Improving the Use of Phosphate in Alfalfa

Phosphate (P) has always been one of the most limiting factors in agricultural soil, and will become more of a constraint as world population grows and environmental concerns gain precedence. It is therefore imperative to improve how efficiently and carefully P fertilizers are applied, as well as how efficiently crop plants use those fertilizers. To explore this, Ma et al. (2012) caused alfalfa plants to express genes that are found in a model legume organism, Medicago truncatula, in the hopes that these genes would improve the use of organic phosphate (Po) by alfalfa plants. Po is generally derived from animal manure, and takes the form of phytate in soil, which is difficult for plants to use. The genes MtPHY1and MtPAP1 produce enzymes that can assist in the digestion of Po, and therefore were selected to express in the roots of transgenic alfalfa. Ultimately, it was concluded that transgenic lines of alfalfa demonstrated significantly better use of Po than control lines when grown in either lab conditions or active farm soil.—Chad Redman
                  Ma, X.F., Tudor, S., Butler, T., Ge, Y., Xi, Y., Bouton, J., Harrison, M., Wang, Z.Y. 2012. Transgenic Expression of Phytase and Acid Phosphatase Genes in Alfalfa (Medicago sativa) Leads to Improved Phosphate Uptake in Natural Soils. Mol Breeding 377, 377–391.
                  Ma et al. inserted genes from Medicago truncatula into the roots of alfalfa

plants in order to promote the use of Po. They chose to use genes that had demonstrated significant ability to increase P uptake in other plants previously, and focused on the use of transgenic plants in the real-world conditions of active farm soil. Specifically, the authors selected phytase (MtPHY1) and purple acid phosphatase (MtPAP1) genes, which both produce phytases, or enzymes that break down phytate into usable P. They also identified two different promoters, or regions of DNA that initiate the process of expressing genes, which could be used for both MtPAP1 and MtPHY1 and tested them independently for each of the genes. These promoters are identified as CaMV35S and MtPT1. Thus, Ma et al.created four different transgenic alfalfa lines, combining one gene with one promoter in all four combinations.

                  Researchers began with two different lab tests, the first growing plants in a medium without any P and the second in a medium supplied with Po. In the first condition, each of the transgenic plant lines along with a control line were grown in sand and fertilized with a solution that did not contain any form of P. After two weeks of growth under P-stressed conditions, the plants were harvested for their roots in order to isolate RNA and measure enzyme activity.
                  The second growth condition also utilized sand, but in this case plants were grown in the presence of fertilizer. The fertilizer composition was standard, except that it contained no inorganic phosphate (Pi), only Po. After six weeks of growth plants were harvested and all above-ground parts of the plant were dehydrated using an oven over the course of one week. The dried biomass of each plant group was recorded to compare use of Po.
                  After these lab-generated medium tests, plants were grown in pots containing soils from active farm ground. Two different soils were tested, one from Texas (soil 1) and the other from Oklahoma (soil 2). Soil 1 was generally less nutrient rich, and also more acidic. Soil 2 had a significantly higher concentration of usable P and a fairly neutral pH. In each soil, all four transgenic plant lines, along with control plants, were grown for three weeks without adding any nutrients to the soil. After three weeks, fertilizer without any form of P was applied in order to isolate the effects of P-stress on the plants. In order to determine biomass, two cuttings of the alfalfa plants were performed, the first after eight weeks and the second after another four weeks. These cuttings were dried and weighed. Additionally, the second cuttings of these plants were used to measure total P contained in the plants.
                  Ma et al. present some intriguing findings. From the RNA and enzyme analysis of plants grown in sand without any added P fertilizer, they find that transgenic plants on the whole produce far greater levels of APase, an enzyme that breaks down forms of phosphorous that plants cannot use into forms that may be digested. This observed difference was highly statistically significant. Moreover, there was an observed significant difference between the enzyme activities induced by the two different promoters. MtPT1 controlled genes had higher levels of APase than CaMV35S controlled genes. However, there was no observed difference between the two genes themselves. These results indicate that the transgenic alfalfa is, in fact, superior to the wild type alfalfa. Furthermore, the MtPT1 promoter is more effective in promoting efficient P use than the CaMV35S promoter.
                  Examination of plant roots revealed that transgenic plants showed higher phytase activity than control plants. Phytase is another enzyme that breaks down P into forms that may be easily used by plants, but phytase is specific to braking down phytate. Phytate is the most abundant source of P in soils, so phytase production is essential to effective use of P fertilizers. Interestingly, MtPHY1 plants exhibited higher phytate activity than MtPAP1 plants, which suggests that MtPHY1 transgene alfalfa is preferable.
                  When it comes to growth performance, transgenic plants consistently outperformed wild type alfalfa. As the above enzyme analyses would suggest, the plants with the MtPT1-MtPHY1 construct had the most vigorous growth and the highest dry biomass when grown in sand with Po readily available. Also, there was a strong correlation between enzyme activity and biomass, with transgenic lines clearly using the Po much more efficiently than the non-transgenic line.
                  In natural soils, those taken from active farm ground, similar patterns were observed. Especially in soil 1, transgenic alfalfa grew as expected, much larger and healthier than the control plants. In soil 2, the transgenes MtPHY1 and MtPAP1 were less effective because the pH of soil 2 was close to neutral, which is too basic for the enzymes to work at optimal levels. Moreover, because soil 2 contained a higher concentration of P to begin with, there was less P stress on wild type plants.
                  What Ma et al. have shown is that transgenic alfalfa can be used to alleviate the economic and environmental costs of applying large amounts of P fertilizer. With more efficient P use, particularly in alfalfa expressing the MtPT1-MtPHY1construct, pasture grounds will require less application of P, resulting in fewer weed control issues and also less runoff into aquatic ecosystems.

