Note: Descriptions are shown in the official language in which they were submitted.
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llrLAS?,KFR FREE TRANSGENIC PLANTS: ENGINEERING THE CHLOROPLAST
GENOME WITHOUT THE
USE OF ANTIBIOTIC SELECTION
CROSS-REFERENCES TO RELATED APPLICATIONS
6 This patent application claims the benefit of U.S. Provisional Application
Nos. 60/208,763, filed
06/06/2000, 60/257,406, filed 12/22/2000 and No. 60/259,154, filed 12/28/2000,
60/186,308, filed
03/02/2000. All applications are hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
11 The work of this invention is supported in part by the USDA-NRICGP grants
95-82770,
97-35504 and 98-0185 to Henry Daniell.
FIELD OF THE INVENTION
This application pertains to the field of genetic engineering of plant plastid
genomes,
16 particularly chloroplasts, and to methods of and engineered plants without
the use of antibiotics.
This application relates in particular to a method of selecting genetically
engineered or
transformed plants without the use of antibiotics as a selectable marker. The
application also relates
to a method of transforming plants to confer drought tolerance and to the
transformed plants which
are drought tolerant.
21
DESCRIPTION OF THE RELATED ART
Publications
Various methods of selection of plants that employ antibiotic-free selectable
marker, or non-
antibiotic selectable markers, have been described in the past.
26 Briggs, in U.S. patent 5,589,611 (December 31,1996) entitled "Disease
resistance gene from
maize and its use for disease resistance as a selectable marker and as a gene
identification probe,"
proposed a method of identifying transformed plants which is disease
resistant. A gene that controls
resistance to both a fungus and a fungal disease toxin is proposed as a
selectable marker to identify
transformed plants, particularly in maize. An expression cassette containing
the DNA sequence of
31 a disease resistance gene, namely the Hml gene in maize, is inserted into
the nucleic genome of the
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1 plant cells. The transformed plants will be capable of producing HC-toxin
reductase. By culturing
the cells in growth medium containing the corresponding toxin produced by the
pathogen, namely
Cocholiobolus carbonum Nelson race 1, the lethal selection of transformed
plants will result.
Ursin, in U.S. patent 5,633,153 (May 27,1997) entitled "Aldehyde dehydrogenase
selectable
markers for plant transformation," proposed a method of using an aldehyde
dehydrogenase as a
6 selectable marker for nuclear transgenic plant cells. A DNA construct coded
for an aldehyde
dehydrogenase through eukaryotic promoters used for nuclear transformation and
culturing such
transformed cells in growth media containing the corresponding phytotoxic
aldehyde, the transformed
plants demonstrate resistance to the phytotoxic aldehyde.
Song, in U.S. patent 5,965,727 (October 12, 1999), entitled "For selectable
markers and
11 promoters for plant tissue culture transformation," proposed transforming
nuclear genome of plant
cells with an expression cassette which contains DNA sequences coded for both
the ASA2 promoter
sequence of Nicotiana tabacum, or fragments thereof, that are capable of
directing tissue culture
specific expression. The ASA2 gene which is substantiallyresistant to
inhibition by free L-Trp or an
amino acid analog of Trp. When such cells are cultured in a medium containing
an amount of an
16 amino acid analog of Trp, successfully transformed plant cells survive.
Several patents have also discussed the conferring of osmoprotection to plants
through plant
transformation. Adams, in U.S. patent 5,780,709 (July 14, 1998) entitled
"Transgenic maize with
increased mannitol content", proposed a method of conferring resistance to
water or salt stress or
altering the osmoprotectant content of a monocot plant by nucleic
transformation. Transformation
21 is accomplished via a vector containing an expression cassette comprised of
a preselected DNA
segment combined with a eukaryotic promoter functional in plant nucleus. Thus,
the preselected
DNA segment that was used to transform the monocot plants was the mtlD gene
which encodes for
the enzyme that catalyzes the synthesis of mannitol. Adams focused on the
osmoprotective properties
of sugar alcohols, specifically mannitol.
26 Wu, in U.S. patent, 5,981,842 (November 9, 1999), proposed that
osmoprotection can be
conferred upon cereal plants by transforming cereal plant cells or protoplasts
with a promoter and
a nucleic acid encoding a group 3 late embryogenesis protein (LEA protein)
such as the HVA1 gene
from barley. The transformed cereal plant accumulates HVA 1 protein in both
leaves and roots. The
2
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1 transformed plants showed an increase tolerance to drought and salt stress
which correlated with the
level of the HVA1 protein accumulated in the transformed plants.
All publications and patent applications are herein incorporated by reference.
BACKGROUND OF THE INVENTION
6 Disadvantages of the antibiotic selectable marker system. Most
transformation techniques co-
introduce a gene that confers antibiotic resistance, along with the gene of
interest to impart a desired
trait. Regenerating transformed cells in antibiotic containing growth media
permits selection of only
those cells that have incorporated the foreign genes as the gene of interest.
Once transgenic plants
are regenerated, antibiotic resistance genes serve no useful purpose but they
continue to produce
11 their gene products. One of the primary concerns of genetically modified
(GM) crops is the presence
of clinically important antibiotic resistance gene products in transgenic
plants that could inactivate
oral doses of the antibiotic (reviewed by Puchta 2000; Daniell 1999A). Another
concern is that the
antibiotic resistant genes could be transferred to pathogenic microbes in the
gastrointestinal tract or
soil rendering them resistant to treatment with such antibiotics. Antibiotic
resistant bacteria are one
16 of the maj or challenges of modern medicine. In Germany, GM crops
containing antibiotic resistant
genes have been banned from release (Peerenboom 2000).
