Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform

Optimization of water blanching and cabinet drying conditions for preservation of nutritional and phytochemical properties of moringa leaves

The perishable nature of moringa leaves leads to its limited shelf life. Drying methods reduce water activity and help extend the shelf life of dried leaves. In this study, the moringa leaves were subjected to water blanching at 50, 60, 70 and 80 oC for 1, 2 and 3 min, followed by drying. Water blanching at 50 °C for 3 min resulted in the highest retention of total carotenoids (24.65 mg/100 g), iron (21.50 mg/100 g), calcium (1413.10 mg/100 g) and colour of dried moringa leaves. To assess their impact on nutrient retention, the water-blanched moringa leaves were then subjected to three different drying methods: cabinet drying, sun drying and shade drying. The drying methods significantly influenced the retention of nutrients, with varying degrees of effectiveness across methods in moringa leaves. The moringa leaves blanched in water at 50oC for 3 min and dried in a cabinet drier retained the higher total carotenoids (24.65 mg/100 g), iron (21.50 mg/100 g) and calcium (1413.10 mg/100 g)

Two different zinc(II)-aqua complexes held up by a metal-oxide based support: synthesis, crystal structure and catalytic activity of [HMTAH]<SUB>2</SUB>[{Zn(H<SUB>2</SUB>O)<SUB>5</SUB>}{Zn(H<SUB>2</SUB>O)<SUB>4</SUB>}{Mo<SUB>7</SUB>O<SUB>24</SUB>}]&#183;2H<SUB>2</SUB>O (HMTAH = protonated hexamethylenetetramine)

An inorganic-organic hybrid material, [HMTAH]2[{Zn(H2O)5}{Zn(H2O)4}{Mo7O24}]&#183;2H2O (1) (where HMTAH = protonated hxamethylenetetramine) has been synthesized and structurally characterized. The compound 1 crystallizes in a monoclinic space group C2/c. The crystal data of 1: &#945; = 43&#183;12(3), b = 12&#183;399(10), c = 16&#183;285(13), &#946; = 111&#183;131(11), Z = 8. Its crystal structure shows that two different Zn(II)-aqua complexes, [Zn(H2O)5]2+ and [Zn(H2O)4]2+ are covalently coordinated to a heptamolybdate anion [Mo7O24]6- resulting in an anionic species of polyoxometalate supported zinc-aqua complexes, [{Zn(H2O)5}{Zn(H2O)4}{Mo7O24}]2-, that is stabilized with two protonated hexamethylenetetramine cations in the title compound 1. In the crystal structure, both lattice water molecules are found to interact with the heptamolybdate cluster anion and the protonated hexamethylenetetramine cation resulting in an intricate three-dimensional hydrogen bonding network. Interestingly, compound 1 exhibits catalytic activity towards oxidation of some primary alcohols

Propagation of acid lime, Citrus aurantifolia (Christm.) swingle and lemon, Citrus limon L. through stem cuttings - A review

Cultivation of acid lime (Citrus aurantifolia (Christm.) swingle) and lemon (Citrus limon L.) holds significant importance in India. Despite its economic importance, propagation faces challenges, primarily due to the recalcitrant nature of seeds, leading to loss of viability and lack of uniformity in seedlings. This necessitates the adoption of vegetative propagation method for producing true-to-type planting materials. Stem cuttings, budding and air layering are the primary methods employed, with stem cuttings being the most suitable for their simplicity and ability to induce precocity in fruit crops. The successful rooting of stem cuttings depends on the maturity of the cuttings, auxins, growing media, nutrition, irrigation water, endogenous food materials, season and microclimatic conditions. The maturity of cuttings influences rooting and uniformity, with virus-free hard, semi-hard, and softwood cuttings being preferred for propagation. The application of Indole Butyric Acid (IBA) significantly influences rooting. Cool season is generally favored for better rooting. Pre-conditioning treatments such as ringing, girdling and wounding influenced rooting. Microclimatic conditions, including temperature, relative humidity and light intensity, are critical for successful rooting. This review is a compilation of up-to-date research and development in stem cuttings of lime and lemon to understand better physiology, the role of auxins, growing media and microclimatic factors.

