LAPORAN PRAKTIKUM KULTUR JARINGAN TUMBUHAN Aklimatisasi Anggrek Dendrobium Anggrek Dendrobium sp. sp.
Disusun oleh :
Cindy Yong Kurnia Putri
(150801578)
LABORATORIUM BIOTEKNOLOGI FAKULTAS TEKNOBIOLOGI UNIVERSITAS ATMA JAYA YOGYAKARTA 2017
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KREDIT NILAI LAPORAN PRAKTIKUM KULTUR JARINGAN TUMBUHAN Judul : Sterilisasi Eksplan dan Kultur Kotiledon KRITERIA
NILAI STANDAR
COVER dan JUDUL I. PENDAHULUAN a. LATAR BELAKANG b. TUJUAN PRAKTIKUM II. TINJAUAN PUSTAKA III. METODE a. ALAT & BAHAN b. CARA KERJA III. HASIL & PEMBAHASAN IV. KESIMPULAN & SARAN DAFTAR PUSTAKA LAMPIRAN JUMLAH Nama NPM Golongan
10 20 15 35 10 10 100
: Cindy Yong Kurnia Kurnia Putri : 150801578 :B
Mengetahui,
Asisten
Hermanto
NILAI ACC
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I.
PENDAHULUAN
a. Latar Belakang
Sterilisasi adalah suatu proses untuk membunuh semua jasad renik yang ada, sehingga jika ditumbuhkan di dalam suatu medium tidak ada lagi jasad renik yang dapat berkembang biak. Sterilisasi harus dapat membunuh jasad renik yang paling tahan panas yaitu spora bakteri. Eksplan adalah bagian tanaman yang diambil dan akan dilakukan dilakukan kultur kultur jaringan tumbuhan. Eksplan yang digunakan tidak mengandung mikroba yang tidak diharapkan baik dipermukaan maupun didalam didalam selnya (Hadioetomo, 2000). Kultur kotiledon adalah teknik kultur jaringan yang menggunakan eksplan
berupa
menghilangkan
embrio. dormansi
Manfaat biji,
dari
kultur
memperpendek
kotiledon
siklus
adalah
perbanyakan,
menyelamatkan pertumbuhan embrio hibrid dan memahami kebutuhan kondisi
lingkungan
dan
nutrisional
untuk
perkembangan
embrio.
Penggunaan eksplan biji pada kultur in-vitro dapat menyerap air lebih cepat sehingga mempercepat induksi tunas. Hal ini disebabkan karena struktur permukaan kotiledon memiliki sel-sel yang berfungsi untuk penyerapan air (Cahyati dkk., 2016). Kultur kotiledon adalah sebuah endosperm dari pembuahan antara gamet jantan yang bersifat haploid dengan inti kandung lembaga yang bersifat diploid, sehingga kotiledon akan bersifat triploid. Kotiledon adalah organ cadangan makanan pada biji didalam suatu tumbuhan yang dimiliki oleh tanaman itu saja saat berkecambah sehingga hanya karbohidrat dan lemak bagi perkembangan embrio . Salah satu teknik kultur jaringan dengan menggunakan sumber eksplan kotiledon yang berasal dari biji yang telah matang. Manfaat dari kultur kotiledon yaitu untuk memberikan asupan cadangan makanan bagi embrio yang tidak dapat bertahan hidup di lingkungan in vitro, vitro, menghasilkan plantlet melalui embriogenesis dan
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Kotiledon yang dikulturkan harus berada dalam kondisi yang menunjukkan masa dormansi yang panjang, embrio hibrida hasil penyilangan interspesifik yang tidak kompatibel dengan endospermnya. Kondisi lingkungan kultur kotiledon yang sesuai untuk mendukung pertumbuhan adalah oksigen, cahaya (14 hari terang kemudian kemudian gelap untuk merangsang pembentukan klorofil), suhu (kadang perlu perlakuan dingin dengan suhu 4 oC untuk memecah dormansi) (Sugito dan Nugroho, 2004). Pada praktikum ini dilakukan kultur kotiledon yang berasal dari jagung ( Zea mays) mays) dengan menggunakan medium MS dengan bantuan hormon 2,4 D yang dapat menstimulasi pertumbuhan dan perkembangan kotiledon yang dapat tumbuh menjadi calon akar, calon tunas bahkan calon daun. Pertumbuhan dan perkembangan diamati selama 14 hari. Kultur ini dikatakan sukses apabila tidak mengalami kontaminasi dan memiliki morfologi yang baik seperti tumbuhnya calon tunas dan calon daun. Kultur kotiledon sangat penting untuk menghasilkan tunas dalam jangka waktu yang singkat serta menghasilkan biakan tanaman yang banyak secara invitro (Hendaryono vitro (Hendaryono dan Wijayani, 1994). b. Tujuan