Plastid genetic engineering as an alternative to nuclear genetic engineering.
Plastid genetic
engineering, particularly chloroplast genetic engineering, is emerging as an
alternative new technology
to overcome some of the environmental concerns of nuclear genetic engineering
(reviewed by
21 Bogorad, 2000). One common environmental concern is the escape of foreign
gene through pollen
or seed dispersal from transgenic crop plants to their weedy relatives
creating super weeds or causing
genetic pollution among other crops (Daniell 1999B). Keeler et al. (1996) have
summarized valuable
data on the weedy wild relatives of sixty important crop plants and potential
hybridization between
crops and wild relatives. Among sixty crops, only eleven do not have congeners
and the rest of the
26 crops have wild relatives somewhere in the world. In addition, genetic
pollution among crops has
resulted in several lawsuits and shrunk the European market of Canadian
organic farmers (Hoyle
1999). Several major food corporations have required segregation of native
crops from those
"polluted" with transgenes. Two legislations have been submitted in the U.S.
to protect organic
farmers whose crops inadvertently contain transgenes via pollen drift (Fox
2000). Maternal
3
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1 inheritance of foreign genes through chloroplast genetic engineering is
highly desirable in such
instances where there is potential for out-cross among crops or between crops
and weeds (Daniell
et al. 1998; Scott and Wilkinson 1999; Daniell 1999C).
Yet another concern in the use of nuclear transgenic crops expressing the
Bacillus thuringiensis
(Bt) toxins is the sub-optimal production of toxins resulting in increased
risk of pests developing Bt
6 resistance. Plant-specific recommendations to reduce Bt resistance
development include increasing
Bt expression levels (high dose strategy), expressing multiple toxins (gene
pyramiding), or expressing
the protein only in tissues highly sensitive to damage (tissue specific
expression). All three
approaches are attainable through chloroplast transformation (Daniell 1999C).
For example,
hyperexpression of several thousand copies of a novel B.t. gene via
chloroplast genetic engineering,
11 resulted in 100% mortality of insects that are up to 40.000-fold resistant
to other B.t. proteins (Kota
et al. 1999). Another hotly debated environmental concern expressed recently
is the toxicity of
transgenic pollen to non-target insects, such as the Monarch butterflies
(Losey et al. 1999; Hodgson
1999). Although pollen from a few plants shown to exhibit maternal plastid
inheritance contains
metabolically active plastids, the plastid DNA itself is lost during the
process of pollen maturation and
16 hence is not transmitted to the next generation (reviewed in Heifetz, 2000,
Bock and Hagmann,
2000). Lack of insecticidal protein in transgenic pollen engineered via the
chloroplast genome with
the cry2A gene has been demonstrated recently, even though chloroplast in
leaves contained as much
as 47% CRY protein of the total soluble protein (De Cosa et al. 2000).
The need for alternatives to the antibiotic selectable marker system. Despite
these advantages,
21 one major disadvantage with chloroplast: genetic engineering in higher
plants may be the utilization
of the antibiotic resistance genes as the selectable marker to confer
streptomycin/spectinomycin
resistance. Initially, selection for chloroplast transformation utilized a
cloned mutant 16S rRNA gene
that does not bind the antibiotic and this conferred spectinomycin resistance
(Svab et al. 1990).
Subsequently, the aadA gene product that inactivates the antibiotic by
transferring the adenyl moiety
26 of ATP to spectinomycin /streptomycin was used (Svab and Maliga 1993).
These antibiotics are
commonlyused to control bacterial infection in humans and animals. The
probability of gene transfer
from plants to bacteria living in the gastrointestinal tract or soil may be
enhanced by the compatible
protein synthetic machinery between chloroplasts and bacteria, in addition to
presence of thousands
4
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1 of copies of the antibiotic resistance genes per cell. Also, most antibiotic
resistance genes used in
genetic engineering originate from bacteria.
Because of the presence of thousands of antibiotic resistant genes in each
cell of chloroplast
transgenic plants and the use of the most commonly used antibiotics in the
selection process, it is
important to develop a chloroplast genetic engineering approach without the
use of antibiotics.
6 Non-obviousness of antibiotic free selection. Despite several advantages
ofplastidtransformation,
one maj or disadvantage with chloroplast genetic engineering in higher plants
is the utilization of the
antibiotic resistance genes as the selectable marker. Initially, selection for
chloroplast transformation
utilized a cloned mutant 16S rRNA gene that did not bind the antibiotic and
this conferred
spectinomycin resistance. Subsequently, the aadA gene was used as a selectable
marker.
11 Aminoglycoside 3'-adenylyltransferase inactivates the antibiotic by
transferring the adenyl moiety of
ATP to spectinomycin /streptomycin. Unfortunately, bacterial infections in
humans and animals are
also controlled by using these antibiotics. The probability of gene transfer
from plants to bacteria
living in the soil or gastrointestinal tract may be enhanced by the compatible
protein synthetic
machinery between chloroplasts and bacteria, in addition to presence of
thousands of copies of the
16 antibiotic resistance genes per cell. Also, most antibiotic resistance
genes used in genetic engineering
originate from bacteria.
