laporan akhir penelitian kerjasama antar perguruan …

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LAPORAN AKHIR

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

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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

LAMPIRAN 2 Bukti Luaran ………………………………………………………………………………17

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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

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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

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Daftar Lampiran

Hal

Lampiran 1. Tabel Daftar luaran 15

Lampiran 2. Bukti Luaran 17

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.

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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

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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

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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

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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.

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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 telah men submit paper yang berjudul : In vitro shoot micropropagation of Gracilaria

verrucosa using dual plant growth regulators. Paper tersebut disubmit pada jurnal AACL

Bioflux (Scopus Q3). Selain itu, peneliti juga telah mempresentasikan paper pada 5th

International Biology Conference (5th IBOC) pada 17 Oktober 2020. Adapun fullpaper yang telah

dipresentasikan tersebut berjudul : A review on silver nanoparticle elicited culture: A

nanotechnological approach to crop improvement and plant metabolite production akan

disubmit ke Agriculture (Switzerland) (Scopus Q2).

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

Berdasarkan hasil penelitian yang diperoleh, perlu dilakukan penelitian lanjutan mengenai

mikropropagasi tanaman bernilai ekonomi tinggi lainnya dengan menggunakan PGRs serta aplikasi

nanopartikel. Penggunaan nanopartikel dalam teknologi kultur in vitro memiliki fungsi penting dalam

mengakselerasi pertumbuhan dan perkembangan eksplan, meminimalisir terjadinya kontaminasi serta

meningkatkan kandungan metabolit sekunder.

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

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13

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14

Fatmawati, T.A, T. Nurhidayati, dan N. Jadid. Pengaruh Kombinasi Zat Pengatur

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https://aip.scitation.org/doi/abs/10.1063/1.4985415





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 shoot micropropagation of

Gracilaria verrucosa using dual

plant growth regulators

AACL Bioflux Submitted

2. A review on silver nanoparticle

elicited culture: A

nanotechnological approach to crop

improvement and plant metabolite

production

Agriculture (Switzerland) draft

*) Status kemajuan: Persiapan, submitted, under review, accepted, published

2. Artikel Konferensi

No Judul Artikel Nama Konferensi (Nama

Penyelenggara, Tempat,

Tanggal)

Status Kemajuan*)

1. A review on silver nanoparticle

elicited culture: A

nanotechnological approach to

crop improvement and plant

metabolite production

5th International Biology

Conference (Department

of Biology, Institut

Teknologi Sepuluh

Nopember, 17 Oktober

2020)

Presented

*) Status kemajuan: Persiapan, submitted, under review, accepted, presented

3. Paten

No Judul Usulan Paten Status Kemajuan

-

*) Status kemajuan: Persiapan, submitted, under review

16

4. Buku

No Judul Buku (Rencana) Penerbit Status Kemajuan*)

-

*) Status kemajuan: Persiapan, under review, published

5. Hasil Lain

No Nama Output Detail Output Status Kemajuan*)

-

*) Status kemajuan: cantumkan status kemajuan sesuai kondisi saat ini

6. Disertasi/Tesis/Tugas Akhir/PKM yang dihasilkan

No Nama Mahasiswa NRP Judul Status*)

1. Iro Datus Soleha 01311740000018 Pengaruh zat

pengatur

tumbuhan pada

kultur in vitro

tanaman herbal

In progress

*) Status kemajuan: cantumkan lulus dan tahun kelulusan atau in progress

17

LAMPIRAN 2 : Bukti luaran

1. AACL Bioflux submission

AACL Bioflux, 201X, Volume X, Issue X.

http://www.bioflux.com.ro/aacl 1090

In vitro shoot micropropagation of Gracilaria

verrucosa using dual plant growth regulators 1Muhammad Evan Nurrahmawan, 2Dwi Oktafitria, 3Hery Purnobasuki, 1Dini

Ermavitalini, 1*Nurul Jadid

1 Department of Biology, Institut Teknologi Sepuluh Nopember, 60111 Surabaya,

Indonesia;2 Department of Biology, Universitas PGRI Ronggolawe 62381 Tuban,

Indonesia; 3 Department of Biology, Universitas Airlangga, Surabaya, Indonesia. *Corresponding author: N. Jadid, [email protected]

Abstract. Gracilaria verrucosa-an Agar producing seaweed-is the highest and most cultivated seaweed in Indonesia. However, its productivity is still low and has not met the global demand for Agar. In addition, the lack of good quality seed stocks of this species becomes serious problem. Therefore, an efficient G.verrucosa in vitro culture could be an alternative to solve this obstacle. However, little is known about this method of cultivation. This study aims to determine the effect of dual plant growth regulators (IAA and BAP) applications on the shoot micropropagation of G.verrucosa. Intercalar explant of G.verrucosa was grown into PES medium supplemented by both IAA and BAP in various concentrations (0; 0.1; 0.3; 0.5 mg/l) for 30 days. Single BAP at 0.5 mg/l treatment showed the best result in all parameters measured, including growth rate (0.42% per day), percentage of explant producing shoot (56%) and the number of shoot per explant (2.64 shoots/explant). Key Words: Gracilaria verrcusoa, micropropagation, plant growth regulators, seaweed, Acropora formosa, coral growth rates, coral nursery, reef restoration, coral transplant.

Introduction. Gracilaria verrucosa is one of the promising red algae (Rhodophyta),

which is commonly cultivated in the tropical regions, including Indonesia. The species

offers important economical interest due to its agar content (Carneiro et al 2011).

Therefore, G. verrucosa is also included as agarophytes, together with other agar-

producing red algae such as Gelidium and Gelidiella (Rocha et al 2019). Agar, the main

product of red algae, is a mixture of agarose and agaropectin. It has been used as

thickeners and emulsifiers in food industry, medicine, cosmetics, paper, textiles,

industrial oils and other biotechnological industries (FAO 2003; Olatunji 2020). Due to an

increase demand of agar worldwide, the cultivation of G. verrucosa has been increasing

significantly in the last decade. According to FAO (2010), Indonesia is considered as the

second largest place for G. verrucosa cultivation, which reaches 253 thousand tons of

dried seaweed or contribute to about 30.02% of the total global Gracilaria production

(Rejeki et al 2018). One of the center of seaweed cultivation in the eastern part of

Indonesia is located in Jabon Subdistrict, Sidoarjo Regency with total production value

reached 1,344 tons of dried seaweed in 2016 (BPS 2016).

