laporan kemajuan penelitian kerjasama antar …
TRANSCRIPT
i
LAPORAN KEMAJUAN
PENELITIAN KERJASAMA ANTAR PERGURUAN TINGGI
DANA ITS 2020
Teknik Mikropropagasi Tunas Mikro Stevia rebaudiana (Bertoni) aksesi Mini secara in vitro
sebagai Upaya Pemuliaan dan Perbanyakan Bibit Unggul Tanaman Pemanis Sehat Alternatif bagi
Penderita Diabetes
Tim Peneliti :
Dr. Nurul Jadid, S.Si., M.Sc/ Biologi / FSAD
Wirdhatul Muslihatin, S.Si., M.Si /Biologi/ FSAD/ ITS Surabaya
Dini Ermavitalini, S.Si., M.Si / Biologi/ FSAD/ ITS Surabaya
Dwi Oktafitria, S.Si., M.Sc / Biologi / FMIPA /Universitas PGRI Ronggolawe Tuban
Christin Risbandini, S.Si / PLP Laboran Biologi / FSAD / ITS Surabaya
DIREKTORAT RISET DAN PENGABDIAN KEPADA MASYARAKAT
INSTITUT TEKNOLOGI SEPULUH NOPEMBER
SURABAYA
2020
Sesuai Surat Perjanjian Pelaksanaan Penelitian No: 964/PKS/ITS/2020
i
Daftar Isi
Daftar Isi .......................................................................................................................................................... i
Daftar Tabel .................................................................................................................................................... ii
Daftar Gambar ............................................................................................................................................... iii
Daftar Lampiran ............................................................................................................................................. iv
BAB I RINGKASAN ..................................................................................................................................... 1
BAB II HASIL PENELITIAN ........................................................................................................................ 2
BAB III STATUS LUARAN……………………………………………………………………………….11
BAB IV PERAN MITRA (UntukPenelitian Kerjasama Antar Perguruan Tinggi)………………………...11
BAB V KENDALA PELAKSANAAN PENELITIAN……………………………………………………11
BAB VI RENCANA TAHAPAN SELANJUTNYA………………………………………………………11
BAB VII DAFTAR PUSTAKA……………………………………………………………………………12
BAB VIII LAMPIRAN…………………………………………………………………………………….15
LAMPIRAN 1 Tabel Daftar Luaran……………………………………………………………………….15
ii
Daftar Tabel
Hal
Tabel 2.1 Respon organogenesis dan kalogenesis setelah 10 minggu diinokulasikan pada kombinasi ZPT yang berbeda
3
Tabel 2.2 Respon jumlah tunas yang dihasilkan oleh eksplan nodus steril dengan perlakuan kombinasi ZPT yang berbeda
6
Tabel 2.3 Respon pembentukan akar dengan perlakuan kombinasi ZPT BA dan Kin yang berbeda
9
iii
Daftar Gambar
Hal
Gambar 2.1 Respon organogenesis 5
Gambar 2.2 Respon organogenesis berupa tunas pada eksplan yang ditanam pada medium 0,5 ppm BA
5
Gambar 2.3 Pengamatan jumlah tunas 10 minggu setelah inokulasi 7
Gambar 2.4 Akar yang terbentuk setelah diberi perlakuan kombinasi ZPT konsentrasi yang berbeda selama 10 minggu
9
iv
Daftar Lampiran
Hal
Lampiran 1. Tabel Daftar luaran 15
1
BAB I RINGKASAN
Tanaman Stevia (Stevia rebaudiana (Bertoni)) merupakan tanaman perdu dari keluarga
Compositae. Budidaya tanaman tersebut memiliki potensi ekonomi tinggi karena memiliki
tingkat kemanisan 200-300 kali lebih tinggi dibanding gula tebu. Namun demikian, kendala
utama dalam budidaya stevia adalah tingkat perkecambahan biji yang rendah. Selain itu,
perbanyakan secara generatif juga menghasilkan mutu bibit stevia yang relatif memiliki
karakteristik fenotip yang beragam. Hal ini menyebabkan rendahnya ketersediaan bibit Stevia
unggul yang memiliki karakteristik seragam. Oleh karena itu, diperlukan teknik budidaya yang
efisien. Salah satu metode perbanyakan dalam upaya penyediaan bibit unggul adalah melalui
teknik mikropropagasi. Teknik tersebut merupakan metode perbanyakan tanaman yang efektif,
dengan penambahan zat pengatur tumbuh (ZPT) sesuai dengan tujuan yang diharapkan secara
in vitro. Teknik ini juga dapat digunakan sebagai salah satu cara dalam pemilihan dan
pemuliaan bibit unggul tanaman. ZPT adalah senyawa organik bukan nutrisi yang
mempengaruhi pertumbuhan dan perkembangan tanaman. Tujuan dari penelitian ini adalah
untuk mendapatkan metode mikropropagasi tanaman stevia aksesi Mini yang efektif dan
efisien melalui modifikasi media kultur dengan kombinasi konsentrasi Kinetin (Kin) dan
Benziladenin (BA) yang berbeda. Beberapa parameter uji yang dianalisis adalah persentase
bertunas, jumlah tunas dan jumlah akar. Penelitian ini didesain menggunakan Rancangan Acak
Lengkap (RAL) faktorial dengan jumlah ulangan sebanyak 5 kali. Hasil awal penelitian ini
menunjukkan bahwa perlakuan penambahan kombinasi BA dan Kin dengan konsentrasi yang
berbeda dapat menginduksi proses organogenesis. Hal ini ditunjukkan dengan terbentuknya
tunas dan organ akar. Selain itu, kombinasi perlakuan ZPT tersebut juga mengakibatkan
terbentuknya massa sel parenkim yang bersifat meristematic (kalus). Perlakuan dengan
penambahan 1 ppm BA menghasilkan rata-rata jumlah tunas terbanyak sebesar 36,05.
Sedangkan perlakuan tanpa pemberian ZPT menghasilkan pembentukan akar paling baik
sebesar 2,6.
Kata Kunci: aksesi Mini, bibit unggul, mikropropagasi, pemuliaan tanaman, Stevia rebaudiana
(Bertoni).
2
Ringkasan penelitian berisi latar belakang penelitian,tujuan dan tahapan metode
penelitian, luaran yang ditargetkan, kata kunci
BAB II HASIL PENELITIAN
2.1. Respon Organogenesis dan Kalogenesis
Zat Pengatur Tumbuh (ZPT) adalah molekul organik selain unsur hara (unsur yang
menyediakan energi atau hara mineral), yang mempengaruhi pertumbuhan dan
perkembangan tanaman. ZPT ini bersifat aktif pada konsentrasi yang relative rendah.
Beberapa jenis ZPT memiliki fungsi untuk mempercepat, menghambat, serta
menyebabkan mutasi pada proses-proses fisiologi yang terjadi pada tanaman (Basra,
2000). Dalam kultur in vitro penggunaan ZPT dalam konsentrasi rendah diperlukan untuk
menginduksi proliferasi sel dan organogenesis (Gaba, 2005). Penggunaan zat pengatur
tumbuh didalam kultur jaringan juga tergantung pada tujuan atau arah pertumbuhan yang
diinginkan. Lestari (2008) mengungkapkan bahwa interaksi antara ZPT yang terdapat
secara alami (endogen) di dalam eksplan (sel, jaringan atau organ tanaman) dan ZPT
eksogen juga berpengaruh pada pertumbuhan dan organogenesis tanaman. Benziladenin
(BA) dan Kinetin (Kin) merupakan ZPT golongan sitokinin yang berfungsi sebagai
pemacu pembelahan sel dan pemecah doninasi apikal oleh auksin (Salisbury dan Ross,
1995).
Penelitian ini menggunakan eksplan berupa nodus dari planlet tanaman stevia steril.
Eksplan steril tersebut umumnya dikenal dengan sebutan secondary explant. Pada
penelitian ini, interaksi BA dan Kin pada konsentrasi yang berbeda menyebabkan respon
organogenesis dan kalogenesis yang berbeda pula (Tabel 2.1). Chishimba (2000)
melaporkan bahwa penambahan BA, Kin dan ZPT golongan sitokinin lain pada kultur in
vitro eksplan tunas pucuk Uapaca kirkiana memberikan respon yang berbeda pula. Hal
tersebut juga dilaporkan oleh Quadri (2012) yang menyatakan bahwa penambahan BA,
Kin dan Thidiazuron pada proliferasi tunas secara in vitro Hyoscyamus niger L. juga
memberikan respon yang berbeda.
