title: Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploidGentiana decumbensL.f.
abstract: Somaclonal variation, often manifested as the increased ploidy of plants observed followingin vitroculture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploidGentiana decumbensL.f. through somatic embryogenesis. A yield of more than 9 × 105protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l-11-naphthaleneacetic acid and 0.1 mg l-1thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l-1kinetin, 0.5 mg l-1gibberellic acid, and 80 mg l-1adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.
authors.affiliation: Department of Experimental Plant Biology, Polish Academy of Sciences Botanical Garden-Center for Biological Diversity in Powsin, Prawdziwka 2, 02-973 Warsaw, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Tomiczak
authors.affiliation: Department of Experimental Plant Biology, Polish Academy of Sciences Botanical Garden-Center for Biological Diversity in Powsin, Prawdziwka 2, 02-973 Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Mikuła
authors.affiliation: Department of Plant Genetics, Physiology and Biotechnology, Laboratory of Molecular Biology and Cytometry, University of Technology and Life Sciences, Kaliskiego Ave. 7, 85-789 Bydgoszcz, Poland.
authors.forename: Elwira
authors.initials: E
authors.lastname: Sliwinska
authors.affiliation: Department of Experimental Plant Biology, Polish Academy of Sciences Botanical Garden-Center for Biological Diversity in Powsin, Prawdziwka 2, 02-973 Warsaw, Poland.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Rybczyński
keywords: Chromosome number; Flow cytometry; Gentian; Protoplast culture; Somaclonal variation