- Email: [email protected]
A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens
Accepted Manuscript Title: A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens Authors: Jingjing Yang, Gaojie Li, Shiqi Hu, Antony Bishopp, P.P.M. Heenatigala, Sunjee Kumar, Pengfei Duan, Lunguang Yao, Hongwei Hou PII: DOI: Reference:
S0304-3770(18)30124-4 https://doi.org/10.1016/j.aquabot.2018.08.004 AQBOT 3058
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
20-5-2018 23-8-2018 23-8-2018
Please cite this article as: Yang J, Li G, Hu S, Bishopp A, Heenatigala PPM, Kumar S, Duan P, Yao L, Hou H, A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens, Aquatic Botany (2018), https://doi.org/10.1016/j.aquabot.2018.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens. Jingjing Yang1, Gaojie Li1, Shiqi Hu1, Antony Bishopp2, P. P. M. Heenatigala 1, Sunjee Kumar1, Pengfei Duan3, Lunguang Yao3, Hongwei Hou1* 1
The State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China. The Key Laboratory of Aquatic
IP T
Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China. University of Chinese Academy of Sciences, Hubei Province, 430072, China Centre for Plant Integrative Biology, University of Nottingham, Nottingham, UK
3
SC R
2
Collaborative Innovation Center of Water Security for Water Source Region of Mid-line of South-to-
North Diversion Project, College of Agricultural Engineering, Nanyang Normal University, Henan,
U
China
N
*Corresponding Author; Tel: (+8627) 68780159; Fax: (+8627) 68780123; Email: [email protected]
Highlights
•22.62 µM 2,4-D and 8.88 µM 6-BA are optimal for callus induction.
•1 % sucrose is the most efficient carbon source.
•2.27 to 4.54 µM TDZ is highly efficient in frond regeneration.
•The stable transformation of S. polyrhiza is established mediated by Agrobacterium.
•TCS::GUS and DR5::GUS constructs are successfully expressed in Spirodela polyrhiza.
A
CC E
PT
ED
M
A
1
ABSTRACT Spirodela polyrhiza represents the largest specie with the smallest genome of all the members of the Lemnoideae. Its genome features have been delineated, revealing its fewest predicted genes of any known plant genome. It is also ideal system for basic biological researches and various practical applications including toxicity testing, bioreactor, biomonitoring and biofuel. In this study, we reported
IP T
the successful induction of S. polyrhiza callus coupled with the efficient stable transformation using the Agrobacterium tumefaciens strain LBA4404 by optimizing each step of the process. We found that the highest callus induction efficiency was achieved with 22.62 µM 2,4-D and 8.88 µM 6-BA, with above
SC R
90 % of fronds forming calli. We also determined that 100 µM acetosyringone in the co-cultivation
medium and the maintenance of pH value at 5.2 were crucial for high transformation efficiency (up to 13±1.5 %). As proof of concept, we transformed S. polyrhiza with the DR5 and TCS synthetic
U
reporters, which have previously been used to report cytokinin and auxin signaling output in the model
N
plant Arabidopsis thaliana. The cytokinin showed highest accumulation at the initial stage of bud
A
formation and the frond apex of S. polyrhiza whilst the expression of auxin was observed highest at
development of
M
frond with middle size. These transformed lines provide an effective way to investigate the S. polyrhiza and may shed light on the interesting way in which this specie
ED
reproduces. This is the first report of highly efficient callus induction and Agrobacterium tumefaciens-
PT
mediated transformation in S. polyrhiza.
Abbreviations
2,4-Dichlorophenoxyacetic acid
CC E
2,4-D
A
Keywords: Spirodela polyrhiza; callus induction; transformation; Agrobacterium tumefaciens; cytokinin; auxin.6-BA AS
6-benzyladenine
Acetosyringone
G418
Geneticin
GUS
β-Glucuronidase
MES
4-Morpholine Ethane Sulfonic Acid 2
MS NAA
Murashige and Skoog medium α-Naphthaleneacetic acid
PB
Phosphate Buffer
TDZ
1-Phenyl-3-(1,2,3,-thiadiazol-5-yl) urea
X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt Yeast Extract Mannitol Broth
A
CC E
PT
ED
M
A
N
U
SC R
IP T
YEB
3
Introduction Duckweeds (Lemnoideae) are monocotyledonous free-floating aquatic plants distributed in various types of habitats throughout the world (Landolt, 1986). There are five genera within the Lemnoideae family (Spirodela, Landoltia, Lemna, Wolffiella and Wolffia) (Appenroth et al., 2013). All plants have highly reduced morphologies, consisting of a frond or thallus (Landolt, 1986). These plants
IP T
rarely reproduce sexually, and instead propagate rapidly in a vegetative manner through the budding of daughter fronds from meristematic pockets (Lemon et al., 2001).The frond of Spirodela, Landoltia and Lemna species produce one or more roots whilst Wolffiella and Wolffia species are rootless (Cao et al.,
SC R
2016).
There has been increased interest in the use of duckweeds, as the high efficiency through which they accumulate biomass raises the possibility of using them in bioreactors (Gasdaska et al., 2003;).
U
Regulating the culture conditions, such as altering either pH or nutrient concentration can affect the
N
starch and protein content, making them promising sources for biofuel (Reid and Bieleski, 1970; Jong
A
and Veldstra, 1971; Porath et al., 1979). The use of duckweeds in bioremediation and biomonitoring
M
has also been explored, as they can absorb and take up contaminants including trace metal elements, organic chemicals, nitrogen and phosphorus (Bergmann et al., 2000; Reinhold and Saunders, 2006;
ED
Hegazy et al., 2009). In addition, duckweeds can synergistically interact with microbiota to degrade contaminants in water (Yamaga et al., 2010; Kristanti et al., 2012). The beneficial symbiotic interaction
PT
between duckweed and P23 belonging to the genera Acinetobacter also attributed to the continuous removal of phenol (Yamaga et al., 2010). Kristanti et al. (2012) found that the strains belonging to the
CC E
genus Cupriavidus accelerated the removal of 3-nitrophenol (100%) in river water samples with S. polyrhiza.
Spirodela polyrhiza (L.) Schleiden is widely distributed and represents the largest specie (1.5 cm
A
long) with the smallest genome size (158 Mb) among all duckweeds measured (Wang et al., 2011). An initial reference genome draft of S. polyrhiza was first published by Wang et al. (2014). Michael et al. (2017) further resolved the 20 chromosomes of S. polyrhiza. Efficient stable transformation protocols have been established for common species of duckweeds including Spirodela punctata (Edelman et al., 1988), Spirodela oligorrhiza (Vunsh et al., 2007; Rival et al., 2008), Lemna gibba (Stomp and Rajbhandari, 2000; Yamamoto et al., 2001), Lemna minor (Gasdaska et al., 2003), Wolffia arrhiza 4
(Khvatkov et al., 2015) and Wolffia globosa (Heenatigala et al., 2018). However, as yet there is no published protocol for the efficient stable transformation of S. polyrhiza. For most plant species Agrobacterium-mediated transformation provides the method for introducing transgenes (Comai, 1993), and has been used to modify plants to increase yield, nutrition and quality, improve stress tolerance and produce protein products (Barton and Brill, 1983; Ma et al., 2005). It also
IP T
provides a way to investigate the functions of genes related to various life activities including metabolism (Bulgakov et al., 2002), the development of leaf (Vu et al., 2008) and shoot apical meristem (Scofield et
al., 2014), plant cell death (Dietrich et al., 1997) and circadian clock (Alabadí et al., 2001). In the current
SC R
study, we developed a protocol for the Agrobacterium-mediated transformation of S. polyrhiza. As proof of concept we introduced two synthetic reporters -TCS::GUS (Müller and Sheen, 2008) and DR5::GUS (Benková et al., 2003), that have previously been used to report cytokinin and auxin output respectively
U
in Arabidopsis (Bai and Demason 2008), tomato (Canellas et al., 2011) and rice (Tao et al., 2017). Such
N
lines will provide an effective way to investigate the effect of auxin and cytokinin during different
A
developmental stages of S. polyrhiza and may shed light on the interesting way in which this specie
transformation in S. polyrhiza. Materials and methods
PT
2.1. Plant cultivation
ED
1.
M
reproduces. This is the first report of high efficiency Agrobacterium tumefaciens-mediated
Spirodela polyrhiza (5543) was collected from East Lake (N 30° 32’, E 114°21’) in the city of
CC E
Wuhan, Hubei Province, China. After sterilization using 0.1 % (m/v) mercuric chloride (HgCl 2) for 2 to 3 min and washing 5 to 8 times with sterilized water, the plants were cultured in Erlenmeyer flasks containing 50 mL half strength Murashige and Skoog (1962) medium (0.5 x MS) supplemented with 1
A
% (m/v) sucrose at pH 5.8, under 25 ± 1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. 2.2. Callus induction and frond regeneration To investigate the optimal conditions for callus induction, fronds were grown on 0.5 x MS medium containing 1 % (m/v) sucrose, 0.6 % (m/v) agar at pH 5.8. This was supplemented with different combinations of hormones. For cytokinins we used either 6-benzyladenine (6-BA) or 5
Thidiazuron (TDZ); for auxins we used either 1-Naphthaleneacetic acid (NAA) or 2,4Dichlorophenoxyacetic acid (2,4-D). We also investigated the effect of carbon source by testing sucrose, maltose, sorbitol and glucose. Around 20 fronds in good condition (4 to 6 colonies) were cultured in sterilized Petri dishes (9 cm diameter) containing 30 mL solid medium for callus induction. To determine the optimal hormones for frond regeneration, calli were cultured on 0.5 x MS
IP T
medium with added 1 % (m/v) sucrose, 0.6 % (m/v) agar, and either 6-BA or TDZ at pH 5.8. Each plate contained 8 calli (2 to 3 mm diameter). All experiments were conducted at 25 ± 1 °C with an irradiance of
85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. Each treatment was performed
SC R
in triplicate and subcultured weekly onto fresh medium. The efficiency of callus induction on each
plate was calculated after one month cultivation, whereas the frond regeneration rate was counted after two weeks. Callus induction and frond regeneration efficiencies in this study are presented by means
U
±SDs.
