Effect of phytohormones on rapid In vitro propagation of dendrobium thyrsiflorum Rchb.f.: An endangered medicinal orchid
Leimapokpam Tikendra, Thoungamba Amom, Potshangbam Nongdam
Department of Biotechnology, Manipur University, Imphal, Manipur, India
|Date of Submission||18-Apr-2018|
|Date of Acceptance||08-May-2018|
|Date of Web Publication||21-Nov-2018|
Department of Biotechnology, Manipur University, Canchipur, Imphal - 795 003, Manipur
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Dendrobium thyrsiflorum is highly vulnerable to extinction in its natural habitats due to overexploitation for the medicinal and horticultural purposes. The poor seed germination in a natural condition and slow plant production through vegetative approach make in vitro micropropagation techniques an ideal choice for effective plant propagation. There are very limited reports on the in vitro culture of this orchid. Objective: The objective of the study is to establish efficient in vitro regeneration protocols for rapid mass propagation of D. thyrsiflorum. Materials and Methods: Five-month-old undehisced capsule was surface sterilized with 0.4% mercuric chloride. To study the influence of growth regulators on in vitro development of shoots and roots, the seeds obtained from the capsule were inoculated on freshly prepared Mitra medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole -3-butyric acid (IBA), indole-3-acetic acid (IAA), and 1-naphthyl acetic acid (NAA). Results: The KN either singly or with IAA produced more effective shooting than BAP at similar concentration and combination. Maximum shoot production (3.83 ± 0.48) was achieved in medium fortified with 1.0 mg/L KN and 2.5 mg/L IAA. IAA was the most influential among auxins tested in promoting rooting and shooting. Prominent shoot development (3.04 ± 0.73) and maximum root formation (6.52 ± 0.37) were noticed in medium supplemented with 2.0 mg/L IAA. The regenerated plantlets were subsequently hardened with 91% survival rate in greenhouse and field condition. Conclusion: The present investigation revealed the possibility of developing effective and reproducible in vitro regeneration protocols for rapid propagation of D. thyrsiflorum. The established in vitro protocols can be employed for fast regeneration of the orchid for useful phytochemical extraction without depending on the already depleted natural population.
Abbreviations used: BAP: 6-benzylaminopurine; KN: Kinetin; IBA: Indole-3-butyric acid; IAA: Indole-3-acetic acid; NAA: 1-naphthyl acetic acid; MT: Mitra medium.
Keywords: Auxin, cytokinin, dhanzyme gold, propagation, protocorm
|How to cite this article:|
Tikendra L, Amom T, Nongdam P. Effect of phytohormones on rapid In vitro propagation of dendrobium thyrsiflorum Rchb.f.: An endangered medicinal orchid. Phcog Mag 2018;14:495-500
|How to cite this URL:|
Tikendra L, Amom T, Nongdam P. Effect of phytohormones on rapid In vitro propagation of dendrobium thyrsiflorum Rchb.f.: An endangered medicinal orchid. Phcog Mag [serial online] 2018 [cited 2021 Oct 21];14:495-500. Available from: http://www.phcog.com/text.asp?2018/14/58/495/245842
Kinetin was found to more effective in inducing shooting than 6-benzylaminopurine.
- Maximum rooting response was produced in Mitra medium supplemented with 2.0 mg/L indole-3-acetic acid (IAA). IAA proved to more efficient in producing rooting as compared to indole-3-butyric acid and NAA
- The in vitro regenerated Dendrobium thyrsiflorum plantlets were successfully hardened with 91% survival rate in greenhouse and field condition.
