Eliminasi Virus Pada Bawang Merah (Allium Cepa Var Aggregatum) Secara In Vitro Menggunakan Termoterapi Dan Kemoterapi

IN VITRO ELIMINATION OF VIRUSES ON SHALLOT
(Allium cepa var. aggregatum ) USING THERMOTHERAPY
AND CHEMOTHERAPY

PRABAWATI HYUNITA PUTRI

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2017

AUTHOR’S STATEMENT ON THESIS AND ITS SOURCE OF
INFORMATION AND DELEGATION OF COPYRIGHT*
I declare that this thesis entitled In Vitro Elimination of Viruses on Shallot
(Allium cepa var. aggregatum) Using Thermotherapy and Chemotherapy is
my own and authentic work under supervision of a thesis commitee and it is not yet
submitted to any universities for any degree fulfillment. Source of information, both
published or unpublished by its authors, used for quotations in this thesis is already
cited appropriately and present in thesis‟s Literature Cited chapter.
I hereby delegate my thesis copyright to the Bogor Agricultural University.
Bogor, January 2017

Prabawati Hyunita Putri
Student ID number: A352130141

______________________
* Copyright delegation of scientific paper from research collaboration with other
organization must be referred on its collaboration agreement.

RINGKASAN
PRABAWATI HYUNITA PUTRI. Eliminasi Virus Pada Bawang Merah (Allium
cepa var. aggregatum) Secara In Vitro Menggunakan Termoterapi dan
Kemoterapi. Dibimbing oleh SRI HENDRASTUTI HIDAYAT dan DINY
DINARTI.
Bawang merah merupakan salah satu komoditas hortikultura yang penting
di Indonesia. Sentra produksi bawang merah di Indonesia terutama di darah Jawa
Tengah, Jawa Timur, Jawa Barat, dan Nusa Tenggara Barat. Sebagian besar
petani di Indonesia menggunakan umbi sebagai bahan perbanyakan vegetatif.
Infeksi virus sangat mudah ditularkan melalui satu generasi ke generasi
selanjutnya dan dari satu daerah ke daerah lainnya sehingga dikhawatirkan
menurunkan kualitas dan hasil produksi. Infeksi virus pada umbi bawang merah
di Indonesia telah dilaporkan, walaupun informasinya masih sangat terbatas.

Penelitian ini bertujuan mengembangkan metode eliminasi virus pada
bawang merah menggunakan kombinasi termoterapi dan kemoterapi dengan
beberapa ukuran eksplan kultur ujung tunas. Penelitian dilaksanakan mulai
Januari 2015 sampai dengan Juni 2016, bertempat di Laboratorium Virologi
Tumbuhan, Departemen Proteksi Tanaman dan Laboratorium Kultur Jaringan 3,
Departemen Agronomi dan Hortikultura, Fakultas Pertanian, Institut Pertanian
Bogor. Umbi benih bawang merah terdiri atas dua kultivar, yaitu „Bima Curut‟
dan „Bima Brebes‟, diperoleh dari penangkar benih di desa Tenguli, Brebes, Jawa
Tengah. Eksplan berupa ujung tunas diisolasi menjadi tiga ukuran yang berbeda
(1 mm, 2 mm, dan 3 mm). Masing-masing eksplan diberi perlakuan suhu (30 oC,
37 oC, dan 25 oC sebagai kontrol) dan diinkubasi selama 4 minggu. Termoterapi
dilakukan pada kondisi homogen dan heterogen. Plantlet hasil termoterapi yang
masih terinfeksi virus kemudian digunakan sebagai sumber eksplan untuk
kemoterapi. Ujung tunas berukuran 2 mm digunakan sebagai eksplan dan
ditumbuhkan pada media yang mengandung ribavirin (10 mg L-1, 25 mg L-1, dan
kontrol 0 mg L-1), selanjutnya diinkubasi pada suhu 30 oC. Deteksi virus setelah
perlakuan menggunakan RT-PCR dengan primer spesifik Carlavirus, Poyvirus,
dan Allexivirus.
Benih umbi bawang merah yang diperoleh dari penangkar benih diseleksi
lebih dahulu melalui deteksi virus dengan metode dot immunobinding assay

(DIBA) menggunakan antibodi OYDV, SLV, dan GCLV. Sebanyak 100 sampel
umbi diambil secara acak pada masing-masing kultivar dan ditanam pada
styrofoam yang telah diberi air di bawahnya. Setelah 7 hari diambil secara acak 50
sampel daun yang tumbuh dari masing-masing kultivar. Hasil deteksi awal umbi
benih bawang merah menunjukkan bahwa kedua kultivar 100 % positif terinfeksi
OYDV. Sampel „Bima Curut‟ yang bereaksi positif terhadap antibodi SLV dan
GCLV berturut- turut sebanyak 100% dan 90%, sedangkan sampel „Bima Brebes‟
berturut- berturut sebanyak 96% dan 96%. Infeksi OYDV dominan dibandingkan
dengan SLV dan GCLV. Hasil deteksi ini mengindikasikan bahwa infeksi virus
pada pertanaman bawang merah di Indonesia sangat tinggi.
Perlakuan termoterapi pada berbagai tingkatan suhu dan ukuran eksplan
menunjukkan bahwa suhu mempengaruhi secara nyata pertumbuhan eksplan

sedangkan ukuran eksplan tidak berpengaruh nyata. Pada kultivar Bima Curut,
kombinasi perlakuan termoterapi terbaik terhadap pertumbuhan eksplan adalah
ukuran eksplan 1 mm dengan kondisi heterogen, sedangkan pada kultivar Bima
Brebes adalah kombinasi ukuran eksplan 2 mm dengan suhu heterogen.
Konfirmasi melalui RT-PCR pada plantlet hasil termoterapi menunjukkan bahwa
semakin kecil ukuran maka tingkat keberhasilan eliminasi semakin tinggi. Seluruh
plantlet (100%) yang berasal dari ukuran eksplan 1 mm bebas virus pada semua

