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ABSTRACT
ILYAS MARZUKI. Study on Morpho-ecotype and Characterization of Essential Oil, Isozyme, and DNA for Banda Nutmeg (Myristica fragrans Houtt),
Moluccas. Advised by H.M.H. BINTORO DJOEFRIE, SANDRA ARIFIN AZIZ,
HERDHATA AGUSTA, and MEMEN SURAHMAN.
Banda nutmeg is an important spice crop economically and ecologically.
The crop has long been growing and adapting well in Moluccas and North Moluccas areas. The objectives of the study are to explore the morphological and agronomic aspects of the plant in two different areas, Moluccas and North Moluccas,
to establish a nutmeg descriptor as a guide for nutmeg description, to extract oil in
nutmeg and characterize its volatile components, and to profile the isozymes and
DNA of the plant. Three locations in Moluccas i.e. Banda, Ambon, and Luhu,
and another three in North Moluccas i.e. Ternate, Tidore, and Bacan were selected
as sampling sites. Nutmeg trees with 25 to 50 cm in girth growing at two elevations, 0 to 50 m above sea level (asl) and 250 to 300 m asl were used as observational samples. For mor-phological and agronomic observations, 21 plant characters and two ripening levels of fruit (immature, 3 to 5 months and mature, > 7
months) were used. Oil in nutmeg and mace was extracted using hydrodistillation
method and the essential components of it were identified and characterized by
Gas Chromatography - Mass Spectrometry (GC-MS). For isozyme identification,
four isozyme systems were applied: peroxi-dase (PER), aspartate aminotransferase (AAT), esterase (EST), and acid phosphatase (ACP). For DNA analysis, two
decamer primers, OPE 10 and OPE 11, were used in PCR amplifications using
Randomly Amplified Polymorphism DNA (RAPD) proce-dure. DNA amplification ran for 45 cycles, and the PCR products were visualized and binary scored (1
for present band, 0 for absence). Isozymes visualized in dendo-gram display.
Similarity index (SI) and Nei’s genetic identity and distance of the DNA were
calculated as well. Morphological data collected were subjected to Bartlett test
and UPGMA cluster analysis, whilst agronomic and proximate data were analyzed with Anova and T test and Dunnett’ test, respectively. Oil contents and
essential components of nutmeg were analyzed with Anova and Bartlett test.
Results showed that Moluccas was characterized by mountainous physiography, tectonic hilly areas, volcanic, and karst with a slope of 10 to 40%. Ecotypes
of Banda and Ambon consisted mainly of volcanic soils, whereas Luhu consisted
of sediment. Generally, Moluccas has climate IIIC type, except Banda has IIB.
Annually, Moluccas receives rainfall 2,029 to 2,951 mm with two patterns, fluctuate (multiple wave) and double (double wave), temperature 22.1 to 31.0 oC, relative humidity 82.1 to 85.5%. It has climate IIIC (Tidore and Bacan) and IVB
(Ternate) types with fluc-tuate and bimodal rainfall patterns with solar radiation
57 to 59%. Meanwhile, North Mouluccas was characterized by volcanic hilly
landscape, tectonic, and karst soils with slope of 25 to 45%. Temperature regime
is between 23.6 and 30.1 oC, relative humidity 83%, and solar radiation 58%. Of
21 morphological plant characters obser-ved, 17 showed a high stability in phenotype. The altered phonotypic characters included fruit colour, shape and length of
peduncles, and fruit size index. Cluster analysis revealed that nutmeg trees could
be grouped into three categories according to their morphology, in which 90% of
the studied trees were similar to Banda type. In general, productions (fruit, seed,
and mace) were not significantly different bet-ween the two elevations. Generally,
nutmeg production in Banda ecotype was higher than other ecotypes. Moreover,
