Journal
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT
Volume 51, Issue 3, Pages 350-359Publisher
SPRINGER
DOI: 10.1007/s11627-015-9674-0
Keywords
Protoplast culture; Somaclonal variation; Gentian; Flow cytometry; Chromosome number
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Funding
- Polish Ministry of Science and Higher Education [3P04C 037 23]
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Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, 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 diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 x 10(5) protoplasts 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(-1) 1-naphthaleneacetic acid and 0.1 mg l(-1) thidiazuron. 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(-1) kinetin, 0.5 mg l(-1) gibberellic acid, and 80 mg l(-1) adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.
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