AGROBACTERIUM-MEDIATED TRANSFORMATION OF … · 2015-12-23 · S. MILOŠEVIĆ: AGROBACTERIUM...

___________________________

Corresponding author: Snežana Milošević,Institute for Biological Research, University of

Belgrade, Bul. despota Stefana 142, 11060 Belgrade, Serbia, tel: + 381 11 2078393, fax: + 381 11

2761433, e-mail: [email protected]

UDC 575

DOI: 10.2298/GENSR1503149M Review paper

AGROBACTERIUM-MEDIATED TRANSFORMATION OF ORNAMENTAL SPECIES:

A REVIEW

Snežana MILOŠEVIĆ*, Aleksandar CINGEL, Angelina SUBOTIĆ

Institute for Biological Research „Siniša Stanković“, University of Belgrade, Belgrade

Milošević S., A. Cingel, A.Subotić (2015): Agrobacterium-mediated

transformation of ornamental species: a review- Genetika, Vol 47, No. 3, 1149-1164.

Integration of desirable traits into commercial ornamentals using genetic engineering

techniques is a powerful tool in contemporary biotechnology. However, these techniques

have had a limited impact in the domain of ornamental horticulture, particularly

floriculture. Modifications of the color, architecture or fragrance of the flowers as well as

an improvement of the plant tolerance/resistance against abiotic and biotic stresses using

plant transformation techniques, is still in its infancy. This review focuses on the

application of Agrobacterium-mediated transformation, a major plant genetic engineering

approach to ornamental plant breeding and the impact it has had to date.

Key words: Agrobacterium rhizogenes; A. tumefaciens; genetic transformation;

ornamental species

Abbreviations: ACC – 1-aminocyclopropane-1-carboxylic acid; Ace-AMP1 –

antimicrobial protein gene; ChiA – chitinase gene; CMV – Cucumber mosaic virus;

dsRNA – double stranded RNA; INSV – Impatiens necrotic spot virus; PAP – pokeweed

antiviral protein; R – resistance genes; RCC2 – rice chitinase gene; RNAi – RNA

interference; rol – root oncogenic loci; TMV – Tobacco mosaic virus; TP – transit

peptides; TSWV – Tomato spotted wild virus; WD – Wasabi japonica defensin gene.

INTRODUCTION

Conventional plant breeding has commonly been utilized in producing new and elite plant

cultivars. However, this is a long and laborious process which can take up to 10 years or more.

Plant transformation technology, developed some 30 years ago, appeared as a promising

1150 GENETIKA, Vol. 47, No 3, 1149-1164, 2015

alternative to classical plant breeding and the first genetically transformed plants reached the

markets during the mid-1990s.

During the recent years, horticulture has become one of the major fields in development

of agriculture sector and the increasing economic importance of ornamentals brings a growing

interest for ornamental plant breeding worldwide. Benefits of horticulture on economic,

environmental or general welfare are accredited and well described (HALL and DICKSON, 2011).

Consumers’ demand triggers continuous development of new and improved genotypes with altered

plant morphology or new flower color, improveds post-harvest life or other novel beneficial traits

(Figure 1). Additionally, growers are continuously interested in superior plant production

performance such as shorter production time and increased resistance to pests and pathogens.

Figure 1. Biotechnological prospects for Agrobacterium-mediated research

Petunia was the first ornamental plant subjected to A. tumefaciens-mediated

transformation (HORSCH et al., 1985; MEYER et al., 1987). Transformation of petunia resulted in

transgenic plants with orange flowers, as a consequence of pelargonidin accumulation (MAYER et

al., 1987), and ever since then the genetic transformation has been employed in ornamental plant

improvement. The objectives for changing a plant's traits by genetic transformation are varied: to

provide resistance to diseases, insects or herbicides; to change the flowering and plant architecture,

as well as expression of genes associated with the biosynthesis of pigments, to attain a long post-

harvest life, especially for cut flowers.

This review focuses on advances in the Agrobacterium transformation of ornamental

species and discusses the prospects for genetic engineering in horticulture.

Disease resistance

Damage caused by pathogens during production, storage or distribution can render ornamentals

unmarketable, and hence viral, bacterial and fungal disease management is paramount in the

production of ornamental than in any other sector of agriculture.