3
Tabel 2.1 Respon organogenesis dan kalogenesis setelah 10 minggu diinokulasikan
pada kombinasi ZPT yang berbeda
Kombinasi ZPT Persentase
eksplan
bertunas
Persentase
eksplan
berkalus
BA
(ppm)
Kin
(ppm)
0 0 100 0
0,5 0 100 0
1 0 100 0
1,5 0 100 0
2 0 100 0
0 2 90 100
0,5 2 100 100
1 2 90 100
1,5 2 100 100
2 2 65 100
0 4 85 100
0,5 4 90 100
1 4 60 100
1,5 4 65 100
2 4 60 100
0 6 75 100
0,5 6 75 100
1 6 50 100
1,5 6 60 100
2 6 85 100
0 8 95 100
0,5 8 75 100
1 8 45 100
1,5 8 75 100
2 8 85 100
4
Analisis data menggunakan Anova – two way menunjukkan bahwa interaksi BA dan
Kin tidak berpengaruh terhadap persentase eksplan bertunas (p value > 0,05). Meskipun
demikian, eksplan yang ditanam pada medium MS dengan perlakuan kombinasi ZPT
yang berbeda hampir seluruhnya seluruhnya memberikan respon pembentukan tunas.
Nilai persentase pembentukan tunas tersebut berkisar 45%-100%. Respon pembentukan
tunas terendah muncul pada medium dengan penambahan ZPT 1 ppm BA + 8 ppm Kin.
Sedangkan respon pembentukan tunas paling tinggi muncul pada medium dengan
peambahan ZPT BA secara tunggal dan kombinasi dengan Kin konsentrasi rendah (0,5
ppm BA + 2 ppm Kin (Gambar 2.1) dan 1,5 ppm BA + 2 ppm Kin). Dari data tersebut
dapat disimpulkan bahwa ZPT BA memberikan respon organogenesis berupa
pembentukan tunas yang lebih baik dibandingkan Kin pada penelitian ini. Rendahnya
persentase bertunas oleh adanya Kin dalam menginduksi tumbuhnya tunas aksilar sesuai
dengan hasil penelitian Thiyagarajan (2012), Razak (2014), Rafiq (2007).
BA memberikan respon pembentukan tunas yang lebih baik karena mempunyai
aktivitas yang lebih kuat (Zaer dan Mapes, 1982) dan menyebabkan pemanjangan yang
lebih nyata dibanding kinetin (Salisbury dan Ross,1995). BA mempunyai struktur dasar
yang sama dengan kinetin tetapi lebih efektif karena BA mempunyai gugus benzil
(George, l984). Flick (1993) menyatakan bahwa pada umumnya tanaman memiliki
respon yang lebih baik terhadap BA dibandingkan terhadap kinetin dan 2-iP sehingga BA
lebih efektif untuk produksi tunas in vitro.
Selain respon eksplan bertunas, pada penelitian ini juga dilakukan pengamatan
terhadap respon eksplan berkalus. Kalus merupakan suatu massa sel tidak berbentuk dan
tidak terorganisasi yang terbentuk pada permukaan potongan jaringan yang terluka
sebagai respon perlindungan untuk menutup jaringan yang terluka (Heryanto, 2014). Pada
penelitian ini 100% kalus terbentuk pada media yang ditambahakan Kin (Gambar 2.1).
Morfologi kalus setelah 10 minggu inokulasi, kalus kompak dan berwarna kecoklatan
sementara dipermukaan atas kalus, kalus bersifat friabele berwarna kehijauan (Ikeuchi,
2013). Gupta (2010) melaporkan bahwa penambahan Kin dengan konsentrasi 3 ppm, 4
ppm dan 5 ppm memberikan respon pembentukan kalus. Pembelahan sel yang terjadi
karena sitokinin dapat memproduksi kalus yang tidak terdiferensiasi (Gaba, 2005).
Anbazhagan (2010) melaporkan bahwa penambahan Kin pada propagasi tunas stevia
dapat menginisiasi tunas pada awal pertumbuhan saja dan hal ini telah didokumentasikan
studi yang lehih awal juga oleh Murashige (1974), Benne dan davies (1986) dan Rogers
(1998). Hasil penelitian yang berbeda dilaporkan oleh ibrahim (2008) bahwa eksplan
5
ujung tunas stevia yang ditanam pada medium dengan penambahan Kin secara tunggal
menghasilkan jumlah tunas yang banyak dibanding jumlah tunas pada penelitian ini,
meskipun respon jumlah tunas yang dihasilkan oleh ZPT BA tetap lebih tinggi dibanding
ZPT Kin. Perbedaan hasil penelitian tersebut dapat disebabkan oleh genotip eksplan yang
digunakan berbeda-beda. Faktor genotip sendiri merupakan faktor yang paling penting
dalam kulur jaringan (Gaba, 2005).
Gambar 2.1 Respon organogenesis berupa tunas (1) dan kalogenesis (2). Eksplan
berumur 10 minggu. Keterangan; A. 0,5 ppm BA + 2 ppm Kin, B. 0,5 ppm BA + 4 ppm
Kin. Garis putih menunjukkan skala 0,5 cm.
Gambar 2.2 Respon organogenesis berupa tunas pada eksplan yang ditanam pada medium 0,5
ppm BA. Eksplan berumur 10 minggu. Keterangan : Garis putih menunjukkan skala 0,5 cm.
2.2 Respon Bertunas
Jumlah tunas yang terbentuk merupakan tujuan utama dalam mikropropagasi
tanaman secara komersial. Multiplikasi tunas diinduksi dengan cara pengaplikasian
sitokinin eksogen kedalam medium pertumbuhan (Gaba, 2005). Hasil analisis anova-two
way menunjukkan bahwa penambahan kombinasi Kin dan BA dengan konsentrasi yang
A B
6
berbeda berpengaruh terhadap jumlah tunas (P value < 0,05). Konsentrasi ZPT yang
menghasilkan jumlah tunas paling banyak adalah 1 ppm BA dengan jumlah tunas yang
dihasilkan sebanyak 36,05 tunas (Tabel 2.2; Gambar 2.3). Sedangkan jumlah tunas
terendah dihasilkan oleh kombinasi ZPT 1 ppm BA + 8 ppm Kin dan 1 ppm BA + 6 ppm
Kin dengan jumlah tunas 0,65. Berdasarkan data tersebut dapat disimpulkan bahwa BA
lebih efektif dalam multiplikasi tunas dibanding Kin. Alhady (2011) dan Anbazhagan
(2010) juga melaporkan bahwa BA lebih efektif untuk multiplikasi tunas stevia. Hal ini
juga terjadi pada spesies yang lain seperti pada Bauhinia veriegate (Ahmed, 2007),
Balanites aegyptiace (Mathur,1992), dan Periploca angustifolia (Abd-Alhady, 2010).
Jumlah tunas yang dihasilkan oleh Alhady (2011) pada perlakuan 2 ppm BA + o,5 ppm
Kin 36,9 tunas (hasil paling tinggi) sedangkan pada perlakuan 0,5 ppm BA + 0,5 ppm
Kin 18.1 tunas (hasil paling rendah). Anbazhagan (2010) menghasilkan jumlah tunas 9,20
pada perlakuan 2 ppm BA dan 4,40 pada 2 ppm Kin.
Tabel 2.2 Respon jumlah tunas yang dihasilkan oleh eksplan nodus steril dengan
perlakuan kombinasi ZPT yang berbeda
* Nilai yang diikuti oleh huruf yang sama pada kolom menunjukkan tidak adanya beda
nyata pada uji Tukey selang kepercayaan 95%.
Kombinasi ZPT Jumlah tunas
Kombinasi ZPT Jumlah tunas
BA (ppm)
Kin (ppm)
BA (ppm)
Kin (ppm)
0 0 1,9e 1,5 4 1,4e
0,5 0 4,95de 2 4 1e
1 0 36,05a 0 6 1,2e
1,5 0 17,2c 0,5 6 1,6e
2 0 26b 1 6 0,65e
0 2 1,9e 1,5 6 0,8e
0,5 2 8d 2 6 1,4e
1 2 3,55de 0 8 1,7e
1,5 2 7,35d 0,5 8 1,05e
2 2 1,05e 1 8 0,65e
0 4 1,55e 1,5 8 1,65e
0,5 4 1,8e 2 8 1,4e
1 4 0,85e
7
Gambar 2.3 Pengamatan jumlah tunas 10 minggu setelah inokulasi. Keterangan; (A)
eksplan yang ditanam pada media MS 0 atau kontrol. (B) eksplan yang ditanam pada
media 0,5 ppm BA. (C) eksplan yang ditanam pada media 1 ppm BA. Garis putih
menunjukkan skala 0,5 cm.
