Clonalin vitromultiplication of grey mangrove and assessment of genetic fidelity using single primer amplification reaction (SPAR) methods

Abstract

An efficient protocol for clonal multiplication of an important mangrove, Avicennia marina, was developed through in vitro culture of nodal segments obtained from a mature plant. The nodal explant induced multiple shoots when cultured on the Murashige and Skoog (MS) basal medium supplemented with varying concentrations and combinations of 6-benzyladenine (BA) and a-naphthalene acetic acid. The highest response in terms of per cent regeneration (73%), average number of shoots/explant (3.25 § 0.25) and maximum shoot length (5.2 § 0.27 cm) was obtained on the MS medium supplemented with BA 5.0 mmol/L C NAA 1.0 mmol/L C 3 g/L activated charcoal after 8 weeks of culturing. The regenerated shoots were rooted well in the MS medium supplemented with 1.0 mmol/L indole-3-butyric acid with an average of 2.9 § 0.24 roots per microshoot. The rooted plantlets were successfully transferred to pots containing normal garden soil with 70% survival rate. The genetic stability of the regenerated plants was evaluated using single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA, directed amplification of minisatellite DNA and intersimple sequence repeat polymorphism. The SPAR analysis revealed monomorphic banding patterns in all in vitro regenerated plantlets of A. marina and similar with that of the mother tree confirming their genetic uniformity and clonal fidelity.

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

“…All RAPD and DAMD-generated bands were monomorphic and identical, confirming the complete absence of somaclonal variations in regenerated plantlets. It has been shown that the use of RAPD and DAMD molecular markers is efficient in determining the genetic stability of regenerated plants in a variety of medicinal plant species, including Withania somnifera [108], Henckelia incana [109], Avicennia marina [110], Ruta graveolens [18], Curcuma zedoaria [72], Artemisia vulgaris [100], Anarrhinum pubescens [111], and Anthurium andraeanum [106]. In recent years, it has been shown that a flow cytometry-based approach for evaluating in vitro raised plantlets is an excellent technique for assessing the clonal integrity and ploidy status in micropropagated plants [34,71,72,112,113].…”

In Vitro Regeneration of Sugar Palm (Arenga pinnata Wurmb Merr.)

“…Well-developed roots in term of length, root tissue density, stem girth and mass of root tips within a plant is a good indicator of acclimatization as it allows plants to regulate the nutrient and water uptake to the surrounding environment [21]. The efficiency of IBA for the induction of roots have been previously reported in other plant species which included mangrove [22], date palm [23], and olive [24]. Meanwhile, a contradictory finding was reported in papaya where the increasing compositions of IBA was affirmed to reduce rooting frequency combined with the growth of unwanted callus [25].…”

Application of In Vitro Plant Tissue Culture Techniques to Halophyte Species: A Review

“…Less used is the addition of activated charcoal, a possible solution to adsorb inhibitory compounds and counteract the negative effects of toxic metabolites and phenolic exudates. It proved to be a key component in the multiplication medium for S. edulis [ 69 ] or A. marina [ 46 ].…”