Iw

SSa

b

a

ARRA

KdINpTV

1

avtsaea(

ttp

panSr

0h

Scientia Horticulturae 164 (2013) 499–506

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

ntroduction of dsRNA-specific ribonuclease pac1 into Impatiensalleriana provides resistance to Tomato spotted wilt virus

nezana Milosevic a,∗, Ana Simonovic a, Aleksandar Cingela, Dragana Nikolic b,lavica Ninkovic a, Angelina Subotic a

Institute for Biological Research, University of Belgrade, Bul. despota Stefana 142, 11060 Belgrade, SerbiaInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 26 April 2013eceived in revised form 30 August 2013ccepted 15 October 2013

eywords:sRNA-specific ribonuclease

mpatiens wallerianaicotiana tabacum

a b s t r a c t

The production of several popular impatiens cultivars in Serbia suffers substantial losses due to highincidence of Tomato spotted wilt virus (TSWV) infections. Since TSWV, like majority of plant viruses,has RNA genome and replicates via double-stranded RNA (dsRNA) intermediates, it is a good targetfor dsRNA-specific endonuclease encoded by pac1 gene from Schizosaccharomyces pombe. In order tointroduce resistance to TSWV, Impatiens walleriana, as well as referent species Nicotiana tabacum, weretransformed with Agrobacterium tumefaciens C58C1pac1 bearing a binary vector pKT-Lpac1. The trans-formation and regeneration was successful in both plant species, but the transformation efficiency washigher in tobacco. The obtained pac1-transformed impatiens and tobacco lines were challenged with

ac1SWVirus resistance

TSWV by manual inoculation in vitro. I. walleriana clones expressing pac1 were completely resistant toTSWV. Some of the transgenic tobacco lines also showed complete resistance, while others were infected,but with lower frequency, prolonged incubation period and milder symptoms in comparison to untrans-formed plants. Comparison of morphological parameters including shoot length, number of nodes, leaflength and number of axillary buds per plant between control and transformed lines revealed that pac1

the m

expression does not alter

. Introduction

Viruses belonging to genus Tospovirus, family Bunyaviridae, suchs tomato spotted wilt virus (TSWV) and impatiens necrotic spotirus (INSV), are known as a very serious pathogens that are easilyransmitted by flower thrips. They infect a number of horticulturalpecies including chrysanthemum, petunia, impatiens, snapdragonnd others (Daughtrey et al., 1997). Vegetative propagation of flow-rs increases the risk of transmitting viral diseases to the progenynd further spread in new production areas via international tradeBraiser, 2008).

The highly polyphagous nature, the efficiency of transmission,

he rapidity with which new variants arise, and difficulties inhe control of the vectors, make TSWV one of the most fearedlant viruses by growers worldwide. The current list of TSWV

Abbreviations: BAP, benzylaminopurine; CPPU, N-(2-chloro-4-pyridyl)-N-henylurea; DAS-ELISA, double antibody sandwich enzyme-linked immunosorbentssay; DPI, days post inoculation; dsRNA, double-stranded RNA; INSV, Impatiensecrotic spot virus; Km, kanamycin; LB, Luria-Bertani medium; MS, Murashighe andkoog basal media; NPT II, neomycin phosphotransferase II; pac1, dsRNA-specificibonuclease; TDZ, thidiazuron; TSWV, Tomato spotted wilt virus.∗ Corresponding author. Tel.: +381 11 2078393; fax: +381 11 2761433.

E-mail address: snezana@ibiss.bg.ac.rs (S. Milosevic).

304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.10.015

orphology of the transformants.© 2013 Elsevier B.V. All rights reserved.

hosts consists of 1090 plants species (Parrella et al., 2003). InSerbia, TSWV is a production constraint to tomato, pepper, potato,tobacco, onion, garlic and ornamentals such as Impatiens (Ðekicet al., 2008; Stankovic et al., 2011; Milosevic, 2010; Milosevicet al., 2011, 2012a,b). The production of several popular Impatienswalleriana and I. hawkerii cultivars grown in private nurseries inSerbia suffered substantial losses due to high incidence of TSWVinfections in 2006. when a high percentage of mother plantsshowed local leaf and tip necrosis, chlorotic rings and mosaic,leaf distortion and death (Milosevic, 2010; Milosevic et al., 2011,2012b).

Several in vitro propagation and regeneration methods havebeen developed for eradicating viruses from infected tissuesand production of virus-free plants, including thermotherapy(Koubouris et al., 2007; Milosevic et al., 2012a), cryotherapy (Wangand Valkonen, 2009) and meristem-tip propagation (Milosevicet al., 2011, 2012a,b). Resistance to viruses may be introduced intocrops by various genetic manipulations (Sudarshana et al., 2007;Clarke et al., 2008). R-proteins, the basis of innate plant immu-nity, may be introduced into sensitive crops either by conventional

breeding or by transgenic approaches. Animal-derived recombi-nant anti-viral antibodies can be ectopically expressed in plantcells granting specific resistance (Safarnejad et al., 2011). Pathogen-derived resistance is also artificial and is based on expression of

5 orticu

sMiiais

wdvcdhhf(e

wovRsiidipmefCehea

2

2

Lsobsmc

2

2tppbpCwTst

00 S. Milosevic et al. / Scientia H

equences that initiate RNA silencing of viral sequences (Simón-ateo and García, 2011) or on expression of viral structural genes

n the host plants, such as wild-type or altered coat proteins thatnterfere with viral particle assembly (Beachy, 1999). All of thesepproaches, however, may provide resistance only to one virus orts close relatives and there is always a possibility that mutated viraltrains escape the established resistance.

The fact that the majority of plant viruses are RNA viruses,hich replicate through double-stranded RNA (dsRNA) interme-iates, allows for targeting dsRNA structures instead of specificiral sequences or proteins by genetic manipulations. pac1 ribonu-lease isolated from Schizosaccharomyces pombe, a highly actives-specific endoribonuclease that cleaves long dsRNAs and smallairpin RNAs (Rotondo and Frendewey, 1996; Rotondo et al., 1997)ave been successfully introduced into several plant species con-

erring a broad resistance or tolerance to viral and viroid pathogensWatanabe et al., 1995; Sano et al., 1997; Ishida et al., 2002; Togurit al., 2003; Ogawa et al., 2005).

