Call: +91-9177734525 | Email: info@opensciencepublications.com

Journal of Plant Science and Research

Research Article


Asymbiotic In vitro Seed Germination andRegeneration of Vanda coerulea Giff. Ex. Lindl., an Endangered Orchid from Northeast India

Lalremsiami Hrahsel and Robert Thangjam*

Corresponding author: Robert Thangjam, Department of Biotechnology, School of Life Sciences, Mizoram University,Aizawl-796004, Mizoram, India. Tel: +919436352386.; E-mail: robertthangjam@gmail.com


Department of Biotechnology, School of Life Sciences, Mizoram University, Aizawl-796004, Mizoram, India


Citation: Hrahsel L, Thangjam R. Asymbiotic In vitro Seed Germination and Regeneration of Vanda coerulea Giff. Ex. Lindl., an Endangered Orchid from Northeast India. J Plant Sci Res. 2015;2(2): 137.


Copyright © 2015 Hrahsel L, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Journal of Plant Science & Research | ISSN: 2349-2805 | Volume: 2, Issue: 2


Submission: 03/09/2015; Accepted: 05/10/2015; Published: 12/10/2015


Reviewed & Approved by: Dr. Gaurab Gangopadhyay, AssociateProfessor, Division of Plant Biology, Bose Institute, India


Abstract


Blue Vanda (Vanda coerulea Giff. ex. Lindl.) is one of the most beautiful orchids found in the northeastern region of India. Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) listed this species in the appendix 1 as a vulnerable one. In the present study asymbiotic in vitro seed germination and regeneration of V. coerulea via protocorm formation was achieved in MS basal medium. Immature seeds cultured on full strength MS basal medium showed higher rate of germination with subsequent formation of ‘protocorm like bodies’ (PLB) in comparison to half strength MS basal medium. Multiplication of the protocorms and differentiation into shoots was attained in MS basal medium supplemented with combination of benzylamino purine (BA) and kinetin (Kn). Highest number of leaf emergence was observed in MS basal medium supplemented with 22.80 μM indole acetic acid (IAA). Supplementation of basal medium with banana extract (75 mg L-1) resulted in highest number of root induction. The standardized protocol for efficient in vitro germination of immature V. coerulea seeds and their subsequent regeneration can be applied for future mass propagation and ex situ conservation of this endangered orchid.


Keywords: Vanda coerulea; In vitro seed germination; Protocorm-like bodies; Regeneration

Introduction


Orchids are famous for their beautiful range of flowers, not only for their aesthetic values but also for pharmaceutical importance.Orchid seeds are dispersed by wind far and wide and if fall intonew environmental condition, they either die or adapt to the newenvironment [1]. Some members of orchids have also been used fortheir therapeutic value owing to their phytochemical constituentssuch as alkaloids, flavanoids, terpenes and glycosides [2]. The orchidseeds are minute and produced in million per capsule but lacksmetabolic machinery and functional endosperm and thus only 0.2 -0.3% of the seeds germinate in nature [3].


Vanda coerulea Giff. ex Lindl., commonly known as Blue Vandaor Autumn lady’s tresses orchid, is a remarkably beautiful plant having large pale to deep liliac-blue flowers (Figure 1A). It produces an erectstem 2 to 3 feet high with the usual distichous loriform channeledcoriaceous leaves, which are unequally truncate with a concave notchand acute lateral lobes. The erect scapes are longer than the leavesand bear dense racemes of 10-15 flowers. The flowers are producedduring September to November, last for 2 to 3 months and showschange in coloration from light blue to darker shades of blue. Thisspecies is distributed in northeast India, China, Burma and Thailand[4]. Due to its high commercial value in the local and internationalflower market it is harvested indiscriminately. The reckless collectionand ever increasing habitat destruction and global warming haveposed direct threats to its existence. V. coerulea has been listed underAppendix II (112/01/05) of CITES [5] and categorized as vulnerable[6]. It has been listed as an endangered species of Red Data Book on Indian Orchidaceae-1 [7] and threatened by International Unionfor Conservation of Nature and Natural Resource [8]. There arenumbers of selected clones of V. coerulea which are urgently neededto conserve for future breeding program [9].


