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Agrobacterium infection of hemp (Cannabis sativa

L.): establishment of hairy root cultures
a a a
Imane Wahby , Juan M. Caba & Francisco Ligero
a
Departamento de Fisiología Vegetal, Facultad de Farmacia , Universidad de Granada ,
18071 , Granada , Spain
Accepted author version posted online: 06 Nov 2012.Published online: 28 Nov 2012.

To cite this article: Imane Wahby , Juan M. Caba & Francisco Ligero (2013) Agrobacterium infection of hemp
(Cannabis sativa L.): establishment of hairy root cultures, Journal of Plant Interactions, 8:4, 312-320, DOI:
10.1080/17429145.2012.746399

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 Journal of Plant Interactions, 2013
Vol. 8, No. 4, 312320, http://dx.doi.org/10.1080/17429145.2012.746399

RESEARCH ARTICLE
Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures
Imane Wahby, Juan M. Caba and Francisco Ligero*

Departamento de Fisiologı´a Vegetal, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain
(Received 10 September 2012; final version received 31 October 2012)

Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with
either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact
seedlings was the most responsive material and the response depended on both bacterial strain and plant variety.
Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic
traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and
abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as
revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed
normal pattern of b-glucuronidase positive staining. To our knowledge, this represents the first reported protocol
for the establishment of Cannabis sativa hairy root cultures.
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Keywords: Agrobacterium; Cannabis sativa; cannabinoids; hairy roots

Introduction phytoremediation of heavy metals (Linger et al. 2005;

Hemp (Cannabis sativa L.) belongs to the Cannaba-
ceae family and the psychotomimetic activity of the
plant has been known since antiquity. Various pre-
parations of hemp under the names of marijuana,
hashish, charas, dagga, and bhang are smoked or
chewed by possibly two or three hundred million
people. Three types of hemp are distinguished based
on the concentration of ^9-tetrahydrocannabinol
(pharmacological active ^9-THC, commonly referred
to as THC) and in cannabidiol (CBD; inactive, but
a good identification marker): the ‘drug’ (resin) type
with high THC concentration (1%) and no CBD;
the ‘hemp’ (fiber) type with very low THC concentra-
tion (B0.3% and in fact B0.03% for the majority of
the ‘textile’ varieties); and the intermediate type, with
high concentrations of both THC and CBD.
Hemp fibers are durable and were used in cloth-
ing, sail making and paper making. Hemp seed oil
has been used for many purposes, from cooking to
cosmetics, and extracts of hemp plants were used
to treat a wide range of ailments (Clarke 1999).
Now, besides the traditional use of hemp fibers novel
applications like composite materials, non-woven and
geotextiles have been developed (Toonen et al. 2004).
High quality oil and proteins are present in hemp
seeds (Johnson 1999) and chemical compounds of
pharmacological implications are found throughout
the plant (Turner et al. 1980). Hemp is a particularly
environmentally friendly crop as it is high yielding
with low requirements for agrochemicals, fits well
in crop rotation schemes improving soil structure
(Toonen et al. 2004) and also has a potential for
Shi & Cai 2010) and biodegradation of weathered
hydrocarbons in contaminated soils (Liste & Prutz
2006).
The use of Agrobacterium rhizogenes as a gene
vector for genetic engineering of higher plants is widely
documented (Hu & Du 2006 and references therein).
Genes incorporated into plants by Agrobacterium-
mediated transformation are normally inherited by
progeny in a Mendelian fashion. However, the trans-
gene may be deleted or impaired by methylation or
mutation in meiotic transmission (Gao et al. 1991).
Agrobacterium rhizogenes and A. tumefaciens are very
closely related microorganisms with a similar infection
process. Therefore, it has been suggested that the
most important A. rhizogenes oncogenes encode pro-
teins involved in the regulation of plant hormone
metabolism. Aux genes provided transformed cells
with an additional source of auxin (Chriqui et al.
1996), but these genes do not seem to be essential
for developing root disease (Palazón et al. 1997).
However, this is not the case for rolA, rolB, and
rolC genes. These genes, located in the TL-DNA of
A. rhizogenes, are responsible for the root induction
process (Palazon et al. 1998, Tiwari et al. 2007). At
present, there is only one report of Agrobacterium
infection on hemp, which describes the transformation
of cell suspension cultures with A. tumefaciens strain
EHA101 carrying the binary vector pNOV3635 with a
gene encoding phosphomannose isomerase (Feeney &
Punja 2003).
This paper describes a hemp transformation
system using either Agrobacterium wild-type strains

*Corresponding author. Email: fligero@ugr.es

Present address: Imane Wahby, MAScIR Biotechnology, Rabatshore, 303 Business Center, 11100 Sala al Jadida, Morocco.
# 2013 Taylor & Francis
 Journal of Plant Interactions 313

(A. tumefaciens and A. rhizogenes harboring Ti and
Ri plasmids, respectively) or disarmed strains of
A. tumefaciens harboring the pRi1855 TL-DNA
genes rolA, rolB, and rolC, alone or all together
cloned in a binary vector pBin19, to establish stably
transformed tissues. We also aimed to investigate
stability of the b-glucuronidase (GUS) gene incorpo-
rated into the genomic DNA of transformed tissues.
100 mM, sucrose 0.5%, MES 30 mM, pH 5.6) and
MI2 (acetosyringone 200 mM, sucrose 2%, sodium
citrate 20 mM, pH 5.5). In each case, induction was
performed by pipetting 1 ml of the treatment solution
on 2-day-old bacterial plates and mixing the cells
with a sterile loop, 5 h before inoculations. Cell
suspensions (milky appearance) in sterile water served
as inoculum.