According to the Indonesian Ministry of Maritime Affairs and Fisheries, the global

demand of Agar in 2016 have reached 550,000 tons and continues to increase every year

(KKP 2016). Various efforts have been made to cover the high demand of Agar such as

the provision of sustainable seeds. To date, the provision of sustainable seeds is still

facing up many obstacles, such as the low growth rate of seaweed and adverse

environmental conditions due to epiphytes attacks and infectious diseases (Sahu et al

2020). The provision of sustainable seeds has been done through conventional

techniques by vegetative propagation using the cutting techniques. This technique

includes the selection of high growth rates seaweed and re-cultivation it into new area

(Masak et al 2011). Nevertheless, frequent vegetative propagation could decrease the

quality of seaweed seed, including low growth rate, reduced Agar content, gel strength

mailto:[email protected]



and increase of seaweed susceptibility in disease (Hurtado and Cheney 2003). Therefore,

the development of an alternative method of propagation is necessarily required.

In vitro micropropagation techniques provides promising prospects for the

development of commercial high yield seaweed. This technique provides good quality

seaweed, since the preliminary selection of high-quality of seeds is done before.

Consequently, the resulting clones should have similar genetic characteristics to the

mother source. In addition, in vitro culture method is not affected by the weather and

uncertain climate factors. Furthermore, the method is not time-consuming and could

produce large amounts of clones (Yong et al 2014).

The efficiency and effectiveness of in vitro shoot micropropagation is mainly

determined by endogenous and exogenous factors. The endogenous factors include

explant characteristics such as age, explant sources, developmental stage and

physiology, whereas exogenous factors includes plant growth regulators (PGRs) (Jadid et

al 2015), salinity, light irradiation, photoperiodism, temperature, pH and media

composition (Yokoya et al 2011). According to Yeong et al. (2014), the use of PGRs play

a role in the success of in vitro shoot micropropagation. It has been demonstrated that

the use of 0.1 mg/L 2,4-D and 0.1 mg/ kinetin L in the PES (Provasoli's Enriched

Seawater) medium stimulates micropropagation of Gracilaria changii shoots explosively.

The in vitro culture of some genus Gracilaria has been reported, for instant G. textorii

(Huang and Fujita 1997), G. vermiculophylla (Yokoya et al 1999), G. chilensis (Collantes

et al 2004), G. tenuifrons (Yokoya 2000), G. tenuistipatata, G. Perplexa (Yokoya et al

2004), G. corticata (Kumar et al 2007). However, studies on the induction and

multiplication of shoots on G.verrucosa are still limited to the use of F2 medium

supplemented with 5 mg/l DPU (Diphenyul urea) (Kaczyna and Megnet 1993) and PES

medium with 5 mg/l kinetin (Gusev et al 1987). Therefore, the present study was

conducted to determine the effect of dual plant growth regulators (IAA and BAP)

applications on the shoot micropropagation of G.verrucosa.

Material and Method

Preparation of explants. Seaweeds were taken using the semi-wet method according

to Sulistiani et al (2012) with modification. The samples were taken from aquaculture

ponds in Kupang Village, Jabon Sub-district, Sidoarjo Regency, East Java (7 ° 31'21.3 "S

112 ° 49'34.0" E). First, samples were cleaned from dirt and attached organisms

(epiphytes). Thereafter, the samples were put into coolbox in a wet condition before

being used in further steps.

Sterilization of explants. Sterilization was done based on Reddy et al (2003) with

modification. First, samples were cut off on the apical thallus (± 5 cm length) in the

laminar air flow and rinsed twice with sterile seawater. Thereafter, explants were

immersed in 0.1% (w/v) of detergent solution for 10 min. After that, explants were

placed into 1% (v/v) povidone iodine solution for 1 min. In each treatment, explants

rinsed with sterile seawater twice. Furthermore, explants were immersed in 3% (v/v) PES

Antibiotic medium solution for 48 hours. The cultures were stored on a rotary shaker with

room temperature 22-25oC, light intensity 1500 lux, and light irradiation (12:12 hours).

Finally, explants were rinsed with sterile seawater twice and dried on sterile tissue.

Sterile explants were ready to use for next treatments.

Inoculation of explants. Inoculation of explants was done based on Sulistiani et al

(2012) with modifications. Sterile explants are cut to a length of ± 0.5 cm (weight ± 0.5

mg) using a sterile scalpel in LAF. After that, the pieces of explants were inserted into a

culture glass containing 2% (v/v) solid PES medium. Each bottle contains 5 explants

arranged circularly. Furthermore, the glass cultures were closed with HDPE plastic and

kept in a culture rack for 4 weeks with room temperature 22-25oC, light intensity 1500

lux, and light irradiation (12:12 hours).



Observation of explants. The observation of explant growth rate was done by using

the formula GR = [LN(Wt/W0)/t] × 100% according to Reddy et al (2003), where W0 is

the initial fresh weight, and Wt is the final fresh weight of the explants after t days of

culture. The observation of the total number of shoot per explant was done by calculating

the percentage of shoot per explants using the formula C = (Et/E0) x 100% according to

Yong et al. (2014). Furthermore, the observation of the total number of shoots was done

by counting the number of shoots per explant that appeared after 30 days of culture.

Data analysis. This study was used Completely Randomized Designs with two factors,

IAA and BAP. Both PGRs have 4 level of concentrations (0; 0.1; 0.3; 0.5 mg/l).

Therefore, 16 treatments and 5 replications were used in this study. Growth rate of

explant, percentage of explant forming shoot and the number of shoots per explant were

observed. The study was conducted using completely randomized design and analyzed

using two-way analysis of variance (ANOVA) and followed by a Tukey post-hoc analysis.

Results and discussion

The effect of IAA and BAP combination on growth rate of G.verrucosa explants.

Combination of IAA and BAP significantly influence the growth rate of G. verrucosa

explants (p <0.05). Meanwhile, BAP alone increases the growth of the explants. Different

results of growth rate might indicate that the effect of the IAA and BAP combination is

concentration dependent. In contrast, the use of IAA alone did not depend on the level of

concentrations. The observation showed that growth rate of the explants varied in this

treatment. In addition, our observation also demonstrated that the balance of IAA dan

BAP concentration resulted in a decrease of G. Verrucosa growth rate along with an

increase of both PGRs concentrations. Highest growth rate performance (0.42% per day)

was obtained when the explants were placed in the PES medium supplemented with 0

mg/l IAA and 0.5 mg/l BAP (Table 1).