| Introduction|| |
Dendrobium thyrsiflorum, an endangered orchid with sympodial growth habit, is found in the deciduous forest of South China, Thailand, Myanmar, Laos, and Vietnam. In India, it is confined to the Northeastern region and Western Ghats of Southern part of India. While it exhibits a graceful floricultural appeal, the species has immense medicinal applications in the treatment of various chronic disorders and as a commercial dye. D. thyrsiflorum is one of the major constituents of Huangcao Shihu, a predominant form of the most precious class of traditional Chinese medicine, Herba Dendrobii. It contains polysaccharides which act as an immunomodulator. There are reports of the presence of scoparone and coumarins in D. thyrsiflorum which help in relaxing smooth muscles and expanding blood vessels and act as anticoagulant of blood., Endophytic fungi obtained from this orchid species are reported to possess potent antimicrobial activity. Fusarium, the predominant genus in antimicrobials, had shown to exhibit inhibitory effect on various bacteria and fungi. The endophytic Epicoccum nigrum also proved to have antibacterial activity against Staphylococcus aureus and Bacillus subtilis, apart from providing inhibitory effects on the growth of other Gram-positive and Gram-negative bacteria with higher potency than that of ampicillin sodium.,
There is a sharp decline in the population of D. thyrsiflorum due to habitat destruction, widespread indiscriminate collection, and overexploitation because of its extensive applications in the field of medicine and horticulture. The orchid is presently listed in Red Data Book of International Union for Conservation of Nature. Furthermore, the whole of Orchidaceae family is included in Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. Devising an effective conservation strategy is highly essential to not only save the endangered orchid from the brink of extinction but also harness its wide range of economic potentials. As typical vegetative propagation methods are time-consuming and commercially not viable, in vitro conservation through plant tissue culture can provide an excellent opportunity for large-scale propagation of orchid without sacrificing rare mother plants. This approach will also overcome the limitation of low seed germination of minute and nonendospermous seeds in nature due to the requirement of mycorrhizal fungi association. Mass propagation through the adoption of micropropagation techniques could minimize the extreme collection pressure imposed on natural populations of D. thyrsiflorum. The reports on in vitro propagation of D. thyrsiflorum are scanty, and in vitro culture approach has not been fully exploited for its conservation. The study was conducted with an aim to mass propagate this endangered medicinal orchid by establishing effective, reliable, and reproducible in vitro regeneration protocols.
| Materials and Methods|| |
Plant materials and explant sterilization
The mature undehisced capsules of about 5months old were collected from the naturally pollinated D. thyrsiflorum orchids grown in the Khonghampat Orchidarium, Manipur, India. The capsules were stored at 4°C until they were used for initiating culture after proper surface sterilization. The sterilization was performed by first washing the capsule thoroughly with running tap water for 5–10 min to remove any dust and soil particles adhered to it. The capsule was subjected to the treatment with 0.4% mercuric chloride for 6–7 min after being washed with 10% Tween 20 for 10 min. The capsule after washing 4–5 times with sterile ultrapure water was flamed sterilized for 3–5 s by dipping in absolute alcohol for 2–3 s.
Culture medium preparation and seed inoculation
The surface sterilized capsule was split opened longitudinally to expose the numerous minute seeds using sterile spatula inside the ultraviolet preilluminated laminar hood. The light exalbuminous seeds obtained from the capsule were inoculated using sterile forceps into sterilized culture tubes (Borosil, 32 mm × 200 mm) containing 40 ml of basal Mitra medium (MT). The medium was supplemented with 3% sucrose (HiMedia, Mumbai) as carbohydrate source and volume makeup were done with double distilled water and the pH adjusted between 5.5 and 5.8 using 1N NaOH and HCl. The medium was incorporated with 0.9% agar (HiMedia, Mumbai) as gelling agent. The boiled media were dispensed in culture tubes, with or without plant growth regulators, and made airtight using aluminum foil. The media-laden culture tubes were finally autoclaved at 121°C for 15 min and were finally allowed to gel in the slanting position. All cultures, after the inoculation of seeds, were maintained at 25°C ± 2°C with proper light illumination at 60 μmol/m2/s for 16/8 h a day using white fluorescent tubes.
Plant growth regulators and transplantation of germinated seeds
To study the influence of growth regulators on in vitro culture development, 6-benzylaminopurine (BAP), kinetin (KN), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and 1-naphthyl acetic acid (NAA) were supplemented either individually or in combinations at different concentrations. The culture vessels were made airtight using aluminum foil after dispensation of definite quantities of growth hormone-laden culture media. The media with different hormonal combinations were finally autoclaved at 121°C for 15 min and were finally allowed to gel in the slanting position. The cultures after inoculation of Dendrobium orchid seeds were maintained at 25°C ± 2°C with proper light illumination at 60 μmol/m/s for 16/8 h a day using white fluorescent tubes.
Hardening of in vitro-raised plantlets
The in vitro-raised seedlings with well-developed shoots and roots were transferred to full strength culture MT devoid of any plant growth regulators for 3–4 weeks. The plantlets were further transferred to half strength MT medium without any sucrose and vitamins and kept for another 3–4 weeks before they were taken out from the culture vessels using sterile forceps. The plantlets were washed with tepid water to remove agar and treated with 0.01% fungicide solution (Sixer, Dhanuka Agritech Ltd., Gujarat, India) for 10–15 min to get rid of fungal contaminants in the seedlings, if any. The plants were then transplanted to perforated small plastic pots containing brick and charcoal pieces along with coconut husk (1:1:1) as potting mixture. The transplanted plants were sprayed with liquid MT medium incorporated with 1 ml/L dhanzyme gold, an organic extract from seaweed (Dhanuka Agritech Limited, Gujarat, India). The potted plantlets were initially kept 3–4 weeks inside the glass house for further acclimatization and finally exposed to normal daylight in greenhouse.
