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.     

In vitro gynogenesis in cotton (<Emphasis Type="Italic">Gossypium</Emphasis> sp.)

The effects of genotype, pollen or growth regulator-pretreatment of pistils, developmental stage of the ovule (embryo sac) and culture media on induction of gynogenesis, and subsequent plantlet regeneration in vitro were assessed in interspecific Gossypium barbadense × G. hirsutum cotton hybrids. Gynogenesis occurred in all genotypes used when the pistils had been pre-treated with pollen from Hibiscus cannabinus and ovaries were harvested 5 or 10 days after anthesis. The use of culture media, SH and MS, showed no significant differences in responding ovules, embryogenic ovules or embryo germination frequency. Recovered progeny were characterized cytogenetically and microscopically to help documenting their reproductive basis. Root tip chromosome counts of 17 plants established from ovule culture revealed that chromosome numbers ranged from 27 to 44. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that gynogenesis in cotton involves some unusual reproductive events. Aneuploids could be useful for functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.     

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.     

Adding gelling agents to cotton ovule culture media leads to subtle changes in fiber development

Summary 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. 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 U.S. Department of Agriculture over others not mentioned.     

Digital image analysis of expansion growth of cultured cotton ovules with fibers and their responses to ABA

Cotton ovule cultures have obvious advantages over whole plants when experimental protocols call for inhibitors, radio-labeled precursors or controlled environmental conditions to be tested. The responses of ovule expansion growth and attached fiber elongation to external factors require accurate measurement techniques. This paper presents a new method for digital image analysis of the growth area of cotton ovules with fibers at high resolution. The method was characterized under constant conditions and during dynamic responses to different levels of ABA (abscisic acid) treatment. The growth area was treated as area occupied within the outline of the Petri dish image of the growing ovule with fibers. Growth area increase showed the same trends as fiber length increase and was significantly correlated with the fiber length increase under different levels of ABA treatment (r ² = 0.97). This new analysis method provides a simple, noninvasive, and more accurate approach for growth analysis in the cotton ovule culture system. Using this method, the effects of ABA on expansion growth of ovule with fibers were characterized.     

Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Differentiation of cotton fibers from single cells in suspension culture

Summary A cotton cell suspension culture has been developed that provides unique opportunities for plant biologists to investigate early developmental events regulating cotton fiber properties, plant cell elongation, and cell wall biogenesis. The suspension culture was derived from cells of cotton (Gossypium hirsutum L.) ovule callus. These cells undergo the stages of fiber development previously described for in vivo fiber development. Fibers range in length up to 11 mm and have secondary walls. Supported by the U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Laboratory, New Orleans, Louisiana, and Cotton Incorporated, Raleigh, North Carolina.     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

In-ovulo embryo culture and seedling development of cotton (Gossypium hirsutum L.)

A convenient and reliable method for culturing cotton embryos is needed to obtain interspecific hybrids of this genus. C.A. Beasley and I.P. Ting (Amer. J. Bot. 60, 130, 1973) developed a phytohormone-supplemented medium (BTP) upon which the growth of ovules was similar that of in situ ovules. This medium was examined for in-ovulo embryo culture. Although good ovule growth occurred on BTP no embryos developed to maturity. However, when the medium was supplemented with NH ₄ ⁺ , more than 50% of the ovules produced mature embryos, and many of these germinated precociously after 8–10 weeks of culture. After germination seedlings were established on a separate medium designed to give balanced root and shoot growth. Subsequently young plants could be transferred to pots for greenhouse culture.     

The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l^-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Induction of homozygous Spathiphyllum wallisii genotypes through gynogenesis

In Spathiphyllum wallisii the production of doubled haploids was attempted. Different combinations of growth regulators were tested, as well as different cultivars. The use of TDZ (0.25–1 M) in ovary cultures of Spathiphyllum was required. On the contrary, cytokinins were not crucial during ovule culture; in fact, the use of a too high TDZ concentration induced diploid parthenogenesis in ovules of `Alfa'. The use of the imidazole fungicides IMA, PRO or TRI during ovary culture was not critical, though they enhanced the swelling of ovules during ovary culture, making the isolation of the ovules easier. The ovule cultures yielded different plantlets. Flow cytometry and AFLP-patterns showed that two doubled haploid genotypes could be obtained from `Stefanie'. These plants showed a normal phenotype. We concluded that the induction of homozygous Spathiphyllum through gynogenesis is possible and is genotype dependent.     

Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber

Cotton fibers, important natural raw materials for the textile industry, are trichomes elongated from epidermal cells of cotton ovules. To date, a number of genes have been shown to be critical for fiber development. In this study, the roles of genes encoding fasciclin-like arabinoglactan proteins (FLAs) in cotton fiber were examined by transforming RNA interfering (RNAi) construct. The RNAi according to the sequence of GhAGP4 caused a significant reduction of its mRNA level, and the expression of other three FLAs (GhAGP2, GhAGP3, GhFLA1) were also partially suppressed. The fiber initiation and fiber elongation were inhibited in the transgenic plants. As for the mature fibers of transgenic cotton, the fiber length became significantly shorter and the fiber quality became worse. In addition, the RNAi of GhAGP4 also affected the cytoskeleton network and the cellulose deposition of fiber cells. Through ovule culture, it was found that the expression of cotton FLA genes were upregulated by GA₃, especially for GhAGP2 and GhAGP4. These results indicate that the FLAs are essential for the initiation and elongation of cotton fiber development.     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

Embryology of theaceae — Anther and ovule development ofAdinandra,Cleyera andEurya

Anther and ovule development of the theaceous Ternstroemioideae is reported for the first time on the basis of eight specles of three generaAdinandra, Cleyera andEurya. Anthers of these three genera are similar and can be characterized by the following traits: tapetum of glandular type, anther dehiscing latrorse-introrse, both connective and anther epidermis heavily tanniniferous, and connective and even anther wall layers having abundant druses. Their ovules are also very similar in being bitegmic and tenuinucellate, and in having a micropyle formed by the inner integument only, three cell-layered integuments, a raphe bundle terminating at chalaza, usually amphitropous or less often campylotropous ovule, embryo sac formation of Polygonum type, ephemeral antipodal cells, and tanniniferous ovule epidermis. Such ovules are readily distinguishable from those of Camellioideae and all other families. It is suggested that the three genera studied are closely related, and that the degree of embryological specialization is highest inAdinandra and lowest inEurya. On the basis of the significant embryological discrepacies, the Ternstroemioideae seem to have diverged rather distantly from the other core-subfamily Camellioideae of the Theaceae.