ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals

8/6/2019 ion of Contained Nicotiana Tabacum Cultivation for the Production of ant Protein Pharmaceuticals.

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O R I G I N A L P A P E R

Optimisation of contained Nicotiana tabacum cultivation

for the production of recombinant protein pharmaceuticals

Richard Colgan Christopher J. Atkinson Matthew Paul Sally Hassan Pascal M. W. Drake Amy L. Sexton Simon Santa-Cruz David James Keith Hamp Colin Gutteridge Julian K-C. Ma

Received: 5 May 2009 / Accepted: 21 June 2009 / Published online: 9 July 2009

Springer Science+Business Media B.V. 2009

Abstract Nicotiana tabacum is emerging as a crop of

choice for production of recombinant protein pharma-

ceuticals. Although there is significant commercial

expertise in tobacco farming, different cultivation

practices are likely to be needed when the objective is

to optimise protein expression, yield and extraction,

rather than the traditional focus on biomass and

alkaloid production. Moreover, pharmaceutical trans-

genic tobacco plants are likely to be grown initially

within a controlled environment, the parameters for

which have yet to be established. Here, the growthcharacteristics and functional recombinant protein

yields for two separate transgenic tobacco plant lines

were investigated. The impacts of temperature, day-

length, compost nitrogen content, radiation and plant

density were examined. Temperature was the only

environmental variable to affect IgG concentration in

the plants, with higher yields observed in plants grown

at lower temperature. In contrast, temperature, supple-

mentary radiation and plant density all affected the

total soluble protein yield in the same plants. Trans-

genic plants expressing a second recombinant protein

(cyanovirin-N) responded differentlyto IgG transgenicplants to elevated temperature, with an increase in

cyanovirin-N concentration, although the effect of the

environmental variables on total soluble protein yields

was the same as the IgG plants. Planting density and

radiation levels were important factors affecting

variability of the two recombinant protein yields

in transgenic plants. Phenotypic differences were

observed between the two transgenic plant lines and

non-transformedN. tabacum, but the effect of different

growing conditions was consistent between the three

lines. Temperature, day length, radiation intensity andplanting density all had a significant impact on biomass

production. Taken together, the data suggest that

recombinant protein yield is not affected substantially

by environmental factors other than growth tempera-

ture. Overall productivity is therefore correlated to

biomass production, although other factors such as

purification burden, extractability protein stability and

quality also need to be considered in the optimal design

of cultivation conditions.

R. Colgan C. J. Atkinson C. Gutteridge

East Malling Research, New Road, East Malling, Kent

ME19 6BJ, UK

M. Paul S. Hassan P. M. W. Drake

A. L. Sexton

J. K-C. Ma (&

)CMM, 2nd Floor Jenner Wing, St. Georges Hospital

Medical School, Cranmer Terrace, London SW17 0RE,

UK

e-mail: [email protected]

S. Santa-Cruz D. James

Empharm Ltd., New Road, East Malling, Kent ME19 6BJ,

UK

K. Hamp

Unigro Ltd., Gay Dawn Offices, Valley Road, Fawkham,

Kent DA3 8LY, UK

123

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DOI 10.1007/s11248-009-9303-y


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Keywords Contained cultivation

Nicotiana tabacum Molecular farming

Recombinant antibody

Introduction

As the first recombinant pharmaceuticals for humans

from transgenic plants approach clinical trials (Ma

et al. 2005), the regulatory framework for manufac-

turing processes of such products is becoming

established (Spok et al. 2008). Of the various stages

in the production process, the early steps involving

plant cultivation, harvest and initial processing rep-

resent the procedures that are specific to plants, and

require the most innovation and development. The

later downstream processing and product purificationsteps are more familiar as they would be essen-

tially similar to those already established for other

recombinant protein expression systems, such as

CHO cells and E. coli.

Tobacco is a major agricultural non-food crop that

is grown worldwide. It is also emerging as a

transgenic production crop of choice for plant-made

recombinant pharmaceuticals (PMPs) (Sparrow et al.

2007) and a number of recombinant proteins, includ-

ing monoclonal antibodies, have been successfully

produced in tobacco (Valdes et al. 2003). Conven-tionally, tobacco is grown in open fields, and the aim

of agricultural practices is to maximise leaf produc-

tion and that of the alkaloid compounds (such as

nicotine). Optimisation for protein expression and its

accumulation has not been considered, and limited

information is currently available. Moreover, molec-

ular farming is most likely to progress, at least

initially, under greenhouse containment. This segre-

gation measure minimises the potential for gene flow,

accidental exposure to animals and humans, contam-

ination of the pharmaceutical crop and also allowsmuch tighter control of environmental conditions for

plant cultivation. However, tobacco is not normally

grown under greenhouse containment; so again, little

horticultural information is available for optimised

growth, particularly with respect to environmental

control.

The primary goal in horticultural management of

plants for PMP production is to maximise recombi-

nant protein production. However, a number of other

factors need to be considered. The stability of the

recombinant protein is important (Stevens et al.

2000). Another key issue is the purification burden

that is determined by the level of indigenous, plant

compounds that are extracted alongside the target

recombinant protein, as well as undesirable degrada-

tion or aggregation products of the target proteinitself. A further important issue is uniformity of

production. An underlying principle of current Good

Manufacturing Practice (cGMP) regulatory oversight

is the achievement of uniformity of product through

consistency of production process. Thus cultivation

procedures for transgenic plants that predispose to

high levels of variability in protein production

between plants are undesirable.

The aim of this study was to identify optimal

environmental conditions for the cultivation of

transgenic Nicotiana tabacum (var. xanthii) for theproduction of recombinant proteins in greenhouse

containment. Two transgenic lines were studied,

expressing either a monoclonal antibody or a cyano-

bacterial protein. The effect of environmental vari-

ables such as temperature, day length, compost

nitrogen availability, radiation intensity (photosyn-

thetically active radiation PAR), and growing density

have been investigated in terms of impact not only on

plant growth and biomass production, but specifically

with regard to the yield of the two recombinant

pharmaceutical proteins. In addition, the plant-to-plant variability of such protein production has been

assessed for each environmental variable.

