Ultrastructure of In Vitro Developed Prtocorm and Naturally Occurring Tuber of Dactylorhiza

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Ultra structure of In Vitro Developed Prtocorm and Naturally Occurring Tuber of Dactylorhiza Hatagirea (D.Don) Soo: A Critically Endangered Medicinal Plant

Anjuli Agarwal*and J.P. Purwar

GBPUA&T Agriculture Research Station, Majhera

PO- Garampani, Nainital (Uttarakhand) 263135 India

Corresponding author Email*: anjulit@rediffmail.com

Received: 19-11-2019                                                                                                        Accepted: 10-02-2020

Dactylorhiza hatagirea (D. Don.) soo (family Orchidaceae) is a critically rare medicinal herb of Indian Himalayan Region. The plant is a terrestrial orchid in alpine meadows and has fleshy tuberous roots which are slightly flattened and palmately lobed. Medicinally, the roots contain glucosides, starch, mucilage, albumen, a trace of volatile oils and ash consisting potassium and lime as the chief constituents. Cultivation of this orchid through seeds is difficult because seeds are minute, has low viability and needs association of mycorrhiza for germination. Therefore, in vitro techniques are used for seed germination and protocorm development. A beverage made from the tubers called ‘Salep’ is obtained from the tuberous roots of D. hatagirea and starch is one of its main constituents. Therefore, aim of the present study was to elucidate and compare the ultrastructure of in vitro developed protocorm with naturally occurring tubers. Transmission electron microscopy revealed many differences and characteristic presence of many subcellular organelles in naturally occurring tuber and in vitro developed protocorm. Presence of amyloplasts in the lower part of protocorm was very distinguished (1-3.3 µ) as compared to the other parts of in vitro grown protocorm and naturally occurring tuber. However, amyloplasts could not be observed in naturally occurring tuber arm.

Key words: Clarias batrachus, leucocytes, lead, pollution.

Dactylorhiza hatagirea (D.Don.) soo, commonly known as Salampanja or hatajari belongs to the family Orchidaceae. The plant has medicinal importance and grows in alpine meadows of the Himalayan region at an altitude from 2800 – 4000m. D. hatagirea has been categorized as critically endangered (CAMP status), critically rare (IUCN status) and is listed under appendix II of CITES4. Medicinally, the roots are used for treating ailments like stomachache, general debility, and as an aphrodisiac18,11. Its tuberous root contains starch as the main constituent along with glucosides, mucilage, albumen and a trace of volatile oils1. The tubers of D. hatagirea are known to yield a high quality ‘Salep’5 which is obtained by grinding the root under water and straining. This is extensively used in local medicine as nervine tonic for its astringent and aphrodisiac properties (Vij et al.,1992; Lal et al., 2004; Baral and Kurmi, 2006).

Paste of tuber is used to cure cuts and wounds by the tribal people of the Himalayan region (Sigh, 2004). Rhizomatous part of D. hatagirea has shown resistance against all Gram positive and Gram-negative bacteria but its aerial part has shown limited resistance against some bacteria (Rampal, 2009).

Being an orchid, seeds of D. hatagirea need mycorrhizal association in nature for germination. Therefore, several studies to standardize protocol for in vitro seed germination and mass multiplication have been performed (Dhyani and Kala, 2005; Giri and Tamta, 2012; Warghat et al., 2014; Agarwal et al., 2015). Seed germination of orchids under in vitro condition leads to the formation of protocorms. Microscopic observations of in vitro seed germination and ultrastructure of protocorm of D. hatagirea through scanning electron microscopy has been reported by our group (Agarwal et al., 2015).  Though, electron microscope observations on the differentiation of D. maculata have been elucidated (Simola, 1982) but information on the transmission electron microscopic (TEM) observations of D. hatagirea protocorm is hardly available. Therefore, the present study was conducted to illustrate and compare the ultrastructure of in vitro developed protocorm and naturally occurring tuber of D. hatagirea through transmission electron microscopy.