Production of virus free ornamentals can increase floral quality and yield, and can reduce

production costs (HAMMOND, 2006; MILOŠEVIĆ et al., 2011a, 2012). At least 125 different viruses

have been identified to cause diseases in ornamental plants (COHEN, 1995). A number of viruses

are transmitted by insect vectors, such as aphids, whiteflies or thrips, while chemicals commonly

S. MILOŠEVIĆ: AGROBACTERIUM MEDIATED TRANSFORMATION OF ORNAMENTAL PLANTS 1151

used against these vectors can have a detrimental impact on the flower quality. These chemicals

are often expensive and toxic to a wide range of organisms including humans (TANAKA et al.,

2005); in addition, viral diseases are more difficult to control by conventional chemical methods.

Planting disease-resistant cultivars is a promising way to control viral diseases. There are

essentially three sources of transgenes for protecting the plants against viruses: natural resistance

genes, genes derived from viral sequences (pathogen-derived resistance), and genes from various

other sources, including R (resistance) genes, enzymes and inhibitors of plant origin and some

mammalian proteins such as 2-5A synthetase and scFv (SUDARSHANA et al., 2006; HSU, 2006;

KYRSHENKO et al., 2007).

In 1925, pokeweed (Phytolacca americana L.) extracts were shown to prevent Tobacco

mosaic virus (TMV) transmission (DUGGAR and ARMSTRONG, 1925). Since then, pokeweed

antiviral protein (PAP) has been recognized as an important antiviral agent. Removing a specific

adenine base from the large ribosomal RNA, PAP acts as a ribosome inactivating protein. LI et al.

(2013) have transferred modified PAP gene into petunia cells with A. tumefaciens and obtained

transgenic plants immune or highly resistant to Cucumber mosaic virus (CMV).

Incorporating viral nucleotide sequences (coat protein, replicase or movement protein

genes) into the host plant genome can enhance plant resistance to viruses (KHAN and LIU, 2009).

Using sense or antisense versions of the coat protein or replicase genes, several research

groups have achieved enhancements in viral resistance in a number of ornamentals, including

chrysanthemum (SHERMAN et al., 1998; YEPES et al., 1999), gladiolus (HAMOND and KAMO, 1995a,

1995b; KAMO et al., 2000a, 2000b), lily (LANGEVELD et al., 1997) and various orchids (DEROLES et

al., 2002).

Also, virus resistance has been documented for Gladiolus transformed with Cymbidium

mosaic virus coat protein or mutated replicase gene (KAMO et al., 2010). SHERMAN et al. (1998)

used A. tumefaciens to transform chrysanthemum with genes of a dahlia isolate of Tomato spotted

wild virus (TSWV), and several lines were shown to be fully resistant to challenge by viruliferous

thrips carrying a virulent chrysanthemum isolate of TSWV. YEPES et al. (1999) utilized the N-

genes of TSWV, and Impatiens necrotic spot virus (INSV) as transgenes to improve plant

resistance. KIM et al. (1995) reported that petunia plants transformed with a cDNA clone of CMV

I17N-satellite RNA exhibited a delay in the emergence of disease symptom and could possibly

offer a protection against infection by CMV and TMV, and other unrelated viruses.

The majority of known plant viruses have RNA genomes. ISHIDA et al. (2002) focused on

methods to inhibit viral RNA replication. Since these viruses replicate via a double stranded RNA

(dsRNA) intermediate, they are thought to be good targets for suppression by dsRNA specific

ribonuclease, pac1 (ISHIDA et al., 2002; TOGURI et al., 2003, 2008; OGAWA et al., 2005;

MILOŠEVIĆ, 2010, 2013; MILOŠEVIĆ et al., 2013). Pac1 gene that was isolated from

Schizosaccharomyces pombe, can confer simultaneous tolerance against virus and viroid diseases.

