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.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers

In animals, the small GTP-binding proteins, Rac and Rho, of theras superfamily participate in the signal rransduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Studies of pollen maturation in cotton: the storage reserve accumulation phase

Summary This study follows the maturation of the pollen grain of cotton (Gossypium hirsutum L.), particularly the development of the vegetative cytoplasm and the various storage products formed. CTEM, HVEM, stereoscopy, and cyto-histochemistry were used to examine the events occurring during the 9 days before anthesis. Starch began to accumulate in plastids at anthesis minus 9 days and reached a peak concentration shortly before anthesis; lipid deposition followed a similar pattern, but started at 6 days before anthesis. Lipid bodies were always seen closely oppressed to the endoplasmic reticulum (ER). Dictyosomes appear active during the entire 9 days; first producing vesicles involved in the formation of the intine and, later, producing vesicles stored in the pollen grain. The dictyosome vesicles appear to contain polysaccharides and concentrate in layers around the lipid bodies. Ribosomes increase in number from 6 days before anthesis and are particularly numerous in the mature pollen. From anthesis minus 6 days until anthesis, the ER cisternae become increasingly inflated and, in the hours immediately before pollen release, form pockets filled with lipid bodies and dictysosome vesicles. The mature pollen has a core region filled with ER pockets and a peripheral cytoplasm in which such pockets are generally lacking.This research was supported in part by NSF Grant BMS575-22-23 and Grant N.RR-00592 from the Division of Research Resources, National Institutes of Health     

Water use efficiency as a function of leaf age and position within the cotton canopy

The gas exchange properties of whole plant canopies are an integral part of crop productivity and have attracted much attention in recent years. However, insufficient information exists on the coordination of transpiration and CO₂ uptake for individual leaves during the growing season. Single-leaf determinations of net photosynthesis (Pn), transpiration (E) and water use efficiency (WUE) for field-grown cotton (Gossypium hirsutum L.) leaves were recorded during a 2-year field study. Measurements were made at 3 to 4 day intervals on the main-stem and first three sympodial leaves at main-stem node 10 from their unfolding through senescence. Results indicated that all gas exchange parameters changed with individual main-stem and sympodial leaf age. Values of Pn, E and WUE followed a rise and fall pattern with maximum rates achieved at a leaf age of 18 to 20 days. While no significant position effects were observed for Pn, main-stem and sympodial leaves did differ in E and WUE particularly as leaves aged beyond 40 days. For a given leaf age, the main-stem leaf had a significantly lower WUE than the three sympodial leaves. WUE's for the main-stem and three sympodial leaves between the ages of 41 to 50 days were 0.85, 1.30, 1.36 and 1.95 μmol CO₂ mmol⁻¹ H₂O, respectively. The mechanisms which mediated leaf positional differences for WUE were not strictly related to changes in stomatal conductance (g_s·H₂O) since decreases in g_s·H₂O with leaf age were similar for the four leaves. However, significantly different radiant environments with distance along the fruiting branch did indicate the possible involvement of mutual leaf shading in determining WUE. The significance of these findings are presented in relation to light competition within the plant canopy during development.     

Physiological responses of cotton to a single waterlogging at high and low N-levels

Surface-irrigated cotton (Gossypium hirsutum L.) grown on slowly draining clay soil is subjected to short-term periods of waterlogging at each irrigation which generally results in reduced productivity. The sequence of above- and below-ground plant responses to transient waterlogging and the role of N availability in modifying the immediate responses were studied. Lysimeters of Marah clay loam (a Natrustalf) were instrumented to monitor soil and plant responses to a 7-day waterlogging event beginning 67 days after sowing. Cotton (‘Deltapine 61’) plants (8 per lysimeter) were grown with two levels of added N (300 kg ha⁻¹ and 30 kg ha⁻¹) and two irrigation treatments (flooded and control). Measured soil-O₂ levels decreased rapidly upon surface flooding because water displaced air and root zone respiration consumed O₂. The rate of O₂ consumption was 2.7 times greater in the high-N treatment than the low-N treatment. This difference was associated with a 1.8 fold difference in numbers of observed roots. Root growth was only slightly affected by flooding. Leaf growth decreased by 28%, foliage temperature increased 2.3% and apparent photosynthesis decreased by 16%. It is suggested that flooding reduced photosynthetic activity within 2 days while other stress symptoms became apparent after about 6 days. Although this stress was reflected in a trend for decreased plant productivity, the effect of flooding on boll dry mass at harvest was not significant at the level of replication used. The single waterlogging did not cause yield reductions comparable to those observed elsewhere when several waterlogging events were imposed. Contribution from the CSIRO, Centre for Irrigation Research, Griffith, NSW, Australia and USDA-ARS, Morris, MI, USA, in cooperation with the univ. of Minnesota.     

