Note: Descriptions are shown in the official language in which they were submitted.
2 .f~ 1~ 3 ~
SELECTION-GENE-FREE TRANSGENIC PLANTS
Technical Field
The invention is directed to materials and
methods which produce transgenic plants containing only
desired foreign genes and which are free of unwanted or
irrelevant selection genes. Advantage is ta~ken of
recombinase systems which permit the exci~ion and
segregation of selection gene DNA from introduced genetic
material.
Background and Related Art
Efforts to obtain transgenic plants with
improved properties have been under way for some time.
Transgenic plants have been obtained which are insect
resistant, for example, due to the presence of a gene
from Bacillus thurinqiensis that confers such insect
resistance. The nutritional value of various plants has
also been improved by insertion of genes encoding
proteins rich in desired amino acids. Introduction of
genes encoding viral resistance mechanisms, herbicide
resistance mechanisms, improved growth characteristics
and the like are all desirable outcomes that can be
` achieved within the parameters of conventional technology
in at least illustrative cases.
Included within the parameters of current
technology is, however, the necessity of transferring,
along with the desired DNA, DNA encoding a selection gene
and means for its expression to permit the efficient
~; recovery of successful transformants. Without these
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selection genes, the succe~sful transformants would be
lost in ~he greatly dominant nontransformed population of
plants or plant cells. The presence of the DNA encoding
the selection genes and the products of these genes is
not necessarily harmful, but does increase the
environmental uncertainty of the distribution of
transgenic plants. The problems introduced by the
uncertainty of the ultimate effects o~ the inclusion and-
propagation of selection genes in plants has a highly
negative effect on the progress of plant improvement. At
- a minimum, reservations expressed at various levels of
intensity and clamor have political e~fects which retard
the progress of obtaining improved plant specles.
The present invention removes this source of
environmental impact uncertainty by providing a method
for excision and segregation of the selection gene which,
after the initial transformation, has outlived its
usefulness. The invention takes advantage of recombinase
systems which are capable of excising marked D~A
sequences.
One efficient system for excision of unwanted
DNA sequences that has been widely employed generally is
the Cre/lox system. This system, illustrative of the
general process, compri~es "lox~ marker sequences that,
when included in a DNA sequence per se, mark the DNA
which they bracket for excision or i~version (depending
on the orientation o~ the lox sequences) by a corres-
ponding "Cre" recombinase enzyme. The operability of
`~ this system in various host cells, including tobacco
cells, has been shown. For example, Sauer, Mol Cell Biol
(1987) 7:2087-2096, showed the operability of the Cre/lox
system in the yeast Saccharomyces cerevisiae. The
operability of the system in mammalian cells wa~ also
shown by Sauer, B., et al., in Proc Natl Acad Sci USA
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(1988) 85:5166-5170; Sauer, B., et al., Nucleic Acids Res
(1989) 17:147 161. The operability of the Cre/lox system
in tobacco cells was demonstrated by Dale, E., et al., in
an abstract published in J Cell Biochem, Supplement 14E,
5 "Abstracts of the l9th Annual UCLA Symposium for
Molecular Strategies for Crop Improvement," page 1978 (16
April 1990), and in an article by Dale, E., et al., Gene
(1990) 91:79-85.
The use of the Cre/lox system in yeast to
v 10 regulate expression was also described by Sauer in
European application 220,009, published 2g April 1987.
Expression of a foreign gene during growth phase was
prevented by strategies that precluded expre99ion in the
absence of the cre enzyme. The gene encoding the
15 recombinase enzyme cre was placed under the control of an
inducible promoter. The expres~ion system for the
desired gene was constructed either to be blocked by a
DNA sequence marked by the lox sequences for excision, to
be repressed by a repressor protein whose gene is marked
20 by the lox sequences for exci~ion, or to ~atisfy the
condition in which the desired gene is designed to be
contained in an inverted position in it~ expression
~ystem and bracketed by lox whereby the cre enzyme
produced when induction take~ place inverts the gene to
25 the correct orientation. In thig last strategy, the lox
sequences bracket the desired sequence in opposite
orientations.
Odell, J., et al., in Mol Gen Genet (1990)
223:369-378, describe the use of the Cre/lox system in
30 higher plant3 to activate the expression of the kanamycin
re~istance gene into the chromosome. The authors further
suggest the use of the sys~em to regulate expression in
higher plants in a manner similar to that described in
the above-referenced EPO application for yeast.
