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Evidence that supports a relation between AOX expression and improvement in plant height, internode length, and total leaf area under cool temperature is shown. Cell expansion and elongation appear to be enhanced when AOX expression was increased. Cotton growth is sensitive to cool temperature during germination and early seedling development. Delayed emergence, seedling damage, and increased risk to disease are common. Late seasonal cool weather is a major factor limiting the consistent production of high-quality cotton lint in West Texas. Alternative oxidase functions in the inner membrane of the mitochondria via an alternative respiration pathway and serves as a multifunctional system for amelioration of abiotic and biotic stresses. Cotton seedling emergence and growth exposed to cool temperatures was examined in plants with enhanced AOX expression. Thirteen T1 seed lines showed 3 to 1 segregation for the T-DNA containing the tobacco AOX1 gene. Two over-expressing, single-copy, homozygous AOX lines (94-20T and 66-6T) and Null line (94-3N) were selected for examination. The transcript levels were ≈ 2 to 6 fold higher in the AOX lines compared to those of the Null line and wild-type in stem, leaf, root and boll tissues. The research examined the hypothesis that transgenic cotton with enhanced AOX expression will have enhanced growth traits under suboptimal cool temperatures. Improved plant height, internode length, plant height and internode length from second node, and total leaf area under cool temperatures were observed in AOX over-expression lines. This may be attributed to improved cell expansion and elongation characteristics in the AOX line.

We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM (∼600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM (∼500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6–11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the “portability” of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.

An understanding of the mechanistic hasis of adverse environmental effects on cotton fiber development is a prerequisite to future improvement through genetic engineering and aids in field management to minimize such adverse effects. In order to clarify previous results from field studies on the effects of cool temperatures on initiation, early elongation, and later elongation of cotton fibers, cotton ovules (Gossypium hirsutum L.) cultured in vitro were used as a manipulable and reproducible experimental system based on previous evidence that they provide a valid model. Culture temperature varied from a control of 34 o C constant to 34/15 o C cycling (12/12 h) to mimic a typical diurnal temperature cycle

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [14C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.

This research was designed to: a) characterize the normal pH changes that occur when cotton cell are grown in culture; b) determine if cotton cells can regulate the pH of their extracellular medium; and c) explore the effects of starting pH on cellular differentiation in culture, including formation of somatic embryos. When an aliquot of cotton cell suspension culture (Gossypium hirsutum L. cv Coker 312) was inoculated into fresh Murashige and Skoog (MS) medium at pH 4.5, the pH stabilized near 5.5 during the log phase of growth and then rose to pH 7.25. Cotton cells actively adjust medium with initial pH between 3 and 8 to near pH 5.5 in the early culture period. By acid/base titration, it has been shown that living cotton cells increase the buffer capacity of water and MS medium. Therefore, the metabolic activity of living cells accounts for the adjustment and stabilization of pH during the log phase of growth. The starting pH of the culture medium affects longterm viability, growth, and differentiation of the cells; pH 3 to 5 is best for cell viability, pH 3 to 4 enhances cell elongation; and pH 4, 7, or 8 stimulates somatic embryogenesis. Cultured cotton cells and the pH of their extracellular medium are in a complex, interactive relationship.

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a "growth ring" for each time the temperature cycled to 22 or 15 degrees C. Rings were rarely detected when the low point was 28 degrees C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34 degrees C/22 degrees C cycling developed similarly to constant 34 degrees C controls, but 34 degrees C/22 degrees C and 34 degrees C/15 degrees C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34 degrees C/22 degrees C cycling, but both parameters were reduced at the end of the experiment under 34 degrees C/15 degrees C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.

Cell suspension cultures of cotton (Gossypium hitirsutum L. cv. Coker 312) were exposed to various temperature:time treatments in order to select cell lines resistant to high temperature stress. Cells were exposed to 45°C for 3 h each day until the total accumulated hours of stress were: 0 h, 10 h, 75 h, 100 h, or 105 h (81 h pulsed then 24 h continuous). After the stress treatments, the cells were plated onto embryo development medium and plants were recovered. The embryogenic calli that were recovered were subcultured monthly for 6 months and tested for increased resistance to the temperature:time treatments previously determined to be lethal and to water stress as imposed by PEG. All of the selected cell lines were more resistant to both types of stress than the control cell lines. Leaf tissue of stress selected (Ro) formed and maintained callus growth when incubated at 38°C; whereas, tissue excised from nonselected controls rarely formed callus and calli which did form quickly became necrotic. These cells and plants will provide a tool for determining the mechanisms involved in resistance to high temperature stress.