Table 1

The effect of IAA and BAP combination on growth rate of G.verrucosa explants

Plant Growth Regulators (mg/L) Growth rate of

G.verrucosa explants (%) IAA BAP

0

0 0.1 0.3 0.5

0.17b 0.23ab 0.32ab 0.42a

0.1

0 0.1 0.3

0.5

0.21ab 0.30ab 0.23ab

0.30b

0.3

0

0.1 0.3 0.5

0.15b

0.22ab

0.24ab 0.16ab

0.5

0

0.1 0.3 0.5

0.23ab

0.13b 0.16b 0.10b

The numbers followed by the same letter do not significantly different according to the Tukey test

with a significant level of 5%.

Meanwhile, treatment of 0.5 mg/l IAA and 0.5 mg/l BAP resulted in a lowest growth rate

response (0.1% per day). Interestingly, PES medium with no PGRs also showed growth

rate response (0.17% per day). This might be due to endogenous hormones that play



role in promoting growth. According to Bradley and Cheney (1991) and Yokoya et al.

(2010), some types of plant hormones are naturally present in seaweed tissues, both

auxin and cytokinin (endogenous auxin / cytokinin) such as IAA, ABA (Abscisic acid), PAA

(isopentenyladenin), and CZ (Cis-zeatin).

Yokoya (2000) reported that the effect of auxin and cytokinin on the growth rate of

Gracilaria tenuifrons was associated with the function of PGRs in the process of cell

division, elongation and differentiation. Auxins play a central role in regulating cell growth

and elongation, while cytokinin regulates plant cell division and morphogenesis (Ooi et al

2013)). Other studies reported that the combination of 1 mg/l IAA and 2.5 mg/l BAP

increased optimally the growth rate of Kappaphycus alvarezii seaweed explants (4% per

day) (Yong et al 2014). The process of growth at the cellular level is defined as an increase of organic

materials (biomass) resulting in an increase of cell size and subsequently affects cell division (Sablowski, 2016). The relationship between growth and the production of new cells (cell cycle) is essential in the development of multicellular organisms (Dewitte and Murray 2003; Jorgensen and Tyers 2004; Jones et al 2017). Growth and cell cycle are controlled by changes in the expression of cyclin-dependent kinase (CDK) and cyclin genes due to the effects of auxin and cytokinin. Auxins play an important role in the process of gene expression dynamics including RNA polymerase activity, ribosomal RNA level, and polyribosome protein mRNA levels enhancement (Sablowski and Dornelas 2014). Meanwhile, cytokinins play a role in the process of protein-specific synthesis during the cell cycle stage (Kieber and schaller 2018).

In cell cycle, auxins induces CDK expression, inhibiting CDK inhibitors (KRP1/KRP2)

(Himanen et al 2002), improving E2FA/B protein stability (Magyar et al 2005),

stimulating SKP2A degradation, and inducing Telomerase expression in phase S through

increased telomerase activity during replication (Tamura et al 1999). Cytokinins are also

reported to induce cdc2 gene expression in the G2-M transition phase, and induce CycD3

expression in the G1-S phase (Sablowski and Dornelas 2014).

The effect of IAA and BAP combination on shoot induction and multiplication of

G.verrucosa explants. Use of PES medium supplemented with combination of IAA and

BAP for 30 days showed shoot induction on the apical, intercalary and basal part of the

explants (Figure 1). Shoot Induction and multiplication are promoted by the PGRs used in

this study. The explants undergo shoot induction in the last month after inoculation,

while explants that did not undergo shoot induction showed bleaching symptoms (Figure

1c), death (Figure 1c) and contamination (Figure 1d).

Figure 1. Condition of explant after 30 days of culture; a) shoots on apical part in 0 mg/l IAA and 0 mg/l BAP treatment; b) shoots on intercalar part in 0 mg/l IAA and 0.5 mg/l BAP treatment; c)

explants experiencing bleaching and death in 0.5 mg/l IAA and 0.5 mg/l BAP treatment; d)



contaminated explant in 0.5 mg/l and 0.3 mg/l treatment (black arrow). (Bar scale = 200 μm; magnification 67.5x)

Combination of IAA and BAP did not significantly induce shoot formation (p<0.05)

but not in shoot multiplication (table 2 and 3). The highest percentage of shoot per

explants and number of shoots were obtained in treatment with 0.5 mg/l BAP alone (56%

and 2.64 shoots per explant, respectively). It seems that the use of BAP alone was more

pronounced compared to the treatment with IAA alone. However, treatment of IAA and

BAP alone did not depend on the level of concentrations and showed diverse response of

shoot induction and multiplication. In contrast, the balance of IAA and BAP concentration

result in a decrease of shoot percentage of G. verrucosa explants, as well as in shoot

multiplication along with an increase of both PGRs concentration. Meanwhile, treatment

of 0.5 mg/l IAA and 0.1 mg/l BAP resulted in lowest shoot percentage (4%). In case of

number of shoot per explant, the lowest result was obtained in treatment with 0.5 mg/l

BAP and 0.5 mg/l IAA. Interestingly, zero PGRs treatments, somehow, induce shoot

formation and multiplication. The later might explained the existance and function of

endogenous hormones in shoot formation and multiplication (Hu et al 2020).

Table 2

The effect of IAA and BAP combination on shoot formation

Plant Growth Regulators (mg/L) Percentage of

Shoot induction (%) Number of shoots per

expants IAA BAP

0

0 0.1 0.3 0.5

24 48 28 56

1.4de

1.96bc

1.76cd 2.64a

0.1

0 0.1 0.3 0.5

40 20 36 16

2.04bc 1.2ef

2.32ab

1.56cde

0.3

0 0.1 0.3 0.5

8 8

8 16

0.72fg 0.76fg 0.72fg 1.16ef

0.5

0 0.1 0.3 0.5

12 4 16 8

1.08ef 0.48g 0.8fg 0.4g

The numbers followed by the same letter do not significantly different according to the Tukey test

with a significant level of 5%.

In vitro shoot Induction and multiplication are influenced by variety of factors,

including PGR interactions, incubation periods, agar-solidifying agents, type of explant, and plant genotypes (Fatima and Anis 2012). The interaction between auxin and cytokinin is very important in controlling the process of plant development such as shoot (Boo et al 2015), root (Jing and Strader 2019) and callus formation (Schaller et al 2015). High level of auxin compared to cytokinin could trigger callus formation. Moreover, auxin alone induces the formation of roots. Finaly, high concentration of cytokinin ratio to auxin induces shoot regeneration (Boo et al 2015).

