Jurnal Biologi Indonesia 7 (2): 251-261 (2011)

Shoot Tip Culture of albomarginata Lobb ex Lindl. In Vitro

Lazarus Agus Sukamto1, Mujiono2, Djukri2, & Victoria Henuhili2

1Research Center for Biology – Indonesian Institute of Sciences. Jl. Raya Jakarta - Bogor Km 46 Cibinong 16911, E-mail: [email protected] 2Universitas Negeri Yogyakarta, Yogyakarta

ABSTRAK

Kultur Pucuk Tanaman Nepenthes albomarginata Lobb ex Lindl. secara In Vitro. N. albomarginata adalah kantong semar kerah putih (white collared pitcher ), salah satu tanaman pemakan serangga yang sangat menarik sebagai tanaman hias. Tanaman ini terancam punah karena pengambilan dan kerusakan habitatnya. Penelitian perbanyakan secara in vitro dilakukan dengan menggunakan pucuk tanaman N. albomarginata pada media formulasi setengah Murashige and Skoog (1/2 MS) dengan tambahan zat pengatur tumbuh 6- benzyladenine (BA) 1 mg l-1 dengan atau tanpa kombinasi dengan á-naphthalene acetic acid (NAA) atau 4 amino 3,5,6, trichloropicolinic acid (Picloram) 0.5, 1, 1.5, and 2 mg l-1. Perlakuan kombinasi BA 1 mg l-1 dengan NAA 0.5 mg l-1 menghasilkan pertambahan tinggi tanaman terbesar. Tanaman menghasilkan jumlah daun terbanyak pada kontrol. Perlakuan BA 1 mg l-1 menumbuhkan tunas aksilar terbanyak, sedangkan kombinasinya dengan NAA 1.5 mg l-1 merupakan perlakuan yang dapat menghasilkan tunas adventif. Kombinasi BA 1 mg l-1 dan NAA 2 mg l-1 menginduksi kalus terbaik. Tanaman yang dihasilkan belum membentuk akar, tetapi pertumbuhan lebih lanjut dapat membentuk perakaran dan dapat hidup di luar botol kultur setelah diaklimasi.

Kata kunci: Nepenthes albomarginata, tanaman pemakan serangga, kantong semar, kultur pucuk tunas, in vitro

INTRODUCTION tips, consists of 129 species, the largest genus of in the world Carnivorous plant has an adaptation (Anonym 2010). There are many new strategy to unfavorable conditions, mostly natural and artificial hybrids among the to low nutrient availability in wet and acid genus Nepenthes (D'Amato 1998; Ano- soils (Adamec 1997), considers having nym 2010; Merbach & Merbach 2010). criteria of catching or trapping prey, ab- The distribution of Nepenthes is restricted sorbs metabolites from prey, and utilizes to tropical areas of the old world. Its habi- these metabolites for growth and devel- tats are very diverse: limestone cliffs that opment (Lloyd 1942). The consist continually damp, sand areas that have of over 600 species, out of a total about wet and dry season, swamps that under 300,000 species of vascular plants in the water part of the year, sea shores, epi- world (Adamec 1997). The genus Ne- phytes on tree plants, and creepers on penthes produce pitchers from its the surface of the soil (Pietropaolo &

