Expression of baculovirus anti-apoptotic p35 gene in tobacco enhances tolerance to abiotic stress

Expression of baculovirus anti-apoptotic p35 gene in plants on biotic stress responses has been well studied but its function on abiotic stress has not been documented. In the present study, the p35 gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed in tobacco. A detached leaf assay was used to test tolerance of p35 transgenic plants to various abiotic stress responses. Expression of p35 gene in tobacco gave tolerance to treatment with methanol and H₂O₂ and also delayed leaf senescence under starvation in the dark. Germination of T₀ seeds on NaCl-containing medium also demonstrated to increase salt tolerance.     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Expression studies of stress responsive genes in cotton Gossypium hirsutum L.

Molecular Biology Reports - Cotton is the world’s richest source of natural fiber. Meanwhile cotton plant is top ranked stress sensitive plant thereby affecting its yield and fiber quality....     

Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Molecular mapping of a new source of Fusarium wilt resistance in tetraploid cotton (Gossypium hirsutum L.)

Fusarium wilt (FW) disease is an economically important disease of cotton worldwide and a major cause of crop losses in Australia and many other cotton-producing countries. Symptoms include wilting, vascular browning and death. Australian races of the causal agent Fusarium oxysporum f. sp. vasinfectum (Fov) are genetically distinct from those in other countries and are thought to have evolved from indigenous races. New sources of resistance for breeding are rare, as cotton cultivars with significant FW resistance against Fov isolates from other cotton-producing regions are usually susceptible to Australian Fov races. MCU-5, an Upland Indian cotton cultivar, has been identified as having improved resistance to Australian Fov and is being used to breed new commercial cultivars with higher resistance to FW. To investigate the genetic basis of the FW resistance in MCU-5, QTL analysis was performed on 244 F3 and 244 F4 families derived from an intraspecific cross between MCU-5 and Siokra 1-4, a cultivar highly sensitive to Australian Fov races. Resistance, as measured by leaf symptoms, vascular browning and survival, showed low to moderate heritability between generations. MCU-5 resistance to FW was found to be complex with three quantitative trait loci (QTL) identified in the F3, and eight in the F4, that explained between 9 and 41% of the phenotypic variation. The QTL were located on four linkage groups including chromosomes A6 (Chr 6), D4 (Chr 22) and D6 (Chr 25), with two QTL located in similar regions to previously identified FW resistance from the Sea Island cultivar Pima 3-79. The QTL identified in this study represent the first targets for marker-assisted selection of FW resistance in Australia.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Characterization of two cotton (Gossypium hirsutum L) invertase genes

Two cotton vacuolar-invertase genes were identified and sequenced. Both genes had seven exons, including an unusually small second exon typical of acid invertases. These genes encode peptides with many features shared by acid invertases from other species including, leader sequences that probably target the peptide to the vacuole, active site motifs and substrate binding motifs. Expression analyses indicated that one of the genes was expressed in roots during the starch filling stage of development. However, expression of the same gene fluctuated during the starch utilization stage of development. Therefore this gene was unlikely to play a role in determining sink strength of this tissue. Both genes were expressed in elongating fibers where they were likely to play a role in cell expansion. The invertase gene uniquely expressed in fiber had a simple sequence repeat (SSR) in the third intron that was polymorphic among various cotton species. An EST was identified with an expansion of the SSR that included the third intron indicating this SSR is associated with a splice variant. The polymorphic SSR may be useful in investigating the function of this gene in fiber development.     

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 g/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Aph IV aminoglycoside phosphotransferase type IV Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 354-89     

A study of heterosis in upland cotton (Gossypium hirsutum L.)

