ORIGINAL PAPER

A passage through in vitro culture leads to efficientproduction of marker-free transgenic plants in Brassicajuncea using the Cre–loxP system

N. Arumugam Æ Vibha Gupta ÆArun Jagannath Æ Arundhati Mukhopadhyay ÆAkshay K. Pradhan Æ Pradeep Kumar Burma ÆDeepak Pental

Received: 27 April 2006 / Accepted: 20 November 2006 / Published online: 12 January 2007� Springer Science+Business Media B.V. 2006

Abstract The Cre–loxP site-specific recombina-

tion system was deployed for removal of marker

genes from Brassica juncea (Indian mustard).

Excision frequencies, monitored by removal of

nptII or gfp genes in F1 plants of crosses

between LOX and CRE lines, were high in

quiescent, differentiated somatic tissues but

extremely poor in the meristematic regions

(and consequently the germinal cells) thus pre-

venting identification and selection of marker-

free transgenic events which are devoid of both

the marker gene as well as the cre gene, in F2

progeny. We show that a passage through

in vitro culture of F1 leaf explants allows effi-

cient development of marker-free transgenics in

the F2 generation addressing current limitations

associated with efficient use of the Cre/loxP

technology for marker gene removal.

Keywords Marker-free plants � Brassica juncea �Cre–loxP � Transgenic crop

Introduction

The use of marker genes conferring resistance to

antibiotics or herbicides is essential for the

selection of transformed cells and tissues in

genetic transformation of plants. However, their

continued presence in transgenics, particularly

crop plants that are used for human and/or animal

consumption, has been a matter of concern for

nutritionists and environmentalists alike, with

member countries of the European Union re-

cently advocating a complete moratorium on the

use of transgenics containing superfluous marker

genes (Konig 2003). Retention of a marker gene

in a transgenic crop plant also prevents its

subsequent use for introducing additional genes

of agronomic value in a transgenic stock. Stacking

agronomically important genes by sexual crosses

using different transgenic stocks raised indepen-

dently using the same marker gene/promoters is

also not a viable proposition as it may lead to

homology-based silencing of the marker gene

N. Arumugam and Vibha Gupta have contributed equallyto this work.

N. Arumugam � V. Gupta � A. Jagannath �A. Mukhopadhyay � A. K. Pradhan � D. Pental (&)Centre for Genetic Manipulation of Crop Plants,University of Delhi South Campus, Benito JuarezRoad, New Delhi 110 021, Indiae-mail: d_pental@hotmail.com

Present Address:N. ArumugamDepartment of Biotechnology, PondicherryUniversity, Pondicherry 605 014, India

A. K. Pradhan � P. K. Burma � D. PentalDepartment of Genetics, University of Delhi SouthCampus, Benito Juarez Road,New Delhi 110 021, India

123

Transgenic Res (2007) 16:703–712

DOI 10.1007/s11248-006-9058-7

and/or the gene of interest cloned in cis (Vauc-

heret 1993; Kumpatla et al. 1998).

With the recent progress in plant genomics, the

development of strategies for marker gene

removal has become very relevant to plant

scientists (Hare and Chua 2002; Halpin 2005).

Several systems such as co-transformation (Math-

ews et al. 2001; Park et al. 2004; Chen et al. 2005),

transposons (Goldsbrough et al. 1993), homolo-

gous recombination (Iamtham and Day 2000),

site-specific recombination (Ow 2002; Lyznik

et al. 2003) and use of P-DNA (Rommens et al.

2004) have been developed for the removal of

marker genes from transgenic plants. Among the

various strategies, site-specific recombination sys-

tems have been tested extensively with the

bacteriophage P1 Cre–loxP system being one of

the most studied systems till date. The Cre–loxP

system consists of two components: (a) two loxP

sites each consisting of 34 bp-inverted repeats

cloned in direct orientation flanking a DNA

sequence and (b) the cre gene encoding a 38-

kDa recombinase protein that specifically binds

to the loxP sites and excises the intervening

sequence along with one of the loxP sites.