The aim of this work is stable integration of pac1 gene into I.alleriana, a valued potted and bedding flower (Balsaminaceae), inrder to protect it from TSWV infections and possibly from otheriral infections as well. TSWV, containing a tripartite ambisenseNA genome (Mandal et al., 2008) is a good target for suppres-ion by pac1 (Ogawa et al., 2005). Along with impatiens, we alsontroduced pac1 into Nicotiana tabacum. More than 20 viruses,ncluding TSWV, occur in tobacco naturally (Gooding, 1991), so viraliseases are one of the most important limiting factors in tobacco

ndustry. Tobacco has already been successfully transformed withac1 and the transformants showed partial tolerance to tomatoosaic virus, cucumber mosaic virus and potato virus Y (Watanabe

t al., 1995). Since tobacco is an extremely versatile model plantor tissue culture and genetic engineering (Ganapathi et al., 2004;lemente, 2006), is hyper-susceptible to viral infections (Glebat al., 2004) and can be experimentally infected with more thanundred of viral species (Gooding, 1991), we used it as a refer-nce to evaluate the efficiencies of transformation, regenerationnd in vitro inoculation protocols used for impatiens.

. Materials and methods

.1. Plant material

The in vitro culture of I. walleriana plants was initiated from Busyizzi Safari mixed F2 seeds (Johnsons Seeds, UK), while the tobaccoeeds cv. Wisconsin 38 were obtained from The Botanical Gardenf Nijmegen. The seeds were surface-sterilized in 10% commercialleach (5% hypochlorite) for 15 min, washed in sterile water andet to germinate on plates with MS (Murashige and Skoog, 1962)edium. All cultures were maintained at 24 ± 2 ◦C under fluores-

ent light of 40 �mol m−2 s−1 16 h light/8 h dark photoperiod.

.2. Bacterial strains and constructs

A binary vector pKT-Lpac1, derived from pBI121 (Toguri et al.,003), was kindly provided by dr Toguri. The T-DNA region ofhe plasmid features kanamycin (Km) resistance, due to neomycinhosphotransferase II (npt II) gene driven by nopaline synthetaseromoter, as well as RNA-virus resistance, conferred in host plantsy RNA-specific ribonuclease pac1 cassette with 35S promoter. TheKT-Lpac1 was introduced into Agrobacterium tumefaciens strain58C1 by elctroporation. The obtained A. tumefaciens C58C1pac1

as used for transformation of both I. walleriana and N. tabacum.

he bacteria were cultured on solid (LB) medium (Bertani, 1951)upplemented with 100 mg l−l Km. Bacterial suspension used forransformation was prepared by transferring a single bacterial

lturae 164 (2013) 499–506

colony to liquid LB medium, and the suspension with OD600 of 0.6was used.

2.3. Transformation of impatiens and tobacco with A.tumefaciens C58C1pac1

I. walleriana nodal segments (∼5 mm long) with one axillary budwere longitudinally cut, needle pricked to inflict epidermal injuries,and immersed into the bacterial suspension. A total of 135 I. wal-leriana explants were inoculated, along with control explants thatwere immersed in sterile LB medium. The explants were shook inbacterial suspensions for 50 min, 2 h or 20 h in darkness. A totalof 72 tobacco leaf discs, approximately 5 mm in diameter, fromfour weeks old in vitro grown plantlets were also inoculated withA. tumefaciens C58C1pac1 for 10 or 30 min. The explants were co-cultivated with bacteria in darkness for 3 days on basal media (MS)supplemented with 100 �M acetosyringone.

After the co-cultivation, the explants were washed with 1 g l−l

cefotaxime solution and blotted on sterile filter paper. Out of 135I. walleriana explants, 75 were transferred onto a shoot-inductionMS medium supplemented with 0.1 �M N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU), while the remaining 60 explants were culturedon media with 0.1 �M thidiazuron (TDZ). All cultures also contained500 mg l−l cefotaxime and 100 mg l−l Km. The medium for tobaccoregeneration (Uzelac et al., 2006) contained 5 �M benzylaminop-urine (BAP), 300 mg l−l cefotaxime and 50 mg l−l Km. The explantswere transferred to a fresh shoot-induction medium containingantibiotics every 3–4 weeks. The regenerated Km-resistant shootswere maintained on the same medium for the next few months.

The success of the transformation was confirmed by thepresence and expression of the pac1 gene in the Km-resistantlines. Genomic DNA was isolated by CTAB method described byZhou et al. (1994). Comparison of different methods for totalRNA isolation from Impatiens tissues rich in phenolics revealedthat the optimal method was CTAB extraction/LiCL precipitationprotocol described by Gasic et al. (2004). A 1000 bp pac1 frag-ment was amplified from genomic DNA samples using specificprimers Fpac1: 5′-GCCGACAGCACCCAGTTCAC and Rpac1: 5′-CCTGCCGTAAGTTTCACCTCACC and GeneAmp® Gold PCR ReagentKit (Applied Biosystems, UK) components according to manufac-turer’s protocol. That the pac1 amplification is a consequence ofits integration into the plant genome and not bacterial contamina-tion was confirmed by the absence of bacterial virG amplificationwith primers FvirG: 5′-GCCGACAGCACCCAGTTCAC and RvirG: 5′-CCTGCCGTAAGTTTCACCTCACC that produce a 390 bp fragment.The PCR program consisted of initial denaturation (95 ◦C/5 min)followed by 38 cycles of denaturation (95 ◦C/1 min), annealing(at 52 ◦C for pac1 or 60 ◦C for virG, for 1 min), and extension(72 ◦C/2 min), with final extension for 10 min at 72 ◦C. The expres-sion of pac1 gene was determined by RT-PCR, using GeneAmp®

Gold RNA PCR Reagent Kit (Applied Biosystems) according to man-ufacturer’s recommendations, with oligo-dT primers in the reversetranscription step and gene-specific primers in the amplificationstep. For both PCR and RT-PCR analyses, DNA or RNA isolated fromnon-transformed plants were used for negative control, while puri-fied plasmids were used as positive control. The amplicons wereanalyzed electrophoretically.

