Indi an Journal of Experimental Biology Vol. 38, February 2000, pp. 149-154

Micropropagation of terrestrial orchids, Anoectochilus sikkimensis and Anoectochilus regalis

A Gangaprasad , P G Latha* & S Seeni

Pl ant Biotechnology Division, Tropical Botanic Garden and Research Institute. Palmle. Thiruvananthapuram 695562, India

Received 7 July 1999; revised II October 1999

Two horticulturally important jewel orchids of the genu s Anoectochilu.1· were successfull y micropropa),!atcd . bo latcd nodes of A. sikkimensis co ll ected from Sikkim in Eastern Himalayas and subsequentl y reared under nursery condition ' and A. regal is coll ected from Western Ghat s in Southern India were cul tured fur 12 weeks un Woody Plant Mediutn 1 WPrvi ) to produce a maximum of 4.8 and 5.6 cal lus- free axi ll ary shoots respectively at 95 and 98% clli ciency. Durin!,! reculturc of the explants from in vitro raised shoots under the same conditions. the total number of shoots obtained from the IH H.Ie' (2 1.4) and shoot tips (8.2) of A. reRalis were signiticantly higher than those hardy and slow ),!l"lliVill!,! shoot s of rl. sikkimensis ( 12.3 and 4.3) respectively. Shoots (4-6 em) were rooted in medium containing NAA (2.70 pMl :111d :tctiv:tted charcoal (0.2%). The rooted plants establi shed at 95-98% rate in community pots after hardening. Arter 6 months. green house adapted communi ty potted plants of A. regalis were transferred to nalllral forest habit at loc<JII y with 9) :~ nd 70',1 survival respect ively after 12 months . The plants. establi shed in communit y pots and native forest habitat were free fnnn :~ n y

morphological and growth defects.

Among orchids, a small group of terres tri als , popularly called Jewel orchids is appreciated for their foliage. Species of the genus Annectochilus are beautiful jewel orc hids of ancient origi n, whose leaves appear as though woven from shimmering silken threads-a blending of cinnamon, purple, olive and moss green 1

• The genus is much more delicate than other groups of jewel orchids like Haemaria and Goodyera and cannot tolerate long periods of drought2

. They are not easy to grow-'.4 Anoectochilus sikkimensis King & Pant!. and A. regalis Bl. are two of the exqui site terrestrial jewel orchids of India .

Being habitat and high altitude specific, and slow growing, propagation and rea ring of both the spec ies of Anoectoc!Jilus pose a major problem. Vegetative propagation by convent iona l means is e ither slow or not poss ible. Extensive co ll ection for commercial purposes has dimini shed their avai lability in native hab itats. Seed capsules are se ldom produced by the plants in nature or under cu lti vati on. Propagation through seeds is uncommon and difficul t.

Embryo culture of these two spec ies was unsuccessful in our laboratory. The major thrust of our research effort, therefore, was to develop suitable

*Correspondent author: Fax :+91 - 04 72- X6964(J

mi cropropagat ion meth ods for bot h th ese orchids using stem node ex plant cultures from mature plant s.

Materials and Methods Nursery-grown, potted plants or /\. sikkilll (' I/Sis

were a gift from Dr. Manmohan Att avar. Indo­American Hybrid Seeds Co .. Bangalore. Flmvering plants of A. regalis were co ll ected from Agasthyamalai in south Western Ghats and maintained at the orchidarium of our In stitu te before use.

Top shoot cuttings (4-5 em long) were washed we ll in tap water, defoliated and dissected to remove nodal segments of I em length. The nodes we re was hed under running tap water for 15 min ;111d soaked in I 11r (v/v) commerc ial Labolene detergent (Cl laxo India Pvt. Ltd ., Bombay) before surface steri l1 sa1 ion hy passage through ethanol (70~/c. ) for 30 sec and diluted Steriliq (Combii Organic Chem., (P) Ltd .. New Delhi ) contai ning 2% hypochl or ite for I 0 min . The ex plants were then washed once in ster ile di stil led water before immersion in aqueous mercur ic chl oride (0.1 %) for 6-7 min and rin sed thurou l! hl y wit h three qui ck changes of steril e distilled water. The ut i ends. already browned we re sliced uri and the e xpl<tn!~

were implanted into nutri ent llll~ dia wi th the nodal porlions immersed in the medi;1.