Materials and methods Plant growth and inoculation conditions

Plant material and bacterial strains
Five varieties of hemp (Cannabis sativa L.) repre-
senting different life cycles and applications were
selected for this study. Futura77, Delta-llosa, and
Delta405 (Semillas Castell S.L., Tarragona, Spain)
are all monoecious and bred for fiber; CAN0111 and
CAN0221 are dioecious drug-type climatic strains
from the Rif region (North of Morocco) and were
obtained from local growers. Nicotiana tabacum cv.
Burley F.13119 (CETARSA, Madrid, Spain) was
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Uniform seeds from each hemp variety were surface
sterilized in 20% v/v commercial bleach containing
0.01% Tween-20 for 15 min, washed five times with
abundant sterile distilled water and germinated on a
wet filter paper pile in Petri dishes in the dark at 258C
for 24 days. Germinated seeds (34 mm radicle)
were transferred to Petri dishes (three per dish) on the
top of agar slopes (½ B5 medium, no sucrose, pH
5.8, 1.2% Bacto-agar; Gamborg et al. 1968) with root
tips dipped into the medium. The dishes, partially

used as positive control. sealed with Parafilm , were incubated in a vertical

Agrobacterium strains listed in Table 1 were
grown at 288C on solid YEM medium (Vincent
1970) with antibiotic as described in Van Haute
et al. (1983).
Stimulation of Agrobacterium virulence
The following treatments were used for the induction
of vir genes in Agrobacterium: distilled water (con-
trol), acetosyringone (20, 100, and 200 mM, aqueous
solutions), and complex media MI1 (acetosyringone
position in a growth chamber (Caba et al. 2000).
Five-day-old plantlets were inoculated at four
tissues by separate: hypocotyls (two sites), cotyledon-
ary node (one site), cotyledons (one site each),
and primary leaves (one site each). For inoculation,
a syringe was used and 12 drops of Agrobacterium
inoculum was applied at the wounds. Hemp material
inoculated as above but with sterile distilled water
served as controls. After inoculation, the dishes were
kept overnight in the dark (208C) and then trans-
ferred to the growth chamber as above. Thirty plants

Table 1. Agrobacterium strains used in this study.

Strain/plasmid Name Characteristics Source/reference

Agrobacterium rhizogenes
477 477 Wild type M. Chamber, Sevilla
478 478 Wild type R. Ordás, Univ. Oviedo
476 476 Wild type R. Ordás, Univ. Oviedo
A4Ti24 A424 Wild type M.Chamber, Sevilla
A4 (Alain) Wt (pRiA4b) A4 Wild type, Rifr M.T. Piñol, Univ. Barcelona/White et al. (1985)
R1601 (pRiA4b, pTUK291) R1601 Kanr, Carbr M.T. Piñol, Univ. Barcelona/Pythoud et al. (1987)
C58C1 (pRi15834b) AR10 His , Rifr J. Stiller, Knoxville/Stiller et al. (1997)
C58C1 (pRi15834b, p35S::gus)a AR10GUS His , Rifr, Kanr J. Stiller, Knoxville/Stiller et al. (1997)
Agrobacterium tumefaciensb
LBA4404 (Bin19::ORF 10) LBA-rolA Kanr D. Mariotti Univ. Rome (Italy), Capone et al. (1989)
LBA4404 (Bin19::ORF 11) LBA-rolB Kanr D. Mariotti Univ. Rome (Italy), Capone et al. (1989)
LBA4404 (Bin19::ORF 12) LBA-rolC Kanr D. Mariotti Univ. Rome (Italy), Capone et al. (1989)
LBA4404 (Bin19::ORF10-11-12) LBA-rolABC Kanr D. Mariotti Univ. Rome (Italy), Capone et al. (1989)
C58 C58 Wild type M.M. López, IVIA, Valencia
IVIA 251-21 IVIA251 Wild type M.M. López, IVIA, Valencia

