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CHAPTER
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Dactylorhiza hatagirea
4
Archit Sood
Institute of Plant Sciences, Volcani Center, Agricultural Research Organization, Rishon LeZion,
Israel
4.1 Introduction
Dactylorhiza hatagirea (D. Don) Soo, an orchid prevalent to North-Western Himalayan region (3000e4200 m above sea level), is a highly valued medicinal herb of
family Orchidaceae. It is commonly called as “Marsh Orchis.” There is existence
of different names of D. hatagirea, region wise. Angmo-Lakpa, Salem Panja,
Hatajari, and Munjataka are some of the specific names attributed to D. hatagirea
in India. It is a terrestrial herb which can reach up to the height of 50e60 cm.
Rhizome of D. hatagirea is tuberous with 2 e 5 finger-like lobes. Its stem is slender
with the presence of leaves all over the surface (Selvam, 2012). Leaves generally
vary from 4 to 6 in number and are erect, subsessile, and lanceolate to rectangular
in shape (Figs. 4.1 and 4.2). Inflorescences of D. hatagirea are spicate racemes
with flowers in proportionate density. Its flowers are generally purple in appearance
and fruits are ellipsosid. Many minute seeds are present which generally give dusty
FIGURE 4.1
Dactylorhiza hatagirea population (plants with violet color flowers) in natural habitat at
Lahaul and Spiti District, Himachal Pradesh, India.
Himalayan Medicinal Plants. https://doi.org/10.1016/B978-0-12-823151-7.00003-9
Copyright © 2021 Elsevier Inc. All rights reserved.
43
44 CHAPTER 4 Dactylorhiza hatagirea
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FIGURE 4.2
Partwise differentiation of Dactylorhiza hatagirea.
appearance (www.bsienvis.nic.in.) The flowering in D. hatagirea generally starts at
the onset of July and remains up to the end of August (Chaurasia et al., 2007).
4.2 Origin and distribution
The distribution of D. hatagirea encompasses Asian countries viz., Afghanistan,
Pakistan, India, China, and Bhutan. As far as India is concerned, this orchid is found
in the Himalayan states of Jammu and Kashmir (former state), Himachal Pradesh,
Uttarakhand, and Sikkim (Dhar and Kachroo, 1983; Samant et al., 2001). Inhabitation of D. hatagirea includes alpine meadows, humus rich soil surrounded by
grasses, near to snowy streams along with other herbs (Bhatt et al., 2005; Pant
and Rinchen, 2012). Unscientific exploitation of D. hatagirea is in practice on
very large scale due to its high demand aimed at therapeutic uses. The specific
pollinator prerequisite conditions and particular mycorrhizal associations for growth
impart D. hatagirea a slow-growing herb. The cultivation of D. hatagirea through
seeds is very problematic as they are very minute structures and also having very
low viability. Its cultivation can be achieved through divisions of the tuber part
which already contain the stem part with bud. In India, only few centers in Himalayan states have been succeeded in making a repository of germplasm of D. hatagirea
for use by the growers as well as scientific community. It has been reported that
taxonomy of orchids like D. hatagirea is generally considered as more complicated
due to morphological variations among species and more levels of hybridization
amid species (Pillon et al., 2007).
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4.3 Medicinal uses
For millenniums, many orchid species have been used as a source of medicine to
treat various disorders and ailments. Orchids have also been used in Chinese medicine system for many years. The Book of Herbs (CE 500) encompassed the application of many medicinal orchids such as Dendrobium species (State Pharmacopeia
Committee of China, 2010). Many species from genus Dactylorhiza have been used
in combating major diseases. The medicinal uses of D. hatagirea range across
various medicine systems like Ayurveda, Unani, Tibetan, and Folk medicine. Mainly
tuber part of this plant is being used as it has neurostimulant, astringent, antibacterial, aphrodisiac, immunomodulator, demulcent, and nutritional properties (Vij
et al., 1992; Thakur and Dixit, 2007). The mucilaginous substance present in the tu-
bers of D. hatagirea is also used to treat aliments like diarrhea, chronic fever, and
dysentery (Selvam, 2012). D. hatagirea is generally considered as med icinal orchid
as there are various reports on health benefits associated with it. Many studies have
been performed in the past which demonstrate its potential in treating various disorders and health ailments. The roots and tubers of D. hatagirea have been found to be
aphrodisiac in nature which denotes its potential in refining the sexual behavior and
performance (Thakur and Dixit, 2007). The antioxidant activity and ameliorative effect of this plant extracts have been nominated as herbal cure for sexual dysfunction
(Thakur et al., 2008). Recently, Sirohi and Sagar (2019) have reported the neuropharamcological properties linked with D. hatagirea by demonstrating the effect
of hydroalcoholic extracts of roots and tubers. Also other parts of plant like leaves
have shown to be associated with antidiabetic activity. The methanol extracts of
leaves of D. hatagirea have shown the antidiabetic activity in various cell lines tested
(Alsawalha et al., 2019).
