Engineering, 2012, 5, 4-7
doi:10.4236/eng.2012.410B002 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
Agrobacterium Tumefaciens Based Transformation of
Pelargonium x Hortrum cv. ‘Samba’ with An-
ti-1-aminocyclopropane-1-carboxylate Synthase cDNA t o Re-
gulate Ethylene Biosynthesis
Rajinder S. R anu1, Jianguo Fan1, Sarada Krishnan1, Pradeep Agarwal1, Amitva Mitra2
1Colorado Sate University, Laboratory of Plant Molecular Biology/Biotechnology, Fort Collins, CO USA
2Department of Plant Pathology, The University of Nebraska, Lincolin, NE USA
Received 2012
Phytohormone, ethylene plays an important role in plant growth and development including fruit ripening and flower senescence.
The synthesis of 1-aminocylo-propane-1- carboxylate (ACC), the immediate precursor of ethylene, from S-adenosyl-methionine is
catalyzed by AC C s ynthase; and which is also a rate limiting step in the ethylene biosynthetic pathway. Therefore, it plays a key role
in ethylene biosynthesis and the genes that code for ACC synthase are of special interest. Moreover, in zonal geraniums, ethylene
bursts rel eased from cuttings can have profound impact on the viability of explants for plant propagation. Biotechnological approach
involving genetic modification that may reduce ethylene levels has potential for increasing the shelf-life of cuttings for plant propa-
gation . These con sid erat ions have led us to clone sever al cDNA of ACC synth ase gen es fro m Pel argon ium x hor toru m cv. ‘Sincerity’.
To transform geranium cells with Agrobacterium tumefaciens an in vitro regeneration system was developed using very young peti-
ole explants. An Antisense construct of ACC synthase cDNA (PHSacc41) ligated into binary vector pAM696 was introduced into A.
tumefaciens EHA 105 cells. Petiole explants were incubated with the Agrobacterium for 15 min and then co-cultivated for several
days on MS medium containing 5 mM BAP and 1 mM IAA in the dark without the antibiotics. Selection for transformants was car-
ried ou t in the presence o f kanamycin and timenti n. Tran sgenic pl antlets gen erated were examin ed for in serted gen e cassette by PCR
and Southern blotting. Recovery of positive transformants that survived selection suggested that it is possible to transform and intro-
duce genes via transformation in hybrid geraniums for genetic modification.
Keywords: ACC Synthase; Ethylene Biosynthesis; Transformation; Geranium Regeneration; Agrob a cteri um Tume faciens
1. Introduction
Pelargonium sp., popularly known as geraniums are grown for
their colorful flowers and to a limited extent for their scented
foliage. They are important ornamental plants that are widely
grown in North America and Europe [1-3]. Because of their
spectacular flowers P. zonale hybrids are some the most
important varieties and command a substantial portion of the
market share of over $300 millions. Unlike seed geraniums,
hybrid varieties are vegetatively propagated [2,3], therefore,
improvements through conventional breeding are difficult and
time-consuming [4]. Contemporary plant biotechnology provides
a potential alternative to conventional breeding through genetic
modification for plant improvement with potential for savings
in time and cost to the growers.
The phytohormone, ethylene plays an important role in plant
growth and development including fruit ripening and flower
senescence [5-10]. In geraniums ethylene bursts released by
plants during shipping (stress) and when cuttings are prepared
for vegetative propagation are a major source of losses to
growers (1). Genetic modification may provide a potential
means by which ethylene bursts in the plants may be regulated
either through antigene technology or through modification of a
key gene(s) promoter(s). In the present investigation we have
attempted to develop an Agrobacterium tumefaciens based
transfor mation system in the vegetatively propagated geranium
with an anti-1-aminocyclopropane-1-carboxylate (ACC) syn-
thase cDNA con str uct in a Agrobacterium binary vector [11,12]
using a plant regeneration system developed for vegetatively
propagated geraniums. Our rationale being, that ACC synthase
catalyses the rate limiting step in the ethylene biosynthetic
pathway [6] and modulation of this step may result in regula-
tion of ethylene biosynthesis. These considerations had previ-
ously led us to the cloning of several cDNA of ACC synthase
genes from P x hortorum cv. ‘Sincerity’ [13,14]. Results from
the present studies show that transformants which survived
kanamycin selection show integration of NPT II gene in trans-
genic plants both by PCR analysis and by southern hybridiza-
2. Materials and Methods
2.1. Explants and Ba c terial Str ains
Petioles of very young leaves were used as explants from P. x
hortorum cv. ‘Samba’ (provided by Pelfi Fisher Geranium USA,
Boulder Colorado). The bacterial strains of Agrobacte- rium
tuma feciens, LBA 4404 or LBG 66 or EHA 105 were used.