A
N
2.3. Agrobacterium strain and vector
The TCS and DR5 elements were synthesized and inserted to the binary vector pKGWFS7.0
M
(http://www.transgen.com.cn/) (Fig. 1) using Gateway technology. Agrobacterium tumefaciens strain
ED
LBA4404 (http://www.transgen.com.cn/) was used as the host strain harboring the binary vector pKGWFS7.0.
PT
2.4. Preparation of Agrobacterium
Agrobacterium harboring the plasmids to be transformed were cultured on solid YEB medium
CC E
(0.6 % (m/v) yeast extract, 0.5 % (m/v) tryptone, 0.5 % (m/v) glucose, 0.6 % (m/v) agar, 2 mM MgSO4, with pH adjusted to 7.0) supplemented with 24.30 μM rifampicin and 246.75 μM spectinomycin and grown at 28 °C for 2 days. The Agrobacterium was then grown in liquid YEB
A
medium containing the same antibiotics until the OD600 reached 0.5 to 1. 2.5. Co-cultivation The green calli were dissected into small pieces (2 to 3 mm diameter) and dipped in Agrobacterium suspension. Calli were shaken for 20 min at 80 to 100 rpm and subjected to vacuum infiltration for
10 min under 0.8 kg/cm2. To investigate the effect of acetosyringone (AS) on 6
transformation efficiency, the above calli were transferred to co-cultivation media at pH 5.2 (0.5 x MS medium, 4.58 mM
4-Morpholine Ethane Sulfonic Acid (MES), 22.62 µM 2,4-D and 8.88 µM 6-
BA) with different concentrations of AS (50, 100 and 200 µM). To evaluate the effect of pH, cocultivation media (0.5 x MS medium, 4.58 mM MES, 22.62 µM 2,4-D, 8.88 µM 6-BA and 100 µM AS) at different pH levels (4.8, 5.2 and 5.6) were prepared.
IP T
Co-cultivation was conducted in Petri dishes (9 cm diameter) containing sterilized filter papers (6 to 8 layers) pre-soaked in 6 to 8 mL liquid co-cultivation medium. Infected calli were incubated for
5
days in the dark at 25±1 °C. Each plate contained 8 calli (2 to 3 mm diameter) and experiments were
SC R
carried out in triplicate. 2.6. Regeneration and selection:
U
Co-cultivated calli were transferred to regeneration medium (0.5 x MS medium supplemented with 1 % (m/v) sucrose, 0.6 % (m/v) agar, 9.99 mM (NH4) 2SO4, 22.62 µM 2,4-D, 8.88 µM 6-BA and
N
0.63 mM cefotaxime, pH 5.8) for 4 days. Calli were then transferred to selection medium (0.5 x MS
A
medium supplemented with 1% (m/v) sucrose, 0.6 % (m/v) agar, 9.99 mM (NH4) 2SO4, 22.62 µM
M
2,4-D, 8.88 µM 6-BA, 0.63 mM cefotaxime, 96.25 µM geneticin (G418), pH 5.8). This selection process lasted for at least one month. The calli that survived this treatment were transferred weekly to
ED
fresh medium. Both regeneration and selection were conducted under 25±1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod.
PT
2.7. Shoot induction
CC E
The calli that survived the selection process were then transferred to shoot induction medium (0.5 x MS medium supplemented with 1 % (m/v) sucrose, 0.6 % (m/v) agar, 4.54 µM TDZ, 0.32 mM cefotaxime, 96.25 µM G418, pH 5.8) and subcultured on fresh medium weekly. G418 was removed after first 2 weeks cultivation. Regenerated fronds belonging to different transformed lines were
A
isolated and cultured separately in solid or liquid 0.5 x MS medium (pH 5.8) containing 1 % (m/v) sucrose under 25±1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. 2.8. PCR analysis Total genomic DNA was extracted from wild-type fronds (to provide a control) and putative transformants using a plant DNA isolation kit (Takara). These genomic DNA extractions were used as 7
a template for PCR analysis to test for the presence of TCS or DR5 element using the specified primers (Table 1). PCR amplification was performed in a thermal cycler (Eppendorf Mastercycler nexus) using the following conditions: initial denaturation at 94 °C for 5 min, then 35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for
5
min. The amplified PCR products were separated by gel electrophoresis using a 1.2 % (m/v) agarose
IP T
gel and imaged using a transilluminator. 2.9. GUS assay
SC R
The histological staining of GUS was performed using the protocol described by Jefferson et al. (1987). Fronds were vacuum-infiltrated in staining solution (100 mM KPO4 buffer (pH 7.0), 5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6]) and 0.96
U
mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt (X-Gluc) for 1 h then incubated overnight in the dark at 37 °C. The following day, fronds were washed with 100 mM phosphate buffer
15 min at 57 °C and another 15 min in a solution of 7 % (m/v) NaOH and 10 % (v/v)
A
methanol for
N
(PB) solution and deionized water. Fronds were washed in a solution of 0.24 N HCl and 20 % (v/v)
M
ethanol. Fronds were rinsed with 40 % (v/v) ethanol then stored in a solution of 5 % (v/v) ethanol and
microscope (Germany). Results
PT
3.
ED
25 % (v/v) glycerin. GUS activity in the fronds of S. polyrhiza was imaged using a Leica Z16
In order to develop an efficient procedure for stable transformation of S. polyrhiza we optimized
CC E
each step of the process, including callus induction, co-cultivation with Agrobacterium, regeneration and selection. To uncover the optimal conditions for callus induction, we first altered the concentrations of either auxin (2,4-D) or cytokinin (6-BA) in the induction media. These experiments
A
revealed that increasing the concentration of 6-BA from 2.22 to 8.88 µM promoted callus induction efficiency
(Fig. 2A), while increasing the concentration further (13.32 µM) resulted in a decrease in
efficiency. Within the optimal concentration of 6-BA (8.88 µM) induction efficiency could be enhanced by altering 2,4-D concentration, with a concentration of 22.62 µM providing the highest efficiency. Increasing the concentration of 2,4-D above 22.62 µM, generally reduced the efficiency of
8
callus induction. In summary, the highest efficiency of callus induction was achieved with 22.62 µM 2,4-D and 8.88 µM 6-BA, with above 90 % of fronds forming calli. We also investigated the effect of changing the auxin to NAA and the cytokinin to TDZ on callus induction of S. polyrhiza (Fig. 2B). For this combination of hormones, the highest callus induction efficiency obtained was with 13.42 µM NAA and 4.54 µM TDZ. However, even with these
IP T
concentrations the efficiency was only 63 %. Increasing the concentrations of hormones to 26.85 µM NAA and 9.08 µM TDZ resulted in a decrease in efficiency to 43 %. With higher concentrations of
NAA (40.28 and 53.70 µM) hardly any callus was formed. In these experiments callus only formed
process of callus formation of S. polyrhiza was shown in Fig. 3.
SC R
when the concentration of auxin was higher or equal to the concentration of cytokinin. The complete
U
We also investigated the effect of carbon source on callus induction of S. polyrhiza. We
substituted sucrose with either maltose, glucose or sorbitol in induction media (Fig. 2C). For these
N
experiments auxin and cytokinin were used at the following concentrations: 22.62 µM 2,4-D and 8.88
A
µM 6-BA. 1% sucrose proved to be the most efficient carbon source with the efficiency of around 80
M
% which was more than double that of the other carbon sources tested. Increasing the concentration of sucrose to 2 % resulted in a lower efficiency (33 %).We finally looked at the effect of altering the
ED
hormonal composition of the regeneration media on frond regeneration (Fig. 2D). We found the highest efficiency of frond regeneration (96 %) was achieved with 4.54 µM TDZ. 6-BA also proved to be
PT
highly efficient with 4.44 µM giving an regeneration efficiency of 83 %.
CC E
To development an efficient transformation system of S. polyrhiza, we first assayed the effect of altering the concentrations of AS on the efficiency of transformation (Fig 4A). We found the highest transformation efficiency with the treatment of 100 µM AS, with 13±1 % for TCS::GUS and 13±4 % for DR5::GUS. We next investigated the effect of altering pH of the co-cultivation media on the
A
transformation efficiency (Fig. 4B). Co-cultivation media with a pH of 5.2 showed the highest transformation efficiency, with 11±4 % for TCS::GUS and 1.2±4 % for DR5::GUS. As an independent method of confirming the transformation, we performed PCR analysis on three randomly selected transgenic lines for both TCS::GUS and DR5::GUS (Fig. 5). We also used GUS assays to confirm that the transgene had been incorporated within the S. polyrhiza genome. Following 9
GUS staining, the blue areas report the domain in which the uidA gene is active (Fig. 6). The TCS::GUS line showed high GUS accumulation at the initial stage of bud formation and the frond apex (Fig. 6A 1-3). As development progressed levels of GUS activity decreased gradually and become more restricted to the frond apex (Fig. 6A 4-7). As the frond became senescent, the GUS activity became increasingly diminished (Fig. 6A 8). DR5::GUS activity was only observed at the tip of
IP T
incipent buds (Fig. 6B 1). As the frond developed, the GUS signal gradually spread over the entire frond (Fig. 6B 2-4). At the later stages of growth, the DR5 signal was much reduced and finally was at a level below our threshold for detection (Fig. 6B 5-8). Discussion
SC R
4.