2.4. Inoculation of test plants with TSWV in vitro

As a source of TSWV for plant inoculation in disease resistancetests, one infected I. hawkerii plant with developed symptoms was

used. The presence of TSWV in I. hawkerii was confirmed by DAS-ELISA, RT-PCR and sequencing of the isolate, as described earlier(Milosevic et al., 2011). Shoot tips of infected plants (2 cm long)were thoroughly washed under running tap water and surface

orticulturae 164 (2013) 499–506 501

srgeKltolitopgitp

3

3w

dieff1bosrindirp

cAvptis

Fig. 1. Sensitivity of I. walleriana explants to Kanamycin. For each indicated Kmconcentration, 10 uniformely sized I. walleriana explants were planted and grownfor four weeks, when the number od roots per explant was recorded as an indicator

TT

Iao

S. Milosevic et al. / Scientia H

terilized with 10% sodium hypochlorite for 15 min before 4–5inses in sterile deionized water. The inoculum was prepared byrinding leaves in a chilled sterile mortar and pestle, and homog-nizing them in 1:2 (w:v) freshly prepared sterile ice cold 0.01 M-phosphate buffer containing 0.01 M Na-sulfite, pH 7.0, under a

aminar hood. Autoclaved Carborundum (600 grit) was added tohe sap as 10% suspension. The saps were used to inoculate leavesf ten I. walleriana and N. tabacum plants of each transformedine, at the two to five-leaf development stages. Fingers with ster-le latex gloves were dipped into the sap and gently rubbed overhe leaves’ surface of each plantlet. The inoculum was maintainedn ice until the inoculation was completed. After inoculation, thelantlets were sprayed with sterile deionized water and kept in arowth chamber. The development of TSWV symptoms was mon-tored daily. The presence of TSWV in the inoculated plants wasested by DAS-ELISA 30 days following inoculation, as describedreviously (Milosevic et al., 2011).

. Results and discussion

.1. Determination of selective kanamycin concentration for I.alleriana

In order to determine suitable Km concentration for reliableiscrimination between the transformed and non-transformed

mpatiens clones, a kanamycin sensitivity test was performed. Forach Km concentration (0, 10, 25, 50 and 100 mg l−l), 10 uni-ormly sized I. walleriana explants were planted and grown in jarsor four weeks. The impatiens explants planted on MS containing0 mg l−l Km grew slower in comparison to the control explants,ut developed roots and axillary shoots normally. Explants grownn 25 mg l−l Km rooted well, but had reduced number of axillaryhoots, while explants on 50 mg l−l Km were retarded, with a singleoot and few axillary shoots that developed with a delay in compar-son to the control. I. walleriana explants set on 100 mg l−l Km didot produce roots at all, and had only a few axillary shoots whicheveloped slower in comparison to other treatments. Sensitivity of

mpatiens explants to Km was presented as a number of developedoots per explant (Fig. 1). Based on these results, it was decided toerform the selection on 100 mg l−l Km.

Dan et al. (2010) found that the concentration of 50 mg l−l Kmompletely inhibited root growth in untransformed I. walleriana cv.ccent Red. This could be assigned to higher sensitivity of the culti-ar used in their study. Since there was no significant difference in

ercentage of shoots producing roots on 25, 35 and 50 mg l−l Km,he autors decided to use non-lethal Km concentration of 35 mg l−l,n order to allow recovery of transgenic plants having low expres-ion of nptII.

able 1ransformation efficiency of I. walleriana and N. tabacum explants with A. tumefaciens C58

Duration ofinoculation

Cytokinin �M No. of inoculatedexplants

No. of lines

passages on

I. walleriana50 min 0.1 TDZ 9 3 (33.33%)

0.1 CPPU 16 6 (37.50%)

2 h 0.1 TDZ 31 17 (54.84%)0.1 CPPU 31 12 (38.71%)

20 h 0.1 TDZ 20 13 (65.00%)0.1 CPPU 28 3 (10.71%)

Total 135 54 (40%) 4/35

N. tabacum10 min 5 BAP 38 25 (65.79%)30 min 34 13 (38.23%)Total 72 38 (52.78%) 15/19

n both species the transformation of the explants depended on duration of inoculation.

ffected the efficiency of transformation. Of Km-resistant lines, 35 out of 54 impatiens and

f tobacco was more efficient in comparison to impatiens.

of sensitivity to antibiotic. Statistical difference at a significance level of P < 0.05 isindicated in different letters.

3.2. Transformation and regeneration of I. walleriana

Duration of inoculation, as one of important factors affectingtransformation and regeneration efficiency, has to be optimized foreach plant species (Karthikeyan et al., 2012). Preliminary resultson nodal segments with axillary buds as explants showed thatinoculation for 10, 20 or 30 min was unfruitful (data not shown).Inoculation of explants with slow shaking in bacterial suspen-sion for 2 h was effective, resulting in PCR positive lines, whilelonger inoculation did not improve the frequency of transformation(Table 1). Prolonged inoculation did not produce any transgenicshoots; many of the explants inoculated for 20 h and grown onCPPU, ceased growing on selective medium within two weeks,became vitrified and eventually wilted. It is possible that Agrobac-terium infectivity decreased, since the exponential growth stagewas over. Longer exposure to Agrobacterium can also lead to over-growth of explants with bacterial cells, resulting in complete loss ofregeneration ability of the transformed cells (Liu and Pijut, 2010).

The composition of shoot growth medium was another deter-minant factor of successful impatiens transformation (Table 1).All positive lines were grown on TDZ containing medium. TDZ is

an efficient plant growth regulator shown to enhance not onlyregeneration (Jones et al., 2007), but also Agrobacterium-mediatedtransformation (Thirukkumaran et al., 2009). TDZ was also superior

C1pac1.

that survived 5 Km

PCR-positive/analyzedlines

RT-PCR-positive lines

0/3 00/5 0

4/7 2 0/7 0

0/10 00/3 02

14/14 14 1/5 1

15

In the case of I. walleriana, the choice of cytokinin apllied for the regeneration also19 out of 38 tobacco lines were further analyzed by PCR and RT-PCR. Transformation

502 S. Milosevic et al. / Scientia Horticulturae 164 (2013) 499–506

Fig. 2. Transformation and regeneration of I. walleriana. Shoots were induced from nodal segments with axillary buds in both control plants (a) and putative transformants(b) on MS medium containing 0.1 �M TDZ, 7 days after innoculation. Tansformed shoots grown for 4 weeks on selective medium (100 mg l−l Km and 500 mg l−l cefotaxime)s ll, whs rmond medi

it2evtp

sadvets5mvnr

vemcupo

upplemented with either 0.1 �M TDZ (c) or 0.1 �M CPPU (d) and (e) thrived weurvived five passages of Km (100 mg l−l) selection spontaneously rooted on solid hoifferences in comparison to control plants (cp) after 4 weeks on hormone-free MS

n comparison to other growth regulators in promoting regenera-ion from I. walleriana cotiledonary nodes (Baxter, 2005; Dan et al.,010). Showing both auxin and cytokinin-like effects (Yanchevat al., 2003), TDZ is highly efficient in regeneration processes inarious plant species by modifying endogenous levels of phy-ohormones and by induction or enhancement of a number ofhysiological and biochemical processes (Guo et al., 2011).