4.4 Dactylorhin
Due to a strong urge of this orchid herb in medicinal industry, its annual demand is
very high, i.e., more than 5000 tonnes. Its economical importance can be assessed by
the fact that the value for its dry tubers ranges across Rs 1800eRs 2000 per Kilogram
(Kala, 2004). Overall, the mature tuber consists of mucilage (up to 45%), glucosides,
starch, volatile oil, albumen, phosphate, and chloride (Chaurasia et al., 2007). The
main medicinal properties of D. hatagirea are generally attributed toward the presence of a glucoside, dactylorhin (Fig. 4.3) in the underground part, i.e., tuber. Dactylorhin is the main active chemical constituent in the mature tubers of plants in the
family Orchidaceae. Overall, five distinct dactylorhin compounds (dactylorhin A,
dactylorhin B, dactylorhin C, dactylorhin D, and dactylorhin E) have been documented from Dactylorhiza species and some other medicinal orchids so far.
4.4.1 Biosynthesis of dactylorhin
Although dactylorhin is considered as the main medicinal constituent of
D. hatagirea, the path way completing its biosynthesis in plant tissues remains still
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FIGURE 4.3
Chemical structure of dactylorhin.
to be elucidated. There are few reports on the isolation and quantification of different
dactylorhin compounds such as dactylorhin A, dactylorhin B, dactylorhin C, dactylorhin D, and dactylorhin E along with other compounds such as dactyloses (Kizu
et al., 1999). Three dactylorhin compounds, i.e., dactylorhin A, dactylorhin B,
and dactylorhin E have been also isolated from other orchids viz., Gymnadenia con-
opsea R. Br. and Coeloglossum viride along with other compounds such as loroglossin and militarine (Li et al., 2009). Using RNA-Seq approach, a comparative
transcriptome-based characterization has been performed to elucidate the biosynthetic pathway of dactylorhin employing different conditions through plant cell culture techniques (Sood).
4.5 Biotechnological interventions in Dactylorhiza
hatagirea
Whenever there is debate on the role of biotechnology for scientific improvement of
orchids, a large repository for the same always exists. A very informative report on
the use of modern biotechnological interventions has been documented where Hos-
sain et al. (2013) have signified the areas like in vitro propagation for mass production
and conservation, functional genomics, genetic transformation, as well as the role of
pharmaceutical biotechnology. For genus Dactylorhiza also, there are adequate number of reports on the application of biotechnology-based tools for various studies in
other species such as Dactylorhiza fuchsii, Dactylorhiza incarnata, Dactylorhiza
majalis, and Dactylorhiza maculate (Hedren et al., 2008; Balao et al., 2017; Devos
et al., 2005). However, very limited biotechnological interventions have been carried
out on D. hatagirea overall as compared to various other herbs endemic to same
growing region, on national and international levels, to date.
4.5.1 Tissue culture strategies for conservation of Dactylorhiza
hatagirea
Tissue culture is an imperative ste p for conservation of terrestrial orchids at ex situ
level (Jakobsone et al., 2007). It is important to know about the morphological and
4.5 Biotechnological interventions in Dactylorhiza hatagirea 47
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physiological characteristics for germination of particular orchid species to establish
effective tissue culture approach.
Like other orchid species, genus Dactylorhiza has not been studied with respect
to employing various tissue culture strategies for mass propagation. However, some
prominent tissue culture approaches have been applied up to some extent for conservation of critically endangered medicinal orchid, D. hatagirea which in turn linked
to low rate of success. This could be attributed to many limitations linked with this
orchid herb such as association of fungus and presence of mucilaginous substanc es
in underground parts such as tubers as well as root s. In vitro propagation of
D. hatagirea was also achieved successfully using various plant growth regulators
(Giri and Tamta, 2012). Different explants such as green pod, shoot bud, leaf
segment, and tuber segment were used for in vitro plantlet regeneration (Fig. 4.4).
However, the success rate was very low. It was observed that Murashige Skoog
(MS) medium added with peptone having concentration of 1.0 g/L, morphoinoethane sulfonic acid with 1.0 g/L concentration, and activated charcoal in ratio of
0.1% showed effectiveness in terms of producing protocorm-like bodies and the
plantlet formation.