Copyright © 2012 SciRes. E NG
2.2. Growth of A. Tumefaciens
A single colony of A. tumefaciens was gro wn in YEP medium
containing 25 µg/ml of kanamycin and 10 µg/ml of tetracycline
containing pAM-41 plasmid and allowed to grow overnight at
28º. The next day one of the cultures was transferred to a fresh
YEP medium (25 ml) and allowed to grow on a shaker to an
O∙D∙ of about O.8-1.0. The cells were harvested by centrifuga-
tion at 6000 rpm at 28º and suspended in 20 ml of sterile dis-
tilled water.
2.3. Preparation of Explants
Young, healthy petiole explants were harvested and sterilized
with 15% Clorox for 15 min with frequent agitation and then
washed three times with sterile distilled water. The explants
were cut into 2-3 mm size and then transferred to a Petri plate
containing suspension of Agrobacterium (prepared previously)
with gentle shaking. The explants were removed and blotted
dry over a sterile paper towel and then transferred to a Mure-
shige and Skoog (MS) medium (15) containing 5 µM benzyl-
aminopurine (BAP) and 1 µM indole-3-acetic acid (IAA); and
were incubated at 25º in the dark for 2-3 days. The explants
were remov ed and wiped cl ean wi th a ster ile pap er towel. Th ey
were then transferred to fresh MS medium containing 5 µM
BAP and 1 µM IAA; and 100 µg/ml of kanamycin and 200
µg/ml of timentin. After three weeks, petiole explants that
showed growth of green buds or shoots were transferred to MS
medium containing 0.44 µM BAP and 0.1 µM IAA; and 100
µg/ml of kanamycin and 200 µg/ml of timentin. After three
weeks, well grown shoots were separated and transferred to
fresh medium containing 0.44 µM BAP and 0.1 µM IAA; and
100 µg/ml of kanamycin and 200 µg/ml of timentin. Shoots that
attain ed a heigh t of abou t 1 cm were transferred to cultu re tubes
containing fresh MS medium and 0.1 µg/ml IAA but without
the antibiotics to allow for rooting to take place. The rooted
plants were moved into pots containing 1:1 ratio of vermiculite
and perlite.
2.4. Preparation of Vector Construction Containing
Anti-ACC synthase cDNA
ACC synthase cDNA (PHSacc41) (13,14) maintained in
pBK-CMV plasmid was at first digested with Not I. The ends
were filled with dCTP and dGTP using Klenow DNA polyme-
rase in buffer containing 0.2 mM Tris-HCl (pH 7.6), 0.2 mM
dCTP and dGTP and 30 units of Klenow DNA polymerase.
Samples were incubated at 30º for 30 minutes. The DNA was
extracted using phenol/chloroform (1:1) and again with chloro-
form. DNA was preci pitated with ethanol in the presence o f 50
mM sodium acetate and finally washed with 70% ethanol.
PHSaac 41 cDNA was then released from the vector by diges-
tion with BamH I and then gel purified. The binary vector
DNA was digested with Hpa I and Bgl II and PHSacc41 was
ligated into the vector and used to transform Escherichia coli
cells. Transformants were isolated and checked for the ACC
synthase cDNA insert by PCR. Results showed successful in-
sertion both by PCR as well as from the size of the DNA in-
serted. The vector was named p AM 696-PHSacc-41 .
2.5. Mobilization of binary vector pAM
696-PHSacc-4 1 into A. tumefaciens
A. tumefaciens strain LB 4044 or EHA 103 was gown in YEP
medium at 28º overnight; next day 1 ml aliquot from it was
transferred to fresh medium (25 ml) and allowed to grow to an
OD of 0.2-0.4. The cells were harvested by centrifugation and
resuspended in 1/10 the volume of fresh YEP medium and 0.1
ml aliquots were transferred to eppendorf tubes and 1 ml of
binary vector construct was added to the solution and it was
then frozen in liquid nitrogen. The sample was allowed to thaw
to room temperature and then platted on YEP agar containing
20 µg/ml of tetracycline. Transformant colonies were identified
and further confirmed to carry plasmid AM-696-PHSacc41 by
PCR using primers designed for NPT II and PHSacc41 genes.
These bacteria were used for plant transformation. The same
procedure was used to introduce plasmid constructs into other
strains of A. tu m efacien s (11).
