Plant Biotechnology 38, 247–256 (2021)

DOI: 10.5511/plantbiotechnology.21.0422b

Original Paper

Transformation of Jatropha curcas L. for production of

larger seeds and increased amount of biodiesel
Wiluk Chacuttayapong1, Harumi Enoki 2, Yusei Nabetani 3, Minami Matsui 4,
Taichi Oguchi 5,6, Reiko Motohashi 2,3,*
1
Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, Shizuoka 422-8529,
Japan; 2 Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, Shizuoka 422-8529, Japan; 3 Graduate
School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, Shizuoka 422-8529,
Japan; 4 Synthetic Genomics Research group, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan;
5
Tsukuba Plant‐Innovation Research Center, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan;
6
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan
* E-mail: motohashi.reiko@shizuoka.ac.jp Tel: +81-54-238-4831

Received January 5, 2021; accepted April 22, 2021 (Edited by K. Mishiba)

Abstract The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a
promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth’s resources.
Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many
other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve
the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic
materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their
seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling
the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote
root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation
medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and
the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana,
which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.
Key words: biodiesel, cDNA overexpression, Jatropha curcas L., rice FOX-hunting.

2014). Jatropha plants can be used for many purposes:

Introduction their latex can be used for wound healing and to treat
Because the energy produced through biofuel is both some skin diseases, their leaves can be used for silkworm
abundant and flexible, research into biofuel production feeding and as anti-inflammatory medicine, their fruit
has rapidly increased. Many organisms are capable of and hull can be used for biogas production and as
naturally producing biofuel; for example, thermophilic combustibles, their seed cake can be used as fertilizer
bacteria (Lin and Xu 2013), cyanobacteria, and and animal feed, and their seed oil can be used for
microalgae (Parmar et al. 2011) as well as land plants soap production, as insecticides and molluscicides, and
such as sugarcane (Saccharum officinarum) and maize importantly, as a resource for biofuel production because
(Zea mays) have been used to produce bioethanol for the Jatropha seeds can serve as partial or full replacements of
purposes of fuel (Byrt et al. 2011). petroleum-based diesel fuel (Forson et al. 2004; Gübitz
Jatropha (Jatropha curcas L.) or physic nut is a et al. 1999). Nonetheless, Jatropha has a long-life cycle;
perennial shrub native to Mexico and Central America; it the period from its sprouting to the yield of flowers and
can also be cultivated in arid or semi-arid environments fruits is long (Achten et al. 2010). However, its seed yield
in Africa, Asia, and South America (Montes Osorio et al. is not stable and is dependent on the growing conditions

Abbreviations: sGFP, synthetic green fluorescent protein; SpR, spectinomycin-resistance gene; ALS, acetolactate synthase gene; JcActin, Jatropha curcas
actin gene; fl-cDNA, full length cDNA; HrcA, heat-inducible transcription repressor; Os03 plant, LOC_Os03g49180 transferred plant; Os04 plant,
LOC_Os04g43210 transferred plant; Os08 plant, LOC_Os08g41910 transferred plant; Os10 plant, LOC_Os10g40934.3 (splicing variant) transferred
plant; Os10L plant, LOC_Os10g40934.11 (splicing variant) transferred plant.
This article can be found at http://www.jspcmb.jp/
Published online June 10, 2021

