Production of fertile hybrid germplasm with diploid Australian Gossypium species for cotton improvement

The 17 wild Australian Gossypium species are distant diploid relatives of the commercial tetraploid cottons, G. barbadense L. and G. hirsutum L. They interest cotton breeders as a source of terpenoid-aldehyde-free seeds, a trait only found in five Australian Gossypium species. They elicit further interest because some species grow near current and projected cotton growing areas in Australia and thus could serve as unintentional recipients of transgenes from genetically engineered cotton cultivars. The utility of the wild Australian Gossypium species in cotton breeding depends on the ability to generate fertile hybrids, and to the extent this is possible under glasshouse conditions, it allows predictions regarding the probability that fertile hybrids between the transgenic cottons and spatially associated populations of wild species will arise without human manipulation. The Australian Gossypium species fall into three morphologically and cytologically distinct groups designated the C, G, and K genomes, The G-genome species hybridize most readily with G. arboretum (a diploid A-genome cultivated cotton), while the C- and K-genome species are more compatible with G. hirsutum (a tetraploid AD-genome cultivated cotton). These intergenomic hybrids are sterile, and the chromosome complement of the hybrids must be doubled prior to backcrossing to G. hirsutum. The only exceptions were four G. hirsutum × K-genome triploids, which exhibited limited female fertility when backcrossed to G. hirsutum. Two of the three diploid species geographically associated with commercial cotton fields (G. australe F. Mueller & G. rotundifolium Fryxell, Craven & Stewart) failed to produce hybrid progeny when pollinated with G. hirsutum pollen; the third species (G. sturtianum J.H. Willis) produced only 5 sterile triploids from 25 pollinations. Thus, the probability that wild species could serve as recipients of transgenes is functionally zero, especially in conjunction with the profound prezygotic barriers that separate the cultivated tetraploid cottons from their wild Australian relatives. Eighteen new fertile synthetic polyploids and 23 self-fertile derivatives of two synthetic hexaploids were produced. Synthetic tetraploids require greater effort to backcross than do synthetic hexaploids. These fertile hybrids represent a new avenue of introgression of genes from wild Australian Gossypium species into commercial cotton cultivars, an avenue limited only by the level of recombination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transformation of α-EXPA1 gene leads to an improved fibre quality in Gossypium hirsutum

The Expansin protein is known for its multifaceted roles in plant growth, especially cell walls. However, very few studies have been done so far to assess the effects of expansin genes on Cotton fibre development. The present study is a successful effort to fill this gap, where the α-EXPA1 gene transformed into a local cotton variety, Gossypium hirsutum, through Agrobacterium-mediated transformation under Gossypium hirsutum Seed coat and Fibre-specific promoter (GhSCFP). The transgenic cotton plants underwent molecular characterization and fibre trait evaluation. Our results indicated that α-EXPA1 showed an up-regulated expression during the transition phase of secondary cell wall synthesis and resulted in improving the fibre parameters, especially micronaire value. Transgenic cotton fibre also showed a finer twisting under the Scanning electron microscope (SEM) as compared to non-transgenic cotton fibre samples. The fibre production is influenced by more than nine thousand genes, and the fibre improvement cannot be just achieved through a single gene transformation. However, α-EXPA1 is one of the potential candidates for cotton fibre research as it significantly improved the cotton fibre.     

Identification of resistance gene analogs in cotton (Gossypium hirsutum L.)

Sequence analyses of numerous plant disease resistance genes have revealed the presence of conserved motifs common to this class of genes, namely a nucleotide binding site (NBS) and leucine rich repeat region. In this study, thirty-three resistance gene analogs (RGAs) were cloned and sequenced from cotton (Gossypium hirsutum L.) following PCR with degenerate primers designed from the conserved NBS motif of plant resistance (R) genes. Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into four distinct classes from which several subgroups were delineated based on nucleic acid sequences. Gene database searches with the consensus protein sequences of each of the four classes and respective subgroups of cotton RGAs revealed their conserved NBS domains and homology to RGAs and known resistance genes from a variety of plant genera. Given the complete lack of knowledge regarding molecular organization of R genes in cotton, the cloned RGAs described here may be useful as probes to map, characterize, and manipulate R genes of the cotton genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterosis in growth and photosynthetic rate in hybrids of cotton

Summary The intraspecific (Gossipium hirsutum) and interspecific (G. hirsutum x G. barbadense) F₁ hybrids of cotton were found to exhibit a high degree of heterosis in the production of fruiting branches, number of bolls (fruits), yield of seed cotton and photosynthetic rates over the parent plants. The developing bolls of the hybrids had significantly higher weights than their parents until the 20th day after anthesis. The patterns of leaf area development among interspecific hybrids differed when compared with the parent plants. The photosynthetic rates of the hybrids were comparable with those of maize and sorghum and much higher than the average rate reported so far for the cotton plant.     

