Chronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells

Cotton fibers are single elongated cells that develop from epidermal cells of the ovule. The chronology of fiber differentiation was investigated using cultured ovules. Epidermal cells differentiate into fiber cells approx. 3 d before anthesis. When ovules were cultured on a defined medium, fiber growth could be initiated on ovules any time between 2 d preanthesis and the time of anthesis by adding indole-3-acetic acid and gibberellic acid to the medium. In the absence of phytohormones, fibers did not grow, and when ovules between 2 d preanthesis and anthesis were cultured without hormones past the day of anthesis and hormones then added, most ovules failed to produce fibers. The results define the timing of fiber differentiation from epidermal cells, and also define a window of time when differentiated cells are capable of further development. During this window, fiber cells are latent awaiting appropriate stimulation which in the intact plant is apparently associated with anthesis.Abbreviations GA₃ gibberellic acid - IAA indole-3-acetic acid     

Growth and mitotic potential of multicelled fibers of cotton (Malvaceae)

We tested the hypothesis that the growth of multicelled cotton fibers of Gossypium hirsutum, cultivar MD51 ne, occurs exclusively within the tip cell. Direct cellular measurements proved the hypothesis incorrect. The results show that all cells within a fiber grow and that the relative growth of the tip cell is reduced as the number of cells per fiber increases. Also, measurements of two- and three-celled fibers show that the two daughter nuclei in two-celled fibers differ. The ability to divide resides primarily, if not exclusively, with the basal cell. Thus, the fate of the tip-cell nucleus is fixed while that of the base cell is not. This rule is unaltered by the presence of IAA (indoleacetic acid) and GA3 (gibberellic acid-3) in the culture medium.     

棉花胚珠纤维发育的研究

将未受精的棉花胚珠漂浮培养在加有不同植物激素的BT培养基上,培养48小时或96小时后,用扫描电镜观察纤维发育情况,以及测定胚珠内IAA氧化酶活性变化及内源ABA的含量变化,并和同一时期的大田生长的胚珠进行比较。结果表明:IAA+GA_3是最佳激素组合。在这种激素组合的培养基中培养的未受精胚珠,在纤维发育、酶活性变化等方面,均与大田生长的胚珠相似。这一激素组合还能抑制离体胚珠内源ABA的增长,但同一时期的大田生长的胚珠,其内源ABA含量却相对要高。     

Changes in biochemical composition of the cell wall of the cotton fiber during development

The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.     

Spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances fiber yield and quality

The capacity of conventional breeding to simultaneously improve the yield and quality of cotton fiber is limited. The accumulation of the plant hormone indole-3-acetic acid (IAA) in cotton fiber initials prompted us to investigate the effects of genetically engineering increased IAA levels in the ovule epidermis. Targeted expression of the IAA biosynthetic gene iaaM, driven by the promoter of the petunia MADS box gene Floral Binding protein 7 (FBP7), increased IAA levels in the epidermis of cotton ovules at the fiber initiation stage. This substantially increased the number of lint fibers, an effect that was confirmed in a 4-year field trial. The lint percentage of the transgenic cotton, an important component of fiber yield, was consistently higher in our transgenic plants than in nontransgenic controls, resulting in a >15% increase in lint yield. Fiber fineness was also notably improved.     

Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA₃) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA₃, which implied that fiber initiation could be promoted by them as additive.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

DEVELOPMENTAL MORPHOLOGY OF COTTON FLOWERS AND SEED AS SEEN WITH THE SCANNING ELECTRON MICROSCOPE

This report assembles and pictorially presents observations on the timing of relatively uniform and well-defined developmental events in the cotton flower and its component parts. The first floral bud occurs on the 7–9th node approximately 35–40 days postemergence; 20–25 additional days elapse until anthesis. Floral parts are morphologically well defined by two weeks preanthesis. In about 85 % of the flowers the basal, abaxial surface of two of the three bracts contains an outer involucral nectary; occasionally, none, one, or three nectaries are found. The maximum rate of increase in floral bud length occurs during the 24 hrs preceding anthesis. Flower opening occurs at about daylight, although light is not required. Multipored pollen grains germinate in about ½ hr after deposition on the stigmatic hairs. Fertilization is accomplished, for most ovules, by the end of the first day postanthesis. Stomata are abundant, particularly at the chalazal ends of ovules. Fiber initials (epidermal cells of the ovule) begin their elongation phase on the morning of anthesis and are bounded by a thin primary wall. Areas of contrast (spots) observed through the scanning electron microscope are speculated to be organelles “seen through” the relatively amorphous fiber wall, which lacks extensive fibrillar orientation of cellulose. Fiber elongation ceases by about 24–28 days postanthesis, and by 50–70 days postanthesis fibers are mature and exhibit a thickened secondary wall and spiral twisting. Concomitant with the time of fiber maturity, the ovary wall splits and opens along locular suture lines.     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Callus Induction by (2-Chloroethyl) Phosphonic Acid on Cultured Cotton Ovules

