Download To assist in plastid inheritance studies, it was necessary to firstly

DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Tissue and plastid targeted transgene expression in a perennial crop,
strawberry
DEFRA project code
HH1031SSF
Contractor organisation
and location
Horticulture Research International
East Malling, West Malling
KENT, ME19 6BJ
Total DEFRA project costs
Project start date
£ 183,528
01/11/00
Project end date
31/03/04
Executive summary (maximum 2 sides A4)
This strategic research focused on developing plastid and nuclear transformation technologies in strawberry,
with an emphasis on improving safety aspects of the genetically modified (GM) crop. It aimed to address
potential and perceived risks in relation to the consumer, non-target species and the environment. This was to
be achieved through reducing the potential for foreign gene escape via pollen, avoiding the use of antibiotic
resistance genes and targeting foreign gene expression to only those tissues where foreign gene products are
required. The work centred on:
i)
Development of the novel use of a non-antibiotic selection system in plastid transformation based on
carbohydrate metabolism. Plastid transformation achieves precise targeting of foreign DNA into the
plastid genome, localisation of foreign gene products within plastids and offers a means of restricting
foreign gene flow through pollen
ii)
Determination of the potential for strawberry tissue-specific promoters to direct foreign gene expression
to specific tissues in GM plants generated by nuclear transformation
iii)
Investigation of the mode of plastid inheritance in strawberry to determine the degree of benefit
afforded, in terms of foreign gene containment, through the use of plastid transformation to generate
GM plants.
Objective 1
Development of the xylose isomerase/xylose selection system for plastid transformation
in strawberry
To develop the plastid transformation system, it was first necessary to a) make a plasmid vector, which is the
vehicle for delivery of the foreign DNA into the recipient plastid genome, and then b) optimise tissue culture
regimes to enable regeneration of GM plants containing the foreign DNA. The vector constructed (called
pFavXG) comprised the xylose isomerase (xylA) gene, whose protein product allows metabolism of the
carbohydrate xylose that is otherwise not utilisable by strawberry and the green fluorescent protein (GFP)
gene, whose protein product fluoresces under UV illumination allowing visualisation of genetically modified
tissues. These genes were joined by a linking DNA sequence (adaptor), surrounded by strawberry gene
CSG 15 (Rev. 6/02)
1
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
regulatory sequences, that control when, and how stably the genes are expressed, and this resulting
expression cassette in turn is surrounded by sequences isolated from the plastid genome, so-called ‘flanking
sequences’. The flanking sequences direct integration of the expression cassette into the recipient plastid
genome at a specific site by a process of homologous recombination, which occurs when like sequences
come into close vicinity. In tissue culture, of the total 30 g/l carbohydrate supplied, a level of 96.6% xylose /
3.4% glucose in the regeneration growth medium was determined as optimal for minimising growth of nontransformed material and was used as the selection level in subsequent transformation experiments. Using
biolistics to introduce plasmid pFavXG into target leaf material, five experiments, totalling 450 shot leaves,
were carried out. A total of 110 putative GM lines were generated that were able to survive the optimised
selection regime. Molecular analyses confirmed that two of the lines were plastid transformed. Additionally,
GFP fluorescence was observed in plastids of a relatively small number of leaf cells in both lines. However, it
was not possible to achieve pure stable lines through tissue culture, even with culture on 100% xylose. Lines
maintained a very low transformed/non-transformed plastid genome ratio.
Objective 2
Tissue culture of plastid transformed lines towards obtaining stable, homoplasmic lines
for future inheritance studies
The intention was to use a plastid transformed line with mixed populations of transformed and nontransformed plastid genomes, previously produced in MAFF project G01001. This line was to be tissue
cultured in the presence of antibiotics to generate a pure, stable line in which all plastid genome copies were
transformed. Progeny derived from this line crossed with a non-transformed plant as the other parent would
subsequently be screened for presence of the foreign introduced genes to determine whether plastids were
inherited from the maternal or paternal parent. However, attempts to achieve stability in the line, transformed
with plasmid pSGA16, containing uidA (GUS) and aadA (confers resistance to spectinomycin and
streptomycin) genes, were unsuccessful. After repeated cycles of tissue culture the line was unable to grow in
the presence of the two antibiotics and the uidA and aadA genes were no longer detectable by PCR.
Instability of the line was speculated to have been due to recombination between the rrn16 promoter sequence
used to control expression of the aadA gene and the identical sequence found in the flanking plastid genome
sequence. Eighteen further putative plastid transformed lines were generated from 297 leaves shot with
plasmid pSGA2, which contains the aadA and uidA genes in the opposite orientation to their position in
plasmid pSGA16, to circumvent problems with recombination between the two rrn16 gene promoters. Of
these, one line was confirmed as being plastid transformed. This tested PCR-positive for the aadA and uidA
genes and GUS protein was observed in plastids of a small proportion its leaf cells. However, attempts to
increase the proportion of transformed plastid genomes through tissue culture with repeat cycles of shoot
regeneration failed. Hence, using either vector it was not possible to generate stable plastid transformed lines
using the aadA gene and selection with spectinomycin and streptomycin antibiotics. In the absence of pure,
stable plastid transformed lines for use in studying plastid inheritance in strawberry, an alternative approach
was devised using non-GM plants (Objectives 5 and 6).
Objective 3
Production of strawberry nuclear transformed transgenic plants containing plasmid
constructs with floral organ and root-specific promoters fused to the uidA (GUS) gene
A total of 47 putative floral organ-specific (FavAP3) and 32 putative root-specific promoter (FavRB7) GUS
fusion containing GM strawberry lines were generated. Control, transgenic lines were also produced that
contained the GUS gene fused to a constitutive promoter (CaMV35S), which should be active in all tissues.
Molecular analyses confirmed presence of the introduced foreign genes and promoters in all lines. Twenty
two of the FavAP3 promoter-containing lines contained the introduced foreign DNA integrated at a single site
within the plant nuclear genome. Whilst 15 lines contained integrations at two or three sites and two lines at
four sites. Eight lines died prematurely, preventing complete analysis. Twenty of the FavRB7 promotercontaining lines contained single site integrations of the foreign DNA within the plant nuclear genome. Whilst
nine lines contained integrations at two sites and one line each at four, five and seven sites. Two of the
CaMV35S promoter lines contained single site insertions whilst a third line contained insertions at two sites.
Objective 4
Analysis of promoter activity in flowering populations of the transgenic plants
transformed with putative tissue-specific promoter constructs
To determine whether the introduced FavAP3 and FavRB7 promoters could direct tissue-specific expression in
the nuclear transformed GM lines, promoter activity was measured in a range of tissues. Since the promoters
controlled expression of GUS, the level of promoter activity was extrapolated from GUS activity, measured
using fluorimetric and histochemical assays. Twenty of the FavAP3 promoter lines, all FavRB7 promoter lines
CSG 15 (Rev. 6/02)
2
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
and the three CaMV35S promoter lines were analysed for promoter activity. The CaMV35S promoter was
shown to be active in all tissues, as expected. The FavAP3 promoter was similarly shown to be active in all
tissues except roots, though the general level of activity was an order of magnitude less than the CaMV35S
promoter. Therefore unpredictably, it was shown not to be floral tissue specific, but constitutive. Promoter
activity in the FavRB7 promoter lines was observed almost exclusively in root tissue, with low-moderate
activity in petioles and it could therefore be considered a near root-specific promoter.
Objective 5
Identification of plastid genome sequence markers in strawberry parental genotypes
using PCR-RFLP and PCR-SSCP
To assist in plastid inheritance studies, it was necessary to firstly identify sequence
differences/polymorphisms, termed markers, between the plastid genomes of different strawberry genotypes.
Eight non-coding regions of the plastid genome within thirteen cultivated Fragaria ananassa and two wild, F.
virginiana and F. chiloensis, strawberry genotypes were investigated for sequence variation. Isolation of the
regions by PCR and comparison of sequences between genotypes revealed a total of seven markers,
occurring within five non-coding intergenic regions. Six of the markers represented nucleotide substitutions
and one as an insertion/deletion. Two of the markers were detected using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) and three by PCR-single strand conformation
polymorphism (PCR-SSCP). Using these techniques to detect specific markers within their plastid genomes it
was possible to differentiate between the strawberry genotypes. Distinction could be made not only at the
interspecific level, between cultivated and wild genotypes, but also intraspecifically within cultivated genotypes.
Objective 6
Determination of whether plastids are transmitted through pollen in strawberry using
plastid genome sequence markers
Five families totalling 648 progeny, in which markers distinguished the parents, were screened using either
PCR-RFLP or PCR-SSCP to determine whether plastids were inherited from the male or female parent. Two
offspring, one each from inter- and intra-specific controlled crosses, were shown to have exclusively inherited
paternal plastids; all other progeny had exclusively inherited plastids from their maternal parent. It is
concluded that plastids are not exclusively maternally inherited in strawberry and that the mode of inheritance,
whether within cultivated genotypes or between cultivated and wild genotypes, can be bi-parental, albeit at low
incidence.
CSG 15 (Rev. 6/02)
3
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
Scientific report (maximum 20 sides A4)
1. INTRODUCTION
Plastid transformation enables targeting of foreign DNA to a precise region within the plastid genome. This is
achieved since incorporation of foreign DNA carrying the genes of interest, into the plastid genome during
transformation occurs via homologous recombination between sequences in the recipient plastid genome and
the donor DNA in the plasmid vector used for transformation. Precise targeting precludes problems such as
genetic instability and gene silencing, as sometimes observed with nuclear transformation, associated with
random, multiple copy insertion of foreign DNA. A typical leaf mesophyll cell contains hundreds of plastids and
these in turn can each contain hundreds of plastid genome copies (6). As a consequence, in a stably plastid
transformed line, where all plastid genome copies are transformed, high, non-variable levels of foreign gene
expression can be achieved.
