Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM8P medium supplemented with 2.685μM α-naphthaleneacetic acid and 0.465μM kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72–81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in cotton.

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pDgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5×108 cells ml)1 (OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19℃) and short co-cultivation duration (48h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50mg l)1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotylderived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence-related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty-nine F2 progeny from the interspecific cross of 'Handan208'×'Pima90' were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty-eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8cM with a mean interlocus space of 9.08 cM. A v2 test for significance of deviations from the expected ratio (1:2:1or3:1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18–28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker-assisted selection.

A new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 mM) and kinetin (KT; 2.32mM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO3 and free of growth regulators. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with L-asparagine (Asn)/L-glutamine (Gln) (7.6/13.6mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis-active Cry1Ac and API-B chimeric gene. Infected embryogenic calli were co-cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7-8 weeks. Then, the kanamycin-resistant calli (Kmr) subcultured in proliferation medium would re-differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100-ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty-five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API-B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium-mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explants: leaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20-50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5-20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 lM/kinetin 0.929 lM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2, 4-D 0.452 lM/kinetin 0.465 lM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.

Asian cotton (Gossypium arboretum L.) was once widely cultivated in China. It has also been a valuable source of genetic variation in modern cotton improvement. In this study, the genetic diversity of selected G. arboreum accessions collected from different regions of China was evaluated by microsatellite (simple sequence repeats, SSRs) analysis. Of the 358 microsatellite markers analyzed, 74 primer pairs detected 165 polymorphic DNA fragments among 39 G. arboreum accessions examined. Twelve accessions could be fingerprinted with one or more SSR markers. With the exception of two accessions, DaZiJie and DaZiMian, genetic similarity coefficients among all accessions ranged from 0.58 to 0.87 suggesting high level of genetic variation in the G. arboreum collections. The UPGMA dendrogram constructed from genetic similarity coefficients revealed positive correlation between cluster groupings and geographic dis-tances. In addition, comparison of the microsatellite amplification profiles of the diploid G. arboreum and tetraploid Gossypium hirsutum L. found that size distribution of amplified products in G. arboreum was dispersive and that of G. hirsutum was relatively concentrated. The information on the genetic diversity and SSR fingerprinting from this study is useful for developing mapping populations for constructing diploid cotton genetic linkage map and tagging economically important traits.

A genetic linkage map of cotton was constructed with a newly developed molecular marker-SRAP (sequence-related amplified polymorphism) using a population consisting of 129 F2 individuals derived from the interspecific cross of "Handan208"