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Pietropaolo 1986). propagation by cutting or layering takes These plants have been used for long time and low results. Vegetative medication, the fluid of unopened pitch- propagation by tissue culture (in vitro) is ers have been used as a laxative, rem- a good choice to get a lot number of uni- edy for burns, coughs, inflamed eyes, bed form plants in relatively short time in or- wetter's, fever, stomachache, dysentery, der to conserve, reduce exploitation on and various skin disorders. The open wild stocks, and replenish declining popu- pitchers have been used as pots to carry lations in the habitat. water and cook food, the vines have been Some carnivorous plants had been used as a cordage; and use for ornamen- propagated successfully by tissue culture, tal purposes (Perry & Metzger 1980; such as Cephalotus follicularis (Adams Pietropaolo & Pietropaolo 1986; Phillips et al. 1979a), Dionaea muscipula & Lamb 1996; D'Amato1998; (Teng1999; Jang et al. 2003), Drosera Puspitaningtyas & Wawangningrum intermedia, D. hilaris, D. brevifolia, D. 2007). rotundifolia, D. capensis, D. binata, N. albomarginata is one of the car- D. omissa, D. peltata, D. indica nivorous plants, a tropical plant native to (Kulkulczansa 1991; Anthony 1992; Borneo, Sumatra, and the Malay Penin- Sukamto 1999; Kim & Jang 2004; sular, characterized by coriaceous leaf in Jayaram & Prasad 2007), Drosophyllum texture and lack of petioles, are lusitanicum (Goncalves & Romano, up to 20 cm long, an attracted white col- 2005), Nepenthes khasiana (Latha & lar of velvety hairs directly beneath the Seeni 1994), Pinguicula moranensis, P. peristome, pitcher size can be up to 15 lusitanica (Adams et al. 1979b; cm high, the color usually is green, but Goncaves et. al. 2008), and Urticularia there are red, purple, and black forms. It inflexa (Ram et al. 1972). The best for- feeds mainly on (D'Amato 1998; mulation of culture medium was ½ MS Moran et. al. 2001; Merbach & Merbach (Anthony 1992; Kim & Jang 2004; 2010). Goncalves et al. 2008). There is no re- Nepenthes spp. in Southeast Asia port on N. albomarginata tissue culture; are in danger of extinction because of over those successes on other carnivorous exploitation, clear-cut deforesting, area plants could be used as references for in conversion to settlements, mount erup- vitro propagation of N. albomarginata. tion, climate change, and pollution (D'Amato 1998; Puspitaningtyas & MATERIALS AND METHODS Wawangningrum 2007). These plants could be propagated by seed, stem cut- Seeds of N. albomarginata origi- ting, air layering, and ground layering nated from West Sumatra, grown in vitro (Pietropaolo & Pietropaolo 1986). Sexual on half strength of macro and micro ele- propagation by seed has pollination prob- ments of MS basal medium formulation lem because male and female flowers (Murashige & Skoog 1962). Plantlets occurred on different plants. Vegetative grown from the seeds in vitro were cut

252 Shoot Tip Culture of Nepenthes albomarginata Lobb ex Lindl. their 1 cm shoot tips with five used period with TL lamp 40 watt. as explants. Each explant was grown on The parameter growth of increas- each culture bottles that contain 20 ml ing plantlet height, increasing leaf num- medium of ten series treatments with 12 ber, axillary shoot number, adventitious replications. There were 120 shoot tip shoot number, percentage of dried leaf, explants of N. albomarginata for experi- percentage of forming calli, quantitative mental study. and qualitative calli, and root formation Media culture based on MS basal were recorded every 2 weeks until 12 medium consisted of Mg SO4.7H2O 185 weeks of culture (WOC). Increasing mg l-1, CaCl2.2H2O 220 mg l-1, KNO3 plantlet height was measured by 950 mg l-1, NH4.NO3 825 mg l-1, substracting plantlet height to plantlet KH2PO4 85 mg l-1, FeSO4.7H2O 13.9 height 2 weeks before. This measure- mg l-1, MnSO4.7H2O 11.15 mg l-1, ment was also same for increasing leaf ZnSO4.7H2O 4.3 mg l-1, H3BO3 3.1 mg number. Axillary shoot is the shoot grown l-1, glycine 2 mg l-1, vitamin B1 0.1 mg l- from node, whereas adventitious shoot is 1, vitamin B6 0.5 mg l-1, nicotinic acid the shoot grown from other parts of the 0.5 mg l-1, KI 0.415 mg l-1, CoCl2.6H2O node. Good callus is white color, wet, and 0.0125 mg l-1, CuSO4.5H2O 0.0125 mg can grow further, whereas bad callus is l-1, NaMoO4.2H2O 0.0125 mg l-1, myo- black color, dry, and did not grow further. inositol 100 mg l-1, sucrose 20 g l-1, and Data were analyzed with analysis of vari- phytagel 2 g l-1 with addition of BA at 1 ance (ANOVA) to know differences mg l-1 with or without NAA or Picloram among treatments, followed with at 0.5, 1, 1.5, 2 mg l-1. The pH was ad- Duncan's multiple range test (DMRT), justed to 5.7 - 5.8, before it was auto- Procedure of Statistical Product and Ser- claved. The medium of 20 ml was poured vice Solution (SPSS) 12.00 for Windows. into 100 ml culture bottle, autoclaved at 1210C and 1 kg cm-2 pressure for ten RESULTS minutes. The experimental design was a ran- Increasing Plantlet Height and Leaf domized complete design (RCD) with one Number factor of hormone doses. There were ten Hormone treatments were not sig- treatments: (1) 0.5 MS (control), (2) BA nificantly different on increasing plantlet 1 mg l-1, (3) BA 1 mg l-1 + NAA 0.5 mg heights of N. albomarginata but the av- l-1, (4) BA 1 mg l-1 + NAA 1 mg l-1, (5) erage of plantlet height was higher in BA 1 mg l-1 + NAA 1.5 mg l-1, (6) BA 1 control than other treatments on 2 WOC. mg l-1 + NAA 2 mg l-1, (7) BA 1 mg l-1 The explants produced the highest plant- + Picloram 0.5 mg l-1, (8) BA 1 mg l-1 + lets on 4 WOC in media with addition of Picloram 1 mg l-1, (9) BA 1 mg l-1 + BA 1 mg l-1 or its combination with NAA Picloram 1.5 mg l-1, (10) BA 1 mg l-1 + 1 mg l-1. Later on, shoot tips of N. Picloram 2 mg l-1. The cultures were in- albomarginata produced the highest cubated at 26 ± 1 0C under 16:8 photo- plantlets on 6 - 12 WOC in media with