perlakuan. Tingkat eliminasi pada kultivar Bima Curut dengan ukuran eksplan 2
mm dan 3 mm pada suhu 30 °C terhadap Potyvirus adalah 67%, sedangkan
terhadap Carlavirus berturut-turut adalah 33% dan 67%. Tingkat eliminasi pada
kultivar Bima Curut dengan ukuran eksplan 2 mm dan 3 mm pada suhu heterogen
terhadap Potyvirus adalah 67%, sedangkan terhadap Carlavirus adalah 50%.
Tingkat eliminasi pada kultivar Bima Brebes dengan ukuran eksplan 2 mm dan 3
mm terhadap Potyvirus pada suhu 30 °C berturut-turut 33% dan 50%, sedangkan
terhadap Carlavirus berturut-turut adalah 50% dan 67%. Tingkat eliminasi pada
kultivar Bima Brebes dengan ukuran eksplan 2 mm dan 3 mm pada suhu
heterogen terhadap Potyvirus adalah 50% dan 67%, sedangkan terhadap
Carlavirus adalah 50% dan 67%. Kultur ujung tunas berukuran 1 mm terbukti
dapat mengeliminasi virus hingga 100%. Namun, kultur ujung tunas ini
memerlukan keahlian dan membutuhkan waktu yang lama dalam mengisolasinya
sebelum dapat digunakan sebagai eksplan. Oleh karena itu, perlakuan
menggunakan ukuran eksplan 2 mm yang dikombinasikan dengan suhu heterogen
merupakan perlakuan terbaik untuk ukuran eksplan yang lebih besar karena dapat
meningkatkan eliminasi virus serta tidak mengganggu pertumbuhan plantlet.
Kombinasi perlakuan termoterapi dan kemoterapi pada konsetrasi
ribavirin lebih dari 10 mg L-1 pada kultivar Bima Curut tidak menghasilkan
plantlet yang hidup. Perlakuan ribavirin 25 mg L-1 pada kultivar Bima Brebes

menghasilkan plantlet yang tumbuh, namun vitrous. Konfirmasi dengan RT-PCR
pada plantlet setelah perlakuan kemoterapi memperlihatkan bahwa Potyvirus dan
Carlavirus masih terdeteksi pada semua plantlet. Hal ini mengindikasikan bahwa
ribavirin 10 mg L-1 yang dikombinasikan dengan termoterapi belum mampu
mengeliminasi virus.
Umbi benih bebas virus diperlukan untuk meningkatkan produksi bawang
merah dan menekan insidensi penyakit di pertanaman bawang merah di Indonesia.
Perlakuaan eliminasi virus menggunakan kultur ujung tunas dengan ukuran
eksplan 1 mm terbukti menghasilkan plantlet bebas virus. Eliminasi virus
menggunakan ukuran eksplan ynag lebih besar (2 mm) membutuhkan kombinasi
dengan termoterapi pada kondisi suhu heterogen. Kedua metode tersebut
merupakan metode yang efisien dalam memperoleh umbi bebas virus.
Kata kunci: Carlavirus, kultur ujung tunas, plantlet bebas virus, Potyvirus, umbi
bebas virus

SUMMARY

PRABAWATI HYUNITA PUTRI. In vitro Elimination of Viruses on Shallot
(Allium cepa var. aggregatum) Using Thermotherapy and Chemotherapy.
Supervised by SRI HENDRASTUTI HIDAYAT and DINY DINARTI.

Shallot is one of important horticultural product in Indonesia. Main
production area in Indonesia are located in Central Java, East Java, Wst Java, and
West Nusa Tenggara. Most shallot‟s farmers in Indonesia use bulb as propagative
material. Viral diseases are easily transmitted via infected bulb from one
generation to the next and from one region to another, causing considerable
quality and yield loss. Viral disease on shallot in Indonesia has been reported,
although the information is very limited.
The objective of this research was to develop method for elimination of
virus infection on shallot bulbs using combination of thermotherapy and
chemotherapy with different explant size in shoot tip culture. Research was
conducted in Laboratory of Plant Virology, Department of Plant Protection and
Laboratory of Tissue Culture 3, Department of Agronomy and Horticulture,
Faculty of Agriculture, Bogor Agricultural University from January 2015 until
June 2016. Two cultivars were used in this study, i.e „Bima Curut‟ and „Bima
Brebes‟. The virus-infected bulbs material used in this study was obtained from
Tenguli village, Brebes, Cental Java. The shoot tips from each samples were
isolated onto three sizes (1 mm, 2 mm, and 3 mm). Each size will then be treated
using 2 level of temperature (30 oC, 37 oC, and control at 25 oC) and incubated for
4 weeks. Thermotherapy was conducted using homogenous and heterogenous
condition. Plantlets from thermotherapy that remained infected by viruses then

used as source of explant for chemotherapy. Explants 2 mm in length were
excised then growth in medium containing ribavirin (0 mg L-1 as control, 10 mg
L-1, and 25 mg L-1) followed by incubation at 30 oC for 4 weeks. Detection of
virus infection after treatment was conducted using reverse transcriptionpolymerase chain reaction (RT-PCR) with universal primer for Potyvirus,
Carlavirus, and Allexivirus.
Screening of bulb samples were done by dot immunobinding assay (DIBA)
using antibody to Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV),
and Garlic common latent virus (GCLV). One hundred samples of each cultivar
were chosen randomly and growth in tray using styrofoam with water below.
Fifty leaf samples from each cultivars were collected randomly for virus detection.
Samples giving positive reaction to viral antibodies were then used as materials
for further in vitro virus elimination. Infection of OYDV was detected 100% from
both cultivars. Samples „Bima Curut‟ were positive to SLV and GCLV 100% and
90%, respectively. Meanwhile, samples „Bima Brebes‟ were positive to SLV and
GCLV 96% and 96%, respectively. These result indicated that virus infection in
shallot has been increasing. In general, the dominant virus infection on shallot
bulbs was OYDV, whereas the least one was GCLV.
Thermotherapy using different temperature and explant size showed that
temperature gave significant effect to plantlets growth, while explant size was not

significant. Based on plantlets growth, the best treatment was using 1 mm-explant
treated at heterogenous condition for cv. Bima Curut, meanwhile for cv. Bima
Brebes the best treatment was using 2 mm-explant treated at heterogenous
condition. Confirmation of virus infection using RT-PCR in plantlets resulted
from thermotherapy showed that smaller explant size enhance the efficiency of
viral elimination. Plantlets derived from 1 mm-explant shown negative result to
viruses for both treated and untreated plantlets from both cultivars. Efficiency of
viral elimination on cv. Bima Curut plantlets derived from 2 mm-explant and 3
mm-explant using 30 °C to Potyvirus was about 67%, while to Carlavirus was
about 33% and 67%, respectively. In heterogenous condition, efficiency of viral
elimination on cv. Bima Curut plantlets derived from 2 mm-explant and 3 mmexplant was 67 dan 50%, respectively. Efficiency of viral elimination on cv.
Bima Brebes plantlets derived from 2 mm-explant and 3 mm-explant using 30 °C
to Potyvirus was about 33%, while to Carlavirus about 33% for both explant size.
Meanwhile, in heterogenous condition efficiency of viral elimination on cv. Bima
Brebes plantlets derived from 2 mm-explant and 3 mm-explant was 50 and 67%
to Potyvirus, and to Carlavirus about 50 and 67%, respectively. In general, shoot
tip culture using 1 mm-explant size could eliminate viruses until 100%. However,
these techniques need expertise and time consuming. Therefore, treatment using 2
mm-explant at heterogenous condition is the best combination for bigger explant
size to decrease viral concentration and did not interfere plantlets growth.

