U.S. patent application number 09/732439 was filed with the patent office on 2001-10-11 for transgenic maize with increased mannitol content.
This patent application is currently assigned to DEKALB Genetics Corporation.. Invention is credited to Anderson, Paul C., Chomet, Paul S., Griffor, Matthew C., Kriz, Alan L..
Application Number | 20010029618 09/732439 |
Document ID | / |
Family ID | 26811181 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010029618 |
Kind Code |
A1 |
Anderson, Paul C. ; et
al. |
October 11, 2001 |
Transgenic maize with increased mannitol content
Abstract
The present invention provides a method for conferring tolerance
or resistance to water or salt stress in a monocot plant, and/or
altering the osmoprotectant content of a monocot plant, by
introducing a preselected DNA segment into the plant. This
invention also relates to the transformed cells and seeds, and to
the fertile plants grown from the transformed cells and to their
pollen.
Inventors: |
Anderson, Paul C.; (West Des
Moines, IA) ; Chomet, Paul S.; (Mystic, CT) ;
Griffor, Matthew C.; (North Stonington, CT) ; Kriz,
Alan L.; (Gales Ferry, CT) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG,
WOESSNER & KLUTH, P.A.
P.O Box 2938
Minneapolis
MN
55402
US
|
Assignee: |
DEKALB Genetics
Corporation.
|
Family ID: |
26811181 |
Appl. No.: |
09/732439 |
Filed: |
December 7, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09732439 |
Dec 7, 2000 |
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08599714 |
Jan 19, 1996 |
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08599714 |
Jan 19, 1996 |
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08113561 |
Aug 25, 1993 |
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Current U.S.
Class: |
800/278 ;
435/320.1 |
Current CPC
Class: |
C12N 15/8207 20130101;
C12N 15/8209 20130101; C12N 15/8241 20130101; C12N 9/88 20130101;
C12N 9/0006 20130101 |
Class at
Publication: |
800/278 ;
435/320.1 |
International
Class: |
C12N 015/82 |
Claims
What is claimed is:
1. An expression cassette comprising a preselected first DNA
segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in a host
cell, wherein the promoter is selected from the group consisting of
the Glb promoter, the AdhI promoter, and the ActI promoter.
2. The expression cassette of claim 1 wherein the osmoprotectant is
a sugar.
3. The expression cassette of claim 2 wherein the osmoprotectant is
a sugar alcohol.
4. The expression cassette of claim 2 wherein the osmoprotectant is
a sugar selected from the group consisting of fructose, erythritol,
sorbitol, dulcitol, glucoglycerol, sucrose, stachyose, raffinose,
ononitol, mannitol, inositol, methyl-inositol, galactol, hepitol,
ribitol, xylitol, arabitol, trehalose, and pinitol.
5. The expression cassette of claim 1 wherein the osmoprotectant is
selected from the group consisting of proline and
glycine-betaine.
6. The expression cassette of claim 1 wherein the enzyme catalyzes
the synthesis of a sugar.
7. The expression cassette of claim 6 wherein the enzyme catalyzes
the synthesis of mannitol.
8. The expression cassette of claim 1 further comprising a second
DNA segment encoding an amino terminal chloroplast transit peptide
which is operably linked to the preselected first DNA segment.
9. The expression cassette of claim 8 wherein the chloroplast
transit peptide is a maize chloroplast transit peptide.
10. The expression cassette of claim 1 which further comprises an
enhancer element.
11. The expression cassette of claim 10 wherein the enhancer
element is subject to tissue-specific regulation.
12. The expression cassette of claim 1 which further comprises a
selectable marker gene or a reporter gene.
13. An expression cassette comprising (a) a preselected first DNA
segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in a host
cell; and (b) a second DNA segment that encodes an untranslated
regulatory element, wherein the second DNA segment separates the
preselected DNA segment from the promoter.
14. The expression cassette of claim 13 wherein the untranslated
regulatory element is the AdhI intron 1.
15. The expression cassette of claim 13 wherein the promoter is
turgor-inducible.
16. The expression cassette of claim 13 wherein the promoter is
abscisic acid inducible.
17. The expression cassette of claim 13 wherein the promoter is
developmentally regulated.
18. The expression cassette of claim 13 wherein the promoter is a
constitutively expressed promoter.
19. The expression cassette of claim 13 wherein the promoter is
subject to tissue-specific regulation.
20. The expression cassette of claim 13 wherein the promoter is
water-stress inducible.
21. An expression cassette comprising (a) a preselected first DNA
segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in a host
cell; and (b) a second DNA segment encoding a maize chloroplast
transit peptide, wherein the second DNA segment is operably linked
to the preselected first DNA segment.
22. A method to increase water stress resistance or tolerance in
monocot plant cells, comprising: (a) introducing into cells of a
monocot plant an expression cassette comprising a preselected first
DNA segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in the
monocot plant cells, to yield transformed monocot plant cells; and
(b) expressing the enzyme encoded by the preselected first DNA
segment in the transformed monocot plant cells so as to render the
transformed monocot plant cells substantially tolerant or resistant
to a reduction in water availability that inhibits the growth of
untransformed cells of the monocot plant.
23. The method according to claim 22 wherein the expression
cassette is introduced into the plant cells by a method selected
from the group consisting of electroporation, protoplast
transformation, and microprojectile bombardment.
24. The method according to claim 22 wherein the cells of the
monocot plant comprise cells of callus, immature embryos, gametic
tissue, meristematic tissue or cultured cells in suspension.
25. The method according to claim 22 wherein the expression
cassette further comprises a second DNA segment encoding an amino
terminal chloroplast transit peptide which is operably linked to
the preselected first DNA segment.
26. The method according to claim 25 wherein the second DNA segment
encodes a maize chloroplast transit peptide.
27. The method according to claim 25 wherein the enzyme is
expressed in the cytosol of the cells of the transformed monocot
plant.
28. The method according to claim 25 wherein the enzyme is
expressed in the chloroplasts of the cells of the transformed
monocot plant.
29. A transformed plant regenerated from the transformed plant
cells obtained by the method of claim 25.
30. A transformed seed of the transformed plant of claim 29.
31. A method to increase water stress resistance or tolerance in a
monocot plant, comprising: (a) introducing into cells of a monocot
plant an expression cassette comprising a preselected DNA segment
encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in the
monocot plant cells, to yield transformed monocot plant cells; and
(b) regenerating a differentiated fertile plant from said
transformed cells, wherein the enzyme encoded by the preselected
DNA segment is expressed in the cells of the plant so as to render
the transformed monocot plant substantially tolerant or resistant
to a reduction in water availability that inhibits the growth of an
untransformed monocot plant.
32. A transformed monocot plant, which plant is substantially
tolerant or resistant to a reduction in water availability, the
cells of which comprise a recombinant DNA segment comprising a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of an osmoprotectant, wherein the preselected DNA segment
is present in the cells of the plant and wherein the enzyme encoded
by the preselected DNA segment is expressed in an amount effective
to confer tolerance or resistance to the transformed plant to a
reduction in water availability that inhibits the growth of the
corresponding untransformed plant.
33. The transformed plant of claim 32 wherein the transformed plant
has an improved osmotic potential when the total water potential of
the transformed plant approaches zero relative to the osmotic
potential of a corresponding untransformed plant.
34. A method for altering the sugar content in a monocot plant,
comprising: (a) introducing into cells of a monocot plant an
expression cassette comprising a preselected DNA segment encoding
an enzyme which catalyzes the synthesis of a sugar, operably linked
to a promoter functional in the plant cells, to yield transformed
plant cells, and (b) regenerating a differentiated fertile plant
from said transformed plant cells, wherein the enzyme encoded by
the preselected DNA segment is expressed in the cells of the
differentiated plant in an amount effective to increase the sugar
content in the cells of the differentiated plant relative to the
sugar content in the cells of an untransformed plant.
35. The method according to claim 34 wherein the sugar is not
detectable in the cells of the untransformed plant.
36. A transformed monocot plant having an altered sugar cellular
content comprising a recombinant DNA segment comprising a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of a sugar, wherein the enzyme encoded by the preselected
DNA segment is expressed in an amount effective to alter the sugar
content of the cells of said plant.
37. The transformed plant of claim 36 wherein the sugar content of
the leaves, seeds, or fruit of the cells of the transformed plant
is greater than the sugar content of the leaves, seeds, or fruit of
the cells of an untransformed plant.
38. A method for altering the mannitol content in a monocot plant,
comprising: (a) introducing into the cells of the monocot plant an
expression cassette comprising a preselected DNA segment encoding
an enzyme which catalyzes the synthesis of mannitol, operably
linked to a promoter functional in the plant cell to yield
transformed plant cells; and (b) regenerating a differentiated
fertile plant from said transformed plant cells, wherein the enzyme
encoded by the preselected DNA segment is expressed in the cells of
the differentiated plant in an amount effective to increase the
mannitol content in the cells of the differentiated plant relative
to the mannitol content in the cells of an untransformed monocot
plant.
39. The method according to claim 38 wherein the mannitol content
of the transformed plant cells is greater than the mannitol content
of the plant cells of step (a).
40. The method according to claim 38 wherein the mannitol content
of the transformed plant cells during a reduction in water
availability is at least about 1.1 to 50 times greater than the
mannitol content in the transformed plant cells during water
availability.
41. A transformed monocot plant having an altered mannitol cellular
content comprising a recombinant DNA segment comprising a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of mannitol, wherein the enzyme encoded by the
preselected DNA segment is expressed so as to alter the mannitol
content of the cells of said plant.
42. The transformed plant of claim 41 wherein the mannitol content
of the seeds, leaves or fruit of the transformed plant is greater
than the mannitol content of the seeds, leaves, or fruit of an
untransformed plant.
43. A fertile transgenic Zea mays plant comprising a recombinant
DNA segment comprising a promoter operably linked to a first DNA
segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, wherein the level of enzyme expressed from the
first DNA segment in the cells of the transgenic Zea mays plant is
substantially increased above the level in the cells of a Zea mays
plant which only differ from the cells of the transgenic Zea mays
plant in which the recombinant DNA segment is absent, and wherein
the recombinant DNA segment is transmitted through a complete
normal sexual cycle of the transgenic plant to the next
generation.
44. The fertile transgenic Zea mays plant of claim 43 wherein the
recombinant DNA segment further comprises a second DNA segment
encoding an amino terminal chloroplast transit peptide operably
linked to the first DNA segment.
45. The fertile transgenic Zea mays plant of claim 43 wherein the
osmoprotectant is a sugar.
46. A seed produced by the transgenic plant of claim 43.
47. A progeny transgenic Zea mays plant derived from the seed of
claim 46.
48. A progeny transgenic Zea mays seed derived from the plant of
claim 43.
49. A method to increase salt stress resistance or tolerance in a
monocot plant, comprising: (a) introducing into cells of a monocot
plant an expression cassette comprising a preselected DNA segment
encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in the
monocot plant cells, to yield transformed monocot plant cells; and
(b) regenerating a differentiated fertile plant from said
transformed cells, wherein the enzyme encoded by the preselected
DNA segment is expressed in the cells of the plants so as to render
the transformed monocot plant substantially tolerant or resistant
to an amount of salt that inhibits the growth of an untransformed
monocot plant.