In the process of de novo organogenesis, explants undergo cavity or cutting process, were cultured in PES medium supplemented with exogenous hormones to produce adventitious roots or shoots (Liu et al 2014). In this study, the induction of G.verrucosa seaweed shoots appeared on the injury part of explant. The ability of cell to regenerate depends on its ability to respond the hormonal signals, both upon entering the cell cycle and in its development (Motte et al 2014; Duclerq et al 2011).

The mechanism of auxin and cytokinin interaction on shoot growth has been reported by Che et al (2008). In their study, shoots were regenerated from root explants



through two stages: preincubation on medium-rich auxin or callus induction medium (CIM) and then cultured into medium-rich cytokines or shoot induction medium (SIM). During the CIM preincubation stage, the pericycle cell of explants undergoes cell division and gain the ability to respond cytokinin signals to form shoots. Cell division activity is needed to obtain the gene's ability to express de novo shoot formation, which occurs only in the SIM medium if root explants are first incubated into CIM media (Che et al 2008; Che et al 2006; Cary et al 2002). After being transfered into SIM medium, explant will develop independent shoots without the presence of cytokinin (Shoot commitment). At that stage, the apical meristem would develop shoots, and the expression of CUP SHAPED COTYLEDON 2 (CUC2) gene would also increase. CUC1 and CUC2 are thought to encode the NAC1-domain transcription factors that are also needed to express SHOOTMERISTEMLESS (STM) genes and the formation of meristem shoots (Aida et al 1997; Takada et al 2001). STM and WUSCHEL (WUS) are also required for the formation of meristem shoots. The role of cytokinin and genes KNOTTED-1 LIKE IN ARABIDOPSIS THALIANA (KNAT1) on shoot development is also associated with the STM gene (Lincoln et al 1994).

Shemer et al (2015) reported that shoot formation of root explants cultured on the CIM medium and then on the SIM medium is also influenced by the DNA methylation. In that study, the chromomethylase (cmt3) mutant showed high capacity to regenerate shoots in SIM-direct medium, without treatment of CIM medium through direct organogenesis. The cmt3 mutants are plants that can reduce CHG methylation. The results showed that shoots were formed directly on the cmt3 explant placed in the SIM-direct medium. It might be caused by the cytokinin signal response in decreasing the level of DNA methylation of explant allowing some related genes such as WUS to be expressed in shoot formation.

Conclusions. This study has successfully documented the effect of IAA and BAP

combination on shoot induction and multiplication of the G.verrucosa in vitro. The best

results were demonstrated in the MS medium supplemented with 0.5 mg/L BAP alone. In

this treatment, 56% of explants displayed shoot formation. Meanwhile, highest shoot

number was also obtained in this treatment (2.64 shoots per explants). Further studies

should be continuously carried out in order to enhance shoot multiplication of the

G.verrucosa.

Acknowledgements. The authors thank the members of plant bioscience and

technology laboratory of Biology Department-Institut Teknologi Sepuluh Nopember (ITS),

Surabaya, Indonesia for their assisstance and supports. This study was partly funded by

Minsitry of Research and Technology, Republic of Indonesia.

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18

2. Draft paper (akan di submit ke agriculture (Switzerland))

Agriculture 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/agriculture

Review 1

A review on silver nanoparticles elicited culture: 2

A nanotechnological approach tofor crop 3

improvement and plant metabolites production 4

Maulidia Rahmawati and Nurul Jadid* 5

Department of Biology, Institut Teknologi Sepuluh Nopember, 60111 Surabaya-Indonesia 6 *Correspondence: [email protected] (N.J.) 7

Received: date; Accepted: date; Published: date 8

Abstract: Plant tissue culture play ans important roles in plant biotechnology due toas its potential 9 forin massive production of improved crop varieties and high yield of important secondary 10 metabolites. Several efforts have been made to improve the effectivenessity and production of plant 11 tissue culture, such as elicitation using biotic and abiotic factors. Nowadays, the addition of 12 nanoparticles as elicitors has gained more interest worldwide because of itsthe success inon 13 microbial decontamination and the enhancement of secondary metabolites. Nanoparticles are small 14 substances with 1-100 nm dimensions that possesshas unique physiochemical properties. Among 15 all of the nanoparticles, silver-nanoparticles (AgNPs) arehas been well-known for theirits 16 antimicrobial and hormetic effects, which in appropriate doses will lead to the improvement of plant 17 biomass as well as its secondary metabolite accumulation. This review aimeds to consolidate all of 18 the progress and findings from the integration of nanotechnology and plant tissue culture, 19 especially highlighting the success of this effort in secondary metabolites enhancement for various 20 purposes. Moreover, its effect on plant growth and biomass accumulation will also be reviewed and 21 presented as well as the possible mechanism of action were also reviewed and presented. Thus, the 22 information provided will be useful for future research in plant improvement and large-scale 23 production of important secondary metabolites through elicitation of silver-nanoparticles. 24

Keywords: crop improvement; secondary metabolites; silver-nanoparticles; tissue culture. 25 26

27

1. Introduction 28

The Plant in vitro culture is considered abelongs to non-GM (genetically modified) 29

biotechnological processy, due toas its role in the improvement of plant growth and products 30

enhancement throughmostly by nutrient and environment modification, without the involvement of 31

gene modification. In addition toDespite its benefits forin basic plant bioscience, this method ishas 32

been also utilizedwell-known for agriculturale and pharmacologicaly purposes [1]. ForIn term of 33

agriculture, in vitro culture has been widely used for massive production of low-proliferation rate 34

crops, production of improved crop varieties, and pathogen-free plants, as well as for maintaining 35

stocks of endangered species as an ex situ conservation approach [1–, 2, 3, 4]. This method can be applied 36

to ain very wide range of plant characteristics and species through different methods and procedures, 37

which isare very helpful at this present given the present state of environmental deterioration. Plant 38

tissue culture has also been used as a major platform for the production of secondary metabolites 39

production, which are widely used for pharmacological as well as industrial purposes [5]. 40

Commented [11]: Perhatikan penyesuaian berikut

Commented [DNJSM(2]: Cek Kembali total kata nya. Maks

200 kata

Commented [DNJSM(3]: Penggunaan singkatan GM

(genetically modified) hanya sekali saja di manuskrip,

sehingga sebaiknya tidak perlu menggunakan singkatan.