150 IND IAN J EX P BIOL. FEBRUAR Y 2000

Nutrient formul ation consisted of the macro and micro salts of Woody Pl ant Medium (WPM)5

containing sucrose (3%) and varying concentrati ons of 6-benzylaminopurine (BAP). The medium was adju sted to pH 5.2 before add ing 0.5 % (w/v) of agar (Oxoid Ltd. , Poole, England). After dissolving agar by heating under constant stirring, the medium was di spensed in 15 ml aliquots into 25x 150 mm culture tubes and autoclaved at 12 1 "C under 1.05 kg cm-2 for 18 min . All the cu ltures were incubated at 25°±2°C under li ght for 12 hr (fl uorescent li ght 25-50 )..!mol m-2

s-1) .

After 12 weeks, regenerated shoots (2-4 em) were harvested and shoot tips and nodes ( I em) were dissected and recultured in fresh medium with same composition. Healthy shoots (4-6 em long) with 3-5 leaves that developed wi thin 15 weeks of culture were detached from th,c exp lants and transferred individually to WPM containing varied concentra­tions of a-naphthaleneacetic ac id (NAA) and 0.2% (w/v) activated charcoa l (E. Merck India Ltd ., Bombay) for rooting.

The rooted plants obtained after 6 weeks were weaned away from the culture tubes, repeated ly washed in tap water to remove traces of agar st icking onto the roots and transp lanted in a potting mi xture of I : I : I charcoa l, broken tiles and river sand in 5x5 em clay community pots . Each pot contained 3-6 plants. The potted plants were well irri gated and hardened for 2 weeks in an improv ised mist chamber, maintaining RH to 70-90%.

After 6 months, estab li shed plants of A. regalis (about I 00) with 3-4 leaves and 4-5 roots were

transferred to the loose humus rich soil of the forest fl oor in a secondary fores t vegetat ion within the inst itute's campus ( 150m) where re lative ly warm and humid conditi ons (22"-34"C, 80-90 Cfr 1<1-1) preva il ed. A second batch of 100 plants was re intmduced intc) a natural forest segment at Ponmud i Hill s. 111 Thiruvananthapuram, Indi a, at an altitude of I 000 m above sea level. The temperature in thi s region ranged from 7°-30°C. Reintroduction of planh in these areas was done during the month of June Jl)CJ6. after the south west monsoon had set in . For planting. one square metre plot was demarcated on tl1e forest fl oor. With minimum disturbance to the ground rlora . the loose soil was dug out to make .'ix ."i c n1 pits in the soil , at a spac ing of IOcm from one another. The plan ts were gentl y removed from the co nm1unity pols without any damage to the groyv in g roo t tips and placed carefull y, one in eac h pit : 1he pit s were then filled with humus, taking ca re to see lhal the plants were in an upright positi on. In order to fac ilitate quick establi shment of the plants. ~l1 e ruots were kept we ll spread out in the pit and he ld in place by tiny pebbles. The plants were all owed to grow under natural condition . o water and ferti I i sv r was given to the plants. Data was co ll ected every month for a period of one year.

Stat isti ca l ana lys is was done and da!<l were compared using ANOV A and LS I) multi ple range test.

Results and Discussion

Nearly 40% of the cu ltures. were bacterial contaminati on within two

los t due to days after

Tabl e 1-EITect or BAP on shoot fo rmation in nodal explants of Anoectochilus regalis and A. sikki111ensi.,· cui Ju red .i11

WPM for 12 weeks.

A. regal is A . . l'ikkillf('/ISi.\

BAP (A) (B) <C) (D) (A) (B) ((') ( I) )

0.0 25 35 25 37

0.44 25 38 1.1 i (78) 2.8 do 25 40 3. 1d'!XC·l 1.-F

1.32 25 35 2.4 1(90) 4 .0h 25 32 :un 1oo ) 3.3 ~·d

2.2 25 38 5.6 "(95) 4.3" 25 35 4 _) .oh( () ;-,) 3. 1 d

4.4 25 35 4.4\89) 3. 1 d 25 37 4.3 1" (X7) ~.4 .-1

8.8 25 37 4 .0 "(96) 2. 1 1 25 3.'i .\.') ""('l--) 2.7 ,.

