ORIGINAL RESEARCH ARTICLE

MORPHOLOGICAL CHARACTERS AND GENETIC VARIABILITY OF CHAMPACA IN BALI

I Made Sukewijaya1*, Made Sudiana Mahendra1, I Nyoman Rai1, and Gede Rai Maya Temaja2

1Laboratory of Agronomy and Horticulture Udayana University, Bali 2Laboratory of Pathology, Udayana University, Bali * Corresponding author : [email protected]

Received : 20th June 2016 | Accepted : 23rd August 2016

ABSTRACT Bali community utilize champaca flower for offering materials and worship, besides beauty salon purposes, the SPA aromatic ingredients, essential oils, perfumes, cosmetics, and drugs. Various champaca plants in Indonesia have not been studied as one of Indonesia's biodiversity that can be used as excellent genetic resources (germplasm). The objective of the study was to determine the genetic diversity of champaca in Bali. The results revealed that 12 (twelve) champaca accession morphologically was characterized. All of accessions obtained from cultivation centers champaca in Bali. Based on the characteristic was observed by morphological characters i.e.: (a) Cempaka Putih Wilis (b) Cempaka Putih Pateh, (c) Cempaka Putih Patemon, (d) Cempaka Putih Sibang, (e) Cempaka Kuning Muda Petemon, (f) Cempaka Kuning Kecil Patemon, (g) Cempaka Kuning Besar Patemon, (h) (i) Cempaka Kuning Kecil Sibang, (j) Cempaka Kuning Tua Sibang, (k) Cempaka Kuning Muda Sibang, and (l) Cempaka Kuning Punah Sibang. Morphologically, champaca in Bali can be grouped into 4 clusters and therefore, and based on RAPD analysis champaca in Bali could be grouped into 5 clusters.

Keywords: champaca, identification, genetic, diversity, Bali

INTRODUCTION aromatic of the flower is to be the Champaca plants have been specific characters of the plant. planted in Indonesia, especially in Bali As a tropical plant, champaca as a commercial commodity, thus it has a wide adaptation range in all support economically for Bali tropical area from low land to highland. communities. These plants have been Usually, this family spread in warm utilized as ceremony purposes and subtropical of East Asia, Southeast worship for Hindu’s people. Others, the Asia, and southeastern part of North champaca flower were used for beauty America, West India, Central America salon, aromatic ingredients, and SPA. (Kundu, 2009). In Bali, usually Taxonomically, champaca were planted champaca was used as landscape in Bali are Magnolia alba and element of yard and roadside, even in Magnolia champaca. The fragrant others part of Indonesia, champaca

ASIA OCEANIA BIOSCIENCES AND BIOTECHNOLOGY CONSORTIUM  29 INTERNATIONAL JOURNAL OF BIOSCIENCE AND BIOTECHOLOGY  VOL. IV NO. 1  SEPTEMBER 2016 ISSN: 9 77230 ISSN ONLINE: 9 772303 337 008 timber was used as building material, Champaca plant that was such as for Balinese Hindu sacred planted in Indonesia has many various building. phenotypic, as exposed by vegetative Beside of that uses, champaca and generative organs. Vary of flower was utilized for industrial raw champaca accession usually named by materials, such as essential oils character of their organ, such as color (Sanimah et al., 2008; Punjee et al., of flower. Diversity of champaca plant 2009; Krisdiana, 2010), parfumes, will continue to increase in accordance cosmetics, and drugs (Zumaidar, 2009; with the trait of champaca plants that Armiyanti et al., 2010). The content of can be propagated by seed. essential compounds that existed at Propagation by seed causes variations plants champaca organs have also been in champaca traits. According to Orwa used as an object of research. Research et al. (2009) champaca cross- by Normansyah et al. (2013) showed pollination can be assisted by beetles. that 80% ethanol extract of yellow Champaca plant varieties needs champaca containing terpenoids and to be identified in order to determine flavonoids compounds. Ahmad et al. their excellence properties that can be (2011) analyzed using HPTLC (high used as the basis of selection on further performance thin layer breeding programs. Generally, chromatography) found that the identification based on morphological quercetin compound (a flavonoid) in traits (shape and structure) easy and champaca leaf is higher than in the fast, however identification by such bark. Flowers of yellow champaca morphological traits has many identified to have the ability of constraints, it can be affected by antioxidant and antimicrobial activity environment, different of plant age, and (Kumar et al., 2011), and flowers of plant tissue (Khanuja et al., 2005). white champaca is also identified to has Plant identification that only base on antioxidant and antifungal activity morphological traits is difficult to (Bawa, 2011), while Ananthi and apply on seedling stage. Chitra (2013) examined the Identification is very important antioxidants of champaca flower by in- to inventory and identify many vitro method. champaca accessions that found and

