ISSN: 1412-033X
PENERBIT:
Jurusan Biologi FMIPA Universitas Sebelas Maret Surakarta,
Puslitbang Bioteknologi dan Biodiversitas Universitas Sebelas Maret Surakarta
ALAMAT PENERBIT/REDAKSI:
LA B O R A T O R I U M PU S A T M I P A
Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375; Tel.: +62-271-646994 Psw. 398, 339; Fax.: +62-271-646655.
E-mail: [email protected]; [email protected]. Online: www.biology.uns.ac.id; www.unsjournals.com
TERBIT PERTAMA TAHUN:
2000
ISSN:
1412-033X
TERAKREDITASI BERDASARKAN KEPUTUSAN DIRJEN DIKTI DEPDIKNAS RI No. 52/DIKTI/Kep/2002
PEMIMPIN REDAKSI/PENANGGUNGJAWAB:
S u t a r n o
SEKRETARIS REDAKSI:
Ahmad Dwi Setyawan, Ari Pitoyo
PENYUNTING PELAKSANA:
Suranto (Biologi Molekuler), Marsusi, Solichatun (Botani),
Edwi Mahajoeno, Sugiyarto (Zoologi), Wiryanto, Kusumo Winarno (Ilmu Lingkungan)
PENYUNTING AHLI:
Prof. Ir. Djoko Marsono, Ph.D. (UGM Yogyakarta) Prof. Dr. Hadi S. Alikodra, M.Sc. (IPB Bogor)
Prof. Drs. Indrowuryatno, M.Si. (UNS Surakarta)
Prof. J.M. Cummins, M.Sc., Ph.D. (Murdoch University Australia) Prof. Dr. Jusup Subagja, M.Sc. (UGM Yogyakarta)
Prof. Dr. R.E. Soeriaatmadja, M.Sc. (ITB Bandung)
Dr. Setijati Sastrapradja (Yayasan KEHATI Jakarta) Dr. Dedi Darnaedi (Kebun Raya Bogor)
Dr. Elizabeth A. Wijaya (Herbarium Bogoriense Bogor) Dr. Yayuk R. Suhardjono (Museum Zoologi Bogor)
BIODIVERSITAS, Journal of Biological Diversitymempublikasikan tulisan ilmiah, baik hasil penelitian asli maupun telaah pustaka (review) dalam lingkup keanekaragaman hayati (biodiversitas) pada tingkat gen, spesies, dan ekosistem. Setiap
naskah yang dikirimkan akan ditelaah oleh redaktur pelaksana, redaktur ahli, dan redaktur tamu yang diundang secara khusus sesuai bidangnya. Dalam rangka menyongsong pasar bebas, penulis sangat dianjurkan menuliskan karyanya dalam Bahasa Inggris, meskipun tulisan dalam Bahasa Indonesia yang baik dan benar tetap sangat dihargai. Hingga nomor
ini, jurnal dikirimkan kepada institusi-institusi yang meminta tanpa biaya pengganti, sebagai bentuk pertukaran pustaka demi mendorong penelitian, perlindungan dan pemanfaatan lestari keanekaragaman hayati. Jurnal ini terbit empat kali
setahun, setiap bulan Januari, April, Juli, dan Oktober.
Format penulisan pada nomor ini merupakan acuan utama bagi para penulis, adapun pedoman ini hanya merupakan ringkasannya.
Setiap naskah harus disertai surat pengantar yang menyatakan bahwa tulisan merupakan hasil karya penulis atau para penulis dan belum pernah dipublikasikan. Penulis diminta mengirimkan dua kopi naskah dan satu disket ukuran 3½” atau compact disc (CD), kecuali naskah yang dikirim melalui e-mail. Pada koreksi terakhir kembali diminta satu disket untuk pencetakan.
Tulisan diketik pada satu sisi kertas putih, ukuran A4 (210x297 mm2), dalam satu kolom, menggunakan spasi ganda, jenis huruf Times New Roman, ukuran 12 point, dengan jarak tepi 2 cm di semua sisi. Program pengolah kata atau jenis huruf tambahan dapat digunakan, namun harus PC compatible dan berbasis Microsoft Word.
Nama ilmiah (genus, spesies, author), dan kultivar atau strain disebutkan secara lengkap pada penyebutan pertama kali. Nama genus dapat disingkat setelahnya penyebutan yang pertama, kecuali menimbulkan kerancuan. Nama author dapat dihilangkan setelah penyebutan pertama. Misalnya pertama kali ditulis Rhizopus oryzae L.
UICC 524, selanjutnya ditulis R. oryzae UICC 524. Nama daerah dapat dicantumkan apabila tidak menimbulkan makna ganda.
Penyebutan nama ilmiah secara lengkap dapat diulang pada bagian Bahan dan Metode. Tatanama kimia dan biokimia mengikuti aturan IUPAC-IUB. Simbol-simbol kimia standar dan penyingkatan untuk nama kimia dapat dilakukan apabila jelas dan umum digunakan, misalnya pertama kali ditulis lengkap butilat hidroksitoluen (BHT) selanjutnya ditulis BHT. Ukuran metrik menggunakan satuan SI, penggunaan satuan lain harus diikuti nilai ekuivalen dengan satuan SI pada penyebutan pertama. Penyingkatan satuan, seperti g, mg, ml, dan sebagainya tidak diikuti titik. Indek minus (m-2, l-1, h-1) disarankan untuk digunakan, kecuali dalam hal-hal seperti “per-tanaman” atau
“per-plot”. Persamaan matematika tidak selalu dapat dituliskan dalam satu kolom dengan teks, untuk itu dapat ditulis secara terpisah.
Angka satu hingga sepuluh dinyatakan dengan kata-kata, kecuali apabila berhubungan dengan pengukuran, sedangkan nilai di atasnya dituliskan dalam angka, kecuali di awal kalimat. Pecahan sebaiknya dinyatakan dalam desimal. Dalam teks digunakan “%” bukannya
“persen”. Pengungkapan ide dengan kalimat yang rumit dan bertele- tele perlu dihindari, sebaiknya digunakan kalimat yang efektif dan efisien. Naskah hasil penelitian diharapkan tidak lebih dari 25 halaman (termasuk gambar dan tabel), naskah telaah pustaka menyesuaikan, masing-masing halaman berisi 700-800 kata, atau sebanding dengan naskah dalam nomor penerbitan ini.