Ditulis lengkap saja.

Commented [DNJSM(4]: bila sitasi berurutan gunakan -.

Missal 1-4. Cek Kembali dan sesuaikan format di jurnal

mailto:[email protected]

Agriculture 2020, 10, x FOR PEER REVIEW 2 of 11

Plant in vitro culture is directed towards the aseptic growth of parts of plants or whole plants 41

under specific nutritional and environmental conditions [6, 7]. This technique depends on the concept 42

of totipotencyt, which refers to the ability of a single cell to express a full genome throughby cell 43

division [6]. Several factors affect the success of in vitro culture, such as the genotype, the physiological 44

status, and type of explants, the disinfection method employed, the culture medium, plant growth 45

regulators (PGRs), light intensity, photoperiod, and temperature. Generally, medium composition 46

strongly influences morphogenesistic inof the explants. The Mmediums are typically composed ofby 47

two kinds of nutrients, which is known as macro- and micronutrients-, as well as amino acids organic 48

supplements, vitamins, carbon sources, PGRs, and solidifying agents [7]. Elicitors are also added for 49

some special purposes, such as enhancing phytochemical production in plants. Elicitors are described 50

as biological or non-biological molecules that have thehas ability toof recognizeing cytoplasmic 51

membrane’s receptors in plant cells. Tthis recognition and binding will leads to signal elicitation, 52

which stimulates the expression of genes related to secondary metabolites production [8, 9]. The 53

elicitors work as plant stressors in a natural environment, and the form of thisthese molecules can be 54

fungi, bacterial, activated enzymes, and any other biological compounds. Other forms of elicitors are 55

from non-biological stressors such as temperature, salinity, heavy metals, and mineral stress [9, 10]. 56

Nanoparticles of heavy metals, as part of nanotechnology, shows high potential as in vitro culture 57

elicitors for theirits tremendous physiochemical properties [9]. 58

Nanotechnology has gained interest recentlythese past years and has rapidly become more 59

prominentemerging rapidly in various scientific disciplines in science [11, 12]. Nanobiotechnology, 60

referring to the application of nanotechnology inon the field of biotechnology, has been widely 61

utilized for drugs and gene delivery, bio detection of pathogens and proteins, disease control, and 62

fluorescent biological labeling, as well as various purposes in plant tissue culture such as seed 63

germination, yield and bioactive compound improvement, and plant protection [7,13]. Nanoparticles 64

arebelongs to three-dimensional nanomaterials, ranging inwith the size range from 1-100 nm. 65

Nanomaterials exhibitprovide unique properties such as low weight, a large surface area-to-volume 66

ratio, the ability to engineer electron exchanges, special electronic and optical attributes, and surface 67

reactive capability [12, 14]. Previous studies have shown that metallic (Ag-, Au-, and Fe- nanoparticles) 68

and metal oxide nanoparticles (nano-ZnO2, TiO2, and -CuO2) are giving positively impact towards 69

plant tissue culture by, such as supporting morphological potential and, propagation, as well asand 70

improving plant resistance to stress [7, 12, 14, 15]. 71

Silver-nanoparticles (AgNPs) havehas gainedgarnered more interest among theany other 72 nanoparticles due toas theirits strong biological activity, resulting in outstanding anti-microbial 73 performance, as anti-microbial and theirits hormetic effects, which in appropriate doses will lead to 74 the improvement of plant biomass as well as its secondary metabolite accumulation. However, the 75 molecules inhibitwill cause inhibition of plant growth in high doses [14, 16]. AgNPs affectexhibit effect 76 on plants at many different levels, which resulting into stimulation of germination, growth 77 invigoration, biomass accumulation, improved shoot induction and proliferation, and pigment 78 content enhancement [14]. NumerousA lot of positive resultsinputs havehas been gained from 79 nanotechnology in plant tissue culture, with involvement of nanoparticles in this field becoming a 80 promising methodway of increasing more effective propagation and production. A rRecent report 81 from Ali et al. [3] showed the effective propagation and secondary metabolites enhancement of the 82 endangered plant species Caralluma tuberculata via AgNPs elicited culture, thus showings the 83 importance of this nanoparticle in advanced plant tissue culture. In-depth and consolidated 84 guidelines of nanoparticles introduction in plant tissue culture areis indeed needed tofor elucidateing 85 the impact of AgNPs in plant tissue culture. This review predominantly focused on the most recent 86

Commented [DNJSM(5]: These molecules : fungi, bacteria.

Cek Kembali apakah elicitors berupa molekul atau bisa

berupa material organik. Bila material organik termasuk,

maka sebaiknya kata molekul juga ditambah dengan

biomaterial.

Commented [16]: Cek cara penulisan Pustaka sesuai format

jurnal


progress and achievement inof AgNPs elicitation in plant tissue culture for the purpose of secondary 87 metabolites enhancement purpose. Their effect on plant growth and biomass accumulation as well 88 as the possible mechanism of action werewill also be reviewed and presented as well as the possible 89 mechanism of action. 90

91 92

2. Silver-nanoparticles in crop improvement 93

Plant tissue culture plays important roles in agrotechnology, as it helps the process of improveing 94

the quality, yield, and growth of important plant species. Plant micropropagation isperforms as one 95

of the best alternatives for crop improvement, andwhich possesses special significance in 96

multiplication through a vegetative approach, minimizing the risk of pathogens accumulation as well 97

as the slow proliferation rate in generative multiplication that causes a loss in both crop quantity and 98

quality. Several studies have shown the positive impacts of AgNPs augmentation for callus 99

induction, shoot generation, and growth (Table 1). In Caralluma tuberculata [3], augmentation withof 100

AgNPs along with 0.5 mg/Ll 2, 4-D + 3.0 mg/Ll BA in Murashige and Skoog (MS) medium showed 101

significant improvement in callus growth and proliferation. This study showed that application of 102

AgNPs alone without any plant growth regulators (PGRs) showed lower biomass ( 0.106 g/L fresh 103

weight (FW); 0.0113 g/L dry weight (DW) as maximum values from application of 90 µg/L AgNPs) 104

than that in the control medium (MS+ 0.5 mg/Ll 2, 4-D + 3.0 mg/Ll BA), in which the values recorded 105

werewas 0.143 g/L FW and 0.140 g/L DW. The application of AgNPs with no PGR added to the 106

medium also ledleaded to the formation of fragile and yellow-brownish colored calli. Meanwhile, 107

augmentation of AgNPs (60 µg/L) along with a PGR s howed maximum callus biomass values of 108

callus biomass ofwith FW (0.780 g/l) and DW (0.051 g/L for FW and DW, respectively). Tthe calli 109

werewas observed as compact and greenish in color. The water content, which is an important 110

parameter for metabolic and physiological status, increased along with the callus’ biomass values [3, 111

