Chhandak Basu

Abstract: The plant secondary metabolite, β-caryophyllene, is a ubiquitous component of many plant resins that has traditionally been used in the cosmetics industry to provide a woody, spicy aroma to cosmetics and perfumes. Clinical studies have shown it to be potentially effective as an antibiotic, anesthetic, and anti-inflammatory agent. Additionally, there is significant interest in engineering phototrophic microorganisms with sesquiterpene synthase genes for the production of biofuels. Currently, the isolation of β-caryophyllene relies on purification methods from oleoresins extracted from large amounts of plant material. An engineered cyanobacterium platform that produces β-caryophyllene may provide a more sustainable and controllable means of production. To this end, the β-caryophyllene synthase gene (QHS1) from Artemisia annua was stably inserted, via double homologous recombination, into the genome of the cyanobacterium Synechocystis sp. strain PCC6803. Gene insertion into Synechocystis was confirmed through PCR assays and sequencing reactions. Transcription and expression of QHS1 were confirmed using RT-PCR, and synthesis of β-caryophyllene was confirmed in the transgenic strain using GC-FID and GC-MS analysis.

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Abstract: Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. Since SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available which can be used free of charge to design desirable SYBR® Green based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design and provides a step-by-step protocol for designing SYBR® Green based primers with free, online software.

Abstract: Weeds play an important role in agriculture and molecular techniques are useful to help understand traits that contribute to weediness and weeds' interactions with the environment. A total of 377 expressed sequence tags (ESTs) from a modest library were arranged into 227 unique fragments and 61 contigs, which consisted of two or more ESTs. From blastx results, we mapped and annotated unigenes using the gene ontology vocabulary according to biological process, cellular component and molecular function. These were then compared to a reference set of Arabidopsis thaliana sequences for statistically significant over- or underrepresented genes. The sequences were also compared against multiple protein databases for similarity of functional domains. Overall, the S. iberica sequences showed high similarity to response to stress, which included salt-induced proteins, betaine aldehydehyde dehydrogenase and calcium binding proteins. Only a modest number of transcripts were sequenced, however the results presented here demonstrate the metabolic versatility of S. iberica in sub-optimal conditions that are likely to contribute to its cosmopolitan distribution. Here we propose that an EST library of an economically important weed species could be used to understand the weed's interactions with the environment.

Abstract: The biosphere is the major source and sink of nonmethane volatile organic compounds (VOCs) in the atmosphere. Gas-phase chemical reactions initiate the removal of these compounds from the atmosphere, which ultimately proceeds via deposition at the surface or direct oxidation to carbon monoxide or carbon dioxide. We performed ecosystem-scale flux measurements that show that the removal of oxygenated VOC via dry deposition is substantially larger than is currently assumed for deciduous ecosystems. Laboratory experiments indicate efficient enzymatic conversion and potential up-regulation of various stress-related genes, leading to enhanced uptake rates as a response to ozone and methyl vinyl ketone exposure or mechanical wounding. A revised scheme for the uptake of oxygenated VOCs, incorporated into a global chemistry-transport model, predicts appreciable regional changes in annual dry deposition fluxes.

Abstract: Copaiba (Copaifera officinalis) is a tropical plant that is also known as the ‘diesel tree’, previously noted for production of diesel-like oleoresin. The advancements in molecular tools such as expressed sequence tag (EST) library construction provide a novel opportunity for insight into the physiology of this tree. We generated a small set of ESTs for a young copaiba, sequencing and annotating a total of 613 unigenes. Of these, 84% showed similarity to the National Center for Biotechnology Information (NCBI) database. Annotation showed 70% of unigenes had at least one associated Gene Ontology (GO) term. We found a majority of ESTs to be associated with heat response genes. Based on these data, this EST library of C. officinalis represents a small but important step in helping to understand the general physiology and heat-response expression patterns. Additionally, this small collection of EST offers a modest starting point in helping to understand this enigmatic tropical plant.

Abstract: Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 microL of labeled probe was sufficient to hybridize onto 1-10 microg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 microL of labeled probe was not able to hybridize with 1 microg of target DNA, although 2 microL of labeled probe was able to detect target DNA ranging from 2 to 10 microg. To test the efficacy of our optimization protocol, we used 1 microL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants.