Previous studies have performed callus induction on species within the Lemnoideae and reported
U
that both the selection and concentration of hormones and carbon sources can have a significant effect on efficiency (Stefaniak et al., 2002; Khvatkov et al., 2014; Huang et al., 2016). These studies used the
N
same range of hormones and carbon sources as in this study (Table 2). In this study we discovered that
A
the optimal balance between auxin and cytokinin for callus induction of S. polyrhiza was a slightly
M
higher concentration of auxin. This result is consistent with the callus induction of other Lemnoideae species including Lemna minor, Lemna gibba, Spirodela punctate, and Landoltia punctata. However,
ED
the optimal hormone balance in this study differed from that in other studies, for example, on the callus induction of L. gibba, L. minor, Spirodela oligorrhiza and Wolffia arrhiza.
PT
In addition to using the correct balance of hormones, the selection and concentration of carbon sources also have a significant effect on callus induction, with different carbon sources having different
CC E
effectiveness in duckweeds (Table 2). Li et al. (2004) reported differences in the optimal carbon source composition for callus induction of S. oligorrhiza (2 % sorbitol, 1 % maltose) and S. punctata (1 % sorbitol). Huang et al. (2016) also reported that the optimal carbon source composition for inducing
A
callus in Landoltia punctata was 1 % sorbitol. The optimal carbon source for inducing W. arrhiza callus was a combination of 0.7 % sorbitol, 0.7 % mannitol and 2.6 % glucose. However, for this specie 2 % sucrose proved more effective for maintaining the long term growth of calli. Treatment with sucrose has a positive effect on both callus induction and growth in L. gibba (1 % and 3 % sucrose) and L. minor (1 %, 1.5 % and 3 % sucrose). In the current study we report that out of maltose, glucose and sorbitol, 1 % sucrose was the most efficient for callus induction of S. polyrhiza. 10
Acetosyringone (AS) has previously been shown to help in the transformation of several plant species including, Arabidopsis (Sheikholeslam and Weeks, 1987), cotton (Afolabi-Balogun et al., 2014), rice (Sahoo et al., 2011) and wheat (Raja et al., 2010). Researchers have also found that adding AS to the medium with a slightly acidic pH was effective in enhancing T-DNA transfer in duckweed species including S. oligorrhiza (Vunsh et al., 2007) and L. minor (Chhabra et al., 2011). Our study
IP T
reported a similar effect with the addition of 100 µM AS in co-cultivation medium at pH 5.2. These conditions were almost the same as those found to be optimal for the transformation of S. oligorrhiza
(Vunsh et al., 2007), L. gibba (Yamamoto et al., 2001) and L. minor (Yamamoto et al., 2001; Chhabra
SC R
et al., 2011; Cantópastor et al., 2014). However, in these studies a pH of 5.6 proved to be optimal for transformation.
Spirodela polyrhiza is the most ancestral duckweed species (Wang et al., 2014). It represents a
U
highly modified structural organization that resulted from the alteration, simplification, or loss of many
N
morphological and anatomical features (Landolt, 1986). Flowering of S. polyrhiza is extremely rare
A
under both natural and laboratory conditions and has been almost replaced by rapid asexual
M
reproduction (Lacor et al., 1968). Wang et al. (2014) proposed that the predominant vegetative reproduction and low flowering frequency as well as the reduced and simple plant body of S. polyrhiza
ED
may due to the re-engineering of the genetic network that controls transitions to the adult and flowering growth phases. Michael et al. (2017) suggested S. polyrhiza streamlines its genome by having fewer
PT
genes to maintain and differentially regulate whilst a few specific gene families involved in ammonium assimilation and light harvesting are maintained or are even amplified. Both auxin and cytokinin
CC E
perform critical role in plant morphogenesis therefore our transformed lines present an excellent opportunity to explore their effect on the development of S. polyrhiza from juvenile to adult. As S. polyrhiza provides us with a unique and fascinating biology, the stable transformation will serve us for
A
future developmental and evolutionary studies among duckweeds. In addition to basic researches, applications of duckweeds including bioreactor, biomonitoring, water remediation and biofuel could also be further improved and optimized. 5.
Conclusion This is the first report of high efficient callus induction and stable transformation in S. polyrhiza.
In our experiments we found that the optimal conditions for induction of S. polyrhiza callus were solid 11
0.5 x MS medium containing 1 % (m/v) sucrose, 22.62 µM 2,4-D and 8.88 µM 6-BA at pH 5.8. We performed stable transformation of this S. polyrhiza callus using Agrobacterium tumefaciens strain LBA4404. As proof of concept we transformed S. polyrhiza with two constructs driving GUS expression under synthetic promoters that have previously been used to show cytokinin and auxin output. The efficiency of this process was around 13 %, which was in line with results from other
IP T
duckweed species. Conflict of Interest
SC R
The authors declare that they have no conflict of interest. Acknowledgments
This work was supported by the State Key Laboratory of Freshwater Ecology and Biotechnology
U
(Grant number 2016FB04) and project of Natural Science Foundation of Hubei Province (Grant
N
number 2015CFB488).
A
Reference
M
Alabadí, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P., Kay, S.A., 2001. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293, 880-883.
ED
Afolabi-Balogun, N.B., Inuwa, H.M., Ishiyaku, M.F., Bakare-Odunola, M.T., Nok, A.J., Adebola, P.A., 2014. Effect of acetosyringone on Agrobacterium-mediated transformation of cotton. J. Agr. Biol. Sci. 9, 284-286.
PT
Appenroth, K.J., Augsten, H., Liebermann, B., Feist, H., 1982. Effects of light quality on amino acid composition of proteins in Wolffia arrhiza (L.) Wimm. using a specially modified bradford method. Biochem. Physiol. Pflanz. 177, 251–258. Bai, F., Demason, D.A., 2008. Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS
CC E
in Arabidopsis thaliana. Am. J. Bot. 95, 133-145.
Barton, K.A., Brill, W.J., 1983. Prospects in plant genetic engineering. Science 219, 671-682. Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G., Friml, J., 2003. Local, Efflux-dependent
A
auxin gradients as a common module for plant organ formation. Cell 115, 591-602.
Bergmann, B.A., Cheng, J., Classen, J., Stomp, A.M., 2000. Nutrient removal from swine lagoon effluent by duckweed. Trans. A.S.A.E. 43, 263-269. Bulgakov, V.P., Kusaykin, M., Tchernoded, G.K., Zvyagintseva, T.N., Zhuravlev, Y.N., 2002. Carbohydrase activities of the rolc-gene transformed and non-transformed ginseng cultures. Fitoterapia. 73, 638-643.
12
Canellas, L.P., Dantas, D.J., Aguiar, N.O., Peres, L.E. P., Zsögön, A., Olivares, F.L., Dobbss, L.B., Facanha, A.R., Nebbioso, A., Piccolo, A., 2011. Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants. Ann. Appl. Biol. 159, 202–211. Cantópastor, A., Mollámorales, A., Ernst, E., Dahl, W., Zhai, J., Yan, Y., Meyers, B.C. Shanklin J.R. Martienssen R., 2014. Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant Biol. 17, 59-65. Cao, H.X., Vu, G.T., Wang, W., Appenroth, K. J., Messing, J., Schubert, I., 2016. The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution. New Phytol. 209, 354-363.
IP T
Chang, W. C., Chiu, P. L., 1978. Regeneration of Lemna gibba, G3 through callus culture. Z. Pjlanzenphysiol. Bd. 89, 91–94.
Chhabra, G., Chaudhary, D., Sainger, M., Jaiwal, P.K., 2011. Genetic transformation of Indian isolate of Lemna minor mediated
SC R
by Agrobacterium tumefaciens, and recovery of transgenic plants. Physiol. Mol. Biol. Plants 17, 129-136. Comai, L., 1993. Impact of plant genetic engineering on foods and nutrition. Annu. Rev. Nutr. 13, 191-215.
Dietrich, R.A., Richberg, M.H., Schmidt, R., Dean, C., Dangl, J.L., 1997. A novel zinc finger protein is encoded by the
U
Arabidopsis LSD1, gene and functions as a negative regulator of plant cell death. Cell. 88, 685-694.
Edelman, M., Perl, A., Flaishman, M., Blumenthal, A., 1988. Transgenic Lemnaceae. Australian Patent Publication AU759570C.
N
Gasdaska, J.R., Spencer, D., Dickey, L., 2003. Advantages of therapeutic protein production in the aquatic plant Lemna. Bio.
A
Process J. 2, 49-56.
M
Heenatigala P.P.M., Yang J.J., Bioshopp A., Sun Z., Li G., Kumar S., Hu S., Wu Z., Lin W., Yao L., Duan P., Hou H., 2018. Development of efficient protocols for stable and transient gene transformation for Wolffia globosa using Agrobacterium.
ED
Frontiers in Chemistry. 6, 227.
Hegazy, A.K., Kabiel, H.F., Fawzy, M., 2009. Duckweed as heavy metal accumulator and pollution indicator in industrial wastewater ponds. Desalin. Water Treat. 12, 400-406.
PT
Huang, M., Fu, L.L., Sun, X.P., Rong, D., Zhang, J.M., 2016. Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata. Acta. Physiol. Plant. 38, 122.
CC E
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W., 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.
Jong, T.D., Veldstra H., 1971. Investigations on cytokinins, I. Effect of 6-benzylaminopurine on growth and starch content of Lemna minor. Physiol. Plantarum 24, 235-238.
A
Khvatkov, P., Chernobrovkina, M., Okuneva, A., Pushin, A., Dolgov, S., 2015. Transformation of Wolffia arrhizal (L.) Horkel ex Wimm. Plant Cell Tiss. Org. 123, 299-307.
Khvatkov, P., Chernobrovkina, M., Okuneva, A., Shvedova, A., Chaban, I., Sergey, D., 2014. Callus induction and regeneration in Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss. Org. 120, 263-273. Kristanti, R.A., Kanbe, M., Hadibarata, T., Toyama, T., Tanaka, T., Mori, K., 2012. Isolation and characterization of 3nitrophenol-degrading bacteria associated with rhizosphere of Spirodela polyrrhiza. Environ. Sci. Pollut. Res. Int. 19, 18521858.