Interaction Between Trichoderma harzianum Bacteria and Sunflower

by Chad Redman

Genetically modified organisms have already revolutionized the global food production industry. However, it is important to continue searching for new opportunities to develop further genetically modified crops and livestock. In that spirit, we will examine this publication by Nagaraju et al. (2012), who test the interaction between three different strains of Trichoderma harzianum bacteria and the seeds of sunflowers. Using a disease susceptible sunflower variety, the researchers coated seeds with these strains of bacteria and observed its effects on disease rate, growth rate, nutrient uptake, and seed production of grown plants. Overall, coating seeds with Trichoderma harzianum proved to significantly benefit the plants, although there was also substantial variation between the different bacterial strains. This finding is highly interesting in the search for more developed genetically modified crops because it introduces many questions. What is it about the bacteria that lead to enhanced plant performance? What genes are responsible? What are the products of those genes? Ultimately, can we isolate and introduce those genes into the plants themselves? This is the beginning of a long line of research to come.

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Drought Tolerance and Recovery in Transgenic and Wild Type Tobacco Plants

                  Genetic engineering of plants that are remarkably adept at flourishing in conditions of drought has become a common practice. A variety of genes have been identified as contributors to this trait in many different plants. However, there is currently great demand for studies measuring the effectiveness of specific genes in specific crop plants with quantitative data. Arif et al. (2012) performed a study on the effect of one gene, Arabidopsis Vacuolar Pyrophosphatase (AVP1), on the drought resistance of tobacco plants. The researchers examined growth performance of transgenic plants overexpressing AVP1 and non-transgenic plants in varying water treatments. Additionally, the cell structures of these two plant types were compared. Interestingly, the data collected on height, mass, seed number, and more from the plants confirmed that transgenic plants overexpressing AVP1 exhibit significantly greater growth under water shortage stress. What’s more, while no statistical significance was established, structural differences were evident between the transgenic and wild-type (WT) plants.—Chad Redman
                  Arif, A., Zafar, Y., Arif, M., Blumwald, E., 2012. Improved Growth, Drought Tolerance, and Ultrastructural Evidence of Increased Turgidity in Tobacco Plants Overexpressing Arabidopsis Vacuolar Pyrophosphatase (AVP1). Molecular Biotechnology
                  Arif et al. examined phenotypic differences between genetically engineered tobacco plants that were designed to over express the gene AVP1 and WT tabacco plants. The objective of this study was to identify the growth difference between this specific transgenic line of tobacco and WT tobacco under differing water stresses. The basic procedural design was to grow both transgenic and WT plants in three different conditions: fully watered, partially limited water, and fully desiccated.
                  The researchers began by breeding their genetically modified tobacco plants, producing several generations so as to ensure that all test plants were expressing the AVP1 gene at the desired level. Tests were conducted to confirm that these plants were producing the correct proteins as an added security against impure transgenic lines. Once these test organisms were prepared, several of the transgenic plants and an equal number of the WT plants were planted in large, identical pots and grown for six weeks under ideal growing conditions. At this point five transgenic tobacco plants and five WT plants were designated as “fully watered” subjects. Similarly, Arif et al.designated five plants of each genotype as “less-watered” and five others as “desiccated.” Over the course of eighteen days, the fully watered plants were maintained at ideal growing conditions, while the less-watered plants received a greatly reduced supply of water. Finally, the desiccated treatment entailed no watering whatsoever. After the eighteen day test period, ideal conditions were resumed for all test groups for five weeks, until harvesting. This period was intended to test for recovery capacity.
                   In order to measure the results of their study, Arif et al. measured the fresh (hydrated) and dry biomass of shoots and capsules from all test tobacco plants, both transgenic and WT. Still more, the mass of seeds produced from all plants was recorded. In order to compare more fundamental differences between the two phenotypes, the researchers used electron microscopy to examine the cell structures of photosynthesizing leaves from each.
                  Results from this straightforward and well-designed experiment were decisive and clear. First, it is important to note that several blotting techniques confirmed the overexpression of the AVP1 gene in test transgenic plants. During the growth period of the experiment, it was clear to researchers that transgenic lines in general produced more numerous capsules and larger leaves than WT plants, and that the two limiting conditions caused far greater wilting and stunting in WT plants. After the recovery period, all plants except the desiccated WT plants survived, and all subjects were harvested. The quantitative data collected on fresh and dry mass of shoots and capsules, dry capsules alone, and dry seeds alone provided Arif et al. with fascinating statistically significant differences. The transgenic and WT plants grown at ideal conditions demonstrated no significant differences, but the other conditions were more disparate. All mass measurements on less-watered and desiccated plants showed that transgenic plants were significantly more successful. In short, the researchers demonstrated that AVP1 overexpression helps tobacco plants cope with drought conditions.
                  Finally, Arif et al. did not identify a significant difference in the number or size of photosynthesizing cells between transgenic and WT tobacco plants. However, they insist that transgenic cells generally displayed larger vacuoles and smoother outline than WT cells, with guard cells boasting thicker cell walls. While this result is not decisive, it provides some notion of what is the structural difference may be that allows plants overexpressing AVP1 to survive water shortage more successfully than WT plants.

