Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W094/03607 . 2 I 2 0 2 5 8 Pcr~EPg3/olg88
D~A C0~8TRUC~ CB~5 A~D PLANT~ D2~I~ED T~BaB~RO~
800p~ o~ the i~tio~
The invention relates to novel DNA constructs~ plant cells
co~taining these constructs and plants derived there~rom, as
well as æeeds obtained ~rom said plants. In particular it
invol~es the use of antisen~e nucleic acid technology to
control gene expression in plants7 Using tissue specific
promoters the control of gene expression can ~e directed to
particular issues of the plant. The invention provides
further, a process for obtaining plants which do not exhibit
enz~matic browning of disrupted tissues.
IntroZuation
Browning and discolouration causes substantial losses in a
wide range of fresh and pro¢essed fruits and vegetables. In
potato~s, internal bruising, also called black spot, is a
physiological response of potato tubers to tissue compaction
and i~ o~e of the most important problems of the potato
industr~. Yearly, large amounts of the potato produc~ion are
valueless for direct consumption or processing due to browning
of the tubers.
i
The biochemistry of browning is relatively well unders~ood
in mammals and bacterial model systems. In plants, however very
little is known about the biochemical processes involved in
browning and the pathways responsible for discolouration in a
wide variety of fruits and vegetables has not been elucidated.
In potatoes, bruising or black spot results from minor physical
damage sustained by the tubers during mechanical harvest and
wo 94/036~ 1 2 0 2 5 8 PCT/EP93~01988
transportation. The colour reaction results from a small number
of cells within the bruise that ~ctually lyse. The grey/black
colour of the bruise i5 caused by the enzymatic production of
melanin, a highly polymerised phenolic compound. The enzyme
polyphenol oxidase (PPO), an oxygenase, catalyses the first two
oxidation reactions of the pathway and converts monophenols
~such as tyrosine) and ortho-diphenols (such as catechol) to
ortho-~uinones (such as dihydroxiphenylalanine-quinone).
Autocatalytic chemical oxidation reactions conv~rt
ortho-quinones into ~elanin via several reactive quinone
intermediates. Although the function of plant polyphenol
oxidases has not been elucidated, several possible roles of the
enzyme have been postulated. These include participation in
electron transport in photosynthesis and participation in the
defence mechanisms of the plant against insect attack or
infections. It is believed that PPO is not involved in the
synthesis of phenolic compounds in healthy, intact cells. Plant
PPo enzymes are nuclear-encoded copper metalloproteins
localised in me~branes of plastids. In potato tubers, PPO has
been localised in the rudimentary thylacoid me~branes of
amyloplasts. The sub-cellular location separates the enzyme
from its substrate which is primarily found in the cell
vacuole. However, in disrupted cells the enzyme is mixed with
substrate leading to oxidation of phenolic compounds by PPO
enzyme.
: : :
Although bruising~has been a severe pro~lem in the potato
industry for many years, no progress has been made until now to
prevent the formation of black spots into potato tubers through
molecular technique~. The term bruising resistance, as used
hereaft2r, refers to resistance against browning and
discolouration upon disruption of plant cells.
We have found that bruising resistance can be obtained
through interfering in the biosynthetic pathway of melanin
production by the expression of an antisense PPO gene. More
particularly, the production of polyphenol oxidase can be
W094/0~7 ~ 2 ~ 2 0 2 5 8 PCT/EPg3/01988
reduced in specific tissues through tissue specific expres~ion
of said antisense PPO gene. Since the expression of anti~nse
PPO may perturb one or more of the postulated functions of PPO,
especially in aerial parts of the plant, limitation of
expression to tubers, by implementation of appropriate tuber
specific promoters, is preferred. Furthermore, we have found
that polyphenol oxidases are encoded by a family of genes
comprising at least S members. We have been able to suppress
the expression of genes, and particularly of all genes,
expressing in tubers through the coordinate expression of only
one antisense PPO gene corresponding to part or entire
sequence, or homologous with one of the genes encoding
polyphenol oxidase. Moreover, we have been able to produce
bruising resistant plants which show a reduced sensitivity to
bruising, especially potato plants of which the tubers show a
reduced sensitivity to bruising.