Chemotherapy using ribavirin in growth medium caused inhibition on
plantlet growth, particularly using concentration of 25 mg L-1. Although plantlets
grew on medium containing 10 mg L-1 ribavirin but they remained contain
Potyvirus and Carlavirus. These results indicated that treatment combination of
ribavirin and thermotherapy could not eliminate viruses.
Virus-free bulbs are needed to decrease disease incidence in the field and
increase yield in Indonesia. Shoot tip culture with 1 mm-explant size resulted in
virus-free plantlets. When bigger size of explants (2 mm) was used, it required
treatment combination of thermotherapy at heterogenous condition.
Key words: Carlavirus, Potyvirus, shoot tip culture, virus-free bulb, virusfree.plantlet

© 2017, Bogor Agricultural University
All Right Reserved
It is prohibited to make any quotations from part or whole of this thesis without citing
the author or the copyright holder. Quotation is allowed as long as for education,
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No part of this thesis may be reproduced or transmitted in any forms or by any means,
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storage and retrieval system without express written permission from Bogor
Agricultural University.

1

IN VITRO ELIMINATION OF VIRUSES ON SHALLOT
(Allium cepa var. aggregatum) USING THERMOTERAPY AND
CHEMOTHERAPY

PRABAWATI HYUNITA PUTRI

Thesis
in partial fulfillment of the requirements for the degree of
Master of Science
at the
Phytopathology Study Program

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR

2017

2

External Examiner on Thesis Defense: Dr Efi Toding Tondok, SP, MScAgr

2

PREFACE
Praise and great gratitude to Allah subhanahu wa ta’ala who always gives
His bless to me for finishing this thesis entitled “In vitro Elimination of Viruses in
Shallot (Allium cepa var. aggregatum) Using Thermotherapy and Chemotherapy”.
This thesis is a requirement in accomplishing the Master of Science Program at
Phytopathology Study Program, Graduate School, Bogor Agricultural University.
I would like to thank all individuals who gave contribution so that this thesis
can be finished. I would like to express my deep gratitude to Prof Sri Hendrastuti
Hidayat, PhD and Dr Diny Dinarti for their support and guidance during lab work
and thesis writing. My sincere grateful to Dr Efi Toding Tondok and Dr Giyanto
for their suggestion during thesis defense.
I would also like to thank Domestic Graduate School Scholarship Organizer
from Ministry of Research, Technology and Higher Education, Republic of
Indonesia for the graduate scholarship in Graduate School of Bogor Agricultural
University. My sincere gratitude to ACIAR who funded this research through
ACIAR Project (HORT 2009/056 Project) “Sustainable productivity
improvements in alliums and solanaceous vegetable crops in Indonesia and subtropical Australia”.
The greatest thanks and appreciation to my beloved parents, Bapak
Kusnanto and Ibuk Sriyani, and Adek Dwi Setia Agusti Putri. They always gave
me their pray, support, love, patience, and motivation during research and
finishing the thesis. Thank you so much.
I addressed sincere gratefulness to big family of Phytopathology class of
2013, Plant Virology Laboratory members, Tissue Culture 3 Laboratory members,
Ngecemes, Gottingers, Sari Nurulita, Aqlima, Tyas Ayu Lestari, Ankardiansyah
Pandu Pradana and Rizki Haerunisa for their support during thesis writing, and for
all relevant parties who I can not mention one by one. May Allah bless you all.
Hopefully this thesis can be useful and give positive contribution to the
development of science.
Bogor, January 2017
Prabawati Hyunita Putri

1

TABLE OF CONTENTS
LIST OF TABLES

vi

LIST OF FIGURES

vi

LIST OF APPENDIXES

vi

1

1
1
2
2
2
3
5
5
6
6
6
7
8
8
8
10
10
10
10

2

3

4

INTRODUCTION
Background
Research Problem
Research Objectives
Research Hypothesis
Research Benefits
LITERATURE REVIEWS
Shallots Production and Its Constraints in Indonesia
Onion yellow dwarf virus (OYDV)
Garlic common latent virus (GCLV)
Shallot latent virus (SLV)
Detection and Quantification of Major Viruses on Shallot
Tehnique for Virus Elimination
Chemoterapy
Thermotherapy
MATERIALS AND METHODS
Screening of Bulb Infected Viruses
Detection of Viruses
Dot immuno binding assay (DIBA)
Reverse Transcription Polymerase Chain Reaction (RT PCR)
method
Shoot Tip with Thermotherapy and Chemotherapy Treatment
Preparation of Tissue Culture Medium
Bulbs and Explants Sterilization
Thermotherapy Treatment
Chemotherapy Treatment
Experimental Design and Variable of Observation
RESULTS AND DISCUSSION
Screening of Bulbs-Infected Virus
The Effect of Thermotherapy and Explant Size to the Number of
Plantlets Survived
The Effect of Thermotherapy and Explant Size to Average Number
of Leaves
The Effect of Thermotherapy and Explant Size to Plantlets Height
The Effect of Thermotherapy and Explant Size to Average Number
of Roots

11
12
12
12
13
13
13
14
14
15
16
18
20

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The Efficiency of Viral Elimination by Thermotherapy

21

The Efficiency of Viral Elimination by Chemotherapy Following
Thermotherapy
General Discussion
5 CONCLUSIONS AND RECOMMENDATIONS
Conclusions
Recommendations
REFERENCES