Several groups are working to introduce virus resistance into various ornamentals, including

Lilium, Gladiolus, Chrysanthemus, Gerbera, Osteospermum, Impatiens, Cyclamen and various

orchids (URBAN et al., 1994; YEPES et al., 1999; ISHIDA et al., 2002; TOGURI et al., 2003, 2008;

OGAWA et al., 2005; BAXTER, 2005; DAN et al., 2010; MILOŠEVIĆ, 2010, 2013; MILOŠEVIĆ et al.,

2010, 2011b, 2011c, 2013). Transgenic Chrysanthemum plants bearing pac1 showed significantly

lower infection frequency with TSWV than control plants (OGAWA et al., 2005). Expression of

pac1 transgene provides complete and effective protection against TSWV in I. walleriana (Figure

2), while transgenic tobacco lines exhibit either complete resistance or enhanced tolerance to

1152 GENETIKA, Vol. 47, No 3, 1149-1164, 2015

TSWV, evident as lower infection frequency (MILOŠEVIĆ, 2010, MILOŠEVIĆ et al., 2013).

Additionally, the introduction of pac1 may protect plants from viroids, such as Potato spindle

tuber viroid that can be digested by pac1 gene product in vitro (SANO et al., 1997; ISHIDA et al.,

2002) or Chrysanthemum stunt viroid (ISHIDA et al., 2002; TOGURI et al., 2003, OGAWA et al.,

2005).

Figure 2. Pac1-transformed and control, untransformed I. walleriana plants challenged with

TSWV by manual in vitro inoculation. Pac1-impatiens were completely resistant to TSWV (a)

while untransformed impatiens plants were exhibited symptoms including chlorotic spots, blisters,

local necrotic lesions and leaf deformations (b).

RNA silencing has been used in transgenic poinsettia where the insertion of the virus

derived hairpin RNA gene has resulted in an increased resistance against Poinsettia mosaic virus

(CLARKE et al., 2008).

The use of the hairpin gene was also chosen for a study in transgenic Chrysanthemum (XU

et al., 2010), where the insertion of bacterial hpaGXoo gene elicited an increased resistance against

the fungi A. tenuissima. Another promising way to increase ornamentals resistance to fungal

diseases is the introduction of the genes encoding chitinases and glucanases, enzymes that

hydrolyze fungal cell wall. The expression of the rice chitinase gene (RCC2) in chrysanthemum

decreased its susceptibility to gray mold (TAKATSU et al., 1999), while the bacterial chitinase gene

(ChiA) expressed in a number of carnation varieties delayed symptoms of the fusarium wilt disease

(BRUGLIERA et al., 2000). Saintpaulia transformed with glucanase-chitinase genes exhibited an

increased resistance against Fusarium oxysporum, Phytophthora sp., Pythium ultimum and Botrytis

sp. (RAM and MOHANDAS, 2003).

In addition, other antimicrobial proteins can be used to introduce the resistance against

fungal diseases in ornamentals. For instance, Petunia hybrida transformed with the Wasabi

japonica defensin gene (WD) showed an increased resistance towards Botrytis cinerea, Alternaria


solani, F. oxysporum and Erysiphe lycopersici (KHAN 2011), while the expression of the

antimicrobial protein gene (Ace-AMP1) in scented geranium and roses resulted in a resistance

against B. cinerea and Sphaerotheca pannosa (BI et al.,1999; LI et al., 2003).

Although transgenic expression of different antimicrobial proteins, of both plant and

animal origin, has proved to be useful against bacterial pathogens, this approach was not

extensively used for ornamentals. Bacterial control in ornamental plants is mostly based on

chemical control of insect disease vectors. In addition, insect pests not only serve as vectors of

plant pathogens, but also reduce the attractiveness and marketability of the ornamentals. Insect-

resistant D. grandiflorum carrying a Bacilus thurigiensis cry1Ab gene was reported by SHINOYAMA

and MOCHIZUKI (2006), while caffeine production in transgenic D. grandiflorum conferred the

resistance against aphids and beet armyworms (KIM et al., 2011).

Flower color and fragrance modification

The floral structure, the color and fragance are not only the determining factors for the

pollinator attraction but also play an important role in consumers acceptance. Hence, the colors of

flowers, leaves and fruits are considered important traits in the ornamental plant breeding.

However, in many ornamentals color range is limited by the genetics of the plant species (DEBNER

and WINKELMANN, 2010). Pigments responsible for the coloration in higher plants are classified in

several groups: chlorophylls, carotenoids (carotenes, xanthophylls), flavonoids (chalcones,

anthocyanins, flavones, flavonols) and betalains (betaxanthin, betacyanin) (MŁODZIŃSKA, 2009).

