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Agrobacterium rhizogenes-induced cotton (Gossypium hirsutum L.) hairy roots were evaluated as a model system for studying molecular cotton-nematode interactions. Hairy root cultures were developed from the root-knot nematode (RKN) (Meloidogyne incognita [Kofoid and White] Chitwood, race 3)-resistant breeding line M315 and from the reniform nematode (RN) (Rotylenchulus reniformis Linford & Oliveira)-resistant accession GB713 (G. barbadense L.) and compared to a nematode-susceptible culture derived from the obsolete cultivar DPL90. M315, GB713, and DPL90 hairy roots differed significantly in their appearance and growth potential; however, these differences were not correlated with transcript levels of the A. rhizogenes T-DNA genes rolB and aux2 which help regulate hairy root initiation and proliferation. DPL90 hairy roots were found to support both RKN and RN reproduction in tissue culture, whereas M315 and GB713 hairy roots were resistant to RKN and RN, respectively. M315 hairy roots showed constitutive up-regulation of the defense gene MIC3 (Meloidogyne Induced Cotton3) compared to M315 whole-plant roots and DPL90 hairy roots. Our data show the potential use of cotton hairy roots in maintaining monoxenic RKN and RN cultures and suggest hairy roots may be useful in evaluating the effect of manipulated host gene expression on nematode resistance in cotton.

Hairy root cultures were induced by inoculating cotyledonary leaves and hypocotyl segments from two cotton species, Gossypium hirsutum and Gossypium barbadense, with Rhizobium rhizogenes 15834. For both species, more hairy roots formed on inoculation sites on cotyledonary leaves than on hypocotyls. The addition of sucrose to basal Murashige–Skoog media increased the frequency of hairy root formation, whereas the addition of naphthalene acetic acid (0.54 μM) did not. After transfer to a liquid culture, hairy root growth was very rapid. After 3 wk in liquid culture, both cotton species produced gossypol, a di-sesquiterpene secondary metabolite with known anticancer activity, and two related methylated derivatives. Most (60–95%) gossypol produced by cultures was retained within the hairy root tissues, but some was found in the media. The average gossypol level observed among 96 different cultures was 15 mg/g of dry culture mass; however, some cultures produced >40 mg/g of dry culture mass. Variation in gossypol levels was greater for cultures arising from different transformation events than for multiple subclones of a single transformant. The high level of gossypol production attained by most of these cultures suggests that they will be valuable for studying the biochemical and molecular aspects of gossypol biosynthesis, capable of producing large amounts of gossypol and related compounds, and useful for generating modified forms of gossypol (e.g., radio-labeled gossypol) for understanding bioactivity mechanisms.

There are only a few cells in the plant kingdom that are as exaggerated in their size or composition as cotton fibers. It is precisely their highly elongated structure and exceptional chemical make-up that establishes cotton fiber as an ideal model for studies of plant cell elongation and cell wall

Nearly 30 years ago the conditions for culturing immature cotton ovules were established to serve as a working research tool for investigating the physiology and biochemistry of fiber development. Not only has this tissue culture method been employed to characterize the biochemistry of plant cell expansion and secondary cell wall synthesis, but ovule cultures have contributed to numerous other aspects of plant cell physiology and development as well. In addition to basic studies on fiber development, cotton ovule cultures have been used to examine plant-fungal interactions, to model low temperature stress responses, to elucidate the pathways responsible for pigment formation in naturally pigmented fiber and to probe how cytoskeletal elements regulate cell wall organization. Success in rescuing Gossypium interspecific hybrids was dependent on ovule culture media formulations that could support early embryo development in ovulo. As tissues produced in culture are analyzed by increasingly more sophisticated techniques, there appear to be some differences between ovule growth in planta and ovule growth in vitro. Discerning how ovule culture fiber development is different from fiber development in field-grown plants can contribute valuable information for crop improvement. Cotton ovule cultures are an especially attractive model system for studying the effects of gravity on cell elongation, cellulose biosynthesis and embryo development and are excellent targets for examining transient expression of introduced gene constructs. With only minor modification, the procedure originally described by C. A. Beasley and I. P. Ting for growing cotton ovules in vitro will continue to be useful research tool for the foreseeable future.

Multiple isoforms of α‐ and β‐tubulin accumulate in higher plant cells, including cotton (Gossypium hirsutum L.) fiber. Isotypes may originate either by transcription of distinct genes or by posttranslational modification of gene products. The existence of two types of posttranslational modification in cotton fiber α‐tubulin has been examined by immunoblotting cotton fiber proteins from two developmental stages and by probing with specific monoclonal antibodies to acetylated (6‐11B‐1) or tyrosinated (YL 1/2 ) α‐tubulin. Control experiments were conducted with an antibody (YOL 1/34) that recognizes a conserved region in plant and animal α‐tubulins. No acetylated forms of α‐tubulin were found in either of two varieties of cotton fiber at 10 or 20 d postanthesis. One isotype of α‐tubulin, isotype 6, failed to cross‐react with YL 1/2 at 10 d postanthesis, which indicated that the protein was detyrosinated, and isotype 8 appeared to be detyrosinated at 20 d postanthesis. Since the carboxyl terminus of higher p...

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate α-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of α-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that α-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total α-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the α-tubulins was specific to fiber cells.