1. Mengetahui cara perbanyakan jagung ( Zea mays) mays) secara vegetatif in vitro menggunakan eksplan calon tunas 2. Menginduksi pembentukan planlet dari calon tunas yang ditumbuhkan secara steril pada jagung ( Zea Zea mays) mays) 3. Mengetahui hasil pertumbuhan dan perkembangan kultur kotiledon jagung ( Zea Zea mays) mays) dari semua botol kultur pada ruang penabur LAF 4. Mengetahui hasil pertumbuhan dan perkembangan kultur kotiledon jagung ( Zea Zea mays) mays) dari semua botol kultur pada ruang penabur enkas 5. Mengetahui perbedaan antara kultur embrio dan kultur kotiledon
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II.
Kultur
jaringan
TINJAUAN PUSTAKA
pada
suatu
tumbuhan
merupakan
suatu
cara
membudidayakan suatu jaringan tumbuhan menjadi tumbuhan kecil yang mempunyai sifat seperti induknya dimana pelaksanaan teknik kultur jaringan ini berdasarkan teori sel seperti yang ditemukan oleh Scheiden dan Scheiden dan Schwann bahwa Schwann bahwa sel mempunyai
kemampuan
autonom,
bahkan
mempunyai
kemampuan
totipotensi. Totipotensi sel adalah kemampuan setiap sel, dari mana saja sel tersebut diambil, apabila diletakan dalam lingkungan yang sesuai akan dapat tumbuh menjadi tanaman yang sempurna. Embriogenesis somatik adalah proses dimana sel somatik (haploid bahkan diploid) berkembang membentuk tumbuhan baru melalui tahap perkembangan embrio yang spesifik tanpa melalui fusi gamet (Hendaryono dan Wijayani 1994). Eksplan adalah bagian atau bahan tanaman yang akan dikultur dalam proses kultur jaringan tumbuhan. Pemilihan eksplan harus pada bagian tanaman muda dan mudah tumbuh, contohnya adalah daun muda, ujung batang dan keping biji. Setiap eksplan memerlukan sterilisasi agar terhindar dari agen kontaminan. Sterilisasi adalah suatu proses untuk membunuh semua jasad renik yang ada, sehingga jika ditumbuhkan di dalam suatu medium tidak ada lagi jasad renik yang dapat berkembang biak. Sterilisasi harus dapat membunuh jasad renik yang paling tahan panas yaitu spora bakteri (Hadioetomo, 2000). Menurut Manullang dkk. (2006), kontaminasi dari bahan tanam sulit diatasi maka diperlukan bahan sterilan yang cocok untuk terhindar dari bahan kontaminan. Apabila kontaminan tidak dihilangkan maka media berupa gula, vitamin bahkan mineral dalam botol akan terpenuhi oleh kontaminan dan menyebabkan eksplan mati. Prinsip sterilisasi eksplan yaitu mensterilkan eksplan dari berbagai mikroorganisme namun eksplan tidak ikut mati. Sterilisasi eksplan dibagi menjadi dua macam metode yaitu: a. Sterilisasi secara mekanis, digunakan untuk eksplan yang keras bahkan
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tiga kali. Contoh bahan yang disterilisasi secara mekanis adalah tebu dan biji salak b. Sterilisasi secara kimiawi, digunakan untuk eksplan yang lunak seperti daun dan tangkai daun. Bahan kimia yang sering digunakan untuk sterilisasi secara kimiawi adalah: 1. Sodium hipoklorit, atau disebut juga clorox dan biasanya digunakan pada konsentrasi 5%-10% selama 5-10 menit dengan perbandingan antara clorox dengan aquades sebesar 1:4. 