Biolistics is commonly used to introduce the donor DNA into target tissue. Introduced foreign DNA
usually comprises the gene(s) of interest and a selectable marker gene, cloned between plastid gene promoter
and terminator sequences, which together make up the expression cassette. This in turn is flanked by
sequence from the target plastome, termed flanking sequence, which is involved in the homologous
recombination process. The most routinely used selectable marker gene has been the aminoglycoside
3’adenyltransferase (aadA) gene (Svab and Maliga, 1993), which confers resistance to the antibiotics
spectinomycin and streptomycin. Since there are multiple copies of the plastid genome within the cell,
antibiotic selection is maintained post-bombardment throughout shoot regeneration to achieve selection and
replication of transformed plastid genome copies and subsequent generation of uniformly transformed pure
lines, termed homoplasmic lines (Maliga, 1993)). Lines containing mixed populations of transformed and nontransformed plastid genomes are termed heteroplasmic and are unstable.
In higher plants, stable plastid transformation has successfully been accomplished in only tobacco
(Svab et al., 1990), Arabidopsis thaliana (Sikdar et al., 1998), potato (Sidorov et al., 1999) and tomato (Ruf et
al., 2001). Heteroplasmic lines have been produced in rice (Khan and Maliga, 1999), Lesquerella fendleri
(Skarjinskaia et al. 2003) and oilseed rape (Hou et al., 2003). Selection systems based on carbohydrate
metabolism have been developed for nuclear transformation. Examples are the use of phosphomannose
isomerase or xylose isomerase genes, of bacterial origin, that have been used successfully to recover sugar
beet, potato, tobacco and tomato nuclear transformed plants (Joersbo et al., 1998; Haldrup et al., 1998a,b).
This study aimed to develop a xylose isomerase selection system for use in plastid transformation. Since the
onset of this study, using tobacco, the species that has most routinely been plastid transformed, other
methods have been developed for avoiding the presence of antibiotic resistance genes in the generated plant.
These have either involved the excision of the antibiotic marker gene following insertion and primary selection,
by using the Cre/lox recombinase system (Corneille et al., 2001; Hajdukiewicz et al., 2001) or by using short
direct homologous DNA repeats to surround the aadA gene (Iamtham and Day, 2000), or by development of
an alternative selection system using betaine aldehyde dehydrogenase (Daniell et al., 2001).
It has been reported that in most angiosperm species, plastids are inherited maternally. The major
impact of this is that where a species that exhibits maternal plastid inheritance is plastid transformed, foreign
genes and their products would not be transmitted via pollen. Containment of transgenes by this means avoids
potential introduction of new environmental allergens through pollen, deleterious effects on beneficial insect
species and also foreign gene transfer by cross-pollination of non-GM crops and weedy relatives. However,
large-scale investigations of plastid inheritance, have extrapolated the mode of inheritance from cytological
studies using DNA staining and fluorescence microscopy to detect plastid DNA in generative or sperm cells
(Corriveau and Coleman 1988; Zhang et al. 2003). The number of angiosperm genera for which mode of
inheritance has been investigated in such studies is relatively few and the proportion characterised using more
conclusive genetic studies, even smaller.
Transmission of plastids during sexual reproduction can be investigated using plastid-transformed
plants. Whereby progenies arising from controlled crosses, using a transformed plant as one of the parents,
can be screened for the introduced foreign genes. Alternatively, plastid inheritance can also be studied using
non-transformed plants and exploiting plastid genome DNA sequence polymorphisms (Chen et al. 2002;
Panda et al., 2003). Techniques such as polymerase chain reaction–restriction fragment length polymorphism
(PCR-RFLP)(Guillon and Raquin, 2000) and PCR-single strand conformation polymorphism (PCRSSCP)(Chen et al., 2002) have been used to detect sequence differences in plastid DNA between parents and
CSG 15 (Rev. 6/02)
4
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
hence to determine contribution of plastids to the progenies. The former technique involves PCR of specific
plastid genome regions, usually non-coding, and restriction enzyme digestion of the PCR products. Sequence
differences can result in either loss or gain of a restriction enzyme recognition sequence, which results in
different fragment lengths being produced upon digestion. The latter utilises PCR in a similar way, however the
PCR products are denatured and the single strands electrophoresed on gels. Here as little as single base
differences within the whole PCR strand can cause differences in mobility, which can be detected. This study
aimed to use these techniques for detection of genetic differences to enable determination of how plastids are
inherited in strawberry. Success would not only reveal the level of sequence conservation within regions of
the strawberry plastid genome but it would also determine the merit of using plastid transformation in
strawberry for containing foreign gene flow in pollen.
Constitutive promoters, such as the CaMV 35S RNA promoter (Lawton et al., 1987), have been used
extensively in GM plants. Their mode of action ensures foreign gene expression throughout the plant, which
has raised concerns over potential harmful effects on non-target organisms. Revelations on the presence of
recombinogenic hotspots within the CaMV 35S RNA promoter sequence (Kohli et al., 1999) have caused
alarm as to the potential for viral recombination of foreign genes and spread to other species. In addition, it
has been shown that transgenic plants susceptible to infection by cauliflower mosaic virus that carry the uidA
(GUS) gene under the control of the CaMV 35S RNA promoter experience foreign gene silencing upon viral
infection (Al-Kaff et al., 1998). These shortcomings highlight the necessity to isolate and use promoters of
plant origin that target foreign gene expression to only those tissues where foreign gene products are required.
Targeted foreign gene expression not only reduces unnecessary use of cellular resources in production of
foreign proteins throughout the plant but it also targets expression of genes, for example those conferring pest
and disease resistance, to specific tissues. This is likely to reduce the impact on non-target species. In this
study, strawberry promoters were analysed, that could potentially target foreign gene expression to ephemeral
floral tissue or to roots. Hence if proven tissue-specific, foreign gene expression would be absent in the
consumed product, the fruit.
This work was an extension of MAFF project G01001, in which plastid transformation was first demonstrated in
strawberry and a heteroplasmic line, containing mixed populations of transformed and non-transformed plastid
genomes, was generated. Putative floral organ and root-specific promoters were also isolated from strawberry
in the same project.
2. PROJECT OBJECTIVES
1. Development of the xylose isomerase/xylose selection system for plastid transformation in strawberry
2. Tissue culture of plastid transformed lines towards obtaining stable, homoplasmic lines for future
inheritance studies
3. Production of strawberry nuclear transformed transgenic plants containing plasmid constructs with
floral organ and root-specific promoters fused to the uidA (GUS) gene
4. Analysis of promoter activity in flowering populations of the transgenic plants transformed with tissuespecific promoter constructs
5. Identification of plastid genome sequence markers in strawberry parental genotypes using PCR-RFLP
and PCR-SSCP
6. Determination of whether plastids are transmitted through pollen in strawberry using plastid genome
sequence markers
3. RESULTS AND DISCUSSION
3.1 Development of the xylose isomerase/xylose selection system for plastid transformation in
strawberry (OBJECTIVE 1)
In strawberry tissue culture, sucrose or glucose are commonly used as carbohydrate sources to sustain
growth. To develop a plastid transformation system based on carbohydrate metabolism, the approach was to
transform the plastids of strawberry with a plasmid vector containing the xylA gene that would allow GM
cultures to regenerate on media containing xylose, which they would not ordinarily be able to utilise. The xylA
gene product, xylose isomerase converts xylose to xylulose, which can be metabolised. The procedure
required construction of a vector containing the xylA gene, in addition to a scorable marker gene that could be
CSG 15 (1/00)
5
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
visualised within the plastid transformed plants, optimisation of the selection regime and plastid transformation
using the vector to regenerate plastid transformed GM plants.
Construction of vector pFavXG
The majority of the individual components required to make up plasmid vector pFavXG (Fig. 1) for plastid
transformation were isolated using PCR and primers (Appendix 1) used were designed to contain restriction
enzyme recognition sequences to aid cloning. Sources of the genetic materials used were i) plasmid vector
pBSplast4.5, generated in MAFF project G01001, containing a 4.5 kb fragment of the plastid genome from the
16S rRNA (rrn16) gene through to the ribosomal protein S7 (rps7) gene, ii) strawberry genomic DNA for the
flanking sequence, the rrn16 promoter (P) and the photosystem II core protein (psbA) gene terminator (T) and
iii) plasmids pVicxylAH (Haldrup et al., 1998) and psmGFP (Davis and Vierstra, 1998) for the xylA and GFP
genes respectively. The RuBisCo large subunit (rbcL) gene 5’ untranslated region (UTR) and the adapter
used to join the xylA and GFP genes were generated by designing synthetic oligonucleotides.
Construction of the vector entailed PCR isolation of the rrn16 promoter (primers RRN16 KPNSGF and
RRN16 SSP), psbA gene terminator (primers PSBABGL and PSBAECOSGF) and xylA (primers XYLAPAG1
and XYLAXHO1) and GFP genes (primers AADA/XYLA:SMGFP and SMGFP BGLAPA).
The
AADA/XYLA:SMGFP primer constituted the joining adapter sequence. The rbcL 5’UTR was generated by
annealing oligonucleotides RBCLc and RBCLd and through a series of ligations and cloning steps all
components were linked to produce the expression cassette. The final cloning step involved release of a
3.179 kb plastid genome fragment from pBSplast4.5 using restriction enzymes Stu I and Psi I, cloning the
fragment into pBluescript and ligation of the expression cassette at a unique internal Bst 1107I site within the
plastid genome fragment, which would serve as flanking sequence, to generate vector pFavXG (Fig. 1).
Fig 1. pFavXG plastid transformation vector
Transformation of vector pFavXG into E. coli and exposure to UV illumination resulted in fluorescence of the
liquid suspension culture, whilst E. coli lacking the plasmid did not fluoresce (Fig. 2). Fluorescence was due to
the presence of GFP, produced from the GFP gene in vector FavXG harboured by the bacteria. This
confirmed that the vector was constructed correctly and that the plastid regulatory elements (rrn16 promoter,
rbcL 5’UTR and psbA terminator), of prokaryotic origin, were functional and were recognised by the bacterial
transcription/translation apparatus resulting in gene expression and protein production.