Young cotton (Gossypium hirsutum) ovules will produce fiber in vitro when floated on a defined culture medium. Our laboratory is interested in examining the effects of altered gravity environments on fiber development as a model for the effects of gravity on cell expansion and cellulose biosynthesis. Since liquid culture media are unsuitable for altered gravity experiments, addition of gelling agents to cotton ovule culture media is necessary. In this study we have systematically examined the effects of four gelling agents at several concentrations on fiber production in culture. A rapid screening method using toluidine blue O staining indicated that after 3 wk in culture, fiber growth on 0.15% (wt/vol) Phytagel™ medium was similar to fiber growth on liquid medium. More detailed analysis of fiber development revealed that fiber length was not influenced by the addition of Phytagel™. Accumulation of cellulose, however, was reduced 50–60% compared with fibers produced in liquid media after 3 wk in culture. The fiber cellulose content rose with additional time in culture for both solid and liquid media treatments. By 4 wk in culture, the difference in cellulose content of fiber cell walls grown on solid versus liquid media was less than 20%. This variance in growth response on gelled media could be due to differences in media matric potential, to the immobility of ions trapped within the gel, or to toxicity of contaminants copurifying with Phytagel™. By identifying why ovule growth and fiber cellulose biosynthesis are reduced in cultures grown on gelled media, it will be possible to reveal new information about these processes in system that is less complicated than physiological systems at the whole plant level.

Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca2+-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.

When cotton (Gossypium hirsutum, DPL 90ne) ovules are removed from the carpel on the day of anthesis and placed on an appropriate medium, fiber cells will differentiatein vitro from the ovule epidermis. Alpha-amanitin, an inhibitor of poly (A)+ RNA synthesis was added to ovule cultures at selected times after culture initiation to determine the timing of events in fiber development. Replicate cultures were initiated from day of anthesis, cotton ovules with α-amanitin being added to media at final concentrations ranging from 0.05μg/ml to 5.0 μg/ml. After 21 d in culture, ovules were evaluated for fiber length, percentage of ovules producing fiber, fiber dry weight, and fiber cellulose content. Cultures treated with α-amanitin on the day of anthesis to 2 d postanthesis were prevented from forming normal numbers of fiber cells. Addition of α-amanitin to cultures between 2 and 10 d postanthesis reduced the length that fiber cells attained in culture after 21 d. Addition of α-amanitin to cultures at 4 d postanthesis had as much an effect on the accumulation of cellulose in the fiber cell wall as the later addition of the inhibitor at 8 to 10 d postanthesis. The inhibitory effect of α-amanitin on fiber growth was reversible when ovules were transferred to amanitin-free medium within 2 d after culture initiation. Ovules placed on media containing α-amanitin for 5 d or longer could not overcome the inhibitory effects when transferred to media lacking the inhibitor. These results indicate critical time periods when RNA transcripts important for specific phases of development are being synthesized.

Our objective was to develop a rapid, specific, and inexpensive method for measuring glucose concentrations in bolls of cotton (Gossypium hirsutum L.). A new procedure was developed, based on a reagent strip for measuring blood glucose in diabetic patients, and compared with three other glucose methods by assaying glucose standard solutions. The strip results were significantly correlated (r = 0.948) with known concentrations of glucose and comparable to other methods in both precision and accuracy. For aqueous extracts of locules (lint and seed) from 15 days-post-anthesis (DPA) cotton bolls, glucose levels were ∼ 10 mg g −1 fresh weight. The raegent strip strongly correlated to glucose hexokinae, glucose oxidase, and anthrone methods with correlation coefficients (r) of 0.983, 0.994, and 0.974, respectively. Thus, the strip technique should be useful for routine glucose determinations in cotton bolls considering the time (∼ 2 min/sample), cost (∼ $0.70/sample), and portability

Gossypium hirsutum cellulose synthase catalytic subunit 4 (GhCesA4) plays an important role in cellulose biosynthesis during cotton fiber development. The transcript levels of GhCesA4 are significantly up-regulated as secondary cell wall cellulose is produced in developing cotton fibers. To understand the molecular mechanisms involved in transcriptional regulation of GhCesA4, β-glucuronidase (GUS) activity regulated by a GhCesA4 promoter (-2574/+56) or progressively deleted promoters were determined in both cotton tissues and transgenic Arabidopsis. The spatial regulation of GhCesA4 expression was similar between cotton tissues and transgenic Arabidopsis. GUS activity regulated by the GhCesA4 promoter (-2574/+56) was found in trichomes and root vascular tissues in both cotton and transgenic Arabidopsis. The -2574/-1824 region was responsible for up-regulation of GhCesA4 expression in trichomes and root vascular tissues in transgenic Arabidopsis. The -1824/-1355 region negatively regulated GhCesA4 expression in most Arabidopsis vascular tissues. For vascular expression in stems and leaves, the -898/-693 region was required. The -693/-320 region of the GhCesA4 promoter was necessary for basal expression of GhCesA4 in cotton roots as well as Arabidopsis roots. Exogenous phytohormonal treatments on transgenic Arabidopsis revealed that phytohormones may be involved in the differential regulation of GhCesA4 during cotton fiber development.

Growth and development of cotton fibers in a developmental mutant, Ligon-lintless, and its near isogenic wild type, Texas Marker-1, were compared in ovule and cell suspension cultures. In both organ and cell cultures the pattern of growth of fiber cells from the two genotypes mimicked the pattern ofin vivo growth. The timing of fiber cell initiation soon after anthesis in Ligon-lintless suggests that the fiber cells on this mutant are analogous to the commercially important lint fibers. Length distributions of elongated cells from cell suspension culture of Ligon-lintless and Texas Marker-1 indicate that the length attained in culture is affected by the genotype of the explant tissue.