High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Abstract

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Development of an Efficient and Rapid Micropropagation Protocol for In Vitro Multiplication of Maerua crassifolia Forssk

“…This result supports the assertion that a proper balance between cytokinins and auxins forms a synergistic effect that induces in vitro proliferation of shoots [26]. Previous studies demonstrated that a relatively higher concentration of cytokinins in combination with lower concentrations of auxins promote in vitro cell division and induce the formation of a greater number of shoots per explant compared to using cytokinins alone [8]. This effect could be explained by the aforementioned synergistic effect between auxins and cytokinins along with their roles in the cell cycle and their significant effects on different phases of cell division and multiplication cycle.…”

“…The overexploitation of M. crassifolia for medicinal purposes and as fodder coupled with the slow growth of the tree raises the need for a rapid and efficient means of propagation. Tissue culture enables the controlled growth and significant multiplication of plants from very small explants of the plant, e.g., hypocotyls, segments of roots, leaves, or stems, on an artificial medium [8], and therefore becomes an integral part of plant biotechnology. This technique has been successfully applied to the propagation of several important and endangered plant species [8][9][10][11][12].…”

Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L.

“…In our study, it was shown that low doses of BA (5.0 µM) had the highest probability of generating shoots from callus, with a greater response and number of induced shoots than higher doses (>5.0 µM). High dosages of cytokinin have been demonstrated to have deleterious effects on the multiplication of shoots in a range of plant species [20,33]. In a number of species, including Ruta graveolens [18], Citrus jambhiri [34], Notopterygium incisum [32], and Mondia whitei [35], BA has been shown to be highly effective in inducing shoot buds from callus mass.…”

“…Faisal, et al [19] reported regeneration through the shoot tip meristems of R. graveolens by cytokinin-auxin synergism. In vitro multiplication of R. chalepensis through direct regeneration was recently reported by Qahtan, et al [20].…”

Micropropagation and Genetic Fidelity of Fegra Fig (Ficus palmata Forssk.) and Grafting Compatibility of the Regenerated Plants with Ficus carica

“…Conversely, the low absorption efficiency of NAA makes it unsuitable for the regeneration of some plant tissues, as it could potentially lower rooting efficiency in culture [71]. In addition, it leads to callus development at the end cut of microshoots of Hebe buchananii, Hebe canterburiensis [72], and Ruta chalepensis [73]. However, NAA showed efficacy in stimulating root formation in vitro in clones of Ficus carica.…”