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PubMed2004

STS markers linked to the Rf1 fertility restorer gene of cotton.

Marker-assisted selection (MAS) can accelerate the process of plant breeding, and sequence-tagged site (STS) markers are highly specific for regions of DNA being used for MAS. The objective of this research was to develop STS markers tightly linked with Rf1, the fertility restoring gene for cytoplasmic male sterility (CMS) in cotton (Gossypium hirsutum L.). Bulked segregant analysis was employed to screen for Rf1-linked RAPD markers in a backcross population. Four RAPD markers were identified, three of which co-segregated with Rf1 (UBC147(1400), UBC607(500), and UBC679(700)). Another fragment, UBC169(800), co-segregated with the previously reported UBC169(700) in repulsion phase at a distance of 4.5 cM from Rf1. A marker published by others (UBC659(1500)) mapped to 2.7 cM from Rf1 and 1.8 cM from UBC169(800). Four sets of STS primer pairs were designed based on the RAPD fragment sequences. The primer pairs from the UBC147(1400) and UBC607(500) fragments both amplified a single fragment specific to fertile plants. The UBC679(700) and UBC659(1500) STS primer pairs each amplified one fragment specific to fertile plants and a monomorphic fragment. These four Rf1-linked STS markers were also present in the Rf1 donor species G. harknessii (D2-2). The three primer pairs that produced co-segregating STS markers also amplified fragments from G. trilobum (D8). However, the D8 fragment amplified by the UBC147(1400) STS primers was larger than that from D2-2, and G. trilobum does not restore fertility to CMS-D2-2 lines. These STS markers will be useful in the development of restorer parental lines in cotton CMS breeding efforts.

Chromosome Mapping↗
PubMed2004

Preconcentration technique for nonylphenol using cellulose cotton with homogenous liquid-liquid extraction for liquid chromatographic analysis.

A powerful preconcentration method for nonylphenol (NP) has been developed for liquid-chromatography by combining the use of cellulose cotton (solid-phase extraction) with homogeneous liquid-liquid extraction. A 100 ml of sample solution was preconcentrated using cotton, and the eluate obtained (acetonitrile; 5 ml) was further preconcentrated to 50 microl within 10 min using a homogeneous liquid-liquid extraction method (volume ratio, 2,000-fold; 100 ml--> 50 microl). The sample concentration increases from preconcentration was 1,599-fold, and NP was extracted into the sedimented phase at 80%. The proposed method was applied to high performance liquid chromatography with fluorescence detection (FL/HPLC); the lowest determination limit obtained was 1.0 x 10(-9) mol l(-1).

Cellulose↗
PubMed2001

Cotton-induced pseudotumor of the femur.

Twenty-five years prior to presentation a 41-year-old man had a femoral fracture stabilized with a 4.5 mm AO/ASIF steel plate. The femur healed uneventfully and the patient was asymptomatic for the following 20 years. He then noticed a slow-growing swelling of the left thigh associated with a degree of weakness. Radiographs of the femur 25 years after fracture stabilization showed a massive expansive osteolytic process surrounded by a rim of bone. Magnetic resonance imaging (MRI) confirmed the presence of a large tumor. Since malignancy could not be excluded the patient underwent incisional biopsy. The histologic findings were nonspecific. Because of persistent symptoms the lesion was marginally excised. Intraoperatively a folded cotton sponge was found adjacent to the femur. Histopathologic investigation confirmed a foreign body reaction probably related to the retained cotton sponge. Reactive, foreign-body-induced change may mimic bone and or soft tissue malignancies.

Adult↗
PubMed2003

Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker 310 FR).

In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were beta-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.

Cells, Cultured↗
PubMed2003

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants.

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Bacillus thuringiensis Toxins↗
PubMed2001

A putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers.