13
Lacor, M.A.M., 1968. Flowering of Spirodela polyrhiza (L.) Schleiden. Plant Biology, 17, 357-359. Landolt E., 1986. The family of Lemnaceae–a monographic study. Veröff. Geobot. Inst. ETH. StiftungRübel (Zürich) 71, 1–566. Lemon, G.D., Posluszny, U., Husband, B.C., 2001. Potential and realized rates of vegetative reproduction in Spirodela polyrhiza, Lemna minor, and Wolffia borealis. Aquat. Bot. 70, 79-87. Li, J., Jain, M., Vunsh, R., Vishnevetsky, J., Hanania, U. Flaishman, M., Perl, A., Edelman, M., 2004. Callus induction and regeneration in Spirodela and Lemna. Plant Cell Rep. 22, 457-464.
IP T
Ma, J.K., Barros, E., Bock, R., Christou, P., Dale, P.J., Dix, P.J., Fischer, R., Irwin, J., Mahoney, R., Pezzotti, M. Schillberg, S., Sparrow, P., Stoger, E., Twyman, R.M., 2005. Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO Rep. 6, 593-599.
SC R
Michael, T.P., Bryant, D., Gutierrez, R., Borisjuk, N., Chu, P., Zhang, H., Xia, J., Zhou, J., Peng, H., Baidouri M.E., et al., 2017. Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies. Plant J. 89, 617-635.
Moon, H.K., Stomp, A.M., 1997. Effects of medium components and light on callus induction, growth, and frond regeneration in
U
Lemna gibba (duckweed). In Vitro Cell. Dev – Pl. 33, 20-25.
N
Müller, B., Sheen, J., 2008. Cytokinin and auxin interplay in root stem-cell specification during early embryogenesis. Nature
A
453, 1094-1097.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.
M
15, 473-497.
Porath, D., Hepher, B., Koton, A., 1979. Duckweed as an aquatic crop: evaluation of clones for aquaculture. Aquat. Bot. 7, 273–
ED
278.
Raja, N.I., Bano, A., Rashid, H., Chaudhry, Z., Ilyas, N., 2010. Improving agrobacterium mediated transformation protocol for
PT
integration of XA21 gene in wheat (Triticum aestivum L.). Pak. J. Bot. 42, 855-865. Reid, M.S., Bieleski, R.L., 1970. Response of Spirodela oligorrhiza to phosphorus deficiency. Plant Physiol. 46, 609-613.
CC E
Reinhold, D.M., Saunders, F.M., 2006. Phytoremediation of fluorinated agrochemicals by duckweed. Trans. A.SA.E 49, 20772083.
Rival, S., Wisniewski, J.P., Langlais, A., Kaplan, H., Freyssinet, G., Vancanneyt, G., Vunsh, R., Perl, A., Edelman, M., 2008. Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res. 17,
A
503-513.
Sahoo, K.K., Tripathi, A.K., Pareek, A., Sopory, S.K., Singla-Pareek, S. L., 2011. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7, 49. Scofield, S., Dewitte, W., Murray, J. A., 2014. STM sustains stem cell function in the Arabidopsis shoot apical meristem and controls KNOX gene expression independently of the transcriptional repressor AS1. Plant Signal Behav. 28, 9. Sheikholeslam, S.N., Weeks, D. P., 1987. Acetosyringone promotes high frequency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol. Biol. 8, 291-298.
14
Stefaniak, B., Woźny, A., Budna, I., 2005. Callus induction and plant regeneration in Lemna Minor L.. Biol. Plant. 45, 469-472. Stomp, A.M., Rajbhandari, N., 2000. Genetically engineered duckweed: US, US6040498. Tao, J., Sun, H., Gu, P., Liang, Z., Chen, X., Lou, J., Xu, G., Zhang, Y., 2017. A sensitive synthetic reporter for visualizing cytokinin signaling output in rice. Plant Methods 13, 89. Vu, H.T., Ondar, U.N., Soldatova, O.P., 2008. Expression of new mutant alleles of AS1, and AS2, genes controlling leaf morphogenesis in Arabidopsis thaliana. Russ J Dev Biol. 39, 6-12.
IP T
Vunsh, R., Li, J., Hanania, U., Edelman, M., Flaishman, M., Perl, A., Wisniewski, J.P., Freyssinet, G., 2007. High expression of transgene protein in Spirodela. Plant Cell Rep. 26, 1511-1519.
Wang, W., Kerstetter, R.A., Michael, T.P., 2011. Evolution of genome size in Duckweeds (Lemnaceae). J. Bot. 570319.
SC R
Wang, W., Haberer, G., Gundlach, H., Gläßer, C., Nussbaumer, T., Luo, M. C., Lomsadze, A. Borodovsky, M. Kerstetter, R.A., Shanklin, J., Byrant, D.W., Mockler, T.C., Appenroth, K.J., Grimwood, J., Jenkins, J., Chow, J., Choi, C., Adam, C., Cao, X. H., Fuchs, J., Schubert, I., Rokhsar, D., Schmutz, J., Michael, T.P., Maye K.F.X., Messing J., 2014. The Spirodela
U
polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat. Commun. 5, 3311. Yamaga, F., Washio, K., Morikawa, M., 2010. Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated
N
from the rhizosphere of duckweed Lemna aoukikusa. Environ. Sci. Technol. 44, 6470-6474.
A
Yamamoto, Y. T., Rajbhandari, N., Lin, X., Bergmann, B.A., Nishimura, Y., Stomp, A.M., 2001. Genetic transformation of
M
duckweed Lemna gibba, and Lemna minor. In Vitro Cell. Dev. Biol. - Plant 37, 349-353. Yang, L., Han, H., Zuo, Z., Zhou, K., Ren, C., Zhu, Y., Bai, Y., Wang, Y., 2014. Enhanced plant regeneration in Lemna minor by
A
CC E
PT
ED
amino acids. Pak. J. Bot. 46, 939-943.
15
Primer
Sequences (5’-3’)
TCS-F
GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCTTTGCTAGCAAAATCTACA
TCS-R
GGGGACCACTTTGTACAAAAAGCTGGGTTGTTATATCTCCTTGGATCGAT
DR5-F
GGGGACAAGTTTGTACAAAAAAGCAGGCTGAATTCGTCGACGGTATCGCAG
DR5-R
GGGGACCACTTTGTACAAGAAAGCTGGGTATCTCCTTGGATCGATCCCCTG
Hormones
Carbon sources
References
Landoltia punctata
2,4-D (67.86 μM), 6-BA (8.87 μM)
1 % sorbitol
Huang et al., 2016
Lemna gibba
2,4-D (45.24 μM) and 2 IP (2.98 μM)
3 % sucrose
Chang and Chiu, 1978
Lemna gibba
20 or 50 μM 2,4-D
3 % sucrose
Lemna gibba
Dicamba (226.24 μM) and 6-BA (8.87 μM)
1 % sucrose
Lemna minor Lemna minor
45 μM 2,4-D 2,4-D (5.0 μM) and 2 IP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) Dicamba (67.87 μM), 2, 4-D (15.83 μM), and 6-BA (4.44 μM) 2,4-D (1 μM) and 6-BA (2 μM) 2,4-D (22.62 μM), 6-BA (8.87 μM) 2,4-D (15.83 μM), dicamba (67.87 μM), and 2 IP (5.96 μM) Picloram (51.77 μM) and 2,4-D (20.62 μM)
3 % sucrose 1 % sucrose
M
A
N
Wolffia arrhiza
A
CC E
PT
ED
Fig. 1. The T-DNA of binary plasmids (pKGWFS7.0) for transformation of S. polyrhiza.
16
Li et al., 2004
SC R
Lemna minor Spirodela polyrhiza Spirodela punctata
Moon and Stomp, 1997
Stefaniak et al., 2002 Chhabra et al., 2011
1.5 % sucrose
Yang et al., 2014
3 % sucrose 1 % sucrose 1 % sorbitol
Canto-Pastor et al., 2014 Li et al., 2004
0.7 % sorbitol, 0.7 % mannitol and 2.6 % glucose
Khvatkov et al., 2014
U
Lemna minor
IP T
Species
IP T SC R U N A
M
Fig. 2. (A) The effect of different concentrations of 2,4-D and 6-BA on callus induction of S. polyrhiza; (B) The effect of different concentrations of NAA and TDZ on callus induction; (C) The effect of different carbon sources on callus induction; (D)
A
CC E
PT
ED
The effect of different concentrations of TDZ and 6-BA on frond regeneration. N = 3. Error bars indicated standard deviation.
Fig. 3. The complete callus formation process of S. polyrhiza. (A-B) Fronds proliferation; (C) Fronds dedifferentiation; (D-F) Callus formation. Bar= 4 mm.
17
IP T SC R
Fig. 4. The effect of different concentrations of AS (A) and pH of co-cultivation medium (B) on transformation efficiency. N = 3.
ED
M
A
N
U
Error bars indicated standard deviation.
Fig. 5. Confirmation of transformed S. polyrhiza by PCR analysis. (M) 2000bp DNA ladder marker; (T1-3) Amplifications of fragment of TCS element in transgenic plants (404 bp); (D1-3) Amplifications of fragment of DR5 element in transgenic plants
A
CC E
PT
(314 bp); (P1) Plasmid containing TCS ; (P2) Plasmid containing DR5 element; (W1-2) Wild plants.
Fig. 6. GUS activity in the frond of S. polyrhiza for TCS::GUS line (A) and DR5::GUS line (B). (A1-3) High GUS accumulation at the initial stage of bud formation and the frond apex; (A4-7) GUS activity decreased gradually and become more restricted to the frond apex; (A8) GUS activity became increasingly diminished; (B1) GUS activity was only observed at the tip of incipent
18
buds; (B2-4) GUS activity gradually spread over the entire frond; (B5-8) GUS activity was much reduced and finally was at a
A
CC E
PT
ED
M
A
N
U
SC R
IP T
level below threshold for detection. Bar=1 mm.
