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. 2021 Feb 16;27(2):341–357. doi: 10.1007/s12298-021-00939-x

RAPD, ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.- an important endangered orchid

Leimapokpam Tikendra 1, Angamba Meetei Potshangbam 1, Abhijit Dey 2, Tongbram Roshini Devi 3, Manas Ranjan Sahoo 3, Potshangbam Nongdam 1,✉
PMCID: PMC7907394  PMID: 33707873

Abstract

Dendrobium fimbriatum is an ornamental and medicinal orchid listed in the Red data book of IUCN. Phytohormones’ effect on the in vitro regeneration of the orchid was studied using Mitra medium supplemented with different growth regulators. KN produced effective shoot formation when present alone or in combination with IBA or NAA. The shooting was gradually increased when KN concentration was increased from 0.8 to 4.8 mg L−1, but the opposite response was observed with BAP at higher concentration (4.8 mg L−1). IBA either in combination with BAP or KN promoted effective root development and multiplication. Micropropagated orchids grown in the basal medium devoid of any phytohormone showed 100% monomorphism, while low genetic polymorphism of 1.52% (RAPD—Random Amplification of Polymorphic DNA), 1.19% (ISSR-Inter Simple Sequence Repeat) and 3.97% (SCoT—Start Codon Targeted) was exhibited among the regenerants propagated in the hormone enriched medium. UPGMA (Unweighted pair group method using arithmetic averages) dendrograms showed the grouping of mother plant (MP) with the in vitro regenerants. The principal coordinate analysis (PCoA) further confirmed the clustering patterns as determined by the cluster analysis. The study reported for the first time the successful in vitro propagation of Dendrobium fimbriatum and their genetic stability assessment using molecular markers.

Keywords: Auxin, Cytokinin, Genetic stability, Genetic distance, Micropropagation

Dendrobium fimbriatum Lindl. var. oculatum Hk. f., belonging to the family Orchidaceae, is an endangered sympodial epiphytic orchid widely distributed in India, Myanmar, China and other Southeast Asian countries (Pradhan 1979; Paul et al. 2017). In addition to its outstanding aesthetic beauty as a popular ornamental plant, the species is known to possess important phytochemicals with its medicinal application for treatment of fractured bones, liver upset and nervous weakness (Bi et al. 2003; Aggarwal and Zettler 2010; Meetei et al. 2012; Jhonson and Janakiraman 2013; Pant 2013). It is also considered as one of the important orchids employed in traditional Chinese medicine (Cheng 2018). Although the plant is protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), the revelation of its various medicinal values and huge contribution to the floriculture market have enhanced tremendously the demand for the plant materials. This leads to the unregulated and undocumented illegal trade of the orchid which ultimately produce an enormous impact on its population (Phelps and Webb 2015; Hinsley et al. 2018). The plant has become endangered and is officially listed in the Chinese Plant Red Book as critically endangered and extinct since 1987 (Fu and Jin 1992). In vitro propagation of the orchid is one of the important strategies for germplasm conservation which circumvents the limitation of mycorrhizal association and thus has the advantage of asymbiotic seed germination and subsequently enhances the regeneration rate of the seedlings (Shin et al. 2011; Pant et al. 2018). However, tissue cultured plants are constantly confronted with stress conditions in vitro and they must cope with high osmotic pressure, abnormal mineral nutrition and high concentration of sugar and plant growth regulators (PGRs) to survive successfully (Desjardins et al. 2009). Somaclonal variation, a random spontaneous genetic variation expressed in the progenies during plant tissue culture can be attributed to genetic and epigenetic modifications in DNA (Sato et al. 2011). The genetic variation occurring in the tissue culture might be due to high concentration of plant growth regulators, explant exposure to sterilizing agents during sterilization, prolonged culture cycle, differential lighting conditions and other nutrient stresses (Jain 2001; Sato et al. 2011). Phytohormones influence somaclonal variation by impacting the cell division cycle and multiplication (Gao et al. 2010), stimulating rapid disorganized growth (Karp 1995) and altering the degree of DNA methylation (Vanyushin 1984). D’Amato (1985) stated that some phytohormones at a particular concentration or in combination with other growth hormones or nutrient constituents may act as mutagens causing genetic variation among the clones. Eeuwens et al. (2002) also opined the significance of the ratio of different growth regulators apart from their concentrations in producing somaclonal variation in oil palm plants. Although, somaclonal variation may provide a valuable source of novel variants for higher yield, improved quality, or disease resistance (Mehta and Angra 2000; Predieri 2001; Unai et al. 2004), the emergence of variation is a major concern if primary regenerants are the required end product for commercialization and conservation of the elite genotypes (Krishna and Singh 2013; Tikendra et al. 2019a). Hence, it has become essential to assess the somaclonal variation for detecting the genetic stability of the in vitro propagated plants. Many PCR based RAPD, ISSR and SCOT molecular markers have been successfully employed to test the genetic homogeneity of several micropropagated plants (Antony et al. 2015; Rathore et al. 2016; Al-Qurainy et al. 2018; Tikendra et al. 2019a). Since conventional DNA markers like RAPD and ISSR have limitation because they target a specific region in the genome, the use of SCoT marker resolves the limitation by targeting the conserved region flanking the start codon (ATG) of a functional gene (Collard and Mackill 2009; Amom and Nongdam 2017).

Micropropagation of D. fimbriatum had been performed with different culture media. But the present investigation employed the Mitra medium (Mitra et al. 1976) as earlier works of D. fimbriatum propagation using other media provided limited information on the shoot and root development. Parmar and Acharya (2016) achieved the in vitro seed germination of D. fimbriatum up to the shoot initiation stage using Murashige and Skoog medium (1962). Sharma et al. (2005) also reported only seed germination and protocorm-like bodies (PLBs) formation of D. fimbriatum on Vacin and Went (1949) medium (VWM) enriched with different growth regulators at different concentrations. Out of the nine hormonal combinations tested, only VWM + 0.1 mgL−1 NAA + coconut water (15% or 25%) induced seed germination and PLBs development ranging from 60 to 90%. The Mitra medium is a nutrient mixture of inorganic salts and vitamins with a higher content of thiamine, riboflavin, biotin, and folic acid for enhancing seed germination, protocorm formation, chlorophyll development, and seedling growth (Mitra et al. 1976). It also contains less potassium nitrate and ammonium sulphate as nitrogen sources, which helps in early protocorm development. The culture medium had been used effectively to propagate several orchid species in vitro (Seeni and Latha 2000; Babbar and Singh 2016; Tikendra et al. 2018).