Fine mapping of open-bud duplicate genes in homoelogous chromosomes of tetraploid cotton

The open-bud mutant plant has a flower bud that is open at its tip due to the shortening of the corollas, resulting in exposure of the stigma and upper anthers. This mutant plant is potentially useful as a parent for producing hybrid cotton seeds. We have identified the open-bud trait to be inherited as recessive duplicate genes and finely mapped them in the homoelogous chromosome (chr) pair of chr. 18 (D13) and 13 (A13). Gossypium hirsutum substitution line 18, which has chr. 18 replaced by its corresponding homozygous chromosome in G. barbadense acc. 3-79 and the genotyped open-bud duplicate genes ob ₁ ob ₁ ob ₂ ob ₂, was crossed with the monomeric genotypic G. hirsutum acc. TM-1 and G. barbadense acc. 3-79 to produce two mapping populations. We then fine-mapped the duplicate open-bud gene ob ₁ in the chr. 18 (D13) and ob ₂ in the chr. 13 (A13) homoelogous chromosome pair in tetraploid cotton. Molecular markers closely linked with ob ₁ and ob ₂ will be useful tools in the development of open-bud lines by marker-assisted selection.     

Genetic control of somatic embryogenesis in cotton petiole callus cultures

Summary Three commercial varieties (Acala SJ-5, Coker 312 and Paymaster 303) and three exotic accessions (T1, T25 and T169) of cotton (Gossypium hirsutum L.) were tested for ability to undergo somatic embryogenesis. Sections of split petiole were cultured on 3 media and evaluated for embryogenesis after 180 days. Embryogenic T25 and Coker 312 plants were selected and crossed in a diallel with non-embryogenic Acala SJ-5, Paymaster 303, T1 and T169 plants. F₁, F₂ and BC₁ populations were generated and tested for embryogenesis on a medium of MS salts and vitamins (1962) plus (per liter) 4.0 mg NAA, 1.0 mg Kn, 30 g glucose, 100 mg myo-inositol, 2.0 g Gelrite and 0.75 g MgCl₂. Segregation for both occurrence and magnitude of embryogenesis was observed, suggesting the action of more than one gene.     

Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids

To evaluate the possible antiaphid function of Amaranthus caudatus agglutinin (ACA) in allogenetic plants, transgenic cotton plants expressing the ACA gene under the control of a phloem‐specific promoter were generated via Agrobacterium‐mediated gene transformation. Based on the results of Southern blot analyses, six plants with single or lower copy transgene and favourable agronomic traits were selected for further studies. ACA expression levels ranged from 0.02% to 0.45% of total soluble protein as determined by Western blot analysis in the six selected transgenic plants. Insect bioassays using nymphs of cotton aphid (Aphis gossypii Glover) showed that five of the six transgenic plants significantly inhibited the population growth of cotton aphid, with the highest inhibition rate of 64.5%. These results shed some new light on the antiaphid function of the ACA gene as well as the promising application of the gene for obtaining aphid‐resistant transgenic cotton plants to reduce the yield loss and honeydew contamination of fibre by aphids.     

DNA fingerprinting studies in coloured cotton genotypes

A collection of 11 coloured cotton Gossypium hirsutum genotypes and four white linted genotypes of different origin were evaluated by randomly amplified polymorphic DNA (RAPD) analysis. These 15 cotton genotypes were evaluated using 32 different 10‐mer primers of arbitrary sequences. All 32 primers were polymorphic ‐ in total, 287 amplified fragments were observed in these patterns. Out of the 287 fragments, 219 were polymorphic accounting for 76.31% of the total number of fragments. Similarity indices were calculated using the Dice coefficient and a dendrogram showing relationships between genotypes was obtained by Unweighted Pair Group Method of Arithmetic Average (UPGMA) cluster analysis. Cluster analysis showed clear‐cut separation of the coloured and white linted genotypes and thus formed three clusters (I, II and III). Among the coloured linted genotypes, all except ‘Parbhani American’ and ‘Lousiana brown’ clustered together. Cluster II contained white linted genotypes orginating from the same breeding station. The results indicate that RAPDs may constitute a relatively simple and efficient method for analysing genetic variation in coloured and white linted G. hirsutum collections.     

Zygosity identification in transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) by real-time quantitative PCR

Successful identification of homozygous and heterozygous transgenic plant with currently available techniques such as southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH) and so on, demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large number of plants differing only by a factor of two in the amount of target sequences. In the present study, we determined zygosity level of transgenes in cotton [Gossypium hirsutum L.] with two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers. TPS, a single copy gene per haploid Gossypium hirsutum genome was used as the endogenous reference to estimate copy number of transgene. Both assays were accurate and reproducible in determination of the number of transgenes present in a cell line. These methods are standard curves and Delta delta C _t method.     