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO3, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA3) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO3. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When ≥5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA3. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.

High frequencies of somaclonal phenotypic and cytogenetic variation have been observed previously among regenerants from cotton (Gossypium hirsutum L., 2n = 4x = 52). In this study we endeavored to determine if cytogenetic abnormalities would be detectable in cotton cell cultures and if so, whether or not the observed abnormalities would parallel those expected on the basis of previous cytogenetic analyses of cotton somaclones. Paired samples from suspension cultures established from 21-month-old 'Coker 312' and 8-month-old 'Coker 315' calli were pretreated or not pretreated with colchicine to detect cytogenetic abnormalities at metaphase or anaphase–telophase, respectively. Cell cultures established from both calli were found to vary in chromosome number. Hypoaneuploidy was common, but hyperaneuploidy and polyploidy were rare. Modal chromosome numbers for the 'Coker 312' and 'Coker 315' cultures were 46 and 50, respectively. Bridges at anaphase and telophase were frequent in the 'Coker 312' cultures but rare in the 'Coker 315'cultures. Cytogenetic differences between the cultures could be due to effects of culture age, genotype, their interaction, or other factors. Very small chromosomes, presumably centric fragments, as well as ring chromosomes and putative bridges between metaphase chromosomes occurred at low frequencies. The prevalence of hypoaneuploidy and rarity of hyperaneuploidy and polyploidy in cultures paralleled previous results on cotton somaclones, indicating that cytogenetic abnormalities arising in vitro probably contribute significantly to cotton somaclonal variation. The occurrence of hypoaneuploidy and bridges, including multiple bridges within single cells, is concordant with the hypothesis that breakage–fusion–bridge cycles may accumulate during in vitro culture of cotton.Key words: cotton, Gossypium, tissue culture, cytogenetics.

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.

Apical meristem explants of three cultivars of potato (Solanum tuberosum L.) formed flowers directly or callused and subsequently formed flower buds when cultured in vitro. Frequency of flower formation was 45%, 59%, and 55% for ‘Red LaSoda’, ‘Viking’, and ‘Norgold “M”’, respectively. Pistils and stamens were reduced in size compared to sepals, and

Three North American potato (Solanum tuberosum L.) cultivars were characterized for callus initiation, shoot regeneration, and efficiency of micropropagation. Primary callus from leaf explants of ‘Viking’ and ‘Norgold-“M”’ formed stolon-like structures; ‘Red LaSoda’ formed both roots and stolon-like structures. After three transfers, 30% of ‘Red LaSoda’ calli eventually regenerated one to three shoots per callus. Micropropagation rates were 5.1, 3.7, and 3.8 shoots per axillary bud of ‘Red LaSoda’, ‘Norgold-“M”’, and ‘Viking’, respectively. No differences in mean number of shoots resulted from different growth conditions for ‘Norgold-“M”’ and ‘Viking’. ‘Red LaSoda’ produced significantly more shoots than ‘Viking’ or ‘Norgold-“M”’- Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); 1-naphthaleneacetic acid (NAA); 6-benzylamino-purine (BA); gibberellic acid (Ga3).

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.

Thirty eight cultivars, strains, and races ofGossypium were screened for somatic embryogenesis with the protocols developed as a model forG. hirsutum L. cv. Coker 312. Four classes of response were identified; high, moderate, low, and non-embryogenic. Four cultivars were further screened with 13 growth regulator regimes to determine if culture environment could change the classification or induce a higher level of response. The classification or level of response did not change. Screening of individual seedlings within a cultivar indicated that genotypic variation for embryogenesis existed. Highly embryogenic individuals were selected from cvs. Coker 312 and Paymaster 303 for use as germplasm sources for transfer of the embryogenic trait to other cultivars and genetic stocks. Only genetically responsive genotypes are amenable to the model developed for Coker 312.