Industrial Crops & Products 132 (2019) 29–40

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Agrobacterium-mediated genetic transformation of ajowan (Trachyspermum T

ammi (L.) Sprague): an important industrial medicinal plant
⁎ ⁎
Mohsen Niaziana, , Seyed Ahmad Sadat-Noorib, , Masoud Tohidfarc, Petr Galuszkad,
Seyed Mohammad Mahdi Mortazavianb
a
Department of Tissue and Cell Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization
(AREEO), 3135933151 Karaj, Iran
b
Department of Agronomy and Plant Breeding Science, College of Aburaihan, University of Tehran, Tehran-Pakdasht, Iran
c
Department of Biotechnology, Faculty of New Technologies and Energy Engineering, Shahid Beheshti University, G. C., Tehran, Iran
d
Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agriculture Research, Palacky University, Olomouc, Czech Republic

A R T I C LE I N FO A B S T R A C T

Keywords: Biotechnology-based breeding methods can improve useful industrial medicinal plants in a faster way. Tissue
Agrobacterium culture-based and in planta Agrobacterium-mediated gene transformation methods were applied to enhance
Ajowan drought and salinity tolerance in ajowan medicinal plant using betaine aldehyde dehydrogenase (BADH) gene.
Betaine aldehyde dehydrogenase The pBI121 binary vector harboring kanamycin resistance gene (nptII) and BADH gene in its T-DNA region was
Dip flora
applied to transfer BADH gene. Different concentrations of the selective antibiotic were tested on 15 day-old
Kanamycin
Thymol
hypocotyl segments and 75 mg/L kanamycin was identified as the minimum inhibitory concentration to screen
for transformed cells of ajowan. The different parameters of gene transformation including Agrobacterium optical
density (OD), Agrobacterium strain, Agrobacterium killing antibiotic, acetosyringone concentration, and in-
oculation duration were tested during tissue culture-based Agrobacterium-mediated transformation. The
OD600 = 0.6-0.8 of LB4404 strain of Agrobacterium × 160 mg/L timentin × 250 μmol/L acetosyringone × 30
min inoculation duration were identified as the best gene transformation parameters. Successful integration and
expression of transferred gene were confirmed in T0 plants using polymerase chain reaction (PCR) and reverse
transcription polymerase chain reaction (RT-PCR) methods. Five transgenic lines with 2.94% transformation
efficiency were achieved by regular tissue culture-based Agrobacterium-mediated gene transformation method,
whereas the dip flora in planta transformation method has led to one transgenic line with 1.42% transformation
efficiency. Bioassay analysis revealed enhanced drought and salinity tolerance in transformed plants in com-
parison with non-transformed plants. The morphological characteristics of plant height and seedling fresh
weight of BADH-transformed plants were improved under salinity stress. Under drought stress, the proline
content and relative water content of transgenic plants were more than non-transformants. The results of gas
chromatography mass spectrometry (GC–MS) revealed significant increase in thymol content of BADH-trans-
formed plants (55.07%) compared with non-transformed plants (39.20%) under drought stress condition. The
obtained transgenic plants are cultivable in unfavorable environments to give an acceptable level of useful
secondary metabolites. The optimized protocol is applicable for gene transformation of other medicinal plants of
Apiacea family for different purposes.

Abbreviations: ANOVA, analysis of variance; BADH, betaine aldehyde dehydrogenase; CRD, completely randomized design; DMRT, Duncan’s multiple range test;
GB, glycine betaine; GC/MS, gas chromatography mass spectrometry; Kin, kinetin; LSD, least significant difference; MS, Murashige and Skoog medium; NOS,
nopaline synthase; nptII, neomycin phosphotransferase; OD, optical density; PCR, polymerase chain reaction; PGRs, plant growth regulators; PPFD, photosynthetic
photon flux density; RT-PCR, reverse transcription-PCR; RWC, Relative water content; 2,4-D, 2,4-dichlorophenoxyacetic acid
⁎
Corresponding authors at: Department of Tissue and Cell Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education
and Extension Organization (AREEO), 3135933151 Karaj, Iran.
E-mail addresses: m.niazian@abrii.ac.ir, mniazian@ut.ac.ir (M. Niazian), noori@ut.ac.ir (S.A. Sadat-Noori).

https://doi.org/10.1016/j.indcrop.2019.02.005
Received 3 June 2018; Received in revised form 3 February 2019; Accepted 4 February 2019
Available online 11 February 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
M. Niazian et al.
1. Introduction
An important group of plants, neglected for many years by humans
and plant breeders, is medicinal plants. The side-effects of chemical
medicines in the past decades, have led to more attention to medicinal
plants and improving them for use in various food, pharmaceutical, and
other industries. Usage of the plant biotechnology and genetic en-
gineering techniques can compensate the occurred delay in the
breeding process of medicinal plants.
Ajowan (Trachyspermum ammi (L.) Sprague.) is one of the valuable
medicinal industrial plants that belongs to Apiaceae family, which
grows in arid and semi-arid regions of the world, including Afghanistan,
Central Europe, Egypt, India, Iran, Iraq, and Pakistan (Ashraf and
Orooj, 2006; Boskabady et al., 2014; Joshi, 2000; Soltani Howyzeh
et al., 2018). The most important limiting factors in arid and semi-arid
regions are environmental stresses, such as water and nutrition defi-
ciency (Akbari et al., 2013). Negative effects of drought and salinity
stresses on yield, yield components and other characteristics of ajowan
and other medicinal plants of Apiaceae family have been reported in the
literature (Akbari et al., 2013; Ashraf and Orooj, 2006; Azhar et al.,
2011; Moosavi et al., 2015; Neffati et al., 2010). It is also well-docu-
mented that abiotic stress can increase the secondary metabolites
content in different medicinal plants (Charles et al., 1993; Jaleel et al.,
2008; Zobayed et al., 2007). These secondary metabolites improve
growth and survival of their carriers plants under stressful environ-
ments (Ashraf et al., 2018). It seems that generation of abiotic resistant
genotypes of a medicinal plant can have double benefit to increase its
secondary metabolite content along with yield and yield components.
Breeders used conventional and modern plant breeding methods to
enhance abiotic stress resistance in different plants.
Gene transformation method is one of the quickest and most reliable
genetic improvement approaches. The development of a standard
transformation method to improve the genome of medicinal plants,
which are rich sources of dynamic secondary metabolites, is crucial
(Sivanandhan et al., 2016).