Carbr, Kanr, Rifr: Carbenicillin, kanamycin and rifampicin resistant, respectively; His : histidine heterotrophy.
a
p35S::GUS-INT: a 2.3 kb BamHI-XhoI fragment from pMC81, including the p35S-CODA-CAMV3? cassette ligated into the integration vector
pAR5 to obtain the pAR5MCCODA which was inserted in the TL region of C58C1 strain via homologous recombination (Stiller et al. 1997).
b
Derivatives of Agrobacterium tumefaciens LBA4404 harboring restriction fragments of the TL-DNA of pRi1855 individually cloned into the
plant binary vector Bin19 (Capone et al. 1989):
LBA-rolABC (EcoRI fragment 15, E15, encompassing ORFs 10, 11 and 12).
LBA-rolA (EcoRI-BamHI fragment 15, EB15, ORF 10).
LBA-rolB (SmaI-HpaI fragment 15, SHp15, ORF 11).
LBA-rolC (HindIIII-EcoRI fragment 15, HE15, ORF 12).
 314 I. Wahby et al.
per each tissue and Agrobacterium strain combination
were used. Each series of inoculation was performed
at least twice.
Other explants (:1 cm fragments of primary
leaves and hypocotyls, and entire cotyledons) were
excised from 7-day-old plantlets grown aseptically
as described above, placed on wet filter paper in
sterile Petri dishes and inoculated with Agrobacterium
R1601 on each cut surface. After overnight in the
dark (208C), the explants (60 of each type) were
cocultured for 2 d, in solid medium (3% sucrose)
and under 60 mmol m 2 s 1 light intensity. Tissues
were then transferred to fresh medium supplemented
with 500 mg/ml cefotaxime (Sigma) for elimination of
bacteria and cultured further for four weeks under the
same conditions. Three independent experiments
were performed.
In another experiment, five-day-old plantlets were
inoculated at hypocotyl with all A. rhizogenes strains
(Table 1) as described above. After 23 d in the
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chamber, the plates were examined for plant survival
(3545 plantlets inspected per bacterial strain and the
experiment was performed at least twice). Moreover,
hemp seedlings were inoculated, under the same
conditions, with several strains of A. tumefaciens
LBA4404 (Table 1) harboring the pRi1855 TL-DNA
genes rolA, rolB, and rolC alone or all together cloned
into the binary vector pBin19 as described by Capone
et al. (1989). Three weeks after inoculation hairy root
response was evaluated for seedlings infected with
each Agrobacterium strain.
Finally, the infection of five hemp varieties
(Futura77, Delta-llosa, Delta405, CAN0111, and
CAN0221) with four Agrobacterium strains (A4,
AR10, C58, and IVIA251) was assayed in this study.
Thirty-five inoculated-healthy plantlets were estab-
lished per plant variety-bacterial strain combination
and the experiment was done in duplicate. Any hairy
roots or tumors emerging from the wounded sites
23 weeks after inoculation were recorded to evaluate
plant response to Agrobacterium infection.
In vitro hairy root and tumor cultures
Single transformed roots, emerging from hypocotyl
wounds, were carefully excised on hormone-free MS
medium, pH 5.8, 3% sucrose, 500 mg ml 1 cefotax-
ime, and 0.6% purified agar (Murashige & Skoog
1962) and cultured at 258C in the dark. New growing
root tips were subcultured to fresh medium with
a 7-day subculturing cycle and the antibiotic in
the medium being successively reduced to 300 and
100 mg ml 1 at 15 days intervals. After repeated
subculturing, actively growing and bacteria-free hairy
root cultures of C. sativa could be obtained.
Tumors were excised 17 days after inoculation,
treated with cefotaxime, transferred to solid MS
medium and subcultured every 23 weeks.
Ethylene determination
Root ethylene evolution was analyzed essentially
following our previously described procedure
(Caba et al. 1998, Ligero et al. 1999). Root samples
(0.5 g FW) representative of lines with thin or thick
morphology, growing on solid MS medium for
four weeks, were placed in 9 ml vacutainers contain-
ing filter paper and 0.5 ml of MS liquid medium.
The vials were stopped with serum caps and after 2 h
incubation, 1 ml aliquots were removed from the
headspace and analyzed by gas chromatography.
Ethylene was measured on 10 replicates and data
are representative of three assays.
b-glucuronidase assays
GUS activity was demonstrated by histochemical
staining as described by Jefferson (1987). Root
tissues were incubated in 5-bromo-4-chloro-3-indolyl
b-D-glucuronic acid (X-Gluc) for 12 h at 378C.
The appearance of a dark blue color was taken as
an indicator of GUS activity.
PCR analysis
Genomic DNA was extracted using the plant tissue
specific Puregene kit (Gentra Systems, Inc.) following
manufacturer instructions as described by Caetano-
Anollés et al. (1999). Primer pairs used, specific to
sequences found in the referred genes, were: rolB1,
5?-AGTTCAAGTCGGCTTTAGGC-3? and rolB2,
5?-TCCACGATTTCAAGTAG-3?; rolC1, 5?-TAAC
ATGGCTGAAGACGACC-3? and rolC2, 5?-AAA
CTTGCACTCGCCATGCC-3?; ipt1, 5?GATCG(G/C)
GTCCAATG(C/T)TGT-3? and ipt2, 5?-GATATCCA
TCGATC(T/C)CTT-3?; virG1, 5?-CGATGACGATG
TCGCTATGC-3?, and virG2, 5?-CAGCACCTCTTG
CAGTCTTG-3?. All primers were purchased from
Sygma-Genosys Ltd. Amplifications were performed
in a DNA Thermal Cycler (PE 2400) following
Caballero et al. (2004). Reaction mixture (50 ml final
volume) consisted of reaction buffer (50 mM KCl,
1.5 mM MgCl2 and 10 mM Tris-HCl, pH 9.0), 150 ng
of template DNA, 50 pmol of each primer, 0.2 mM
of dNTPs and 0.5 U of Taq DNA polymerase (Amer-
sham Biosciences). Cycling conditions were: 5 min at
958C followed by 45 cycles of 30 s at 948C, 1 min at
578C, and 1 min at 728C. Finally, PCR products were
separated on agarose gel (2%) in tris-borate-EDTA
(TBE) buffer and stained with ethidium bromide.
Results
Transgenic nature of in vitro cultures
Transgenic nature of our hemp cultures was
confirmed by (1) PCR analysis for the presence,
in plant genome, of rolB and rolC genes (roots),
ipt gene (tumor), and virG gene (for the absence
of Agrobacterium in transformed material) and (2)
histochemical localization of GUS activity in root
 Journal of Plant Interactions 315

