Abstract
Efficient protocols, safe from somaclonal variation, were developed for regeneration of Iris sibirica plants via organogenesis and somatic embryogenesis from leaf-base explants cultivated on Murashige and Skoog media supplemented with thidiazuron (TDZ, 1.0mg/l) or 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0mg/l). The morphogenic response and callus formation efficiency differed significantly between 2,4-D (80.9%) and TDZ (67%) morphogenesis induction treatments. TDZ induced only organogenic calli, while calli obtained with 2,4-D were composed of three types differing in color and consistency: white, friable — embryogenic calli (4.5%, 3.8mg/explant), green, compact — organogenic calli (12.4%, 48.4mg/explants) and yellow — non-regenerative calli (77.3%, 254.4mg/explant). The cultivation of embryogenic calli on medium with 2,4-D and Kinetin resulted in further development of somatic embryos (54embryos/g of calli) which germinated with a frequency of 62% after being transferred to a medium without plant growth regulators. Stable shoot cultures were established by transferring organogenic calli with shoot primordia to media with 0.1mg/l α-naphthaleneacetic acid (NAA) and 1.0mg/l 6-benzylaminopurine (BA), while further cultivation on media of the same composition (TDZ or 2,4-D) resulted in the reduced growth and rhizogenesis, respectively. The TDZ induction treatment resulted in higher number of shoots per explant (7.9) than the 2,4-D treatment (4.3). After successful rooting and ex vitro acclimatization, plants were grown in the field and flowered to seed production. Flow cytometry, chromosome counting and random amplification of polymorphic DNA (RAPD) analysis indicated no evidence of genetic variation in plants regenerated via somatic embryogenesis or organogenesis. The results suggest that established protocols are safe for use in genetic transformation procedures or large-scale production of true-to-type I. sibirica plants.
Highlights
Siberian Iris (Iris sibirica L.) is a perennial monocotyledonous plant and a horticulturally valuable member of the Iridaceae family of Monocot plants (Goldblatt and Manning, 2008)
It is native to wet fields and mountain regions of the Northern Hemisphere and listed as an endangered and protected species in Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BA, 6-benzyladenine; BM, basal medium; DAPI, 4,6-diamidino-2-phenylindole-2HCl; MS, Murashige and Skoog mineral solution; NAA, α-naphthaleneacetic acid; OI-2,4-D, organogenesis induced by 2,4-D; OI-TDZ, organogenesis induced by TDZ; PGR, plant growth regulators; RAPD, random amplification of polymorphic DNA; Scanning electron microscopy (SEM), scanning electron microscopy; TDZ, thidiazuron (1-phenyl-3(1,2,3-thiadiazol-5-yl) urea)
After 6 weeks of culture, leaf-base explants of I. sibirica cultivated on BM supplemented with 2,4-D or TDZ developed calli with a frequency of 80.9% and 66.7% respectively, while no callus formation was observed on BM without PGR (Fig. 1)
Summary
Siberian Iris (Iris sibirica L.) is a perennial monocotyledonous plant and a horticulturally valuable member of the Iridaceae family of Monocot plants (Goldblatt and Manning, 2008). It is a rhizomatous plant, up to 120 cm tall with long narrow leaves and monochasial cymose inflorescence with 1–7 blue-liliac actinomorphic flowers (Szöllősi et al, 2010, 2011). I. sibirica plants, like Iris pallida and Iris germanica, accumulate in their roots essential oils, whose constituent terpenoid ketone-irone with strong violet-like scent is widely used in perfumery and cosmetics (Aslanyants and Marshavina, 1979; Gozu et al, 1993; Jéhan et al, 1994). I. sibirica is widely grown as ornamental garden plant or cut flower
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