A novel plant gene CFL1 was cloned from cotton (Gossypium hirsutum L.) fibers by expressed sequence tag (EST) database searching and 5'-RACE (rapid amplification of cDNA ends). This gene shows sequence homology with FKS1 which has been identified as the putative catalytic subunit of the yeast beta-1,3-glucan synthase. It encodes a protein (CFL1p) of 219 kDa with 13 deduced transmembrane helices and 2 large hydrophilic domains, one of which is at the N-terminus and the other in the internal region of the polypeptide. CFL1 displays 21% identity and 41% similarity to FKS1 at the amino acid level over its entire length, with 31% identity and 52% similarity for the hydrophilic central domain. Using RNA and protein blot analysis, CFL1 was found to be expressed at higher levels in cotton fibers during primary wall development. CFL1 also had a strong expression in young roots. Using a calmodulin (CaM)-gel overlay assay, the hydrophilic N-terminal domain of CFL1p was shown to bind to CaM, while the hydrophilic central domain did not. A putative CaM-binding domain, 16 amino acids long, was predicted in the hydrophilic N-terminal domain. Moreover, a product-entrapment assay demonstrated that a protein associated with an in vitro-synthesized callose pellet could be labeled by anti-CFL1 antibodies. Our finding suggests that CFL1 is a putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 and could be involved in callose synthesis.

Calmodulin-Binding Proteins↗
PubMed2001

Heat stress induces the synthesis of a new form of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in cotton leaves.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activase mRNA and protein synthesis were measured in the leaves of cotton (Gossypium hirsutum L.) plants under control (28 degrees C) or heat-stress (41 degrees C) conditions. A decline in activase transcript abundance occurred rapidly during the photoperiod and was unaffected by heat stress. In response to high temperature, de novo protein synthesis rapidly shifted from mainly expression of Rubisco large and small subunits to the major heat-shock proteins, while de novo synthesis of the constitutively expressed 47- and 43-kDa activase polypeptides was not appreciably altered. However, heat stress induced the synthesis of a 46-kDa polypeptide that immunoprecipitated with antibodies monospecific to activase. Expression of the 46-kDa polypeptide ceased within 1 h of the return of heat-stressed plants to control conditions. Activase precursors of 55 and 51 kDa were detected among the in vitro translation products of RNA from control and heat-stressed plants. In addition, a 53-kDa polypeptide that also immunoprecipitated with anti-activase IgG was among the in vitro translation products of RNA from heat-stressed plants. This putative activase precursor did not occur among the in vitro translation products of RNA from plants that had recovered from heat stress. The levels of the constitutive 47- and 43-kDa activase polypeptides were similar in control and heat-stressed plants, based on immunoblotting with antibodies to activase. However, a 46-kDa cross-reacting polypeptide was also present in heat-stressed plants and constituted about 5% of the total activase after 48 h at high temperature. The identity of the heat-induced 46-kDa polypeptide as activase was confirmed by protein sequencing, which showed that its N-terminal sequence was identical to that of the constitutive 47-kDa activase polypeptide. The presence of multiple isoforms for both the 47- and 43-kDa activase polypeptides on immunoblots of two-dimensional gels and the complex banding pattern on Southern blots together suggest the existence of more than one activase gene and the possibility that the synthesis of the heat-induced activase polypeptide may be regulated transcriptionally. Induction of a new form of activase may constitute a mechanism of photosynthetic acclimation to heat stress in cotton.

Antibodies↗
PubMed2005

Wide-cross whole-genome radiation hybrid mapping of the cotton (Gossypium barbadense L.) genome.

Whole-genome radiation hybrid mapping has been applied extensively to human and certain animal species, but little to plants. We recently demonstrated an alternative mapping approach in cotton (Gossypium hirsutum L.), based on segmentation by 5-krad gamma-irradiation and derivation of wide-cross whole-genome radiation hybrids (WWRHs). However, limitations observed at the 5-krad level suggested that higher doses might be advantageous. Here, we describe the development of an improved second-generation WWRH panel after higher dose irradiation and compare the resulting map to the 5-krad map. The genome of G. hirsutum (n = 26) was used to rescue the radiation-segmented genome of G. barbadense (n = 26) introduced via 8- and 12-krad gamma-irradiated pollen. Viable seedlings were not recovered after 12-krad irradiation, but 8-krad irradiation permitted plant recovery and construction of a 92-member WWRH mapping panel. Assessment of 31 SSR marker loci from four chromosomes revealed that the 8-krad panel has a marker retention frequency of ca. 76%, which is approximately equivalent to the rate of loss in a low-dose animal radiation hybrid panel. Retention frequencies of loci did not depart significantly from independence when compared between the A and D subgenomes, or according to positions along individual chromosomes. WWRH maps of chromosomes 10 and 17 were generated by the maximum likelihood RHMAP program and the general retention model. The resulting maps bolster evidence that WWRH mapping complements traditional linkage mapping and works in cotton, and that the 8-krad panel complements the 5-krad panel by offering higher rates of chromosome breakages, lower marker retention frequency, and more retention patterns.