17]. However, application of AgNPs beyond the optimum level of 60 µg/L along with caused ashowed 112

significant decline in callus biomass. 113

In sugarcane [19], the addition of 35 ± 15 nm- sized AgNPs resulted into significantly higher shoot 114

multiplication and elongation at AgNP concentrations ofwith 50 and 100 mg/L AgNPs concentration,. 115

wWhile 25 mg/L AgNPs on the medium displayed nodid not show any effect onof the growth. This 116

studyresearch exhibited the hormetic effect of AgNPs in plant tissue culture, as it was clearly shown 117

that the application of 200 mg/L AgNPs in the medium resulted in inhibition of shoot multiplication 118

and elongation. This studyresearch also revealedprovided information that the addition of AgNPs 119

increased the important nutrients in the medium, such as N, Mg, and Fe. Thus, it could be inferred 120

that increasing of the levels of those nutrients that is related to chlorophyl biosynthesis leads to higher 121

photosyntheticsis activity, while higher concentrations of AgNPs may lead to phytotoxicity into plant 122

cells. Theseis results were similar to those ofshowed similarity with previous studiesresearches, such 123

as those onin Cucurbita pipo[20] (500 mg/L AgNPs reduced plant biomass bying 57% plant biomass) 124

and Raphanus sativus[21] (500 mg/L AgNPs reduceding shoot length by 47.,7% shoot length). Addition 125

of the same size of AgNPs (35 ± 15 nm) to the MS culture medium of Stevia rebaudiana B. [16] exhibited 126

an increase inincreasing of shoot number and length in all of the AgNPs treatments compared to 127

those in the control. However, the highest concentration (200 mg/L) of AgNPs showed a lower 128

amountnumber of shoot multiplication and elongation. 129

Commented [17]: 47.7%


The in vitro culture of Prunella vulgaris L., which is known as self-healing, using medium 130

containinged NAA + AgNPs alone or augmented with AuNPs showed a significant increase inof 131

callus proliferation to 100% in 30 µg/L AgNPs, AgAu (1:2), and AgAu (2:1) compared to that in the 132

control (95%) [22]. In Lavandula angustifolia shoot propagation [9], the media enrichment with AgNPs 133

and AuNPs showed significantly improved plant development compared to that in the control. 134

Regardless of the components and concentration, the addition of those nanoparticles (NPs) was 135

reported to enhance the formation of lateral shoots. Generally, addition of AgNPs even atin the 136

highest level (50 mg/dm3) did not exhibit any visible toxic effects such as necrosis;, however, higher 137

levels of AuNPs (50 mg/dm3) exhibited a slight color change in thely leaf blades color changes to 138

yellowish. 139

140


141

Table 1. Summary of recent studies in AgNPs elicitation for Crop Improvement and Secondary Metabolites enhancement 142

Plant Species Size (nm) Concentration Effects References

Caralluma tuberculata 40 nm 30, 60, 90 µg/L Enhanced Calli callus biomass increasing

TPC and TFC content improvement

[3]

Sugarcane 35 ±15 nm 0, 25, 50, 100, 200

mg/L

Improved shoot number and length

Phenolic compound antioxidant improvement

[19]

Capsicum sp. - 50 ppm Capsaicin content enhancement [28]

Stevia rebaudiana 35 ±15 nm 0, 25, 50, 100, 200

mg/L

Shoot growth and proliferation number and length

improvement

[16]

Bitter gourd 2 – 12 nm 0, 1, 5, 10 mg/L Flavonoid and phenolic content enhancement

Increased anti-microbials and bioactivity level

[29]

Prunella vulgaris 25– 35 nm 30µg/L of NPs

(AgNPs/AuNPs 1:2,

1:3, 2:1, and 3:1)

Increased callus proliferation to 100%

Increased TPC and TFC content on cell suspension culture

[22, 30]

Lavandula angustifolia Mill. 27.5 ± 4.8 nm 1, 2, 5, 10, 20 and

50 mg/dm3

Increased shoot multiplication and oil gland [9]

Arabidopsis thaliana 10, 40, 100 nm 0.5, 1, 5 mg/L Increasing Increased of camalexin accumulation content in

plant

[26]

Oryza sativa cv. IR64 1 – 50 nm 0, 5, 10, 15, 20 mg/L

Callus induction improvement [23]

Nicotiana tabacum AgIAA: 20 – 140

nm; AgIBA: 6 – 40

nm

0,02 mg/L Rooting improvement [25]

Chrysanthemum morifolium

< 20 nm 0; 0,5; 1; 1,5; 2; 3; 5;

7; 10 ppm.

Improved number of shoots, plant heigh, and biomass [24]

143

Commented [18]: Tambah lagi referensinya

Commented [19]: Dimana letak improvement nya?


Manickavasagam et al. [23] investigated the effect of AgNPs augmentation along with 2.,5 mg/L 2,4-D 144

in callus induction of Oryza sativa cv. IR64;, the highest callus induction was observed at ain 145

concentration of 10 mg/L AgNPs in the medium. The callus induction increased 20% compared to 146

that in the control medium without AgNPs (62%). Nghia et al. [24] studied the optimum concentration 147

of AgNPs (< 20 nm inof size) for three possible propagation methods of Chrysanthemum morifolium 148

Ramat cv. Jimba:, which were in solid medium, liquid medium, and microponics. As Ffor in vitro 149

culture, the optimum concentration of AgNPs for both for solid and liquid mediums waswere 1.,5 150

ppm. The addition of AgNPs to the culture medium significantly improved the number of shoots as 151

well as, the plant height, and biomass. Furthermore, the addition of thesethis nanoparticles atin 152

higher levels was observed suppresseding the growth index of the plant. Another approach was 153

examined by Thangavelu et al. [25], whoby synthesizeding hormone-stabilized AgNPs, resulting ined 154

AgIBA and AgIAA. The examination of both of the nanobullets in Nicotiana tabacum showed 155

improvedbetter rooting results in AgIBA, probably due to the smaller size of the NPs produced (6- 156

