The Development and a Cytogenetic Study of Monosomics of Gossypium Hirsutum L.

Monosomics of cotton (Gossypium hirsutum L.) were obtained by irradiation of pollen by -rays and by irradiation of seeds by thermal neutrons. Many monosomics were derived directly from irradiation, but a number of monosomics were also recovered in the progeny of plants with translocations and of desynaptic plants. Only 28 primary monosomics showed normal pairing at metaphase-1 of meiosis. The others formec rare trivalents or additional univalents. Partial desynapsis was detected in some monosomics. The pollen fertility levels of monosomics are presented. New morphological characters were detected among the monosome plants of cotton.     

Morphometric Analysis of Chloroplasts of Cotton Leaf and Fruiting Organs

We examined morphological and ultrastructural differences in chloroplasts of cotton leaves and the fruiting organs, bract, and capsule wall to advance our understanding of their commonly observed differences in photosynthetic efficiency. Chloroplasts from leaves were large (7.1 μm long in cross section), lens shaped with a well developed membrane system differentiated into grana and stroma lamellae that occupied the large cross-sectional area (12.3 μm²) of the organelle. A few small plastoglobuli and starch grains were scattered in the stroma region. The bract chloroplasts were correlative of leaf chloroplasts in size (6.8 μm in length) and shape with the exception that the bract chloroplasts exhibited greater thylakoid number per granum (15.8) than the leaf chloroplasts (10.5). In contrast to leaf and bract, the capsule wall chloroplasts were smaller in size (4.3 μm) and cross sectional area (6.8 μm²) than either the leaf or bract. The most intriguing feature of the capsule wall chloroplasts was its domination by large starch granules (5.3 μm²) in the stroma which filled the whole chloroplast coercing the membrane system to move towards the periphery of the organelle. Grana number and thylakoids per granum were lowest in the capsule wall chloroplasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Frequency and Diversity of Fungi Colonizing Tissues of Upland Cotton

A study of the mycoflora of upland cotton in Alabama was conducted throughout the 2000 and 2001 growing seasons. Plants were sampled at seedling, first bloom, full bloom, and maturity stages of development. Thirty-seven genera representing 58 species of fungi were isolated, including 9 species of Fusarium. Fusarium oxysporum, F. solani, and F. equiseti were the most common members of this genus occurring at all four sampling stages in both years. Eight species accounted for 67% of the total fungi isolated during the two-year study. Alternaria alternata was the most common fungus encountered, accounting for 19 and 10% of the total fungi isolated in 2000 and 2001, respectively. Twenty species of fungi are reported for the first time colonizing upland cotton tissues.     

A Chlorophyll Fluorescence Analysis of the Allocation of Radiant Energy Absorbed in Photosystem 2 Antennae of Cotton Leaves During Exposure to Chilling

When dark-acclimated cotton (Gossypium hirsutum L. cv. Coker 312) leaves, pre-treated with lincomycin to inhibit chloroplast protein repair processes, were exposed to 10 °C and a PPFD of 500 mol m^-2 s^-1, the proportion of excitation energy entering photochemistry (P) increased, but only to 5 % of the total energy absorbed at steady state levels of P, which were reached at 40 min of irradiation. Thermal dissipation (D) of absorbed energy increased throughout the 360 min irradiation period and accounted for the greatest portion of absorbed energy at 10 °C. When D was partitioned into constitutive (D_CON), regulated (D_REG), and photoinhibitory (D_PI) components, it was primarily composed of D_REG, the readily reversible portion of D. However, the induction of D was slow at 10 °C. Sixty minutes were required for D to reach 70 % of the energy absorbed. Considerable absorption of energy in excess of that utilized in photochemistry or dissipated thermally (designated as E) occurred, especially during induction of P and D. Over the irradiation period, the time-dependent averaged E exhibited an inverse, linear relationship with the ratio of variable (F_v) to maximum (F_m) fluorescence (PS2 efficiency) and a linear relationship with D_PI. We propose that time-dependent averaged E may be useful for estimating the potential for damage to PS2 under stressful environmental conditions.     

Some Physiological Characteristics in Resistant and Susceptible Cotton Cultivars Infected with Cotton Leaf Curl Virus

Eight cultivars/lines of Gossypium hirsutum (CIM-443, CIM-448, CIM-1100, FH-634, S-111, S-113, Cedix, and LRA 5166) resistant to cotton leaf curl virus (CLCuV), one moderately-resistant (cv. NIAB-Krishma), and one susceptible (cv. S-12) were used. All the resistant lines remained free of all disease symptoms, whereas in moderately-resistant and susceptible cvs. leaf curling and vein thickening occurred. Cultivars with varying degree of CLCuV-resistance had different pattern of accumulation of macronutrients. Leaf N content was lowest in S-12, but increased considerably due to disease. Leaf K and Ca contents of S-12 were lower in the diseased leaves than in healthy ones. Chlorophyll a and b contents were highest in lines S-111, S-113 and S-12. A marked reduction in chlorophyll b content was observed in the diseased leaves of S-12. Leaf water potential in S-12 and NIAB-Krishma was also decreased due to disease. The most distinctive characteristic to differentiate between lines was epicuticular wax content, since all the resistant lines had considerably higher wax content on their leaf surfaces than the moderately-resistant or susceptible cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Rapid and Simple Method for in vitro Plant Regeneration from Split Embryo Axes of Six Cultivars of Cotton

Plant regeneration was achieved from the seed derived decotyledonated split embryo axes of six Indian cultivars of cotton (Gossypium hirsutum L.). Explants were cultured on Murashige and Skoog's basal medium supplemented with 2 % sucrose and 0.65 % agar. Incorporation of 0.25 % charcoal in the medium and incubation of the cultures at 30 ± 2 °C had synergistic effect on the frequency of shoot and root formation. The method employed is genotype independent, simple and rapid.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Interaction of Rotylenchulus reniformis,Soil Salinity,and Cotton

Rotylenchulus reni]ormis occurred equally in relatively non-saline (4.0 mmhos/cm) and highly-saline (16.5 mmhos/cm) soils in sampling transects across zones of depressed plant growth in six Texas cotton fields.Results from greenhouse pot experiments indicated progressive positive interaction of salinity and R. reni[ormis pathogenicity in the range 6-18 mmhos/cm.     