After three days of co-cultivation, I. walleriana explants wereelected on MS medium supplemented with 100 mg l−l Km, withddition of CPPU or TDZ. Axillary buds were developed within sevenays both in control (Fig. 2a) and inoculated (Fig. 2b) explants. Indi-idual shoots were excised and transferred to the fresh mediumvery 4 weeks. The efficiency of axillary buds formation in poten-ially transformed tissue depended on the applied cytokinin: thehoots elongated faster on selective media (100 mg l−l Km and00 mg l−l cefotaxime) containing 0.1 �M TDZ (Fig. 2c), but wereore numerous on 0.1 �M CPPU (Fig. 2d and e), as descibed pre-

iously (Subotic et al., 2008). The control explants completelyecrotized after 5 passages on selective media with 100 mg l−l Kmegardless of the applied cytokinin (Fig. 2f).

Out of 135 inoculated I. walleriana explants, 54 (40%) sur-ived five subcultures of Km selection (Table 1). The Km-resistantxplants were then transferred to antibiotic- and hormone-free MSedium. At first these explants grew slower in comparison to the

ontrol plants that were cultured without antibiotics, but caughtp in the next passage and spontaneously rooted (Fig. 2g). Theresence of pac1 in I. walleriana genome was confirmed in 4 outf 35 analyzed lines (i3, i6, i8 and i10, Fig. 3) showing a 1000-bp

ile the control plants (f) did not survive the Km treatment. Transformed shootse-free MS medium (g). Transformed lines i6 and i8 showed no visible morphologicalum (h).

amplified fragment. All of the pac1-positive lines were inoculatedfor 2 h and regenerated on TDZ (Table 1). This procedure gave riseto 23.52% of surviving shoots being true transformed shoots, withfinal transformation efficiency of 12.81% (four positive lines out of31 explants).

None of the tested lines was contaminated with residual A. tume-faciens that survived the cefotaxime treatment, as confirmed by theabsence of virG amplification (Fig. 3), meaning that the amplifiedpac1 was not of bacterial origin. RT-PCR amplification of a 1000-bp pac1 segment revealed that pac1 is expressed in lines i6 and i8(Fig. 3), so the transformation was successful, but with low effi-ciency. Negative results for i3 and i10 lines suggest either verylow pac1 expression or possibly its silencing, due to inadequateinsertion site.

It is hard to explain how 40% of the explants survived the Kmselection, and yet only 4 lines had pac1 successfully amplified, butit is possible that the integration and expression of nptII was moreeffective in comparison to pac1 gene. Independent integration ofselectable marker and a gene of interest cassettes from the samevector were shown in many cases, for example for nptII and chal-cone synthase integration into Bidens pilosa genome (Wang et al.,2012). Notably, it has been reported that up to 90% of kanamycin-survived shoots could be non-transformed (Dan et al., 2010).

Even though the regeneration protocol starting with I. walleriana

nodal segments with axillary buds was successful, the frequencyof transformation was low (12.81%). Other research groups alsofound I. walleriana to be recalcitrant to tissue culture and transfor-mation manipulations (Baxter, 2005; Dan et al., 2010). Successful

S. Milosevic et al. / Scientia Horticulturae 164 (2013) 499–506 503

Fig. 3. PCR and RT-PCR confirmation of impatiens and tobacco transformation with pac1. Presence of pac1 gen was confirmed by PCR amplification of a 1000 bp fragment infour I. walleriana lines (i3, i6, i8 and i10), as well as in 15 N. tobacco lines (t2–t4 and t8–t12 are shown). That the pac1 amplification was not due to bacterial contamination wasconfirmed by the absence of virG amplification product of 390 bp in all pac1-positive lines. As determined by RT-PCR analyses, pac1 is expressed in two impatiens lines (i6and i8), as well as in 15 tobacco lines (t2–t4 and t8–t14, but with very faint t8 band, are shown). N – Untransformed control plants as a negative control; i1–i11 – I. wallerianapotential transformants; t2–t15 – N. tabacum potential transformants; At – A. tumefaciens C58C1pac1 used as a positive control in PCR reactions.

Fig. 4. Transformation and regeneration of N. tabacum. Calli were induced from leaf explants on MS medium with 5 �M BAP in contol untransformed culture (a) and intransformed culture on selective medium containing 50 mg l−l Km and 300 mg l−l cefotaxime, (c) 7 days after inoculation. The control leaf explant culture did not survive ons both i5 n MS

m enic pt lants

Iaotrt

3

pl

elective medium. (b) Shoot regeneration on MS medium with 5 �M BAP occured

0 mg l−l Km and 300 mg l−l cefotaxime. (e) Regenerated plants after four weeks oedium (g) and transformed plants on selective medium. (h) Rooting of the transg

13 and t16 showed no visible morphological differences in comparison to control p

. walleriana regeneration was achieved from cotiledonary nodes,fter elaborate optimization, but no stable transformation has beenbtained with these explants (Baxter, 2005). A high-frequencyransformation protocol with A. tumefaciens bearing a GFP binaryeporter vector has been eventually developed using in vitro mul-iple bud cultures as explants (Dan et al., 2010).

