A rapid dehydration procedure with acidified 2,2-dimethoxypropane (DMP) has been successfully applied to immature or wet cotton fibers. The ultrastructural integrity and fiber quality parameters such as cell wall area and perimeter of developing and mature cotton fibers were well-preserved using this procedure as demonstrated by light, scanning and electron microscopy of whole fibers and cross-sections. Embedding in resin following DMP dehydration also prevented the collapse of the immature fibers due to drying of lumen.
Key words Cell wall, 2,2-dimethoxypropane, fiber quality, maturity, moisture, thin sections
Microscopic studies of cotton fibers should be undertaken ideally without altering standard fiber quality parameters such as perimeter and cell wall area during specimen preparation. This has been successfully accomplished on well-dried mature cotton fiber samples [1-4]. However, fiber quality parameters associated with growth and development of fibers have not been addressed hitherto due to problems such as rapid drying and collapse of immature fibers during sample preparation. Embedding and processing of immature as well as wet, mature fibers pose problems due to lengthy dehydration procedures and, as a result, fibers collapse because of drying of the cytoplasm in the lumen and possible alterations of cell wall properties occur . Evaluation of fiber quality parameters such as perimeter, cell wall thickness and other fiber traits due to genetic engineering or textile applications is essential to understand the impact of introduced transgenes or postharvest treatments in cotton fiber. For example, the mechanism of the effect of moisture on the quality of naturally-hygroscopic cotton fiber in ginning and textile processing is not well understood . A rapid, chemical dehydration procedure, originally developed for transmission electron microscopy of biological samples [7, 8], was applied to immature or mature and wet fibers. According to this procedure, acidified 2,2-dimethoxypropane (DMP) was used for rapid dehydration of biological samples followed by embedding in methyl butyl methacrylate  or other embedding resins  for sectioning. Instant hydrolysis of DMP by water results in gradually increasing concentrations of methanol and acetone . This technique has been successfully applied in our laboratory to immature or mature and wet cotton fiber samples so that the integrity of the fibers is maintained without collapsing and cellular structures are well preserved for evaluation by image analysis and light or electron microscopy.
Materials and Methods
Cotton plants (Gossypium hirsutum cv. Coker 312) were grown under natural light and day/night conditions in greenhouse in 2004. Mature cotton fibers were passed through a fine-tooth metal comb and grouped tightly parallel to make small bundles [3, 11]. One-half of the samples were placed in water for 5 minutes with constant stirring, and a drop of the wetting agent (Tween-20) was added to aid water absorption. Next, the bundle was removed from the water and excess fluid was drained from the bundle before dehydration and embedding. Immature fibers were collected from 16 or 40 day old bolls.
Fiber Sample Preparation and Microscopy
Cotton bolls of 16 days-post-anthesis (dpa) and 40 dpa were excised and placed in a beaker of water and immediately returned to the laboratory for fixation. Immature cotton fibers were fixed according to Yatsu and Jacks [12, 13]. Briefly, the fixative 3% gluteraldehyde in 0.05 Na-cacodylate pH 7.0 containing 1 drop of Triton X-IOO per 50 mL, was injected into one end of each locule and allowed to egress out of a small incision at the other end. A total of 4 mL of fixative passed through each locule before it was cut open. seeds were placed into a fresh beaker of fixative and vacuum infiltrated at 380 mm-Hg for 2 hours followed by several rinses in distilled water to remove residual gluteraldehyde. Fibers were post-fixed in 2% osmium tetroxide in water for 2 hours at room temperature and rinsed in several exchanges of distilled water. The mature, air-dried fibers did not require fixation.
Dehydration procedures were carried out as described before  in acidified DMP, followed by three exchanges for 15 minutes each and two exchanges of 100% acetone for 15 minutes each. Samples were stored overnight at 4�C.
(c) Light Microscopy
Immature fibers fixed in gluteraldehyde but not post-fixed, were placed on glass slides and stained in 0.1% aqueous solution of basic fuchsin. Fiber samples were also fixed as described for transmission electron microscopy, but were sectioned at 2 �m, affixed to glass slides and stained with toluidine blue.
Cross-sections of mature fibers were obtained as follows: After the DMP dehydration procedure , the fiber bundles were removed from the DMP solution vial, drained of excess DMP, and placed in a vial of methacrylate medium . Vials were then placed inside a standard bell jar dessicator under vacuum for 20 minutes to aid exchange of DMP with the methacrylate resin. After 20 minutes, the fiber bundle was pulled into "Tygon" tubing as previously described [3, 11, 15]. The bundles were exposed to ultra-violet radiation for 40 minutes, and allowed to cool for 20 minutes. Thick sections (2-3 �m) were cut using an ultramicrotome (Porter-Blum MT-I) for light microscopy , These seclions were placed in 10% ethanol drops on a slide covered with an albumin-glycerin mixture. Chloroform was used to expand the embedding medium to spread the cotton fiber sections on the slide. The slide was then placed on a hot plate for drying and subsequent image analysis.