The present work aims to investigate the effects of phytohormones on in vitro growth and development of D. fimbriatum, which leads to the successful establishment of effective regeneration protocols. The study also seeks to assess the genetic variation that might have occurred among the clones due to the stress effect of high plant growth regulator concentration by employing fast and effective RAPD, ISSR and SCoT molecular markers. The objectives of the present study were accomplished through successful in vitro propagation of the orchid using different combinations and concentrations of phytohormones, followed by greenhouse acclimatization and molecular genetic homogeneity assessment of the regenerated orchids using molecular markers.

Materials and methods

Micropropagation

Explant source and sterilization

Mature undehisced capsules of D. fimbriatum grown in the natural population of State Orchidarium, Khonghampat (24.76′ N, 93.86′ E), Manipur, were used as explant for initiation of in vitro culture and micropropagation. Sterilization was started by washing the capsule thoroughly with running tap water for 5–10 min to remove any dust and soil particles stuck to them. The capsules were then treated with 10% (v/v) Tween-20 (Merck) for 10 min followed by washing and surface sterilization with 0.4% (w/v) mercuric chloride (Hi media) for 6–7 min. Mercury chloride from the capsule’s surface was eliminated by washing them 4–5 times with sterile ultra-pure water. Finally, the capsules were dipped in absolute alcohol and flamed for 2–4 s.

Culture medium preparation and hormonal combination

To study the in vitro growth and phytohormone stress effect on the D. fimbriatum, Mitra medium (Mitra et al. 1976) supplemented with 3% (w/v) sucrose (Himedia, Mumbai) as carbohydrate source and 0.9% agar (Himedia, Mumbai) as the gelling agent was used. The pH of the medium was adjusted at 5.5–5.8 using 1 N NaOH and HCl. Further, plant growth regulators such as cytokinin (BAP and KN) and auxin (IBA and NAA) at different concentrations and combinations were incorporated in the medium while other environmental factors and salts concentration were uniformly maintained throughout the experiment. Regenerants are represented hereafter with symbols;  MP for mother Plant and P1–P9 for in vitro regenerated plants [P1 = 4.8 mg L−1 BAP; P2 = 0.8 mg L−1 BAP; P3 = 4.8 mg L−1 KN; P4 = 0.8 mg L−1 KN; P5 = 2.4 mg L−1 BAP + 1.2 mg L−1 IBA; P6 = 2..4 mg L−1 BAP + 3.6 mg L−1 IBA; P7 = 2.4 mg L−1 KN + 1.2 mg L−1 IBA; P8 = 2.4 mg L−1 KN + 3.6 mg L−1 IBA; and P9 = 2.4 mg L−1 KN + 1.2 mg L−1 NAA].

Explant inoculation, acclimatization and field transfer

Initially, the exalbuminous seeds scooped out from the surface-sterilized capsules were allowed to germinate on basal medium. The in vitro growth was examined by transferring the germinated seeds to freshly prepared basal medium (control treatment) and culture medium containing various combinations and concentrations of plant growth hormones. Each hormonal treatment had 10 replicates and the experiments were performed thrice. All the cultures were maintained at 25 ± 2 °C with proper light illumination at 60 μmol m−2 s−1 for 16/8 h day/night photoperiods using white fluorescent tubes.

The well-developed seedlings were transferred to full strength Mitra medium devoid of any plant growth regulators for 3–4 weeks followed by subculture to half-strength Mitra medium without plant growth regulators, sucrose and vitamins for another 3–4 weeks. Seedlings adapted to these differential culture media were taken out from culture vessels, washed with lukewarm water for removal of any solidified agar from the media. Fungal contaminants, if any, were removed by treating with 0.01% fungicide solution (Sixer, Dhanuka Agritech Ltd. Gujarat) for 15 min followed by washing with distilled water. The plants were then transplanted to side-perforated small plastic pots containing coconut husk, pieces of bricks and charcoal (1:1:1) as a potting mixture. The transplanted plants were sprayed every alternate day with 1/4th Mitra salt solution incorporated with 1ml L−1 Dhanzyme gold (Dhanuka Agritech Limited, Gujarat), an organic extract from seaweed, to prevent from dehydration. After 3–4 weeks in the glasshouse for further acclimatization the potted plantlets were finally exposed to normal daylight in the greenhouse.

Culture data recording and statistical analysis

All experiments were conducted in a completely randomized manner. The in vitro growth responses with regards to the development of shoots and roots were recorded at regular intervals. The data were subjected to statistical analysis using analysis of variance (ANOVA, p ≤ 0.05), and the mean values of different treatments were compared using Duncan’s Multiple Range Test (DMRT) at (P ≥ 0.05). All statistical analysis was performed using the SPSS (Version 16.0; SPSS Inc., Chicago, USA).

Genetic stability assessment

DNA extraction

Genomic DNA was extracted from the leaves of acclimatized well-grown in vitro raised plants of basal medium, randomly selected hormone incorporated media and mother plant using modified CTAB (Cetyl-trimethyl-ammonium bromide) method (Doyle and Doyle 1990). The qualities and quantities of the isolated DNA samples were determined by using a spectrophotometer (Perkin-Elmer Lambda 35) at 260 and 280 nm, respectively. The purity and integrity of the amplified DNA were later checked by performing 0.8% agarose gel electrophoresis and comparing the intensity of the resultant bands with 1 kb DNA ladder (Hi-Media). The DNA samples were finally diluted to 50 ng/µl and stored at − 20 °C for further use. The DNA samples were then assessed for homogeneity using PCR based RAPD, ISSR and SCoT markers.

PCR amplification

An initial screening of 25 (10 mers) RAPD primers (Eurofins), 34 ISSR primers of 16–18 nucleotide bases in length (Integrated DNA Technology, Banglore, India) and 18 SCoT primers of 17–18 nucleotide bases in length (Integrated DNA Technology, Banglore, India) were performed to select the primers which produced the scorable and reproducible bands. Similar to our earlier published work (Tikendra et al. 2019a), all the PCR reactions were performed in a total volume of 25 μL. The reaction mixture contained 20 ng of template DNA, 2.5 μL of 10 X PCR buffer having 15 mM MgCl2, 0.02 mM dNTPs, 1unit of Taq polymerase (Bangalore Genei, India) and 20 ng primer. The final volume make-up was done with molecular grade ultra-pure water. The PCR condition consisted of initial denaturation at 94 °C for 4 min followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 5 °C less than the melting temperature (Tm) of the respective primer for 1 min and 2 min extension at 72 °C with a final extension at 72 °C for 10 min.