.3. Transformation and regeneration of N. tabacum

Transformation of tobacco with A. tumefaciens C58C1pac1 waserformed using leaf discs as target explants. This is a well estab-

ished protocol used widely for rapid evaluation of transgenes in

n control explants (d) and in transformed culture on medium supplemented withmedium: control plants without antibiotics, (f) control plants grown on selectivelants occured spontaneously on hormone-free MS medium. (i) Transformed lines

(cp) after two weeks on hormone-free MS medium (j).

higher plants (Clemente, 2006), including pac1-derived resistance(Watanabe et al., 1995). Both control explants (Fig. 4a) and inoc-ulated leaf discs (Fig. 4c) developed calli on 5 �M BAP within 7days following inoculation. The inoculation for 10 min was moreeffective than longer inoculation for 30 min (Table 1). While theinoculated explants thrived on medium containing 50 mg l−l Kmand 300 mg l−l cefotaxime (Fig. 4c), the control explants did notsurvive if placed on selective medium (Fig. 4b). Shoots regener-

ated from calli in both control (Fig. 4d) and transformed explants(Fig. 4e) on inductive medium supplemented with 5 �M BAP, andone shoot from each leaf disk was further propagated. Resistanceto Km was confirmed in 4-weeks old shoots by comparing control

504 S. Milosevic et al. / Scientia Horticulturae 164 (2013) 499–506

Fig. 5. pac1-transformed impatiens and tobacco are resistant to TSWV infection. Control and pac1-transformed impatiens and tobacco plants were challenged with TSWVb four wl I. wallen oculat

(s

sfitf

3

wptcarpuasbTiMucdcegasea

t1s

y manual in vitro inoculation. The symptoms of the infection were observed after

eaf distortion and necrosis as visible symptoms of infection; (b) pac1-transformed

ervature on control N. tabacum plants and (d) transformed N. tabacum, line t19, in

Fig. 4f and g) and transformed shoot culture (Fig. 4h). Rooting waspontaneous on hormone-free MS medium (Fig. 4i).

From 72 inoculated discs e.g. regenerants, 38 lines survived fiveubcultures of Km selection, of which 19 Km-resistant lines wereurther analyzed (Table 1 and Fig. 3). The presence of pac1 as well asts expression was confirmed in 15 transgenic lines (t2–t4, t8–t17,19 and t21), as presented in Fig. 3, indicating higher transformationrequency than in impatiens plants.

.4. Screening for TSWV resistance in vitro

To test whether the transformed impatiens and tobacco linesere resistant to TSWV, all I. walleriana lines where presence of

ac1 gene was confirmed (i3, i6, i8 and i10) and four selectedobacco lines that express pac1 gene (t3, t4, t15 and t19) werehallenged with TSWV. The plants were inoculated manually withbrasive, which is a routine technique for the evaluation of virusesistance, but the entire inoculation and screening process waserformed in vitro. Only a few papers describe similar in vitro inoc-lation protocols. Russo and Slack (1998) used glass rods withbrasive silicon carbide for in vitro inoculation of three Solanaceaepecies, potato, tomato and tobacco, with Potato virus Y and Cucum-er mosaic virus. The method we developed for inoculation withSWV is similar to simple and efficient in vitro method for test-ng Lettuce mosaic virus resistance in lettuce culture, developed by

azier et al. (2004). Putative virus-resistant transgenic plants aresually transferred from tissue culture to a greenhouse or a growthhamber to screen for virus resistance and to monitor symptomsevelopment (Mandal et al., 2008). This is a costly and time-onsuming process, constrained by environmental safety issues,specially when handling recombinant or quarantine viruses andenetically modified plants. Regardless of the method, additionaldvantages of in vitro inoculation and screening include decreasedpace requirements for propagation and maintenance of lines andasier controlling of the environment including light, temperaturend other pathogens and pests (Russo and Slack, 1998).

The inoculated plants were observed daily for development ofhe symptoms. The majority of control impatiens plants (7 out of0 inoculated, Table 2) developed symptoms including chloroticpots, blisters, local necrotic lesions and leaf deformations (Fig. 5a),

eeks: (a) control I. walleriana plants inoculated with TSWV display chlorotic spots,riana line i6 appear healthy four weeks following inoculation; (c) mottle along leafed with TSWV with no signs of infection.

12–21 days post inoculation (DPI). The presence of the virus wasconfirmed in all symptomatic plants by DAS-ELISA test, performed30 DPI. All but one (9/10) control tobacco plants initially producedchlorotic spots (8–10 DPI), while leaf deformation and mottlesalong veins (Fig. 5c) developed 8–16 DPI. Two control plants weresystemically infected (with necrotic spots on newly developedleaves), and two plants died a week following symptoms recording.Serological analyses, however, confirmed the TSWV presence in allthe remaining plants, including the asymptomatic one (Table 2).Since the majority of challenged impatiens plants and all con-trol tobacco plants were infected, we could conclude that themethod of in vitro inoculation was successful. Manual transmis-sion of TSWV is not very efficient in certain host species, resultingin many ‘escapes’, as reported for mechanical transmission of TSWVto peanut (Mandal et al., 2006, 2008). Plant growth environment,growth stage, source of inoculum, antioxidants and abrasives werefound to influence the rate of mechanical transmission of viruses,but manual inoculation also depends on the experimenter’s skill,as uneven hand pressure while rubbing lamina may cause dam-age to the leaves. The higher inoculation efficiency of tobacco incomparison to impatiens may be due to different leaves’ size, sur-face and softness, which can influence the inoculum coverage andpenetration (Mandal et al., 2008).

Transformed I. walleriana lines i6 and i8, which accumulatedpac1 transcript (Fig. 3) were completely resistant to TSWV, asno symptoms were developed within 30 DPI, and the ELISA testwas negative (Fig. 5b and Table 2). Interestingly, even i3 and i10lines with undetectable pac1 expression showed lower incidenceof infection, delayed symptom development and milder symptomsin comparison to control plants (Table 2). Expression of 35S-driventransgenes may vary not only among independent transformants orindividuals of the same line, but also between leaves on the sameplant and within a leaf (van Leeuwen et al., 2001), so it is possi-ble that i3 and i10 lines actually had some (low) pac1 expressionthat was hardly detectable by RT-PCR. All of the tobacco lines chal-lenged with TSWV accumulated the pac1 transcript (Fig. 3), but

only t3 and t19 lines showed complete resistance (Fig. 5d), while t4and t15 were infected, but with lower frequency, prolonged incu-bation period and somewhat milder symptoms in comparison tountransformed plants (Table 2). The presence of the symptoms was

S. Milosevic et al. / Scientia Horticulturae 164 (2013) 499–506 505

Table 2Screening for TSWV resistance in vitro.