Genetic characterization of reniform nematode resistance for Gossypium arboreum accession PI 417895

Rotylenchulus reniformis is an important root pathogen of cotton in the south‐eastern United States, and management is hindered by the lack of host‐plant resistance in upland cotton (Gossypium hirsutum). The G. arboreum accession PI 417895 is highly resistant to R. reniformis, and a segregating population of 300 F₂ plants was developed for phenotypic characterization of resistance. The population showed quantitative variation for nematode infection. Twenty plants showed no infection and were classified as escapes. Fifty‐four plants were classified as resistant or moderately resistant, whereas, 226 were classified as moderately susceptible or susceptible based on the nematode response of the susceptible parent, indicating resistance is a recessive trait, but these data did not support the single recessive gene model. Alternatively, this model would be supported if the 77 plants with a similar nematode response as observed for PI 417895 were classified as resistant. Twelve plants showed high levels of resistance and these data would support a two recessive gene model. Accession PI 417895 represents a new source of R. reniformis resistance with two major genes conferring resistance. Introgression of multiple resistance genes into G. hirsutum will require the development of larger populations to recover the resistant phenotype.     

Over-expression of Gastrodia anti-fungal protein enhances Verticillium wilt resistance in coloured cotton

Currently there are no adequate control measures for the cotton fungal diseases Verticillium wilt and Fusarium wilt, which are important factors limiting yield under certain conditions. In this study the gene encoding a Gastrodia anti‐fungal protein was introduced into three cultivars of coloured cotton using the method of pollen‐tube pathway transformation, with the purpose of obtaining transgenic plants with improved resistance to wilt. Of the 121 herbicide‐resistant cotton plants two, LB‐5‐8 and ZB‐1‐49, were scored as transgenic based on Southern blot, RT‐PCR analysis and in vitro anti‐fungal activity assay. Field analysis demonstrated that the transgenic lines LB‐5‐8 and ZB‐1‐49 possess an increased resistance to wilt. After 2 years of breeding, the progeny of LB‐5‐8 and ZB‐1‐49 lines still showed a stable and strong resistance to Verticillium wilt. Lines with high levels of resistance to Verticillium wilt obtained from the present study may be widely planted and help to reduce the future impact of cotton wilt on cotton production resulting in increased yields.     

Effects of the dominant glandless gene Gl2e on agronomic and fibre characters of Upland cotton

Seven pairs of near‐isogenic lines (glandless vs. glanded) and the recurrent parents were used to determine the effects of the dominant glandless gene from ‘Hai 1’(Gossypium barbadense) on agronomic, fibre, and seed characters in Upland cotton, Gossypium hirsutum, backgrounds. The results showed that there were no apparent linkage associations of the glandless gene on most agronomic, fibre and seed characters of Upland cotton, except for seed quality. The glandless line derived from ‘Liaomian 7’had significantly more protein (489.6 g/kg), and that from H237 had significantly more oil (362.4 g/kg) and had the largest oil index (2.70 g) and protein index (3.03 g). The gossypol content of seed in dominant glandless lines in Upland cotton was very low (<0.04 g/kg). Therefore, it is suggested that the glandless gene can play an important role in breeding glandless or low seed‐gossypol Upland cotton cultivars.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Inheritance of somatic embryogenesis using leaf petioles as explants in upland cotton

Somatic embryogenesis (SE) is a critical step leading to plant regeneration in tissue culture of many plant species. The objective of the present study was to analyze the inheritance of SE in cotton (Gossypium hirsutum L.) using leaf petioles as explants. A high embryogenic callus (HEC)—producing line, W10, was selected by petiole callus culture from a commercial Chinese cotton cultivar CRI24 and crossed with a non embryogenic line, TM-1 and a low embryogenic (LEC) commercial Chinese cotton cultivar, CRI12, respectively. The parental lines, F₁ and F₂ were grown in field conditions for sources of leaf petioles as explants. The F₁ plants were similar to the HEC parent in embryogenic callus (EC) induction, indicating that high EC ability is dominant. The classical Mendelian analysis showed that the high EC ability in the HEC line W10 is controlled by two independent dominant genes with complementary effect, designated Ec ₁ and Ec ₂ , while the LEC line CRI12 contains one dominant gene Ec ₂ . A joint segregation analysis confirmed that SE ability in cotton is controlled by two major genes with epistatic effects along with other polygenes. A SSR marker analysis identified three quantitative trait loci (QTLs) on two linkage groups, one of which harbored a major QTL (qEc1) which is assigned to the major gene Ec ₁ . This qualitative and quantitative genetic study has provided an incentive to fine map the genes responsible for SE towards the isolation of the SE genes in cotton.     