Long Term Effects of GM Maize and Roundup on Rats

Genetic modifications to crops raise many concerns about possible health threats to humans and other animals. Extensive testing is required to show that a particular modification does not produce a substance or alter a biological process that may lead to health concerns for consumers. While studies of this nature have become commonplace, results are hardly consistent across the board, and truly long-term procedures are rare. Séralini et al. (2012) conducted an experiment to measure the effects of both Roundup-tolerant maize feed and the herbicide Roundup itself on rats over a two year time span, using many different concentrations and variations of these variables. The researchers find that rats exposed to GM maize feed and/or Roundup herbicide demonstrate higher levels of tumor development as well as earlier death as compared to control groups. However, most of their results are of limited impact due to statistical insignificance.—Chad Redman
                  Séralini, G. E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., Hennequin, D., Vendomois, J. S., 2012. Long Term Toxicity of a Roundup Herbicide and a Roundup-Tolerant Genetically Modified Maize. Food and Chemical Toxicology 50, 4221–4231.

                  Séralini et al. raised 200 rats over the span of two years, 100 males and 100 females. For feed, the researchers grew three distinct plots of maize, one being an isogenic, non-GM strain, one being Roundup-tolerant but not sprayed with Roundup, and the other being Roundup-tolerant and sprayed regularly with Roundup. In order to test for adverse effects of both the genetically modified Roundup-tolerant maize and the herbicide Roundup itself, both the males and females were randomly separated into ten distinct treatment groups. Six of these groups were fed the GM maize, three the Roundup treated stock and three the untreated stock. For each stock, three different diets were fed, one group a diet of 11% maize, another group a diet of 22% maize, and the third a diet of 33% maize. In addition, these rats were given access to plain water. The next three groups of rats were fed non-GM maize diets, but given water containing differing concentrations of Roundup; the three percentages of Roundup in the drinking water, by weight, were 1.1 x 10–8%, 0.09%, and 0.5% Roundup. Finally, each sex also had a control group of ten rats that were fed isogenic maize and plain water. These rats were kept in tightly controlled conditions at a comfortable temperature and humidity, a twelve hour light-dark cycle, and absolutely consistent feeding patterns.
                  The findings of Séralini et al. are interesting and somewhat alarming. Average lifespans of the rats were determined to be 624±21 days for males, while the females lived an average of 701±20 days. The mortality rate of rats that were outside of the control group was generally higher, and the first rats that died over the course of the procedure were males in the groups fed GM maize. Unfortunately, it is unclear if the differences observed in time of death for control rats versus any of the test group rats were statistically significant. Nonetheless, the trend is easily observable that rats in the control groups lived the longest. As for specific tissue testing and tumor development, the rats had blood, urine, and after death, organ samples taken and tested for nutrient content and cell structure similarity. Here again, the researchers failed to establish statistical significance with their data. Trends show that test groups, especially females, developed oddities in cell structure as well as demonstrating high propensity for tumor development. In short, although these researchers presented interesting data and conducted a thorough, long term experiment on the health risks of GM maize, they fall short on making a groundbreaking discovery because the observed trends were not statistically significant.