tissues. Also, hairy root and tumor cultures were

tested for their ability to grow in hormone-free
medium.
Primers specific for rolB and rolC genes of
A. rhizogenes and for ipt gene of A. tumefaciens
amplified fragments of the expected size (780, 540,
and 427 bp, respectively) in PCR reactions with DNA
from hairy roots (Figure 1a, lanes 110), tumor lines
(Figure 1b, lanes 12) and the positive controls
A. rhizogenes (Figure 1a, lane 11), and A. tumefaciens
(Figure 1b, lanes 34). The apparent absence of rolB
PCR product in Lane 5, probably because of DNA
degradation, could not be confirmed with a new
DNA sample. Hence we assume that the correspond-
ing hairy root line contains the rolB gene. These
PCR products were absent in untransformed tissues
(Figure 1, lane C). All transformed tissues were negative
for the presence of the virG gene (Figure 1). Moreover,
expression pattern of GUS reporter did not differ
from those in hairy roots of other species, i.e. primary
expression in growing root tips and vascular tissues of
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young (Figure 2f) and mature rolB and rolC-positive

hairy roots (data not shown).

Characterization of transformed cultures

Under optimized experimental conditions, several
transformed callus and root cultures of C. sativa
were initiated, hypocotyls of intact seedlings being

Figure 2. Hemp response to Agrobacterium infection.

Hairy root development from wounded hypocotyls of hemp
(a, b) and tobacco (c) seedlings on solid B5 ½, four weeks
after inoculation with A. rhizogenes R1601 strain. Tobacco
was known to be highly susceptible to Agrobacterioum
strains. Tumor development from wounded hypocotyls
of tobacco (d) and hemp (e) seedlings three weeks after
inoculation with A. tumefaciens C58 strain on the same
medium. (f) Histochemical staining of hemp hairy roots
tissue transformed with the A. rhizogenes strain AR10GUS.
Axenic transformed hemp root cultures on solid MS
medium for 4 weeks exhibiting the thin [typical thin, long
and highly branched hairy roots (g)] or thick [some shorter
and thicker branched roots with extreme abundance of root
hairs (h)] morphology.

Figure 1. PCR analysis of the transformed tissues of
C. sativa for the presence of rolB, rolC and virG genes in
genomic DNA from roots transformed with A. rhizogenes
(a) or ipt and virG genes in genomic DNA from A. tumefaciens
induced tumors (b). For primer sequences, DNA isolation
and PCR conditions, see materials and methods section.
(a) Lanes 110, ten independent hairy root lines; lane 11,
A. rhizogenes R1601 DNA; lane C, untransformed control;
lane M: molecular mass markers (100 bp DNA ladder,
Sigma). (b) Lanes 12, tumor lines induced by A. tumefaciens
strains C58 and IVIA251; lanes 34, DNA from A.
tumefaciens strains C58 and IVIA251. Expected PCR product
sizes are indicated in parenthesis. The presence of rolB
product in lane 5 could be confirmed when a new sample of
DNA was used.
the most responsive material. Plant response to
Agrobacterium was found to be dependent on bacter-
ial strain and, to some extent, on plant variety.
A short general characterization of the established
cultures is given below.
Wild-type transformed roots
As shown in Table 2, hypocotyls of intact seedlings
were the most susceptible material responding in
a way typical of hairy roots, with small root galls,
fleshy in appearance (Figure 2a), emerging from
wounds. Aseptically wounded plantlets, as control
for callusing, produced no outgrowths (data not
 316 I. Wahby et al.
Table 2. Response of hemp tissues to in vivo (intact Hairy roots from all A. rhizogenes-plant variety
seedlings) or in vitro (tissue explants) infection with combinations could be cultured in vitro and over
A. rhizogenes strain R1601 pretreated for 5 h with 20 mM 20 actively growing root lines were stabilized. They
acetosyringone. all displayed the typical transformed phenotype
Response Response of plagiotropic growth, high incidence of lateral
In vivo frequencya In vitro frequencyb branching and abundance of root hairs (Figure 2).
Untransformed roots showed poor growth, no branch-
Hypocotyl 88.396.7 Hypocotyl 67.197.2 ing and premature senescence and death (data not
Cotyledonary 58.795.8 Cotyledon 0 shown). Two morphological phenotypes, on the basis
node of gross morphology, were distinguished in these root
Cotyledon 0 Leaf 0
lines: the thin morphology (Figure 2g) and the thick
Leaf 0
morphology (Figure 2h). The first one was much
Note: Aseptically grown seedlings of the accession CAN0221 more frequent and both remained stable over more
or explants derived from them were infected as described in than two years. Further these phenotypes apparently
Materials and methods. Data represent mean values9SE of three
did not relate with bacterial strains or plant variety.
independent experiments. Analysis was performed three weeks
after infection, with 30 seedlings or 60 explants per organ examined Root lines with thick morphology grew faster than
for each measurement. those of thin morphology, the growth rate (measured
a
Percentage of infection sites with root induction as fresh weight) being on average almost 2-fold higher
b
Percentage of explants with root formation for the former (Table 5), after four weeks of culture,
with an increase of inoculum FW by 100 and 53 folds,
shown). Nicotiana tabacum, known to be highly respectively. These differences, however, did not
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susceptible to Agrobacterium was used as positive
control for root formation (Figure 2c). As the
different induction media used (see Materials and
methods) had little effect on strain virulence (between
12 and 20% over controls), the simplest treatment
(acetosyringone 20 mM) was used with satisfactory
results.
All A. rhizogenes strains used were able to induce
hairy roots on hemp seedlings in short time, although
with different frequency which ranged between
43% for AR10GUS and 98% for R1606 (Table 3).
Moreover, the addition of the GUS-I plasmid to the
strain AR10 did not appreciably alter the virulence
(Table 3). All hemp varieties considered were effec-
tively infected by both A. rhizogenes strains and
quantitative variability in their responses were also
observed (Table 4). CAN0221 and Delta405 achieved
higher frequency of root induction, CAN0111 gave
the highest root number and Futura77 attained
transformed roots with better growth.
correlate with their capacity to synthesize ethylene
(Table 5).
Wild-type transformed calli
An effective compatible interaction between all the
hemp varieties and A. tumefaciens strains used was
observed 58 days after inoculation as tumor-like
growths at infection sites. Tumors, only one per
infection site, were green in color, compact in texture
and 310 mm in diameter (Figure 2e), similar to
galls formed on Nicotiana tabacum (positive control;
Figure 2d). Differences between plant varieties or
bacterial strains in tumor induction frequencies were
moderate (Table 6). However, higher differences were
apparent in tumor growth, suggesting some differ-
ential response to Agrobacterium within this C. sativa
germplasm. Finally, tumors could be cultured in vitro
and vigorous tumor lines were obtained after six
weeks (data not shown).