50. A transformed monocot plant, which plant is substantially salt
tolerant or resistant, the cells of which comprise a recombinant
DNA segment comprising a preselected DNA segment encoding an enzyme
which catalyzes the synthesis of an osmoprotectant, wherein the
preselected DNA segment is present in the cells of the plant and
wherein the enzyme encoded by the preselected DNA segment is
expressed in an amount effective to confer tolerance or resistance
to the transformed plant to an amount of salt that inhibits the
growth of the corresponding untransformed plant.
51. The method according to claim 31, 34, 38, or 49 further
comprising (c) obtaining progeny from said fertile plant of step
(b), which comprise said preselected DNA segment.
52. The method according to claim 51 wherein said progeny are
obtained by crossing said fertile plant of step (b) with an inbred
line.
53. The method according to claim 51 comprising obtaining seed from
said progeny and obtaining further progeny plants comprising said
preselected DNA segment from said seed.
54. The method according to claim 53 wherein seeds are obtained
from said further progeny plants and plants comprising said
preselected DNA segment are recovered from said seed.
55. The method according to claim 52 comprising obtaining seed from
said progeny and obtaining further progeny plants comprising said
preselected DNA segment from said seed.
56. The method according to claim 55 wherein seeds are obtained
from said further progeny plants and plants comprising said
preselected DNA segment are recovered from said seed.
57. The method according to claim 52 wherein the progeny obtained
in step (c) are crossed back to the inbred line, to obtain further
progeny which comprise said preselected DNA segment.
58. The method according to claim 57 wherein said further progeny
are crossed back to the inbred line to obtain progeny which
comprise said preselected DNA segment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
currently pending U.S. application Ser. No. 08/113,561, filed Aug.
25, 1993, which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Unpredictable rainfall, increases in soil salinity, and low
temperature at the beginning or end of the growing season often
result in decreased plant growth and crop productivity. These three
environmental factors share at least one element of stress and that
is water deficit or dehydration.
[0003] Drought is a significant problem in agriculture today. Over
the last 40 years, for example, drought accounted for 74% of the
total U.S. crop losses of corn (Agriculture, U.S. Department of,
1990. Agricultural Statistics. US Government Printing Office,
Washington, D.C.). To sustain productivity under adverse
environmental conditions, it is important to provide crops with a
genetic basis for coping with water deficit, for example by
breeding water retention and tolerance mechanisms into crops so
that they can grow and yield under these adverse conditions.
[0004] When the rate of transpiration exceeds that of water uptake
or supply, water deficit occurs and wilting symptoms appear. The
responses of plants to water deficits include leaf rolling and
shedding, stomata closure, leaf temperature increases, and wilting.
Metabolism is also profoundly affected. General protein synthesis
is inhibited and significant increases in certain amino acid pools,
such as proline, become apparent (Barnett et al., Plant Physiol.
41, 1222 (1966)). During these water deficit periods, the
photosynthetic rate decreases with the ultimate result of loss in
yield (Boyer, J. S., In: Water deficits and plant growth, T. T.
Kozlowski (ed.)., Academic Press, New York., pp. 154-190 (1976)).
If carried to an extreme, severe water deficits result in death of
the plant.
[0005] Several mechanisms appear to enable water deficit-tolerant
plants to survive and produce. For example, a comparison of
drought-resistant and drought-sensitive lines of Zea mays indicates
that higher levels of abscisic acid (ABA), which is known to
regulate stomata opening and perhaps other signal responses are
correlated with resistance (Milborrow, B. V., In: The physiology
and biochemistry of drought resistant plants, Paleg and Aspinall
(eds.), Academic Press, NY, pp.348-388 (1981)). In addition,
ABA-insensitive mutants and ABA-deficient mutants of Arabidopsis
are prone to wilting (Koorneef et al., Theoret Appl Genet., 61, 385
(1982); Finkelstein et al., Plant Physiol. 94, 1172 (1990)).
[0006] Of the mechanisms employed by water deficit-tolerant plants
to grow and yield, those with major impact on plant productivity
are osmotic adjustment through the increased synthesis of
osmoprotective metabolites, control over ion uptake and
partitioning within the plant, ability to increase water intake,
and acceleration of ontogeny. Examples of osmoprotective
metabolites include sugars, such as sugar alcohols, proline, and
glycine-betaine (Bohnert et al., The Plant Cell, 7, 1099 (1995);
McCue et al., Tibtech, 8, 358 (1990)). Sugar alcohols, or polyols,
such as mannitol and sorbitol, are major photosynthetic products
of, and are known to accumulate to high levels in, various higher
plant species. While mannitol is the most abundant sugar alcohol in
at least 70 plant families, it is not produced at detectable levels
in any important agricultural field or vegetable crop, other than
celery (Apiaceae), coffee (Rubiaceae), and olive (Oleacea). Other
sugar alcohols, such as ononitol and pinitol, are known to be
produced in some plants under conditions of stress from drought,
salt, or low temperature.
[0007] To produce a plant with a genetic basis for coping with
water deficit, Tarczynski et al. (Proc. Natl. Acad. Sci. USA, 82,
2600 (1992); WO 92/19731, published Nov. 12, 1992; Science, 2, 508
(1993)) introduced the bacterial mannitol-1-phosphate dehydrogenase
gene, mtlD, into tobacco cells via Agrobacterium-mediated
transformation. Root and leaf tissues from transgenic plants
regenerated from these transformed tobacco cells contained up to
100 mM mannitol. Control plants contained no detectable mannitol.
To determine whether the transgenic tobacco plants exhibited
increased tolerance to water deficit, Tarczynski et al. compared
the growth of transgenic plants to that of untransformed control
plants in the presence of 250 mM NaCl. After 30 days of exposure to
250 mM NaCl, transgenic plants had decreased weight loss and
increased height relative to their untransformed counterparts. The
authors concluded that the presence of mannitol in these
transformed tobacco plants contributed to water deficit tolerance
at the cellular level.
[0008] While Tarczynski et al. (WO 92/19731, published Nov. 12,
1992)) disclose that the same methodology might be applied to other
higher plants, such as field crops, the introduction of exogenous
DNA into monocotyledonous species and subsequent regeneration of
transformed plants expressing useful phenotypic properties has
proven much more difficult than transformation and regeneration of
dicotyledonous plants.
[0009] Thus, there is a need for transgenic monocot plants that are
resistant or tolerant to a reduction in water availability. Also, a
method to produce transgenic monocot plants with increased levels
of osmoprotectants is needed.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method to increase water
stress resistance or tolerance in a monocot plant cell or monocot
plant, comprising introducing an expression cassette into-the cells
of a monocot plant to yield transformed monocot plant cells.
Monocot plant cells include cells of monocotyledenous plants such
as cereals, including corn (Zea mays), wheat, oats, rice, barley,
millet and the like. The expression cassette comprises a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of an osmoprotectant, operably linked to a promoter
functional in the monocot plant cell. The enzyme encoded by the DNA
segment is expressed in the transformed monocot plant cells to
increase the level of the osmoprotectant so as to render the
transformed cells substantially tolerant or resistant to a
reduction in water availability that inhibits the growth of
untransformed cells of the plant.
[0011] As used herein, an "osmoprotectant" is an osmotically active
molecule which, when that molecule is present in an effective
amount in a cell or plant, confers water stress tolerance or
resistance, or salt stress tolerance or resistance, to the cell or
plant. Osmoprotectants include sugars such as monosaccharides,
disaccharides, oligosaccharides, polysaccharides, sugar alcohols,
and sugar derivatives, as well as proline and glycine-betaine. A
preferred embodiment of the invention is an osmoprotectant that is
a sugar. A more preferred embodiment of the invention is an
osmoprotectant that is a sugar alcohol. Thus, useful
osmoprotectants include fructose, erythritol, sorbitol, dulcitol,
glucoglycerol, sucrose, stachyose, raffinose, ononitol, mannitol,
inositol, methyl-inositol, galactol, hepitol, ribitol, xylitol,
arabitol, trehalose, and pinitol. A preferred osmoprotectant of the
invention is mannitol.
[0012] Genes which encode an enzyme that catalyzes the synthesis of
an osmoprotectant include genes encoding mannitol dehydrogenase
(Lee and Saier, J. Bacteriol., 153 (1982)) and
trehalose-6-phosphate synthase (Kaasen et al., J. Bacteriol., 174,
889 (1992)). Through the subsequent action of native phosphatases
in the cell or by the introduction and coexpression of a specific
phosphatase, these introduced genes result in the accumulation of
either mannitol or trehalose, respectively, both of which have been
well documented as protective compounds able to mitigate the
effects of stress. Mannitol accumulation in transgenic tobacco has
been verified and preliminary results indicate that plants
expressing high levels of this metabolite are able to tolerate an
applied osmotic stress (Tarczynski et al., cited supra (1992),
(1993)).
[0013] Also provided is an isolated transformed monocot plant cell
and an isolated transformed monocot plant comprising said
transformed cells, which cell and plant are substantially tolerant
or resistant to a reduction in water availability. The cells of the
transformed monocot plant comprise a recombinant DNA segment
comprising a preselected DNA segment encoding an enzyme which
catalyzes the synthesis of an osmoprotectant. The preselected DNA
segment is present in the cells of the transformed plant and the
enzyme encoded by the preselected DNA segment is expressed in those
cells to yield an amount of osmoprotectant effective to confer
tolerance or resistance to said cells to a reduction in water
availability that inhibits the growth of the corresponding cells of
the untransformed plant. A preferred embodiment of the invention
includes a transformed monocot plant that has an improved osmotic
potential when the total water potential of the transformed plant
approaches zero relative to the osmotic potential of a
corresponding untransformed plant.
[0014] Another preferred embodiment of the invention is an isolated
transgenic Zea mays cell or plant, comprising a recombinant DNA
segment comprising a promoter operably linked to a first DNA
segment encoding an amino terminal chloroplast transit peptide
operably linked to a second DNA segment encoding an enzyme which
catalyzes the synthesis of an osmoprotectant. The enzyme encoded by
the DNA sequence is expressed in the transgenic Zea mays plant or
cell so that the level of the osmoprotectant in the cells of the
transgenic Zea mays plant is substantially increased above the
level in the cells of a Zea mays plant which only differ from the
cells of the transgenic Zea mays plant in that the DNA segment is
absent. The DNA segment is transmitted through a complete normal
sexual cycle of the transgenic plant to its progeny and to further
generations.
[0015] A further embodiment of the invention is a method for
altering the sugar content in a monocot plant, such as a Zea mays
plant, or monocot cell. The method comprises introducing an
expression cassette into the cells of a monocot plant so as to
yield transformed monocot plant cells. The expression cassette
comprises a preselected DNA segment encoding an enzyme which
catalyzes the synthesis of a sugar, operably linked to a promoter
functional in the plant cells. A differentiated plant is
regenerated from the transformed plant cells. The enzyme encoded by
the preselected DNA segment is expressed in the cells of the
differentiated plant in an amount effective to increase the sugar
content in the cells of the differentiated plant relative to the
sugar content in the cells of the untransformed differentiated
plant.
[0016] Yet another embodiment of the invention is an isolated
transformed monocot plant cell or transformed monocot plant, having
an altered sugar cellular content. The transformed monocot
comprises a recombinant DNA segment comprising a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of a
sugar. The enzyme encoded by the DNA segment is expressed in an
amount effective to alter the sugar content of the cells of the
plant.
