AXILLARY SHOOTS DERIVED FROM SHOOT TIPS IN IN VITRO MASS PROPAGATION OF ANOECTOCHILUS FORMOSANUS HAYATA

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1 Tedusrtiklid / Reserch rticles 121 Agrrtedus 2 * XXIX * Journl of Agriculturl Science 2 * XXIX * AXILLARY SHOOTS DERIVED FROM SHOOT TIPS IN IN VITRO MASS PROPAGATION OF ANOECTOCHILUS FORMOSANUS HAYATA Budi Winrto 1,2, Smijn 2 1 Indonesin Ornmentl Crops Reserch Institute, Jln. Ry Ciherng, Pcet-Cinjur 43253, West Jv Indonesi 2 Centrl Jv Assessment Institute for Agriculture Technology, Jl. Soekrno Htt KM.26 No.10, Kotk Pos 124, Teglsri, Bergs Lor, Bergs, Semrng, Centrl Jv, Indonesi Sunud: Received: Aktsepteeritud: Accepted: Avldtud veeis: Pulished online: Vstutv utor: Budi Winrto Corresponding uthor: E-mil: udi.winrto67@yhoo.co.id Keywords: ccession, explnt,, tissue culture, Anoectochilus formosnus. doi: /js ABSTRACT. Axillry shoot prolifertion in in vitro mss propgtion of Anoectochilus formosnus ws successfully estlished vi selection of different explnt types, ccessions nd culture medi to plntlet cclimtiztion. In the initition stge, shoot tips nd Murshige nd Skoog (MS) contining 1.5 mg l -1 N6-enzylminopurine (BAP) nd 0.25 mg l -1 α-nphthlene cetic cid (NAA) were determined s high potentil explnt nd for xillry shoot regenertion of A-1 nd A-2 ccessions of A. formosnus compred to others. High xillry shoots up to 7.0 shoots per explnt with 1.0 cm shoot height nd 9.8 leves per explnt derived from shoot tip explnts of A-1 ccession were signifycntly induced nd proliferted in MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA. Higher root formtion up to 2.4 roots per shoot nd 1.0 cm root length of A-1 ccession ws esily prepred on Hyponex (2 g l -1 20N:20P:20K) contining 150 ml l -1 coconut wter (CW). While high survivl rte of cclimtized plntlets s high s 90.4 % ws successfully done y plnting them in plstic oxes contining mixture of urned rice-husk nd orgnic mnure (1:1, v/v) fter 2 months. In the study, it ws lso reveled tht in in vitro culture of A-1 ccession of A. formosnus showed higher response compred to A-2 ccession in ll in vitro culture stges. The successful estlished protocol expected cn e pplied for prepring high-qulity plnting mterils for commercil purposes nd developing new route of in vitro mss propgtion for other species of A. formosnus Akdeemiline Põllumjnduse Selts. Kõik õigused kitstud Estonin Acdemic Agriculturl Society. All rights reserved. Introduction Anoectochilus is one of Orchidcee fmily memers nd elong to group of terrestril orchids tht re generlly known s "Jewel Orchids" (Cvestro, 1994; Shiu et l., 2002). The genus consists of 40 species widespred throughout Southest Asi, New Cledoni nd Hwii (Ket et l., 2004). Anoectochilus formosnus is one of jewel nd terrestril orchids grown primrily for its eutiful folige s well s for medicine enefits (Tseng et l., 2006; Wu et l., 2007; Gutiérrez, 2010). The A. formosnus, normlly grow t elevtion rnge etween 800 nd meter ove se level in Lnyu Islng in Tiwn, Ryukyu Islnd in Jpn nd Chin s Fujin Province (Refish et l., 2015), is n herl plnt widely used s dietry supplement nd folk remedy in Asi (Kun et l., 2011). For helth purposes, it cn e pplied s heptoprotection (Du et l., 2003; Wu et l., 2007; Fng et l., 2008), nti-ftigue (Ikeuchi et l., 2005), ntioxidtive (Wng et l., 2002; Wng et l., 2005), nti-hyperglycemi (Shih et l., 2002; Ho et l., 2018), ntihyperliposis (Fng et l., 2008), ntiosteoporosis (Msud et l., 2008), nti-tumor (Shyur et l., 2004; Tseng et l., 2006), nd immune modultion (Kun et l., 2011). The species lso hs high potentil widely cultivted in severl res of Indonesi, however, developing the plnt is constrined y vilility of high-qulity plnting mterils. A. formosnus is slow-growing perennil her. Growth dn development of its seedlings to rech mturity nd er seed tke 2 3 yers (Tsy, 2002). The plnt is trditionlly propgted y seeds, ut their

2 122 Budi Winrto, Smijn germintion rte is very low (Ket et l., 2004). It flowers only once yer, especilly in winter (Octoer to Decemer) (Tsy, 2002). Nowdys due to overcollection from nturl resources, in one hnd nd less conservtion efforts, this orchid is under the thret of extinction (Belitsky, Bersenev, 1999). Therefore, to overcome ll these limittions, to mintin nd prepre the vilility of high qulity of plnting mterils oth for conservtion nd commercil purposes, propgtion of the plnts vi tissue culture works is importntly ddressed. Severl works in in vitro propgtion on Anoectochilus were pulished previously. Severl species of Anoectochilus developed vi the in vitro cultures were A. eltus (Sherif et l., 2012; Sherif et l., 2016; Rj, 2017), A. formosnus (Chng, Chen, 1987; Shiu et l., 2002; Tsy, 2002; Ket et l., 2004; Refish et l., 2015), A. sikkimensis nd A. reglis (Gngprsd et l., 