Fungsi dasar sitokinin adalah memacu sitokinesis atau pembelahan sel. Kajian
terhadap pembelahan sel yang diaktifkan oleh sitokinin di meristem apikal, Houssa, dkk
(1990) memperoleh hasil yang sebagian besar sejalan dengan kajian Fosket dkk (1981).
Mereka menemukan bahwa benziladenin sangat mempersingkat waktu berlangsungnya
fase S dalam daur sel (dari G2 ke mitosis, yaitu tahap sintesis DNA dan protein
pembelahan sel). Fosket, dkk (1981) menyimpulkan bahwa sitokinin mendorong
pembelahan sel dalam biakan jaringan dengan cara meningkatkan peralihan dari G2 ke
mitosis dan hal tersebut terjadi karena sitokinin menaikkan laju sintesis protein. Beberapa
protein tersebut berupa protein pembangun atau enzim dibutuhkan untuk mitosis. Protein
bisa ditingkatkan dengan cara memacu pembentukan mRNA yang menyandikan protein
tersebut.
A
C
E
B
D
F
8
Sitokinin dapat menginduksi pembentukan tunas dengan jalan terbentuknya sinyal
sitokinin kerena regulasi positif dari WIND1 yang muncul akbat adanya pelukaan.
WINDs merupakan faktor transkripsi dan mendukung dedifferensiasi (memiliki fungsi
yang sama dengan WUS). Setelah sinyal sitokinin muncul, WUS menghambat ekspresi
dari ARRs untuk mempertahankan populasi sel induk pada jaringan meristem tunas.
Setelah adanya regulasi positif dari sinyal sitokinin ke WUS, WUS meregulasi positif
CLV3. CLV3 berfungsi meregulasi proliferasi sel di SAM, adanya regulasi timbalbalik
antara CLV3 dan WUS untuk mempertahankan populasi sel induk dan ukuran jaringan
mengakibatkan differensiasi sel kemudian terbentuk penyusunan organ lateral (Ikeda,
2014).
4.3 Respon Perakaran
Pembentukan akar secara in vitro perlu dilakukan untuk mengubah tunas menjadi
menjadi plantlet utuh yang bisa dipindahkan ke greenhouse (Gaba,2005). Inisiasi
perakaran tanaman secara in vitro dapat dipacu dengan menambahkan ZPT golongan
auksin seperti Indole-3-Acetic Acid (IAA), Naphtalene Acetic Acid (NAA) dan Indole-
3-Butyric Acid (IBA) (Arlianti, 2013). Beberapa eksplan pada menelitian ini memberikan
respon pembentukan akar (Tabel 2.3).
Analisis data menggunakan Anova – two way menunjukkan bahwa interaksi BA dan
Kin berpengaruh terhadap jumlah akar (p value > 0,05). Jumlah akar paling tinggi
dihasilkan oleh perlakuan MS 0 atau tanpa tambahan ZPT. Perlakuan pemberian sitokinin
dalam konsentrasi tinggi dapat menghambat pembentukan akar. Perlakuan pelukaan saat
penanaman tunas dapat menginduksi sintesis auksin untuk pembentukan akar. Pada
beberapa kasus tertentu, akar bisa diinduksi dengan cara pemindahan tunas pada medium
tanpa ZPT sebanyak satu atau duakali untuk menurunkan konsentrasi sitokinin.
Pilihan metode yang lain adalah penambahan auksin dengan konsentrasi rendah untuk
menginduksi akar (Gaba, 2005). Arlianti (2013) melakukan percobaan induksi perakaran
Stevia, menghasikan konsentrasi ZPT terbaik untuk induksi akar adalah 0,2 ppm NAA
dengan jumlah akar 8 buah. Sedangakan Anbazhagan (2010) melaporkan ZPT 1 ppm
IAA menghasilkan akar 11 buah, Alhady (2011) dengan ZPT 2 ppm IBA menghasilkan
akar 8,4 buah dan Das (2011) dapat menghasilkan akar tanpa penambahan ZPT (MS 0)
dengan jumlah akar 15,58 buah.
9
Tabel 2.3 Respon pembentukan akar dengan perlakuan kombinasi ZPT BA dan Kin yang
berbeda.
Gambar 2.4 Akar yang terbentuk setelah diberi perlakuan kombinasi ZPT konsentrasi yang
berbeda selama 10 minggu. Keterangan; A: Akar yang terbentuk pada media MS 0, B: Akar
yang terbentuk pada media 0,5 ppm BA. Tanda panah menunjuk bagian akar. Garis putih
menunjukkan skala 0,5 cm.
A1 B
Kombinasi ZPT Jumlah
akar
Kombinasi ZPT Jumlah
akar BA
(ppm) Kin
(ppm) BA
(ppm) Kin
(ppm)
0 0 2,6a 1,5 4 0,0b
0,5 0 0,15b 2 4 0,0b
1 0 0,0b 0 6 0,0b
1,5 0 0,0b 0,5 6 0,0b
2 0 0,0b 1 6 0,0b
0 2 0,05b 1,5 6 0,0b
0,5 2 0,0b 2 6 0,0b
1 2 0,0b 0 8 0,0b
1,5 2 0,0b 0,5 8 0,0b
2 2 0,0b 1 8 0,0b
0 4 0,0b 1,5 8 0,0b
0,5 4 0,0b 2 8 0,0b
1 4 0,0b * Nilai yang diikuti oleh huruf yang sama pada kolom menunjukkan
tidak adanya beda nyata pada uji Tukey selang kepercayaan 95%.
10
Auksin sendiri memiliki banyak peran dalam kultur jaringan, tergantung dari struktur kimia,
konsentrasi dan respon jaringan tanaman itu sendiri. Auksin menyebabkan pembentukan kalus
dan akar serta pertumbuhan ekstensi batang. Auksin secara umum memiliki fungsi menstimulasi
pemanjangan sel, pembelahan sel pada jaringan kambium dan bersama sitokinin menstimulasi
differensiasi xilem dan floem. Penambahan auksin eksogen yang cukup tinggi dapat
menginduksi somatik embrio genesis. Perbandingan rasio auksin lebih tinggi dibanding sitokinin
akan menginduksi pembentukan akar pada tunas, inisiasi kalus pada tanaman monokotil, dan
inisiasi embriogenesis somatik. Perbandingan rasio auksin dan sitokinin yang hampir seimbang
akan menginduksi terbentuknya akar tambahan dari kalus dan inisiasi kalus pada tanaman
dikotil. Perbandingan rasio auksin yang lebih rendah dibanding sitokinin akan menginduksi
tunas tambahan dan produsksi tunas aksiler. Untuk itu interaksi antara auksin dan sitokinin
sangat penting untuk mengontrol banyak proses pertumbuhan dan perkembangan secara in vitro
(Gaba, 2005).
11
BAB III STATUS LUARAN
Luaran wajib dari penelitian ini berupa jurnal ilmiah internasional terindeks scopus. Saat ini tim peneliti
sedang mempersiapkan manuskrip untuk di submit ke jurnal. Manuskrip tersebut rencananya akan
disubmit ke jurnal peerJ (terindeks scopus Q1) atau jurnal Heliyon (terindeks scopus Q2). Adapun draft
manuskrip jurnal tersebut terlampir.
BAB IV PERAN MITRA (UntukPenelitian Kerjasama Antar Perguruan Tinggi)
Mitra penelitian ini adalah Universitas PGRI Ronggolawe, Tuban, Jawa Timur. Mitra berperan dalam
membantu dalam proses drafting manuskrip jurnal ilmiah dan memberikan masukan terkait pilihan
jurnalnya. Selain itu, mitra juga berperan penting dalam memberikan jejering dalam mendapatkan
sumber atau material tanaman yang digunakan dalam penelitian ini (Stevia rebaudiana aksesi Mini).
BAB V KENDALA PELAKSANAAN PENELITIAN
Kendala utama dalam pelaksanaan penelitian ini adalah akses yang terbatas pada laboratorium yang
saat ini menjalankan protocol covid. Namun demikian, sebagian besar penelitian dapat diselesaikan
dengan baik.
BAB VI RENCANA TAHAPAN SELANJUTNYA
Sebagian besar penelitian eksperimental di laboratorium telah selesai dilaksanakan. Beberapa rencana
aktivitas selanjutnya lebih pada persiapan manuskrip seperti Analisa hasil, editing gambar, dan
proofreading manuskrip.