Abstract: Transcription factors (TFs) are proteinaceous complex, which bind to the promoter regions in the DNA and affect transcription initiation. Plant TFs control gene expressions and genes control many physiological processes, which in turn trigger cascades of biochemical reactions in plant cells. The databases available for plant TFs are somewhat abundant but all convey different information and in different formats. Some of the publicly available plant TF databases may be narrow, while others are broad in scopes. For example, some of the best TF databases are ones that are very specific with just one plant species, but there are also other databases that contain a total of up to 20 different plant species. In this review plant TF databases ranging from a single species to many will be assessed and described. The comparative analyses of all the databases and their advantages and disadvantages are also discussed.

Abstract: Cryopreservation is a way to store elite quality plant germplasms. The exact mechanism of stress tolerance during cryopreservation is unknown. Unavavailability of a detailed protocol for understanding the molecular genetics of plant cryostress is a major obstacle in plant cryobiology research. This paper describes the methods of extraction of total RNA from cryogenically stored plant tissues accompanied by successful amplication of cDNAs by reverse transcriptase PCR. The whole process can be completed in two to three days. Through this protocol, several genes were identified which were differentially expressed during cryostress. This protocol will help researchers to pursue further research in the field of molecular genetics of plant cryostress. Interesting genes identified via these processes can be cloned and plants can be transformed for the purpose of trait enhancement and modification.

Abstract: The importance of terpenes in both nature and human application is difficult to overstate. Basic knowledge of terpene and isoprene biosynthesis and chemistry has accelerated the pace at which scientists have come to understand many plant biochemical and metabolic processes. The abundance and diversity of terpene compounds in nature can have ecosystem-wide influences. Although terpenes have permeated human civilization since the Egyptians, terpene synthesis pathways are only now being understood in great detail. The use of bioinformatics and molecular databases has largely contributed to analyzing exactly how and when terpenes are synthesized. Additionally, terpene synthesis is beginning to be understood in respect to the various stages of plant development. Much of this knowledge has been contributed by the plant model, Arabidopsis thaliana. Considering the advances in plant terpene knowledge and potential uses, it is conceivable that they may soon be used in agrobiotechnology.

Abstract: Terpenes are defense chemicals found in wide groups of plants. Terpenoids play a large role in plant development and stress response. The terpene synthase family comprises a diverse set of genes, all which contribute to production of terpenoids. We have used tools of bioinformatics and performed an in silico analysis of developmental and tissue specific terpene synthase gene expression in Arabidopsis thaliana, as well as those expressed due to biotic and abiotic environmental stimuli. Using software tools from Genevestigator, a powerful microarray analyzer, we used multiple tool sets to better understand terpene synthase expression in Arabidopsis, which will hopefully open the genetic door to further wet laboratory investigations. The data can be used to predict roles of terpene synthase genes in plant cell division and growth. The data presented here can be used to model for terpene synthesis expression in other plant species and can also be used to integrate basic plant physiology, and ‘omics’ disciplines.

Abstract: The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha(-1) of glyphosate (i.e. Roundup) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 microM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F(1) hybrids between GR and GS horseweed. Thirty GSxGR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GSxGFP/GR F(1) hybrids produced F(2) seeds, and F(2) plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype.

Abstract: Bacterial wilt of Poa annua has been seen increasingly in the Northeast and mid-Atlantic United States in the past few years. The disease causes severe injury to putting greens and can kill large stands of turfgrass. For some time, however, both the bacterial origin of the disease and the causal agent were in doubt. In order to investigate the identity of the causal agent, isolation of the pathogen was undertaken and pathogenicity was confirmed using Koch's postulates on P. annua. Additional pathogenicity trials then were undertaken to determine the host range of the causal bacterium. Ability of the bacterium to cause disease was restricted to P. annua var. annua and P. attenuata. However, the bacterium was able to survive asymptomatically in vascular systems of P. annua var. reptans and P. trivialis. Experiments to determine the optimal growth temperature of the organism demonstrated that the bacterial growth peaked between 30 and 35°C. Fatty acid analysis suggested that the bacterium might be a species of Xanthomonas but was inconclusive. Ribosomal RNA analysis demonstrated significant similarity to the American Type Culture Collection isolate of Xanthomonas translucens pv. poae at 99.8%. Comparison of the host range to previously reported data agrees with our molecular findings and indicates that the likely casual organism of bacterial wilt of annual bluegrass is X. translucens pv. poae.