Long Term Effects of GM Maize Feeding on Cows

As the use of genetically modified crops becomes more practical, health concerns become increasingly relevant, both for humans as well as other organisms. While some research has focused on the effects of feeding genetically modified diets to organisms such as pigs and mice, little work has been done to observe the long term effects of feeding GM maize to dairy cows. Guertler et al. (2012) investigated the presence of nucleic acid or protein residue from GM maize in blood, milk, urine and feces, as well as testing for differences in apoptosis, inflammation, and cell cycle pathways between cows on GM and isogenic diets. The study finds no significant differences in any of these cell pathways, nor in the production or consumption of the cows. However, residue of the Cry protein produced in the GM maize was detected in feces.—Chad Redman
            Guertler, P., Brandl, C., Meyer, H. H. D., Tichopad, A., 2012. Feeding Genetically Modified Maize (MON810) to Dairy Cows: Comparison of Gene Expression Pattern of Markers for Apoptosis, Inflammation and Cell Cycle. Consumer Protection and Food Safety 7, 195–202.

            Guertler et al. noted the lack of long term analysis of the effects of GM crops being fed to livestock, leading them to perform a study on how a diet of GM maize can impact dairy cows in the long run. The process began simply, feeding 36 cows identical diets, with the exception that half were fed GM maize and the other half near perfectly isogenic maize. This stage of the procedure was conducted using crops that were grown in controlled conditions, as were animals, effectively controlling for all variables save the condition of eating GM maize.
            During this 25 month period, the subject cows were monitored closely, having samples of feces, blood, milk, and urine taken throughout. The researchers were testing for the presence of Cry proteins and nucleic acids which are only present in GM maize. After the time period of 25 months, ten cows from the experimental group and seven from the control group were slaughtered, and tissue samples from the liver, rumen, abomasum, small intestine, large intestine, and appendix were removed and tested for the same Cry residue. The specific technique used to detect Cry residue was quantitative polymerase chain reaction (qPCR).
            In order to access the cell pathways of apoptosis, inflammation, and cell cycle, Guertler et al. chose a reference and a target gene for each. The reference genes histone, ubiquitin and GAPDH were chosen because they were highly expressed in the sample tissues, giving researchers a base level to go by. The target genes, on the other hand, were chosen because their activation would indicate the activity of a cell pathway. Several specific target factors were analyzed for each of the three pathways in question using qPCR.
            The results of this study are promising for the safety of GM crops. Guertler et al. did not discover any remnants of nucleic acids in any tested product of the experimental group of dairy cows. They did find traces of Cry protein in the feces of the cows, but this is not as concerning as it might have been had residue been discovered in milk or blood. The same holds for the sample tissues from the test group of cows; none of the organs showed a significant trace of the GM maize. These results basically show that, on a long run scale, GM maize and its products are not detectable in the cow that consumed it. What’s more, none of the target genes were expressed significantly differently from the reference genes. That means that, for these three major cell functions, there was no detectable impact from the GM maize diet. This clear paper does an outstanding job of presenting data that are promising for the future use of GM crops.

Exploring the Mechanism Behind Nematode Resistance in Soybean Plants

Soybean crops are becoming increasingly important throughout the world as a source of renewable oil as well as protein. However, they are also a crop that poses many great challenges during production. Perhaps the most destructive of these, a pest called the cyst nematode (Heterodera glycines Ichinohe), has been controlled using resistant soybean plants for many years. However, very little is known about the actual mechanism behind this resistance, and new understanding is desperately needed as old resistant strains of soybean lose their effectiveness. Liu et al.(2012) set out to clone the specific gene that provides resistance to cyst nematodes, and find what proteins it produces to accomplish this feat. The researchers used complicated methods to isolate a gene within the Rhg4 (resistance to Heterodera glycines 4) section of soybean DNA as the gene of interest, and determine that it is responsible for the production of an enzyme that interconverts serine and glycine, two different amino acids. In the future, this knowledge will lead to genetically modified soybean strains and increased production.—Chad Redman
            Liu, S., Kandoth, P. K., Warren, S. D., Yeckel, G., Heinz, R., Alden, J., Yang, C., Jamai1, A., El-Mellouki, T., Juvale, P. S., Hill, J., Baum, T. J., Cianzio, S., Whitham, S. A., Korkin, D., Mitchum, M. G., Meksem, K., 2012. A Soybean Cyst Nematode Resistance Gene Points to a New Mechanism of Plant Resistance to Pathogens. Nature 492, 256–262.