Data recording and statistical analysis
The experiments were conducted thrice using ten replicates per treatment. The first subculture was performed in 10 weeks followed by consecutive subculture to freshly prepared medium for every 4 weeks. The culture responses with regard to the development of shoots and roots were recorded at the regular intervals. The data were subjected to analysis of variance, and the significant differences were determined by employing Duncan's multiple range test at P = 0.05. The statistical data analysis was performed using SPSS software program (SPSS Inc., Chicago, IL, USA).
| Results|| |
The seeds started swelling at 2–3 weeks of culture indicating successful initiation of seed germination [Figure 1]a. This was followed by rupturing of testa and formation of spherical-shaped chlorophyllous and protocorms in 5 and 7 weeks, respectively [Figure 1]b. Subsequent development after protocorm stage resulted in apical shoot meristem and leaf primordia formation [Figure 1]c and [Figure 1]d. Further growth led to successful in vitro generation of seedlings with leaves and roots.
|Figure 1: In vitro seed germination and protocorm-like bodies formation: swelling of seeds indicating initiation of seed germination (a); young chlorophyllus protocorm formation (b); development of shoot meristem (c); formation of shoot and leaf primordia (d)|
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Individual hormonal effect on culture growth
Multiple shoot induction was observed in either BAP or KN-supplemented medium [Figure 2]a. Elevating the concentration of BAP or KN from 0.5 mg/L to 2.0 mg/L gave more shoots and longer shoots length [Table 1]. However, KN at 2.0 mg/L was more effective in inducing multiple shooting (3.16 ± 0.47) [Figure 2]b as compared to BAP generating lesser shoot number (2.18 ± 0.54) at a similar concentration. Production of greater shoot number and longer shoots was also witnessed with increased concentration of individual auxins (IAA, IBA, and NAA) from 0.5 mg/L to 2.0 mg/L. The presence of 2.0 mg/L IAA in medium promoted vigorous root formation with maximum rooting response (6.52 ± 0.37) witnessed in culture [Figure 2]c. While favorable root formation was also evidenced with IBA supplemented medium, NAA seemed to be the least influential among the auxins in inducing rooting.
|Figure 2: In vitro development and propagation of Dendrobium thyrsiflorum: in vitro shooting after protocorm stage on medium fortified with cytokinin (a); induction of multiple shooting in medium enriched with 2.0 mgL-1 kinetin (b); proliferous root formation on medium augmented with 2.0 mg/L indole-3-acetic acid (c); hardening of in vitro propagated plants in potting mixture of brick pieces, charcoal pieces, and coconut husk (1:1:1) (d)|
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|Table 1: Effect of individual growth hormones on the shoot and root development at 30 weeks of culture|
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Combine effect of auxin and cytokinin on the culture development
The combined effects of cytokinin (BAP or KN) and auxins (IAA, IBA, or NAA) at a similar concentration (1.0 mg/L) were tested to determine best in vitro shoot and root development response [Table 2]. Maximum shoot number (3.49 ± 0.96), shoot length (2.45 ± 0.29 cm), root number (4.85 ± 0.75), and root length (2.81 ± 0.15 cm) were recorded in MT + 1.0 mg/L KN + 1.0 mg/L IAA. The shoot production was also prominent in MT + 1.0 mg/L KN + 1.0 mg/L IBA and MT + 1.0 mg/L KN + 1.0 mg/L NAA with the formation of 2.78 ± 0.92 and 2.44 ± 0.72 shoots, respectively. Cultures responded effectively to MT + 1.0 mg/L KN + 1.0 mg/L IBA and MT + 1.0 mg/L KN + 1.0 mg/L NAA for root formation yielding 2.59 ± 0.87 and 3.16 ± 0.24 roots, respectively [Table 2]. The combination of BAP and NAA exhibited least response to shoot (1.92 ± 0.85) and root (1.01 ± 0.48) formation, followed by the association of 1.0 mg/L BAP and 1.0 mg/L IBA producing 2.16 ± 0.84 shoots and 0.79 ± 0.32 roots, respectively. KN proved to be more effective than BAP in inducing multiple shoots and root formation when associated with IAA, IBA, or NAA at a similar concentration.
|Table 2: Combine effect of cytokinin and auxin on the shoot and root formation at 30 weeks of culture|
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Effect of addition of auxins on root and shoot growth
A parallel study was conducted after third passage (18 weeks of culture) to determine the effects of auxins on shoot and root development in seedlings by transferring ten seedlings each from the initial culture condition to freshly prepared media containing new hormone combinations. A comparative study was conducted on shoot and root development in seedlings grown in the initial plant growth hormone combinations and new combinations.