Prior to this invention, there was no report of modi ing_the plastid ~enome
without the use
of antibiotic selection. Daniell et al. (2001) reported the first genetic
engineering of the higher plant
chloroplast genome without the use of antibiotic selection. The betaine
aldehyde dehydrogenase
21 (BADH) gene from spinach was used as a selectable marker. The selection
process involves
conversion of toxic betaine aldehyde (BA) by the BADH enzyme to nontoxic
glycine betaine, which
also serves as an osmoprotectant. While it was known earlier that BADH was a
plant enzyme, it
could not be conclusively demonstrated that this was a chloroplast enzyme
because it lacked the
typical transit peptide found in all chloroplast proteins imported from the
cytosol.
26 The absence of a typical transit peptide raised several questions about
proper cleavage of
BADH enzyme in the stroma within plastids to be fully functional. It was not
known whether the
BADH enzyme would be catalytically active without proper cleavage within
plastids.
The nuclear BADH cDNA with high GC content was never anticipated to express
well in the
AT rich prokaryotic plastid compartment because the codon usage is very
different between the
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1 prokaryotic chloroplast compartment and the eukaryotic nuclear compartment.
Therefore, it was not
obvious to express a nuclear gene in the plastid compartment.
When the chloroplast transformation system was developed, it was hypothesized
that the
transformationprocess is possible onlyundernon-lethal selection. Accumulation
ofbetaine aldehyde
is toxic and lethal to plant cells. Therefore, it was not clear whether non-
lethal selection was required
6 for chloroplast transformation. This invention has confirmed that the only
requirement was that the
selection process should be specific to plastids, particularly chloroplasts.
Rapid regeneration of chloroplast transgenic plants obtained under BA
selection was never
anticipated or suggested in any prior art. Chloroplast transformation
efficiency was 25 fold higher in
BA selection than spectinomycin and this was never anticipated in anyprevious
investigations. Higher
11 efficiency of betaine aldehyde selection compared to spectinomycin should
facilitate chloroplast
transformation of many economically important crops, including cereals that
are naturally resistant
to spectinomycin, in addition to conferring salt/drought tolerance.
Use of genes that are naturally present in spinach for selection, in addition
to gene
containment, should ease public concerns regarding GM crops.
16
SUMMARY OF THE INVENTION
The invention provides for a method to circumvent the problem of genetic
pollution through
plastid transformation and the use antibiotic-free selectable markers.
Antibiotic-free phytotoxic
agents and their corresponding detoxifying enzymes or proteins are used as a
system of selection.
21 In particular, the betaine aldehyde dehydrogenase (BADH) gene from spinach
has been used as a
selectable marker. This enzyme is present only in chloroplasts of a few plant
species adapted to dry
and saline environments. The selection process involves conversion of toxic
betaine aldehyde (BA)
by the chloroplast BADH enzyme to nontoxic glycine betaine (GB), which also
serves as an
osmoprotectant.
26 The preferred embodiment of this invention provides a method of selecting
plant
transformants using a plastid vector that includes a promoter targeted to the
plastid, a DNA sequence
encoding a gene of interest, another DNA sequence encoding a selectable marker
such as an aldehyde
dehydrogenase, and a terminator sequence. The transformed plants are selected
by allowing
transformed plants to grow in medium with the effective amount of a phytotoxin
which is detoxified
31 by the encoded aldehyde dehydrogenase. Lethal selection of the plants
transformants will result.
6
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1 It is another embodiment of this invention, the vector is targeted to plant
chloroplasts. This
embodiment can be carried out using both the universal chloroplast vector and
a vector which is
universal. Preferably, the vector includes a ribosome binding site and a 5'
untranslated region (5'
UTR. A promoter functional in green or non-green plastids is to be used in
conjunction with the
5'UTR.
6 The invention provides the application of a wide variety of plants species
and plant parts,
including flowers, fruits, cereals, and all major crop plants.
The invention also provides for the plants transformants engineered and
selected a antibiotic-
free selectable marker with preferably a target heterologous DNA sequence.
The invention also provides for a method of conferring drought tolerance to
plants with a
11 antibiotic-free selectable marker. The plants or plant cells are
transformed through the chloroplast
by a vector containing a promoter targeted to the chloroplast, a DNA sequence
encoding betaine
aldehyde dehydrogenase, DNA sequences encoding at least one gene of interest,
and a terminator
sequence. The transformed plants are selected by allowing transformed plants
to grow in medium
with the effective amount of a phytotoxin which is detoxified by the encoded
aldehyde
16 dehydrogenase. Lethal selection of the plants transformants will result.
The plants so transformed
will be capable of glycine betaine production that leads to enhanced drought
tolerance.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the chloroplast universal vector pLD BADH. Primer 3P lands on
the native
21 chloroplast genome (in the 5' end region of 16-S r DNA gene). 3M lands on
the aadA gene
generating a 1.6 kb fragment. Restriction enzyme cut site are located on the
map.
Figure 2 shows BADH enzyme activity in E.coli. Cells harvested from overnight
grown cultures
were resuspended in a minimal volume of the assay buffer. Sonicated cell
homogenate was
desalted in G-25 columns and 50 ~,g total protein was used fr each assay. NAD+
dependent
26 BADH enzyme was analyzed for the formation of NADH by increase in the
absorbency at 340
nm.
Figure 3 shows a comparison of betaine aldehyde and spectinomycin selection.
A. N. tabacum
Petit Havana control in RMOP medium containing spectinomycin after 45 days. B.
Bombarded
leaf discs selected on spectinomycin in RMOP medium after 45 days. C.