30  ASIA OCEANIA BIOSCIENCES AND BIOTECHNOLOGY CONSORTIUM MORPHOLOGICAL CHARACTERS AND GENETIC VARIABILITY OF CHAMPACA IN BALI | I Made Sukewijaya, Made Sudiana Mahendra, I Nyoman Rai, and Gede Rai Maya Temaja cultivated in Bali and it is an effort to thermometer, scissors, knife, ice box, genetic germplasm conservation and and stationary. plant development in the future. First Leaves were washed by tap step is morphological identification and water until clean and dried by tissue, genetic identification. and 0.1 g fresh sample were used. The aim of the study is to find Sample was blended by mortar and out genetic variability of many added 800 µl CTAB buffer solution. champaca accessions and to assess the CTAB buffer solution consist of CTAB relationship between morphological 2%, 1,4M NaCl, 100 mM Tris HCl pH and genetic traits of champaca 8, 20 mM EDTA pH 8, 1% PVP-40 1, accessions. 1% mercaptoethnol, and kept under 65oC for 30 minutes. Mix solution MATERIALS AND METHODS This research was carried out at incubated under 65˚C for 60 minutes. central of cultivation area in Bali, that To homogenize the mixture was mixed are Sibang Gede and Sibang Kaja, for 10 minutes. Badung Regency and some areas that After incubated for 60 minutes champaca was found. Morphological mixtures were kept for 2 minutes then identification was done by inventory added for every 500 µl samples by 24 and observes important plant parts, chloroform mixture : 1 isoamil alcohol (CIAA) and vortex for 5 minutes then such as leaf and flower. The plant parts were collected and measured their centrifuged for 15 minutes at 12,000 entire dimension. rpm. Supernatan was taken and added After identification of cold isopropanol by 2/3 total morphological trait was done, genetic (supernatant + sodium acetate), then identification was carried out by using mixed by shake and kept at -20˚C for RAPD method. Young leaves were minimum 1 hour. Afterward, it used as material of RAPD analysis. centrifuged at 12,000 rpm for 10 Centrifuge, digital balance, freezer, minutes. Supernatant was taken and micro pipet, PCR machine, DNA sediment washed by 500 µl electrophoresis machine, UV ethanol 70% then centrifuged for 5 transilluminator, digital camera, vortex, minutes at 12,000 rpm. Supernatant was taken and DNA sediment was

ASIA OCEANIA BIOSCIENCES AND BIOTECHNOLOGY CONSORTIUM  31 INTERNATIONAL JOURNAL OF BIOSCIENCE AND BIOTECHOLOGY  VOL. IV NO. 1  SEPTEMBER 2016 ISSN: 9 77230 ISSN ONLINE: 9 772303 337 008 dried. After dry, DNA sediment was based on primer. Primers were utilized diluted again by 50 – 100 µl TE buffer in this experiment as shown in Table 1. + 1% RNAse in waterbath at 37˚C for PCR was carried out at 10 µl 60 minutes. The incubation product total volume on every PCR tube. Each was kept at refrigerator at 4oC. PCR consist of 5 µl PCR mix Go Taq® DNA purification was then Green (Promega), 0.25 µl 100 µM quantified by GeneQuant to detect primer (Sigma-Proligo), 2.5 µl DNA DNA concentration. Reference was set sample (template) and 2.25 steril by used psdH2O. Dilution factor aquabidest. (psdH2O) about 1998 µl and 2 µl DNA Tabel 1. Concentration and purity poured into cuvette, then put into the DNA GeneQuant. DNA DNA No. Sample concentration Dilution was carried out to purity (ng/ µl) detect the concentration of DNA that 1. Putih Wilis 2250 1.731 suitable for amplification process. To 2. Putih Pateh 1800 1.333 Putih get final volume of dilution, DNA 3. Patemon 2050 1.206 solution was added of psdH2O 4. Putih Sibang 3650 1.352 Kuning therefore the DNA concentration Muda 5. Patemon 1850 1.542 suitable for PCR Kuning Kecil process.ndnndndndndn 6.. Patemon 4000 1.455 Kuning Dilution formula: Besar V1. M1 = V2. M2 7. Patemon 1800 1.565 M1= concentration of DNA sample Kuning Kecil 8. Sibang 950 1.462 V1= initial volume of sample will be Kuning Tua diluted 9. Sibang 1100 1.692 Kuning M2= 2.5 ng/µl 10. Besar Sibang 300 1.200 V2= final volume Kuning 11. Muda Sibang 1400 1.867 Dilution volume = V2 - V1 Kuning DNA amplification was carried Punah 12. Sibang 1100 2.000 out by PCR to amplify DNA sequent