19
S0304-3770(18)30124-4 https://doi.org/10.1016/j.aquabot.2018.08.004 AQBOT 3058
To appear in:
Aquatic Botany
Received date: Revised date: Accepted date:
20-5-2018 23-8-2018 23-8-2018
Please cite this article as: Yang J, Li G, Hu S, Bishopp A, Heenatigala PPM, Kumar S, Duan P, Yao L, Hou H, A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens, Aquatic Botany (2018), https://doi.org/10.1016/j.aquabot.2018.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens. Jingjing Yang1, Gaojie Li1, Shiqi Hu1, Antony Bishopp2, P. P. M. Heenatigala 1, Sunjee Kumar1, Pengfei Duan3, Lunguang Yao3, Hongwei Hou1* 1
The State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China. The Key Laboratory of Aquatic
IP T
Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China. University of Chinese Academy of Sciences, Hubei Province, 430072, China Centre for Plant Integrative Biology, University of Nottingham, Nottingham, UK
3
SC R
2
Collaborative Innovation Center of Water Security for Water Source Region of Mid-line of South-to-
North Diversion Project, College of Agricultural Engineering, Nanyang Normal University, Henan,
U
China
N
*Corresponding Author; Tel: (+8627) 68780159; Fax: (+8627) 68780123; Email: [email protected]
Highlights
•22.62 µM 2,4-D and 8.88 µM 6-BA are optimal for callus induction.
•1 % sucrose is the most efficient carbon source.
•2.27 to 4.54 µM TDZ is highly efficient in frond regeneration.
•The stable transformation of S. polyrhiza is established mediated by Agrobacterium.
•TCS::GUS and DR5::GUS constructs are successfully expressed in Spirodela polyrhiza.
A
CC E
PT
ED
M
A
1
ABSTRACT Spirodela polyrhiza represents the largest specie with the smallest genome of all the members of the Lemnoideae. Its genome features have been delineated, revealing its fewest predicted genes of any known plant genome. It is also ideal system for basic biological researches and various practical applications including toxicity testing, bioreactor, biomonitoring and biofuel. In this study, we reported
IP T
the successful induction of S. polyrhiza callus coupled with the efficient stable transformation using the Agrobacterium tumefaciens strain LBA4404 by optimizing each step of the process. We found that the highest callus induction efficiency was achieved with 22.62 µM 2,4-D and 8.88 µM 6-BA, with above
SC R
90 % of fronds forming calli. We also determined that 100 µM acetosyringone in the co-cultivation
medium and the maintenance of pH value at 5.2 were crucial for high transformation efficiency (up to 13±1.5 %). As proof of concept, we transformed S. polyrhiza with the DR5 and TCS synthetic
U
reporters, which have previously been used to report cytokinin and auxin signaling output in the model
N
plant Arabidopsis thaliana. The cytokinin showed highest accumulation at the initial stage of bud
A
formation and the frond apex of S. polyrhiza whilst the expression of auxin was observed highest at
development of
M
frond with middle size. These transformed lines provide an effective way to investigate the S. polyrhiza and may shed light on the interesting way in which this specie
ED
reproduces. This is the first report of highly efficient callus induction and Agrobacterium tumefaciens-
PT
mediated transformation in S. polyrhiza.
Abbreviations
2,4-Dichlorophenoxyacetic acid
CC E
2,4-D
A
Keywords: Spirodela polyrhiza; callus induction; transformation; Agrobacterium tumefaciens; cytokinin; auxin.6-BA AS
6-benzyladenine
Acetosyringone
G418
Geneticin
GUS
β-Glucuronidase
MES
4-Morpholine Ethane Sulfonic Acid 2
MS NAA
Murashige and Skoog medium α-Naphthaleneacetic acid
PB
Phosphate Buffer
TDZ
1-Phenyl-3-(1,2,3,-thiadiazol-5-yl) urea
X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt Yeast Extract Mannitol Broth
A
CC E
PT
ED
M
A
N
U
SC R
IP T
YEB
3
Introduction Duckweeds (Lemnoideae) are monocotyledonous free-floating aquatic plants distributed in various types of habitats throughout the world (Landolt, 1986). There are five genera within the Lemnoideae family (Spirodela, Landoltia, Lemna, Wolffiella and Wolffia) (Appenroth et al., 2013). All plants have highly reduced morphologies, consisting of a frond or thallus (Landolt, 1986). These plants
IP T
rarely reproduce sexually, and instead propagate rapidly in a vegetative manner through the budding of daughter fronds from meristematic pockets (Lemon et al., 2001).The frond of Spirodela, Landoltia and Lemna species produce one or more roots whilst Wolffiella and Wolffia species are rootless (Cao et al.,
SC R
2016).
There has been increased interest in the use of duckweeds, as the high efficiency through which they accumulate biomass raises the possibility of using them in bioreactors (Gasdaska et al., 2003;).
U
Regulating the culture conditions, such as altering either pH or nutrient concentration can affect the
N
starch and protein content, making them promising sources for biofuel (Reid and Bieleski, 1970; Jong
A
and Veldstra, 1971; Porath et al., 1979). The use of duckweeds in bioremediation and biomonitoring
M
has also been explored, as they can absorb and take up contaminants including trace metal elements, organic chemicals, nitrogen and phosphorus (Bergmann et al., 2000; Reinhold and Saunders, 2006;
ED
Hegazy et al., 2009). In addition, duckweeds can synergistically interact with microbiota to degrade contaminants in water (Yamaga et al., 2010; Kristanti et al., 2012). The beneficial symbiotic interaction
PT
between duckweed and P23 belonging to the genera Acinetobacter also attributed to the continuous removal of phenol (Yamaga et al., 2010). Kristanti et al. (2012) found that the strains belonging to the
CC E
genus Cupriavidus accelerated the removal of 3-nitrophenol (100%) in river water samples with S. polyrhiza.
Spirodela polyrhiza (L.) Schleiden is widely distributed and represents the largest specie (1.5 cm
A
long) with the smallest genome size (158 Mb) among all duckweeds measured (Wang et al., 2011). An initial reference genome draft of S. polyrhiza was first published by Wang et al. (2014). Michael et al. (2017) further resolved the 20 chromosomes of S. polyrhiza. Efficient stable transformation protocols have been established for common species of duckweeds including Spirodela punctata (Edelman et al., 1988), Spirodela oligorrhiza (Vunsh et al., 2007; Rival et al., 2008), Lemna gibba (Stomp and Rajbhandari, 2000; Yamamoto et al., 2001), Lemna minor (Gasdaska et al., 2003), Wolffia arrhiza 4
(Khvatkov et al., 2015) and Wolffia globosa (Heenatigala et al., 2018). However, as yet there is no published protocol for the efficient stable transformation of S. polyrhiza. For most plant species Agrobacterium-mediated transformation provides the method for introducing transgenes (Comai, 1993), and has been used to modify plants to increase yield, nutrition and quality, improve stress tolerance and produce protein products (Barton and Brill, 1983; Ma et al., 2005). It also
IP T
provides a way to investigate the functions of genes related to various life activities including metabolism (Bulgakov et al., 2002), the development of leaf (Vu et al., 2008) and shoot apical meristem (Scofield et
al., 2014), plant cell death (Dietrich et al., 1997) and circadian clock (Alabadí et al., 2001). In the current
SC R
study, we developed a protocol for the Agrobacterium-mediated transformation of S. polyrhiza. As proof of concept we introduced two synthetic reporters -TCS::GUS (Müller and Sheen, 2008) and DR5::GUS (Benková et al., 2003), that have previously been used to report cytokinin and auxin output respectively
U
in Arabidopsis (Bai and Demason 2008), tomato (Canellas et al., 2011) and rice (Tao et al., 2017). Such
N
lines will provide an effective way to investigate the effect of auxin and cytokinin during different
A
developmental stages of S. polyrhiza and may shed light on the interesting way in which this specie
transformation in S. polyrhiza. Materials and methods
PT
2.1. Plant cultivation
ED
1.
M
reproduces. This is the first report of high efficiency Agrobacterium tumefaciens-mediated
Spirodela polyrhiza (5543) was collected from East Lake (N 30° 32’, E 114°21’) in the city of
CC E
Wuhan, Hubei Province, China. After sterilization using 0.1 % (m/v) mercuric chloride (HgCl 2) for 2 to 3 min and washing 5 to 8 times with sterilized water, the plants were cultured in Erlenmeyer flasks containing 50 mL half strength Murashige and Skoog (1962) medium (0.5 x MS) supplemented with 1
A
% (m/v) sucrose at pH 5.8, under 25 ± 1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. 2.2. Callus induction and frond regeneration To investigate the optimal conditions for callus induction, fronds were grown on 0.5 x MS medium containing 1 % (m/v) sucrose, 0.6 % (m/v) agar at pH 5.8. This was supplemented with different combinations of hormones. For cytokinins we used either 6-benzyladenine (6-BA) or 5
Thidiazuron (TDZ); for auxins we used either 1-Naphthaleneacetic acid (NAA) or 2,4Dichlorophenoxyacetic acid (2,4-D). We also investigated the effect of carbon source by testing sucrose, maltose, sorbitol and glucose. Around 20 fronds in good condition (4 to 6 colonies) were cultured in sterilized Petri dishes (9 cm diameter) containing 30 mL solid medium for callus induction. To determine the optimal hormones for frond regeneration, calli were cultured on 0.5 x MS
IP T
medium with added 1 % (m/v) sucrose, 0.6 % (m/v) agar, and either 6-BA or TDZ at pH 5.8. Each plate contained 8 calli (2 to 3 mm diameter). All experiments were conducted at 25 ± 1 °C with an irradiance of
85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. Each treatment was performed
SC R
in triplicate and subcultured weekly onto fresh medium. The efficiency of callus induction on each
plate was calculated after one month cultivation, whereas the frond regeneration rate was counted after two weeks. Callus induction and frond regeneration efficiencies in this study are presented by means
U
±SDs.