The invention is based on the application of antisense
nucleic acid technology. Although the mechanism of antisense is
~not elucidated, it~is believed that the inhibition of the
expression of the target gene is obtained either through the
formation of a complex of the antisense RNA and the endogenous
RNA which are complementary, preventing the translation, or
through~ the formation of a complex structure with the DNA
sequence, preventing the transcription.
The invention provides~a method for producing bruising
resistant plants showing a reduced PPO enzyme activity and
which also have been shown to have a significantly reduced
~ browning phenotype in the field.
; The invention can be used to control bruising upon
disruption of tuber, vegetable, or fruit tissues in which the
browning is the resuIt of PPO enzyme activity in or around the
damaged plant tissue. It is understood that the invention is
not restricted to potatoes but that it can also be used for the
reduction of browning upon physical tissue disruption in other
WOg4/03607 ~ PCT/EP93/019BX
~,120Z58 ,.1~,
plants like leaf vegetables, i.e. lettuce, chicory or fruits,
where ~he browning is ~he result of the expression of genes
encoding PPO.
Det~iled ~es~riptio~ of the invention
The present invention provides DNA constructs comprising i~
a promoter fu~ctional in plants, ii) a DNA se~uence
corresponding to an antisense polyphenol oxidase ~ene or part
thereof, or a DNA sequence homologous thereto and iii) a
terminator functional in plants.
The term antisense polyphenol oxidase gene refers to a
double stranded DNA sequence which sequence corresponds to or
is homologous with the endogenous gene encoding PFO or part
thereof, but which has a reversed orientation with respect to
the said PPO ge~e. In a antisense DNA sequence the coding
strand becomes the~template strand and vice versa.
The term homologous sequence refers to a sequence in which
the aver~age nucleotide sequence homology with the contiguous
gene is~ at least 65 %. ~ ~
The term DNA sequence refers to a double stranded DNA
sequence which can be~expressed in piant cells if said seguence
is under:the expression control of a promoter functional in
plants and having a terminator functional in plants.
The term promoter used herein refers to a transcription
regulation region upstream from the coding sequence containing
the regulatory se~uences required for the transcription of the
adjacent coding se~uence and includes the S' non-translated
:
region or so calied leader sequence of mRNA. The promoters used
in DNA ~constructs according to the invention have to be
functional in plant cells.
WOg4/03607 212 0 Z 5 PCT/EI93/01 ~
A further aspect of the invention comprises DNA constructs
in which the promoter is a tissue specific promoter. A tissue
specific promoter directs the transcription of the adjacent
sequence in specific tissues of the plants. Suitable promoters
for use in DNA constructs according to the invention include
but are not limited to~the cauliflower mosaic virus 35S (CaMV
35S) promoter, or potato tuber specific promoters like the
promoter of the ~lass I Patatin gene or preferably the granule
bound starch synthase gene.
The term terminator refers to a region downstream of the
coding sequence which~ directs the termination of the
transcription, also call~ed the 3' non-translated region, which
includes the poly-adenylation signal. Terminator sequence
functional in plants are well known and described in the
literature. ~
The DNA sequence ~aocording to the invention comprises at
least 25~nucleotides. In~general, a suitable length of the DNA
sequence comprises lOO~to 1000, up to 2000, nucleotides. In a
pre~ferred embodiment of~ the invention the DNA sequence
;comprises the entiré~ coding~sequence of the endogenous PP0
gene, or a sequence~ ho:mologous thereto, in inverted
orientation.
The DNA sequence~according to the invention can be derived
from~genomic DNA~or cDNA~encoding PPO. The preparation of DNA
constructs according to~ the invention is described in more
detail below. The invention also provides vectors comprising
the DNA construct accordlng to the invention.