            Liu et al. investigated the genes responsible for making certain strains of soybean resistant to Heterodera glycines (rgh), a prevalent and damaging pest commonly known as the cyst nematode. While the general locations, known as quantitative trait loci, of these genes have been mapped for many years, the genes themselves have not before been precisely defined, cloned, or examined for their specific functions. The genes in question have been tracked down to chromosomes 18 (rgh1) and 8 (Rgh4) in the soybean genome, with Rgh4 being the more significant and important. Therefore, the team of researchers began by breeding a variety of soybean called Forrest, which exhibits resistance to cyst nematodes thanks to rgh1 and Rgh4 working in tandem. Because the resistance alleles of the mystery genes are not completely dominant, the lines of Forrest soybean had to be inbred for several generations, and comparing the resistant and nonresistant offspring that were produced gave Liu et al. the opportunity to track down exactly what the differences were. In short, by breeding nematode resistant soybean plants, researchers could define exactly what genes at the rgh1 and Rgh4 quantitative trait loci were causing certain plants to exhibit resistance.
            After bounding the gene that they suspected to be linked to nematode resistance, Liu et al. devised several ways of confirming that they had identified the correct gene. First, they used a technique called TILLING, which essentially searches for mutations in DNA strands. Through this technique, the researchers were able to identify the absolute pinpoint differences between the genes of interest in resistant versus nonresistant plants.
            After using TILLING, Liu et al. sequenced the gene which they suspected contributed cyst nematode resistance to soybean plants in 81 fully distinct varieties of soybean. They were testing to find out if the lines which exhibited a resistant phenotype also possessed the gene with the correct mutations that had been revealed through the use of TILLING. Simillarly, they examined the nonresistant plants to test for the opposite allele of their gene suspect.
            In order to further strengthen the connection between the identified gene and soybean resistance to cyst nematodes, the researchers implemented a technique known as virus-induced gene silencing. Fortunately, this name says it all; a virus is designed to effectively “turn off” the targeted gene. Liu et al. infected soybean plant tissue with this virus and exposed previously resistant plants to nematodes.
            Next, researchers wanted to confirm that they had found a gene that was acting from the Rgh4 site of the soybean genome. This was done relatively simply; a nematode-susceptible plant had the Rgh4 section of DNA from a resistant plant introduced into its gnome, and then its resistance was measured relative to its original level of resistance.
            As a final step in their procedure, Liu et al. sought a picture of what protein the gene they had isolated was coding for. Using yet another complicated technique called homology modeling, researchers were able to determine what protein was being produced and what its function was. Moreover, they ascertained how proteins varied from resistant to nonresistant soybean plants. Once a suspect protein was recognized, the different forms of this protein were injected into E. coli strains that needed the same protein to survive. The functionality of these E. coli was telling of the function served by the protein in question.
            Certainly, Liu et al. conducted a procedure with many complicated facets. However, they remained wonderfully focused and their results are easily summarized, as they all point to the same conclusions. The early work produced lines of resistant and nonresistant soybean which exhibited one difference in particular that interested researchers; a gene called SHMT was found to have five point mutations that differentiated resistant and nonresistant soybean plants. From here, the team of researchers was able to prove that this gene was causing nematode resistance using TILLING, which allowed them to identify the precise difference between the two gene alleles. What this allowed for was the testing of each specific mutation for its effect on resistance. Additionally, by sequencing SHMT in 81 different varieties of soy bean, Liu et al.found that resistant strains had the SHMTallele which they had predicted would lead to resistance from the TILLING method. Still more, silencing the SHMT gene removed nematode resistance from Forrest soybean plants, indisputable proof of its role in providing such resistance. In their attempt to show that SHMT is in the Rgh4 locus, the researchers succeeded, finding that transplanting this section of DNA from a resistant plant to a nonresistant plant would transfer resistance over. Essentially, this finding merely affirms the importance of Rgh4 over rgh1. And finally, Liu et al. uncovered just what SHMT is responsible for producing. They found that it codes for an enzyme which reversibly converts glycine to serine. In fact, they suspect the resistant allele of this protein interrupts this process, a surprising finding given that this protein is conserved across a huge number of species, including humans, and is essential to metabolism. However, there was some uncertainty as to whether or not there may be other mechanisms being affected that were not described. In sum, what this enlightening paper found was that SHMT produces a protein which inhibits the ability of the cyst nematode to feed off the roots of soybean plants, potentially solving a huge problem for the agricultural industry.

Engineering Crops to Use Alternate Forms of Phosphorous May Slow Phosphorous Depletion and Provide Weed Control

The limited stores of phosphorous (P) here on earth have been a concern for the agricultural industry for many years. Consequently, a great deal of research is being done to find ways to use P more efficiently. One proposed system for managing P use in agricultural crops is to genetically engineer them to use a form of P that weeds and common bacteria cannot consume, or even find poisonous (López-Arredondo and Herrera-Estrella 2012). Not only would such a mechanism ensure that almost all P applied to agricultural crops is used by the desired crop, but also noxious weeds would be stunted and potentially killed by the same chemical. Numerous benefits may be reaped if these modified crops can be implemented, including reduced fertilizer and pesticide use, lower food prices, and a smaller chemical load contaminating aquatic ecosystems.—Chad Redman
            López-Arredondo, D. L., Herrera-Estrella, L., 2012. Engineering Phosphorus Metabolism in Plants to Produce a Dual Fertilization and Weed Control System. Nature Biotechnology 30, 889–895.