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While the Cre/lox system has been demonstrated
to be particularly useful, other such recombinase systems
exist and are derived from a variety of sources. For
example, O'Gorman, S., et al., in Science (1991)
251:1351-1355, describe the FLP recombinase system from
S. cerevisiae as operable in mammalian cells; Golic,
K.G., et al., Cell (1989) 59:499-509, report the
operability of this system in Drosophila. Backman, K.C.;
U.S. Patent 4,673,640, describes a marker/recombinase
system from bacteriophage lambda, and Matsuzaki, H., et
al., J Bacteriol (1990) 172:610-618 describe an analogous
system from the yeast C. rouxii.
Thus, it appears that recombinase/marker
systems are available from a variety of sources and are
functional in a number of hosts. The pre~ent invention
specifically takes advantage of such recombinase/marker
systems to excise and segregate selection genes in higher~
plants.
` 20 Disclosure of the Invention
The invention includes a means to control the
nature of transgenic plants so a~ to provide plants which
contain only the desired transgenic material and lack the
selection genes useful in conducting the transformations
necessary for their preparation. The re3ulting plants
thus have more predictable environmental effects than
~" those previously available in the art. The invention
methods take advantage of recombinage/marker systems for
excision and segregation of selection marker genes.
Thus, in one aspect, the invention is directed
: to vectors suitable for transformation of higher plants
which comprise selection genes marked by DNA marker
sequences which form a part of a recombina~e/marker
system. The selection gene i~ further linked to control
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sequences which are capable of effecting its expression
so that it can be used in the selection of transformed
plants prior to excision. The invention also includes
these vectors that further contain an expression system
for a desired gene. The invention i9 also directed to
plant cells or cultures in regenerated plants transformed
with the vectors of the invention and to plant cells and
plants which are further transformed with expression
vectors that include the gene for the recombinase enzyme
associated with the recombinase marker system. The
invention is also directed to methods to produce such
transformants and to segregate the selection gene from
the desired gene in progeny.
Brief Description of the Drawinqs
Figure 1 is a schematic 3howing the
construction of pED37 and pED53. As indicated in Figure
l, the pED37 oriented as shown will be inserted into the
SalI site of pED53 in the recombined vector pE~53::pED37.
Figure 2A show~ the po~itioning of the insert
from pED53::pED37 into the host genome and the expected
sizes of PCR products obtained from primers as described
in the examples hereinbelow.
Figure 2B shows the expected orientation of
this insert in the gene after excision of the hpt
selection gene. The expected PCR product size is also
shown.
Figure 3 shows the sequence of the amplified
; portions indicated in Figures 2A and 2B.
Modes of Carryinq Out the Invention
The invention is directed to method~ which
result in transgenic plants that are capable of
expre3sing a deslred gene but that are free of genes
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associated with the transformation process as ~selectable
markers." The invention methods provide, therefore,
plants having conferred characteristics whlch are llmited
to those desired to be achieved by the transformation and
not associated with unrelated, and perhaps unwanted
selection characteristics.
- In general, these plants are obtained by elther
of two related methods. Both methods require that the
selection gene associated with the initial transformation
with the desired gene be operably linked to marker DNA
sequences that mark the gene for excision mediated by a
corresponding recombinase enzyme. In both methods,
therefore, plants or plant cells are initially
transformed with a recombinant vector which contains a
selection characteristic gene operably linked to marker
DNA sequences that mark the gene for excision, the marked
gene being contained in an expression system 90 that
unless excised, the gene is expressed. The vector
further includes an expression sy3tem which comprises the
desired gene operably linked to appropriate control
sequences. Of course, the expression systems must be
operable in higher plant cells. ~nitial transformants
are selected by virtue of the presence of the selection
gene contained in the recombinant vector and can be
verified to express, as well, the gene for the desired
characteristic.
In one approach, initially tran~formed plant
cells or plant~ can be further transformed in a second
round of transformation with a second recombinant vector
which contains the recombinase gene that corresponds to
the DNA sequences that mark the selection gene for
excision. In order to verify the second tran~formation,
of course, a second selection gene needs to be included
so that the second round of transformation can be
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verified. The successful second transformant~, for the
most part, will be free of the first selection mar~er due
to the operation of the recombinase product of the
recombinase gene. Among those transformants free of the
first selection marker, progeny are screened for freedom
from the second selection marker as well. As the second
selection marker is not linked to the desired gene
construct, it is segregated from the desired gene in the-
progeny.
Typically, this first approach is conducted by
transformation of plant cells, and regenerating the
screened progeny into intact plants. Alternatively, the
second transformants can first ~e regenerated into plants
and self-pollinated to produce the segregated progeny.
