Saint Katherine is considered a “biodiversity hotspot” because of the high level of endemism of reported plant species. In this study, conservation of the endangered endemic plant; Phlomis aurea of Saint Katherine, Southern Sinai, Egypt, was carried out through micropropagation and DNA barcoding. The first efficient micropropagation protocol for Phlomis aurea was established as a mean of ex situ conservation of the plant. Shoot tips and nodal segments of in vitro germinated seedlings were established on Murashige and Skoog medium supplemented with 0.54 µM β-naphthalene acetic acid (NAA) and 2.46 µM N6-(2-isopentenyl) adenine (2iP) in combination with 6-benzylaminopurine (BA) or kinetin (Kin). The medium supplemented with 3.48 µM Kin considered optimum for both explants. For multiplication, BA was the most efficient cytokinin. The percentage of rooted explants reached 100% at the concentration of 14.7 µM indolebutyric acid (IBA), whereas the highest number of roots was recorded for 4.90 µM, which considered the optimum concentration with a percentage of 80% of rooting. Rooted plantlets were transplanted in the greenhouse with 75% survival rate. The present study also aimed to carry out DNA barcoding of Phlomis aurea for accurate identification to provide a database for establishing an efficient conservation program for the plant. Three chloroplast DNA markers were used [ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), maturase K (matK) and RNA polymerase C1 (rpoC1)] and all were successful in amplifying target regions, however the performance of both rbcL and matK markers seemed to be species‐specific. The similarity percentage was maximum for rbcL (99.81%) and matK (100%) compared to the database of the same species.
E * n effective protocol for micropropagation of the endangered Searsia tripartita plant (Anacardiaceae) was developed. Shoot tips and stem node sections from the mature plant and diverse explants from seedlings were used as explants. Murashige and Skoog (MS) medium supplemented with 0.1 mg L -1 naphthalene acetic acid (NAA) and 1.5 mg L -1 benzyl adenine (BA) induced the highest mean length of shoots 5.8, 6.6 and 5.3 cm for shoot tips from mature plants, shoot tips and stem node sections from seedlings, respectively. This medium also provided the highest percentage of growth induction with these three explants. The medium supplemented with 0.1 mg L -1 NAA and BA at 1.0 mg L -1 induced the maximum mean length of shoots (3.4 cm) with the stem sections from mature plants. A length of the shoot of 4.7 cm was gained from the hypocotyl explant with MS medium free from plant growth regulators (PGRs). MS medium supplemented with several concentrations of either BA or kinetin (Kin) were used to determine their influence on shoot multiplication. The highest rooting percentage recorded 70% on MS medium supplemented with 3.0 mg L -1 IBA with 100 mg L -1 phloroglucinol (PG) and 1 g L -1 activated charcoal (AC). An average of 50-60% of the in vitro derived transplants were survived after transferring into greenhouse conditions. Five phenolic compounds with numerous health benefits were detected by HPLC in a comparative study between mother plant, in vitro multiplied plants and acclimatized plants to determine which of them contains high amount of phenolic compounds.
Ephedra alata is an important Egyptian medicinal plant with enormous applications in pharmaceutical industry. Shake-flask suspension cultures of E. alata were established from internodal segment-derived callus. The suspension cultures were treated with two biotic elicitors, namely Aspergillus niger and yeast extract, to enhance the synthesis of ephedrine -the bioactive constituent of E. alata. Ephedrine was quantified using the high performance liquid chromatography technique. Ephedrine concentration was not directly related to biomass production. Aspergillus niger and yeast extract enhanced the production of ephedrine by 2.5-to 7-fold after different durations of incubation as compared to ephedrine content in the mother plant. Treatment with the fungal elicitor A. niger extract induced the maximum accumulation of ephedrine at the concentration of 1.67%; it also increased ephedrine biosynthesis by 7-fold in 24-day-old culture as compared to the ephedrine content in the mother plant and caused higher ephedrine biosynthesis than that in the control treatment without elicitors. The present protocol could be applicable for the large-scale production of ephedrine from E. alata.
Background: Anthropogenic activity, climate change, pollution, and exploitation of natural resources are some reasons that cause threatening of plant diversity. Silene schimperiana is an endangered plant species in Egypt and is endemic to the high mountain of Saint Katherine Protected Area in southern Sinai. The purpose of the study was the ex situ conservation of Silene schimperiana through in vitro propagation and DNA barcode analysis. Results: To develop an efficient ex situ conservation program of the plant, in vitro propagation protocol has been achieved from shoot tip and stem nodal segment explants of in vitro germinated seedlings. Explants were established in vitro on Murashige and Skoog (MS) medium supplemented with 2.89 μM gibberellic acid (GA 3) , 1.08 μM α-naphthaleneacetic acid (NAA), and 1.16 μM kinetin (Kin). The highest number of axillary shoots (9.27) was obtained when they were transferred to MS medium supplemented with 4.48 μM 6-benzyl adenine (BA). Hundred percent of multiple axillary shoots were rooted on quarter-strength MS medium supplemented with 4.92 μM indole-3-butyric acid (IBA) and 10.75 μM NAA. Rooted plants were transferred to pots containing a soil-peat mixture (1: 2 v/v) and successfully acclimatized in the greenhouse. Plant identification is a crucial aspect to understand and conserve plant diversity from extinction. DNA barcode analysis of Silene schimperiana was carried out using two chloroplast DNA markers (cpDNA): 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) and RNA polymerase subunit (rpoC1) and a nuclear ribosome DNA marker (ncDNA), internal transcribed spacer (ITS). Phylogenetic analysis revealed a successful identification of Silene schimperiana on the species and genus levels and supported the inclusion of Silene schimperiana in genus Silene. Conclusions: In this study, a relevant in vitro propagation method was established to facilitate the recovery of Silene schimperiana, in addition to DNA barcoding of the plant as a tool for effective management and conservation of plant genetic resources.
ilene schimperiana (Caryophyllaceae) is an endemic plant of Saint Katherine Protectorate, Sinai, Egypt. This genus has various medicinal properties. Due to over exploitation, S. schimperiana is at risk of extinction at the vulnerable stage in Egypt. Therefore, its conservation is important. An efficient protocol for S. schimperiana synthetic seeds production was developed in the present study. Shoot tips and nodal segments obtained from in vitro proliferated shoots were encapsulated in calcium alginate beads for medium term conservation and germplasm exchange. Encapsulated propagules were stored for five months at 4°C in three different matrixes of calcium alginate; water, Murashige and Skoog (MS) medium and MS medium containing 3% sucrose. The most efficient gel complex for formation of identical beads was 3% Na-alginate and 100 mM CaCl2.2H2O. The optimum temperature for storage was 4°C with 100% viability during the period of five months of storage with all tested alginate matrix compositions. However, the matrix containing MS medium with 3% sucrose was the best for the encapsulated propagules. The regrowth frequency of encapsulated propagules and mean number of shoots were decreased by increasing in storage duration. Rooting was achieved on ¼ MS medium supplemented with 1.0 mg L-1 indole-3-butyric acid (IBA) and 2 mg L-1 α-naphthalene acetic acid (NAA). About 90% of regenerated plantlets were successfully acclimatized under greenhouse conditions. This method can be successfully applied for in vitro preservation of S. schimperiana propagules for five months at 4°C to conserve this valuable plant.
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