Spectinomycin
7
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1 resistantclones cultured again (sound round) to obtain homoplasmy. D. Petit
Havana control in
RMOP medium containing betaine aldehyde after 12 days of culture. E.
Bombardedleaf discs
selected on betaine aldehyde in RMOP medium after 12 days of culture; arrow
indicates
unbombarded leaf disc as control. Note that 23 shoots are formed on a disc
selected on betain
aldehyde against 1-2 shoots per disc on spectinomycin. F. Betaine aldehyde
resistant clones
6 cultureed again (second round) to obtain homoplasmy. G. Selection on lOmM
betaine aldehyde
of untransformed (1) and transgenic (2-4) leaf discs. Note shoots from
transgenic leaf discs and
death of untransformed leaf disc.
Figure 4 shows the PCR analysis of DNA extracted from transformed plants run
on a 0.8%
agarose gel. Lane M lkb ladder, lane 1, untransformed Petit Havana control,
lane 17 is positive
11 control and lanes 2through 16 are transgenic clones. Except lanes 10, 13,
15 and 16 all other
lanes show the integration of aadA gene into the chloroplast genome.
Figure 5 shows the Southern analysis of transgenic plants. A: Probe P1 was
used to confirm
chloroplast integration of foreign genes. The 0.81 kb fragment was cut with
BamHl and Bglll
contains the flanking sequence used for homologous recombination.
Untransformed control
16 plants shuold generate 4.47 kb fragment and transformed plants should
generate a 7.29 kb
fragment. B: Lanes 1, untransformed Petit Havana; Lanes 7 pLD-BADH plasmid DNA
or
purified DNA or purified 1.0 kb Eco Rl BADH gene fragment. Lanes 2 through 6
of transgenic
plants. Probe (P2) was used t confirm the integration of BADH gene.
Figure 6 shows BADH enzyme activity in different ages of leaves of transgenic
tobacco plant.
21 Proteins were extracted from 1-2 g leaves. Extracts were centrifuged at
10,000xG for 10 minutes
and the resulting supernatant was desalted in small G-25 columns, and tested
for assay (50 ~,g
protein per assay). NAD+ dependent BADH enzyme was analyzed for the formation
of NADH.
Y, D, M and O represent young, developing, mature and old leaves,
respectively.
Figure 7 shows the phenotypes of control (A) and chloroplast transgenic plants
(B).
26 Figure 8 shows the germination of control untransformed (a) and chloroplast
transgenic (b) seeds
on MS medium containing 500 ~g/ml spectinomycin.
Figure 9 A and B show the vectors for BADH selection in other plants.
Table 1 shows the comparison of spectinomycin and betaine aldehyde as the
selectable marker for
the first round of selection.
8
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DETAILED DESCRIPTION OF THE INVENTION
The invention discloses a novel way of selecting transformed plants, wherein
the plant's
plastid genome is transformed via a vector targeted to the plastid, and the
selectable markers used
for such transformation is a antibiotic-free marker. The invention further
consists of the plants
transformed and selected using the present method. The invention also
discloses a method to confer
6 osmoprotection to plants through chloroplast transformation.
The present invention is applicable to all plastids of plants. These include
chromoplasts which
are present in the fruits, vegetables and flowers; amyloplasts which are
present in tubers like the
potato; proplastids in roots; leucoplasts and etioplasts, both of which are
present in non-green parts
of plants.
11 The Vectors. This invention contemplates the use of vectors capable of
plastid transformation,
particularly of chloroplast transformation. Such vectors would include
chloroplast expression vectors
such as pUC, pBR322, pBlueScript, pGEM, and all others identified by Daniell
in U.S. patent no.
5,693,507 and U.S. patent no. 5,932,479. Included are also vectors whose
flanking sequence is
located outside the inverted repeat of the chloroplast genome. These
publications and patents are
16 herein incorporated by reference to the same extent as if each individual
publication or patent was
specifically and individually indicated to be incorporated by reference
A preferred embodiment of this invention utilizes auniversal integration and
expression vector
competent for stably transforming the chloroplast genome of different plant
species (Universal
a
Vector). A universal vector is described in WO 99/10513 which was published on
March 4, 1999,
21 which is herein incorporated in its entity.
The vector pLD-BADH was constructed by generating a PCR product using spinach
cDNA
clone as the template. The 5' primer also included the chloroplast optimal
ribosome binding site
(GGAGG). PCR product was subcloned into the EcoRl site of pLD-CtV, resulting
in pLD-BADH.
BADH is one of the few proteins targeted to the chloroplast that lacks a
definite transit peptide
26 (Rathinasabapathi et al 1994). Authors suggest that information for
transport may be contained
within the mature protein. Even if a transit peptide was present, it should be
cleaved in the stroma
by the stromal processing peptidase (Keegstra and Cline, 1999). Furthermore,
nuclear encoded
cytosolic proteins with transit peptides have been successfully expressed
within chloroplasts and
9
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1 found to be fully functional (Daniell et al. 1998). Therefore there was no
need to delete any transit
peptide.
The universal vector, pLD-BADH, as shown in Figure l,~ integrates the aadA and
BADH
genes into the 16S-23S-spacer region of the chloroplast genome. Expression
cassettes of the
chloroplast integration vector contain the chimeric aadA gene and the BADH
gene driven by the
6 constitutive 16S rRNA promoter and regulated by the 3' untranslated region
of the plastid psbA gene.
The chimeric aadA gene encoding aminoglycoside 3'adenyltransferase confers
spectinomycin
resistance in chloroplasts enabling selection of the transformants on
spectinomycin dihydrochloride.