The invention also includes plant cells comprising DNA
constructs according to the invention, plants derived therefrom
:
and seeds of such plants. Transformed plants can be obtained
using standard transformation techniques like Ti-plasmid
mediated transformation,~ direct transformation methods as
particle bom~ardment and the like. The preferred transformation
W094/03607 PCT/EP93/0198B
2 ~2 02 S ~ ` 6
technique to be used depends on the plant tissue and is known
to the person skilled in the art.
The scope of the invention will be illustrated below,
employing the construction of bruising resistant potatoes as an
example. It is understood that similar approaches, according to
the invention, can be used for obtaining other bruising
resistant crops.
Browning of potato tubers upon physical damage can be
r~duced when potato plants are transformed by co-cultivation
with A~robacterium tumefaciens, comprising the DNA construct
ac~ording to the i~vention, using internode explants and
following standard protocols. In the transformed plants a
reduced level of PPO activity has been measured in the tubers,
whether a full length or partial sequence has been inserted in
the construct in antisense orientation. The use of the tuber
specific promoters preceding the DNA sequences according to the
invention results in a reduction of PPO expression in the tuber
but does not significontly~effect the expression of PPO genes
in other tissues of the~plant.
In~order to make~DNA constructs according to the invention
which will specifi~al~ly target tuber expression of PPO genes,
a tuber cDNA clone bank was made. Screening this bank with a
probe corresponding to the se~uence of a leaf PPO gene (Shahar
T. ,: Hening N., et al., The Plant Cell, 4, 135-147) or part
thereof, resulted in the isolation of several different tuber
specific PPO genes. At least fiv~ different genes are expressed
in the potato tuber. ~
Further sequence analysis of the different isolated genes
revealed that these genes share a nucleotide homology of at
least 71,7 %. Comparison of the sequences of the tuber
expressed PPO genes and PPO genes expressed in leaves showed
that they are diffe~ent from each other. Homology analysis with
PPO genes expressed in other organisms like, bacterial, fungal
W094/0~07 - 2 1 2 0 2 5 8 PCT/~P93~019~8
-
or animal species revealed that.no significant homology could
be found. Only conserved regions homo}ogous to ~he Cu-binding
sites could be identified. From these observations the
conclusion can be drawn that the PPO genes of potato and in
particular those expresæed in the potato tuber are distinct
from those identified in other species.
For obtaining br~ising resistance the expression of all PPO
genes ~xpressed in the tuber s~ould be substanti~lly reduced.
Depending on the potato cultivar used the expression should be
reduced 5 to 50 fold. We have been able to obtain tubers
according to the invention in which no PPO enzyme activity
could be measured, using detection protocols as described
hereafter, indicating that the expression of the occurring PPO
genes has been inhibited in these transgenic plants. Also
tubers were obtained with a reduced PPO activity with respect
to the activity in the tubers of the non transformed control
plants. These tubers were derived from plants transformed with
antisens~ constructs acrording to the invention. Consequently
the invention provides DNA constructs which causes at least a
reduction of the expression of the entire PPO gene family. This
multi-gene inhibition can be obtained using one antisense
sequence homologous to~any DNA sequence coding for PPQ. We have
demonstrated that at least a reduction of PPO expression can be
achieved regardless of whether tuber or leaf genes were used
for the construction of the antisense PPO constructs. We have
also demonstrated that the antisense sequence does ~ot have to
correspond to the entire length of the coding sequence but also
that partial sequences with a length of about 1200 nucleotides
starting from the 5' end and including 800 nucleotides of the
coding sequence, in antisense orientation, reduces the
expression of the PPO genes in the tuber. Furthermore, the
tubers showing a reduced PPo activity are less susceptibie to
bruising. In order to determine the extent of browning one can
perform standardised bruising tests in which the sensitivity to
~ruising is expressed as a Browning Index. One test is given
for instance in the following examples.