Judul ditulis secara padat, jelas, dan informatif, maksimum 20 kata. Judul ditulis dalam bahasa Indonesia dan Inggris untuk naskah dalam bahasa Indonesia atau bahasa Inggris saja untuk naskah dalam bahasa Inggris. Naskah yang terlalu panjang dapat dibuat berseri, tetapi naskah demikian jarang diterbitkan jurnal ini. Judul pelari (running title) sekitar lima kata. Nama penulis atau para penulis pada naskah kelompok ditulis secara lengkap dan tidak disingkat. Nama dan alamat institusi ditulis lengkap dengan nama dan nomor jalan (lokasi), kode pos, nomor telepon, nomor telepon genggam, nomor faksimili, alamat e-mail, dan website. Pada naskah kelompok perlu ditunjukkan penulis untuk korespondensi beserta alamat dengan urutan seperti di atas. Abstract sebaiknya tidak lebih dari 200 kata, ditulis dalam bahasa Indonesia dan Inggris untuk naskah dalam bahasa Indonesia (teks dalam bahasa Indonesia hanya untuk kepentingan keredaksian) atau bahasa Inggris saja untuk naskah dalam bahasa Inggris. Kata kunci (Keywords) sekitar lima kata, meliputi nama ilmiah dan daerah (apabila ada), topik penelitian dan metode-metode khusus yang digunakan. Pendahuluan (Introduction) sekitar 400-600 kata, meliputi latar belakang, tinjauan pustaka dan tujuan penelitian. Bahan dan Metode (Materials and Methods) sebaiknya ditekankan pada cara kerja dan cara analisis data. Hasil dan Pembahasan (Results and Discussion) ditulis sebagai satu rangkaian, pada tulisan yang cukup panjang sebaiknya dibuat beberapa sub judul. Pembahasan merupakan jawaban pertanyaan mengapa dan bagaimana hasil penelitian dapat terjadi, bukan sekedar mengungkapkan kembali hasil penelitian dalam bentuk kalimat. Pembahasan yang lengkap dan menyeluruh lebih disukai dari pada pembahasan yang tidak tuntas. Naskah telaah pustaka tanpa sub judul Bahan dan Metode, serta Hasil dan Pembahasan.
Kesimpulan (Conclusion) sebaiknya tetap diberikan, meskipun biasanya sudah terungkap pada Hasil dan Pembahasan. Ucapan terima kasih (Acknowledgments) apabila diperlukan ditulis secara singkat. Gambar dan Tabel maksimum tiga halaman, dapat dibuat dengan tinta cina atau printer laser. Judul gambar ditulis di bawah gambar, sedangkan judul table ditulis di atas tabel. Foto dicetak pada kertas glossy dan diberi keterangan. Gambar berwarna dapat diterima apabila informasi ilmiah dalam naskah dapat hilang tanpa gambar tersebut. Setiap gambar dan foto sebaiknya menyertakan file digital.
Penulis dianjurkan menyertakan foto atau gambar untuk sampul depan, meskipun tidak dimuat dalam naskah sendiri. Tidak ada lampiran, semua data atau analisis data dimasukkan dalam Hasil dan Pembahasan.
Pustaka dalam naskah ditulis dalam bentuk nama belakang penulis dan tahun. Pada kalimat yang diacu dari beberapa penulis, maka nama penulis diurutkan berdasarkan kebaharuan pustaka.
Naskah yang ditulis oleh dua penulis, maka nama keduanya disebutkan, sedang naskah yang ditulis oleh tiga penulis atau lebih, maka hanya nama penulis pertama ditulis diikuti et al. atau dkk., misalnya: Sprent dan Sprent (1990) atau (Smith 1982a, b; Baker and Manwell, 1991; Suranto et al., 1998). Pada sitasi bertingkat digunakan kata citatau dalam, misalnya (Gyorgy, 1991 cit Coward, 1999) atau Gyorgy (1991, dalam Coward, 1999).
Daftar Pustaka diketik dengan spasi ganda. Sitasi mengikuti CBE-ELSE-Vancouver style dengan modifikasi sebagai berikut:
Jurnal:
Suranto, S., K.H. Gough, D.D. Shukla, and C.K. Pallaghy. 1998. Coat protein sequence of Krish-infecting strain of Johnson-grass mosaic potyvirus. Archives of Virology 143: 1015-1020.
Buku:
Sprent, J.l., and P. Sprent. 1990. Nitrogen Fixing Organisms: Pure and Applied Aspects. London: Chapman and Hall.
Bab dalam buku:
Baker, C.M.A. and C. Manwell. 1991. Population genetics, molecular markers and gene conservation of bovine breeds. In: Hickman, C.G. (ed.). Cattle Genetic Resources. Amsterdam: Elsevier Science Publishers B.V.
Abstrak:
Liu, Q., S. Salih, J. Ingersoll, R. Meng, L. Owens, and F.
Hammerschlag. 2000. Response of transgenic ‘Royal Gala’ apple (Malus x domestica Borkh.) shoots, containing the modified cecropin MB39 gene to Erwinia amylovora [084]. Abstracts of 97th Annual International Conference of the American Society for Horticultural Science. Lake Buena Vista, Florida, 23-26 July 2000.
Prosiding:
Alikodra, H.S. 2000. Keanekaragaman hayati bagi pembangunan dae- rah otonom. Dalam: Setyawan, A.D. dan Sutarno (ed.). Menuju Taman Nasional Gunung Lawu, Prosiding Semiloka Nasional Konservasi Biodiversitas untuk Perlindungan dan Penyelamatan Plasma Nutfah di Pulau Jawa. Surakarta, 17-20 Juli 2000.
Skripsi, Tesis, Disertasi:
Purwoko, T. 2001. Biotransformasi Isoflavon oleh Rhizopus oryzae UICC 524 dan Aktivitas Antioksidan Isoflavon Aglikon dari Tempe terhadap Oksidasi Minyak Kedelai. [Tesis]. Jakarta: Universitas Indonesia.
Informasi dari Internet:
Rosauer, D. 1998. Forest Disturbance and Succession. http://
www.anu.edu.au/ Forestry/silvinative/ daniel/chapter1/1.1.html Naskah publikasi “in press” dapat disitasi dan dicantumkan dalam daftar pustaka. “Komunikasi pribadi” dapat disitasi, tetapi tidak dapat dicantumkan dalam daftar pustaka. Penelitian yang tidak dipublikasi- kan atau sedang dalam tahap pengajuan publikasi tidak dapat disitasi.
Beberapa catatan tambahan. Naskah diketik tanpa tanda hubung (-), kecuali kata ulang. Penggunaan huruf “l” (el) untuk “1” (satu) atau “O”
(oh) untuk “0” (nol) perlu dihindari. Simbol D, E, F, dan lain-lain dimasukkan melalui fasilitas insert, bukan mengubah jenis huruf.
Kata-kata dan tanda baca sesudahnya tidak diberi spasi.
Kemajuan Naskah. Pemberitahuan naskah dapat diterima atau ditolak akan diberitahukan sekitar satu bulan setelah pengiriman.