Copyright © 2021 Japanese Society for Plant Biotechnology

248 Transformation of Jatropha for production of larger seeds

and environment, due to which it cannot meet human membrane and are involved in important processes in
expectations. plants, such as the development of kernels in sunflower
Many studies have attempted to address these seeds (Salas et al. 2011). Additionally, the oil content of
obstacles, particularly studies of Jatropha flowers, fruits, the seed is partially composed of sphingolipids (Wang
and seeds, such as those focusing on the promotion of et al. 2006). The human alkaline ceramidase-like protein
flower production by increasing the concentrations of in Arabidopsis is related to biotic and abiotic stresses in
endogenous cytokinins involved in flower development plants (Wu et al. 2015).
(Ming et al. 2020) and ovule development-related gene LOC_Os04g43210 is the gene that encodes probable
expression (Xu et al. 2019). Seed quality (i.e., size, oil inositol transporter 2. In Arabidopsis, the protein
content, and oil component) is important with regards encoded by this gene is localized at the plasma
to biofuel production (Ruan et al. 2012). The ability to membrane and mediates the symport of H+ and several
increase Jatropha seed size is a promising sign for biofuel inositols, such as myoinositol, scyllo-inositol, D-chiro-
production. The manipulation of auxin response factor inositol, and muco-inositol (Schneider et al. 2007). The
19 (ARF19) in Jatropha has been shown to lead to larger inositol phospholipids play an important role in signaling
seeds (Sun et al. 2017). Additionally, the ability to alter and transportation between the plasma membrane
the lipid content of Jatropha seeds by increasing the and the endoplasmic reticulum, which regulate cell
expression of transcription factors related to specific lipid growth and proliferation (Stevenson et al. 2000).
synthesis (Ye et al. 2018) and lipid composition at each Arabidopsis seeds contain inositol, such as phosphatidyl
stage of maturation has also been studied (Jonas et al. inositol, phytoglycolipids, and ceramide-phosphate-
2020; Sinha et al. 2015). polysaccharide (Carter and Kisic 1969).
Previously, the expression and function of genes have LOC_Os08g41910 is the gene that encodes the Sua5/
been studied in Arabidopsis thaliana using the loss-of- YciO/YrdC/YwlC family protein, which is a putative
function method; however, some genes play a critical role translation factor involved in translation, ribosomal
in plant survival and the loss-of-function of these genes structure, and biogenesis in Shewanella oneidensis
is lethal. Alternatively, the gain-of-function method has and in the psychrophile Colwellia psychrerythraea 34H
been used to support the study of mutants that cannot (Heidelberg et al. 2002; Methé et al. 2005); it is also
be isolated via the loss-of-function method (Nakazawa essential for translation regulation in yeast (Lin et al.
et al. 2003). This has led to the development of a novel 2010). In Shewanella piezotolerans, the Sua5/YciO/YrdC/
gain-of-function system known as the full-length YwlC family protein has functions related to the assembly
cDNA overexpressing gene hunting (FOX-hunting) of ribosomes under low temperature conditions (Li et al.
system (Ichikawa et al. 2006). Sakurai et al. established 2008b).
the rice FOX-hunting system by introducing rice full- LOC_Os10g40934 is the gene that encodes a putative
length cDNAs into Arabidopsis plants via Agrobacterium flavonol synthase, flavone 3-hydroxylase, or 2OG-
tumefaciens-mediated transformation; approximately Fe(II) oxygenase. Flavonols are present in plant parts
30,000 independent Arabidopsis transgenic lines that are colored, such as fruits and flowers (Mol et al.
expressing rice full-length cDNAs (rice FOX Arabidopsis 1998). The accumulation of flavonoids has also been
mutant lines) were generated (Sakurai et al. 2011). reported in Arabidopsis seeds (Routaboul et al. 2006).
These rice FOX Arabidopsis lines were systematically Flavonoids are effective antioxidant agents that can
categorized on the basis of various criteria such as interrupt glucose and lipid metabolism by increasing
morphology, physiology, and stress tolerance. glucose and lipid oxidation. Additionally, the relative
The present study focuses on the larger seed size compounds of flavonol, such as quercetin, help regulate
of rice FOX Arabidopsis lines. To produce larger seeds, auxin transportation, possess antioxidant properties,
the rice cDNAs of FOX Arabidopsis lines that exhibited and are involved in signaling (Owens et al. 2008). Due
larger seed phenotypes, which were identified as LOC_ to splicing, LOC_Os10g40934 has many transcript sizes.
Os03g49180 (Os03), LOC_Os04g43210 (Os04), LOC_ Therefore, in this study, we selected the two longest
Os08g41910 (Os08), and LOC_Os10g40934 (Os10), were sizes to develop two types of overexpressing constructs
transferred into Jatropha. of LOC_Os10g40934.3 (Os10, Os10 is the LOC_
LOC_Os03g49180 is the gene that encodes ceramidase, Os10g40934.3 splicing variant with a 645-bp CDS) and
an enzyme that hydrolyzes ceramide into sphingosine LOC_Os10g40934.11 (Os10L, has a larger transcript size
and fatty acids. Ceramidase is a key regulator enzyme than Os10; Os10L is the LOC_Os10g40934.11 splicing
of sphingolipid homeostasis (Mao et al. 2000; Wu et al. variant with a 1074-bp CDS) to transfer into Jatropha.
2015). Arabidopsis Turgor regulation defect 1 is a Golgi- Initially, the transformation procedure for Jatropha
localized alkaline ceramidase associated with turgor was established via Agrobacterium tumefaciens
pressure regulation (Chen et al. 2015). Sphingolipids and infection of cotyledon explants using the herbicide
phospholipids are the major lipid structures of the cell phosphinothricin as the selection agent (Li et al. 2008a).

