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Biotechnology Letters ISSN 0141-5492 Biotechnol LettDOI 10.1007/s10529-011-0710-9

Hairy roots cultures from differentSolanaceous species have varyingcapacities to produce E. coli B-subunitheat-labile toxin antigen

Giorgio De Guzman, AmandaM. Walmsley, Diane E. Webster & JohnD. Hamill

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ORIGINAL RESEARCH PAPER

Hairy roots cultures from different Solanaceous specieshave varying capacities to produce E. coli B-subunitheat-labile toxin antigen

Giorgio De Guzman • Amanda M. Walmsley •

Diane E. Webster • John D. Hamill

Received: 20 June 2011 / Accepted: 15 July 2011

� Springer Science+Business Media B.V. 2011

Abstract The gene encoding enterotoxigenic

Escherichia coli B-subunit heat-labile toxin (LTB)

antigen was co-transformed into hairy root cultures of

Nicotiana tabacum (tobacco), Solanum lycopersicum

(tomato) and Petunia parodii (petunia) under the

CaMV35S promoter. Tobacco and petunia roots

contained *65–70 lg LTB g-1 tissue whilst hairy

roots of tomato contained *10 lg LTB g-1. Antigen

at *600 ng ml-1 was detected in growth medium of

tobacco and petunia. Tobacco roots with higher LTB

levels showed growth retardation of *80% whereas

petunia hairy roots with similar levels of LTB showed

only *35% growth retardation, relative to vector

controls. Regeneration of plants from LTB-containing

tobacco hairy roots was readily achieved and re-

initiated hairy roots from greenhouse-grown plants

showed similar growth and LTB production charac-

teristics as the original hairy root cultures.

Keywords Hairy root � E. coli toxin � Molecular

pharming � Plant-made vaccine � Rhizosecretion

Introduction

Improved viral vector systems have increased the

efficacy of transient systems to allow large amounts

of immunogenic proteins to be produced in plants

(Gleba et al. 2005; Hefferon 2010). Despite the many

advantages of these approaches, they still have some

limitations with on-going requirements for re-infec-

tion/infiltration of plants and extensive batch-to-batch

testing for accurate dosage purposes. The use of

stable transgenic plants as a potential alternative for

vaccine production is an attractive proposition but the

technology has been slow to become established, due

in part to relatively low levels of vaccine production

in vivo and also the need for high levels of bio-

containment to avoid inadvertent dispersal of trans-

genic plants. Rhizosecretion of foreign proteins from

roots has also been noted as being a potentially

valuable feature of transgenic plants and has been

shown to be possible for both small (e.g. 4E10) and

large (e.g. Guy’s 13) functional proteins at relatively

high levels, particularly when roots of transgenic

plants are treated with the phytohormone auxin

(Drake et al. 2009).

G. De Guzman � A. M. Walmsley � D. E. Webster �J. D. Hamill (&)

School of Biological Sciences, Monash University,

Melbourne, VIC, Australia

e-mail: John.hamill@monash.edu.au

G. De Guzman

e-mail: Giorgio.deguzman@monash.edu.au

A. M. Walmsley

e-mail: Amanda.walmsley@monash.edu.au

D. E. Webster

e-mail: Diane.e.webster@monash.edu.au

D. E. Webster

Faculty of Medicine, Nursing & Health Sciences, Monash

University, Melbourne, VIC, Australia

123

Biotechnol Lett

DOI 10.1007/s10529-011-0710-9

Author's personal copy

Axenic hairy root cultures represent a genetically

stable transgenic tissue culture system which grows

rapidly in vitro in simple and inexpensive media,

without a requirement for exogenous phytohormones

(Hamill and Lidgett 1997; Guillon et al. 2006). Of

particular relevance for foreign protein production,

hairy roots are non-photosynthetic and thus easily

bio-contained and can be scaled up to produce large

amounts of biomass in industrial scale bio-reactors

(Wilson 1997; Boehm 2007; Shih and Doran 2009).

Recombinant proteins, such as GFP and SEAP, are

secreted into the surrounding media raising the

prospect of low-cost downstream purification proto-

cols for valuable therapeutic proteins produced by

hairy roots (Gaume et al. 2003).

LTB production by transgenic plants has been

reported (Mason et al. 1998; Walmsley et al. 2003).