2000), A. roxurghii (Zhng et l., 2015). These works generlly used severl explnt sources such s nodes, internodes, leves nd shoot tips; Murshige nd Skoog (1962; MS) used s sic ; N6-enzyldenine (BA), N6-enzylminopurine (BAP, thidizuron (TDZ), kinetin (Kin), N6-(2-isopentenyl) denine (2-iP), Zetin (Ze), 2,4-diclhorophenoxycetic cid (2.4-D), α-nphthlene cetic cid (NAA), indole-3- cetic cid (IAA), indole-3-utyric cid (IBA), nd 4- mino-3,4,6-trichloro picolinic cid (Pic) pplied s chosen hormones either individully or in comintions; nd citric cid, trisodium citrte, peptone, coconut wter, potto extrct, nd nn pulp utilized s potentil dditives pplied in these cultures. Vried ppliction nd comintion of them stimulted vriedresults, however, ech in vitro culture stge needs specific tretment nd condition. Estlishment of new nd relile protocol route of in vitro propgtion of A. formosnus s min ojective of the study ws successfully determined. The protocol ws initited y culturing shoot tips of A-1 ccession s explnt source for initition nd prolifertion of xillry shoots, followed y plntlet preprtion nd its cclimtiztion. Detil nd interesting finding in ech step were discussed in this pper. Mterils nd Methods Plnting mteril nd its preprtion Mterils used in the study were two ccessions of A. formosnus viz, A-1 nd A-2 ccessions collected y frmer t Jgkrs, Jkrt. The hrvested shoots with 4 5 leves nd 5 7 cm in length derived from the two ccessions were explnts sources utilized in the experiments. Before their steriliztion, the explnts were prepred y cutting leves remining petiole slt prts for 1 2 mm in length. The prepred explnts were redy for steriliztion stge. Explnt Steriliztion nd Preprtion All explnts with short sl prts of petioles were pre-treted y plcing them under running tp wter for 60 minutes (min), followed y immersing them in 1% sop solution for 30 min, 1% pesticide solution (50% enomil nd 20% knmycin sulphte) for 30 min with mnul shking nd then rinsing 4 5 times (@ 3 min ech) using distilled wter until ll remin disinfection mterils totlly removed. The explnts were then rought to lminr ir flow cinet for steriliztion. They were soked in 0.05% mercury chloride (HgCl 2) solution dded y 5 drops of Tween 20 for 5 min, followed y rinsing them with sterile distilled wter (SDW) 2 times (@ 3 min ech) with mnul shking. The remining petiole slt prts were then removed crefully using tissue culture lde. The shoot tips with one short node nd the third node were sliced nd put in different 25 ml Erlenmeyers contining 5 ml SDW. After ll explnts successfully prepred, ll shoot tip nd node explnts were further sterilized using 0.01% HgCl 2 for 3 min nd rinsed with SDW 4 6 times (@ 3 min ech) with the sme tretment. The explnts were then cultured in different initition medi. Culture medi Bsic medi used in the study were full strength MS nd hlf strength MS. All medi components oth mcro, micro nd vitmin were from Merck (Drmstdt, Germny), BAP nd NAA from Sigm-Aldrich (Drmstdt, Germny), Swllow gr (Jkrt-Indonesi). Initition, prolifertion nd rooting medi were prepred y mixing stock solution of mcro, micro, Fe chelte, nd vitmin. The ph of medi ws djusted to 5.8 using Model 420A ph meter of Thermo Orion (Beverly, USA) using 1N NOH or HCl. After ph djustment, the medi were dded y 30 g l -1 sucrose (except s tretment) nd 7 g l -1 Swllow gr; mixed homogenously, oiled, poured in jm ottles (7 cm in dimeter, 13 cm in height; 30 ml medi per ottle) nd sterilized for 20 min t 121ºC nd 15 kp using Pressure Stem Sterilizer Verticl Cylindricl LS. 001, SMIC (Shnghi, Chin). Culture incution Culture incution pplied in the study ws light incution. The light incution ws performed y plcing explnt cultures under cool fluorescent lmps for 12 h photoperiod with ~13 µmol m 2-1 s -1 light intensity. Incution period for ll experiments ws pproximtely 2 months. Effect of explnt types nd initition medi on xillry shoot regenertion nd prolifertion In the step, there were two types of explnts tested i.e. shoot tips nd nodes. While initition medi (IM) pplied in the study were MS contining (1) 1.5 mg l -1 BAP nd 0.5 mg l -1 NAA (IM-1), (2) 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA (IM-2), (3) 1.5 mg l -1 BAP nd 0.1 mg l -1 NAA (IM-3), (4) 1.5 mg l -1 BAP nd 0.05 mg l -1 NAA (IM-4) nd (5) 1.5 mg l -1 BAP nd mg l -1 NAA (IM-5). The experiment ws rrnged in split plot design with three replictions, where explnt types of shoot tips nd nodes were min plot; nd 5 initition medi s suplot. Ech tretment consisted of 3 ottles nd ech ottle ws cultured 3 explnts. Totl explnts used in the experiment were 270 explnts. Two ccessions of A-1 nd A-2 of A. formosnus in the step were studied in different