On the other Band, BADH converts the toxic betaine aldehyde in cells to
glycine betaine. When
present, this pathway is compartmentalized within chloroplasts (Nuccio, et al.
1999). To facilitate
11 translation of the dicistronic mRNA, independent Shine-Dalgarno (SD)
sequences were provided to
the aadA and BADH genes upstream of the initiation codons. In order to
accurately compare
transformation efficiency ofboth selectable markers under identical
bombardment and transformation
conditions, aadA and BADH genes were inserted into the same vector, at the
same site. Bombarded
leaves were treated in identical manner except the addition of selection
reagent.
16 Other plant specific vectors can be used to transform the plastids,
particularly chloroplast, of
various crops for betaine aldehyde selection. Some examples of~these include
the pLD-Alfa-BADH
is for transforming the chloroplast genome of Alfalfa using betaine aldehyde
selection; the pLD-Gm-
utr-BADH is for transforming the chloroplast genome of Soybean (Glycine max)
with betaine
aldehyde; this contains the psbA promoter and untranslated region (UTR) for
enhanced expression;
21 the pLD-St-BADH is for transforming the chloroplast genome of potato
(Solanum tuberosum) using
betaine aldehyde selection; pLD-St-utr-BADH is for transforming the
chloroplast genome of potato
(Solanum tuberosum) with betaine aldehyde; this contains the psbA promoter and
untranslated region
(UTR) for enhanced expression; and the pLD-Tom-BADH is for transforming the
chloroplast
genome of tomato using betaine aldehyde selection.
26 Promoters. For transcription and translation of the DNA sequence encoding
the gene of interest,
the entire promoter region from a gene capable of expression in the plastid
generally is used. The
promoter region may include promoters obtained from green and non-green
chloroplast genes that
are operative upon the chloroplast, such as the psbA gene from spinach or pea,
the rbcL, atpB
promoter region from maize, the accD promoter and 16S rRNA promoter. Competent
promoters
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1 are also described in U.S. patent 5,693,507, and the other literature
sources contained therein. These
publications and patents are herein incorporated by reference to the same
extent as if each individual
publication or patent was specifically and individually indicated to be
incorporated by reference.
Selectable markers. The preferred embodiment of this invention teaches the use
of the spinach
BADH gene as a selectable marker; wherein a plant is transformed via the
chloroplast with the
6 spinach BADH gene along with another nucleotide sequence encoding a
desirable trait. The BADH
gene product - betaine aldehyde dehydrogenase - will oxidize the betaine
aldehyde in the growth
medium allowing for the lethal selection of transformed plants.
Other forms of Antibiotic-Free Selection. Enzymes and proteins that function
in plastids can be
used as antibiotic-free phytotoxic agents. In case of amino acid biosynthesis,
the synthesis is
11 regulated by the substrate. When adequate amino acid is made, it binds to
one of the enzymes in the
pathway to block further synthesis (feed back inhibition). Mutant genes are
available for many
enzymes that are insensitive to such feed back inhibition. Such enzymes are
expressed in the
chloroplast by engineering feed back insensitive mutant genes via the
chloroplast genome. Putative
transgenic shoots are regenerated in a growth medium lacking specific amino
acids. True transgenic
16 plants will be regenerated in the growth medium. Thus, antibiotic free
selection is accomplished.
Pigment biosynthesis can also be used in antibiotic free selection in
plastids. While ancient
plants (including pines) have the ability to synthesize chloroplhyll in the
dark, flowering plants lost
this capacity. This is because of the last step in chlorophyll biosynthesis is
controlled by the enzyme
protochlorophyllide reductase. This enzyme can function in the dark in
primitive land plants and
21 certain algae but is light dependent in higher plants. That is why
ornamental plants kept inside the
house requires light to synthesize chlorophyll. It is known that the
chloroplast gene (chlB) for
protochlorophyllide reductase in the green alga Chlamydomonas is required for
light independent
protochlorophyllide reductase activity (Plant Cell 5: 1817-1829). Therefore,
chlB gene from the
Chlamydomonas chloroplast is introduced into the chloroplast genome of
higherplants and transgenic
26 green shoots appearing in the dark is selected. Thus, pigment biosynthesis
genes are used as
antibiotic free selectable markers.
Anotherpossibilityisherbicideselection. Several
methodscanbeusedtogeneticallyengineer
herbicide resistance via the chloroplast genome. The target enzyme or protein
is overproduced with
10,000 copies of foreign genes per transformed cell. This results in binding
of all herbicide molecules
11 .
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1 thereby facilitating regeneration of transgenic shoots. Another approach is
the use of modified
enzyme or proteins (mutant) that does not bind the herbicide. The third
approach is to use enzymes
or proteins to breakdown the herbicide.
Drought tolerance likewise can be used as a selectable marker. Expression of
the BADH
enzyme or trehalose phosphate synthase via the chloroplast genome enables
cells to tolerate drought.
6 Drought conditions are created in culture plates by the addition of
polyethylene glycol to the growth
medium (3-6%). Only cells that express BADH or TPS are capable of drought
tolerance and grows
in the presence of polyethylene glycol. Thus, antibiotic free chloroplast
transgenic plants are
obtained.
Other Aldehyde Dehydrogenases. Other genes that code for an aldehyde
dehydrogenase capable
11 of detoxifying other phytotoxic aldehydes can be used in this novel
selection system. These include,
and are not limited to, genes that encode acetaldehyde dehydrogenase,
formaldehyde dehydrogenase,
proprionaldehyde dehydrogenase, and butyraldehyde dehydrogenase.