If given proper environment and suitable conditions, seed embryos can also
develop into entities like protocorm for mass propagation. In a different study, a positive attempt was made to culture immature seed embryos of D. hatagirea to develop
into protocorms and also in shoot regeneration (Warghat et al., 2014). Out of various
media combinations, MS medium with 3 mg/L of indole butyric acid and 1 mg/L of
kinetin produced significant results in terms of production of shoots and roots from the
culture of protocorms with leaf primordia. Further propagation of in vitro plants was
also significant in the potting mixture of cocopeat þ perlite þ vermiculite (1:1:1).
Also, for enhancing the rate of callus induction, in vitro propagation of tubers/
roots was also achieved where young shoots from mature plants were taken as explants (Fig. 4.5) (Sood). The shoots explants were cultured in various media combinations with various plant growth regulators (Table 4.1), and it was observed that
they served as an alternate for better callus induction as compared to direct callus
formation.
4.5.2 Role of molecular markers
There is a sharp decline in the natural populations of D. hatagirea due to which this
species has been recorded as critically endangered species in CAMP (Conservation
Assessment and Management Plan), endangered by CITES (Convention on International Trade in Endangered Species) under appendix II and critically rare by IUCN
(International Union for Conservation of Nature and Natural Resources) (Uniyal
et al., 2002; Bhatt et al., 2005). There is scarcity of information available with
respect to study the genetic diversity and population structure of D. hatagirea at molecular- or DNA-based markers. It is anticipated that efficient and robust molecular
markers should be used for the conservation and genetic improvement programs at
very significant level.
48 CHAPTER 4 Dactylorhiza hatagirea
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FIGURE 4.4
(a) Seeds swelling after 9 weeks of inoculation. (b) Development of protocorm after
20 weeks of seed germination. (c) Enlarged view of protocorm showing distinct shoot apex
and rhizoids after 22 weeks of seed germination. (d) In vitroeraised plantlets after
91 weeks of seed inoculation on MS medium. (e) Mother tuber and daughter tubers. (f)
Daughter tubers having shoot buds used as an explants. (g) and (h) Shoots developed
from the sprouted buds and attained an average length of 2.72 cm on MS medium
supplemented with TDZ (10.0 mM). (i) Sprouted plantlet inside the poly house after
12 weeks of planting. (j) Root emergence in apical segments of tuber after 16 weeks of
planting. mM, Micro molar; MS, Murashige and Skoog; TDZ, Thiadiazuron.
Courtesy: Giri, D., Tamta, S., 2012. Propagation and conservation of Dactylorhiza hatagirea (D. Don) Soo, an
endangered alpine orchid. Afr J Biotechnol 11 (62), 12586e12594.
Inter Simple Sequence Repeats markers were employed to estimate the level of
genetic diversity among D. hatagirea populations in cold desert of Ladakh region of
Indian Himalayas (Fig. 4.6)(Warghat et al., 2013). It was observed that a moderate
level of genetic variations among populations exists. Also, random amplified
4.5 Biotechnological interventions in Dactylorhiza hatagirea 49
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FIGURE 4.5
(a) In vitro plantlet regeneration and (b) tuber/root induction in Dactylorhiza hatagirea.
Table 4.1 Media combinations used for in vitro plant propagation and tuber/
root induction.
Media composition Response
BM2 e
BM2 þ IBA (1 mg/L) þ
MS þ IBA (1 mg/L) e
BM2, orchid medium with vitamins, sucrose, casein hydrolysate, 6-BAP; IBA, indole-3-butyric acid;
mg/L, milligram per liter; MS, Murashige and Skoog basal medium.
polymorphic DNA markers were used to assess the genetic diversity among nine
populations of D. hatagirea in Ladakh region which revealed the occurrence of moderate genetic variations among population (Fig. 4.7)(Warghat et al., 2012).
Overall low level of genetic diversity among populations supports the theory that
endangered or threatened species with narrow level of distribution are usually
depauperate. The low genetic variations are gener ally linked with the less ability
of the species to cope up with environmental fluctuations as well as at higher risk
of being extinct. This can also lead to increase the phenomenon of inbreeding
(Frankham et al., 2010).