            López-Arredondo and Herrera-Estrella set out to genetically modify plants, giving them the capability of using a form of P that most plants are incapable to metabolizing. For now, both crop plants and weeds use a form of P called orthophosphate (PO4–3) as an essential nutrient. However, some bacteria digest phosphite (PO3–3) and produce orthophosphate. Previous studies have identified a gene called ptxD as the reason they can utilize phosphite, and López-Arredondo and Herrera-Estrella proceeded to implant this gene in Arabidopsis plants, a model organism commonly known as mouse-ear cress. After producing these transgenic plants (plants with the ptxD gene engineered into them), the researchers attempted to grow them, along with control wild-type plants, in a medium completely lacking orthophosphate but supplemented with phosphite. They were interested in the height each type of seedling achieved over a few days, and how the root systems developed over a longer time window.
            Next, López-Arredondo and Herrera-Estrella tested the growth ability of these transgenic plants against wild-type plants in greenhouse conditions. That is, in a greenhouse, both transgenic and wild-type plants were grown in sandy soils containing either orthophosphate or phosphite as their sole source of P. After a period of time, the biomass and root length of each plant in each condition were measured.
            Of course, López-Arredondo and Herrera-Estrella were interested in the effectiveness of the ptxD gene in more than just mouse-ear cress. As a further step, they produced transgenic tobacco plants and grew them alongside wild-type plants in unfertilized, orthophosphate fertilized, and phosphite fertilized conditions. Importantly, all of these different soil types were sterilized so as to control for the effects of bacteria. Furthermore, the researchers measured the rate of photosynthesis of wild-type and transgenic tobacco plants under similar fertilization conditions. Photosynthesis rate is relevant because plants use P in their respiration processes.
            As any genetically modified food crop inevitably raises health questions, López-Arredondo and Herrera-Estrella preformed measurements to detect the presence of phosphite in the leaves, flowers and fruits of transgenic plants. This was merely a precaution, as the US Food and Drug Administration has declared phosphite safe for animal consumption.
            The next step in this procedure was to test the ptxDtransgenic plants in more realistic soil taken from active farm ground. This soil varied most from previous experiments because it contained all the microorganisms normally found in soil. Both alkali and acidic soils were used, taken from farms in Mexico, and transgenic and wild-type mouse-ear cress was raised using varied concentrations of both orthophosphate and phosphite fertilizer.
            Finally, López-Arredondo and Herrera-Estrella investigated the viability of phosphite as a weed control mechanism. They tested two common weeds, false brome grass and tall morning-glory, to determine if these plants were capable of utilizing phosphite as a source of P. Upon discovering that they were not, the researchers used these plants in a greenhouse competition test against transgenic plants. The plants were grown in soil that was directly extracted from an active farm, and again various fertilizer applications were tried. Bear in mind that both the weeds and the crop were raised in the same planter in this trial. Fertilizer conditions were unfertilized, orthophosphate fertilized, and phosphite fertilized. The same procedure was conducted with transgenic mouse-ear cress and transgenic tobacco.
            The many results of this study were all in line with the notion that ptxD transgenic crops may be highly beneficial if used on a large scale. The initial test to determine if transgenic plants would grow normally in the absence of orthophosphate proved successful, with the wild-type plants completely failing in the same medium. This result set up the rest of the López-Arredondo and Herrera-Estrella experiments, showing that their transgenic plants had acquired the ability to metabolize phosphite. Ensuing greenhouse tests had similar results, with transgenic and wild-type plants responding similarly to orthophosphate fertilization and no fertilization, but with the transgenic plants developing normally with phosphite as the sole source of P and wild-type plants completely dying out. Identical results were recorded for tobacco plants as well. These data are more strong evidence that genetically engineered crops with the ptxD gene are well suited to replace traditional crops using either orthophosphate or phosphite as a source of P. Moreover, there was no detectable amount of phosphite on the leaves, fruit, or flowers of the tobacco transgenic plants.
            Testing of transgenic plants is non-sterile soils produced results that were in line with previous greenhouse testing. Transgenic plants matched the growth of wild-type plants over many different concentrations of orthophosphate fertilizer while demonstrating this same phenotype growing in phosphite fertilizer. Impressively, equal biomass and root length was achieved with a substantially lower concentration of phosphite fertilizer as compared to orthophosphate fertilizer.
            Lastly, the greenhouse competition tests between crops and weeds ended with crops being outstripped when the planter was fertilized with orthophosphate, but transgenic crops completely smothered out all tested weed forms when treated with phosphite. These results suggest that phosphite could not only conserve the finite supply of P, but also reduce the necessity of additional herbicide chemicals given its disruptive effects on most wild-type plants.