The holotoxin consists of two subunits, LTA

(28 kDa) and LTB (11.6 kDa) (Sixma et al. 1993).

LTB forms a *60 kDa pentamer which facilitates

binding to the GM1 ganglioside receptors on host

epithelial cells allowing the LTA to endocytose and

cause toxicity (Mason et al. 1998). In the present

study, we quantify the capacity of hairy roots from

three species in the Solanaceous family to produce

and secrete LTB protein. We also explore the ability

of hairy roots to be regenerated into transgenic plants

from which LTB-producing hairy roots can be re-

initiated, thereby providing a low-technology alter-

native to repeated subculturing or more expensive

techniques such as cryopreservation for long-term

perpetuation of hairy root lines with desirable anti-

genic protein producing properties.

Materials and methods

Introduction of expression constructs

into Agrobacterium rhizogenes

The pBin? 35sLTB construct was made using the

pTH210 vector (Mason et al. 1998) where the

expression cassette was ligated into appropriate sites

of the pBinPlus binary vector described by van

Engelen et al. (1995). The native pBinPlus and the

pBI121 vector (containing the GUS gene) were used

as controls (Jefferson et al. 1987). Each construct was

separately transformed into Agrobacterium rhizoge-

nes strain LBA9402 by high voltage electroporation

(Wen-Jun and Forde 1989). Bacteria were selected on

YMB agar containing appropriate antibiotics and

single colonies screened by PCR to confirm the

presence of the genes of interest (Hamill et al. 1991;

Hamill and Lidgett 1997).

Establishment of hairy root cultures

Nicotiana tabacum var. NC 95 (tobacco; Cane et al.

2005), Solanum lycopersicum var. Roma (tomato;

Yates, Australia) and Petunia parodii (petunia; a gift

from Prof E.C. Cocking and Dr J.B. Power, Notting-

ham University, UK) were grown in a greenhouse

under a 16 h photoperiod as described previously by

Cane et al. (2005). Young leaves were infected with

A. rhizogenes containing pBin ? 35SLTB, pBI121 or

vector control, and clonal hairy root cultures selected

as described previously by Hamill and Lidgett

(1997). After *2 weeks, DNA from healthy prolif-

erating roots was extracted and PCR-screened for

the presence of LTB sequence using primers

FWD 50-GCCATGGTGAAGGTGAAGTGCTA-30 REV

50-CCATGGTGAAGGTGAAGTGCTA-30. RolB and

VirD primers were also used to show that the hairy

roots contained A.rhizogenes T-DNA without residual

Agrobacterium in the cultures (Hamill et al. 1991).

Similar PCR-screening was performed on regener-

ated plant tissues to confirm their transgenic status.

Growth

Culture flasks were seeded with 0.1 g healthy roots

using C5 selected independent clonal hairy root lines

of each species. Triplicate root cultures for each

independent line were harvested. Blotted dry weights

(BW) were recorded after repeated blotting to remove

extracellular moisture (Chintapakorn and Hamill

2003) as preliminary experiments (data not shown)

showed that levels of pentameric LTB protein

extracted from dried root material were greatly

reduced when compared to that of blotted plant

material. Separate analysis showed that the ratio

between BW and dry weight was 10:1 (P \ 0.05) for

roots of all species.

Extraction of total soluble protein

Total soluble protein (TSP) extractions of root

(100 lg BW) and leaf (500 lg) material were

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performed as described by Wang et al. (2003) with

addition of 29 Roche Complete Protease Inhibitor

Cocktail solution to extraction buffer and homogeni-

sation with a 3 mm tungsten bead for 1 min at

28 Hz/s in a Qiagen Mixer Mill. Concentrations of

TSP were determined using a Bradford based com-

mercial kit method (Bio-Rad Protein Assay).

SDS PAGE—Western blot

Equal amounts of TSP (100 lg) were used for SDS-

PAGE (Walmsley et al. 2003). The primary antibody

used was a rabbit anti-LTB monoclonal antibody

(1/5,000 in 0.5% PBSTM) (Benchmark Biolabs) and

the secondary antibody was an anti-rabbit fluorescent

antibody ALEXA 700 (Rockland inc.) (1/5,000 in

0.5% PBSTM). Blot was detected on a Licor Odyssey

imaging system.