Table 3. Response of CAN0221 hemp seedlings (drug variety) to in vivo infection with different A. rhizogenes strains.

Strain Response timea Response frequencyb Root numberc Root sized Root mean lifee

477 8.8 64 8.8 8.0 12.9

476 10.4 60 7.3 6.7 10.2
A424 11.3 52 6.7 6.9 13.4
AR10GUS 10.6 43 7.6 7.4 13.3
A4 9.8 63 8.6 9.0 13.4
478 9.4 67 8.1 8.5 14.0
AR10 10.3 53 9.7 7.8 14.9
R1601 9.4 98 7.9 7.9 8.7
LSD(0.05) 1.0 13 0.9 1.2 1.8

Note: Values are means of two independent experiments, with 3545 seedlings per strain infected at hypocotyls (two sites) in each experiment.
Data were subjected to analysis of variance and means were compared by the LSD test.
a
Days since inoculation till root emergence at inoculation sites.
b
Percentage of inoculated sites with hairy root induction.
c
Mean root number per infected site.
d
Mean length of roots (mm) emerged from each per infection site.
e
Days since roots emergence at infection sites till their death.
 Journal of Plant Interactions 317
Table 4. Response of hemp varieties of drug-type cannot utilize them as a source of carbon and
(CAN0111 and CAN0221) or fiber-type (Futura77, Delta- nitrogen because unlike Agrobacterium, they do not
405, Delta-llosa) to infection with A. rhizogenes strains. possess the genes for opine catabolism (Palazón et al.
A. rhizogenes Hemp Response Root Root 1997).
strain variety frequency number size

A4 CAN0111 38.0 9.2 9.1 Discussion

CAN0221 63.0 8.6 9.0 The interaction of A. rhizogenes with plants delivers a
Futura77 49.0 8.5 10.2 by-product, the hairy root, of high value for research
Delta-405 60.0 7.2 8.1 and practical purposes. Agrobacterium rhizogenes
Delta-llosa 42.2 6.8 6.8
infection of hemp has only been reported in a
AR10 CAN0111 26.0 9.1 7.8
CAN0221 59.0 6.2 6.7
preliminary assay carried out in our laboratory
Futura77 41.1 7.2 13.4 (unpublished) and the present work represents the
Delta-405 47.4 6.4 10.8 first reported efficient protocol for transformation of
Delta-llosa 33.0 5.9 5.9 C. sativa resulting in hairy roots cultures. It was
LSD(0.05) 8.7 0.8 1.4 found that hemp, a difficult to transform plant, was
susceptible to a number of A. rhizogenes wild-type
Note: Aseptically grown seedlings were inoculated at hypocotyls
(two sites) with the agropine type A4 and AR10 strains. Root
strains, which induced transformed roots in a short
number and size were recorded three weeks post infection. Values time, high number and with good vigor, and survival
are means of two independent experiments with 35 healthy rate (Tables 2 and 3, Figure 2a, 2b), but with different
inoculated plantlets per plant-bacterial combination. Data were frequency. The virulence of the strain (response
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subjected to analysis of variance and means were compared by the
LSD test. Other details as in Table 3.
RolABC, rolA, rolB, and rolC transgenic roots
The A. tumefaciens LBA4404 harboring rolABC,
rolA, rolB, or rolC genes (Table 1) cloned in the
binary vector pBin19 (which has a neomycin
phosphotransferase gene, nptII) was used to infect
hypocotyls of intact five-day-old plantlets grown
aseptically. The strains harboring the rolA, rolB,
and rolC alone induced weak plant responses, with
roots that were few in number and did not survive
(Table 7) and thus were not further considered in
this study. The construction including the three rolA,
B, and C genes, however, induced transgenic roots
with a frequency and other characteristics similar to
those observed with the complete T-DNA (Table 7).
Interestingly, in this case only the morphology
referred as thin was observed (Figure 2g). These
rolABC transgenic roots were shown to offer, relative
to wild-type transformed roots, a further advantage
of not accumulating opines. Opines, a group of
aminoacid derivatives, are synthesized in great
amounts in transformed cells, although these cells
Table 5. Growth (fresh weight), growth index (harvest FW
per inoculum FW) and ethylene evolution of hemp hairy
roots lines after four weeks of culture on MS medium.
FW Ethylene evolution
Root (mg Growth (pmol C2H4 g
morphology plate 1) index DW 1 h 1)
Thick 403953 100 178.3946.0
Thin 214937 53 172.3939.7
Note: Inoculum FW was 4 mg (ca. 15 mm length). For growth,
10 plates per root line were analyzed and averaged for each root
phenotype. For ethylene evolution, 10 representative samples of
each phenotype were considered. Data presented are mean
values9SE.
frequency), however, was not always related to the
good performance of the induced root clone, which is
consistent with results of Thimmaraju et al. (2008)
for red beet (Beta vulgaris) hairy roots. Seedling
hypocotyls proved to be the hemp tissue most
susceptible to the infection with A. rhizogenes, while
young leaves and cotyledons did not respond to the
infection, even when the bacteria were stimulated
with acetosyringone. Similar differences between
organs and/or explants have also been reported
for Taraxacum platycarpum (Lee et al. 2004),
Gentiana macrophylla (Tiwari et al. 2007), and
Whitania somnifera (Murthy et al. 2008). Host tissues
associated with actively dividing cells, i.e. nodes
and internodes, are postulated as good target sites
for successful transformation by Agrobacterium
(Chauduri et al. 2005). Moreover, since TL-DNA
confers the competence to respond to auxin (Shen
et al. 1988), plant material capable of auxin synthesis
(intact seedlings with shoot tips and young leaves) are
expected to show higher transformation rates.
In the course of this study over 20 hairy root lines,
which displayed the T-DNA-borne traits of fast and
phytohormone-independent growth, have been estab-
lished. Moreover hairy roots induced by AR10GUS
strain, harboring the binary p35SGUS intron plas-
mid, showed normal pattern of GUS positive staining
(Figure 2f), demonstrating cotransfer of the binary
vector and the expression of the foreign GUS gene.
All the above provides unequivocal proof that
hemp is transformable by Agrobacterium, supporting
and extending results of Feeney and Punja (2003),
who reported the transformation of cell suspensions
with A. tumefaciens. As previously indicated, two
morphological phenotypes were distinguished within
hairy root lines, referred as thin and thick mor-
phology (Figure 2g and 2h, respectively), which
were stable for more than two years. Similar mor-
phological phenotypes have also been described for
 318 I. Wahby et al.
Table 6. Response of hemp varieties of drug-type (CAN0111 and CAN0221) or fiber-type (Futura77, Delta-405, Delta-llosa)
to infection with A. tumefaciens strains C58 and IVIA251.