A Dehydration Method for Immature or Wet Cotton Fibers for Light and Electron Microscopy2
Textile Research Journal, Jun 2006 by Rajasekaran, Kanniah, Muir, Alan J, Ingber, Bruce F, French, Alfred D
(d) Scanning Electron Microscopy
Both whole fibers and cross sections were imaged by scanning electron microscopy (SEM). Thick sections (2-3 �m) were cut from the cotton fiber embedments and placed on 12-mm-diameter glass coverslips. These were placed on glass slides inside gla'ss Petri dishes sitting atop several layers of cheese cloth. Methyl ethyl ketone was added to the Petri dish in a fume hood to dissolve the methacrylate embedding medium. The solvent solution was exchanged several times to ensure complete removal of the resin. Next, the coverslips were adhered to standard 0.5 inch SEM stubs using double-sided sticky adhesive tabs. The samples were sputter-coated with gold/palladium to a thickness of approximately 200 nm before high vacuum SEM imaging with a XL30 ESEM (FEI Instruments, Hillsboro, OR, U.S.A.). Uncut fibers were imaged at low vacuum (6 Torr) with the XL30 ESEM or critical point dried  and coated with gold/palladium before imaging at high vacuum (3.7 � 10^sup -6^ mbar).
(e) Transmission Electron Microscopy
For transmission electron microscopy (TEM), dehydrated fibers were gradually infiltrated with Spurr's resin  as follows: 1 : 3 (Spurr's : acetone) for 8 hours; 1 : 1 overnight, 3 : 1 for 8 hours, 100% Spurr's overnight, followed by two more exchanges of 100% for 4 hours each. During infiltration, the samples were slowly agitated on a rotating platform to enhance the infiltration of resin. Samples were embedded in BEEM capsules as well as 0.5 mL centrifuge tubes and polymerized overnight under a vacuum of 300 mm-Hg at 68�C. Ultrathin sections (approx. 60-90 nm) were cut with a diamond knife (Delaware Diamond Knives, Wilmington, DE, U.S.A.) on an ultramicrotome (Reichert Ultramicrotome, Research and Manufacturing Company, Inc., Tucson, AZ, U.S.A.) and collected on uncoated 200-mesh nickel grids. sections were stained for 10 minutes in 2% (w/v) uranyl acetate and 5 minutes in Reynolds lead citrate  before examining with a Philips CM-120 TEM operating at 80 kV.
Results and Discussion
Immature cotton fibers are long single cells with an active cytoplasm. They collapse soon after isolation from the seed epidermis. This prevents proper observation of the developing fibers and their cell wall deposition details. In-situ fixation of immature cotton fibers with gluteraldehyde preserved the integrity of the cell wall and cytoplasm (Figure Ia and b) and the long fiber cells did not plasmolyze and collapse. The advantages of in-situ fixation of cotton fibers have been reported previously [12, 13]. Post-fixation with osmium tetroxide was also helpful in not only preserving the fiber cell structure but also in obtaining high resolution in ultrathin sections by TEM. The rapid dehydration procedure with DMP, originally developed for other biological samples by Muller and Jacks , was successfully applied to immature, developing cotton fibers, as documented in the light micrograph of a fiber cross-section (Figure Ic). Dehydration with DMP was accomplished within 75 minutes for fiber samples in comparison with several days using the conventional procedure, which uses a graduated ethanol or acetone series. Observation of ultrathin sections under the transmission electron microscope revealed well-preserved cytoplasm and cell wall details in young, developing fibers (Figures 2a-e). Cross-sections of these fibers were round and did not show plasmolysis and collapse of the fibers.
The authors have also demonstrated the effectiveness of the DMP-embedding procedure in mature, wet cotton fibers. Air-dried (or boll-dried) fibers were harvested as long convoluted fibers with collapsed lumen (Figure 3a). The swollen fibers, after soaking in water, retained their shape after rapid dehydration with DMP (Figures 3b and c). DMP dehydration thus appears to be very effective for studying cell wall morphology and the cytoplasmic contents of the fiber and this will be useful in studying subtle changes in fiber structure due to genetic, physiological or textile manipulations. Fiber quality determinations, as affected by moisture uptake, can also be made from cross-sections of immature or mature fibers using this procedure. The authors have also successfully analyzed immature and wet fiber cross-sections by image analysis as described previously [3, 11, 15].
This manuscript is dedicated to our colleague, the late Dr. Tom Jacks, who offered initial advice in developing this technique. Expert assistance from Andr� Varnado and Jeannine Moraitis with regard to sectioning and image analysis, respectively, is gratefully acknowledged. We thank Dr. Zuzana Hruska, Mr Wilton R. Goynes and Ms MarieAlice Rousselle for their suggestions for improvement of the manuscript.
2 Disclaimer: names of companies or commercial products are given solely for the purpose of providing specific information. Their mention does not imply recommendation or endorsement by the United States Department of Agriculture over others that are not mentioned
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About the author:
Kanniah Rajasekaran1, Alan J. Muir, Bruce F. Ingber and Alfred D. French
USDA, ARS. Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, U.S.A.
1 Corresponding author: USDA, ARS, SRRC, 1100 Robert E. Lee Blud., New Orleans, LA 70124, U.S.A. Tel.: +1504 286 4482; Fax: +1504 286 4419; e-mail: krajahitsrrc.ars.usda.gov
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