In a second approach, regenerated plants from
the first and second transformations (which are conducted
independently) are cros~-pollinated to effect the
excision of the first selection marker. The progeny of
the product~ of this cross-pollination that show
expression of the desired gene can then be screened for
~ the absence of the second selection gene. As the desired
i gene and the second selection gene are not linked,
; segregation occurs in these second generation progeny.
As used herein, a ~selection characteristic
gene" or "selection gene" refers to a gene which produces
a product that confers on a host containing it a
;` selectable property such a~ herbicide or antibiotic
resistance. Suitable selection genes for method~ of the
invention include genes encoding hygromycin resi~tance
(the hpt gene) or kanamycin resistance (nptII gene) and
any other gene which confers such characteristics on
higher plant cells.
As used herein, a "desired gene" i9 a gene that
may encode a protein or an RNA, the presence of which is
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desired in the finished plant. Such genes may include
~ those encoding 3eed storage protein3, those encoding
insect resistance, or any other gene which i5 thought to
confer desired characteristics on the plant containing
it.
As used herein, "expression system" has its
usual meaning--i.e., DNA which contains the coding
sequence of a gene operably linked to control systems
which effect the expression of the gene in the higher
plant cellq. Typically, such control 3equences include a
promoter, and optionally, additional sequences which aid
in expression such as polyadenylation sites, or other
appropriate control sequences.
"Marker DNA sequences" refer to sequences that,
when present in proper orientation with respect to
included DNA, mark the gene for excision or inversion
when in the presence of an appropriate corresponding
enzyme. "Corresponding recombinase" refers to the enzyme
which is capable of recognizing the marker DNA sequences
and excising or inverting the included DNA. Illustrated
herein is the system that includes the lox DNA sequence9
corresponding to the cre recombina~e enzyme, but a
multiplicity of such systems are known. These are
reviewed, for example, by Craig, Annual Review of
Genetics (1988) 22:77-105. Any such system operable in
higher plants may be used.
In constructing the vectors containing
expression systems useful in the in~ention, control
regions which are functional either constitutively are
employed. Transcription initiation regions, for example,
include the various opine initiation regions, such as
octopine, mannopine, nopaline and the like. Plant viral
promoters can also be used, such as the cauliflower
moqaic viru~ 35S promoter.
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A large number of ~uitable control systems are
available. For example, the cauliflower mosaic virus
(~aMV) 35S promoter has been shown to be highly active in
many plant organs and during many stages of development
when integrated into the genome of transgenic plants
including tobacco and petunia, and has been shown to
confer expression in protoplasts of both dicots and
monocots.
Organ-specific promoters are also well known,
but may be less convenient. For example, the E~ promoter
is only transcriptionally activated during tomato fruit
ripening, and can be used to target gene expression in
ripening tomato fruit (Deikman and Fischer, EMBO ~ (1988)
7:3315; Giovannoni et al., The Plant Cell (1989) 1:53)-
The activity of the E8 promoter i5 not limited to tomatofruit, but is thought to be compatible with any system
wherein ethylene activates biological processes.
Either a constitutive promoter (such as the
CaM~ or Nos promoter illustrated above) or a de~ired
organ-specific promoter (such as the ~8 promoter from
tomato) i9 then ligated to the gene to be expressed using
standard techniques now common in the art. The
expression system may be further optimized by employing
supplemental elements such as transcription terminators
and/or enhancer elements.
Thus, for expression in plants, the recombinant
expression cassette will contain in addition to the
coding sequence, a plant promoter region, a transcription
initiation site (if the coding sequence to be transcribed
lack~ one), and a transcription termination sequence.
Unique restriction enzyme site~ at the 5~ and 3' end~ of
the cassette may also be included to allow for easy
insertion into a pre-existing vector.
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Sequences controlling eucaryotic gene
expression have been extensively studied. Promoter
sequence elements include the TATA box consensus sequence
(TATAAT), which is usually 20 to 30 base pairs (bp)
upstream of the transcription start site. In most
instances the TATA box is required for accurate
transcription initiation. By convention, the start site
is called +1. Sequences extending in the 5~ (upstream)
direction are given negative numbers and sequences
extending in the 3' (downstream) direction are given
positive numbers.
