30. viki's paper iceea

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2010 Ittl fc vtl gg Applct E2010

Micropropagation of Vanda coerulea Griff ex Lindl.: A Study of Regeneration

Competence of Roots in vitro

Viki Manners

lant iotechnology aboratory, Department of otany,Centre for Advanced Studies

North Easte Hill UniversityShillong, ndia

E-mail: manersv@gmai!.com

-A rapid and ecient procedure is outlined for propagation of d Gri. ex Lind!., an endangeredepiphytic orchid. Root segments (0.5-1.0 cm long) from plantsgrowing both and were cultured on MS

(Murashige and Skoog, 1962) medium supplemented withauxins [Indole 3-acetic acid (IAA)], cytokinins [6-benzylaminopurine (HAP), 6-furfuryl amino purine (KN)] andcoconut water (CW). The root explants from sourcesfailed to respond whereas those from grown plantletsregenerated protocorm-ike bodies (PLHs). An optimum of 16.2 PLHs developed from root explants on mediumsupplemented with 30M HAP and 15M IAA. The resultingplant lets were successfully transferred to pots containing brickpieces, charcoal chunks and decaying litter (1:1:1) + a top layerof moss. Ninety-ve percent of the plantlets survived in theglass house aer two months of transplantation.

ydd ; ; dd; ;

I. NTRODUCTION

Orchids are commercially valuable plants which aremarketed both as plants as well as cut owers and theirproduction has increased magnicently worldwide [1, 2].However, the orchids are fast depleting om their naturalhabitats due to deforestation, urbanization, utilization of landfor agriculture and over-exploitation for commercialpurposes. Tissue culture methods have been extensivelyexploited, not only for rapid and large-scale propagation of orchids, but also for their ex situ conservation. rotocolshave been developed for a number of orchid species throughin vitr culture of various plant parts [3, 4, 5].

Vand a coeruleaGriff ex.

ind!.(Fig.J a), popularly known as the blue Yanda of Asia is a perennial epiphyte

growing at an elevation of 10001500m in Northeast ndiaand in the northe ranges of Thailand and urma. Due to itsclear blue owers it has progenated a vast variety of oriculturally signicant hybrids and it has been bred forsuch qualities as ower size, oriferousness, vigour and cold tolerance in mode vandaceous hybrids. The species is alsoimportant ethnobotanically. The juice om its leaves is used to cure diarrhoea, dysentery, and dermal disorders [6].Habitat destruction and overexploitation are the twoimportant factors threatening its survivability in India [7]. Itis listed in the Appendix of the Committee for nteational Trade in Endangered Species of Wild Fauna and Flora. t is

97844486/$6 © 1

Suman Kumaria and Pramod Tandon

lant Biotechnology aboratory, Department of otany,Centre for Advanced Studies

North Easte Hill UniversityShillong, ndia

E-mail: [email protected]

also included in the list of Threatened lants of Indiapublished by the nteational Union for Conservation of Nature and Natural Resources. Therefore, there is an urgentneed to conserve this endangered taxon. The present paperdescribes a successl protocol devised for the regenerationof Vand a coerulea Gri ex. ind!. om root explants so as to obtain ue-to-type plantlets for conservation.

II. ATERALS AND ETHODS

A. Plant Material

The young and actively growing roots (1.5-2.0cm long)om mature greenhouse-grown plants (in vivo) and 5-months old in vitro cultures were harvested. The roots were then segmented to prepare 0.5-1.0 cm long explants.

Culture Med ium

Murashige and Skoog [8] medium supplemented with

groth regulators like 6-benzyl aminopurine (A), indole3-acetic acid (AA) and 6-rryl amino purine (Kinetin,KN) were added to the medium singly and in combinations.esides, the eect of organic supplement, coconut waterincorporated in MS basal medium was also tested in variouscombinations. The medium was incorporated with 3% (WV)sucrose and solidied with 0.8% (WV) agar. The pH of themedia was adjusted to 5.8 ± 0.02 using IN NaOH prior toautoclaving at 121°C.

Culture Cond itions

Cultures were maintained at 25 ± 2°C for 12hphotoperiod under 50mol mZs light intensity. There were10 replicates for each treatment and experiments were

repeated thrice. Statistical Analyss

The data was subjected to statistical analysis using oneway Anova and comparisons between the mean values of

treatment were made by Fisher's SD test [9].

Acclimatization ofplantlets

The plantlets with well-dened roots (2-3 cm in length)were transplanted into pots containing brick pieces, charcoalchunks and decaying litter (1: 1: 1) + a top layer of moss. Thepercentage survival of plants was calculated aer twomonths of transfer.



