Micropropagation of Stevia rebaudiana Betroni and assessment of genetic stability of in vitro regenerated plants using inter simple sequence repeat (ISSR) marker

Abstract

Stevia rebaudiana Bertoni has become a new sweetener crop in different regions in the world. Stevia produces few seeds, therefore, micropropagation is a prevalent method to obtain sufficient amount of uniform plants. In the present study, in vitro propagation of Stevia was attempted through multiple shoot regeneration from nodal segments cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), α-napthyalene acetic acid (NAA), indole acetic acids (IAA) and thidiazuran (TDZ). The maximum of number axillary shoots per explant (3.24) and highest shoot length (3.12 cm) were observed with MS medium supplemented with 1.0 mg l −1 BA+0.05 mg l −1 NAA and 2.0 mg l −1 BA+0.05 mg l −1 NAA, respectively. Roots were produced within two weeks and the highest percentage (98.72%) of root induction, the maximum number of roots (9.46 roots/shoot) and root length (9.87 cm) were recorded on MS medium fortified with 0.5 mg l −1 IAA and 1.5 mg l −1 IAA, respectively. The ex-vitro plantlets were successfully acclimatized with a survival rate of 70% at the hardening phase. Inter simple sequence repeat (ISSR) markers were used to analyze the genetic stability of micro-propagated and mother plants of Stevia. Four ISSR primers generated clear, distinct and reproducible bands. All ISSR profiles from micro-propagated plants were monomorphic and similar to mother plants, while low variation was induced in the next sub-culture. The results indicated that Stevia plantlets regenerated using micropropagation techniques standardized at our lab were genetically stable especially in the early sub-cultures.

Micropropagation and Assessment of Genetic Stability of Acclimatized Streptocarpus x hybridus Voss Plantlets Using RAPD Markers

“…Our findings confirm that auxins have an important role also in Streptocarpus tissue culture development as stimulators of root formation. Similar results have been reported in other plant species as well (Soliman et al, 2014). Regarding cytokinins, if they are not used in theoptimum concentration in combination with auxins, at low concentrations they can have an insignificant stimulatory effect but in higher concentrations they can inhibit or antagonize the stimulatory effect of auxins on plant organ formation.…”

“…Despite the many advantages of micropropagation through tissue culture, the presence of somaclonal variation is a major problem encountered in the in vitro culture (Soliman et al, 2014). Factors that can cause somaclonal variations during in vitro culturing may include chromosomal rearrangements, changes at molecular level, such as single-nucleotide changes, deamplification and amplification of genes, alterations in DNA methylation patterns and transposable element activations (Ilczuk and Jacygrad, 2016).…”

Impact of application of zinc oxide nanoparticles on callus induction, plant regeneration, element content and antioxidant enzyme activity in tomato (Solanum lycopersicum Mill.) under salt stress

“…of callus survived/total no. of calli cultured x 100 according to [25]; (iii) CRGR: Callus relative growth rate = callus final weight -callus initial weight/callus initial weight according to [26]; (iv) CWC: Callus water content (%) = Callus fresh weight -Callus dry weight/Callus fresh weight x 100 according to the equation described previously [27]. Callus dry weight was gravimetrically determined after drying at 80 o C for 48 h.…”

“…The cultures were maintained in the growth chamber as above. After 4 weeks, the plant regeneration percentage (PRP) was obtained according to [25].…”

ISSR markers to assess genetic diversity of cultivated populations from artificial selection of <i>Stevia rebaudiana</i> (Bert.) Bertoni

“…ISSR primers have been used to evaluate the genetic fidelity of in vitro propagated plants of Stevia rebaudiana (Bert.) ( Deshmukh et al 2017 , Lata et al 2013 , Singh et al 2014 , Soliman et al 2014 ) and to assess genetic similarity among collections ( Sharma et al 2016 ) and different accessions ( Garro-Monge et al 2014 , Heikal et al 2008 , Othman et al 2018 ) of S. rebaudiana .…”