Figure 1: In vitro seed germination and regeneration of Vanda coerulea Giff.ex. Lindl. - A) V. coerulea in natural habitat. B) Immature capsule. C) Invitro germination of seeds in full strength MS basal media. D) Protocormlikebodies (PLBs) formation in full strength MS basal medium. E) Shootinduction in full strength MS basal medium. F) Induction of leaf formation infull MS basal medium supplemented with combination of 4.44 μM BA + 4.70μM Kn. G) Regenerated plantlet with fully formed leaves in full MS basalmedium supplemented with 22.8 μM IAA. H) Regenerated plants ready foracclimatization.


Plant tissue culture technology helps to propagate plants ofeconomic importance such as orchids and other ornamental plantsin large numbers through meristem culture or by other in vitromethods. The earliest report of orchid tissue culture dates back to1949 when it was demonstrated that Phalaenopsis plantlets coulddevelop from the buds of inflorescence stalks [10]. However thecredit of achieving mass clonal propagation of orchids goes to Morelwho in 1960 [11] was successful in culturing Cymbidium shoot apiceson Knudson C agar medium. Further successes in other speciesthrough shoot tip culture are reported in Aranda [12], Aranthera[13], Rhynchostylis [14], Dendrobium [15,16] and Vanda [12]. The invitro technique of asymbiotic seed germination could be useful forconservation and reintroduction of V. coerulea like other orchids[17,18]. Knudson in 1946 [19] revolutionized the technique of invitro asymbiotic seed germination for orchid cultivation. Variousexplants have been used for the in vitro regeneration of plantlets [20].However, shoot tip based micro propagation system, which is themain mode of propagation for sympodial orchids, is not optimal forthis monopodial species because only a few axillary buds develops atthe lower half of the plant and the continual growth from the apicalmeristem inhibits development of axillary buds into offshoots, and hence resulting in low rate of vegetative multiplication under naturalconditions [22]. The removal of meristem tips also greatly endangersthe survival of the mother plant [20,21]. Thus the present study of invitro regeneration of V. coerulea found in Mizoram, northeast Indiawas undertaken using asymbiotic seed germination techniques.


Materials and Methods


Plant material


Immature green pods of V. coerulea (Figure 1B) were collectedfrom Mission Veng, Aizawl, India during February and kept at 4 °Cuntil use.


In vitro germination


The immature green pods were removed and washed thoroughlyusing few drops of detergent (labolene) in running tap water for 10-15 min. The pods were surface sterilized by immersion in 1% Bavistin(Carbendazim 50% w/w) for 10-15 min and rinsed with runningtap water for 15 min. They were then brought to the Laminar AirFlow Chamber and treated with 0.1% Mercuric chloride for 15 min,70% ethanol (v/v) for 10 minutes and rinsed thoroughly with steriledistilled water 4-5 times. They were then dried with a sterile blottingpaper and cut longitudinally into two halves using a sterile blade. Theimmature seeds together with cotton fibres in between were scoopedout into sterile petriplates. After careful separation of the immatureseeds from the fibres with the help of a pair of sterile forceps, theimmature seeds were transferred to petriplates containing variablestrength of MS basal medium [23]. All the cultures were maintained at25±1 °C continuous dark and 60-70% relative humidity. Observationswere recorded every week for the production of protocorm-likebodies (PLBs) as a successful sign of in vitro germination.