Materials and methods

Transgenic plant lines

Two well established homozygous transgenic plant

lines were used (Ma et al. 1994, Sexton et al. 2006).

The Guys 13 line expresses a murine IgG1 monoclo-nal antibody (MAb) that binds specifically to the SA I/

II protein ofStreptococcus mutans (Smith et al. 1984).

The CV-N plant line expresses cyanovirin-N (CV-N),

an 11 kDa protein from Nostoc ellipsosporum, which

has potent neutralising activity against HIV (Boyd

et al. 1997).

The Guys 13 line seeds are from the T4/F3

generation, that is they result from the sexual cross

between T0 transgenic plants individually expressing

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the immunoglobulin light and heavy chains and are

the third generation seed containing both transgenes.

The CV-N line seeds are from the T2 generation

that is the third generation seed containing the single

transgene.

Controlled environment cultivation

The GroDome facility is a secure, contained, double-

skinned, polycarbonate building, divided into six

independent walk-in growth rooms. Each room has a

controlled environment, that allows accurate control

of carbon dioxide concentration with controllable

pressure and air movement, temperature control

(within a 1C tolerance in a preset range of

530C) and ambient or supplementary lighting, with

integrated control software monitors for conditions in

each room. Supplementary radiation was supplied viasodium lamps (Osram Vialox Navcson_T) to deliver

*3,400 W per chamber over the growing area. The

facilities are designed to meet level II containment

requirements and are controlled and monitored by a

building management system which provides full

data storage of internal conditions.

Five environmental variables were investigated

day-time temperature (32/21 vs. 23/21C), day length

(18/6 vs. 12/12 h), compost nitrogen levels (180 vs.

108 g/m3), radiation intensity (ambient vs. 660 lmol/

m2 /s) and planting density (69 vs. 6/m2). Radiationintensity was measured over the plant growing area

within the chamber and is an average. Two identical

growth chambers were used, under the same condi-

tions with the exception of the test variable. In

addition, CO2 levels were maintained at [twice

ambient, i.e. 800 ll CO2 l-1.

Plant cultivation

Seeds of wild type, Guys 13 and CV-N tobacco lines

were sown on the top of Levington ProfessionalMedia (seed and modular compost F2 ? S (sand) pH

5.56) and germinated, with bottom heat, under

containment glass at ambient conditions. After

emergence, supplementary lighting (*150 lmol/m/

s) was provided and seedlings were pricked out into

12 cm square plastic pots in compost mixture (Rich-

moor compost, Whitemoss, Kirby, UK enriched with

osmacote (N:P:K 18:10:11) 4.4 g/l, ScottsMiracle-

Gro, The Netherlands). Seedlings were placed in a

controlled growth chamber at set temperature of 20C

with ambient radiation intensities and day length and

allowed to grow until plants had produced two true

expanded leaves, then transferred into another

GroDome chamber(s) set for experimental parame-

ters. Each individual experiment depending on con-

ditions utilised a GroDome chamber and at least 50replicate plants of each type were grown per exper-

iment. Plants were cultivated, unless density was

variable, at around nine plants per m2 to avoid any

self-shading. The plants were watered twice a day.

There was no regulation of humidity.

Plant sampling

Duplicate leaf discs were sampled either side of the

mid vein from the tip region of leaves avoiding any

secondary veins, using a sharp 7 mm cork-borer. Discswere taken from the top most fully expanded leaf and

from the lowest, visually non-senescent leaf. Pairs of

leaf discs were placed immediately into 1.5 ml

Eppendorf tubes and flash frozen in liquid nitrogen

prior to storage at -80C. For each experimental

growing condition, at least 10 replicate plants were

sampled from both bottom and topleaves of each plant.

After sampling at the end of each experiment,

stems were cut off at soil level. Leaf and stem

material was separated and placed into a drying oven

at 80C for 48 h (until dry) and dry leaf and stemsweights were measured individually.

For specific protein determination and total soluble

protein measurement, frozen leaf discs were homog-

enised in 300 ll ice cold phosphate buffered saline

(PBS) using a mill (Retsch, UK; MM301). The

samples were centrifuged at 20,000g for 3 min and

the supernatants analysed.

Recombinant protein assays

Plant extract supernatants were transferred to micro-titre plates (Immulon, Nunc, UK) pre-coated with a

specific ligand to determine the extractable levels of

functional recombinant protein.

For detection of fully assembled and functional

(antigen-binding) IgG, the microtitre plates were

coated with a recombinant fragment of streptococcal

antigen I/II at 1:5,000 dilution (van Dolleweerd et al.

2003) and blocked with 5% (w/v) non-fat dry milk in

PBS. For detection of functional cyanovirin-N, the

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microtitre plates were coated with recombinant HIV

gp120 at 1 lg/ml (EVA 607, NIBSC CFAR) and

blocked with 2.5% bovine serum albumin (Sigma

UK). Plant samples were assayed in duplicate and in

serial dilutions. Incubation was for 2 h at 37C after

which the plates were washed with 0.1% Tween 20

(dH2O).Bound recombinant immunoglobulin was detected

by incubation with a horseradish peroxidase-labelled

goat anti-mouse IgG-gamma chain antiserum

(1:1,000, The Binding Site), for 2 h at 37C, followed

by addition of TMB (3,30,5,50-tetramethylbenzidine,

Sigma, UK) as the substrate. The colour reaction was

stopped with 2 M sulphuric acid, and the absorbance

was determined at 450 nm (Sunrise, Tecan, UK).

Antibody concentration was calculated by compari-

son of binding curves with a pre-existing standard

(Guys 13 hybridoma supernatant).Bound recombinant cyanovirin was detected by

incubation with a CV-N specific polyclonal rabbit

antiserum (courtesy of Dr. Barry OKeefe) at 1:1,000

dilution, followed by a horseradish peroxidase

labelled anti-rabbit IgG antiserum (Sigma, UK) at

1:1,000 dilution. These incubations were for 2 h at

37C or overnight at 4C. The addition and detection

of substrate was as described above.

Total soluble protein assay

Total protein concentrations in soluble plant extracts

were determined by the bicinchonic acid assay (BCA

Protein Assay, Pierce Chemical Co., Rockford, IL).