proximate analysis showed that water and pectin contents of the nutmeg from
Banda ecotype were significantly different from the remaining. Protein, fat contents, and EP, however, were not significantly diffe-rent. Based on climatic, soil,
and nutmeg characteristics, Moluccas and North Moluccas can be divided into
four morpho-ecotypes, i.e. Banda (covering Banda and Ambon), Luhu, Ternate
(covering Ternate and Bacan), and Tidore ecotypes. In this study, four nutmeg descriptors representing each ecotype have been established. Another results
showed that nutmeg originated from Banda, Ambon, Luhu, Ternate, and Bacan produced uncolored nutmeg oils, whereas that from Tidore was pale yellow. Oil contents of mature nut of Banda, Ambon, and Luhu were 11.69, 11.92, and 9.99%, respec-tively; meanwhile those in Ternate, Tidore, and Bacan were 7.9, 59.61, and
8.64%, consecutively. In addition, the immature seeds showed a higher level of oil
content. Oil contents of Banda, Ambon, and Luhu were respectively 13.07, 12.82, and
11.27%, whilst those of Ternate, Tidore, and Bacan were 13.32, 11.99, and 11.03%,
res-pectively. Furthermore, nutmeg oil of Moluccas has specific gravity ranged from
0.897 to 0.909 g/ml; refraction index, 1.489 to 1.491; optical rotation, +11.40 to
+16,30; and evapora-tion residue, 0.7 to 0.9%. For North Moluccas, the specific
gravity was 0.884 to 0.910 g/ml, the refraction index, 1.486 to 1,491, the optical rotation, +18.2 to +40.0, and the evaporation residue, 0.9 to 1.0%. GC-MS analysis revealed that nutmegs from Moluccas consisted of 28 to 31 components of essential
oils, and North Moluccas, 29 to 31 compo-nents. The analysis also showed that nutmeg from Banda ecotype comprised 52.8% monoterpene hydrocarbon (MH), 21.11%
oxygenated monoterpene (OM), and 18,04% aromatic compound (AC); Ambon’s,
consisted of 45.12% MH, 24.51% OM, and 16.97% AC; and Luhu’s was composed
of 56.06% MH, 27.34% OM, and 13,62% AC. The same analysis revealed that North
Moluccas’ nutmegs contained 29 to 31
essential oil components. Ternate’s nutmeg comprised 37.22% MH, 20.12% OM, and 2.80% AC; Tidore’s, 57.99% MH,
13.50% OM, and 24.27% AC; and Bacan’s, 50.45% MH, 11.64% OMT, and 34.15%
AC. Further analysis revealed that there were four important volatile oils fractions in
nutmegs, namely myristicin, elemicin, safrole, and eugenol. In Moluccas, it was
found that nutmeg contained 5.57 to 13.76% myristicin, whereas in North Moluccas,
myristicin content was 5.97 to 11.42%. In addition, it was found that safrole content
ranged from 0.97 to 2.46% in Moluccas, and 1.49 to 3.37% in North Moluccas. In
conclusion, Oil of Banda nutmeg shows a high stability in all physico-chemical
properties, except the optical rotation. Besides, Banda’s nutmeg has a higher content
in aromatic compound myristicin. Isozyme analysis showed that AAT of Moluccas
nutmeg produced zymogram with monomorphic bands, whereas that of North Moluccas revealed a slightly polymorphic. Low level of polymorphic in ACP was also detected. However, in EST isozyme, nutmeg of Moluccas and North Moluccas showed
monomorphic patterns. In RAPD analysis, Moluccas nutmeg showed monomorphic
DNA bands, whilst North Moluccas revealed a low level of polymorphism. With
OPE-11, Moluccas nutmeg has similarity index, SI, ranged from 0.83 to 1.00, and
North Moluccas from 0.73 to 0.86. With OPE-10, SI ranged from 0.95 to 1.00 for
Moluccas, and 0.78 to 0.83 for North Moluccas. In summary, it can be stated that
Banda nutmeg generally has a relatively high stability in morphological, isozymes
and DNA charac-teristics.
ABSTRAK
ILYAS MARZUKI. Studi Morfo-ekotipe dan Karakterisasi Minyak Atsiri,
Isozim, dan DNA Pala Banda (Myristica fragrans Houtt) Maluku. Di bawah
bimbingan H.M.H. BINTORO DJOEFRIE, SANDRA ARIFIN AZIZ,
HERDHATA AGUSTA, dan MEMEN SURAHMAN.
