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     

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.     

Effects of root restriction and growth regulator treatments on the growth of carrot (Daucus carota L.) seedlings

The growth of carrot (Daucus carota L.) seedlings in clear polystyrene vials (7.5 × 2.5 cm diameter) which were either perforated or unperforated and suspended in a large container containing nutrient solution was compared with that of similar seedlings grown directly in the main container. After 28 days plants grown in the perforated, aerated vials had smaller shoots, tap roots (c 20 percent reduction) and fibrous roots (c 60 percent reduction) than unrestricted plants. In the absence of adequate aeration, the effects of root restriction were greater. Foliar applications of ethephon to the leaves of unrestricted plants reduced shoot weights but had little effect on either tap root or fibrous root weights, or even increased tap root weight at lower concentrations. Silver thiosulphate, aminovinylglycine and a highly mobile cytokinin (SD8339) all slightly enhanced shoot growth of restricted plants but had no effect on either tap roots or fibrous root development. It is suggested that although root restriction effects may be partly mediated by endogenous hormones, it seems unlikely that such effects are due solely to stimulation of ethylene production or a reduction in endogenous cytokinin levels.Abbreviations AVG Aminovinylglycine - BA N⁶-Benzyladenine - Ethephon 2-Chloroethylphosphonic Acid - SD8339 6-Benzylamino-9-(Tetrahydropyran-2-yl)-Adenine - STS Silver Thiosulphate     

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.     

Effects of plant growth regulators on secondary wall thickening of cotton fibres

The effect of plant growth regulators on the secondary wall thickeningof cotton fibre was studied. The results indicated that the GA_S andiP+iPA levels in the fibre of field-grown cotton plantsremained almost constant but the IAA and ABA levels changed considerably duringfibre development. Although the change in both IAA and ABA levels seemed not tobe closely related with the rate of cellulose accumulation, there was still arelationship between the ratio of ABA to IAA and secondary wall thickening. Inin vitro studies, ABA (50mol·L^–1) markedly enhanced theaccumulation of dry matter and cellulose in the fibre cell wall duringsecondarywall thickening, but no similar effect was observed with NAA, GA₃ orkinetin treatments. The role of ABA in secondary wall thickening of cottonfibreis discussed.     

The effects of elevated atmospheric CO2 and soil P placement on cotton root deployment

Root proliferation into nutrient rich zones is an important mechanism in the exploitation of soil nutrients by plants. No studies have examined atmospheric CO₂ effects on cotton (Gossypium hirsutum L.) root distribution as affected by localized phosphorus (P). Cotton plants were grown in a Troup sand (loamy, thermic Grossarenic Kandiudults) using 17.2-l containers placed in open top field chambers (OTC) under ambient (360 mol mol^–1) or enriched (720 mol mol^–1) atmospheric CO₂ concentrations for 40 days. Equivalent amounts of P were added (150 mg P per kg of soil) to 100, 50, 25, 12.5, and 6.25% of the total soil volume; control containers with no added P were also included. Under extremely low P (controls), cotton was unresponsive to CO₂ enrichment. In treatments with both fertilized and unfertilized soil volumes, root proliferation was greater in the unfertilized soil under elevated CO₂ conditions. Stimulation of root growth occurred in the P-fertilized soil fraction; the pattern of stimulation was similar under both CO₂ levels. Under ambient CO₂, cotton plant response was positive (shoot mass, and total root mass and length) when soil P was confined to relatively small proportions of the total soil volume (6.25 and 12.5%). However, elevated CO₂ grown plants tended to respond to P regardless of its distribution.     

Establishment of molecular markers and linkage groups in two F2 populations of Upland cotton

