Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Share Embed

Descrição do Produto

Annals of Botany 110: 479 –489, 2012 doi:10.1093/aob/mcs086, available online at


Composite Cucurbita pepo plants with transgenic roots as a tool to study root development Elena L. Ilina1, Anton A. Logachov1, Laurent Laplaze3, Nikolay P. Demchenko1, Katharina Pawlowski2 and Kirill N. Demchenko1,* 1

Komarov Botanical Institute, Russian Academy of Sciences, Prof. Popova 2, 197376, St.-Petersburg, Russia, 2Department of Botany, Stockholm University, 10691 Stockholm, Sweden and 3Institut de Recherche pour le De´veloppement, UMR DIADE (Agro.M/INRA/IRD/UM2), Equipe Rhizogene`se, 911 Avenue Agropolis, F-34394 Montpellier cedex 5, France * For correspondence. E-mail [email protected] Received: 1 December 2011 Returned for revision: 2 February 2012 Accepted: 5 March 2012 Published electronically: 2 May 2012

Key words: Agrobacterium rhizogenes, auxin, CaMV 35S enhancer, composite plants, Cucurbita pepo, Cucurbitaceae, DR5, hairy roots, root, squash.

IN T RO DU C T IO N The Cucurbitaceae family includes three genera of economically important crop species whose habitats extend from temperate to tropical zones: Cucumis (cucumber, melon), Citrillus (watermelon) and Cucurbita [winter and summer squash, pumpkins, marrows, zucchini (courgettes) and gourds] (Gaba et al., 2004). Cucurbits are well known for their relatively fast accumulation of biomass and high yields in conditions of moderate extra nutrition. In most plant species, initiation of lateral root primordia occurs above the elongation zone (reviewed by Lloret and Casero, 2002). However, in cucurbits and some other species (Fagopyrum esculentum, Ipomea purpurea, Pistia stratiotes and Eichhornia crassipes), lateral root primordia initiation and development take place in the apical meristem of the parental root (Gulyaev, 1964; O’Dell and Foard, 1969; Mallory et al., 1970; Seago, 1973; Clowes, 1985; Dubrovsky, 1986, 1987; Demchenko and Demchenko, 2001).

By using fusions of phytohormone-responsive promoters and reporter genes, the involvement of the plant hormones in developmental processes can be studied (Ulmasov et al., 1997; D’Agostiono et al., 2000; Vernoux et al., 2011). To use these tools to examine the physiological and molecular mechanisms of lateral root initiation and development, a plant transformation method is required. Techniques for the transformation of Cucurbitaceae family members (cucumber and squash) by Agrobacterium tumefaciens have been described (Smarrelli et al., 1986; Chee, 1990; Vasudevan et al., 2007). However, these transformation techniques are labour-intensive, expensive and take a long time. For the study of root development, A. rhizogenesmediated transformation offers an alternative approach. Agrobacterium rhizogenes is a soil bacterium able to induce the development of so-called ‘hairy roots’ on a range of dicotyledonous plants. Infection of wounded plants by A. rhizogenes results in the transfer, integration and expression of T-DNA from the root-inducing (Ri) plasmid. Hairy roots

# The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected]

Downloaded from at Stockholms Universitet on July 11, 2012

† Background and Aims In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash. † Methods The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′ -deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or b-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively. † Key Results Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots. † Conclusions The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.


Ilina et al. — Use of composite Cucurbita plants to study root development

M AT E R IA L S A ND M E T HO DS Plasmids and vectors

The modified pMDC162-GFP vector was obtained by replacing the hygromycin resistance marker with 35S::gfp. For this purpose, pMDC162 (Curtis and Grossniklaus, 2003) was digested by XhoI to remove the hygromycin resistance marker, purified on a gel and treated with calf alkaline phosphatase. The 35S::gfp cassette from pHKN29 (Kumagai and Kouchi, 2003) was amplified by PCR using primers 5′ -CCCCTCGAGTTATCTGGGAACTACTCACA-3′ and 5′ ATTCTCGAGTTTGACAGCTTATCATCGG-3′ to introduce an XhoI restriction site (underlined) on both ends. Purified PCR products were digested by XhoI and ligated to the XhoI-digested pMDC162. The pKGW-RR-MGW vector (Op den Camp et al., 2011) containing a Egfp-gusA fusion reporter (Karimi et al., 2002) as well as pAtUBQ10::DsRED1 as a selectable marker (Limpens et al., 2004) was kindly provided by Dr Erik Limpens (Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands). The DR5 promoter cloned in pBI101.3 was kindly provided by Dr Tom J. Guilfoyle (University of Missouri, Columbia, USA). To create pKGW-RR-MGW-DR5 and pMDC162GFP-DR5, the DR5 promoter was PCR-amplified using the pBI101.3 construct with DR5 as template, and primers including EcoRI restriction sites (underlined): 5′ -CGAATTCGGTA TCGCAGCCCCCTTTTGTCTCC-3′ and 5′ -CGAATTCTGT TGTTTGTTGTTTGTTGTTGTTGGTAATTGTTG-3′ . The DR5 PCR product was cloned in pJET1.2 (Fermentas, Thermo Fisher Scientific, Schwerte, Germany), excised by

EcoRI digestion and subsequently cloned in pBluescript II KS(+) (Stratagene, La Jolla, CA, USA). DR5 was excised from pBluescript II KS(+) using BamHI and XhoI, and cloned in the pUC18-entry8 GATEWAY Entry vector (Hornung et al., 2005). DR5 was transferred into pKGWRR-MGW and the pMDC162-GFP destination binary vectors by LR clonase reaction (Gatewayw LR ClonaseTM II Enzyme Mix, Life Technologies, Gaithersburg, MD, USA). The resulting fusions DR5::Egfp-gusA ( pKGW-RR-MGW-DR5) and DR5::gusA ( pMDC162-GFP-DR5) were verified by PCR amplification of fragments with a forward primer for DR5 and a reverse primer for gusA (DR5_5′ -CGAATTCGGTAT CGCAGCCCCCTTTTGTCTCC-3′ and Ec_GUS_seqrev_5′ TCCCACCAACGCTGATCAAT-3′ ) and sequencing of the products. Bacterial strains

Agrobacterium rhizogenes strains R1000 and MSU440 were used for transformation of squash seedlings. Agrobacterium rhizogenes strain R1000 contains the pRiA4b Ri plasmid from A. rhizogenes strain A4T (Moore et al., 1979; White et al., 1985), while MSU440 contains the Ri plasmid pRiA4 (Sonti et al., 1995). The binary vectors pKGW-RRMGW-DR5 and pMDC162-GFP-DR5, and original vectors were introduced into agrobacterial cells by electroporation (MicroPulserTM , Bio-Rad Laboratories). Each strain was used with four different plasmids, pKGW-RR-MGW-DR5, pKGW-RR-MGW, pMDC162-GFP-DR5 and pMDC162GFP. Transformed agrobacteria were grown for 2 d on YEB plates under spectinomycin selection (50 mg mL21) for pKGW-RR-MGW-DR5 or under kanamycin selection (50 mg mL21) for pMDC162-GFP-DR5. The presence of the vectors in the transformed agrobacteria was confirmed by colony PCR with the same primers as described above. The integrity of the constructs was confirmed by DNA isolation and digestion with EcoRV for pKGW-RR-MGW-DR5, and with KpnI and EcoRI for pMDC162-GFP-DR5. For plant transformation, the transformed strains were grown on YEB agar with the appropriate antibiotics at 25 8C for 36 h. Plant material