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Tabel 2. Primers were used to DNA seconds, annealing at 37oC for 1 amplification of champaca minute, and elongation at 72˚C for 1 Primer Nucleotide base sequent OPA 5 5’ AGGGGTCTTG 3’ minute and 30 seconds, then followed OPA 7 5’ GAAACGGGTG 3’ by final elongation at 72oC for 7 OPA 8 5’ GTGACGTAGG 3’ OPB 3 5’ CATCCCCCTG 3’ minutes. OPB 5 5’ TGCGCCCTTC 3’ DNA of PCR result then OPB 7 5’ GGTGACGCAG 3’ OPB 8 5’ GTCCACACGG 3’ electrophoreses by 1,0 % (b/v) agarosa OPC 1 5’ TTCGAGCCAG 3’ that was added ethidium bromide, in OPC 2 5’ GTGAGGCGTC 3’ OPC 4 5’ CCGCATCTAC 3’ TBE buffer (consist of 0,45 M Tris- DNA amplification was done HCl pH 8, 0,45 M Boric acid, 20 mM by PCR System BOECO equipment. EDTA) 100 volt in voltage for 45 First denaturation was done at 95oC for minutes. Subsequently, it visualized by 1 minute, then followed by 45 cycles UV light, as shown in Figure 1. with temperature at 95˚C for 45

OPA 5 OPA 7

OPA 8 OPA 3

OPB 5 OPB 7

OPB 8 OPC 1

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OPC 2 OPC 4 Figure 1. DNA amplification result of champaca by ten primers

Data was found by scoring of are white champaca and yellow electrophoresis result to each sample at champaca. Twelve variances were certain size, if presence of band was found as follows on Figure 2 and Table scored 1 and if absence scored 0. 3. Binary data then analyzed by NTSYS- White champaca (Magnolia pc ver. 2.2. alba) has morphology trait as follows: up to 30 meters in high. On the RESULT AND DISCUSSION branches of white champaca usually Based on the results of field covered with grayish fine hairs. The observations found that champaca leaves are oval and green. Petiole is plants cultivated as a cultivation plants quite long, reaching almost half the are in Sibang Gede and Sibang Kaja, length of the leaves. The white Badung Regency and Patemon, champaca also has white flowers that Buleleng Regency. Champaca spread have a distinctive fragrance (Orwa, all over Bali, sporadically from low 2009; Flora of China, 2008). land to highland. Morphological trait of yellow Based on inventory result, champaca plants (Magnolia champaca) champaca plant named based on flower is as follows (Orwa, 2009; Flora of color and their location. According to China, 2008). Yellow champaca trees similarity and difference of champaca can reach a height of 15-25 m. Ends of characters that were observed, it be twigs are hairy. The leaves are ovate discovered 12 champaca variances. lanceolate shape, with a pointed tip and Champaca plants grouped into 2 base, 10-28 x 4.5 to 11 cm, thin as the species that depend on color flower that skin.