The DNA amplification of RAPD, ISSR and SCoT analysis was performed in a thermal cycler (Himedia, India). The amplified DNA fragments were separated on a 2.0% (w/v) agarose gel in 1X Tris–acetate EDTA buffer stained with 0.5 μg L−1 ethidium bromide. A 1 kb DNA ladder (Himedia, India) was employed to determine the size of unknown DNA fragments on the agarose gels. The gel was finally visualized and photographed using the Gel Documentation system (Syngene, UK). The amplification reactions of RAPD, ISSR and SCoT primers were repeated twice to ensure the reproducibility of the banding pattern.

Data analysis

The consistent, unambiguous and reproducible bands generated by RAPD, ISSR and SCoT primers were scored. The band intensity was not considered while scoring. The data were pooled into a binary matrix based on the presence (1) or absence (0) of the selected bands. The genetic similarity matrices between the mother and in vitro regenerated plants were calculated according to Nei and Li (Nei and Li 1979). These similarity coefficients obtained were used to construct dendrograms through GenAlex 6.502 (Peakall and Smouse 2012) software using an unweighted pair-group method with arithmetic mean (UPGMA). PCoA implemented with GenAlEx 6.502 was performed to spatially distribute the in vitro clones and the mother plant based on the relative genetic distances and to detect the consistency of clustering pattern as defined by the UPGMA dendrograms. Mantel test (Mantel 1967) with 999 permutations was also performed to determine the correlation between the RAPD, ISSR and SCoT markers.

Results and discussion

Micropropagation

Protocorm and leaf primordium development

Although phytohormones are effective in removing seed dormancy (Hidayati et al. 2012), seed germination in the present study occurred in the basal Mitra medium (M) (Mitra et al. 1976) devoid of any plant growth hormones. The orchid seeds that underwent successful in vitro germination showed swelling and rupturing of the testa through the absorption of water and nutrients (Fig. 1a). Successful protocorm formation was observed when the germinated seeds were transferred in newly prepared basal medium and the media incorporated with different combinations and concentrations of phytohormones. The protocorms are unique structures formed after seed germination before a plantlet is formed. The primary function of the protocorms is to facilitate nutrient absorption and form a shoot apical meristem (SAM) for plantlet growth (Yeung 2017). Time taken for protocorm formation from the seed germination stage was recorded with the earliest protocorm development (16.33 ± 0.52 days) observed in M +  4.8 mg L−1 KN, while the longest (29.33 ± 1.51 days) was noticed in the basal M medium (Table 1). The basal M medium deprived of any phytohormones incurred longer duration for protocorm formation compared to other media with different hormone concentrations indicating the positive hormonal influence on rapid protocorm development. In our study, the duration of protocorm formation was shortened by two weeks as compared to protocorm development observed in the sixth week by Sharma et al. (2005). The necessity of auxins and or cytokinins for the neo-formation of protocorms and plantlet development in orchids were also reported earlier (Kalimuthu et al. 2007; Roy et al. 2011; Tikendra et al. 2019b). The protocorm formation percentage was high in M ± 2.4 mg L−1 KN (90.92 ± 0.74%) and M ± 0.8 mg L−1 KN (89.70 ± 1.41%) while it was lower in M + 2.4 mg L−1 BAP (83.79 ± 1.91%) and M + 2.4 mg L−1 BAP + 3.6 mg L−1 NAA (83.83 ± 1.02%) (Fig. 2). Protocorm formation was higher with KN in the media when incorporated alone or in combination with IBA or NAA than with BAP. Earlier studies also reported the successful protocorm generation in the media supplemented either with a single growth regulator or combinations of various auxins and cytokinins (Sujjaritthurakarn and Kanchanapoom 2011; Nongdam and Tikendra 2014). The protocorms gave rise to shoot apical meristems (SAM) which showed enlargement forming the first leaf primordium from the cells occupying the peripheral regions (Nongdam and Tikendra 2014). The SAM developed into dome-shaped structure when they became mature and bigger producing leaf primordia at regular intervals. By providing suitable nutritional and growth conditions through 1st subculture cycle, the protocorms preceded to the next development stage of SAM formation and generation of the first leaf primordium (Fig. 1b). The first leaf primordium formation was evident early in M + 0.8 mg L−1 KN, M + 2.4 mg L−1 KN, M + 4.8 mg L−1 KN and M + 2.4 mg L−1 KN + 1.2 mg L−1 NAA (Table 1) which suggested the promotive effect of KN on early leaf formation. The influence of BAP was low compared to KN with longer time duration for the first leaf primordium formation in M + 2.4 mg L−1 BAP + 1.2 mg L−1 NAA; M + 2.4 mg L−1 BAP + 3.6 mg L−1 NAA; M + 2.4 mg L−1 BAP + 1.2 mg L−1 IBA and M + 2.4 mg L−1 BAP + 3.6 mg L−1 IBA (Table 1). A similar observation of higher efficiency of KN over BAP in initial leaf formation in vitro was earlier reported in Dendrobium chrysotoxum (Nongdam and Tikendra 2014).

Fig.1.

Fig.1

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Asymbiotic seed germination and seedling development of D. fimbriatum. a Germinated seeds in Mitra medium fortified with 2.4 mg L−1 KN after 2–3 weeks of culture. b Protocorm with first leaf primordium in medium incorporated with 2.4 mg L−1 KN + 1.2 mg L−1 NAA. c Leaf development and shoot multiplication in medium supplemented with 2.4 mg L−1 KN + 3.6 mg L−1 IBA. d Robust root multiplication and elongation in M + 2.4 mg L−1 KN + 3.6 mg L−1 IBA. e In vitro development of seedling complete with healthy leaves and roots. f Hardening of in vitro raised mature D. fimbriatum

Table 1.