Effect of Growth Regulators on Photosynthesis, Transpiration and Related Parameters in Water Stressed Cotton

Gas exchange in Gossypium hirsutum L. cv. H-777 as affected by water deficit and growth regulators (IAA, GA₃, BAP, ABA, ethrel) was examined. Sixty days after sowing, growth regulators in concentration 50 µM were applied as foliar spray and irrigation was withheld to get desired (moderate and severe) water deficit. All the parameters were measured on the third leaf from the top between 10:00 and 11:00. Net photosynthetic rate (P_N), transpiration rate (E), stomatal conductance (g_s), carboxylation efficiency (CE), and water potential (_w) decreased significantly with the increasing water stress, however, water use efficiency (WUE) was unaffected. Foliar spray with IAA, GA₃ and BAP partially counteracted the effect of water deficit on the above parameters except _w, which became more negative. ABA and up to some extent ethrel increased WUE and maintained higher _w, however, caused further decrease in P_N, E, and g_s.     

Effects of Subsoiling and Nematicides on Hoplolaimus columbus Populations and Cotton Yield

Subsoiling to a depth of 35 cm under the planting row for 3 consecutive years increased annual yields of seed cotton by 50 to 200%. Annual subsoiling was essential for maximum yields. The application of a nematicide, 1,2-dibromo-3-chloropropane (DBCP) or aldicarb, reduced the population of Hoplolaimus columbus but did not increase seed-cotton yields over subsoiling alone. Subsoiling reduced H. columbus in the top 20 cm of soil since the treatment favored deeper penetration by much of the root syslem and, consequently, less root colonization of the upper soil zone.     

Physiologic Response of Cotton to the Insecticide Imidacloprid under High-Temperature Stress

The insecticide imidacloprid (tradename Trimax™) has been shown to increase cotton (Gossypium hirsutum L.) yield in the absence of insects, but the explanation for this is not clear. Growth room studies were designed to investigate changes in the physiology and biochemistry of imidacloprid-treated cotton plants and provide information on the mode of action of yield enhancement. Imidacloprid was applied at the pinhead square growth stage at the rate of 52.3 g ai/ha and plants were exposed to day temperatures of 30, 33, 36, and 39°C. Increased levels of photosynthesis and higher values of chlorophyll fluorescence yield, measured two days after imidacloprid application, showed an advantage of imidacloprid-treated over untreated plants. The effect of imidacloprid was greater at the higher temperatures of the growth chamber studies. The results suggested that the imidacloprid-treated plants suffered less temperature stress. This suggestion was supported by findings of reduced glutathione reductase in the imidacloprid-treated plants in the growth chamber, indicating that the untreated plants were experiencing more stress, necessitating the activation of this defense mechanism.     

Influence of Incubation Solution on the Rate of Recovery of Pratylenchus brachyurus from Cotton Roots

The rate of recovery of Pratylenchus brachyurus from cotton roots was enhanced when the tissue was incubated in solutions containing 10 ppm ethoxyethyl mercuric chloride, 50 ppm dihydrostreptomycin sulfate, 50, 100, or 1,000 ppm diisobutylphenoxethyl dimethyl benzyl ammonium chloride, or mixtures of these compounds. Incubation in 10 or 100 ppm zinc sulfate, zinc chloride, or magnesium chloride also enhanced the rate of recovery. Incubation solutions containing 1 or 1,000 ppm zinc chloride or magnesium chloride had no influence on this phenomenon, whereas, 10,000 ppm zinc sulfate, zinc chloride, or magnesium chloride retarded the rate of recovery. A t all incubation intervals during the first 21 days after the roots were removed from soil, the P. brachyurus population consisted of approximately 25% second-stage juveniles, 44% third and fourth-stage juveniles, and 31% females. At least 88% of the second-stage juveniles and 51% of the third and fourth-stage juveniles passed through a single 325-mesh sieve, whereas, 84% of the females collected were retained on a sieve of this mesh.     

Cotton somatic embryo morphology affects its conversion to plant

Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed. The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown control plants regardless of somatic embryo morphology.     

Cotton benzoquinone reductase: Up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris

Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.     

Effect of Growth Regulators on Photosynthetic Metabolites in Cotton under Water Stress

The contents of several photosynthetic metabolites — 3-phosphoglyceric acid (3-PGA), pyruvate, nicotinamide adenine dinucleotide phosphate (NADP) and adenosine triphosphate (ATP) — were determined in leaves of cotton plants (Gossypium hirsutum L. cv. H-777) subjected to waterlogging at vegetative stage, and/or drought at the reproductive stage. In controls, soil moisture contents was kept at field capacity. One day prior to stress, the plant shoots were sprayed with 5 M aqueous solution of indole-3-acetic acid (IAA), gibberellic acid (GA₃), benzylaminopurine (BAP), abscisic acid, and ethrel. In control plants, various growth regulators reduced contents of 3-PGA and ATP while increased contents of NADP and pyruvate. During waterlogging IAA promoted 3-PGA content, and BAP enhanced pyruvate content. During drought, GA₃ enhanced ATP and 3-PGA contents, while IAA enhanced pyruvate content.     

Characterization of the Brassinosteroid Insensitive 1 Genes of Cotton

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.     

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     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.