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 W. Chacuttayapong et al. 249

Subsequently, Kajikawa et al. (2012) developed a novel either vectors A2 or A3 (PalSelect system; Kumiai Chemical
method of Agrobacterium-mediated transformation Industry Co. Ltd., Tokyo, Japan) was used. Vectors A2 and A3
using the herbicide bispyribac-sodium; however, the are binary vectors that were used to transfer green fluorescent
explant regeneration efficiency was relatively low. The protein (GFP) gene and rice fl-cDNA into Jatropha. Both A2
high efficiency of callus regeneration associated with and A3 contain the spectinomycin-resistance (SpR) gene as
Agrobacterium-mediated transformation at an early a selection marker for Agrobacterium and the A. thaliana
phase of regeneration, followed by shoot and root acetolactate synthase (ALS) gene between the left border and
regeneration, is essential and could determine the success right border regions as a selection marker gene for plants. The
of the transformation. cloning sites of A2 and A3 are located between the left and right
In this study, we examined the favorable concentration border: A2 has multiple cloning sites with various restriction
and ratio of each phytohormone in Murashige and sites, whereas A3 utilizes Gateway® cloning technology. The
Skoog (MS) medium (Murashige and Skoog 1962) for constructs to be transferred into Jatropha were prepared using
Jatropha callus and shoot induction. When inducing Gateway® cloning technology (from pENTR/D-TOPO vector to
root regeneration, we cultured shoot explants in A3 vector cloning, followed by LR reaction). The GFP gene was
Gamborg’s B5 medium (Gamborg et al. 1968) containing cloned into the A2 vector at multiple cloning sites, whereas the
sucrose and 3-indolebutyric acid (IBA) to improve root five rice fl-cDNAs were cloned into the A3 vector separately at
induction. the gateway cloning site.

Agrobacterium culture and infection buffer

Materials and methods preparation
Plant materials and media Agrobacterium containing the designed vectors was cultured
Wild-type Tanzania line J. curcas L. seeds (Sekisui Chemical in lysogeny broth liquid medium containing rifampicin (final
Co., Ltd. Kyoto, Japan) were used as the transformation concentration, 50 mg/l), spectinomycin (final concentration,
material; only the cotyledons inside the seeds were used for 80 mg/l), and kanamycin (final concentration, 50 mg/l). Next,
Agrobacterium infection. The infected explants, including 10–14-h cell cultures of Agrobacterium were selected, and the
the calli, shoots, and rooting plants, were incubated under optical density (OD) was measured at 600 nm to produce 15 ml
controlled conditions with 16 h of light at 60 µmol/m2/s of infection buffer with a final OD600 of 0.2 using MS liquid
and 8 h of dark per day. The temperature and humidity were medium and acetosyringone (final concentration, 100 µM).
maintained at 26°C and 80%, respectively. MS medium was
used as the basal formula for callus induction media (CIM) and Explant preparation and Agrobacterium infection
shoot induction media (SIM). We found that MS medium with Jatropha seed shells were removed using pliers. The kernels
1.0 mg/l 6-benzyladenine (BA) and 0.5 mg/l IBA was suitable were soaked in tap water for 8–14 h to soften the endosperm
for induction of callus formation, whereas MS medium with and allow water uptake to the cotyledons. The kernel
3.0 mg/l BA and 0.1 mg/l IBA was suitable for induction of endosperms were removed and cotyledons were collected.
shoot formation (Enoki et al. 2017). For root induction media The collected cotyledons were dipped in a sterilization buffer
(RIM), we used Gamborg’s B5 as the basal formula instead of (MilliQ water with 4% sodium hypochlorite and one drop of
MS medium. In a previous study, we used MS medium as the Tween 20) for 2 min. After sterilization, the cotyledons were
basal medium for RIM, with some phytohormones added to washed with autoclaved water three–five times or until bubbles
the medium to improve the chances of inducing root formation; did not appear. The cotyledons were placed on a wet paper or
however, it did not improve root initialization and inhibited inside a falcon tube to prevent drying and were cut into small
the cut shoots from producing roots. Gamborg’s B5 medium pieces using a sterilized blade on a glass petri dish (one pair of
contains less NH+4 than MS medium and contains KNO3 as cotyledons was cut into 12–16 small pieces of explants). The
the main source of nitrogen, which is reported to increase the embryo was discarded because plants regenerated from the
percentage of root induction (Sriskandarajah et al. 1990). The embryo are likely to resist the antibiotics that were used for the
plant phytohormones IBA (0.5 mg/l) and powder-dipped IBA selection of the transgenic plants (escaping plants). The explants
were added to Gamborg’s B5 media and were used for a short were dipped into the infection buffer and then placed inside an
period (3–7 days) to enhance root initialization. They were aspirator and vacuumed at −50 kPa for 5 min. Infected explants
subsequently removed because the presence of phytohormones were transferred to the co-cultivation agar medium supported
tends to hinder the rooting of cut shoots. by filter paper and incubated at 26°C under dark conditions for
4 days.
A. tumefaciens strains and vectors
A. tumefaciens strain EHA101 contains a C58 chromosomal In vitro tissue culture of J curcas L
background that is resistant to the antibiotic rifampicin; it also After incubation for 4 days, the infected explants gradually
contains the pEHA101 Ti plasmid, which is resistant to the changed into yellowish curling explants. Only yellow and
antibiotic kanamycin. A. tumefaciens strain EHA101 containing curled explants were selected and transferred to a new