Fig 2. GFP fluorescence in E. coli liquid culture harbouring vector pFavXG illuminated with UV light
CONTROL
pFavXG
Determination of the optimal xylose levels as selection agent in plastid transformation
The concentration of xylose to be used as selective agent for strawberry cultures transformed with vector
pFavXG was determined empirically through a series of regeneration experiments using non-transformed
tissue. These involved culture of 0.5 cm3 in vitro generated cut leaf pieces on ZN102 regeneration medium
(Murashige and Skoog (MS) medium including vitamins, 1 mg/l TDZ, 0.2 mg/l NAA, solidified with 2.5 g/l
Gelrite, at pH 5.7), supplemented with varying levels of xylose as a percentage of the total 30 g/l carbohydrate
supplied. Glucose was used to make up the remaining carbohydrate.
Initially, duplicate experiments were carried out where xylose percentages assessed were 100, 83, 66,
50, 34, 17 and 0% and cultures were grown under a 16 hr photoperiod at 22 ºC. The ability of 100 explants
CSG 15 (1/00)
6
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
Project
title
DEFRA
project code
HH1031SSF
cultured at each xylose concentration to produce callus and regenerate shoots was analysed. In these
experiments culture with pure xylose was too stringent and callus and shoot production were both severely
restricted. At all other concentrations of xylose there was variation between replicates and no correlation
observed between percentage of xylose and ability to form callus and regenerate shoots. Additionally, shoot
regeneration was generally high, which was likely due to residual photosynthetic capability of the cultures.
The experiment was modified to include culture in the dark to circumvent this.
The third experiment thus assessed growth of cultures in the dark with xylose at 100, 98.3, 96.6, 91.6,
83.3, 75, 66.6 and 0% in the regeneration medium (Fig. 3)
Fig 3. Sensitivity of strawberry callus (white bars) and shoot (black bars) regeneration to D-xylose under dark growth
conditions
100
% r e g e n e r a ti o n
90
80
70
60
50
40
30
20
100
100
9 8 .3
9 6 .6
98.3 96.6
0
0
8 3 .3
0
1 00
Plastid transformation of strawberry using vector pFavXG
The BioRad Biolistic PDS-1000/He system particle gun was used to bombard five leaflets per Petri dish, either
abaxial or adaxial side up, at 1100 psi with 0.6 µm gold particles coated with approximately 1.7 µg vector
pFavXG. Prior to bombardment, leaves were cultured on ZN102 medium for one or two days and two
distances of donor material from the firing platform were used. In one experiment, half the tissues were
subjected to an osmotic treatment with mannitol to reduce the impact of the bombardment. Post
bombardment leaf tissue was cut into 0.5 cm2 pieces and regenerated on ZN102 medium containing 96.6%
xylose under low-level light. After six-weekly subculture on the same medium, regenerated shoots were
removed from the original explant and transferred to SMI shoot proliferation medium (MS medium including
vitamins, 0.224 mg/l BAP, 0.1 mg/l GA3, 0.104 mg/l IBA, solidified with 7.5 g/l Agar, at pH 5.7) containing 30 g/l
xylose as the sole carbohydrate source. Cultures were maintained on this medium, with subculture at sixweekly intervals and cutting back the material to single crowns each round to encourage selection and
replication of transformed plastid genomes. Five experiments were carried out as detailed in Table 1.
Table 1 Summary of pFavXG plastid transformation experiments
No. plates
bombarded
Preculture
time
FavXG 1
18
1d
ZN102
FavXG 2
18
2d
ZN102
FavXG 3
18
1d
ZN102
Experiment
number
CSG 15 (1/00)
Pre-culture
medium
Orientation and position of
donor material
Replicated either adaxial or
abaxial side up, 7.5 cm or
11 cm below firing platform.
Replicated either adaxial or
abaxial side up, 7.5 cm or
11 cm below firing platform.
Replicated either adaxial or
7
No. explants
generated
No. lines
regenerated
540
55
540
7
540
10
5
9 6 .6
B
N
2
2
0
5
Maximal callus production was only affected at xylose percentages greater that 96.6%, with ability to produce
any shoots also affected at this concentration. Shoot regeneration was inversely proportional to the amount of
xylose in the medium. However, only 40% shoot regeneration was observed when grown on pure glucose,
which is in contrast to the 100% shoot regeneration routinely observed when cultures are grown on pure
glucose in the light. On the basis of these results and also since shoots grown in the dark were etiolated and
vitrified, it was decided to culture plastid transformation experiments under low level light (1-3 µmol m-2 s-1) and
with 96.6 % xylose in the medium.
N
2
9 8 .3
D - x y l o s e a s a p e r c e n t a g e o f t o ta l c a r b o h y d r a te s u p p li e d
B
N
B
N
B
0
0
5
0
2
6 6 .6
2
0
2
2
N
B
N
2
0
2
75
B
N
2
0
2
8 3 .3
B
N
2
0
2
9 1 .6
B
N
2
0
2
9 6 .6
B
N
B
N
B
9 8 .3
2
0
2
0
2
1 00
2
0
5
2
N
B
N
1
0
5
6 6 .6
Z
N
1
0
5
75
Z
N
1
0
5
9 1 .6
Z
N
1
0
5
9 6 .6
Z
Z
N
1
0
5
0
5
1
Z
Z
9 8 .3
N
1
0
5
100
D-xylose as percentage of total carbohydrate supplied
N
1
0
2
N
Z
N
1
0
2
0
6 6 .6
66.6
Z
N
1
75
75
Z
N
Z
N
Z
8 3 .3
83.8
1
0
2
2
0
2
9 1 .6
91.6
1
0
2
N
1
0
Z
N
1
0
1
Z
N
Z
Z
N
1
0
2
0
2
10
91.
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
abaxial side up, 7.5 cm or
11 cm below firing platform.
Replicated either adaxial or
abaxial side up.
All 11 cm below firing platform.
FavXG 4
18
2d
ZN102
FavXG 5
18
1d
ZN102, half plates
substituted with
0.2M mannitol
All adaxial side up all 5 cm
below firing platform.
HH1031SSF
540
35
540
3
All 110 putative plastid transformed regenerated lines were screened for GFP fluorescence under UV
illumination using a stereomicroscope. This level of screen was unreliable since background fluorescence was
high in stressed or necrotic tissue. Therefore, all lines were analysed by PCR to determine whether they
contained the inserted foreign genes. Analyses confirmed the presence of the xylA gene and its linkage to the
GFP gene in only two of these lines. Far more non-transformed lines survived selection than were anticipated
from the results of the xylose sensitivity experiments carried out on non-transformed material. The two PCRpositive lines, named Sx1 and Sx2, originated from experiment FavXG1 and grew well on pure xylose (Fig.
4A). PCR carried out to span the plastome insertion site revealed that a very high proportion of nontransformed plastomes remained in these lines and a fragment indicating the transformed plastomes could not
be detected under the conditions used. However, using high power microscopy, it was possible to confirm
presence of the foreign genes in the plastome since GFP fluorescence could be detected in plastids of some
cells of both lines. An example is shown for line Sx2 in Fig. 4B.
Fig 4. (A) Plastid transformed Sx1 and Sx2 lines on SMI shoot proliferation medium containing 100% xylose as the
carbohydrate source. (B) Bright field (1), autofluorescence (2), UV GFP (3) and GFP fluorescence (4) filter set images of leaf cells of
line Sx2, obtained using a Zeiss Axiophot microscope equipped with Neofluar lenses
A
B
Sx1
SX1
Line Sx2
Sx2
SX2
1
2
3
4
Red autofluorescence of plastids was observed in all cells, whilst generally GFP fluorescence in plastids was
observed in a relatively very small proportion of cells. This indicated that although, plastid transformed, the
level of transformed plastid genomes was very low in each line, which confirmed the PCR data.
Repeated cycles of tissue culture on SMI medium containing 100% xylose did not increase the extent
to which the cultures were transformed. Attempts to regenerate callus and shoots from transformed cells in
leaves of these primary transformants cultured on ZN102 medium containing xylose were also unsuccessful.
The persistence of heteroplasmic lines could have been due to poor selection pressure. Whereby
perhaps either the xylose was being broken down over the course of tissue culture into metabolites that could
sustain growth or strawberry may potentially have an endogenous xylose isomerase activity. In fact, a xylose
isomerase gene has been isolated from barley (Kristo et al., 1996) and there have been reports of other plant
species expressing the enzyme weakly (Haldrup et al., 1998a,b). A similar vector to pFavXG was made,
pNtXG, in which the strawberry flanking sequences were substituted for the corresponding sequences from
the tobacco plastid genome. Similar to the pFavXG vector, when transformed in E. coli, pNtXG worked
efficiently and large amounts of GFP protein were produced. However, plastid transformed plants were not
generated when this vector was used to plastid transform tobacco. A recent report by Magee et al. (2004),
which shows that expression levels from plastid transformation vectors in E. coli may not be reflected in vivo in
the plant, may be of significance to this study. In strawberry (and tobacco), expression of the xylA gene may
CSG 15 (1/00)
8
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
have been low, which in addition to the postulated afore mentioned contributory factors, could have caused the
problems in achieving homoplasmy.
Conclusion
The plastid transformation system developed, based on xylose selection, was inefficient since only two plastidtransformed lines were generated from 450 shot leaves and selection regimes were unable to produce pure
lines where all plastid genomes were transformed.
3.2 Tissue culture of plastid transformed lines towards obtaining stable, homoplasmic lines for future
inheritance studies (OBJECTIVE 2)
The aim of this objective was to produce a pure plastid transformed strawberry line, through tissue culture, that
could subsequently be used to determine the mode of plastid inheritance in strawberry. Presence of foreign
genes within plant plastids would facilitate inheritance studies since they would provide a means of
distinguishing between plastids of plant parents that could be used in a controlled cross. Progeny raised from
a cross between plastid transformed and non-transformed parents could easily be screened for presence of
the foreign genes and the parent responsible for transmitting plastids determined.