Two F₂ populations of cotton (Gossypium hirsutum L.) from the crosses of HS46 x MARCABUCAG8US-1-88 (MAR) and HS46 x Pee Dee 5363 (PD5363) were characterized for restriction fragment length polymorphisms (RFLPs) using DNA probes. Seventy-three probe/enzyme combinations were used in the HS46 x MAR population analysis, which resulted in 42 informative polymorphic fragments. These 42 moleclar markers represented 26 polymorphic loci, which consisted of 15 codominant and 11 dominant (+/-) genotypes. Chi-square analyses of these loci fit expected genotypic ratios of 121 and 31, respectively An analysis of these loci with the MAPMAKER program resulted in the establishment of four linkage groups A, B, C, and D with 4,2,2, and 2 loci, respectively, as well as 16 unlinked loci. Six probe-enzyme combinations were assayed on the HS46 x PD5363 population, which resulted in 11 informative polymorphic fragments. These 11 fragments represented 6 polymorphic loci, 1 dominant (+/-) and 5 codominant genotypes. The MAPMAKER analysis of these loci yielded 2 linked loci. Thus, a total of 53 polymorphic fragments and 32 polymorphic loci, representing five linkage groups, were identified among the two families.Contribution of the USDA-ARS in cooperation with the Miss Agric For Exp Stn.     

Top and root growth and nutrient absorption of Prunus avium L. at two soil pH and P levels

One-year-old Prunus avium L. were grown under greenhouse conditions in a Countesswells soil in all combinations of 2 pH and 2 P levels. The soil, obtained from a long-term liming and fertilizer experiment, provided pH values throughout the experiment of 3.75–3.99 (pH 1) and 4.81–5.41 (pH 2). The P treatments had 0.43% acetic acid extractable P of 31–44 g g^-1 (P1) and 145–173 g g^-1 (P2). The trees were harvested 92 (H1), 134 (H2), and 168 (H3) days after initiation of growth.Top (leaf+new stem) dry weight was significantly increased for pH 2 and P2 at H2 and H3. P2 also increased leaf weight (H1), the weight of the original stem-root (H2 and H3), and root length but decreased root diameter at both soil pHs (H2 and H3). Total tree uptake of N, P, K, Ca, and Mg was also increased by pH-P combinations which had significantly greater dry matter production and root length. Total Mn uptake decreased at pH2. Root nutrient inflows (uM m^-1 day^-1) were increased for Ca at pH2 and for P at P2. Mn inflow decreased at pH2 and at pH1 P2 although the increased root length associated with the latter treatmen resulted in increased total tree Mn uptake. In general, high nutrient inflows occurred in all trees at H1 and in severely stunted trees at pH1 P1; both had larger than average root diameters.     

Growth and nutrient concentrations of alfalfa and common bean as influenced by soil acidity

Growth and nutrient utilization of alfalfa (Medicago sativa L. cv. Arc) and common bean (Phaseolus vulgaris L. cv. Carioca) were studied in an acid soil adjusted to eight levels of soil acidity by lime addition. Application of lime significantly (P<0.05) increased shoot and root growth for both species. However, common bean was far less sensitive to soil acidity than alfalfa. Maximum alfalfa growth was obtained at a soil pH of 5.8 and maximum bean growth was achieved at pH 5.0. Root and shoot growth of both legumes was positively correlated (P<0.01) with soil pH, exchangeable Ca and exchangeable Mg and negatively correlated (P<0.01) with soil exchangeable Al. Common bean had a lower internal P requirement for maximum growth and was more efficient than alfalfa in taking up Ca and Mg. These characteristics would contribute to the favorable growth of common bean in acid-infertile soils.     

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     

Phenolic compounds and somatic embryogenesis in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Studies of phenolic compounds were performed during cell suspension cultures in relation with the induction of embryogenic structures in two cultivars of cotton. Coker 312 produced embryogenic structures, unlike R405-2000 which was found to be a non-embryogenic cultivar. Embryogenesis induction in Coker 312 was strongly linked to a higher content of caffeic, ferulic and salicylic acids and to the appearance of p-coumaric acid, benzoic acid, trans-resveratrol, catechin and naringenin.     

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.     

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     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

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.     

The rate of cellulose increase is highly related to cotton fibre strength and is significantly determined by its genetic background and boll period temperature

This experiment was conducted to study the relationship between the increase in cellulose content in developing cotton bolls and their final cotton fibre strength. The rate of cellulose increase over time was estimated using logistical regression, and the logistic equation parameters were then used to compare different cotton cultivars in different temperature environments. The increase in cellulose content followed a typical “S” curve, with the boll period time divided into slow-fast-slow stages. In different cultivars, the final fibre strength was closely related to the characters of the fast cellulose content increasing stage, negatively related to the maximal cellulose increasing rate (P < 0.05), and positively related to the duration of the fast cellulose content increasing stage (P < 0.01). In the same cultivar, low temperature reduced the maximal cellulose increasing rate and prolonged the duration of the fast cellulose increasing stage. The results indicate that, in diverse genetic background, long-lasting and tempered cellulose growth during the rapid cellulose increasing stage is of significant benefit to high strength fibre development. For closely related cotton cultivars, decreasing the maximal cellulose increasing rate and the termination of rapid cellulose increasing stage reduced fibre strength that often occurs when temperatures are low.     