The squash (Cucurbita pepo L. var. giromontia) cultivar ‘Beloplodniy’ was used in this study. Seeds were purchased from a local provider. The young seedlings used for the transformation procedure were prepared as follows. Seeds were surface sterilized in a mix (2:1:1) of 96 % ethanol/33 % hydrogen peroxide/sterile double-distilled water (ddH2O) for 8 min, rinsed 3 – 4 times in copious amounts of sterile ddH2O and plated on 0.8 % water agar ( pH 5.6) slopes. The Petri dishes with agar slopes were oriented vertically to enable the roots to grow downward. Petri dishes were sealed with SilkofixTM adhesive tape to ensure sufficient gas exchange. Seeds were germinated at 25 8C in the darkness for 6 d. Afterwards, healthy etiolated seedlings with uncovered cotyledons and 2.0 – 2.5 cm hypocotyls were selected for further use. For 5-ethynyl-2′ -deoxyuridine (EdU) incorporation experiments, seeds were sterilized as described above, planted in

Downloaded from at Stockholms Universitet on July 11, 2012

emerge due to expression of the rolA, rolB, rolC and rolD genes (Gelvin, 1990; Christey and Braun, 2005). If A. rhizogenes harbours a binary vector in addition to the Ri plasmid, transgenic roots can be co-transformed with both the T-DNA from the Ri plasmid and the T-DNA cassette from the binary vector. Composite plants with wild-type shoots and transgenic roots obtained by A. rhizogenes transformation are widely used for the investigation of nodulation and plant – nematode interactions (Quandt et al., 1993; Limpens et al., 2004; Alpizar et al., 2006; Kereszt et al., 2007). Previous studies reported success in establishing hairy roots for Cucumis sativus, Cucumis melo and Cucurbita pepo (Katavic´ et al., 1991; Sanita di Toppi et al., 1997; Pak et al., 2009; Anuar et al., 2011), based on the infection of excised cotyledons. However, so far no protocol has been established for the production of composite transgenic plants of squash. Moreover, Katavic´ et al. (1991) found it impossible to induce hairy roots on intact cucurbit plants. Thus, in order to examine root branching mechanisms in squash, a protocol had to be developed for the production of composite plants with transgenic hairy root systems. In this paper, a procedure for A. rhizogenes-mediated transformation of squash is described. Our protocol is based on the approach developed for Arabidopsis thaliana and Medicago truncatula (Limpens et al., 2004), but differs in the use of agrobacterial strains, inoculation site and culture conditions.

Ilina et al. — Use of composite Cucurbita plants to study root development sterile vermiculite moistened with 1/4 strength Hoagland’s medium (Hoagland and Arnon, 1938) and incubated at 25 8C for 6 d with a light period of 16 h. Agrobacterium rhizogenes-mediated transformation

Detection of DNA replicating cells

Healthy 6-day-old squash seedlings were incubated in aerated 1/4 strength Hoagland’s medium supplemented with 50 mM EdU (Life Technologies; Salic and Mitchison, 2008) for 4 h in darkness. Then hypocotyl and cotyledons were cut into 3– 4 mm segments. The hypocotyl segment closest to the root basis was referred to as the basal segment and the one closest to the cotyledons was referred to as the upper segment. Plant material was infiltrated with fixative (3 %

paraformaldehyde, 0.25 % glutaraldehyde, 0.1 % Tween-20, 0.1 % Triton X-100 in 0.3× MTSB; Vitha et al., 1997; Stumpe et al., 2006) under vacuum (– 1 atm), fixed overnight at 4 8C and rinsed with 0.3× MTSB buffer (50 mM PIPES, 5 mM MgSO4, 5 mM EGTA). The hypocotyl segments were embedded in 3 % agarose (SeaKem LE agarose, Cambrex, Karlskoga, Sweden) and sectioned with a vibrating blade microtome HM 650V (Microm, Walldorf, Germany). Sections of 50 mm were RNaseA treated [0.1 % RNaseA (Sigma-Aldrich, Taufkirchen, Germany) in 2 × SSC buffer] at 37 8C for 1 h, subsequently rinsed with Tris-buffered saline (TBS: 50 mM Tris – HCl, 150 mM NaCl, pH 7.3) and blocked in blocking buffer [5 % bovine serum albumin (BSA; Roche, Mannheim, Germany), 1 % goat serum (Life Technologies), 0.2 % cold fish gelatine (Life Technologies) in TBS, pH 7.3]. The Click-iT reaction for EdU visualization was performed according to the manufacturer’s instructions (Click-iTTM EdU AlexaFluor 488 Imaging Kit, Life Technologies). Nuclei were counterstained with 2 mM TO-PRO3 iodide (Life Technologies). Sections were mounted on slides in ProLong Gold antifade reagent (Life Technologies) under cover slips. PCR analyses

In order to confirm the transfer of the T-DNA from the binary vector, total DNA was purified from DsRED1-positive or GFP-positive roots, respectively, using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). To assess the quality of purified DNA, PCRs for the plant ubiquitin gene were carried out with the following primers: 5′ -ATGCGATYTTTGTGA AGAC-3′ and 5′ -ACCACCACGRAGACGGAG-3′ . The cycling conditions for ubiquitin amplification were as follows: 90 s at 94 8C, followed by 35 cycles of 30 s at 94 8C, 30 s at 54 8C and 30 s at 72 8C. In order to confirm that the plant DNA was not contaminated with agrobacterial DNA, PCRs with primers VCF/VCR for the virC operon on the Ri plasmid were carried out (Sawada et al., 1995). PCR conditions were 3 min at 95 8C, followed by 30 cycles of 30 s at 95 8C, 30 s at 55 8C and 60 s at 72 8C. The following primers were used for the amplification of a 1115 bp fragment of the DR5::gfp-gusA fusion from pKGW-RR-MGW-DR5 T-DNA or a 450 bp fragment of the DR5::gusA fusion from pMDC162-GFP-DR5, respectively: DR5_5′ -CGAATTCGGT ATCGCAGCCCCCTTTTGTCTCC-3′ and Ec_GUS_seqrev_ 5′ -TCCCACCAACGCTGATCAAT-3′ . PCR conditions were 3 min at 95 8C, followed by 30 cycles of 30 s at 95 8C, 30 s at 52 8C, and 90 or 60 s at 72 8C, respectively. DreamTaqTM Green DNA polymerase (Fermentas) was used for all amplifications (1.25 U per 50 mL reaction). PCR products were separated by electrophoresis on 1.5 % LE SeaKem agarose gels stained with ethidium bromide in TAE (Tris-acetate/EDTA) electrophoresis buffer, pH 8.0. Images were obtained using the GelDoc XR imaging system (Bio-Rad Laboratories) and processed using Quantity One software. Histochemical GUS staining