A. tumefaciens strain Hemp variety Response frequency Tumor FWa Tumor DWb Tumor sizec

C58 CAN0111 50.9 65.4 6.2 5.5

CAN0221 47.8 60.5 4.9 5.8
Futura77 40.6 60.5 4.5 4.5
Delta-405 63.0 25.4 2.1 3.9
Delta-llosa 50.9 49.9 4.3 5.4
IVIA251 CAN0111 54.8 76.4 6.9 5.9
CAN0221 33.7 90.0 9.4 6.4
Futura77 40.6 63.1 5.2 4.7
Delta-405 55.9 27.4 3.2 4.1
Delta-llosa 50.9 41.3 4.1 4.6
LSD(0.05) 8.7 11.7 1.6 0.8

Note: Tumor weight and size (only one tumor developed at wounded sites) were recorded three weeks after infection of hemp hypocotyls.
Data were subjected to analysis of variance and means were compared by the LSD test. Other details as in Table 3.
a,b
Mean fresh and dry weight (mg) of tumors per plant.
c
Mean size (mm of diameter) of tumors per plant.

Catharanthus roseus hairy roots, which further were rolA and rolC gene products can by themselves
correlated with the capacity for ethylene biosynthesis promote root formation in tobacco but not in
Downloaded by [86.13.167.155] at 00:55 21 June 2014

and the level of rolC gene expression (Palazon et al.
1998). On the other hand, in hemp hairy roots
showed higher growth rate (Table 5), branching and
root hair density (Figure 2g, 2h), but no correlation
between ethylene biosynthesis and root morphology
(Table 5) was observed. Finally, the dedifferentiation
ability leading to the so-called callus-like morphology
described for hairy roots of some Solanaceae plants
(Moyano et al. 1999), Pennax gingsen (Mallol et al.
2001), Withania somnifera (Bandyopadhyay et al.
2007), and Gentiana macrophilla (Tiwari et al. 2007),
have not been observed in this work.
It is well known that T-DNA-rol genes of Ri
plasmid are responsible for hairy root induction in
many plants. Furthermore, individual rol genes or
their combination have also been able to induce
transformed roots in a number of species including
Daucus carota (Capone et al. 1989), Nicotiana
tabacum (Palazón et al. 1997), and Atropa belladonna
(Bonhomme et al. 2000) among others. In hemp,
however, only the combination of the three rolABC
loci evoqued biological responses similar to those
elicited by the strains with the complete set of T-DNA
genes in the corresponding Ri plasmid, which showed
that the effect of these three rol genes was synergistic.
These results are consistent with the observation that
kalanchoe (Spena et al. 1987). According to these
authors, the products of rol loci must also interact
with plant factors controlling organ differentiation.
If these factors were similar or identical to those
responsible for the effects of growth hormones in
plants, it is understandable why they could be
different in different host species.
In conclusion, with the optimized experimental
conditions stably transformed tissues from hemp,
a difficult to transform plant, could be established.
An important aspect is that wild-type transformed
and rolABC transgenic root cultures are generally
characterized by high biosynthetic capacity and
genetic as well as biochemical stability (Toivonen
1993; Palazón et al. 1997). This feature, coupled with
rapid growth in simple media lacking phytohor-
mones, makes them especially suitable for biochem-
ical studies not easily undertaken with roots of intact
plants. Therefore, they can be considered to offer
better prospects for the investigation of biosynthetic
pathways and commercial production of secondary
metabolites including pharmacologically active THC.
Recently, biosynthetic pathways of the cannabinoids
have been successfully established. Moreover, a gene
which encodes for enzyme involved in THC biosynth-
esis, tetrahydrocannabinolic acid (THCA) synthase,

Table 7. Response of hemp seedlings of CAN0221 (drug variety) to in vivo infection with different A. tumefaciens LBA4404
harboring rolA, rolB, rolC or rolABC genes cloned in the binary vector pBin19.