[0017] The present invention also provides an isolated transgenic
Zea mays cell or plant, comprising a recombinant DNA segment
comprising a promoter operably linked to a preselected DNA segment
encoding an enzyme which catalyzes the synthesis of a sugar. The
enzyme encoded by the recombinant DNA segment is expressed so that
the level of sugar in the cells of the transgenic Zea mays plant is
substantially increased above the level in the cells of a Zea mays
plant which only differ from the cells of the transgenic Zea mays
plant in which the recombinant DNA segment is absent. The
recombinant DNA segment is transmitted through a complete normal
sexual cycle of the transgenic plant to its progeny and further
generations.
[0018] A preferred embodiment of the invention is a method for
altering the mannitol content in a monocot plant cell or plant,
such as a Zea mays plant. The method comprises introducing an
expression cassette into the cells of the monocot plant so as to
yield transformed plant cells. The expression cassette comprises a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of mannitol, operably linked to a promoter functional in
the plant cell. A differentiated plant is regenerated from the
transformed plant cells. The enzyme encoded by the DNA segment is
expressed in the cells of the differentiated plant in an amount
effective to increase the mannitol content in the cells of the
differentiated plant relative to the mannitol content in the cells
of an untransformed differentiated monocot plant.
[0019] Also provided is an isolated transformed monocot plant
comprising an altered mannitol cellular content. The plant
comprises a recombinant DNA segment comprising a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of
mannitol. The enzyme encoded by the DNA is expressed in an amount
effective to alter the mannitol content of the cells of the
plant.
[0020] Another embodiment of the invention is a method to increase
salt stress resistance or tolerance in a monocot plant. The method
comprises introducing an expression cassette into the cells of a
monocot plant. The expression cassette comprises a preselected DNA
segment encoding an enzyme which catalyzes the synthesis of an
osmoprotectant, operably linked to a promoter functional in a
monocot plant cell, to yield transformed monocot plant cells. These
transformed cells are regenerated to form a differentiated monocot
plant. The enzyme encoded by the DNA segment is expressed so as to
render the transformed monocot plant substantially resistant to an
amount of salt that inhibits the growth of an untransformed monocot
plant. Also provided is a transformed monocot plant which is salt
stress tolerant or resistant. The cells of the plant comprise a
recombinant DNA segment comprising a preselected DNA segment
encoding an enzyme which catalyzes the synthesis of an
osmoprotectant. The enzyme is expressed in the cells of the plant
in an amount effective to confer tolerance or resistance to the
transformed plant to an amount of salt that inhibits the growth of
the corresponding untransformed plant.
[0021] As used herein, the term "salt" includes, but is not limited
to, salts of agricultural fertilizers and salts associated with
alkaline or acid soil conditions. A preferred salt of the invention
is sodium chloride (NaCl).
[0022] The present invention also provides an expression cassette
comprising a preselected DNA segment encoding an enzyme which
catalyzes the synthesis of an 6smoprotectant, operably linked to a
promoter functional in a host cell. The promoter in the expression
cassette is selected from, but not limited to, the group consisting
of the Glb promoter, the AdhI promoter, and the ActI promoter.
[0023] Also provided is an expression cassette comprising a
preselected first DNA segment encoding an enzyme which catalyzes
the synthesis of an osmoprotectant, operably linked to a promoter
functional in a host cell, wherein a second DNA segment separates
the first preselected DNA segment encoding the enzyme from the
promoter. A preferred second DNA segment is the AdhI intron 1.
[0024] Further provided is an expression cassette comprising a
preselected first DNA segment encoding an enzyme which catalyzes
the synthesis of an osmoprotectant, operably linked to a promoter
functional in a host cell, wherein a second DNA segment encoding a
maize chloroplast transit peptide is operably linked to the
preselected first DNA segment encoding the enzyme.
[0025] As used herein, a "preselected" DNA sequence or segment is
an exogenous or recombinant DNA sequence or segment that encodes an
enzyme which catalyzes the synthesis of an osmoprotectant, such as
a sugar. The enzyme preferably utilizes a substrate that is
abundant in the plant cell. More preferably, the substrate is
present in either, or both, the cytosol and chloroplasts of the
plant cell. It is also preferred that the preselected DNA segment
or sequence encode an enzyme that is active without a co-factor, or
with a readily available co-factor. For example, the mtlD gene of
E. coli encodes a mannitol-1-phosphate dehydrogenase (M1PD). The
only co-factor necessary for the enzymatic activity of M1PD in
plants is NADH and the substrate for M1PD in plants is
fructose-6-phosphate. Both NADH and fructose-6-phosphate are
plentiful in higher plant cells.
[0026] As used herein, "substantially increased" or "elevated"
levels of an osmoprotectant in a transformed plant cell, plant
tissue, plant part, or plant, are greater than the levels in an
untransformed plant cell, plant part, plant tissue, or plant, i.e.,
one where the genome has not been altered by the presence of a
preselected DNA sequence. In the alternative, "substantially
increased" or "elevated" levels of an osmoprotectant in a
water-stressed transformed plant cell, plant tissue, plant part, or
plant, are levels that are at least about 1.1 to 50 times,
preferably at least about 2 to 30 times, and more preferably about
5-20 times, greater than the levels in a non-water-stressed
transformed plant cell, plant tissue, plant part or plant.
[0027] For example, the levels of mannitol in a monocot plant
transformed with a preselected DNA sequence encoding an enzyme
which catalyzes the synthesis of mannitol, are compared to the
levels in an untransformed plant. In the alternative, the levels of
mannitol in a homozygous backcross converted inbred plant
transformed with a preselected DNA sequence encoding an enzyme
which catalyzes the synthesis of mannitol, are compared to the
levels in a recurrent inbred plant. A homozygous backcross
converted inbred transformed plant is a transformed plant which has
been repeatedly crossed to the recurrent inbred parent until the
transformed plant is substantially isogenic with the recurrent
inbred parent except for the presence of the preselected DNA
sequence, and is then self-pollinated (selfed) at least once, and
preferably 5 or more times.
[0028] As used herein, "substantially isogenic" means that the
genomic DNA content of a homozygous backcross converted inbred
transformed plant is at least about 92%, preferably at least about
98%, and most preferably at least about 99%, identical to the
genomic DNA content of a recurrent inbred parent of the transformed
plant.
[0029] As used herein, a plant cell, plant part, plant tissue or
plant that is "substantially resistant or tolerant" to a reduction
in water availability is a plant cell, plant part, plant tissue, or
plant that grows under water-stress conditions, e.g., high salt,
low temperatures, or decreased water availability, that normally
inhibit the growth of the untransformed plant cell, plant tissue,
plant part, or plant, as determined by methodologies known to the
art. Methodologies to determine plant growth or response to stress
include, but are not limited to, height measurements, weight
measurements, leaf area, plant water relations, ability to flower,
ability to generate progeny, and yield. For example, a homozygous
backcross converted inbred transformed plant of the invention has a
superior osmotic potential during a water deficit relative to the
corresponding, i.e., substantially isogenic, recurrent inbred
plant.
[0030] As used herein, an "exogenous" gene or "recombinant" DNA is
a DNA sequence or segment that has been isolated from a cell,
purified, and amplified.
[0031] As used herein, the term "isolated" means either physically
isolated from the cell or synthesized in vitro in the basis of the
sequence of an isolated DNA segment.
[0032] As used herein, a "native" gene means a DNA sequence or
segment that has not been manipulated in vitro, i.e., has not been
isolated, purified, and amplified.
[0033] As used herein, "altered" levels of an osmoprotectant in a
transformed plant, plant tissue, plant part, or plant cell are
levels which are different, preferably greater, than the levels
found in the corresponding untransformed plant, plant tissue, plant
part, or plant cells. In the alternative, altered levels of the
osmoprotectant in a backcross converted inbred transformed plant
are different, preferably greater, than the levels found in the
corresponding recurrent inbred plant.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1. A schematic diagram of plasmid pDPG451.
[0035] FIG. 2. A schematic diagram of plasmid pDPG165.
[0036] FIG. 3. A schematic diagram of plasmid pDPG480.
[0037] FIG. 4. A schematic diagram of plasmid pDPG493.
[0038] FIG. 5. A schematic diagram of plasmid pDPG586.
[0039] FIG. 6. A schematic diagram of plasmid pDPG587.
[0040] FIG. 7. A time course of leaf osmotic potential values
collected from a population of transgenic maize plants. All plants
were derived from AT824 cells bombarded with pDPG165 and pDPG480
which were subsequently selected on bialaphos-containing medium.
(A) S80HO-5201, (B) S80HO-5205, and (C) S80HO-5208.
[0041] FIG. 8. Leaf temperature data from Glufosinate.RTM.
sensitive (mtlD negative) and resistant (mtlD positive) plants
grown under water stress conditions in the field.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The identification and characterization of plants that are
resistant or tolerant to water deprivation has long been a goal of
agronomy. However, it has not been possible to accomplish the
identification and isolation of genes that can provide resistance
or tolerance to water stress. The insertion of such genes into
monocots has the potential for long term improvement in, and
expansion of, agriculture world-wide.
[0043] The ability of a plant to adapt to changes in water and salt
concentrations is dependent on the ability of the plant to
osmotically adjust its intracellular environment by altering the
concentration of osmoprotectants within the cells of the plant.
These osmoprotectants include, but are not limited to, various
sugar molecules, such as monosaccharides, disaccharides,
oligosaccharides, polysaccharides, sugar alcohols, and sugar
derivatives. Thus, to provide a plant that is tolerant or resistant
to a reduction in water availability, a preselected DNA segment or
"gene" or "transgene" encoding an enzyme which catalyzes the
synthesis of a particular osmoprotectant can be introduced into the
genome of the plant. The osmoprotectant may be one that is not
normally synthesized by the plant, but one which can be synthesized
from a substrate that is abundant in the cells of the plant after
the introduction of the preselected DNA segment. In the
alternative, the osmoprotectant may be one that is naturally
synthesized by the plant but the levels of the osmoprotectant in
the plant are insufficient to render the plant tolerant to a
reduction in water availability.
[0044] The accumulation of a non-naturally occurring osmoprotectant
in a plant, plant cell, plant part, or plant tissue, could result
in a detrimental effect because the substrate employed to
synthesize the osmoprotectant is being depleted and a non-naturally
occurring product is produced, which most likely would not be
degraded. Moreover, a single introduced preselected DNA segment in
the transgenic maize plant resulted in a beneficial effect to the
transgenic maize plant when it is placed under water stress, i.e.,
the plant became more water stress-tolerant than its untransformed
counterpart. Furthermore, the expression of the preselected DNA
segment in the transgenic plant did not substantially affect the
reproduction or growth of the plant, relative to its untransformed
counterpart.
[0045] Thus, the present invention provides a method of genetically
engineering monocot plants so as to produce altered agronomic or
physiologic changes in the plants by the alteration in the levels
of an osmoprotectant, such as a sugar, or more preferably a sugar
alcohol, within the tissues of the plant. Alterations in these
levels result in more negative osmotic water potentials in
transformed plant tissues under either, or both, water stress or
non-water stress conditions relative to the osmotic potentials in
untransformed plant tissues.
[0046] Yet another embodiment of the invention is a method to
confer tolerance or resistance to a reduction in water availability
to a monocot plant, plant tissue, plant part or plant cell. Methods
and compositions are provided for producing callus cultures, plant
tissues, plants and seeds that are tolerant and/or resistant to a
reduction in water availability under conditions that normally
inhibit the function or growth of these cultures, tissues, plants
or seeds. Such plants and seeds sexually can transmit this trait to
their progeny.
[0047] The methods provided in the present invention may be used to
produce increased levels of osmoprotectants, such as a sugar in
monocots and other cereal crops including, but not limited to,
maize, rice, rye, millet, wheat, barley, sorghum, and oats.