It was intended to use a plastid-transformed line previously generated in MAFF project G01001, that
was transformed with vector pSGA16 (Fig. 5) that in addition to the uidA gene, contained the aadA gene. Less
than 5% of the plastid genome copies within this line were transformed (as indicated by Southern analysis)
and tissue culture regimes were carried out over a period of two years, to try to increase these levels so that
all copies were transformed. Whether shoot proliferation cultures were maintained on media containing 500
mg/l spectinomycin, on more stringent selection of 1000 mg/l spectinomycin, or on a mixture of 200 mg/l
spectinomycin and 200 mg/l streptomycin, over the course of time, the foreign genes were no longer
detectable and all clones subsequently produced bleached leaves. This was indicative that they were no
longer resistant to the antibiotics and hence confirmed that they did not contain the aadA gene that afforded
the protection. Attempts to initiate shoot proliferating cultures from meristems isolated from shoot tips of
earlier clones that had been proven to contain the foreign genes by PCR failed, as did regeneration of new
shoot cultures from leaves of confirmed transformed clones. It was postulated that loss of the foreign genes
was due to recombination between the rrn16 gene promoter used to control expression of the aadA gene in
the transformation vector and the same promoter, controlling the 16S gene, nearby in the flanking sequence
surrounding the insertion site. This would cause excision of the aadA gene and eventual loss of the uidA gene
since it would no longer be under selective pressure.
It was decided to initiate new transformations to obtain a stable, pure plastid transformed line. These
utilised vector pSGA2 (Fig. 5), which contained the expression cassette in opposing orientation to the one in
pSGA16, to circumvent problems with recombination between the two rrn16 promoters.
Fig 5. pSGA16 and pSGA2 plastid transformation vectors. Strawberry flanking sequences are shown as black boxes. P and T
denote promoters and terminators respectively. Arrows show the direction of the rrn16 promoters within the expression cassettes and
flanking sequences
 pSGA16
16S /trnV
TrbcL
aadA Prrn16 TpsbA
uidA
PpsbA
3’rps12 / rps7
16S /trnV
PpsbA
uidA
aadA
TrbcL
3’rps12 / rps7
 pSGA2
TpsbA Prrn16
A total of 297 leaves were bombarded with vector pSGA2 and after culture on ZN102 regeneration medium
containing 20 mg/l spectinomycin, for six subculture rounds of six weeks each, eighteen putative plastid
transformed shoots were regenerated. These original regenerated lines were called the RI generation.
Sixteen of these shoots were PCR-negative for the foreign genes and were presumed spectinomycin resistant
mutants. Shoot proliferation cultures, on SMI media containing 50 mg/l spectinomycin, were initiated from the
two RI generation shoots in which the uidA and aadA genes could be detected by PCR. One line, named
CSG 15 (1/00)
9
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
P6d, only produced bleached shoots, which were PCR-negative for the foreign genes. It is likely that the PCR
result for the original regenerated shoot was a false positive, resulting from cross-contamination.
The second transformed lined, named P18b, grew well and produced green shoots when cultured on
SMI shoot proliferation media with spectinomycin levels increased to 1000 mg/l. Leaves were taken from this
RI regenerant and cultured on ZN102 regeneration medium containing 500 mg/l spectinomycin to produce
new shoots, which were termed the RII generation. This procedure of regenerating shoots and taking leaves
from these to regenerate new shoots was continued until the RVI generation was produced, which took
approximately 18 months. This was carried out to encourage selective shoot regeneration from cells of a leaf
containing transformed plastid genomes to enable enrichment of the transformed plastid genomes in the
cultures and subsequent generation of a pure line. All cultures generated were clones of the original P18b
line. Shoot proliferation cultures were initiated from clones of each generation and maintained on SMI medium
containing 1000 mg/l spectinomycin. Selected clones from the generations were confirmed PCR positive for
the aadA and uidA genes. Resistance to spectinomycin in these clones was proved to be due to the presence
of the aadA gene since green shoots regenerated on ZN102 medium containing 200 mg/l of both
spectinomycin and streptomycin. Regeneration of bleached shoots would have indicated that shoots were
spectinomycin resistant mutants. Leaves of these clones were subjected to GUS histochemical assay, by
incubation in 5-bromo-4-chloro-3-indolyl--D-glucuronic acid (X-Gluc) substrate at 37˚C overnight, to detect
presence of GUS protein. GUS expression in a leaf of one of the clones of line P18b is shown in Fig. 6. Of 27
leaves screened from this clone, this was the leaf in which highest GUS expression was observed.
Fig 6. GUS expression observed in leaves of an RVI regenerant of plastid-transformed line P18b. Tissue was incubated in XGluc substrate and chlorophyll cleared by washing in 70% ethanol. (A) Whole leaf viewed under the stereomicroscope at 10X
magnification and (B) High power magnification (400X) of mesophyll cells in an unsectioned leaf
A
B
A
Though all of the original 18 putative plastid-transformed lines were screened by GUS histochemical assay,
line P18b was the only one to exhibit GUS staining. GUS expression was still maintained in the RVI
generation after 18 months in tissue culture. However, expression was not always detected in all leaves of a
clone and where it was detected, it was not present throughout the leaf. Comparison of expression in RIII
through to RVI regenerants did not reveal an increase in the extent of GUS expression in the tissues. This
suggested that the proportion of transformed plastomes was not being increased with each repeat
regeneration cycle. Even though the line was repeat regenerated through to the sixth generation, it was hence
not possible to generate a stable, pure line. The level of selection used here was higher than has been used
in plastid transformation of other species, which range between 40 – 500 mg/ml spectinomycin (Svab et al.,
1990; Sikdar et al., 1998; Sidorov et al., 1999; Ruf et al., 2001). Positive selection, using aadA may provide
cross protection to non-transformed plastid genomes in a heteroplasmic population, thus reducing the
selection stringency and slowing progression towards homoplasmy (Dix and Kavanagh, 1995). It is worth
noting that achieving homoplasmy in plastid transformed tomato using the aadA gene and spectinomycin
selection took almost two years (Ruf et al., 2001) and in rice and oilseed rape, it was not possible to achieve
homoplasmic lines (Hou et al., 2003; Khan and Maliga, 1999). Therefore, the results reported here for
strawberry are not without precedent.
CSG 15 (1/00)
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Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
Project
title
DEFRA
project code
HH1031SSF
Conclusion
Attempts to generate a stable plastid transformed line with all copies of its plastid genome transformed,
through tissue culture with antibiotic selection, were unsuccessful. Consequently, in the absence of a stable
pure plastid transformed line for use in inheritance studies, it was necessary to adopt an alternative approach
to study plastid inheritance in strawberry. This is covered in sections 3.5 and 3.6
3.3 Production of strawberry nuclear transformed transgenic plants containing plasmid constructs
with floral organ and root-specific promoters fused to the uidA (GUS) gene (OBJECTIVE 3)
The aim of this objective was to generate transgenic strawberry plants containing putative strawberry floral
organ (FavAP3) and root-specific (FavRB7) promoters fused to the uidA (GUS) gene. Subsequent analysis of
these plants, for distribution of GUS expression, would determine the extent to which the promoters were able
to direct tissue specific expression.
Nuclear transformation of strawberry with promoter-GUS fusion constructs
Vectors pSCVFavAP3 and pSCVFavRB7, in addition to pSCV1.6 that contains the constitutive CaMV 35S
promoter used as a control (Fig. 7), were transformed into Agrobacterium tumefaciens strain EHA 101.
Fig 7. Schematics of promoter-GUS fusion vectors used in strawberry nuclear transformation. (A) pSCVFavAP3, containing the
FavAP3 promoter (FavAP3 P), (B) pSCVFavRB7, containing the FavRB7 promoter (FavRB7 P) and (C) pSCV1.6, containing the
CaMV 35S constitutive promoter (35S P). T represents the terminator regions, RB and LB, right and left borders respectively and the
intron within the uidA gene is chequered. OD represents an overdrive T-DNA enhancer and a scale size bar is given.
A
B
LB
LB
FavAP3 P
FavRB7 P
LB
35S P
uidA
35S T
35S P
uidA
35S T
35S P
uidA
35S T
35S P
npt II
nos
T
RB
npt II
nos
T
RB
npt II
nos
T
RB
C
OD
OD
OD
1 kb
To achieve nuclear transformation, the transformed Agrobacteria were used to infect strawberry leaf discs in
two experiments.
Experiment 1 involved transformation of Fragaria vesca with pSCVFavAP3 and
regeneration of transformed shoots on ZD102 medium (MS including vitamins, 30 g/l sucrose, 1 mg/l TDZ, 0.2
mg/l 2,4-D, solidified with 7.5 g/l Oxoid Agar No. 3, pH 5.7) containing 25 mg/l kanamycin. Experiment 2
involved transformation of F. ananassa cv Calypso with pSCVRB7 and regeneration of transformed shoots on
ZN102 medium containing 50 mg/l kanamycin. Controls were included in both experiments, where discs were
transformed with pSCV1.6 and transformed shoots recovered on selective medium and non-transformed
shoots recovered on medium lacking kanamycin. All shoots surviving selection and negative controls were
weaned onto soil and all established plants were phenotypically normal.
Molecular analysis of transgenic lines
PCR to detect the promoter, uidA and nptII genes confirmed presence of all three in all transformed lines and
a total of 47 FavAP3, 32 FavRB7 and 3 CaMV35S promoter transgenic lines were generated. pSCV1.6 3 and
pSCV1.6 D CaMV35S promoter lines were generated in experiment 1 and the pSCV1.6 CaMV35S promoter
line generated in experiment 2. The foreign genes were not detected in the three negative control lines
generated for each experiment.
Southern analysis was carried out on all lines to determine the number of foreign gene integration sites.
Genomic DNA extracted from leaf tissue of each line was digested with Kpn I, to determine integration of uidA
and nptII genes in FavAP3 and CaMV 35S promoter lines. DNA was digested with Kpn I and Sma I to
determine integration of nptII and uidA genes respectively, in FavRB7 promoter lines. Digestion products were
electrophoresed on agarose gels, transferred to nylon membranes and probed with nptII and uidA-specific
Digoxigenin-11-dUTP (DIG)-labelled probes. Southern data obtained for those plants taken forward for
promoter activity studies are shown in Table 2.