Lines No. of symptomaticplants

Pac1expression

Incubationperiod (DPI)

Type ofsymptoms

No. TSWV positiveplants by DAS-ELISA

I. wallerianacontrol 7/10 – 12–21 cs, bs, nll, d, ld 7/10i3 2/10 − 18–24 cs, nll 2/10i6 0/10 + - - 0/10i8 0/10 + - - 0/10i10 3/10 − 19–20 cs, nll 3/10

N. tabacumcontrol 9/10 – 8–16 mnn, ld, si 8a/8t3 0/10 + - - 0/10t4 3/10 + 11–19 cs, d, mnn 3/10t15 4/10 + 12–19 mo, da, d 4/10t19 0/10 + - - 0/10

TSWV was mechanically transmitted in vitro to 10 plantlets of each of I. walleriana and N. tabacum transformed lines and controls. In some plants the inoculation causeddevelopment of the following symptoms: bs, blister and sunken part; cs, chlorotic spots; d, distortion; da, dark areas; ld, leaf deformation; mnn, mottle near leaf’s nerves;mo, mottle; nll, necrotic local lesion; si, systemic infection. Incubation period, e.g. days post inoculation (DPI) when the symptoms developed is indicated. All the plants weretested for the presence of TSWV by DAS-ELISA at 30 DPI.

a Two of the control plants died from infection before the DAS-ELISA test performed, so 8 plantlets were analyzed.

Table 3Morphological traits of I. walleriana and N. tabacum pac1 transformants.

Line Shoot length(mm)

Number ofnodes per plant

Number ofleaves per plant

Leaf length(mm)

Number of axillarybuds per plant

I. wallerianaControl 93.33 ± 0.11a 10.67 ± 0.33a 12.33 ± 0.18a 29.20 ± 0.67b 1.67 ± 0.33ai6 89.20 ± 0.10a 10.40 ± 0.40a 13.00 ± 0.14a 27.42 ± 0.41ab 1.60 ± 0.20ai8 78.67 ± 0.09a 10.00 ± 0.36a 11.83 ± 0.10a 24.76 ± 0.17a 1.83 ± 0.18aN. tabacumControl 9.26 ± 0.24b 11.00 ± 0.58bcd 7.23 ± 0.50cd 1.33 ± 0.13abt3 9.20 ± 0.53b 10.33 ± 0.88bc 7.33 ± 0.56cd 1.67 ± 0.03abct4 8.93 ± 0.38b 8.67 ± 0.33ab 7.50 ± 0.36cd 1.67 ± 0.33abct9 9.27 ± 0.47b 10.33 ± 0.33bc 7.20 ± 0.15cd 1.67 ± 0.11abct10 10.50 ± 0.32b 14.67 ± 0.27d 6.87 ± 0.18d 1.67 ± 0.23abct11 11.50 ± 0.48b 11.67 ± 0.18bcd 7.63 ± 0.19d 2.33 ± 0.09cdt12 10.80 ± 0.46b 12.00 ± 0.59bcd 7.33 ± 0.14cd 2.00 ± 0.17bct13 10.37 ± 0.79b 13.67 ± 0.5cd 6.50 ± 0.56bcd 3.00 ± 0.31dt15 5.80 ± 0.17a 7.00 ± 0.17a 4.40 ± 0.12a 2.33 ± 0.16cdt16 9.07 ± 0.28b 12.00 ± 0.63bcd 5.70 ± 0.19bc 2.00 ± 0.13bct17 10.3 ± 0.15b 13.00 ± 0.7cd 6.70 ± 0.59bcd 1.30 ± 0.09ab

M e andt differ

ipsmu1n(todbdeTwt2l

3t

c

t19 5.23 ± 0.34a 8.33 ± 0.33ab

orphometric parameters were determined for 10 plants of each transformed linransformants. Statistical difference at a significance level of P < 0.05 is indicated in

n accordance with ELISA results. Our results are consistent withreviously reported results for pac1-transformed tobacco, whichhowed a decrease in lesion numbers when challenged with Tomatoosaic virus, and a delay in the appearance of symptoms when inoc-lated with Cucumber mosaic virus or Potato virus Y (Watanabe et al.,995). Transgenic Chrysanthemum plants bearing pac1 showed sig-ificantly lower infection frequency with TSWV than control plantsOgawa et al., 2005). These effects of pac1 dsRNase-mediated resis-ance in transformed plants are probably due to (partial) inhibitionf virus replication in each cell (Watanabe et al., 1995). The intro-uction of pac1 may protect plants not only from a variety of viruses,ut also from viroids, such as Potato spindle tuber viroid that can beigested by pac1 gene product in vitro (Sano et al., 1997; Ishidat al., 2002) or Chrysanthemum stunt viroid (Ishida et al., 2002;oguri et al., 2003, Ogawa et al., 2005). Since pac1 introductionas shown to confer broad spectrum of resistance against mul-

iple plant viruses and viroids (Watanabe et al., 1995; Ishida et al.,002; Ogawa et al., 2005), the obtained I. walleriana transgenes are

ikely resistant to some other viruses beside TSWV.

.5. Morphological traits of pac1-transgenic I. walleriana and N.

abacum

Transformed I. walleriana and N. tabacum that express pac1 wereompared with control plants in morphological traits including

4.90 ± 0.10ab 1.00 ± 0.07a

control in order to evaluate the effect of pac1 expression on morphology of theent letters.

shoot length, number of nodes per plant, number of leaves, leaflength, and the number of axillary buds (Table 3). For all therecorded parameters there were no statistically significant differ-ences between the transgenic and control impatiens plants, exceptthat the leaves were somewhat shorter in transformed plants(Table 3). Most of the tobacco lines also did not differ signifi-cantly from the untransformed lines, except that lines t15 and t19were smaller than control plants. It can be concluded that the pac1expression does not significantly alter morphology of the regener-ants. The obtained results are in concordance with earlier findingsthat pac1 gene expression caused no detrimental effects on tobaccoregeneration, physiology, morphology or fertility (Watanabe et al.,1995). Also, the pac1 transgenic chrysanthemums did not look dif-ferent from the non-transgenic plants (Ishida et al., 2002).