A
N
2.3. Agrobacterium strain and vector
The TCS and DR5 elements were synthesized and inserted to the binary vector pKGWFS7.0
M
(http://www.transgen.com.cn/) (Fig. 1) using Gateway technology. Agrobacterium tumefaciens strain
ED
LBA4404 (http://www.transgen.com.cn/) was used as the host strain harboring the binary vector pKGWFS7.0.
PT
2.4. Preparation of Agrobacterium
Agrobacterium harboring the plasmids to be transformed were cultured on solid YEB medium
CC E
(0.6 % (m/v) yeast extract, 0.5 % (m/v) tryptone, 0.5 % (m/v) glucose, 0.6 % (m/v) agar, 2 mM MgSO4, with pH adjusted to 7.0) supplemented with 24.30 μM rifampicin and 246.75 μM spectinomycin and grown at 28 °C for 2 days. The Agrobacterium was then grown in liquid YEB
A
medium containing the same antibiotics until the OD600 reached 0.5 to 1. 2.5. Co-cultivation The green calli were dissected into small pieces (2 to 3 mm diameter) and dipped in Agrobacterium suspension. Calli were shaken for 20 min at 80 to 100 rpm and subjected to vacuum infiltration for
10 min under 0.8 kg/cm2. To investigate the effect of acetosyringone (AS) on 6
transformation efficiency, the above calli were transferred to co-cultivation media at pH 5.2 (0.5 x MS medium, 4.58 mM
4-Morpholine Ethane Sulfonic Acid (MES), 22.62 µM 2,4-D and 8.88 µM 6-
BA) with different concentrations of AS (50, 100 and 200 µM). To evaluate the effect of pH, cocultivation media (0.5 x MS medium, 4.58 mM MES, 22.62 µM 2,4-D, 8.88 µM 6-BA and 100 µM AS) at different pH levels (4.8, 5.2 and 5.6) were prepared.
IP T
Co-cultivation was conducted in Petri dishes (9 cm diameter) containing sterilized filter papers (6 to 8 layers) pre-soaked in 6 to 8 mL liquid co-cultivation medium. Infected calli were incubated for
5
days in the dark at 25±1 °C. Each plate contained 8 calli (2 to 3 mm diameter) and experiments were
SC R
carried out in triplicate. 2.6. Regeneration and selection:
U
Co-cultivated calli were transferred to regeneration medium (0.5 x MS medium supplemented with 1 % (m/v) sucrose, 0.6 % (m/v) agar, 9.99 mM (NH4) 2SO4, 22.62 µM 2,4-D, 8.88 µM 6-BA and
N
0.63 mM cefotaxime, pH 5.8) for 4 days. Calli were then transferred to selection medium (0.5 x MS
A
medium supplemented with 1% (m/v) sucrose, 0.6 % (m/v) agar, 9.99 mM (NH4) 2SO4, 22.62 µM
M
2,4-D, 8.88 µM 6-BA, 0.63 mM cefotaxime, 96.25 µM geneticin (G418), pH 5.8). This selection process lasted for at least one month. The calli that survived this treatment were transferred weekly to
ED
fresh medium. Both regeneration and selection were conducted under 25±1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod.
PT
2.7. Shoot induction
CC E
The calli that survived the selection process were then transferred to shoot induction medium (0.5 x MS medium supplemented with 1 % (m/v) sucrose, 0.6 % (m/v) agar, 4.54 µM TDZ, 0.32 mM cefotaxime, 96.25 µM G418, pH 5.8) and subcultured on fresh medium weekly. G418 was removed after first 2 weeks cultivation. Regenerated fronds belonging to different transformed lines were
A
isolated and cultured separately in solid or liquid 0.5 x MS medium (pH 5.8) containing 1 % (m/v) sucrose under 25±1 °C, an irradiance of 85 μmol photons PAR m-2 s-1, and a 16 h photoperiod. 2.8. PCR analysis Total genomic DNA was extracted from wild-type fronds (to provide a control) and putative transformants using a plant DNA isolation kit (Takara). These genomic DNA extractions were used as 7
a template for PCR analysis to test for the presence of TCS or DR5 element using the specified primers (Table 1). PCR amplification was performed in a thermal cycler (Eppendorf Mastercycler nexus) using the following conditions: initial denaturation at 94 °C for 5 min, then 35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for
5
min. The amplified PCR products were separated by gel electrophoresis using a 1.2 % (m/v) agarose
IP T
gel and imaged using a transilluminator. 2.9. GUS assay
SC R
The histological staining of GUS was performed using the protocol described by Jefferson et al. (1987). Fronds were vacuum-infiltrated in staining solution (100 mM KPO4 buffer (pH 7.0), 5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6]) and 0.96
U
mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt (X-Gluc) for 1 h then incubated overnight in the dark at 37 °C. The following day, fronds were washed with 100 mM phosphate buffer
15 min at 57 °C and another 15 min in a solution of 7 % (m/v) NaOH and 10 % (v/v)
A
methanol for
N
(PB) solution and deionized water. Fronds were washed in a solution of 0.24 N HCl and 20 % (v/v)
M
ethanol. Fronds were rinsed with 40 % (v/v) ethanol then stored in a solution of 5 % (v/v) ethanol and
microscope (Germany). Results
PT
3.
ED
25 % (v/v) glycerin. GUS activity in the fronds of S. polyrhiza was imaged using a Leica Z16
In order to develop an efficient procedure for stable transformation of S. polyrhiza we optimized
CC E
each step of the process, including callus induction, co-cultivation with Agrobacterium, regeneration and selection. To uncover the optimal conditions for callus induction, we first altered the concentrations of either auxin (2,4-D) or cytokinin (6-BA) in the induction media. These experiments
A
revealed that increasing the concentration of 6-BA from 2.22 to 8.88 µM promoted callus induction efficiency
(Fig. 2A), while increasing the concentration further (13.32 µM) resulted in a decrease in
efficiency. Within the optimal concentration of 6-BA (8.88 µM) induction efficiency could be enhanced by altering 2,4-D concentration, with a concentration of 22.62 µM providing the highest efficiency. Increasing the concentration of 2,4-D above 22.62 µM, generally reduced the efficiency of
8
callus induction. In summary, the highest efficiency of callus induction was achieved with 22.62 µM 2,4-D and 8.88 µM 6-BA, with above 90 % of fronds forming calli. We also investigated the effect of changing the auxin to NAA and the cytokinin to TDZ on callus induction of S. polyrhiza (Fig. 2B). For this combination of hormones, the highest callus induction efficiency obtained was with 13.42 µM NAA and 4.54 µM TDZ. However, even with these
IP T
concentrations the efficiency was only 63 %. Increasing the concentrations of hormones to 26.85 µM NAA and 9.08 µM TDZ resulted in a decrease in efficiency to 43 %. With higher concentrations of
NAA (40.28 and 53.70 µM) hardly any callus was formed. In these experiments callus only formed
process of callus formation of S. polyrhiza was shown in Fig. 3.
SC R
when the concentration of auxin was higher or equal to the concentration of cytokinin. The complete
U
We also investigated the effect of carbon source on callus induction of S. polyrhiza. We
substituted sucrose with either maltose, glucose or sorbitol in induction media (Fig. 2C). For these
N
experiments auxin and cytokinin were used at the following concentrations: 22.62 µM 2,4-D and 8.88
A
µM 6-BA. 1% sucrose proved to be the most efficient carbon source with the efficiency of around 80
M
% which was more than double that of the other carbon sources tested. Increasing the concentration of sucrose to 2 % resulted in a lower efficiency (33 %).We finally looked at the effect of altering the
ED
hormonal composition of the regeneration media on frond regeneration (Fig. 2D). We found the highest efficiency of frond regeneration (96 %) was achieved with 4.54 µM TDZ. 6-BA also proved to be
PT
highly efficient with 4.44 µM giving an regeneration efficiency of 83 %.
CC E
To development an efficient transformation system of S. polyrhiza, we first assayed the effect of altering the concentrations of AS on the efficiency of transformation (Fig 4A). We found the highest transformation efficiency with the treatment of 100 µM AS, with 13±1 % for TCS::GUS and 13±4 % for DR5::GUS. We next investigated the effect of altering pH of the co-cultivation media on the
A
transformation efficiency (Fig. 4B). Co-cultivation media with a pH of 5.2 showed the highest transformation efficiency, with 11±4 % for TCS::GUS and 1.2±4 % for DR5::GUS. As an independent method of confirming the transformation, we performed PCR analysis on three randomly selected transgenic lines for both TCS::GUS and DR5::GUS (Fig. 5). We also used GUS assays to confirm that the transgene had been incorporated within the S. polyrhiza genome. Following 9
GUS staining, the blue areas report the domain in which the uidA gene is active (Fig. 6). The TCS::GUS line showed high GUS accumulation at the initial stage of bud formation and the frond apex (Fig. 6A 1-3). As development progressed levels of GUS activity decreased gradually and become more restricted to the frond apex (Fig. 6A 4-7). As the frond became senescent, the GUS activity became increasingly diminished (Fig. 6A 8). DR5::GUS activity was only observed at the tip of
IP T
incipent buds (Fig. 6B 1). As the frond developed, the GUS signal gradually spread over the entire frond (Fig. 6B 2-4). At the later stages of growth, the DR5 signal was much reduced and finally was at a level below our threshold for detection (Fig. 6B 5-8). Discussion
SC R
4.
