In vitro mass propagation and conservation of a rare medicinal plant, Zhumeria Majdae Rech.f & Wendelbo (Lamiaceae)
Introduction
In situ and ex situ strategies are well-known technologies for the plant diversity conservation, which have been widely used to establish effective conservation programs. One of the useful methods of the ex situ conservation for plant diversity is in vitro culture (Fay, 1994). Micropropagation of endangered plants can be beneficial for the rapid cultivation of this species, which exist in threatened habitats and have a limited reproductive capacity (Fay, 1992). In vitro propagation (IVP) protocols have already been established for different endangered medicinal plants such as Siphonochilus aethiopicus (Ngwenya et al., 2010), Celastrus paniculatus Willd. (Senapati et al., 2013) and Thymus persicus (Bakhtiar et al., 2014). IVP methods have also been established for the genus of Lamiaceae family such as Lavandula (Dias et al., 2002), Salvia (Bassolino et al., 2015) and Thymus(Marco-Medina and Casas, 2015).
Zhumeria majdae Rech.f & Wendelbo (Lamiaceae) commonly known as “Mohrekhosh” is an endangered perennial fragrant shrub, native to the southern tropical regions of Iran (Asareh, 2005, Jalili and Jamzad, 1999). This unique plant found in Hormozgan province in the south of Iran. The people use aerial part of this plant in folk medicine as anticonvulsant, anti-spasmodic and for dysmenorrhoea. Several reports are available about the antileishmanial and antiplasmodial (Moein et al., 2008), antibacterial (Mahboubi and Kazempour, 2009), anti-inflammatory (Sharififar et al., 2012), anticonvulsant (Mandegary et al., 2012) and antioxidant activities of the essential oil and extract of the plant. The antioxidant activity of the plant extract has been attributed to the presence and accumulation of phenolic compounds (Moein and Moein, 2010, Sharififar et al., 2008). The aerial parts of Z. majdae plants are harvested from their natural habitats, and also there is an increase in both internal and external market demands for their raw materials and valuable metabolites (Soltanipoor et al., 2007). Destructive harvesting procedures and over exploitation from its natural dry-arid habitats (Fig. 1) along with poor seed germination and low vegetative multiplication ratio lead to extinction of this species (Jalili and Jamzad, 1999) Therefore, a highly effective micropropagation technique would be useful for cultivation of the species. Although, seed germination and organogenesis of this plant from the callus cultures have already been reported (Aghazadeh and Behboodi, 2015), it is very necessary to introduce an effective protocol for the mass propagation of the plant due to protecting and preserving its natural habitats.
The development of micropropagation has been influenced by a wide range of difficulties such as discoloration of the medium, browning and hyperhydricity (vitrification) of the explant. Hyperhydricity is a limiting factor in the IVP of the plants which has previously been reported in the species of Lamiaceae family such as Lavandula dentate (Echeverrigaray et al., 2005), Salvia officinalis (Gostin, 2008) and Salvia guaranitica (Echeverrigaray et al., 2010). In hyperhydric plantlets, leaves have large vacuolated mesophyll cells, fewer stomata and less photosynthetic capacity, which decrease quantities of chlorophyll and protein (Kevers et al., 2004, Leshem, 1983, Sharma and Mohan, 2006). Their stem and leaves are often rigid, thick and breakable. During acclimatization, the ability of hyperhydric plants to grow normally is reduced and they have shown disorders (Yadav et al., 2003). Therefore, the present study was aimed to investigate the effects of various concentrations of plant growth regulators (PGRs), explant source (in vitro grown seedlings, pot-grown plants and wild plants) and explant type (shoot tips and nodal segments associated to their position along the shoot axis) on the IVP of Z. majdae, especially to control of hyperhydricity. The essential oil of in vitro regenerated plant (IVRP) was also analyzed and compared to that of wild plants (WP) in order to ensure that biosynthetic ability was not altered as a consequence of the regeneration protocol. Our findings could be utilized for in vitro and ex vitro conservation, large-scale multiplication and production of rich linalool essential oils of the plant.
Section snippets
Plant materials and chemicals
Mature seeds of Z. majdae, collected from the plants growing in the natural habitat were obtained from the Department of Natural Resources, Yazd University. In addition, mature plant shoot segments (10–12 cm) were collected from wild growing plants in the Geno Mountain (27° 23′ 10″ N, 56° 11′ 55″ E at an altitude of 800 m), Bandar-Abbas, Hormozgan Province in the south of Iran (Fig. 1). One-year-old pot-grown plants were also used to evaluate the explant source on the initial in vitro
Seed germination and establishment of explants
As the extreme aim of this work is the conservation of Z. majdae, seeds are the ideal beginning material to initiate in vitro cultures because they allow the maintenance of a wider genetic base (Fay, 1992). Z. majdae seeds produce a thick layer of mucilage around the pericarp within minutes after hydration. The mucilage was removed by hand. Mucilage removal did not inhibit seed germination under ideal laboratory conditions. After mucilage removing, 70% of the collected seeds from the habitat
Conclusions
The present work establishes an efficient method to control hyperhydricity and in vitro mass propagation of Z. majdae, a monotypic endangered medicinal plant, from different explants by application of exogenous BAP and KN. The aseptic seeds of the plant germinated in the distilled sterile water on a shaker within four weeks. Upper nodal explants taken from in vitro grown seedlings were found to be the best starting material for in vitro regeneration. From normal shoots, basal nodal segments
Acknowledgments
The authors thank the Shahid Beheshti University Research Council for financial support of this project. We also thank Mr. Gholam Hossein Khosravifar and Dr. Asghar Mosleh Arany for their kind collaboration in plant materials and seed collection, respectively. This work was a part of Maryam Fallah M.Sc. thesis.
Conflict of interest
The authors declare no conflicts of interest.
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