To assay b-glucuronidase (GUS) reporter activity, whole DsRED1- or GFP-positive roots were infiltrated with staining

Downloaded from at Stockholms Universitet on July 11, 2012

The roots of 6-day-old squash seedlings were cut off at the root – hypocotyl transition site using a sterile scalpel. Each wounded surface was inoculated with A. rhizogenes strain R1000 or MSU440 harbouring the pKGW-RR-MGW-DR5 or pMDC162-GFP-DR5 binary vector, respectively, which had been scratched off the plate. In the first control experiment, agrobacterial paste was substituted with sterile ddH2O. In the second control experiments, wounded hypocotyls were inoculated with R1000 or MSU440 strains harbouring the binary vectors pKGW-RR-MGW or pMDC162-GFP, respectively, without the DR5 insert. Inoculated seedlings were transferred to agar slopes in square Petri dishes consisting of 0.5× Murashige and Skoog (MS) salts (Murashige and Skoog, 1962; Duchefa, Haarlem, The Netherlands), 1 % sucrose and 0.8 % Microagar (Duchefa). The agar slopes were covered by filter paper to prevent the seedlings from sliding down. Seedlings were co-cultivated with agrobacteria for 7 d at 20 8C and a 16 h light period, then rinsed twice with excessive amounts of sterile ddH2O and plated on a 0.5× MS agar slope supplemented with the antibiotic cefotaxime (500 mg mL21) and silver nitrate (5 mg mL21) as antiseptic. Putatively transformed squash seedlings were incubated at 25 8C and a 16 h light period until the first roots had emerged from the calli that developed at the wound site (typically for 3 – 5 d). Rooted transformants were transferred to sterile plastic vessels halffilled with autoclaved vermiculite wetted with 1/4 strength Hoagland’s medium. Further cultivation of the transformed plants took place in non-axenic conditions. During the first week of incubation, Hoagland’s medium was supplemented with silver nitrate (5 mg mL21) to prevent any contamination with bacteria or fungi. The vessels were covered with transparent lids in order to maintain a high humidity. At 7 – 14 d after transfer, new roots developed which were potentially co-transformed with the DR5 construct. Transformation was confirmed via analysis for DsRED1 fluorescence in the case of the pKGW-RR-MGW vector, and for GFP fluorescence in the case of the pMDC162-GFP vector. No fluorescence in the red spectrum was observed in control roots of plantlets infected with sterile ddH2O, while they showed background levels of green fluorescence which could easily be distinguished from the strong fluorescence of green fluorescent protein (GFP) in pMDC162-GFP-DR5-transformed seedling roots.



Ilina et al. — Use of composite Cucurbita plants to study root development

solution (1 mM EDTA, 0.2 % Triton X-100, 0.2 % Tween-20 in TBS, pH 7.3) containing 1 mM 5-bromo-4-chloro-3-indolylb-D-glucuronide (X-Gluc). Ferricyanide (0.25 mM) was added to prevent indigo precursor migration. The chelator EDTA was added to the staining solution to prevent any gene expression during the staining procedure. Vacuum (– 1 atm) was applied twice for 5 min to improve infiltration. Roots were incubated in staining solution at 37 8C until sufficient blue staining had been developed. Immunolocalization

Root tip segments (approx. 6 mm long) of co-transformed [A. rhizogenes strain R1000 ( pKGW-RR-MGW-DR5)] and non-transformed roots were fixed on ice as described above and embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. Longitudinal sections (7 mm thick) were obtained on a rotation microtome HM 360 (Microm) equipped with a tungsten carbide knife type D (Thermo Fisher Scientific, Walldorf, Germany) and attached to SuperFrostw slides by heating (70 8C). The cell walls were stained with 0.01 % Ruthenium red (Sigma-Aldrich) solution in 0.1 M borate buffer at pH 9.2 for 5 min (Gutierrez-Gonzalvez et al., 1984). The sections were mounted with EUKITT mounting medium (Sigma-Aldrich) under cover slips and examined under an AxioImager.Z1 (Carl Zeiss) microscope using the Plan-Apochromat ×10/0.45 and ×20/0.80 objectives (×100 and ×200 total magnification, respectively). Counting the number of cells in three outer cortical layers between two of the most proximal lateral root primordia in each rank was performed for the region 2 –6 mm from the root tip behind the cortical dividing zone (Demchenko and Demchenko, 2001). The index of lateral root initiation (Dubrovsky et al., 2009) was determined as the number of lateral root primordia corresponding to 100 cortical cells in one cell layer. Statistical analyses

Data were analysed using Microsoft Office Excel 2010 and Sigma Plot v.12.2 software. Means and standard errors for DsRED1-positive roots were calculated. R E S U LT S Selection of squash seedling site for inoculation with agrobacteria


Examination and imaging of composite plants was performed using a SteREO Lumar.V12 fluorescent stereomicroscope equipped with a MRc5 digital camera (Carl Zeiss, Go¨ttingen, Germany). For observation of DsRED1 fluorescence (Limpens et al., 2004), the filter set 43 HE (EX BP 550/25, EM BP 605/70) was used, while for GFP fluorescence, the filter set 38 HE (EX BP 470/40, EM BP 525/50) was used. Total root systems and roots stained for GUS activity were examined with the same stereomicroscope using bright field microscopy. Images were processed using AxioVision 4.8.2 software (Carl Zeiss) and Adobe Photoshop CS5. Examination of the nuclei in segments of hypocotyls and cotyledons labelled with the DNA synthesis precursor EdU and with TO-PRO3 was performed under a confocal laser scanning microscope LSM 510 META (Carl Zeiss) using 488 nm for the AlexaFluor 488 and 633 nm for the TO-PRO3 laser lines, respectively. Examination and imaging of immunolocalized GFP and GUS was performed under a confocal laser scanning microscope LSM 780 (Carl Zeiss) using a 488 nm laser line for the AlexaFluor 488. For background elimination, a linear spectral unmixing technique and ZEN 2010b software (Carl Zeiss) were used.