4. Conclusions

A successful transformation and regeneration protocol startingwith nodal segments with axillary buds has been developed for I.walleriana, a plant known to be recalcitrant to tissue culture manip-ulations and transformation. However, the protocol may be further

optimized in order to increase its efficiency. The developed manualin vitro method for inoculation of plants with TSWV is very effec-tive, as most of the inoculated plants were infected. Expression ofpac1 transgene provides complete and effective protection against

5 orticu

Tcltewap

A

eW“sD

R

B

B

B

B

C

C

D

D

Ð

G

G

G

G

G

I

J

K

K

L

06 S. Milosevic et al. / Scientia H

SWV in I. walleriana, while transgenic tobacco lines explain eitheromplete resistance or enhanced tolerance to TSWV, evident asower incidence of infection, delayed symptoms and milder symp-oms in comparison to the untransformed plants. Importantly, pac1xpression does not affect the morphology of the transformants. Itould be interesting to challenge the obtained transgenic I. walleri-

na lines with other viruses, especially with INSV, since pac1 shouldrovide a very broad resistance to viral and viroid diseases.

cknowledgments

We thank Dr. Toshihiro Toguri, Plant Laboratory, Kirin Brew-ry Co., Japan, for providing vector pKT-Lpac1 used in this work.e also thank Dr. Branka Uzelac, Institute for Biological Research

Sinisa Stankovic” for the tobacco in vitro culture. This study wasupported by the Ministry of Education, Science and Technologicalevelopment of the Republic of Serbia, Project TR-31019.

eferences

axter, A., 2005. Regeneration and Transformation of Impatiens walleriana usingCotyledonary Node Culture. Master of Science in Horticulture, State University,Blacksburg.

eachy, R.N., 1999. Coat-protein-mediated resistance to tobacco mosaic virus: dis-covery mechanisms and exploitation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354(1383), 659–664.

ertani, G., 1951. Studies on lysogenesis, I. The mode of phage liberation by lysogenicEscherichia coli. J. Bacteriol. 62, 293–300.

raiser, C.M., 2008. The biosecurity threat to the UK and global environment frominternational trade in plants. Plant Pathol. 57, 792–808.

larke, J.L., Spetz, C., Haugslien, S., Xing, S., Dees, M.W., Moe, R., Blystad, D.R., 2008.Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbiapulcherrima, with virus-derived hairpin RNA constructs confers to Poisettiamosaic virus. Plant Cell Rep. 27, 1027–1038.

lemente, T., 2006. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana). In:Wang, K. (Ed.), Agrobacterium protocols. Methods in Molecular Biology 343,Vol. 1, 2nd ed. Humana Press Inc., New Jersey, pp. 143–154.

an, Y., Baxter, A., Zhang, S., Pantazis, C.J., Veilleux, R.E., 2010. Development ofefficient plant regeneration and transformation system for impatiens usingAgrobacterium tumefaciens and multiple bud cultures as explants. BMC PlantBiol. 10, 165.

aughtrey, M.L., Jones, R.K., Moyer, J.W., Daub, M.E., 1997. Tospoviruses strike thegreenhouse industry – INSV has become a major pathogen on flower crops. PlantDis. 81, 1220–1230.

ekic, I., Bulajic, A., Vucurovic, A., Ristic, D., Krstic, B., 2008. Influence of Tomato Spot-ted WiltVirus uneven distribution on its serological detection in tomato, PepperOrnamentals. Pestic. Phytomed. (Belgrade) 23, 225–234 (in Serbian).

anapathi, T.R., Suprasanna, P., Rao, P.S., Bapat, V.A., 2004. Tobacco (Nicotianatabacum L.) – a model system for tissue culture interventions and genetic engi-neering. Indian J. Biotechnol. 3, 171–184.

asic, K., Hernandez, A., Korban, S.S., 2004. RNA extraction from different appletissues rich in polyphenols and polysaccharides for cDNA library construction.Plant Mol. Biol. Rep. 22, 437a-437g.

ooding, G.V., 1991. Disease caused by viruses. In: Shew, H.D., Lucas, G.B. (Eds.),Compendium of Tobacco Diseases. American Phytopathological Society, St. Paul,MN, pp. 41–47.

leba, Y., Marillonnet, S., Klimyuk, V., 2004. Engineering viral expression vectors forplants: the full virus and the deconstructed virus strategies. Curr. Opin. PlantBiol. 7, 182–188.

uo, B., Haider, A.B., Zeb, A., Xu, L.L., Wei, Y.H., 2011. Thidiazuron: a multi-dimensional plant growth regulator. Afr. J. Biotechnol. 10 (45), 8984–9000.

shida, I., Tukahara, M., Yoshioka, M., Ogawa, T., Kakitani, M., Toguri, T., 2002. Pro-duction of anti-virus, viroid plants by genetic manipulations. Pest Manage. Sci.58, 1132–1136.

ones, M.P.A., Cao, J., O’Brien, R., Murch, S.J., Saxena, P.K., 2007. The mode of actionof thidiazuron: auxins, indoleamines and ion channels in the regeneration ofEchinacea purpurea L. Plant Cell Rep. 26, 1481–1490.

arthikeyan, A., Valarmathi, A., Nandini, S., Nandhakumar, M.R., 2012. Geneticallymodified crops: insect resistance. Biotechnology 11, 119–126.

oubouris, G.C., Maliogka, V.I., Efthimiou, K., Katis, N.I., Vasilakakis, M.D., 2007. Elim-ination of Plum pox virus through in vitro thermotherapy and shoot tip culture

compared to conventional heat treatment in apricot cultivar Bebecou. J. Gen.Plant Pathol. 73 (5), 370–373.

iu, X., Pijut, P., 2010. Agrobacterium-medicated transformation of mature Prunusserotina (black cherry) and regeneration of trangenic shoots. Plant Cell TissueOrgan Cult. 101, 49–57.

lturae 164 (2013) 499–506

Mandal, B., Csinos, A.S., Martinez-Ochoa, N., Pappu, H.R., 2008. A rapid and effi-cient inoculation method for Tomato spotted wilt virus. J. Virol. Methods 149,195–198.

Mandal, B., Pappu, H.R., Csinos, A.S., Culbreath, A.K., 2006. Response of peanut, pep-per, tobacco, and tomato cultivars to two bilogically distinct isolates of Tomatospotted wilt virus. Plant Dis. 90 (9), 1150–1155.

Mazier, M., German-Retana, S., Flamain, F., Dubois, V., Botton, E., Sarnette, V., LeGall, O., Candresse, T., Maisonneuve, B., 2004. A simple and efficient methodfor testing Lettuce mosaic virus resistance in in vitro cultivated lettuce. J. Virol.Methods 116 (2), 123–131.