Effect of addition of indole-3-acetic acid to initial cytokinin-containing media
Incorporation of higher auxin concentration of 3.5 mg/L IAA and 4.0 mg/L IAA to initial 0.5 mg/L BAP and 2.0 mg/L BAP, respectively, produced less number of shoots and roots [Figure 3]. However, the new combination of MT + 0.5 mg/L KN + 3.5 mg/L IAA showed better multiplication of shoots (2.50 ± 0.54) and roots (2.82 ± 0.19) than initial medium-enriched only with 0.5 mg/L KN [Figure 3]. Addition of higher concentration of IAA (4.0 mg/L) to the earlier medium with 2.0 mg/L of KN generated higher shoot number (3.31 ± 0.81) though shoot length (1.34 ± 0.26 cm) and root formation (1.98 ± 0.73) was diminished prominently.
|Figure 3: Comparison of shoot and root after their transfer from cytokinin (6-benzylaminopurine or kinetin) containing medium to medium supplemented with both cytokinin and auxin. (→) indicates the transferring of seedlings from initial cytokinin only containing media to new combination with high auxin concentration|
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Effect of intensified auxin concentration
When the auxin concentration was increased from the initial 1.0 mg/L to 2.5 mg/L in new combinations, there was notable reduction in shoot formation though root development was slightly improved. While initial hormonal combinations of MT + 1.0 mg/L BAP + 1.0 mg/L IAA, MT + 1.0 mg/L BAP + 1.0 mg/L IBA, and MT + 1.0 mg/L BAP + 1.0 mg/L NAA recorded 2.18 ± 0.36, 2.16 ± 0.84, and 1.92 ± 0.85 number of shoots, respectively, the new combinations with higher auxin concentration of MT + 1.0 mg/L BAP + 2.5 mg/L IAA, MT + 1.0 mg/L BAP + 2.5 mg/L IBA, and MT + 1.0 mg/L BAP + 2.5 mg/L NAA produced reduced shoot number of 1.23 ± 0.51, 0.83 ± 0.75, and 0.78 ± 0.43, respectively [Table 3]. Except for 2.5 mg/L NAA which showed lesser roots (2.50 ± 1.04) with 1.0 mg/L KN, higher root development was witnessed when KN at 1.0 mg/L was associated with 2.5 mg/L of IAA or IBA. Elevated level auxins (IAA, IBA, and NAA) from 0.5 mg/L to 3.5 mg/L gave profound impact on in vitro shoot and root development of the culture. Improvement in shoot formation was evident in MT + 3.5 mg/L IAA, MT + 3.5 mg/L IBA, and MT + 3.5 mg/L NAA with the production of 2.66 ± 1.03, 2.08 ± 0.75, and 2.12 ± 0.41 shoots, respectively [Figure 4]. The rooting response was more intense in new culture conditions as compared to initial combinations with lower auxin concentration. Substantial increase in root formation was noticed in MT + 3.5 mg/L IAA, MT + 3.5 mg/L IBA, and MT + 3.5 mg/L NAA yielding 6.17 ± 0.14, 5.66 ± 2.06, and 4.86 ± 0.68 roots, respectively [Figure 4].
|Table 3: Effect of increased auxin concentration on shoot and root development at 30 weeks of culture|
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|Figure 4: Comparison of shoot and root of seedlings after their transfer from lower to higher auxin containing media at 30 weeks of cultures. (→) indicates the transfer of seedlings from initial low auxin containing media to media with higher auxin concentration|
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The well-grown seedlings were finally acclimatized in greenhouse condition using brick pieces, charcoal pieces, and coconut husk (1:1:1) as potting mixture with survival rate of 91% after 16 weeks of hardening [Figure 2]d.