Quantification of LTB antigen

Equal amounts of TSP (25 lg) from each test sample

were analysed by ELISA using the GM1 ELISA

protocol (Walmsley et al. 2003). Standard curves

were produced using recombinant bacterial (rLTB)

protein (Benchmark Biolabs) in twofold serial dilu-

tions starting at 5 ng. Plates were incubated overnight

at 4�C. The primary antibody used was anti-LTB-

rabbit (Benchmark Biolabs) at 1/2,000 dissolved in

0.5% skim milk PBST and the secondary antibody

(goat anti rabbit—HRP) (Sigma Aldrich) was used at

a dilution of 1/15,000 in 0.5% skim milk PBST.

Tetra-methyl-benzedine (TMB) (Bio-Rad Laborato-

ries) was used for detection at 620 nm on a ‘Labsys-

tems Multiscan’ plate reader. Data obtained was

standardised to the rLTB standard curve.

Re-initiation of hairy roots from regenerated

transgenic plants

Regeneration of plants from selected hairy root

cultures of tobacco and transfer of plants to soil for

growth under greenhouse conditions was undertaken

as reported previously (Chintapakorn and Hamill

2003). Re-initiation of hairy roots was achieved by

culture of *2 cm pieces of surface sterilised upper

leaves from non-flowering plants grown on standard

MS agar medium containing 30 g l-1 sucrose. Roots

typically formed within 1–2 weeks of culture and

were easily transferred to liquid culture and grown

and per original hairy roots as noted above.

Results

Following selection in media containing kanamycin,

hairy roots of all species produced by A. rhizogenes

containing pBin? 35sLTB contained LTB using

western analysis (Fig. 1). Oligomer sizes detected in

these extracts reflect that of the known LTB oligomer

sizes of the crystallised pentamer (Sixma et al. 1993).

Fifteen independently derived hairy root cultures were

assessed for each species at the mid-point in a typical

culture cycle. Mean LTB levels for each species are

shown in Fig. 2. To determine the extent to which

growth of individual root lines in each species varied

in relation to capacity for LTB production, C5

cultures representing high (above mean) and also

median levels of LTB within that species, were

monitored at regular time periods over a typical hairy

root culture growth cycle of *4 weeks (Fig. 3a, b, c).

Lines containing GUS protein served as controls, as

the presence of GUS did not alter growth relative to

lines transformed with A. rhizogenes alone (Fig. 3d).

However, hairy roots containing GUS protein typi-

cally produced more biomass per unit time than

comparable hairy roots of each species containing

LTB. Furthermore, a pattern was observed in each

species whereby roots containing high levels (above

mean) of LTB produced less biomass over time than

those which contained median levels of LTB. This

observation was most noticeable for hairy roots of

tobacco, showing *80% growth retardation relative

to the controls (Fig. 3a).

In the case of petunia, deleterious effects of LTB

production upon biomass produced per unit time were

noticeably less than in tobacco, with only *35%

growth retardation relative to their vector controls

(Fig. 3b). In the case of tomato hairy root cultures,

lines containing LTB grew only slightly slower than

their equivalent GUS controls (Fig. 3c). However, it is

important to note that the maximum level of LTB

observed in tomato hairy roots was 5–6 times lower

than the mean level observed in either petunia or

tobacco hairy roots. It is possible that any tomato hairy

root lines possessing high levels of LTB were either not

viable or grew so slowly that they were out-competed

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during the initial stages of culture in vitro by faster

growing roots with low capacity for LTB production.

Overall, therefore, in terms of maximising LTB

production of hairy roots per unit mass, these results

suggested that petunia was the most promising of the

three Solanaceous members represented in this study.

This was further confirmed in subsequent experi-

ments whereby a selection of root lines from each

species possessing the highest LTB production

capacities at day 15 of the culture cycle was

monitored for both growth and LTB content through-

out a complete growth cycle of *4 weeks. Results

presented in Fig. 4a showed that whilst all hairy root

lines contained progressively higher levels of LTB

over the growth cycle, in terms of LTB produced with

the largest amount of biomass in a culture cycle of

*4 weeks, hairy root lines of petunia were by far the

most promising (Fig. 4). In addition to containing

progressively higher levels of LTB per unit mass of

tissue as they proceeded throughout their growth

cycle, hairy roots of each species were observed to be

increasingly capable of releasing detectable levels of

LTB into surrounding media (Fig. 4b). Again, tomato

hairy roots were the least promising in this respect

with tobacco and petunia roots both secreting

noticeably higher levels, throughout the growth cycle.