Hairy root transformation starts with the transfer of T-DNA from the Ri plasmid to plant nuclei (Gelvin, 1990). In order to achieve callus formation, cells that have received T-DNA inserts have to proliferate. Thus, for hairy root formation, cells that are capable of proliferating after T-DNA insertion have to be transformed. The incorporation of the DNA synthesis precursor EdU in 6-day-old squash seedlings was used to label cells in the S-phase of the cell cycle. The distribution of labelled cells was analysed in different segments of squash seedlings, i.e. in the cotyledons and in the upper and lower parts of the hypocotyl. We found that all segments of 6-day-old squash seedlings contained cells in the S-phase that were able to go through the cell cycle. The majority of labelled cell nuclei were in the vascular cylinder (including the pericycle), the endoderm and in the inner layer of the cortex (Fig. 1). The frequency of labelled cells was lower in the outer cortical files. The presence of DNA-synthesizing cells in the pericycle and surrounding cell files is very important because adventitious roots, including roots caused by A. rhizogenes-mediated transformation, initiate in the pericycle. These results demonstrate that the base of the hypocotyl of 6-day-old squash seedlings is an appropriate site for inoculation by A. rhizogenes.

Downloaded from at Stockholms Universitet on July 11, 2012

To localize GFP and GUS in the transgenic root tissues, DsRED1-positive roots were harvested, immediately cut into 4 – 5 mm segments with a razor blade and fixed on ice as described above. Fixed root segments were rinsed three times with 0.3× MTSB, dehydrated in a graded ethanol series and embedded in Steedman’s wax (Steedman, 1957; Vitha et al., 1997). Serial longitudinal sections (16 mm) were obtained on a rotation microtome HM 360 (Microm) equipped with single-use knives 35SEC-p. Sections were attached to egg white-coated SuperFrostw slides (Carl Roth, Karlsruhe, Germany). Slides were de-waxed using 96 % ethanol, rehydrated in a graded ethanol series and transferred to TBS. Sections were blocked in blocking buffer for 1 h (Stumpe et al., 2006). Briefly, sections were then incubated with the primary rabbit anti-GFP (Life Technologies, #A6455) or with rabbit anti-GUS (Life Technologies, #A-5790) antibodies in a 1:200 dilution at 4 8C overnight; rinsed thoroughly with 0.2 % BSA in TBS and incubated in the secondary AlexaFluorw 488 goat anti-rabbit antibodies for 1 h at room temperature. Sections were rinsed twice with TBS and mounted in ProLong Gold antifade reagent (Life Technologies) under cover slips.

Estimation of lateral root initiation index

Ilina et al. — Use of composite Cucurbita plants to study root development A











Construction of the pMDC162-GFP-DR5 and pKGW-RR-MGW-DR5 binary vectors

Agrobacterium rhizogenes-mediated transformation results in the development of numerous adventitious roots. These roots may be co-transformed with both the T-DNA from the Ri plasmid and the T-DNA from the binary vector. They may also be transformed only with the Ri T-DNA, or their binary vector T-DNA might have inserted in a position where expression is repressed. Therefore, it is necessary to identify roots containing the binary vector T-DNA and express its genes using a non-destructive selection method, e.g. the detection of fluorescent proteins. For successful identification of co-transformed roots, the binary vectors pMDC162-GFP-DR5 and pKGW-RR-MGW-DR5 were prepared (Fig. 2). The pMDC162-GFP-DR5 vector contains the gene for GFP under the control of the Cauliflower mosaic virus (CaMV) 35S promoter, providing the option to identify transgenic roots based on fluorescence of GFP. It was important to find out whether the CaMV 35S promoter had an influence on the expression of the adjacent DR5::gusA cassette. pKGW-RR-MGW-DR5 contains the open reading frame of the red fluorescent protein (DsRED1) under the control of the constitutively active ubiquitin promoter of A. thaliana (AtUBQ10::DsRED1; Limpens et al., 2004), providing the option to identify transgenic roots based on the fluorescence of DsRED1. Agrobacterium rhizogenes-mediated transformation of squash seedlings: analysis of transformation efficiency

For the construction of composite squash plants, a protocol developed for A. thaliana and M. truncatula was modified (Limpens et al., 2004). Approximately 3 weeks after inoculation with a strain containing vector pKGW-RR-MGW-DR5, the first co-transformed roots were observed based on DsRED1 fluorescence (Fig. 3A–C). Roots that developed earlier mostly were either chimeric, i.e. consisted of DsRED1-fluorescent and non-fluorescent sectors, indicating that transformed and non-transformed cells had

contributed to their initiation, or represented non-transgenic or not co-transformed adventitious roots, i.e. they did not show DsRED1 fluorescence. DsRED1 fluorescence was not observed in control roots emerging from water-inoculated plants (Fig. 3E). Morphologically, most of the DsRED1-positive roots were indistinguishable from adventitious roots formed on control plants inoculated with sterile ddH2O (Fig. 3D). The index of lateral root initiation (LRI; Dubrovsky et al., 2009) was determined for co-transformed and for control squash roots (Table 1). Since the LRI indices did not differ statistically between both types of roots, it can be concluded that co-transformed adventitious roots have the same branching pattern as control adventitious roots. Fluorescent transgenic roots did not display features associated with hairy roots, such as plagiotropic growth, abundant root hairs or high frequency of branching (Yoko et al., 1998; Shanks and Morgan, 1999; Christey and Braun, 2005). However, all fluorescent transgenic roots were slightly thinner than wild-type roots (data not shown). Roots from individual composite plants demonstrated heterogeneity in their diameters, and there was some variability in the level of DsRED1 fluorescence between roots of different composite plants (data not shown). Roots harbouring pMDC162-GFP-DR5 T-DNA (35S::gfp, DR5::gusA) as well as control roots (harbouring only 35S::gfp without the DR5 insert) were identified based on GFP fluorescence. These roots also had wild-type morphology (Fig. 6A, B). Squash seedlings showed different levels of susceptibility to the two A. rhizogenes strains R1000 and MSU440, yet transgenic roots were obtained with both strains. If an inoculated squash seedling gave rise to at least one co-transformed (i.e. fluorescent) root it was counted as a transformation event. High transformation efficiencies were achieved using A. rhizogenes strain R1000, i.e. 85 % of surviving squash composite plants inoculated by strain R1000 developed co-transformed roots (n ¼ 35). In contrast, only 33 % of squash seedlings transformed using strain MSU440 strain developed co-transformed transgenic roots (n ¼ 20). On average, each R1000-transformed plant formed 6 (6.4 + 0.85)

Downloaded from at Stockholms Universitet on July 11, 2012

F I G . 1. Distribution of DNA-synthesizing cells in longitudinal sections (50 mm) of the hypocotyl base of squash seedlings. The picture represents the maximum intensity projection of 22 confocal optical sections; the total depth is 15 mm. (A) Green channel, 5-ethynyl-2’-deoxyuridine (EdU; a nucleoside analogue of thymidine, incorporated into DNA during active DNA synthesis) in nuclei is labelled with AlexaFluor 488. (B) Red channel, DNA in cell nuclei stained with TO-PRO-3 iodide with background staining of the xylem vessel. (C) Overview of tissues in the middle position of depth; differential interference contrast. (D) Overlay of the three channels; most DNA-synthesizing cells are localized in the vascular cylinder and in the inner layers of the cortex. C, cortex; p, pericycle; x, xylem. Scale bar ¼ 100 mm.


