The substantive equivalence of transgenic (Bt and Chi) and non-transgenic cotton based on metabolite profiles

Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T₂ transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its non-transgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves.     

Enhanced yield performance of <Emphasis Type="Italic">Bt</Emphasis> rice under target-insect attacks: implications for field insect management

The rapid development of transgenic biotechnology has greatly promoted the breeding of genetically engineered (GE) rice in China, and many GE rice lines are in the pipeline for commercialization. To understand field performances of GE rice, key agronomic traits of two insect-resistant Bt rice lines that have been granted biosafety certificates for commercial production in China were evaluated together with their nontransgenic counterparts under environmental conditions with significant differences in insect pressure. Results from the experiments showed enhanced field performances of the Bt GE rice lines compared with the non-GE counterparts for yield-related traits such as number of panicles and filled seeds per plant, under environmental conditions with no insecticide application. No detectable underlying cost of the Bt transgene was observed in the two insect-resistant GE rice lines, particularly in the GE hybrid rice line. Results further indicated significantly greater yield performances of the two insect-resistant GE rice lines under environmental conditions with non-target insect control compared with no insect control. It is concluded from this study that insect-resistant Bt GE rice, particularly the hybrid line, has great potential to maintain its high yield when ambient insect pressure is high. In addition, proper application of insecticides to control non-target insects will guarantee optimal performance of insect-resistant Bt GE rice.     

The effect of root exudates from two transgenic insect-resistant cotton lines on the growth of Fusarium oxysporum

The attenuation of disease resistance in transgenic insect-resistant cotton has become one of the important factors restricting cotton production in China. Two transgenic insect-resistant cotton lines and their parental conventional cotton lines were used as the testing materials. The effects of root exudates of these cotton lines on the spore germination and mycelial growth of Fusarium oxysporum were studied and the components, contents of amino acids and sugars were determined. The results showed that the resistance of the two insect-resistant cotton lines to F. oxysporum was inferior to the parental lines, and that their root exudates promoted fungal spore germination and mycelial growth. Considerable differences in the components and contents of both, amino acids and sugars were found between the root exudates of transgenic cotton lines and their parental lines, where the disease indices were highly correlated with the total amount of sugars in the root exudates.     

Unintended polar metabolite profiling of carotenoid-biofortified transgenic rice reveals substantial equivalence to its non-transgenic counterpart

Substantial equivalence is a critical concept for biosafety assessment of genetically modified (GM) crops. To investigate substantial equivalence among carotenoid-biofortified GM rice and five conventional rice cultivars having common white (three) and red (two) grain colors, profiles of 52 polar metabolites were analyzed using gas chromatography time-of-flight mass spectrometry. The results were compared to evaluate the differences among GM and non-GM rice cultivars using principal components analysis. The GM rice is more comparable to its non-transgenic counterpart rice variety according to the closer co-separation than for other cultivars tested. This suggests that profiling of unintended polar metabolites could be a useful tool to reveal substantial equivalence of GM rice.     

Field evaluation of effects of transgenic <Emphasis Type="Italic">cry1Ab/cry1Ac,cry1C</Emphasis> and <Emphasis Type="Italic">cry2A</Emphasis> rice on <Emphasis Type="Italic">Cnaphalocrocis medinalis</Emphasis> and its arthropod predators

The impacts of transgenic Bt rice on target pests and their predators need to be clarified prior to the commercialization of Bt rice. In this study, the percentages of folded leaves of three transgenic Bt rice lines and non-transgenic parental rice line caused by Cnaphalocrocis medinalis were studied over two successive growing seasons. In addition, the population densities, relative abundance and population dynamics of C. medinalis and four species of its natural arthropod predators were investigated at three sites in China. The results showed that rice line significantly affected the percentages of folded leaves and population densities of C. medinalis larvae. Significantly higher percentages of folded leaves were observed on the non-transgenic rice compared with the three transgenic Bt rice on most sampling dates. Significantly higher densities of C. medinalis larvae and higher relative abundance of C. medinalis within phytophages were found on non-transgenic rice compared with three transgenic Bt rice at different sites across the study period. The population dynamics of C. medinalis larvae were significantly affected by rice line, rice line×sampling date, rice line×year, rice line×sampling date×year. However, there was little, if any, significant difference in the relative abundance, population density and population dynamics of the four arthropod predators between the three Bt rice lines and non-transgenic rice. The results of this study indicate that the Bt toxin in transgenic Bt rice can effectively suppress the occurrence of C. medinalis, but has no significant effects on the occurrence of the four predatory arthropod species.     