Naskah dapat ditolak apabila materi yang dikemukakan tidak sesuai dengan misi jurnal, kualitas materi rendah, format tidak sesuai, gaya bahasa terlalu rumit, terjadi ketidakjujuran keaslian penelitian, dan korespondensi tidak ditanggapi. Penulis atau penulis pertama pada naskah kelompok akan mendapatkan satu eksemplar jurnal yang memuat tulisannya selambat-lambatnya sebulan setelah naskah diterbitkan. Penulis akan kembali mendapatkan satu eksemplar jurnal nomor penerbitan berikutnya.
PENTING: Penulis atau para penulis dalam naskah kelompok setuju memindahkan hak cipta (copyright) naskah yang diterbitkan BIODIVERSITAS, Journal of Biological Diversitykepada Jurusan Biologi FMIPA UNS Surakarta. Penulis tidak lagi diperkenankan menerbitkan naskah secara utuh tanpa ijin penerbit. Penulis atau pihak lain diperkenankan memperbanyak naskah dalam jurnal ini selama tidak untuk tujuan komersial. Untuk penemuan baru, penulis disarankan mengurus hak patennya sebelum mempublikasikan dalam jurnal ini.
Pengantar Terbitan
Tidak dalam setiap terbitan Dewan Redaksi memberikan Pengantar Terbitan, namun pada pergantian tahun ini ada kejadian sangat luar biasa yang menguji ketahanan masyarakat Indonesia sebagai suatu bangsa, yang telah disatukan oleh Soekarno-Hatta, dengan proklamasi 17 Agustus 1945. Kejadian tersebut adalah gempa bumi diikuti gelombang tsunami yang memporak-porandakan kawasan Nangroe Aceh Darussalam dan sebagian Sumatera Utara. Bencana alam dengan episentrum tidak jauh dari pulau Simeuleu itu telah merenggut lebih dari 100.000 jiwa warga negara Indonesia, serta sejumlah hampir sama di negara-negara tetangga. Kejadian ini merupakan bencana paling dahsyat yang pernah terjadi di nusantara, bahkan lebih dahsyat dari meletusnya gunung Krakatau pada abad ke-18, dengan korban 30.000 jiwa;
bencana alam yang selama ini dikenal paling mematikan. Bencana ini menggugah rasa kemanusiaan setiap jiwa manusia.
Ucapan bela sungkawa dan simpati tidak akan cukup untuk menghapuskan rasa sedih, bahkan andaikan seluruh kata-kata di bumi ini ditumpahkan, tetap tidak akan cukup untuk menunjukkan betapa dalamnya duka ini. Bantuan material berapapun jumlahnya tidak akan cukup untuk menghapuskan ingatan dari kegetiran dan kengerian ini. Namun rasa berserah diri dengan tulus ikhlas kepada Allah SWT, serta tidak putus asa atas ujian yang diberikanNya, akan membantu untuk tetap tegar, terus beribadah dan menjalankan hidup ini dengan sungguh-sungguh, sebagaimana surat yang dikirim seorang sahabat, seorang penulis pada terbitan ini:
“Syukur alhamdulillah, saya selamat dari musibah tsunami di Aceh, namun keluarga saya (istri, dua anak, dan adik) terbawa arus dan jenazahnya tidak ditemukan, dan kondisi rumah hancur total tanpa bekas, selain itu juga ada dua keponakan saya yang hilang... Saat ini saya berada di Medan untuk mencari sanak keluarga yang mungkin (masih) ada yang selamat dan mengungsi di sana... Salam.” SENIN, 17 JANUARI 2005 Semoga Allah SWT memberi al-jannah kepada yang dipanggilNya, dan memberikan ganti yang lebih baik kepada yang ditinggalkan. Amin.
Dewan Redaksi perlu pula melaporkan adanya perubahan jadwal penerbitan. Apabila pada tahun-tahun sebelumnya jurnal ini terbit dua kali setahun, maka mulai tahun ini – insya Allah – akan terbit empat kali setahun setiap bulan Januari, April, Juli, dan Oktober. Hal ini dilakukan mengingat banyaknya artikel yang masuk, sehingga banyak di antaranya yang terpaksa dikembalikan, bukan karena tema yang tidak sesuai dengan misi jurnal, atau kualitas tulisan yang buruk, namun semata-mata karena terbatasnya jumlah halaman.
Dalam terbitan ini materi keanekaragaman pada aras spesies dan ekosistem, masih jauh lebih banyak dari pada aras genetik. Keanekaragaman genetik merupakan the ultimate dalam kajian keanekaragaman hayati; mengingat kesempatan untuk menjelajahi dunia hayati terbuka luas hampir tanpa batas. Hal-hal baru dengan sumbangan kuat terhadap kemajuan ilmu pengetahuan dan peradaban sering ditemukan, namun kajian ini membutuhkan sarana dan prasarana khusus, sehingga pihak yang dapat terlibat cenderung terbatas. Di negara-negara maju yang umumnya terletak di belahan bumi utara atau selatan dengan keanekaragaman hayati terbatas, kajian keanekaragaman genetik menjadi prioritas. Di negara berkembang seperti Indonesia – tanpa menyebutnya latah – kajian ini diharapkan juga akan menjadi pandu dalam pengembangan kajian hayati, meski pada kenyataannya terdapat keterbatasan yang sangat dalam teori dan metode, yang harus diimport hampir-hampir completely built up. Akibatnya kajian keanekaragaman genetik, masih sering dikaitkan dengan istilah-istilah etalase, mercu suar, lipstik atau bahkan kemayu; para ilmuwan menghambur-hamburkan dana yang besar dengan hasil yang dipertanyakan.
Di sisi lain, terdapat ruang yang sangat luas untuk kajian keanekaragaman pada aras spesies dan ekosistem. Teori dan metode kajian ini dengan mudah dapat diterapkan oleh siapapun dengan pelatihan sederhana. Namun karena sifatnya yang lumrah, kajian ini cenderung dinomorduakan; meskipun diyakini masih terdapat ribuan spesies tumbuhan, hewan, dan mikroba Indonesia yang belum teridentifikasi, baik di hutan dan terutama di lautan. Jurnal ini tidak bermaksud menganakemaskan salah satu kajian keanekaragaman hayati, hatta keanekaragaman budaya manusia yang secara nyata mempengaruhi keanekaragaman hayati. Banyaknya publikasi keanekaragaman spesies dan ekosistem dalam jurnal ini merupakan pilihan sadar untuk memberi kesempatan kajian yang biasa-biasa itu terbaca masyarakat luas, di samping – sejujurnya – karena terbatasnya naskah kajian genetik.