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250 Transformation of Jatropha for production of larger seeds

Figure 1. Effects of auxin and dark incubation treatment on Jatropha root induction. RIM: root induction medium with bispyribac-sodium at a
final concentration of 20 nM. RIM-D: RIM with auxin (IBA; 0.5 mg/l). Dark incubation: cultivated under dark conditions for 24 h (placed inside a
box). Light incubation: cultivated under a light/dark cycle (16-h light [60 µmol/m2/s]/8-h dark cycle). The dashed-line box inside represents different
experiments of the improved method for root regeneration before the shoots were moved to basal RIM. More than 30 separate explants were used in
each experiment. Experiments included three–five replicates.

medium for sequential cultivation in CIM, SIM, RIM, and should be considered because young and mature explants have
soil acclimation. The infected explants were cultured in CIM different antibiotic tolerances. We delayed the selection of
for 2 weeks, and then the calli were transferred to SIM. Shoots transgenic plants by adding bispyribac-sodium to the media at
that emerged from the calli with a length of more than 10 mm the following time periods: the beginning of the culture after
were cut and transferred to RIM. Regenerated shoots from the co-cultivation of the explant, 14 days after transformation, and
calli were treated in RIM in the presence or absence of IBA 28 days after transformation (Table 3).
(0.5 mg/l) under light (16 h of light at 60 µmol/m2/s and 8 h of
dark) or dark conditions (24 h of dark) for 3 or 7 days before PCR-based analysis for screening candidate
subculturing back to RIM without auxin. Dark incubation transgenic plants
involved placing the shoot, after being cut from the callus, Four types of primers were employed to screen candidate
on RIM containing 0.5 mg/l IBA, which was then placed in transgenic plants (see Supplementary Table 1 for primer
an incubator without light for 3 or 7 days. Light incubation sequences). DNA was extracted from 100 mg (fresh weight)
involved shoot cultivation under normal light conditions. of young leaf tissue from growing plants using the DNeasy®
Auxin was applied to the shoot by either directly dipping Plant mini kit (QIAGEN, Hilden, Germany) according to
the shoot that was cut from the callus into the auxin powder the kit protocols. The JcActin gene was used as a control and
mixture (Oxyberon; Bayer Cropscience, Leverkusen, Germany: the ALS gene was used for selection by bispyribac-sodium
IBA mixture) before placing it on basal RIM or the shoot was from the A2 and A3 vectors. GFP, rice fl-cDNA, and the heat-
transferred to RIM containing 0.5 mg/l IBA. Next, the shoots inducible transcription repressor (HrcA) gene were used to
were incubated in dark or light conditions for 3 or 7 days before detect possible contamination of Agrobacterium DNA in
being transferred again to basal RIM. Healthy rooted plantlets the extracted DNA (if the DNA samples of the young leaves
were transferred to sterile soil (autoclaved vermiculite and soil of these plants showed HrcA expression, the DNA of different
at a ratio of 1 : 1) and incubated under controlled conditions young leaves were extracted again to confirm the expression
with 16 h of light at 60 µmol/m2/s and 8 h of dark per day of the transferred gene) via amplification by PCR using RBC
(Figure 1). Taq DNA polymerase (RBC Bioscience, New Taipei, Taiwan).
The PCR reaction mixture was created according to the kit
Selection of transgenic plants using instructions using 90 ng of extracted DNA and the following
bispyribac-sodium procedure: initial denaturation at 94°C for 2 min; 30 cycles
Infected explants were transferred from the co-cultivation of denaturation at 94°C for 30 s, annealing for 1 min (at the
medium to the selection medium using 20 nM bispyribac- temperatures listed in Supplementary Table S1), and extension
sodium as a selection agent. Some of the calli from the co- at 72°C for 1 min; and final extension at 72°C for 7 min.
cultivation medium were moved to CIM containing 20 nM
bispyribac-sodium for the selection of resistant calli (delayed Expression of transferred genes
selection treatment), whereas the other calli obtained from the The expression of transferred genes in the transgenic plants
co-cultivation medium were moved to CIM without bispyribac- was analyzed by the detection of transcripts using RT-PCR.
sodium for 2 or 4 weeks (delayed screening treatment) before Total RNA was extracted from 100 mg (fresh weight) of young
being moved to CIM or SIM containing 20 nM bispyribac- leaf tissue obtained from growing transgenic plants using
sodium. The tolerance of the plant to the selection marker the RNeasy® Plant mini kit (QIAGEN, Hilden, Germany),