2. Merkuri klorit, atau disebut juga sublimat 0,05% yang memiliki prinsip kinerja yang sama dengan sodium hipoklorit namun hanya memerlukan waktu yang lebih pendek karena merkuri klorit bersifat keras apabila terlalu lama maka menyebabkan kerusakan pada eksplan. 3. Alkohol 70%, dimana bahan kimia ini sering digunakan sebagai agen sterilan dan konsentrasi alkohol yang paling baik untuk membunuh agen kontaminan adalah alkohol 70%. Menurut George dan Sherrington (1984), berdasarkan letak kontaminan maka kon taminasi eksplan dibagi menjadi dua yaitu kontaminasi internal dan eksternal. Kontaminasi internal merupakan kontaminan yang berasal dari jaringan tanaman tersebut maka diperlukan fungisida sistemik bahkan antibiotik. Kontaminasi eksternal adalah kontaminasi yang berasal dari permukaan luar bahan tanam sehingga dapat disterilisasi hanya bagian permukaan luar eksplan saja. Teknik kultur in-vitro dapat berhasil apabila pemilihan eksplan yang baik dan bebas dari kontaminan, pengaturan udara yang baik (terutama pada kultur cair) dengan benar seperti sirkulasi udara dengan eksplan harus bersih dan steril, keadaan aseptik dimana dalam kultur in-vitro harus menjaga sterilitas dan memiliki prinsip aseptis agar terhindar dari agen kontaminan serta penggunaan medium yang cocok agar memperoleh hasil kultur yang baik dan dapat disubkultur untuk kepentingan kultur in-vitro. Apabila menggunakan embrio bahkan bagian biji sebagai eksplan maka
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eksplan yang baik), waktu inhibisi (waktu dimana tanaman mengalami pertumbuhan dan perkembangan dengan cepat lalu nanti akan berhenti namun tergantung pada kondisi lingkungan), temperatur (eksplan yang tumbuh akan dipengaruhi oleh temperatur yang sesuai pada eksplan) dan dormansi (keadaan berhenti bertumbuh pada tumbuhan sebagai adanya keadaan keadaan lingkungan yang tidak mendukung) (Yuniarti dan Djaman, 2015). Menurut Ardiansyah dkk. (2014), teknik sterilisasi permukaan eksplan banyak digunakan untuk menghilangkan kontaminan pada permukaan eksplan dengan merendam eksplan pada larutan desinfektan dengan konsentrasi tertentu. Contoh agen sterilisasi permukaan pada permukaan eksplan yaitu AgNO 2 (perak nitrat), HgCl2 (merkuri klorida), H2O2 (hidrogen peroksida), alkohol dan etanol. Sterilan yang biasa dipakai untuk sterilisasi permukaan adalah natrium hipoklorit (NaOCl) atau kalsium hipoklorit (Ca(OCl) 2) yang dapat mengurangi agen kontaminan dengan cara mikropropagasi dimana Ca(OCl) 2 memiliki pH yang tidak stabil dan merusak jaringan sedangkan NaOCl tidak dapat merusak jaringan dan pH stabil. Berdasarkan konsentrasi bahan dan waktu yang diperlukan, sterilisasi eksplan dibagi menjadi 3 yaitu: 1. Sterilisasi ringan, dengan cara eksplan direndam dalam clorox 20% selama 10 menit dan dibilas dengan air bersih, lalu eksplan direndam dengan clorox 15% selama 10 menit dan dibilas dengan air bersih serta dilanjutkan kembali eksplan direndam dengan clorox 10% selama 10 menit dan dibilas kembali dengan air bersih 2. Sterilisasi sedang, dengan cara eksplan direndam dalam HgCl 2 0,1-0,5
