Production Of Interspecific Cybrid, Symmetric Somatic Hybrid In Nicotiana Via Protoplast Fusion And Their Identification By RAPD And SCAR Markers

Somatic hybridization has been a newly elaborated alternative to sexual crossing in higher plants, as it allows the creation of novel plant forms that are genetically distinct from analogical sexual hybrids. Creation of new CMS and achievement of symmetric somatic hybrid are of great interests and prime importance in genus Nicotiana.In present study, three experiments in relation to somatic hybridization in tobacco were carried out: rapid creation of a new CMS source via protoplast fusion between N.tabacum and N.repanda based on a single inactivation procedure with Rhodamine-6G to nuclear donor protoplast; the morphological, cytological and molecular characterization of a novel symmetric somatic hybrid between Nicotiana tabacum and N. glauca, and the field evaluation for agronomic traits of advanced generation progenies of somatic hybrids with tobacco varieties. The main results were as follows:1. Division of mesophyll protoplasts isolated from N.tabacum var. K326 were significantly restrained after inactivation by Rhodamine 6G, a reported potent inhibitor of mitochondrial oxidative phosphorylation, at the dose of 5 u g/ml for 15 minutes.2. Thirty three plants were regenerated from fusion-derived protoplast cultures between Rhodamine 6G-treated protoplasts of N.tabacum var. K326 and those of N.repanda.3. All 33 regenerated plants displayed uniform characteristics identical to K326 except flower morphology and male sterility. Morphological, cytological, biochemical investigation and RAPD analysis for nuclear genomes, as well as PCR amplification using primers for mitochondria atpA gene sequence of N.repanda revealed that they were cybrids with nuclei of K326 and cytoplasm carrying mitochondrial factors from N.repanda.4. The cybrid was further sexually backcrossed with K326 and other tobacco cultivars for three successive seasons. No revertants were observed among the progenies, showing a strict maternal inheritance of the male-sterile phenotype. The creation of the new cytoplasmic male sterile source is of much value for tobacco breeding programs in China.5. The methodology described in this project is a simple and effective method forinterspecific somatic cybridization in terms of rapid creation of tobacco CMS source with mitochondrial factors from wild species. It also seems to have a wide applicability, as there is no requirement for fusion partners.6. A symmetric somatic hybrid was obtained via fusion of protoplasts between N. tabacum var. Gexin No.1 and N.glauca Grah. The hybrid has 72 chromosomes, the total sum of its parents, its morphology was found distinct from its parents and other species in genus Nicotiana. The significance of this symmetric somatic hybrid from previously reported analogical amphidiploids by chromosome duplication treatment of the sexual amphihaploid Fl hybrids of N. tabacum x N. glauca is probably its fertility, though low, it is endowed with more potential utility in practical breeding programs.7. Results of RAPD analysis using 50 random primers showed that this hybrid exhibited bands specific to its parents respectively, but not always the sum type of total from both sides.8. Two polymorphic bands generated using primer S18 in parental genomic DNA analysis were separated, cloned and sequenced. The two sequences were submitted to Genbank and assigned with accession number AY437884 and AY437885, respectively.9. According to the sequences, two pairs of primers, 20bp in length, were synthesized, and used for testing genomic DNA of hybrid. RAPD markers were successfully transformed to SCAR markers.10. Field evaluation for agronomic traits and disease-resistance of 8 advanced generation progenies (or breeding lines) derived from continuously backcrossing the somatic hybrids with tobacco cultivars showed that these breeding lines had typical morphology identical to N. tabacum varieties, improved disease resistance and harmonious chemical components. Three breeding lines were highlighted for mu...