Although the Cre–loxP system has been tested

in several plants including Arabidopsis (Zuo et al.

2001), Nicotiana (Odell et al. 1990; Dale and Ow

1991; Gleave et al. 1999), Zea mays (Zhang et al.

2003) and Oryza sativa (Hoa et al. 2002; Sreekala

et al. 2005), none of these studies have reported

successful development of marker-free transgen-

ics, which are devoid of the marker as well as the

cre genes but contain the gene of interest (GI), in

the F2 progeny.

In this study, we tested the efficiency of the

Cre–loxP system for marker gene removal in

Brassica juncea (Indian mustard), a major oilseed

crop of the Indian subcontinent, which requires

introduction of many desirable agronomic traits

using transgenic technology to enhance its yield

potential (Grover and Pental 2003). Independent

transgenic lines homozygous for the marker gene

(NPT-LOX plants with the kanamycin resistance-

conferring nptII gene between the loxP sites) and

the recombinase source (CRE plants with the cre

gene driven by the CaMV35S promoter) were

developed in B. juncea. PCR analysis of the

marker gene status in NPT-LOX · CRE F1

hybrids revealed efficient excision of the marker

gene in leaf tissues. However, no marker-free

transgenics could be obtained in the F2 progeny of

such marker-free F1 plants. Studies on the

dynamics of Cre-mediated excision using the gfp

gene (GFP-LOX plants containing the gfp gene

within the loxP sites) showed efficient excision in

the quiescent somatic cells of cotyledons, leaves

and roots of GFP-LOX · CRE F1 hybrids. Exci-

sion frequencies were, however, very poor in

actively dividing meristematic tissues including

the shoot apical meristems, which are the pro-

genitors of germinal cells. We tested the possibil-

ity of obtaining marker-free individuals by

in vitro regeneration of plants from marker-free,

somatic (leaf) tissues of F1 events. We report that

these regenerants (designated ‘‘F1LR’’ events)

showed unambiguous segregation of marker-free

individuals in the F2 progeny thereby enabling

successful deployment of the Cre–loxP system in

the crop.

Materials and methods

Development of transformation vectors

pCGMCP-npt(lox)

A new binary vector (pCGMCP22; Fig. 1a) was

constructed in this study by modification of the

binary vector pPZP200 (Hajdukiewicz et al.

1994). The BclI-ScaI fragment of pPZP200 was

replaced with a 1.4-kb synthetic T-DNA se-

quence comprising the 25 bp consensus left and

right border repeats of T-DNA, a number of

unique restriction sites, loxP recombination sites

in direct orientation and a consensus overdrive

sequence from an octopine Ti plasmid outside

the right border. The synthetic DNA sequence

was designed to maintain an overall GC-content

of 42% in consonance with the GC-content of a

large number of dicot genomes (Salinas et al.

1988). Methylation-prone sequences (CGs and

CNGs) were also removed to the possible extent.

Keeping in view the polar transfer of T-DNA

from right to left border, the loxP sites for the

removal of marker genes were placed towards

the left border. The 1.4-kb sequence was

704 Transgenic Res (2007) 16:703–712

123

assembled using five sub-fragments (300–350 bp

in length) with unique restriction sites at their

ends for directional assembly. These sub-frag-

ments were constructed by recursive PCR (Dil-

lon and Rosen 1990) using six overlapping

oligonucleotides each of which was 60–80 nucle-

otides in length. The 1.4-kb synthetic sequence

was cloned as a BclI-ScaI fragment at the

corresponding sites of pPZP200 to generate the

plasmid pCGMCP22 (Fig. 1a).

The Pnos-nptII-ocspA fragment (used as the

selectable marker) was cloned at the EcoRV site

between the loxP sites of pCGMCP22. The

CaMV35S-gus-35SpA cassette was cloned in the

Ecl136II site (outside the loxP site) and used as a

reporter as well as the passenger gene of interest.