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addition of BA 1 mg l-1 + NAA 0.5 mg l- axillary shoot on 2 WOC. They started 1. Increasing plantlet height decreased by to produce axillary shoots at BA 1 mg l-1 increasing NAA doses combined with treatment or it's combined with NAA 0.5 BA; even combination of BA and Piclo- mg l-1 and NAA 1.5 mg l-1 on 4 WOC. ram at 1 - 2 mg l-1 did not have any in- Axillary shoot number at BA 1 mg l-1 creasing plantlets height (Figure 1 A; Table was significantly higher compared to 1). those other treatments on 6 - 12 WOC, N. albomarginata shoot tips pro- its combination with NAA inhibited pro- duced the highest leaf number in control ducing axillary buds. The combination of on 2 - 4 WOC. The addition of BA 1 mg BA and Picloram did not produce any l-1 + NAA 0.5 mg l-1 produced the high- axillary shoot (Figure 2 A; Table 1). est leaf number on 6 - 10 WOC. The Shoot tip explants of N. addition NAA at higher dose than 0.5 mg albomarginata started to produce adven- l-1 inhibited leaf growth (Figure 1 B). titious shoots in media with addition of Later on, N. albomarginata produced BA 1 mg l-1 + NAA 1.5 mg l-1 on 6 WOC the highest leaf number in control on 12 (Figure 2 B). The other treatments did WOC (Table 1). not produce any adventitious shoot (Table 1) Axillary and Adventitious Shoot Numbers Dried leaf Shoot tip explants of N. Explants had been cut and grown in albomarginata have not produced any media culture, became stress and showed

Table 1. Effects of BA, combined with NAA or Picloram on growth, dried leaf and callus formation of N. albomarginata shoot tip on 12 WOC Hormone Increasing Shoot Numbers Dried Callus Formation Treatments Leaf Height Leaf Axillary Adventi. % Quan Qualit. (l-1) (%) (mm) Number tit. 1/2 MS (Control) 3.50 ab 5.58 a 0.00 b 0.00 a 24.12 de 0.00 c - - BA 1.0 3.50 ab 4.92 ab 1.58 a 0.00 a 9.20 e 0.00 c - - BA 1.0+NA 0.5 4.08 a 5.42 a 0.17 b 0.00 a 12.02 e 8.33 bc + bad BA 1.0+NA 1.0 3.58 ab 4.92 ab 0.00 b 0.00 a 39.25 d 8.33 bc + bad BA 1.0+NA 1.5 2.83 b 3.83 bc 0.08 b 0.25 a 59.67 c 8.33 bc + good BA 1.0+NA 2.0 1.67 c 3.58 c 0.00 b 0.00 a 61.37 c 41.67 a ++++ good BA 1.0+Pic 0.5 1.18 c 1.64 d 0.00 b 0.00 a 80.62 b 0.00 c - - BA 1.0+Pic 1.0 0.00 d 0.27 e 0.00 b 0.00 a 94.15 ab 36.36 ab +++ good BA 1.0+Pic 1.5 0.00 d 0.00 e 0.00 b 0.00 a 100.00 a 25.00 abc ++ bad BA 1.0+Pic 2.0 0.00 d 0.00 e 0.00 b 0.00 a 100.00 a 33.33 ab ++ bad Notes: Means in the same group followed by the same letter in the columns are not significantly different based on DMRT test at the 5% level

254 Shoot Tip Culture of Nepenthes albomarginata Lobb ex Lindl.

A) B)

Figure 1. Effect of hormone treatments on increasing plantlet height (A) and increasing leaf number (B) of N. albomarginata every 2 WOC A) B)

Figure 2. Effect of hormone treatments on axillary shoot number (A) and adventitious shoot number (B) of N. albomarginata every 2 WOC A) B)