Statistical analyses

Data were subject to analysis of variance (ANOVA)

using Genstat version 10, least significance difference

(LSD) values for treatment interactions were calcu-

lated at P\ 0.05 level. For testing environmental

effects on inter-plant variability, data was subject toBartletts test of homogeneity.

Results

Sampling variability

An initial requirement was to assess the variability in

sampling from individual plants as a basis for the

ensuing studies. IgG transgenic plants were grown

under a 28C/21C day/night cycle with 18 h day

length, 660 lmol supplementary radiation and 800 ll

CO2 l-1, until they developed five leaves and were

approximately 15 cm high. Ten samples (2 leaf discs

each) were taken from a bottom leaf and a further ten

samples were taken from a top leaf from each of fourplants. The concentrations of functional IgG and total

soluble protein were determined and are shown in

Fig. 1.

The most striking observation was the difference

in IgG concentrations seen between the samples taken

from bottom and top leaves of the same plants. It has

previously been observed that recombinant IgG

concentrations were higher in the uppermost leaves

of transgenic tobacco plants (Stevens et al. 2000) and

this was confirmed here (Fig. 1a). In all four plants,

the differences between the mean extracted IgG ofsamples taken from the top and the bottom leaves

were statistically significant (P\ 0.001). The same

trends between top and bottom leaves were observed

in total soluble protein (TSP) concentrations

(Fig. 1b), although this only reached statistical sig-

nificance for plants 2 and 3 (P\ 0.001).

The difference between top and bottom leaf

concentrations was a consistent finding throughout

this study and is correlatively linked with the stage of

maturation of the plant tissue. It is not due to

significant differences in the mass of the plantsamples, as the bottom leaf discs were invariably

thicker than those taken from top leaves.

The data also indicates the degree of plant to plant

variation in antibody yields (ie comparing the top

leaves from all four plants, or the bottom leaves).

This variation was generally less than the variability

between samples taken from visually similarly loca-

tions on different parts of a single leaf (Fig. 1a),

indicating that plants from this transgenic line

expressed a relatively uniform level of IgG. This

notion was supported by similar findings for totalsoluble protein concentrations between plants, and

between samples from the same leaf (Fig. 1b). The

variability within samples taken from the same leaf

could be due to a number of reasons, the most likely

being anatomical differences across the leaf with

varying amounts of palisade, spongy mesophyll, and

vascular tissues, within a relatively small tissue

sample. The variability would of course be eliminated

at scale-up when whole leaves are sampled. In all

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(a) Extracted IgG (ng/ml)

800.00

1000.00

1200.00

1400.00

1600.00

ng/ml

0.00

200.00

400.00

600.00

800.00

IgGn

Bottom TopBottom TopBottom TopBottom Top

Bottom Top Bottom TopBottom Top Bottom Top

(b) Extracted TSP (g/ml)

2000.00

2500.00

Plant 4

Plant 4

Plant 3Plant 2Plant 1

Plant 1

500.00

1000.00

1500.00

TSPug/ml

S l ( 10) M

0.00

Plant 3Plant 2

Samp e n= Mean sd

Plant 1 Bottom leaf 0.033 0.012

Plant 1 Top leaf 0.078 0.013



(c) Extracted IgG (%TSP)





Fig. 1 Variability of IgG

and total soluble protein

TSP yields due to sampling.

a IgG concentration in

processed samples from

each four individual plants.

Data is shown from bottom

andtop

leaves (Eachcolumn contains 10 data

points from one plant. The

mean of those points is

shown as a black square in

each case); b TSP

concentration in the same

processed samples from the

same plants (10 samples

each, and the mean is

shown as a black square);

and c IgG expressed as a

percentage of TSP. Data is

shown as mean and sd for

the top and bottom leafsamples from the same four

plants

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subsequent experiments an attempt was made to

minimise this variability by sampling in a reproduc-

ible way from a standard position on each leaf.

Expressing the recombinant IgG expression in

terms of a percentage of the TSP may mitigate the

effects of the sampling variability arising from

differences in sample mass. It also gives an indicationof the purification burden for later downstream

processing steps (Fig. 1c, Hassan et al. 2008). The

results confirmed that the yield of IgG was highest

from the top leaves, and this was statistically

significant for all four plants (P\ 0.001).

Environmental variables have little impact

on recombinant antibody expression

and accumulation

Five environmental variables have been assessed withregards their potential impacts on expression and

accumulation of recombinant IgG MAb. These were

air temperature, day length, compost nitrogen con-

tent, radiation intensity (PAR) and plant density (pot

number per m2) (Table 1). These studies were

possible due to the sensitivity and reproducibility of

environmental control achievable between individual

growth chambers in the GroDome.

For each experiment, all but one of the five

parameters was kept constant. In addition, the CO2

concentration was maintained at 800 ll CO2 l-1

throughout. The experimental duration was varied

between experiments only if the experimental vari-

able under investigation enhanced, or reduced the rate

of biomass accumulation and maturity dramatically.

Experimental duration did not however vary within

each separate experiment. Of necessity, these exper-

iments were performed at different times of the year

(Table 1) and so ambient light and day length varied

across experiments.

In all the experiments, as shown earlier, the yield

of MAb from top leaves was consistently greater thanthat from lower leaves. Yield in these studies is

represented by the concentration of IgG recovered

from each two leaf disc sample. However, with the

exception of the temperature experiment, no signif-

icant differences were observed in expression levels

between plants grown under the different conditions.

In the temperature experiment, plants grown in a day/

night temperature cycle of 23/21C accumulated

approximately double the amount of MAb, in both

bottom and top leaves compared to plants grown on a

32/21C cycle; these differences were significant

(P\0.05).

Environmental variables affect total soluble

protein accumulation differently to IgG

accumulation in transgenic plants

In contrast to the findings for IgG, the concentration

of total soluble protein (TSP) were not consistently

elevated in top leaves as compared with bottom

leaves (Table 2). Indeed, this was only observed

when plants were grown at high temperatures (32/

21C) or at high density (69 plants/m2) irrespective of

the lighting supplied.