[0048] In accord with the present invention, a preselected DNA
segment is identified, isolated, and combined with at least a
promoter functional in a plant cell to provide a recombinant
expression cassette. Once formed, an expression cassette comprising
a preselected DNA segment can be subcloned into a known expression
vector. Suitable known expression vectors include plasmids that
autonomously replicate in prokaryotic and/or eukaryotic cells.
Specific examples include plasmids such as pUC, pSK, pGEM, pBS and
pSP-derived vectors, the pBI121 or pBI221 plasmid constructed as
described by Jefferson (Pl. Mol. Biol. Repr., 5, 387 (1987)), or a
binary Ti plasmid vector such as pG582 as described by An (Plant
Cell, 1, 115 (1989)), and the like.
[0049] An expression cassette of the invention can be subcloned
into an expression vector by standard methods. The expression
vector can then be introduced into prokaryotic or eukaryotic cells
by currently available methods including, but not limited to,
protoplast transformation, Agrobacterium-mediated transformation,
electroporation, microprojectile bombardment, tungsten whiskers
(Coffee et al., U.S. Pat. No. 5,302,523, issued April 12, 1994) and
liposomes. The vector can be introduced into prokaryotic cells such
as E. colt or Agrobacterium. Transformed cells can be selected
typically using a selectable or screenable marker encoded on the
expression vector.
[0050] The expression cassette or vector can be introduced into
monocot plant cells. Plant cells useful for transformation include
callus, immature embryos, meristematic tissue, gametic tissue, or
cultured suspension cells. Optionally, other preselected DNA
segments encoding enzymes which catalyze the synthesis of
osmoprotectants can be introduced into the plant cell. The
transformed plant cell can then be regenerated into a plant and the
plant tested for its ability to grow or thrive under stress
conditions, such as high salinity or reduced water availability.
Depending on the type of plant, the level of gene expression, and
the activity of the enzyme encoded by the preselected DNA segment,
introduction of the preselected DNA into the plant can confer the
phenotype of tolerance or resistance to water deficit to the
plant.
[0051] The introduced preselected DNA segments can be expressed in
the transformed monocot plant cells and stably transmitted
(somatically and sexually) to the next generation of cells
produced. The vector should be capable of introducing, maintaining,
and expressing a preselected DNA segment in plant cells, wherein
the preselected DNA can be obtained from a variety of sources,
including but not limited to plants and animals, bacteria, fungi,
yeast or virus. Additionally, it should be possible to introduce
the vector into a wide variety of cells of monocot plants. The
preselected DNA segment is passed on to progeny by normal sexual
transmission.
[0052] Introduction and expression of foreign genes in
dicotyledonous (broad-leafed) plants such as tobacco, potato and
alfalfa has been shown to be possible using the T-DNA of the
tumor-inducing (Ti) plasmid of Agrobacterium tumeffaciens. Using
recombinant DNA techniques and bacterial genetics, a wide variety
of foreign DNAs can be inserted into T-DNA in Agrobacterium.
Following infection by the bacterium containing the recombinant Ti
plasmid, the foreign DNA is inserted into the host plant
chromosomes, thus producing a genetically engineered cell and
eventually a genetically engineered plant. A second approach is to
introduce root-inducing (Ri) plasmids as the gene vectors.
[0053] While Agrobacterium appear to attack only dicots, many
important crop plants including maize, wheat, rice, barley, oats,
sorghum, millet, and rye are monocots and are not known to be
susceptible to transformation by Agrobacterium. The Ti plasmid,
however, may be manipulated in the future to act as a vector for
monocot plants. Additionally, using the Ti plasmid as a model
system, it may be possible to artificially construct transformation
vectors for monocot plants. Ti-plasmids might also be introduced
into monocots by artificial methods such as microinjection, or
fusion between monocot protoplasts and bacterial spheroplasts
containing the T-region, which can then be integrated into the
plant nuclear DNA.
[0054] Transformation of plant cells with a preselected DNA segment
may also be accomplished by introducing a preselected DNA into
other nucleic acid molecules that can transfer the inserted DNA
into a plant genome, e.g., plant pathogens such as DNA viruses like
CaMV or geminiviruses, RNA viruses, and viroids; DNA molecules
derived from unstable plant genome components like extrachromosomal
DNA elements in organelles (e.g., chloroplasts or mitochondria), or
nuclearly encoded controlling elements; DNA molecules from stable
plant genome components (e.g., origins of replication and other DNA
sequences which allow introduced DNA to integrate into the
organellar or nuclear genomes and to replicate normally, to
autonomously replicate, to segregate normally during cell division
and sexual reproduction of the plant and to be inherited in
succeeding generations of plants) or transposons.
[0055] A preselected DNA may be delivered into plant cells or
tissues directly by microorganisms with infectious plasmids,
infectious viruses, the use of liposomes, microinjection by
mechanical or laser beam methods, by whole chromosomes or
chromosome fragments, electroporation, and microprojectile
bombardment.
[0056] I. Recipient Cells
[0057] Practicing the present invention includes the generation and
use of recipient cells. As used herein, the term "recipient cells"
refers to monocot cells that are receptive to transformation and
subsequent regeneration into stably transformed, fertile monocot
plants.
[0058] A. Sources of Cells
[0059] Recipient cell targets include, but are not limited to,
meristem cells, Type I, Type II, and Type III callus, immature
embryos and gametic cells such as microspores pollen, sperm and egg
cells. Type I, Type II, and Type III callus may be initiated from
tissue sources including, but not limited to, immature embryos,
seedling apical meristems, microspores and the such. Those cells
which are capable of proliferating as callus are also recipient
cells for genetic transformation. The present invention provides
techniques for transforming immature embryos followed by initiation
of callus and subsequent regeneration of fertile transgenic plants.
Direct transformation of immature embryos obviates the need for
long term development of recipient cell cultures. Pollen, as well
as its precursor cells, microspores, may be capable of functioning
as recipient cells for genetic transformation, or as vectors to
carry foreign DNA for incorporation during fertilization. Direct
pollen transformation would obviate the need for cell culture.
Meristematic cells (i.e., plant cells capable of continual cell
division and characterized by an undifferentiated cytological
appearance, normally found at growing points or tissues in plants
such as root tips, stem apices, lateral buds, etc.) may represent
another type of recipient plant cell. Because of their
undifferentiated growth and capacity for organ differentiation and
totipotency, a single transformed meristematic cell could be
recovered as a whole transformed plant. In fact, it is proposed
that embryogenic suspension cultures may be an in vitro
meristematic cell system, retaining an ability for continued cell
division in an undifferentiated state, controlled by the media
environment.
[0060] In certain embodiments, cultured plant cells that can serve
as recipient cells for transforming with desired DNA segments
include maize cells, and more specifically, cells from Zea mays L.
Somatic cells are of various types. Embryogenic cells are one
example of somatic cells which may be induced to regenerate a plant
through embryo formation. Non-embryogenic cells are those which
will typically not respond in such a fashion. An example of
non-embryogenic cells are certain Black Mexican Sweet (BMS) maize
cells. These cells have been transformed by microprojectile
bombardment using the neo gene followed by selection with the
aminoglycoside, kanamycin (Klein et al., Plant Physiol., 91, 440
(1989)). However, this BMS culture was not found to be
regenerable.
[0061] The development of embryogenic maize calli and suspension
cultures useful in the context of the present invention, e.g., as
recipient cells for transformation, has been described in Gordon et
al. (U.S. Pat. No. 5,134,074, issued Jul. 28, 1992, incorporated
herein by reference).
[0062] The present invention also provides certain techniques that
may enrich recipient cells within a cell population. For example,
Type II callus development, followed by manual selection and
culture of friable, embryogenic tissue, generally results in an
enrichment of recipient cells for use in, e.g., microprojectile
transformation. Suspension culturing, particularly using the media
disclosed herein, may also improve the ratio of recipient to
non-recipient cells in any given population. Manual selection
techniques which employed to select recipient cells may include,
e.g., assessing cell morphology and differentiation, or may use
various physical or biological means. Cryopreservation is also
contemplated as a possible method of selecting for recipient
cells.
[0063] Manual selection of recipient cells, e.g., by selecting
embryogenic cells from the surface of a Type II callus, is one
means employed in an attempt to enrich for recipient cells prior to
culturing (whether cultured on solid media or in suspension). The
preferred cells may be those located at the surface of a cell
cluster, and may further be identifiable by their lack of
differentiation, their size and dense cytoplasm. The preferred
cells will generally be those cells which are less differentiated,
or not yet committed to differentiation. Thus, one may wish to
identify and select those cells which are cytoplasmically dense,
relatively unvacuolated with a high nucleus to cytoplasm ratio
(e.g., determined by cytological observations), small in size
(e.g., 10-20 .mu.m), and capable of sustained divisions and somatic
proembryo formation.
[0064] It is proposed that other means for identifying such cells
may also be employed. For example, through the use of dyes, such as
Evan's blue, which are excluded by cells with relatively
non-permeable membranes, such as embryogenic cells, and taken up by
relatively differentiated cells such as root-like cells and snake
cells (so-called due to their snake-like appearance).
[0065] Other possible means of identifying recipient cells include
the use of isozyme markers of embryogenic cells, such as glutamate
dehydrogenase, which can be detected by cytochemical stains (Fransz
et al., Plant Cell Rep., 8, 67 (1989)). However, it is cautioned
that the use of isozyme markers such as glutamate dehydrogenase may
lead to some degree of false positives from non-embryogenic cells
such as rooty cells which nonetheless have a relatively high
metabolic activity.
[0066] B. Media
[0067] In certain embodiments, recipient cells are selected
following growth in culture. Where employed, cultured cells will
preferably be grown either on solid supports or in the form of
liquid suspensions. In either instance, nutrients may be provided
to the cells in the form of media, and environmental conditions
controlled. There are many types of tissue culture media comprised
of amino acids, salts, sugars, growth regulators and vitamins. Most
of the media employed in the practice of the invention will have
some similar components (see, e.g., Table 1 hereinbelow), the media
differ in the composition and proportions of their ingredients
depending on the particular application envisioned. For example,
various cell types usually grow in more than one type of media, but
will exhibit different growth rates and different morphologies,
depending on the growth media. In some media, cells survive but do
not divide.
[0068] Various types of media suitable for culture of plant cells
have been previously described. Examples of these media include,
but are not limited to, the N6 medium described by Chu et al.
(Scientia Sinica, 18, 659 (1975)) and MS media described by
Murashige & Skoog (Plant Physiol., 15, 473 (1962)). Media such
as MS which have a high ammonia/nitrate ratio are counterproductive
to the generation of recipient cells in that they promote loss of
morphogenic capacity. N6 media, on the other hand, has a somewhat
lower ammonia/nitrate ratio, and is contemplated to promote the
generation of recipient cells by maintaining cells in a
proembryonic state capable of sustained divisions.