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dilanjutkan dengan eksplan direndam dengan clorox 20% selama 10 menit dan dibilas dengan air bersih kembali. Kultur kotiledon adalah sebuah endosperm dari pembuahan antara gamet jantan yang bersifat haploid dengan inti kandung lembaga yang bersifat diploid, sehingga kotiledon akan bersifat triploid. Kotiledon adalah organ cadangan makanan pada biji didalam suatu tumbuhan yang dimiliki oleh tan aman itu saja saat berkecambah sehingga s ehingga hanya karbohidrat dan lemak bagi perkembangan embrio. Salah satu teknik kultur jaringan dengan menggunakan sumber eksplan kotiledon yang berasal dari biji yang telah matang. Manfaat dari kultur kotiledon yaitu untuk memberikan asupan cadangan makanan bagi embrio yang tidak dapat bertahan hidup di lingkungan in vitro, vitro, menghasilkan plantlet melalui embriogenesis dan mempermudah regenerasi pada tanaman (Bhojwani dan Razdan, 1989). Kotiledon atau endosperm yang digunakan dalam penelitian ini merupakan endosperm yang telah dikecambahkan sehingga hanya berupa sel-sel lemak dan karbohidrat sebagai cadangan makanan bagi perkembangan embrio atau hipokotil, hasil yang diperoleh dalam kultur in-vitro ini in-vitro ini hanya berupa sel-sel kalus. Indikasi perbedaan genetik yang akan mengakibatkan perbedaan kemampuan kompetensi regenerasi atau kondisi fisiologis dari jaringan eksplan. Eksplan yang digunakan merupakan kotiledon muda yang masih dalam proses pengisian biji, jadi memungkinkan ada sel embrio dalam eksplant tersebut, sehingga memungkinkan menghasilkan planlet melalui proses embriogenesis (Gunawan, 1995). Kotiledon yang dikulturkan harus berada dalam kondisi yang menunjukkan masa dormansi yang panjang, embrio hibrida hasil penyilangan interspesifik yang
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dibandingkan dengan kuncup lateral karena kuncup apeks memiliki pertumbuhan langsung
ke
permukaan
dengan
cepat.
Ujung
kuncup
dipotong
agar
pertumbuhannya lebih cepat serta s erta pemotongan nodus menghasilkan tunas aksiler. aksil er. Kultur tunas juga dapat menggunakan eksplan dari kotiledon bahkan akar tanaman karena memiliki tingkat regenerasi dan pertumbuhan yang lebih cepat dan baik. Regenerasi tanaman dari kalus melalui embriogenesis somatik lebih menguntungkan karena embrio somatik mempunyai struktur bipolar yang mengandung meristem tunas dan akar. Sel somatik berkembang melalui pembelahan untuk untuk membentuk embrio embrio sempurna sama seperti pada embrio zigotik. Pada embriogenesis somatik dapat digunakan zat pengatur tumbuh dalam konsentrasi yang memadai (Priadi dkk., 2014). Penggunaan eksplan kotiledon pada kultur in-vitro dapat in-vitro dapat menyerap air lebih cepat sehingga mempercepat induksi tunas. Hal ini disebabkan karena struktur permukaan kotiledon memiliki sel-sel yang berfungsi untuk penyerapan penyerapan air (Cahyati dkk., 2016). Tanaman jagung ( Zea mays) mays) dari kingdom (Plantae), divisi (Spermatophyta), kelas (Monocotyledone), ordo (Graminae), famili (Graminaceae), genus (Zea) dan species ( Zea Zea mays). mays). Jagung adalah tanaman berkeping tunggal atau monokotil, akar jagung berupa berupa akar serabut yang dapat mencapai kedalaman 8 m tapi rata rata pada kisaran 2 m. Jagung termasuk tanaman semsim, semsi m, sebagai bahan pokok jagung hidup dengan penyelesaian umur antara 80 – 150 150 hari (Martin, 1989). Jagung merupakan tanaman pangan beriklim panas dan pada dasarnya tumbuh pada temperatur antara 21-30 oC (70-86 F) namun optimum pada suhu 1821 oC (64-58 F). Paruh pertama dari siklus merupakan tahap pertumbuhan vegetatif