Terbitan ini secara khusus didedikasikan untuk para kolega dari Universitas Syiah Kuala (Unsyiah) Banda Aceh dan sekitarnya, untuk terus mengeksplorasi ayat-ayat qouliyah dankauniyahNya dalam segala keterbatasan yang ada.
Selamat membaca.
Wassalam, Dewan Redaksi
B I O D I V E R S I T A S ISSN: 1412-033X
Volume 6, Nomor 1 Januari 2005
Halaman: 1-5
jAlamat korespondensi:
Jl. Raya Bogor Km. 46, Cibinong-Bogor 16911.
Tel.: +62-21-8754587 Fax.: +62-21-8754588 e-mail: [email protected]
Storage and the Use of Peroxydase Enzyme to Detect Germination Capability of Sandoricum koetjape Merr. Seeds-
A Neglected Tropical Fruit Species
USEP SOETISNAj, DODY PRIADI, SRI HARTATI, ENNY SUDARMONOWATI Research Centre for Biotechnology, The Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911.
Received: 17 September 2004. Accepted: 14 October 2004.
ABSTRACT
Sandoricum koetjape which belongs to the group of mahogany, possesses seeds with sticky white aril, is a neglected local fruit species that might extinct if conservation efforts are not made. Besides preserving the embryos and embryonic axis on different periods of storage (0, 3, 5, 7 days) on a vacuum glass container containing silica gel, the latter organs were also preserved in liquid nitrogen to study the possibility of long-term storage. The water content of the preserved organs was measured in relation to the length of storage and the germination rate.
To determine the role of peroxidase in the germination rate of preserved zygotic embryos, the level of peroxidase was measured. Seeds of control and of 3-day storage were mostly germinated at day-6. The average rate of germination was reduced to 23.33% when the seeds were desiccated with silica gel for 7 days which resulted in 27.69% water content. This germination capability and the length of hypocotyls seem to correlate with peroxidase activity in the seeds. In general, the higher the percentage of germination, the longer the length of hypocotyls, the higher the peroxidase activity, except for seeds desiccated for 7 days. The range of peroxidase activity was 6.81-3856.20 'A/2min/mg. When the seeds were desiccated for 7 days, they still could germinate at day-18 which indicated by a very high peroxidase activity. Peroxidase activity assay could detect the viability within 15 seconds while the TTZ requires 15 minutes. Although the highest percentage of survived embryonic axis after storage in liquid nitrogen was only 23.42%, the results showed that soaking in 10-20% DMSO for 20 minutes of prerequisite as without DMSO led to no survival. These results offer an alternative procedure to detect the germination ability of seeds at early stage and longer period of preservation which could contribute to future ex situ conservation.
2005 Jurusan Biologi FMIPA UNS Surakarta Keywords: recalcitrant, seeds, embryos, desiccation, preservation, peroxidase, Sandoricum koetjape.
INTRODUCTION
Sandoricum which belongs to the family of Meliaceae comprises 5 species, four of which are restricted to western Malesia. The fifth, S. koetjape, is commonly cultivated mainly for its fruit and frequently naturalized from India, Burma (Myanmar) and Indo-China to Thailand, the whole of Malesian region, and tropical Australia and even in the New World Tropics. Timber plantations of S. koetjape have been established in Burma (Myanmar). The wood of this species is used for house construction, furniture, cabinet work, joinery, interior construction, shop fitting, paneling, planking and decking of boats, scantlings, carvings, butcher chopping blocks, packing cases, household implements, and sandals. The wood is also used for the production of veneer and plywood, blackboard, and for pulp and paper. It yields a good-quality charcoal and is used as firewood in Indonesia. It yields a lightweight to medium-weight hardwood with a density of 290-590 kg/m3 at 15% moisture content. The texture is moderately fine to slightly coarse and even. In Europe, the timber has been applied for furniture and interior finishing. In Malaysia, the timber is traded in mixed consignments of medium-weight hardwood.
Thailand which partly exports to Great Britain, considers this timber is potential (Sosef et al., 1998).
This species is a well-known fruit tree, the fruits being eaten fresh or processed into jam and chutney. The fruits of the other Sandoricum are also edible but less palatable. S.
koetjape is also an excellent shade tree with ornamental value, is planted as an avenue tree, and is suitable for use in shelter-belts. It has medicinal use as its pounded leaves are sudorific when applied to the skin and are used to make a decoction against diarrhea and fever. The powdered bark is an effective treatment for ringworm, shows anti-cancer activity, and has been used for tanning fishing nets. The roots are employed as an anti-diarrheic, anti-spasmodic, carminative, stomachic and are prescribed as a general tonic after childbirth. Limonoids isolated from the seeds showed insecticidal activity (Sosef et al., 1998).
It is semi-deciduous, small to large trees, up to 45-50 m tall and occurs scattered in primary or sometimes secondary rain forests, up to 1200 m altitude. S. koetjape has been reported from lowland dipterocarps forest but also from kerangas on podzolic soils in both perhumid and seasonal climates. It can be propagated by seed, but also by vegetative means like budding, grafting, inarching and marcotting. Seeds however, can not be stored for any length of time. The seed with or without the adhering pulp have 90-95% germination in 16-31 days. Various cultivars of this fruit tree exist including the tetraploid ones. Important tree collections are held in the Philippines, Malaysia and Thailand (Sosef et al., 1998).
There are three main categories of seed storage that have been recognized. Roberts (1973) defined orthodox and recalcitrant seeds, as those that survive long term dry storage and those that can not withstand dehydration,
respectively. More recently, a third category, intermediate, was identified comprising seeds that can withstand dehydration to a certain extent but have reduced longevity.
The first one could be further dried to low moisture contents (< 5%) without losing viability. The second one are characterized by the absence of maturation drying and are shed at moisture contents > 50% (on a Fresh Weight basis).
The so-called intermediate seeds survive drying to moderately low moisture contents (8-10%) but are often injured by low temperatures (Ellis, 1991).
Zygotic embryos/embryonic axes have been quite widely used in attempts at conserving germplasm of plants with recalcitrant seeds or those with seeds in the intermediate category. Cryopreservation system which is based on the reduction and subsequent detention of the metabolic function, including the cellular division of the explants, is accomplished when materials are brought to the temperature of liquid nitrogen (-196°C). Successful cryopreservation of plant material should be achievable by the appropriate balance between tissue water content and freezing rate making the use of cryoprotectants a secondary consideration. Thus, flash-drying and very fast freezing rate could be a solution to achieving successful cryopreservation method as suggested by Berjak et al. (1989).
Peroxidase has been implicated in a various physiological processes in plants. Plant peroxidases have been related to several processes such as cell growth, lignin biosynthesis (Gross, 1977), auxin metabolism (Gaspar, 1986), disease resistance, and wound healing (Espelie et al., 1986). Research concerning plant peroxidase activity has been reported in several species such as in mung bean (Chabanet et al., 1993), peanut (Zheng and van Huystee, 1992) and in pedicel of grape (Perez and Gomez, 1998).