2.6. Polymerase Chain Re action and So uthern
Hybridizat ion
Polymerase chain reaction (PCR) was used to detect the inte-
gration of cassette containing NPT II gene and ACC synthase
into the plant genome. DNA from young leaves of transgenic
and control plants was isolated according to Taylor (16 ). The
NPT II primer sequences used were: 5’-CTG AAT GAA CTG
TA GC-3’ which is expected t o yield a fragment of 50 0 bp from
the NTP II coding sequence. The primers used to detect inte-
grated an ti ACC synth ase gen es were: 5 ’-CCC AAA TTT GGG
from the 5’end which is part of the 35’s CaMV promoter an d is
expected to amplify a DNA fragment of 600 bp. The DNA from
transgenics and control plants were also used for southern hy-
bridization. Southern transfers were prepared by digestion of
DNA (10 µg) with Bam HI and electorphoresed in 0.8% aga-
rose gel. Hybridizations were carried out with [32p] labeled
NTP II probe developed using the random primer labeling Kit
from Amersham according to manufacturer’s directions.
3. Results and Discussion
In order to transform geranium cells with A. umefaciens an in
vitro regeneration system has been developed using very young
petiole explants in the presence of zeatin or BAP and IAA
(Figures 1 and 2). These studies showed that the regeneration
efficiencies vary considerably with different cultivars; varying
from 10-60% (see also17) but with cultivar Samba nearly
90-100% of the explants regenerated. It should be noted that
with zeatin regeneration efficiencies in general were a little
better than BAP but differences were not that significant. Sam-
ba with nearly 100% regeneration efficiency thus provides a
system that could be used to determine whether genetic trans-
formation of hybrid geraniums with Agrobacterium is feasible.
Antisense constructs of ACC synthase cDNA (pPHSacc41)
(14) were ligated into binary vector pAM696 (Figure 3) and
Lanes fro m left to right are: Lane 1, DN A ladder; l ane 2, DNA
from control plant and lanes 3-11 DNA from transformants.
introduced into A. tumefaciens EHA105 cells. Petiole explants
Copyright © 2012 SciRes. ENG
were incubated with Agrobacterium for15 min and then co-
cultivated for several days on MS medium containing 5 mM
BAP and 1mM IAA in the dark without the antibiotics. Selec-
tion for transformants was carried out in the presence of kana-
mycin and timentin. After about three weeks, petioles with
Figure 1. Growth and expansion of shoots from petiole explants
in the presence and abs ence of horm on e zeat in.
Figure 2. Rooting and growth of plantlets in the presence of IAA.
Shoot buds separated from mother explants were cultured indivi-
dually. Within 3-4 weeks it developed a healthy root system.
Figure 3. A diagrammatic representation of antisense of ACC syn-
thase cDNA (pPHSacc41 ) construc t in plas mid vector.
Figure 4. PCP amplification of NTP II gene from DNA of control
and transformed plantl ets.
green buds or shoots were transferred to fresh MS medium
containing 0.44 mM BAP and 0.1 mM IAA and finally on me-
dium containing 0.1 mM IAA for rooting (Figure 2). Plantlets
generated after transformation were examined for the insertion
of gene cassette i nto the plant by PCR and Southern hybridiza-
tion. Results showed nearly 50% of the tranformants that sur-
vived selection were positive by P CR (Figure 4) as well as by
Southern hybridization for the NTP II gene (results not shown);
suggesting that a successful transformation of geranium in cul-
ture has been established. These plants are being evaluated
further in the greenhouse.
Previously, transformation of other types of geraniums has
been carr ied out with variable success (18-20). Our findings on
the development of a regeneration and Agrobacterium based
transformation system with hybrid geraniums (sometimes also
called zo nale geraniums) on e of the most desirable of the gera-
nium species, now provides a system for further genetic mod-
ifications of these ornamental plants. Future modification may
include flower color change, introduction of desirable flower
scents and enhanced disease resistance to bacterial or viral pa-
thogens and others, limited only by imagination.
4. Acknowledgements
This research was supported by a research grant from Tagawa
Greenhouses, Inc of Brighton, Colorado.
[1] White, J. (1993) Geranium, IV. Geneva, IL: Ball Publishing.
[2] Wood, H.J. (1966) Pelargoniums: a complete guide to their
cultivation. UK: Wheato n & Co.
[3] Moore, H.E. Plant classification 3: Taxonomy in cultivation .In:
Mastalerz, J.M., ed. Geraniums, a Penn State manual.University
Park, Pennsylvania: Pennsylvania Flower Growers. 1971:14-15.