[0069] C. Cell Cultures
[0070] 1. Initiation
[0071] In the practice of the invention it is sometimes, but not
always, necessary to develop cultures which contain recipient
cells. Suitable cultures can be initiated from a number of whole
plant tissue explants including, but not limited to, immature
embryos, leaf bases, immature tassels, anthers, microspores, and
other tissues containing cells capable of in vitro proliferation
and regeneration of fertile plants. In one exemplary embodiment,
recipient cell cultures are initiated from immature embryos of Zea
mays L. by growing excised immature embryos on a solid culture
medium containing growth regulators including, but not limited to,
dicamba, 2,4-D, NAA, and IAA. In some instances it will be
preferred to add silver nitrate to culture medium for callus
initiation as this compound has been reported to enhance culture
initiation (Vain et al., Plant Cell, tissue and Organ Culture., 18
143 (1989)). Embryos will produce callus that varies greatly in
morphology including from highly unorganized cultures containing
very early embryogenic structures (such as, but not limited to,
type II cultures in maize), to highly organized cultures containing
large late embryogenic structures (such as, but not limited to,
type I cultures in maize). This variation in culture morphology may
be related to genotype, culture medium composition, size of the
initial embryos and other factors. Each of these types of culture
morphologies is a source of recipient cells.
[0072] The development of suspension cultures capable of plant
regeneration may be used in the context of the present invention.
Suspension cultures may be initiated by transferring callus tissue
to liquid culture medium containing growth regulators. Addition of
coconut water or other substances to suspension culture medium may
enhance growth and culture morphology, but the utility of
suspension cultures is not limited to those containing these
compounds. In some embodiments of this invention, the use of
suspension cultures will be preferred as these cultures grow more
rapidly and are more easily manipulated than callus cells growing
on solid culture medium.
[0073] When immature embryos or other tissues directly removed from
a whole plant are used as the target tissue for DNA delivery, it
will only be necessary to initiate cultures of cells insofar as is
necessary for identification and isolation of transformants. In an
illustrative embodiment, DNA is introduced by particle bombardment
into immature embryos following their excision from the plant.
Embryos are transferred to a culture medium that will support
proliferation of tissues and allow for selection of transformed
sectors, at about 0-14 days following DNA delivery. In this
embodiment of the invention it is not necessary to establish stable
callus cultures capable of long term maintenance and plant
regeneration.
[0074] 2. Maintenance
[0075] The method of maintenance of cell cultures may contribute to
their utility as sources of recipient cells for transformation.
Manual selection of cells for transfer to fresh culture medium,
frequency of transfer to fresh culture medium, composition of
culture medium, and environment factors including, but not limited
to, light quality and quantity and temperature are all important
factors in maintaining callus and/or suspension cultures that are
useful as sources of recipient cells. It is contemplated that
alternating callus between different culture conditions may be
beneficial in enriching for recipient cells within a culture. For
example, it is proposed that cells may be cultured in suspension
culture, but transferred to solid medium at regular intervals.
After a period of growth on solid medium cells can be manually
selected for return to liquid culture medium. It is proposed that
by repeating this sequence of transfers to fresh culture medium it
is possible to enrich for recipient cells. It is also contemplated
that passing cell cultures through a sieve, e.g., a 1.9 mm sieve,
is useful in maintaining the friability of a callus or suspension
culture and may be beneficial is enriching for transformable
cells.
[0076] 3. Cryopreservation
[0077] Additionally, cryopreservation may effect the development
of, or perhaps select for, recipient cells. Cryopreservation
selection may operate due to a selection against highly vacuolated,
non-embryogenic cells, which may be selectively killed during
cryopreservation. There is a temporal window in which cultured
cells retain their regenerative ability, thus, it is believed that
they must be preserved at or before that temporal period if they
are to be used for future transformation and regeneration.
[0078] For use in transformation, suspension or callus culture
cells may be cryopreserved and stored for periods of time, thawed,
then used as recipient cells for transformation. An illustrative
embodiment of cryopreservation methods comprises the steps of
slowly adding cryoprotectants to suspension cultures to give a
final concentration of 10% dimethyl sulfoxide, 10% polyethylene
glycol (6000 MW), 0.23 M proline and 0.23 M glucose. The mixture is
then cooled to -35.degree. C. at 0.5.degree. C. per minute. After
an isothermal period of 45 minutes, samples are placed in liquid
N.sub.2 (modification of methods of Withers et al., Plant Physiol.,
64, 675 (1979); and Finkle et al., Plant Sci., 42, 133 (1985)). To
reinitiate suspension cultures from cryopreserved material, cells
may be thawed rapidly and pipetted onto feeder plates similar to
those described by Vaeck et al. (Nature, 328, 33 (1987)).
[0079] II. DNA Sequences
[0080] Virtually any DNA composition may be used for delivery to
recipient monocotyledonous cells to ultimately produce fertile
transgenic plants in accordance with the present invention. For
example, a preselected DNA segment encoding a gene product whose
expression confers an increase in intracellular mannitol levels, or
drought resistance, in the form of vectors and plasmids, or linear
DNA fragments, in some instances containing only the DNA element to
be expressed in the plant, and the like, may be employed.
[0081] In certain embodiments, it is contemplated that one may wish
to employ replication-competent viral vectors in monocot
transformation. Such vectors include, for example, wheat dwarf
virus (WDV) "shuttle" vectors, such as pW1-11 and PW1-GUS (Ugaki et
al., Nucl. Acid Res., 19, 391 (1991)). These vectors are capable of
autonomous replication in maize cells as well as E. coli, and as
such may provide increased sensitivity for detecting DNA delivered
to transgenic cells. A replicating vector may also be useful for
delivery of genes flanked by DNA sequences from transposable
elements such as Ac, Ds, or Mu. It has been proposed (Laufs et al.,
Proc. Natl. Acad. Sci. USA, 87, 7752 (1990)) that transposition of
these elements within the maize genome requires DNA replication. It
is also contemplated that transposable elements would be useful for
introducing DNA fragments lacking elements necessary for selection
and maintenance of the plasmid vector in bacteria, e.g., antibiotic
resistance genes and origins of DNA replication. It is also
proposed that use of a transposable element such as Ac, Ds, or Mu
would actively promote integration of the desired DNA and hence
increase the frequency of stably transformed cells.
[0082] Vectors, plasmids, cosmids, YACs (yeast artificial
chromosomes) and DNA segments for use in transforming such cells
will, of course, generally comprise the preselected cDNA(s),
preselected DNA(s) or genes which one desires to introduce into the
cells. These DNA constructs can further include structures such as
promoters, enhancers, polylinkers, or even regulatory genes as
desired. The DNA segment or gene chosen for cellular introduction
will often encode a protein which will be expressed in the
resultant recombinant cells, such as will result in a screenable or
selectable trait and/or which will impart an improved phenotype to
the regenerated plant. However, this may not always be the case,
and the present invention also encompasses transgenic plants
incorporating non-expressed transgenes related to
drought-resistance or mannitol expression.
[0083] DNA useful for introduction into maize cells includes that
which has been derived or isolated from any source, that may be
subsequently characterized as to structure, size and/or function,
chemically altered, and later introduced into maize. An example of
DNA "derived" from a source, would be a DNA sequence or segment
that is identified as a useful fragment within a given organism,
and which is then chemically synthesized in essentially pure form.
An example of such DNA "isolated" from a source would be a useful
DNA sequence that is excised or removed from said source by
chemical means, e.g., by the use of restriction endonucleases, so
that it can be further manipulated, e.g., amplified, for use in the
invention, by the methodology of genetic engineering. Such DNA is
commonly referred to as "recombinant DNA."
[0084] Therefore useful DNA includes completely synthetic DNA,
semi-synthetic DNA, DNA isolated from biological sources, and DNA
derived from RNA. It is within the scope of the invention to
isolate a preselected DNA segment from a given maize genotype, and
to subsequently introduce multiple copies of the preselected DNA
segment into the same genotype, e.g., to enhance production of a
given gene product such as a protein that confers tolerance or
resistance to water deficit.
[0085] The introduced DNA includes, but is not limited to, DNA from
plant genes, and non-plant genes such as those from bacteria,
yeasts, animals or viruses. The introduced DNA can include modified
genes, portions of genes, or chimeric genes, including genes from
the same or different maize genotype. The term "chimeric gene" or
"chimeric DNA" is defined as a gene or DNA sequence or segment
comprising at least two DNA sequences or segments from species
which do not combine DNA under natural conditions, or which DNA
sequences or segments are positioned or linked in a manner which
does not normally occur in the native genome of untransformed
maize, or other monocot.
[0086] The introduced DNA used for transformation herein may be
circular or linear, double-stranded or single-stranded. Generally,
the DNA is in the form of chimeric DNA, such as plasmid DNA, that
can also contain coding regions flanked by regulatory sequences
which promote the expression of the recombinant DNA present in the
resultant maize plant For example, the DNA may itself comprise or
consist of a promoter that is active in maize which is derived from
a non-maize source, or may utilize a promoter already present in
the maize genotype that is the transformation target.
[0087] Generally, the introduced DNA will be relatively small,
i.e., less than about 30 kb to minimize any susceptibility to
physical, chemical, or enzymatic degradation which is known to
increase as the size of the DNA increases. As noted above, the
number of proteins, RNA transcripts or mixtures thereof which is
introduced into the maize genome is preferably preselected and
defined, e.g., from one to about 5-10 such products of the
introduced DNA may be formed.
[0088] A. Regulatory Elements
[0089] The construction of vectors which may be employed in
conjunction with the present invention will be known to those of
skill of the art in light of the present disclosure.
[0090] Ultimately, the most desirable DNA segments for introduction
into a monocot genome may be homologous genes or gene families
which encode a desired trait (e.g., increased yield per acre) and
which are introduced under the control of novel promoters or
enhancers, etc., or perhaps even homologous or tissue-specific
(e.g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or
leaf-specific) promoters or control elements. Indeed, it is
envisioned that a particular use of the present invention will be
the targeting of a preselected DNA segment in a tissue- or
organelle- or turgor-specific manner.
[0091] Vectors for use in tissue-specific targeting of a
preselected DNA segment in transgenic plants will typically include
tissue-specific promoters and may also include other
tissue-specific control elements such as enhancer sequences.
Promoters which direct specific or enhanced expression in certain
plant tissues will be known to those of skill in the art in light
of the present disclosure. These include, for example, the rbcS
promoter, specific for green tissue; the ocs, nos and mas promoters
which have higher activity in roots or wounded leaf tissue; a
truncated (-n90 to +8) 35S promoter which directs enhanced
expression in roots, an a-tubulin gene that directs expression in
roots and promoters derived from zein storage protein genes which
direct expression in endosperm. It is particularly contemplated
that one may advantageously use the 16 bp ocs enhancer element from
the octopine synthase (ocs) gene (Ellis et al., supra (1987);
Bouchez et al., supra (1989)), especially when present in multiple
copies, to achieve enhanced expression in roots.
[0092] B. Marker Genes
[0093] In order to improve the ability to identify transformants,
one may desire to employ a selectable or screenable marker gene as,
or in addition to the expressible preselected DNA segment. "Marker
genes" are genes that impart a distinct phenotype to cells
expressing the marker gene and thus allow such transformed cells to
be distinguished from cells that do not have the marker. Such genes
may encode either a selectable or screenable marker, depending on
whether the marker confers a trait which one can `select` for by
chemical means, i.e., through the use of a selective agent (e.g., a
herbicide, antibiotic, or the like), or whether it is simply a
trait that one can identify through observation or testing, i.e.,
by `screening` (e.g., the R-locus trait). Of course, many examples
of suitable marker genes are known to the art and can be employed
in the practice of the invention.
[0094] Included within the terms selectable or screenable marker
genes are also genes which encode a "secretable marker" whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes which can be detected by their catalytic
activity. Secretable proteins fall into a number of classes,
including small, diffusible proteins detectable, e.g., by ELISA;
and proteins that are inserted or trapped in the cell wall (e.g.,
proteins that include a leader sequence such as that found in the
expression unit of extensin or tobacco PR-S).