The objectives of the study were to investigate possible storage method for seeds and embryonic axes and to determine alternative assay employing peroxidase to detect the viability of seeds of S. koetjapi.
MATERIALS AND METHODS
Source of plant materials
Seeds were collected from two mature trees grown in the Germplasm Garden of Research Centre for Biotechnology-Indonesian Institute of Sciences in Cibinong, Bogor District, West Java, Indonesia. The trees were originated from seeds which were collected from Serpong, Banten Province. The size of the fruits from where the seeds and embryonic axis were excised was ranging from 5.5 cm (small fruits) to 8.0 cm (larger fruits) in diameter in average. The aril was removed from the seeds by scrapping with ash washed and then mixed with fungicide Dithane and followed with natural drying for 1 hour.
Water content determination and germination test
Fifteen seeds were placed in a container in a desiccator which also contains 200 g silica gel and kept for either 3, 5 or 7 days. The number of replicates was three. Water content was measured on day 0 and 3-7 days after storage by weighing before and after drying in the oven at 100oC for 5 hours. Germination was conducted by placing the seeds on a wetted tissue placed in a germinator at 37oC.
Maintenance was conducted by spraying the tissues containing samples with water every day.
Growth of seeds after desiccation in growing media
Having been maintained in a germinator for one month during the germination test, the desiccated seeds were sown either in the soil or in the sand placed in a plastic tray.
They were watered every other day and maintained at room temperature.
Cryopreservation of embryonic axis
Embryonic axes of S. koetjape were soaked for 20 minutes in either 10% or 20% DMSO (dimethyl-sulfoxide) or 6% sucrose. Prior to freezing, they were placed in cryo tubes and followed with 2 drops of each related cryoprotectant solution. After thawing, the axes were germinated in wetted tissue and watered every day.
Viability test using peroxidase
Enzyme extraction. Enzyme extraction was carried out by grinding approximately 0.02 g to 0.1 g embryonic axis with 1 ml extraction buffer consisting of 25 mM Tris-HCl pH 7.5 using a plastic grinder. The homogenate was centrifuged at 10,000 rpm at 4oC for 15 minutes. The supernatant was used as crude peroxidase.
Protein determination. Protein content was determined with Bradford solution using the method developed by Grey (1990) using Bovine Serum Albumin as a standard.
Enzyme assay. Total peroxidase activity was estimated colorimetrically according to Campbell and Ellis (1992). The activity was determined by adding 720 Pl of 50 mM potassium phosphate pH 7.0 to 80 Pl enzyme extract and 2000 Pl of 0.5% guaiacol. The reaction was initiated by adding 2000 Pl of 0.075% hydrogen peroxidase followed by rapid inversion. The absorbance of the mixture was monitored at 470 nm after 2 minutes. Peroxidase activity was expressed as 'A/2min/mg protein.
Qualitative viability test
Tetrazolium test was conducted by applying 23.4 mM TTC solution in 0.05 M phosphate buffer pH 7.5 to both half-cut seeds and embryonic axis. Both germinated and non germinated ones were soaked in the solution for 15 to 60 minutes. Peroxidase solution used was the same as that for quantitative assay. Both materials (half-cut seeds and embryonic axis of germinated and non-germinated) were either soaked in 1 ml solution or given 20 Pl drop of solution.
RESULTS AND DISCUSSION
Critical water content and germination pattern
Water content of seeds before desiccation was 44.63%
which led to 86.67% germination in average. Desiccating the seeds for 3 days (35.20% moisture content) did not seem to have an effect on the germination rate as 90% of seeds still could germinate. Reducing the water content to 31.19% has caused the germinated rate declined 56.67%
which then dropped to 23.33% when the water content was reduced to 27.69% which was achieved by 7 days desiccation. The reduction of the water content not only caused the decline of the germination rate but also the delay in the germination. Without desiccation, seeds would germinate within 6 days while those subjected to desiccation germinated in 10-18 days depending on the water content (Table 1.). Figure 1. shows the effect of desiccation period on water content and germination rate of S. koetjape seeds.
SOETISNA et al. – Germination of Sandoricum koetjape 3 Table 1. Effect of desiccation period and water content on
germination rate of S. koetjape seeds.
Desiccation period (days)
Moisture content
(%)
Average germination
rate (%)*
Peak of germination
time (days) 0
3 5 7
44.63a 35.20ab 31.19ab 27.69b
86.67a 90.00a 56.67ab
23.33b
6 10 14 18 Note: * Observation was conducted at day-20. Figures followed with different letters indicate significant different at 1% based on Duncan Multiple Range Test.
Statistical analysis on the relation between water content and the percentage of germinated seeds showed that 7 days desiccation has reduced the germination rate significantly as the reduction of water content dropped from 44.6 to 27.7% (Table 1).
0 10 20 30 40 50 60 70 80 90 100
0 3 5 7
Desiccation Period (days)
Germination and Water Content (%)
Figure 1. Effect of desiccation period on water content and germination rate of S. koetjape seeds.
The range of water content level which is considered high in order to have high germination rate suggests that this species could be grouped either intermediate or recalcitrant which requires further confirmation through series of research in the future. Critical water content of seeds under this category is varied so preliminary information has to be searched supported with experiments prior to drying or desiccation. In certain recalcitrant seeds, the reduction rate of humidity affected further storage.
Azadirachta indica of Thailand provenance could maintain viability over 60% after 6 weeks storage if the seeds were subjected to sun drying for 2-3 days (Chaisurisri et al., 1986). Seeds of one plant species could behave differently as reported in neem. This plant seed has been designated as being recalcitrant (Ezumah, 1986) but also as orthodox (Tompsett, 1994), while according to Sacande et al. (1997) the seeds had intermediate storage behavior and were cold sensitive. The viable seed showed red color after treated by TTC solution (Figure 2.).
Effect of desiccation and water content on germination in growing media
The desiccated seeds could still grow and produce roots and shoots after 64-72 days depending on the desiccation treatments and growing media. Most seeds could germinate in the soil regardless desiccation treatments given as the percentage of seeds producing roots was ranging from 89.9 -100%. Unlike in the soil, those planted in the sand was ranging from 0-100% depending on desiccation periods
(Table 2.). Although the germination percentage in the sand was lower than in the soil, the number of the roots was higher and they were longer in size (Figure 3.). Sand also resulted shoots to emerge and elongate at 35 days after planting (64-72 days after desiccation and viability test in a germinator). Figure 4 shows the shoot growth of S. koetjape seeds which have been desiccated for 5 days at 2 months after sewing in sand placed in a plastic container. Those planted in the soil produced shoot slower as the shoots emerged not earlier than 55 days after planting. This indicates that seeds of S. koetjape were still viable for more than 2 months as long as the growing media is appropriate.