Assessment of Gossypium sturtianum and G. australe as potential sources of Fusarium wilt resistance to cotton

When challenged with Fusarium oxysporum f. sp. vasinfectum (Fov) from vegetative compatibility groups (VCGs) 01111 and 01112 in glasshouse tests, Gossypium australe Mueller and Gossypium sturtianum Willis accessions showed a variety of disease responses ranging from highly resistant to highly susceptible. Under high disease pressure G. sturtianum accession Gos-5275 was significantly more resistant than the commercial G. hirsutum cultivars that are designated standards for Fusarium resistance by Australian cotton breeders. Under low disease pressure G. sturtianum accession Gos-5250 was more susceptible than a highly susceptible commercial cultivar. A series of glasshouse tests was performed at two locations (Indooroopilly, QLD. and Canberra, ACT), and under low and high disease pressure. In these tests, a hexaploid cross (Gos-5271) generated from a Fusarium-resistant G. sturtianum (Gos-5275) and a Fusarium-susceptible G. hirsutum L. (CPI-138969) was significantly more resistant to Fusarium wilt than its G. hirsutum parent. Thus G. sturtianum, with a diploid genome and a range of responses to Fov challenge, has the potential to provide the basis for the elucidation of the genetic basis of resistance to Fusarium wilt in cotton species. In addition, resistant accessions of G. sturtianum are identified as a potential source of Fusarium wilt resistance genes for cotton breeding. In the glasshouse tests used to assess the resistance of various Gossypium accessions to Fusarium wilt disease, the scoring of vascular browning was found to give a more reliable indication of disease severity than the scoring of foliar symptoms. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improved salt tolerance and seed cotton yield in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) by transformation with <Emphasis Type="Italic">betA</Emphasis> gene for glycinebetaine synthesis

Homozygous transgenic cotton (Gossypium hirsutum L.) plants that accumulated glycinebetaine (GB) in larger quantities were more tolerant to salt than wild-type (WT) plants. Four transgenic lines, namely 1, 3, 4, and 5, accumulated significantly higher levels of GB than WT plants did both before and after salt stress. At 175 and 275 mM NaCl, seeds of all the transgenic lines germinated earlier and recorded a higher final germination percentage, and the seedlings grew better, than those of the WT. Under salt stress, all the lines showed some characteristic features of salt tolerance, such as higher leaf relative water content (RWC), higher photosynthesis, better osmotic adjustment (OA), lower percentage of ion leakage, and lower peroxidation of the lipid membrane. Levels of endogenous GB in the transgenic plants were positively correlated with RWC and OA. The results indicate that GB in transgenic cotton plants not only maintains the integrity of cell membranes but also alleviates osmotic stress caused by high salinity. Lastly, the seed cotton yield of transgenic lines 4 and 5 was significantly higher than that of WT plants in saline soil. This research indicates that betA gene has the potential to improve crop’s salt tolerance in areas where salinity is limiting factors for agricultural productivity.     

Molecular characterization of cytoplasmic male sterility conditioned by <Emphasis Type="Italic">Gossypium harknessii</Emphasis> cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F₁ seed lots.     

Promoter anchored amplified polymorphism based on random amplified polymorphic DNA (PAAP-RAPD) in cotton

Non-coding sequences account for a majority of the higher plant genome, some of which have important effects in gene regulation and plant development. In an effort to develop molecular marker systems to search for polymorphisms associated with high fiber yield and quality in cotton, we have developed a methodology that could specifically target the regulatory regions of the cotton genome. In this study we designed 10-nucleotide degenerate promoter primers based on conserved core promoter sequences and tested their applicability in PCR amplifications in combination with 10-mer random amplified polymorphic DNA (RAPD) primers. The amplified markers are called promoter anchored amplified polymorphism based on RAPD (PAAP-RAPD). Forty cotton genotypes with diverse genetic and geographical backgrounds were used to test the PAAP-RAPD system using polyacrylamide gel electrophoresis. Based on PAAP-RAPD markers amplified from 12 primer combinations, the 40 genotypes were classified into five distinctive groups: two Upland cotton (Gossypium hirsutum) groups from China, another two Upland cotton groups from the USA, and one group from American Pima cotton (G. barbadense). The groupings are in general consistent with their genetic and geographical origins. Thirty-six PAAP-RAPD and RAPD fragments were cloned and four of them were further subjected to sequence analysis. Signal scanning using software PLACE confirmed that they contained an array of cis-regulatory sequences in addition to the core promoter sequences. The results demonstrate the potential application of PAAP-RAPD as a new marker system specifically targeting regulatory regions of the plant genome.