[0095] With regard to selectable secretable markers, the use of a
gene that encodes a protein that becomes sequestered in the cell
wall, and which protein includes a unique epitope is considered to
be particularly advantageous. Such a secreted antigen marker would
ideally employ an epitope sequence that would provide low
background in plant tissue, a promoter-leader sequence that would
impart efficient expression and targeting across the plasma
membrane, and would produce protein that is bound in the cell wall
and yet accessible to antibodies. A normally secreted wall protein
modified to include a unique epitope would satisfy all such
requirements.
[0096] One example of a protein suitable for modification in this
manner is extensin, or hydroxyproline rich glycoprotein (HPRG). The
use of the maize HPRG (Steifel et al., The Plant Cell, 2, 785
(1990)) is preferred as this molecule is well characterized in
terms of molecular biology, expression, and protein structure.
However, any one of a variety of extensins and/or glycine-rich wall
proteins (Keller et al., EMBO J., 8, 1309 (1989)) could be modified
by the addition of an antigenic site to create a screenable
marker.
[0097] Elements of the present disclosure are exemplified in detail
through the use of particular marker genes, however in light of
this disclosure, numerous other possible selectable and/or
screenable marker genes will be apparent to those of skill in the
art in addition to the one set forth hereinbelow. Therefore, it
will be understood that the following discussion is exemplary
rather than exhaustive. In light of the techniques disclosed herein
and the general recombinant techniques which are known in the art,
the present invention renders possible the introduction of any
gene, including marker genes, into a recipient cell to generate a
transformed monocot.
[0098] 1. Selectable Markers
[0099] Possible selectable markers for use in connection with the
present invention include, but are not limited to, a neo gene
(Potrykus et al., Mol. Gen. Gent. 122, 183 (1985)) which codes-for
kanamycin resistance and can be selected for using kanamycin, G418,
and the like; a bar gene which codes for bialaphos resistance; a
gene which encodes an altered EPSP synthase protein (Hinchee et
al., Biotech., 915 (1988)) thus conferring glyphosate resistance; a
nitrilase gene such as bxn from Klebsiella ozaenae which confers
resistance to bromoxynil (Stalker et al., Science, X, 419 (1988));
a mutant acetolactate synthase gene (ALS) which confers resistance
to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
(European Patent Application 154,204, 1985); a
methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem.,
263, 12500 (1988)); a dalapon dehalogenase gene that confers
resistance to the herbicide dalapon; or a mutated anthranilate
synthase gene that confers resistance to 5-methyl tryptophan. Where
a mutant EPSP synthase gene is employed, additional benefit may be
realized through the incorporation of a suitable chloroplast
transit peptide, CTP (European Patent Application 0,218,571,
1987).
[0100] An illustrative embodiment of a selectable marker gene
capable of being used in systems to select transformants is the
genes that encode the enzyme phosphinothricin acetyltransferase,
such as the bar gene from Streptomyces hygroscopicus or the pat
gene from Streptomyces viridochromogenes (U.S. patent application
Ser. No. 07/565,844, which is incorporated by reference herein).
The enzyme phosphinothricin acetyl transferase (PAT) inactivates
the active ingredient in the herbicide bialaphos, phosphinothricin
(PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol.
Gen. Genet., 205 42 (1986); Twell et al., Plant Physiol., 91, 1270
(1989)) causing rapid accumulation of ammonia and cell death. The
success in using this selective system in conjunction with monocots
was particularly surprising because of the major difficulties which
have been reported in transformation of cereals (Potrykus, Trends
Biotech., 7, 269 (1989)).
[0101] 2. Screenable Markers
[0102] Screenable markers that may be employed include, but are not
limited to, a .beta.-glucuronidase or uidA gene (GUS) which encodes
an enzyme for which various chromogenic substrates are known; an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et
al., in Chromosome Structure and Function, pp. 263-282 (1988));
.beta.-lactamase gene (Sutcliffe, PNAS USA, 75, 3737 (1978)), which
encodes an enzyme for which various chromogenic substrates are
known (e.g., PADAC, a chromogenic cephalosporin); a xyle gene
(Zukowsky et al., PNAS USA, 8, 1101 (1983)) which encodes a
catechol dioxygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikuta et al., Biotech., 8, 241 (1990)); a
tyrosinase gene (Katz et al., J. Gen. Microbiol., 129, 2703 (1983))
which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to form the easily detectable
compound melanin; a 13-galactosidase gene, which encodes an enzyme
for which there are chromogenic substrates; a luciferase (lux) gene
(Ow et al., Science, 234, 856 (1986)), which allows for
bioluminescence detection; or even an aequorin gene (Prasher et
al., Biochem. Biophys. Res. Comm., 126, 1259 (1985)), which may be
employed in calcium-sensitive bioluminescence detection, or a green
fluorescent protein gene (Niedz et al., Plant Cell Reports, 14, 403
(1995)).
[0103] Genes from the maize R gene complex are contemplated to be
particularly useful as screenable markers. The R gene complex in
maize encodes a protein that acts to regulate the production of
anthocyanin pigments in most seed and plant tissue. Maize strains
can have one, or as many as four, R alleles which combine to
regulate pigmentation in a developmental and tissue specific
manner. A gene from the R gene complex was applied to maize
transformation, because the expression of this gene in transformed
cells does not harm the cells. Thus, an R gene introduced into such
cells will cause the expression of a red pigment and, if stably
incorporated, can be visually scored as a red sector. If a maize
line is carries dominant alleles for genes encoding the enzymatic
intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2,
Bz1 and Bz2), but carries a recessive allele at the R locus,
transformation of any cell from that line with R will result in red
pigment formation. Exemplary lines include Wisconsin 22 which
contains the rg-Stadler allele and TR112, a K55 derivative which is
r-g, b, Pl. Alternatively any genotype of maize can be utilized if
the C1 and R alleles are introduced together.
[0104] It is further proposed that R gene regulatory regions may be
employed in chimeric constructs in order to provide mechanisms for
controlling the expression of chimeric genes. More diversity of
phenotypic expression is known at the R locus than at any other
locus (Coe et al., In: Corn and Corn Improvement, Sprague et al.
(eds.) pp. 81-258 (1988)). It is contemplated that regulatory
regions obtained from regions 5' to the structural R gene would be
valuable in directing the expression of genes, e.g., insect
resistance, drought resistance, herbicide tolerance or other
protein coding regions. For the purposes of the present invention,
it is believed that any of the various R gene family members may be
successfully employed (e.g., P, S, Lc, etc.). However, the most
preferred will generally be Sn (particularly Sn:bol3). Sn is a
dominant member of the R gene complex and is functionally similar
to the R and B loci in that Sn controls the tissue specific
deposition of anthocyanin pigments in certain seedling and plant
cells, therefore, its phenotype is similar to R.
[0105] A further screenable marker contemplated for use in the
present invention is firefly luciferase, encoded by the lux gene.
The presence of the lux gene in transformed cells may be detected
using, for example, X-ray film, scintillation counting, fluorescent
spectrophotometry, low-light video cameras, photon counting cameras
or multiwell luminometry. It is also envisioned that this system
may be developed for populational screening for bioluminescence,
such as on tissue culture plates, or even for whole plant
screening.
[0106] C. Transgenes for Maize Modification
[0107] Improvement of the ability of maize to tolerate various
environmental stresses including, but not limited to, drought,
excess moisture, chilling, freezing, high temperature, salt, and
oxidative stress, can be effected through expression of
heterologous, or overexpression of homologous, genes.
[0108] Expression of novel preselected DNA segments that favorably
effect plant water content, total water potential, osmotic
potential, and turgor can enhance the ability of the plant to
tolerate drought. As used herein, the terms "drought resistance"
and "drought tolerance" are used to refer to a plants increased
resistance or tolerance to stress induced by a reduction in water
availability, as compared to normal circumstances, and the ability
of the plant to function and survive in lower-water environments,
and perform in a relatively superior manner. In this aspect of the
invention it is proposed, for example, that the expression of a
preselected DNA segment encoding the biosynthesis of
osmotically-active solutes can impart protection against drought.
Within this class of preselected DNA segments are DNAs encoding
mannitol dehydrogenase (Lee and Saier, J. Bacteriol., 153 (1982))
and trehalose-6-phosphate synthase (Kaasen et al., J. Bacteriol.,
174, 889 (1992)). Through the subsequent action of native
phosphatases in the cell or by the introduction and coexpression of
a specific phosphatase, these introduced preselected DNAs will
result in the accumulation of either mannitol or trehalose,
respectively, both of which have been well documented as protective
compounds able to mitigate the effects of stress. Mannitol
accumulation in transgenic tobacco has been verified and
preliminary results indicate that plants expressing high levels of
this metabolite are able to tolerate an applied osmotic stress
(Tarczynski et al., cited supra (1992), (1993)).
[0109] Similarly, the efficacy of other metabolites in protecting
either enzyme function (e.g. alanopine or propionic acid) or
membrane integrity (e.g., alanopine) has been documented (Loomis et
al., J. Expt. Zool., 252, 9 (1989)), and therefore expression of a
preselected DNA segment encoding the biosynthesis of these
compounds can confer drought resistance in a manner similar to or
complimentary to mannitol. Other examples of naturally occurring
metabolites that are osmotically active and/or provide some direct
protective effect during drought and/or desiccation include sugars
and sugar derivatives such as fructose, erythritol (Coxson et al.,
Biotropica, 24, 121 (1992)), sorbitol, dulcitol (Karsten et al.,
Botanica Marina, 35, 11 (1992)), glucosylglycerol (Reed et al., J.
Gen. Microbiol., 1, 1 (1984); Erdmann et al., J. Gen. Microbiol.,
118, 363 (1992)), sucrose, stachyose (Koster and Leopold, Plant
Physiol., 88, 829 (1988); Blackman et al., Plant Physiol., 100, 225
(1992)), ononitol and pinitol (Vernon and Bohnert, EMBO J., 11,
2077 (1992)), and raffinose (Bemal-Lugo and Leopold, Plant
Physiol., 98, 1207 (1992)). Other osmotically active solutes which
are not sugars include, but are not limited to, proline (Rensburg
et al., 1993) and glycine-betaine (Wyn-Jones and Storey, In:
Physiology and Biochemistry of Drought Resistance in Plants, Paleg
et al. (eds.), pp. 171-204 (1981)). Continued canopy growth and
increased reproductive fitness during times of stress can be
augmented by introduction and expression of preselected DNA
segments such as those controlling the osmotically active compounds
discussed above and other such compounds, as represented in one
exemplary embodiment by the enzyme myoinositol
0-methyltransferase.
[0110] It is contemplated that the expression of specific proteins
may also increase drought tolerance. Three classes of Late
Embryogenic Proteins have been assigned based on structural
similarities (see Dure et al., Plant Mol. Biol., 12, 475 (1989)).
All three classes of these proteins have been demonstrated in
maturing (i.e., desiccating) seeds. Within these 3 types of
proteins, the Type-II (dehydrin-type) have generally been
implicated in drought and/or desiccation tolerance in vegetative
plant parts (i.e. Mundy and Chua, EMBO J., 7, 2279 (1988);
Piatkowski et al., Plant Physiol., 94, 1682 (1990);
Yamaguchi-Shinozaki et al., Plant Cell Physiol., 33 217 (1992)).