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 W. Chacuttayapong et al. 251

according to the kit protocols, with a modification using almost all infected explants had fully developed into the
the cetyltrimethylammonium bromide extraction method second stage, called a callus. On the other hand, from
(Sangha et al. 2010). The JcActin gene served as a control, and day 6 onward, some of the developed calli were oozing
GFP and rice fl-cDNA were used to detect the expression of Agrobacterium, which worsened with time.
the transferred genes, which were amplified by RT-PCR with The developed calli were large, and two different types
two types of primers (see Supplementary Table S2 for primer of calli were identified. The first type was a rough and
sequences) using the Prime Script High Fidelity RT-PCR Kit bumpy callus; this type of callus was likely to produce
(TaKaRa, Shiga, Japan). PCR was performed as follows: 30 shoots as the third stage of development. The second
cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, type of callus had a smoother surface and a sponge-like
and extension at 72°C for 1 min. characteristic; it rarely produced any shoots. Regenerated
shoots usually elongated up to 2 cm within 7 to 14 days.
Acclimation and cultivation of transgenic plants Shoots longer than 1 cm were transplanted into RIM to
Transgenic plants that expressed the transferred genes, as induce the shoot to progress to the fourth stage, a rooted
confirmed using PCR and RT-PCR, were grown in a sterile soil plant. Shoots with adventitious roots were taken from
mixture of vermiculite and soil. Wild-type Tanzania Jatropha RIM, residual medium was washed away, and they were
plants and transgenic plants were kept inside a specific netted transplanted into sterile soil media. At this stage, most
house for the genetic engineering quarantine experiment at the of the rooted plants successfully produced root hair and
University of Tsukuba. The fruits and seeds from transgenic functioned normally like germinated plants.
plants were collected, and their width, length, thickness
(volume was calculated using width, length, and thickness data Auxin-improved root regeneration
using the ellipsoid shape formula: V=4/3*π*a*b*c, where a The root regeneration percentage for RIM (Gamborg’s
is the width, b is the length, and c is the thickness), and fresh B5 formula) was relatively low (9.91%±9.60%)
weight were measured. compared with that for RIM containing auxin (IBA;
33.42%±9.11%). This suggests that the addition of auxin
Ploidy levels of transgenic plants to RIM promotes root regeneration from the cut shoot.
The transgenic plants, confirmed using PCR and RT-PCR, The powder-dipped auxin treatment and the RIM
were further analyzed using a Beckman coulter Gallios flow containing auxin (IBA) treatment had a greater effect on
cytometer and software v.1.1 (Beckman coulter, Inc., California, the root regeneration percentage of Jatropha shoots than
USA) to verify the ploidy levels of each plant. the treatment of RIM without auxin (IBA; Table 1).

Shoot culture in dark and light conditions affect

Results root regeneration
Explant developmental process Shoots were incubated at different dark and light
The regeneration of infected explants was classified conditions to investigate their effects on root
into the following four progressive stages: infected regeneration. Dark incubation led to noticeably higher
explant, callus, shoot, and rooted plant. Infected root regeneration percentages (29.40%±11.70%) than
explants were curled and initiated callus formation at 4 light incubation (14.31%±12.21%).
days post infection (DPI). The callus was enlarged and The 3-day dark condition appeared to induce the
differentiated into primordial shoots within 21–84 DPI. highest root regeneration percentage (35.20%), followed
Regenerated shoots were cut and placed into either RIM by the 7-day dark condition (27.62%) and the light
or the auxin root induction treatment. Depending on the condition (14.31%; Table 2).
treatment, regenerated roots were noted between 91 and
147 DPI. Bispyribac-sodium selection delays improved in
The length of the newly infected explants that had vitro Jatropha tissue culture
been incubated on co-cultivation media was assessed The shoot regeneration percentages were not affected by
starting at 1 day and up to 7 days. From day 4 onward, the timing of bispyribac-sodium treatment (13.11–14.88%;

Table 1. Effect of the application of auxin on Jatropha root regeneration.