3 Axillry shoots derived from shoot tips in in vitro mss propgtion of Anoectochilus formosnus Hyt 123 experiments seprtely due to optiml hndling nd results expected. Effect of prolifertion medi on xillry shoot production of two ccessions of A. formosnus In the stge, promising initition estlished in the previous experiment i.e. MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA ws optimized y reducing MS strength from full to hlf-strength; vrying BAP concentrtion from 1.5 mg l -1 incresed to 1.75 mg l -1 nd reduced to 1.25 mg l -1 ; nd pplying MS fortified y 1.5 mg l -1 BAP, 0.25 mg l -1 NAA nd 60 mg l -1 denine sulphte (AS) promising from different study. Prolifertion medi (PM) tested in the stge were (1) MS fortified y 1.5 mg l -1 BAP, 0.25 mg l -1 NAA nd 60 mg l -1 AS (PM-1), (2) MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA (PM- 2; control), (3) MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA (PM-3), (4) MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA (PM-4), (5) Hlf-strength MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA (PM-5); control), (6) Hlf-strength MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA (PM-6), nd (7) Hlf-strength MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA (PM-7). Shoot tips were etter explnt source shoot regenertion determined from the previous study were used to investigte response of A-1 nd A-2 in the prolifertion experiment. The experiment ws rrnged in split plot design with three replictions, where two ccessions of A-1 nd A-2 were pplied s min plot; nd 7 PM s suplot. Ech tretment consisted of 3 ottles nd ech ottle ws cultured 3 explnts. Totl explnts used in the experiment were 378 explnts. Shoot rooting Prepring plntlets for cclimtiztion purpose were crried out y culturing shoots with 2 3 leves nd ± 2.5 cm in height on different rooting medi. The rooting medi (RM) tested in the stge were (1) MS supplemented with 60 g l -1 sucrose (RM-1), (2) MS supplemented with 60 g l -1 sucrose nd 1.5 g l -1 ctivted chrcol (AC) (RM-2), (3) Hlfstrength MS with full vitmin (RM-3), (4) Hlf-strength MS with full vitmin nd 1.5 g l -1 AC (RM-4), (5) MS contining 0.2 mg l -1 BAP nd 0.02 mg l -1 NAA (RM-5), (6) MS supplemented with 0.25 mg l -1 BAP nd 1.5 g l -1 AC (RM-6), nd (7) Hyponex (2 g l -1 20N:20P: 20K) contining 150 ml l -1 coconut wter (CW) nd 1.5 g l -1 AC. The experiment ws rrnged in split plot design with three replictions, where two ccessions of A-1 nd A-2 were pplied s min plot; nd 7 RM s suplot. Ech tretment consisted of 3 ottles nd ech ottle ws cultured 5 explnts. Totl explnts used in the experiment were 210 shoots. Plntlet cclimtiztion Plntlets of A-1 nd A-2 ccessions with 5 8 leves, 3 5 cm in height nd 2 3 roots derived from the previous experiment were used for cclimtiztion step. The plntlets were then pulled out from culture ottles gently using lunt forceps. The roots of plntlet were put under running tp wter to remove remins of gr ttching them. The plntlet roots were then immersed in 1% pesticide solution (50% enomil nd 20% knmycin sulphte) for 3 min, ir-dried them on pper for while, then cultured on plstic oxes contining mixture of urned-rice husk nd orgnic mnure (1:1, v/v) wtered sufficiently. The plstic oxes were then covered y plstic trnsprent for 30 dys. Ech week, the trnsprent plstic ws opened, then the cclimtized plntlets were spryed y 1 g l -1 Growmore high N solution then covered gin. Ech plstic ox ws plnted ± 40 plntlets for two replictions. The experiment ws rrnged in complete rndomized design (CRD) with 8 replictions. Ech tretment consisted of 20 plntlets. Totl plntlets cclimtized in the step were 320 plntlets. Vriles Vriles oserved in the study were (1) Numer of shoots per explnt, (2) height of shoot (cm), (3) numer of leves per explnt, (4) numer of roots per shoot, (5) length of roots (cm), (6) percentge of survivility (%), nd (7) Numer of survivl plntlets nd Qulity of plnts. Periodicl oservtion in ech experiment ws crried out to know nd oserve response nd ltertion of explnt during incution period. Finl oservtion nd vriles mesurement were recorded ± 2.0 months fter culture. Anlysis of Dt Collected dt generted from these experiments were crried out y nlysis of vrince (ANOVA) using SAS Relese Windows If there were significnt differences etween mens, the men vlues were further nlyzed using Tukey test, P = 0.05 (Mttjik, Sumertjy, 2006). Results Effect of explnt types nd initition medi on xillry shoot regenertion of A-1 nd A-2 ccessions Under periodicl oservtion it ws clerly known tht development of new shoots nd initil proliferted shoots ws noted 7 10 dys fter culture, while initil new leves were oserved dys fter culture. The initil proliferted shoots nd leves grew continully nd incresed in numer nd size of shoots following incution time. In the end of experiment, numer of shoots ws in rnge of 1 3 shoots per explnt with cm shoot height nd 1 4 leves per shoot.