Effect of different plant growth regulators on in vitro protocorm and leaf primordium formation of Dendrobium fimbriatum

Mitra medium (M) + PGRs (mg L−1) Time taken in days
BAP KN NAA IBA Protocorm formation 1st leaf primordium formation
0 0 0 0 29.33 ± 1.51h 34.16 ± 0.89d
0.8 24.52 ± 0.84e 30.06 ± 0.98c
2.4 25.17 ± 1.17efg 31.10 ± 0.96ac
4.8 25.06 ± 0.63ef 39.23 ± 1.26e
0.8 18.08 ± 1.36ab 27.36 ± 1.01ab
2.4 18.67 ± 1.51bc 27.60 ± 0.26ab
4.8 16.33 ± 0.52a 28.20 ± 0.41ab
2.4 1.2 27.17 ± 0.75h 44.23 ± 1.29f
2.4 3.6 26.83 ± 0.98fgh 43.47 ± 1.27f
2.4 1.2 25.16 ± 0.75ef 40.13 ± 0.45e
2.4 3.6 25.50 ± 1.38efg 40.90 ± 0.79e
2.4 1.2 20.26 ± 0.43cd 29.07 ± 0.47ac
2.4 3.6 21.23 ± 0.75d 29.93 ± 0.61c
2.4 1.2 17.83 ± 1.33a 29.53 ± 0.25c
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Mean values (± SD) within a column followed by the same letter are not significantly different by Duncan Multiple Range Test (P < 0.05). Values are based on ten replicates per treatment in three independent experiments

Fig.2.

Fig.2

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Effect of BAP and KN and their combination with auxins on the protocorm development

Individual effect of cytokinins on shoot and root development

The phytohormones at various concentrations induced protocorm formation which are composed of meristematic cells having distinct cell fates developing into multiple numbers of shoots. Protocorm cultured on the basal Mitra medium which was deprived of any growth hormones produced the lowest number of shoots (1.99 ± 0.43) per explants. The addition of BAP and KN, individually, at three different concentrations (0.8 mg L−1, 2.4 mg L−1 and 4.8 mg L−1) influenced shoot multiplication and regular sub-culturing under the same culture conditions further increased the shoot proliferation rate. Except for the basal medium, the other media containing BAP and KN individually at different concentrations produced good shoot numbers (> 3), with the highest shoot number of 8.53 ± 2.03 recorded in M + 4.8 mg L−1 KN (Fig. 3). In contrast to the observation made by Panwar et al. (2012) in Eulophia nuda, the increased concentration of KN in the present study favored in vitro shoot development and multiplication. The longest shoot length (4.40 ± 0.44 cm) was observed in the M + 2.4 mgL−1 BAP followed by M + 4.8 mgL−1 KN (3.42 ± 0.37 cm), while very short shoot of 0.75 ± 0.18 cm and 0.79 ± 0.22 cm were produced in basal medium and M + 4.8 mg L−1 BAP, respectively (Table 2). Shoot formation and growth were reduced when BAP concentration was increased and this repressive effect of high BAP concentration on leaf development was also reported in Dendrobium nobile (Sana et al. 2011). Root growth and multiplication were satisfactorily observed when BAP and KN were appended alone in the medium. A similar observation of effective root development in cytokinin enriched medium was made in Dendrobium aqueum (Parthibhan et al. 2015). High root number (5.19 ± 1.24) and the longest root length (5.23 ± 1.06 cm) were recorded in medium supplemented with 2.4 mg L−1 BAP (Fig. 3 and Table 2). The root number decreased when the concentration of BAP increased from 2.4 to 4.8 mg L−1, while there was an improvement in root multiplication when the KN concentration was elevated gradually from 0.8 to 4.8 mg L−1. Such an inhibitory effect of BAP on root multiplication and elongation when present at higher concentration was also reported in Dendrobium primulinum (Pant and Thapa 2012). The basal medium produced the lowest root multiplication (2.26 ± 0.33) while the medium incorporated with 4.8 mg L−1 KN generated the highest root number (5.28 ± 0.41) (Fig. 3).

Fig.3.

Fig.3

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Individual effect of BAP and KN on shoot and root multiplication of D. fimbriatum after 26 weeks of culture

Table 2.

Individual effect of BAP and KN on shoot and root length of Dendrobium fimbriatum after 26 weeks of culture

Mitra medium (M) + PGRs (mg L−1) Shoot length (cm) Root length (cm)
BAP KN
0 0 0.75 ± 0.18d 1.99 ± 0.60cd
0.8 1.42 ± 0.25c 3.52 ± 0.37b
2.4 4.40 ± 0.44a 5.23 ± 1.06a
4.8 0.79 ± 0.22d 2.51 ± 0.82bc
0.8 1.56 ± 0.19c 1.30 ± 0.42d
2.4 1.62 ± 0.22c 1.44 ± 0.50cd
4.8 3.42 ± 0.37b 3.53 ± 0.54b
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Mean values (± SD) within a column followed by the same letter are not significantly different by Duncan Multiple Range Test (P < 0.05). Values are based on ten replicates per treatment in three independent experiments

Synergetic effects of cytokinin and auxin on the shoot and root development

The combinations of cytokinin and auxin at different concentrations prompted varied growth effects on in vitro shoot and root development. High shoot number and longer shoot length were recorded in M + 2.4 mg−1 L KN + 1.2 mg−1 L IBA ( 9.23 ± 0.92; 4.68 ± 0.18 cm) and M + 2.4 mg−1 L KN + 3.6 mg−1 L IBA ( 9.34 ± 1.71; 4.39 ± 0.12 cm) (Figs. 1c, 4). The occurrence of high shoot multiplication and shoot length increment was earlier noticed in KN containing media in Dendrobium aqueum (Parthibhan 2015) and Dendrobium hybrid (Martin et al. 2005). However, the shoot development was suppressed in M + 2.4 mg−1 L KN + 1.2 mg−1 L NAA (4.31 ± 0.58) and M + 2.4 mg−1 L KN + 3.6 mg−1 L NAA (4.06 ± 0.62). While NAA showed inhibitory effect on shoot formation, IBA proved to be more favorable for shoot multiplication when associated with KN. The promotive effect of IBA on shoot development was earlier demonstrated in several orchids (Aktar et al. 2007; Riva et al. 2016).

Fig.4.