Auxin treatment Root regeneration (%)
Powder-dipped and placed on root induction media (Oxyberon: IBA mixture) 34.38±11.15a
Root induction media containing auxin 0.5 mg/l (IBA) 26.01±11.38a
Root induction media 9.55±8.67b
Each replicate comprised more than 30 separate explants. Experiments included three–five replicates. Average rooting and errors were calculated using SPSS software
by comparing means method using one-way analysis of variance with a significance level of 95%. Data with different superscript letters are significantly different
(Duncan’s test at p≤0.05).

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252 Transformation of Jatropha for production of larger seeds

Table 3). Conversely, root regeneration was affected by the Transgenic plant detection and selection
timing of treatment, in which postponement of selection efficiency
until after 28 days of transformation led to the highest A total of 242 rooted plants were evaluated to
percentage of rooted plantlets (42.19%), followed by determine whether they were transgenic; 15 plants
selection at 14 days (25.58%), with the lowest percentage were Agrobacterium-free transgenic plants, 6 were
of rooted plantlets noted when selection was performed at false-positive transgenic plants from Agrobacterium
the beginning of the culture (17.02%; Table 3). contamination, and 227 were false-positive. The
efficiency of transgenic plant production using our
method ranged from 2.78 to 10.53%, with a total efficacy
Table 2. Effect of growing conditions on Jatropha root regeneration. of 6.20% (Table 4).
Root
Growing condition Expression of transferred genes in transgenic
regeneration (%)
lines
3 days of dark condition and then light condition 35.20±14.13a
7 days of dark condition and then light condition 27.62±7.02a, b
The expression of the transferred genes was examined
Light condition 14.31±12.21b in the transgenic plants; each transgenic plant showed
Each replicate comprised more than 30 separate explants. The experiment
expression of the correct target gene (Figure 2 and
included three–five replicates. The average rooting and errors were calculated Supplementary Figure S1).
using SPSS software by the comparing means method using one-way analysis of
variance with a significance level of 95%. Data with different superscript letters
are significantly different (Duncan’s test at p≤0.05).

Table 3. Regeneration of explants, according to stage, as a response to different selection times.

Selection timing Number of calli Number of shoots (%) Number of rooting plants (%)
Start of study 353 47 (13.31) 8 (17.02)
14 days after transformation 328 43 (13.11) 11 (25.58)
28 days after transformation 430 64 (14.88) 27 (42.19)

Table 4. PCR-based screening of rooting plants.

Number of transferred Number of
Number of Number of
Number of gene false-positive samples false-positive plants
Explant transferred gene Agrobacterium-free
rooting plants from Agrobacterium not containing
positive samples (%) transgenic plants (%)
DNA (%) the transferred gene (%)
sGFP 32 6 (18.75) 3 (9.38) 3 (9.38) 29 (90.63)
Os03 42 3 (7.14) 1 (2.38) 2 (4.76) 40 (95.24)
Os04 53 3 (5.66) — 3 (5.66) 50 (94.34)
Os08 36 1 (2.78) — 1 (2.78) 35 (97.22)
Os10 38 6 (15.79) 2 (5.26) 4 (10.53) 34 (89.47)
Os10L 41 2 (4.88) — 2 (4.88) 39 (95.12)
Total 242 21 (8.68) 6 (2.48) 15 (6.20) 227 (93.80)

Figure 2. RT-PCR analysis of the transferred genes in transgenic Jatropha plants. a. Amplification of sGFP gene. b. Amplification of LOC_
Os03g49180 (Os03) gene. c. Amplification of LOC_Os04g43210 (Os04) gene. d. Amplification of LOC_Os08g41910 (Os08) gene. e. Amplification of
LOC_Os10g40934 (Os10) gene. f. Amplification of LOC_Os10g40934.11 (Os10L: larger transcript size than Os10) gene. Lanes: bp, molecular size
marker; W, wild-type plant; Number refers to independent transgenic plants.

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 W. Chacuttayapong et al. 253

Figure 3. Ploidy level of Jatropha plants. Left: wild-type plant. Right: transgenic Os10-4 plant. 2× shows the diploid plant calibrated to the wild-type
Jatropha plant.

Table 5. Size and weight of wild-type Tanzania Jatropha seeds, wild-type Tanzania Jatropha seeds grown in Japan, and Os10-4 transgenic Jatropha
seeds.
Seed width Seed length Seed thickness Seed volume Seed weight
(mm) (mm) (mm) (mm3) (g)
Original wild-type seeds from Tanzania 12.28±0.58 19.02±0.05 9.61±0.45 1176.46±85.88a 0.74±0.06a
Seeds from wild-type Tanzania planted in Japan 7.12±0.28 14.95±0.32 6.73±0.65 374.53±40.03c 0.31±0.04c
Seeds of transgenic Os10-4 plant 10.84±0.18 16.21±0.44 8.46±0.14 779.18±41.48b 0.58±0.03b
Data with different superscript letters are significantly different (Duncan’s test at p≤0.05).