4 124 Budi Winrto, Smijn 2,0 1,5 1,0 Shoot tips Nodes A 2,5 2,0 1,5 1,0 IM-1 IM-2 IM-3 IM-4 IM-5 c B c c 0,5 0,5 0,0 Numer of shoots per explnt Height of shoots (cm) Numer of leves per explnt 0,0 Numer of shoots per explnt Height of shoots (cm) Numer of leves per explnt Figure 1. Effect of explnt types nd initition medi on xillry shoot regenertion. IM-1, MS contining 1.5 mg l -1 BAP nd 0.5 mg l -1 NAA, IM-2, MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, IM-3, MS with 1.5 mg l -1 BAP nd 0.1 mg l -1 NAA, IM-4, MS fortified y 1.5 mg l -1 BAP nd 0.05 mg l -1 NAA, nd IM-5, MS ugmented with 1.5 mg l -1 BAP nd mg l -1 NAA. A. Effect of explnt types on xillry shoot regenertion, B. Effect of culture medi on xillry shoot regenertion. Verticl histogrm rs followed y the sme letter in the sme cluster re not significntly different sed on Tukey test, P = Different types of explnts nd culture medi tested in the first experiment, in fct, gve significnt effect on xillry shoot regenertion sttisticlly, P = Shoot tips were the most pproprite explnt source for otining higher xillry shoot regenertion thn tht of the node explnts. The explnts induced 1.5 shoots per explnt with 1.1 cm shoot height nd 1.7 leves per explnt (Figure 1A). While IM-2, MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, ws the suitle for the experiment purpose. The successfully stimulted 1.6 shoots per explnt with 1.2 cm in height of shoots nd 1.6 leves per explnt (Figure 1B). The IM-3 ws the second est, while the lowest results were performed y IM-5. Investigting effect of explnt types nd culture medi ws lso gve significnt interction effect in ll vriles oserved, where explnt types exhiited higher effect thn culture medi. Shoot tip explnts cultured on IM-2 were etter comintion tretment in otining high xillry shoot regenertion thn other comintions. The comintion successfully induced xillry shoots per explnt up to 2.0 shoots (Tle 1) with 1.2 cm shoot height (Tle 2) nd 2.1 leves per explnt (Tle 3). The second est tretment ws performed y culturing of the explnt on the IM-3. While the lowest results were indicted y culturing the similr explnt on the IM-5. Node explnts in comintion with ll medi tested generlly regenerted lower numer of shoots nd leves per explnt, however, they generlly produced higher shoot performnces (Tle 1, 2 nd 3). In the second experiment, testing explnt types nd culture medi for A-2 ccession lmost gve similr results s performed y A-1 ccession. Shoot tips were etter explnt type for inducing xillry shoots thn the nodes. Tle 1. Interction effect of type of explnts nd initition medi on numer of shoots per explnt explnt Shoot tips Nodes IM IM IM IM IM CV, % Notes: IM-1, MS contining 1.5 mg l -1 BAP nd 0.5 mg l -1 NAA, IM-2, MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, IM-3, MS with 1.5 mg l -1 BAP nd 0.1 mg l -1 NAA, IM-4, MS fortified y 1.5 mg l -1 BAP nd 0.05 mg l -1 NAA, nd IM-5, MS ugmented with 1.5 mg l -1 BAP nd mg l -1 NAA. Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Tle 2. Interction effect of type of explnts nd initition medi on height of shoots (cm) explnt Shoot tips Nodes IM IM IM IM IM CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Tle 3. Interction effect of type of explnts nd initition medi on numer of leves per explnt explnt Shoot tips Nodes IM IM IM IM IM CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = 0.05.

5 Axillry shoots derived from shoot tips in in vitro mss propgtion of Anoectochilus formosnus Hyt 125 The explnt produced the shoots up to 1.7 shoots per explnt with 1.1 cm in height of shoots nd 2.0 leves per explnt (Tle 4). IM-2 ws lso the pproprite tht successfully stimulted 2.2 shoots per explnt with 1.2 cm shoot height nd 2.0 leves per explnt (Tle 5). Though the explnt types nd culture medi showed significnt effect on xillry shoot formtion sttisticlly, there ws no interction effect of the two tretments. Tle 4. Effect of explnt types on xillry shoot regenertion of A-2 ccession explnt Numer of shoots per explnt Height of shoots (cm) Numer of leves per explnt Shoot tips Nodes CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Tle 5. Effect of explnt types on xillry shoot regenertion of A-2 ccession Numer of shoots per explnt Height of shoots (cm) Numer of leves per explnt IM IM IM IM IM CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Improvement of xillry shoot production of A-1 nd A-2 ccessions on different prolifertion medi Bsed on periodicl oservtion, initil xillry shoot nd lef formtion ws lmost similr noted s the previous experiments with different vlues on numer of shoots produced per explnt, height of shoots nd numer of leves per explnt. In the stge, two A. formosnus ccessions nd prolifertion medi generted significnt effect on xillry shoot production sttisticlly, P = A-1 ccession kept the most responsive ccession on xillry shoot production with 4.9 shoots per explnt, 1.1 cm in height of shoots nd 9.2 leves per explnt. While PM-7, MS fortified y 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA, ws the most optiml nd produced 4.3 shoots per explnt with 1.1 cm shoot height nd 7.0 leves per explnt (Figure 2B). PM-1 ws the