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diambil pelan-pelan dengan pinset agar kotiledon yang terambil tidak rusak dan tidak melukai kotiledon yang akan ditanam nantinya (Badami dan Amzeri, 2010). Pada pemberian unsur hara perlu dilakukan sesuai dengan dosis dan kebutuhan jagung, sedangkan untuk pemeliharaan dilakukan secara rutin, termas uk dalam hal pengairan dan pemberantasan organisme pengganggu tanaman yang bisa mengurangi hasil produksi jagung. Pada proses perkecambahan biji jagung, radikula akan keluar terlebih dahulu kemudian diikuti oleh pertumbuhan plumula. Proses perkecambahan tersebut dapat dipengaruhi oleh berbagai hal seperti kadar air, cahaya, temperatur, oksigen, dan salinitas. Proses perkecambahan juga dapat diinisiasi dengan skarifikasi (Manuhara dkk., 2010). Menurut Cahyani (2009), kotiledon yang lebih muda membutuhkan media yang lebih kompleks dibandingkan dengan kotiledon yang lebih tua. Perpindahan embrio dari lingkungan normal dalam biji akan mengatasi hambatan yang ditimbulkan oleh kulit biji yang sulit ditembus. Biji jagung disebut kariopsis, dinding ovari atau perikarp menyatu dengan kulit biji atau testa, membentuk dinding buah. Biji jagung terdiri atas tiga bagian utama yaitu pericarp (l apisan luar yang tipis, berfungsi mencegah embrio dari organisme pengganggu dan kehilangan air), endosperm, sebagai cadangan makanan, mencapai 75% dari bobot biji yang mengandung 90% pati dan 10% protein, mineral dan minyak) dan embrio (sebagai miniatur tanaman yang terdiri atas plamule, akar radikal, scutelum, dan koleoptil). Perbedaan kultur kotiledon dan kultur embrio adalah kultur embrio menggunakan eksplan pada seluruh bagian biji dimana endosperm pun juga ikut dikulturkan sedangkan kultur kotiledon hanya memakai eksplan berupa embrio saja
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menjadi inhibitor dalam proses perkecambahan seperti tidak tersedianya air, temperatur yang rendah, dan adanya glukosa atau sukrosa yang dapat bertindak sebagai sinyal molekul yang mengontrol ekspresi gen dan proses perkembangan dalam perkecambahan (Manuhara dkk., 2010). Zat pengatur tumbuh memegang peranan penting dalam keberhasilan induksi tunas diantaranya dari golongan sitokinin adalah BAP ( Benzil ( Benzil Amino Purin)/BA Purin )/BA ( Benziladenin) Benziladenin) kinetin dan zeatin. Pada kultur in vitro vitro biji jagung, media yang digunakan adalah media MS setengah sete ngah padat dan mengandung zat pengatur tumbuh 2,4-D. Sumber eksplan berupa kotiledon dengan ukuran antara 1,0-1,5 mm diambil dari biji jagung (Manuhara dkk., 2010). Menurut Manuhara dkk. (2010), 2,4-D merupakan auksin terbaik untuk menginduksi pembentukan embrio somatik dari eksplan dibandingkan dengan tipe auksin lain (IAA, IBA, dan NAA). Sifat 2,4-D yang mudah diserap sel tanaman, tidak mudah terurai dan menjadi tidak aktif, berfungsi sebagai auksin kuat dan mampu mendorong aktifitas morfogenetik yang merupakan beberapa faktor pendukung, pendukung, sehingga 2,4-D lebih baik dari tipe auksin lain. 2,4-D juga dapat merangsang pertumbuhan biji dengan prosentase perkecambahan sebesar 66,6%. Pengaruh suatu hormon bergantung pada jenis tanaman, jaringan, organ maupun konsentrasi hormon yang diberikan. Semakin tinggi konsentrasi perlakuan yang diberikan akan mempercepat waktu tumbuh kalus namun harus secukupnya karena sifat 2,4-D itu sendiri merupakan hormon yang mudah diserap, stabil tidak mudah diurai, mampu mendorong perkembangan ekplan namun semakin tinggi konsentrasi 2,4 D dengan konsentrasi berlebih yang diberikan maka akan