WOg4/03607 . - PCT/EP93/019~
~,~ ?,oZ5~ 8
In a preferred embodiment of the invention the DNA sequence
is derived from tuber~;PPo genes, and preferably from the PPO
clones isolated from the tuber cDNA clone bank which seguences
are li~ted in figure la and lb.
In another preferred embodimeilt of the invention the DNA
sequenc~s in inverted orientation are preceded by a tuber
specific transcription initiation region as has been set forth
above. Particular criteria for the choice of a prefered tuber
specific promoter which directs tuber specific expression may
be summarised as follows: Expression of the antisense gene
should temporally precede or at least coincide with the
expression of the endogenous PPO genes. In a preferred
embodiment of the invention expre~sion of the antisense gene
should be in the s~me tissue location as the endogenous gene.
Furthermore, levels of expression should be at an appropriate
level to obtain the antisense effect.
This invention can:be used to control bruising upon tissue
disruption of tuber, vegetable or fruit tissue in which the
browning is the result of PPO enzyme activity in or around the
damaged plant tissue, due to cell damage. It is understood that
the invention is~not restricted to potatoes but that it can
also be used for the reduction browning upon bruising in other
plants like leaf vegetables, i.e. Iettuce, chicory or-fruits,
.
: where the brownin~ is the result of the expression of genes
encoding PPO. ~
The invention will now be described further in the examples
with reference to the accompanying drawings, in which:
Figure la depicts:the nucleotide sequence of a PPO tuber
gene. pKG45-8. This nucleotide sequence is referred to as
SEQ ID NO:1.
Insertion sites :
~ site A at the level of nucleotide 1
- site B at the level of nucleotide 1213
W~94/03607 PCT/EP93/019~8
21202~8
- site C at the~level of nucl otide 1833.
Figure lb depicts the nucleotide sequence of another P~O
tuber gene. pK~59 4. This nucleotide cequence is referred
to as SEQ ID NO: 2.
Insertion sites :
- site A at the level of nucleotide 1
site E at the ~evel of nucleotide 1173
- site ~ a~ the level of nucleotide 1645A
Figure 2a is a diagra~matic repre~entation of the
co~truction of differe~t recombinant vectors.
Figure 2b is a diagrammatic representation of the
construction of different reco~binant DNA constructs used
for the transformation of potato varieties.
Figure 3 is a ~ar diagram showing the enzyme activities of
all plants transformed with antisense PPO constructs in
comparison to untransformed controls. The va~iants denoted
as 1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, trahsformants comprising GBSS
promoter constructs, untransformed control, respectivily.
Figure 4 is a bar diagram showing the bruising indexes of
selected plants transformed antisense PPO constructs in
comparison to untransformed controls. The varian~s denoted
as ~1, 2 and 3 are: transformants comprising CaMV 35S
promoter constructs, transformants comprising GBSS promoter
constructs, untransformed control, respectivily.
The following examples illustrate the aspects of the
invention, but does not restrict the scope of the
invention.
WOg4/03607 PCT/EP93/01gBX
2,120258 ' 10 " "
Exampl~3
Example 1: I~olation, characterisation and cloning of PPO
clones from a tuber specific cDNA library
In order to make antisense constructs which will
specifically inhibit expression of PPO genes in the tuber; a
cDNA clone bank made from tuber RNA as starting material in the
vector lambda ZAPII (Stratagene Cloning systems, 11099 North
~orrey Pines Rd, La Jolla, CA 92037). The bank, comprising of
5 x 104 individual plaques, was screened with 32P labelled
clone coding for a leaf PPO gene ~Shahar et al. 1992). A total
of 16 positively reacting plaques were taken through three
rounds of plaque-purification and were chose~ for further
analysis
'~
Using PCR primers directed to the termini of the lambda
Z~PII vec r~cloning sites, the inserts of the 16 clones ware
amplified and analyzed for their length and were typed
according ~to:their~restriction sites.~ Six inserts were just
ovex 2 kb in length,~ making them likely candidates for full
length clones, based on work indicating that the PPO protein
has a molecular weight of 4S000-59000:.