The Occurrence of Renin Inhibitor in Rice: Isolation,Identification, and Structure-Function Relationship

We found renin inhibitory activity in rice. The physico-chemical data on the isolated inhibitors were identical to those of oleic acid and linoleic acid. Oleic acid and linoleic acid competitively inhibited renin activity, with K_i values of 15.8 and 19.8 μM respectively. Other unsaturated free fatty acids also inhibited renin activity, but saturated fatty acids had no effect on it.     

Insect-resistant Bt-maize response to the short-term non-target mite-pest infestation and soil drought

Transgenic lepidopteran insect-resistant maize expressing the cry1Ab gene (Bt) and its non-transgenic counterpart at the 12-leaf-stage (V₁₂) were infested by the two-spotted spider mite or dehydrated by cessation of soil watering to check Bt-maize capacity to respond to other stresses than those assured by the presence of Cry protein. Since the antioxidant enzymes are key components of plant defence against biotic and abiotic stresses, the engagement of leaf superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) in response to 6-day mite feeding and soil drought has been investigated. The reduction of leaf hydration and soluble protein content in the fully expanded 8th leaf was independent of genotype and more pronounced in response to water cessation than mite infestation. Similarly, the changes in enzyme activities depended more on the kind of stress than the presence of the transgene. Water shortage in the soil enhanced the activity of all enzymes, whereas mite feeding decreased the activity of SOD and CAT, and markedly increased POX in the 8th leaf of both cultivars. In mite-infested leaves of the non-transgenic plant, the CAT activity remained unaffected, whereas decreased in leaves of Bt maize due to the hampered activity of CAT-2. In comparison to the control, all enzyme activity in the 10th non-infested leaf of mite-infested non-transgenic maize decreased, whereas it changed in the 10th leaf of Bt maize in the same way as in the 8th mite-infested leaf. The results suggest that SOD, CAT and POX can strongly confer short-term drought-stress response in both maize cultivars, whereas POX is the only responsive enzyme in mite-infested Bt maize.     

Effect of transgenic alfalfa plants with introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial community composition and physiological profile

The aim of this study was to evaluate the effect of transgenic alfalfa (Medicago sativa L.) plants, in comparison to their non-transgenic counterpart, on the density and physiological profiles of aerobic bacteria in the rhizosphere. Plants of transgenic alfalfa expressing the AMVcp-s gene coding for Alfalfa Mosaic Virus coat protein were cultivated in a climatic chamber. Two methods were used to determine the microbial diversity in rhizospheres of transgenic plants. First, the cultivation-dependent plating method, based on the determination of the density of colony-forming bacteria, and second, a biochemical method using the Biolog™ system, based on the utilization of different carbon sources by soil microorganisms. Statistically significant differences in densities of rhizospheric bacteria between transgenic and non-transgenic alfalfa clones were observed in ammonifying bacteria (GTL4/404-1), cellulolytic bacteria (GTL4/404-1, GTL4/402-2, A5-3-3), rhizobial bacteria (GTL4/402-2), denitrifying bacteria (A5-3-3) and Azotobacter spp. (GTL4/402-2). The highest values of substrate utilization by microbial communities and average respiration of C-sources were determined in non-transgenic alfalfa plants of the isogenic line SE/22-GT2. Carbohydrates, carboxylic acids and amino-acids were the most utilized carbon substrates by both Gram-negative and Gram-positive bacteria. Both, the community metabolic diversity and the utilization of C-sources increased in all alfalfa lines with culture time and regardless of transgenic or non-transgenic nature of lines.     

Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria

Root exudates represent an important source of nutrients for microorganisms in the rhizosphere and seem to participate in early colonization inducing chemotactic responses of rhizospheric bacteria. We characterized the root exudates collected from rice plantlets cultured under hydroponic conditions and assessed their effects on the chemotaxis of two strains of endophytic bacteria, Corynebacterium flavescens and Bacillus pumilus, collected from the rice rhizosphere. We compared these chemotactic effects on endophytic bacteria with those on two strains of plant-growth-promoting bacteria, Azospirillum brasilense (isolated from the corn rhizosphere) and Bacillus sp. (from the rice rhizosphere). The root exudates were collected at different time intervals. The highest concentration and diversity of amino acids and carbohydrates were found during the first 2 weeks after seeding. Histidine, proline, valine, alanine, and glycine were the main amino acid residues identified during the 4 weeks of culture. The main carbohydrates identified were glucose, arabinose, mannose, galactose, and glucuronic acid. The chemotactic responses of the analyzed endophytic bacteria to root exudates were 3.9 to 5.1 times higher than those of A. brasilense and 2.2 to 2.8 times higher than Bacillus sp. Our results indicate that rice exudates may induce a higher chemotactic response for endophytic bacteria than for other bacterial strains present in the rice rhizosphere.     