Previous studies have performed callus induction on species within the Lemnoideae and reported
U
that both the selection and concentration of hormones and carbon sources can have a significant effect on efficiency (Stefaniak et al., 2002; Khvatkov et al., 2014; Huang et al., 2016). These studies used the
N
same range of hormones and carbon sources as in this study (Table 2). In this study we discovered that
A
the optimal balance between auxin and cytokinin for callus induction of S. polyrhiza was a slightly
M
higher concentration of auxin. This result is consistent with the callus induction of other Lemnoideae species including Lemna minor, Lemna gibba, Spirodela punctate, and Landoltia punctata. However,
ED
the optimal hormone balance in this study differed from that in other studies, for example, on the callus induction of L. gibba, L. minor, Spirodela oligorrhiza and Wolffia arrhiza.
PT
In addition to using the correct balance of hormones, the selection and concentration of carbon sources also have a significant effect on callus induction, with different carbon sources having different
CC E
effectiveness in duckweeds (Table 2). Li et al. (2004) reported differences in the optimal carbon source composition for callus induction of S. oligorrhiza (2 % sorbitol, 1 % maltose) and S. punctata (1 % sorbitol). Huang et al. (2016) also reported that the optimal carbon source composition for inducing
A
callus in Landoltia punctata was 1 % sorbitol. The optimal carbon source for inducing W. arrhiza callus was a combination of 0.7 % sorbitol, 0.7 % mannitol and 2.6 % glucose. However, for this specie 2 % sucrose proved more effective for maintaining the long term growth of calli. Treatment with sucrose has a positive effect on both callus induction and growth in L. gibba (1 % and 3 % sucrose) and L. minor (1 %, 1.5 % and 3 % sucrose). In the current study we report that out of maltose, glucose and sorbitol, 1 % sucrose was the most efficient for callus induction of S. polyrhiza. 10
Acetosyringone (AS) has previously been shown to help in the transformation of several plant species including, Arabidopsis (Sheikholeslam and Weeks, 1987), cotton (Afolabi-Balogun et al., 2014), rice (Sahoo et al., 2011) and wheat (Raja et al., 2010). Researchers have also found that adding AS to the medium with a slightly acidic pH was effective in enhancing T-DNA transfer in duckweed species including S. oligorrhiza (Vunsh et al., 2007) and L. minor (Chhabra et al., 2011). Our study
IP T
reported a similar effect with the addition of 100 µM AS in co-cultivation medium at pH 5.2. These conditions were almost the same as those found to be optimal for the transformation of S. oligorrhiza
(Vunsh et al., 2007), L. gibba (Yamamoto et al., 2001) and L. minor (Yamamoto et al., 2001; Chhabra
SC R
et al., 2011; Cantópastor et al., 2014). However, in these studies a pH of 5.6 proved to be optimal for transformation.
Spirodela polyrhiza is the most ancestral duckweed species (Wang et al., 2014). It represents a
U
highly modified structural organization that resulted from the alteration, simplification, or loss of many
N
morphological and anatomical features (Landolt, 1986). Flowering of S. polyrhiza is extremely rare
A
under both natural and laboratory conditions and has been almost replaced by rapid asexual
M
reproduction (Lacor et al., 1968). Wang et al. (2014) proposed that the predominant vegetative reproduction and low flowering frequency as well as the reduced and simple plant body of S. polyrhiza
ED
may due to the re-engineering of the genetic network that controls transitions to the adult and flowering growth phases. Michael et al. (2017) suggested S. polyrhiza streamlines its genome by having fewer
PT
genes to maintain and differentially regulate whilst a few specific gene families involved in ammonium assimilation and light harvesting are maintained or are even amplified. Both auxin and cytokinin
CC E
perform critical role in plant morphogenesis therefore our transformed lines present an excellent opportunity to explore their effect on the development of S. polyrhiza from juvenile to adult. As S. polyrhiza provides us with a unique and fascinating biology, the stable transformation will serve us for
A
future developmental and evolutionary studies among duckweeds. In addition to basic researches, applications of duckweeds including bioreactor, biomonitoring, water remediation and biofuel could also be further improved and optimized. 5.
Conclusion This is the first report of high efficient callus induction and stable transformation in S. polyrhiza.
In our experiments we found that the optimal conditions for induction of S. polyrhiza callus were solid 11
0.5 x MS medium containing 1 % (m/v) sucrose, 22.62 µM 2,4-D and 8.88 µM 6-BA at pH 5.8. We performed stable transformation of this S. polyrhiza callus using Agrobacterium tumefaciens strain LBA4404. As proof of concept we transformed S. polyrhiza with two constructs driving GUS expression under synthetic promoters that have previously been used to show cytokinin and auxin output. The efficiency of this process was around 13 %, which was in line with results from other
IP T
duckweed species. Conflict of Interest
SC R
The authors declare that they have no conflict of interest. Acknowledgments
This work was supported by the State Key Laboratory of Freshwater Ecology and Biotechnology
U
(Grant number 2016FB04) and project of Natural Science Foundation of Hubei Province (Grant
N
number 2015CFB488).
A
Reference
M
Alabadí, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P., Kay, S.A., 2001. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293, 880-883.
ED
Afolabi-Balogun, N.B., Inuwa, H.M., Ishiyaku, M.F., Bakare-Odunola, M.T., Nok, A.J., Adebola, P.A., 2014. Effect of acetosyringone on Agrobacterium-mediated transformation of cotton. J. Agr. Biol. Sci. 9, 284-286.
PT
Appenroth, K.J., Augsten, H., Liebermann, B., Feist, H., 1982. Effects of light quality on amino acid composition of proteins in Wolffia arrhiza (L.) Wimm. using a specially modified bradford method. Biochem. Physiol. Pflanz. 177, 251–258. Bai, F., Demason, D.A., 2008. Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS
CC E
in Arabidopsis thaliana. Am. J. Bot. 95, 133-145.
Barton, K.A., Brill, W.J., 1983. Prospects in plant genetic engineering. Science 219, 671-682. Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G., Friml, J., 2003. Local, Efflux-dependent
A
auxin gradients as a common module for plant organ formation. Cell 115, 591-602.
Bergmann, B.A., Cheng, J., Classen, J., Stomp, A.M., 2000. Nutrient removal from swine lagoon effluent by duckweed. Trans. A.S.A.E. 43, 263-269. Bulgakov, V.P., Kusaykin, M., Tchernoded, G.K., Zvyagintseva, T.N., Zhuravlev, Y.N., 2002. Carbohydrase activities of the rolc-gene transformed and non-transformed ginseng cultures. Fitoterapia. 73, 638-643.
12
Canellas, L.P., Dantas, D.J., Aguiar, N.O., Peres, L.E. P., Zsögön, A., Olivares, F.L., Dobbss, L.B., Facanha, A.R., Nebbioso, A., Piccolo, A., 2011. Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants. Ann. Appl. Biol. 159, 202–211. Cantópastor, A., Mollámorales, A., Ernst, E., Dahl, W., Zhai, J., Yan, Y., Meyers, B.C. Shanklin J.R. Martienssen R., 2014. Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant Biol. 17, 59-65. Cao, H.X., Vu, G.T., Wang, W., Appenroth, K. J., Messing, J., Schubert, I., 2016. The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution. New Phytol. 209, 354-363.
IP T
Chang, W. C., Chiu, P. L., 1978. Regeneration of Lemna gibba, G3 through callus culture. Z. Pjlanzenphysiol. Bd. 89, 91–94.
Chhabra, G., Chaudhary, D., Sainger, M., Jaiwal, P.K., 2011. Genetic transformation of Indian isolate of Lemna minor mediated
SC R
by Agrobacterium tumefaciens, and recovery of transgenic plants. Physiol. Mol. Biol. Plants 17, 129-136. Comai, L., 1993. Impact of plant genetic engineering on foods and nutrition. Annu. Rev. Nutr. 13, 191-215.
Dietrich, R.A., Richberg, M.H., Schmidt, R., Dean, C., Dangl, J.L., 1997. A novel zinc finger protein is encoded by the
U
Arabidopsis LSD1, gene and functions as a negative regulator of plant cell death. Cell. 88, 685-694.
Edelman, M., Perl, A., Flaishman, M., Blumenthal, A., 1988. Transgenic Lemnaceae. Australian Patent Publication AU759570C.
N
Gasdaska, J.R., Spencer, D., Dickey, L., 2003. Advantages of therapeutic protein production in the aquatic plant Lemna. Bio.
A
Process J. 2, 49-56.
M
Heenatigala P.P.M., Yang J.J., Bioshopp A., Sun Z., Li G., Kumar S., Hu S., Wu Z., Lin W., Yao L., Duan P., Hou H., 2018. Development of efficient protocols for stable and transient gene transformation for Wolffia globosa using Agrobacterium.
ED
Frontiers in Chemistry. 6, 227.
Hegazy, A.K., Kabiel, H.F., Fawzy, M., 2009. Duckweed as heavy metal accumulator and pollution indicator in industrial wastewater ponds. Desalin. Water Treat. 12, 400-406.
PT
Huang, M., Fu, L.L., Sun, X.P., Rong, D., Zhang, J.M., 2016. Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata. Acta. Physiol. Plant. 38, 122.
CC E
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W., 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.
Jong, T.D., Veldstra H., 1971. Investigations on cytokinins, I. Effect of 6-benzylaminopurine on growth and starch content of Lemna minor. Physiol. Plantarum 24, 235-238.
A
Khvatkov, P., Chernobrovkina, M., Okuneva, A., Pushin, A., Dolgov, S., 2015. Transformation of Wolffia arrhizal (L.) Horkel ex Wimm. Plant Cell Tiss. Org. 123, 299-307.
Khvatkov, P., Chernobrovkina, M., Okuneva, A., Shvedova, A., Chaban, I., Sergey, D., 2014. Callus induction and regeneration in Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss. Org. 120, 263-273. Kristanti, R.A., Kanbe, M., Hadibarata, T., Toyama, T., Tanaka, T., Mori, K., 2012. Isolation and characterization of 3nitrophenol-degrading bacteria associated with rhizosphere of Spirodela polyrrhiza. Environ. Sci. Pollut. Res. Int. 19, 18521858.