12
BAB VII DAFTAR PUSTAKA
Geuns J. M. C., 2003. Molecules of Interest – Stevioside. Phytochem. 64, 913-921.
Jeppesen PB, Gregersen S, Alstrup KK. 2002. Stevioside induces antihyperglycaemic,
insulinotropic and glucagonostatic effects in vivo: studies in the diabetic Goto-Kakizaki
(GK) rats. Phytomed. 9:9-14.
Gregersen S, Jeppesen PB, Holst JJ, Hermansen K. 2004. Antihyperglycemic effects of
stevioside in type 2 diabetic subjects. Metabolism. 53:73-76.
Brandle JE, Starrtt AN, Gijzen M. 1998. Stevia rebaudiana: international agriculturalf,
biological chemical properties. Can J Plant Su 78:527-536.
Megeji NW, Kumar JK, Singh V, Kaul VK and Ahuja PS. 2005. Introducing Stevia
rebaudiana, a natural zero-calorie sweetener. Curr Sci. 88:801-805.
Rodiansah, Asep. 2007. Induksi Mutasi Kromosom dengan Koklisin pada Tanaman Stevia
(Stevia rebaudiana Bertoni) Klon Zweeteners Secara In Vitro. Skripsi. Bogor : Progam
Studi Hortikultura Fakultas Pertanian Institut Pertanian Bogor.
Hendaryono, Daisy P. Dan Wijayani, A. 1994. Teknik Kultur Jaringan. Kanisus
:Yogyakarta.
Ibrahim, I. A., M. I. Nasr, B. R. Mohammed, M. M. El-Zefzafi. 2008. Plant Growth
regulators affecting in vitro Cultivation of Stevia rebaudiana. Sugar Tech. 10(3) : 254-
259.
Anbazhagan, M., M. Kalpana, R. Rajendran, V. Natarajan dan D. Dhanavel. 2010. In Vitro
Production of Stevia rebaudiana Bertoni. Emir J. Food Agric. 22(3) : 216-222.
Sami, W., Ansari, T., Butt, N. S., & Hamid, M. (2017). Effect of diet on type 2 diabetes
mellitus: A review. International journal of health sciences, 11(2), 65–71.
Ramírez-Mosqueda M.A., Iglesias-Andreu L.G.Direct organogenesis of Stevia
rebaudiana Bertoni using Thin Cell Layer (TCL) method. Sugar Tech, 18 (2016), pp.
424-428.
Sharma, Saurabh, Swati W., Bikram S., dan Rakesh K.. 2016. Comprehensive review on
gro technologies of low-calorie natural sweetener stevia (Stevia rebaudiana Bertoni) : a
boon to diabetic patients. J Sci Food Agric. 96: 1867-1879.
Shaffert, E.E. and Chebotar, A.A. (1994) Development of the female gametophyte in
Stevia rebaudiana, after introduction in the south coast of the Crimea. Buletinul
Academiei de Stiinte a Republicii Moldova Stiinte Biologice Si Chimice. 2, 3–9.
Yadav, A. K., S. Singh, D. Dhyani dan S. Ahuja. 2011. A Review on The Improvement of
Stevia (Stevia rebaudiana (Bertoni)). Can. J. Plant Sci. 91: 1-27.
13
Lemus-Mondaca, R., A. Vega-Galves, L. Zurabravo dan K. Ah-Hen. 2012. Stevia
rebaudiana Bertoni, source of a high-potency natural sweetener: A comprehensive review
on the biochemical, nutrional and functional aspect. Food Chemistry. 132:11211132.
Singh, H. P., Seema D., dan Sarwan K. Dhir. 2008. Stevia Compendium of Transgenic
Crop Plants: Transgenic Sugar, Tuber and Fiber Crops. Blackwell Publishing Ltd.
ISBN 978-1-405-16924-0.
Sumaryono dan Masna Maya Sinta. Petunjuk Teknis Budidaya Tanaman Stevia. Bogor
: Pusat Penelitian Bioteknologi dan Bioindustri (PPBBI).
Merindasya, M., T. Nurhidayati dan Parnidi. 2013. Induksi Tunas Tiga Aksesi Stevia
rebaudiana Bertoni pada Media MS dengan Penambahan BAP dan IAA secara In Vitro.
Skripsi. Institut Teknologi Sepuluh Nopember.
Gasmalla, M., A., A., Yang., R., Amadou dan Hua X. 2014. Nutritional Composition of
Stevia rebaudiana Bertoni Leaf: Effect of Drying Method. Trop J Pharm Res,;13 (1).
Afandi, A., Sarijan, S., dan Shaha, R., K.2013. Optimization of Rebaudioside a Extraction
from Stevia Rebaudiana (Bertoni) and Quantification by High Perfomance Liquid
Chromatography Analysis. Journal of Tropical Resources and Sustainable Science.
Vol. 1 (1):62-70.
Sirshendu D, Mondal S, Banerjee S. 2012. Stevioside : Technology, Applications and
Health. John Wiley & Sons Inc.
Rukmana, R. 2003. Budi Daya Stevia Bahan Pembuatan Pemanis Alami. Yogyakarta
: Penerbit Kanisius.
Departemen Pertanian. 1984. Mengenal pemanis alami Stevia rebaudiana Bertoni M.
Bogor : BPP Ciawi.
Karjadi, Asih K. 2016. Kultur Jaringan dan Mikropropagasi Tanaman Kentang (Solanum
tuberosum L). Iptek Tanaman Sayuran.
Rahardja, P.C. dan W. Wiryanto. 2004. Aneka Cara Memperbanyak Tanaman.
Agromedia Pustaka, Jakarta.
Campbell, Neil A., J. B. Reece, and L.G. Mitchell. 2003. Biologi. Edisi Kelima Jilid 2
(diterjemahkan oleh Wasmen Manalu). Erlangga, Jakarta.
Salisbury, Frank B. Dan Cleon W Roos. 1995. Fisiologi Tumbuhan Jilid 3- Edisi
keempat. Bandung : Penerbit ITB.
Lestari, Endang G., 2008. Kultur Jaringan. Akademia : Bogor.
Gupta, Pratibha, Satyawati Sharma, dan Sanjay Saxena. 2010. Callusing in Stevia
rebaudiana (Natural Sweetener) for Steviol Glycoside Production. World Academy of
Science, Engineering and Technology.
14
Fatmawati, T.A, T. Nurhidayati, dan N. Jadid. Pengaruh Kombinasi Zat Pengatur
Tumbuh IAA dan BAP pada Kultur Jaringan Tembakau Nicotiana tobacum L.
VAR. Prancak 95. Pogam Studi Biologi Fakultas Matematika dan Ilmu Pengetahuan
Alam Institut Teknologi Sepuluh Nopember, Surabaya.
Nurul Jadid, priyono, Tutik Nurhidayati. Pengaruh Indole Acetic Acid (IAA) dan
Kinetin pada Kultur in vitro Nodus Tunas Mikro Vanili (Vanilla planifolia). Pogam
Studi Biologi Fakultas Matematika dan Ilmu Pengetahuan Alam Institut Teknologi
Sepuluh Nopember, Surabaya.
Nower, A.A. In Vitro Propagation and Synthetic Seeds Production: An Efficient Methods
for Stevia rebaudiana Bertoni. Sugar Tech 16, 100–108 (2014).
Muslihatin M, Jadid N, Puspitasari IK, Safitri CE. Growth of vegetative explant
Moringa oleifera on different composition of auxin and cytokinin and its synthetic
seed germination. AIP Conference Proceedings 1854, 020024 (2017).
Das, Arpita, Saikat G. dan Nirmal M., 2011. Micropropagation of an Elite Medical Plants:
Stevia rebaudiana Bert.. International Journal of Agricultural Research. 6(1): 40-48.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassay with
tobacco tissue cultures. Physiol. Plant 15:473–497.