This construct, termed pCGMCP-npt(lox), is

schematically represented in Fig. 1b.

pPZP200-cre (Fig. 1c)

The cre gene was PCR amplified from bacterio-

phage P1 and mobilized between the CaMV35S

promoter and the 35S polyA signal. The

CaMV35S-cre-35SpA cassette was cloned at the

HindIII site of pPZP200. The nos-nptII-ocspA

BamHI(6182) SphI(7645)

T-DNA Region

LB RBlox lox PacI(7058)PflMI(6803)

PmeI(7310)

ScaI(4)

SpeI(7041)SspI(7029)

XbaI(7291)

AvrII(6721)

BclI(6161)

Ecl136II(7279)

EcoRV(6708)HindIII(6689)

SfiI(6443)

SfiI(7462)

AflII(6562) AflII(7400)BglII(6574)

BstXI(6466) BstXI(7482)

MscI(6552)

MscI(7390)

MspI(6540)

MspI(7412)

T-DNA Region

SwaI(7363)XmnI(6590) BglII(7376)

a

d

LB

bar probegfp probe

HindIIISpeI

HindIII

EcoRVEcoRV

RB

EcoRV

Amv loxP loxP

P35S gfp 35SpA P35S bar 35SpA

c

cre probe

RB

HindIII

LB

EcoRI

P4

P5

HindIII

EcoRV EcoRV

BamHI

EcoRI

XbaINcoI NcoI

Pnos nptII OcspA P35S cre 35SpA

XbaI

RB

gus probenptII probe P3

b

LB

EcoRVHindIII

P2

NcoINcoI

P1

EcoRVXbaI

XhoII

EcoRV

loxPloxP

P35S gus 35SpAPnos nptII OcspA

Fig. 1 Constructs usedfor testing the Cre–loxPsystem for marker generemoval in B. juncea. (a)pCGMCP22, (b)pCGMCP-npt(lox), (c)pPZP200-cre and (d)pCGMCP-gfp(lox).Unique restriction sitesare shown in bold inFig. 1(a). LB and RB:Left and Right borders,respectively of the T-DNA, P1 to P5 are PCRprimers specific foramplification of pre-excision (P2–P3), post-excision (P1–P3) and cre(P4–P5) gene products

Transgenic Res (2007) 16:703–712 705

123

cassette was cloned at the EcoRI site of pPZP200

and used as the in vitro selection marker.

pCGMCP-gfp(lox) (Fig. 1d)

The CaMV35S-gfp-35SpA cassette was cloned at

the HindIII site between the two loxP sites of

pCGMCP22 and used for monitoring Cre-medi-

ated excision in plant tissues. The CaMV35S-bar-

35SpA cassette (conferring phosphinothricin

resistance) was cloned as a blunt-ended fragment

at the Ecl136II site (outside the loxP site) towards

the right border and used as an in vitro selection

marker.

Transformation of Brassica juncea

The pCGMCP-npt(lox), pCGMCP-gfp(lox) and

pPZP200-cre constructs were mobilized indepen-

dently into the Agrobacterium strain GV3101 by

electroporation (Mattanovich et al. 1989). B.

juncea was transformed using hypocotyl explants

following Mehra et al (2000). Regenerated shoots

were transferred for rooting on a selection

medium containing 50 mg/l kanamycin (for

pCGMCP-npt(lox) and pPZP200-cre constructs)

or 20 mg/l phosphinothricin (for pCGMCP-

gfp(lox) construct). Only one T0 transformant

per explant was selected. Transgenics were trans-

ferred to soil and grown under containment in

accordance with guidelines of the Department of

Biotechnology, Government of India.

Development of F1LR events: shoot

regeneration from leaf explants of NPT-

LOX · CRE F1 progeny

Five mm2 segments of healthy leaves from 15-

day-old F1 shoot cultures were placed on a

regeneration medium consisting of MS salts

supplemented with 1 mg/l NAA, 1 mg/l BAP

and 20 lM AgNO3. Culture conditions for

obtaining regenerants were as described by Me-

hra et al (2000). The regenerated shoots (F1LRs)

were maintained on MS medium with 2 mg/l IBA,

transferred to soil under containment and self-

pollinated to raise F2 progeny.