Fig.4

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Synergetic effect of BAP or KN with IBA and NAA on shoot and root multiplications of D. fimbriatum after 26 weeks of culture

In the present study, IBA with either BAP or KN in the medium was dominant over NAA in inducing root development and multiplication (Fig. 1d). M + 2.4 mg−1 L KN + 1.2 mg−1 L IBA, M + 2.4 mg−1 L KN + 3.6 mg−1 L IBA and M + 2.4 mg−1 L BAP + 1.2 mg−1 L IBA produced higher root number and longer root length of 5.23 ± 0.92, 6.04 ± 0.89 cm; 6.12 ± 0.90, 6.57 ± 0.86 cm; 5.25 ± 0.76, 3.41 ± 0.38 cm, respectively as compared to M + 2.4 mg−1 L KN + 1.2 mg−1 L NAA, M + 2.4 mg−1 L KN + 3.6 mg−1 L NAA and M + 2.4 mg−1 L BAP + 1.2 mg−1 L NAA which generated low root number and root length of 3.93 ± 0.68,2.65 ± 1.39 cm; 4.47 ± 0.68, 2.74 ± 1.28 cm; 3.94 ± 0.51, 2.23 ± 0.26 cm, respectively (Fig. 4, Table 3). The more effectiveness of IBA over NAA in imparting positive influence on root induction was earlier reported in Cymbidium dayanum, Cymbidium mastersii, Dendrobium densiflorum, Dendrobium officinale and Dendrobium thyrsiflorum (Nongdam and Nirmala 2012; Mohanty et al. 2012; Pradhan et al. 2013; Song et al. 2013; Tikendra et al. 2018). The in vitro regenerated plantlets of D. fimbriatum with healthy leaves and roots were successfully acclimatized and hardened with an 81% survival rate (Fig. 1e, f).

Table 3.

Synergetic effect of BAP or KN with IBA and NAA on shoot and root length of Dendrobium fimbriatum after 26 weeks of culture

Mitra medium (M) + PGRs (mg L−1) Shoot length (cm) Root length (cm)
BAP KN IBA NAA
2.4 1.2 2.94 ± 0.24c 3.41 ± 0.38b
2.4 3.6 1.42 ± 0.07c 5.71 ± 0.26a
2.4 1.2 0.67 ± 0.13f 2.32 ± 0.26b
2.4 3.6 0.72 ± 0.09f 2.43 ± 0.41b
2.4 1.2 4.68 ± 0.18a 6.04 ± 0.89a
2.4 3.6 4.39 ± 0.12a 6.57 ± 0.86a
2.4 1.2 3.56 ± 0.25b 2.65 ± 1.39b
2.4 3.6 2.49 ± 0.26c 2.74 ± 1.28b
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Mean values (± SD) within a column followed by the same letter are not significantly different by Duncan Multiple Range Test (P < 0.05). Values are based on ten replicates per treatment in three independent experiments

Genetic stability assessment using molecular markers

Molecular marker polymorphism

Due to diverse explant sources, varied culture conditions, media component imbalances relating to high phytohormone concentration, different regeneration methods and prolonged sub-cultural passages, somaclonal variation appeared in micropropagated plants (Chatterjee and Prakash 1996; Goto et al. 1998; Haisel et al. 2001; Krishna et al. 2016). Hence, assessing the somaclonal variation to determine the genetic stability of the micropropagated plants is essential when the primary regenerants are the preferred end products for commercial applications (Lakshmanan et al. 2007; Tikendra et al. 2021). In the present study, RAPD, ISSR and SCoT markers were used to assess the possibility of any genetic variation that may arise due to the effect of phytohormones in the culture media. Genetic stability of the plantlets grown in the hormone free basal media was first assessed using 33 selected primers belonging to RAPD, ISSR and SCoT markers. A total of 185 monomorphic and reproducible bands were scored exhibiting 100% monomorphism between the mother plant and in vitro clones raised in the basal medium (Fig. 5). The size of the monomorphic bands observed ranged between 250 and 2500 bp and an average of 5.61 bands per primer was recorded (Table 4). However, assessment of genetic stability between nine regenerated plants of D. fimbriatum raised in phytohormones incorporated medium exhibited a low degree of polymorphism when assessed using RAPD, ISSR and SCoT markers. A total of 25 arbitrary RAPD primers were screened for the study, of which 11 primers generated a total of 53 reproducible and scorable bands with an average of 4.82 bands per primer. The sizes of amplified bands ranged between 250 and 2500 bp (Table 5). While a maximum of 7 monomorphic scorable bands were observed when genomic DNA was amplified using OPA-01 followed by 6 monomorphic bands for OPC-05 and 5 monomorphic bands for OPE-07 and OPD-08 (Fig. 6a), and a minimum of 2 monomorphic bands for OPA-13. Primer OPA-07, OPA-10, OPA-13; OPC-07, OPC-08 and OPG-15 also exhibited a monomorphic banding pattern indicating 100% genetic similarity between the in vitro regenerants and the mother plant (Table 5). Analysis with RAPD marker revealed low polymorphism (1.52%) among the in vitro regenerated D. fimbriatum and the mother plant. Our results corroborate the earlier reports of successful application of RAPD markers in genetic stability assessment of in vitro propagated Dendrobium nobile and Dendrobium chrysotoxum (Bhattacharyya et al. 2014; Tikendra et al. 2019a). While molecular assessment of genetic variation using RAPD technique had been explicably used for its easy and rapid results, it failed to disclose changes in repetitive sequences in the genomes of some plants (Palombi and Damiano 2002). The weakness of the RAPD markers can be overcome by substantiating the RAPD analysis with another more advanced markers (Dey et al. 2019). Hence, the result of RAPD analysis was further affirmed by employing ISSR and SCoT primers. From a total of 34 ISSR primers screened, 12 primers were successful in amplifying with genomic DNA producing 67 reproducible and scorable bands giving an average of 5.58 bands per primer. The sizes of observed bands ranged between 250 and 2000 bp. Out of these 67 amplified bands, 65 bands were monomorphic displaying a high degree of monomorphism (97.42%) among the mother plant and the in vitro regenerants (Table 6). Such high genetic identity and low polymorphism were similarly reported among the micropropagated Vanilla planifolia, Anoectochilus formosanus and Dendrobium moschatum using ISSR markers (Sreedhar et al. 2007; Zhang et al. 2010; Tikendra et al. 2019b). A minimum of 3 bands was scored for UBC-807 while a maximum of 9 bands was observed for UBC-801 followed by 7 bands each for UBC-810 and UBC-830 (Fig. 6b). UBC-810 and UBC-835 exhibited one polymorphic band each resulting in low polymorphism of 2.58% (Table 6). The result of ISSR analysis was further validated by SCoT markers which detected genetic variation based on the short conserved region flanking the ATG start codon in plant genes (Antony et al. 2015). After an initial screening with 18 SCoT primers, 10 primers produced 65 unambiguous and reproducible bands with 6.5 bands per primer and band size ranging from 500 to 2000 bp (Table 7). The maximum of 9 bands were observed for SCoT-S3 and S5 (Fig. 6c), while the least number of 2 bands was witnessed for S6. SCoT-S1 and S17 detected one polymorphic band each resulting in 3.93% polymorphism (Table 7). The analysis revealed high genetic uniformity (96.07%) and low genetic variation (3.93%) among the in vitro regenerants and the mother plant. Earlier report on D. nobile using SCoT markers revealed similar observation of 3.50% of polymorphism among the micropropagated orchids (Bhattacharyya et al. 2014). The pooled data of RAPD, ISSR and SCoT analysis showed a total of 180 monomorphic and 5 polymorphic bands from a total of 185 scorable bands revealing low polymorphism (2.70%) and high genetic stability (97.30% monomorphism) among the micropropagated orchids. The prevailing low degree of polymorphism witnessed through RAPD (1.52%), ISSR (2.58%) and SCoT (3.93%) analysis could be attributed to the hormonal effects as the plants regenerated in the basal medium devoid of phytohormones showed 100% monomorphism.

