i 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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i
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
Sesuai Surat Perjanjian Pelaksanaan Penelitian No: 964/PKS/ITS/2020
i
Daftar Isi
Daftar Isi .......................................................................................................................................................... i
Daftar Tabel .................................................................................................................................................... ii
Daftar Gambar ............................................................................................................................................... iii
Daftar Lampiran ............................................................................................................................................. iv
BAB I RINGKASAN ..................................................................................................................................... 1
BAB II HASIL PENELITIAN ........................................................................................................................ 2
BAB III STATUS LUARAN……………………………………………………………………………….11
BAB IV PERAN MITRA (UntukPenelitian Kerjasama Antar Perguruan Tinggi)………………………...11
BAB V KENDALA PELAKSANAAN PENELITIAN……………………………………………………11
BAB VI RENCANA TAHAPAN SELANJUTNYA………………………………………………………11
BAB VII DAFTAR PUSTAKA……………………………………………………………………………12
BAB VIII LAMPIRAN…………………………………………………………………………………….15
LAMPIRAN 1 Tabel Daftar Luaran……………………………………………………………………….15
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
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).
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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
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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).
(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)
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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
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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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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
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
Agriculture 2020, 10, x FOR PEER REVIEW 3 of 11
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%
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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
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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
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
(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
Agriculture 2020, 10, x FOR PEER REVIEW 2 of 11
[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
Agriculture 2020, 10, x FOR PEER REVIEW 3 of 11
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
Agriculture 2020, 10, x FOR PEER REVIEW 4 of 11
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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