Ploidy level of transgenic Jatropha plants particularly at the most critical point, i.e., the induction
Analysis via flow cytometry revealed that all transgenic of root regeneration.
plants were diploid (Figure 3). Adventitious root formation from excised shoots
is promoted by auxin, the main component in many
Seed size of transgenic Jatropha plants root-inducing products. The cut shoot is unique in that
Transgenic plants were grown in a controllable allows the adventitious root to form more efficiently
greenhouse; however, they hardly produced flowers or than normal, particularly under conditions of altered
seeds; only 1 out of 15 transgenic lines (Os10-4 plant: the nutrition resources and endohormone homeostasis
transgenic plant of LOC_Os10g40934.3) produced seeds. (Druege et al. 2019). Explants cultured in RIM and auxin
The original wild-type seeds from Tanzania were larger had a three-times higher percentage of root regeneration
and heavier than those of wild-type Tanzania Jatropha than explants with no-auxin treatment, suggesting that
plants grown in Japan, with 2–4 times the seed volume auxin is indeed crucial for Jatropha adventitious root
and 1.5–2 times the seed weight (Table 5). The seeds of regeneration. Rooting may be induced by condensed
transgenic Os10-4 plants were larger and heavier than auxin, which is normally synthesized in the shoot
those of wild-type Tanzania Jatropha plants grown in tip and transported to the base of the shoot, where it
Japan, with almost twice the seed volume and 1.5 times accumulates, rather than being transported to the calli, in
the seed weight. addition to auxin uptake from the media.
The effect of auxin on root induction has been
investigated both in vitro (Purkayastha et al. 2010)
Discussion and in vivo (Camellia et al. 2009). The role of auxin
Auxin application increases root induction may be related to the adventitious rooting of plants
efficiency over hormone cross-talks (Pacurar et al. 2014). For
Jatropha in vitro tissue culture and transformation were instance, genes involved in auxin homeostasis are widely
performed to determine their most optimal growth differentially expressed in cuttings of tea (Camellia
conditions. Many factors affect explant regeneration; sinensis) with and without IBA treatment (Wei et al.
these can be both biological factors, such as the genetics 2019). Moreover, auxin and auxin-derivative compounds
of the plant, the explant condition, or the vector strain, such as melatonin can promote a stronger effect on the
and nonbiological factors, such as the phytohormone rooting of plants (Sarropoulou et al. 2012).
combination, incubation environment, or transformation
procedure (Kalimuthu et al. 2007; Kumar and Reddy Dark condition and auxin application
2010; Sharma et al. 2011; Singh et al. 2010; Sujatha and As shown in Table 2, shoots that were incubated in the
Dhingra 1993; Sujatha and Mukta 1996; Sujatha et al. dark for 3 days had a greater chance of root formation
2005). This study identified some factors that improve than those incubated in the dark for 7 days, but the
the likelihood of successful Jatropha transformation, shoots that continued to be incubated in the dark

