Small Amounts Of Plant Tissue Research Articles

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing

BackgroundAcidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants.ResultsA high-throughput sample preparation method for liquid chromatography–tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports.ConclusionThe method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry.

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson's disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one-two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g-1 FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.

A Rapid Method for Quantifying RNA and Phytohormones From a Small Amount of Plant Tissue.

Phytohormones are involved in most plant physiological processes and the quantification of endogenous phytohormone levels and related gene expressions is an important approach to studying phytohormone functions. However, the quantification of phytohormones is still challenging due to their extremely low endogenous level in plant tissues and their high chemical diversity. Therefore, developing a method to simultaneously quantify phytohormone levels and RNA would strongly facilitate comparative analyses of phytohormones and gene expression. The present work reports a convenient extraction protocol enabling multivariate analysis of phytohormones and RNA from small amounts of plant material (around 10 mg). This high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method demonstrates quantification of phytohormones and their related metabolites from four plant hormone classes: cytokinin, auxin, abscisic acid, and gibberellin. The UPLC-MS/MS method can quantify thirteen phytohormones and their metabolites simultaneously in 14 min. To validate the developed method, we determined the dynamic profiles of phytohormones and gene expressions in small axillary shoot buds in garden pea. This new method is applicable to quantification analysis of gene expression and multiple phytohormone classes in small amounts of plant materials. The results obtained using this method in axillary buds provide a basis for understanding the phytohormone functions in shoot branching regulation.

Application of a simplified method of chloroplast enrichment to small amounts of tissue for chloroplast genome sequencing.

Premise of the study:High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms.Methods:Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing.Results:Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully.Discussion:This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species).

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmolg-1 fresh weight). We have developed a simple microscale magnetic immunoaffinity-based method for selective one-step isolation of CKs from very small amounts of plant tissue (less than 0.1mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one-step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30-80% depending on individual CKs. Immunoaffinity extraction using group-specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry-based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N9 -glucosides) in 1.0-mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.

Standard Addition Quantitative Real-Time PCR (SAQPCR): A Novel Approach for Determination of Transgene Copy Number Avoiding PCR Efficiency Estimation

Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T0 tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis.

A fluorometric assay for trehalose in the picomole range.

BackgroundTrehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.ResultsA kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20–30 nmol g-1FW of trehalose, increasing to about 50–60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.ConclusionThe fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Ice Core Genetics Reveals Palaeo- Ecosystems in Greenland and Antarctica

Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal–plant feeding interactions in the soil.

Rapid and sensitive detection of auxins and flavonoids in plant samples by high‐performance liquid chromatography coupled with tandem mass spectrometry

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2-10 mg of the plant material was required to obtain quantitative results.

Detecting ingested plant DNA in soil-living insect larvae.

Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal–plant feeding interactions in the soil.

Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography–electrospray tandem mass spectrometry

We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites – phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7′-hydroxy-ABA (7′-OH-ABA), 9′-hydroxy-ABA (9′-OH-ABA), ABAaldehyde, and ABAalcohol – before analysis by a novel technique for these substances, ultra-performance liquid chromatography–electrospray ionisation tandem mass spectrometry (UPLC–ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol–water–acetic acid (10:89:1, v/v), simple purification by Oasis ® HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01–0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC–ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1–5 mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC–ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.

Extraction and labeling methods for microarrays using small amounts of plant tissue