Polyoxometalates: toward new materials

This article describes an account of some of our polyoxometalate (POM)-based research, we have been doing in our laboratory last several years. There are several well-defined POM cluster anions, that are structurally characterized. We have chosen Anderson-type of heteropolyanion [Al(OH)6Mo6O18]3- and explored its linking propensity in different dimensions using 's', 'd' and 'f' block elements as linkers.We have demonstrated how a lanthanide linker provides a new pathway in forming a two-dimensional linked {As8V14} system [{Ln(H2O)6}2As8V14O42(SO3)]n · 8nH2O, that is derived from discrete {As8V14} cluster containing compound (NH4)6[As8V14O42(SO3)]. A polyoxometalate compound has been described in which a reduced tungstovanadate-heteropolyanion clusters get linked via capped V = O groups into one-dimensional chains. All these systems have already been reported elsewhere. The last portion of this article will be described by a new system [3-ampH]6[V10O28] · 2H2O having discrete molecular structure and extended supramolecular structure

Development and ergonomic evaluation of hand held vegetable seedling transplanter

Efficient vegetable production is essential to meeting growing food demands, requiring innovative solutions to reduce labor intensity and enhance productivity. Vegetable cultivation, especially for small farmers, demands innovative and affordable solutions to enhance productivity and reduce labor-intensive practices. The mechanization of agricultural processes has become increasingly important in reducing the physical strain and time-intensive nature of traditional farming operations, paving the way for innovative tools that enhance efficiency and user comfort. Transplanting of seedlings, a highly drudgery-oriented operation, is mainly performed by women. A hand held seedling transplanter has been designed and developed for transplanting vegetables seedlings based on the anthropometric dimensions of the user population. The number of seedlings transplanted per min using the handheld seedling transplanter ranged from 19 to 21, compared to 16 to18 using the conventional method. By using the hand held seedling transplanter, a significant reduction in the cost of operation was achieved, with 11.44 to 16.52% savings in cost reported. The average working heart rate while transplanting using the seedling transplanter was 103 bpm, compared to 120 bpm for conventional transplanting. The average energy expenditure while transplanting the seedlings using the seedling transplanter was 7.72 kJ min-1, compared to 10.94 kJ min-1. The performance evaluation of the designed transplanted indicted an increase in human productivity and a decrease in musculoskeletal discomfort during the transplanting process. The introduction of ergonomic and cost-effective solutions, such as the handheld seedling transplanter, not only improves productivity and reduces operational costs but also promotes sustainable practices while prioritizing the well-being of farmers

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

Citation: Chapman, J. A., Mascher, M., Buluç, A., Barry, K., Georganas, E., Session, A., . . . Rokhsar, D. S. (2015). A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome. Genome Biology, 16(1). doi:10.1186/s13059-015-0582-8Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population. © 2015 Chapman et al. licensee BioMed Central.Additional Authors: Muehlbauer, G. J.;Stein, N.;Rokhsar, D. S

Cloning and Characterization of a Putative TAC1 Ortholog Associated with Leaf Angle in Maize (Zea mays L.)

BACKGROUND: Modifying plant architecture to increase photosynthesis efficiency and reduce shade avoidance response is very important for further yield improvement when crops are grown in high density. Identification of alleles controlling leaf angle in maize is needed to provide insight into molecular mechanism of leaf development and achieving ideal plant architecture to improve grain yield. METHODOLOGY/PRINCIPAL FINDINGS: The gene cloning was done by using comparative genomics, and then performing real-time polymerase chain reaction (RT-PCR) analysis to assay gene expression. The gene function was validated by sequence dissimilarity analysis and QTL mapping using a functional cleaved amplified polymorphism (CAP). CONCLUSIONS: The leaf angle is controlled by a major quantitative trait locus, ZmTAC1 (Zea mays L. Leaf Angle Control 1). ZmTAC1 has 4 exons encoding a protein with 263 amino acids, and its domains are the same as those of the rice OsTAC1 protein. ZmTAC1 was found to be located in the region of qLA2 by using the CAP marker and the F(2:3) families from the cross between Yu82 and Shen137. Real-time PCR analysis revealed ZmTAC1 expression was the highest in the leaf-sheath pulvinus, less in the leaf and shoot apical meristem, and the lowest in the root. A nucleotide difference in the 5'-untranslated region (UTR) between the compact inbred line Yu82 ("CTCC") and the expanded inbred line Shen137 ("CCCC") influences the expression level of ZmTAC1, further controlling the size of the leaf angle. Sequence verification of the change in the 5'-UTR revealed ZmTAC1 with "CTCC" was present in 13 compact inbred lines and ZmTAC1 with "CCCC" was present in 18 expanded inbred lines, indicating ZmTAC1 had been extensively utilized in breeding with regard to the improvement of the maize plant architecture

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples