8
BAB II METODE PERBANYAKAN
Vanilla planifolia Andrews
A. Vanilla planifolia Andrews Panili merupakan sejenis tumbuhan famili Orchidaceae yang berasal dari Mexico dan kini banyak ditanam di kawasan tropika (Gambar 2.1). Tanaman panili muncul di Indonesia pada 1819 dibawa masuk oleh Merchal dan kemudian meluas ke sebagian Indonesia dan mulai ditanam di Pulau Jawa kemudian ke Bali, Aceh dan Sumatera Barat. Panili dari Pulau Jawa istimewa karena terkenal dengan kualitasnya yang mengandung kadar vanilin tinggi dan dikenal sebagai Java Vanilla Bean (Ackerman, 2003).
Gambar 2.1 Daerah Penyebaran Tanaman Panili. (Sumber : www.vanillareview.com)
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9
Klasifikasi tanaman panili menurut Correll, (1953) adalah : Kingdom
:
Plantae
Divisi
:
Liliopsida
Ordo
:
Aspargales
Family
:
Orchidacheae
Sub Family
:
Vanilloideae
Genus
:
Vanilla
Spesies
:
Vanilla planifolia Andrews
Tanaman ini memiliki batang monopodial dan habitusnya berupa semak yang merambat pada pohon berkayu yang ada disekitarnya (Gambar 2.2).
Gambar 2.2 Habitus Panili.
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10
Batang panili berbentuk silindris, lunak, beruas-ruas, tumbuh akar berupa akar adventitif yang berwarna putih dan muncul pada ruasnya, diameter batang ± 1,5 cm, berwarna hijau berbintik putih (Gambar 2.3) (Ackerman, 2003).
. Gambar 2.3 Morfologi Batang Panili yang Beruas-Ruas dengan Akar yang Tumbuh pada Tiap Ruasnya. (Sumber: www.uni-graz.at)
Anatomi batang menunjukkan, berkas pembuluh terletak menyebar di daerah meristem. Tidak mempunyai kambium vaskular sehingga tidak mempunyai jaringan sekunder. Pembuluh di jaringan ini disebut tipe berkas kolateral tertutup. Batasan antara empulur dan korteks tidak bisa dibedakan. Jaringan penguat terdiri dari serabut-serabut dengan dinding lignin yang tebal. Berkas Pengangkut terdiri atas xilem dan floem. Xilem terdiri atas serabut-serabut, trakeid dan parenkima. Xilem selalu terletak dalam berkas menghadap ke tengah batang. Floem terdiri atas sel tapisan dan sel pengiringnya kecil (biasanya persegi atau segi empat pada penampang
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11
melintang) Sel floem terletak dengan pola yang teratur di luar xilem. Berkasnya biasanya dikelilingi oleh serabut lignin dan membentuk sarung pembuluh (Gembong, 2005). Anatomi akar menunjukkan, pada jaringan akar terdapat epidermis, korteks (bagian terdalam korteks disebut endodermis, pada endodermis terdapat pita kaspari), perisikel, dan berkas pembuluh. Stele akar pada monokotil, floem terpisah berselangseling dan terletak di luar xilem tapi tidak sampai ke tengah akar sehingga dijumpai adanya empulur. Empulur akar ini merupakan derivat prokambium (Gembong, 2005). Daun panili termasuk daun tunggal, bentuknya lonjong, berseling, pangkal membulat dan ujungnya meruncing, panjang 15-19 cm dengan lebar 5-8 cm, pelepah pendek, tepi daun rata, pertulangan sejajar, dan warnanya hijau (Gambar 2.4). Anatomi daun terdiri atas: epidermis (pada epidermis terdapat stomata yang dilindungi sel penutup), mesofil (terdiri dari parenkim palisade dan parenkim spon) (Ackerman, 2003).
Gambar 2.4 Morfologi Daun Panili (Sumber: www.uni-graz.at)
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12
Bunga panili merupakan bunga majemuk, letaknya di ketiak daun, memiliki tangkai berbentuk silindris, panjangnya kurang lebih 0,5 cm, bermahkota, bunganya berwarna putih (Gambar 2.5). Kandungan vanilin berada dalam buah yang merupakan hasil dari penyerbukan bunga panili. Satu bunga hanya akan membentuk satu buah (Ackerman, 2003).
Gambar 2.5 Morfologi Bunga Panili (Sumber: www.uni-graz.at)
Bunga panili berumah satu dimana alat kelamin jantan (benang sari) dan alat kelamin betina (putik) berada dalam satu bunga. Terdapat selaput yang memisahkan kedua organ tersebut sehingga penyerbukan bunga secara alami sangat sulit dilakukan (Ackerman, 2003).. Penyerbukan bunga secara alami hanya dapat dilakukan dengan bantuan lebah Melipone yang hanya hidup di Mexico. Untuk itu satu-satunya cara penyerbukan bunga panili yang bisa dilakukan di negara lain yaitu hanya melalui penyerbukan buatan dengan bantuan tangan manusia (Ackerman, 2003).
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13
Buah panili termasuk buah polong dan bentuknya lonjong (Gambar 2.6), ketika masih muda buahnya berwarna hijau dan setelah tua akan berubah warna menjadi kuning kemerah-merahan. Buah panili yang sudah matang pada pohonnya lama-kelamaan akan membuka dan pada akhirnya mengering. Di dalamnya terkandung senyawa fenol yang menyebabkan timbulnya aroma khas pada buah panili (Ackerman, 2003).
Gambar 2.6 Morfologi Buah Panili (Sumber: www.uni-graz.at)
Dalam buah panili terkandung serbuk biji panili. Disebut serbuk karena ukurannya yang sangat kecil, bijinya berbentuk bulat, agak pipih dan berwarna hitam (Gambar 2.7). Seperti halnya biji pada kelompok anggrek-anggrekan yang lain, biji panili tidak dapat berkecambah tanpa bantuan fungi (Ackerman, 2003).
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14
Gambar 2.7 Morfologi Biji Panili. (A) Biji Dalam Buah Bu ah Panili Kering. (B) Kumpulan Biji yang Sudah Dipisahkan. (C) Struktur Biji Panili di Bawah Mikroskop. (Sumber www.javaorganicvanillabean.blogspot.com)
Pada sayatan melintang buah panili (Gambar 2.8), terlihat epidermisnya mengandung kloroplas. Pada sel epidermis ini terkandung kristal kalsium oksalat yang merupakan penyusun dinding buah. Buah panili dipanen pada usia sekitar 8-10 bulan dimana buah masih berwarna hijau dan kandungan vanilinnya relatif sedikit. Untuk meningkatkan kadar vanilin, diperlukan proses pengeringan sekitar kurang lebih 3-6 bulan (Frenkel, 2004).
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15
Pada sayatan melintang buah panili (Gambar 2.9) plasenta menyerupai rambut yang mengandung enzim untuk memproduksi vanilin. Sel ini juga menghasilkan lemak berbentuk globular (Gambar 2.9.a). Terdapat pula sel parenkim (Gambar 2.9.b) yang nantinya akan membentuk penyusun dinding buah berwarna putih sementara jaringan buah yang lainnya akan menghasilkan enzim-enzim lain untuk produksi vanillin (Frenkel, 2004).
Gambar 2.9 Sayatan Melintang (x 400) Buah Panili. (a) Plasenta, (b) Parenkim (Sumber : Frenkel, 2004) Vanilla planifolia Andrews merupakan tumbuhan CAM (Crassulacean Acid Metabolism). Tumbuhan ini membuka stomata pada malam hari dan menutupnya
pada siang hari. Tujuan menutup stomata pada siang hari adalah mencegah
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16
dilepaskan dari asam organik yang dibuat pada malam hari sebelum dimasukkan dalam kloroplas (Gembong, 2005).
B. Perbanyakan Panili Perbanyakan tanaman panili umumnya dilakukan secara vegetatif melalui stek batang ataupun melalui kultur jaringan. Penggunaan metode perbanyakan panili secara vegetatif menjadi salah satu metode perbanyakan tanaman panili yang paling umum dilakukan karena tanaman ini memerlukan waktu 3 hingga 4 tahun untuk menghasilkan bunga, dan bunganya pun hanya muncul sekali dalam setahun (Frenkel, 2004). Sejauh ini perbanyakan tanaman panili melalui metode kultur jaringan sudah banyak diupayakan dimana pada awalnya kultur panili menggunakan tunas aksilar sebagai eksplan (George, (George, 1997). Seiring
dengan berkembangnya metode metode kultur kultur
jaringan, saat ini perbanyakan tanaman panili secara in vitro sudah memanfaatkan eksplan lain berupa kultur kalus, protocorm, akar, dan organ lainnya (Ravishankar, 2004).
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17
maka di negara-negara lain metode penyerbukan bunga panili dilakukan dengan cara polinasi buatan yaitu dengan bantuan tangan manusia (Gambar 2.10) (Elizabeth, 2002).
Gambar 2.10 Penyerbukan bunga panili dengan bantuan tangan manusia. (Sumber : www.vanillareview.com)
Panili banyak dimanfaatkan dalam bidang industri dan merupakan komoditas ekspor penghasil devisa negara, 95 % diusahakan oleh perkebunan rakyat dan berpotensi besar untuk dikembangkan. Panili banyak digunakan sebagai pengharum dalam industri makanan, minuman dan confectionary products yang digunakan dalam bentuk utuh, bubuk, atau ekstrak. Untuk keperluan farmasi digunakan dalam bentuk tincture, sedang untuk parfum dalam bentuk tincture dan absolut (Nurjanah dan
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18
C. Kultur Jaringan Menurut Suryowinoto (1991 dalam Hendaryono & Wijayani, 1994), kultur jaringan dalam bahasa asing disebut sebagai tissue culture, weefsel cultuus atau gewebe kultur . Kultur adalah budidaya dan jaringan adalah sekelompok sel yang
mempunyai bentuk dan fungsi yang sama. Kultur jaringan berarti membudidayakan suatu jaringan tanaman menjadi tanaman kecil yang mempunyai sifat seperti induknya. Metode kultur jaringan dikembangkan untuk membantu memperbanyak tanaman, khususnya untuk tanaman yang sulit dikembangbiakkan secara generatif yang akan dieksploitasi secara besar-besaran (seperti vanilla, lada, jahe, pisang, jati, kapolaga, abaka), berbagai tanaman obat dan tanaman hortikultura, pada tanaman tahunan penyerbuk silang, (seperti jambu mente, cengkeh, melinjo, asam dan kapuk), pada berbagai tanaman tahunan seperti tanaman kehutanan (jati, cendana) dan tanaman buah-buahan (Hendaryono & Wijayani, 1994). Teknik ini dilakukan dengan cara mengisolasi bagian tanaman seperti biji, daun, akar atau mata tunas, yang kemudian ditumbuhkan ke dalam media buatan
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19
diperbanyak dalam jumlah yang besar sehingga tidak terlalu membutuhkan tempat yang luas, mampu menghasilkan bibit dengan jumlah besar dalam waktu yang singkat, kesehatan dan mutu bibit lebih terjamin, dan kecepatan tumbuh bibit lebih cepat dibandingkan dengan perbanyakan konvensional (Hendaryono & Wijayani, 2000).
D. BIJI Biji merupakan sumber makanan yang penting bagi hewan dan manusia. Biji dibentuk dengan adanya perkembangan bakal biji. Struktur biji terbagi menjadi tiga, yang pertama berupa plumula yang terdiri atas dua daun embriogenik, yang akan menjadi daun-daun sejati yang pertama tumbuhan bibit dan tunas terminal (apikal). Tunas ini ialah meristem dan padanyalah akan terjadi pertumbuhan batang yang
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20
Gambar 2.11 Struktur Biji (Sumber: www.wikipedia.com)
Pertumbuhan dan diferensiasi bakal biji, kantung embrio, serta endosperm dan embrio berlangsung menurut stadium yang saling terkait serta mengikuti urutan yang khas. Setelah pembuahan, pertumbuhan bakal biji segera diikuti oleh pertumbuhan
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21
sejumlah besar bahan makanan. Pada waktu perkecambahan, sel yang sama akan membalikkan proses metabolisme dan akan menghidrolisis zat hara yang tersimpan. Dalam pembalikan metabolisme yang mendasar itu diduga terlibat aktivasi dan deaktivasi gen (Hidayat, 1995).
E. Organogenesis dan Embriogenesis Organogenesis secara in vitro telah dicapai pada lebih dari 1000 jenis tanaman melalui seleksi empiris pada eksplan, komposisi medium, kontrol lingkungan fisik. Pada semua percobaan, proses organogenik dimulai dengan perubahan sel parenkim tunggal atau sekelompok kecil sel, dimana selanjutnya membelah menghasilkan suatu masa sel globuler atau meristemoid, besifat kenyal dan berkembang menjadi primordium pucuk atau akar. Kejadian ini dapat terjadi langsung pada eksplan atau
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22
yang sama tanpa melalui pemindahan ke medium baru. Tahap multiplikasi juga merupakan tahap pembentukan tunas adventif dan tunas aksiler yang tumbuh dari mata tunas adventif bersama-sama (George & Sherrington, 1984). Organogenesis tidak langsung untuk inisiasi tunas melalui kalus. Di sini tunas adventif maupun akar akan terbentuk dengan diawali terjadinya kalus. Kultur kalus memiliki potensial morfogenetik bervariasi. Kalus dari beberapa jenis tanaman atau dari beberapa eksplan, sering gagal beregenerasi membentuk tunas atau hanya membentuk akar. Perbanyakan tanaman melalui kalus akan menghasilkan tanaman dengan genetik yang bervariasi, dan ini sangat dikehendaki oleh pemulia tanaman sebagai sumber keragaman genetik (George & Sherrington, 1984). Embriogenesis adalah proses perkembangan embrio yang berasal dari zigot. Tetapi banyak tanaman yang mempunyai kemampuan untuk membentuk embrio
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23
sel-sel epidermal, namun ada juga dari jaringan petiola atau sel-sel korteks. Embrio somatik juga dapat terbentuk dari pucuk atau akar tanaman. Embrio dari jaringan nucelus terbentuk dari jaringan nucelus dari kultur ovul. Kemampuan embriogenetik dari nucelus tertingi bila jaringan tersebut lebih dahulu diinduksi menjadi kalus (George & Sherrington, 1984). Embriogenesis tidak langsung, embrio somatik terbentuk dari kalus dan kultur suspensi telah banyak dilakukan. Kalus yang diperoleh dari inisiasi awal akan memiliki kemampuan untuk beregenerasi membentuk embrio somatik yang tinggi dibanding kalus hasil subkultur. Seperti pada embriogenesis langsung, kemampuan ini tergantung pada eksplan awal yang dikulturkan. Eksplan memiliki kemampuan untuk menghasilkan embriogenesis langsung (misalnya nucelus poliembrionik pada Citrus), tetapi dapat juga membentuk kalus embrionik. Kadang embriogenesis dapat
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24
Embriogenesis dari kultur kalus sekunder tidak mempunyai kemampuan untuk morfogenetik, tetapi dapat diinduksi melalui subkultur ke medium yang menginduksi morfogenesis. Kalus yang dihasilkan dari eksplan daun muda yang tidak memiliki daya morfogenesis, bila di subkultur ke medium yang tepat, maka beberapa sel akan membentuk kalus dengan laju yang cepat dan dapat diinduksi membentuk struktur yang menyerupai embrioid dan selanjutnya membentuk planlet setelah subkultur. Hasil perbanyakan dengan cara ini ternyata seragam, dan secara morfologi
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25
Kultur embrio (embryo culture), yaitu penanaman embrio dewasa pada media buatan secara aseptis. Aplikasi kultur embrio ini antara lain perbanyakan tanaman, pematahan dormansi untuk mempercepat program pemuliaan serta perbanyakan tanaman yang sulit berkecambah secara alami, misalnya anggrek dan vanilla. Embryo culture atau kultur embrio adalah isolasi steril dari embrio muda ( immature embryo)
atau embrio dewasa/tua (mature embryo) secara in-vitro dengan tujuan untuk memperoleh tanaman yang lengkap.
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26
pengatur tumbuh. Komponen lain seperti senyawa nitrogen organik, berbagai asam organik, metabolit dan ekstrak tambahan tidak mutlak, tetapi dapat menguntungkan ketahanan sel dan perbanyakannya (Wetter & Constabel, 1991). Medium yang dikembangkan oleh Murashige dan Skoog (MS) merupakan salah satu medium kultur yang dilaporkan cocok digunakan pada berbagai macam tanaman (Bhojwani & Razdan, 1983). Keistimewaan medium MS adalah kandungan nitrat, kalium, dan amoniumnya yang tinggi, selain itu jumlah hara anorganik pada
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27
yang lain dengan inti polar sehingga akan terjadi endosperm (endospermium). Endosperm ini mengandung zat makanan dan segera setelah endosperm terbentuk, maka inti endosperm akan membelah diri berulang kali dengan cepat sehingga endosperm dapat menjadi bertambah besar (Hendaryono & Wijayani, 1994). Pertumbuhan embrio di dalam biji pada permulaannya berjalan dengan sangat lamban. Tetapi, setelah embrio itu dapat menyerap zat makanan yang tertimbun di dalam endosperm, maka pertumbuhannya akan menjadi bertambah cepat. Pada
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28
seperti asam sitrat, asam nikotinat, asam pantotenal, asam folat, niacin, riboflavin, dan thiamin. Terdapat pula dua hormon alami yaitu auksin dan sitokinin sebagai pendukung pembelahan sel embrio kelapa (Sunaryo, 2006). Air kelapa mengandung makronutrien, mikronutrien, protein, mineral, dan vitamin baik pada air kelapa yang berada dalam kelapa dewasa dan air kelapa yang berada dalam kelapa yang masih muda (Lampiran). 2. Medium Tomat
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29
Tabel 2.1 Kandungan Gizi Tomat Per 100 g Kandungan Gizi Tomat / 100 g Kalori 20 kal Protein
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30
Tumbuh adalah senyawa organik bukan nutrisi yang dalam konsentrasi rendah (<1mM) mendorong, menghambat atau secara kualitatif mengubah pertumbuhan dan perkembangan tanaman (Wetherell, 1976).
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31
Auksin pertama kali diisolasi pada tahun 1928 dari biji-bijian dan tepung sari bunga yang tidak aktif. Dari hasil isolasi didapatkan rumus kimia auksin (IAA = Asam Indolasetat) atau C H O N. Setelah ditemukan rumus kimia auksin, maka
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32
pertumbuhan tunas lateral oleh pengaruh auksin yang diproduksi tunas apikal ini dikenal dengan istilah dominansi apikal (McMahon et al, 2002). 2. Sitokinin
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33
3. Growmore Growmore adalah pupuk daun lengkap dalam bentuk kristal berwarna biru, sangat mudah larut dalam air. Dapat diserap dengan mudah oleh tanaman baik itu