The fact that the growth of roots was excessive which tends to repress the growth of shoots, suggest that during germination a growth regulator to stimulate the growth of shoots such as GA3 is required.
Table 2. Effect of desiccation period and growing media on the production of roots and shoots of planted seeds of S. koetjape.
Desiccation Period (days)
Growing media
Seeds producing
roots (%)
Number of roots
per seeds*
Seeds producing
shoots (%)
Length of shoots
(cm)*
0 3 5 7
Soil 100 100 89.9 100
5 16.5 11.5 15
0 16.7 14.6 0
- 3.0**
1.0**
0.5**
0 3 5 7
Sand 100 50 83.3
0
50 14 19 -
100 16.7 33.3 0
3.0 1.0 4.0 - Note: *Observation at day 61 after sewing. ** All leaves were not yet opened.
Cryopreservation of embryonic axis
Embryonic axis which was not treated with DMSO or high sucrose (control) could not survive after storage in liquid nitrogen for 6 days. It seems that the effect of the concentration of DMSO was different to different types of S.
koetjape. The survival rates were 19.67% and 23.42%
which seeds were treated with either 10 or 20% DMSO, respectively. DMSO seems to be more effective than 6%
sucrose as the percentage of survival was slightly higher i.e. 23.42% and 10%, respectively (Table 3.).
The results which showed very low survival, suggest that treatments applied were not yet optimum. DMSO alone did not seem to be sufficient to protect embryonic axis to withstand ultra low temperature especially when rapid/direct freezing in liquid nitrogen is applied. Combination of DMSO with other cryoprotectants as has been applied to other plant species might solve the problems. Vitrification or encapsulation dehydration might be one alternative method for preserving embryonic axis of S. koetjape. Vitrification solution which employs 30% glycerol, 15% ethylene glycol, and 15% DMSO in liquid medium with 0.4 M sucrose could result in up to 80% survival of tropical tree species tried after immersion in liquid nitrogen (Sudarmonowati, 2000).
Other cryopreservation protocol has been developed mostly for various plant species but mainly temperate species such as Allium cepa (Lakhanpaul et al., 1996). The highest percentage (62.5%) of surviving embryos of citrus “Garut”
after storage in liquid nitrogen was obtained by dehydrating the naked embryos for 4 hours in laminar air flow and soaked in vitrification solution (a mixture between 0.8 M sucrose and 1.0 M glycerol) for 18 hours (Sudarmonowati et al., 1998). In other plant species such as Ribes nigrum (Benson et al., 1996), encapsulation-dehydration has given a better result than vitrification as it gave a higher survival.
germination rate
water content
On the other hand, embryo axes of “Garut” citrus and longan fruit were more suitable with vitrification technique for preservation. This might be because the composition of vitrification solution tried (0.8 M sucrose + 0.1 M glicerol + 200 ppm citric acid) might not be sufficient. Other causes might be the high content of phenolic compounds in the zygotic embryos which triggered with the would due to the formation of ice crystal which causes the phenolic compounds accumulated. As different species seems to require different technique, optimum one for each species needs to be developed.
Water content of seeds which embryonic axis was excised was still around 44.63% which considered still high.
Reduction of water content to the optimum one is, therefore, needed which implies that a technique for a more
appropriate dehydration needs to be optimized. Storage of the axis at -196oC has reduced the survival; further study is required to optimize the desiccation and freezing procedures. Embryonic axis will still be used as the use of this intact organ has the advantage that if cryopreservation procedures are successful, it should merely be a matter of manipulating post-freezing conditions to produce vigorous plantlets.
Peroxidase for detecting viability of stored seeds
The level of peroxidase declined in line with the reduction of germination rate, although in certain cases it was deviated. The highest level of peroxidase which was approaching 4000 U was obtained from seeds that have been desiccated for 7 days (Figure 5.).
0 500 1000 1500 2000 2500 3000 3500 4000
A/2 min/mg protein
0 3 5 7
Desiccation Period (days)
1a 1b 1c 2a 2b 2c 3a 3b 3c
Figure 5. Peroxidase activity of S. koetjape seeds after desiccation up to 7 days.
0 10 20 30 40 50 60 70 80 90 100
Hyocotyl Length (mm) or Percentage of Seeds Showing Peroxidase over 1000 U
0 3 5 7
Desiccation Period (days)
1a Peroxidase over 1000 U
1b 1c
2a 2b
2c 3a
3b 3c
Figure 6. Correlation between desiccation periods, hypocotyls length, and the percentage of germinated seeds of S. koetjape showing over 1000 U peroxidase level.
This treatment also led to the highest percentage of seeds containing peroxidase at the level of over 1000 U (Figure 6.). This enzyme also indicates the ability of rooting as the highest content was obtained from organs possessing the longest roots. The higher the hypocotyls of germinated seeds were indicated by higher peroxidase Figure 2. The assay of viability using TTC (left to right:
germinated, not yet germinated but viable, dead).
Figure 3. The growth of roots and shoots from seeds of S.
koetjape which have been desiccated for 0, 3, 5 and 7 days (left to right)
Figure 4. The shoot growth of S. koetjape seeds which have been desiccated for 5 days at 2 months after sewing in sand placed in a plastic container.
SOETISNA et al. – Germination of Sandoricum koetjape 5 level. Peroxidase is considered to be the main enzyme
responsible for the catabolism of the phytohormone IAA in higher plants, thereby, the suggestion of its participation in the regulation of plant growth as quoted by Zheng and van Huystee (1992). In terms of IAA catabolism, it can proceed in the absence (IAA oxidase) or presence (IAA peroxidase) of H2O2. The IAA oxidase and peroxidase catalyzed IAA catabolism was considered as part of auxin regulation in vivo, hence, a part of growth regulation. The correlation between auxin, in this case, IAA with peroxidase content has been proven in peanut as peroxidase content was twofold in hypocotyls cultured on medium containing high IAA (4 mg/l) as compared to control (Zheng and van Huystee, 1992). The fact that the growth of roots was considered excessive in seeds of S. koetjape after desiccation which resulted in the repression of shoot emergence indicated that the concentration of IAA is high which related to a high peroxidase level.