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Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
Table 2 Summary of foreign gene integration sites in promoter-GUS fusion transgenic lines
FavAP3
promoter
line
Fav 5A
Fav 5D
Fav 9
Fav 15B
Fav 16
Fav 23
Fav 40
Fav 41
Fav 42A
Fav 47A
Fav 48C
Fav 70
Fav 75B
Fav76F
Fav 82C
Fav 86
Fav 94A
Fav 95A
Fav 115
Fav 168
CaMV35S
promoter line
pSCV1.6 3
pSCV1.6 D
uidA
nptII
1
3
4
1
2
1
2
3
1
1
1
3
1
1
3
1
1
1
1
2
1
2
2
1
2
1
2
3
1
1
1
2
1
1
3
1
1
1
1
2
uidA
nptII
1
1
1
2
FavRB7
promoter
line
1
5b
5c
6
13
14
37
38
39
41
42
43
44
46
48
49
53a
53b
55
58b
CaMV35S
promoter line
PSCV1.6
uidA
nptII
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
4
3
1
1
2
2
1
1
1
1
1
1
1
7
2
1
1
1
2
1
5
4
2
2
uidA
nptII
1
2
FavRB7
promoter
line
61
64
65
66
67
71
72
75
76
77
78
102
uidA
nptII
1
1
1
1
2
1
2
1
1
1
2
2
1
1
1
1
1
1
2
1
1
1
2
2
The majority of the transgenic lines contained simple integrations at one or two sites. In some lines,
integrations were more complex with incomplete integration of the T-DNA region of the vector as indicated by
unequal integrations of uidA and nptII genes. However, good populations of FavAP3 and FavRB7 promotercontaining lines were generated for further analysis.
3.4 Analysis of promoter activity in flowering populations of transgenic plants transformed with tissuespecific promoter constructs (OBJECTIVE 4)
Assessment of GUS activity as a measure of promoter activity in different tissues of transgenic plants
Since the promoters regulate expression of the uidA gene to produce GUS enzyme, promoter activity was
extrapolated from the amount of GUS enzyme activity in different tissues of the transgenic plants. GUS
enzyme activity was assessed quantitatively by fluorometric assay, which measures the amount of 4methylumbelliferone (4-MU) fluorescent product produced as a result of cleavage of methylumbelliferyl
glucuronide (MUG) substrate by GUS enzyme. Fluorometric assays to detect GUS activity (Jefferson, 1987),
were carried out as described in Gittins et al. (2000), with the modifications that 10 mM -mercaptoethanol and
20% (v/v) methanol were added to the extraction buffer. One plant of each line generated in experiment 1 was
sampled for analysis. Duplicate plants were generated, from runner populations, for each line generated in
experiment 2 and both were sampled for analysis. From each line, for each tissue type, material was taken
from six individual organs or was dissected from six individual flowers, ground under liquid nitrogen and 10 –
100 mg used per assay. Fluorescence was quantified using a FLUOstar Galaxy multiwell plate reader with
excitation and emission filters set at 360 nm and 460 nm respectively. Protein concentrations were
determined by BioRad assay and GUS specific activity expressed as pmol 4-MU produced per mg total soluble
protein per minute.
Analysis of GUS activity in 12 different vegetative and floral tissue types of the lines generated in
experiment 1 revealed that both FavAP3 and CaMV35S promoters exhibited constitutive expression; no
activity was detected in the negative control lines as expected. In the CaMV35S promoter lines, average GUS
activities for each tissue type were 140,374 (leaves), 84,912 (petioles), 10,375 (roots), 95,514 (pedicels and
peduncles), 45,861 (floral buds), 88,608 (sepals), 42,151 (petals), 28,110 (stamens), 30,274 (carpels), 6,411
(receptacles), 15,399 (green fruit) and 15,726 (red fruit) pmol 4-MU per mg protein per minute. Though
variation in the level of activity was observed between tissues, the CaMV35S promoter exhibited strong,
constitutive expression. The constitutive nature of the FavAP3 promoter is illustrated in Fig. 8, which shows a
CSG 15 (1/00)
12
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
Box-and-Whisker plot of GUS specific activity data, grouped by tissue type, obtained from the 20 independent
FavAP3 promoter transgenic lines.
Fig 8. GUS specific activity data, from different tissue types of 20 independent FavAP3 promoter transgenics, represented by
Box-and-Whisker plot. Tissues tested were Le, leaves; Pt, petioles; Ro, roots; P/P, pedicels and peduncles; B, floral buds; Se,
sepals; Pe, petals; St, stamen; Ca, carpels; Re, receptacles Gf, green fruit and Rf, red fruit. All 20 lines are represented by the data for
each tissue type except red fruit, where fruit from only 14 lines were analysed. The box spans the interquartile range of the GUS
activity values, the horizontal line indicates the median and the whiskers extend to the minimum and maximum values. Outliers are
plotted with a closed circle with the representing individual transgenic line numbers indicated, whilst far-outliers are plotted with a
closed triangle.
20000
FavAP3P
pmol 4-MU (mg protein)-1 min-1
17500
15000
12500
5D
168
115
76F
5D
10000
76F
7500
5000
2500
5D
5D
15B
5D
23
5A
0
Le Pt R P/P B Se Pe St Ca Re Gf Rf
Highest GUS activity, and hence FavAP3 promoter activity, was observed in petals and carpels, with relatively
moderate activity in green fruit, floral buds and receptacles and lower but considerable activity in leaves,
sepals, pedicels and peduncles. Activity in petioles, stamens and red fruit was relatively weak and the lowest
activity was observed in roots. Promoter activity in lines Fav 9, Fav 16 and Fav 95A was negligible in all
tissues. Generally, the FavAP3 gene promoter was found weaker, by approximately one order of magnitude,
than the CaMV 35S RNA promoter. Additionally, RT-PCR was carried out to detect presence of FavAP3
mRNA transcripts in the different tissues, which would give an indication of endogenous FavAP3 gene
promoter activity. Transcripts were detected in all tissues.
FavAP3 is a homologue of the Arabidopsis gene AP3, a floral organ homeotic gene that is specifically
expressed in petals and stamens (Jack et al., 1994). Unlike AP3 and homologues from other species that
exhibit similar floral organ specificity (Irish, 2003), FavAP3 is expressed in all tissues and may have an
additional role(s) to determining floral organ identity in strawberry.
GUS specific activity in leaf, whole flowers, petioles and roots of lines generated in experiment 2 were
analysed and results are represented graphically in Fig. 9.
Fig 9. GUS specific activity in different tissues of FavRB7 and CaMV35S promoter transgenics.
CSG 15 (1/00)
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Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
pmol 4-MU
-1
-1
mg protein min
160 000
140 000
120 000
100 000
80 000
60 000
40 000
20 000
0
35S -
71 66 75 6 5 102 49 72 53b 67 39 5c 44 13 46 48 61 37 76 58b 6 14 64 4 2 5b 78 1 41 38 4 3 77 53 a 5 5
leaf
root
petiole
flowers
Line
GUS was shown to be constitutively expressed in the CaMV35S promoter line (35S) whilst in the FavRB7
promoter lines (represented by numbers), expression was predominantly in roots. In many lines, the FavRB7
promoter was stronger in root tissue than the CaMV35S promoter. Using median GUS specific activity values
from the 32 FavRB7 promoter lines analysed, root:petiole, root:leaf and root:floral ratios were 1:0.3, 1:0.04 and
1:0.009 respectively. GUS activity was also analysed in mature fruit of FavRB7 promoter lines and it was
shown to be negligible. FavRB7 is a homologue of the tobacco gene TobRB7, which is a root-specific
tonoplast intrinsic protein that similarly exhibits root-specific expression in tobacco (Conkling et al., 1990;
Yamamoto et al., 1990).
Worthy of mention in this report is the finding, in parallel research to this study, that the strawberry
FavRB7 promoter was constitutive when transformed into tobacco and used to drive expression of GUS. This
suggests that there are nuclear factors present in strawberry that are involved in the regulation of root-specific
promoter activity which are absent in tobacco. This highlights the importance of not making assumptions that
promoters will behave in heterologous species as they do in the species from which they are isolated.
Tissue samples from negative control and FavAP3, FavRB7 and CaMV35S promoter strawberry lines
were also subjected to qualitative GUS histochemical analysis. Whereby tissues were incubated in X-Gluc
substrate, which is converted to a blue product by GUS activity. Observations of blue staining in tissues,
which represented GUS activity and hence promoter activity, reflected the data obtained from GUS
fluorometric analysis.
Detection of GUS mRNA by reverse transcription-polymerase chain reaction (RT-PCR) as an indication of
promoter activity
Non-quantitative RT-PCR was carried out to detect presence of nptII and uidA mRNA in different tissues of six
FavAP3 and FavRB7 promoter transgenic lines containing single integrations of the foreign DNA. CaMV35S
promoter and negative control lines were also analysed. Presence of transcripts are an indicator of promoter
activity.
Total RNA was extracted from each tissue type using an RNeasy® Plant Mini kit (Qiagen), from which
mRNA was extracted using an OligotexTM mRNA kit (Qiagen). The mRNA was reverse transcribed to cDNA
using an oligo(dT)22 primer with OmniscriptTM reverse transcriptase (Qiagen). Using the cDNA as template
multiplex PCR was carried out using Taq DNA polymerase (Invitrogen) with Npt II A/Npt II B and Gus 27/Gus
392 primer pairs (Appendix 1). Amplification products were electrophoresed on 0.8% (w/v) agarose gels and
visualised by UV illumination after staining with ethidium bromide.
Messenger RNA transcripts for both genes were detected in all tissues of CaMV35S promoter
transgenic lines, where both genes were controlled by the constitutive CaMV35S promoter and were absent
from the negative control lines. Npt II and uidA mRNAs were detected in all tissues of the FavAP3 promoter
lines tested (Fig. 10).