In plants, further upstream from the TATA box,
at positions -80 to -100, there i9 typically a promoter
element with a series of adenines surrounding the
`~ 15 trinucleotide G(or T)NG (Messing, J. et al., in Genetic
Enqineering in Plants, Kosage, Meredith and Hollaender,
eds. (1983) pp. 221-227). Other sequences conferring
tissue specificity, response to environmental signals, or
maximum efficiency of transcription may also be found in
the promoter region. Such sequences are often found
within 400 bp of the transcription initiation site, but
may extend as far as 2000 bp or more.
In the construction of heterologous
promoter/structural gene combinations, the promoter is
preferably positioned about the same distance ~rom the
heterologous transcription start site as it is from the
transcription start site in its natural setting. As is
known in the art, however, some variation in this
distance can be accommodated without los~ of promoter
; 30 function.
As stated above, any of a number of promoters
which direct transcription in plant cells is ~uitable.
The promoter can be either constitutive or inducible.
Promoters of bacterial origin include the octopine
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synthase promoter, the nopaline synthase promoter and
- other promoters derived from native Ti plasmids
(Herrera-Estrella et al., Nature (1983) 303:209-213).
Viral promoters include the 35S and l9S RNA promoters of
cauliflower mosaic virus (O'Dell et al., Nature (1985)
313:810-812). Plant promoters include the
ribulose-1,3-disphosphate carboxylase small subunit
promoter and the phaseolin promoter.
In addition to a promoter sequence, the expres-
sion cassette should also contain a transcription
termination region downstream of the ~tructural gene to
; provide for efficient termination. The termination
region may be obtained from the same gene as the promoter
`. sequence or may be obtained from different genes.
If the mRNA encoded by the structural gene is
to be efficiently processed, DNA sequences which direct
polyadenylation of the RNA are also commonly added to the
vector construct (Alber and Kawasaki, Mol and Appl Genet,
(1982) 1:419 434). Polyadenylation i~ of importance for
expression of the transcription product RNA in plant
cells. Polyadenylation sequences include, but are not
limited to the Agrobacterium octopine synthase signal
; (Gielen et al., EMBO J, (1984) 3:835-846) or the nopaline
- synthase signal (Depicker et al., Mol and Appl Genet
~5 (1982) 1:561-573).
The resulting expression system or cassette is
ligated into or otherwise constructed to be included in a
recombinant vector which is appropriate for higher plant
; transformation.
~` 30 As is particularly relevant herein, the vector
`~ will also contain an expression system for a selection
gene by which transformed plant cells can be identified
in culture. Usually, the selection gene will encode
antibiotic resistance, e.g., resi tance to G418,
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hygromycin, bleomycin, kanamycin, and gentamicin. After
transforming the plant cells, those cells having the
vector will be identified by their ability to grow on a
medium containing the particular antibiotic. Replication
sequences, of bacterial or viral origin, are generally
- also included to allow the vector to be cloned in a
bacterial or phage host, preferably a broad hos~ range
procaryotic origin of replication is included. A
selection gene suitable for bacteria should also be
included to allow selection of bacterial cells bearing
the desired construct. Suitable procaryotic selection
genes also include resistance to antibiotics such as
kanamycin or tetracycline.
Other DNA sequences encoding additional func-
tions may also be pre~ent in the vector, as is known inthe art. For instance, in the case of Agrobacterium
transformations, T-DNA sequences will also be included
for subsequent transfer to plant chromo~omes.
In addition, vectors can also be constructed
that contain in-frame ligations between the coding
- sequence of the desired gene and ~equences encoding other
molecules of interest resulting in fusion proteins, by
techniques well known in the art.
When an appropriate vector i9 obtained,
transgenic plants are prepared which contain the desired
expression system. A number of techniques are available
~` for transformation of plants or plant cell3. All types
of plants are appropriate subjects for "direct"
transformation; in general, only dicots can be
transformed using Agrobacterium-mediated infection.
In one form of direct transformation, the
- vector is microinjected directly into plant cells by use
of micropipettes to mechanically transfer the recombinant
DNA (Crossway, Mol Gen Genetics (1985) 202:179-185). In
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another form, the genetic material i9 transferred into
the plant cell using polyethylene glycol (Kren~, et al.,
Nature (19~2) 296:72-74), or high velocity ballistic
penetration by small particles with the nucleic acid
either within the matrix of small beads or particles, or
; on the surface, is used (Klein, et al., Nature (1987)
327:70-73). In still another method protoplasts are
fused with other entities which contain the DNA whose
introduction is desired. These entities are minicells,
cells, lysosomes or other fusible lipid-~urfaced bodies
(Fraley, et al., Proc Natl Acad Sci USA (1982)
79:1859-1863).
