Effects of fusicoccin on intact cotton plants (Gossypium hirsutum L.)

Fusicoccin (FC) was applied as a spray to shoots of intact field- and glasshouse-grown cotton plants. Distortions of shoot morphology resulted. Stems and petioles of FC-treated plants were irregular in diameter and twisted, whereas leaf laminae were curled and crinkled. Shoot elongation was inhibited by FC; the effect was dependent upon the concentration and timing of the applications.Abbreviation FC fusicoccin     

Microcolinearity and genome evolution in the AdhA region of diploid and polyploid cotton (Gossypium)

Genome sizes vary by several orders of magnitude, driven by mechanisms such as illegitimate recombination and transposable element proliferation. Prior analysis of the CesA region in two cotton genomes that diverged 5–10 million years ago (Ma), and acquired a twofold difference in genome size, revealed extensive local conservation of genic and intergenic regions, with no evidence of the global genome size difference. The present study extends the comparison to include BAC sequences surrounding the gene encoding alcohol dehydrogenase A ( AdhA ) from four cotton genomes: the two co-resident genomes (A_T and D_T) of the allotetraploid, Gossypium hirsutum , as well as the model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). In contrast to earlier work, evolution in the AdhA region reflects, in a microcosm, the overall difference in genome size, with a nearly twofold difference in aligned sequence length. Most size differences may be attributed to differential accumulation of retroelements during divergence of the genome diploids from their common ancestor, but in addition there has been a biased accumulation of small deletions, such that those in the smaller D genome are on average twice as large as those in the larger A genome. The data also provide evidence for the global phenomenon of 'genomic downsizing' in polyploids shortly after formation. This in part reflects a higher frequency of small deletions post-polyploidization, and increased illegitimate recombination. In conjunction with previous work, the data here confirm the conclusion that genome size evolution reflects many forces that collectively operate heterogeneously among genomic regions.     

An efficient grafting system for transgenic plant recovery in cotton (Gossypium hirsutum L.)

A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacterium infection and kanamycin selection was developed. Various aspects of in vitro grafting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitro grafting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitro seedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitro grafting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors.     

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.     

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     

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications. An erratum to this article is available at .     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

Somatic embryogenesis and plant regeneration from different wild diploid cotton (Gossypium) species

Calli were successfully induced from hypocotyls of eight wild diploid cotton species (Gossypium) on MSB (MS salts and B₅ vitamins) medium supplemented with 0.09 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.32 μM KT (kinetin). Plant growth regulator (PGR) combinations, adding GA₃ (Gibberellic acid), high inorganic salt stress, and PGR-free media were used to induce embryogenic calli from nonembryogenic calli. Embryogenic cultures were induced from G. aridum S. (D₄ genome), G. davidsonii K. (D₃-d genome), G. klotzschianum A. (D₃-k genome), G. raimondii U. (D₅ genome), and G. stocksii M. (E₁ genome). We then observed somatic embryogenesis in the five species while calli of G. africanum V. (A₁-2 genome), G. anomalum W. (B₁ genome), and G. bickii P. (G genome) remained nonembryogenic. Somatic embryogenesis was adjusted by changing sugar sources, regulating combinations of PGRs, and using cell suspension culture. Embryos at various developmental stages produced mature and germinating embryos when cultured on filter paper placed on the media containing different sugar sources. The utility of different sugar sources promoted globular embryos developing into cotyledonary stage and increased the frequency of cotyledonary embryos developing into normal plants. Normal plantlets were regenerated from G. davidsonii, G. klotzschianum, G. raimondii, and G. stocksii. Only abnormal plantlets were obtained in G. aridum. This work will contribute to broadening the number of regenerable cotton species and provide foundations for somatic hybridization in cotton to create new germplasm.     

Life history divergence of sympatric diploid and polyploid populations of brine shrimp Artemia parthenogenetica

In order to study how polyploidy affects life history patterns in animals, we have examined sympatric diploid and polyploid brine shrimp (Artemia parthenogenetica) from China, Italy and Spain under laboratory conditions. At optimal temperature and salinity (25°C and 90 ppt), diploids from the three populations had much higher intrinsic rates of increase, higher fecundity, faster developmental rates, and larger brood sizes than their sympatric polyploids. The Chinese and Italian populations were selected for further analysis to determine the life history responses of diploids and polyploids to temperature and salinity changes. Under intermediate and high salinities, Chinese and Italian polyploids produced most of their offspring as dormant cysts while their sympatric diploids produced most of their offspring as nauplii. This relationship is reversed in the Spanish diploid-polyploid complex. For the Chinese population at 25° C, pentaploid clones had higher developmental rates than diploid clones at 35 ppt; at 90 ppt, diploid clones had higher developmental rates than the pentaploids. Italian diploids and tetraploids had different responses to variation in both temperature (25° C and 31° C) and salinity (30 ppt and 180 ppt). Our results demonstrate that relative fitness of the two cytotypes is a function of environmental conditions and that sympatric diploids and polyploids respond differently to environmental changes. Chinese and Italian polyploids are expected to have lower fitness than their sympatric diploids when the physical environment is not stressful and when intraspecific competition is important. However, polyploids may have advantages over sympatric diploids in stressful habitats or when they encounter short-term lethal temperatures. These results suggest that polyploid Artemia have evolved a suite of life-history characteristics adapting them to environments that contrast to those of their sympatric diploids.     