There are many gene transformation methods but Agrobacterium-
mediated gene transformation is the most effective method among all,
under laboratory conditions (Niazian et al., 2017b). Agrobacterium-
mediated gene transformation efficiency depends on several factors,
such as strain and concentration of Agrobacterium, usage of phenolic
material in medium such as acetosyringone, plant species and genotype,
plant growth regulators (PGRs), type of explant, light and temperature,
temperature of co-cultivation, antibiotics, wound treatment in plant
tissue, and proper method to identify transformed cells from non-
transformed cells (Niazian et al., 2017b). In Agrobacterium-mediated
gene transformation, optimization of the regeneration system and
aforementioned factors can lead to more regenerated plants and in-
crease the efficiency of transformation method. These factors are dif-
ferent in various plants according to genetics and many other criteria.
Finding the best combination of these parameters is critical in reaching
successful and durable gene transformation.
There are three defense responses that plants adopt under stress
conditions, including morphological (escape, avoidance, phenotypic
flexibility), physiological (cell and tissue water conservation, anti-
oxidant defense, cell membrane stability, plant growth regulators,
compatible solutes and osmotic adjustment) and molecular mechanisms
(aquaporins, stress proteins, signaling and drought stress tolerance)
(Farooq et al., 2009). Osmoprotectants, carbohydrates, polyols, amino
acids or Krebs cycle intermediates are the main plant metabolism
changes in response to abiotic stresses. Plants can stabilize their pro-
teins and membranes, detoxify reactive oxygen species, and keep the
cell osmotic pressure under abiotic stress by accumulation of osmo-
protectants (compatible solutes) (Zandalinas et al., 2018). Glycine be-
taine (GB) is one of these osmotically active molecules that plants ac-
cumulate in response to abiotic stresses. Plants have different
capabilities in accumulating GB in response to stress condition.
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Industrial Crops & Products 132 (2019) 29–40
Therefore, some of them are classified as accumulators and some as
non-accumulators (Castiglioni et al., 2018). A two-step oxidation re-
action leads to synthesis of GB from choline: the first step includes
oxidation of choline to betaine aldehyde catalyzed by choline mono-
oxygenase (CMO), and the second step includes the dehydration of
betaine aldehyde to GB, catalyzed by betaine aldehyde dehydrogenase
(BADH) (Chen and Murata, 2002). Introduction of the genes involved in
synthesis of GB can enhance tolerance to abiotic stresses in non-GB-
accumulating plant species (Masood et al., 2016). There are different
studies that report introduction of BADH gene to increase tolerance to
different abiotic stresses in transgenic plants (Yang et al., 2007; Li et al.,
2011, 2014; Yu et al., 2017). In addition, there is other study that
suggests that the transfer of a drought resistance gene can affect the
secondary metabolite accumulation in medicinal plants (Liu et al.,
2017).
The enhancement of GB, through metabolic engineering, in an im-
portant medicinal plant like ajowan, is valuable, since beside faster
production of transgenic plants, which will be able to survive in
drought and saline lands with higher content of valuable secondary
metabolites, the optimized Agrobacterium-mediated gene transforma-
tion protocol is valuable for metabolite engineering of other medicinal
plants and for produce custom-designed medicinal plant through tar-
geted genome editing method of clustered regularly interspaced short
palindromic repeats (CRISPR)-associated protein (CRISPR/Cas9) (Zhou
et al., 2018). The present study was conducted to (i) optimize the BADH
Agrobacterium-mediated gene transformation of ajowan in both in vitro
and in planta methods, (ii) enhance drought and salinity tolerance of
ajowan through gene transformation, and (iii) assess the effect of op-
timized transformation protocol on its developmental, physiological,
and essential oil composition.
2. Materials and methods
2.1. Plant materials
Sterile seeds of ajowan from Shiraz ecotype (12,313), procured from
the Research Institute of Forests and Rangelands of Iran, were culti-
vated in ½ MS medium (Murashige and Skoog, 1962) to generate sterile
explants in a phytotron with 24 °C and 16/8 h (light/dark) photoperiod.
Fifteen-day-old hypocotyls were used as explant for gene transforma-
tion experiments.
2.2. Plant tissue culture and in vitro regeneration process
All in vitro studies, such as optimization of the studied factors and
regeneration of transformed plants, were conducted based on our pre-
vious optimized indirect somatic embryogenesis protocol (Niazian
et al., 2017a).
2.3. The binary vector construction
The pBI121-BADH binary vector containing neomycin phospho-
transferase (nptII), which is a kanamycin resistance gene and acts as a
selectable marker, and betaine aldehyde dehydrogenase (BADH) gene,
located between T-DNA left and right borders was used (Fig. 1). In this
construct, nptII was cloned under nopaline synthase promoter and ter-
minator, whereas BADH gene was under CaMV-35S promoter and NOS
terminator (Fig. 1).
2.4. Determination of kanamycin threshold level
In the first step of experiment, kanamycin threshold of ajowan was
evaluated to determine the minimum inhibitory concentration of the
selection agent. For this purpose, different concentrations of kana-
mycin, including 0, 25, 50, 70, and 100 mg/L were applied on 15-d old
hypocotyls of ajowan. The experiment was conducted in the form of
M. Niazian et al.
Fig. 1. The schematic presentation of binary vector pBI121 harboring BADH
gene and nptII selectable marker in its T-DNA region.
CRD design with three Petri dishes for each treatment as replications.
Hypocotyl segments were cultured in Petri dishes containing 30 ml MS
medium supplemented with aforementioned concentrations of kana-
mycin plus 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) + 0.5 mg/L
Kinetin (Kin) (Niazian et al., 2017a). All cultures were incubated in
phytotron at 24 °C with 60–65% relative humidity under 16/8 (light/
dark) photoperiod with a 40 μmol m−2s-1 PPFD provided by cool white
fluorescent lamps.
2.5. Agrobacterium-mediated gene transformation factors
Agrobacterium’s cell concentration and strain, types of Agrobacterium
killing antibiotics, concentrations of acetosyringone, and different in-
oculation durations were the Agrobacterium-mediated transformation
factors investigated in the present study. In the first experiment, the
effect of Agrobacterium’s cell density and strain, and Agrobacterium
killing antibiotics factors on number of regenerated plants were in-
vestigated in a factorial experiment using a CRD design with three re-
plications as the following:
A: Agrobacterium cell density (OD600 = 0.2, 0.6–0.8, and 1–1.2)
B: Agrobacterium strains (LB4404, and GV3101)
C: Agrobacterium killing antibiotics (Timentin (160 mg/L), and
Cefotaxime (250 mg/L))
After 20 min inoculation with aforementioned factors, five 15-d old
hypocotyls segments were placed in each Petri dish containing 30 mL of
MS medium supplemented with 1.5 mg/L 2,4-D + 0.5 mg/L
Kin + 75 mg/L kanamycin. After 7 weeks, proliferated calli were
transferred to PGR-free MS medium + 75 mg/L kanamycin. Finally,
number of differentiated embryos from each combination of factors was
recorded.
