Clonal fidelity of Iris sibirica plants regenerated by somatic embryogenesis and organogenesis in leaf-base culture — RAPD and flow cytometer analyses

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, 1.0 mg/l) or 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/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%, 3.8 mg/explant), green, compact -organogenic calli (12.4%, 48.4 mg/explants) and yellow -nonregenerative calli (77.3%, 254.4 mg/explant). The cultivation of embryogenic calli on medium with 2,4-D and Kinetin resulted in further development of somatic embryos (54 embryos/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 0.1 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/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.

Assessment of somaclonal variation in indirect morphogenesis-derived plants of Arracacia xanthorrhiza

“…These results are similar to those obtained by Stanišić et al (2015), who found that Iris sibirica L. plants regenerated simultaneously via organogenesis and somatic embryogenesis from a callus previously induced on a medium supplemented with 1.0 mg L -1 2,4-D. Rocha et al (2015) attributed the parallel induction of shoot organogenesis and somatic embryogenesis to specific concentrations and types of PGRs, affecting the callus culture. These authors also stressed that the manipulation of in vitro culture conditions might allow these morphogenic pathways to be induced alternatively or simultaneously.…”

Somaclonal variations and their applications in horticultural crops improvement

“…A wide variety of tools are available for the detection and characterization of somaclonal variants which are primarily based on the differences in morphological traits (Pérez et al 2009, 2011; Nhut et al 2013), cytogenetical analysis for the determination of numerical and structural variation in the chromosomes (Clarindo et al 2012; Currais et al 2013; Abreu et al 2014), biochemical (Vujovic et al 2010; Kar et al 2014), molecular DNA markers (Krishna and Singh 2007; Pathak and Dhawan 2012; Hossain et al 2013; Bello-Bello et al 2014) or their combinations (Horáček et al 2013; Dey et al 2015; Stanišić et al 2015). The best test for assessing somaclonal variation is to fruit out the plants and conduct an extensive horticultural evaluation, which is unfortunately a long-term endeavor with woody fruit crops, particularly (Grosser et al 1996).…”

In vitro propagation of Silene bolanthoides Quézel, Contandr. & Pamukç. and assessment of genetic stability by flow cytometry

“…It was successfully used to assess genetic stability in micropropagated Puya berteroniana through adventitious shoots [41], in various tissue culture-derived plants of Citrus limon Burm. [13], in Iris sibirica plants regenerated by somatic embryogenesis and organogenesis [42], and in regenerated plants of Eryngium planum L. performed through axillary bud proliferation [17]. Cytogenetic studies and chromosome counts can provide information about abnormal mitosis or changes in ploidy levels of plants [43].…”