Diversity of Orchids in Terms of Their Distribution, Uses and Conservation in Annapurna Conservation Area of Nepal

The Annapurna Conservation Area (ACA), the first conservation area and the largest protected area (PA) in Nepal, is incredibly rich in biodiversity. Notwithstanding this, orchids in the ACA have not been explored enough yet thus making the need for ambitious research to be carried out. Previous study only included 81 species of orchids within ACA. This study aims to update the record of species and genera richness in the ACA. In total 198 species of orchids, belonging to 67 genera (40% and 62% of the total recorded orchid species and genera in Nepal) has been recorded in ACA. This represents an increase of 144% in species and 56% in genera over the previous data. Out of the 198 species, 99 were epiphytes, 6 were holomycotrophic and 93 were terrestrial. Among the 67 genera, Bulbophyllum (17) species were dominant, followed by Dendrobium (16), Herminium (10), Coelogyne, Plantanthera (9 each), Eria, Habenaria, Oberonia (8 each), Calanthe (7), and Liparis (6). Fifty-six species were found to be ornamentally significant and 85 species medicinally significant.

In vitro germination and propagation of a threatened medicinal orchid,Cymbidium aloifolium(L.) Sw.through artificial seed

Objective:To study the in vitro germination and plantlet regeneration from artificial seeds of Cymbidium aloifolium(C.aloifolium),a highly threatened medicinal orchid of Nepal.Methods:Artificial seeds were produced in vitro by encapsulation of protocorms with 4%sodium alginate and 0.2 mol/L calcium chloride solution.In vitro germination and plantlet regeneration of the artificial seeds were tested by culturing them on different strength of Murashige and Skoog(MS) liquid media(0.25,0.5 and 1.0) and MS liquid medium supplemented with 0.5 mg/L benzyl amino purine and 0.5 mg/L naphthalene acetic acid.Freshly produced artificial seeds were stored up to 28 d at 4 ℃.In order to check the viability,storage artificial seeds were treated with five different sterilization techniques(T_1 T_2,T_3 T_4,T_5) and inoculated on full strength(1.0) of MS liquid medium after each 7 d of interval upto28 th days.Results:The highest percentage of germination(100%) of artificial seed was obtained on quarter(0.25),half(0.5) and full(1.0) strength of MS liquid medium.Experimentally,full strength of MS liquid medium was more effective for earlier seedling development of C.aloifolium.Artificial seeds were successfully stored at 4 ℃ till 28 th days.Treatments T_1 and T_2showed 97.5%viability of storage artificial seeds and hence considered as the most effective sterilization techniques to recover the plant from storage artificial seeds.Plantlets developed from artificial seeds were successfully acclimatized in potting mixture containing cocopeat,litter and sphagnum moss with 85%survival rate.Conclusions:The present study revealed that artificial seeds are the good alternative explants for in vitro mass propagation and short term conservation of C.aloifolium.

Efficient Plant Regeneration of Cymbidium aloifolium (L.) Sw., a Threatened Orchid of Nepal through Artificial Seed Technology

Artificial seed technology is a method of considerable potential for mass propagation and conservation of rare, endangered and threatened species. In the present investigation, artificial seeds were obtained through encapsulation of three weeks old protocorm (3.0 ± 1.0 mm diameter) of Cymbidium aloifolium with calcium alginate. Artificial seeds were cultured on liquid Murashige and Skoog (MS) or Knudson (Kn C) medium at different strength (×1.0, 0.5, 0.25) and full strength (1.0) of both media supplemented with 0.5 mg/l 6-benzyl aminopurine (BAP) and 0.5 mg/l α-naphthalene acetic acid (NAA). Full strength of MS medium without plant growth regulators was found to be the most favourable condition for efficient plantlet regeneration of C. aloifolium (9.83 shoot and 2.66 roots per culture). The storage potential of artificial seed was tested at 4°C and room temperature (RT, 21°C ± 2°C) for up to 90 days on both media and found 83.33% viability at 4°C storage on MS media. Eighty five percent of plantlets regenerated from artificial seeds culture were successfully hardened in a potting mixture of cocopeat, clay and sphagnum moss (2:1:1). Hence, the methodology can be used for propagation and conservation of C. aloifolium through artificial seed system.

<i>In Vitro</i>Propagation of Endangered Orchid, <i>Vanda pumila</i>Hook.f. through Protocorms Culture

The Vanda pumila is a monopodial orchid with beautiful flowers that are native to Thailand but now found across South Asia. The immature seeds of Vanda pumila were used for in vitro culture and then the protocorms developed were used as explants for seedling development and mass propagation. Protocorms were cultured on 1/2 MS (Murashige and Skoog, 1962) medium fortified separately with Kinetin (Kn), 6-Benzyl amino purine (BAP) and Gibberellic Acid (GA3) each in different concentrations as (0.5 mg/L, 1.0 mg/L and 2.0 mg/L) well as each on each concentrations of each medium supplemented with 5% and 10% coconut water (CW) respectively. The greatest number of shoots (9.50 ± 0.29 shoots per culture) was developed on 1/2 MS medium fortified with 1.0 mg/L Kn plus 10% CW and the longest shoots (0.78 ± 0.07 cm per culture) developed on 1/2 MS medium fortified with 2.0 mg/L BAP plus 10% CW. The shoots derived from protocorms were then developed on 1/2 MS medium fortified with three different rooting hormones viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and α-Naphthalene acetic acid (NAA), each in four concentrations (0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2.0 mg/L) as well as 1.0 mg/L of each hormone supplemented with 10% CW. The 1/2 MS medium fortified with 0.5 mg/L IAA was found to be the most effective condition for the development of maximum number of root (5 ± 0.0 roots per culture) and root length (0.93 ± 0.07 cm). Hence, the present study could be useful for standardizing the protocol for mass propagation of the endangered orchid V. pumila.

The antioxidant activity of selected wild orchids of Nepal

Objective:To assess the antioxidant activity as well as the total polyphenolics and flavonoids content of thirteen extracts from nine wild orchids of Nepal.Methods:First,a preliminary phytochemical screening of thirteen extracts of nine orchids was carried out using established protocols.Then,the total polyphenolic and flavonoid contents of each extract were evaluated using the Folin-Ciocalteu's phenol reagent and aluminium chloride methods respectively.Finally,antioxidant activity was determined by 2,2-diphenyl-1-picryl hydrazyl free radical scavenging assay and expressed in terms of half inhibition concentrations(IC50 values).Results:All the tested parameters showed significant variation at P=0.05.The total flavonoids varied with the greatest amounts found in the leaves of Rhynchostylis retusa and the smallest amounts found in the roots of Gastrochilus acutifolius(G.acutifolius).The amount of total polyphenolics was highest in the stems of Vanda cristata and lowest in the leaves of G.acutifolius.Antioxidant activity was highest in the stems of Vanda cristata and lowest in the leaves of G.acutifolius.Conclusions:Some of the orchid extracts studied exhibited levels of antioxidant activity which were comparable to or even higher than those found for extracts of medicinal plants and orchids studied previously.It is suggested that drugs and natural food additives can be extracted from those wild orchids with high levels of antioxidant activity.