After finding the best combination of aforementioned factors, two
separate experiments were conducted to find the best concentrations of
acetosyringone and different inoculation duration. In the second ex-
periment, the effect of four concentrations of acetosyringone (200, 250,
300, and 400 μmol/L) was tested on number of somatic embryogenesis.
In the third experiment, the effect of three inoculation durations (1, 20,
and 30 min) on number of differentiated embryos was assessed. Both
second and third experiments were conducted using completely ran-
domized design with three replications. Callus induction and somatic
embryo regeneration in these two experiments were the same as in the
first experiment.
2.6. Floral dip transformation
The BADH gene was also transferred to ajowan using tissue culture-
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Industrial Crops & Products 132 (2019) 29–40
independent method of floral dip. For this purpose, seeds of Shiraz
ecotype of ajowan were cultivated in plastic pots in greenhouse con-
dition (25 °C with 60% relative humidity under 16/8 (light/dark)
photoperiod). After three months, Agrobacterium was applied on in-
florescence of the plants at the early stages of flowering by the im-
mersion of shoots in Agrobacterium suspension (Tague and Mantis,
2006).
For preparation of Agrobacterium culture, a single colony of
Agrobacterium tumefaciens strain LB4404 harboring pBI121 plasmid was
grown in 100 ml LB liquid medium (5 g/L yeast extract + 10 g/L tryp-
tone + 10 g/L NaCl) supplemented with 75 mg/L rifampicin and
50 mg/L kanamycin. Erlenmeyer flasks (DURAN®, Germany) were kept
in an orbital shaker-incubator (IKA®KS 4000, USA) at 28 °C overnight.
In the next step, bacteria were collected by centrifugation at 4000 rpm
for 5 min at room temperature (Jiang et al., 2015). The supernatant was
discarded, and the collected bacteria were then re-suspended in liquid
½ MS medium (pH = 5.8) (OD600 = 0.8) supplemented with 5% (w/v)
sucrose and 0.4% (v/v) Silwet-77 surfactant (Clough and Bent, 1998;
Zale et al., 2009). To remove the remaining LB medium, centrifugation
was repeated another time and then the collected bacteria were sus-
pended in half strength liquid MS medium with aforementioned ad-
ditives. The final concentration of bacteria cells was adjusted to
OD600 = 0.8–1.0 (Zale et al., 2009). The umbel inflorescences of
ajowan plants (Fig. 2a) were submerged in the 50 ml falcons (Fig. 2b)
containing the achieved Agrobacterium suspension for 2 min (Zale et al.,
2009). After labeling the transformed inflorescences, the supplemen-
tary transformations were repeated on them two more times with 1-
week interval (Liu et al., 2012). To reduce the adverse effect of the
Agrobacterium on transformed flowers, the transformed plants were
covered with transparent plastic bags and kept in the greenhouse con-
dition at 20 °C with 70% relative humidity and regular irrigation. The
emerging non-transformed inflorescences were removed with small
scissors to prevent cross-pollination. At the end of growing season (˜two
months), the mature seeds of labeled inflorescences were collected and
transferred to laboratory to assess their transformation. Seeds were then
surface-sterilized with 70% (v/v) ethanol for 3 min, followed by one
time washing with sterile distillated water and immersion in sodium
hypochlorite (1.5%, v/v) for 5 min and finally washed for three times
with autoclaved distillated water (Niazian et al., 2017a). In final,
sterilized seeds of transformed plants along with seeds of non-trans-
formed plants germinated on ½ MS medium solidified with 0.3%
phytagel (w/v) and supplemented with 75 mg/L kanamycin + 160 mg/
L timentin. All culture vessels were kept in phytotron under afore-
mentioned condition for other in vitro experiments.
Fig. 2. Floral dip transformation of ajowan medicinal plant. (a) The umbel
inflorescences of ajowan plants. (b) submerging of the umbel inflorescences in
the 50 ml falcons containing the Agrobacterium suspension.
M. Niazian et al.
Table 1
Characteristics of used primers for amplification of virG and BADH genes.
Gene Primer Primer sequence PCR product size
name (bp)
BADH Forward 5’-ATGATTGTACATCCTTCACG-3’ 1166
Reverse 5’-TGCTGTTTTTATCAGTTGAG-3’
virG Forward 5' –CACCACCGCAACTTCCAC-3' 750
Reverse 5' -CAGGAAACAGCTATGAC-3'
2.7. DNA extraction and polymerase chain reaction
Genomic DNA of kanamycin-resistant plants was extracted ac-
cording to Sika et al. (2015). The standard PCR was carried out using
the BADH gene specific primers for amplification of 1166 bp fragment
(Table 1). The PCR reaction was performed in 25 μl reactions con-
taining 12.5 μl 2X PCR Master Mix (Sinaclon, Iran) + 0.33 μM of each
forward and reverse primers + 10 ng template DNA (1 μl) + 9.5 μl nu-
clease-free water. PBI121 plasmid DNA and nuclease-free water were
used as positive and negative control samples in PCR reaction. The
amplification was carried out in a thermal cycler (MyCycler, BIO-RAD,
USA), which was programed as the following: initial denaturation at
94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min,
annealing at 57 °C for 1 min, extension at 72 °C for 1 min, the last cycle
was followed by a final extension at 72 °C for 5 min. The PCR products
were analyzed on a 1% agarose gel and visualized by ethidium bromide
staining and then documented using an E-Box gel documentation
imaging (VILBER, France).
2.8. Agrobacterium infection test
Agrobacterium infection was assessed by PCR testing using virG gene
specific primers. Primers sequences and PCR product size of this gene
are presented in Table 1. The PCR program was performed with an
initial denaturation at 94 °C for 5 min, followed by 35 cycles of dena-
turation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at
72 °C for 1 min, and then last cycle followed by a final extension at 72 °C
for 7 min.
2.9. Reverse Transcription-PCR (RT-PCR)
Total RNA was extracted from leaf tissue (˜100 mg) of putative
transformed and non-transformed plants of ajowan using TRIzol® re-
agent (Invitrogen™ -Thermo Fisher Scientific, Wilmington, DE), ac-
cording to instruction of manufacturer. Five micrograms of the DNase-
treated extracted RNA were used for synthesis of complementary DNA
(cDNA) using 2-steps RT-PCR KiT (RTPL-12) (Sinaclon, Iran) by fol-
lowing the manufacturer’s protocol. After obtaining the cDNA, PCR was
carried out using the aforementioned PCR program with primers for
BADH gene (Table 1). The PCR products were electrophoresed (Pow-
erPac, BIO-RAD, USA) for 30 min on a 1% agarose gel stained with
ethidium bromide and then observed under UV irradiation.