As stated in the ‘‘Introduction’’, hairy roots of

several genera and families can often be induced to

regenerate transgenic plants possessing the Ri T-DNA

of A. rhizogenes (Tepfer 1984; Christey 2001;

250150

75

50

kDaM gTOM tTOM rLTBtTOB tPETgTOB gPET

1 2 3 4 5 6 7 8

3730

25

15

Fig. 1 Western blot

analysis using TSP extracts

from control hairy roots

containing the GUS gene

(g) and transgenic (t) hairy

roots containing the

CaMV35S LTB construct of

tomato (TOM) (tracks 2 and

5), tobacco (TOB) (tracks 3and 6) and petunia (PET)

(tracks 4 and 7). Track 1(M) contains molecular

weight markers as indicated

and track 8 (rLTB) contains

authentic recombinant LTB

from E. coli. Hairy roots are

able to produce each

oligomer of the LTB

pentamer and show a

similar banding pattern to

the native bacterial derived

LTB shown in lane 8

Fig. 2 Quantification of LTB in separate clonal hairy root

cultures of each Solanaceous species at day 15 of the growth

cycle. Mean levels of LTB are significantly (P B 0.05) higher

in tobacco and petunia cultures than in tomato but are not

significantly different from each other (n = 5). Error barsindicate SEM

Biotechnol Lett

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Chintapakorn and Hamill 2003). In the current study,

although repeated attempts employing a large range of

phytohormone combinations were unsuccessful in

regenerating plantlets from all hairy root lines of

petunia and tomato, hairy roots of tobacco expressing

LTB readily regenerated into plantlets in vitro when

cultured on basic medium containing a high cytoki-

nin:auxin ratio. Following transfer to compost, these

hairy root-derived transgenic tobacco plants were

observed to possess the slightly wrinkled leaf pheno-

type typical of plants containing Ri-T-DNA (Tepfer

1984; Chintapakorn and Hamill 2003) but overall

were healthy and contained LTB in both roots and

leaves (data not shown). Plants were maintained in a

healthy state in a bio-containment greenhouse for

more than 2 years. Culture of surface-sterilised leaf

material from these transgenic plants resulted in

prolific adventitious root formation when tissue was

cultured in vitro on standard medium lacking phyto-

hormones. Following removal from parental leaf

tissue, axenic hairy root cultures were easily estab-

lished which possessed growth rates and LTB con-

tents comparable to the parental hairy root culture

from which their source plants were derived (Fig. 5).

Discussion

Results noted above illustrate the potential value of

analysing a range of species to maximise potential for

vaccine production in cultured hairy roots. In the

current study, we focussed on species belonging to

three genera within the family Solanaceae and

observed that whilst all were highly receptive to

transformation with A. rhizogenes, Petunia parodii

(petunia) is the most capable of producing healthy

and rapidly growing hairy roots also expressing

high levels of LTB protein. LTB protein has been

produced at various levels in a range of other trans-

genic plant systems. For example transgenic carrots

contained 3 lg LTB g-1 FW (Rosales-Mendoza

et al. 2008), transgenic potato tubers contained up

to 17 lg g-1 FW (Mason et al. 1998), fruits from

transgenic tomato plants contained 38 lg g-1 DW

(Walmsley et al. 2003) and transiently-transformed

leaf tissues of N. benthamiana contained 75 lg g-1

FW (Wagner et al. 2004) Levels of LTB in hairy

roots produced in the current study compare favour-

ably therefore with those observed in other plant

systems.

0 5 10 15 20 25 300

1

2

3

4

5

Tom GusPet GusTob GusTob NegPet NegTom Neg

Time (Days)

Wei

gh

t (g

)0 5 10 15 20 25 30

0

1

2

3

4

5

Tom High LTB

Tom Medium LTB

Tom Gus (Neg)

Time (Days)

Wei

gh

t (g

)

0 5 10 15 20 25 300

1

2

3

4

5

Pet High LTB

Pet Medium LTB

Pet Gus (Neg)

Time (Days)

Wei

gh

t (g

)

0 5 10 15 20 25 300

1

2

3

4

Tob Medium LTBTob High LTB

Tob Gus (Neg)

Time (Days)

Wei

gh

t (g

)

A B

C D

Fig. 3 Growth of representative hairy root cultures of tobacco

(a), tomato (b) and petunia (c) expressing LTB compared to

GUS producing control hairy root cultures of each species.