Strain Response time Response frequency Root number Root size Root mean life

LBA-rolA 18.091.5 3.490.4 1.290.3  a

LBA-rolB 17.092.3 4.590.3 2.190.1  
LBA-rolC 18.191.9 1.490.4 2.090.3  
LBA-rolABC 10.590.5 74.495.0 7.190.6 7.590.7 12.790.7

Note: Data represent mean values9SE of two independent experiments with 3545 seedlings per strain infected at hypocotyls (two sites) in
each experiment. Other details as in Table 3.
a
Roots did not grow beyond 1 mm and died.
 Journal of Plant Interactions
have been isolated from C. sativa plants and its
molecular cloning and characterization have been
reported (Sirikantaramas et al. 2007). This opens the
way for biotechnological production of pharmacolo-
gically active THC using wild-type transformed or
rolABC transgenic root cultures of C. sativa.
Acknowledgements
The authors would like to thanks Drs Rafael Salto
and Natalia Sevillano (Department of Biochemistry and
Molecular Biology II, University of Granada) for assistance
with PCR analysis. Financial support was obtained
from Junta de Andalucı́a (PAI Group AGR-205, Project
A42/00).
References
Bandyopadhyay M, Jha S, Tepfer D. 2007. Changes in
morphological phenotypes and withanolide composi-
tion of Ri-transformed roots of Withania somnifera.
Plant Cell Rep. 26:599609.
Downloaded by [86.13.167.155] at 00:55 21 June 2014
Bonhomme V, Laurain-Mattar D, Lacoux J, Fliniaux M-A,
Jacquin-Dubreuil A. 2000. Tropane alkaloid produc-
tion by hairy roots of Atropa belladonna obtained after
transformation with Agrobacterium rhizogenes 15834
and Agrobacterium tumefaciens containing rolA, B, C
genes only. J Biotechnol. 81:1518.
Caba JM, Recalde L, Ligero F. 1998. Nitrate-induced
ethylene biosynthesis and the control of nodulation in
alfalfa. Plant Cell Environ. 21:8793.
Caba JM, Centeno ML, Fernández B, Gresshoff PM,
Ligero F. 2000. Inoculation and nitrate alter phyto-
hormone levels in soybean roots: differences between
a supernodulating mutant and wild type. Planta.
211:98104.
Caballero JJ, Girón MD, Vargas AM, Sevillano N,
Suárez MD, Salto R. 2004. AU-rich elements in the
mRNA 3?-unstranslated region of the rat receptor for
advanced glycation end products and their relevance to
mRNA stability. Biochem Biophys Res Comm.
319:24755.
Caetano-Anollés G, Schlarbaum SE, Trigiano RN. 1999.
DNA amplification fingerprinting and marker
screening for pseudo-testcross mapping of flowering
dogwood (Cornus florida L). Euphytica. 106:20922.
Capone I, Spanò L, Cardarelli M, Bellincampi D, Petit A,
Costantino P. 1989. Induction and growth properties
of carrot roots with different complements of
Agrobacterium rhizogenes T-DNA. Plant Mol Biol.
13:4352.
Chauduri KN, Ghosh B, Tepfer D, Jha S. 2005.
Genetic transformation of Tylophora indica with
Agrobacterium rhizogenes A4: growth and tylophorine
productivity in different transformed root clones. Plant
Cell Rep. 24:2535.
Chriqui D, Guivarc’h A, Dewitte W, Prinsen E, Onkelen H.
1996. Rol genes and root initiation and development.
Plant Soil. 187:4755.
Clarke RC. 1999. Botany of the genus Cannabis. In: Rinalli
P, editor. Advances in Hemp Research. New York:
Haworth Press; p. 118.
Feeney M, Punja ZK. 2003. Tissue culture and
Agrobacterium-mediated transformation of hemp
319
(Cannabis sativa L.). In Vitro Cell Dev Biol Plant.
39:57885.
Gamborg OL, Miller RA, Ojima K. 1968. Nutrient
requirements of suspension cultures of soybean root
cells. Exp Cell Res. 50:1518.
Gao J, Lee JM, An G. 1991. The stability of foreign protein
production in genetically modified plant cells. Plant
Cell Rep. 10:5336.
Hu Z-B, Du M. 2006. Hairy root and its application
in plant genetic engineering. J Integr Plant Biol.
48:1217.
Jefferson RA. 1987. Assaying chimeric genes in plants:
the GUS gene fusion system. Plant Mol Biol Rep.
5:387405.
Johnson P. 1999. Industrial hemp: a critical review of
claimed potentials for Cannabis sativa. Tappi J.
82:11323.
Lee MH, Yoon ES, Jeong JH, Choi YE. 2004. Agro-
bacterium rhizogenes-mediated transformation of
Taraxacum platycarpum and changes of morphological
characters. Plant Cell Rep. 22:8227.
Ligero F, Poveda JL, Gresshoff PM, Caba JM. 1999.
Nitrate- and inoculation-enhanced ethylene biosynth-