Figure 3. Effect of hormone treatments on dried leaf percentage (A) and calli percentage (B) of N. albomarginata every 2 WOC 0.5 - 2 mg l-1 increased substantially the drying their leaves 24.12%. The addition dried leaf, even combination of BA 1 mg of BA 1 mg l-1 and its combination with l-1 with Picloram 0.5 - 2 mg l-1 caused NAA 0.5 mg l-1 could decrease dried leaf deadly the explants, especially all explants 9.20% and 12.02% consecutively, even died at Picloram 1,5 - 2 mg l-1 (Figure though it was not significantly different. 3a; Table 1). Combination of BA 1 mg l-1 with NAA

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Figure 4. N. albomarginata culture in vitro (left) and . N. albomarginata ac climatized in vivo (right)

Callus formation DISCUSSION Shoot tip explants of N. albomarginata started to produce cal- The average of plantlet height of N. lus in BA 1 mg l-1 + NAA 1.5 mg l-1 albomarginata was higher in control than on 6 WOC. Then, producing calli oc- other treatments on 2 weeks of culture. curred in BA 1 mg l-1 combined with It could be the shoot tip explants have NAA 1 - 2 mg l-1 or Picloram 1 - 2 mg not adapted on media with addition of exogenous hormones. Later on, shoot tips l-1 on 8 WOC (Figure 3b). The best calli of N. albomarginata produced the high- production occurred on combination BA est plantlets in media with addition of BA 1 mg l-1 with NAA 2 mg l-1 (Table 1). 1 mg l-1 + NAA 0.5 mg l-1 on 6 - 12 Resulted calli of N. albomarginata were weeks of culture. It showed the shoot tips wet, white color, and compact on BA 1 have adapted to BA and NAA hormones. mg l-1 + NAA 2 mg l-1, but white color Increasing plantlet height decreased by and friable on BA 1 mg l-1 + Picloram 1 increasing NAA doses combined with mg l-1 treatments. BA. It indicated that combination of BA 1 mg l-1 and low dose of NAA (0.5 mg l- Root formation 1) caused synergism effect. This result All plantlets derived from the treat- was agreed with Windasari (2004) on ments did not produce any root. How- shoot tip culture of Chrysanthemum ever, eventually, plantlets grew further morifolium; Goleniowski et. al. (2003) on could produce pitchers and roots in vitro meristem culture of Origanum vulgare x (Figure 4). These plantlets were accli- applii. The high dose of NAA greatly matized and could survive in mixture stimulate ethylene formation, which it in- media of soil, sand, compost, coco peat, hibits elongation of stems and roots and burned rice hulls in vivo. (Salisbury & Ross, 1978). Combination of BA and Picloram at 1 - 2 mg l-1 did

256 Shoot Tip Culture of Nepenthes albomarginata Lobb ex Lindl. not have any increasing plantlets height. also inhibited producing axillary buds. It It because Picloram considers as hor- related to the ratio cytokinin to auxin that mone and also strong herbicide that is is an important to control morphogenesis, stronger than other auxins for inhibiting high values of ratio cytokinin to auxin organogenesis (Davis & Olson 1993) and stimulate shoot formation (Skoog & Picloram disturbs DNA transcription and Miller 1957). Combination of BA and Pi- RNA translation that inhibits producing cloram did not produce any axillary shoot. enzyme for growth regulator (Salisbury It showed that Picloram is stronger auxin & Ross 1978). than NAA that inhibited axillary shoot N. albomarginata plantlets pro- formation of N. albomarginata. duced the highest leaf number in control Plantlets of N. albomarginata pro- on 2 - 4 weeks of culture, but the addi- duced adventitious shoots only in media tion of BA 1 mg l-1 + NAA 0.5 mg l-1 with addition of BA 1 mg l-1 + NAA 1.5 produced the highest leaf number on 6 - mg l-1. It showed that combination of 10 weeks of culture. It showed the plant- BA 1 mg l-1 and NAA 1.5 mg l-1 was lets have adapted to BA and NAA hor- the right dose for initiating adventitious mones and caused synergism effect to shoots. This result has agreed with improve increasing leaf number on lower Bidwell (1979) that interaction of cytoki- dose of NAA. The same result had been nin with auxin at the right dose could in- reported by Chairunnisa (2004) on in- duce organ formation. creasing leaf number of Chrysanthemum. The addition of BA 1 mg l-1 and its NAA is an auxin that could increase leaf combination with 0.5 mg l-1 NAA inhib- number (Lyndon 1990). However, the ited leaf senescence, because cytokinins addition NAA at dose higher than 0.5 mg including BA could prevent chlorophyll l-1 inhibited leaf growth. The similar re- loss (Bidwell 1979) and inhibits leaf se- sult also reported by Windasari (2004) on nescence (Salisbury & Ross1978; De Chrysanthemum morifolium. Later on, Klerk 2010). Dried leaf of N. N. albomarginata produced the highest albomarginata explants decreased over leaf number in control on 12 weeks of twice by addition of BA 1 mg l-1. Differ- culture. It could be plantlets had been ex- ent result was obtained by Sukamto posure long enough to the hormones that (2001), which BA mg l-1 increased twice inhibited producing leaf number. dead explants. It happened because of Plantlets of N. albomarginata pro- media, micro-environment (light & tem- duced axillary shoots that was signifi- perature) and culture period differences. cantly higher at BA 1 mg l-1 compared Combination of BA 1 mg l-1 with NAA to those other treatments on 6 - 12 weeks or Picloram 0.5 - 2 mg l-1 increased sub- of culture. It showed that BA is an im- stantially the dried leaf. It happened be- portant cytokinin that releases apical cause auxins (NAA & Picloram) could dominance and stimulates outgrowth of increase ethylene, high ethylene accumu- axillary shoot (Bidwell 1979). The addi- lation in the bottle culture caused leaf tion of NAA inhibited activity of BA and senescence (George 1996; De Klerk

