Epigenomic Insight of Lingonberry: Health Promoting Trait Under Micropropagation

Epigenetic variation plays a role in developmental gene regulation and responses to the environment. An efficient interaction of zeatin induced cytosine methylation and secondary compounds has been displayed for the first time in tissue-culture shoots of lingonberry (Vaccinium vitis-idaea) in vitro, in vivo and its cutting-cultivar Erntedank. Through MSAP assay, we observed highest methylated sites in leaf regenerants (LC1) from all primer combinations (108 bands), with their highest variation in secondary metabolites. We measured that four tissue-culture plants showed higher methylation bands than cutting propagated donor plants (ED) which exhibited 79 bands of methylation, which is comparatively low. On the other hand, we observed the highest total phenolic content in node culture-derived greenhouse grown plants, NC3 but leaf culture-derived greenhouse grown plants, LC1 represented low phenolic content. Our study showed more methylation in micropropagated plants (NC1, NC2, NC3, LC1) than those derived from cutting propagated ED plants, where methylation was not present. On the contrary, we observed higher secondary metabolites in ED plants but comparatively less in micropropagated shoots (NC1, NC2) and plants (NC3, LC1). Our study displayed that higher methylation sites observed in micropropagated plants possessed less amount of secondary metabolites.

Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents, and Antioxidant Activity of Micropropagated Lycium schweinfurthii Plants

Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L−1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L−1 Indole-3-butyric acid (IBA). The Jaccard’s similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL−1 of MiP dried leaves’ methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.

Coverage effect on hardening of arrow cane (Gynerium sagitatum Aubl.) micropropagated plants

To evaluate the effect of plastic film coverage on ex vitro acclimatization of arrow cane (Gynerium sagitatum Aubl.), shoots of “Criolla”, Martinera” and “Costera” cultivars were in vitro micropropagated in a medium supplied with 6-Benzylaminopurine and half of them were rooted in a medium with 1-Naphtalene acetic acid. Rooted and unrooted shoots were transplanted in a shade house with fog irrigation, into plastic trays (72 clusters per tray), using peat as substrate and half of them was covered with translucent plastic film during 5 days after transplant while the other half was maintained uncovered. The experiment consisted of a three-way factorial arrangement with 12 treatments distributed with a split-plot design where tray coverage was the main plot, cultivars were the split, and rooting condition was the split-plot. Each treatment (36 clusters) was repeated three times for a total of 1296 experimental units. After 40 days in the shade house, the survival rate was calculated, and plant heigth, number of shoots and number of roots data were analyzed by ANOVA (P<0.05) and means were separated by Tukey test (P<0.05). Plant survival was complete (100%) regardless of genotype, rooting, or coverage condition. Transferring plant into uncovered trays statistically resulted in higher levels for plant height, number of shoots, and number of roots. Ex vitro adaptation of micropropagated arrow cane plants without plastic film covers increased plant growth and reduced labor.

Micro-Structural Stability of Micropropagated Plants of Vitex negundo L.

Micropropagation techniques allow producing large numbers of clones of genetically identical plants. However, there is evidence of disorders in internal structures due to sophisticated in vitro conditions. Such variations are responsible for the mortality of plantlets in the field and cause huge loss to the tissue culture industry. Anatomical evaluation at different growth conditions allows for understanding structural repair of in vitro raised plantlets. Therefore, the present study was aimed to identify the structural changes that occurred in micropropagated plants of Vitex negundo under heterotrophic, photomixotrophic, and photoautotrophic conditions. To achieve this, structural variations were analyzed in the plantlets obtained from in vitro, greenhouse and field transferred stages using light microscopy. Underdeveloped dermal tissues, palisade cells, intercellular spaces, mechanical tissues, vascular bundles, and ground tissues were observed with the plants growing under in vitro conditions. The self-repairing of structural disorders and transitions in vegetative anatomy was observed during hardening under the greenhouse environment. Field transferred plantlets were characterized by well-developed internal anatomy. These findings showed that the micropropagated plantlets of V. negundo were well-adapted through a series of self-repairing the in vitro induced structural abnormalities at the subsequent stages of plant development.

Micropropagation of Ginger (Zingiber officinale Roscoe) ‘Bentong’ and Evaluation of Its Secondary Metabolites and Antioxidant Activities Compared with the Conventionally Propagated Plant

‘Bentong’ ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied in many plant species to produce their disease-free planting materials. As ‘Bentong’ ginger is less known for its micropropagation, this study was conducted to investigate the effects of Clorox (5.25% sodium hypochlorite (NaOCl)) on explant surface sterilization, effects of plant growth regulators, and basal media on shoots’ multiplication and rooting. The secondary metabolites and antioxidant activities of the micropropagated plants were evaluated in comparison with conventionally propagated plants. Rhizome sprouted buds were effectively sterilized in 70% Clorox for 30 min by obtaining 75% contamination-free explants. Murashige and Skoog (MS) supplemented with 10 µM of zeatin was the suitable medium for shoot multiplication, which resulted in the highest number of shoots per explant (4.28). MS medium supplemented with 7.5 µM 1-naphthaleneacetic acid (NAA) resulted in the highest number of roots per plantlet. The in vitro-rooted plantlets were successfully acclimatized with a 95% survival rate in the ex vitro conditions. The phytochemical analysis showed that total phenolic acid and total flavonoid content and antioxidant activities of the micropropagated plants were not significantly different from the conventionally propagated plants of ‘Bentong’ ginger. In conclusion, the present study’s outcome can be adopted for large-scale propagation of disease-free planting materials of ‘Bentong’ ginger.