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.     

Storage protein accumulation patterns in somatic embryos of cotton (Gossypium hirsutum L.)

Summary The storage protein content of somatic embryos of Gossypium hirsutum L. cv. Coker 201 was determined using extinction level, antigen/antibody association detection methods. Mature storage protein was first detected in early globular-stage somatic embryos at a total concentration of 0.36% of the embryo protein mass. Tulip-stage and mature somatic embryos were comprised of 3.0% and 1.3% mature storage protein, respectively. Maximum storage protein synthesis was found to occur during early globular- and early heart-stages. During this period of development, significant levels of protein precursors were found also to accumulate. The pattern of storage protein synthesis, processing and accumulation paralleled the pattern that has been reported for the zygotic system, although somatic embryos accumulate storage protein at much earlier stages and to a lesser degree. The possibility of using complex biochemical pathways to monitor embryogenic systems in vitro is discussed.     

Effect of crop residues on root growth and phosphorus acquisition of pearl millet in an acid sandy soil in Niger

The effect of long-term (1983–1988) applications of crop residues (millet straw, 2–4 t ha^-1 yr^–1) and/or mineral fertilizer (30 kg N, 13 kg P and 25 kg K ha^-1 yr^-1) on uptake of phosphorus (P) and other nutrients, root growth and mycorrhizal colonization of pearl millet (Pennisetum glaucum L.) was examined for two seasons (1987 and 1988) on an acid sandy soil in Niger. Treatments of the long-term field experiment were: control (–CR–F), mineral fertilizer only (–CR+F), crop residues only (+CR–F), and crop residues plus mineral fertilizer (+CR+F).In both years, total P uptake was similar for +CR–F and –CR+F treatments (1.6–3.5 kg P ha^-1), although available soil P concentration (Bray I P) was considerably lower in +CR–F (3.2 mg P kg^-1 soil) than in –CR+F (7.4) soil. In the treatments with mineral fertilizers (–CR+F; +CR+F), crop residues increased available soil P concentrations (Bray I P) from 7.4 to 8.9 mg kg^-1 soil, while total P uptake increased from 3.6 to 10.6 kg P ha^-1. In 1987 (with 450 mm of rainfall), leaf P concentrations of 30-day-old millet plants were in the deficiency range, but highest in the +CR+F treatment. In 1988 (699 mm), leaf P concentrations were distinctly higher, and again highest in the +CR+F treatment. In the treatments without crop residues (–CR–F; –CR+F), potassium (K) concentrations in the leaves indicated K deficiency, while application of crop residues (+CR–F; +CR+F) substantially raised leaf K concentrations and total K uptake. Leaf concentrations of calcium (Ca) and magnesium (Mg) were hardly affected by the different treatments.In the topsoil (0–30 cm), root length density of millet plants was greater for +CR+F (6.5 cm cm^-3) than for +CR–F (4.5 cm cm^-3) and –CR+F (4.2 cm cm^-3) treatments. Below 30 cm soil depth, root length density of all treatments declined rapidly from about 0.6 cm cm^-3 (30–60 cm soil depth) to 0.2 cm cm^-3 (120–180 cm soil depth). During the period of high uptake rates of P (42–80 DAP), root colonization with vesicular-arbuscular mycorrhizal (VAM) fungi was low in 1987 (15–20%), but distinctly higher in 1988 (55–60%). Higher P uptake of +CR+F plants was related to a greater total root length in 0–30 cm and also to a higher P uptake rate per unit root length (P influx). Beneficial effects of crop residues on P uptake were primarily attributed to higher P mobility in the soil due to decreased concentrations of exchangeable Al, and enhancement of root growth. In contrast, the beneficial effect of crop residues on K uptake was caused by direct K supply with the millet straw.