` DNA may also be introduced into the plant cell~
by electroporation (Fromm et al., Proc Natl Acad Sci USA
(1985) 82:5824). In this technique, plant protoplasts
are electroporated in the presence of plagmidg containing
the expression cassette. Electrical impulses of high
field strength reversibly permeabilize biomembranes
allowing the introduction of the plasmids.
~` 20 Electroporated plant protoplasts reform the cell wall,
divide,`and regenerate.
For transformation mediated by bacterial infec-
tion, a plant cell is infected with Aqrobacterium
~ tumefaciens or A. rhizogenes previously transformed with
i~ 25 the DNA to be introduced. Agrobacterium is a
representative genu~ of the gram-negative family
b Rhizobiaceae. Its species are responsible for crown gall
.~ (A. tumefaciens) and hairy root disease (A. rhizogene~).
The plant cells in crown gall tumors and hairy roots are
30 indùced to produce amino acid de~ivatives known as
opines, which are catabolized only by the bacteria. The
bacterial genes re~ponsible for expression of opines are
a convenient source of control element~ for chimeric
expression cassettes. In addition, assaying for the
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presence of opines can be used to identify transformed
tissue.
Heterologous genetic sequences can be
introduced into appropriate plant cellg, by means of the
Ti plasmid of A. tumefaciens or the Ri plasmid of A.
rhizoqenes. The Ti or Ri plasmid i9 transmitted to plant
cells on infection by Agrobacterium and is stably
integrated into the plant genome (Schell, J., Science
(1987) 237:1176-1183). Ti and Ri plasmids contain two
regions essential for the production of transformed
cells. One of these, named transferred DNA (T-DNA), is
transferred to plant nuclei and induces tumor or root
formation. The other, termed the virulence (vlr) region,
is es~ential for the trangfer of the T-DNA but is not
itself transferred. The T-DNA will be transferred into a
plant cell even if the vlr region i~ on a different
plasmid (Hoekema, et al., Nature (1983) 303:179-189).
The transferred DNA region can be increased in size by
`~the insertion of heterologous DN~ without its ability to
i20 be transferred being affected. Thus a modified Ti or Ri
plasmid, in which the disease-causing genes have been
`~deleted, can be used as a vector for the transfer of the
``~`gene constructs of this invention into an appropriate
plant cell.
Construction of recombinant Ti and Ri plasmids
in general follows methods typically used with the more
common bacterial vectors, such as pBR322. Additional use
" can be made of accessory genetic elements sometimes found
with the native plasmids and ~ometimes constructed from
foreign sequences. These may include but are not limited
; to "shuttle vectors,~ uvkum and Ausubel, Nature (1981)
298:85-88), promoters (Lawton et al., Plant Mol Biol
(1987) 9:31S-324) and structural genes for antibiotic
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re3istance a3 a selection factor (Fraley et al., Proc
- Natl Acad Sci (1983) 80:4803-4807).
There are two clas~es of recombinant Ti and Ri
plasmid vector systems now in use. In one class, called
"cointegrate," the shuttle vector containing the gene of
interest is inserted by genetic recombination into a non-
oncogenic Ti plasmid that contains both the cis-acting
and trans-acting elements required for plant
transformation as, for example, in the pMLJ1 shuttle
10 vector o~ DeBlock et al., EMBO J (1984) 3:1681-1689 and
the non-oncogenic Ti plasmid pGV3~50 de~cribed by
Zambryski et al., EMBO J (1983) 2:2143-2150. In the
second class or "binary~ system, the gene of interest is
inserted into a shuttle vector containing the cis-acting
elements required for plant transformation. The other
necessary functions are provided in trans by the non-
- oncogenic Ti plasmid as exemplified by the pBIN19 shuttle
vector described by ~evan, Nucleic Acids Research (1984)
` 12:8711-a721 and the non-oncogenic Ti plasmid PAL4404
20 described by Hoekema, et al., Nature (1983) 303:179-180.
Some of these vectors are commercially available.
There are ~wo common ways to transform plant
cells with Agrobacterium: co-cultivation of
Agrobacterium with cultured isolated protoplasts, or
~ 25 ~ransformation of intact cells or tissues with
`i Agrobacterium. The first re~uires an established culture
system that allows for culturing protoplasts and
subsequent plant regeneration from cultured protoplasts.
The second method requires (a) that the intact plant
tissues, such as cotyledons, can be transformed by
Agrobacterium and (b) that the transformed cells or ~i9-
sues can be induced to regenerate into whole plants.