LTB production has a negative effect on the growth capacity of

roots of each species, particularly in tobacco. GUS expression

within a species does not significantly have an effect on root

growth compared to roots containing only empty vector

T-DNA (d). Error bars indicate SEM

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Rapidly growing hairy roots can be readily estab-

lished for species belonging to a wide range of

dicotyledonous species (Hamill and Lidgett 1997)

thus opening up the possibility of yet higher total

yields than have been observed using petunia as

outlined in the current study. From a bio-containment

point of view however, use of this species for

production of foreign proteins by hairy roots cultured

in vitro has the potential advantage that plantlets do

not readily regenerate from hairy roots, unlike

Fig. 4 a LTB produced by hairy roots of tobacco (panel A),

petunia (panel B) and tomato (panel C) harvested at different

stages of the growth cycle is shown for all species. Highest

levels of LTB in roots of each species at the point of harvest

were observed to be *3 weeks after subculture. Petunia

cultures are able to produce greater biomass while expressing

LTB levels at similar levels to tobacco hairy roots (P C 0.05).

Tomato hairy roots show rapid growth capacity but much lower

LTB levels. b LTB detected in media of tobacco, petunia and

tomato hairy root cultures harvested at discrete stages of the

growth cycle is shown for all species. Concentration of LTB in

the media was seen to be highest *3 weeks into the growth

cycle (*600 ng ml-1), with petunia and tobacco cultures

secreting greater amounts than that of tomato. Error barsindicate SEM

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tobacco. It also is not in the food chain, unlike tomato

and thus the possibility of inadvertent contamination

of food stuffs with transgenic material is extremely

remote. Furthermore, it is also related to widely grown

ornamental petunia (Wijsman 1982) and does not

produce toxic pyridine alkaloids (data not shown) and

thus its use to produce therapeutic proteins may have

minimal negative connotations from a public percep-

tion perspective. These features may be helpful in the

context of acquiring regulatory approval for proteins

produced using hairy roots of P. parodii in future. In a

recent study by Pelosi et al. (2011) the same petunia

hairy root lines from these experiments were shown to

be able to elicit an immune response in mice, further

giving credence to usefulness of these cultures.

For long term maintenance of transgenic hairy

roots possessing useful foreign protein production and

acceptable growth attributes, a variety of approaches

may be used. For species which do not readily

regenerate into plants with otherwise potentially

desirable bio-containment, growth and biomass pro-

ductivity features, such as petunia as identified in the

current study, long-term storage solutions may be the

cryopreservation of tissues in liquid N2 (Benson and

Hamill 1991; Hirata et al. 2002; Lambert et al. 2009).

This approach does of course require a moderate-high

level of expertise and technological infrastructure.

For species which readily regenerate plants from

hairy roots (Christey 2001), maintenance of trans-

genic plants in a bio-contained greenhouse or as

axenic plantlets in vitro in an approved tissue culture

facility may also represent a feasible, cheap and

technologically simple way to maintain germplasm

from which hairy roots may be re-initiated. This

assumes of course that roots thus formed from such

transgenic plants have similar growth and productiv-

ity attributes as the original hairy root cultures, which

was demonstrated in this study to be feasible for

tobacco hairy roots. Moreover, although regenerated

transgenic plants did show a reduction in capacity for

seed set as has been reported previously for trans-

genic tobacco plants regenerated from hairy root

cultures (Tepfer 1984), in the present case we

observed that ample seed-production was possible

when stigmae of flowers were manually fertilised

with their own pollen. Further work has shown that

hairy roots can also be established from offspring of

these transgenic plants (De Guzman unpublished)

allowing for the possibility of long term storage of

therapeutic protein-producing hairy root germplasm

in a seed bank.

Acknowledgments We are grateful to Elena Virtue and

Dr Raelene Pickering, for providing unpublished data and

advice on hairy root culture; Dr Kathleen De Boer and Suzy

Ryan for general technical advice and laboratory support. GDG

acknowledges receipt of a graduate support scholarship from

Monash University. This work was supported by Monash

University support grants to JDH and DW and an Australian

Research Council (ARC) Linkage grant involving AMW and

JDH.

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