Scientists Have Determined Which Gene Promotes Phosphorus-Starvation Survival in Kasalath Rice

Phosphorus (P) is an absolutely critical nutrient for plant growth. This has large implications for the agricultural industry; fertilizers must be applied where soils do not contain sufficient P. However, the world’s store of P is finite, and costs of extraction are steadily rising (Gamuyao et al. 2012). Therefore, it is of vital importance to pursue species of crops, such as Kasalath rice, that are well suited for growth in low-P environments. While scientists have known for years that Kasalath rice is capable of producing high yields under the stress of minimal P and other nutrients, they have not before identified the specific gene that contributes this attribute. Gamuyao et al. conducted an investigation to discover what genetic trait Kasalath possess that is absent in other varieties of rice, and what physical effects this trait ultimately conveys. They concluded that a gene dubbed phosphorus-starvation tolerance 1 (PSTOL1) promotes the extended development of roots, allowing plants to absorb higher quantities of all nutrients, including P.—Chad Redman
            Gamuyao, R., Chin, J. H., Pariasca-Tanaka, J., Pesaresi, P., Catausan, S., Dalid, C., Slamet-Loedin, I., Tecson-Mendoza, E. M., Wissuwa, M., Heuer, S., 2012. The Protein Kinase Pstoll from Traditional Rice Confers Tolerance of Phosphorus Deficiency. Nature 488, 535–541.

            Gamuyao et al. searched for the genetic mechanism behind the P-starvation survival trait of certain types (aus-type) of rice, specifically the variety Kasalath. Kasalath has become vital to the agricultural industry throughout Southeast Asia due to its ability to produce high yields in poor soil. Originating in eastern India, this rice has been implemented for years in a poverty stricken region of the world where rice can be the only source of calories and 60% of farmland is composed of poor and problem soils (Gamuyao et al.). However, it has remained a mystery as to how Kasalath rice could survive in such nutrient-deprived ground; that is the mystery Gamuyao et al.set out to uncover.
            While the actual investigative techniques were quite technical, the basic notion of what these researches did can be understood in simpler terms. They began by sequencing the region of Kasalath DNA that has been associated previously with P-starvation tolerance, named Pup1. That is to say, they read what instructions the DNA was giving the rice. From this analysis, Gamuyao et al. were able to identify four specific instructions that they thought were possible contributors to Kasalath’s resistance to P-starvation. From here, the scientists narrowed the possible candidates down from four to just one using a process known as quantitative polymerase chain reaction (qPCR). In a nut shell, using qPCR allowed researchers to decide what piece of Kasalath DNA exists in the Pup1 region that does not exist in other varieties of rice. Ultimately, the gene that was singled out was called phosphorus-starvation tolerance 1 (PSTOL1). After isolating the bit of DNA that they suspected was responsible for Kasalath’s resilience, Gamuyao et al.performed several experiments to test the physical impact of PSTOL1, beginning by artificially inserting the gene into rice varieties that do not naturally have P-starvation tolerance. The real test was to observe the root development of these transgenic specimens. Next, researchers placed these transgenic plants into solutions varying in P concentration (100µM and 10µM), with controls that did not possess the gene in question. Again, root development was the main subject of study.
            The following procedures were conducted to confirm the hypothesis of the researchers that their mystery gene, PSTOL1, impacts root development. First, Gamuyao et al. used RNA interference to down-regulate the gene in Kasalath plants; they made it so the rice plant could not use its PSTOL1 gene. Next, the scientists analyzed the expression of PSTOL1 during the root development of a plant by “watching” for the presence of a marker in the roots of a plant that indicate PSTOL1 activity. Additionally, both transgenic plants (plants that have had the P-starvation gene artificially implanted in them and which express this gene at a very high rate) and control plants (Kasalath) were grown in dirt, and root samples were taken from them for an Affymetrix gene-array. This analysis essentially checks for how cells in the plant are using PSTOL1to make the structures that provide the plant with P-starvation resistance. Finally, using hydroponics, Gamuyao et al. performed more qPCR on root samples from plants to detect the presence of more molecules that indicate PSTOL1is active in root development.
            Clearly, this procedure was a highly comprehensive one. Similarly, the results yielded were convincing and complete. Original sequencing of the Pup1 region revealed four possible P-starvation tolerance sites, and further qPCR found all but the PSTOL1gene to be shared to some extent between Kasalath and other, non-resistant varieties of rice.
            The results of the initial transgenic plant analysis, where rice plants not possessing PSTOL1 were implanted with the gene, were promising; these artificially created varieties also exhibited P-starvation resilience. This was a good indication that PSTOL1 was the missing link from non-resilient varieties of rice. When the transgenic plants were grown in mediums containing varied concentrations of P, they developed longer roots with more surface area than control plants, regardless of their treatment. This result demonstrates that PSTOL1 is an active gene independent of P concentration in the environment.
            On to the procedures that were used as confirmation of the Gamuyao et al. hypothesis that PSTOL1 works through affecting root growth, when the researchers inhibited PSTOL1, they found significantly less root mass was produced by the plant. In the marker experiment, while the actual observations were rather complex, the conclusion was that PSTOL1 promotes early root development in Kasalath and the transgenic plants. Moreover, similar results were acquired in the Affymetrix gene-array. Gamuyao et al. found 23 genes within the transgenic plants that were expressed at different rates as compared to the control rice plants regardless of stress situations, namely P-starvation. This data suggests that PSTOL1 is, in effect, regulating the expression of these 23 other genes, all of which are related to stress responses such as low P and flooding. Putting the proverbial cherry on top, Gamuyao et al.conducted one last qPCR analysis on root material from these same transgenic plants, ultimately reinforcing the independence of PSTOL1 and its associated genetic impacts from levels of P in the soil.