2010 Ittl fc vtl gg Applct E2010

III. ESUL TS AND ISCUSSION

In vitro micropropagation using root tips has beensuccesslly used for propagation of a number of orchids

either for conservation or for commercial production [10, II. Tissue culture production of oamental plants in general andorchids in particular forms the basis for the entirehoricultural industry. Several workers using various culturemedia introduced tissue culture methods for regeneration of orchids and dfferent explant have been used for in vitroorchid propagation, the most common being shoot tip andaxillary buds. Attempts to produce protocorm-like bodies(PLBs) om roots have not been common in orchids. Unlike to shoot meristems, the root apices are considered to behighly recalciant in terms of bud formation both in vivo andin vitro [12]. n our study, root segments om in vivo plantsfailed to respond whereas those om in vio grown plantletsregenerated Ls. The differential responses of the explants

om mature and juvenile roots under identical nutritionalconditions seems to indicate the importance of their sourceand physiological age of explants for in vitro regeneration[13, 14]. Oen in vitro regeneration in orchids is achieved

through either callus or PLBs formation. Since many orchidspecies require auxins and/or cytokinins for PLB formationand plantlet development; the combinations, concentrationsand the ratio between them are usually critically important[I]. n the present investigation, the root segments directlydeveloped into the PLBs and the equency of the LBsproduced was markedl inuenced by the quality andcombination of growth hormones in the nuient medium. Acombination of 30�M BA and I�M AA in MS medium(Table 1) proved to be the best treatment for eplants

response(98.2%) wherein 16.2±0.37 PLBs were formed withan average of I.8±0.37 shoots emerging out of these(Fig. 1b). The similar stimulatory eect of auxins and cytokinins onroots for proto corm multiplication as well as shoot formationin Cymbid ium hybrid has been reported [16]. AP at 30�Min MS medium also showed good explants response (96.8%)in terms of PLBs formation and shoot formation. Increasedconcenations of both auxins and cytokinins in the mediumwere inhibitory for response as indicated by the decrease inmultiplcation and proliferation of PLs and shoots. Mediumsupplemented with coconut water as well as KN alsoinduced LBs regeneration to plantlets but the response wasquite low comparatively (Table 1). The emerging shootsdeveloped roots in 3-4 weeks (Fig. 1 c) on being transferred

om treatment medium to basal medium. However, plantletswere also able to root eventuall in all BAP and AAcombinations. The rooted plantets were transplanted to potscontaining brick pieces, charcoal chunks and decaying litter(1:1 :1) + a top layer of moss (Fig. 1 d), and 9% of plantletswere successfully grown in the greenhouse. The layer of

1 1

moss on top proved to be benecial due to higher retentionof moisture content. Feeding the plantlets with diluted (10 times) MS nutrient salt solution fornightly proved benecialfor the healthy growth as it provided essential nutrients to thedeveloping plantets [17].

TABLE . FEE OF DIFFERET ROWTH RETORS DOT WTER (C O DEVEOMET OF ROTOORM-IE ODIES

(PLB) FROM ROOT EXTS OF VD OERE

Grwth Cncentratin Percentag Number Numberregulatr s e (% f fPLBs f shts

s respnsive(IM) explants

Control 0 0 0 0. 0 0 0I. 63.2±0.8 .4±. 04.4±0.24

30.0 96.8±0.37 09.8±0.37 09 ± 37. 31.2±0.58 02.4±0.24 02.2±0.20

. 2.4±0.81 02.4±0.24 o .2±0.37AA I.0 .4±0.60 03.6±0.24 03.0±0.26

30.0 78.0±0.4 07.4±0.37 06.8±0.31. 33.6±0. 02.6±0.26 01.0±0.24. 38.6±0.40 01.8±0.37 01.0±0.37

I.0 42.2±0.37' 03 0±0.31 02.4±.24

30.0 9.2±.37 .4±0.24 .0±0.20. 26.4±0. 01.4±0.24 01.0±0.24

.+ . .8±0.37 07.6±0.24 07.2±0.20BAP+ I.0 + I.0 3.8±0. 12.0±0.31 12.4±0.31

AA

30.0 + I.0 98.2±0.37 16.2±0.37 I.8±0.3730.0 + 30.0 63.2±0.32 .4±0.24 04.9±0.24

BAP+ .+ I.0 0 0 0 I.0+ 30.0 26.8±0.83 03.4±0.24 02.4±0.24. 0 0 0

CW 10.0 36.4±0. 04.4±0.24 04.0±0.24(%v/v) I.0 39.4±0. 02.4±0.24 02.0±0.24

Basal medium used is MS. Data recorded aer 6 weeks Values are mean±S.E. Means followed bysame leter in the column are not signicantly different as indicated by Fisher's LSD (p 0.05)

V. ONCLUSON

n conclusion, the results clearly indicate that the rootsegments can be protably used for micropropagation of Vand a coerulea. Since this orchid is endangered in the wild,

the root explants acts as an effective alteative to shootmeristem for micropropagation as it does not require thesacrice of the mother plant, and oers excitingoppotuntes to ase large numers of tre-to-ype plantlets. Thus, this protocol can be used both for restoring thedwindling populations of Vand a coerulea in nature and masspropagation for its oamental or pharmaceutical imporance.