23
25
27
27
27
28

APPENDIX

33

BIOGRAPHY

40

3

LIST OF TABELS
1
2
3
4
5

Research of elimination in the plant using meristem culture for
period 1991-2010
Primers used for amplification of Potyvirus, Carlavirus, and
Allexivirus
The average of virus infection (%) on shallots leaves samples
Virus incidence on plantlets cv. Bima Curut and cv. Bima Brebes
after thermotherapy
Effect of chemotherapy to plantlets growth

9
12
14
22
24

LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Research flow chart
Growing bulb samples in plastic tray using styrofoam
Nitrocellulose membrane showing the reaction of samples to
antibodies (purple dots) in dot immunobinding assay.
Numbers of plantlets survived after thermotherapy on different
explant size (1, 2, 3 mm) of two shallot cultivars : (A) cv. Bima
Brebes and (B) cv. Bima Curut
Plantlets growth on the thermotherapy treatment
The effect of thermotherapy on average number of leaves cv. Bima
Curut (A and B) four weeks during treatment and (C and D) four
weeks after treatment
The effect of thermotherapy on average number of leaves cv. Bima
Brebes (A and B) four weeks during treatment and (C and D) four
weeks after treatment
The effect of temperature (A) and explant size (B) on plantlets
height of cv. Bima Curut
Plantlets height and number of shoots following thermotherapy
application on cv. Bima Curut explants derived from different sizes
The effect of temperature (A) and explant size (B) on plantlets
height of cv. Bima Brebes
The effect of thermotherapy on average number of roots cv. Bima
Brebes (A and B) four weeks during treatment and (C and D) four
weeks after treatment
The effect of thermotherapy on average number of roots cv. Bima
Curut (A and B) four weeks during treatment and (C and D) four
weeks after treatment
Virus detection of Potyvirus in plantlets derived from 2 mmexplant (A) and Carlavirus in plantlets derived from 3 mmexplants (B) cv. Bima Curut and cv. Bima Brebes
Number of plantlets survived after chemotherapy. (A) cv. Bima
Curut (A); (B) cv. Bima Brebes

4
10
14
15
16
17
18
19
19
19
20
21
23
23

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15 DNA amplification of Potyvirus (A) and Carlavirus (B) from
plantlets of cv. Bima Curut and cv. Bima Brebes following
thermotherapy and chemotherapy

24

LIST OF APPENDIXES
1
2
3
4
5
6
7

Description of shallot cultivars used in this research
Analysis of t-test the effect of thermotherapy on life percetage
plantlets cv. Bima Curut and cv. Bima Brebes
Analysis of the effect of temperatures, explant size, and interaction
to an average number of leaves and plantlets height cv. Bima Curut
Analysis of the effect of temperature, explant size, and interaction
to an average number of leaves and plantlets height cv. Bima
Brebes
Analysis of effect of temperature, explant size, and interaction to an
average number of roots cv. Bima Brebes
Analysis of effect of temperature, explant size, and interaction to an
average number of roots cv. Bima Curut
Comparison between cv. Bima Curut and cv. Bima Brebes

34
34
35
36
37
38
39

1

1 INTRODUCTION
Background
Shallot (Allium cepa var. aggregatum) is one of important horticultural
comodity in Indonesia. Production center of shallot in Indonesia is located in
West Java, Central Java, East Java, Yogyakarta, and West Nusa Tenggara.
Although shallot can be propagated through generative or vegetative stages using
true seeds or bulbs, respectively, most farmers in Indonesia use bulbs as
vegetative propagative material. This is due to difficulties to produce true shallot
seed (TSS) in Indonesia because of climate condition. Several pathogens,
including viruses, can be transmitted through vegetative materials, such as shallot
bulbs (Sharaeen et al. 2008). There are three genera of viruses that has been
reported to infect shallot, i.e. Carlavirus (Shallot latent virus / SLV and Garlic
common latent virus / GCLV), Potyvirus (Onion yellow dwarf virus / OYDV,
Shallot yellow stripe virus / SYSV, and Leek yellow stripe virus / LYSV), and
Allexivirus (Garlic virus B / GV-B, Garlic virus C / GV-C), and Garlic virus D /
GV-D) (Sharaeen et al. 2008; Sevik and Ackura 2013).
Infection of LYSV, SLV, and OYDV on shallot in Indonesia has been
reported from Lembang and Subang, West Java (Duriat and Sukarna 1990;
Wulandari et al. 2002). SYSV was detected in shallot samples varieties Jawa and
Brebes from Brebes, Central Java (Kurniawan and Suastika 2013). Recently,
Kadwati and Hidayat (2015) reported, the infection of Potyvirus, Carlavirus, and
Allexivirus on shallot from Brebes (Central Java) and Cirebon (West Java).All of
these reports indicated that shallot disease caused by viruses has increasing
recently in Indonesia.
Little is known about yield loss on Allium, shallots in particular, caused by
virus infection. Walkey et al. (1989) reported the potency of OYDV infection to
cause yield loss on garlic up to 88%. In France, weight of garlic clove was
reduced about 56-84% as a result of mixed infection of OYDV and LYSV (Lot et
al. 1998). Infection of OYDV in garlic and shallot in Turkey caused weight
reduction up to 25-54% compared with healthy bulb (Sevik and Ackura 2013). All
of these reports indicated that virus infection could reduce yield and decrease
quality of the bulbs. Therefore, information regarding yield loss in shallot due to
virus infection is important to know especially to determine its affect on bulbs
quality.
Viral disease control based on chemical application is not effective,
although many farmers spray insecticides to reduce insect vectors (Ahmed and
Elhassan 2013; Taskin et al. 2013). Cultural practices, such as controlling
inoculum sources (Zitter and Simons 1980) is less effective because it is difficult
to identify virus symptoms on shallot in the field. Therefore, virus-free bulbs are
essentially needed because it will protect the plants in the early growing stage.
Unfortunately, virus-free bulbs are difficult to obtain using propagative method
since the viruses can be transmitted through vegetative material. One of solution is
using in vitro shoot tip culture to produce virus-free bulbs (Al Maarri et al. 2012).
Shoot tip culture can be combined with several techniques to improve efficiency
of virus elimination, such as thermotherapy (heat treatment), chemotherapy