2.10. Bioassay of transformed plants
Morphological and physiological assessments were applied to
evaluate the BADH transformed plants against intact plants of ajowan.
Drought stress was applied using water holding method (Bao et al.,
2009). Plastic pots (8 cm in diameter and 10 cm in height) were filled
with perlite-cucurbit: farm soil (1:1) and then covered with black mulch
plastics (Fig. 3). The plastic pots were irrigated to field capacity for four
weeks with two-day intervals and then irrigation was cut off until ob-
servation of wilting symptoms in the plants. Drought tolerance response
of wild type and BADH transformed plants were recorded on the 6th day
and the 8th day after water-withhold (Bao et al., 2009). Relative water
content (RWC) and proline content were measured as physiological
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Industrial Crops & Products 132 (2019) 29–40
responses on the 6th day of drought stress. Proline content was mea-
sured using Bates et al. (1973) method. For measurement of the relative
water content, one piece of leaf tissue (˜0.1 g) was excised and the fresh
weight was measured immediately. Next, the excised leaves were
floated at 4 °C overnight and then their rehydrated weight was re-
corded. In final, the rehydrated leaves were kept at 80 °C in an oven and
after 48 h their dry weight was measured. The RWC was measured
according to Gaxiola et al. (2001):
RWC= (fresh weight - dry weight) / (rehydrated weight - dry weight).
(1)
2.11. Essential oil extraction and GC/MS analysis
A 20 g seed sample of each transformed and intact plants was used
for essential oil extraction. Seed samples were crushed using an electric
grinder, then the achieved fine powder was added to 500 ml distilled
water on top of a heater at 100 °C and oil was extracted using a
Clevenger-type 5 apparatus (Niazian et al., 2017c; Noori et al., 2017)
for 2.5 h. For GC/MS, 1 μL of the essential oil samples was injected into
the GC split-less with the injection port. A GC–MS apparatus using the
HP (Agilent Technology): 6890 Network GC System gas chromatograph
connected to a mass detector (5973 Network Mass Selective Detector)
was used for GC–MS analysis. Helium gas was a carrier and its flow rate
was 1.0 ml/min. The oven temperature was linearly programmed from
40 °C to 250 °C at a rate of 3 °C/min. The composition of the essential
oil was identified through comparing their retention indices (RI), mass
spectra fragmentation with NIST (National Institute of Standards and
Technology) Adams library spectra, Wiley 7 n.1 mass computer library,
and with those reported in literatures (Adams, 1997). Ultimately, area
under the curve of GC/MS spectra was used to calculate the relative
percentage of each component (Noori et al., 2017).
2.12. Statistical analyses
All statistical analyses in experimental designs were carried out
using SAS® (SAS Institute Inc., Cary, NC) software. Means were com-
pared by LSD and DMRT at 5% probability level. All graphs were
plotted using Excel 2010 software.
3. Results and discussion
3.1. Kanamycin threshold
To evaluate the effect of different concentrations of kanamycin and
determine the minimum inhibitory dosage of selective agent for the
selection of putative transformants, non-infected hypocotyls segments
were cultured on callus induction medium (Niazian et al., 2017a)
supplemented with aforementioned concentrations of kanamycin and
the number of survived explants was recorded in each subculture. The
evaluations were finished in the second month of the experiment, when
all explants died in the medium that contained the minimum con-
centration of kanamycin (25 mg/L). The means comparison analysis
using LSD test showed that there was no significant difference between
25 mg/L of kanamycin and control treatment for percentage of callus
induction at 5% probability level (Fig. 4). The highest mean of callus
induction was related to 25 mg/L kanamycin with 100% callus induc-
tion (Fig. 4). Callus induction was prevented with 50, 75, and 100 mg/L
of kanamycin, but there was no significant difference between 75 and
100 mg/L concentrations of kanamycin for percentage of callus induc-
tion in ajowan medicinal plant (Fig. 4). According to these results,
75 mg/L of kanamycin was sufficient for selection of putative trans-
formants in ajowan, and after three weeks, cultured explants in this
concentration became bleached and died (Fig. 5). The Kanamycin
killing curve also showed that both 75 and 100 mg/L concentrations of
M. Niazian et al.
Fig. 4. The results of means comparison analysis of the effect of different
concentration of kanamycin selective agent on callus induction percentage of
ajowan using Duncan’s multiple range test at 5% probability level (Means fol-
lowed by the same letters within columns are not significantly different at the
5% level).
Fig. 5. The effect of kanamycin antibiotic on callus induction percentage in 15-
d old hypocotyl segments of ajowan. (a) 25 mg/L kanamycin (b) 50 mg/L ka-
namycin (c) 75 mg/L kanamycin (d) 100 mg/L kanamycin (in all cases the
bar = 3 cm).
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Industrial Crops & Products 132 (2019) 29–40
Fig. 3. The effect of water holding drought
stress on growth characteristics of ajowan. (a)
The appearance of BADH-transformed (left)
and non-transformed (right) plants of ajowan
in the first day of experiment with field capa-
city irrigation. (b) The appearance of BADH-
transformed (left) and non-transformed (right)
plants of ajowan in the 6th day of water
holding. (c) The appearance of BADH-trans-
formed (left) and non-transformed (right)
plants of ajowan in the 8th day of water
holding (in all cases bar = 5 cm).
Fig. 6. Kanamycin killing curve in ajowan medicinal plant.
kanamycin led to the highest means of non-callus induced plants
(Fig. 6).
The selection of transformants is a critical step in gene transfor-
mation studies. Therefore, finding the optimized concentration of the
antibiotic used as a selection agent have major effect on transformation
efficiency (Baranski, 2008). The high concentration of selection agent
led to reliable selection while on the other hand, it led to reduction of
somatic embryogenesis and plant development potential (Baranski,
2008). Therefore, determination of the minimum inhibitory con-
centration of selective agents in target plants is a key factor to screening
of transformed tissues and prevention of non-transformed cells growth
(Kim et al., 2016). The selectable marker, efficient gene delivery, and
responsive tissue culture are the three important factors that successful
transformation system depends on (Che et al., 2018). Neomycin phos-
photransferase is one of the most widely used selectable markers and
also recently used in gene transformation of different medicinal plants
(Gao et al., 2015; Sivanandhan et al., 2015; Subramanyam et al., 2015;
Vaidya et al., 2016). In the present study, 75 mg/L kanamycin was
sufficient to prevent callus formation in non-transformed hypocotyls
(Fig. 5) as it led to the death of explants showing the toxicity of this
concentration of kanamycin on ajowan. Different kanamycin thresholds
have been reported in various gene transformation studies and it is
obvious that kanamycin threshold depends on the species of the plant of
interest and also many other external factors. Sivanandhan et al. (2016)
also reported 75 mg/L of kanamycin as minimum inhibitor concentra-
tion for Agrobacterium-mediated gene transformation of Hybanthus en-
neaspermus medicinal plant (Sivanandhan et al., 2016).