`' Most dicot species can be transformed by
Aqrobacterium as all species which are a natural plant
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host for Agrobacterium are transformable ln vitro.
Monocotyledonous plants, and in particular, cereals, are
not natural hosts to Aqrobacterium. Attempts to
tran3form them using Aqrobacterium have been unsuccessful
until recently (Hooykas-Van Slogteren et al., Nature
(1~84) 311:763-764). However, there i~ growing evidence
now that certain monocots can be transformed by
A~robacterium. Using novel experimental approaches
cereal species such as rye (de la Pena et al., Nature
(1987) 325:274-276), maize (Rhodes et al., Science (1988)
240:204-207), and rice (Shimamoto et al., Nature (19~9)
338:274-276) may now be transformed.
Identification of transformed cells or plants
is generally accomplished by including a selectable
mar~er in the transforming vector, or by obtaining
evidence of successful bacterial infection.
Plant cells which have been transformed can
-`` also be regenerated using known techniques.
~ Plant regeneration from culturèd protoplasts is
20 described in Evans et al., Handbook of Plant Cell
Cultures, Vol. 1: (MacMillan Publishing Co. New York,
1983); and Vasil I.R. (ed.), Cell_Culture and _omatic
Cell Genetics of Plants, Acad. Press, Orlando, Vol. I,
- 1984, and Vol. II, 1986). It is known that practically
25 all plants can be regenerated from cultured cells or
tissue~, including but not limited to, all major species
of sugarcane, sugar beet, cotton, fruit trees, and
legumes.
~ Means for regeneration vary from species to
} 30 species of plants, but generally a suspension of
transformed protoplasts or a petri plate containing
~- transformed explants is first provided. Callus tissue is
formed and shoots may be induced from callus and
subsequently rooted. Alternatively, somatic embryo
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formation can be induced in the callus tissue. These
somatic embryo~ germinate a~ natural embryos to form
plants. The culture media will generally contain various
amino acids and plant hormone~, such as auxin and
cytokinins. It is also advan~ageous to add glutamic acid
and proline to the medium, especially for such species as
corn and alfalfa. Efficient regeneration will depend on
the medium, on the genotype, and on the history of the
culture. If these three variables are controlled, then
regeneration is usually reproducible and repeatable.
After the expression cassette is stably incorporated into
regenerated transgenic plants, it can be transferred to
~` other plants by sexual crossing. Any of a number of
standard breeding techniques can be used, depending upon
the species to be crossed.
~` The following examples are intended to
` illustrate but not to limit the invention.
Example 1
Construction of Plasmids
i Plasmid pED23 which contains the expres~ion
system for the cre gene was constructed as described by
Dale, E.D. and Ow, D.W., Gene (1990) 91:79-85. This
plasmid uses a pUC19 host vector (Yanich-Perron, C. et
25 al. Gene (1985) 33:103-119) and utilizes a 35S promoter
to transcribe a Cre-nos3' fusion.
The plasmid pED53 is constructed from the
Agrobacterium gene transfer vector p~INl9, the
construction of which was described by Bevan, M., Nucleic
- 30 Acids Res (1984) 12:8711 8721. To convert pBINl9 to
pED53, a 1.3 ~b PstI fragment was dele~ed. The deletion
removes part of the T-DNA including the coding region of
the kanamycin resistance gene (nptII) along with HindIII
and SphI sites.
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Plasmid pED37 was constructed from pED26, which
contains the luciferase expression system and has been
described by Dale, E.C. and Ow, D.W. (supra) by inserting
a 2.0 kb HindIII fragment containing the 35S-hpt-nos3'
chimeric gene. The hpt gene i9 described by Kaster, K.R.
et al. Nucleic Acids Res (1983) 11:6895 6911.
The co-integrate plasmid pED53::pED37 was
formed by linearizing pED37 at an XhoI site adjacent to
one of the lox 3ites and inserting it into the SalI site
10 of pED53. As shown in Figure 1, pED37 contains the 35S
promoter operably linked to the luciferase gene which is
~` in turn terminated by the nos3' sequence. Bracketed by
the lox sites are ampicillin resistance, and the hpt
expression system. The pED37 is co-integrated into pED53
between the right and left border (RB and LB) regions in
the orientation shown.
The co-integrate plasmid which supplie~ the cre
gene, pBINl9::pED23 was obtained by ligating the HindIII
linearized pED23 into the HindIII site of pBIN19 so that
the transcription of the cre gene is directed toward the
Agrobacterium LB.