Example~2: Se ~ ence analysis and detailed characterisation of
potato tuber PPO cDNA clones
On the basis of the preliminary analysis of the PPO cDNA
clones, two representative cDNA clones, thought to be full-
length, were chosen for sequence analysis using standard
sequencing protocols ~(pKG45-8 and pKG59-4; see Figure 1).
Substrates for s ouencing were produced using the in vivo
excision protocol on lambda ZAPII clones tstratagene). The
remaining clones were partially sequenced in order to obtain
more information about the number of individual genes being
expressed in the tuber. The nucleotide sequence data revealed
W0~4/03607 - PCT/EP93/Olg88
2120-2S8
11
two categories of cDNA clones which, although homologous, fell
into two distinct classes: Class I clones (pKG45-8; Table 1)
and Class II clones (pKG59-4; Table 1). The classification of
the clones given in Table 1 is based on sequence homology.
Interestingly th~ class II clones were not similar to any of
the potato cDNA clones isolated and are therefore deduced to ~e
uniquely expressed in tubers.
: Amongst the 16 clones the most abundant transcript was found
to belong to the same gene family ~B-E) of which 10 independent
cDNA clones were identified. Within this group, the B gene
transcript was found most often (6 independent clones). All
other transcripts (A, D and E ) were present in the cDNA bank
at lower levels (,see Table 1).
;~ Tahle 1
~: ~
Genes ~lass I (size in bp) Class II (siz~ in bp)
: : A pnG45-5 (~B76
pKG45-8 (185Q)~
~: B _ ~ pKG59-4 ~1900)
: pKG45-~ (1850)
, ~
: pKG45-4 ~1600)
_ . ~
. ~ pKG45-7 (1600) .- . ~
~: _ pKG45-9 (1400)
~:~ pKG59-1 (500)
C : pKG45-11 ~1800)
pKG45-3 (1500)
.
D ~ pKG45-10 (1300
E pKG59-2 (800)
- _ _ ,. . _ _
:
Example 3: Construction of a Ti expression-vector family for
constitutive and tuber specific expression.
To achieve tuber specific expression the GBSS-G28 (Rohde et
W094/03607 . PCT/EP93/01988
2~,~02~ "
12
al~ 1990. J. Genet. and Breed. 44: 311-315) promoter was
chosen. Th~ GBSS promoter~used was i~olated from genomic DNA of
the potato variety B~n~e (from seguence data of the genomic
clone G28; Rohde, W. et al. 1990.)
Fragments containing all sequences necessary to direct
tissue specificity were isolated using PCR with standard
protocols. Included in the PCR primers were restriction sites
to facilitate cloning into the Ti-vect~rs.
The GBSS promoter used (from genomic clone G28; Rohde et al.
1990), contained DNA from -1184 to -8. A HindIII site ~5') and
a BamHI site (3') were inserted at the termini by inclusion of
the recognition sites in th~e PCR primers. This fragment was
inserted into the gel purified Ti-vector (pKG1001; described
below) after treatment of both fragment and vector with HindIII
and BamHI.
The CaMV 35S expression vector was constructed from the
vector pBI121 (Jefferson et al. 1987. EMB0 J. 6: 3901-3907~.
The modifications;include replacement of the mutant NPTII gene
in pBI121 and the deletion of the GUS coding region; the
resulting vector ~pKG1001; see Figure 2a) was also the basis
for the other expression vectors described below. The tuber
specific promoter was inserted into pKG1001 resulting in
pKG1001/GBSS containing the GBSS promoter. Figure 2a shows a
diagrammatic representation of the construction of
Ti-expression vectors used to make the antisense constructs~
Example 4: Cloning of antisense tuber PP0 constructs using
the CaMV 35S promoter and tuber specific expression Ti-vectors
Antisense constructs were made, using each of the
full-length PP0 genes (from p~G45-1 and pKG5~-4). Another set
of constructs were made using a shorter region around the
translation initiation site (see Figure 1).