Ilina et al. — Use of composite Cucurbita plants to study root development EcoRI (1) nos terminator


EcoRV (10803) gusA


EcoRV (1354) EcoRV (1585) LB

attR2 EcoRV (2451) pMDC162-GFP-DR5 11697 bp

Kan R

DR5 attR1 KpnI (2735)

EcoRV (8179)

NotI (4007) pBR322 origin NotI (6829) NotI (5539) Sm/Sp R

LB Kpn I (1748)


Sma I (1750) Nde I(11850)

Xba I(1759) XhoI (1771) Eco RI(1804) Bgl II(2777) pKGW-RRMGW-DR5 13999 bp

Bgl II(2850) Xbe I(2853) pAtUBQ10-DsRED1

Nde I(9361)

Xbe I(3596) Eco RI(3600) RB gfp-gusA

Nde I(7628) attR 1

EcGUS3 primer

DR5 attR 2 F I G . 2. Maps of binary vectors (A) pMDC162-GFP-DR5 and (B) pKGW-RR-MGW-DR5 used for A. rhizogenes-mediated transformation of squash seedlings. (A) pMDC162-GFP-DR5 vector, containing GFP under the control of the CaMV 35S promoter as a screenable marker and a DR5::gusA fusion within the T-DNA borders. (B) pKGW-RR-MGW-DR5 vector, containing DsRED1 under control of the AtUbq10 promoter as a screenable marker, and a DR5::gfp-gusA fusion within the T-DNA borders.

DsRED1-positive roots and each MSU440-transformed plant formed 3 (2.7 + 0.54) DsRED1-positive roots. The portion of co-transformed roots relative to the total number of adventitious roots formed (chimeric or non-fluorescent) was also higher

(23 %; Fig. 4A) in R1000-transformed squash plants than in MSU440-transformed plants (8 %; Fig. 4B). These data demonstrate that the transformation efficiency of A. rhizogenes R1000 on squash was higher than of that of strain MSU440.

Downloaded from at Stockholms Universitet on July 11, 2012


Ilina et al. — Use of composite Cucurbita plants to study root development A







F I G . 3. Squash composite plants harbouring pKGW-RR-MGW-DR5 T-DNA (AtUBQ10::DsRED1 and DR5::gfp-gusA). (A) Overview of a 2-week-old squash composite plant. The arrow indicates callus developed in the inoculation site and roots emerging from the callus. Scale bar ¼ 1 cm. (B, C) The base of the hypocotyl with developed co-transformed roots of a 3-week-old composite squash plant: (B) bright field illumination; (C) fluorescence of DsRED1 in co-transformed roots. Scale bars ¼ 2.5 mm. (D, E) Co-transformed roots are morphologically indistinguishable from adventitious roots on water-inoculated plants (nontransformed control roots, see arrow): (D) bright field illumination; (E) fluorescence of DsRED1 in co-transformed squash root, absence of DsRED1 in nontransformed root. Scale bars ¼ 3 mm. (F –I) Root tip of a co-transformed squash root: (F) bright field illumination; (G) DsRED1 fluorescence; (H) GFP fluorescence (DR5::gfp) indicating auxin response in the columella and in the region above it (black arrow) as well as in a lateral root primordium (white arrow); (I) histochemical b-glucuronidase reporter activity staining (DR5::gusA) indicates more auxin responses in the columella (white arrow) and in the region above it, as well in numerous lateral root primordia (black arrows). Scale bars ¼ 1 mm.

PCR analysis of transformed roots

To confirm the presence of the T-DNA from the binary vectors in the putative transgenic roots, total DNA was purified from DsRED1-positive roots and GFP-positive roots and from non-fluorescent roots of ddH2O-inoculated plants (control roots). PCRs with primers for DR5::gfp-gusA and DR5::gusA were carried out. Amplification based on DNA from putatively transgenic roots showing DsRED1 fluorescence yielded PCR products of the expected size (1115 bp) identical to that of the positive control (amplification based on pKGWRR-MGW-DR5 plasmid DNA; Fig. 5A). In the same way, amplification based on DNA from GFP-positive roots yielded PCR products of the expected size (450 bp) identical to that of the positive control (amplification based on pMDC162-GFP-DR5

plasmid DNA; data not shown). No PCR product was obtained using DNA from control roots. Additionally, PCR for the ubiquitin gene was performed to analyse the quality of purified DNA, always yielding several fragments of the expected sizes and confirming DNA quality (Fig. 5B). As a test for the contamination of the DNA from putative transgenic roots (DsRED- or GFP-positive) with agrobacterial DNA, PCR for the virC operon on the Ri plasmid was carried out using the VCF/VCR primers (Sawada et al., 1995). Amplification based on total DNA from A. rhizogenes strains R1000 or MSU440 yielded a PCR product of the expected size (730 bp), but no PCR product was obtained using DNA from putative transgenic or control roots (Fig. 5C), confirming the absence of any contamination of the root DNA with agrobacterial DNA.

Downloaded from at Stockholms Universitet on July 11, 2012





Ilina et al. — Use of composite Cucurbita plants to study root development

TA B L E 1. The index of lateral root initiation (no. of lateral root primordia per 100 cortical cells) in transformed (AtUBQ10::DsRED1, DR5::gfp-gusA) and non-transformed roots of Cucurbita pepo L

Transformed roots, n ¼ 14 Non-transformed roots (inoculation with sterile ddH2O), n ¼ 18 P

1 2 3 4 5 6 7 8 9 10

Outer cortical layer II*

Outer cortical layer III*

4.6 + 0.18 4.6 + 0.14

4.4 + 0.21 4.2 + 0.11

4.5 + 0.18 4.4 + 0.13

1000 bp




C + C W M 1 2 3 4 5 6 7 8 9 10

23 %

R1000 77 % 8%


Total number of transgenic roots Total number of non-transgenic roots

F I G . 4. Transformation efficiency of A. rhizogenes strains R1000 and MSU440. The percentage of co-transformed (dark sector) and not co-transformed (light sector) roots obtained via R1000 (A) and MSU440 (B) strain transformation of squash seedlings. The total number of inoculated plants was 35 for strain R1000 and 20 for strain MSU440. A total of 546 roots were examined for strain R1000 (127 DsRED-positive and 419 DsRED-negative) and 358 for strain MSU440 (27 DsRED-positive and 331 DsRED-negative).