Fig.5.

Fig.5

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Banding profiles obtained for in vitro raised plants of Dendrobium fimbriatum in hormone free basal medium. a RAPD profile for OPA-03, b ISSR profile for UBC-871, c SCoT profile for S1. L: 1 kb DNA ladder; M: Mother Plant; B1–B7: Randomly selected in vitro regenerated plants in basal medium

Table 4.

RAPD, ISSR and SCoT primers used, amplified fragment generated and the monomorphism detected in the micropropagated D. fimbriatum grown in hormone free basal medium

RAPD, ISSR and SCoT primers Primer sequence (5′ → 3′) No. of scorable bands No. of bands Percentage of Band size (bp)
Monomorphic Polymorphic Monomorphism Polymorphism
OPA-01 5′-CAGGC3TTC-3′ 07 07 – 100 – 1500–500
OPA-03 5′AGTCAGCCAC-3′ 06 06 – 100 – 2000–500
OPA-07 5′-GA3CG3TG-3′ 04 04 – 100 – 1500–500
OPA-10 5′-GTGATCGCAG-3′ 03 03 – 100 – 1500–250
OPA-13 5′-CAGCAC3AC-3′ 02 02 – 100 – 2000–500
OPC-05 5′-GATGACCGCC-3′ 06 06 – 100 – 1500–250
OPC-07 5′-GTCCCGACGA-3′ 05 05 – 100 – 1000–250
OPC-08 5′-TGGACCGGTG-3′ 05 05 – 100 – 2000–250
OPD-08 5′-GTGTGCCCCA-3′ 06 06 – 100 – 2500–500
OPE-07 5′-AGATGCAGCC-3′ 06 06 – 100 – 2500–250
OPG-15 5′-ACTG3ACTC-3′ 03 03 – 100 – 1500–250
UBC-801 5′-(AT)8 T-3′ 09 09 – 100 – 1500–500
UBC-807 5′-(AG)8 T-3′ 03 03 – 100 – 2000–250
UBC-810 5′-(GA)8T-3′ 07 07 – 100 – 2000–500
UBC-814 5′-(CT)8A-3′ 05 05 – 100 – 2000–250
UBC-824 5′-(TC)8G-3′ 05 05 – 100 – 1500–250
UBC-827 5′-(AC)9G-3′ 04 04 – 100 – 1500–250
UBC-828 5′- (GT)8A-3′ 06 06 – 100 – 2000–250
UBC-830 5′-(TG)8G-3′ 07 07 – 100 – 2000–500
UBC-835 5′-(AG)8YC-3′ 06 06 – 100 – 2000–250
UBC-841 5′-(GA)8Y*C-3′ 04 04 – 100 – 2000–500
UBC-848 5′-(CA)7CR*G-3′ 05 05 – 100 – 1500–250
UBC-871 5′-(TAT)6-3′ 06 06 – 100 – 2000–250
S1 5′-KCCA-3′ 04 04 – 100 – 1500–750
S3 5′-KCCG-3′ 09 09 – 100 – 2000–500
S5 5′-KCGA-3′ 09 09 – 100 – 2000–500
S6 5′-KCGC-3′ 02 02 – 100 – 1000–750
S12 5′-LACG-3′ 08 08 – 100 – 2000–500
S17 5′-MGAG-3′ 07 07 – 100 – 1500–500
S25 5′- MGGG-3′ 04 04 – 100 – 1500–500
S26 5′-MGTC-3′ 07 07 – 100 – 2000–750
S30 5′-NGCG-3′ 08 08 – 100 – 2000–500
S33 5′-NCAG-3′ 07 07 – 100 – 2000–500
Total 185 185 – 100 – –
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K = CAACAATGGCTACCA; L = ACGCATGGCGACCA M = ACCATGGCTACCACC; N = CCATGGCTACCACCG

Table 5.

RAPD primers used, amplified fragment generated and the polymorphism detected in micropropagated D. fimbriatum grown in hormone enriched medium

RAPD primer Primer sequence (5′ → 3′) No. of scorable bands No. of bands Percentage of Band size (bp)
Monomorphic Polymorphic Monomorphism Polymorphism
OPA-01 5′-CAGGC3TTC-3′ 07 07 – 100 – 1500–500
OPA-03 5′AGTCAGCCAC-3′ 06 05 01 83.33 16.67 2000–500
OPA-07 5′-GA3CG3TG-3′ 04 04 – 100 – 1500–500
OPA-10 5′-GTGATCGCAG-3′ 03 03 – 100 – 1500–250
OPA-13 5′-CAGCAC3AC-3′ 02 02 – 100 – 2000–500
OPC-05 5′-GATGACCGCC-3′ 06 06 – 100 – 1500–250
OPC-07 5′-GTCCCGACGA-3′ 05 05 – 100 – 1000–250
OPC-08 5′-TGGACCGGTG-3′ 05 05 – 100 – 2000–250
OPD-08 5′-GTGTGCCCCA-3′ 06 06 – 100 – 2500–500
OPE-07 5′-AGATGCAGCC-3′ 06 06 – 100 – 2500–250
OPG-15 5′-ACTG3ACTC-3′ 03 03 – 100 – 1500–250
Total 53 52 01 98.48 1.52 –
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Fig.6.