Recently, expression of a Type-III LEA (HVA-1) in tobacco was found
to influence plant height, maturity and drought tolerance
(Fitzpatrick, Gen. Engineering News, 22, 7 (1993)). Expression of
structural genes from all three groups may therefore confer drought
tolerance. Other types of proteins induced during water stress
include thiol proteases, aldolases and transmembrane transporters
(Guerrero et al., Plant Mol. Biol., 15, 11 (1990)), which may
confer various protective and/or repair-type functions during
drought stress. The expression of a preselected DNA segment that
effects lipid biosynthesis and hence membrane composition can also
be useful in conferring drought resistance on the plant.
[0111] Many genes that improve drought resistance have
complementary modes of action. Thus, combinations of these genes
might have additive and/or synergistic effects in improving drought
resistance in maize. Many of these genes also improve freezing
tolerance (or resistance); the physical stresses incurred during
freezing and drought are similar in nature and may be mitigated in
similar fashion. Benefit may be conferred via constitutive
expression of these genes, but the preferred means of expressing
these novel genes may be through the use of a turgor-induced
promoter (such as the promoters for the turgor-induced genes
described in Guerrero et al. (Plant Molecular Biology, 15, 11
(1990)) and Shagan et al., Plant Physiol, 101, 1397 (1993), which
are incorporated herein by reference). Spatial and temporal
expression patterns of these genes may enable maize to better
withstand stress.
[0112] It is proposed that expression of genes that are involved
with specific morphological traits that allow for increased water
extractions from drying soil would be of benefit. For example,
introduction and expression of genes that alter root
characteristics may enhance water uptake. It is also contemplated
that expression of DNAs that enhance reproductive fitness during
times of stress would be of significant value. For example,
expression of DNAs that improve the synchrony of pollen shed and
receptiveness of the female flower parts, i.e., silks, would be of
benefit. In addition it is proposed that expression of genes that
minimize kernel abortion during times of stress would increase the
amount of grain to be harvested and hence be of value. It is
further contemplated that regulation of cytokinin levels in
monocots, such as maize, by introduction and expression of an
isopentenyl transferase gene with appropriate regulatory sequences
can improve monocot stress resistance and yield (Gan et al.,
Science, 27, 1986 (1995)).
[0113] Given the overall role of water in determining yield, it is
contemplated that enabling maize to utilize water more efficiently,
through the introduction and expression of novel genes, will
improve overall performance even when soil water availability is
not limiting. By introducing genes that improve the ability of
maize to maximize water usage across a full range of stresses
relating to water availability, yield stability or consistency of
yield performance may be realized.
[0114] D. Preparation of an Expression Cassette
[0115] An expression cassette of the invention can comprise a
recombinant DNA molecule containing a preselected DNA segment
operably linked to a promoter functional in a host cell. A
preselected DNA segment can be identified and isolated by standard
methods, as described by Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, NY (1989). The preselected
DNA segment can also be obtained from water stress-tolerant cell
lines. The preselected DNA segment can be identified by screening
of a DNA or cDNA library generated from nucleic acid derived from a
particular cell type, cell line, primary cells, or tissue.
Screening for DNA fragments that encode all or a portion of the
preselected DNA segment can be accomplished by screening plaques
from a genomic or cDNA library for hybridization to a probe of the
DNA from other organisms or by screening plaques from a CDNA
expression library for binding to antibodies that specifically
recognize the protein encoded by the preselected DNA segment. DNA
fragments that hybridize to a preselected DNA segment probe from
other organisms and/or plaques carrying DNA fragments that are
immunoreactive with antibodies to the protein encoded by the
preselected DNA segment can be subcloned into a vector and
sequenced and/or used as probes to identify other cDNA or genomic
sequences encoding all or a portion of the preselected DNA
segment.
[0116] Portions of the genomic copy or copies of the preselected
DNA segment can be sequenced and the 5' end of the DNA identified
by standard methods including either DNA sequence homology to other
homologous genes or by RNAase protection analysis, as described by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Once portions
of the 5' end of the preselected DNA segment are identified,
complete copies of the preselected DNA segment can be obtained by
standard methods, including cloning or polymerase chain reaction
(PCR) synthesis using oligonucleotide primers complementary to the
preselected DNA segment at the 5' end of the DNA. The presence of
an isolated full-length copy of the preselected DNA can be verified
by hybridization, partial sequence analysis, or by expression of
the preselected DNA segment.
[0117] The construction of such expression cassettes which may be
employed in conjunction with the present invention will be known to
those of skill in the art in light of the present disclosure (see,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, New York (1989); Gelvin et al., Plant Molecular
Biology Manual, (1990)).
[0118] 1. Promoters
[0119] Once a preselected DNA segment is obtained and amplified, it
is operably combined with a promoter to form an expression
cassette.
[0120] Most genes have regions of DNA sequence that are known as
promoters and which regulate gene expression. Promoter regions are
typically found in the flanking DNA sequence upstream from the
coding sequence in both prokaryotic and eukaryotic cells. A
promoter sequence provides for regulation of transcription of the
downstream gene sequence and typically includes from about 50 to
about 2,000 nucleotide base pairs. Promoter sequences also contain
regulatory sequences such as enhancer sequences that can influence
the level of gene expression. Some isolated promoter sequences can
provide for gene expression of heterologous DNAs, that is a DNA
different from the native or homologous DNA. Promoter sequences are
also known to be strong or weak or inducible. A strong promoter
provides for a high level of gene expression, whereas a weak
promoter provides for a very low level of gene expression. An
inducible promoter is a promoter that provides for turning on and
off of gene expression in response to an exogenously added agent or
to an environmental or developmental stimulus. Promoters can also
provide for tissue specific or developmental regulation. An
isolated promoter sequence that is a strong promoter for
heterologous DNAs is advantageous because it provides for a
sufficient level of gene expression to allow for easy detection and
selection of transformed cells and provides for a high level of
gene expression when desired.
[0121] The promoter in an expression cassette of the invention can
provide for expression of the preselected DNA segment. Preferably,
the preselected DNA segment is expressed so as to result in an
increase in tolerance of the plant cells to water deficit, or to
increase the content of an osmoprotectant in the plant cells. The
promoter can also be inducible so that gene expression can be
turned on or off by an exogenously added agent. For example, a
bacterial promoter such as the P.sub.tac promoter can be induced to
varying levels of gene expression depending on the level of
isothiopropylgalactoside added to the transformed bacterial cells.
It may also be preferable to combine the preselected DNA segment
with a promoter that provides tissue specific expression or
developmentally regulated gene expression in plants.
[0122] Preferred constructs will generally include, but are not
limited to, a plant promoter such as the CaMV 35S promoter (Odell
et al., Nature, 313, 810 (1985)), or others such as CaMV 19S
(Lawton et al., Plant Mol. Biol., 2, 31F (1987)), nos (Ebert et
al., PNAS USA, 84, 5745 (1987)), Adh (Walker et al., PNAS USA, 84,
6624 (1987)), sucrose synthase (Yang et al., PNAS USA, 87, 4144
(1990)), .alpha.-tubulin, ubiquitin, actin (Wang et al., Mol. Cell.
Biol. 12, 3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet.,
215, 431 (1989)), PEPCase (Hudspeth et al., Plant Mol. Biol., 12,
579 (1989)) or those associated with the R gene complex (Chandler
et al., The Plant Cell, 1, 1175 (1989)). Other promoters useful in
the practice of the invention are known to those of skill in the
art, including, but not limited to, water-stress, ABA and
turgor-inducible promoters.
[0123] A preselected DNA segment can be combined with the promoter
by standard methods as described in Sambrook et al., cited supra.
Briefly, a plasmid containing a promoter such as the 35S-CaMV
promoter can be constructed as described in Jefferson, Plant
Molecular Biology Reporter, 5, 387 (1987) or obtained from Clontech
Lab in Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, these
plasmids are constructed to provide for multiple cloning sites
having specificity for different restriction enzymes downstream
from the promoter. The preselected DNA segment can be subcloned
downstream from the promoter using restriction enzymes to ensure
that the DNA is inserted in proper orientation with respect to the
promoter so that the DNA can be expressed. In a preferred version,
a bacterial MIPD gene is operably linked to a 35S CaMV promoter in
a plasmid. Once the preselected DNA segment is operably linked to a
promoter, the expression cassette so formed can be subcloned into a
plasmid or other vectors.
[0124] 2. Optional Sequences in the Expression Cassette
[0125] The expression cassette can also optionally contain other
DNA sequences. Transcription enhancers or duplications of enhancers
can be used to increase expression from a particular promoter.
Examples of such enhancers include, but are not limited to,
elements from the CaMV 35S promoter and octopine synthase genes
(Last et al., U.S. Pat. No. 5,290,924, issued Mar. 1, 1994). For
example, it is contemplated that vectors for use in accordance with
the present invention may be constructed to include the ocs
enhancer element. This element was first identified as a 16 bp
palindromic enhancer from the octopine synthase (ocs) gene of
Agrobacterium (Ellis et al., EMBO J., 6, 3203 (1987)), and is
present in at least 10 other promoters (Bouchez et al., EMBO J., 8,
4197 (1989)). It is proposed that the use of an enhancer element,
such as the ocs element and particularly multiple copies of the
element, will act to increase the level of transcription from
adjacent promoters when applied in the context of monocot
transformation. Tissue-specific promoters, including but not
limited to, root-cell promoters (Conkling et al., Plant Physiol.,
93, 1203 (1990)), and tissue-specific enhancers (Fromm et al., The
Plant Cell, 1, 977 (1989)) are also contemplated to be particularly
useful, as are inducible-promoters such as water-stress-, ABA- and
turgor-inducible promoters (Guerrero et al., Plant Molecular
Biology 15: 11-26), and the like.
[0126] Tissue specific expression may be functionally accomplished
by introducing a constitutively expressed gene (all tissues) in
combination with an antisense gene that is expressed only in those
tissues where the gene product is not desired. For example, a
preselected DNA segment encoding an enzyme which catalyzes the
synthesis of an osmoprotectant, may be introduced so that it is
expressed in all tissues using the 35S promoter from Cauliflower
Mosaic Virus. Expression of an antisense transcript of this
preselected DNA segment in a maize kernel, using, for example, a
zein promoter, would prevent accumulation of the gene product in
seed. Hence the protein encoded by the preselected DNA would be
present in all tissues except the kernel.
[0127] Alternatively, one may wish to obtain novel tissue-specific
promoter sequences for use in accordance with the present
invention. To achieve this, one may first isolate cDNA clones from
the tissue concerned and identify those clones which are expressed
specifically in that tissue, for example, using Northern blotting.
Ideally, one would like to identify a gene that is not present in a
high copy number, but which gene product is relatively abundant in
specific tissues. The promoter and control elements of
corresponding genomic clones may then be localized using the
techniques of molecular biology known to those of skill in the
art.
[0128] Expression of some genes in transgenic plants will occur
only under specified conditions. For example, it is an object of
the present invention that expression of preselected DNA segment
that confer resistance to environmental stress factors such as
drought will occur only under actual stress conditions. Expression
of such genes throughout a plants development may have detrimental
effects. It is known that a large number of genes exist that
respond to the environment. For example, expression of some genes
such as rbcS, encoding the small subunit of ribulose bisphosphate
carboxylase, is regulated by light as mediated through phytochrome.
Other genes are induced by secondary stimuli.