In vitro regeneration, rooting and acclimatization


Protocorms which have developed from the immature seedscultured on half and full strength MS medium were collected and thentransferred to fresh basal MS medium supplemented with variousconcentrations of plant growth regulators (PGRs) viz., 6-Benzylaminopurine (BA) and Kinetin (Kn) either singly or in combinations. Thevarious concentrations of BA and Kn used are BA (4.44 μM), BAP(8.88 μM), Kn (4.70 μM), Kn (9.40 μM), BA (4.44 μM) + Kn (4.70μM), BA (4.44 μM) + Kn (9.40 μM) and BA (8.88 μM) + Kn (4.70μM). Similarly, PLBs were also cultured on half and full strength basalMS media without PGRs as control. The PLBs which were culturedon half MS and full strength MS basal media showing multiplicationof protocorms and formation of shoots were transferred intodifferent rooting media containing MS basal medium supplementedwith indole acetic acid (IAA) and banana extract (75 mg L-1). Theconcentration of IAA added in the MS basal medium varies from 5.70μM to 28.5 μM. The pH of the medium was adjusted to 5.8 priorto autoclaving at 15 psi and 121 °C for 20 min and was maintainedat 25±2 °C under a 16 h photoperiod with a photosynthetic photonflux density of 35 μmol/m2/s provided by cool white fluorescenttubes (Philips, India). They were sub-cultured every 2 weeks into afresh media containing the same composition. For each treatment 3replicates were taken. The individual and combined effects of thesePGRs were analyzed through parameters such as the multiplicationof protocorms and formation of shoots and roots. All the chemicals used in this study were of HiMedia, Mumbai, India. The regeneratedplantlets with well-developed roots were removed from the culturemedium, washed gently under running tap water and transferred toplastic pots containing charcoal chips, brick pieces and sphagnummosses. The pots containing the plantlets were then covered withtransparent polyethylene bags to maintain adequate moisture for aweek and transferred to the greenhouse (temperature: 25± 2 °C, lightintensity: 4000 lux; photoperiod: 14-h; humidity: 70%). After 4 weeks,the plastic covering was removed and the plantlets were maintainedin the greenhouse in plastic pots containing normal garden soil untilthey were transplanted to the nursery.


Statistical analysis


All the experiments had three replicates per treatment and wererepeated at least twice. The differences in means were contrastedusing Duncan’s new multiple range test following ANOVA. Allstatistical analysis was carried out using SPSS statistical softwarepackage version 16.0.


Results


In vitro seed germination and PLB formation


In vitro seed germination and their subsequent regeneration of V.coerulea cultured on half and full strength MS media showed variableresponse. Seeds cultured on half strength MS basal medium showedfirst response i.e. swelling of immature seeds by the 4th week whereas inthe full strength MS medium the initial response was observed by the7th week, which was followed by pigment synthesis (Figure 1C). Thecolour change observed in the seeds were observed as follows: creamyyellow within 9th week in the half strength MS medium whereas in fullstrength MS medium it was observed within 6th week then greenishyellow within the 12th week in the half strength MS medium and 7thweek in the full strength MS medium. The formation of protocormlikebodies (PLBs) was observed in the 14th week in half strength MSmedium whereas it was observed in the 12th week in full strengthMS medium. PLBs are outgrowths that arise from seeds which act astransitory bodies leading to in vitro regeneration of orchids (Figure 1D). Thus, the immature seeds cultured in full MS medium showedhigher response than those cultured in half strength MS medium.This showed that the seedling development of V. coerulea was betteron full MS medium.


The in vitro germinated seeds of V. coerulea on subsequenttransfer to half and full strength MS medium supplemented with various concentrations of BA and Kn either singly or in combinationsshowed variable response. The half MS derived PLBs showed theirfirst response i.e. multiplication of PLBs within the 2nd week in mediacontaining 4.44 μM BA, 4.70 μM Kn, 9.40 μM Kn, 4.44 μM BA + 4.70μM Kn, 4.44 μM BA + 9.40 μM Kn and 8.88 μM BA + 4.70 μM Kn.In the control (full strength MS without PGRs) the same observationwas recorded by the 4th week in control. On the other hand the fullstrength MS media derived PLBs showed the first response in the 2ndweek in all the media composition.