M. Niazian et al.
Table 2
Analysis of variance of interaction effects of Agrobacterium concentration, Agrobacterium strain, and Agrobacterium killing antibiotic on percentage of callus induction
and number of somatic embryos in ajowan medicinal plant.
Source of variation
Agrobacterium concentration (OD600)
Agrobacterium strain
Agrobacterium killing antibiotic
Agrobacterium concentration × Agrobacterium strain
Agrobacterium concentration × Agrobacterium killing antibiotic
Agrobacterium strain× Agrobacterium killing antibiotic
Agrobacterium concentration × Agrobacterium strain× Agrobacterium killing antibiotic
Error
Coefficient of Variation (%)
a ** *
degree of freedom. , Significant at 1% and 5% probability level, respectively.
3.2. Optimization of gene transformation parameters (Agrobacterium’s
parameters, concentration of Acetosyringone, and inoculation duration)
3.2.1. Agrobacterium’s parameters
The analysis of variance from factorial experiment to study the ef-
fects of Agrobacterium OD, Agrobacterium strain and Agrobacterium
killing antibiotic on number of regenerated plants is presented in
Table 2. According to the results of ANOVA, the main effects of three
investigated factors on percentage of callus induction and number of
regenerated somatic embryo were significant at 1% probability level
(p ≤ 0.01), whereas their three-way interaction effect was significant
on percentage of callus induction and number of somatic embryo at 5%
probability level (Table 2). The results of means comparison analysis
using multiple ranges Duncan’s test at 5% probability level showed that
highest means of callus induction percentage and number of somatic
embryos were related to OD600 = 0.6-0.8 × LB 4404 × timentin
(160 mg/L) (Fig. 7a). The means comparison analysis revealed that
other Agrobacterium’s optical densities in both LB 4404 and GV3101
strains of Agrobacterium, can prevent somatic embryogenesis in ajowan
(Fig. 7b). Accordingly, OD = 0.6-0.8 of LB 4404 strain of Agrobacterium
along with 160 mg/L of timentin Agrobacterium killing antibiotic were
identified as optimized gene transformation parameters in ajowan
medicinal plant. Obviously, two important factors that can highly affect
the Agrobacterium-mediated gene transformation efficiency in Apiaceae
family are plant genotype and Agrobacterium’s strain. Type of vir
plasmid, binary plasmid, and chromosomal background of bacteria are
the Agrobacterium’s strain factors that can affect the efficiency of gene
transformation (Baranski, 2008). The interaction effect of plant geno-
type and bacterial strain is a complicated issue in Agrobacterium-
mediated transformation studies. Therefore, selecting the right Agro-
bacterium’s strain in different plant genotypes is important. The
Fig. 7. The results of means comparison analysis of interaction effect of Agrobacterium strain × Agrobacterium concentration × Agrobacterium killing antibiotic on (a)
callus induction percentage and (b) number of somatic embryo for BADH gene transformation of ajowan using Duncan’s multiple range test at 5% probability level
(Means followed by the same letters within columns are not significantly different at the 5% level).
34
Industrial Crops & Products 132 (2019) 29–40
dfa Mean squares
Percentage of callus induction No. of somatic embryo
2 0.7** 385.75**
1 0.36** 354.69**
1 0.16** 164.69**
2 0.09** 81.19**
2 0.02* 18.69**
1 0.04* 90.25**
2 0.02* 9.25*
24 0.055 2.13
13.54 12.85
bacterium concentration is another crucial parameter in gene trans-
formation studies. The overgrowth of Agrobacterium suspension can
lead to high level of contamination and reduction of plant regeneration
(Jin et al., 2005). On the other hand, reaching to the most active log
phase of Agrobacterium growth is very effective for transformation
(Yadav et al., 2012). Therefore, finding the optimized bacterial con-
centration is critical not only for improvement of the transformation
but also to avoid the contamination from bacteria’s abundant pro-
liferation (Jin et al., 2005). In the present study, OD600 = 0.6-0.8 was
determined as the best Agrobacterium optical density value for gene
transformation of ajowan and it seems that the most active phase of
growth and log phase of investigated strains of Agrobacterium is located
in this cell concentration (Zobayed et al., 2007) and proper for gene
transformation of ajowan medicinal plant. Pandey et al. (2013) eval-
uated the effect of different Agrobacterium cell concentrations including
values of OD600 0.2, 0.4, 0.6, 0.6, and 1 to optimize GUS gene trans-
formation in cumin (Cuminum cyminum L.) medicinal plant and re-
ported OD600 = 0.6 as the best Agrobacterium optical density for
transformation of cumin. These results indicate that Agrobacterium op-
tical density in the range of OD600 = 0.6 -0.8 can be considered as
optimum bacterial concentration for Agrobacterium–mediated transfor-
mation of medicinal plants of Apiacea family.
The selection of appropriate Agrobacterium killing antibiotic to
eliminate Agrobacterium after co-cultivation is another important factor
that can affect number of transformed regenerated plants and thus the
transformation efficiency. In the present study, it is revealed that
160 mg/L of timentin was better than 250 mg/L cefotaxime to eliminate
Agrobacterium from medium. Sivanandhan et al. (2016) also compared
the effect of different concentrations of timentin and cefotaxime anti-
biotics on the number of regenerated plants in Hybanthus enneaspermus
and reported that number of regenerated plants in medium
M. Niazian et al.
supplemented with timentin was more than when cefotaxime was used.
They also reported that the regenerated shoots from the medium con-
taining timentin were greener, healthier and grew actively, in com-
parison with the medium containing cefotaxime. Therefore, the result
of the present study was consistent with the results of Sivanandhan
et al. (2016). In a transformation study using GUS gene in Chry-
santhemum, Naing et al. (2014) compared the effect of carbenicillin,
cefotaxime, and clavamox antibiotics on number of regenerated plants
and reported less adverse effect of carbenicillin and clavamox com-
pared with cefotaxime. They observed the highest plant growth in
shoots that have been treated with 125 mg/L Clavamox. These ob-
servations point to the importance of finding the best type and con-
centration of Agrobacterium killing antibiotic in each desired plant. Most
of the previous studies evaluated the effect of Agrobacterium killing
antibiotic in control condition on non-transformed explants, but in the
present study, the effect of different types of Agrobacterium killing an-
tibiotics was examined on inoculated explants and in the presence of
Agrobacterium, so it seems that these results can be more reliable.