15
BAB VIII LAMPIRAN
LAMPIRAN 1 Tabel Daftar Luaran
Program : Penelitian Kerjasama Antar Perguruan Tinggi
Nama Ketua Tim : Dr. Nurul Jadid, M.Sc
Judul : Teknik Mikropropagasi Tunas Mikro Stevia rebaudiana
(Bertoni) aksesi Mini secara in vitro sebagai Upaya
Pemuliaan dan Perbanyakan Bibit Unggul Tanaman Pemanis
Sehat Alternatif bagi Penderita Diabetes
1.Artikel Jurnal
No Judul Artikel Nama Jurnal Status Kemajuan*)
1 In Vitro Propagation of Stevia
rebaudiana (Bertoni) using Axenic
Auxillary Nodes
PeerJ draft
*) Status kemajuan: Persiapan, submitted, under review, accepted, published
In Vitro Propagation of Stevia rebaudiana (Bertoni) 1
using Axenic Auxillary Nodes 2
3
4
Nurul Jadid1*, Suci Anggraeni1, Wirdhatul Muslihatin1, Dini Ermavitalini1, Dwi Oktafitria2, 5
Supiana Dian Nurtjahyani2, Christin Risbandini1 6 7 1 Department of Biology, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia 8 2 Department of Biology Universits PGRI Ronggolawe, Tuban, Indonesia 9
10
11
Corresponding Author: 12
Nurul Jadid* 13
Kampus ITS Sukolilo, Surabaya, 60111 Indonesia 14
Email address: [email protected] 15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Abstract 42
Stevia cultivation (Stevia rebaudiana) has high economic potential because of its sweetness level 43
200-300 times, which is higher than sugar cane. However, the main obstacles in the stevia 44
cultivation is the low seed germination rate. Therefore, an efficient cultivation technique is 45
required. Micropropagation is an effective method of plant propagation, with the addition of plant 46
growth regulator (PGR) in accordance with the expected objectives. PGR is an organic compound 47
not a nutrient that affects plant growth and development. This research aims to analyse the effect 48
of different combination of Kinetin (Kin) and Benzyladenine (BA) on stevia micropropagation. Data 49
were analyzed by Anova at 95% confidence level followed by Tukey test. Different responses on 50
organogenesis (shoot and root formation) and callogenesis of stevia were observed. Intrestingly, 51
all treatment with Kin alone demonstrated callogenesis response. Treatment with the addition of 52
1 ppm BA resulted the best average of shoot number (36,05). While the treatment without PGR 53
resulted the best root formation (2,6). 54
55
Introduction 56
Stevia (Stevia rebaudiana) is a shrub in the Compositae family from Paraguay. Stevia 57
leaves produce a sweet taste caused by the presence of glycosides with a sweetness level of 200-58
300 times higher than sugar cane or sucrose (Geuns, 2003). Stevia leaf glycosides do not contain 59
calories and have a glycemite index of almost zero so it is suitable for diabetics and someone who 60
is losing weight (Jeppesen et al., 2002; Gregersen et al., 2004). Stevia sugar is widely used in the 61
food, soft drink, toothpaste, antibacterial and antioxidant industries. The sweet taste derived from 62
Steviosida is not digested in the body's metabolism so it is highly recommended for people with 63
diabetes, hypertension, obesity and fungal infections (Brandle et al., 1998; Megeji et al., 2005). 64
The use of stevia as a sweetener has developed in developed countries such as America and Japan. 65
A
b
s
t
r
a
c
t
G
u
i
In Japan, 5.6% of the sugar marketed is stevia sugar or what is known as sutebia (Rodiansah, 66
2007). 67
The number of industries that have applied stevia plants as sweeteners, makes stevia plant 68
cultivation have promising opportunities and has the potential to have quite high economic value. 69
However, stevia cultivation has a weakness in the propagation of stevia plants itself. This is due 70
to the percentage of seed germination of only 10%. Propagation of stevia by stem cuttings also 71
requires a lot of parent plants, so cultivation is largely less efficient. Due to the low rate of seed 72
germination and less efficient propagation through stem cuttings, a more effective propagation 73
method is needed. One method that can be applied for the propagation of stevia is through 74
micropropagation. 75
Micropropagation or also called plant tissue culture is the propagation of plants in sterile 76
conditions with take advantage of the totipotential properties of plant cells. The type and 77
concentration of growth regulators (ZPT) added to the culture media have an effect on the ability 78
of shoot regeneration. For shoot formation, the most commonly used ZPT is cytokinins such as 6-79
Benzyladenine (BA), kinetin, isopentenyl adenine (2-ip), zeatin and thidiazuron (TDZ) (Lestari, 80
2008). Apart from growth regulators, another important thing that influences the response of plant 81
explants is depending on the plant species, variety, accession or plant from which the explants 82
come from. Species, varieties, accessions and plant origins of the explants encode different genes, 83
so that when interacting with the environment, they will have different effects. The influence of 84
genes is closely related to the factors that influence explant growth, such as nutritional 85
requirements, growth regulators, and the culture environment (Hendaryono and Wijayani, 1994). 86
Ibrahim, et al (2008) and Anbazhagan, et al (2010) have succeeded in conducting 87
micropropagation experiments of stevia shoots with ZPT 6-Benzyladenine (BA) and Kinetin 88
(Kin). Based on this research, the utilization of 6-Benzyladenine (BA) and kinetin (Kin) produced 89
quite a lot of shoots, so it is necessary to experiment with the application of these hormones with 90
a lower limit concentration of BA 0 mg / L and an upper limit of 2 mg / L while the lower limit of 91
Kin 0 concentration. mg / L and an upper limit of 8 mg / L for Kin in the hope of producing more 92
shoots because the needs and types of ZPT used for each genotype are not the same (Lestari, 2008). 93
In Indonesia, there are several accessions, namely green accessions, jumbo accessions, 94
purple accessions, yellow accessions and mini accessions. In this study, mini accessions were used. 95
Therefore, this research was carried out by developing several previous studies in order to obtain 96
the optimum combination of ZPT for the micropropagation of miniature Stevia rebaudiana shoots. 97
98
Materials & Methods 99
Plant Material 100
Stevia acc. Mini was obtained from Malang's Sweetener and Fiber Crops Research Institute. The 101
explants used were in the form of nodes or segments of uniform size, located in the order of 1-4, 102
counted from the shoots and not yet flowering. 103
Preparation and Sterilization of Media 104
Preparation of the media begins with dissolving sucrose with a concentration of 30 g / L in distilled 105
water with a volume of half the volume of media to be made. After dissolving, MS instant was 106
added with a concentration of 4.43 g / L then dissolved in the sugar solution that had been made 107
previously until it was homogeneous. Once homogeneous, add distilled water to reach the volume 108
of media you want to make. The combination of the growth regulator Kinetin (concentration range 109
0 ppm to 8 ppm) and Benzyladenine (concentration range 0 ppm to 2 ppm) is added according to 110
the type of medium to be made. The pH of the media was measured and conditioned in the range 111
of 5.6-5.8 (1N NaOH or 1N HCl added to reach this pH) with a pH meter. To be added with a 112
concentration of 8.2 g / L after the pH has reached 5.6-5.8. In order to be dissolved on a magnetic 113
stirrer with a hot plate, after dissolving the media it is poured into a culture bottle of 25-30 ml. The 114
culture bottle containing the medium was covered with aluminum foil. Media is sterilized at a 115
temperature of 121˚C with a pressure of 1 atm for 20 minutes by autoclave. The media is allowed 116
to stand at room temperature for 2-3 days to see whether the media made is sterile or not. 117
Sterilization of Equipment and Materials 118
Sterilization of the equipment begins by sterilizing the culture bottles by immersing them in 0.3% 119