Chromosome Mapping↗
PubMed2004

Chlorfenapyr resistance in two-spotted spider mite (Acari: Tetranychidae) from Australian cotton.

The responses of Tetranychus urticae Koch from Australian cotton to chlorfenapyr has been monitored since the 1997--1998 growing season. Resistance was first detected in the 2001--2002 season and then increased quickly in both level and proportion of resistant strains detected. In response, the resistance management strategy for chlorfenapyr use in cotton was altered and now recommends a further restriction of use from two to one spray per season. There was no evidence of negative cross-resistance to the pyrethroid bifenthrin, but chlorfenapyr was associated with an undefined negative cross-resistance.

Animals↗
PubMed2006

Verticillium lecanii spore formulation using UV protectant and wetting agent and the biocontrol of cotton aphids.

Verticillium lecanii spores (10(8 )spores ml(-1)) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.

Animals↗
PubMed2006

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker.

The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T(0) plants and T(1) progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T(0) and T(1 )transgenic plants towards glyphosate was also observed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Population structure of the boll weevil in cotton fields and subtropical forests of South America: a bayesian approach.

The main goal of this contribution is to investigate the genetic structure of boll weevil populations from South America (Argentina and Brazil) and to make further comparisons with a putative source population from USA. Samples were collected in a Paranaense forest under reserve protection, cotton fields and non-cultivated areas. Data from anonymous molecular markers were analysed using both traditional methods of population genetics and Bayesian approaches. Results help to support a previous hypothesis on the presence of two lineages of boll weevil populations in South America: one with characteristics of recent invaders and the other with characteristics of ancient populations. The sample from Urugua-í Provincial Park (Misiones, Argentina) shows the highest percentage of polymorphic loci, the highest values of mean heterozigosity, and the largest number of population-specific alleles, all being typical features of ancient populations. Furthermore, the Urugua-í sample shows two gene pools occurring in sympatry, probably as a consequence of a secondary contact. The remaining samples reveal not only lower percentages of polymorphic loci and heterozygosity values, but also an almost negligible presence of specific alleles. Bayesian methods also suggest the occasional migration of some individuals of ancient lineages from their natural habitats in fragments of the Paranaense forest into cotton fields, and vice versa.

Animals↗
PubMed2004

Evaluation of transgenic cotton varieties and a glyphosate application on seedling disease incidence.

A study was conducted to determine whether stand densities of transgenic cotton (Gossypium hirsutum) varieties, with or without glyphosate, were similar to conventional varieties of the same lineage group in Georgia and Mississippi. Transgenic and conventional cotton varieties were placed into five lineage groups of related varieties and seedling disease was evaluated in three greenhouse tests and a field trial using Rhizoctonia solani AG-4. Seed vigor was determined by standard germination studies were conducted evaluating conventional and transgenic varieties of similar lineage. Results showed that no interactions occurred for the heights and dry weight data across treatments within the lineage groups in any of the experiments. No interactions were shown between stand densities at different inoculum rates and inoculated versus uninoculated pots (plots). Across all greenhouse studies, stand counts of PM 1220 were similar to the transgenic varieties PM 1220 RR and PM 1220 B/RR with or without a glyphosate application. In the field trial, PM 1220 B/RR + glyphosate had significantly lower stands than all other treatments expect PM 1220 RR (no glyphosate treatment) prior to and after glyphosate application. Stand densities for varieties within the lineage group DPL 5415 were also inconsistent when compared between the greenhouse and field trials with no apparent trends occurring. However, the Coker 312 varieties containing glyphosate tolerance had consistently lower stand counts compared to the conventional variety of Coker 312 but only during the greenhouse studies. Seed germination of Coker 312 could not be correlated with either the greenhouse or field trial data. In general, the commercially available varieties such as PM 1220, DPL 5690, DPL 5415, and DPL 50 with glyphosate tolerance had similar stand count, height, and dry weight data when compared to the conventional varieties from the same lineage group regardless of a glyphosate application. When differences did occur, no consistent trends could be determined within these four lineage groups.