Molecular analysis: Southern hybridization

and tissue PCR

Single copy transgenics were identified by South-

ern hybridization. Total DNA was isolated from

leaf tissues of putative T0 transgenics by the

CTAB method (Rogers and Bendich 1994). For

NPT-LOX plants, 5 lg of total DNA was inde-

pendently restricted with HindIII and XbaI,

electrophoresed and blotted on to nylon mem-

branes following standard procedures (Sambrook

et al. 1989). The HindIII and XbaI blots were

hybridized with gus and npt gene probes (Fig. 1b),

respectively. For CRE plants, blots were prepared

after digestion of genomic DNA with EcoRI and

BamHI and hybridized with the cre and npt gene

probes (Fig. 1c), respectively. The copy number

of T-DNA in GFP-LOX plants was determined

by digesting the total DNA with SpeI and

hybridization with bar and gfp gene probes

(Fig. 1d). Plants showing single copy integrations

on both flanks of the T-DNA were taken further

for subsequent studies.

For confirming removal of the marker gene,

genomic DNA isolated from the leaf tissues of

relevant F1, F2 and homozygous parent transgenic

lines was digested with HindIII and the blots were

hybridized with nptII or gus gene probes (Fig. 1b)

for confirming the presence or absence of the

respective genes.

PCR primers were designed for specific ampli-

fication of pre-excision (presence of the marker

gene), post-excision (presence of empty donor

site after removal of the marker gene from NPT-

LOX plants) and cre gene products (Fig. 1b, c).

The primer sequences and the expected sizes of

PCR products are given in Table 1. Template

DNA for tissue PCR was prepared from leaf

tissues and/or shoot tips of in vitro grown trans-

genic plants and relevant controls following Kli-

myuk et al (1993). PCR conditions for

amplification of pre-excision products were: ini-

tial denaturation at 94oC for 5 min followed by 40

cycles each of 94oC, 1 min, 55oC, 1 min and 72oC,

1 min. A final extension at 72oC for 5 min was

given. PCR conditions for amplification of post-

excision and cre gene products were: initial

denaturation at 94oC for 5 min followed by 40

706 Transgenic Res (2007) 16:703–712

123

cycles each of 94oC, 1 min, 54oC, 1 min and 72oC,

1 min. A final extension at 72oC for 5 min was

given.

Confocal microscopy

F1 seeds of the GFP-LOX · CRE cross were

germinated on MS medium and seedlings were

transferred to a medium consisting of MS salts

supplemented with 2 mg/l IBA, 20 mg/l phosph-

inothricin and 50 mg/l kanamycin. Seedlings

which rooted on the above double selection

medium were grown further for 15 days in a liquid

medium of the same composition but without any

selection pressure. The parental lines were also

grown on respective selection media and subse-

quently on the liquid medium. Shoot tips were

dissected under an Olympus Stereo Zoom micro-

scope and mounted on slides in PBS buffer (80 g/l

NaCl, 2 g/l KCl, 6.09 g/l Na2HPO4, 2 g/l KH2PO4

and pH 7.4) as whole mounts. Five to ten

millimeter long root segments along with the root

tips were also whole mounted in PBS buffer. GFP

imaging was done using a LSM5 Pascal Confocal

Laser Scanning Microscope set at 488 nm excita-

tion and 510 nm emission. Photographs were

taken using Image pro-plus 4.0 software.