34  ASIA OCEANIA BIOSCIENCES AND BIOTECHNOLOGY CONSORTIUM MORPHOLOGICAL CHARACTERS AND GENETIC VARIABILITY OF CHAMPACA IN BALI | I Made Sukewijaya, Made Sudiana Mahendra, I Nyoman Rai, and Gede Rai Maya Temaja

Figure 2. Twelve variances of champaca found in Bali

Table 3. Twelve variances of champaca found in Bali and their traits No. Name Specific Trait Variance 1. Cempaka Putih Wilis Flower color white; small sized 2. Cempaka Putih Pateh Flower color white; small sized, huge habitus 3. Cempaka Putih Patemon Flower color white; medium sized, leaf as Cempaka Putih Sibang 4. Cempaka Putih Sibang Flower color white; large sized 5. Cempaka Kuning Muda Patemon Flower color yellowish white; smooth tepal (Putih Cenana) 6. Cempaka Kuning Kecil Patemon Flower color yellow; small sized 7. Cempaka Kuning Besar Patemon Flower color yellow; large sized 8. Cempaka Kuning Kecil Sibang Flower color yellow; small sized 9. Cempaka Kuning Tua Sibang Flower color dark yellow; large sized 10. Cempaka Kuning Besar Sibang Flower color yellow; large sized 11. Cempaka Kuning Muda Sibang Flower color yellowish white; smooth tepal 12. Cempaka Kuning Punah Sibang Flower color pale yellow; unsmooth tepal

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Scared stipule of the petiole i.e Cempaka Putih Sibang. Cluster-4 length is more than half of the consist of 4 accessions i.e. Cempaka petiole.On the base of the pole-shaped Kuning Kecil Patemon, Cempaka flowers, ovaries and stamens clearly Kuning Kecil Sibang, Cempaka separated by a space. Ovary is more Kuning Tua Sibang, and Cempaka than 20, squeezed, flattened egg shape, Kuning Besar Sibang. Cluster 5 consist hairy, each with some ovules. Fruit of 2 accessions i.e. Cempaka Kuning shape is spherical elongated, slightly Besar Patemon and Cempaka Kuning bent, first green then pale gray, covered Punah Sibang. The dendrogram of with acne. Ripe seeds are dark red morphological trait as shown on Figure hanging out on the beam that extends 3. into a slender thread. After identify genetically by Morphologically, champaca can RAPD method by using 10 primers, be grouped into 5 clusters. First cluster champaca found in Bali can be consist of 1 accession i.e. Cempaka clustered into 4 clusters. Cluster-1 Putih Wilis, Cluster-2 consist of 4 consist of 1 accession i.e. Cempaka accession i.e. Cempaka Putih Pateh, Putih Wilis. Cluster-2 consist of 4 Cempaka Putih Patemon, Cempaka accessions i.e. Cempaka Putih Sibang, Kuning Muda Sibang, and Cempaka Cempaka Kuning Muda Patemon, Kuning Muda Patemon. Cempaka Kuning Muda Sibang, and Cluster-3 consist of 1 accession Cempaka Kuning Punah Sibang.

Figure 3. Dendrogram based on morphological trait of champaca found in Bali

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Coefisien

Figure 4. Dendrogram of genetic distance of 12 champaca found in Bali

Cluster-3 consist of 5 quercetin in Michelia champaca L. (Champa) leaves and stem accessions i.e. Cempaka Kuning Kecil bark by HPTLC. International Patemon, Cempaka Kuning Besar Journal of Pharma and Bio Sciences, 2(4): 388 – 397. Patemon, Cempaka Kuning Tua Ananthi, T. and M. Chitra. 2013. In Sibang, Cempaka Kuning Kecil vitro evaluation of antioxidant activity of Michelia champaca Sibang, and Cempaka Kuning Besar (L.) flowers. American Journal Sibang. Cluster-4 consist of 2 of Advanced Drug Delivery, 1(5): 734 – 742. accessions i.e Cempaka Putih Pateh Armiyanti, M. A. Kadir, S. Kadzimin, and Cempaka Putih Patemon. dan S. B. Panjaitan. 2010. Plant regeneration of Michelia Dendrogram of genetic analysis by champaca L., through somatic RAPD method shown in Figure 4. embryogenesis. African Journal of Biotechnology, 9(18), pp. Morphologically, champaca in Bali can 2640 – 2647. be grouped into 4 clusters and Bawa, I G. A. G. Aktivitas antioksidan dan antijamur senyawa atsiri therefore, based on RAPD analysis bunga cempaka putih (Michelia champaca in Bali could be grouped into alba). Jurnal Kimia, 5(1): 43 – 50. 5 clusters. Flora of China. 2008. Michelia Linnaeus, Sp. Pl. 1:536.1753. Flora of China 7: 77-90 REFERENCES Khanuja, S. P. S., Shasany, A. K.,