second est, ut PM-6 stimulted the lowest results compred to others. In the study, it ws lso reveled tht improving xillry shoot production ws estlished y lowering BAP concentrtion from 1.5 to 1.25 mg l -1. In the study, it ws lso reveled tht two A. formosnus ccessions nd seven prolifertion medi hd significnt interction effect in ll vriles oserved, where the ccessions gve higher effect thn the prolifertion medi. Shoot tip explnts of A-1 ccession cultured on PM-7 were the est comintion tretment in resulting high xillry shoots compred to others. The comintion tretment successfully regenerted xillry shoots per explnt s high s 7.0 shoots (Tle 6) with lower shoot height down to 1.0 cm (Tle 7) nd higher numer of leves per explnt up to 9.8 leves (Tle 8) thn other comintions. The second est comintion ws recorded on the shoot tips of A-1 ccession with PM-2, while the lowest comintion results were determined on the explnt nd ccession with PM-6. Wheres the shoot tip explnts of the A-2 ccession indicted higher results when they cultured on the PM-1. Culturing them in other medi reduced the xillry production results significntly. The results lso reveled tht ech explnt source nd genotype hd specific ehvior in tissue culture condition A Numer of shoots per explnt A-1 A-2 Height of shoots (cm) Numer of leves per explnt B Numer of shoots per explnt PM-1 PM-2 PM-3 PM-4 PM-5 PM-6 Height of shoots (cm) Numer of leves per explnt Figure 2. Effect of different A. formosnus ccessions nd prolifertion medi on xillry shoot production. PM-1, MS fortified y 1.5 mg l -1 BAP, 0.25 mg l -1 NAA nd 60 mg l -1 AS, PM-2, MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-3MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-4, MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-4, Hlf-strength MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-6Hlfstrength MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-7, Hlf-strength MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA. A. Response of two A. formosnus ccessions on xillry shoot production, B. Effect of prolifertion medi on xillry shoot production. Verticl histogrm rs followed y the sme letter in the sme cluster re not significntly different sed on Tukey test, P = 0.05 PM-7

6 126 Budi Winrto, Smijn Tle 6. Interction effect of type of explnts nd initition medi on numer of shoots per explnt ccession A-1 A-2 PM PM PM PM PM PM PM CV, % Notes: PM-1, MS fortified y 1.5 mg l -1 BAP, 0.25 mg l -1 NAA nd 60 mg l -1 AS, PM-2, MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-3MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-4, MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-4, Hlf-strength MS supplemented with 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA, PM- 6Hlf-strength MS supplemented with 1.75 mg l -1 BAP nd 0.25 mg l -1 NAA, PM-7, Hlf-strength MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA. Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Shoot Rooting Culturing shoots derived from A-1 nd A-2 ccessions on different rooting medi lso exhiited vried responses on root formtion. Under periodicl oservtion it ws known tht initil roots immersing in sl prts of interclry meristem res of nodes were clerly oserved 6 10 dys fter culture. The initil roots grew continully nd developed in numer, size nd length following the incution time. In the end of experiment, numer of roots per shoot ws vried from 1 4 roots with cm root length. In the study, different ccession of A. formosnus nd rooting medi gve significnt effect of root formtion sttisticlly, P = A-1 ccession indicted higher responses thn A-2 ccession. The ccession induced 1.8 roots per shoot nd 0.9 cm root length (Figure 3A). Furthermore, though higher numer of roots per shoot determined of RM-4 nd RM-5 (Figure 3B), there were no significnt differences compred to others. While optiml root formtion of A-1 ccession shoots ws estlished on RM-7, Hyponex (2 g l -1 20N:20P: 20K) contining 150 ml l -1 CW nd 1.5 g l -1 AC. The comintion tretment stimulted 2.4 roots per shoot (Tle 9) nd 1.0 cm root length (Tle 10). Wheres mximl root formtion of A-2 ccession shoots ws determined on RM-4, hlf-strength MS with full vitmin nd 1.5 g l -1 AC nd RM-5, MS contining 0.2 mg l -1 BAP nd 0.02 mg l -1 NAA. Tle 7. Interction effect of type of explnts nd initition medi on height of shoots (cm) ccession A-1 A-2 PM PM PM PM PM PM PM CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Tle 8. Interction effect of type of explnts nd initition medi on numer of leves per explnt ccession A-1 A-2 PM PM PM PM PM PM PM CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = ,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 A-1 A-2 A 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 RM-1 RM-2 RM-3 RM-4 RM-5 RM-6 0,0 0,0 Numer of roots per Length of roots (cm) Numer of roots per shoot Length of roots (cm) shoot Figure 3. Response of two A. formosnus ccessions nd effect of seven rooting medi on root formtion. RM-1, MS supplemented with 60 g l -1 sucrose, RM-2, MS supplemented with 60 g l -1 sucrose nd 1.5 g l -1 AC, RM-3, hlf-strength MS with full vitmin, RM-4, hlf-strength MS with full vitmin nd 1.5 g l -1 AC, RM-5, MS contining 0.2 mg l -1 BAP nd 0.02 mg l -1 NAA, RM-6, MS supplemented with 0.25 mg l -1 BAP nd 1.5 g l -1 AC, nd RM-7, Hyponex (2 g l -1 20N:20P:20K) contining 150 ml l -1 CW nd 1.5 g l -1 AC. Verticl histogrm rs followed y the sme letter in the sme cluster re not significntly different sed on Tukey test, P = 0.05 c c cd RM-7 c B c cd