13 .2 25 35 2.0g(90) 1. 3 gh 25 40 .\.I "' !'! I ) 2.-+ '1

17.6 25 38 I ;(85) 1.2 gh 25 36 :i.:: I~ ( X_l ) . ~-~ I

22.0 25 40 I; (84) I.Oh 25 3.'i 1.0 '()-IX ) I .X I

Figures in parantheses indicate % caulogenic respo nse o f the infection free explan ts. Mean va lues follo wed hy th e ': 11nc supcr,cripl> arc not significantly diflerenl at <P<0.05) according to ANOVA and LSD multiple range tes t. (A)-No. of ex plan ts cultured; (B)-% loss of explants; (C)-means shoot no./noclc; and (D)-mean shoot kn~Jh tL 111 1.

GANGA PRASAD e1 a/.: M ICROPROPAGATION OF TERRESTRI AL ORCI-1 1 DS 15 I

inoculation . The remaining exp lants swe lled in size, the swelling being pronounced at nodal joints. Though the cu t ends al so swelled in size, there was no exudation and callusing.

Swelling was pronounced at noda l joints and formation of axi ll ary buds ( 1-3) free of callusing and protocorm-like bodies was noticed in 3-4 weeks (Figs I , 2). Bud · formati on was dependent on exogenous supply of cytokinins with more than 90% of nodes producing shoots/shoot buds. Differences in cau logenic response between the two spec ies were

negligible. Average number or shoots/node was 4.6 and 5.6 in A. sikkimensis (Fi g. 3) and A. regulis respecti vely after 12 weeks of culture (Table I ).

While the percentage response of the ex plants was hi gh over a range of concentrati ons. the number of shoots formed were drastical ly red uced at concentrations exceeding 17.6 ~JM or BAP. The shoots so formed were stunted vvith ca llo id outgrowth s.

In variabl y all the shoot tips and nodes or in t•itro formed shoots responded we ll durin ~ rec ulturc with

Figs 1-9-Formation of 1-3 axillary buds from stem nodes of ( I )-Anoeclochilus sikki111ensis: (2)-A. rl'galis cultured in t'ilm for .f weeks; (3)-Multiple shoots obtained from stem nodes of A. sikkimensis after 12 weeks of cu l ture: (4)-Multiplt; shoot s oht;tined from stem nodes of A. regal is after 8 weeks of reculture: Root ing of the shoots (5)-A. sikki111 ensis: (6\-A. regalis after 6 week;, in medium containing 2.2 !lM of NAA and charcoal (0.2%); Micropropagated plants (7)-A . sikki111ensis: 0:0-A. rcgalis establi shed in communit) pots after 6 weeks o f transplantation: (9)-Micropropagated plants of A. regalis established in the forest of Ponmudi. l nd1 ;1 altcr I~

months (Bars represent I em for Figs 1-4; and 2 em for Figs 5-9).

152 I DIAN J EXP BIOL, FEBRUARY 2000

Table 2-Relative response or recultured nodes and shoot tips of A. regal is and A. sikki111en.1is in WPM for X wee b.

BAP Mean no . or shoot s/node Mean shoot length (ern ) Mean no. or shoots/ shoot tip Mc;Hl .< IH•ntlcni! til ll'll l) (JlM) AR AS AR AS AR AS AR AS

0.0

0.~8 9.4 c 6 .X I 4.3 d 3.7 de 6.6 ah J.5 d 3.X ,,,· 3.1 1

2.2 21.4 a 12.3 nl 5.X a 4. 1 d ~- 2" 4 .] '"' 5.2 . -+A <

4.4 12.2 d X. 6 <~ 3.<Jd 3.7 'k 6.4 < 4. 1 ,. -+ .-+ 3.6 .