Shoot induction and leaf formation


The half MS derived PLBs showed shoot induction in 3 weeksin the media containing 4.44 μM BA, 4.44 μM BAP + 4.70 μM Knand 8.88 μM BA + 4.70 μM Kn whereas it took 4 weeks in mediacontaining 9.4 μM Kn, 4.44 μM BA + 9.40 μM Kn. Shoot inductionwas not observed in the control media as well as those supplementedwith 8.88 μM BA and 4.70 μM Kn. While the full strength MS derivedPLBs showed shoot induction by 3rd week in control as well in themedia supplemented with 4.44 μM BA, 4.70 μM Kn, 9.40 μM Kn, 4.44μM BA + 4.70 μM Kn, 4.44 μM BA + 9.40 μM Kn, 8.88 μM BA +4.70 μM Kn. But in the 9.40 μM Kn and 4.44 μM BA + 9.40 μM Knsupplemented MS media it was observed by the 4th week. No shootinduction was observed in the media supplemented with 8.88 μMBA only. Leaf formation for the half MS medium derived PLBs wasobserved in 4 weeks only in one media combination i.e. MS mediacontaining combination of 8.88 μM BA + 4.7 μM Kn. Whereas theleaf formation for the full strength MS medium was observed in 4weeks in the control as well as in the media supplemented with 4.44μM BA + 9.40 μM Kn and 3 weeks in the media containing 4.44 μMBA + 4.70 μM Kn. For the development and multiplication of leaves,the shoots were sub-cultured in a fresh media containing the samecomposition. Details of the shoot induction and leaf formation areprovided in Table 1 and Figure 1E, 1F, 1G & 1H.


Table 1: Effect of half and full strength MS media supplemented with indole acetic acid (IAA) and banana extract on leaf and root multiplication V. coerulea.


Leaf multiplication, rooting and hardening of regeneratedplantlets


The regenerated shoots which resulted in the formation of leaveswere sub-cultured to different rooting media containing differentconcentrations of Indole Acetic Acid (IAA) and banana extract(75 mg L-1). After 4 weeks of culture the multiplication of leavesand induction of roots started (Table 1). The total number of leafformation observed after 8 weeks of culture was found to be variablein the different media compositions. In the half strength MS media the mean number of leaves ranged from 2.66 (control) to 5.33 (22.8μM IAA) while in the full strength MS media, it ranged from 3.33(control) to 7.33 (22.8 μM IAA). The supplementation of bananaextracts to MS media containing IAA did not show any incrementaleffect to the multiplication of leaves as evident by the lesser numberof leaves recorded after 8 weeks of culture. The mean number of rootformation observed after 8 weeks of culture in half and full strengthMS basal media containing banana extract (75 mg L-1) was 12.33 and14.33 respectively (Table 1).


The fully rooted plantlets with at least 3 roots were taken outfrom the culture tubes, washed thoroughly to remove any remains of medium, and planted in small plastic pots containing charcoal chips,brick pieces and sphagnum mosses. The pots containing the plantletswere then covered with transparent polyethylene bags to maintainadequate moisture for a week and transferred to the greenhouse(temperature: 25± 2 °C, light intensity: 4000 lux; photoperiod: 14-h; humidity: 70%). After 4 weeks, the plastic covering was removedand the plantlets were maintained in the greenhouse in plastic potscontaining normal garden soil until they were transplanted to thenursery. About 80% of the hardened plants survived in the nursery.The overall protocol standardized for the asymbiotic in vitro seedgermination and its subsequent regeneration of V. coerulea in thepresent study are described in Figure 2.


Figure 2: Schematic flow chart describing the protocol for in vitro seed germination and regeneration of Vanda coerulea Giff. ex. Lindl.


Discussion


The present investigation indicated that in vitro germinationof V. coerulea and their subsequent regeneration can be achievedsuccessfully with minimum use of plant growth regulators. PLBformation was more suitable with full strength MS basal mediawithout growth regulators. However, the multiplication of the PLBsand differentiation into shoots and leaves was favourable in basalMS supplemented with combination of BA and Kn in comparisonto either BA or Kn alone. Highest number of leaf multiplicationwas observed in MS basal media supplemented with 22.80 μM IAAwhile supplementation with banana extract (75 mg L-1) showed thehighest number of root induction. The present standardized protocolfor efficient in vitro germination and subsequent regenerationof immature V. coerulea seeds can be applied for future massmultiplication and conservation of this endangered orchid.