W094/03607 2 ~ 2 0 2 5 8 PCT/EP93/01988
As a general strategy for cloning PPQ genes into Ti-vectors,
sequence specific PCR primers were designed against the
required sites of the PP0 cDNAs ~see sequence, Figure 1, for
precise location of sites). Incorporated into these primers
were recognition sites for restriction enzymes to be used in
the cloning (BamHI and BqlII, 5' and 3' termini respectively).
Tuber PP0 sections from pKG59-4 and pKG45-8 were inserted
into the expression~ vectors described (pKGlOOl and
pKGlOOl/GBSS). In these~experiments both the 5' segment and the
full length sections from the two cDNAs were used (See
sequence, Figure 1, for precise location of sites). Figure 4b
shows a diagrammatic representation of the construction of the
antisense PP0 constructs. The names and description of the 12
resulting antisense PPO clones is shown in Table 2. The table
lists the promoters used and the antisense genes inserted.
Where 5' denotes the use of the fixst approximately 1200
nucIeotides of the corresponding c~NA clone.
:.:
~ :, .
Table 2 ~ ~
conse:~ct Rrn ~ter Gene
pKG60 ~ -CaMV 35S~ -;p~G45-8
KG6l ~ p-CaMV 35S~ - DKG45-8/5'
pKG62 - ~ ~-CaMV 35S ~- pKG5s-4
KG63 ~ P-CaMV 35S - DKG59-4/5'
pKG70 ~ p-GBSS - ~KG45-8
PKG7l~ ~P-GBSS - ~KG45-8/5'
pKG72 p-GBSS - ~KG59-4
pKG73 p-GBSS - pKG59-4/5'
Example 5: Transformation of antisense PP0 constructs into
potato plants
All potato PP0 constructs were introduced into Aqrobac$erium
tumefaciens strain GV3lOl via electroporation and their
integrity was rechecked by restriction enzyme analysis.
W094/0~07 PCT/EPg3/01988
Subsequently, the descri~ed constructs were introduced into two
lines of potato (van Gogh and Diamant) by co-cultivation using
internode explants. Standard protocols were used in these
procedures. Circa 50 regenerants per construct and cultivar
were grown on selective media and analyzed further.
Example 6: Analysis of PPO enzyme activities in transformed
tubers
From a total of 1500 regenerants 882 were propagated and
used to produce in vitro micro-tubers. From about 5 g of
micro-tubers of each line a crude protein extract was produced
with which PPO enzyme assays were performed (after the method
of Flurkey, W. 1986 Plant Physiol. 8~: 614-618). Two assays
were done on ea?~h transgenic line using 50 ~ catechol as a
substratc for the assay in an ex~ract volume of 1 ml. Enzyme
activity is expressed as the rate of change of OD at 520 nm
over 1 min at 25C (slope~. Two independent repeat measurements
were performed on each line and thè mean of the results were
taken for a~alysis. On average 74% and 72% of antisense
transformants in Diamant and van Gogh, respectively, gave lower
PPO enzyme activity than the controls. 32 lines, harb~uring the
antisense PPO constructs, were found to ha~e no detectable PPO
activity with this substrate. In Diamant: 7 lines with the 35S
CaMV-promoter constructs and 10 lines with the GB5S- promoter
constructs showed no PPO activity. In van Gogh: 10 lines with
the 35S ca?Mv- promoter constructs and 5 lines with the
GBSS-promoter constructs had undetectable PPO activity levels.
No statistically significant differences were found between
the different antisense PPo genes (Class I and II~ or with
respect to the size of the PPo gene-sections used in the
constructs.