Establishment of embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.)

Summary Maintainable, highly embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.) have been obtained. Callus with no apparent organization was used to establish the liquid culture. Callus growth conditions as well as suspension medium composition were optimized. A visual selection scheme was beneficial for the maintenance of the embryogenic suspension. These liquid cultures have been maintained for over 10 mo. with no loss in embryogenic capacity. The somatic embryos developed after transfer of the embryogenic tissues to a hormone-free liquid medium. Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. This is journal article No. 71-87.     

Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors

The seasonal cycle and persistence of a plant is governed by a combination of the determinate or indeterminate status of shoot and root apical meristems. A perennial plant is one in which the apical meristem of at least one of its shoot axes remains indeterminate beyond the first growth season.TERMINAL FLOWER1 (TFL1) genes play important roles in regulating flowering time, the fate of inflorescence meristem and perenniality. To investigate the role of TFL1-like genes in the determination of the apical meristems in an industrially important crop cultivated for its fibers, we isolated and characterized two TFL1 homologs (TFL1a and TFL1b) from tetraploid cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii). All isolated genes maintain the same exon–intron organization. Their phylogenetic analysis at the amino acid level confirmed that the isolated sequences are TFL1-like genes and collocate in the TFL1 clade of the PEBP protein family. Expression analysis revealed that the genes TFL1a and TFL1b have slightly different expression patterns, suggesting different functional roles in the determination of the meristems. Additionally, promoter analysis by computational methods revealed the presence of common binding motifs in TFL1-like promoters. These are the first reported TFL1-like genes isolated from cotton, the most important crop for the textile industry.     

Line x tester analysis for fixed effect model in cotton (Gossypium hirsutum L.)

Summary The data from an experiment in cotton consisting of three testers and 12 lines selected deliberately have been analysed. The investigation showed higher specific combining ability variance for yield of seed cotton and number of bolls, indicating the predominance of non-additive gene action. Of parental lines, H777 was found to possess high g.c.a. effects for seed cotton yield, number of bolls and number of sympodes. Parent H842 contributed only for boll weight, whereas H655 was good general combiner for number of monopodes. There appeared to be better chances for increasing the yield by exploiting hybrid vigour for the number of bolls and boll weight. The presence of marked non-additive gene effects, in addition to additive gene effects, indicated the need for exploiting both the fixable and non-fixable components of genetic variance for increasing productivity in cotton.     

Genetics of allozyme variation in Gossypium arboreum L. and Gossypium herbaceum L. (Malvaceae)

Summary Seed protein extracts from 90 accessions of Gossypium arboreum and 70 accessions of Gossypium herbaceum were electrophoretically analyzed for isozyme variation. Eighteen enzyme systems were resolved, ten of which were polymorphic among accessions. No within accession isozyme variation was observed within these highly inbred lines. A minimum of 24 genes encode the isozymes resolved and data is presented for codominant inheritance at 13 loci. Tests for non-random joint segregation in 63 of the 78 possible two-locus combinations from the 13 characterized loci give evidence for four pairs of linked genes (Lap2/Me1 [r=0.160+/-0.027], Lap2/Pgi1 [r= 0.285+/-0.055], Mdh6/Tpi1 [r= 0.197+/-0.028], and 6Pgd2/6Pgd3[r 0.000]. Numerous presumptive duplicate isozyme loci were observed and these were usually expressed as patterns of nonsegregating heteromultimers within accessions. Single gene expression was also observed at several loci. The observed results are in agreement with those of previous cytological investigations which have proposed the polyploid origin of the diploid Old World Gossypiums.     