The co-integrate plasmids were selected by
ability to confer resistance to both kanamycin and
ampicillin in bacteria. This selection i9 facilitated by
transformation into the E. coli polymerase I-deficient
host JZ294 (argH, strA, polA::TnlO) which permits
replication of the wide host range replicons of pED53 and
pBIN19 but not the ColE1 replicons of pED37 and pED23.
Example 2
Production of Selectable Marker-Free Plants
The co-integrate plasmid~ pED53::pED37 and
pBINl9::pED23 were mobilized into Agrobacterium
tumefacien~ strain GV3111(pTiB6S3SE) for infection of
.
~ ~ r~;~p~
- 19 -
Nicotiana tabacum (Wisconsin-38 cultivar) leaf explants
as described by Horsch, R.~. et al. Science (1985)
:~ 227:229-231. HygR and KanR plants were scored for the
ability of leaf explants to form shoots on shoot-
inducing MS media containing antibiotics (20 ~g/ml
hygromycin sulfate or 100 ~g/ml kanamycin sulfate). Luc+
plants were assayed for luciferase activity as described
by Ow, D.W. et al., Science (1986) 34:356-859.
Initially, hygromycin-resistant plants were
obtained from transformation with pED53::pED37. These
are designated "ntED5337" plan~s. These plants contain
the lucifera3e expression sygtem and the hpt expression
system incorporated into the genome in the configuration
.. ~ shown in Figure 2A. This wa~ verified by genetic
analysis as described below. The ntED53~7 plants express
the luciferase gene, and are hygromycin-resistant as
required by the selection protocol.
The ntED5337 plants were transfected, using the
protocol described above, with pED23. Successful
: 20 transformants will be selectable for kanamycin resistance
and are capable of production of the cre enzyme. The
``' resulting transformants were thus selected by kanamycin
resistance and the kanamycin-resistant plants were then
tested for hygromycin resistance as described above.
Most of them, designated ntED5337-23, were hygromycin-
sensitive. This showed that the introduction of the cre
enzyme catalyzed recombination o~ the lox sites flanking
the hygromycin resistance gene. The deleted DNA, no
longer linked to the replicating host chromosome, is lost
in the progeny cells deriving from the primary cell where
the excision event occurred. The ntEDs337-23 plant cells
or the regenerated plants express the gene encoding
luciferase, however.
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In addition to the protocol described above,
plants regenerated from independent transformations by
pED23::pED37 and pBINl9::pED23, respectively, were cross-
pollinated to obtain progeny. Of 316 progeny examined,
78 produced luciferase and were kanamycin-resistant.
`; Among these, 42 were hygromycin-sensitive. These 42
plants are substantially equivalent to those regenerated
from the cellular progeny ntED5337-~3.
Example 3
Genetic ~nalysis
To verify the genetic status of the
transformants, DNA was prepared by grinding leaf tissues
~ in liquid N2~ extracting with 100 mM Tris/1% SDS/50 mM
`~ 15 EDTA/500 mM NaCl/10 mM ~-mercaptoethanol at 65C, 10
min., followed by adding potassium acetate to 1.3 M,
chilling to 0C and removing the debris by
centrifugation. The DNA was then precipitated with
.; isopropanol, wa~hed with 70~ ethanol and re~uspended in
10 mM Tris/1.0 mM EDTA. Polymerase chain reactions were
carried out under standard conditions as described by
Saiki, R.K. et al., Science (1988) 239:487-491 with
denaturation, annealing, and extension at 94C, 55C and
72C, respectively for 1 min. each during 30 cycles.
Reaction products were resolved using a 1.5% agarose gel.
The sequence of the PCR primers in Figures 2A and 2B are:
A, 5'-GAGCTCGGTACCCGGGGATC-3'; }3, 5'-
GAGTGCACCATATGCGGTGT-3~; C, 5'-GACGCCCCAGCACTCGTCCG-3';
D, 5'-GGTACCCGGGATCCTCTAG-3'; E, 5'-GTTCATTTCATTTGGAGA~G-
3'; F, 5'-CAGTGATACACATGGGGATC-3'. The two primers for
detection of the cre gene (5'-ATGTCCAATTTACTGACCGT-3' and
5'-CTAATCGCCATCTTCCAGCA-3~) represent the N and C-
terminal cre coding sequence and the expected PCR product
size is 1.O kb. To determine the sequence of the lox
.. . . .