In the temperature experiment, a lower TSP at

higher temperature was observed in the bottom

leaves, unlike the fall in IgG concentration at highertemperatures which was also observed in the top

leaves (Table 1).

In the density experiment, plants grown at low (6/

m2) density had consistently higher TSP levels than

those grown at high (69/m2). In this experiment,

supplementary radiation also caused a significant

increase in TSP, as compared with ambient lighting.

It is notable that this experiment was performed in the

summer, when ambient day light and length is

maximal.

This finding confirmed the result in the radiationexperiment in which 660 lmol/m2 /s PAR also

induced higher TSP concentrations than ambient

light.

Day length and compost nitrogen content had no

significant effect on TSP concentration.

The effect of environmental variables

on cyanovirin-N accumulation in transgenic

plants differs from IgG

In a second series of experiments, the effects oftemperature, compost nitrogen content and radiation

intensity on expression and accumulation of a second

recombinant protein, cyanovirin-N (CV-N) were

studied (Table 3).

A notable finding was that the extractable CV-N

concentration was not lower in the bottom leaves in any

of the three experiments, as observed for IgG. Indeed,

in all cases, the concentrations were either the same or

higher, and the differences reached significance after

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5 days in the temperature experiment and in the

nitrogen experiment.

In contrast to the IgG transgenic plants, higher

concentrations of CV-N were recovered after 5 days

from plants grown at the higher day temperature

(32C). This was observed in both bottom and top

leaf samples (0.89 vs. 0.63 and 0.82 vs. 0.49;

P\ 0.05). In this experiment, a second series of

Table 1 The effect of five environmental parameters on IgG yield from transgenic plants

IgG Temperature

(C)

Day/night

cycle

(hours)

Radiation

(lmol/

m2/s)

Density

(plants/

m2

)

Nitrogen

(g/m3

)

Time of year Expt.

duration

(days)

Bottom

(IgG

lg/ml)

Top

(IgG

lg/ml)

LSD0.05

Temperature 23/21 18/6 660 16 180 January

February

2008

5 0.13b 0.16a 0.04 on

72 df

*

32/23 18/6 660 16 180 January

February

2008

5 0.05c 0.07c

23/21 18/6 660 16 180 January

February

2008

14 0.09d 0.15ab

32/23 18/6 660 16 180 January

February

2008

14 0.02ce 0.01e

Day-length 28/21 18/6 660 16 180 October

December

2006

35 0.64 0.94 0.45 on

36 df

*

28/21 12/12 660 16 180 October

December

2006

35 0.63 1.092

N2 28/21 18/6 660 16 180 December

March 2007

83 1.62a

4.07b

1.56 on

33 df

*

28/21 18/6 660 16 108 December

March 2007

83 1.48a

2.91ab

Radiation 28/21 18/6 660 16 180 AprilMay

2007

19 1.92a

6.61b

2.46 on

36 df

*

28/21 18/6 Ambient 16 180 AprilMay

2007

19 2.27a

7.20b

Density 28/21 18/6 660 69 180 AugustSeptember

2007

23 0.20a

0.29c

0.05 on72 df

28/21 18/6 660 6 180 August

September

2007

23 0.22ab

0.21a

*

28/21 18/6 Ambient 69 180 August

September

2007

23 0.17a

0.25bc

28/21 18/6 Ambient 6 180 August

September

2007

23 0.17a

0.20a

The cultivation conditions are described for each experiment, and the single modified parameter in each case is identified. The time ofyear the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the

specific environmental conditions). The mean IgG yield results are shown individually for bottom and top leaves. Data that are

statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ae), with different

letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown. The asterisks in the final column

denote groups that were cultivated under the same conditions, except for the experimental duration and time of year

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plants was also sampled at maturity on day 14. There

were higher concentrations in the more mature plant

samples, although this only reached significance in

the plants grown at the lower (23C) day-time

temperature (0.63 vs. 1.02 and 0.49 vs. 0.99;

P\ 0.05).

However like IgG, no significant differences were

observed in CV-N concentrations between transgenic

plants grown under different compost nitrogen levels,

or under different radiation intensities.

Environmental conditions affect TSP

accumulation in CV-N transgenic plants

in the same way as IgG transgenic plants

The results for TSP accumulation were consistent

with those found for the IgG plants (Table 4). Thus,

at a higher temperature, the TSP was reduced in the

bottom leaves of plants after 5 days. Surprisingly,

this was not seen after 14 days. There was also a

statistically higher TSP concentration in top leaves

from plants grown under more intense lighting in the

Radiation experiment, but in the bottom leaves there

was no significant reduction in TSP levels.

The effect of environmental variables

on transgenic plant biomass and leaf area

Data for total leaf area and plant biomass in each

experiment are shown for IgG transgenic, CV-N

transgenic and wild-type plants in Table 5. In gen-eral, IgG transgenic plants and wild-type non-trans-

genic plants performed in the same way, in terms of

biomass production. At high temperature (32/21C),

long day length (18/6 h) and ambient radiation, there

was no significant difference in final dry tissue

weights between the two plant lines. However, at low

temperature, short day length and with supplementary

lighting there were statistically significant lower

biomass yields in the IgG plants.

Table 2 The effect of five environmental parameters on TSP yield from IgG transgenic plants

IgG-TSP Time of year Expt. duration

(days)

Bottom

(TSP mg/ml)

Top

(TSP mg/ml)

LSD0.05

Temperature

23/21C February 2008 5 2.12a 2.37a 0.32 on 72 df

32/21C February 2008 5 1.55a

2.15a

23/21C 14 1.90a 2.07a

32/21C 14 1.65ab 1.84a

Day-length

18/6 h OctoberDecember 2006 35 3.11 2.93 1.00 on 36 df

12/12 h OctoberDecember 2006 35 2.41 3.51

N2

180 g/m3

DecemberMarch 2007 83 1.97 2 0.47 on 36 df

108 g/m3

DecemberMarch 2007 83 2.07 1.94

Radiation

660 lmol/m2 /s AprilMay 2007 19 2.75

a 3.67c 0.51 on 36 df

Ambient AprilMay 2007 19 1.95b 2.35ab

Density

660 lmol/m2 /s 69 plants/m

2AugustSeptember 2007 23 0.22

ad0.8

b0.14 on 72 df

660 lmol/m2 /s 6 plants/m

2AugustSeptember 2007 23 1.14

c 1.16c

Ambient 69 plants/m2

AugustSeptember 2007 23 0.10d 0.26a

Ambient 6 plants/m2

AugustSeptember 2007 23 0.36ae 0.43e

The cultivation conditions are the same as shown in Table 1, here only the modified parameter in each case is shown. The time of

year the experiment was performed is shown along with the duration of the experiment (time that the plants were subjected to the

specific environmental conditions). The mean TSP yield results are shown individually for bottom and top leaves. Data that are

statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ae), with different

letters denoting statistical significance (P\0.05). The least significant difference (LSD) is shown