2

(antiviral), electrotherapy (electrical current), and cryotherapy (cold temperature).
Among them, thermotherapy and chemotherapy are the most common method
used for virus elimination (Rout et al. 2006).
Thermotherapy consisted of two techniques i.e hot water treatment and hot
air treatment. Ali et al. (2013) reported the use of meristem tip culture in
combination with thermotherapy to eliminate viruses in potatoes and the
technique had efficiency about 24.55%. Chemotherapy is a virus elimination
technique using a substance named antiviral, such as ribavirin. Neelamathi et al.
(2014) reported that the elimination of Sugarcane mosaic virus in sugarcane using
ribavirin 10 mg L-1 had an efficiency up to 95%. Shoot tip culture - based
techniques combined with thermotherapy and chemotherapy need to be developed
as a method to get virus-free bulbs in order to improve the quality of shallot seeds
in Indonesia.
Problem Statement
Most of shallot growers in Indonesia using bulbs as propagative material.
High virus infestation was detected from seed bulbs collected from shallot‟s
production center in Indonesia. Bulb borne viruses may infect the plants since
early stage and cause yield loss. The actual yield loss caused by virus infection
has not been studied because virus-free bulbs is very difficult to obtain. Among
other techniques available, virus-free bulb can be obtained using shoot tip culture.
Tissue culture in combination with thermotherpy and chemotherapy may increase
efficiency of virus elimination. These treatment has been known to eliminate
viruses in some horticultural plant succesfully. Therefore, thermotherapy and
chemotherapy might have a good potential to be applied on shallots. The use of
virus-free bulbs as seeds source can suppress disease incidence on shallots in
Indonesia.
Research Objectives
Objective of this research is to develop in vitro virus elimination method for
shallot through thermotherapy (30 oC dan 37 oC) and chemotherapy (10 mg L-1
and 25 mg L-1) using different cultivars („Bima Curut‟ and „Bima Brebes‟) and
explant size (1 mm, 2 mm, and 3 mm) (Figure 1).
Research Hypothesis
1. Modification of explant may effect virus elimination efficiency in tissue culture
method.
2. Combination of thermotherapy and chemotherapy could improve efficiency of
virus elimination method.
3. Thermotherapy and chemotherapy will not interfere with plantlets growth.

3

Research Benefits
The expected output of this research is to obtain an efficient methods to
eliminate viruses from shallot bulbs based on thermotherapy and chemotherapy
treatment. A succesfull regeneration of planlet-free viruses will support
government program to improve seeds quality. Good quality of shallot seeds with
virus free characters may suppres virus transmission and disease incidence in the
field.

4

Field survey and samples screening

Detection of viral infection in shallots
(DIBA)

Treatment : three explant sizes (1
mm, 2 mm, dan 3 mm) with two
temperatures (30 oC and 37 oC) and
incubated in homogenous condition.
Control at temperatures 25 oC.

Treatment : three explant sizes (1
mm, 2 mm, dan 3 mm) with two
temperatures (30 oC and 37 oC) and
incubated in heterogenous condition.
Control at temperatures 25 oC.

RT-PCR

Virus-infected Plantlets

Virus-free Plantlets

Thermotherapy
combined
with
chemotherapy (ribavirin, 10 mg L-1
and 25 mg L-1), as a control 0 mg L-1
RT-PCR

Virus-infected Plantlets

Virus-free plantlets

In vitro elimination
methods

: method

: result

: detection

: process

Figure 1 Research flow chart of “Elimination of Viruses on Shallots (Allium cepa
var. aggregatum) Using Thermotherapy and Chemotherapy

5

2 LITERATURE REVIEWS
Shallot Production in Indonesia and Its Constraint
Shallot is a member of plant in family : Lilliaceae; belongs to genus :
Allium L.; species : Allium cepa var. aggregatum. Different variety of shallot is
grown by farmers across Indonesia. The most popular varieties grown in West
Java are „Lodra‟, „Sumenep‟, „Batu‟, „Maja Merah‟, „Cigugur Merah‟, and
„Ciniru‟; in Central Java are „Bima Curut‟, „Bima‟, „Bima Timor‟, „Bima Arjuna‟,
„Kuning Tablet‟, „Kuning Gombong‟, and „Filipina‟ (Gunaeni et al. 2011).
Shallot production areas are found in all across Indonesia, but mainly in
Java, mostly in the lowland. Shallots are grown at the beginning and at the end of
the rainy season (October – November and March – July, respectively). A high
bed of soil was built with 50 – 60 cm in height and 1.2 – 2 m in width, with a
ditch between beds. The ditch is filled with water throughout growing period with
water level 25 – 30 cm below the surface of the bed with water level 25 – 30 cm
below the surface of the bed. Watering was done twice a day until 1 week before
harvest (Permadi 1994).
Propagation of shallot can be done using generative and vegetative materials.
Generative is using seeds produced by flower as propagative material, meanwhile
vegetative is using bulbs. Seed-propagated shallots were difficult may be due to
unfavourable condition of climate so that many farmers in Indonesia use bulbs as
propagative materials. Farmers produce their own seeds from selected bulbs
which are dried in the sun after harvested. They are stored in bunches called
“para-para” in the kitchen above the stove. Seed producers were built special
storagehouse which seeds were hung on bamboo racks. Below bamboo racks were
built artificial drier which wood is burned to further dry and smoke the bulbs.
Bulbs which is used as propagative material commonly stored for 2 until 3 months
before planted in the field (Grubben 1994; Permadi 1994).
Shallots farmers in Indonesia has to deal with several contraints, among
others are yield and quality of shallots production among farmers, poor
propagation material, pest and disease control, storage losses, inappropriate
fertilizer, and marketing. The health of the planting bulbs is an important factor
that affect shallots production. As described earlier, production of seedpropagated shallot were not enough and about 70% of bulbs were proved to be
infested with viruses. Bulbs-infested viruses caused vigour declining of the
planting materials (Van Dijk 1993; Grubben 1994).
According to Mishra et al. (2014) all kind of pathogens were commonly
found infecting shallot. Bacteria that infected shallot are Pseudomonas aeruginosa
causes brown rot disease on the bulbs, Pseudomonas cepacia causes bulb rot
disease, Erwinia carotovora and Pseudomonas allicola causes soft rot disease.
Fungus that known infected shallot are Peronospora destructor (Berk) Casp.
causes powdery mildew, black rot disease caused by Aspergillus niger, and
Fusarium spp. causes wilt disease or bulb rot. Colletotrichum gloeosporioides
causes anthracnose disease. Alternaria porri has been reported as a causal agent of
purple blotch disease in shallot (Permadi 1994; Madhavi et al. 2012)