These results clearly demonstrate the integration of the DR5::gfp-gusA cassette into the genome of DsRED1-positive transgenic roots and of the DR5::gusA cassette into the genome of GFP-positive transgenic roots. Expression of the gfp-gusA fusion, and of gusA, in transgenic squash roots

GFP fluorescence and histochemical localization of GUS reporter activity were analysed in DsRED1-positive roots harbouring the T-DNA from pKGW-RR-MGW-DR5, and in control roots formed on ddH2O-inoculated plants. GFP fluorescence (Fig. 3H) from the GFP – GUS fusion protein could be

1000 bp

B c1 c2 W M 1 2 3 4 5 6 7 8 9 10

750 bp

F I G . 5. PCR analyses of DsRED1-positive co-transformed squash roots. (A) PCR with primers for DR5 and gusA (1115 bp PCR product). (B) PCR with primers for ubiquitin. Lanes: +, positive control: pKGW-RR-MGW-DR5 plasmid DNA; C1, negative control 1: DNA of wild-type roots (inoculation with sterile dd H2O); C2, negative control 2: DNA from roots transformed with pKGW 243 GGRR; W, water used as a template; 1 –10, DNA from different DsRED1-positive roots (AtUBQ10::DsRED1, DR5::gfp-gusA); N, molecular weight marker (200 bp O’RangeRuler, Fermentas; reference bands are 1000, 2000 and 3000 bp). (C) PCR with the primer set VCF/VCR for detection of the virC operon on the Ri plasmid (730 bp; Sawada et al., 1995). Lanes: +, positive control [total DNA from A. rhizogenes strain R1000 ( pKGW-RR-MGW-DR5)]; C, negative control (DNA of non-transformed roots; inoculation with sterile ddH2O); W, ddH2O used as a template; 1– 10, DNA from different DsRED1-positive roots (AtUBQ10::DsRED1, DR5::gfp-gusA); N, molecular weight marker (1 kb O’RangeRuler, Fermentas; reference bands are 1000, 3000 and 6000 bp, respectively).

seen only in the DsRED1-positive roots with the brightest fluorescence (approx. 50 %; Fig. 3G), while approx. 90 % of all DsRED1-positive roots showed GUS staining (Fig. 3I). No GFP fluorescence or GUS activity staining was detected in control roots from ddH2O-inoculated plants. Histochemical GUS activity staining was also performed on roots induced by A. rhizogenes strains harbouring both different binary vectors without the DR5 promoter insert, to test for possible background GUS activity [obtained using R1000 (pMDC162-GFP) or R1000 (pKGW-RR-MGW) as well as MSU440 (pMDC162-GFP) or MSU440 (pKGW-RR-MGW), respectively]. No, or in approx. 10 % of the roots, only very weak background staining was detected in roots co-transformed with pKGW-RR-MGW T-DNA (data not shown). However, very strong and extensive background activity was obtained in approx. 80 % of the roots containing the pMDC162-GFP T-DNA (Fig. 6C). This GUS activity is likely to be due to the influence of the enhancer of the CaMV 35S promoter on the DR5::gusA construct located within 3 kb of the CaMV 35S promoter (Fig. 2). These results demonstrate that of both binary vectors, only pKGW-RR-MGW is suitable for the analysis of promoter::reporter gene fusions in squash because the AtUbq10::DsRED fusion does not seem to affect the expression of the reporter gene. In the parental squash root, GFP and indigo (the product of the histochemical GUS reaction) could be detected in the root

Downloaded from at Stockholms Universitet on July 11, 2012


92 %

+ c1c2W M

Outer cortical layer I*

Values are the mean + s.e. for at least two independent experiments. Data evaluation for each parameter was done using the Mann–Whitney rank test and one-way ANOVA on ranks; P-values are indicated. * No significant differences were found between the groups (P . 0.05).



Ilina et al. — Use of composite Cucurbita plants to study root development A










F I G . 6. Co-transformed squash roots harbouring pMDC162-GFP T-DNA without a DR5 insert. (A) Bright field microscopy; (B) GFP fluorescence in co-transformed root; (C) excessive background b-glucuronidase activity in the absence of a DR5 promoter. Scale bar ¼ 2 mm.

cap and in the region of the quiescent centre (Fig. 3F, G, H, I). Apart from the main auxin response maximum in the root tip, numerous points of auxin response could be observed, marking developing lateral root primordia. The distribution of lateral root primordia in the co-transformed transgenic roots (Fig. 3I) was the same as in wild-type roots (data not shown). To verify the presence of GFP and GUS in co-transformed root tissues, immunolocalization of the proteins was carried out. Immunolocalization was performed on longitudinal root sections using primary antibodies against GFP and GUS, respectively. As shown in Fig. 7, GFP and GUS have a similar distribution in roots co-transformed with pKGW-RR-MGWDR5. The data on GFP and GUS distribution provided by immunolocalization are in agreement with the GFP fluorescence pattern and the indigo pattern obtained during the examination of whole roots (Fig. 3H, I). These data confirm that co-transformed DsRED1-positive roots are able to express the DR5::gus-gfpA cassette. Both GFP and GUS are reliable reporter proteins for the visualization of DR5 promoter activity, with GUS displaying a lower detection limit.

D IS C US S IO N This study presents a procedure for A. rhizogenes-mediated transformation of squash plants that reliably and efficiently leads to the production of composite transgenic plants. Furthermore, the transgenic roots obtained by this method

were nearly indistinguishable from wild-type roots, showing no differences in branching pattern or root hair formation, which should render them an excellent system for the study of the molecular mechanisms of lateral root formation. In the study by Katavic´ et al. (1991), the stems of 3-week-old squash plants grown in vivo were inoculated with A. rhizogenes. In these plants, the cells of the tissues surrounding the infection site most probably had stopped progressing through the cell cycle. So presumably, in these mature plants, there were no cells able to respond to agrobacterial stimulus, while the same study showed that 6- to 8-day-old cotyledons could give rise to hairy roots. Sanita` di Toppi et al. (1997) used 2-week-old seedlings grown in vitro. The transformation yielded hairy roots from either stems or cotyledons. These investigations clearly demonstrate the importance of the developmental stage of the plant material used for A. rhizogenes-mediated transformation. First, the age is related to the seedling’s ability to survive the agrobacterial infection. In our study, seedlings younger than 6 d did not survive the co-cultivation with agrobacteria (data not shown). Secondly, the age of the seedlings is indirectly linked to the presence of proliferating cells in the pericycle and the surrounding cell layers that will give rise to A. rhizogenes-induced adventitious roots. In our study we have shown the presence of DNA-synthesizing cells in the basal parts of the hypocotyls of 6-day-old squash seedlings. Based on the quantity of cells with EdU incorporation, we assumed that the labelled DNA-synthesizing (proliferating) cells kept progressing through the cell cycle, i.e. were not performing a last DNA replication before exiting the cell cycle, an assumption verified by their ability to form adventitious roots in response to inoculation with A. rhizogenes. In summary, seedlings suited for transformation have to be old enough to survive the wounding and the subsequent agrobacterial infection and young enough to contain cells progressing through the cell cycle in the pericycle and adjacent layers.

Downloaded from at Stockholms Universitet on July 11, 2012

F I G . 7. Immunolocalization of GFP (A–C) and GUS (D–E) in the tip of a co-transformed squash root (AtUBQ10::DsRED1, DR5::gfp-gusA). (A– C) Immunolocalization of GFP. (A) An overview of a root tip; differential interference contrast. (B) Distribution of GFP (in green). (C) An overlay of the two channels; the GFP protein is located in the columella and in the region of the quiescent centre. (D– F) Immunolocalization of GUS. (D) Overview of a root tip; differential interference contrast. (E) Distribution of GUS (in magenta). (F) Overlay of the two channels; GUS is located in the columella and in the region of the quiescent centre. Scale bars ¼ 100 mm.