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The CV-N plants had a very different growing

habit that was obvious visually. Overall, they took

considerably longer to germinate and establish as

seedlings (usually 4 weeks, compared with 2 weeks

for IgG and WT plants). The established plants were

also more compact and slower growing, which is

reflected in the biomass data. In all experiments, the

CV-N plant biomass at harvest were significantly

lower than WT and IgG plants cultivated under the

same conditions (P\ 0.05).

Table 3 The effect of three environmental parameters on CV-N yield from transgenic plants

CV-N Temperature

(C)

Day/night

cycle

(hours)

Radiation

(lmol/

m2/s)

Density

(plants/

m2

)

Nitrogen

(g/m3

)

Time of year Expt.

duration

(days)

Bottom

(CV-

N lg/ml)

Top

(CV-

N lg/ml)

LSD0.05

Temperature 23/21 18/6 660 16 180 February 2008 5 0.63a 0.49b 0.14 on

72 df

32/21 18/6 660 16 180 February 2008 5 0.89c 0.82c

23/21 18/6 660 16 180 February 2008 14 1.02c 0.99c

32/21 18/6 660 16 180 February 2008 14 0.92c 0.87c

N2 28/21 18/6 660 16 180 December

March 2007

83 0.80a

0.69b

0.08 on

38 df

28/21 18/6 660 16 108 December

March 2007

83 0.81a 0.64b

Radiation 28/21 18/6 660 16 180 AprilMay

2007

19 2.1 2.03 0.35 on

36 df

28/21 18/6 Natural 16 180 AprilMay

2007

19 1.87 1.96

The cultivation conditions are described for each experiment, and the single modified parameter in each case is identified. The time of


specific environmental conditions). The mean CV-N yield results are shown individually for bottom and top leaves. Data that are

statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ac), with different


Table 4 The effect of three environmental parameters on TSP yield from CV-N transgenic plants

CV-N-TSP Time of year Expt. duration

(days)

Bottom

(TSP mg/ml)

Top

(TSP mg/ml)

LSD0.05

Temperature

23/21C February 2008 5 1.39a

1.45a

0.34 on 72 df

32/21C February 2008 5 0.72b

1.17a

23/21C February 2008 14 1.65ac

1.79c

32/21C February 2008 14 1.38a

1.06ab

N2

180 g/m3 DecemberMarch 2007 83 0.55 0.54 0.26 on 36 df

108 g/m3

DecemberMarch 2007 83 0.78 0.8

Radiation

660 lmol/m2 /s AprilMay 2007 19 0.94

a0.80

a0.33 on 36 df

Ambient AprilMay 2007 19 0.91a

0.48b

The cultivation conditions are the same as shown in Table 3, here only the modified parameter in each case is shown. The time of


specific environmental conditions). The mean TSP yield results are shown individually for bottom and top leaves. Data that are

statistically significant from other data in that experiment are in italics and underlined and marked with a letter (ac), with different


Transgenic Res (2010) 19:241256 249

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With regards the effect of the different environ-

mental variables on each plant line, day time temper-

ature had a variable effect on leaf area in the juvenile

phase over the first 5 days in IgG and WT plants, but

the observed differences were not significant. In the

CV-N plants, a significantly higher leaf area was

achieved in plants grown at the 32/21C regime bothover 5 and 14 days. These differences did not

however, translate to significant differences in leaf

dry weight. In all cases, the higher temperature regime

led to a significant increase in stem dry weight.

Indeed, it was visually evident that the plants grown at

higher temperature reached maturity earlier, which

can be explained by a re-partitioning of carbohy-

drate from leaves into stem extension growth and

flower initiation. Thus growing plant lines at lower

temperatures may slow development and allow pro-

duction of plants with higher leaf:stem ratios, thereby

increasing the proportion of leaf biomass available for

extraction of recombinant protein and reducing the

amount of waste, particularly stem biomass.

Day length had a variable impact on leaf area, but

the differences did not reach significance. However,there was a consistent and significantly higher leaf

and stem dry weight for all the plant lines under the

18/6 h day length schedule.

Leaf area data was not collected from the compost

nitrogen experiment. However, no significant differ-

ences were observed in leaf or stem biomass

measurements, except for the wild-type line which

unexpectedly did not respond to additional compost

nitrogen.

Table 5 The effect of environmental parameters on dry weight and leaf area in IgG and CV-N transgenic and WT plants

Variable Leaf area per plant (cm2

) Dry leaf (g) Dry stem (g)

IgG CV-N WT IgG CV-N WT IgG CV-N WT

Temperature 32/21 (C) 890 979 1,124 7.54 4.70 7.97 0.96 0.77 1.09

5-day sample 23/21 (C) 983 746 998 6.98 4.96 8.76 0.43 0.31 0.68

LSD0.05 54 df 142.3 1.01 0.17

Temperature 32/21 (C) 1,755 1,799 1,815 11.30 9.70 12.62 6.51 4.97 7.71

14-day sample 23/21 (C) 1,903 1,313 1,708 16.10 9.06 15.49 6.49 2.00 6.50

LSD0.05 54 df 276 1.45 0.88

Day-length 18/6 h 2,433 1,467 2,234 16.20 10.70 16.00 11.50 7.24 11.52

12/12 h 2,289 1,657 2,394 11.40 8.60 12.80 7.00 5.72 8.43

LSD0.05 54 df 289.0 1.56 1.22

Nitrogen 180 (g/m3

) 7.20 2.50 4.00 4.80 2.30 3.10

108 (g/m3

) 7.10 2.60 9.30 4.90 2.50 6.50

LSD0.05 54 df 2.10 1.43

Radiation 660 (lmol/m

2

/s) 1,644 1,636 1,941 12.10 9.40 14.30 4.20 2.41 4.80Ambient 1,842 1,552 1,999 5.30 3.40 5.60 2.10 1.06 2.80