Carotenoids, essential component of photosynthesis, are ubiquitously distributed in plants and

confer a yellow or red flower color. Flavonoids and their colored class, anthocyanins, contribute to

a wide range of colors; pale yellow, orange, red, magenta, violet, blue. Betalains also result in

yellow and red color, but only the families of Caryophyllales produce betalains (TANAKA et al.,

2010). Using genetic engineering, expression of the genes involved in the pigment biosynthetic

pathways may be altered in different ways: by introducing new genes to create new biosynthetic

pathways, by redirecting biosynthetic pathways through up-regulation of the genes already present

or by suppressing biosynthetic pathways through the down-regulation of genes (CHANDLER and

BRUGLIERA, 2011).

Identification and cloning of the key genes of the flavonoid (TANAKA et al., 2005, 2010;

NISHIHRA and NAKATSUKA, 2011; TOGAMI et al., 2011), anthocyanin (TANAKA et al., 2005, 2010)

and carotenoid (CAZZONELLI and POGSON, 2010; SANDMANN et al., 2006; TANAKA et al., 2005;

JEKNIĆ et al., 2012) biosynthesis pathways has allowed engineering novel color traits. The first

successful isolation of a gene involved in flavonoid biosynthesis (HOLTON et al., 1993) and the

development of genetic transformation (MEYER et al., 1987) method brought the

commercialization of transgenic D. caryophyllus and R. x hybrida plants with modified color

(CHANDLER and SANCHEZ, 2012). Modified D. caryophyllus (developed by Florigene Pty.

Ltd./Suntory Ltd.), firstly marketed in Australia in 1997 (TANAKA et al., 2005), are now also

cultivated in South America and Japan (CHANDLER and SANCHEZ, 2012). The color-altered

transgenic carnations lines, vegetatively propagated and grown under greenhouse regime, have

been stable over a period of more than 15 years, while the phenotype of transgenic petunia was not

stable and diverse and variably colored flowers have appeared in the outdoor grown plants due to

promoter methylation (MEYER et al., 1992). It has been suggested that the differences in

phenotypic stability between carnation and petunia could be due to the differences in either the

transgene promoter or growing conditions: the plants outside are subjected to much higher

1154 GENETIKA, Vol. 47, No 3, 1149-1164, 2015

temperatures and higher light intensity levels than those grown inside, which could be a reason for

the phenotypic instability (TANAKA et al., 2010).

Another potential problem is that anthocyanidins often go through a number of color-

changing glycosylations, acylations, and methylations before being stored in the vacuoles, which

makes it very difficult to control the synthesis of a specific anthocyanin (TANAKA et al., 2010).

Different factors, such as vacuolar pH (QUATTROCCHIO et al., 2006; YOSHIDA et al., 2009), co-

pigmentation (TAKEDA et al., 2010), metal ion-complexation (SHIONO et al., 2008; MOMONOI et al.,

2009) as well as the cell size and shape (KAY et al., 2003; DYER et al., 2007), determine

anthocyanin color so that even if the specific anthocyanin was accumulated, the expected color

was not always achieved.

Production of novel flower colors through the carotenoid biosynthesis modification, using

crtW gene from A. auranticum fused to different transit peptides (TP), was demonstrated by

UMEMOTO et al., (2006) and UMEMOTO and TOGURI (2012). The resulting change in color of the

transgenic petunia was due to differential accumulation of astaxanthin, which depended on a TP

used to target crtW. Similarly, the flowers of crtW transformed Lotus japonicus accumulated novel

carotenoids, including astaxanthin (SUZUKI et al., 2007). JEKNIĆ et al. (2014) transformed iris with

the crtB gene from Pantoea agglomerans, where an increased influx of the metabolites into the

carotenoid biosynthetic pathway lead to elevated levels of lycopene.

Another strategy for flower color modification is manipulation with pigment genes

regulators. For instance, an increase in anthocyanin levels in transgenic petunia has been achieved

by overexpression of genes encoding transcriptional factors that regulate structural genes in the

biosynthetic pathways (BRADLEY et al., 1998). However, multiple interactions between

transcription factors can have unpredicted impact on genetic elements, posing the limitations for

broader application of this approach (TANAKA et al., 2005).