Results

Transformation of B. juncea and development

of homozygous LOX and CRE lines

Transformants were generated in B. juncea using

the pCGMCP-npt(lox) and pPZP200-cre con-

structs. Ten NPT-LOX and eight CRE transgen-

ics with single copy integrations of the T-DNA

were identified by Southern analysis of 64 NPT-

LOX and 40 CRE plants, respectively using the

restriction enzymes and probes described in

Materials and methods (data not shown). Single

copy transgenics were self-pollinated to obtain T1

seeds. T1 seedlings of most of the selected T0

plants segregated in the expected 3:1 ratio for

kanamycin resistance:sensitivity. T1 kanamycin-

resistant plants were transferred to soil, self-

pollinated and the T2 progeny of those plants that

did not segregate for kanamycin resistance and

susceptibility were identified as homozygous

NPT-LOX and CRE lines (data not shown).

Analysis of marker gene (nptII) excision in F1

and F2 progeny of NPT-LOX · CRE crosses

Seven homozygous CRE lines were crossed with

seven homozygous NPT-LOX lines in 49 different

combinations. Screening a representative popula-

tion of the F1 progeny of each of the above

crosses by Tissue-PCR of leaf tissue using primers

specific for pre-excision, post-excision and cre

gene products (Table 1), indicated excision of the

marker gene in ~53% of F1 plants which showed

no pre-excision product but tested positive for

post-excision and cre gene products. To obtain

marker-free transgenics (which are also devoid of

the cre gene), F2 plants were generated from two

selected F1 lines (NPT-LOX1 · CRE2 showing

maximum and NPT-LOX1 · CRE4 showing least

expression levels of the cre protein). These two F1

lines lacked the marker gene within the loxP sites

as revealed by PCR analysis of leaf tissues.

However, PCR analysis did not show any

marker-free plants among the analyzed F2 prog-

eny (Table 2). Amplification of post-excision

PCR products (indicating excision of the marker

gene) was always observed to be associated with

the presence of the cre gene. We also observed

Table 1 Primers used for PCR analyses and expected size of PCR products

Product Primer Primer code Sequence Expected sizeof PCR product

Pre-excision Forward P2 5¢-GCTAGCTGATAGTGACCTTAGGCGACTT-3¢ 745Reverse P3 5¢-GCTACTTGGAGGTTCAACATTGTAGGACA-3¢

Post-excision Forward P1 5¢-ATGCATTCATGATGGTCACTCTCTCA-3¢ 292Reverse P3 5¢-GCTACTTGGAGGTTCAACATTGTAGGACA-3¢

cre gene Forward P4 5¢-CAGCAACATTTGGGCCAGCTAAAC-3¢ 368Reverse P5 5¢-TCTCTACACCTGCGGTGCTAACCAG-3¢

Transgenic Res (2007) 16:703–712 707

123

two new classes of plants: chimeric plants (con-

taining pre-excision, post-excision and cre PCR

products) and plants containing only the pre-

excision product without the cre gene (Table 2).

PCR analysis of excision profiles in apical meri-

stematic tissues (which are the progenitors of

germinal cells) of the F1 plants showed the

presence of both pre- and post-excision products

(Fig. 2) indicating poor efficiency of marker gene

excision in the same. We also analyzed by tissue

PCR, shoot tips of 48 randomly selected F2 plants

showing post-excision and cre products in the leaf

tissues. All these events were also found to

contain pre-excision product in their shoot tips

(data not shown).

Analysis of cre-mediated excision pattern in

plants using a reporter gene (gfp)

To monitor the excision pattern in plant tissues

more accurately, we generated transgenics in B.

juncea using a construct containing the green

fluorescent protein-encoding gfp gene between

the loxP sites (pCGMCP-gfp(lox); Fig. 1d). Sin-

gle copy GFP-LOX transgenics were identified

and crossed with the CRE lines used earlier.

Analysis of the fluorescence pattern of GFP in the

F1 progeny of the above crosses showed strong

fluorescence in the shoot as well as in the root

apical meristems (indicating non-excision of the

gfp gene) while no fluorescence could be detected

in any of the other somatic tissues adjacent to the

meristematic regions (Fig. 3).