This result suggests that not only the viability, the length of hypocotyls of germinated seeds seems to correlate with the level of peroxidase in the seeds. The length of hypocotyls of germinated seeds desiccated for 3 days (35.2% of water content) was almost uniform while the others varied. In general, the activity of peroxidase was higher in non desiccated seeds and lower when the exposure was longer up to 5 days. In mung bean, the decrease of growth rate of hypocotyls was in parallel with the loss of cell wall extensibility which suggests changes in cell wall structure. The cessation of growth might result from cell wall stiffening processes related to the integration of diphenyl phenolic cross-linked wall polymer subunits into the polysaccharide network which is thought to be catalyzed by specific cell wall peroxidases. It was noted that in mung bean, the development of peroxidase activities in epidermal cell wall just at the onset of growth decrease (Cabhanet et al., 1993). This might explain the increase of peroxidase level when the seeds of S. koetjape were desiccated for 7 days.
Giving the peroxidase solution only 2 drops seems to be sufficient for detecting the viability of seeds. The time required using peroxidase for detecting the viability of seeds was much shorter than using TTZ, i.e. 15 seconds versus 15 minutes. The viable and germinated seeds had result much darker solution than the non germinated one (Figure 6.). This technique offers an alternative protocol for detecting the viability of stored tissues as well as other growth function. The role of peroxidase in plant species has been reported such as in Glycine max (Gillikin and Graham, 1991) and in peanut (Zheng and van Huystee, 1992).
CONCLUSIONS
Drying seeds up to 35.20% water content could still maintain the germination rate of S. koetjape to 90%, although they have been maintained for 2 months. With this condition, the longest storage period will be investigated in the future to confirm whether this water content level is the most optimum one for seed storage. It seems that there was a correlation between the viability of S. koetjape seeds and the level of peroxidase in the seeds. In addition, peroxidase activity might correlate with the growth of this species seeds. Detection of viability with peroxidase assay offers an alternative method to others as it could give faster result. Effort to cryopreserve the seeds or embryonic axis in the future would provide a long-term storage of this
recalcitrant species. Various factors affecting the success of this technique, are, therefore, need to be conducted.
ACKNOWLEDGEMENTS
The authors would like to thank the technical assistance of Ms. Nurchaedar Rahman and Mr. Nanang Taryana. The assistance of Mr. Jitno Rijadi in the documentation of the results was greatly acknowledged.
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Sacande, M., S.P.C. Groot, F.A. Hoekstra, R. De Castro, and R.J. Bino.
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Volume 6, Nomor 1 Januari 2005 Halaman: 6-11
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Biodiversity of Soil Microbes from Rhizosphere at Wamena Biological Garden (WBiG), Jayawijaya, Papua
SRI WIDAWATIj, SULIASIH, H.J.D. LATUPAPUA, ARWAN SUGIHARTO Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences, Bogor 16002
Received: 30 November 2004. Accepted: 26 Desember 2004.
ABSTRACT
The isolation, identification and population of soil microbes from rizosphere at WBiG had been done in the Soil Laboratories Microbiology, Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences (LIPI), Bogor. The soil was collected randomly from 16 sites in WBiG, and taken from 0-15 cm depth. Isolates of microbes were identified by Bergeys manual method for bacteria; Ellis method for fungi, and the morphology of isolate method for Actinomycetes. The population of microbes was estimated by plate count method. The result of isolation, identification and population soil microbes from 16 samples in WBiG showed that 20 isolates of bacteria (Azotobacter sp., Accinetobacter sp. , Bacillus sp., Citrobacter sp., Flavobacterium sp., Klebsiella sp., Nitrosomonas sp., Pseudomonas sp., Rhizobium sp., Thiobacillus sp., Azospirillum sp., Azotobacter chrococcum, Bacillus panthothenticus, Chromobacterium violaceum, C.lividum, Escherrrichia coli, Flavobacterium breve, Klebsiella aerogenes, Spaerotillus natans, and Staphylococcus epidermidis); nine isolates of fungi (Aspergillus niger,Bisporomyces, Monilia sp., Cephalospharium sp., Verticillum sp., Giocladium sp., Penicillium sp., Nelicocephalum sp., and Cuninghamella sp.), and seven isolates of Actinomycetes (Streptomyces, Streptosporangium, Nocardia, Thermomonospora, Thermoactinomyces, Micromonospora, Mycobacterium). The population of Bacillus (108-109),Rhizobium (106-107),Azospirillum (106-107), andThiobacillus (104 -109 ) were founded all of soil samples.
2005 Jurusan Biologi FMIPA UNS Surakarta Key words: soil microbes, Wamena Biological Garden (WbiG).
INTRODUCTION
Biodiversity of soil microbes has been regarded as human and vegetation life resource, especially the one connected with biological and environment resources. This can be achieved by developing a conservation system of ex-situ, like the one being pioneered by Indonesian Institute of Sciences (LIPI) in Kabupaten Jayawijaya called Wamena Biological Garden (WBiG). This is the first conservation area of ex-situ mountain biota in the Eastern part of Indonesia. It is located on Susu mountain and its surround- ings with the height of l595-1670 m above sea level. The temperature is 15-26qC with a rainfall of 1500-1900 mm. It has a wet tropical climate with vague difference between rainy and dry seasons (SW, 2004, personal observation).
WBiG is hilly with steep and gradual slopes having various typical soil colors which make it possible for the diversity of microbes, especially in rhizosphere area. WBiG environment is still virgin and has not been touched by the cruelty of chemical fertilizers and pesticides, and is an advantage and gives a positive impact to vegetation and indigenous microbes. Especially photosphere area, it is rich in biological activities as microbes feed on the carbon compounds exuded by root. Plants may exude compounds that attack certain species to the rhizosphere that protect the root from diseases (SW, 2004, personal observation).
Soil is a unity of subsistence that includes the varieties
of microbes, because microbes community is one of the important components of soil, therefore, the microbial activity and species compositions are generally influenced by the physical characteristic and soil chemical properties, climate and vegetation (Jha et al., 1992).
Soil microbes are one of biota communities, which are very interesting to be studied in order to find out their existence and uses. So, soil microbes have an important role to the subsistence on earth, because it has the role on biological and chemical cycling among the flora, fauna, and life of microbes itself. Nevertheless, not every soil microbe is suitable and compatible with the habitat and its host, and it is well known that they can perform symbiotic and com- mensalisms. Each type of microbes fills as a unique niche and plays a different role in nutrients cycling and soil structure.
Microorganism living in the soil can be grouped into bacteria, fungi, actinomycetes, algae, and protozoa (Rao, 1994). Some groups of soil microbes are useful as biofer- tilizer and biocontrol. They were Klebsiella, Nitrosomonas, Thiobacillus, Lactobacillus, Azotobacter, Azospirillum, Rhizobium, Bacillus, Pseudomonas, and Frankia (one of actinomycetes group). The other actinomycetes group is Streptomyces. It is potential as a source of various bioactive compounds used in pharmaceutical industry, agriculture and for other purpose. These Streptomyces were found to have high biodiversity and can be used as source of germ plasm. The work was also done to find candidate of bio- pesticide from Streptomyces that can be applied together with Rhizobium and phosphate solubilizing bacteria as bio- fertilizer (Lestari et al., 2002). Thus the soil microbes perform a wide range of function in the ecosystem.