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umum digunakan untuk kultur embrio adalah medium MS dalam ½ konsentrasi garam-garamnya (Zulkarnain, 2010). Menurut Razdan (2002), faktor-faktor yang mempengaruhi pertumbuhan tunas: a. Faktor kimia Pertumbuhan kuncup menjadi tunas pada kultur jaringan sangat dipengaruhi oleh perbandingan auksin dan sitokinin yang ada pada medium. Auksin akan mempercepat proliferasi sel dan diferensiasi akar, sementara tingginya sitokinin akan meningkatkan laju diferensiasi kuncup menjadi tunas. b. Faktor fisika Tingginya intensitas cahaya maka dapat menghambat pembentukan tunas. Temperatur dapat mempengaruhi proses pertumbuhan dan diferensasi dimana proses pembentukan tunas dan diferensiasi berada pada suhu optimal sebesar 18oC Menurut Suryowinoto (1988), kontaminasi berasal dari kontaminan eksternal baik berupa jamur maupun bakteri yang tumbuh di dalam jaringan tanaman. Kontaminasi adalah terdapatnya senyawa atau organisme asing dalam suatu media
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eksplan dalam jangka waktu yang singkat. Hal-hal yang harus diperhatikan dalam sterilisasi eksplan adalah: a. Kondisi bahan eksplan, bahan eksplan yang digunakan diambil dari tanaman yang sehat tidak mengalami kotaminasi yang berasal dari jaringan
yang
masih
muda
(jaringan
yang
sel-selnya
bersifat
meristematik) b. Kondisi lingkungan, berupa kelembaban (kelembaban yang terlalu rendah meyebabkan eksplan mudah terkontaminasi, dan kelembaban yang tela lu tinggi membuat media cepat mongering), suhu (suhu yang dibutuhkan sekitar 250C untuk menyimpan eksplan) dan intensitas cahaya (pencahayaan umumnya membutuhkan intensitas 800 – 3000 3000 lux untuk kondisi bercahaya atau bahkan gelap total). c. Alat-alat dissecting set, yang digunakan harus disterilisasikan dengan alkohol 90% dan dilewatkan dengan api. Alat – alat alat yang digunakan harus dipastikan benar – benar benar steril. d. Keadaan ruangan dan kondisi laminar air flow, harus dalam keadaan steril dimana dalam LAF harus di UV terlebih dahulu se belum digunakan agar
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perkembangannya perkembangannya baik), komposisi nutrisi dalam media (untuk pertumbuhan embrio harus mengandung unsur makro, mikro dan gula), intensitas caha ya (untuk perkembangan embrio membutuhkan tempat gelap selama 7-14 hari) dan temperatur optimum (tergantung dari jenis tumbuhan yang digunakan dimana temperatur paling tinggi adalah 22-28̊C) (Yuliarti, 2010). Menurut Gunawan (1995), larutan clorox merupakan clorox merupakan larutan sterilan dalam kategori sterilisasi ringan yang dilakukan agar tetap menjaga kesterilan pada eksplan biji yang akan dipakai. Penggunaan konsentrasi clorox tidak clorox tidak diperkenankan terlalu rendah bahkan tinggi disebabkan karena jaringan eksplan akan mati tergantung pada morfologi pada suatu eksplan serta pada umumnya penggunaan clorox pada clorox pada biji sekitar 5-10%. Konsentrasi clorox yang clorox yang tepat untuk pertumbuhan biji secara bertahap agar pertumbuhan eksplan biji menjadi lebih optimal dan terbebas
dari
agen
kontaminan
yang
dapat
mengganggu
kelangsungan
perkembangan kotiledon. Menurut Suryowinoto (1988), penggunaan enkast dan LAF digunakan sebagai ruang penabur untuk melakukan kultur baik kalus bahkan eksplan serta untuk sterilisasi bahan yang digunakan. Sterilisasi enkast dengan formalin tablet
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serta blower ditekan, meja kerja LAF disemprotkan dengan larutan alkohol dan dilap satu arah dengan menggunakan tissue. tissue. Bunsen sebanyak 2 buah dimasukkan dalam LAF dan dinyalakan selama sterilisasi. Alat-alat yang akan digunakan selama pelaksanaan kerja dimasukkan ke dalam LAF dan dijenuhkan setelah disterilisasi (Wijayanto, 2011).