8.8 8.0 <1' 6 ~I .J 3.6 l: 3.2 cf 5.4 cd 3.3 I _u · 2.-F

13.2 4.2 g 1? h 3. 1 r 3.6 c 5. 1'1 2.X I ~ :2,(, 1" 2.1 h

22.0 3. 1 h 2.3 i I ~ ii . . 1 3.6 c 5. 1 d 2.X I~ 2J> ~~ - :2. I h

Mean values followed by the same superscripts are not significantly different at (P_~0 . 05) accord ing to A OV A and LSD multiple range tes t. AR-A. regalis; AS-A. sikki111ensis

the emergence of addi ti on~ ! buds in a week followed by the ir growth into 3.0-3.5 em shoots in 8 weeks. As new buds continued to form from the base of the ex isting shoots even after 12 weeks, the shoots formed were highly heterogenous in size and stage of development (Fig. 4). Apical portion of the shoot tips in cu lture continued to show longi tudina l growth, but formation of ax illary shoots was confined to the basa l part. There was evidence of apica l meristem contributing to add iti ona l shoot formation. After 8 weeks of reculture, s ignificantly large r number of shoots were formed in nodes than in shoot tip cultures (Table 2). Besides, the rate of shoot multiplication in both node and shoot tip cultures was higher in A. regalis than in A. sikkimensis . Better growth as evidenced from increased mean length and soft tex ture of the shoots were also characte ri st ic of the

former spec ies. Root initiati on in shoots tran sferred to rooting

medium occurred within 2 weeks at a frequency of 85-90% . Differences in rhi zogenic response between the two species were marginal. A maximum of 3.8 roots/shoot with a mean length of 2.8 em, and 3. 1 roots/shoot with a mean length of 2.4 em were obtai ned afte r 6 weeks of culture for A. regalis and A. sikkimen.sis respecti ve ly in media containing 2.7 f.l-M of NAA and 0.2% (w/v) of charcoa l (Table 3, Figs 5,

6). Auxin (exceedi ng I 0 .8f.1M ) induced formation of few roots at low frequ ency ( 40%) and root formation was invari ably preceded by callu s forma ti on at cut ends of shoots.

Rooted plants free from ca llus ing were hardened in the mist chamber for 2 weeks and showed hi gh percentage establi shment (Figs 7, 8) . Differences in estab li shment rates between two spec tes were

Table ]-Effect of NAA on rooting of in l'i /II J r ;~i ~cd ~ ilont ~ nf A. regal is and A. sikki111ensis in WPivl with ().:2'/r ( w/v)

NAA (JlM)

0.0

1.08

1.62

2.70

5.4

10.8

16.2

ch<li'C O<ll . Mean no. of roots/ shoot

AR AS

1. 8d 1.2 ~

2.6 ' 2.2 nl

3.!:! a 2.X " 2.9 ah 2.(1 l '

1. 8d 1.3 gh

I.O g 1.6 <

Mc;rn root length (C ill )

AR AS

1 ) h I -'!7 ,.

2.~ d· 1 .., I'll. '

3.1 :I :2.-+ h

') - h ') , I'll.·

' ' :2.-+ h :2.1 '

1.:.: d I.'J'

Observations were made after 6 wcds or culture. IVIc;1n v; i/u cs followed by the same supcrscript ~ arc not signil icant ly different at (?~0 . 05) according to ANOV 1\ ;md LSD mu lti pi c range test. AR-A. regalis . AS-A. sikki111ensi_,.

neglig ible. The hardened plants tran s fe rred to gree n

house showed signs o r growth as ev idenced by formation of new leaves after 6 weeks. T he plant s so ra ised were free from morpho log i ·a l and growth defects. The micropropagated plant ~ oJ' A. regulis subsequently transferred to forest ha b itat s within the institute's campus and at Ponmudi hi lls prod uced new leaves I month after transfer and survived at 95 and 70% rate respect ive ly afte r 12 months (Table 4 .

F ig. 9). Jewe l orchids are exquisite ho rticu ltural taxa

recommended for breeding app li c.tt ions in vo lvin g somatic hybridi sati on6 Publi shed re port s indi ca te successfu l multipli cat ion through rhi zome node cu lture of Haemeria discolor and Heweriu cri.,·wft /'_ Spec ies of Annec fo chilus di stribut ed in pan .~ o J' As ia have se ldom been a subj ec t o r s imil ar research . In the

GANGAPRASAD eta/.: MICROPROPAGATION OF TERRESTR IAL ORCI-11 DS 15~

Table 4-Rein troduction or Anoectuchilus regalis plants into ra rest segment s of Pon mucli and also in the institute campus

No. or plants re introduced

25 25 25 25

25 25 25 25

% surviva l 6 months 12 months

Pon111udi

9 1 70

Institute Ca111p 11.1'

95 95 .

only study of its kind , Anoectochilus fo nnosanus was multiplied from seedling expl ant cultures9

. The results of the present study confirm the amenability of Anoectochilus from both the megadi versity centres of India, vi z. Himalayas (A. sikkimensis) and Western Ghats (A. regalis) fo r clonal multiplication in vitro .