13
Lacor, M.A.M., 1968. Flowering of Spirodela polyrhiza (L.) Schleiden. Plant Biology, 17, 357-359. Landolt E., 1986. The family of Lemnaceae–a monographic study. Veröff. Geobot. Inst. ETH. StiftungRübel (Zürich) 71, 1–566. Lemon, G.D., Posluszny, U., Husband, B.C., 2001. Potential and realized rates of vegetative reproduction in Spirodela polyrhiza, Lemna minor, and Wolffia borealis. Aquat. Bot. 70, 79-87. Li, J., Jain, M., Vunsh, R., Vishnevetsky, J., Hanania, U. Flaishman, M., Perl, A., Edelman, M., 2004. Callus induction and regeneration in Spirodela and Lemna. Plant Cell Rep. 22, 457-464.
IP T
Ma, J.K., Barros, E., Bock, R., Christou, P., Dale, P.J., Dix, P.J., Fischer, R., Irwin, J., Mahoney, R., Pezzotti, M. Schillberg, S., Sparrow, P., Stoger, E., Twyman, R.M., 2005. Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO Rep. 6, 593-599.
SC R
Michael, T.P., Bryant, D., Gutierrez, R., Borisjuk, N., Chu, P., Zhang, H., Xia, J., Zhou, J., Peng, H., Baidouri M.E., et al., 2017. Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies. Plant J. 89, 617-635.
Moon, H.K., Stomp, A.M., 1997. Effects of medium components and light on callus induction, growth, and frond regeneration in
U
Lemna gibba (duckweed). In Vitro Cell. Dev – Pl. 33, 20-25.
N
Müller, B., Sheen, J., 2008. Cytokinin and auxin interplay in root stem-cell specification during early embryogenesis. Nature
A
453, 1094-1097.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.
M
15, 473-497.
Porath, D., Hepher, B., Koton, A., 1979. Duckweed as an aquatic crop: evaluation of clones for aquaculture. Aquat. Bot. 7, 273–
ED
278.
Raja, N.I., Bano, A., Rashid, H., Chaudhry, Z., Ilyas, N., 2010. Improving agrobacterium mediated transformation protocol for
PT
integration of XA21 gene in wheat (Triticum aestivum L.). Pak. J. Bot. 42, 855-865. Reid, M.S., Bieleski, R.L., 1970. Response of Spirodela oligorrhiza to phosphorus deficiency. Plant Physiol. 46, 609-613.
CC E
Reinhold, D.M., Saunders, F.M., 2006. Phytoremediation of fluorinated agrochemicals by duckweed. Trans. A.SA.E 49, 20772083.
Rival, S., Wisniewski, J.P., Langlais, A., Kaplan, H., Freyssinet, G., Vancanneyt, G., Vunsh, R., Perl, A., Edelman, M., 2008. Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res. 17,
A
503-513.
Sahoo, K.K., Tripathi, A.K., Pareek, A., Sopory, S.K., Singla-Pareek, S. L., 2011. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7, 49. Scofield, S., Dewitte, W., Murray, J. A., 2014. STM sustains stem cell function in the Arabidopsis shoot apical meristem and controls KNOX gene expression independently of the transcriptional repressor AS1. Plant Signal Behav. 28, 9. Sheikholeslam, S.N., Weeks, D. P., 1987. Acetosyringone promotes high frequency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol. Biol. 8, 291-298.
14
Stefaniak, B., Woźny, A., Budna, I., 2005. Callus induction and plant regeneration in Lemna Minor L.. Biol. Plant. 45, 469-472. Stomp, A.M., Rajbhandari, N., 2000. Genetically engineered duckweed: US, US6040498. Tao, J., Sun, H., Gu, P., Liang, Z., Chen, X., Lou, J., Xu, G., Zhang, Y., 2017. A sensitive synthetic reporter for visualizing cytokinin signaling output in rice. Plant Methods 13, 89. Vu, H.T., Ondar, U.N., Soldatova, O.P., 2008. Expression of new mutant alleles of AS1, and AS2, genes controlling leaf morphogenesis in Arabidopsis thaliana. Russ J Dev Biol. 39, 6-12.
IP T
Vunsh, R., Li, J., Hanania, U., Edelman, M., Flaishman, M., Perl, A., Wisniewski, J.P., Freyssinet, G., 2007. High expression of transgene protein in Spirodela. Plant Cell Rep. 26, 1511-1519.
Wang, W., Kerstetter, R.A., Michael, T.P., 2011. Evolution of genome size in Duckweeds (Lemnaceae). J. Bot. 570319.
SC R
Wang, W., Haberer, G., Gundlach, H., Gläßer, C., Nussbaumer, T., Luo, M. C., Lomsadze, A. Borodovsky, M. Kerstetter, R.A., Shanklin, J., Byrant, D.W., Mockler, T.C., Appenroth, K.J., Grimwood, J., Jenkins, J., Chow, J., Choi, C., Adam, C., Cao, X. H., Fuchs, J., Schubert, I., Rokhsar, D., Schmutz, J., Michael, T.P., Maye K.F.X., Messing J., 2014. The Spirodela
U
polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat. Commun. 5, 3311. Yamaga, F., Washio, K., Morikawa, M., 2010. Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated
N
from the rhizosphere of duckweed Lemna aoukikusa. Environ. Sci. Technol. 44, 6470-6474.
A
Yamamoto, Y. T., Rajbhandari, N., Lin, X., Bergmann, B.A., Nishimura, Y., Stomp, A.M., 2001. Genetic transformation of
M
duckweed Lemna gibba, and Lemna minor. In Vitro Cell. Dev. Biol. - Plant 37, 349-353. Yang, L., Han, H., Zuo, Z., Zhou, K., Ren, C., Zhu, Y., Bai, Y., Wang, Y., 2014. Enhanced plant regeneration in Lemna minor by
A
CC E
PT
ED
amino acids. Pak. J. Bot. 46, 939-943.
15
Primer
Sequences (5’-3’)
TCS-F
GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCTTTGCTAGCAAAATCTACA
TCS-R
GGGGACCACTTTGTACAAAAAGCTGGGTTGTTATATCTCCTTGGATCGAT
DR5-F
GGGGACAAGTTTGTACAAAAAAGCAGGCTGAATTCGTCGACGGTATCGCAG
DR5-R
GGGGACCACTTTGTACAAGAAAGCTGGGTATCTCCTTGGATCGATCCCCTG
Hormones
Carbon sources
References
Landoltia punctata
2,4-D (67.86 μM), 6-BA (8.87 μM)
1 % sorbitol
Huang et al., 2016
Lemna gibba
2,4-D (45.24 μM) and 2 IP (2.98 μM)
3 % sucrose
Chang and Chiu, 1978
Lemna gibba
20 or 50 μM 2,4-D
3 % sucrose
Lemna gibba
Dicamba (226.24 μM) and 6-BA (8.87 μM)
1 % sucrose
Lemna minor Lemna minor
45 μM 2,4-D 2,4-D (5.0 μM) and 2 IP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) Dicamba (67.87 μM), 2, 4-D (15.83 μM), and 6-BA (4.44 μM) 2,4-D (1 μM) and 6-BA (2 μM) 2,4-D (22.62 μM), 6-BA (8.87 μM) 2,4-D (15.83 μM), dicamba (67.87 μM), and 2 IP (5.96 μM) Picloram (51.77 μM) and 2,4-D (20.62 μM)
3 % sucrose 1 % sucrose
M
A
N
Wolffia arrhiza
A
CC E
PT
ED
Fig. 1. The T-DNA of binary plasmids (pKGWFS7.0) for transformation of S. polyrhiza.
16
Li et al., 2004
SC R
Lemna minor Spirodela polyrhiza Spirodela punctata
Moon and Stomp, 1997
Stefaniak et al., 2002 Chhabra et al., 2011
1.5 % sucrose
Yang et al., 2014
3 % sucrose 1 % sucrose 1 % sorbitol
Canto-Pastor et al., 2014 Li et al., 2004
0.7 % sorbitol, 0.7 % mannitol and 2.6 % glucose
Khvatkov et al., 2014
U
Lemna minor
IP T
Species
IP T SC R U N A
M
Fig. 2. (A) The effect of different concentrations of 2,4-D and 6-BA on callus induction of S. polyrhiza; (B) The effect of different concentrations of NAA and TDZ on callus induction; (C) The effect of different carbon sources on callus induction; (D)
A
CC E
PT
ED
The effect of different concentrations of TDZ and 6-BA on frond regeneration. N = 3. Error bars indicated standard deviation.
Fig. 3. The complete callus formation process of S. polyrhiza. (A-B) Fronds proliferation; (C) Fronds dedifferentiation; (D-F) Callus formation. Bar= 4 mm.
17
IP T SC R
Fig. 4. The effect of different concentrations of AS (A) and pH of co-cultivation medium (B) on transformation efficiency. N = 3.
ED
M
A
N
U
Error bars indicated standard deviation.
Fig. 5. Confirmation of transformed S. polyrhiza by PCR analysis. (M) 2000bp DNA ladder marker; (T1-3) Amplifications of fragment of TCS element in transgenic plants (404 bp); (D1-3) Amplifications of fragment of DR5 element in transgenic plants
A
CC E
PT
(314 bp); (P1) Plasmid containing TCS ; (P2) Plasmid containing DR5 element; (W1-2) Wild plants.
Fig. 6. GUS activity in the frond of S. polyrhiza for TCS::GUS line (A) and DR5::GUS line (B). (A1-3) High GUS accumulation at the initial stage of bud formation and the frond apex; (A4-7) GUS activity decreased gradually and become more restricted to the frond apex; (A8) GUS activity became increasingly diminished; (B1) GUS activity was only observed at the tip of incipent
18
buds; (B2-4) GUS activity gradually spread over the entire frond; (B5-8) GUS activity was much reduced and finally was at a
A
CC E
PT
ED
M
A
N
U
SC R
IP T
level below threshold for detection. Bar=1 mm.
19