6

Major Viruses in Shallot
Major group of viruses that infect shallot are Potyvirus, Carlavirus,
Allexivirus, and Nepovirus. There are four important species of viruses that has
been reported infecting shallot i.e. OYDV, SLV, GCLV, and SYSV (Diekmann
1997; EPPO 2007).
Onion yellow dwarf virus (OYDV)
Onion yellow dwarf virus is a member of the genus Potyvirus, with
flexious, filament-shaped particle. Virus particle approximately 772-823 nm in
length and weight of coat protein about 34 kDa. Onion yellow dwarf virus is
transmitted through insect vector, Myzus persicae (Aphididae), with non
persistent manner. The viruses is also transmitted mechanically through vegetative
propagation material, and can not be transmitted through pollen (Takaichi et al.
2001).
On shallots, OYDV produces symptom of a mild yellow chlorotic
irregular stripes to bright yellow stripes. Infected leaf become curling downward,
and shrinken. In addition, infected shallot become stunted and the bulb size
smaller than healthy bulb. Mechanical inoculation of OYDV can cause local
lession on Chenopodium murale. The common method for detection of OYDV is
using serological method, i.e. indirect enzyme-linked immunosorbent assay (IELISA). At the beginning of plant growth OYDV is difficult to detect because
the concentration of viruses is very low (Mishra et al. 2014).
Garlic common latent virus (GCLV)
One member from genus Carlavirus that infect shallots is Garlic common
latent virus (GCLV). These virus belongs to the family Betafleviridae, and ordo
Tymovirales. The virus particle is filamentous and flexious. The nucleic acid is
single stranded RNA approximately 8.6 kb. Garlic common latent virus spread
across several region around the world but it is not endemic (Barg et al. 1994,
Barg et al. 1997). Single infection of GCLV produce no symptom or symptomless.
Severe yellowing and mosaic was produced when shallot and garlic infected by
combination of GCLV and Potyvirus, such as OYDV and LYSV. Carlavirus and
Potyvirus may have a synergitic effect on symptom expression when infecting
garlic and shallot (Takaichi et al. 1998).
Decoration tests in France showed the general presence in French garlic of a carlavirus not

Shallot latent virus (SLV)
Shallot latent virus (SLV) is a member of the genus Carlavirus. Virus
particles formed flexious filament-shaped about 650 nm in length (Diekmann
1997). Shallot latent virus has a wide range of host in the family Alliaceae and
spread worldwide. The virus is transmitted mechanically and through Aphids
(Myzus ascalonicus) with a non persistent manner. However, SLV transmission
through Aphids are rare and less efficient than Potyvirus. Therefore, SLVs
transmission are more efficient through vegetative propagation material. Single
infection of SLV on shallot, garlic, onions, and leeks are asymptomatic and not
significant. However, SLV infection accompanies with other viruses from genus

7

Potyvirus will cause serious damage and yield losses. SLVs showed local
symptomatic lesion in some indicator plants i.e Chenopodium spp., Celosia
argentea, and Vicia faba. Systemic symptoms were showed on Nicotiana
occidentalis and N. hesperis (Van Dijk 1993).
Detection And Quantification of Major Viruses on Shallots
Detection of viral diseases in the plant seldom using symptoms observation.
The methods is not always reliable because more than one virus can cause similar
symptoms in the plant and many non-biotics disorder, such as nutritional
deficiency or drought, may causes symptoms similar to virus diseases.
Furthermore, the diseases symptoms also depends on cultivars and growth stages
of host plants (Naidu and Hughes 2009).
Nowadays, detection and quantification of virus particles in the plant can be
done through two approach i.e. serological and nucleic acid-based methods. These
two methods are reliable to identify viruses at all times. Serological methods for
virus identifications are based on the virus coat protein properties. The basic
principle in which the virus antigens are recognized by their specific antibodies
(IgG) in association with colorimetric properties. This method is able to detect
viruses in very low concentrations and can be used with viruses of different
particle morphology. Because of its adaptability, high sensitivity, and economy in
the use of reagents, serological methods are used in a wide range of situations,
especially for detection a large number of samples in a relatively short period of
time. Several serological methods that used in virus detection, i.e enzyme linked
immunosorbant assay (ELISA) and dot immunobinding assay (DIBA) (Webster et
al. 200; Naidu and Hughes 2009). DIBA methods was used in this research to
detect virus diseases in samples.
Quantification and detection of virus particles using serology method had
several weakness i.e cross reaction between antigens which closely related so give
unspecific result, diversity among virus strain, and pH of reagent affected test
result.
Molecular methods based on nuleid acid are widely used for detection and
quantification viruses in the field. PCR is a techniques that has specifity and
sensitivity to detect plant pathogens (Naidu and Hughes 2009). Molecular
methods for detecting plant pathogens are based on the accurate design of primers
and target sequences. Specific nucleotide regions are selected and primers specific
for DNA or RNA targets can be easily designed. The target sequences then
amplified using dNTP (nucleotide triphospate) and enzyme Taq polymerase.
Revers transription polymerase chain reaction (RT-PCR) is a modification of PCR
technique for enable amplification of RNA instead of DNA. For the case of RNA
viruses, RNA were reversed into cDNA using enzyme transcriptase then amplified
using appropriate primers (Naidu and Hughes 2009; Martinelli et al. 2015).
Molecular methods based on nucleic acid are efficient, specific, and able to
detect a single target in omplex mixture. However, it had several weakness, i.e
false negatives can occur when the DNA target sequence is degraded or reagents
are of insufficient quality, the sensitivity of PCR-based methods or RT-PCR based
methods are often lower than expected due to inhibitors of transcriptases and/or