Plastid Transformation
The transformation of this invention maybe accomplished by any methods of
transformation
16 known in the art. Such methods include, but are not limited to PEG
treatment, Agrobacterium
treatment, and microinjection. Methods of transformation are described by
Daniell et. al., "New
Tools for Chloroplast Genetic Engineering," Nat. Biotechnology, 17:855-857
(1999). This
publication is hereby incorporated by reference in its entirety. In the
preferred embodiment, the
method for transformation is by bombardment.
21 The BADH gene expression was tested in E. coli cell extracts by enzyme
assays before
proceeding with bombardment. The universal vector pLD-BADH was transformed
into the E. coli
strain XL-1 Blue and grown in Terrific Broth (Guda et al. 2000) in the
presence of ampicillin (100
~.g/ml) at 37°C for 24 hours. In E. coli, the level of expression by
the chloroplast Prrn promoter is
equivalent to that of the highly efficient T7 promoter and both systems have
highly compatible
26 protein synthetic machinery (Brixey et al. 1997). Therefore, BADH enzyme
activity was tested in
untransformed cells and cells transformed with pLD-BADH, a high copy number
plasmid (Figure 2).
Crude sonic extracts isolated from transformed cells showed 3-5 fold more BADH
activity than the
untransformed control, confirming that the expression cassette is fully
functional. This result also
12
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
1 suggests that codon preference of the nuclear BADH gene is compatible with
expression in the
prokaryotic chloroplast compartment.
Tobacco (Nicotiana tabacum var. Petit Havana) was grown aseptically by
germination of
seeds in MSO medium. This medium contains MS salts (4.3 g/liter), BS vitamin
mixture
(myoinositol,100 mg/liter; thiamine-HCI,10 mg/liter; nicotinic acid, 1
mg/liter; pyridoxine-HCI, 1
6 mg/liter), sucrose (30 g/liter) and phytagar (6 g/liter) at pH 5.8. Fully
expanded, dark green leaves
of about two month old plants were used for bombardment.
Leaves were placed abaxial side up on Whatman No. 1 filter papers laying on
the RMOP
medium (Daniell 1993) in standard petri plates (100x15 mm) for bombardment.
Microprojectiles
were coated with plasmid DNA (pLD-BADH) and bombardments were carried out with
the biolistic
11 device PDS 1000/He (Bio-Rad) as describedbyDaniell (1997).
Followingbombardment, petri plates
were sealed with parafilm and incubated at 24°C under 16 hour
photoperiod. Two days after
bombardment, leaves were chopped into small pieces of ~5 mm2 in size and
placed on the selection
medium (RMOP containing 500 ~.g/ml of spectinomycin dihydrochloride or 5-10 mM
betaine
aldehyde) with abaxial side touching the medium in deep ( 100x25 mm) petri
plates. The regenerated
16 resistant shoots were chopped into small pieces (~2mm2) and subcIoned into
fresh deep petri plates
containing the same selection medium. Resistant shoots from the second culture
cycle were
transferred to the rooting medium (MSO medium supplemented with IBA, 1
mg/liter containing
appropriate selectable marker). Rooted plants were transferred to soil and
grown at 26°C under 16
hour photoperiod.
21 Selection and heightened, rapid regeneration of homoplasmic transgenic
plants.
The entire process of regeneration, starting from bombardment until transfer
to soil, takes
about 3-6 months for spectinomycin selection and 2-3 months for betaine
aldehyde selection. Figure
3 and Table 1 show differences between the two selection processes. Under
spectinomycin selection,
leaf discs continued to grow but pigments were bleached; resistant clones
formed green shoots in
26 about 45 days (Figure 3B). On the other hand, under betaine aldehyde
selection, growth of the leaf
discs was completely inhibited and photosynthetic pigments were degraded
(Figure 3 G-1 ), resistant
clones formed green shoots within 12 days (Figure 3E). Leaf disks in Figure 3
under betaine
aldehyde selection appear partially green because they were photographed 12
days after the initiation
of the selection process whereas the disc photographed on spectinomycin were
45 days after initiation
13
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
1 of the selection process. In spite of the short period of selection one leaf
disk was almost bleached
(Fig 3D) and all of them were killed after 30 days. Under l OmM betaine
aldehyde selection, control
untransformed samples were killed (turned black, 3G-1) whereas transgenic
leaves produced new
shoots (Figure 3G, 2-4).
When the leaf discs were selected for spectinomycin resistance, only 15% of
the discs
6 responded and an average of one resistant shoot per plate was observed after
45 days. From each
callus, all resistant shoots are considered to represent an individual clone.
Under betaine aldehyde
selection 80% of the discs responded and an average of 25 resistant shoots per
plate was observed.
Responding leaf disks formed one or two resistant shoots under spectinomycin
selection whereas
under betaine aldehyde selection, as many as 23 shoots were observed from a
single leaf disk.
11 Overall,10 resistant shoots were regenerated from ten bombardments under
spectinomycin selection
while more than 150 shoots were recovered from six bombardments underbetaine
aldehyde selection.
Therefore, the efficiency of transformation is 25 fold higher in betaine
aldehyde selectiomthan
spectinomycin selection. Additionally, the latter procedure results in rapid
regeneration.