7 Axillry shoots derived from shoot tips in in vitro mss propgtion of Anoectochilus formosnus Hyt cm 3.2 cm 0.70 cm A B C 0.47 cm 0.55 cm 0.55 cm D E F 2.5 cm 5.0 cm 8.7 cm G H I 8.7 cm 2.3 cm 4.3 cm J K L Figure 4. In vitro propgtion process of A. formosnus sed on selection of explnt types, ccessions nd culture medi. A Hrvested shoots of A-1 ccession ccepted from Jgkrs frmer. B Prepred explnts of A-1 ccession redy for steriliztion step. C Shoot tips nd nodes used s explnt sources used in the initition experiments. D Initil regenerted shoots derived from shoot tips of A-1 ccession on MS contining 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA ± 30 dys fter culture. E Regenerted shoots derived from shoot tips of A-1 ccession on the sme ± 2 months fter culture. F Regenerted shoots derived from nodes of A-1 ccession on the sme ± 2 months fter culture. G Plntlets prepred on Hyponex (2 g l -1 20N:20P:20K) contining 150 ml l -1 CW nd 1.5 g l -1 AC ± 30 dys fter culture. H Immersing of plntlet roots in 1% pesticide solution (50% enomil nd 20% knmycin sulphte) for 3 min. I Plntlets of A-1 ccession plnted in plstic ox contining mixture of urned-rice husk nd orgnic mnure (1:1, v/v). J Covering plntlets of A-1 ccession plnted in plstic ox contining with trnsprent plstic for one month. K Morphologicl performnces of A-1 ccession ± 2 months fter cclimtiztion. L Morphologicl performnces of A-2 ccession ± 2 months fter cclimtiztion

8 128 Budi Winrto, Smijn Tle 9. Interction effect of ccession types nd rooting medi on numer of roots per shoot ccession A-1 A-2 RM RM cd 1.3 RM RM c 2.2 RM c 2.2 RM d 1.2 RM CV, % Notes: RM-1, MS supplemented with 60 g l -1 sucrose, RM- 2, MS supplemented with 60 g l -1 sucrose nd 1.5 g l -1 AC, RM-3, hlf-strength MS with full vitmin, RM-4, hlfstrength MS with full vitmin nd 1.5 g l -1 AC, RM-5, MS contining 0.2 mg l -1 BAP nd 0.02 mg l -1 NAA, RM-6, MS supplemented with 0.25 mg l -1 BAP nd 1.5 g l -1 AC, nd RM-7, Hyponex (2 g l -1 20N:20P: 20K) contining 150 ml l - 1 CW nd 1.5 g l -1 AC. Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Tle 10. Interction effect of ccession types nd rooting medi on numer of root length (cm) ccession A-1 A-2 RM d 0.5 RM e 0.5 RM RM RM RM cd 0.6 RM c 0.7 CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Plntlet cclimtiztion Acclimtiztion plntlets, though in some plnt tissue culture works to e criticl point, in the study plntlets were esily trnsferred to ex vitro condition successfully under grdul process s descried in mterils nd methods. Percentge of survivility ws vried from 80 to 95% or survivl plntlets in mixture of urned-rice husk nd orgnic mnure (1:1, v/v) were noted in ech repliction. A-1 ccession kept resulting in higher responses thn A-2 ccession with percentge of survivility up to 90.4% nd 18.1 survivl plntlets in verge (Tle 11). Plntlets cclimtized derived from A-1 ccession generlly hd helthy nd vigour growth compred to the A-2 ccession. Tle 11. Different responses of A. formosnus ccessions in cclimtiztion ccession Percentge of survivility (%) Numer of survivl plntlets A A CV, % Mens followed y the sme letter in the sme column re not significnt difference sed on Tukey test, P = Discussion In vitro propgtion protocol inititing from xillry shoot regenertion, prolifertion, shoot rooting nd cclimtiztion of plntlet on A. formosnus ws successfully estlished. The new findings cn e used s new lterntive method in prepring high qulified plnting mterils in developing the plnt commercilly in lrge scle. The protocol cn lso complete nd improve other studies pulished previously (Chng, Chen, 1987; Shiu et l., 2002; Ket et l., 2004; Refish et l., 2015). Results of the study cn lso e utilized s comprison method nd improving ides for other Anoectochilus in vitro studies of A. sikkimensis nd A. reglis (Gngprsd et l., 2000), A. roxurgii (Zhng et l., 2015), A. eltus (Sherif et l., 2012; Sherif et l., 2016; Rj, 2017). Successful initition stge in in vitro culture of plnt will led to high potentil in chieving the next step. Ket et l. (2004) recorded tht high xillry shoots derived from shoot tip explnts up to 11.2 shoots per explnt with 3.8 cm shoot length of A. formosnus ws estlished on Hyponex supplemented with 2 mg l -1 TDZ nd 1.0 g l -1 AC, while lower results down to shoots were recorded on MS contining 3 mg l -1 BA nd 0.5 mg l -1 NAA (Chng, Chen, 1987). In the study, MS contining 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA s potentil ws improved y decresing concentrtion of BAP from 1.5 to 1.25 mg l -1 to produce 7.0 xillry shoots per explnt with 1.0 cm shoot height nd 9.8 leves per explnt derived from shoot tips of A-1 ccession of A. formosnus. While in other studies, 5.6 shoots per explnt of A. reglis nd 4.5 shoot per explnt of A. sikkimensis were