In vitro shoot regeneration from leaf explants

of NPT-LOX · CRE F1 plants and generation

of marker-free transgenics

To obtain marker-free individuals, we regener-

ated plants from the leaf (somatic) tissues of the

same NPT-LOX1 · CRE2 and NPT-

LOX1 · CRE4 F1 plants that were used earlier

for development of F2 progeny (see above).

Tissue PCR analysis of the F1 regenerants thus

obtained (designated ‘‘F1LR’’) showed the pres-

ence of only post-excision and cre products

indicating that these shoots have regenerated

from marker-free cells of the F1 plant. Such

regenerants were transferred to the field and self-Ta

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708 Transgenic Res (2007) 16:703–712

123

pollinated to obtain F2 progeny (designated

‘‘LRF2’’). Tissue PCR analysis of LRF2 individ-

uals from both the independent events revealed

the presence of marker-free plants among the

segregating progeny in the expected ratio

(Table 2).

Removal of the marker gene in marker-free

plants was confirmed by Southern hybridization

using the npt and gus gene probes. The NPT-LOX

parent and F2 plants that contained the marker

gene (without cre) showed a 6.5-kb hybridization

band using both the probes (Fig. 4a, b; lanes 1,

5–7). The hybridization pattern of

NPT-LOX · CRE F1 plants (Fig. 4a, lane 3) with

the nptII probe showed the presence of the cre

gene (due to a hybridization signal at 4.5 kb

corresponding to the nptII locus derived from the

CRE parent; Fig. 4a, lane 2) and the absence of

marker gene at the NPT-LOX locus (due to

absence of the 6.5 kb band as was observed in the

NPT-LOX parent). In the marker-free LRF2

plants, no hybridization signal was observed with

the nptII probe (Fig. 4a, lanes 8–14) indicating

successful excision of the marker gene as well as

absence of the cre gene due to genetic segrega-

tion. The presence of a 4.2-kb hybridization band

in all the marker-free LRF2 plants using the gus

gene as a probe confirmed the presence of the

passenger gene of interest (Fig. 4b).

Discussion

In general, two approaches have been used for

expression of the cre gene in lox plants for

marker-gene removal. In the first approach, the

marker-containing lox plant is crossed with a

transgenic plant containing the cre gene and an

F2 population is generated for obtaining marker-

free plants (Dale and Ow 1991; Russel et al.

1992; Hoa et al. 2002; Zhang et al. 2003). In the

other approach, the recombinase is expressed

either by retransformation of the lox plant with

the cre gene (Vergunst et al. 1998; Cornielle

et al. 2001) or in a transient manner by induction

Fig. 2 Marker gene removal in transgenic B. juncea. Gelphotograph showing amplified PCR products using prim-ers specific for (a) pre-excision, (b) post-excision and (c)cre gene. Lane 1, 100 bp marker DNA; lane 2, untrans-formed control; lane 3, NPT-LOX1 parent; lane 4, CRE2parent; lane 5, CRE4 parent; lane 6, NPT-LOX1 · CRE2F1 (leaf); lane 7, NPT-LOX1 · CRE4 F1 (leaf); lane 8,NPT-LOX1 · CRE2 F1 (shoot tip); lane 9, NPT-LOX1 · CRE4 F1 (shoot tip)

Fig. 3 Fluorescence images of shoot (a) and root (c)segments of GFP-LOX parent and corresponding organsfrom an F1 plant (b, d) of the cross between the GFP-LOXand CRE parents (Bar: a, b—50 lm; c, d—100 lm). Themeristematic regions show strong fluorescence while non-dividing cells lack the same indicating removal (and hencelack of expression) of the gfp gene

Transgenic Res (2007) 16:703–712 709

123

using physical and chemical agents (Zuo et al.

2001; Zhang et al. 2003; Yuan et al. 2004; Sree-

kala et al. 2005). In cases wherein the recom-

binase was expressed by external induction, the

cre gene was cloned as a part of the construct

containing the marker gene. Although previous

studies have shown efficient excision of the

marker gene in F1 plants (Hoa et al. 2002;

Zhang et al. 2003), none of them provide data

on the production of marker-free transgenics

(which lack the marker as well as the cre genes

but contain the passenger gene of interest) in the

F2 generation.