Fig 10. Detection of npt II (700 bp) and uidA (366 bp) partial cDNAs in different tissues of a representative FavAP3 promoter
transgenic line, line Fav76F. The upper and lower fragments (arrowed) represent npt II and uidA respectively. Tissues tested are Le,
leaves; Pt, petioles; Ro, roots; P/P, pedicels and peduncles; Gf, green fruit; Rf, red fruit; B, floral buds; Se, sepals; Pe, petals; St,
stamen; Ca, carpels and Re, receptacles. H, depicts water negative control, P, plasmid pSCV1.6 positive control and M, molecular size
standards.
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Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
Le
Pt
Ro P/P Gf
Rf
B
Se Pe
St
Ca Re
H
DEFRA
project code
HH1031SSF
P M kb
- 0.85
- 0.65
- 0.5
- 0.4
Fav76f
F
In FavAP3 promoter lines the npt II gene is controlled by the constitutive CaMV35S promoter and the uidA
gene by the FavAP3 promoter. Detection of mRNA for the uidA gene in all tissues indicated that the promoter
was active in all tissues, which corroborated the GUS activity data. In the FavRB7 promoter lines, npt II was
detected in all tissues, whilst uidA was shown to be present in only root and petiole tissues. This was also as
expected from the biochemical data.
Conclusion
Biochemical and molecular analyses of strawberry transgenic lines containing FavAP3 promoter::uidA and
FavRB7 promoter::uidA constructs show that the FavAP3 promoter is a constitutive, weak-moderate promoter
and that the FavRB7 promoter is a moderate-strong promoter exhibiting near root-specific regulation.
3.5 Identification of plastid genome sequence markers in strawberry parental genotypes using PCRRFLP and PCR-SSCP (OBJECTIVE 5)
The aim of this objective was to determine whether there were any sequence differences, known as
polymorphisms, within specific regions of the plastid genomes of different octoploid strawberry genotypes and
to use PCR-RFLP and PCR-SSCP analyses to detect these. This would enable differentiation between the
different genotypes on the basis of their plastid genomes, which could be exploited subsequently for
determination of the mode of plastid inheritance in strawberry.
The fifteen strawberry genotypes analysed included 10 Fragaria ananassa cultivars, three F. ananassa
breeding lines and two wild accessions are shown in Table 3. The 13 cultivated genotypes were selected due
to their diverse genetic backgrounds.
Table 3 Octoploid strawberry genotypes screened for plastid genome sequence polymorphisms
Reference
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Genotype
F. ananassa cv. Alice
F. ananassa cv. Bolero
F. ananassa cv. Ciflorette
F. ananassa cv. Diamante
F. ananassa cv. Everest
F. ananassa cv. Florence
F. ananassa cv. Mara Des Bois
F. ananassa cv. Nida
F. ananassa cv. Onda
F. ananassa cv. Symphony
F. ananassa breeding line EM0965
F. ananassa breeding line EM0980
F. ananassa breeding line EMR154
F. chiloensis
F. virginiana
Isolation of plastid genome regions and detection of sequence polymorphisms
Eight plastid genome non-coding regions were targeted and amplified from each genotype using PCR.
Intergenic and intron regions were chosen since these are under less evolutionary constraint than gene coding
sequences and were thus more likely to have incurred sequence changes. Sequences of newly synthesised
primers are listed in Appendix 1 or where published primers were used, references are cited.
Total genomic DNA was extracted from young leaf tissue and used as template in PCR reactions using
KOD HotStart DNA polymerase. Primers used to amplify trnT – trnL (5GR/5GF) and trnL – trnF (6IR/6IF)
intergenic regions and the trnL intron (5HF/5HR) were as designed by Taberlet et al. (1991). To amplify trnH –
CSG 15 (1/00)
15
Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
psbA (2CF/2CR), trnS – trnG (3DF/3DR) and rpl20 – 5’rps12 (7JF/7JR) intergenic regions primers were as
designed by Hamilton (1999), with the exception of the primer designed to anneal in the psbA gene. Due to
sequence divergence between strawberry and tobacco psbA gene sequences, the psbA (2CR) primer was redesigned. Primers 1AF/1AR and 3EF/3ER were newly designed to amplify rpl2 and trnG introns respectively.
To obtain sequence information for a proportion of the amplified fragments, selected PCR products were
cloned into the plasmid pCR®-Blunt II-TOPO and sequenced using dye-terminator cycle sequencing.
Sequencing of fragments representing each region, from a random selection of eight genotypes for
each, indicated that no sequence polymorphisms were present within trnG, rpl2 and trnL gene introns, of
lengths 764, 757 and 494 bp respectively. For each trnH – psbA and trnS – trnG intergenic regions, of lengths
350 bp and 680 bp respectively, a single nucleotide insertion/deletion (IN/DEL) was observed in one of the
eight genotypes studied. These polymorphisms were not investigated further. Five polymorphic loci,
nominated marker one through to marker five, that could be exploited in PCR-RFLP and PCR-SSCP analyses,
were shown to be present within the three remaining intergenic regions (Fig. 11).
Fig 11. Schematic representation of PCR-amplified plastid genome regions characterised for polymorphisms. A, B and C
represent the three different regions studied. Horizontal lines depict intergenic regions where the nucleotide sequence is identical in all
genotypes and surrounding genes are illustrated by hatched boxes. Sites within the intergenic region displaying polymorphism are
indicated in their relative positions as markers (M), with the sequence differences observed at these positions shown. Primers used to
amplify specific fragments are represented by arrows. PCR fragments are shown as dashed lines and lengths indicated.
A
5GF
G179F
1005 bp
181 bp
G179R
M1
trnT
M2
A/C
A/C
trnL
C
B
6IF
5GR
498 bp
6IR
7JF
837 bp
M3
trnL
7JR
J2XF
180 bp
J2XR
M4 M5
C/T trnF
A/T A8/A9
rpl20
5’rps12
Markers one and two, within the trnT – trnL intergenic region at 179 and 423 bp from the 5’ end of the PCR
fragment respectively, were both shown to be nucleotide substitutions. At marker one, genotypes Bolero,
Florence, Nida, Symphony and EM0980 are represented by an A, whist all other genotypes by a C. At marker
two, Bolero, Florence, Nida, Symphony, EM0980 and EM0965 genotypes have an A, whilst all others have a
C. When an A is present, a Psi I restriction enzyme recognition sequence is created, allowing distinction of
marker two by RFLP analysis. Marker three, a nucleotide substitution within the trnL – trnF region at 423 bp
from the 5’ end of the PCR fragment, similarly enables detection by RFLP. Presence of a C, as seen with
Bolero, Florence, Nida, Symphony, EM0980 and EM0965, creates an EclHK I recognition sequence and when
a T is present, as with the remaining genotypes, a Bse GI recognition sequence is created. Within the rpl20 –
5’rps12 region markers four and five, at loci 551 bp and starting at 552 bp from the 5’ end of the fragment
respectively, were shown not to result in creation or destruction of a restriction enzyme recognition sequence,
and like marker one, could be exploited by SSCP analysis. At the marker four locus Alice, Ciflorette,
Diamante, Everest, Mara Des Bois, Onda and EMR154 are represented by a T whilst all other genotypes have
an A. The marker five locus consists of an IN/DEL where F. chiloensis has a run of nine As, whilst all other
genotypes have a run of eight.
Detection of plastid genome sequence polymorphisms using PCR-RFLP
PCR products from each genotype, representing trnT --- trnL and trnL --- trnF intergenic regions, were
digested at 37 ˚C for 20 hours with Psi I and EclHK I restriction enzymes respectively. Restriction fragments
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were separated on 1.5 % (w/v) agarose gels and stained with ethidium bromide. The results of the PCR-RFLP
analysis are shown in Figure 12.
Fig 12. PCR-RFLP analysis of octoploid strawberry genotypes. Genotypes are numbered according to the key shown in Table 3.
Restriction fragment analysis of PCR amplified trnT --- trnL and trnL --- trnF intergenic regions from each genotype, containing markers
M2 and M3 respectiviely and digested with restriction enzymes Psi I and EclHK I respectively. Markers are shown with surrounding
sequence.
Marker M2: TTA/CTAA
1 2 3 4 5 6 7 8 9101112131415
Marker M3: GAC/TGGTGCGTC
M bp
1 2 3 4 5 6 7 8 9 101112131415 M bp
850
650
500
400
300
200
850
650
500
400
300
200
100
Using PCR-RFLP, it was possible to detect each marker. All genotypes contain a Psi I recognition sequence
located 276 bp from the 3’ end of the trnT – trnL fragment. For detection of marker 2, in genotypes where
there was an A at the locus, the fragment was further cleaved by Psi I to produce three fragments in total. For
detection of marker 3, the trnL – trnF fragment was cleaved by EclHK I to liberate two fragments only in those
genotypes that possessed a C at the locus; those with a T remained uncut. The trnL – trnF fragments were
also digested with restriction enzyme Bse GI and the opposite results were observed for detection of marker 3.
Detection of plastid genome sequence polymorphisms using PCR_SSCP
Since resolution of single nucleotide differences by PCR-SSCP is more optimal when shorter PCR products
are analysed, PCR fragments of 181 bp and 180 bp were generated within the trnT --- trnL and rpl20 --- 5’
rps12 intergenic regions respectively. Primers designed and used to amplify an internal fragment of each
region were G179F/G179R and J2XF/J2XR respectively (Fig. 11, Appendix 1). PCR products from each
region for each genotype were denatured in denaturation solution (deionised formamide containing 1 M NaOH
and a few grains bromophenol blue). The sample mixture was heated at 95 ˚C for 5 min and immediately
placed on ice for 3 min. Gel electrophoresis of samples was carried out on Gene Mutation Analysis (GMA TM)
gels (Elchrom Scientific). Different temperatures, voltages and run times were tested to achieve optimal
resolution of DNA sequence polymorphisms. For optimal separation, electrophoresis was carried out at 72V at
7 ˚C for 12 hours using Elchrom’s SEA 2000® gel apparatus. Post-electrophoresis, gels were stained with
SYBR® Gold (Molecular Probes Inc.). The results of the PCR-SSCP analysis are shown in Figure 13.