Furthermore, antisense and co-suppression approaches have been utilized for down-

regulation of gene expression in plants. Accordingly, a suppression of genes involved in pigment

biosynthetic pathways is an important strategy in flower color modification. For example, using

RNA interference (RNAi), the key gene in flavonoid biosynthesis (chs) has been silenced, altering

the color of Torenia hybria flower from blue to white (FUKUSAKI et al., 2004). Similarly, an

insertion of a transposon into a flavonoid biosynthetic pathway gene or a regulatory gene of the

flavonoid biosynthesis has resulted in the occurrence of white sectors in otherwise colored flowers

(TANKA et al., 2005). Additional strategies to modify color include manipulations of co-pigments,

vacuole acidity (pH) and metal ion transportation (CHANDLER and SANCHEZ, 2012).

Floral scent plays an important role in attracting pollinators. It is also important to

consumers’ choice in flower purchase due to its sensual associations. Floral scent is made up of

various compounds, over 700 of which have been identified in 60 families of plants. They are fatty

acid derivatives such as benzenoids, phenylpropanoids and terpenoids. Floral scent compounds are

synthesized de novo in petals (TANKA et al., 2005). The possibility of fragrance modification,

using genetic transformations of ornamentals has been reviewed by several authors (DUDAREVA

and PICHERSKY, 2008; DUDAREVA et al., 2006; UNDERWOOD and CLARKE, 2011; YU and UTSUMI,

2009). A detailed understanding of mechanisms underlying floral fragrance occurrence is still

necessary for its successful commercial exploitation of this trait.


Abiotic stress tolerance

Drought, light intensity, humidity and temperature changes have an impact on

maintaining ornamentals quality in distribution before they reach consumers, as well as, on spatial

and seasonal ornamental plants cultivation and availability, which is of the great interest for

growers and consumers. For instance, our result suggested that exogenous application of salicylic

acid can generally counteract the effects of drought on all growth, physiological and biochemical

parameters in I. walleriana (LOJIĆ et al., 2015). However, the introduction of genes involved in

plant response to abiotic stress in ornamentals is still mostly in conception.

Post-harvest life

The post-harvest life of potted plants and cut flowers is often limited by their inability to

maintain photosynthesis under the light conditions of the interior environment where they are held.

The quality and post-harvest life of many potted plants are often reduced by the presence of

ethylene in the environment. A large amount of ethylene is produced, mostly in the petals, several

days after the full opening of the flower (WOODSON et al., 1992) and increased ethylene production

promotes the in-rolling of petals resulting in wilting of the flower. Also, cut flowers must have the

capability to endure a few weeks in the distribution chains, so that the flower resistance to

senescence promotes and vase-life reducing factors such as ethylene and bacterial infection have to

be managed (CHANDLER and SANCHEZ, 2012).

Following harvesting, carnations are typically treated with silver thiosulfate which

effectively interferes with the perception of ethylene. Silver thiosulfate is classified as a toxic

chemical and there is an increasing pressure on the industry to reduce the usage of such chemicals

(TANAKA et al., 2005). In the ethylene biosynthetic pathway, there are two key enzymes: ACC

synthase and ACC oxidase, involved in the ACC cyclization and subsequent conversion to

ethylene (CROIZER et al., 2000). Regulation of ethylene production in carnation flowers was

achieved via post-transcriptional floral-specific gene silencing of the gene encoding ACC oxidase

(SALVIN et al., 1995) or ACC synthase (Florigene Ltd., published results in TANAKA et al., 2005).