successfully regenerted on Woody Plnt Medium supplemented with 0.5 mg l -1 BAP (Gngprsd et l., 2000), 2.7 shoots per explnt with 2.2 cm shoot length of A. eltus were recorded on MS fortified y 1.0 mg l -1 BA (Sherif et l., 2012), 7 shoots per explnt with 6.2 cm shoot length on MS 2.5 mg l -1 TDZ nd 5% CW (Rj, 2017); 1 10 shoots per explnt nd cm shoot length of A. roxurghii ws noted on MS fortified y 0.2 mg l -1 2,4-D, 0.9 mg l -1 NAA, 1 mg l -1 BA, 0.25 mg l -1 Zetin, 0.6% gr nd 4.5% sucrose (Refish et l., 2015), high prolifertion rte of shoots up to 4.33 on hlf-strength MS fortified y 3.0 mg l -1 BA, 1.0 mg l -1 Kinetin, 0.5 mg l -1 NAA nd dditives (Zhng et l., 2015). Shoot rooting ws importnt step in prepring high successful cclimtiztion of plntlets. In the study, high root formtion with 2.4 roots per shoot nd 1.0 cm root length of A-1 ccession of A. formosnus ws estlished on Hyponex (2 g l -1 20N:20P:20K) contining 150 ml l -1 CW. Almost similr results with 2.3 roots per shoot nd 2.5 cm root length were noted on Hyponex with 2% sucrose AC free (Ket et l., 2004). In other Anoectochilus species, 4.3 roots per shoot nd 2.2 cm in length of roots of A. eltus were recorded on MS contining 0.3 g l -1 AC (Sherif et l., 2012), 3.2 roots per shoot with 2.1 cm root length on Mitr mended with 1.0 mg l -1 AgNO 3 (Sherif et l., 2016), 86.6% root formtion nd 4.4 cm root length on MS fortified y 7.0 mg l -1 IBA (Rj, 2017). Well plntlet preprtion will e nothing nd in in vitro culture works when the prepred plntlets were

9 Axillry shoots derived from shoot tips in in vitro mss propgtion of Anoectochilus formosnus Hyt 129 fil to e cclimtized. High percentge of plntlets survivility of A. formosnus round 90% ws estlished on mixture of pet moss nd vermiculite fter 2 months (Shiu et l., 2002), while in the study, percentge of survivility up to 90.4% nd 18.1 survivl plntlets ws determined on mixture of urned-rice husk nd orgnic mnure (1:1, v/v) fter 2 months. In other Anoectochilus species, 95% survivl plntlets of A. sikkimensis nd 70% for A reglis were recorded t loose humic rich soil fter 12 months (Gngprsd et l., 2000), 100% survivility ws proved in greenhouse fter 4 weeks (Ket et l., 2004); 80% survivility of A. eltus cclimtized plntlets ws noted on coconut choir, AC nd commercil fertilizers (3:1:1, v/v/v) (Sherif et l., 2012), 72.3% survivl rte ws noted t mixture of grden soil, snd, vermicompost nd te wste (8:4:2:1) (Sherif et l., 2016). 88% survivl plntlets were oserved in Kulivlvu regions of Kolli Hill fter 60 dys (Rj, 2017). Wheres high survivl rte up to 90.2% of A. roxurghii plntlets ws noted on plstic cups continning sterile snd nd pet soil mixture in rtio of 1:2 tht their surfces were covered y live moss (Zhng et l., 2015). Specific ehviour of ech explnt nd genotype on different types of culture medi in in vitro culture of A. formosnus ws lso successfully reveled in the study. Shoot tip ws more responsive thn node explnt; A-1 ccession etter thn A-2 ccession. The two explnts nd ccessions of A. formosnus indicted different growth nd performnces in ech step of culture. Ket et l. (2004) lso found the similr results in A. formosnus tht shoot tip explnts produced higher xillry shoots thn the node explnts. In A. eltus, node explnt ws more productive thn shoot tip explnts on shoot regenertion (Sherif et l., 2012), internode > node > lef explnts on cllus prolifertion nd regenertion (Sherif et l., 2016). Gngprsd et l. (2000) recorded tht node explnt of A. reglis hd high numer nd fster growth of shoots thn node derived from A. sikkimensis. Conclusions The seril experiments crried out in the study were successfully estlished n in vitro propgtion protocol for A, formosnus vi xillry shoot regenertion nd prolifertion. In the initil step, shoot tips s explnt sources nd MS contining 1.5 mg l -1 BAP nd 0.25 mg l -1 NAA were determined s high potentil explnt nd for xillry shoot regenertion for A-1 nd A-2 ccessions of A. formosnus. High xillry shoot production up to 7.0 shoots per explnt with 1.0 cm shoot height nd 9.8 leves per explnt derived from hoot tip explnts of A-1 ccession ws resulted in MS supplemented with 1.25 mg l -1 BAP nd 0.25 mg l -1 NAA. The regenerted shoots were esily rooted on Hyponex (2 g l -1 20N:20P:20K) contining 150 ml l -1 CW with 2.4 roots per shoot nd 1.0 cm root length of A-1 ccession. The well growth plntlets were successfully cclimtized on plstic oxes contining mixture of urned rice-husk nd orgnic mnure (1:1, v/v) with survivl rte s high s 90.4 %. In ll step of in vitro culture of A. formosnus it ws lso reveled tht A-1 ccession showed higher response compred to A-2 ccession. Acknowledgements We express our grtitude to Jgkrs frmer in supplying hrvested fresh shoots of A-1 nd A-2 ccessions s mterils nd explnt sources in the study. We would like lso to express our gret pprecition to Euis Rohyti nd Nin Mrlin for their coopertion nd helps during reserch ctivities conducted t Tissue Culture Lortory of the Indonesin Ornmentl Crops Reserch Institute. Conflict of interest We declre tht there is no conflict of interest deling with Jgkrs frmer who supplied reserch mterils, uthors nd