MATERIALS AND METHODS

Protocorms of D. hatagirea were developed in vitro from the seeds. Seed pods were collected from district Pithoragarh, Uttarakhand state of India. Detached Seeds from the pods were stored at 4ºC. Seeds were surface sterilized for 1 min in 70% ethanol followed by 0.1% HgCl2 solution for 2 min and then for 3 min in 2 ml of 4% NaOCl mixed with 6 ml sterile distilled water. Seeds were rinsed 3 times with sterile distilled water for 1 min each under aseptic conditions and cultured in vitro in Knudson C medium (Knudson, 1946). Cultures were maintained in continual darkness at 25 ± 2 ºC for 5 weeks and after that exposed to 16 h photoperiod under the white light intensity of 3000 lux. Seed cultures showed swelling as the first symptom of germination, then forming a protocorm like bodies. In terrestrial orchids like D. hatagirea seeds are very minute (200 – 1700 µm) and testing of seed viability is tedious and delicate procedure (Warghat et al., 2014). However, Protocorms survival and multiplication was standardized using various media formulations (Agarwal et al., 2015). For protocorm multiplication MS (Murashige and Skoog, 1962) half strength basal media supplemented with peptone, tryptone, casein hydrolysate (0.1% each) and activated charcoal (0.2%) along with the addition of growth hormones at a concentration 2.0 mgl-1 BA  & 1.0 mgl-12-iP was used (Agarwal et al., 2015).  For transmission electron microscopy, the in vitro developed protocorm was divided in to three different parts as shown in the figure 1a. Part ‘a’ was considered as upper portion, part ‘b’ as lower and part ‘c’ as roots. Comparative study was performed using naturally occurring tuber available in the local market for transmission electron microscopy. Arm of the naturally occurring tuber was taken for the TEM as per the figure 1b. Protocol for the fixation of samples was as per Simola (1982) with minor modifications. Sections of 1-1.5 mm thickness of each sample were fixed in 2.5% glutaraldehyde & 2.0% p-formaldehyde for 12 h at 4°C. Samples were washed with 0.1 M phosphate buffer (pH 7.2-7.4) three times at 15 min interval at 4°C. Then post fixed in 1% osmium tetraoxide for 2 h at 4°C, dehydrated through a series of acetone (30, 50, 70, 80, 90 & 100%) and embedded in pure resin. Further, the material was sectioned (60-90 nm thickness) and stained with 2% uranyl acetate and lead citrate (Reynolds, 1963). The stained sections were examined with JEOL microscope (JEM 1011). TEM work was performed at College of Veterinary Science, G.B.P.U.A.&T., Pantnagar, India. Size of the organelles was extrapolated on the basis of microscopic scale bar.

RESULTS AND DISCUSSION

Transmission electron microscopy of three parts of in vitro grown protocorm and the arm of naturally occurring tuber of D. hatagirea showed the presence of amyloplasts, mitochondria, vacuoles, vesicles and rough endoplasmic reticulum along with starch and reserve material. Comparison of in vitro developed protocorm with the tuber formed in nature clearly revealed many differences and characteristic presence of some subcellular organelles in the ultrastructure of cells. Presence of amyloplasts in the lower part of protocorm was very distinguished as compared to the other parts of in vitro grown protocorm and naturally occurring tuber. Size of the amyloplasts in the lower part of in vitro grown protocorm ranged from 1- 3.3 µ (Fig.2a & b). However, amyloplasts could not be observed in naturally occurring tuber arm. Compound amyloplasts as the characteristic structure in young protocorms of D. maculata were also observed in the other studies (Simola, 1982). In the young aseptically cultivated protocorms of Cypripedium reginae, the presence of compound amyloplasts and smooth endoplasmic reticulum as the most characteristic structures has also been reported (Simola, 1980). The cells of these protocorms were not highly vacuolated and lipid & protein stores were rarely seen. In the present study, similar structural set up was observed in the lower portion of D. hatagirea protocorm. The cells of lower part of the protocorm seem to store much starch in compound amyloplasts, whereas upper part of the protocorm was more meristematic and showed the presence of mitochondria (Fig.2d). Mitochondria in naturally occurring tuber arm were smaller in size (0.23-0.38 µ) when compared with in vitro cultured protocorm (Fig.2e). However, size of mitochondria varied from 0.83-1.16 µ and 0.8-2.0 µ in roots and upper portion of in vitro cultured protocorm respectively (Fig. 2f & d).

Presence of rough endoplasmic reticulum with irregular pattern was noted in the root cells of in vitro developed protocorm (Fig. 2c). Accumulation of reserve material, possibly some secondary metabolites was also seen in the vacuoles (0.75-1.00 µ) of root cells (Fig. 2h). Reserve material in the vacuoles of naturally occurring tuber was dense and starch was visible in the cells but no amyloplasts could be observed (Fig. 2g). Similarly, accumulation of secondary metabolite in the vacuoles as a characteristic feature of several microspore cultures of Picea abies has been reported (Simola, 1986) Vesicles are also considered to play important role in the subcellular framework. Proteins synthesized in the endoplasmic reticulum are packaged in to vesicles which then fuse with the golgi apparatus. In D. hatagirea, presence of vesicles was noted in the upper part of protocorm only (Fig. 2i).      

This ultrastructural study indicated that starch being the main constituent of tuber of D. hatagirea is synthesized and stored in the amyloplasts during its active growth period. Comparison of ultrastructure of naturally occurring tuber and in vitro grown protocorm revealed the difference in size of mitochondria and presence of amyloplasts. Size of mitochondria in protocorms was observed larger, possibly due to active phase of growth.  For the production of starch, in vitro produced protocorms can be used as an alternative of naturally occurring tuber of D. hatagirea.

ACKNOWLEDGEMENTS

The authors thank Life Sciences Research Board, DRDO, New Delhi, India for financial support of the project and Director Experiment Station, Govind Ballabh Pant University of Ag. & Tech, Pantnagar, India for providing the necessary facilities.

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