3.2.2. Acetosyringone concentration
With previously optimized gene transformation parameters
(OD600 = 0.6–0.8 of LB4404 strain of Agrobacterium and 160 mg/L
timentin), in this step, the effects of four different concentrations of
acetosyringone, including 200, 250, 300, and 400 μmol/L, were eval-
uated as the percentage of callus induction and number of somatic
embryos regenerated in MS medium supplemented with 75 mg/L ka-
namycin. The results of the means comparison analysis using Duncan’s
test at 5% probability level showed that the highest means of the callus
induction percentage and number of somatic embryo were related to
250 μmol/L of acetosyringone in the inoculation medium (Fig. 8a, b).
However, there was no significant difference between 200 and 250 μm/
L of acetosyringone for the percentage of callus induction, whereas the
number of kanamycin resistant regenerated somatic embryos in the
inoculation medium containing 250 μm/L of acetosyringone was more
than with 200 μm/L and this difference was significant at 5% prob-
ability level (Fig. 8b). The results of means comparison analysis showed
that application of asetosyringone at concentration higher than
250 μmol/L, can lead to significant reduction in callus induction per-
centage and number of regenerated somatic embryos and therefore can
reduce the gene transformation efficiency of ajowan medicinal plant
(Fig. 8a, b).
Chemical stimulants such as asetosyringone and p-hydroxybenzoic
acid or usage of physical wounding are usually critical for a successful
plant transformation (Baranski, 2008), yet there are other chemical and
mechanical additives including vacuum infiltration, sonication, and
silwet-L77 surfactant that were used in various Agrobacterium-mediated
gene transformation studies (Arun et al., 2015; Ding and Yuan, 2016;
Fig. 8. The results of means comparison analysis of effect of different concentrations of asetosyringone on (a) callus induction percentage and (b) number of somatic
embryo for BADH gene transformation of ajowan using Duncan’s multiple range test at 5% probability level (Means followed by the same letters within columns are
not significantly different at the 5% level).
35
Industrial Crops & Products 132 (2019) 29–40
Fister et al., 2016). Phenolic compound acetosyringone is a persuasive
vir gene inducer that is involved in relocation of T-DNA to plant cells
(Sivanandhan et al., 2016). Optimal concentration of acetosyringone is
crucial for gene transformation efficiency because its lower con-
centration is not sufficient to induce the virulence of Agrobacterium and
high concentration has toxic effect on Agrobacterium and plant cells (De
Clercq et al., 2002). Different concentrations of acetosyringone have
been reported as optimal concentration in different plant transforma-
tion studies and in different steps of gene transformation (pre-culture,
immersion or co-cultivation) (Fernand et al., 2016; Sivanandhan et al.,
2016). It is obvious that optimal concentration is different from plant to
plant and can be also affected by other gene transformation factors such
as Agrobacterium strain, pre-culture treatment, inoculation duration,
etc.
3.2.3. Immersion time
To find the best duration of inoculation with Agrobacterium, three
immersion times including 1, 20 and 30 min were applied on hypocotyl
explants. Other gene transformation parameters were fixed in this ex-
periment (OD600 = 0.6–0.8 of LB4404 strain of
Agrobacterium + 160 mg/L timentin + 250 μmol/L acetosyringone).
The results of means comparison analysis using Duncan’s test at 5%
probability level revealed that the highest and lowest percentage of
callus induction and number of regenerated somatic embryos in MS
medium supplemented with 75 mg/L kanamycin were related to 30 min
and 20 min immersion durations, respectively (Fig. 9a, b). Increase of
inoculation duration from 1 min to 20 min led to a significant reduction
in callus induction percentage and kanamycin resistant somatic em-
bryos, whereas increase of this parameter from 20 min to 30 min led to
a significant increase in the callus induction percentage and number of
kanamycin resistant somatic embryos (Fig. 9a, b), and therefore could
effectively lead to increase in gene transformation efficiency.
Undoubtedly, inoculation duration is an important factor in
Agrobacterium-mediated gene transformation because it provides re-
quired time for bacteria connection to plant cell wall and penetration of
T-DNA to plant cell. Gene transformation efficiency obviously depends
on Agrobacterium’s ability to cross from cuticle layer (Tokuji and
Fukuda, 1999). The cuticle of fresh hypocotyls is a difficult barrier for
bacterial cells (Baranski, 2008); therefore, finding the optimized in-
oculation duration is very crucial for efficient and durable gene trans-
formation. However, structure and thickness of cuticle layer is different
in different plants and as a result, this gene transformation parameter
varies from plant to plant. Based on the results of the present study,
30 min was the best inoculation duration for transformation of BADH
gene to ajowan medicinal plant. Depending on explant age and its
nature, Agrobacterium strain, type and composition of medium, mode of
infection, and temperature, inoculation duration can be variable from
M. Niazian et al.
Fig. 9. The results of means comparison analysis of effect of different inoculation durations on (a) callus induction percentage and (b) number of somatic embryo for
BADH gene transformation of ajowan using Duncan’s multiple range test at 5% probability level (Means followed by the same letters within columns are not
significantly different at the 5% level).
2 min to 60 min (Sivanandhan et al., 2016). Jha et al. (2011) suggested
30 min as the optimal infection time for gene transformation of Penni-
setum glaucum. Rajesh et al. (2013) reported 20 min as the optimal in-
fection time for gene transformation of Podophyllum hexandrum med-
icinal plant. Yadav et al. (2012) compared the effect of different
infection times including 10, 15, 20, and 30 min to transfer annexin 1bj
gene to Vignaradiate and reported 15 min as the best infection time for
gene transformation of this medicinal plant (Yadav et al., 2012). These
reports reveal the variable nature of inoculation time in Agrobacterium-
mediated gene transformation in different medicinal plants. However,
based on the results of the present study, 30 min inoculation duration
was optimal for ajowan medicinal plant and it can be considered for
gene transformation of other medicinal plants of Apiacea family, with
respect to other parameters of gene transformation.
3.3. Molecular confirmation of BADH insertion and Agrobacterium
contamination
The polymerase chain reaction using BADH gene specific primers
confirmed the integration of the expected transgene in the kanamycin
resistant T0 plants (Fig. 10). Strong band with the same size as the
BADH gene in the PCR amplification of the plasmid (positive control)
was observed (Fig. 10). No amplification in the control PCR without
template DNA showed that PCR was free from contamination (Fig. 10).
In addition, the absence of the transgene band in the negative control
with non-transformed template DNA revealed that this gene does not
exist in ajowan and proved the accuracy of material and PCR procedure
(Fig. 10). Overall, integration of at least one copy of BADH gene was
confirmed in five plants (lanes 1–5) from all 170 tested plants with
2.94% efficiency (Fig. 10). The achieved gene transformation efficiency
Fig. 10. The PCR amplification of BADH gene (expected size 1166 bp) in reg-
ular Agrobacterium-mediated transformation and dip flora method: M: DNA
marker (1100 bp marked), 1-6: putative transformants, P: plasmid (positive
control), C: non-transformed plant DNA (negative control), H2O: negative
control without template.