The response of cultured cotton (Gossypium hirsutum L.) ovules to the ethylene releasing growth regulator, (2-chloroethyl) phosphonic acid (ethephon), was measured. Control ovules grown on a complete liquid medium without hormones continued the differentiation normal for cotton, although at a slower rate than in vivo. When ethephon was added to the medium, normal organ and fiber development was inhibited but callus was induced from the micropylar end of the ovule. The callus was extremely friable and produced many free cells in the culture medium. Dry weight of the callused ovules increased over 4-fold and total cell number increased 56% over the controls. Apparently ethylene released from the ethephon stimulated both cell division and cell expansion in forming the callus.     

A Quantitative Procedure for Estimating Cotton Fiber Growth

An improved procedure for quantitation of cotton fiber development, the “stain-destain” method, is reported. Toluidine blue 0 was used to selectively stain fibers subsequently destained in an acid-alcohol solution. Absorbance of the dyecontaining destaining solution was used as a measure of fiber development, and expressed in terms of total fiber units (TFU), one OD unit at 624 nm having been assigned the value of one TFU. Optimum conditions for the procedure, including staining and destaining times and solution to ovule ratios were determined: (1) 20 ovules with associated fibers stained for 15 sec in 80 ml 0.018% toluidine blue O, (2) nonabsorbed dye removed by 60 sec wash, (3) ovuls destajned in 100 ml glacial acetic acid-ethanol-water (10:95:5), (4) absorbance determined after one hr destaining. The procedure is deemed accurate and precise for the purpose intended—quadtation of fiber development as modified by phytohormones or other treatments. Data are shown correlating TFU with fiber length through 14 days postanthesis and an example is given in which the method was used to determine the effect of combined application of gibberellic acid and indoleacetic acid on in vitro cotton fiber development.     

Production of micronucleoli and onset of cotton fiber growth

Experiments focused on the early development of fiber cells of cotton (Gossypium hirsutum L. cv. MD51 ne) ovules produced two novel findings: one biological, the other methodological. The first concerns a micronucleolus in the nucleus of fibers. This developmental marker appears at or a little before 4 days postanthesis (dpa) in about 10% of the fibers and increases thereafter to nearly 80% provided the fibers are growing on fertilized ovules. Micronucleoli are neither seen in nuclei of fibers at 0–2 dpa nor in nuclei of non-fiber cells. Consequently, it is postulated that they are the product of specific developmental genes associated with fiber growth. The second, methodological finding, involves a cytological means of directly counting the number of fibers produced on young ovules at 1–4 dpa. The method provides quantitative data unavailable in the past. We used this method to show that emasculation caused a temporary 24-h delay in the initiation of fibers, that 30% of the fibers are affected, and that at 3 dpa both fertilized and unfertilized ovules have about 14 500 fibers. These data indicate that the fibers on fertilized and unfertilized ovules represent the same cell populations, a finding heretofore unknown. Received: 24 October 1997 / Accepted: 5 January 1998     

Characterization of cell-wall polymers from cotton ovule culture fiber cells by gel permeation chromatography

Summary Cotton (Gossypium hirsutum, Texas Marker-1) fiber cells originating from ovule culture have been analyzed by gel permeation chromatography of dimethyl acetamide/lithium chloride-soluble components and compared within planta-grown fibers. The profile of cell-wall polymer molecular weights indicated that fibers grown for 21 d in culture more closely resembled fibers growingin planta for 30 d post-anthesis than fully mature fibers. The weight average molecular weight was 3 400 000 and number average molecular weight of polymers from ovule culture fibers was 109 000. Analysis of the polymer weight fraction distribution revealed that ovule culture fibers were similar to 30 d post-anthesis immature fibers but lacked a low molecular weight (log M 3–4) polymer fraction. Assessment of the polymer branching frequency showed that ovule culture fibers were intermediate in branching between 30 d post-anthesis fiber and maturein planta fiber. In summary, polymers deposited in cell walls of ovule culture fibers appear to grossly mimic the polymers accumulated during normal fiber biogenesisin planta, yet subtle differences may explain why ovule culture fibers rarely reach their full genetic potential in length.     