Media and gelling agent effect on cotton callus initiation from excised seed hypocotyls

A factorial experiment was performed to develop a medium which would support initiation and proliferation of callus in a diverse group of exotic lines of Gossypium hirsutum. Seed hypocotyls of T1, T25 and T133 were cultured on Linsmaier and Skoog (LS) basal medium (1965) with NAA or 2,4-D tested in combination with BA or kinetin. The best medium from this study was then compared to five published media for support of callus initiation and growth of the varieties Acala 1517-75, Coker 500, Dunn 120, Paymaster 303 and TM1. Furthermore, the effects of two gelling agents, Difco-Bacto agar and Kelco Gelrite, were investigated with each of the six media. Significantly more callus was initiated on media solidified with Gelrite than with agar. The best callus production occurred on LS medium supplemented with 30gl^-1 glucose, 0.1 mgl^-1 BA and 0.1 mgl^-1 2,4-D.     

Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton

Improvement in somatic embryogenesis has been achieved in several cotton lines (Gossypium hirsutumL.) from the Georgia and Pee Dee germplasm with culture media containing various Putrescine concentrations. The best results were obtained with the -naphthalene acetic acid (NAA)-based treatments, S15 g.05 NAA and EMMS₂, as compared to the 2,4-dichlorophenoxyacetic acid (2,4-D)-based culture medium, EMMS₄. Inclusion of 0.5 mg l^–1 Putrescine improved somatic embryo (SE) induction for most treatments and lines tested. An 8-and 2-fold improvement was achieved in SE production on the EMMS₂-0.5 Putrescine treatment as compared to EMMS₂ alone for cotton lines PD 97019 and GA 98033, respectively. A significant increase in SE number (53-fold) was obtained with the addition of 0.5 mg l^–1Putrescine to EMMS₂ for PD 97021, which was essentially recalcitrant without Putrescine treatment. Conversion of SEs into plants was both genotype- and culture medium-dependent.     

Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis

Summary Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by culturing these explants on callus-inducing solid medium [Murashige and Skoog salts plus vitamins of Gamborg's B₅ medium, 30 g l⁻¹ glucose, 100 mg l⁻¹ myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected over filter mesh 40 subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Photosynthesis of individual field-grown cotton leaves during ontogeny

Photosynthetic characteristics of field-grown cotton (Gossypium hirsutum L.) leaves were determined at several insertion levels within the canopy during the growing season. Single-leaf measurements of net photosynthesis (Pn), stomatal conductance to CO₂ (g_s·CO₂), substomatal CO₂, leaf area expansion, leaf nitrogen, and light intensity (PPFD) were recorded for undisturbed leaves within the crop canopy at 3–4 day intervals during the development of all leaves at main-stem nodes 8, 10, and 12. Patterns of Pn during leaf ontogeny exhibited three distinct phases; a rapid increase to maximum at 16–20 days after leaf unfolding, a relatively short plateau, and a period of linear decline to negligible Pn at 60–65 days. Analysis of the parameters which contributed to the rise and fall pattern of Pn with leaf age indicated the primary involvement of leaf area expansion, leaf nitrogen, PPFD, and g_s·CO₂ in this process. The response of Pn and g_s·CO₂ to incident PPFD conditions during canopy development was highly age dependent. For leaves less than 16 days old, the patterns of Pn and g_s·CO₂ were largely controlled by non-PPFD factors, while for older leaves Pn and g_s·CO₂ were more closely coupled to PPFD-mediated processes. Maximum values of Pn were not significantly different for any of the leaves monitored in this study, however, those leaves at main-stem node 8 did possess a significantly diminished photosynthetic capacity with age compared to upper canopy leaves. This accelerated decline in Pn could not be explained by age-related variations in g_s·CO₂ since all leaves showed similar changes in g_s·CO₂ with leaf age.Abbreviations g_s·CO₂ stomatal conductance to CO₂ - Pn net photosynthesis - PPFD photosynthetic photon flux density     

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.     

Integrative placement and orientation of non-redundant SSR loci in cotton linkage groups by deficiency analysis

A combination of previously mapped and unmapped non-redundant SSR loci, using 381 primer pairs were chromosomally and sub-chromosomally localized by deficiency analysis of two sets of quasi-isogenic interspecific Gossypium hirsutum L. hypoaneuploid F₁ hybrids involving Gossypium barbadense L. and Gossypium tomentosum (Nuttall ex Seemann). Polymorphisms were detected for 369 SSR primer pairs. A total of 318 SSR loci were rendered deficient by the available hypoaneuploid stocks, which included primary monosomics (2n = 51), monotelodisomics and duplication-deficient (segmental trisomic–monosomic) (2n = 52) types. Chromosomal associations were newly determined for 123 SSR loci, of which 90, 106 and 73 were polymorphic in G. tomentosum, G. barbadense, and both sets, respectively. The deficiency tests independently confirmed the recent identifications of linkage groups (LG) A01, A02, A03 and D08 to be chromosome (Chr)-13, Chr-8, Chr-11 and Chr-19, respectively, and collectively delimited LG D02 and D03 to Chr-21 and 24, and their homeologs to Chr-8 and 11. Segmental homeology was detected between Chr-2 and Chr-17 loci, adding to evidence of segmental homeology between Chr-2 and 3 versus Chr-14 and 17. The 318 non-redundant SSR loci localized in this study will enhance the construction of linkage maps and QTL identification in molecular marker assisted selection since the confirmed and newly discovered SSR loci can serve as anchor loci for their respective chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ATG-anchored AFLP (ATG-AFLP) analysis in cotton

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.