2010 Ittl fc vtl gg ApplctIA 2010

Figure I. Regeneration competence of root segments of Vanda coeruleaGri ex Lind!: (a) Blooming plants of Vanda coerulea (bar= 3cm); (b)

Multiple shoots obtained from root segments of V. coerulea cultured in MSwith BAP (30M) and AA (15 M) (bar = Icm); (c) Development of rootson MS basal medium aer one month (bar = I cm); (d) Regenerated plantsin pots containing brick pieces, charcoal chunks and decaying litter (I: I: I)

+ a top layer of moss (bar = 2 cm).

CKNOWLEDGMENT

Financial support received om UC vide grant no. F.

32-355/2006 (SR) is gratelly acknowledged.

EFERNCES

[I] K. Tokuhara and M. Mii, "Micropropagation of Phalaenopsis andDoritaenopsis by culturing shoot tips of ower stalk buds, Plant CellRep., 1993, vol. 13, pp. 7.

[2] C. Chang and W. C. Chang, "Micropropagation of Cymbidiumensifolium var. Misericors through callus-derived rhizome, n VitroCell. Dev.Biol., 2000, vol. 36, pp. 517-520.

[3] J Arditti and R. Est, "Micropropagation of orchids, New Yorohn Wiley and Sons, 1993, pp. 682.

[4] S. Kumaria and P Tandon, "Orchids The world's most wondrousplant. in P. Pathak, R. N. Shekhar, M. Sharma and A. Sood, Eds.

12

Orchids: Science and Commerec, Bishen Singh Mahendrapal Singh,New Connaught Place, Deharadun, nda. 2001, pp. 17-28.

[5] S. Kumaria, S. Corrie, S. Sharma, and P. Tandon, "n vitroconservation of some commercially important orchids of Northeast

ndia, nt. J For. Usu Mngt, 2005, vol. 6 (2), pp. 36-40.[6] A K Nadkai, ndian Materia Medica, 3rd ed , vol. 1. Popular Book

Depot, Bombay, ndia, 1954.

[7] U C. Pradhan, "Red Data Sheet on ndian Orchidaceae I andacoerulea Grif ex Lindl, nd. Orchid , 1985, vol. I pp. 54.

[8] T. Murashige and F. Skoo, "A revised medium for rapid rowth andbioassays for tobacco cultures, Physiol. Plant. 1962, vol. 15, pp.473-497.

[9] R. A Fisher, he design of experiment, Oliver and Boyd,Edinburgh-London. 1935.

[10] S.Y. Park, H. N. Murhy, and K.Y. Paek, "Protocorm-ike bodyinduction and subsequent plant regeneration from root tip cultures of Doritaenopsis, Plant Sci ., 2003, vol. 164, pp. 919923.

[II] S. P. Vij, S. Aggarwal, and P. Pathak, "Regeneration competence of Cymbidium Great Waltz x valley ower roots: A study in vitro, J

Orchid Soc. ndia, 2004, vol. 18(1-2) pp. 109-115.[12] R.L. Peterson, "The initiation and development of root buds, in TheDevelopment and Function of Roots. JG. Torrey and D.T. Larkson,Ed. London, Academic Press. 1975, pp. 125-161.

[13] S. P. Vij, "Regeneration response of Orchid roots: A study in vitro, .Orchid Soc. ndia, 1993, vol. 7(1-2) pp. 61-72.

[14] P. Pathak and S. P. Vij, "On the regeneration potential of orchid roots A study in vitro, Proc. Natl. Seminar on a Decade of OrchidResearch and Development. The Orchid Society of ndia, PunjabUniversity, Chandigarh, ndia. 1994

[15] . Arditti and R. Est, "Micropropagation of orchids, New York,ohn Wiley and Sons, 1993, pp. 682.

[16] S. Yasugi, K. Sakamoto, K. Onodera, and M. Tamashiro, "Plantletregeneration in root segment culture of Cymbidium Kenny winecolour', P. Tiss. Cult. Leters, 1994, vol. II (2), pp. 150-152.

[17] S. Kumaria and P. Tandon, "Clonal propagation and establishment of Dendrobium mbriatum var. oculatum Hk. F., in Advances in PlantCell Tissue Culture n ndia, P Tandon, Ed. ndia PragatiPrakashan,. 1994, pp.218-231.

30. viki's paper iceea

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