sodium hypolorite (NaOCl) for 24 hours, then washing them with soap and drying them in the sun. 120
Scalpels, spatulas and tweezers are sterilized by spraying with 70% alcohol. The materials that 121
need to be sterilized are tissue, distilled water and black cloth. Material sterilization is carried out 122
by heating the diauoclave at a temperature of 121˚C at a pressure of 1 atm for 20 minutes. 123
Sterilization of the Inoculation Room 124
Before doing explant inoculation, the inoculation room needs to be sterilized first. First, spray the 125
glass walls of Laminar Air Flow (LAF) with 70% alcohol and then wipe them with a tissue. Media, 126
sterile tissue, tweezers, scalpels, spatulas and LAF distilled water. The UV lamp is turned on for 127
5 minutes, then the blower is turned on and left for 3 minutes. LAF is ready to use. 128
Explant Sterilization 129
This research was conducted using explant nodes or segments in the order of 1-4 counted from the 130
shoots. Sterilization of nodes from parent plants to produce sterile plantlets that will be used for 131
shoot micropropagation. Stems containing 1-4 nodes of the parent plant are cut into pieces per 132
node. Sterilization begins by rinsing the explants under running water for 1 minute. Drain the node 133
so that no water drips. The nodes are soaked and shaken with 70% alcohol for 1 minute. 134
Sterilization was followed by soaking in 1.5% NaOCl for 5 minutes while shaking. The final step 135
is to rinse the expanant with sterile aquadest 4 times, each rinse for 4 minutes in LAF. 136
137
Explant Inoculation 138
Node explants from sterilized broodstock plants are cut at each end on sterile tissue. The explants 139
were inoculated into MS 0 media, with a total of 5 explants 140
every bottle. Before opening and closing the culture bottle, the lip of the bottle is heated through a 141
bunsen fire. The explant is inoculated with the bud facing upwards. After planting, the culture 142
bottles were closed using aluminum foil which was previously passed through the fire. After the 143
culture bottle is covered with aluminum foil, the tip of the bottle is heated in a bunsen area. 144
Growth of Inoculation Result Explants 145
Culture bottles containing media and explants were placed in a culture room with a temperature of 146
approximately 25˚C on culture racks lit with 40 watts per square meter of TL lamps, approximately 147
1000 lux with 16 hours of light and 8 hours of darkness. . Maintenance is carried out by checking 148
the contamination level of the observed material every 2x a week. 149
Observation Variables 150
The observation variables of this study are: 151
a. Percentage of explants sprouting, which is calculated by the formula: 152
153
154
b. Percentage of callous explants: 155
𝛴 𝐸𝑘𝑠𝑝𝑙𝑎𝑛 𝑦𝑎𝑛𝑔 𝑏𝑒𝑟𝑡𝑢𝑛𝑎𝑠
𝛴 𝐸𝑘𝑠𝑝𝑙𝑎𝑛 𝑦𝑎𝑛𝑔 𝑡𝑒𝑟𝑠𝑒𝑑𝑖𝑎 𝑝𝑎𝑑𝑎 𝑠𝑒𝑡𝑖𝑎𝑝 𝑝𝑒𝑟𝑙𝑎𝑘𝑢𝑎𝑛 x 100%
𝛴 𝐸𝑘𝑠𝑝𝑙𝑎𝑛 𝑦𝑎𝑛𝑔 𝑏𝑒𝑟𝑘𝑎𝑙𝑢𝑠
𝛴 𝐸𝑘𝑠𝑝𝑙𝑎𝑛 𝑦𝑎𝑛𝑔 𝑡𝑒𝑟𝑠𝑒𝑑𝑖𝑎 𝑝𝑎𝑑𝑎 𝑠𝑒𝑡𝑖𝑎𝑝 𝑝𝑒𝑟𝑙𝑎𝑘𝑢𝑎𝑛 x 100%
156
c. Number of shoots and roots. 157
Calculation of the number of shoots, roots and callus formed was carried out after 10 weeks from 158
planting time. 159
Research Design and Data Analysis 160
This research was conducted using a completely randomized design (CRD) with 5 repetitions. 161
Data analysis was performed using the Minitab 17 - Anova two way program. If there is a 162
significant difference, then proceed with the Tukey test with a confidence level of 95%. The data 163
analyzed included the percentage of sprouting parameters, 164
number of shoots, and number of roots. 165
Results and Discussion 166
Organogenesis and Callogenesis Responses 167
Plant growth regulators (PGRs) are organic molecules other than nutrients (elements that provide 168
energy or mineral nutrients), which affect plant growth and development. PGRs is active at 169
relatively low concentrations. Several types of PGRs have functions to accelerate, inhibit, and 170
cause mutations in physiological processes that occur in plants (Basra, 2000). In in vitro culture 171
the use of PGRs in low concentrations is required to induce cell proliferation and organogenesis 172
(Gaba, 2005). The use of growth regulators in tissue culture also depends on the goal or direction 173
of growth desired. Lestari (2008) revealed that the interaction between PGRs that occurs naturally 174
(endogenously) in explants (cells, tissue or plant organs) and exogenous PGRs also affects plant 175
growth and organogenesis. Benzyladenine (BA) and Kinetin (Kin) are PGRs cytokinins that 176
function as a stimulant for cell division and breakdown of apical donations by auxins (Salisbury 177
and Ross, 1995). This study used explants in the form of nodes from sterile stevia plantlets. This 178
sterile explant is generally known as a secondary explant. In this study, the interaction of BA and 179
Kin at different concentrations caused different responses to organogenesis and callogenesis 180
(Table 1). Chishimba (2000) reported that the addition of BA, Kin and PGRs other cytokinins to 181
the in vitro culture of shoot explants of Uapaca kirkiana shoots gave a different response. This 182
was also reported by Quadri (2012) who stated that the addition of BA, Kin and Thidiazuron to 183
the in vitro shoot proliferation of Hyoscyamus niger L. also gave a different response. 184
Data analysis using Anova - two way showed that the interaction between BA and Kin had 185
no effect on the percentage of explants sprouting (p value> 0.05). However, explants grown on 186
MS medium with different combination treatment of ZPT almost entirely responded to shoot 187
formation. The value of the percentage of shoot formation ranged from 45% -100%. The lowest 188
shoot formation response appeared in the medium with the addition of ZPT 1 ppm BA + 8 ppm 189
Kin. While the highest response to shoot formation appeared in the medium with the addition of 190
ZPT BA singly and in combination with low concentrations of Kin (0.5 ppm BA + 2 ppm Kin 191
(Figure 2.1) and 1.5 ppm BA + 2 ppm Kin). From these data it can be concluded that BAZ ZPT 192
provides an organogenetic response in the form of better shoot formation than Kin in this study. 193
The low percentage of sprouting due to the presence of Kin in inducing the growth of axillary 194
shoots is in accordance with the results of research by Thiyagarajan (2012), Razak (2014), Rafiq 195
(2007). 196
BA provides a better response to shoot formation because it has stronger activity (Zaer and 197
Mapes, 1982) and causes more significant elongation than kinetin (Salisbury and Ross, 1995). BA 198
has the same basic structure as kinetin but is more effective because BA has a benzyl group 199
(George, l984). Flick (1993) stated that in general, plants have a better response to BA than to 200
kinetin and 2-iP so that BA is more effective for in vitro shoot production. 201
In addition to the response of sprouted explants, this study also observed the response of callous 202
explants. Callus is an amorphous and unorganized mass of cells that is formed on the surface of 203
the injured tissue piece as a protective response to cover the injured tissue (Heryanto, 2014). In 204
this study, 100% callus was formed on media supplemented with Kin (Figure 2.1). The 205
morphology of the callus after 10 weeks of inoculation, the callus is compact and brownish while 206
on the top surface of the callus, the callus is friabele greenish in color (Ikeuchi, 2013). Gupta 207
(2010) reported that the addition of Kin with a concentration of 3 ppm, 4 ppm and 5 ppm gave a 208
response to callus formation. 209
Cell division that occurs due to cytokinins can produce undifferentiated callus (Gaba, 210
2005). Anbazhagan (2010) reported that addition of Kin to stevia shoot propagation can initiate 211
shoots at the beginning of growth only and this has documented earlier studies also by Murashige 212
(1974), Benne and Davies (1986) and Rogers (1998). Different research results were reported by 213
ibrahim (2008) that the shoot tip explants of stevia planted on the medium with the addition of Kin 214