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III.
METODE
A. Alat dan Bahan
Alat-alat yang digunakan pada saat praktikum yaitu skalpel, pinset, gelas beker berukuran 500 ml, enkast , tabung konikel, tisu, LAF ( Laminar Air Flow Flow), ), alumunium foil, plastic wrap, wrap , kertas saring, gelas beker berukuran 250 ml dan cawan petri. Bahan-bahan yang digunakan pada saat praktikum adalah clorox (5% clorox (5% dan 10%), biji jagung muda, medium MS dengan hormon 2,4 D dan aquades steril. steril .
B. Cara Kerja
1. Sterilisasi ruang penabur (LAF) Tombol lampu dinyalakan dan pintu LAF dibuka (tidak perlu terlalu lebar). Tangan praktikan disemprot dengan alkohol dan tisu serta alkohol 70% dimasukan kedalam laminar air flow (LAF). flow (LAF). Meja LAF disemprot dengan alkohol 70% dan dikeringkan (di lap) dengan tisu secara searah menuju bagian luar LAF. Pintu LAF ditutup dan tombol lampu
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3. Sterilisasi eksplan biji dan kultur kotiledon jagung muda ( Zea ( Zea mays) mays) Ruang penabur disterilisasi terlebih dahulu. Diluar ruang penabur, biji jagung muda dalam keadaan tenggelam diambil sebanyak 30 biji dan dibilas dengan air filtrasi ( pure it ). ). Air filtrasi dibuang dan ditutup dengan alumunium foil serta dimasukkan kedalam enkas / LAF. Didalam ruang penabur (enkast ( enkast / LAF), eksplan biji berada didalam enkast / / LAF. Biji jagung digojog dengan clorox 10% clorox 10% sebanyak 50 ml selama 5 menit dan clorox 5% clorox 5% sebanyak 50 ml selama 5 menit didalam botol flakon. Biji jagung muda dibilas dengan dengan aquades steril sebanyak 3 kali dan dikeluarkan dari botol flakon. Biji jagung muda diambil embrio (kotiledon) dengan cara biji jagung tersebut ditekan dengan pinset dan
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N2 = Jumlah larutan stok yang akan dipakai
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IV.
HASIL DAN PEMBAHASAN
Kultur jaringan tumbuhan adalah cara membudidayakan jaringan tumbuhan menjadi tumbuhan kecil yang memiliki sifat seperti dengan induknya berdasarkan teori totipotensi sel Schleiden dan Schwann dimana sel memiliki kemampuan untuk tumbuh dan berkembang dari tanaman kecil menjadi tanaman dewasa dan dapat tumbuh dilingkungan dili ngkungan ex-vitro. Embriogenesis somatik merupakan sel somatik yang berkembang menjadi tanaman baru tanpa melewati fusi gamet. Eksplan adalah bagian atau bahan tanaman yang akan dikultur dalam proses kultur jaringan tumbuhan dimana eksplan yang digunakan adalah tanaman muda dan mudah tumbuh (Hadioetomo, 2000).