36
Industrial Crops & Products 132 (2019) 29–40
(the ratio of the PCR positive plants to all tested kanamycin resistant
plants), does not only depend on the studied gene transformation fac-
tors, but also it depends on many other parameters involved in tissue
culture procedure, including type and age of explant, and the compo-
sition of the used PGR for plan regeneration (Baranski, 2008). Some of
these plant tissue culture parameters were optimized in the previous
study (Niazian et al., 2017a). The results of the PCR analysis for T0
kanamycin-resistant plants of floral dip method confirmed the presence
of the transgene in only one line (lane 6) from 75 tested plants (Fig. 10).
As is shown, the efficiency of the in planta method was less than the
regular tissue culture-based Agrobacterium-mediated transformation
method. Anyway, in planta method was much faster and more eco-
nomical than the regular method. These results revealed that applica-
tion of in planta method is possible in valuable medicinal plants of
Apiacea family (Baranski, 2008, Niazian et al., 2017b), and more re-
search is needed for further development of this method.
All of the six PCR confirmed plants were analyzed by PCR using virG
specific primers to evaluate their Agrobacterium contamination before
they were subjected to RT-PCR analysis. The absence of expected band
of virG (750 bp) in all six transgenic lines revealed that these lines were
free from Agrobacterium contamination (Fig. 11). The presence of
Agrobacterium in the transgenic lines is one the potential problems in
Agrobacterium-mediated gene transformation studies that can lead to
systemic contamination (Tohidfar and Mohsenpour, 2010). Potentially,
Agrobacterium cells can survive antibiotic selection and persist for 24
months in the cells in leaves, stems, and roots of transgenic plants (Yang
et al., 2006; Palla and Pijut, 2015). The expression of the transgene can
be tested after confirmation of the absence of Agrobacterium tumefaciens
in transformed plants (Kalbande and Patil, 2016).
Fig. 11. The PCR amplification of VirG gene (expected size 750 bp) using gene-
specific primers to identify of Agrobacterium contamination in putatively
transformed ajowan plants: M: DNA marker (700 bp marked), 1-6: putative
transformants, P: plasmid (positive control), C: non-transformed plant DNA
(negative control), H2O: negative control without template.
M. Niazian et al. Industrial Crops & Products 132 (2019) 29–40

Fig. 12. RT-PCR analysis of BADH expression (expected size 1166 bp) in
transgenic plants of ajowan: M: DNA marker (1100 bp marked), 1-6: trans-
formed plants, P: plasmid (positive control), C: non-transformed plant DNA
(negative control), H2O: negative control without template.

3.4. RT-PCR

The results of RT-PCR analysis using two microliters of synthesized

c-DNA from transformed and non-transformed plants and specific pri-
mers of BADH gene confirmed the stable expression of the transgene in
all six transgenic lines (Fig. 12). The amplification of the 1166 bp
fragment of the BADH gene was found in all six transgenic lines and
plasmid control (Fig. 12, lanes 1–6), but it was absent in non-trans-
formed plant and the negative control (Fig. 12, lanes 7,8). The suc-
cessful heterologous expression of transformed BADH gene has been
reported in Arabidopsis using RT-PCR analysis (Yu et al., 2017).
Fig. 14. The effect of applied concentrations of NaCl on growth characteristics
of ajowan. (a) The first day of applying 150 mM NaCl on non-transformed
(right) and BADH-transformed (left) plants of ajowan (b) the 20th day of ap-
plying 150 mM NaCl on non-transformed (right) and BADH-transformed (left)
plants of ajowan (c) The first day of applying 200 mM NaCl on non-transformed
(right) and BADH-transformed (left) plants of ajowan (d) the 20th day of ap-
plying 200 mM NaCl on non-transformed (right) and BADH-transformed (left)
plants of ajowan (in all cases the bar = 2 cm).

concentrations of NaCl showed significant difference between trans-

3.5. Salt and drought tolerance assessment in transformed and wild type
formed and non-transformed plants for growth characteristics (Fig. 14).
plants
On the twentieth day of experiment, the difference of BADH trans-
formed and non-transformed plants of ajowan was obvious, especially
The transformed plants (four weeks old with ˜2–3 cm height) along
in 200 mM concentrations of NaCl, at which non-transformed plants
with non-transformed plants were cultivated in MS medium containing
completely lost their color and died (Fig. 14d, right), whereas BADH
different concentration of NaCl including 150 and 200 mM, and then all
transformed plants were alive and survived in the presence of salt stress
vessels were kept in a phytotron with aforementioned condition for 20
(Fig. 14d, left).
days. The results of means comparison analysis using Duncan’s test at
After assessment of salinity tolerance, the survived transgene plants,
5% probability level revealed that the increasing level of NaCl con-
with 10–12 cm height, were transferred to plastic pots and hardened to
centration led to the reduction of plant height and plantlet fresh weight
evaluate their drought tolerance. The results of means comparison
in both transformed and non-transformed plant. However, this reduc-
analysis using Duncan’s test at 5% probability level revealed that the
tion was more visible in non-transformed plants (Fig. 13a, b). In both
mean of proline content in BADH transformed plants was higher,
150 and 200 mM concentrations of NaCl, plant height and plantlet fresh
comparing to the non-transformed (Fig. 15a). Also means comparison
weight of BADH transformed plants were more than wild type plants
analysis showed that RWC of BADH-transformed plants was higher,
(Fig. 13a, b). The morphological assessment of transgenic and wild type
comparing to the non-transformed plants of ajowan (Fig. 15b). The
plants of ajowan under salinity stress of 150 and 200 mM

Fig. 13. The results of means comparison analysis of effect of different concentrations of NaCl on (a) seedling fresh weight and (b) plant height of BADH-transformed
and non-transformed plants of ajowan using Duncan’s multiple range test at 5% probability level (Means followed by the same letters within columns are not
significantly different at the 5% level).

37
M. Niazian et al.
Fig. 15. The results of means comparison analysis of the effect of water holding drought stress on (a) proline content, and (b) relative water content of BADH-
transformed and non-transformed plants of ajowan using Duncan’s multiple range test at 5% probability level (Means followed by the same letters within columns are
not significantly different at the 5% level).
higher content of proline and RWC in BADH transformed plants of
Arabidopsis, in comparison with non-transformed plants, has been also
reported (Yu et al., 2017).