Milosevic, S., Cingel, A., Jevremovic, S., Stankovic, I., Bulajic, A., Krstic, B., Subotic, A.,2012a. Virus Elimination from Ornamental Plants Using In Vitro Culture Tech-niques. Pestic. Phytomed. (Belgade) 27 (3), 203–211.

Milosevic, S., Simonovic, A., Cingel, A., Jevremovic, S., Todorovi c, S., Filipovic, B.,Subotic, A., 2012b. Response of antioxidative enzymes to long-term Tomato spot-ted wilt virus infection and virus elimination by meristem-tip culture in twoImpatiens species. Physiol. Mol. Plant Pathol. 79, 79–88.

Milosevic, S., Subotic, A., Bulajic, A., Ðekic, I., Jevremovic, S., Vucurovic,A., Krstic, B., 2011. Elimination of TSWV from Impatiens hawkeriiBull and regeneration of virus-free plant. Electron J. Biotechnol. 1, 4,http://dx.doi.org/10.2225/vol14-issue-1-fulltext/5.

Milosevic, S., 2010. Regeneration and Genetic Transformation of Plants Impatienswalleriana L. and Impatiens hawkerii Bull. in vitro culture (in Serbian). Universityof Belgrade, Serbia, Doctoral dissertation.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassayswith tobacco tissue culture. Physiol. Plant 15, 473–497.

Ogawa, T., Toguri, T., Kudoh, H., Okamura, M., Momma, T., Yoshioka, M., Kato, K.,Hagiwara, Y., Sano, T., 2005. Double-stranded RNA-specific ribonuclease conferstolerance against Chrysanthemum Stunt Viroid and Tomato Spotted Wilt Virus intransgenic Chrysanthemum plants. Breed. Sci. 55, 49–55.

Parrella, G., Gognalons, P., Gebre-Selassie, K., Vovlas, C., Marchoux, G., 2003. Anupdate of the host range of Tomato spotted wilt virus. J. Plant Pathol. 85,227–264.

Rotondo, G., Frendewey, D., 1996. Purification and characterization of the Pac1ribonuclease of Schizosaccharomyces pombe. Nucleic Acids Res. 24 (12),2377–2386.

Rotondo, G., Huang, J.Y., Frendewey, D., 1997. Substrate structure requirements ofthe Pac1 ribonuclease from Schizosaccharmyces pombe. RNA 3 (10), 1182–1193.

Russo, P., Slack, S.A., 1998. Tissue culture methods for the screening and analysis ofputative virus-resistant transgenic potato plants. Phytopathology 88, 437–441.

Safarnejad, M.R., Jouzani, G.S., Tabatabaei, M., Twyman, R.M., Schillberg, S., 2011.Antibody-mediated resistance against plant pathogens. Biotechnol. Adv. 29 (6),961–971.

Sano, T., Nagayama, A., Ogawa, T., Ishida, I., Okada, Y., 1997. Transgenic potatoexpressing a double-stranded RNA-specific ribonuclease is resistant to potatospindle tuber viroid. Nat. Biotechnol. 15 (12), 1290–1294.

Simón-Mateo, C., García, J.A., 2011. Antiviral strategies in plants based on RNA silenc-ing? Biochim. Biophys. Acta 1809 (11–12), 722–731.

Stankovic, I., Bulajic, A., Vucurovic, A., Ristic, D., Milojevic, K., Berenji, J., Krstic,B., 2011. Status of tobacco viruses in Serbia and molecular characterization oftomato spotted wilt virus isolates. Acta Virol. 55, 337–347.

Subotic, A., Jevremovic, S., Cingel, A., Milosevic, S., 2008. Effect of urea-typecytokininon axillary shoots regeneration of Impatiens walleriana L. Biotechnol.Biotechnol. 22 (3), 817–819.

Sudarshana, M.R., Roy, G., Falk, B.W., 2007. Methods for engineering resistance toplant viruses. Methods Mol. Biol. 354, 183–195.

Thirukkumaran, G., Ntui, V.O., Khan, R.S., Mii, M., 2009. Thidiazuron: an efficientplant growth regulator for enhancing Agrobacterium-mediated transformationin Petunia hybrida. Plant Cell Tissue Organ Cult. 99, 109–115.

Toguri, T., Ogawa, T., Kakitani, M., Tukahara, M., Yoshioka, M., 2003. Agrobacterium-mediated transformation of Chrysanthemum (Dendranthema grandiflora) plantswith a disease resistence gene (pac1). Plant Biotechnol. 20 (2), 121–127.

Uzelac, B., Atanackovic, J., Janosevic, D., Ninkovic, S., Budimir, S., 2006. Shoot api-cal meristem structure in transgenic tobacco. In: IV Balkan Botanical Congress:Plant, Fungal and Habitat Diversity Investigation and Conservation, Sofia, Bul-garia, p. 148.

van Leeuwen, W., Ruttink, T., Borst-Vrenssen, A.W., van der Plas, L.H., van der Krol,A.R., 2001. Characterization of position-induced spatial and temporal regulationof transgene promoter activity in plants. J. Exp. Bot. 52 (358), 949–959.

Wang, C.-H., Hsu, S.-Y., Chen, P.-Y., To, K.-Y., 2012. Transformation and characteri-zation of transgenic Bidens pilosa L. Plant Cell Tiss. Organ Cult. 109, 457–464.

Wang, Q., Valkonen, J.P.T., 2009. Cryotherapy of shoot tips: novel pathogen eradica-tion method. Trends Plant Sci. 14 (3), 119–122.

Watanabe, Y., Ogawa, T., Takahashi, H., Ishida, I., Takeuchi, Y., Yamamoto, M., Okada,Y., 1995. Resistence against multiple plant viruses in plants mediated by a doublestranded-RNA specific ribonuclease. FEBS Lett. 372, 165–168.

Yancheva, S.D., Golubowicz, S., Fisher, E., Lev-Yadun, S., Flaishman, M.A., 2003. Auxin

type and timing of application determine the activation of the developmentalprogram during in vitro organogenesis in apple. Plant Sci. 165, 299–309.

Zhou, X., Guangcheng, C., Rufa, L., Yongru, S., Wenbin, L., 1994. A rapid and efficientDNA extraction method of genus Fagopyrum for RAPD analysis. In: Proceedingsof IPBA, Rogla, pp. 171–175.