alone produced a higher number of shoots compared to the number of shoots in this study, although 215
the response to the number of shoots produced by BA ZPT was still higher than ZPT. Kin. The 216
differences in the results of these studies could be caused by different explants' genotypes. The 217
genotype factor itself is the most important factor in tissue culture (Gaba, 2005). 218
Shoot Formation 219
The number of shoots formed is the main purpose in commercial micropropagation of plants. 220
Multiplication of shoots is induced by the application of exogenous cytokines to the growth 221
medium (Gaba, 2005). The results of the two-way anova-analysis showed that the addition of the 222
combination of Kin and BA with different concentrations had an effect on the number of shoots 223
(P value <0.05). The concentration of ZPT that produced the highest number of shoots was 1 ppm 224
BA with the number of shoots produced as many as 36.05 shoots (Table 2.2; Figure 2.3). While 225
the lowest number of shoots was produced by a combination of ZPT 1 ppm BA + 8 ppm Kin and 226
1 ppm BA + 6 ppm Kin with a number of shoots of 0.65. Based on these data, it can be concluded 227
that BA is more effective in shoot multiplication than Kin. Alhady (2011) and Anbazhagan (2010) 228
also reported that BA was more effective for stevia shoot multiplication. This also occurs in other 229
species such as Bauhinia veriegate (Ahmed, 2007), Balanites aegyptiace (Mathur, 1992), and 230
Periploca angustifolia (Abd-Alhady, 2010). The number of shoots produced by Alhady (2011) in 231
the treatment of 2 ppm BA + o, 5 ppm Kin 36.9 shoots (highest yield) while in treatment 0.5 ppm 232
BA + 0.5 ppm Kin 18.1 shoots (lowest yield) . Anbazhagan (2010) produced the number of shoots 233
9.20 at 2 ppm BA treatment and 4.40 at 2 ppm Kin. 234
The basic function of cytokinins is to stimulate cytokinesis or cell division. A study of 235
cytokinin-activated cell division in the apical meristem, Houssa et al. (1990) obtained results 236
largely consistent with the study of Fosket et al. (1981). They found that benzyladenine greatly 237
shortened the time that the S phase took place in the cell cycle (from G2 to mitosis, the DNA and 238
protein synthesis stage of cell division). Fosket, et al (1981) concluded that cytokinins promote 239
cell division in tissue cultures by increasing the transition from G2 to mitosis and this occurs 240
because cytokinins increase the rate of protein synthesis. Some of these proteins are in the form of 241
building proteins or enzymes needed for mitosis. Protein can be increased by stimulating the 242
formation of mRNA that encodes these proteins. 243
Cytokinins can induce shoot formation by generating cytokinin signals due to positive 244
regulation of WIND1 that occurs due to injury. WINDs are transcription factors and support 245
dedifferentiation (have the same function as WUS). After the cytokinin signal appeared, WUS 246
inhibited the expression of ARRs to maintain the stem cell population in the shoot meristem tissue. 247
After positive regulation of cytokinin signals to WUS, WUS positively regulates CLV3. CLV3 248
functions to regulate cell proliferation in SAM, the presence of reciprocal regulation between 249
CLV3 and WUS to maintain stem cell population and tissue size resulting in cell differentiation 250
and forming lateral organ arrangement (Ikeda, 2014). 251
Root Formation 252
In vitro root formation is necessary to convert shoots into whole plantlets that can be 253
transplanted into a greenhouse (Gaba, 2005). In vitro plant root initiation can be stimulated by 254
adding ZPT auxin groups such as Indole-3-Acetic Acid (IAA), Naphtalene Acetic Acid (NAA) 255
and Indole-3-Butyric Acid (IBA) (Arlianti, 2013). Several explants in this study responded to root 256
formation (Table 2.3). Data analysis using Anova - two way shows that the interaction between 257
BA and Kin affects the number of roots (p value> 0.05). The highest number of roots was produced 258
by MS 0 treatment or without the addition of ZPT. Treatment of cytokinins in high concentrations 259
can inhibit root formation. The wound treatment during shoot planting can induce auxin synthesis 260
for root formation. In certain cases, roots can be induced by transplanting the shoots to the medium 261
without ZPT once or twice to reduce the cytokinin concentration. 262
Another method option is the addition of auxin at low concentrations to induce roots (Gaba, 263
2005). Arlianti (2013) conducted an experiment on root induction of Stevia, which resulted in the 264
best ZPT concentration for root induction of 0.2 ppm NAA with 8 roots. While Anbazhagan (2010) 265
reported ZPT 1 ppm IAA produced 11 roots, Alhady (2011) with 2 ppm IBA ZPT produced 8.4 266
fruits and Das (2011) was able to produce roots without the addition of ZPT (MS 0) with 15 roots. 267
58 pieces. 268
Auxin itself has many roles in tissue culture, depending on the chemical structure, 269
concentration and response of the plant tissue itself. Auxins cause callus and root formation as 270
well as the growth of stem extensions. Auxins in general have the function of stimulating cell 271
elongation, cell division in the cambium tissue and together with cytokinins stimulating xylem and 272
phloem differentiation. The addition of high enough exogenous auxin can induce somatic embryo 273
genesis. A higher ratio of auxin to cytokinins will induce root formation in shoots, callus initiation 274
in monocot plants, and initiation of somatic embryogenesis. A nearly balanced ratio of auxin to 275
cytokinin will induce the formation of additional roots from callus and callus initiation in dicot 276
plants. A lower ratio of auxin to cytokinin will induce additional shoots and axillary shoot 277
production. For this reason, the interaction between auxin and cytokinins is very important to 278
control many growth and development processes in vitro (Gaba, 2005). 279
Conclusions 280
We report the use of secondary explants derived from axenic auxillary nodes of stevia accession 281
Mini. Different organogenesis and callogenesis responses resulted from the use of different 282
combination of cytokinins (Kin and BA) concentration. The percentage of shoot formation ranged 283
from 45-100%. The lowest shoot formation was obtained from the MS media with 1 ppm BA and 284
8 ppm Kin. Whereas, the highest percentage of shoot formation appeared in the MS with BA only 285
and in combination with low concentration of Kin. Meanwhile, callogenesis responses were mostly 286
obtained from the MS media containing Kin. Explant placed in the MS medium with 1 ppm BA 287
alone showed highest shoot proliferation (36,05 shoots). Overall, these results indicate that the 288
comprehensive selection of PGRs types and concentration contribute to better organogenesis 289
responses. Also, these findings might be used for accelerating breeding and genetic improvement 290
of stevia cultivars. 291
Acknowledgements 292
The authors would like to thank all members of the laboratory of plant bioscience and technology 293
for all technical supports. We also acknowledge Indonesian Sweetener and Fiber Crops Research 294
Institute for providing plant materials. 295
References 296
Geuns J. M. C., “Molecules of Interest – Stevioside,” Phytochem. 64, (2003) 913-921. 297
Jeppesen PB, Gregersen S, Alstrup KK., “Stevioside induces antihyperglycaemic, insulinotropic 298
and glucagonostatic effects in vivo: studies in the diabetic Goto-Kakizaki (GK) rats,” 299
Phytomed. (2002) 9:9- 14. 300
Gregersen S, Jeppesen PB, Holst JJ, Hermansen K., “Antihyperglycemic effects of stevioside in 301
type 2 diabetic subjects,” Metabolism, (2004) 53:73-76. 302
Brandle JE, Starrtt AN, Gijzen M., “Stevia rebaudiana: international agriculturalf, biological 303
chemical properties,” Can J Plant Su, (1998) 78:527-536. 304
Megeji NW, Kumar JK, Singh V, Kaul VK and Ahuja PS., “Introducing Stevia rebaudiana, a 305
natural zero-calorie sweetener,” Curr Sci, (2005) 88:801-805. 306