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

Optimising DNA isolation for medicinal plants

In African traditional health care systems medicinal plants have long been known to contain pharmacologically active compounds. This has led to an excessively high demand of these plant products resulting in the extinction of some plant species. With the application of molecular techniques in plant diversity conservation becoming increasingly popular, the isolation of PCR amplifiable genomic DNA becomes an important pre-requisite. However, medicinal plants are known to contain high levels of polyphenols and polysaccharides posing a major challenge in the isolation of high quality DNA. The objective of our research was to optimize a cetyl-trimethyl ammonium bromide (CTAB)-based protocol for the extraction of genomic DNA from a range of medicinal plant species, namely Sclerocarya birrea (tree), Barleria greenii (shrub), Aloe polyphylla and Huernia hystrix (both succulent plants). The quantity of DNA (µg/g) isolated using the modified CTAB protocol was higher for the lower plant tissue amounts (0.1 and 0.2 g) per 500 µl of extraction buffer. The spectral quality of DNA as measured by the A 260/ A 280 ratio ranged from 1.76 to 2.14 for S. birrea, B. greenii and A. polyphylla and 1.39 to 1.74 for H. hystrix. The DNA purity was further confirmed by restriction endonuclease digestion and PCR gel electrophoresis using operon arbitrary decamer primers (OPB-05, OPB-06 and OPG-07). The results show that the optimization of the amount of plant tissue per extraction buffer volume is a critical factor in genomic DNA isolation. In all cases the isolated DNA yielded high quantities from small amounts of plant tissue, and had good spectral qualities amenable to restriction endonuclease digestion and PCR amplification.

Simultaneous Determination of Multiple Phytohormones in Plant Extracts by Liquid Chromatography−Electrospray Tandem Mass Spectrometry

A rapid multiresidue method to quantify three different classes of plant hormones has been developed. The reduced concentrations of these metabolites in real samples with complex matrixes require sensitive techniques for their quantification in small amounts of plant tissue. The method described combines high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Deuterium-labeled standards were added prior to sample extraction to achieve an accurate quantification of abscisic acid, indole-3-acetic acid, and jasmonic acid in a single run. A simple method of extraction and purification involving only centrifugation, a partition against diethyl ether, and filtration was developed and the analytical method validated in four different plant tissues, citrus leaves, papaya roots, barley seedlings, and barley immature embryos. This method represents a clear advantage because it extensively reduces sample preparation and total time for routine analysis of phytohormones in real plant samples.

Inexpensive, High Throughput Microplate Format for Plant Nucleic Acid Extraction: Suitable for Multiplex Southern Analyses of Transgenes

To achieve high throughput analysis of genomic DNA from small amounts of plant tissue, a standard nucleic acid extraction was formatted to microplates by linking a novel series of interlocking plates. The protocol integrates steel pellet tissue homogenization and unique liquid handling and phase separation techniques with minimal special equipment. The method was validated for difficult plant DNAs, such as hop (Humulus lupulus L.), soybean [Glycine max (L.) Merr.], and tobacco (Nicotiana tabacum L.), with agarose gel separations of restriction fragments and Southern blot analysis of transgene integrants in soybean. The range of success rates for Southern blots was 73 to 92% per sample per plate (n = 2016 samples). Variation in absolute yield was quantified by PicoGreen microplate flourimetry. The variation in yield among samples per plate was small enough to obviate individual sample concentration adjustment before Southern analysis. Average absolute yield for the barley (Hordeum vulgare L.) mapping population based on flourimetry was 6.8 μg ± 0.85 SE, n = 96; providing enough pure, stable DNA for many individual polymerase chain reaction (PCR) reactions. Intersample cross‐contamination and suitability for PCR analysis were validated by simple sequence repeat (SSR) display of a previously characterized barley mapping population. Cost calculation from a materials detail was U.S. $0.17 per sample extraction. Although the implementation requires some skill and practice, we find it to be a robust, adaptable, and versatile alternative to expensive commercial kits.

A mass spectrometry method for measuring 15N incorporation into pheophytin

The 15N content of pheophytin, the magnesium-free derivative of chlorophyll, can be measured with great accuracy and precision using positive-ion atmospheric pressure ionization electrospray mass spectroscopy following a simple solvent extraction of small amounts of plant tissue. The molecular weight of pheophytin prepared from Chlamydomonas reinhardtii grown in different ratios of 14N/ 15N showed linear regression with the isotopic input, with a precision of 0.5–1%. Using an isotope dilution strategy, we have shown that nitrogen fixation can contribute substantial 14N to pheophytin isolated from Medicago truncatula plants grown in symbiosis with Sinorhizobium meliloti. The assay is sensitive, precise, rapid, simple, and robust. These features suggest that it could become an important tool for measuring the contribution of symbiotic and associative nitrogen fixation to plant metabolism.