LSD0.05 54 df 257.5 1.28 0.72

Density 660 (lmol/m2/s)

69 plants/m2

3.00 3.30 0.90 1.30

660 (lmol/m2/s)

6 plants/m2

7.20 7.90 1.20 1.70

Ambient light

69 plants/m2

0.80 1.40 0.20 0.40

Ambient light

6 plants/m2

1.70 2.60 0.30 0.50

LSD0.05 42 df 0.97 0.25

The cultivation conditions are shown. Dry weights are shown for leaves and stems and represent the total mass for the ten plants

studied. Leaf area data also represents total area for the ten plants combined. The least significant difference (LSD) is shown for each

data set, and those results which are significantly different (P\0.05) are in italics and underlined

250 Transgenic Res (2010) 19:241256

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In the radiation experiment, supplementary light-

ing at *660 lmol/m2 /s is sufficient to saturate

the photosynthetic assimilation of tobacco plants

(Henkes et al. 2001) and the effect was to more than

double total dry biomass of IgG, WT and CV-N

plants through a large increase in leaf biomass and a

smaller increase in stem dry weight, leading to higherthan average leaf to shoot ratios. No significant

differences however, were seen in mean total plant

leaf areas and the differences in biomass were

attributable to differences in leaf thickness.

A related issue was apparent in the density

experiment with supplementary radiation, where

plants grown at a density of 6 plants/m2 had

consistently higher leaf and stem biomass than their

counterparts grown at 69 plants/m2, with the increase

in leaf biomass being more prominent than that of the

stems. A similar tendency was observed in the plantsgrown under ambient radiation, but this did not reach

significance. At both densities, the plants grown with

supplementary radiation had significantly higher

biomass compared with those grown under ambient

lighting. The addition of supplementary lighting

reduced the impact of planting at higher densities.

Although the highest biomass per plant was achieved

with supplementary lighting and low density plant-

ing, on a batch basis, the overall increase in yield per

area attained with increased planting densities would

compensate for the reduction of yield per plant.

Temperature, day length and nitrogen do not

influence the variability of expression within

transgenic plants, but plant density and radiation

are important

The impact of environmental conditions on the

variability of recombinant protein accumulation and

extraction in different transgenic plants was investi-

gated. In this case, data representing recombinant

protein as a percentage of total soluble protein wasused to minimise any impact of variation in sampling

and processing.

The results for IgG transgenic plants are shown in

Fig. 2. The shaded box contains data between 25 and

75% and the outer bars represent data at the 10 and

90% levels. The mean and median data are repre-

sented by a bold line and a line within the shaded box

respectively. In the temperature experiment (Fig. 2a),

there is a tendency for greater inter-plant variability

in the top leaves at 23/21C as compared with top

leaves grown at 32/21C. This difference did not

however, reach significance. No such difference was

observed in the bottom leaves.

In the day length experiment, a significant differ-

ence (P = 0.002) was observed with higher variabil-

ity observed in bottom or top leaves grown at 12/12 hday length regime (Fig. 2b). Under high nitrogen

levels (Fig. 2c), the variability in top leaves was

significantly greater than in the three other samples

(P = 0.04).

In the density/radiation experiment, more substan-

tial differences were observed (Fig. 2d). Under both

660 lmol supplementary radiation and ambient light-

ing conditions, the inter-plant variability was sub-

stantially decreased under less dense conditions in

both bottom and top leaves (compare columns 1 vs. 3,

2 vs. 4, 5 vs. 7 and 6 vs. 8 P\ 0.001). In the highdensity planting, there was little difference in the IgG

yield in bottom leaves under different radiation

regimes (compare columns 1 vs. 5), but in the top

leaves there was less variation in the plants grown

with supplementary lighting (compare columns 2 vs.

6; P\ 0.001). It was also noteworthy, that under

conditions that favoured limited inter-plant variation,

the mean yield was much lower than achieved under

other conditions.

The results from IgG transgenic plants was

supported by the CV-N transgenic plants (Fig. 3).In the temperature experiment, at the 5-day time

point (columns 14) no differences in variability were

observed. At the 14-day sample however, (columns

58), there was increasing variability in the plants

grown under the higher temperature regime (compare

columns 5 vs. 7 and 6 vs. 8 P\ 0.05). These data

also demonstrate an increase in variation in top leaves

at the higher temperature with time (compare

columns 4 vs. 8; P\0.05).

In the CV-N radiation experiment, supplementary

lighting reduced variability in the CV-N yield in topleaves (P\ 0.05), but no difference was observed in

the bottom leaves.

Discussion

The instinctive approach to growing plants is to

optimise conditions for plant health and vigour (bio-

mass accumulation) based on visual phenotype.

Transgenic Res (2010) 19:241256 251

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12/16

However, although this may lead to larger healthylooking plants, the conditions may not be optimal for

recombinant protein production where other parame-

ters may be important. These include the levels of

protein expression, variability of the protein, the

stability and accumulation of the protein and finally

the extractability of the protein. In this study, we have

focused on recombinant IgG, as this is a major

commercial target for molecular farming (Ma et al.