| Discussion|| |
A symbiotic seed germination had been employed as suitable propagation technique to increase population size of many declining orchid species mainly due to their slow and low seed germination rate in nature.,, The numerous nonendospermous orchid seeds need specific nutrition under certain environmental conditions as they possess very limited food reserves. The medium comprising inorganic and organic constituents, additives, and exogenous plant hormones furnishes the essential nutrients for the germinating seeds under in vitro condition. The same was found true for exalbuminous seeds of D. thyrsiflorum when they were grown on nutrient medium. KN influenced in vitro shoot proliferation by inducing quick cell division to form numerous shoots in Dendrobium huoshanense. Similarly, incorporation of KN in medium enhanced shoot formation in D. thyrsiflorum indicating the significance of exogenous cytokinin in promoting multiple shooting. In the present investigation, maximum shoot formation was reported on medium enriched with 2 mg/L KN. The longest shoot length (3.36 ± 0.37 cm) was also recorded in the same hormone combination defying the general observation that shoot length was always shorter in culture with higher shoot numbers. This is because all the available nutrients might have been utilized in the formation of multiple shoots. Srivastava et al. earlier reported the formation of highest shoot number and longest root length in Aerides ringens when grown on KN-enriched medium. Asghar et al. also encountered longest shoot formation in Dendrobium nobile on medium fortified with 1.5 mg/L KN.
The present study showed BAP to be less effective as compared to KN in initiating in vitro shooting response. This result was in agreement with the earlier reports of inhibitory response of BAP on the shoot development in D. nobile, Dendrobium aqueum, and Doritis pulcherrima.,, The combination of BAP with IAA, IBA, or NAA at different concentrations proved less favorable in comparison to KN with similar auxin combinations. The more effectiveness of KN over BAP in producing better shoots and roots when combined with other auxins have also been previously demonstrated. The presence of IAA at 2.0 mg/L generated higher shooting response than cytokinins (BAP and KN) at different concentrations. Similar observations of higher shoot production under the influence of auxins were earlier reported in Dendrobium pendulum, Dendrobium primulinum, Dendrobium heterocarpum, D. aqueum, Dendrobium microbulbon, and Dendrobium chrysanthum.,,, The effect of auxin in promoting in vitro rooting in plant was well studied. The present research also revealed the promontory effect of auxins in rooting development in the culture. The maximum root formation was recorded in IAA-supplemented medium in 30 weeks. Similarly, the highest root formation in IAA augmented medium was earlier noted for D. primulinum and Dendrobium aggregatum., Increase in concentration of IAA from 0.5 mg/L to 3.5 mg/L after the third passage remarkably improved root formation. Enhancement in root development was also observed with a higher concentration of IBA at 2.0 mg/L (4.93 ± 0.82) and 3.5 mg/L (5.66 ± 2.06). Bhattacharyya et al. also recorded high root formation (5.5 ± 0.5) in D. thyrsiflorum when medium was fortified with 2.0 mg/L of IBA. Similar observation of higher root multiplication was encountered with increased auxin concentration in culture medium in A. ringens and Alcantarea odorata., Of the three auxins (IAA, IBA, and NAA) tested at different concentrations in the present study, NAA was found to be least effective in inducing rooting. Lesser influence of NAA in in vitro rooting induction was also observed in Cymbidium masterii. Root formation though usually occurs with most of the auxins in plants, IAA and IBA are especially the most effective in many orchids.,,, Incorporation of higher auxin (IAA) concentration into the initial cytokinin-containing medium did not prove favorable for shoot and root development as root and shoot number reduced prominently. Similarly, higher concentration of auxins in the presence of cytokinin exhibited poor shooting and rooting response in D. nobile.
The results from the present study demonstrated the effectiveness of KN and IAA in promoting in vitro shoot and root development in culture of D. thyrsiflorum. KN proved to be more efficient than BAP in inducing shooting while IAA was the most influential among the auxins tested for vigorous root formation. The rapid plant propagation with high survival rate posthardening process revealed the prospect of utilizing the established in vitro protocols for large-scale production of plant materials. The in vitro propagated medicinal orchid may be utilized for the preparation of phytomedicines and floricultural-related applications without further exploiting its fast diminishing natural populations.
| Conclusion|| |
The present investigation revealed the possibility of developing effective and reproducible in vitro regeneration protocols for rapid propagation of D. thyrsiflorum. The established in vitro protocols can be employed for fast regeneration of the orchid for useful phytochemical extraction without depending on the already depleted natural population.
Financial support and sponsorship
The authors are thankful to University Grant Commission, New Delhi, India, for the financial support.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]