Acknowledgements


The authors gratefully acknowledge the University GrantsCommission, Government of India, New Delhi, for funding theproject (MRP-MAJ-BIOT-2013-921) which enable establishment ofthe facility utilized for the works carried out.


References

  1. Linthoingambi L, Ghosh SK, Das AK, Singh PK (2014) Conservation of orchids in Imphal valley, Manipur. J Energy Rese Environ Technol 1: 11- 13.
  2. Devi J (1999) Conservarion strategy for orchids of India. Everyday Sc 34: 12-14.
  3. Singh F (1992) In vitro propagation of orchids ‘state of the art’. J Orchid Soc India 6: 11-14.
  4. Williams B (1984) Orchids for everyone. A practical guide to the home cultivation of over 200 of the world’s most beautiful varieties. Treasure Press, 59 Grosvenor street, London W.I. pp 11-13.
  5. https://cites.org/eng/gallery/species/orchid/vanda_coerulea.html.
  6. https://cites.org/sites/default/files/ndf_material/WG4-CS4.pdf.
  7. Pradhan UC (1985) Red Data sheet on Indian Orchidaceae - I Vanda Coerulea Griff. ex Lindl. Ind Orchid J 1: 54.
  8. Seeni S, Latha PG (2000) In vitro multiplication and eco-rehabilitation of the endangered Blue Vanda. Plant Cell Tissue Organ Cult 64: 1-8.
  9. Wannakrairoj S (1998) In Vitro preservation of Vanda coerulea Giff. protocorm like bodies. Kasetsart J 32: 329-332.
  10. Rotor G (1949) A method of vegetative propagation of Phalaenopsis in vitro. Amer Orchid Soc Bull 18: 738-739.
  11. Morel G (1964) Tissue Culture - A new means of clonal propagation of orchids. Amer Orchid Society Bulletin 33: 473- 478.
  12. Goh CJ (1970). Tissue Culture of Vanda Miss Joaquim. J Singapore Nat Acad Sci 2: 31-33.
  13. Irawati HSS, Suseno H, Idris S (1977) Tissue Culture of Aranther James Storie. Orchid Rev 85: 138-142.
  14. Vajrabhaya M, Vajrabhaya T (1970) Tissue Culture of Rhynchostylis gigantean, a monopodial orchid. Amer Orchid Soc Bull 39: 907-910.
  15. 15.Kim KK, Kunisaki JT, Sagawa Y (1970) Shoot tip culture of Dendrobium. Amer Orchid Soc Bull 39: 1077-1080.
  16. Sagawa Y, Kumisaki JT (1984) Clonal propagation: orchids. In: Vasil I.K. (ed.) Cell Culture and somatic cell Genetics of Plants, Academic Press, New York, 1: 61-67.
  17. Pedroza-Manrique J, Fernandez-Lizarazo C, Suarez-Silva A (2005) Evaluation of the effect of three growth regulators in the germination of Comparettia falcata seeds under in vitro conditions. In Vitro Cell Dev Biol - Plant 44: 838-843.
  18. Stewart SL, Kane MK (2006) A symbiotic seed germination and in vitro seedling development of Habernaria macroceratitis (Orchidaceae) a rare Florida terrestrial orchid. Plant Cell Tiss Org Cult 86: 147-158.
  19. Knudson L (1946) A new nutrient solution for germination of orchid seed. Am Orchid Soc Bull 15: 214–217.
  20. Vij SP, Aggarwal S (2003) Regenerative competence of Foliar explants: Vanda coerulea Griff. J Orchid Soc India 17: 73-78.
  21. Roy AR, Patel RS, Patel VV, Sajeev S, Deka BC (2011) Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex. Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid. Sci Hortic 128: 325-331.
  22. Arditti J, Ernst R (1993) Micropropagation of Orchids. John Wiley and sons, New York.
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473-497.