16 Lethal Selection. The prior art suggests that chloroplast transformation
system is possible only
under non-lethal selection (Svab and Maliga 1993). This invention distinctly
shows that this is not
the case. Non-lethal selection was defined in the chloroplast transformation
literature as Iack of
suppression of growth on the selection medium and that this was an absolute
requirement for plastid
transformation (Staub and Maliga 1993).. It is known that accumulation of
betaine aldehyde is toxic
21 and lethal to plant cells (Rathinasabapathi et. a1.1994). This invention
confirm earlier observations
that betaine aldehyde is toxic to plant cells and inhibits growth. Therefore,
this invention teaches that
non-Iethal selection is not a requirement for plastid transformation. The only
requirement is that the
selection process should be specific to plastids.
Confirmation of chloroplast integration, homoplasmy and copy number.
26 Integration of a foreign gene into the chloroplast genome was confirmed by
PCR screening
of chloroplast transformants (Figure 4). Primers were designed to eliminate
mutants, nuclear
integration and to determine whether the integration of foreign genes had
occurred in the chloroplast
genome at the directed site by homologous recombination. The strategy to
distinguish between
nuclear and chloroplast transgenic plants was to land one primer (3P) on the
native chloroplast
14
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
1 genome adjacent to the point of integration and the second primer (3M) on
the aadA gene (Figure
1). This primer set generated 1.6 kb PCR product in chloroplast transformants
(Figure 4). Because
this product cannot be obtained in nuclear transgenic plants, the possibility
of nuclear integration can
be eliminated. PCR screening for chloroplast transformants after the first
culture cycle showed that
11 out of 15 betaine aldehyde resistant clones integrated foreign genes into
the chloroplast genome.
6 The rest of the resistant shoots may be either escapes or nuclear
transformants. Hence, only PCR
positive clones were advanced to further steps of regeneration. In contrast,
nearly 60% of the
spectinomycin resistant clones were mutants. Other labs have recently reported
as high as 90%
mutants among spectinomycin resistant clones (Eibl et al. 1999; Sidorov et al.
1999).
Southern blot analysis was performed using total DNA isolated from transgenic
and wild type
11 tobacco leaves. Total DNA was digested with a suitable restriction enzyme.
Presence of a BgllI cut
site at the 3' end of the flanking 16S rRNA gene and the trnA intron allowed
excision of predicted
size fragments in the chloroplast transformants and untransformed plants. To
confirm foreign gene
integration and homoplasmy, individual blots were probed with the flanking
chloroplast DNA
sequence (probe 1, Figure SA). In the case of the BADH integrated plastid
transformants, the border
16 sequence hybridized with a 7.29 kbp fragment while it hybridized with a
native 4.47 kbp fragment
in the untransformed plants (Figure SB). The copy number of the integrated
BADH gene was also
determined by establishing homoplasmy in transgenic plants (Daniell et al.
1998; Guda et al. 2000).
Tobacco chloroplasts contain about 10,000 copies of chloroplast genomes per
cell. If only a fraction
of the genomes was transformed, the copy number should be less than 10,000. By
confirming that
21 the BADH integrated genome is the only one present in transgenic plants, it
could be established that
the BADH gene copy number could be as many as 10,000 per cell.
DNA gel blots were also probed with the BADH gene coding sequence (P2) to
confirm
specific integration into the chloroplast genomes and eliminate transgenic
plants that had foreign
genes also integrated into the nuclear genome. In the case of the BADH
integrated plants, the
26 BADH coding sequence hybridized with a 7.29 kbp fragment which also
hybridized with the border
sequence in plastid transformant lines (Figure SB). This shows that the BADH
gene was integrated
only into the chloroplast genome and not the nuclear genome in transgenic
lines examined in this blot.
Also, this confirms that the tobacco transformants indeed integrated the
intact gene expression
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
cassette into the chloroplast genome and that no internal deletions or loop
outs during integration
occurred via homologous recombination.
Osmoprotection.
In higher plants accumulation of osmoprotectants during salinity and drought
stress is a
common phenomenon in their metabolic adaptation. Osmoprotectants help to
protect plant
6 organelles from osmotic shock as well as the cellularmembranes from damage
during stress (Nuccio
et al. 1999). Among the osmoprotectants, glycine betaine is the most effective
and is commonly
present in a few families, including Chenopodiaceae and Poaceae. But most of
the crop species
including tobacco do not accumulate glycine betaine. Since synthesis and
localization of glycine
betaine is compartmentalized in chloroplasts, engineering the chloroplast
genome for glycine betaine
11 synthesis may provide an added advantage for chloroplast transgenic plants.
BADH converts toxic
betaine aldehyde to non-toxic glycine betaine which is the second step in the
formation of glycine
betaine from choline. By analyzing BADH enzyme activity, the expression of
introduced BADH
gene can be monitored. Since BADH is a NAD+ dependent, enzyme activity is
analyzed for the
formation NADH. The reaction rate is measured by an increase in absorbency at
340 nm resulting
16 from the reduction of NAD+.
BADH enzyme activity was assayed in crude leaf extracts of wild type and
transgenic plants.
Unlike previous reports, no purification with ammonium sulfate was necessary
in order to perform
the BADH assay. Crude extracts from chloroplast transgenic plants showed
elevated activity (15-18
fold) compared to the untransformed tobacco (Figure 6). The wild type tobacco
showed low
21 endogenous activity as reported previously (Rathinasababathy et al. 1994).
BADH enzyme activity
was investigated from young (top 3-4 leaves), mature (large well developed),
developing leaves (in
between young and mature) and bleached old leaves from transgenic plants.