For efficient markers to be used in conservation genetic studies among closely
related species, some features like absence of genotypic linkage disequilibrium
within populations and also cross-specific amplification are anticipated. Later in
2014, a study reported development of simple sequence repeat (SSR) markers to
assess the genetic diversity within and between Chinese populations of
D. hatagirea (Lin et al., 2014). This resulted in the identification of 14 SSR markers
of different repeats which can be employed to for population-level studies to assess
genetic diversity among D. hatagirea and its closely related other orchid species as
50 CHAPTER 4 Dactylorhiza hatagirea
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Mean of esL Ln prob of data
C
–14000
–15000
–16000
–17000
–18000
–19000
–20000
–21000
L(K) (mean +- SD)A
Delta K
2
4 6
K
8 2
250
200
150
100
DeltaK = mean(|L"(K)|)/ sd(L(K))B
50
0
43 5 76
K
8
FIGURE 4.6
Structure packageebased analysis of Dactylorhiza hatagirea population (Based on ISSR
analysis). (a) The relationship between K and Ln P D; (b) the relationship between K and
dK; (c) the grouping when K ¼ 2. Location code: PdBogdang; QdSkampuk; RdSkurru;
SdHunder; TdTurtuk; UdTirith; VdSumur; WdChanglung; XdStaksha.
Courtesy: Warghat, A.R., Bajpai, P.K., Srivastavaa, R.B., et al., 2013. Population genetic structure and con-
servation of small fragmented locations of Dactylorhiza hatagirea in Ladakh region of India. Sci Hortic 164,
448e454.
well. Keeping in mind the same objective for Indian populations from Western Himalayan region, (Sharma et al. (2015)) reported characterization of novel polymorphic
SSR markers, out of which seven were further identified to be linked with studying
the extent of genetic diversity and population genetics in D. hatagirea. Later, a
different marker approach, i.e., isoenzymes, was also used along with morphological
and biochemical parameters to study variability among populations of D. hatagirea
which indicates a significant level of intrapopulation diversity (Chauhan et al., 2014).
There is an immense need to employ more robust and specific molecular markers
along with screening of large number of populations for all the habitats of
D. hatagirea in Himalayan region to enhance the conservation management strategies on a large scale.
4.5.3 Role of “omics” technologies
“Omics” refers to the systematic analysis at specific level in any living system. In the
past, various “omics” technologies have paved the way forward for understanding of
organism at cellular, gene, protein, and metabolite level (Witzel et al., 2015). During
the last decade, plant science has witnessed the use of “omics” approaches at very
4.5 Biotechnological interventions in Dactylorhiza hatagirea 51
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FIGURE 4.7
Dendrogram of 96 individuals of 9 population of Dactylorhiza hatagirea based on UPGMA
analysis of RAPD polymorphism.
Courtesy: Warghat, A.R., Bajpai, P.K. , Murkute, A.A., Sood, H., Chaurasia, O.P., Srivastava, R.B., 2012. Genetic
diversity and population structure of Dactylorhiza hatagirea (Orchidaceae) in cold desert Ladakh region of India. J
large and in an efficient manner and perhaps the most implemented for it. Modernday “omics” approaches are anticipated for full-level understanding of unexplored
mechanisms linked with various pathways and processes associated with plants.
The most potential application of “omics” for plant research currently is genomics
which has enabled techniques like genetic transformation, genome-assisted
breeding, and development of transgenic/genome-edited plants (Hruz et al., 2008;
Tsai et al., 2017). More or less it is imperative that techniques like next-
generation sequencing (NGS) have commissioned the role of “omics” for betterment
of an organism at various developmental stages and proce sses.
Med Plants Res 6, 2388e2395.
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4.5.4 Next-generation sequencing to characterize transcriptome
For genus Dactylorhiza, the use of ultramodern and advanced NGS platforms is well
documented for applications like genome and transcriptome sequencing to characterize chloroplast genome, phylogenomic relationship, and ecological divergence
among various species (Balao et al., 2017; Brandrud et al., 2020; May et al.,
2019; Paun et al., 2011).
As D. hatagirea is concerned, there are very few reports on employing NGS platforms to study any genomic and transcriptomic characterization. Recently,
Nova-Seq sequencing platform from Illumina Inc. was employed to perform
RNA-Seq analysis of various in vitro cultured and wild tissues of D. hatagirea to
elucidate biosynthetic pathway of important secondary metabolite, i.e., dactylorhin
(Fig. 4.8) (Sood). Also another study reported the use of another NGS platform, i.e.,
Genome Analyzer IIx (Illumina Inc.) to elucidate biosynthetic mechanism of various
secondary metabolites such as resveratrol and stilbenes in the tuber of D. hatagirea
(Dhiman et al., 2019). Also molecular cues linked with freezing stress and other
environmental factors were identified using this transcriptomic characterization.
FIGURE 4.8
Methodology followed to characterize transcriptome for the elucidation of dactylorhin
biosynthesis pathway in Dactylorhiza hatagirea. cDNA, Complementary deoxy ribonucleic
acid; DEGs, Differentially expressed genes; RNA, Ribonucleic acid.
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