Pala Banda adalah tanaman rempah yang memiliki peranan penting baik
secara ekonomi maupun ekologi. Pala tumbuh dan beradaptasi sangat baik di
daerah Malu-ku. Penelitian bertujuan mengeksplorasi lebih jauh aspek ekologi,
morfologi dan agronomi pala Banda di Maluku dan Maluku Utara; membuat
deskriptor sebagai pedoman pendeskripsian sifat-sifat pala; mengekstraksi minyak
pala dan karakteri-sasi komponen atsirinya; dan mengidentifikasi isozim dan
DNA pala Banda. Tiga sampling di Maluku, yaitu Banda, Ambon, dan Luhu, serta
tiga lainnya di Maluku Utara: Ternate, Tidore, dan Bacan dipilih sebagai lokasi
penelitian. Pohon pala produktif berlingkar batang 25 sampai 50 cm, tumbuh di
dua elevasi, 0 sampai 50 m di atas permukaan laut (dpl) dan 250 sampai 300 m
dpl, digunakan sebagai bahan pengamatan. Untuk studi morfologi dan agronomi,
21 sifat morfologi tanaman dan dua tingkat kemasakan buah (muda, 3 sampai 5
bulan dan tua, > 7 bulan) dipelajari. Minyak biji dan fuli pala diekstrak secara
hidrodistilasi, sementara isolasi dan iden-tifikasi komponen atsiri minyak pala
dilakukan dengan metode Gas Kromatografi-Spektometri Massa (GC-MS).
Analisis isozim menggunkan empat jenis enzim: peroksidase (PER), aspartat
aminotransferase (AAT), esterase (EST), dan asam fosfatase (ACP); dan analisis
DNA dengan RAPD. Dalam analisis isozim digunakan bufer Tris-HCL dengan
pH 7,5 dan gel pati 10%. Elektroforesis dilakukan pada arus tetap 18 mA selama
5 jam. Gel diwarnai sesuai sistem pewarna tiap isozim dan difik-sasi dalam
larutan fiksatif. Pada analisis RAPD, DNA daun pala beku diekstrak kemudian
diamplifikasi menggunakan primer dekamer OPE-10 (GACTCTCAGG) dan
OPE-11 (GAGTCTCAGG). PCR berlangsung 45 siklus. Produk amplifikasi
dipisahkan dalam 1,5% gel elektroforesis dan diwarnai etidium bromida. Pita
DNA divisualisasi dan diskor secara biner, 1 bila ada pita dan 0 bila tidak ada.
Indeks kesamaan (SI) DNA dihitung termasuk identitas dan jarak genetik.
Hasil penelitian menunjukkan agroekologi Maluku dicirikan oleh fisiografi
pegunungan, perbukitan tektonik, vulkanik, dan karst dengan kelerengan 10-40%.
Wilayah Ambon dan Banda (Maluku) sebagian besar tersusun dari tanah berbahan
induk vulkanik, sementara Luhu adalah sedimen. Sebagian besar wilayah Maluku
bertipe iklim IIIC kecuali kepulauan Banda, IIB. Wilayah Maluku menerima
curah hujan 2.029-2.951 mm/tahun dengan pola fluaktuatif (multiple wave) dan
ganda (double wave), suhu udara 22,1- 31,0 0C, kelembaban udara 82,1-85,5%,
dan penyi-naran matahari 57- 59%. Maluku Utara dicirikan oleh tanah yang
berbahan induk sedimen dan plutonik dan didominasi fisiografi
perbukitan/pegunungan vulkan, tektonik, dan karst dengan kelerengan 25-45%.
Iklim Maluku Utara bertipe IIIC (Tidore dan Bacan) dan IVB (Ternate) dengan
pola hujan fluktuatif dan ganda. Suhu udara 23,6-30,1 0C, kelembaban udara 83%
dan penyinaran matahari 58%. Diantara 21 karakter morfologi tanaman yang
diamati, 17 memperlihatkan fenotipe yang stabil. Sifat-sifat morfologi yang
cenderung berubah menurut ekotipe adalah warna buah, bentuk pangkal buah,
panjang tangkai buah, dan indeks ukuran buah. Analisis klaster menunjukkan
pohon-pohon pala menurut karakter morfologinya mengelom-pok ke dalam tiga
kelas yang mana 90% mengikuti karakteristik pala dari ekotipe Banda. Secara
umum produksi pala di ekotipe Banda lebih tinggi daripada ekotipe lainnya, dan
tidak memperlihatkan perbedaan berarti antara dua elevasi. Analisis proksimat
memperlihatkan kadar air dan pektin daging buah pala ekotipe Banda ber-beda
secara nyata dengan lainnya, tetapi kadar protein, lemak, dan EP tidak berbeda.