In our studies on deployment of the Cre–loxP

system in B. juncea, we found that although

excisions occurred with very high efficiency in

quiescent, differentiated somatic tissues of F1

plants, no marker-free transgenics could be

obtained in the F2 progeny. We hypothesized

that this could be due to lack of excisions in the

apical meristems, which are the progenitors of

germinal cells. Experiments using gfp as a

reporter gene to track Cre-mediated excision

and PCR analysis of meristematic tissues of F1

plants conclusively established that excision fre-

quencies are poor in meristematic tissues. Similar

observation in F2 plants (containing pre-excision

product in meristematic tissues and post-excision

and cre products in leaf tissues) also indicates that

it may not be possible to obtain marker-free

transgenics in the F3 generation also. To address

the above limitation, we regenerated plants from

marker-free, quiescent, differentiated somatic

cells of F1 progeny (F1LR) and successfully

generated marker-free segregants among the F2

progeny of such regenerants.

Recent studies on marker gene removal using

the Cre–loxP system in Arabidopsis (Zuo et al.

2001), maize (Zhang et al. 2003) and tobacco

(Wang et al. 2005) describe the use of constructs

containing both the cre and the marker genes

within the same loxP sites. In these studies,

simultaneous removal of cre and the marker

genes was effected by transient, chemical or

heat-inducible expression of the recombinase

under in vitro conditions. Based on our observa-

tions, it is evident that the success of this

approach is not due to the use of an inducible

system per se but because of regeneration of

plants through in vitro culture following transient

induction of the recombinase in transgenic ex-

plants. However, such inducible systems lack

stringent regulatory controls, which lead to de-

regulated auto-excision of the marker gene and

consequently, a drastic reduction in transforma-

tion frequencies (Wang et al. 2005). Using such

Fig. 4 Hybridization profile of HindIII-digested totalDNA using (a) nptII and (b) gus gene probes. Lane 1,NPT-LOX1 parent; lane 2, CRE4 parent; lane 3, (NPT-LOX1 · CRE4) F1 plant without the marker gene; lane 4,untransformed control; lanes 5–7, marker-containing F2

plants obtained by direct self-pollination of F1 plants; andlanes 8–14, marker-free plants obtained by self-pollinationof F1LR plants (obtained by in vitro regeneration from

leaf explants of F1 plants). The absence of hybridizationwith nptII probe in lanes 8–14 indicates that these plantslack both the cre gene and the nptII marker. The presenceof a low molecular weight (4.2 kb) hybridization signal inthese plants as compared to that in the NPT-LOX1 parent(6.5 kb) on hybridization with the gus gene furtherconfirmed the marker-free status of these transgenic plants

710 Transgenic Res (2007) 16:703–712

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an approach, generation of transformed chimeras

also cannot be ruled out.

The introduction of the recombinase through

pollen transfer (sexual crosses) is preferable over

inducible expression in tissue culture as it avoids

repeated use of the cre gene in subsequent

transformation experiments. Development of a

single cre stock for a crop will suffice. Our results

clearly suggest that unambiguous segregation of

the marker-free trait and cre gene in F2 plants can

be obtained only by in vitro regeneration of

plants from the quiescent, somatic (leaf) tissues

of F1 (NPT-LOX · CRE) plants followed by self-

pollination of the regenerants. If our observations

on B. juncea hold true in general, removal of

marker genes from transgenic stocks could

become a routine affair addressing all the current

bottlenecks in the development of marker-free

transgenics.

Acknowledgements Financial support for this researchwas provided by DOFCO, a subsidiary of the NationalDairy Development Board (NDDB). B. S. Yadavprovided technical support. The gfp gene was a kind giftfrom Prof Jen Sheen. We are grateful to the Departmentof Botany, University of Delhi for extending use of theconfocal microscope facility.

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