As shown in Figure 3, both potato varieties used showed
reduced means in enzyme activity in all cases where either the
35S CaMV or GBSS promoters were used. The highest average
W094~03607 . . ~ 2 ~ z 5 ~ PCT/EP93/01988
suppression of PPO activity was found in lines which were
transformed with constructs under CaMV 35S promoter control.
Example 7: Molecular analysis of transformants
200 of the low PPO activity potato lines were further
analyzed on a molecular level. Using the Southern hybridization
technique it was shown that aI1 contained T-DNA inserts of the
expected sizesO Insertion copy-number ranged from 1 to l0 where
50~ had copy numbers < 3. Also PPO mRNA levels were shown to be
reduced when compared to untransformed controls. Assays fsr PPO
~RNA were performed using a PCR protocol.
Example 8: Analysis of ~ruising resistance in the
transformants
On the basis of the data from the enzyme activity, 50 lines
from each variety, including all variants of antisense-PPO
constructions r were selectQd for field trails. The selected
lines represent plants with the lowest PPO enzyme activities.
In conventional breeding practice, standardised tests are
carried out to determine the~ex~ent of browning in tubers from
breeding lines. An index is calculated for bruising sensitivity
which takes into account the level of tuber browning after
subjecting the tubers o standardised mechanical damage and the
specific gravity of the tubers in the test~ The resultinq
Browning Index tBI) ranges from 0 to 50.
BI's of field grown tubers from the selected lines are shown
in Figure 4. Statistical tests show significant differences in
: the CaMV 35s promoter series and the GBSS promoter series for
both varieties when compared to the mean of the controls.
The data from these trials clearly demonstrates a
significant improvement of the bruising phenotype under field
WOg4/03607 PCT/EP93/01988
~o ~5~ 16
conditions as ~ direct result of the expression of the
constructs provided by the invention.
Mat~ri~l a~ M~tho~
Potato plants (Solanum Tuborosum cv. van Gogh and Diamant)
were grown ln ~ on MS medium (Murishige, T. and Skovg, F.
1962. Physiol. Plant 15: 473 - 497) suppleme~ted with 30g/1
sucrose.
Potato internode explants were transformed with
Aqrobacterium tumefaciens (~strain GV3101; van Larebeke, N. et
al. 1974. Nature 2S2: 169 - 170) containing the antisense-PPO
Ti-plasmid constructs using the co-cultivation method
essentially according to protocols described (Ooms, G. 1989. et
al. Theor. Appl.:Genet 73: 744 -750).
Molecular biological methods, including Southern, Northern
and Western analysis, PCR and other DNA manipulations were as
des*ribed by Maniatis ~:(~aniatis, et al. 1982. Molecular
Cloning: A Laboratory Manual. Cold Spri~g Harbor Laboratory,
Cold Spring Harbor NY.).~:
Substrates for sequencing were produced using the in vivo
excision protocol on lambda ZAPII clones (Stratagene3 isolated
from a sink tuber cDNA.
The bruising test and the determination of the Browning
Xndex were conducted as follows: Potatoes harvested from each
line, grown in separate plots, were subjected to bruising under
standard conditions. 2 - 3 kg of potatoes were placed in a
shaking device comprising of a wooden box with padded walls.
The box, is mechanically ag_~ated for 2 mins. After the
bruising procedure, tubers are stored for 4 days at 10C ~+
1C). Subsequently the potatoes are mecl~anically peeled until
80% of the skin is removed and the degree of browning is scored
W094/03607 . 21 2 0 2 ~ 8 PCT/EP93/01~88
17
in terms of percent of the surface area effected by
discolouration. The percentages are categorised into four
classes and the number of tubers in ea~h class, together with
the specific gravity of the tubers is entered into the
following formula from which the BI is determined:
SG x (CI+l) x (CII+2) x (CIII+3) x (CIV+4)
6 x ~CI+CII+CIII+CIV)
Where SG is the specific gravity of the whole sample and CI
- CIV are the number of tubers categorised in a given class.
" ~
;~ .
~ ~ .
:~ ' '
`'.