Indonesin Ornmentl Crops Reserch Institute nd Centrl Jv Assessment Institute for Agriculture Technology tht fcilitted nd funded the reserch ctiveties. Author contriutions All uthors ply importnt roles eqully in designing, crrying out nd nlysing ll dt regenerting from the reserch till writing, editing nd pproving the finl mnuscript. References Cvestro, W Cultivting Anoectochilus, Dossini, Mcodes nd other jewel orchids. Americn Orchid Society Bulletin, 63: Chng, H.C.K., Chen, Z.Z Tissue culture nd cclimtiztion in Anoectochilus formosnus hy. Bulletin Tiwn Forestry Reserch Institute New Series, 2(2): Belitsky, I., Bersenev, V.N.A Jewel Orchids. Mgzine Americn Orchid Society, Du, X.M., Sun, N.Y., Hyshi, J., Chen, Y., Sugiur, M., Shoym, Y Heptoprotective nd ntihyperliposis ctivities of in vitro cultured Anoectochilus formosnus. Phytother. Res. 17:30 33, doi: /ptr Fng, H.L., Wu, J.B., Lin, W.L., Ho, H.Y., Lin, W.C Further studies on the heptoprotective effects of Anoectochilus formosnus. Phytother. Res., 22: , doi: /ptr Gngprsd, A., Lth, P.G., Seeni, S Micropropgtion of terrestril orchids, Anoectochilus sikkimensis nd Anoectochilus reglis. Indin J. Exp. Biol., 38: Gutiérrez R.M.P Orchids: review of uses in trditionl medicine, its phytochemistry nd phrmcology. J. Med. Plnts Res., 4: , doi: /JMPR Ho, Y., Chen, Y.F., Wng, L.H., Hsu, K.Y., Chin, Y.T., Yng, Y.C.S.H., Wng, S.H., Chen, Y.R., Shih, Y.Y., Liu, L.F., Wng, K., Whng-Peng, J., Tng, H.Y., Lin H.Y., Liu, H.L., Lin, S.J Inhiitory Effect of

10 130 Budi Winrto, Smijn Anoectochilus formosnus Extrct on Hyperglycemi-Relted PD-L1 Expression nd Cncer Prolifertion. Front. Phrmcol., 9:807, doi: / fphr Ikeuchi, M., Ymguchi, K., Nishimur, T., Yzw, K Effects of Anoectochilus formosnus on endurnce cpcity in mice. J. Nutr. Sci. Vitminol.. 51: Ket, N.V., Hhn, E.J., Prk, S.Y., Chkrrty, D., Pek, K.Y Micropropgtion of n endngered orchid Anoectochilus formosnus. Biol. Plntrum, 48 (3): , doi: /B:BIOP Kun, Y.C., Wu, T.J., Kuo, C.Y., Hsu, J.C., Chng, W.Y., Sheu, F Moleculr Cloning of New Immunomodultory Protein from Anoectochilus formosnus which Induces B Cell IgM Secretion through T-Independent Mechnism. PLoS ONE 6(6):e21004, doi: /journl.pone Msud, K., Ikeuchi, M., Koym, T., Ymguchi, K., Woo, J.T., Nishimur, T., Yzw, K Suppressive effects of Anoectochilus formosnus extrct on osteoclst formtion in vitro nd one resorption in vivo. J. Bone Miner. Met., 26: , doi: /s Mttjik, A.A., Sumertjy, I.M Experimentl design with ppliction of SAS nd minit. IPB Press, Bogor, 334 p. Murshige, T., Skoog, F A revised for rpid growth nd iossy with tocco tissue cultures. Physiol. Plntrum, 15: Rj, D.H Effect of Cytokinins on Micropropgtion of Anoectochilus eltus Lindl. from shoot tip explnts An Endngered Medicinl Orchid. IOSR J. Biotechnol. Biochem., 3(3):73 76, doi: / 264X Refish, N.M.R., Fu, C.H., Wng, M.G Comprtive Study of the Chemicl Components of Anoectochilus Roxurghii nd Anoectochilus Formosnus Tissue Culture. Int. J. Life Sci. Res., 3(2): Shiu, Y.J., Sgre, A.P., Chen, U.C., Yng, S.R., Tsy, H.S Conservtion of Anoectochilus formosnus Hyt y rtificil cross-pollintion nd in vitro culture of seeds. Bot. Bull. Acd. Sinic, 43: Sherif, N.A., Benjmin, J.H.F., Muthukrishnn, S., Kumr, T.S., Ro, M.V Regenertion of plntlets from nodl nd shoot tip explnts of Anoectochilus eltus Lindley, n endngered terrestril orchid. Afr. J. Biotechnol., 11(29): , doi: /AJB Sherif, N.A., Kumr, T.S., Ro, M.V In vitro regenertion y cllus culture of Anoectochilus eltus Lindley, n endngered terrestril jewel orchid. In Vitro Cell. Dev. Biol.-Plnt, 52(1):72 80, doi: /s Shih, C.C., Wu, Y.W., Lin, W.C Antihyperglycemic nd nti-oxidnt properties of Anoectochilus formosnus in dietic rts. Clin. Exp. Phrmcol. Physiol., 29: Shyur, L.F., C.H. Chen, C.P. Lo, S.Y. Wng, P.L. Kng, S.J. Sun, C.A. Chng, C.M. Tzeng nd N.S. Yng Induction of poptosis in MCF-7 humn rest cncer cells y phytochemicls from Anoectochilus formosnus. J. Biomed. Sci., 11: , doi: / Tsy, H.S Use of Tissue Culture for the Mss Propgtion of Pthogen-Free Plnts. Deprtment of Applied Chemistry, Choyng University of Technology 168 Gifeng E. Rd., Wufeng, Tichung 41301, Tiwn, 8 p. Tseng, C.C., Shng, H.F., Wng, L.F., Su, B., Hsu, C.C., Ko, H.Y., Cheng, K.T Antitumor nd immunostimulting effects of Anoectochilus formosnus Hyt. Phytomedicine, 13: , doi: /j.phymed Wng, S.Y., Kuo, Y.H., Chng, H.N., Kng, P.L., Tsy, H.S., Lin, K.F., Yng, N.S., Shyur, L.F Profiling nd chrcteriztion ntioxidnt ctivities in Anoectochilus formosnus Hyt. J. Agric. Food Chem., 50: , doi: /jf Wng, L.F., Lin, C.M., Shih, C.M., Chen, H.J., Su, B., Tseng, C.C., Gu, B.B., Cheng, C Prevention of cellulr oxidtive dmge y n queous extrct of Anoectochilus formosnus. Ann. N. Y. Acd. Sci., 1042: , doi: /nnls Wu, J.B., Lin, W.L., Hsieh, C.C., Ho, H.Y., Tsy, H.S., Lin, W.C The heptoprotective ctivity of kinsenoside from Anoectochilus formosnus. Phytother. Res., 21:58 61, doi: /ptr Zhng, A., Wng, H.Z., Sho, Q.S., Xu, M.J., Zhng, W.S., Li, M.Y Lrge scle in vitro propgtion of Anoectochilus roxurghii for commercil ppliction: Phrmceuticlly importnt nd ornmentl plnt. Ind. Crop. Prod., 70: , doi: / j.indcrop