Fig.6

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Banding profiles obtained from in vitro raised plants of Dendrobium fimbriatum. a RAPD: OPD-08, b ISSR: UBC-830, c SCoT-S5. L: 1 kb DNA ladder; MP—Mother Plant; P1–P9: In vitro regenerated plants. [P1 = 4.8 mg L−1 BAP; P2 = 0.8 mg L−1 BAP; P3 = 4.8 mg L−1 KN; P4 = 0.8 mg L−1 KN; P5 = 2.4 mg L−1 BAP + 1.2 mg L−1 IBA; P6 = 2..4 mg L−1 BAP + 3.6 mg L−1 IBA; P7 = 2.4 mg L−1 KN + 1.2 mg L−1 IBA; P8 = 2.4 mg L−1 KN + 3.6 mg L−1 IBA and P9 = 2.4 mg L−1 KN + 1.2 mg L−1 NAA]

Table 6.

ISSR primers used, amplified fragment generated and the polymorphism detected in micropropagated D. fimbriatum grown in hormone enriched medium

ISSR primer Primer sequence (5′ → 3′) No. of scorable bands No. of bands Percentage of Band size (bp)
Monomorphic Polymorphic Mono morphism Polymorphism
UBC-801 5′-(AT)8T-3′ 09 09 – 100 – 1500–500
UBC-807 5′-(AG)8T-3′ 03 03 – 100 – 2000–250
UBC-810 5′-(GA)8 T-3′ 07 06 01 85.71 14.29 2000–500
UBC-814 5′-(CT)8A-3′ 05 05 – 100 – 2000–250
UBC-824 5′-(TC)8G-3′ 05 05 – 100 – 1500–250
UBC-827 5′-(AC)9G-3′ 04 04 – 100 – 1500–250
UBC-828 5′- (GT)8A-3′ 06 06 – 100 – 2000–250
UBC-830 5′-(TG)8G-3′ 07 07 – 100 – 2000–500
UBC-835 5′-(AG)8YC-3′ 06 05 01 83.33 16.67 2000–250
UBC-841 5′-(GA)8Y*C-3′ 04 04 – 100 – 2000–500
UBC-848 5′-(CA)7CR*G-3′ 05 05 – 100 – 1500–250
UBC-871 5′-(TAT)6-3′ 06 06 – 100 – 2000–250
Total 67 65 02 97.42 2.58 –
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Table 7.

SCoT primers used, amplified fragment generated and the polymorphism detected in micropropagated D. fimbriatum grown in hormone enriched medium

SCoT primer Primer sequence (5′ → 3′) No. of scorable bands No. of bands Percentage of Band size (bp)
Monomorphic Polymorphic Mono morphism Polymorphism
S1 5′-KCCA-3′ 04 03 01 75 25 1500–750
S3 5′-KCCG-3′ 09 09 – 100 – 2000–500
S5 5′-KCGA-3′ 09 09 – 100 – 2000–500
S6 5′-KCGC-3′ 02 02 – 100 – 1000–750
S12 5′-LACG-3′ 08 08 – 100 – 2000–500
S17 5′-MGAG-3′ 07 06 01 85.71 14.29 1500–500
S25 5′-MGGG-3′ 04 04 – 100 – 1500–500
S26 5′-MGTC-3′ 07 07 – 100 – 2000–750
S30 5′-NGCG-3′ 08 08 – 100 – 2000–500
S33 5′-NCAG-3′ 07 07 – 100 – 2000–500
Total 65 63 02 96.07 3.93 –
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K = CAACAATGGCTACCA; L = ACGCATGGCGACCA M = ACCATGGCTACCACC; N = CCATGGCTACCACCG

Genetic distance and cluster analysis

A genetic distance matrix developed from the pooled RAPD-ISSR-SCoT data showed Nei’s genetic distance values of 0.000, 0.011 and 0.023, and Nei’s genetic identity values of 0.977, 0.989 and 1.000 indicating high genetic identity among the regenerants and the mother plant. While in vitro regenerated P1 differed from P2, P3, P4, P5, P6, P7, P8, P9 and MP by an identity matrix value of 0.977, the genotypes of P2 to P9 differed from MP by a close Nei’s genetic distance matrix value of 0.989 each. The study also revealed the total genetic homogeneity (0.000 or 1.000) between the genotypes of P2, P3, P4, P5, P6, P7, P8 and P9 (Table 8). The observed genetic homogeneity among the in vitro regenerants and the mother plant was also reported in earlier studies of Nepenthes khasiana and D. nobile (Devi et al. 2013; Bhattacharyya et al. 2014).

Table 8.

Genetic relationship matrices between mother plant (MP) and in vitro regenerants (P1–P9) of Dendrobium fimbriatum based from pooled data of RAPD-ISSR-SCoT analysis

MP P1 P2 P3 P4 P5 P6 P7 P8 P9
**** 0.977 0.989 0.989 0.989 0.989 0.989 0.989 0.989 0.989 MP
0.023 **** 0.977 0.977 0.977 0.977 0.977 0.977 0.977 0.977 P1
0.011 0.023 **** 1.000 1.000 1.000 1.000 1.000 1.000 1.000 P2
0.011 0.023 0.000 **** 1.000 1.000 1.000 1.000 1.000 1.000 P3
0.011 0.023 0.000 0.000 **** 1.000 1.000 1.000 1.000 1.000 P4
0.011 0.023 0.000 0.000 0.000 **** 1.000 1.000 1.000 1.000 P5
0.011 0.023 0.000 0.000 0.000 0.000 **** 1.000 1.000 1.000 P6
0.011 0.023 0.000 0.000 0.000 0.000 0.000 **** 1.000 1.000 P7
0.011 0.023 0.000 0.000 0.000 0.000 0.000 0.000 **** 1.000 P8
0.011 0.023 0.000 0.000 0.000 0.000 0.000 0.000 0.000 **** P9
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Above diagonal represents Nei’s genetic identity and below diagonal represents Nei’s genetic distance