Berdasarkan karakteristik iklim, tanah, dan tanaman pala, Maluku dan Maluku
Utara dapat dibagi ke dalam empat morfo-ekotipe: Banda (yang mencakup Banda
dan Ambon), Luhu, Ternate (yang meliputi Ternate dan Bacan), dan Tidore. Pada
pene-litian telah dihasilkan deskriptor pala Banda yang masing-masing mewakili
tipikal ekotipe. Hasil lainnya menunjukkan pala yang berasal dari ekotipe Banda,
Ambon, dan Luhu menghasilkan minyak pala yang tidak berwarna, sedangkan
minyak pala Tidore berwarna kuning pucat. Kadar minyak pala tua ekotipe
Banda, Ambon, dan Luhu berturut-turut adalah 11,69; 11,92; dan 11,03%. Hasil
selanjutnya memperli-hatkan biji pala muda mengandung minyak yang lebih
tinggi. Kadar minyak pala ekotipe Banda, Ambon, dan Luhu masing-masing
13,07; 12,82; dan 11,27%; dan Ternate, Tidore, dan Bacan adalah 13,32; 11,99;
dan 11,03%. Analisis fisiko-kimia menunjukkan minyak pala Maluku memiliki
bobot jenis 0,897-0,909 g/ml; indeks bias 1,489-1,491; putaran optik (+11,4)(+16,3); dan sisa penguapan 0,7-0,9%. Pala Maluku Utara memiliki bobot jenis
0,884-0,910 g/ml; indeks bias 1,486-1,491; putaran optik (+18,2)-(+40,0); dan
sisa penguapan 0,9-1,0%. Analisis GC-MS menunjukkan pala dari Maluku
tersusun dari 28-31 komponen atsiri, dan Maluku Utara 29-31 komponen. Minyak
atsiri pala ekotipe Banda tersusun atas 52,8% hidrokarbon monoterpen (HM);
21,11% monoterpen teroksigenasi (MT), dan 18,04% senyawa aromatik (SA);
pala ekotipe Ambon mengandung 45,12% HM; 24,51% MT; dan 16,97% SA; dan
pala Luhu, memiliki 56,06% HM, 27,34% MT, dan 13,62% SA. Analisis yang
sama menunjukkan pala Maluku Utara terdiri atas 29-31 komponen. Komponen
atsiri pala ekotipe Ternate adalah 37,22% HM; 20,12% MT; dan 2,80% SA; pala
dari Tidore tersusun dari 57,99% HM; 13,50% MT; dan 24,27% SA; dan pala
ekotipe Bacan mengandung 50,45% HM; 11,64% MT; dan 34,15% SA. Empat
komponen utama senyawa aromatik minyak atsiri pala Banda, yaitu miristisin,
elemisin, safrol, dan eugenol. Pala dari Maluku mengandung miris-tisin 5,5713,76%; sementara Maluku Utara 5,97-11,42%. Kadar safrol pala dari Maluku
0,97-2,46% dan Maluku Utara 1,49-3,37%. Hasil secara umum menunjuk-kan
pala Banda di enam ekotipe stabil dalam sifat fisiko-kimia kecuali putaran optik.
Kadar senyawa aromatik miristisin pala ekotipe Banda adalah yang tertinggi.
Anali-sis isozim menunjukkan bahwa zimogram AAT pala Maluku monomorfik,
sedang-kan Maluku Utara agak polimorfik. Isozim EST pala kedua daerah
monomorfik. Pada analisis RAPD, pala dari Maluku memperlihatkan pola pita
DNA monomorfik, tetapi sedikit polimorfik untuk pala Maluku Utara. Dengan
primer OPE-11, pala Maluku memperlihatkan indeks kesamaan (SI) 0,83-1,00;
dan Maluku Utara 0,73-0,86. Dengan OPE-10, SI pala Maluku 0,95-1,00; dan
0,78-0,83 untuk Maluku Utara. Dengan demikian dapat dinyatakan bahwa secara
umum pala Banda Maluku dan Maluku Utara memiliki stabilitas yang cukup
tinggi dalam sifat morfologi, isozim, dan DNA.