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2010). NAA and Picloram at high con- could be affected by inhibition of BA cy- centration could kill plants and catego- tokinin to auxin's function or MS strength, rize as effective herbicides (Salisbury & and the auxin type. This result confirmed Ross 1978). by Goncalves & Romano (2005) that the Shoot tip explants of N. albomar- highest rooting frequency was 0.25 MS ginata could produce callus at BA 1 mg and indole-3-butyric acid (IBA) at 0.2 mg l-1 + NAA 1.5 mg l-1, BA 1 mg l-1 + l-1 on Drosophyllum lusitanicum cul- NAA 1 - 2 mg l-1 or Picloram 1 - 2 mg l- ture; Goncalves et al. (2008) reported 1 but did not produced any callus on con- that 0.25 MS and indole acetic acid (IAA) trol, BA 1 mg l-1, BA + NAA or Piclo- at 0.2 mg l-1 produced the highest root- ram at doses 0.5 mg l-1. It showed shoot ing frequency, NAA at 0.5 mg l-1 sup- tip tissues of N. albomarginata did not pressed completely rooting response on have enough auxin endogen. Ratio cyto- Pinguicula lusitanica culture. kinin to auxin is an important to control This experiment revealed that shoot morphogenesis; low values of ratio cyto- tip culture of N. albomarginata could kinin to auxin stimulate callus formation grow on 0.5 MS media with or without (Skoog & Miller 1957). Plant tissues addition of hormones, but axillary and formed callus because of wounded tis- adventitious shoots could not formed on sue or stress (Kyte 1990). Resulted calli control. The highest increasing height of of N. albomarginata from BA 1 mg l-1 plantlets occurred on combination BA 1.0 + NAA 2 mg l-1 were wet, white color, mg l-1 and NAA 0.5 mg l-1, whereas the and compact, whereas from BA 1 mg l-1 highest increasing leaf number on con- + Picloram 1 mg l-1 were white color and trol. The best formation of axillary shoots friable. Calli structures are important for occurred on BA 1.0 mg l-1, whereas ad- further morphogenesis. Compact calli ventitious shoot only occurred on BA 1 structures are usually produce adventi- mg l-1 + NAA 1.5 mg l-1. The best cal- tious shoot, whereas friable structure in- lus occurred on BA 1 mg l-1 + NAA 2 dicated embryogenic calli (Torres 1989). mg l-1. All plantlets did not produce root The similar results of Picloram effect but eventually, plantlets grew further could were reported by Vaverde et al. (1987) produce pitchers and roots in vitro, also in embryogenesis of pejibaje palm; Omar survived after acclimatization in vivo. This & Novak (1990) in callogenesis of date successful propagation of N. palm; Davis & Olson (1993) in organo- albomarginata could be used for con- genesis of spurge. servation and increasing population. All plantlets derived from the treat- ments did not produce any root. This ex- ACKNOWLEDGMENTS periment was only use 0.5 MS and BA + NAA or Picloram but did not use NAA The authors thank to Dra. Hartuti- alone or other auxins. Adventitious root ningsih M. Siregar and Dr. Irawati who development is usually promoted by auxin gave fruits of N. albomarginata for ex- treatment (Salisbury & Ross 1978). It periments.

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Received: January 2011 Accepted: Aprl 2011

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