Fig 13. PCR-SSCP analysis of octoploid strawberry genotypes. Genotypes are numbered according to the key shown in Table 3.
SSCP analysis of trnT --- trnL intergenic region, containing marker M1, and rpl20 --- 5’ rps12 intergenic region, containing markers M4
and M5, PCR amplified fragments from each genotype, denatured and electrophoresed on GMA TM gels. Markers are shown with
surrounding sequence.
Marker M1: AATA/CTCA
1 2 3 4 5 6 7 8 9 10 1112131415
Markers M4 & M5:
TTTA/TA8/A9TTTC
1 2 3 4 5 6 7 8 9 101112131415
Following optimisation of the electrophoresis conditions, it was possible to reproducibly resolve differences in
single-stranded DNA of the fragments according to nucleotide composition at each marker. Marker one was
shown to give rise to two different SSCP profiles, determined by whether genotypes harboured an A or C at
position 179. Three different profiles were observed for genotypes for the rpl20 – 5’rps12 region that contains
two markers. All cultivated genotypes and the wild accession F. chiloensis exhibited profiles according to
whether marker 4 is an A or T. The profile for the wild accession F. virginiana differed further since at marker
5 it contains an extra A compared to all the other genotypes.
Both PCR-RFLP and PCR-SSCP analyses enabled sequence attributes at each marker to be
distinguished and each also confirmed the sequence data.
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Conclusion
Seven sequence polymorphisms, markers, were shown to be present within five plastid genome intergenic
regions of 13 octoploid strawberry genotypes, which could be detected by PCR-RFLP and PCR-SSCP
analyses. The data show that Fragaria genotypes are distinguishable by plastid genome markers at both
intra- and inter-specific levels.
3.6 Determination of whether plastids are transmitted through pollen in strawberry using plastid
genome sequence markers (OBJECTIVE 6)
The aim of this objective was to screen progenies from controlled crosses, derived from parents with
distinguishable plastid genome markers, to determine which markers were present on their plastid genomes.
This would reveal whether plastid genomes, and hence plastids, had been inherited from the paternal or
maternal parent and would hence allow the mode of plastid inheritance in strawberry to be determined.
Progenies from both intra-specific and inter-specific crosses were investigated.
Raising of progenies from controlled crosses
Progenies raised from controlled crosses between cultivated genotypes, grown in the field, were kindly
provided by Dr David Simpson, leader of the strawberry breeding programme at Horticulture Research
International, East Malling, UK.
Progenies from the cross between cultivated and wild genotypes were raised from a cross between F.
ananassa cv. Everest and F. virginiana plants. A day prior to pollination sepals, petals and stamens, from
flowers with closed petals on F. virginiana plants, were removed. Recently opened flowers were removed from
F. ananassa cv. Everest plants, the petals removed and the flowers incubated at 30 ºC for 7-8 hr until the
anthers dehisced. Pollen from the anthers was brushed against receptive stigmas of F. virginiana plants to
facilitate pollination and fruit subsequently allowed to mature. Seeds from fruit were removed, surface
sterilised and plated on 0.75 % (w/v) agar plates containing 3 % (w/v) sucrose and Murashige and Skoog
medium including vitamins at pH 5.7. Seedlings were grown at 22 ºC under a 16 hr photoperiod and once
established with 6 – 8 leaves, weaned onto compost and grown under the same growth conditions in a growth
room.
Five families totalling 648 progeny, in which markers distinguished the parents, were investigated
(Table 4).
Table 4 Families used to determine plastid inheritance in strabwerry intra- and inter-specific crosses
Family
ID
Paternal parent x maternal
parent
No. of
progeny
F. ananassa x F. ananassa
104
Alice x EM0965
163
Bolero x Diamante
199
Alice x Bolero
102
EM0980 x EM0965
56
147
75
237
F. ananassa x F. virginiana
CW
Everest x F. virginiana
133
Determination of the mode of plastid inheritance in strawberry
Genomic DNA was extracted from leaf tissue of parents and progenies from each family and was subjected to
either PCR-RFLP or PCR-SSCP analysis to detect specific markers.
Analysis of family 199, using PCR-RFLP to detect marker 2, revealed that all bar one of the progeny
exhibited identical restriction fragments to Bolero the female parent. Individual number 19 was shown to have
inherited plastids from Alice, the paternal parent (Fig. 14).
Fig 14. PCR-RFLP analysis of progeny raised from a controlled cross between cultivated strawberry (F. ananassa)
genotypes. PCR fragments representing the trnT --- trnL intergenic region containing marker M2, generated from progeny (labeled 1 –
75) and digested with restriction enzyme Psi I are shown alongside similarly produced fragments from the male, Alice (M), and female,
Bolero (F), parental genotypes used in the cross.
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27 28
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kb
850
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200
All progeny from families 104 and 163 totalling 203 individuals, were shown to have contained plastid genome
DNA derived from the maternal parent using PCR-RFLP to detect markers 2 and 3 respectively.
Analysis of family 102 using PCR-SSCP to detect marker one, the largest family studied with 237
progeny, also revealed exclusive maternal plastid inheritance. Paternal chloroplast inheritance was also
detected in progeny derived from the cross between wild and cultivated genotypes. PCR-SSCP profiles for 23
of the 133 progeny screened, to detect markers four and five, are shown in Figure 15. Offspring number 105
was the sole sibling, from the total 133 screened, to have exhibited an identical PCR-SSCP profile to the male
parent Everest.
Fig 15. PCR-SSCP analysis of progeny raised from a controlled cross between cultivated and wild strawberry genotypes.
PCR fragments within the rpl20 – 5’rps12 intergenic region containing markers M4 and M5, generated from progeny (labeled 93 – 115)
and the male, F. ananassa cv. Everest (M), and female, F. virginiana (F), parental genotypes used in the cross, denatured and
electrophoresed on a GMATM gel.
93
115 M F
Strawberry is not unique in displaying incomplete maternal inheritance of plastids. Paternal plastid
transmission has also been observed in other angiosperm species, such as tobacco (Medgyesy et al., 1986).
Similarly, in gymnosperms, which generally exhibit a paternal mode of plastid inheritance, a low frequency of
maternal transmission has been observed (Shiraishi et al., 2001).
Conclusion
Two offspring, one each from inter- and intra-specific controlled crosses, were shown to have exclusively
inherited paternal plastids; all other progeny inherited plastids from the maternal parent. It is concluded that
plastids are not exclusively maternally inherited in strawberry and that the mode of inheritance, whether within
cultivated genotypes or between cultivated and wild genotypes, can be bi-parental, albeit at low incidence.
4. MAIN IMPLICATIONS OF FINDINGS
Utilising strawberry and focusing on safety and precision, this strategic research aimed to develop plastid and
nuclear transformation technologies for the production of GM plants. A parallel study was conducted to
elucidate the mode of plastid inheritance in strawberry. This would enable the potential for the use of plastid
transformation to engineer GM strawberry as a means of eliminating gene flow through pollen to be
determined.
Whilst plastid transformed plants were successfully generated utilising selection with the carbohydrate
xylose, production of stable lines, where all plastid genome copies were transformed, proved problematic. The
same difficulty was experienced with a plastid-transformed line that was subjected to selection with the
antibiotic spectinomycin. Selection of tissue cultures with antibiotics often promotes the generation of
antibiotic resistant mutants, which survive the selection process along with plastid-transformed plants that are
resistant since they carry an antibiotic resistance marker gene. It was hoped that developing a system that
was based on carbohydrate metabolism, in addition to eliminating the use of antibiotic resistance genes in the
system, would reduce the number of non-transformed regenerants that survived the selection process.
However, the xylose selection system generated a large proportion of non-transformed lines, indicating the
poor effectiveness of xylose as a selection agent under the growth conditions used. Even when cultured in the
presence of solely xylose, the proportion of transformed plastid genomes in partially transformed lines could
not be increased. It is clear that although strawberry plastid transformed plants have been generated using
both antibiotic and non-antibiotic selection systems, both systems have their limitations, and further
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development is necessary to engineer stable strawberry plastid transformed lines. This is likely to involve
consideration of alternative selection systems and construction of new improved plastid transformation vectors
carrying regulatory elements that ensure efficient transcription and translation in planta.
Study of the mode of plastid inheritance revealed bi-parental inheritance in strawberry. No previous
studies on the mode of plastid inheritance in strawberry have been reported. The finding that there was a low
frequency of paternal inheritance has implications on engineering the strawberry plastid genome for
containment of foreign genes. Whilst foreign gene escape via pollen would be significantly reduced in
strawberry plastid transformed plants, in comparison to those transformed in their nuclear genome, there may
still be potential for a small degree of escape. There would be potential for paternal plastid transmission within
octoploid strawberry at both intra-specific and inter-specific levels. It is worth noting that the data obtained
from this work emanated from controlled crosses and the frequencies of paternal plastid transmission under
natural hybridisation conditions may differ. The markers and techniques developed in this work could be used
in further studies investigating inheritance in natural populations arising from open pollination. Additionally,
since sequence polymorphisms occur at low frequency within the strawberry plastid genome, the ones
identified in this study will be powerful tools for phylogenetic and population genetic studies. The finding that
plastids can be paternally inherited is of significance for population genetic studies where strict maternal
inheritance is often assumed. There have been limited studies investigating plastid genome sequence
variation in Fragaria species and the markers detected in this work add to the current knowledge.
The FavAP3 promoter was investigated for its ability to drive floral organ specific gene expression with the
longer-term goal of using it to drive expression of anti-fungal protein genes. The target in strawberry being the
fungus Botrytis cinerea, which enters the plant through floral tissue. Therefore gene expression could be
specifically targeted to the tissues requiring the foreign protein, which would be absent in the consumed part of
the plant, the fruit, where its presence was not necessary. Unfortunately, though FavAP3 promoter activity
was highest in floral tissue, it was present throughout all plant tissues and could not be considered tissue
specific. It therefore would offer little benefit for restricting foreign gene expression to particular tissues.