40 nm) compared to those in AgIAA (20-140 nm). 157

Along with their effects on growth improvement, AgNPs also possessprovide anti-microbials 158

properties. Sarmast & Salehi [13] reported that the addition of AgNPs in culture media postponed the 159

appearance of internal contamination of Araucaria excelsa. In this study, 18 nm-sized AgNPs were 160

added to the medium atin concentrations of 25, 50, 100, 200, 400, 800, and 1600 lg/mLl. According to 161

the report, the addition of 400 lg/mLl AgNPs addition to the medium could reduce bacterial 162

contamination fromof 81.,25% in the control to 18.,75% in the treated media after four weeks of 163

treatment. Another report from Lee et al. [25] also showed that application of AgNPs (3 nm) 164

successfully controls pathogenic fungal strains. 165

Although several studiesevidences have shown the positive effectsinputs of AgNPs onin plant 166 propagation, its exact mechanism has not been understood in details. From the data presented, the 167 effect of AgNPs effect on plant growth in vitro and media decontamination were dependented on 168 various factors, such as plants species, nature, explant type, and age, as well asand also the AgNPs 169 size and concentration [3,13]. Thus, the results of the examinations suggested that the effect of AgNPs 170 iswere dose-dependent, in which application of high concentrations of these nanomaterials leads to 171 growth inhibitionory and necrosis. It was suggested that tThe increase inof callus biomass was 172 suggested as the effect of whichdue to AgNPs mutilatinged the plant cell wall, possibly allowing 173 thethus plant cells tocould possibly uptake more water and nutrientsnutrition from the medium [3]. 174 This suggestion was based on the structural impact of the cell wall in plant cells, compared to that in 175 the animal cells [18]. 176

177

3. Silver-nanoparticles in plant secondary metabolites enhancement 178

Plants are rich in secondary metabolites, which naturally help them surviveing underin 179

challenging environmental conditions. FurthermoreIn other hands, plant secondary metabolites are 180

also well known for their benefits tofor humans, as in pharmacological or any other industries. In 181

vitro plant cell and organ culture has been proven to be advantageous for plant secondary metabolites 182

production [7]. Application of elicitors, such as AgNPs, has beenwas provend to have a positive effect 183

ongood inputs in secondary metabolites enhancement, such as AgNPs in several studiesexperiments. 184

In Caralluma tuberculata, application of AgNPs (90 µg/L) alone in the MS medium showed the highest 185

result forin tTotal pPhenolic cCompounds (TPC, 3.0 mg), tTotal fFlavonoid contents (TFC, 1.8 mg), 186

phenylalanine ammonia lyase (PAL, 5.8 U/mg), and DPPH free radical activity (90%). Even though 187

Commented [110]: Cek penulisan “et al.” sesuai format

jurnal

Commented [111]: Koma diganti titik

Commented [112]: Cek satuan…sepertinya kelebihan “l”.

apa yang benar g ?

Commented [113]: Tidak perlu diberi singkatan. Karena

hanya sekali dipakai


no significant increase was observed from the augmentation of AgNPs and PGRs, the data showed 188

that application of 30 µg/L AgNPs with PGR resulted inwas resulting the second highest secondary 189

metabolites production in C. tuberculata culture [3]. This showed that the addition of AgNPs alone in 190

the medium was more supportive for secondary metabolites enhancement. 191

Bello-Bello et al. [19] observed an increaseing inof total phenolic compounds on sugarcane after 192

media elicitation with AgNPs (35 ± 15 nm). Thise studyresult showed that the optimum concentration 193

for phenolic compound enhancement in sugarcane was 50 mg/L. AtIn 100 and 200 mg/L AgNPs, 194

phenolic compound and antioxidant production decreased while the production of reactive oxygen 195

species (ROS) continued to rise. Application of different sizes of nanoparticles (10, 40, and 100 nm) 196

atin various concentrations (0.5, 1, and 5 ppm) to the plant model Arabidopsis thaliana exhibited a 197

significant change in camalexin content compared to that in the control. Application of 10 nm AgNPs 198

at a concentration ofin 5 ppm concentration displayed theshowing highest result forin camalexin 199

accumulation. Pathway network analysis on the AgNPs in A. thaliana revealed the increases inof the 200

TCA-cycle, sugar metabolism, and shikimate phenylpropanoid metabolism, which are also 201

associated withpart of plant resistance to abiotic stress. The ability of AgNPs to induce production of 202

phytoalexins and other secondary metabolites isare promising for enhancement of plant immunity 203

against pathogens if applied in an optimized manner [26]. 204

The effort of Ccapsaicin enhancement trough suspension culture of Capsicum sp. augmented with 205

50 ppm AgNPs showed promising results, as the capsaicin levels werewas observed higher than 206

those in Capsicum fruit even without cell immobilization. The capsaicin content of the suspension 207

culture increased about two-fold between the third andto sixth days, resulting ined high yield of 208

capsaicin in the culture harvested after six daysfrom six-day culture [27]. The cCell suspension culture 209

(CSC) approach was also employed by Chung et al. [28] for important secondary metabolites 210

production from bitter gourds. Total phenolic and flavonoid compounds were examined, 211

revealinged that CSC with 5 mg/L AgNPs accumulated elevated total phenolic and flavonoid 212

contents. Ultra-HPLC assessment was performed to identify individual phenolic compounds, 213

resulting in 20twenty individual detected individual phenolic compounds. AgNPs elicitation ofto the 214

culture increased the levelsaffected the increase of several phenolic compounds such as 215

hydroxybenzoic acid (1713.40 µg/g), hydroxycinnamic acid (1080.10 µg/g), and flavanols 216

(1822.37 µg/g). Elicitation of AgNPs affectedwas observed to affect the pharmacological activity of 217

the bitter gourd extracts, with the elicited-CSC extract showing a higher inhibitory effect of 75.5% 218

towards α-amylase enzyme by 75,5% compared to that of the non-elicited CSC extract (61%). This 219

result was important as bitter gourd extract iswas used for postprandial hyperglycemia in diabetes 220

patients [28, 29]. The elicited-CSC extract also showed higher anti-microbial activity as well as a higher 221

inhibitory effect onof MCF-7 and HT-29 cells propagation. Fazal et al. [22, 30] conducted stabilized cell 222

suspension culture of Prunella vulgaris using Au- and AgNPs, both augmented and applied alone. 223