Pawar, A., Lal, R. K., Darokar, Ahmad, H., A. Mishra, R. Gupta, dan M. P., Naqvi,A. A., Rajkumar, S. A. Saraf. Determination of S., Sundaresan, V., Lal, N. and

ASIA OCEANIA BIOSCIENCES AND BIOTECHNOLOGY CONSORTIUM  37

MORPHOLOGICAL CHARACTERS AND GENETIC VARIABILITY OF CHAMPACA IN BALI | I Made Sukewijaya, Made Sudiana Mahendra, I Nyoman Rai, and Gede Rai Maya Temaja

Kumar, S. 2005. Essential oil Jawa dan Sumatera Barat constituents and RAPD markers menggunakan teknik RAPD. to establish species relationship Zuriat, 14 (1): 35 – 44. in Cymbopogon Spreng. Normansyah, A., Ariantari, N. P., (Poaceae). Biochemical Astuti, K. W. 2013. Profil Systematics and Ecology, kandungan kimia ekstrak etanol 33(2): 171 – 186. 80% kulit batang Michelia Krisdiana, D. 2010. Isolasi, champaca L. dengan Karakterisasi, Identifikasi kromatografi lapis tipis dan Komponen, dan Uji Aktivitas pereaksi pendeteksi. Jurnal Minyak Atsiri Bunga Cempaka Farmasi Udayana, 2(3): 153 – Putih (Michelia alba) terhadap 156. Escherichia coli dan Orwa C, Mutua A , Kindt R , Staphylococcus aureus. Jamnadass R, Simons A. 2009. [http://karya- Agroforestry Database: a tree ilmiah.um.ac.id/index.php/ reference and selection guide kimia/article/view/6168]. version 4.0 (http://www. Diakses pada tanggal 18 worldagroforestry.org/af/treedb/ Agustus 2013. ) Diakses tanggal 18 Pebruari Kumar, R. V., S. Kumar, S. 2014. Shashidhara, S. Anitha, dan M. Punjee, P. U. Dilokkunanant, U. Manjula. 2011. Antioxidant Sukkatta, S. Vajrodaya, V. and antimicrobial activities of Haruethaitanasan, P. various extracts of Michelia Pitpiangchan, and P. champaca Linn flowers. World Rakthaworn. 2009. Scented Applied Sciences Journal, 12 extracts and essential oil (4): 413-418. extraction from Michelia alba Kundu, M. 2012. Michelia champaca D.C. Kasetsart J. (Nat. Sci.), 43: Linn. Seed Leaflet, No. 158 197 – 203. Agustus 2012. Tropical Forest Sanimah, S., R. Suri, R. Nor Azizun, Research Institute. Forest & A. Hazniza, M. Radzali, I. Landscape Denmark, University Rusli, and M.D. Hassan. 2008. of Copenhagen. Volatile compounds of essential Kundu, S. R. 2009. A synopsis on oil from different stages of distribution and endemism of Michelia alba (cempaka putih) Magnoliaceae s.l. in Indian flower development. J. Trop. subcontinent. Thaiszia - J. Bot., Agric. and Fd. Sci., 36(1): 109 19: 47 – 60. – 119. Mansyah, E., Baihaki, A., Zumaidar. 2009. Kajian cempaka Setiamihardja, R., Darsa, J. S. kuning (Michelia champaca L.) dan Sobir. 2003. Analisis sebagai tumbuhan obat. J. variabilitas genetik manggis Floratek, 4: 81 – 85. (Garcinia mangostana L.) di

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