8

polymerases, sample cross-contamination can give false positives, and the cost of
equipment and reagents must be considered when selecting a molecular detection
method (Naidu and Hughes 2009; Martinelli et al. 2015)
Technique for Virus Elimination
Tissue culture is a method to isolate parts of plant (protoplasm, cell, tissue,
and organ) based on totipotency and can be cultured in aseptic condition. Culture
plant is carried out under control condition so that the parts of plant can be
regenerated became a whole plant. Tissue culture widely used nowadays because
it can give some benefits, i.e produce plant in large quantities in a short time and
disease-free, seed production with desirable properties, and need a narrow place
(Altman et al. 2005)
Variety of tissue culture techniques is available, among others is shoot tip
culture. Initial explants in shoot tip culture is shoot tips which consisted of leaf
primordia and one meristem dome. Those type of culture usually used to get
virus-free plant. Some leaf primordia could be included because if explant is too
small it difficult to do root regeneration (Rout et al. 2006; Panattoni et al. 2013).
The successful of virus elimination determined by explant size used for tissue
culture. In order to increase efficiency of virus elimination shoot tip culture
usually is combined with some other treatments, such as thermoteraphy,
cryotherapy, electrotherapy, and chemotherapy (Milasevic et al. 2012; Panattoni
et al. 2013).
Chemotherapy
A method for virus elimination by application of drugs or chemical
substances as antiviral agent is known as chemotherapy. There are several
antiviral agents that may inhibit virus replication and used in chemotherapy, such
as ribavirin, acylguanocine, acidotimidin, green malachit, 2-thiouracil (Milasevic
et al. 2012). The most common antiviral used in tissue culture technique is
ribavirin. Ribavirin can be used in plant meristem that cultured in vitro or cultured
before shoot tip or meristem isolation (Panattoni et al 2013).
Ribavirin is a synthetic chemical that analog with guanocyne. Added 10-50
-1
mg L of ribavirin in the selective culture media prevent several viruses infection
such as PVX and PVY in potato, CMV in meristem of Nicotiana rustika (Hadidi
et al. 1998). Soliman et al. (2012) reported that 20 mg L-1 dose of antiviral can
eliminate OYDV in garlic about 85%. Ribavirin interferes formation of nucleid
acid so that inhibit virus replication. Active form of ribavirin is triphospate which
inhibit capping process at 5‟ RNA virus. Moreover, ribavirin inhibits virus
replication in the initial phase by interfering RNA-dependant RNA polymerase
and RNA helicase synthesis. In the final phase, ribavirin is known to interfere coat
protein synthesis (Neelamathi et al. 2014; Panattoni et al. 2013).
Thermotherapy
Thermotherapy is a common techniques for eliminating
concentration of pathogens in the plant by the application
temperature). Thermotherapy can eliminate viruses because
replication and virus translocation in the plant. Temperatures

or decreasing
of heat (high
inhibit virus
that given in

9

thermotherapy depends vary on explant and target of pathogens. In principal,
thermotherapy could decrease concentration of pathogens but did not inhibit plant
growth. Generally, type of explants used for thermotherapy are seeds, bulb, and
shoots which are more stable at high temperature (Milasevic et al. 2012; Panattoni
et al 2013).
There are two types of thermotherapy i.e hot water treatment and hot air
treatment. Hot water treatment is a common method used for seeds.
Thermotherapy that applied in growing plant often used temperatures around 3554°C for several days or weeks. Thermotherapy is usually combined with shoot
tip and meristem culture to increase propability of pathogens elimination, such as
viruses. Many successful research of thermotherapy has been reported for period
1991-2010 (Table 1) (Panattoni et al 2013).
Table 1 Research of virus elimination in the plant using thermotherapy during
1991-2010 (Panattonni et al. 2013)
Plant
Grape

% Successfulness1
34.5

Other plant

13.8

Sugarcane
Garlic
Potato
Others

13.8
10.3
6.9
20.7

1

percentage of trial out of total

Virus Target
Closteroviridae; Comoviridae;
Flexiviridae; Tymoviridae
Bromoviridae; Caulimoviridae;
Ophioviridae; Potyviridae
Luteoviridae; Potyviridae
Potyviridae
Potyviridae
Bromoviridae; Caulimoviridae;
Fleviviridae; Potyviridae

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3 MATERIALS AND METHODS
Research activities were conducted in Laboratory of Tissue Culture 3,
Department of Agronomy and Horticulture; and Laboratory of Plant Virology,
Departement of Plant Protection, Faculty of Agriculture, Bogor Agricultural
University from January 2015 to June 2016. The research was consisted of 4 main
activities, i.e (1) collection of bulb samples from Tenguli village (Brebes, Central
Java), (2) detection of viruses from bulb samples, (3) tissue culture with
thermotherapy and chemotherapy treatment, and (4) detection of viruses in tissue
culture plantlets after treatment.
Screening of Bulbs-infected Viruses
Bulb samples were collected from seed grower in Tenguli village, Brebes,
Central Java. There are two cultivars that commonly used as seeds in Brebes, i.e.
cv. Bima Curut and cv. Bima Brebes (Appendix 1). Bulb samples has been stored
for 60 until 70 days in a dry place with low humidity named “para-para”. One
hundred bulbs for each cultivars were grown in styrofoam with water below (Fig
2). The bulbs were then placed in a cool room with temperatures of 16 oC. After
one week, leaf samples were collected randomly, i.e. 50 leaves for each cultivar,
and tested for virus incidence by dot immunobinding assay (DIBA) using three
antibodies, i.e. OYDV, SLV, and GCLV. Bulbs gave positive reactions were then
used as materials for tissue culture.

Figure 2 Growing bulb samples in plastic tray using styrofoam
Detection of Viruses
Two methods were used, i.e dot immunobinding assay (DIBA) for early
detection of viruses from shallots bulb, and reverse transcription polymerase chain
reaction (RT-PCR) for detection of viruses from plantlets after in vitro treatment.
DIBA Method
The assay was conducted following method described by Asniwita (2012)
using antibodies for OYDV, GCLV, and SLV. Leaf samples were weighed 0.1 g,
then ground in Tris Buffer Saline (TBS, Tris-HCl 0.02 M dan NaCl 0.15 M, pH
7.5) 1:10 (w/v). Plant sap was spotted onto nitrocellulose membrane about 2 μL.
The membrane was air dried, then it was submerged in solution containing 2%
non-fat milk and TBST buffer (Tris buffer saline with Triton X-100). The
membranes was incubated at room temperature and shaked at 50 rpm for 60 min.