In addition, a number of potted plants exhibit petal abscission in response to ethylene and

amelioration of this effect can be achieved by suppressing the expression of the ACC oxidase

gene. Transgenic Torenia fournieri with an introduced fragment of ACC oxidase gene in either

sense or antisense orientation exhibited an extended flower life and produced more flowers

simultaneously per stem than the wild-type plants (AIDA et al., 1998). However, plants with

engineered ethylene production are still susceptible to exogenous ethylene. Thus, the modification

of ethylene perception is shown to be more effective for reduced sensitivity to ethylene and

delayed flower senescence in ornamental plants. For example, introduction of mutated ethylene

receptor genes (SHAW et al., 2002; SRISKANADARAJAH et al., 2007; SANIKAHANI et al., 2008)

and/or regulators of ethylene response (SHIBUYA et al., 2004) improved flower life in Petunia,

Carpanula and Kalanchoë. Leaf yellowing is also a negative attribute in both cut flowers and

potted plants. To inhibit leaf senescence, modification using mutated ethylene receptor genes has

proved potentially very useful, as demonstrated for D. grandiflorum (SATOH et al., 2008).

Knowing that cytokinins are associated with delayed senescence and reduction in

ethylene biosynthesis, another effective strategy for the improvement of the post harvest life of

ornamentals is introduction of the IPT, gene encoding protein that catalyzes initial step in

cytokinin production. Using this approach, CHANG et al. (2003) have demonstrated that increase in

1156 GENETIKA, Vol. 47, No 3, 1149-1164, 2015

cytokinin concentration, obtained by integration of the IPT gene, lead to a significant ethylene

tolerance and prolonged flower life in petunia.

Application of rol-genes in floriculture

A. rhizogenes T-DNA, that is known to be transferred and integrated into the plant

genome, carries four rol genes (rolA, rolB, rolC and rolD) that induce the formation of hairy roots

(VEENA and TAYLOR, 2007). Plant inoculation with A. rhizogenes usually shows high

transformation efficiency and the hairy root cultures are feasible to establish (MILOŠEVIĆ et al.,

2009a, 2009b, 2015; LOJIĆ et al., 2013). Hairy root cultures can have a diverse range of

biotechnological uses, as illustrated in Figure 1. High stability of the production of secondary

metabolites (GUILLON et al., 2006; TIWARI et al., 2008) or recombinant proteins holds immense

potential for pharmaceutical industry. Hairy roots of various plant species can significantly

detoxify pesticides, antibiotics and various toxic industrial effluents (GUILLON et al., 2006; VEENA

and TAYLOR, 2007) and have been used to remove or increase tolerance to high levels of phenols

(SANTOS DE ARAUJO et al., 2006; GONZÁLEZ et al., 2012; TADIĆ et al., 2014).

Plants regenerated from the hairy roots exhibit a modified phenotype, which can be used

for the production of new ornamental varieties with improved characteristics. It has been shown

that the rol genes affect the morphology and number of flowers, flower pigment biosynthesis,

biosynthetic pathways of secondary metabolites, as well as the vase life of cut flowers

(ZDRAVKOVIĆ-KORAĆ et al., 2004; CASANOVA et al., 2005; CHANDLER and LU, 2005).

Heterologous expression of rolA results in a dwarf or semi-dwarf phenotype characterized by

reduced internodes, whereas plants transformed with rolB alone often exhibit morphological

alterations such as reduced apical dominance and altered leaf morphology. Expression of rolC

exerts a cytokinin-like effect, so that plants transformed with rolC alone often exhibit reduced and

suppression of apical dominance, increase in the number of lateral shoots, early flowering and

reduction in flower size and pollen production. Little is known about the function of rolD,

although some reports documented dwarf phenotype or earlier flowering (CHRISTENSEN and

MÜLLER, 2009a). It has also been noted that the rol genes can increase the resistance of plants to

biotic or abiotic stress (CASANOVA et al., 2005).

In the commercial production of different ornamental species elongated inflorescence are

considered undesirable. Short and compact plants with good maintaining attributes and ornamental

assets are preferred by consumers. Similarly, compared to more elongated plants, smaller and

compact plants are preferred as they tolerate mechanical handling and transport far better, require

less room in production facilities and distribution facilities and are easier to handle (MÜLLER,

2011). Compact plants are hence favored by retailers. Compact growth in ornamental plants is

commonly achieved through the application of chemical growth retardants (CHRISTENSEN and

MÜLLER, 2009a, b). During recent years, such chemicals have been extensively criticized as

hazardous to human health and to the environment (ANDERSEN et al., 2002; FUJIMOTO et al., 1997).

