Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Abstract

A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (1) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos± kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (1) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum-and N. rotundifolia-like hybrids had nuclear DNA contents near that of N. tabacum 9X40^0X24 pg or N. rotundifolia 5X29^0X36 pgY respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels ± N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8X90^0X30 to 19X57^0X33 pgX The data from the morphological and cytological analysis provided evidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.

Effect of enzyme concentrations on protoplast isolation and protoplast culture of Spathiphyllum and Anthurium

“…An integrated approach was reported for Poaceae (Zhou et al 2001;Xiang et al 2003;Xu et al 2003), Solanaceae (Parokonny et al 1994;Rasmussen et al 1997;Rutgers et al 1997;Ilcheva et al 2001) or Brassicaceae (Forsberg et al 1998;Yamagishi et al 2002), but also in Helianthus (Binsfeld et al 2000;Binsfeld and Schnabl 2002) and Citrus (Liu and Deng 2002). Within ornamentals, good results were obtained in Liliaceae (Saito and Nakano 2002;Horita et al 2003).…”

Somatic Cell Cybrids and Hybrids in Plant Improvement

“…Several schemes have been developed for somatic hybrid selection. These schemes include selective media (Hossain et al, 1994); metabolic inhibitors (Shimonaka et al, 2002;Xiang et al, 2003); complementation systems such as chlorophyll deficiency complementation (Dragoeva et al, 1999), auxotroph complementation (Wolters et al, 1995), resistance markers (Ilcheva et al, 2001) and double mutants (Conner et al, 1995); individual selection and culture (Saha et al, 2001); and application of the green fluorescent protein (GFP) marker gene (Olivares-Fuster et al, 2002).…”

“…Other molecular markers AFLP analysis was applied by several researchers to characterize somatic hybrids in higher plants (Brewer et al, 1999;Guo et al, 2002;Ilcheva et al, 2001;Wang et al, 2003). Inter-simple sequence repeat (ISSR) -PCR is a rapid and sensitive method for detecting the introgression of alien DNA into cultivars via somatic hybridization.…”