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hidup di lingkungan in vitro, memecah dormansi embrio yang mengalami dormansi dalam jangka waktu yang panjang dan meningkatkan tingkat germinasi pada biji. Kultur kotiledon adalah sebuah endosperm dari pembuahan antara gamet jantan yang bersifat haploid dengan inti kandung lembaga yang bersifat diploid, sehingga kotiledon akan bersifat triploid. Kotiledon adalah organ cadangan makanan pada biji didalam suatu tumbuhan yang dimiliki oleh tanaman it u saja saat berkecambah sehingga s ehingga hanya karbohidrat dan lemak bagi perkembangan embrio. Manfaat dari kultur kotiledon yaitu untuk memberikan asupan cadangan makanan bagi embrio yang tidak dapat bertahan hidup di lingkungan in vitro, vitro, menghasilkan plantlet melalui embriogenesis dan mempermudah regenerasi pada tanaman (George dan Sherrington, 1984).
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menurunnya berat basah kalus yang diperoleh. 2,4-D kurang berperan dalam regenerasi kalus kotiledon kecambah kedelai dalam kultur in vitro (Budisantoso vitro (Budisantoso dan Prayoga, 2012). Pada praktikum kultur tunas digunakan bahan ekplan yang berasal dari embrio jagung, dimana embrio jagung digunakan karena mudah didapatkan dengan mudah serta mempunyai daya regenerasi yang tiggi untuk membentuk tunas yang lebih cepat. Tanaman jagung ( Zea mays) mays) dari famili Gramineae yang memiliki proses perkecambahan dengan radikula akan keluar terlebih dahulu lalu mengalami pertumbuhan plumula yang dapat dipengaruhi oleh berbagai hal seperti kadar air, cahaya, temperatur, oksigen, dan salinitas. Skarifikasi pada embrio dalam biji jagung adalah mekanisme dormansi pada biji tanaman agar biji cepat mengalami
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dilanjutkan digojog dengan clorox clorox 5% sebanyak 50 ml selama 5 menit didalam botol flakon untuk mencegah kontaminasi dan sebagai desinfektan pada biji jagung. jagung. Biji jagung muda dibilas dengan aquades steril ste ril sebanyak 3 kali dan dikeluarkan dari botol flakon agar mengurangi mengurangi bahkan menghilangkan clorox hasil dari penggojogan yang sudah dilakukan sebelumnya.
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Tabel 1. Hasil pengamatan kultur kotiledon jagung dengan ruang penabur enkas Botol ke1
Pengamatan tanggal 22 September 26 September 28 September 2017 2017 2017 Tumbuh calon Tumbuh calon Tumbuh calon akar, tidak ada akar dan tunas, akar dan tunas ,
3 Oktober 2017 Tumbuh calon akar dan tunas,
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kontaminasi dan tumbuh 3 tunas berwarna hijau Berdasarkan percobaan yang telah dilakukan, didapatkan hasil berupa tabel 1, diketahui bahwa botol kultur ke 1 tidak mengalami kontaminasi, tumbuh tunas
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adalah alat-alat yang digunakan dalam proses kultur tidak disterilkan terlebih dahulu, kondisi bahan eksplan yang diambil kurang baik yang dapat mempengaruhi jaringan yang masih muda, kondisi lingkungan seperti kurangnya kelembaban dan suhu yang kurang optimal saat proses kultur dapat mengganggu pertumbuhan eksplan, banyak komunikasi antar praktikan didalam ruang kultur sebagai
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dilakukan, didapatkan hasil tabel 2. berupa hasil pengamatan kotiledon jagung dengan ruang penabur LAF sebagai berikut: Tabel 2. Hasil pengamatan kotiledon jagung dengan ruang penabur LAF Botol ke-
22 September
Pengamatan Hari ke26 September 28 September
3 Oktober
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