Shreeti Pradhan

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

ABSTRACT 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 oC. In order to check the viability, storage artificial seeds were treated with five different sterilization techniques (T1, T2, T3, T4, T5) and inoculated on full strength (1.0) of MS liquid medium after each 7 d of interval upto 28th 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 oC till 28th days. Treatments T1 and T2 showed 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.

Production of virus-free orchid Cymbidium aloifolium (L.) Sw. by various tissue culture techniques.

Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV) is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼) with or without 0.5 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (Naphthalene acetic acid). To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother) plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%). The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.

In vitro Mass Propagation of an Epiphytic Orchid, Cymbidium aloifolium (L.) Sw., through Protocorm Culture

Aim: To develop a protocol for in vitro propagation of Cymbidium aloifolium, a threatened orchid highly used for medicinal purpose through protocorm culture. Place and Duration of Study: Tissue culture Laboratory, Plant Biotechnology Unit, Department of Botany, Tribhuvan University, Kirtipur, Nepal, between November 2013 to December 2014. Methodology: Small, green and globular protocorms with 0.1-0.3 cm diameter were subjected to grow individually on solidified Murashige and Skoog (MS) basal medium and MS medium supplemented with various concentration of plant growth regulators, 6-Benzylaminopurine (BAP, 0.5; 1; 1.5; 2 mg/l) or α-Naphthalene Acetic Acid (NAA, 0.5; 1 mg/l) or their combination. Six replicates were used for each concentration. The data for development of shoot and root from each protocorm culture were recorded in every two weeks for upto six month. Results: Almost all conditions favoured multiplication but MS medium fortified with BAP (1 mg/l) and NAA (1 mg/l) resulted in maximum induction of rootless healthy shoots with an average value of 8-9 shoots per culture. On this medium, shoot multiplication was initiated after 9 weeks of culture Original Research Article Regmi et al.; BJI, 19(1): 1-6, 2017; Article no.BJI.34891 2 whereas MS medium fortified with BAP (2 mg/l) and NAA (0.5 mg/l) was found to be most effective condition for the shoot multiplication along with well developed roots. Conclusion: MS medium supplemented with high concentration of BAP and low concentration of NAA was found to be efficient for maximum multiplication of shoot and root. The in vitro developed healthy rooted plantlets of C. aloifolium were successfully acclimatized in green house on potting mixture containing cocopeat and moss in the ratio of 2:1. On this condition, nearly 70% of the plantlets were successfully survived. Hence, this protocol might be useful for mass propagation and ex situ conservation of this orchid through protocorm culture.