Copyright © 2021 Japanese Society for Plant Biotechnology

254 Transformation of Jatropha for production of larger seeds

gradually lost their green color from 7 days onward. Nonetheless, the high number of rooting plants suggests
This result suggests that a short period of darkness is a good protocol, but the selection for true transgenic
important for root formation and regeneration. A study plants requires more attention as the number of false-
of rice adventitious roots from the stem found that the positive plants also increases. Nevertheless, we still
dark condition is necessary and improves adventitious strongly believe that the chances of producing successful
root formation through auxin transportation and transgenic plants, even when there are many false-
ethylene-related growth promotion (Lin and Sauter positive plants, is better than if strictly selecting using
2019). In petunias (Petunia hybrida), dark incubation the selection marker with the loss of transgenic plants.
accelerates the root formation associated with the Notably, we noted a case of a transgenic sGFP control
accumulation of indole-3-acetic acid in the base of the shoot that never produced any roots; hence, we could not
stem (Yang et al. 2019). In Jatropha, auxin improves the transfer the plants into the soil.
rooting quality of the cut stem (Camellia et al. 2009);
however, for in vitro culture, the effect of auxin on root Transgenic plant characteristics
formation in Jatropha has not yet been investigated. The transcription and expression of both endogenous
In this study, we examined the effects of IBA and dark and exogenous genes can affect the productivity of the
conditions on Jatropha root induction. The IBA and plant. We investigated the expression of transferred
dark treatments led to a higher percentage of rooting genes to confirm that the transferred genes were properly
than the light treatment (Tables 1, 2), suggesting that expressed.
dark treatment applied to the in vitro tissue culture of Inducing polyploidy in Jatropha is a widely employed
Jatropha can improve its rooting efficiency. The 3-day strategy to produce larger seeds for increased biofuel
treatment led to a higher percentage of rooting than production (de Oliveira et al. 2013; Niu et al. 2016;
the 7-day treatment. These results may be due to the Premjet et al. 2019). Some polyploid Jatropha lines have
morphology of Jatropha. Regenerated shoots usually vegetative parts with different sizes and characteristics as
had one–three leaves, which were small at the root well as different fruits and seeds due to changes in their
induction stage. When shoots are incubated in the dark genetic material. Our transgenic plants were confirmed
condition, photosynthesis cannot occur, and the shoots to be diploid plants; thus, we can conclude that the
gradually reduce energy production. Therefore, even results cannot be due to the effect of polyploidization.
though adventitious root regeneration can be promoted The transgenic Jatropha plants hardly produced
by a carbon source in the shoot area, the reserved flowers. Only one transgenic plant managed to produce
energy inside the cells cannot be replenished, resulting fruits and seeds: the Os10-4 plant that expressed the
in a lower rooting percentage in the 7-day treatment. rice LOC_Os10g40934 gene. Transgenic Os10-4 plant
These data suggest that a short dark period facilitates the seeds were smaller than those from Tanzania plants.
initialization of root regeneration in Jatropha. Given that similar plants grown in Japan also produce
seeds smaller than those produced by Tanzania plants,
Delayed bispyribac-sodium selection the environment in Japan may affect fruit and seed
As expected, the number of regenerated shoots and production. On the positive side, the transgenic seeds
rooting plants increased with delays of the selection produced by the Os10-4 plant were larger than those
marker. Explants in the cotyledon phase may have produced by the wild-type plant grown in Japan, which
lower viability than callus and shoot explants; therefore, suggests that our transgenic plants could fulfill the
the survivability of explants may be improved when objectives required for biofuel production.
screening begins at a later explant phase. Similar In the present study, we successfully applied our
experiments have been conducted using kanamycin as modified methods to create transgenic Jatropha
a selection marker (Fu et al. 2015); however, the results plants that had a higher chance of survival than those
demonstrated that growing explants without kanamycin produced in past studies, mainly by improving the
for 1 week could produce more regenerated shoots induction of roots from shoot explants. Additionally,
than growing explants without kanamycin for 2 or 3 we found an interesting effect of auxin application and
weeks. We assume that the difference in the selection dark incubation for in vitro Jatropha tissue culture. Our
mechanism of bispyribac-sodium and kanamycin method is simple, practical, and can be used in most
leads to a difference in the timing of screening and that laboratories. In the future, we hope that our transgenic
including all plant samples can also affect the results. plants will eventually produce fruits and seeds; these
could be valuable resources for biofuel and plant lipid
Transgenic plant efficiency research as well as for the production of green energy to
The Agrobacterium-free transgenic plants were estimated mitigate the issues related to global warming.
as roughly one true transgenic plant for every 6–36
rooting plants, with a high number of false positives.

Copyright © 2021 Japanese Society for Plant Biotechnology

 W. Chacuttayapong et al. 255

Jonas M, Ketlogetswe C, Gandure J (2020) Variation of Jatropha

Acknowledgements
curcas seed oil content and fatty acid composition with fruit
We thank Prof. Mitsuo Omura (Shizuoka University) and Prof. maturity stage. Heliyon 6: e03285
Takashi Nakatsuka (Shizuoka University) for valuable discussion Kajikawa M, Morikawa K, Inoue M, Widyastuti U, Suharsono S,
on the technical aspects of this study. This work was supported Yokota A, Akashi K (2012) Establishment of bispyribac selection
in part by a Cooperative Research Grant of the Plant Transgenic protocols for Agrobacterium tumefaciens- and Agrobacterium
Design Initiative Program, Gene Research Center, University of rhizogenes-mediated transformation of the oil seed plant Jatropha
Tsukuba (Grant no. 1722, 1823, 1923, and 2027) (TO, WC, and curcas L. Plant Biotechnol 29: 145–153
RM). Kalimuthu K, Paulsamy S, Senthilkumar R, Sathya M (2007) In
vitro propagation of the biodiesel plant Jatropha curcas L. Plant
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