Mineral element distribution in organs of dual-toxin transgenic (Bt+CpTI) cotton seedling

Abstract

Being a new cultivar, the physiology of transgenic cotton, especially dual-toxin transgenic (Bt+CpTI) cotton, is not yet completely understood. Twelve elements in three organs of dual-toxin transgenic cotton seedlings were analyzed by ICP-MS. The distributions of the 12 elements were substantially different from those of non-transgenic cotton. In particular, the contents of B, Mg, P, K and Ca were the highest in leaves, while those of Si, Fe, Rb and Cu were the highest in roots; other elements had similar contents in the two organs, which were higher than those in the stem. Compared with non-transgenic cotton, the 12 elements could be classified into four groups according to their contents and distributions in the three organs: (a) P, K and Cu: their contents in transgenic cotton were remarkably lower, especially contents of P and K in leaves that were one times lower than those in leaves of non-transgenic cotton; (b) B, Mg and Mo: their contents in leaves and roots of transgenic cotton were higher, but lower in stems, compared with non-transgenic cotton; (c) Si, Mn, Fe, Rb and Zn: compared with non-transgenic cotton, these were lower in leaves and stems, but higher in roots of transgenic cotton; and (d) Ca: compared with non-transgenic cotton, its content was higher in all three organs of the transgenic counterpart. The decrease in soluble proteins and the expression of Bt and CpTI genes could be responsible for these changes. Further studies are needed to verify this hypothesis.     

Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modified rice: its implication in ecological biosafety assessment

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F₁ hybrids with common wild rice and F₂ progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F₁ hybrids and F₂ progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.     

Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T₅) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.     

Effects of Bt transgenic rice line on stripe stem borer,Chilo suppressalis and its consequences on egg parasitoid,Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) in the laboratory

Interactions among Chilo suppressalis (Walker), its larval parasitoid Trichogramma brassicae Bezdenko and insect-resistant transgenic rice lines were evaluated under laboratory conditions. The results showed that all neonates and second larvae of stem borer were dead regardless of being fed rotationally or permanently on Bt rice, but 18% and 28% of the third and fourth instar larvae could complete development and turned to adults, respectively, when fed rotationally. In the second year of project, third instar larvae of stem borer were used to evaluate Bt rice impact on T. brassicae. The result showed that emergence rate was lower, the abnormal female rate increased and adult female longevity of the wasp decreased significantly in comparison with the conventional variety. There was no significant difference in gender ratio and parasitism rate under Bt rice treatment in comparison with conventional variety.     

Production of trans-10, cis-12 Conjugated Linoleic Acid in Rice

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.     

RNAi mediated silencing of lipoxygenase gene to maintain rice grain quality and viability during storage

Lipoxygenase (LOX) is a common enzyme which catalyzes lipid peroxidation of seeds and consequently enhances seed quality deterioration and decreases seed viability. During seed storage, peroxidation of unsaturated fatty acids occur due to enhancement of LOX activity which directly leads to reduction in seed vigour and deterioration of grain nutritional quality. This study was undertaken to overcome these problem during rice seed storage by attenuating LOX activity using RNAi technology. To improve seed storage stability, we down regulated LOX gene activity by using a functional fragment of the LOX gene under the control of both constitutive (CaMV35S) and aleurone-specific (Oleosin-18) promoter separately. To understand the storage stability, RNAi–LOX seeds and non-transgenic control seeds were subjected to accelerated aging at 45 °C and 85 % relative humidity for 14 days. Our studies demonstrate that down regulation of LOX activity reduces the seed quality deterioration under storage condition. In addition GC–MS analysis revealed that reduction of fatty acid level in non-transgenic seeds during storage was higher when compared with that of transgenic rice seeds. Furthermore, the transgenic rice seeds with reduced LOX activity exhibited enhanced seed germination efficiency after storage than that of non-transgenic rice seeds. This study will have direct impact on nutritional stability of quality rice grains.