Terres tri al orchids in general prefer dilute media for germinati on 10 and therefore, such medi a would be most suited fo r ti ssue culture of these spec ies. Yam and Weatherhead8 have used Knop's medium with low ionic concentrations to initi ate pl antlets iH vitro

of the jewel orchid , Hetaeria cristata . In the present study too, the nutrient fo rmul ation (WPM) of Lloyd and McCown5 containing low ionic concentrati ons was used for micropropagation of two other jewel orchids.

In the present study about 40% of the nodal explants were lost due to bacteri al infec tion, even though the explants were decontaminated in two steps with ethanol, sod ium hypochl orite and mercuric chloride. It appeared th at the so iled nature of the shoots compounded the di sinfestation process. The stems of these two terrestri als creep on the ground and hence the chances of lodging microbial contaminants are very high. Therefore a furth er refinement of the sterili sati on procedure is needed to reduce the percentage loss of the ex plants particularl y in the rare taxa.

As expected, cytokinin (BAP) effec ti vely induced multiple axill ary buds in the nodal ex plants of both the species and BAP alone at a concentrati on of 2.21-LM was sufficient to initiate and sustain the growth of the buds in to shoots. The observed earl y emergence of buds in the recultured explants is in

compliance with the find ings in other cu ltu re II I" 'I . . I I systems · -. t was tn terest tng wweve r. t 1at

di ffe rences in the response or the nodal ex plants between the two spec ies , unn oti ced at culture initi ati on became signifi ca nt during rec ulture. In Firm raised shoots of A. regolis were so ft textured and relative!' fast growing and multipli ca ti on of th is spec ies was achieved more rap idly th an A.

sikkimensis (Table 2). The source plan ts being qualitati vely different and the exp lant s used for culture initiati on being eq uall y hard. diffe rences in the caulogenic responses of the ex plants between the spec ies during culture initi ati on were neg li gible. The differences were pronounced and spec ie.s-spec ifi c responses were trul y revea led when shoots rai sed under uniform conditi ons served as the source or ex plants used in reculture experiments.

Root initiati on was achi eved in WPM medi um with 2.70!-lM NAA, preferabl y in conjuncti on with 0.2% (w/v) acti vated charcoa l. These resul ts are in agreement with the publi shed work on Renwllhem

imschootiana 11 where co mplete pl ants read y for community pots · were obta ined in basa l medium containing NAA. NAA as the so le hormone produces roots in several Hong Kong orchids1

; In Vundu

crista/a, rhi zogenes is remains e lusive un ti l the m~dium is supplemented with NAA 1 ~, As observed in the present study, presence of act ivated charcoal in the nutrient promotes root growth in certain other orchids15

·16

• Although some plant spec ies do not survive direct transfer from the culture vesse ls to so il. es tabli shment of deflaskecl. roo ted A. sikk i11 1ensis and A. regalis plantlets in the communi ty pots was readi ly achieved by maintaining hi gh humid ity aroun d the plantlets during the first two weeks hy frequ ent watering under diffused li ght in a gree n house. Under these conditions, most of the mi crop ropaga tcd plant s of both the species survived and res umed norma l growth in the pots, and later when transp lanted into the humus rich fo res t undercover within the institute's campus. Even . more stri kin g was the observation that micropropagated pl ants or A. reguli.l .

reintroduced into Ponmudi , a constituent segment or the Western Ghats got estab li shed at 7!Ji/r effic iency. It shoul d be fruitful to reintroduce the mi cropropagated pl ants of both the spec ies ro various segments of Western Ghats and Eas tern Him ; tl ay a~

and find out their restorati on through mu lt ilocation trials.

154 I 1DIAN J EXP BIOL, FEB RUAR Y 2000

Acknowledgement We thank Dr. Palpu Pushpangadan, the Director of

the Institute for laboratory fac ilities, Mr K P Pradeep Kumar for photography aass istance and the Ministry of Environment and Forests, Government of India, fo r fin ancial support.

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