2005). However in order to determine whether our

observations are specific for IgG or generic forrecombinant proteins, we have also investigated a

second non-immunoglobulin protein that is also

targeted to the plant secretory pathway. Both trans-

genes were expressed from the same vector, using the

same controlling elements including the Cauliflower

mosaic virus 35S promoter, and a murine leader

peptide which directs the transgene product to the

endomembrane system. However, unlike IgG,

which is a large (150 kDa) multimeric glycosylated

1.0

1.2

1.4

(b) Day length(a) Temperature

0.014

0.016

0.018

LSD0.05

IgG(%

TSP)

0.2

0.4

0.6

0.8 LSD0.05Plot 1

IgG%

TSP

0.004

0.006

0.008

0.010

0.012

18hours

: Bott

omleaf

18hours

: Top

leaf

12hours

: Bott

omleaf

12hours

: Top

leaf

0.0

23/21

CBotto

mleaf

23/21

CTop l

eaf

32/21

CBotto

mleaf

32/21

CTop l

eaf

0.000

0.002

(d) Density / Radiation(c) Nitrogen

0.5

0.6

0.7

LSD0.05

0.3

0.4

0.5

LSD0.05

IgG(%TSP)

0.1

0.2

0.3

0.4

Plot 1

IgG(%TSP)

0.1

0.2

High N

2,Bottom lea

f

High

N2, Top lea

f

Low

N2, Bott

om leaf

Low

N2, Top lea

f

0.0

H.D.

660

mol,

Bot le

af

H.D.

660

mol,

Top

leaf

L.D. 660

mol,

Bot le

af

L.D.

660

mol,

Top

leaf

H.D.

ambie

nt, Bo

t leaf

H.D.

ambie

nt,Top

leaf

L.D.

ambie

nt, Bo

t leaf

L.D.

ambie

nt,Top

leaf

0.0

Fig. 2 Effect of cultivation conditions on functional IgG

expression in transgenic tobacco plants grown: a under two

temperature regimes; b under two day length regimes; c with

two different compost nitrogen contents; and d under two

different planting density and two different radiations intensi-

ties. In each case, data is shown for bottom and top leaf

samples (n = 10 in each case). The shaded box represents the

25th and 75th interquartile range, whiskers delineate the 10th

and 90th percentile. The dark line within the box marks the

mean and the fainter line the median. Outlying data points are

represented by dots. LSD0.05 for the interaction between

temperature regime and leaf position is 0.0014 on 36 df.

LSD0.05 for the interaction between day length and leaf

position is 0.097 on 36 df. LSD0.05 for the interaction between

leaf position and nitrogen regime is 0.099 on 36 df. LSD0.05 for

density and lighting regime and leaf position is 0.0621 at 71 df

252 Transgenic Res (2010) 19:241256

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13/16

mammalian protein, CV-N is a small (11 kDa) single

non-glycosylated bacterial polypeptide. Reliable

assays exists in both cases to confirm antigenicity as

well as molecular functionality, and it is important

here that we have always measured functional pro-

tein activity rather than total recombinant protein

concentration.

The post-translational events that affect the con-

centration of protein expression include efficiency of

protein folding, assembly and sub-cellular localisa-

tion. Perhaps surprisingly, environmental parameters

associated with plant stress such as temperature and

day-light hours, which might up-regulate the indig-

enous stress response did not have a consistent and

significant impact on protein concentraton, neither for

IgG nor CV-N.

The stability and accumulation of recombinant

proteins in planta is another important factor. Growing

plants at higher day time temperatures reduced the

amount of IgG that could be recovered by a significant

amount. This is in agreement with earlier findings,

where transgenic tobacco plants grown at 25C

yielded less recombinant IgG than plants grown at

15C (Stevens et al. 2000). This is counter-intuitive, as

tobacco thrives in hot climates, but this is an example

where conditions that are optimal for biomass pro-

duction and alkaloid levels are clearly not suited for

protein production. A likely explanation is that at

higher temperatures, the turnover or degradation of

accumulated IgG increases. Similarly, the reason for

greater IgG levels in top leaves as compared with

bottom leaves is probably due to this mechanism, with

degradative processes increased in bottom leaves,

which are likely to have entered a senescence process.

Indeed, Stevens et al. have demonstrated higher in

vitro degradation of IgG using crude plant extracts

from bottom leaves as compared with higher leaves in

the same plant (Stevens et al. 2000).

This hypothesis is also supported by the findings

from the TSP analysis. No significant differences

were observed between bottom and top leaves, nor

did temperature appear to make an important differ-

ence. TSP represents a mixture of proteins throughout

the plant cell, but at least 50% of the TSP comprises

RUBISCO (Geada et al. 2007) which is compart-

mentalised in the relatively stable environment of the

chloroplast and not exposed to the same array of

proteases encountered by secreted IgG.

1.80.30

(a) Temperature 5/ noitaidaR(b)syad41

1.2

1.4

1.6

LSD0.05

0.20

0.25 LSD0.05

CV-N(%

TSP)

0.4

0.6

0.8

1.0

CV-N(%TSP)

0.05

0.10

0.15

0 mo

l, bott

omleaf

660

mol, t

opleaf

mbien

t, bott

omleaf

Ambie

nt,top

leaf

0.0

0.2

botto

mleaf

,S1

1C

tople

af,S1

botto

mleaf

,S1

1Cto

ple

af,S

1

botto

mleaf

,S2

1C

tople

af,S

2

botto

mle

af,S2

1C

tople

af,S

20.00

1 2 3 4 5 6 7 8

660 6 Am

b

23/21C

bo

23/21C

32/21C

bo

32/2

23/21Cbo

23/21C

32/21Cbo

32/21C

Fig. 3 Effect on cultivation conditions on functional CV-N

expression in transgenic tobacco plants grown: a under two

temperature regimes sampled after 5 (S1) and 14 (S2) days;

and b under supplementary (660 lmol, m2

, s-1

) or ambient

light. In each case, data is shown for bottom and top leaf

samples (n = 10 in each case). The shaded box represents the

25th and 75th interquartile range, whiskers delineate the 10th

and 90th percentile. The dark line within the box marks the

mean and the fainter line the median. Outlying data points are

represented by dots. In a, numbers 18 are provided for ease of

reference to the text; LSD0.05 for the interaction between

temperature regime and leaf position is 0.0295 on 72 df.

LSD0.05 for the interaction between nitrogen regime and leaf

position is 0.2486 on 36 df

Transgenic Res (2010) 19:241256 253

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It is interesting that CV-N yields were affected

differently by alterations in temperature, but this may

also be explained on the basis of protein stability.