esis in soybean roots as a possible mediator of
nodulation control. J Plant Physiol. 154:4828.
Linger P, Ostwald A, Haensler J. 2005. Cannabis sativa L.
growing on heavy metal contaminated soil: growth,
cadmium uptake and photosynthesis. Biol Plant.
49:56776.
Liste H-H, Prutz I. 2006. Plant performance, dioxygenase-
expressing rhizosphere bacteria, and biodegradation
of weathered hydrocarbons in contaminated soils.
Chemosphere. 62:141120.
Mallol A, Cusidó RM, Palazón J, Bonfill M, Morales C,
Piñol MT. 2001. Ginsenoside production in different
phenotypes of Panax ginseng transformed roots.
Phytochem. 57:36571.
Moyano E, Fornalé S, Palazón J, Cusidó RM, Bonfill M,
Morales C, Piñol MT. 1999. Effect of Agrobacterium
rhizogenes T-DNA on alkaloid production in
Solanaceae plants. Phytochem. 52:128792.
Murashige T, Skoog F. 1962. A revised medium for rapid
growth and bioassay with tissue culture. Plant Physiol.
15:47397.
Murthy HN, Dijkstra C, Anthony P, White DA, Davey
MR, Power JB, Hahn EJ, Paek KY. 2008. Establish-
ment of Withania somnifera hairy root cultures for
the production of Withanolide A. J Integr Plant Biol.
50:97581.
Palazón J, Cusidó RM, Roig C, Piñol MT. 1997. Effect of
rol genes from Agrobacterium rhizogenes TL-DNA on
nicotine production in tobacco root cultures. Plant
Physiol Biochem. 35:15562.
Palazon J, Cusidó RM, Gonzalo J, Bonfill M, Morales C,
Piñol MT. 1998. Relation between the amount of rolC
gene product and indole alkaloid accumulation in
Catharanthus roseus transformed root cultures. J Plant
Physiol. 153:7128.
Pythoud F, Sinkar VP, Nester EW, Gordon MP. 1987.
Increased virulence of Agrobacterium rhizogenes
conferred by the vir region of pTiBo542: application
to genetic engineering of poplar. Biotechnology.
5:13237.
 320 I. Wahby et al.
Shen WH, Petit A, Guern J, Tempé J. 1988. Hairy roots are
more sensitive to auxin than normal roots. Proc Natl
Acad Sci USA. 85:341721.
Shi G, Cai Q. 2010. Zinc tolerance and accumulation in
eight oil crops. J Plant Nutr. 33:98297.
Sirikantaramas S, Taura F, Morimoto S, Shoyama Y. 2007.
Recent advances in Cannabis sativa research: biosyn-
thetic studies and its potential in biotechnology. Curr
Pharm Biotechnol. 8:23743.
Spena A, Schmülling T, Koncz T, Schell J. 1987. Indepen-
dent and synergistic activity of rolA, B and C loci in
stimulating abnormal growth in plants. EMBO J.
6:38919.
Stiller J, Martirani L, Tuppale S, Chian RJ, Chiurazzi M,
Gresshoff PM. 1997. High frequency transformation
and regeneration of transgenic plants in the model
legume Lottus japonicus. J Exp Bot. 48:135765.
Thimmaraju R, Venkatachalam L, Bhagyalakshmi N. 2008.
Morphometric and biochemical characterization of
red beet (Beta vulgaris L.) hairy roots obtained after
single and double transformations. Plant Cell Rep.
27:103952.
Tiwari RK, Trivedi M, Guang ZC, Guo G-Q, Zheng G-C.
Downloaded by [86.13.167.155] at 00:55 21 June 2014
2007. Genetic transformation of Gentiana macrophylla
with Agrobacterium rhizogenes: growth and production
of secoiridoid glucoside gentiopicroside in transformed
hairy root cultures. Plant Cell Rep. 26:199210.
Toivonen L. 1993. Utilization of hairy root cultures for
production of secondary metabolites. Biotechnol Prog.
9:1220.
Toonen MAJ, Maliepaard C, Reijmers TH, Van der Voet
H, Mastebroek HD, Van den Broeck HC, Ebskamp
MJM, Kessler W, Kessler RW. 2004. Predicting the
chemical composition of fibre and core fraction of
hemp (Cannabis sativa L). Euphytica. 140:3945.
Turner CE, Elsohly MA, Boeren E. 1980. Constituents of
Cannabis sativa L. XVII. A review of the natural
constituents. J Nat Prod. 43:169234.
Van Haute E, Joos H, Maes M, Warren G, Van Montagu
M, Schell J. 1983. Intergeneric transfer and exchange
recombination of restriction fragments cloned in
pBR322: a novel strategy for the reversed genetics of
the Ti plasmids of Agrobacterium tumefaciens. EMBO
J. 2:4117.
Vincent JM. 1970. A manual for the practical study of root
nodule bacteria. IBP Handbook No. 15. Oxford:
Blackwell.
White FF, Taylor BH, Huffman GA, Gordon MP,
Nester EW. 1985. Molecular and genetic analysis of
the transferred DNA regions of the root-inducing
plasmid of Agrobacterium rhizogenes. J Bacteriol.
164:3344.