MICROCALORIMETRY: A NOVEL APPROACH TO DECISION MAKING IN CUT ROSE PRODUCTION

Since its innovative use by Lavoisier at the end of the eighteen-century, calorimetry has been one of the most important foundations of modern science. However, only recently, with the advent of modern and accurate sensors, has it to play a central role in the life sciences. The combination of calorimetric and respirometric studies resulted in a thermodynamic model that represents the rate of storage of chemical energy in structural biomass or Specific Growth Rate (RSG) that serves as an indicator for potential growth rate (PGR) of the tested tissue. Simultaneous measurements of metabolic heat rate (q) and respiratory heat production (RCO2) at the relevant range of environmental conditions such as temperature provides an insight into plant’s response to these factors and allow us to define their optimal range. The actual measurement is destructive in nature but with microcalorimetry only a small amount of plant tissue (2-3 mg DM) is required for each observation. This report demonstrates the use of calorespirometric analyses for two processes, using Rosa x hybrida ‘Kardinal’: • RSG as a function of temperature of young rose leaflets and sprouting axillary buds. • RSG as a function of moisture tension of young rose leaflets. Optimal temperature for young leaflets was found to be 25°C while that of young buds was below 15°C. Optimal temperature control in rose greenhouses is discussed in view of these results. The effect of physical characteristics of the root zone, such as moisture tension and unsaturated hydraulic conductivity K(h) on (Specific Growth Rate) RSG of young leaflets was evaluated at the optimal temperature. It was found that the higher the tension, the lower was the RSG. However, based on comparing the physical properties of 2 different media: UC mix and coir, it can be assumed that at high moisture tension, K(h) is more effective than moisture tension per se in moderating RSG.

Mini-scale method for nuclear run-on transcription assay in plants

We present a mini-scale method for nuclear run-on transcription assay. In our method, all the centrifuge steps can be carried out by using micro-tubes for short time (5 min each) throughout the process, including isolation of transcriptionally active nuclei and purification of labeled RNA after synthesis of RNA in isolated nuclei. The assay can be performed using a small amount of plant tissue, which enables analysis of developmental changes in transcriptional status of given genes in a single individual plant. Successful results were obtained using the tissues of flower and leaf of petunia and embryo of pea, suggesting that the method is potentially applicable to a variety of plant tissues.

Quantification of Bacillus thuringiensis Insect Control Protein Cry1Ac Over Time in Bollgard Cotton Fruit and Terminals

A sensitive quantitative bioassay was developed for the measurement of Bacillus thuringiensis Berliner α-endotoxin, CrylAc, in Bollgard cotton plant tissue. Cotton tissue samples were evaluated in a sensitive Heliothis virescens (F.) growth inhibition bioassay, with Cry1Ac levels being quantified by comparison with concurrently run concentration-response curves using purified Cry1Ac protein. The assay was amenable to large numbers of samples, used small amounts of plant tissue, and avoided some of the concerns associated with immunobased quantitative assays. Field studies were conducted during the 1995 growing season in which Cry1Ac levels were quantified in the Bollgard cotton line 531 (in Coker 312 background). Analyses were conducted to test for the effects of environment (field site differences), plant age, and tissue type on Cry1Ac concentrations. The small foliage of main terminals and the primary, or proximal, fruiting structure from node 9 (9th main stem branch from the bottom of the plant) were sampled at various intervals throughout the growing season at 6 field sites. Mean CrylAc levels in terminals and fruiting tissue differed significantly, with terminal foliage levels higher than node 9 fruit. Within each tissue type, significant differences among sites were also seen, suggesting environmental influence on either CrylAc production or stability. Mean Cry1Ac values in fruiting structures declined significantly from 57.1 μg/g dry weight at 53 d after planting to 6.7 μg/g at 116 d after planting. Mean terminal Cry1Ac levels also declined significantly from 163.4 μg/g to 34.5 μg/g at 53 d after planting and 116 d after planting, respectively. In general, Cry1Ac levels in the fruit and terminals of Bollgard cotton declined steadily as the growing season progressed, with greater activity always occurring in the terminal foliage.

Subtractive hybridization of cDNA from small amounts of plant tissue.

A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.