The use of some growth retardants, such as paclobutrazol and daminozide, has been banned in

many countries, and more will probably be forbidden in the future. Alternatively, compact

phenotypes can be effectively achieved using genetic engineering techniques, by transformation

with rol genes (reviewed by CASANOVA et al., 2005 and CHRISTENSEN and MÜLLER, 2009a).

Besides the desirable compact phenotype, it has been documented that ornamentals with

inserted rol genes can exhibit earlier flowering (SUGINUMA and AKIHAMA, 1995), more flowers


(HOSOKAWA et al., 1997), increased fragrance (PELLEGRINECHI et al., 1994) or changed flower

shape (HANDA, 1992).

Although the biochemical functions of rol genes in transformed plants remain to be fully

elucidated, they proved to be highly useful tools for the improvement of ornamental species. A

number of rol-transformed plants, especially those with rolC and rolD genes, exhibited new and

beneficial traits. It can be expected that in the future, with increased knowledge about the function

of these genes, the ornamentals with introduced rol sequences will be more numerous.

Limitations of genetic modifications of ornamentals

The most significant limitation for the full use of genetic engineering in ornamental plant

improvement is the cost associated with the technology. These expenses include time, facilities,

personnel, and expenses for developing the technology for generation of new modified genotypes

(e.g., regeneration methods, gene discovery and isolation, etc.), licensing of existing technology,

regulatory approvals, and the evaluation of transgenic plants. A typical genetic engineering project

resulting in a single new genotype can require the use of 17 patented technologies (ADKINS et al.,

2003). A relatively small market for ornamental crops makes many costly breeding strategies less

profitable for breeders within floricultural industries. Expensive licensed transformation methods,

as well as registration and approvals for genetically transformed plants are still a determining

factor for commercial use of genetic engineering in biotechnology (MÜLLER, 2011). The

regulations for production and use of GM ornamental plants, on a global level, are considerably

diverse: USA and China are comparatively permissive; EU and Japan are rather restrictive,

whereas countries like Australia fall into the middle (AUER, 2008; CHANDLER and SANCHEZ, 2012).

However, the acceptance of genetically modified ornamentals, as non-food crops, should be

relatively less demanding and concerning, compared to the crops for human consumption. Genetic

engineering has the potential to create ornamental plants suitable for environmentally friendly

production, nevertheless consumers’ and retailers’ skepticism against GMOs as well as costly

patents and registration procedures are still an important limiting factor for its extensive

acceptance.

ACKNOWLEDGMENTS

The authors are grateful to the Serbian Ministry of Education, Science and Technological Research

(Project TR 31019) for financial support of this work. Special thanks are due to Dr. Branka Uzelac

(Institute for Biological Research „Siniša Stanković“, University of Belgrade), who made English

language corrections to the text.

Received August 23h, 2015

Accepted October 29h, 2015

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AGROBACTERIUM-POSREDOVANA TRANSFORMACIJA UKRASNIH BILJNIH

VRSTA: PREGLED

Snežana MILOŠEVIĆ*, Aleksandar CINGEL, Angelina SUBOTIĆ

Institut za biološka istraživanja „Siniša Stanković“, Univerzitet u Beogradu, Beograd

Izvod

UvoĊenje korisnih osobina u komercijalne ukrasne biljke primenom tehnika genetiĉkog

inžinjeringa je moćno oruĊe savremene biotehnologije. Ipak ove tehnike imaju ograniĉenu

primenu u hortikulturi, posebno u cvećarstvu. Promena boje, graĊe ili mirisa cvetova, kao i

poboljšanje tolerantnosti/rezistentnosti biljaka na abioiĉki i biotiĉki stres primenom tehnika

genetiĉke transformacije je još uvek u povoju. Ovaj pregled je fokusiran na primenu

Agrobacterium-posredovane transformacije, glavnog pristupa genetiĉkog inženjeringa biljaka, u

proizvodnji ukrasnih vrsta i uticaju koji je, na ovu oblast, imao do sada.

.

Primljeno 23. VIII .2015.

Odobreno 29.X. 2015.

AGROBACTERIUM-MEDIATED TRANSFORMATION OF … · 2015-12-23 · S. MILOŠEVIĆ: AGROBACTERIUM...

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