Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i> Bert. Using ISSR Marker

Abstract

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog's (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg•L −1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L −1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT) 3 M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

Evaluation of genetic homogeneity of Jatropha curcas L. hybrid at an early stage of shoot bud formation from petioles using ISSR marker

“…The total number of 96 marker bands amplified during the assay in this study is sufficient to detect any somaclonal variation. This is comparable to the 40, 45 and 51 bands amplified in other previous studies for various plant species employing ISSR-based markers to evaluate genetic stability [32][33][34]. The ISSR analysis in this study showed that the shoot buds generated from tissue culture were completely uniform, suggesting a high level of genetic homogeneity among them.…”

Thidiazuron Induced Direct Shoot Organogenesis in Stevia rebaudiana and Assessment of Clonal Fidelity of Regenerated Plants by RAPD and ISSR

“…Micropropagation protocols of Stevia have been established using leaf (Sivaram and Mukundan, 2003;Sreedhar et al, 2008;Jain et al, 2009), nodal (Sung, 2006;Ahmed et al, 2007;Laribi et al, 2012;Modi et al, 2012;Thiyagarajan and Venkatachalam, 2012;Hassanen and Khalil, 2013;Lata et al, 2013;Singh and Dwivedi, 2013;Singh et al, 2014) and shoot tips explants (Tamura et al, 1984;Ibrahim et al, 2008a;Ibrahim et al, 2008b;Hassanen and Khalil, 2013), however, a major problem associated with in vitro culture is the possible occurrence of somaclonal variation through indirect organogenesis (Modi et al, 2012;Singh et al, 2014). Literatures revealed the occurrence of variability in tissue culture raised plants which did not produced true-to-type plants.…”

Hydrangea DNA Methylation Caused by pH Substrate Changes to Modify Sepal Colour is Detected by MSAP and ISSR Markers

“…The aim of using this type of molecular marker was to assess the genetic stability of hydrangea plants after a pH substratum change. This type of marker has been previously used to check the genetic fidelity among mother and micropropagated plants in, for example, bamboo [44], Stevia rebaudiana [37], or Ochreinauclea missionis [36]. ISSR that Agronomy 2019, 9, 871 9 of 11 amplified by PCR inter-SSR sequences, are able to detect polymorphisms among different samples both when the priming sites are present/absent or when the length of the amplified sequence varied [45].…”

“…On the other hand, Inter Simple Sequence Repeat (ISSR) are molecular markers, based on microsatellite regions, which have been used in plants to register genetic fingerprints, phylogenetic analysis, diversity and genetic variability, and the identification of cultivars [33][34][35]. They have also been used to document genetic fidelity in plants that come from micropropagation [36,37]. It is a simple and fast method that combines most of the advantages of microsatellites (SSR) and amplified fragment length polymorphism (AFLP) to the universality of random amplified polymorphic DNA (RAPD) [33,35].In this work, the colour change of the sepal of 28 hydrangea plants was evaluated after being treated with different culture conditions by modifying the pH.…”