The FavRB7 gene promoter was shown to be near root-specific and moderately strong. It could be used
for near root-specific expression of genes that confer resistance to pests and diseases, such as Otorynchus
sulcata (vine weevil) larvae, Verticillium dahliae (Verticillium wilt) and Phytophthora fragariae (red core) that
attack the root. With very limited gene expression in the aerial parts of the GM plant, the potential risks to
consumers and beneficial insects would be reduced. With the possible future use of GM technology to
combat pests and diseases, this promoter will become increasing valuable for targeting anti-pest/fungal
proteins to roots, especially with the gradual withdrawal of soil sterilants such as chloropicrin, which is currently
used to combat soil-borne diseases. The finding, from work carried out in a parallel study, that the FavRB7
gene promoter, different to the situation in strawberry, directs constitutive expression in a heterologous host
stresses the importance of not assuming that a promoter will behave the same way when placed in a
heterologous host as it does in the species from which it has been isolated.
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Guillon J-C, Raquin C (2000) Maternal inheritance of chloroplasts in the horsetail Equisetum variegatum (Schleich). Curr. Genet. 37:5356
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effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol. Biol. 37:287-296
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8:513-525
Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transgenic Res.
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APPENDIX 1 PRIMERS
Primers used in the contruction of pFavXG plastid transformation vector
RRN16SSP 5’ AAATTAATATTCGCCTGGAGTTCGCT 3’
RRN16 KPNSGF 5’ ATTATGGTACCGCGATCGCTTTCCATTTTTATTTGC 3’
RBCLc 5’ CAGGCCTGTTGTAGGGAGGGACA 3’
RBCLd 5’ GTACACAGGGAGGGATGTTGTCCGGACCCGG 3’
PSBABGL 5’ ACTGCAGATCTCCATCTATCAATGGATAAGGCTT 3’
PSBAECOSGF 5’ TATAGAATTCGCGATCGATTACTTGTAAAAAAGAATACAACAT 3’
XYLAPAG1 5’TTAATCATGAATAAATATTTTGAGAACG 3’
XYLAXHO1 5’ ATTATCTCGAGTTATTCTGCAAACAAAT 3’
SMGFPBGLAPA 5’ TATAGGGCCCAGATCTTATTTGTATAGTTCATCCATG 3’
AADA/XYLA:SMGFP 5’
ATCTGCAGCTCGAGATGACTAATTAGAAGGGAGGGAATCATGAGTAAAGGAGAAGAACTTTTCACTG 3’
Primers used in RT-PCR to detect marker genes in transgenic lines
Npt II A 5’ GAGGCTATTCGGCTATGACTG 3’
Npt II B 5’ ATCGGGAGCGGCGATACCGTA 3’
Gus 27 5’ CCTGTAGAAACCCCAACCCGTG 3’
Gus 392 5’ CCCGGCAATAACATACGGCGTG 3’
Primers used in the plastid inheritance study to amplify regions of the plastid genome
2CR 5’ TGAAGTTCCGTCTATCAATGG 3’
1AF 5’ AAAATGGGAAATGCCCTACCT 3’
1AR 5’ TTCCAAGTGTKATTTCTATGTT 3’
3EF 5’ GCGGGTATAGTTTAGTGGTAA 3’
3ER 5’ AGCGGGTAGCGGGAATCGAA 3’
G179F 5’ ATGATATAAATGTAGAAAAATTCAAC 3’
G179R 5’ ATATGTAATGTAATAGTCAAAAAATC 3’
J2XF 5’ CTGAGTAGCTGACCCTGTTAGT 3’
J2XR 5’ AAATGTATGGTTCCCGTTGGTG 3’
6. APPENDIX 2 TECHNOLOGY/KNOWLEDGE TRANSFER ACTIVITIES
Papers
Massiah, A.J. (2004) Genetic modification of plants. Oxford Series, Oxford Companion to the Garden. Oxford University
Press. In press
Massiah, A.J., Baker, S.A., Theodoridou, S. and James, D.J. (2004) A Strawberry (Fragaria x ananassa Duch.)
APETALA3 ortholog (FavAP3) regulates petal and stamen formation and is expressed during reproductive and vegetative
plant development. Submitted to Journal of Experimental Botany
Massiah, A.J., Baker, S.A and Passey, A.J.(2004) Incomplete maternal inheritance of chloroplasts in the genus Fragaria
detected using inter- and intraspecific sequence polymorphisms of chloroplast DNA. In preparation. To submit to
Molecular Genetics and Genomics journal (June 2004)
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Vaughan, S.P., James, D.J, Lindsey, K. and Massiah, A.J. (2004) Isolation and characterisation of the gene and promoter
of FavRB7, a tonoplast intrinsic protein homologue from strawberry (Fragaria x ananassa Duch.). In preparation,
submission planned for August 2004
PhD Thesis: Simon Vaughan (2003) Tissue and organelle targeted transgene expression in plants. Student registered
with University of Durham
James, D. J. and Massiah, A. J. (2003) Genetic modification of temperate fruit crops – why we do it and why it’s
necessary. Forum contribution towards topics 1. GM Food and Feed Safety and 2. Future Developments, UK
Government GM Science Review. http://www.gmsciencedebate.org.uk/topics/forum/0029.htm
James, D. J., Gittins, J. R., Pellny, T, K., Massiah, A. J., Hiles, E. R., Biricolti, S., Vaughan, S. P. and Passey, A. J. (2001)
Using heterologous and homologous promoters to study tissue-specific transgene expression in fruit crops. Acta
Horticulturae. No560: 55-62
Patents
UK Patent Office Application in the name Horticulture Research International, number 0312449.2: Novel Promoters
(FavRB7 promoter for root-specific expression in strawberry and constitutive expression in heterologous hosts).
Inventors: Vaughan, S.P., Massiah, A.J. and James, D.J. Filed 30/05/03
GenBank DNA sequence accessions submitted
AY429427 – Full length FavAP3 sequence
AY429428 – Partial length FavAP3 sequence with 3’ UTR
AY429429 – FavAP3 gene promoter sequence with 5’ end of gene attached
Posters
Vaughan, S. P., Massiah, A. J. and James, D. J. (2001) Plastid transformation in strawberry. Presented at Horticulture
Research International Graduate Student Symposium 2001
Conference proceedings
James, D., Massiah, A.J., Blakesley, D., Passey, A.J. and Bulley, S. (August 2002) Using Genetic Modification in apple
and strawberry to improve fruit quality – benefits for the grower, the consumer and the environment. ISHS Conference
2002 – Toronto, Canada. Session: Biotechnology of Horticultural Crop Improvement: Achievements, Opportunities and
Limitations
James, D. and Massiah, A. (September 2001) Regulation and targeting of transgene expression in fruit crops.
Presentation at the Greater London Molecular Plant Sciences Symposium
James, D. and Massiah, A. (July 2001) Strawberry biotechnology for genetic improvement and healthcare.
International RUBUS / RIBES Symposium
8 th
James, D. and Massiah, A. (June 2001) Strawberry biotechnology programmes at East Malling. Presentation to Meiosis
at the Soft Fruit Conference Day
Oral presentations
Vaughan, S.P., Massiah, A.J. and James, D. J. (June 2003) Strawberry GM research at HRI-EM. Oral presentation at
East Malling Research Association – Soft Fruit Research Day.
James, D. J. and Massiah, A. J. (September 2002) GM technology at East Malling. Oral presentation to invited VIPs for
opening of East Malling Conference Centre and East Malling VIP Open Day
Massiah, A. J., Passey, A. J., Vaughan, S. P., Baker, S. and James, D. J. (September 2002) Presentations: ‘GM
technology, the public debate’ and ‘GM plants-how they are made’ East Malling Public Open Day
CSG 15 (1/00)
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Project
title
Tissue and plastid targeted transgene expression in a
perennial crop, strawberry
DEFRA
project code
HH1031SSF
Massiah, A, Passey and A, Blakesley, D (March 2002) Production of foreign proteins in GM plants. Presentation to
Enterprise Hub Directors
Vaughan, S. P., James, D. J. and Massiah, A. J. (February 2002) Towards root specific transgene expression in
strawberry. Presentation a HRI Annual Student Symposium (Vaughan awarded prize for best presented talk)
Massiah, A., Passey, A. and Bulley, S. (February 2002) GM crop technologies. Presentation to the Parliamentary Fruit
Group.
Massiah, A.J. (October 2001) Plant biotechnology. Presentation for Team CI 5, Molecular Pharming and Gene Delivery,
at Horticulture Research International Institute Assessment Exercise 2001
Massiah, A.J. (October 2001) Genetic modification of fruit crops. Presentation to Dr Emma Hennessey, DEFRA
Massiah, A.J. (May 2001) Transgene expression. Presentation to Dr David Jones, DEFRA
Massiah, A. (March 2001) Genetic engineering in fruit crops. Presentation to three members of the Ethical Investment
Advisory Group to the Church of England
Massiah, A. J., Vaughan, S. & James, D. J. (2001) Plastid transformation in the fruit crop strawberry. Invited speaker,
Department of Biology, National University of Ireland, Maynooth, Ireland.
Massiah, A. (February 2001) Tissue and plastid targeted transgene expression in strawberry. Horticulture Research
International Soft Fruit Research Review meeting
James, D. and Massiah, A. (February 2001) Regulation and targeting of transgene expression in fruit crops. Lecture to
final year honours students, University of Greenwich
James, D. and Massiah, A. (January 2001) Genetically Modified Fruit. Lecture to MSc students at JIC, Norwich
James, D. and Massiah, A. (November 2000) Regulation and targeting of transgene expression in fruit crops.
Presentation to Dr Seppo Sovari plus four other visitors from MTT Horticulture, Finland
Technical reports
Massiah, A.J., Vaughan, S.P. and James, D. J. (June 2003) Strawberry GM research at HRI-East Malling. East Malling
Research Association – Soft Fruit Research Day report.
Massiah, A. (2002) Production of GM plants using chloroplast transformation. Horticulture Research International Annual
Report 2001-2002
Walden, R.M., James, D.J., Blaskesley, D. and Massiah, A. J. (2000) GM fruit workshop East Malling Research
Association (EMRA) report
CSG 15 (1/00)
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