Unlike IgG which is known to be readily degraded in

tobacco plants (Ma et al. 1995), CV-N is extremely

stable and remarkably resistant to proteolytic degra-

dation under a variety of conditions (Colleluori et al.2005). Thus we suggest that the higher CV-N levels

found in plant samples grown at higher temperature

result from the combination of higher recombinant

protein synthesis and lower degradation of accumu-

lated product. Unlike IgG, no significant difference

was observed between the CV-N expression levels in

bottom and top leaves. If the hypothesis for IgG

degradation in ageing leaves is correct, this too could

be explained by the greater stability of CV-N.

As the rate of degradation of accumulated recombi-

nant protein determines the extractable yield, theselection of optimal harvest time is an important

consideration. Here, we have not yet formally assessed

the optimal time of harvest. These are complicated and

lengthy experiments requiring very large amounts of

greenhouse space and will be the subject of future

studies. However, a preliminary indication can be

gained by comparing the results from different exper-

imental groups (marked with an asterisk in Table 1)

that were subject to near identical conditions. For both

IgG and CV-N, the data suggest maximal expression

after 19 days in the environmental chamber. Thisfinding needs to be interpreted with caution and

supported by further studies however, as of practical

necessity, the experiments were performed during

different parts of the year (as indicated). This had an

impact on the germination and development time for

the plantlets before they were introduced into the

controlled environment chamber. In addition, although

the radiation was theoretically saturating at 660 lmol/

m2 /s, there would have been variability in ambient

radiation wavelengths between experiments (although

the variation would have been very small during asingle experiment).

The evaluation of extracted total soluble protein

concentration provided a useful reference against

which to compare IgG and CV-N yield. Most notably,

plants grown under supplementary radiation consis-

tently yielded higher TSP, however this was not

linked to a parallel increase in recombinant protein

yield. Similarly, low density planting increased TSP

yield, but had no effect on IgG yield. Stevens et al.,

reported a parallel effect of climatic conditions on

IgG as compared with TSP (Stevens et al. 2000), but

this was not the case in our studies.

Although day length, radiation and planting den-

sity had little effect on recombinant protein yield,

they did, as expected, have significant impacts on

plant biomass production. Our data indicate thatincreasing day length would favour protein manu-

facture by improving biomass, whilst having little

effect on IgG or TSP yield. In the case of supple-

mentary radiation and low density planting, which

also increase biomass production per plant, more

careful consideration needs to be given, as the

biomass gains must be balanced by the recombinant

protein yield as well as the increased purification

burden (extractability) resulting from a higher rela-

tive burden of total soluble protein.

In addition to studying the effect of differentenvironmental factors on recombinant protein levels,

we were also interested to study the possibility that

different environmental factors might either increase

or decrease the inter-plant variation in these levels.

This is an important factor when considering the

design of a facility for establishment of a uniform and

highly reproducible cGMP compliant production

process. It is also an important consideration for the

definition of batches during manufacture. The effect

of senescence on the glycosylation profile of endog-

enous tobacco proteins has been reported, but it wasalso shown that different growth conditions had no

impact (Elbers et al. 2001). Both the IgG and CV-N

seed lines were homozygous for each transgene locus

and should have been genetically homogeneous. The

uniformity of conditions achievable in the GroDome

allowed us to investigate inter-plant variability under

different cultivation conditions. The results suggest

that inter-plant variation is limited, which contrasts

with the findings of Hassan et al., (Hassan et al. 2008),

but is probably explained by the level consistency of

environmental conditions that is achieved in theGroDome, as compared with a standard laboratory

growth facility. This variation was largely unaffected

by temperature and compost nitrogen content. How-

ever, short day lengths and high plant density (69/m2)

significantly increase variability. The effect of high

plant density could be mitigated to some extent in top

leaves by supplementary radiation, but not in bottom

leaves. Together these results point to light availabil-

ity being the most important factor for ensuring

254 Transgenic Res (2010) 19:241256

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uniformity of recombinant protein yield in transgenic

plants.

Overall, the selection of optimal cultivation con-

ditions for protein production in transgenic plants is

complex. The only significant effect on IgG and CV-N

yield was observed under different day-time temper-

ature conditions. In the case of IgG, although higheryields were observed at a lower growing temperature,

using higher temperatures may reduce the amount of

time required for plants to attain sufficient maturity

and allow increased numbers of crops to be grown

each year. Extractability and purification burden are

important considerations, as the ratio of recombinant

protein to total protein needs to be compatible with

purification processes. Thus, the use of supplementary

lighting should be balanced in view of the finding that

it resulted in a significant increase in TSP, but not of

IgG or CV-N. Increasing the photo-period from 12/12to 18/6 h had an important effect on increasing leaf

biomass per plant without affecting purification

burden and in decreasing inter-plant variability.

However, although increasing planting density also

increased biomass per unit area, and the addition of

supplementary lighting mitigated the impact of higher

density planting on the yield per plant, these condi-

tions also increased inter-plant variability in terms of

recombinant protein yield.

An important challenge to be faced in using

tobacco for PMP production will be to control stemelongation and flower development. It is envisaged

that only leaves will be harvested for PMP extraction,

therefore without control of stem growth and flower

formation a diversion of resources into the growth of

non-usable plant product will impact on leaf biomass

production and lead to an increase in the amount of

GM plant waste requiring disposal through autoclav-

ing or other approved channels.

Finally, although the IgG transgenic plants

behaved broadly similarly to the non-transgenic wild-

type plants, a clear difference was observed with CV-N transgenic plants. It is likely that subtle phenotypic

alterations would result from the demands of

constitutive protein expression, particularly under

sub-optimal cultivation conditions. The additional

differences seen in CV-N transgenic plants may be

due to transgene positional effects, or due to direct

effects of CV-N in planta. Given its mannose-binding

properties, it is quite possible that the expression of

CV-N in the plant endomembrane system could affect

the synthesis and/or targeting of other plant

glycoproteins.

Acknowledgments The authors would like to acknowledge

DTI (TP/3/BIO/6/I/17346), Plant Vaccines Ltd., the EU Pharma-

Planta Integrated Project and the Hotung Foundation for

supporting the project. Also, Dr. Barry OKeefe for providing

CV-N specific antiserum, and Dr. Gillian Arnold for statisticalsupport. We also thank Mick Buss for supervising the

horticultural operations and Mike Davies for technical support

at EMR.

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