FIBER INITIATION ON THE COTTON OVULE (GOSSYPIUM HIRSUTUM)

Cotton ovules at the stage of fiber initiation were studied with a scanning electron microscope. On the day before anthesis the anatropous ovule had many anomocytic stomata, especially on the chalazal end. At anthesis, fibers first appeared at the crest of the funiculus and then around the lateral circumference of the ovule. Fiber initials were delayed for a few hours at the chalazal cap and for 3 or 4 days at the extreme end of the micropylar region. The morphological differentiation of a fiber began when an epidermal cell rounded up and protruded. After a brief period of diametric expansion, the initial began elongation toward the micropylar end. Between 2 and 3 days of age the fibers segregated into small groups, developed tapered tips, and began spiral growth. Fiber density was about 3,300 fibers per mm², and the ratio of fiber initials to total epidermal cells was 1:3.7 at anthesis.     

Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development

The contents of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), and cytokinins were determined in ovules of normal cotton (Tm-1) and a kind of fiber differentiation mutant (Xin) before and after flowering by enzyme-linked immunosorbent assays. It was found that 24 h before flowering, a peak of IAA content was observed in ovules of Tm-1, whereas in ovules of Xin, a low level of IAA was determined. From –1 day (1 day before flowering) to +3 days (3 days after flowering), GA₁₊₃ levels in ovules of Xin were 40–70% lower than those of Tm-1; GA₄₊₇ levels were very low, and there was no visible difference in GA₄₊₇ content between normal and mutant cotton. The ABA content in ovule of Tm-1 decreased by 70% 3 days after flowering, whereas that of Xin only decreased by 20%. The levels of cytokinins in ovules of Tm-1 decreased after flowering, and those of Xin kept up a steady increase.Abbreviations IAA indole-3-acetic acid - GA gibberellin - ABA abscisic acid - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - iPA isopentenyladenosine - ZR zeatin riboside - DHZR dihydrozeatin riboside - CTK cytokinin     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE ZYGOTIC EMBRYOS OF MUSCADINE GRAPE (VITIS ROTUNDIFOLIA) CULTIVARS

Embryogenic cell lines of Vitis rotundifolia were produced from immature zygotic embryo explants obtained by culturing ovules, harvested at 20 d postanthesis, for 8 wk and then dissecting embryos from them. Ovules cultured on Nitsch and Nitsch medium with naphthoxyacetic acid and benzyladenine (BA) produced a brown exudate, necessitating three transfers to fresh medium at 2-wk intervals during the 8-wk culture cycle. Zygotic embryos that were subsequently isolated from cultured ovules and placed on the same medium produced a heterogenous callus from which eventually emerged embryogenic cell lines. A higher percentage of ovules from cultivars ‘Dixie’, ‘Fry’, ‘Nesbitt’, and ‘Welder’ produced zygotic embryos (31%–39%) than did those from ‘Carlos’ (3%). A higher percentage of ‘Fry’ ovules produced embryogenic lines from cultured zygotic embryos (6.3%) than did those of the other four cultivars (1%–1.6%). Embryogenic cell lines were white and composed of variably sized cell clusters, somatic embryos, and embryonic tissue embedded in a watery matrix. These lines were maintained for over 1 yr on modified Murashige and Skoog (MS) medium lacking growth regulators by transfer of selected cell clusters every 6 wk. White, opaque somatic embryos grew directly from cell clusters and passed through recognizable developmental stages. Germination was induced by transfer of somatic embryos to MS medium with BA. Although 80%–100% of embryos germinated, plant recovery was low due to poor shoot development.     

EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM UNFERTILIZED COTTON OVULES

Fertilization of cotton ovules was prevented by removal of styles and stamens on the morning of anthesis. Forty-eight hr later ovaries were harvested and ovules were aseptically transferred to liquid culture medium supplemented with various plant growth substances. In the absence of phytohormones, ovules browned and failed to increase in size or produce fibers. Indoleacetic acid and gibberellic acid provided for ovule growth and fiber development. Kinetin provided for ovule growth only. The ovule's capacity for indoleacetic acid- or gibberellic acid-stimulation of fiber development was reduced by high concentrations of kinetin or abscisic acid. Low concentrations of kinetin partially reversed the inhibitory effect of abscisic acid.