Abstract: In spite of the large yield losses that weeds inflict on crops, we know little about the genomics of economically important weed species. Comparative genomics between plant model species and weeds, map-based approaches, genomic sequencing and functional genomics can play vital roles in understanding and dissecting weedy traits of agronomically important weed species that damage crops. Weed genomics research should increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make weeds a successful group of plants. Here, we propose specific weed candidates as genomic models, including economically important plants that have evolved herbicide resistance on several occasions and weeds with good comparative genomic qualities that can be anchored to the genomics of Arabidopsis and Oryza sativa.

Abstract: The beta-glucuronidase (GUS) gene has been successfully used as a reporter gene in innumerable number of plant species. The functional GUS gene produces blue coloration in plants upon integration into the plant genome. Because of the ease it provides to analyze the gene expression (as no expensive equipment is needed), GUS gene is surely plant biotechnologist's first choice as a reporter gene. The turfgrass family contains the world's most economically important horticultural crops. There is a world-wide drive for genetic modification of grasses due to its huge economic importance. GUS gene can be transiently or stably expressed in grasses for the purpose of promoter analysis and to study tissue-specific and developmental gene expression. This paper summarizes the use of GUS gene for transient and stable expression studies in various turfgrass species.

Abstract: Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When beta-glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus.

Abstract: Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.

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Abstract: Parameters for use of the PDS-1000/He (Bio-Rad, USA) particle delivery system were optimized for routine delivery of transgenes into turfgrass cells in an attempt to standardize methods for routine and efficient genetic transformation of turfgrass species (creeping bentgrass, cv. Penn A4). Among various callus types produced on callus induction medium, embryogenic callus was identified using electron microscopy. Suspension cultures of Penn A4 were bombarded using a ubiquitin rice promoter-GUS gene construct. Based on the number of ‘GUS hits’, the following conditions were determined to be optimal for transfer of foreign gene constructs into turfgrass cells: 1.0 μm gold, 7 μl DNA/ shot and 1100 psi rupture disc type. Transient GUS expression patterns in turfgrass suspension culture cells driven with three different promoters (PGEL1, ubiquitin rice-GUS and ubiquitin corn-GUS) were also evaluated. The ubiquitin rice promoter was determined to be an ideal promoter for driving high levels of foreign gene expression in turfgrass cells. This observed variation in reporter gene (GUS) expression provides important information regarding variability of promoter strengths and expression patterns that will be important when choosing a strong promoter for gene transfer experiments targeted for trait modification or enhancement in turfgrass.

Abstract: Embryogenic callus was generated from seeds of turfgrass species (creeping bentgrass, cv. Penn A4 and Providence) on tissue culture medium. After 12 weeks, embryogenic calli were identified and used for bombardment with chimeric gene constructs that confer phosphinothricin resistance (bar) as a selectable marker and β-glucuronidase (GUS) as a reporter gene. After bombardment, the calli were transferred to selection medium and then to regeneration medium. Putative transgenic turfgrass plants were generated and molecular analysis (PCR) was performed to confirm presence of the transgene (bar, GUS). We analyzed reporter gene (GUS) expression in turfgrass cells over a developmental time period of 15 days following microprojectile bombardment with a plasmid containing a GUS reporter gene driven by the ubiquitin rice promoter. Varietal differences for tissue developmental stage specific patterns of reporter gene expression have been observed. We observed transient expression patterns of reporter genes (GUS) driven by four different promoters (ubiquitin rice, ubiquitin corn, ubiquitin-3 potato and CaMV 35S) in calli, root and leaf tissue of turfgrass. The ubiquitin rice promoter driving GUS produced the highest number of GUS spots compared to three other promoters both in young and mature leaves. We analyzed GUS expression at the two different developmental stages of the leaves, young leaf vs. fully mature leaf. Dramatically higher GUS expression was observed in mature leaves compared to young leaves following microprojectile bombardment. From these observations we can say that transient expression of a reporter gene (GUS in this case) does change considerably with the developmental stage of the creeping bentgrass plant. Fully expanded mature leaves were found to be much more efficient in GUS expression than young leaves. We have successfully inhibited in vitro endogenous nucleolytic activity in turfgrass cells by different nuclease inhibitors (0.6% heparin, aurintricarboxylic acid, polyethylene glycol and spermidine). We have observed more than a 1000-fold enhancement of GUS expression in 0.6% heparin treated 14-month-old Penn A4 callus compared to equivalent aged untreated callus. These results will lead to development of efficient strategies of use of biotechnology for trait enhancement and modification of turfgrass.