Nei’s genetic distance based UPGMA dendrogram established between the in vitro regenerants and the mother plant revealed the presence of two clusters each for RAPD, ISSR and SCoT markers (Fig. 7a–c). In RAPD based dendrogram, all the in vitro regenerated plants were grouped in one major cluster while the MP existed separately. The P1 however, remained isolated in a subcluster from the rest of the genotypes in the main cluster. In ISSR based dendrogram, the single major cluster had MP and P1 positioning at separate subclusters, while P2, P3, P4, P5, P6, P7, P8 and P9 were clubbed together in a group (Fig. 7b). In the dendrogram analysis of SCoT, the in vitro regenerants P2, P3, P4, P5, P6, P7, P8, P9 and MP were clustered together while P1 genotype was positioned alone in a separate cluster (Fig. 7c). Dendrogram obtained from the pooled RAPD-ISSR-SCoT data showed two separate clusters. In one major cluster, the in vitro regenerants (P2, P3, P4, P5, P6, P7, P8, P9) and MP were grouped together while P1 alone was segregated in a minor cluster indicating its genetic divergence from the rest of the genotypes (Fig. 7d). P1 which was raised in M + 4.8 mg L−1 BAP showed the highest genetic variation from the MP as compared to other plants developed in medium with different hormone combinations. As other culture conditions were uniformly maintained for all the clones except for the differences in the hormonal content and composition in the medium, the depiction of genetically variant P1 and existence of low polymorphism could be ascribed due to the stress effect of high BAP concentration (4.8 mg L−1). Cytokinin at higher concentration was known to enhance somaclonal variation in in vitro propagated plants by inducing adventitious shoot multiplication and increasing the polyploidy cell frequency (Zakhlenyuk and Kunakh 1987; Damasco et al. 1996). Smith (1988) also reported the failure in the production of true-to-type plants due to the occurrence of various chromosomal abnormalities under high growth hormone concentration conditions.

Fig.7.

Fig.7

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UPGMA dendrogram of a RAPD, b ISSR, c SCoT and d Pooled RAPD-ISSR-SCoT showing the genetic relationship between mother plant (MP) and selected in vitro regenerants (P1–P9) of D. fimbriatum

Two-dimensional principal coordinate analysis (PCoA) of pooled data sets of RAPD, ISSR and SCoT markers revealed the spatial distribution of MP and P1 in the first and fourth quadrants, respectively, while P2 to P9 were nested together in the third quadrant (Fig. 8). The grouping pattern of the mother plant and the regenerants substantially corresponded with the clustering pattern generated by UPGMA dendrograms.

Fig.8.

Fig.8

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PCoA plot depicting the distribution of the mother plant (MP) and nine in vitro raised plants of D. fimbriatum (P1–P9) obtained from pooled RAPD-ISSR-SCoT data

Mantel test was conducted to perform correlation studies between RAPD, ISSR and SCoT markers. The test between RAPD and ISSR revealed a coefficient of correlation (r) at 0.96 (p = 0.02), which indicated a strong correlation between the two markers (Fig. 9a). Also, positive correlations were observed between the genetic matrices of RAPD and SCoT markers (r = 0.67, p = 0.04) (Fig. 9b) and ISSR and SCoT markers (r = 0.64, p = 0.03) (Fig. 9c). The observation of close correspondence between RAPD-ISSR (r = 0.96, p = 0.02) as compared to RAPD-SCoT and ISSR-SCoT markers indicated functional similarity between the RAPD and ISSR markers which targeted mostly the non-coding regions in the genome (Wolfe and Liston 1998). But the SCoT markers which recognize the conserve region flanking the start codon (ATG) of a functional gene are more reliable and reproducible than the arbitrary markers like RAPD and ISSR in determining the genetic diversity and identification of genotypes (Collard and Mackill 2009; Etminan et al. 2016). Similar observation of positive correlation between the genetic matrices of RAPD, ISSR and SCoT markers was earlier reported (Amom et al. 2020). Positive correlations were observed between the genetic matrices of RAPD, ISSR and SCoT with pooled RAPD-ISSR-SCoT matrix, suggesting the effectiveness of RAPD, ISSR and SCoT markers in determining genetic polymorphism and somaclonal variation, albeit very low between the in vitro regenerants and the mother plant (Fig. 9d–f).

Fig.9.

Fig.9

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Mantel test showing the correlation between a RAPD and ISSR, b RAPD and SCoT, c ISSR and SCoT, d RAPD and pooled RAPD-ISSR-SCoT, e ISSR and pooled RAPD-ISSR-SCoT and f SCoT and pooled RAPD-ISSR-SCoT markers

Conclusion

The present study reported a reliable and efficient regeneration protocol for in vitro propagation of genetically stable D. fimbriatum orchids which can be exploited for commercial and conservational proposes. Also, the present investigation provided a strong evidence of maintaining high genetic stability amongst the in vitro propagated D. fimbriatum, and molecular detection of low genetic variation due to stress caused by high hormone concentration. To our knowledge this is the first report of molecular assessment of genetic stability of the micropropagated D. fimbriatum orchids using RAPD, ISSR and SCoT markers.

Author contributions

The experiment is conceived and designed by LT and PN. Funding acquisition: PN; Experimentation: LT; Materials and result analysis: LT, TRD and AMP; Writing initial version of the manuscript: LT; Reviewing and editing: AD, MRS and PN; Supervision: PN. All authors have read and agreed to the published version of the manuscript.

Funding

The research work is funded by Department of Science and Technology (DST), New Delhi, India.

Availability of data and material

Not applicable.

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Footnotes

Publisher's Note

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Contributor Information

Leimapokpam Tikendra, Email: tikenleimapokpam@gmail.com.

Angamba Meetei Potshangbam, Email: angambameetei@gmail.com.

Abhijit Dey, Email: abhijitbio25@yahoo.com.

Tongbram Roshini Devi, Email: roshnidevibt@gmail.com.

Manas Ranjan Sahoo, Email: manas.sahoo@icar.gov.in.

Potshangbam Nongdam, Email: nongpuren@gmail.com.

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