Rodiansah, Asep., “Induksi Mutasi Kromosom dengan Koklisin pada Tanaman Stevia 307
(Stevia rebaudiana Bertoni) Klon Zweeteners Secara In Vitro,” Skripsi, Bogor : Progam 308
Studi Hortikultura Fakultas Pertanian Institut Pertanian Bogor (2007). 309
Lestari, Endang G., “Kultur Jaringan,” Akademia : Bogor (2008). 310
Hendaryono, Daisy P. Dan Wijayani, A., “Teknik Kultur Jaringan,” Kanisus :Yogyakarta (1994). 311
Ibrahim, I. A., M. I. Nasr, B. R. Mohammed, M. M. El-Zefzafi. “Plant Growth regulators 312
affecting in vitro Cultivation of Stevia rebaudiana,” Sugar Tech, (2008) 10(3) : 254-259. 313
Anbazhagan, M., M. Kalpana, R. Rajendran, V. Natarajan dan D. Dhanavel, “In Vitro Production 314
of Stevia rebaudiana Bertoni,” Emir J. Food Agric, (2010) 22(3) : 216-222. 315
Basra, A. S., Plant Growth Regulators in Agriculture and Horticulture: Their Role and 316
Commercial Uses, Haworth Press : New York (2000). 317
Gaba, V. P., “Plant Growth Regulators,” Plant Tissue Culture and Development. CRC Press : 318
London. (2005) p. 87-99. 319
Salisbury, Frank B. Dan Cleon W Roos, Fisiologi Tumbuhan Jilid 3- Edisi keempat, Bandung : 320
Penerbit ITB (1995). 321
Kolb, N., J. L. Herrera, D. J. Ferreyra dan R. F. Uliana. 2001. Analysis of Sweet Diterpene 322
Glycosides from Upaca kirkrana Improved HPLC Method. J. Agric Food Chem, (2000) 323
49, 4538-4541. 324
Quadri, R. R., Jaime A. T. S., Azra N. K. dan Ali M, “Effect of 6- Benzyladenine, Kinetin and 325
Thidiazuron on in Vitro Shoot Proliferation of Hyoscyamus niger L.,” Medical and 326
Aromatic Plant Science and Biotechnology, (2012) 6(1), 81-83. 327
Thiyagarajan, M dan P. Venkatachalam, “Large Scale In vitro Propagation of Stevia rebaudiana 328
(bert) for Commercial Application : Pharmaceutically Important and Antidiabetic Medical 329
Herb,” Industrial Crops and Products, (2012) P. 111-117. 330
Razak, U. N. A. A., Chong B. O., Tiew S. Y. Dan Li Kiaw L., “ In vitro Micropropagation of 331
Stevia rebaudiana Bertoni in Malaysia,” Brazilian Archives of Biology and Technology – 332
An Interntional Journal. Vol. 57, n.1. (2014) pp. 23-28. 333
Rafiq, Muhammad, M. Umar D., Sher M., Mangrio, Habib A. N. Dan Iqbal A. Qarshi, “In vitro 334
Clonal Propagation and Biochemical Analysis of Field Establishment Stevia rebaudiana 335
Bertoni,” Pak. J. Bot.,(2007) 39(7): 2467-2474. 336
Zaer dan Mapes, Action of Growth Regeneration in Bonga and Durzan (eds.) Tissue Culture in 337
Forestry. Martinus Nijhoff : London. (1982) P. 231-235. 338
George, E. F. Dan P. D. Sherington, Plant Propagation by Tissue Culture. Handbook and 339
Directory of Commercial Laboratories. Exegetic : England. (1984) P. 709. 340
Flick, C. E., D. A. Evans dan W.R. Sharp, Organogenesis. Handbook of Plant Cell Culture and 341
Development, CRC Press : London. (1993) P. 87-100. 342
Heryanto, Aditya F., C. J. Soegihardjo, L.M. Ekawati, “Optimasi Produksi Steviosida dari Kalus 343
Daun Stevia rebaudiana Bertoni dengan Variasi Kombinasi Zat Pengatur Tumbuh,” Jurnal 344
Teknobiologi, (2014) P. 1-13. 345
Ikeuchi, Momoko, Keiko S. dan Akira I., “REVIEW – Plant Callus: Mechanisms of Induction and 346
Repression,” The Plant Cell. Vol. 25 (2013) 3159-3173. 347
Gupta, Pratibha, Satyawati Sharma, dan Sanjay Saxena, “Callusing in Stevia rebaudiana (Natural 348
Sweetener) for Steviol Glycoside Production,”World Academy of Science, Engineering 349
and Technology (2010). 350
Murashige, T. and F. Skoog, “ A revised medium for rapid growth and bioassay with tobacco 351
tissue cultures,” Physiol. Plant, (1962)15:473– 497. 352
Benne, L. K. and F. T. Davies, “In vitro propagation of Quercus shumardii seedling,” Hort. Sci, 353
(1998) 21:1045–1047. 354
Rogers, D. S., J. Beech, and K. S. Sharma, “Shoot regeneration and plant acclimatization of the 355
wetland monocot Cattail (Typha latifolia),” Plant Cell Rep. (1998) 18:71–75. 356
Abd-Alhady, M.R., M.M Abd Alla, G. A. Hegazi dan M. F. Gabr, “Rapid Propagation of 357
Periploca angustifolia Labill. By Tissue Culture,” International J. Plant Development 358
Biol. 4(1 (2010):15-18. 359
Ahmed, M.R. dan M. H. Bekhit, “In vitro Propagation of Balanites aeygptical (L) Del. An 360
Endangered Medical Plant,” J. Agri. Sci. Mansoura Univ.. 32(4) (2007) :2907-2915 361
Mathur, J. Dan Mukunthakumar, “Micropripagation of Bauhinin variagate dan Parkinsoma 362
aceuleaa from nodal explant of mature trees,” Plant Cell Tiss. Organ Cult. 28 (1992) 363
:199-121. 364
Ikeda, Miho, dan Masaru Ohme-Takagi, “TCPs, WUSs, and WINDs: Families of Transcription 365
Factors that Regulate Shoot Formation, Stem Cell Maintenence, and Somatic Cell 366
Differentiation,” Frontiers in PLANT SCIENCE – Mini Review Article. Vol. 5 (2014). 367
Arlianti, Tias, Aitti F. S., NN Kristina, dan Otih R, “Pengaruh Auksin IAA, IBA dan NAA 368
Terhadap Induksi Perakaran Tanaman Stevia (Stevia rebaudiana) Secara In vitro,” Bul. 369
Littro. Vol. 24, No. 2 (2013). 370
Das, Arpita, Saikat G. dan Nirmal M., “Micropropagation of an Elite Medical Plants: Stevia 371
rebaudiana Bert.,” International Journal of Agricultural Research. 6(1) (2011): 40-48. 372
373
374
375
Tabel 2.1 Organogenesis and callogenesis responses of stevia in MS containing different 376
concentration of PGRs 377
PGRs Combination Explants
forming
shoot (%)
Callogenesis
(%) BA
(ppm)
Kin
(ppm)
0 0 100 0
0,5 0 100 0
1 0 100 0
1,5 0 100 0
2 0 100 0
0 2 90 100
0,5 2 100 100
1 2 90 100
1,5 2 100 100
2 2 65 100
0 4 85 100
0,5 4 90 100
1 4 60 100
1,5 4 65 100
2 4 60 100
0 6 75 100
0,5 6 75 100
1 6 50 100
1,5 6 60 100
2 6 85 100
0 8 95 100
0,5 8 75 100
1 8 45 100
1,5 8 75 100
2 8 85 100
378
Tabel 2.2 The response to the number of shoots produced by sterile node explants was 379
treated with different combinations of PGRs 380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
Kombinasi ZPT Jumlah tunas
Kombinasi ZPT Jumlah tunas
BA (ppm)
Kin (ppm)
BA (ppm)
Kin (ppm)
0 0 1,9e 1,5 4 1,4e
0,5 0 4,95de 2 4 1e
1 0 36,05a 0 6 1,2e
1,5 0 17,2c 0,5 6 1,6e
2 0 26b 1 6 0,65e
0 2 1,9e 1,5 6 0,8e
0,5 2 8d 2 6 1,4e
1 2 3,55de 0 8 1,7e
1,5 2 7,35d 0,5 8 1,05e
2 2 1,05e 1 8 0,65e
0 4 1,55e 1,5 8 1,65e
0,5 4 1,8e 2 8 1,4e
1 4 0,85e
Table 2.3 Response to root formation with different combinations of ZPT BA and Kin treatment 420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
Kombinasi ZPT Jumlah
akar
Kombinasi ZPT Jumlah
akar BA
(ppm) Kin
(ppm) BA
(ppm) Kin
(ppm)
0 0 2,6a 1,5 4 0,0b
0,5 0 0,15b 2 4 0,0b
1 0 0,0b 0 6 0,0b
1,5 0 0,0b 0,5 6 0,0b
2 0 0,0b 1 6 0,0b
0 2 0,05b 1,5 6 0,0b
0,5 2 0,0b 2 6 0,0b
1 2 0,0b 0 8 0,0b
1,5 2 0,0b 0,5 8 0,0b
2 2 0,0b 1 8 0,0b
0 4 0,0b 1,5 8 0,0b
0,5 4 0,0b 2 8 0,0b
1 4 0,0b * Nilai yang diikuti oleh huruf yang sama pada kolom menunjukkan
tidak adanya beda nyata pada uji Tukey selang kepercayaan 95%.
460
461
462
463
464
465
466
467
468
469
Figure 2.1 Organogenesis responses are in the form of shoots (1) and callogenesis (2). 470
The explant is 10 weeks old. Information; A. 0.5 ppm BA + 2 ppm Kin, B. 0.5 ppm BA 471
+ 4 ppm Kin. The white line shows the 0.5 cm scale. 472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
A1 B1
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
Figure 2.2 Organogenetic response in the form of shoots on explants planted on 0.5 ppm BA 517
medium. The explant is 10 weeks old. Note: The white line indicates the 0.5 cm scale. 518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
Figure 2.3 Observation of the number of shoots 10 weeks after inoculation. Information; (A) 570
explants grown on MS 0 medium or control. (B) explants grown on 0.5 ppm BA media. (C) 571
explants grown on 1 ppm BA medium. The white line shows the 0.5 cm scale. 572
573
574
575
576
A1
C
E
B1
D1
F1
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
Figure 4.4 Roots formed after being treated with a combination of ZPT with different 598
concentrations for 10 weeks. Information; A: Roots formed on MS media 0, B: Roots formed on 599
0.5 ppm BA media. The arrows point to the roots. The white line shows the 0.5 cm scale. 600
601
A1 B