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-21-
sites, fragments from PCR reactions were purified,
digested with appropriate restriction enzymes (~+B;
~: BamHI/NdeI, C+D; PstI/BamHI, A+D; ClaI/BamHI) and ligated
- into either pUC19 (for A+B and C+D) or pBR322 (for A+D).
The nucleotide sequences of the regions surrounding the
lox site~ derived form PCR reactions from plants nt~D5337
and ntED5337-23 were determined by the dideoxy method as
modified by U.S. Biochemical (Sequenase kit).
~ Five of the hygromycin-sensitive, kanamycin-
e~ 10 resistant and luciferase-producing plants derived from
the secondary transformation described in Example 2 were
analyzed to confirm site-specific recombination at the
lox sites. Both parental ntED5337 and derivative
ntED5337-23 plants were examined. The predicted
resultants are shown in Figure 2A and 2B. As confirmed
by gel electrophoresis, a fragment of the predicted size
of 1.1 kb for primers A~3 and 0.71 kb for primers C~D was
obtained from the parent genome, but not from ntED5337-
23. However, primer A+C produced a 0.87 kb band from
20 ntED5337-23, but not from the parent. This band
corresponds to the fragment expected from the joining of
the chimeric luciferase gene with the sequence adjacent
to the LB. Primers E+F were used to assay for the
possibility that the hygromycin resistance gene might
have translocated elsewhere in the genome; a fragment
corresponding to the expected 0.56 kb band was found in
the parent, but not in the descendent ntED5337-23. Thus,
the excised hygromycin gene is not present in the genome
at all. The excision event also removes the silent
ampicillin resistance marker from the plant genome.
Of five plants examined, the PCR profiles
showed no indication of harboring both excised and intact
copies as shown in Figure 2A and 2B suggesting that the
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-22-
excision must have occurred early between introduction o~
the cre-expressing construct and plant organogenesi~.
For seguencing plant ntEDs337 derived frasments
produced from PCR amplification by primers A+B and
primers C+D, as well as the ntEDs337-23 derived fragment
from primers A+D were cloned into plasmid vectors.
Figure 3 shows the experimentally obtained sequences of
the three different lox-containing regions. The lox
regions derived form primers A+}3 and C+D share 80 bp of
identity that include the 34 kb lox sequence, but diverge
outside of this segment. As shown in Figure 3, each
; parental lox sequence i3 adjacent to one characteristic
restriction site, either SalI or BamHI, but on opposite
.' ~
sides of the lox sequence. The lox sequence of the PCR
product derived from primers A+D from ntED5337-23 DNA,
^ however, is flanked by both restriction sites and by
:~ 3equences found in opposing gideg of each of the parental
ntED5337 lox sites. As in bacteria, the recombination
event within plant chromatin was conservative, i.e.,
without 109s or alteration of the lox sequence or its
flanking DNA. To our knowledge, this is the first
description at the nucleotide sequence level of cre/lox-
catalyzed recombination in eucaryotic chromosomal DNA.
~ .
Example 4
Segregation
Two ntED5337-23 plant~ were self-pollinated to
allow segregation of the luciferase gene from the cre
locus which also harbory the linked nptII selection gene.
Approximately 100 R1 germinated seedling~ from each self-
pollinated plant were scored for lucifera~e activity. In
both cases, about 3/4 of the total progeny produced
luciferase and among these, approximately 1/4 were
expected to be sensitive to kanamycin. This was the case
' ` "
.
.
-23-
- with the progeny from one plant. From the second plant,
however, only 1 out of approximately every luciferase
producer progeny was sensitive to kanamycin. This
. indicated either genetic linkage between the cre-nptII
and the luc loci or that there were two unlinked copies
of the cre-nptII construct. It is conceivable that more
than a single gene transfer event occurred during the
second round of Aqrobacterium infection. PCR analysis of
DNA prepared from luciferase-producing seedlings using
primers internal to the cre gene sequence also showed
independent segregation of the cre-nptII locus at the
same time frequencies obtained in the screen for the
~anamycin sensitive phenotype. The loss of the cre-
nptII locus, as determined by the PC~ analysis, was
confirmed phenotypically by an inability of leaf explants
- to form shoots in the presence of kanamycin. A more
extensive PCR analysis of the luc locus (as de~cribed
above) of three representative Luc+HygSKans Rl plants
showed that they have the same profile of fragments as
described for the Ro ntED5337-23 plants. Hence, in two
generations (Ro and Rl), we have shown the feasibility of
transferring a gene into the plant genome without
incorporating a selectable marker.
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