The aim of the research is to know the biodiversity of soil microbes from rhizosphere at WBiG.
WIDAWATI et al. – Soil microbes at Wamena Biological Garden 7 MATERIALS AND METHODS
The soil was collected randomly (sampling square method/stratification) from 16 sites from rhizosphere area at WBiG such as Susu mountain, Dapuk hill, Dapuk valley and near Dapuk river area. Soil sample number 1 to 11 was collected from Susu mountain area. This area was dominated by Imperata cylindrica. Whereas number 12 to 16 were collected from Koliken valley, Koliken hill and surround of Dapuk river. This area was dominated by I.
cylindrica and seno (Castanopsis accuminattisima). The soil sample was taken from rhizosphere (0-15 cm depth). There are many different color types, physical element, and soil chemistry. It founded 11 type soil samples from WBiG (Table 1. and 2.).
One kg soil sample from 16 sites at WBiG was kept in black plastic bags (still in fresh condition) and in the Soil Microbiological Laboratory, Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences, Bogor these samples were air dried before the analysis of physic element and soil chemistry. The identification of microbes population used fresh soil. The population of microbes was determined by serial dilution plate count method (Thompson, 1989; Ravina et al., 1993). Isolation, identification, and the count of population of microbes used a selective medium, i.e. Pikovskaya, tauge agar, mannitol ashby, okon, yema congo red, DPY, PY, Na, LB, TA, etc.
Identification of soil microbes was estimated by morpho- logy, physiological test, microscopic and chemistry test.
Isolation, identification and population procedure of bacteria Ten grams fresh soil was suspended into 90 ml distilled water solution. Mix on wrish action shaker for one hour to provide mechanical desegregation of bacterial cell.
Subsequent dilutions were prepared by manually shaking the suspension for 10 seconds to resuspend the soil. Then transfer 1 ml an aliquot with a sterile pipette to 9 ml sterile distilled water in a test tube. This suspension was shacked manually for 10 seconds, and subsequent serial dilutions were prepared as method as noted above 10-1 to 10-7. Spread 0.2 ml of soil suspension from each serial dilution onto isolation selective agar medium (Pikovskaya, okon, manitol ashby, yema congo red, DPY, and PY). The number of bacteria colony was estimated after 3-7 days of incubation at 28oC by plate count method. Pick up the colonies to the same isolation medium, 8 strains per petridish. Select and transfer the different colonies to nutrient agar or LB (culture collection medium) or selective agar medium. The isolates of bacteria were identified by using morphological characteristic as observation of cell shape by monstaining (coccus, rod, short
rod/filamentous and spore formation), gram, stain, observation of living cell (motility, spore formation, and single, paired or chain) (Krieg and Holt, 1984).
Isolation, identification, and population procedure of fungi
Ten grams soil sample was suspended onto 90 ml of distilled water (in erlenmeyer glass), than mix on writh action shaker for one hour at 120 rpm. The soil extract was diluted from 10-1 to 10-7 . Spread 0.2 ml soil sample suspension from each serial dilution onto isolation TA medium with antibiotic. It was inoculated at 28oC for 3 days.
Pick up the colonies to the same isolation medium. Select and transfer the different
colonies to be identified by Charmichael et al. (1980), Domasch and Gans (1980), and Ellis (1993).
Isolation, identification, and population procedure of actinomycetes
Soil samples were dried at room temperature for 3 to 5 days. Then they were heated at 90 to 110oC for 10 to 60 minutes. The soil samples were spreaded onto isolation agar medium. They were incubated at 28oC for 7 to 14 days. Pick up the colonies to the same agar medium (8 colonies/petridish). The isolates were selected and transferred colonies to humic acid containing medium and YS medium. The isolation of medium for actinomycetes was humic acid-Vitamin agar medium: Humic acid 0.3 g, Na2HPO4 0.5 g; KCl 1.7 g, MgSO4.7H2O 0.05 g, FeSO4 7H2O 0.01 g, CaCO3 0.02g, B-Vitamins solution 2 ml, 4N- NaOH 0.6 ml, agar 18 g, pH 7.2. Humic acid-gellan gum medium: Humic acid 0.3 g, MOPS 1 g, CaCL2 0.44 g, 4 N- NaOH 0.6 ml, trace element solution 0.5 ml, gellan gum 7 g/liter, pH 7.0. Identification of actinomycetes isolates were used morphological characteristic i.e. observation of colony (growth, color of aerial and substrate mycelium, and diffusible pigment), on ISP No. 2, ISP No. 3, ISP No. 4, and YS agar media in petridish, microscopic morphology (spore, sporangium, aerial mycelium and substrate mycelium) (Miyadoh, 2003).
RESULT AND DISCUSSION
The typical characteristics of soil and vegetation at WBiG influence soil microbes diversity, such as bacteria, fungi and actinomycetes population (table 3, 4, and 5). In an aerobe condition, bacteria dominated the area and carried out some microbiological activities in the soil because fungi and actinomycetes could not grow well without oxygen (Rao, 1994).
Physical and chemical analyses show that, in general, the soil condition at WBiG is acid with pH range of 4 to 6.
The soil texture is sand clay. According to Rao (1994) the soil texture depended on the percentage of sand, dust, and clay. In the case of sand clay or dust clay, its particles came together to form an aggregate. The stability of an aggregate depended on both the content of organic matter in each type of the soil (Table 1.) and the nature condition of microbes which tied the soil particles to become one. Soil texture, therefore, is important for microbes and vegetation to survive in their habitat.
Table 1. Soil physic analysis from 11 sites in Wamena Biological Garden.
Soil Texture (%) Soil
samples Soil color Vegetation
Sand Clay Dust 1 Black Imperata cylindrica 18.03 38.06 43.01 2 Brown Imperata cylindrica 15.65 35.74 48.61 3 Gray Imperata cylindrica 10.89 38.82 49.28 4 Red Imperata cylindrica 11.51 65.75 22.69 5 Brown reddish Imperata cylindrica 16.36 43.45 40.19 6 Yellow Imperata cylindrica 17.51 41.42 41.06 7 Lime particle Imperata cylindrica 35.30 16.06 45.61 8 Black Pittosporum ramiflorum 5.79 51.37 42.84 9 Brown Vaccinium varingiaefolium 16.73 28.01 55.26 10 Brown Castanopsis accuminattisima 20.17 18.56 61.25 11 Dark brown Grevillea papuana 7.38 52.08 40.54