Crude leaf extracts from
different developmental stages of the same transgenic plant showed
differential activitywith the most
activity observed in mature leaves (18 fold over control) and least activity
in older leaves (15 fold
26 over control, as seen in Figure 6). Unlike nuclear transgenic lines, crude
extracts from different
chloroplast transgenic lines did not show significant variation in BADH
activity (data not shown).
Lack of pleiotropic effects. Expression of BADH and resultant accumulation of
glycine betaine did
not result in any pleiotropic effects; transgenic plants are morphologically
indistinguishable from
control untransformed plants (Figure 7). They grew normally, flowered and set
seeds. Germination
16
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
1 of seeds from untransformed plants in the presence of spectinomycin resulted
in complete bleaching
whereas seeds from the chloroplast transgenic plants germinated and grew
normally (Figure 8).
Because untransformed seeds germinated in very high concentrations of betaine
aldehyde (10-15
mM), no comparison between control and transgenic seeds could be made during
germination on
betaine aldehyde. This may be due to the presence of an active endogenous BADH
or similar
6 enzymatic activity in non-green plastids during germination. These results
demonstrate that the
introduced trait is stably inherited in the subsequent generation and that it
is safe to use betaine
aldehyde selection because of the lack of pleiotropic effects.
Application to Other Plants. This invention applies to any higher plants, such
as
monocotyledonous and dicotyledonous plant species. The plants that may be
transformed via the
11 universal vector with a antibiotic-free selectable marker maybe solanacious
plants orplants that grow
underground. Most importantly, this invention is applicable to the major
economically important
crops such as maize, rice, soybean, wheat, and cotton. A non-exclusive list of
examples of higher
plants which maybe so transformed include cereals such as barley, com, oat,
rice, and wheat; melons
such as cucumber, muskmelon, and watermelon; legumes such as bean, cowpea,
pea, peanut; oil
16 crops such as canola and soybean; solanaceous plants such as tobacco; tuber
crops such as potato
and sweet potato; and vegetables like tomato, pepper and radish; fruits such
as pear, grape, peach,
plum, banana, apple and strawberry; fiber crops like the Gossypium genus such
as cotton, flax and
hemp; and other plants such as beet, cotton, coffee, radish, commercial
flowing plants, such as
carnation and roses; grasses, such as sugar cane or turfgrass; evergreen trees
such as fir, spruce, and
21 pine, and deciduous trees, such as maple and oak.
The invention is exemplified in the following non-limiting examples.
Example 1
A. Betaine Aldehyde Selection of Tobacco Chloroplast Transformation. Tobacco
plant chloroplasts were transformed by the universal vector containing both a
targeted gene of
26 interest and the spinach betaine aldehyde dehydrogenase gene. The
transformed cells were cultured
in growth medium containing betaine aldehyde, a phytotoxic~aldehyde. Since
betaine aldehyde is
lethal to all untransformed cells, such cells will not be regenerated in the
growth medium. Therefore,
all cells which grow in the growth medium containing betaine aldehyde are
successfully transformed
17
CA 02401954 2002-09-03
WO 01/64023 PCT/USO1/06275
1 with the BADH gene. More importantly, such cells are successfully
transformed with the targeted
gene of interest.
B. Other possible plants. Other than tobacco, this invention can be practiced
upon
other monocotyledonous and dicotyledonous plants, including maize, rice,
soybean, wheat, cotton,
oat, barley, cucumber, muskmelon, watermelon, bean, cowpea, pea, peanut,
canola, potato and sweet
6 potato; tomato, pepper, radish, pear, grape, peach, plum, banana, apple,
strawberry, flax, hemp, beet,
coffee, radish, commercial flowing plants, such as carnation and roses;
grasses, such as sugar cane
or turfgrass; fir, spruce, and pine, maple and oak.
C. Other antibiotics that can be replaced. This example provides that the
11 invention can replace all antibiotics as a selectable marker, including
those listed in Molecular
Biotechnology by Glick and Pasternak, page 437, Table 17.4.
D. Other targeted genes of interest. This invention provides that genes of
interest
expressing desirable traits are encoded by the targeted DNA sequence in the
expression cassette.
16
Example 2
Other antibiotic-free phytotoxic agents include phytotoxic aldehydes such as
acetaldehyde, formaldehyde, proprionaldehyde, and butyraldehyde; herbicides
such as triazines
and cyanamide, including those listed in Molecular Biotechnology by Glick and
Pasternak, page
21 459, Table 18.4. Also useful is light selection.
Example 3
Other Genes of interest may be isolated from other organisms such as Sugar
Beet and E.
Coli.
26
Example 4
Other Promoters can be used to drive expression of the genes, including the
psbA
promoter, the accD promoter, the l6SrRNA promoter, and those listed in U.S.
patent 5,693,507
and International Publication No. W099/10513, both to Daniell.
18
CA 02401954 2002-09-03
1
WO 01/64023 PCT/USO1/06275
Example 5
Other chloroplast vectors may be used in lieu of the universal vector,
including those
listed in U. S. patents 5693507 and 5932479 to Daniell.
6
Example 6
Targeted Genes of Interest include: Polypepide pro-insulin, PBP synthetic
polymer,
Insulin, Human Serum Albumin, and Herbicide glyphosate. Other genes of
interest include, but
are not limited to the aminoglycosides listed in "Aminoglycosides: A Practical
Review" by
11 Gonzalez, L. S. and Spencer, J.P., American Family Physician, No. 8,
58:1811.
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