[4] Horn, W. (1994) Interspecific crossability and inheritance in
Pelargonium. P l an t Bree d. 113:3-17.
[5] Mattoo AK, Suttle JC (1991) Plant Hormone Ethylene,CRCPress,
Boca Rato n, FL, 352 pp
[6] Yang, S.F. & Hoffman, N.E.(1984) Ethylene biosynthesis andits
regulation in higher plants. Annu. Rev. Plant Physiol.
[7] Ranu, RS., Wang D. Fan J. Role of
1-aminocyclopropane-1-caboxylate (ACC) synthase genes and
genes involved in ethylene signal transduction and rose flower
Copyright © 2012 SciRes. E NG
senescence. Floriculture and Ornamenral Biotechnology Spe-
cia l is s ue on roses. 3, 104-110, 2009.
[8] Ranu RS. Chakrabarti, S. Lysa-Anne M. Volpe L-A. M., Brown
B., Wang D., Fan, F. 1-Aminocyclopropane-1-carb oxylat e (AC C)
Synthases of Rosa hybrida: Analysis of Genomic Gene Structure
and the Cis-Acting Regulatory Elements in their Promoters.
Genes , Gen o mes a nd Genetics. 2, 68-76, 2008
[9] Ma N, Cai L, Lu WJ, Tan H, Gao JP (2005) Exogenous ethylene
influences flower opening of cut roses (Rosa hybrida) by regu-
lating the genes encoding ethylene biosynthesis enzymes.
Scie nce in Ch i na , Ser ies C 48, 434-444
[10] Ma N, Tan H, Liu X, Li Y, Gao J (2006) Transcriptional regula-
tion of ethylene receptor and CTR genes involved in ethy-
lene-induced flower opening in cut rose (Rosa hybrida) cv. Sa-
mantha. Journal of Experimental Botany 57, 2763-2773
[11] An, G., Ebert, P.R., Mitra, A. & Ha, S.B. (1988) Binary vectors
in Plant M olecula r Biology Manual, Ed, Klawcr Academic Pub-
lisher s , Dor drect, Bel g ium A 3, p p. 1-19.
[12] Mitra, A. & Higgins, D.W. (1994) The Chlorella virus adenine
methyltransferase gene promoter is a strong promoter in plants.
Plant Mol. Biol. 26:85-93
[13] Fan, J., Wang, D. & Ranu, R.S. (1996) Clon ing an d cha ract eri za-
tion of cDNA encoding 1-aminocyclopropane-1-carboxylate
synthases from Pelargonium x hortorum cv. Sincerity. FASEB
[14] Fan J, Wang D, Ranu RS (2007) Characterization and expression
of 1-aminocyclopropane-1-carboxylate(ACC) synthase from ge-
ranium (Pelargonium x hortorum cv Sincerity). Journal o f Plant
Bioc hemistry and Biotec hnology 16, 87-96
[15] Murashige, T. & Shook, F. (1962) A revised medium for
rapid growth and bioassays with tobacco tissue cultures. Physiol.
Plant. 15:473-497.
[16] Taylor, B.H., Manhart, J.R. & Amasino, R.R. (1993) Isolation
and char acteriza tion of plant DNA. In: Glick, B.R. & Thomp-
son, C.R. (eds) Methods in plant molecular biology and biotech-
nology. C RC Press , Boca Rat on, pp 37-38.
[17] Ag arwal, P. Ranu, RS.(2000) Regenration of plantlets from leaf
and p etiole exp la nt s of Pelargonium x hortorum. In viro CellDev
Biol-Plant 3 6,392-396
[18] Boase, M.R., Deroles, S.C., Winefield, C.S., Butcher, S.M.,
Borst, N.K. & Butler, R.C. (1996) Genetic transformation of
regal pelargoinium (Pelargonium x domesticum Dubonnet) by
Agrobacterium tumefaciens. Plant Sci. 121:47-61.
[19] Krishnaraj, S., Bi, Y.-M. & Saxena, P.K. (1997) Somatic em-
bryogenesis and Agrobacterium-mediated transformation system
from scented geranium (Pelargonium sp. Frensham). Planta.
[20] Pellegrineschi, A., Damon, J.P., Valtorta, N., Paillard, N. &
Tepfer, D. (1994) Improvement of ornamental characters and
fragrance production in lemon scented geranium through genetic
transformat ion by Agrobacterium rhizogenes. Biotech. 12 :6 4-68.