Summary Heterosis (over mid parent) and useful heterosis (over commercial variety H14) estimates were obtained from a line x tester analysis of crosses involving thirteen diverse female parents with two locally adapted varieties H14 (local standard) and J34. Marked heterosis was observed for seed cotton yield, boll number and halo length. The values of positive heterosis and useful heterosis for seed cotton yield ranged from 28.1 to 87.0% and 20.1 to 45.5%, respectively. The overall study of heterosis revealed that female parents PRS-72 (USSR), 5904F (USSR) and MCU-5 (Madras Cambodian Uganda Selection, Coimbatore) were among the top three females, showing considerable heterosis in crosses with H14 and J34 for seed cotton yield and fibre properties. The practical difficulties in exploiting the phenomenon of heterosis and possible experimental approaches in upland cotton are discussed.     

Wide-cross whole-genome radiation hybrid mapping of cotton (Gossypium hirsutum L.)

We report the development and characterization of a "wide-cross whole-genome radiation hybrid" (WWRH) panel from cotton (Gossypium hirsutum L.). Chromosomes were segmented by gamma-irradiation of G. hirsutum (n = 26) pollen, and segmented chromosomes were rescued after in vivo fertilization of G. barbadense egg cells (n = 26). A 5-krad gamma-ray WWRH mapping panel (N = 93) was constructed and genotyped at 102 SSR loci. SSR marker retention frequencies were higher than those for animal systems and marker retention patterns were informative. Using the program RHMAP, 52 of 102 SSR markers were mapped into 16 syntenic groups. Linkage group 9 (LG 9) SSR markers BNL0625 and BNL2805 had been colocalized by linkage analysis, but their order was resolved by differential retention among WWRH plants. Two linkage groups, LG 13 and LG 9, were combined into one syntenic group, and the chromosome 1 linkage group marker BNL4053 was reassigned to chromosome 9. Analyses of cytogenetic stocks supported synteny of LG 9 and LG 13 and localized them to the short arm of chromosome 17. They also supported reassignment of marker BNL4053 to the long arm of chromosome 9. A WWRH map of the syntenic group composed of linkage groups 9 and 13 was constructed by maximum-likelihood analysis under the general retention model. The results demonstrate not only the feasibility of WWRH panel construction and mapping, but also complementarity to traditional linkage mapping and cytogenetic methods.     

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     

Chilling injury in cotton (Gossypium hirsutum L.) Prevention by abscisic acid

Cotton (Gossypium hirsutum L.) intact seedlings and isolatedcotyledonary discs were exposed to chilling (4?C) under humidconditions which prevented dehydration. The damage resultingfrom chilling was estimated by means of electrolyte leakageand survival in whole seedlings and by the electrolyte leakageand necrotic areas in isolated cotyledonary discs. Also, theeffect of chilling on membrane phospholipids and cellular reducedglutathione was determined. Within the first two and three daysof chilling, there was a marked reduction in the reduced glutathioneand membrane phospholipid levels without electrolyte loss andnecrosis. This reduction was completely prevented by pretreatmentwith abscisic acid. Prolonging the chilling period resultedin decreased survival in whole seedlings and in progressiveincrease in electrolyte leakage and necrosis in isolated cotyledonarydiscs. Pretreatment with abscisic acid prior to chilling almostcompletely prevented this chilling injury when exposure to 4?Cwas less than 5 days. Even with longer chilling periods, theabscisic acid pretreatment greatly reduced the damage. ³Incumbent of the Seagram Chair in Plant Science. (Received July 21, 1979; )     

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.     

Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Summary Seventeen cultivars of cotton (Gossypium hirsutum L.) were evaluated for callus initiation and maintenance using 3 initiation media and 3 maintenance media. After a series of transfers of a 3% glucose media, calli were placed on a 3% sucrose medium. After several weeks calli were observed for the presence of embryo-like structures. Cultivars Coker 201 and Coker 315 were identified as embryogenic. Embryogenic callus has since been routinely obtained within 6 weeks by initiating callus on glucose media for 3–4 weeks followed by transfer to sucrose media. Histological examination has shown that embryos are derived from isodiametric, densely cytoplasmic cells and follow predictable patterns of development. Upon maturity, transfer to auxin-free media with reduced sucrose levels results in embryo germination. Regenerated plants can be transferred to greenhouse within 90 days of callus initiation.The senior author is presently a Research Geneticist, USDA-ARS, and Assistant Professor Present address