Changes in the non-structural carbohydrate content of cotton (Gossypium spp.) fibres at different stages of development

The neutral sugars (glucose, fructose, and sucrose) and the sugar phosphates (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) soluble in hot aqueous 80% methanol from the fibres of cotton — Gossypium arboreum L., G. barbadense L., and G. hirsutum L. — were determined at various stages of fibre development. In addition, the (13)--D-glucan content was measured and in the case of G. arboreum the rate of (13)--D-glucan and cellulose synthesis was determined with [¹⁴C]sucrose as the precursor. For each of the species a similar chronology was obtained for the changes in content of the various non-structural carbohydrates. At the early stages of secondary wall formation, glucose and fructose exhibited a maximum which was closely followed by a maximum in the (13)--D-glucan content and in the sugar phosphates. On the other hand, the sucrose content increased regularly until fibre maturity. The rates of synthesis of (13)--D-glucan and of cellulose were highest following the maximum in the (13)--D-glucan content, when the latter was being depleted.Abbreviations DMSO dimethyl-sulphoxide - DPA days post anthesis - UDP-glucose uridinediphosphoglucose     

Measuring gene flow in the cultivation of transgenic cotton (Gossypium hirsutum L.)

Transgenic Bt cotton NewCott 33B and transgenic tfd A cotton TFD were chosen to evaluate pollen dispersal frequency and distance of transgenic cotton (Gossypium hirsutum L.) in the Huanghe Valley Cotton-producing Zone, China. The objective was to evaluate the efficacy of biosafety procedures used to reduce pollen movement. A field test plot of transgenic cotton (6×6 m) was planted in the middle of a nontransgenic field measuring 210×210 m. The results indicated that the pollen of Bt cotton or tfd A cotton could be dispersed into the environment. Out-crossing was highest within the central test plot where progeny from nontransgenic plants, immediately adjacent to transgenic plants, had resistant plant progeny at frequencies up to 10.48%. Dispersal frequency decreased significantly and exponentially as dispersal distance increased. The flow frequency and distance of tfd A and Bt genes were similar, but the pollen-mediated gene flow of tfd A cotton was higher and further to the transgenic block than that of Bt cotton (χ² = 11.712, 1 degree of freedom, p<0.001). For the tfd A gene, out-crossing ranged from 10.13% at 1 m to 0.04% at 50 m from the transgenic plants. For the Bt gene, out-crossing ranged from 8.16% at 1 m to 0.08% at 20 m from the transgenic plants. These data were fit to a power curve model: y=10.1321x ^−1.4133 with a correlation coefficient of 0.999, and y=8.0031x ^−1.483 with a correlation coefficient of 0.998, respectively. In this experiment, the farthest distance of pollen dispersal from transgenic cotton was 50 m. These results indicate that a 60-m buffer zone would serve to limit dispersal of transgenic pollen from small-scale field tests.     

Relationships among violaxanthin deepoxidation,thylakoid membrane conformation,and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Z; peak at 506 nm) and by light scattering (S; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (S_g) for absorbance changes around 540 nm that are due to a spectral overlap with Z. This protocol isolated the component which is caused by light-scattering changes alone (S_n). In control leaves, the kinetics and temperature dependence of the initial rate of rise in S_n that takes place upon illumination, closely matched that of Z. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both S_n and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on A_n and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of S_n and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of Z upon darkening was much slower than the relaxation of S_n and NPQ. The relaxation of S_n and NPQ showed quantitatively similar kinetics and temperature dependencies (Q₁₀=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause S_n or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by S_n. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained S_n and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.Abbreviations A antheraxanthin - DTT dithiothreitol - F, F_m chlorophyll fluorescence yield at actual, full closure of the PSII centers - NPQ nonphotochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - V violaxanthin - Z zeaxanthin - S_n, Z spectral absorbance change caused by light-scattering, violaxanthin deepoxidation We thank Connie Shih for skillful assistance in growing the plants, and for conducting HPLC analyses. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W. B. is gratefully acknowledged. This work was supported in part by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B. This is C. I. W. — D. P. B. Publication No. 1094.     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Correlation studies on yield and fibre traits in upland cotton (Gossypium hirsutum L.)

Summary Two diverse parents of upland cotton namely J.34 and I.C. 1926 were crossed. A comparison between biparental intermated progenies and F₃ families indicated alteration of correlation coefficient between yield and halo length. The significant negative correlation in F₃ population between these two attributes changed to a positive but non significant one in biparental intermated progenies. A change in correlation coefficients was expected due to breakage of linkage upon intermating. An increase in the correlation coefficients could also be expected when linkages are predominantly in the repulsion phase. It is suggested that intermating in early generations coupled with selection of desirable segregants may prove a useful method for improving yield and quality simultaneously. The diallel selective mating system may also supplement intermating to improve yield and quality in cotton.Part of Ph.D. Thesis submitted to the Haryana Agricultural University. Hissar-125004, India     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.