The TPC and TFC of the suspended cell showed significantly higher results in the NPs- elicited-CSC,. 224

with the hHighest result was observed in the combined AgAuNP treatment of combined AgAuNPs 225

(1:3). 226

4. Possible mechanism of AgNPs in plant cells 227

Several suggestions have been made to elucidate the possible mechanism of AgNPs to growth 228

improvement and biomass accumulation due to AgNP addition inof plant culture. Bello-Bello et al. 229


[19] reported a significant increase inof some macronutrients contents in the shoots of sugarcane 230

exposed to AgNPs. Those shoots accumulated larger amounts of N, Mg, and Fe compared to those 231

in the control. This finding indicates theleads to possibility of those elements contributingon of those 232

elements to to the increaseding shoot number and length. Nitrogen is a constituent of chlorophyll, 233

proteins, nucleic acid, and hormones. Magnesium iswas in a porphyrin moiety inof chlorophyll and 234

is involved in adenosine triphosphate reactions. Moreover,And Fe is an important molecules in redox 235

enzyme and chlorophyll biosynthesis. Several reports have shownexhibited that the use of AgNPs in 236

culture induces improvement in theof photosynthetic machinery in plants [16]. Since photosynthetic 237

activity leads to the formation of biomass in plants, it is possible that this mechanism helps improve 238

growth inimprovement of plants exposed to AgNPs. There is still insufficient information 239

regardingabout how AgNPs affect nutrient absorption [19];, it may be speculated that AgNPs can 240

enhance the plant cell’s uptake by cell wall mutilation. Thus, the absence of cell wall structure will 241

leadaffect to higher uptake of nutrients and water [3]. 242

Another possible mechanism of growth improvement by AgNPs elicitation on tissue culture is 243

that this supplementation affects PGR- responsive genes expressions. Further investigation by 244

Manickavasagam et al. [23] revealed that addition of AgNPs decreasedaffected to the expression 245

decrease inof ethylene- and ABA- responsive genes, which increased the callusi regeneration 246

frequency. UnderIn culture conditions, with explants were cultured in a closed container, plantthe 247

development will be highly influenced by plant-produced gases such as ethylene, which possibly 248

regulate senescence. AgNPs acted as ethylene perception inhibitors by downregulatinge the genes 249

involved in ethylene productions. A pPrevious study also reported that AgNPs affect root growth 250

improvement through the upregulation of auxin synthesis [31]. 251

Even though the direct biophysical and biochemical interactions betweenof nanoparticles and the 252

biological system haveare not yet been explained, it has been established that the initial response of 253

plants exposed to NPs might include increased levels of ROS, cytoplasmic Ca2C, and mitogen-254

activated protein kinase (MAPK) cascades;, these responses were the same as those in the biotic and 255

abiotic stress defensce mechanisms [3, 7, 12, 30, 32]. AgNPs could enter cells through plasmodesmata, 256

causing damage to the plasma membrane. The initial recognition of AgNPs by plasma membrane- 257

bound receptors triggered Ca2+ burst and ROS induction in A. thaliana [33]. NPs also induced an ROS- 258

mediated MAPPAK cascade and a calcium spike, which possibly enhanced the secondary metabolites 259

production tofor copeing with oxidative stress, resultinged in a higher quality and quantity of 260

secondary metabolites during AgNPs exposure [30] (Figure 1). 261


262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

Figure 1. Possible mechanism involved in plant cell during nanoparticles enhancement 277

278

279

Kruszka et al. [26] reported that both nanoparticles and the ionic form of silver could stimulate 280

changes in theof secondary metabolites profile, which suggested that AgNPs affect plant metabolism 281

via ions releasing to the medium. In stevia, high doses of AgNPs werewas reported to be accumulated 282

in plant cells and tissues. These molecules were reported to be internalized in the plant through the 283

vascular bundles and are translocated to neighbourhood cells via apoplasts. The transfer mechanism 284

via apoplasts requires NPs to cross a membrane;, this indicates that the NPs uptake must be size- 285

specific. Nanomaterials could also be transferred via plasmodesmata;, this channel inhas 286

approximately 40 nm in diameter, so it is possible for 40 nm- sized AgNPs to be transferred via 287

plasmodesmata. Furthermore, a study byfrom Syu et al. [34] revealed that the morphology and size of 288

the AgNPs affected the plant response during exposure. The study exhibited that smaller size of 289

AgNPs showeding higher levels of anti-microbials activity and CSD2 proteins that respondse to 290

increasesing inof oxidative stress. 291

5. Conclusion and persepectives 292

Elicitation of plant tissue culture medium with AgNPs disclosed positive impacts on plant 293

growth and secondary metabolites enhancement. From the relevantdisclosed reports, it can be 294

concluded that AgNPs affects callus proliferation, shoot multiplication, rooting, and secondary 295

metabolites by altering antioxidant enzyme activities, gene expression, ethylene inhibition, and ROS 296

production. From the reports presented, Vvarious sizes and concentrations of AgNPs were employed 297

in the presented reports. Different responses and hormetic effects were observed depending on the 298

plant species and explants type. Thus, the optimization of AgNPs properties is required for each plant 299

species tofor improvebetter result of secondary metabolites enhancement. However, AgNPs shows 300

high potential to be employed for those improvement purposes in plants. Large-scale approaches, 301

such as nano-integrated suspension culture, areis a promising method ofway for secondary 302

metabolites production in the future. The fFindings on the growth improvement effects of AgNPs 303


could also providebe a basic knowledge to employ this nanomaterial for plant propagation, which 304

could possibly support the effort of crop improvement efforts as well as plant conservation. Further 305

assessment and evaluation are required to understand the mechanism behind the effects of AgNPs 306

in plant culture. 307

308

Author Contributions: Conceptualization, N.J.; data curation, M.R.; writing—original draft preparation, M.R.; 309 writing—review and editing, N.J. and M.R.; supervision, N.J.; funding acquisition, N.J. All authors have read 310 and agreed to the published version of the manuscript. 311

Funding: This research was funded by Institut Teknologi Sepuluh Nopember, Surabaya, grant number 312 964/PKS/ITS/2020. 313

Acknowledgments: Authors are grateful to Research Center for Agri-Food and Biotechnology of Institut 314 Teknologi Sepuluh Nopember, Surabaya-Indonesia and to members of the Laboratory of Plant Bioscience and 315 Technology for the supports. 316

Conflicts of Interest: The authors declare no conflict of interest. 317

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