Ilina et al. — Use of composite Cucurbita plants to study root development promoter, so the distribution of the reporter proteins reflects the distribution of responses to auxin (Ulmasov et al., 1997). The distribution of auxin responses in root tissues is well known for A. thaliana (Sabatini et al., 1999), so we can use this pattern as a reference point to understand the activity of the DR5 promoter in squash root tissues. By analysis of GFP fluorescence and GUS enzyme activity driven by the DR5 promoter, the distribution of auxin responses has been visualized for the first time in C. pepo, i.e. in a plant with a non-standard type of lateral root initiation. As the pattern of DR5 activity in the root tip of squash was similar to that in A. thaliana which displays the standard type of lateral root initiation, we conclude that the transformation technique presented here is a suitable tool to investigate further the physiological and molecular genetic mechanisms of root development in cucurbits, and to compare both types of lateral root initiation. ACK N OW L E DG E M E N T S The authors are grateful to Dr Tom J. Guilfoyle (University of Missouri, Columbia, USA) for kindly providing us with the pBI101.3 derivative containing the DR5 promoter, and to Dr Erik Limpens (Department of Plant Sciences, Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands) for the pKGW-RR-MGW vector. We appreciate the Core Centrum ‘Cell and Molecular Technologies in the Plant Science’ in the Komarov Botanical Institute (St.-Petersburg, Russia) and the Core Centrum ‘Genomic Technologies and Cell Biology’ in the All-Russia Institute for Agricultural Microbiology (Pushkin, St.-Petersburg, Russia) for provision of equipment. This work was financially supported by the Russian Foundation for Basic Research (grant: 11-04-02022), the Focus Program of Presidium of the Russian Academy of Sciences ‘Wildlife: current status and problems of development’, and the Ministry of Education and Sciences of the Russian Federation (16.552.11.7047, 14.740.11.1226, 14.740.11.1196). L I T E R AT U R E C I T E D Alpizar E, Dechamp E, Espeout S, et al. 2006. Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots. Plant Cell Reports 25: 959– 967. Anuar MR, Ismail I, Zainal Z. 2011. Expression analysis of the 35S CaMV promoter and its derivatives in transgenic hairy root cultures of cucumber (Cucumis sativus) generated by Agrobacterium rhizogenes infection. African Journal of Biotechnology 10: 8236–8244. Baranski R, Klocke E, Schumann G. 2006. Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation. Plant Cell Reports 25: 190– 197. Chee PP. 1990. Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants. Plant Cell Reports 9: 245– 248. Christey MC, Braun RH. 2005. Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation. Methods in Molecular Biology 286: 47–60. Clowes FAL. 1985. Origin of epidermis and development of root primordia in Pistia, Hydrocharis and Eichhornia. Annals of Botany 55: 849 –857. Curtis MD, Grossniklaus U. 2003. A Gateway cloning vector set for highthroughput functional analysis of genes in planta. Plant Physiology 133: 462–469. D’Agostino IB, Deruere J, Kieber JJ. 2000. Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiology 124: 1706–1717.

Downloaded from at Stockholms Universitet on July 11, 2012

As a result of the correctly determined age of squash seedlings for transformation, high efficiencies of transformation were obtained (up to approx. 85 %). There is evidence for host specificity of agrobacteria (Smarrelli et al., 1986; Baransky et al., 2006). The specificity of the interaction is believed to depend on the signal molecules secreted by wounded plants and perceived by the agrobacteria. Indeed, we obtained similar differences in the susceptibility of squash seedlings to A. rhizogenes strains R1000 and MSU440 independent of the vector used (85 % vs. 33 % of co-transformed plants). R1000 and MSU440 had been chosen as strains that were equally suitable for both vectors, pKGW-RR-MGW and pMDC162-GFP, based on their antibiotic sensitivities. In a previous study, we had tested other A. rhizogenes strains (ARqua1, A4RS and LBA1334) with pMDC162-GFP (data not shown). Strains Arqua1 and A4RS were very virulent on the squash seedlings, while LBA1334 which had been shown to be very efficient on many plant species (Baransky et al., 2006; Markmann et al., 2008) was almost unable to induce transgenic roots on squash (data not shown). Altogether, it can be stated that strains R1000, ARqua1 and A4RS were most efficient for the transformation of squash seedlings (data not shown). Non-axenic conditions for plant cultivation were chosen for several reasons. First, they offer the option to grow the plants in semi-natural conditions with normal aeration and on soil or vermiculite. Secondly, squash plants are considerably larger than most model plants (A. thaliana, Lotus japonicas, M. truncatula, etc.) which makes sterile culture more difficult and expensive, so it is more desirable to cultivate them nonaxenically. More natural growth conditions should help to prevent artefacts linked to the cultivation of plants in axenic culture or under hydro- or aeroponic conditions. This is especially important when an environmentally regulated process such as lateral root initiation is to be studied. It was shown in this study that the identification of co-transformed roots containing the T-DNA of pKGW-RR-MGW based on DsRED1 fluorescence is adequate for squash, in that no autofluorescence that could interfere with DsRED1 fluorescence was observed in wild-type roots. DsRED1 fluorescence also allows discrimation between chimeric roots, of which not all cells contain the T-DNA of the binary vector, indicating that they arose from a co-transformed cell and another cell in the wound callus, and roots of which all cells were co-transformed with the binary vector T-DNA. Our data underline that in order to obtain adequate results it is necessary to avoid interaction of promoters on the T-DNA of the binary vector. In previous studies it was shown that the use of a strong promoter such as CaMV 35S for the expression of the selectable marker gene resulted in the ectopic expression of the reporter gene construct (Yoo et al., 2005; Zheng et al., 2007). In the present study, we detected GUS activity in co-transformed roots containing 35S::gfp linked to gusA without promoter, while the AtUBQ10::DsRED1 fusion did not affect the expression of the DR5::gfp-gusA fusion. Hence, if a promoter::reporter gene construct is to be cloned in the vicinity of a CaMV 35S cassette, proper controls have to be introduced to identify the effects of the CaMV 35S enhancer. In our study, squash plants were transformed with a DR5::gfp-gusA cassette. DR5 is an artificial auxin-responsive