11

Membranes was then washed 5 times with H2O, each washed process was shaked
at 100 rpm for 5 min. After washing, the membranes were soaked into 2.5 μL first
antibody solution (with 2% non-fat milk-TBST), then incubated at 4 oC overnight.
After washing 5 times for 5 min using TBST, the membranes was soaked on 2.5
μL conjugate antibody (with 2% non-fat milk-TBST), then incubated for 60 min.
The membranes was soaked on 10 mL AP-buffer (Tris-HCl 0.1 M, NaCl 0.1 M,
MgCl2 5 mM, H2O) containing 1 tablet nitro blue tetrazolium (NBT) and bromo
chloro indolil phosphate (BCIP). Positive reaction was qualitatively indicated by
appearance of purple colour in nitrocelullose membrane on the spotted area.
Reaction was stoped by soaking the membranes onto dH2O.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) Method
Leaf samples was collected from each plantlet, then it was bulked, i.e. one
sample consisted of 10 leaves. The RT-PCR method started with total RNA
extraction, followed by cDNA synthesis, cDNA amplification, and visualization
using gel electrophoresis.
Total RNA Extraction. Extraction of total RNA was conducted following
Doyle and Doyle (1987) method with some modification. Samples was weighed
and 0.1 g of samples was ground in extraction buffer containing 1%
merchaptoethanol (1:5 w/v). The samples were incubated at 65 °C for 30 min and
inverted every 10 min. Chloroform/isoamyl alcohol (24:1, v/v, 500 µL) was added,
then the tube was mixed well. The samples was centrifugated at 11 000 rpm for 15
min. The supernatant was transferred into a clean 1.5 mL tube and isopropanol
(2/3 volume supernantant) was added for precipitation, then incubated in -80 °C
for 2 hr. The supernatant containing isopropanol was sentrifugated at 11 000 rpm
for 15 min. The pellets were washed with 500 µL 70% ethanol, then centrifugated
at 8000 rpm for 5 min. The supernatant was discarded, and pellets was dried under
room temperature. Dried pellets containing RNA were dissolved in 50 µL TE
buffer (pH 8).
cDNA Synthesis. The total RNA was used for reverse transcription
reaction to form cDNA (complementary DNA) using reverse transcriptase enzyme.
Reagent and volume per tube that used for this reaction consisted of: 2 µL 5x
Buffer RT, 1 µL 0.1 M DTT, 1 µL dNTP mix, 0.5 µL RNAse inhibitor (40 U/µL),
0.5 µL M-MuLV (200 U/µL), 1 µL Poty 1 primer, and 1 µL nuclease-free water.
The mixtures were added into clean tube, then 3 µL of total RNA was added.
Reverse transcription reaction was done using automated thermal cycler (Gene
Amp PCR System 9700; PE Applied Biosystem, USA) with one cycle program at
temperature 65 oC for 5 min, 42 oC for 60 min, and 70 oC for 10 min to inactivate
RNAse inhibitor. The synthesized cDNA was then used as DNA template in the
amplification reaction.
cDNA Amplification and Visualization. Amplification reaction per tube
contains 12.5 µL Go Taq Green (Thermoscientific), 9.5 µL nuclease-free water,
10 µM for each primer (forward and reverse), and 1 µL of cDNA. Amplification
of cDNA viruses was performed ini GeneAmp PCR System 9700 started with preheating cycle for 5 min at 94 oC, followed by 35 cycles of denaturation (20 sec at
94 oC), annealing (1 min at 54 oC), and extension (3 min at 72 oC). The last stage
was ended at 72 oC for 1 min and cooled down to 4 oC. The amplicon then
visualized by electrophoresis using 1% agarose gel in 0.5x TBE (Tris-Boric acid-

12

EDTA) buffer. The electrophoresis was performed at 50 V for 50 min, then the
gel was soaked on to 0.1% EtBr for 10 min, washed with H2O for 5 min, and
visualized under UV transilluminator. Three pairs of primers was used separately
to amplify Potyvirus (U341/Poty1), Allexivirus (pGV3t/Poty1), and Carlavirus
(AlcarF/Poty1) (Chen et al. 2001; Langeveld et al. 1991) (Table 2).
Table 2 Primers used for amplification of Potyvirus, Allexivirus, and Carlavirus
Sequences1

Primers

Amplicon
(bp)
600-750
Potyvirus

U341
Poty1

F: 5‟-CCGGAATTCATGRTITGGTGYATIGAIAAYGG-3‟
R: 5‟-GGATCCCGGGTTTTTTTTTTTTTTTTTV-3‟

AlcarF
Poty1

F: 5‟-TGCTGCYTTTGATACYTTCGAT-3‟
R: 5‟-GGATCCCGGGTTTTTTTTTTTTTTTTTV-3‟

715
Carlavirus

pGV3t
Poty1

F: 5‟-TGGNCNTGCTACCACAANGG-3‟
R: 5‟-GGATCCCGGGTTTTTTTTTTTTTTTTTV-3‟

950
Allexivirus

1

F : forward; R : reverse

Shoot Tip Culture with Thermotherapy and Chemotherapy Treatment
Thermotherapy was applied through hot air treatment by incubating
explants in growth chamber. Ribavirin (Virozole, Sigma Aldrich) was used as
antiviral agent in chemotherapy.
Preparation of Tissue Culture Medium
Tissue culture medium consisted of shoot-inducing medium and rootinducing medium. Shoot-inducing medium was made by MS (Murashige-skoog)
medium modified with B5 vitamin. Growth regulator hormones such as 2ip (2 mg
L-1) and GA3 (0.3 mg L-1) was added onto MS medium. Distilated water was
added up to 1 L and pH was checked up to 5.8 - 6. Gelrite 2 g L-1 was added onto
medium and stired well. The medium was poured into 8 mL sterilized culture tube.
Culture tube contains medium was autoclaved at 121 °C, 1 atm, for 15 min. The
medium was then incubated for 3-6 days at 25 °C, and only medium-free
contaminant were later used.
Bulbs and Explants Sterilization
The bulbs were prepared before treatment. The outer protective skin and
necrotic stem base of the mature bulbs were removed. The bulbs were washed
using detergent and rinsed with tap water. The bulbs was soaked into 2 g L-1
fungicide (Dithane 80 WP) and 2 g L-1 bactericide (Agrept 20 WP) and incubated
overnight. Bulbs was then soaked into 20% sodium hypochlorite and shaked at
150 rpm for 20 min. These was followed by rinsing 3 times in sterile distilated
water in clean bench, two outer layers were removed, then soaked into 10%
sodium hypochlorite for 10 min and rinsed for 3 times. The bulbs were excised to
get 5 mm-shoot tip explants, the explants were dipped into 5% sodium
hypochlorite for 5 min then planted temporary in MS0 medium to prevent explant

13

damage. The 5 mm-explants were excised into 3 mm, 2 mm, and 1 mm using
sterile needles under binocular microscope. Three sizes of explant were used as a
treatment for thermotherapy experiment. Each size of explants were cultured on
basic MS medium and incubated for 4 days. Explants that contaminant-free were
transferred into medium to induce shoot development and used later for
thermotherapy.
Thermothe