylation efficiency. The parameters A, E and A/Ci showed a positive correlation as the doses increased, while Ci declined, in both cultivars. The application of glyphosate on these soybean cultivars causes different injuries according to the sensitivity. In general, RR2 soybeans have the ability to recover from visual intoxication injuries and reestablish the normal chlorophyll production and photosynthetic parameters after glyphosate application.     

Herbicide-resistant, transgenic zonal pelargoniums produced by step-down glyphosate selection and plantlet recovery in the presence of aromatic amino acids

A genetic transformation protocol was developed using the transfer of a synthetic CP4 EPSPS transgene, as a conditional positive selectable marker, into commercially relevant zonal pelargoniums using an Agrobacterium tumefaciens strain in combination with a novel step-down glyphosate selection system. The transformation efficiency based on independent T-DNA integration events averaged 1.9?% over 10 experiments. Some 273 independent transformants were produced within an average time of 6?mo from explant inoculation with Agrobacterium to plantlet recovery. For plantlet recovery, three aromatic amino acids were incorporated into the rooting medium to ameliorate the accumulative effects of glyphosate selection. The T-DNA also contained a mutant ethylene receptor (etr1-1) cDNA from Arabidopsis thaliana, under control of the petunia flower-specific, floral-binding protein promoter, to confer ethylene insensitivity. However, delayed flower senescence was not obtained. The transformation protocol provides a reliable method to add herbicide resistance and other traits to zonal pelargonium.     

Mycorrhizal and Rhizobial Colonization of Genetically Modified and Conventional Soybeans

We grew plants of nine soybean varieties, six of which were genetically modified to express transgenic cp4-epsps, in the presence of Bradyrhizobium japonicum and arbuscular mycorrhizal fungi. Mycorrhizal colonization and nodule abundance and mass differed among soybean varieties; however, in no case was variation significantly associated with the genetic modification.