The Widespread Use of Bt Cotton Allows for Reduced Pesticide Usage and Promotes Biocontrol Services

Insecticide and other artificial chemical use in the field of agriculture is a hotly debated subject and is generally regarded to entail, at a minimum, some negative effects. Certainly much research has focused on uncovering avenues to reduce the prolific use of such chemicals, including work with genetically modified organisms (GMOs). Bt cotton is one widely implemented and successful example of a GMO; with more than 66,000,000 hectares (ha) of Bt cotton cultivated worldwide in 2011, it has certainly proved popular with both farmers and governments (Lu et al. 2012). Essentially, Bt cotton allows farmers to apply less pesticide to a crop throughout a season. The research performed by Lu et al. in north China focused on two main questions: first, whether Bt cotton implementation on a large scale resulted in an increase of generalist arthropod predators in Bt cotton fields as well as other neighboring crops. Second, this research looked for increased biocontrol in China as a potential result of increased predator populations. Ultimately, the research concluded that Bt cotton implementation, and more importantly the reduced usage of insecticides, positively affected predator density, negatively affected pest density, and produced some evidence that these effects spill over to neighboring fields.—Chad Redman
            Lu, Y., Wu, K., Jiang, Y., Guo, Y., Desneuz, N., 2012. Widespread Adoption of Bt Cotton and Insecticide Decrease Promotes Biocontrol Services. Nature 487, 362–367.

            Lu et al investigated the effects of using Bt cotton in order to reduce the need for pesticides on biological control services. Modern farming techniques are continually moving farther and farther away from biological control services to manage increasingly valuable and productive crops. However, manufactured chemicals may not always be the answer. Lu et al. surveyed arthropod predators and cotton aphid populations in northern China from 1990 to 2010 and performed experiments at the Langfang experimental station of the Chinese Academy of Agricultural Science (CAAS) from 2001 to 2011. Their research was geared at monitoring predators and aphids in Bt and non-Bt cotton fields, treating some of each with pesticide and leaving some of each without pesticide. The researchers also kept track of the predator abundance and insecticide use in cotton in 36 locations throughout northern China from 1990 to 2010. Additionally, aphid density was recorded in 24 places over the same time period in order to measure the biocontrol services rendered by predators. Finally, Lu et al. performed exclusion cage trials in the years 2010 and 2011.
            Regarding the experiment in which researchers compared Bt and non-Bt cotton both with and without pesticide control, there was no significant difference found between the two crops when similar management methods were used; that is, the genetically modified cotton was no different than the unmodified cotton in this particular experiment. However, there was a striking difference between the predator and aphid populations in the insecticide treated crops versus the non-treated crops; those crops that were not treated had much lower pest populations. This is an exciting result because it means that the biological services of predators out performed our chemical control methods.
            From 1990 to 2010, the cotton industry underwent many changes. Due to governmental mandates, Bt cotton became common in 1997 and this resulted in a spike in the predator population throughout north China. This also coincided with a drastic fall in the use of pesticides as well as the population of pests; more strong evidence that biocontrol is very effective. Moreover, the exclusion cage experiments further confirmed the hypotheses of Lu et al..
            This study came to several conclusive realizations. It is evident that overusing pesticides can actually result in higher relative populations of pests compared to allowing several predator species to thrive and produce a biocontrol service. Less convincing, yet still compelling, are the data showing trends for these arthropod predators to migrate into neighboring fields, regardless of the particular crop in that adjacent field.