The emergence of wilting symptoms in non-transformed plants was
faster than in transgenic plants (Fig. 3b), and at the eighth day of water
deficit stress the wilting of non-transformed plants was irreversible
(Fig. 3c right). One of the most important plant responses to the
changing environmental conditions, which guarantees their survival, is
the accumulation of osmolytes (Serraj and Sinclair, 2002; Masood et al.,
2016). Glycine betaine is one of the osmotic protectants that accumu-
late rapidly in plants in response to abiotic stresses, such as salinity,
drought, heat, and cold stresses (Yu et al., 2017). Indeed, this amino
acid derivative, with osmotic adjustment during abiotic stresses, pro-
tects membranes, photosystem II, and many of functional macro-
molecules from stress damage (Li et al., 2014). Chloroplast is the main
accumulation site of GB, which can protect and maintains the phyto-
chemical efficacy of thylakoid membrane (Genard et al., 1991). In the
abiotic stress condition, glycine betaine can resolve the inhibition of
protein biosynthesis role therefore, enhances the photosystem II repair,
which finally leads to increase of stress tolerance (Chen and Murata,
2008). Li et al. (2014) introduced a BADH gene from spinach (Spinacia
oleracea L.) into tomato (Lycopersicon esculentum cv. ‘Money maker’) via
Agrobacterium-mediated transformation method and reported that
transgenic plants had higher photosynthetic capacities and D1 protein
content comparing to the wild type plants, which led to accelerated
repair of photosystem II and decrease of the degree of membrane injury
under heat stress (42 °C) in transformed plants. The three different
possible ways that GB can protect transgenic plant under stress condi-
tion include: (i) maintenance of the stability of the quaternary struc-
tures of complex proteins and enzymes, which provide stable photo-
synthetic (ii) increase of the antioxidant defense mechanisms in plants,
and (iii) conservation of transcriptional and translational procedures
(Ke et al., 2016).
3.6. The essential oil profile of BADH-transformed versus wild type plants of
ajowan
The drought stress led to increase of thymol content in both wild
type and transformed plants of ajowan (39.2 and 55.07%, respectively)
(Table 3), in contrast to normal irrigation condition (38%)
(Mirzahosseini et al., 2017). The results of GC–MS analysis revealed
significant differences between essential oil profile of BADH-trans-
formed and wild type plants of ajowan under drought stress condition
(Table 3). The percentage of all essential oil components in transgenic
plants were more than non-transformed plants, except percentages of α-
Thujene, β-Pinene, α-Terpinene, and γ-Terpinene, which were in-
creased in non-transformed plants (Table 3). Alfa-thujene has
38
Industrial Crops & Products 132 (2019) 29–40
pharmaceutical toxicity (Sanco, 2002; Mirzahosseini et al., 2017);
therefore, decrease of this component can increase pharmaceutical
quality of the essential oil of ajowan.
In the present study, thymol, γ-terpinene, and p-cymene were the
main components of essential oil of BADH-transformed and wild type
plants of ajowan (Table 3). This is in line with the results of previous
study on essential oil composition of Iranian ecotypes of ajowan
(Mirzahosseini et al., 2017). The most impressive finding in the present
study was that thymol content of BADH-transformed plants was in-
creased (Table 3) that is very valuable for agricultural and medical
purposes.
The overexpression of the involved genes in the synthetic pathway
of secondary metabolites through Agrobacterium-mediated gene trans-
formation is a common way to enhance the percentage of desired sec-
ondary metabolites in different medicinal plants (Verpoorte and
Memelink, 2002; Sharma et al., 2017), but in the present study,
transformation of an agronomical important gene, that enhance
drought and salinity stress, led to increase of thymol content of ajowan
plants under drought stress condition. It is obvious that using plant
biotechnology methods can lead to faster progress in breeding of
medicinal plants than conventional breeding methods. Inthima et al.
(2017) reported that introduction of GA20ox gene from Torenia four-
nieri (TfGA20ox2) to Artemisia annua led to the double content of ar-
temisinin in transgenic plants in comparison to wild type plants. Noori
et al. (2017) reported positive and significant effect of in vitro tetra-
ploidy induction on thymol content of ajowan.
4. Conclusion
The world food security is seriously under the threat of drought
(Farooq et al., 2009); therefore, the development of fundamental and
applied research is critical to dealing with this threat. To our best
knowledge, until today no previous study has described stable trans-
formation of T. ammi and optimization of its Agrobacterium-mediated
transformation factors. There are huge numbers of Agrobacterium-
mediated gene transformation studies in medicinal plants but most of
them just optimized the transformation of a selectable or reporter gene,
instead of an applicable gene with valuable agronomic characteristics
(Mishra et al., 2013; Gao et al., 2015; Khan et al., 2015; Fernand et al.,
2016; Sivanandhan et al., 2016). Agrobacterium-mediated transforma-
tion is a multivariable biological system with complex nature. However,
there are some useful computational methods that can help researchers
to overcome the complex nature of biological systems, such as artificial
neural network method (Abdipour et al., 2018; Niazian et al.,
2018a,b,c). The development of an optimized gene transformation
protocol can help metabolic engineering and/or transfer of usable
agronomic traits in medicinal plants. Transformed plants obtained in
M. Niazian et al.
Table 3
Essential oil profile of non-transformed and BADH-transformed plants of ajowan under drought stress condition.
No. Essential oil component Retention time (min)
non-transformed
1 α-Thujene 8.24
2 β-Pinene 8.36
3 β-Myrcene 10.32
4 α-Terpinene 10.41
5 p-Cymene 11.65
6 β-Phellandrene 12.33
7 γ-Terpinene 13.10
8 Terpinene-4-ol 14.22
9 Thymol 20.36
a
The percentage of essential oil content = 3.43%.
b
The percentage of essential oil content = 3.40%.
the present study can be used for cultivation in infertile and marginal
lands. The developed gene transformation protocol can be used for
overexpression of genes involved in biosynthesis of secondary meta-
bolites. In the present study both tissue culture-based and dip flora in
planta Agrobacterium-mediated transformation methods were optimized
for transfer of BADH gene to ajowan. The results of the present study
are not only useful for genetic transformation of other plants of Apiacea
family but also led to abiotic resistance ajowan plants that had higher
level of thymol content, which is valuable for food and pharmacy in-
dustries. In addition, the optimized Agrobacterium-mediated transfor-
mation protocol is applicable in new genome editing method of
CRISPR/Cas9 (Niazian et al., 2017b).
Acknowledgements
This work was financially supported by Biotechnology Development
Council of the Islamic Republic of Iran [grant number: 950620]. The
authors are thankful to the Research Institute of Forests and Rangelands
of Iran for procuring the seeds and also grateful to Dr. M.H Asare, the
secretary of science and technological development staff of medicinal
plants and traditional medicine of Islamic Republic of Iran for his kind
support of ajowan project.
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