Ilina et al. — Use of composite Cucurbita plants to study root development

whole genome duplication yields symbiotic cytokinin type-A response regulators. Plant Physiology 157: 2013–2022. Pak H-K, Sim J-S, Rhee Y, et al. 2009. Hairy root induction in Oriental melon (Cucumis melo) by Agrobacterium rhizogenes and reproduction of the root-knot nematode (Meloidogyne incognita). Plant Cell, Tissue and Organ Culture 98: 219 –228. Quandt HJ, Puhler A, Broer I. 1993. Transgenic root-nodules of Vicia hirsuta – a fast and efficient system for the study of gene expression in indeterminate-type nodules. Molecular Plant– Microbe Interactions 6: 699–706. Sabatini S, Beis D, Wolkenfelt H, et al. 1999. An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99: 463– 472. Salic A, Mitchison TJ. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proceedings of the National Academy of Sciences, USA 105: 2415–2420. Sanita` di Toppi L, Pecchioni N, Durante M. 1997. Cucurbita pepo L. can be transformed by Agrobacterium rhizogenes. Plant Cell, Tissue and Organ Culture 51: 89–93. Sawada H, Ieki H, Matsuda I. 1995. PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology 61: 828–831. Seago JL. 1973. Developmental anatomy in roots of Ipomoea purpurea. 2. Initiation and development of secondary roots. American Journal of Botany 60: 607– 618. Shanks JV, Morgan J. 1999. Plant ‘hairy root’ culture. Current Opinion in Biotechnology 10: 151– 155. Smarrelli J, Watters MT, Diba LH. 1986. Response of various cucurbits to infection by plasmid-harboring strains of Agrobacterium. Plant Physiology 82: 622– 624. Sonti RV, Chiurazzi M, Wong D, et al. 1995. Arabidopsis mutants deficient in T-DNA integration. Proceedings of the National Academy of Sciences, USA 92: 11786–11790. Steedman HF. 1957. Polyester wax: a new ribboning embedding medium for histology. Nature 179: 1345– 1345. Stumpe M, Gobel C, Demchenko K. 2006. Identification of an allene oxide synthase (CYP74C) that leads to formation of alpha-ketols from 9hydroperoxides of linoleic and linolenic acid in below-ground organs of potato. The Plant Journal 47: 883– 896. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ. 1997. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. The Plant Cell 9: 1963–1971. Vasudevan A, Selvaraj N, Ganapathi A, Choi CW. 2007. Agrobacterium-mediated genetic transformation in cucumber (Cucumis sativus L.). American Journal of Biochemistry and Biotechnology 3: 24–32. Vernoux T, Brunoud G, Farcot E, et al. 2011. The auxin signalling network translates dynamic input into robust patterning at the shoot apex. Molecular Systems Biology 7: 508. Vitha S, Baluska F, Mews M, Volkman D. 1997. Immunofluorescence detection of F-actin on low melting point wax sections from plant tissues. Journal of Histochemistry and Cytochemistry 45: 89–95. White FF, Taylor BH, Huffman GA, Gordon MP, Nester EW. 1985. Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. Journal of Bacteriology 164: 33– 44. Yoko A, Masahiro M, Hiroyuki D. 1998. Morphological alterations and root nodule formation in Agrobacterium rhizogenes-mediated transgenic hairy roots of peanut (Arachis hypogaea L.). Annals of Botany 81: 355– 362. Yoo SY, Bomblies K, Yoo SK, et al. 2005. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221: 523– 530. Zheng XL, Deng W, Luo KM, et al. 2007. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Reports 26: 1195–1203.

Downloaded from at Stockholms Universitet on July 11, 2012

Demchenko KN, Demchenko NP. 2001. Changes of root structure in connection with the development of lateral root primordia in wheat and pumpkins. In: Gasparikova O, Ciamporova M, Mistrik I, Baluska F eds. Recent advances of plant root structure and function. Dordrecht, The Netherlands: Kluwer Academic Publishers, 39– 47. Dubrovsky JG. 1986. Origin of tissues of embryonic lateral root in the cucumber, tissue interactions, and positional control in development. Ontogenez 17: 176–189. (English translation from Russian appeared in Soviet Journal of Developmental Biology, New York, Consultant Bureau 17: 119– 128.) Dubrovsky JG. 1987. The latent embryonic root system of the cucumber. Botanicheskii Zhurnal (Botanical Journal) Leningrad 72: 171–176. Dubrovsky JG, Soukup A, Napsucialy-Mendivil S, Jeknic Z, Ivanchenko MG. 2009. The lateral root initiation index: an integrative measure of primordium formation. Annals of Botany 103: 807– 817. Gaba V, Zelcer A, Gal-On A. 2004. Cucurbit biotechnology – the importance of virus resistance. In vitro Cellular and Developmental Biology – Plant 40: 346–358. Gelvin SB. 1990. Crown gall disease and hairy root disease: a sledgehammer and a tackhammer. Plant Physiology 92: 281– 285. Gulyaev VA. 1964. Initiation and formation of lateral radicles in several species within the Cucurbitaceae family. Botanicheskii Zhurnal (Botanical Journal, Leningrad) 49: 1482–1485. Gutierrez-Gonzalvez M, Stockert J, Ferrer J, Tato A. 1984. Ruthenium red staining of polyanion containing structures in sections from epoxy-resin embedded tissues. Acta Histochemica 74: 115–120. Hoagland DR, Arnon DT. 1938. The water-culture method for growing plants without soil. In: California Agriculture Experiment Station Circular 347. Berkeley, USA: University of California. Hornung E, Krueger C, Pernstich C, Gipmans M, Porzel A, Feussner I. 2005. Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds. Biochimica et Biophysica Acta 1738: 105– 114. Karimi M, Inze D, Depicker A. 2002. GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science 7: 193– 195. Katavic´ V, Jelaska S, Bakran-Petricioli T, David C. 1991. Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes. Plant Cell, Tissue and Organ Culture 24: 35–42. Kereszt A, Li D, Indrasumunar A, et al. 2007. Agrobacterium rhizogenesmediated transformation of soybean to study root biology. Nature Protocols 2: 948– 952. Kumagai H, Kouchi H. 2003. Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Molecular Plant–Microbe Interactions 16: 663– 668. Limpens E, Ramos J, Franken C, et al. 2004. RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Botany 55: 983– 992. Lloret PG, Casero PJ. 2002. Lateral root initiation. In: Waisel Y, Eshel A, Kafkafi U eds. Plant roots – The hidden half. New York: Marcel Dekker, Inc., 127–156. Mallory TE, Chiang S-H, Cutter EG, Gifford EM. 1970. Sequence and pattern of root formation in five selected species. American Journal of Botany 57: 800–809. Markmann K, Giczey G, Parniske M. 2008. Functional adaptation of a plant receptor-kinase paved the way for the evolution of intracellular root symbioses with bacteria. PLoS Biology 6: e68. journal.pbio.0060068. Moore L, Warren G, Strobel G. 1979. Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2: 617– 626. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473– 497. O’Dell DH, Foard DE. 1969. Presence of lateral root primordia in the radicle of buckwheat embryos. Bulletin of the Torrey Botanical Club 96: 1–3. Op den Camp R, De Mita S, Lillo A, Cao Q, Limpens E, Bisseling T, Geurts R. 2011. A phylogenetic strategy based on a legume-specific


Lihat lebih banyak...


Copyright © 2017 DADOSPDF Inc.