Crops, Agricultural↗
PubMed2006

Isolation and characterization of genes associated to cotton somatic embryogenesis by suppression subtractive hybridization and macroarray.

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and pre-globular somatic embryos, as the tester, and cDNA from non-embryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 uni-genes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, post-transcription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from pro-embryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

Blotting, Northern↗
PubMed2004

Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura.

A hybrid delta-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known delta-endotoxins. The hybrid delta-endotoxin was created by replacing amino acid residues 530-587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated delta-endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the best known delta-endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC50 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid delta-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1-0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid delta-endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC50 58 ng/ml diet) compared to expression in E. coli.

Amino Acid Sequence↗
PubMed2002

Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice.

The majority of the human population has been infected with herpes simplex virus type 1 (HSV-1). During a typical primary episode, HSV-1 spreads from the oral pharynx to the trigeminal ganglia, where a latent HSV-1 infection is established. Cold sores at the mucocutaneous junction of the lip are the typical manifestation of recurrent HSV-1. We investigated whether HSV-1 also infects the brain during the primary infection. We used HSV-1 infected BALB/c mice and inbred cotton rats as models. While both species were susceptible to HSV-1 infection, the time course of lesion formation and healing in the cotton rat more closely reflected that seen in humans. In both species, HSV-1 replicated in the brainstem and cerebellum, as well as the trigeminal ganglia during a primary infection of the lip. The brain infection was produced by a low inoculation dose, and did not cause observable neurologic signs or mortality. Using PCR and RT-PCR techniques, we demonstrated HSV-1 thymidine kinase in the absence of infectivity in the brains of both species 30-40 days after primary infection.

Animals↗
PubMed1992

Further characterization of reversal of signs of induced cotton effects of dicumarol derivatives-alpha 1-acid glycoprotein systems by protriptyline.

The interaction of dicumarol derivatives and protriptyline with respect to the binding to alpha 1-acid glycoprotein (AGP) has been investigated by circular dichroism (CD), equilibrium dialysis and ultrafiltration. Investigation of the induced CD spectra of dicumarol derivatives bound to AGP indicated that the conformations of these compounds were different when bound to AGP. Though all the dicumarol derivatives, protriptyline and AGP formed a ternary complex, interaction modes were different, depending upon the substituent groups at position 3 of the dicumarol molecule. On the basis of the protriptyline effect on the CD spectra of all dicumarol derivatives bound to AGP, the compounds were classified in the following way: (1) Dicumarol, ethylidenebis 4-hydroxycoumarin and propylidenebis 4-hydroxycoumarin caused reversal of the sign of ellipticity. This interaction was explained by cooperative binding. (2) Butylidenebis 4-hydroxycoumarin and pentylidenebis 4-hydroxycoumarin generated new band and disappeared ellipticity of the original Cotton effect. This interaction was also explained by the cooperative binding mode. (3) Ethylbiscoumacetate which generated the CD band similar to that of dicumarol in the absence of protriptyline, reversed the sign of the CD spectrum only at 325 nm. The interaction was anticooperative in nature. (4) Benzylidenebis 4-hydroxycoumarin represented type four which had no change in the CD spectrum by the addition of protriptyline. This interaction was explained by the two-state model accompanying the conformational change of AGP. These results suggested that all compounds, except for benzylidenebis 4-hydroxycoumarin, induced negative Cotton effects at 325 nm by taking the same asymmetrical perturbation by the addition of protriptyline and the interaction was carried out according to model 2. An attempt to study the interaction mechanism of two or more drugs with regard to the binding to protein using these models is thought to help in understanding drug-protein interactions.

Binding Sites↗
PubMed1983

Effects of anti-inflammatory drugs on urinary excretion of prostaglandin metabolites in cotton pellet inflammation.

The influence of cotton pellet inflammation in rats and the effect of prednisolone and indomethacin on the excretion of the main urinary metabolite (MUM)s of prostaglandin (PG) F and E types were studied. After cotton pellet implantation, the excretion of PGE-MUM and F-MUM was progressively increased. In the early phase of inflammation, the excretion of PGE-MUM and PGF-MUM was suppressed by administration of indomethacin. However, prednisolone suppressed only PGF-MUM excretion in the early phase of inflammation. These results indicate that no direct interaction was obtained between the inhibition of the inflammatory response by prednisolone and indomethacin and PGs-MUM excretion in inflamed rats.

Animals↗