[0129] For example, synthesis of abscisic acid (ABA) is induced by
certain environmental factors, including, but not limited to, water
stress. A number of genes have been shown to be induced by ABA
(Skriver et al., Plant Cell, 2, 503 (1990)). Therefore, inducible
expression of a preselected DNA segment in transgenic plants can
occur.
[0130] In some embodiments of the present invention expression of a
preselected DNA segment in a transgenic plant will occur only in a
certain time period during the development of the plant.
Developmental timing is frequently correlated with tissue specific
gene expression. For example, expression of zein storage proteins
is initiated in the endosperm about 15 days after pollination.
[0131] As the DNA sequence inserted between the transcription
initiation site and the start of the coding sequence, i.e., the
untranslated leader sequence, can influence gene expression, one
can also employ a particular leader sequence. Preferred leader
sequence include those which comprise sequences selected to direct
optimum expression of the attached gene, i.e., to include a
preferred consensus leader sequence which can increase or maintain
MRNA stability and prevent inappropriate initiation of translation
(Joshi, Nucl. Acid Res., 15, 6643 (1987)). Such sequences are known
to those of skill in the art. Sequences that are derived from genes
that are highly expressed in plants, and in maize in particular,
will be most preferred.
[0132] Regulatory elements such as Adh intron I (Callis et al.,
Genes Develop., 1, 1183 (1987)), sucrose synthase intron (Vasil et
al., Plant Physiol., 91, 5175 (1989)) or TMV omega element (Gallie
et al., The Plant Cell 1, 301 (1989)) can also be included where
desired. Other such regulatory elements useful in the practice of
the invention are known to those of skill in the art.
[0133] Additionally, expression cassettes can be constructed and
employed to target the gene product of the preselected DNA segment
to an intracellular compartment within plant cells or to direct a
protein to the extracellular environment. This can generally be
achieved by joining a DNA sequence encoding a transit or signal
peptide sequence to the coding sequence of the preselected DNA
segment. The resultant transit, or signal, peptide will transport
the protein to a particular intracellular, or extracellular
destination, respectively, and can then be post-translationally
removed. Transit or signal peptides act by facilitating the
transport of proteins through intracellular membranes, e.g.,
vacuole, vesicle, plastid and mitochondrial membranes, whereas
signal peptides direct proteins through the extracellular membrane.
By facilitating transport of the protein into compartments inside
or outside the cell, these sequences can increase the accumulation
of gene product.
[0134] The preselected DNA segment can be directed to a particular
organelle, such as the chloroplast rather than to the cytoplasm.
Thus, the expression cassette can further be comprised of a
chloroplast transit peptide encoding DNA sequence operably linked
between a promoter and the preselected DNA segment (for a review of
plastid targeting peptides, see Heijne et al., Eur. J. Biochem.,
180, 535 (1989); Keegstra et al., Ann. Rev. Plant Physiol. Plant
Mol. Biol., 40, 471 (1989)). This is exemplified by the use of the
rbcS (RuBISCO) transit peptide which targets proteins specifically
to plastids.
[0135] An exogenous chloroplast transit peptide can be used. A
chloroplast transit peptide is typically 40 to 70 amino acids in
length and functions post-translationally to direct a protein to
the chloroplast. The transit peptide is cleaved either during or
just after import into the chloroplast to yield the mature protein.
The complete copy of the preselected DNA segment may contain a
chloroplast transit peptide sequence. In that case, it may not be
necessary to combine an exogenously obtained chloroplast transit
peptide sequence into the expression cassette.
[0136] Exogenous chloroplast transit peptide encoding sequences can
be obtained from a variety of plant nuclear genes, so long as the
products of the genes are expressed as preproteins comprising an
amino terminal transit peptide and transported into chloroplast.
Examples of plant gene products known to include such transit
peptide sequences include, but are not limited to, the small
subunit of ribulose biphosphate carboxylase, ferredoxin,
chlorophyll a/b binding protein, chloroplast ribosomal proteins
encoded by nuclear genes, certain heatshock proteins, amino acid
biosynthetic enzymes such as acetolactate acid synthase,
3-enolpyruvylphosphoshikimate synthase, dihydrodipicolinate
synthase, and the like. Alternatively, the DNA fragment coding for
the transit peptide may be chemically synthesized either wholly or
in part from the known sequences of transit peptides such as those
listed above.
[0137] Regardless of the source of the DNA fragment coding for the
transit peptide, it should include a translation initiation codon
and an amino acid sequence that is recognized by and will function
properly in chloroplasts of the host plant. Attention should also
be given to the amino acid sequence at the junction between the
transit peptide and the protein encoded by the preselected DNA
segment where it is cleaved to yield the mature enzyme. Certain
conserved amino acid sequences have been identified and may serve
as a guideline. Precise fusion of the transit peptide coding
sequence with the preselected DNA segment coding sequence may
require manipulation of one or both DNA sequences to introduce, for
example, a convenient restriction site. This may be accomplished by
methods including site-directed mutagenesis, insertion of
chemically synthesized oligonucleotide linkers, and the like.
[0138] Once obtained, the chloroplast transit peptide sequence can
be appropriately linked to the promoter and the preselected DNA
segment in an expression cassette using standard methods. Briefly,
a plasmid containing a promoter functional in plant cells and
having multiple cloning sites downstream can be constructed as
described in Jefferson, cited supra. The chloroplast transit
peptide sequence can be inserted downstream from the promoter using
restriction enzymes. The preselected DNA segment can then be
inserted immediately downstream from and in frame with the 3'
terminus of the chloroplast transit peptide sequence so that the
chloroplast transit peptide is linked to the amino terminus of the
protein encoded by the preselected DNA segment. Once formed, the
expression cassette can be subcloned into other plasmids or
vectors.
[0139] Targeting of the gene product to an intracellular
compartment within plant cells may also be achieved by direct
delivery of a preselected DNA segment to the intracellular
compartment. For example, an expression cassette encoding a
protein, the presence of which is desired in the chloroplast, may
be directly introduced into the chloroplast genome using the method
described in Maliga et al., U.S. Pat. No. 5,451,513, issued Sep.
19, 1995, incorporated herein by reference.
[0140] It may be useful to target DNA itself within a cell. For
example, it may be useful to target an introduced preselected DNA
to the nucleus as this may increase the frequency of
transformation. Within the nucleus itself, it would be useful to
target a gene in order to achieve site-specific integration. For
example, it would be useful to have a gene introduced through
transformation replace an existing gene in the cell.
[0141] When the expression cassette is to be introduced into a
plant cell, the expression cassette can also optionally include 3'
nontranslated plant regulatory DNA sequences that act as a signal
to terminate transcription and allow for the polyadenylation of the
resultant mRNA. The 3' nontranslated regulatory DNA sequence
preferably includes from about 300 to 1,000 nucleotide base pairs
and contains plant transcriptional and translational termination
sequences. Preferred 3' elements are derived from those from the
nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al.,
Nucl. Acid Res., 11 369 (1983)), the terminator for the T7
transcript from the octopine synthase gene of Agrobacterium
tumefaciens, and the 3' end of the protease inhibitor I or II genes
from potato or tomato, although other 3' elements known to those of
skill in the art can also be employed. These 3' nontranslated
regulatory sequences can be obtained as described in An, Methods in
Enzymology, 153, 292 (1987) or are already present in plasmids
available from commercial sources such as Clontech, Palo Alto,
Calif. The 3' nontranslated regulatory sequences can be operably
linked to the 3' terminus of the preselected DNA segment by
standard methods.
[0142] An expression cassette of the invention can also be further
comprise plasmid DNA. Plasmid vectors include additional DNA
sequences that provide for easy selection, amplification, and
transformation of the expression cassette in prokaryotic and
eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9,
pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors,
pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.
The additional DNA sequences include origins of replication to
provide for autonomous replication of the vector, selectable marker
genes, preferably encoding antibiotic or herbicide resistance,
unique multiple cloning sites providing for multiple sites to
insert DNA sequences or genes encoded in the expression cassette,
and sequences that enhance transformation of prokaryotic and
eukaryotic cells.
[0143] Another vector that is useful for expression in both plant
and prokaryotic cells is the binary Ti plasmid (as disclosed in
Schilperoort et al., U.S. Pat. No. 4,940,838, issued Jul. 10, 1990)
as exemplified by vector pGA582. This binary Ti plasmid vector has
been previously characterized by An, cited supra, and is available
from Dr. An. This binary Ti vector can be replicated in prokaryotic
bacteria such as E. coli and Agrobacterium. The Agrobacterium
plasmid vectors can be used to transfer the expression cassette to
plant cells. The binary Ti vectors preferably include the nopaline
T DNA right and left borders to provide for efficient plant cell
transformation, a selectable marker gene, unique multiple cloning
sites in the T border regions, the colE1 replication of origin and
a wide host range replicon. The binary Ti vectors carrying an
expression cassette of the invention can be used to transform both
prokaryotic and eukaryotic cells, but is preferably used to
transform plant cells.
[0144] III. DNA Delivery
[0145] Following the generation of recipient cells, the present
invention generally next includes steps directed to introducing a
preselected DNA segment or segment, such as a preselected cDNA,
into a recipient cell to create a transformed cell. The frequency
of occurrence of cells receiving DNA is believed to be low.
Moreover, it is most likely that not all recipient cells receiving
DNA segments or sequences will result in a transformed cell wherein
the DNA is stably integrated into the plant genome and/or
expressed. Some may show only initial and transient gene
expression. However, certain cells from virtually any monocot
species may be stably transformed, and these cells developed into
transgenic plants, through the application of the techniques
disclosed herein.
[0146] An expression cassette of the invention can be introduced by
methods of transformation especially effective for monocots,
including, but not limited to, microprojectile bombardment of
immature embryos (U.S. patent application Ser. No. 08/249,458,
filed May 26, 1994, incorporated by reference herein; U.S. patent
application Ser. No. 08/112,245, filed Aug. 25, 1993, incorporated
by reference herein) or Type II embryogenic callus cells as
described by W. J. Gordon-Kamm et al. (Plant Cell, 2, 603 (1990)),
M. E. Fromm et al. (Bio/Technology, 8, 833 (1990)) and D. A.
Walters et al. (Plant Molecular Biology, 18, 189 (1992)), or by
electroporation of type I embryogenic calluses described by
D'Halluin et al. (The Plant Cell, 4 1495 (1992)), or by Krzyzek et
al. (U.S. Pat. No. 5,384,253, issued Jan. 24, 1995).
[0147] A. Electroporation
[0148] Where one wishes to introduce DNA by means of
electroporation, it is contemplated that the method of Krzyzek et
al. (U.S. Pat. No. 5,384,253, issued Jan. 24, 1995, incorporated
herein by reference) will be particularly advantageous. In this
method, certain cell wall-degrading enzymes, such as
pectin-degrading enzymes, are employed to render the target
recipient cells more susceptible to transformation by
electroporation than untreated cells. Alternatively, recipient
cells are made more susceptible to transformation, by mechanical
wounding.
[0149] To effect transformation by electroporation one may employ
either friable tissues such as a suspension culture of cells, or
embryogenic callus, or alternatively, one may transform immature
embryos or other organized tissues directly. One would partially
degrade the cell walls of the chosen cells by exposing them to
pectin-degrading enzymes (pectolyases) or mechanically wounding in
a controlled manner. Such cells would then be recipient to DNA
transfer by electroporation, which may be carried out at this
stage, and transformed cells then identified by a suitable
selection or screening protocol dependent on the nature of the
newly incorporated DNA.
[0150] B. Microprojectile Bombardment
[0151] A further advantageous method for delivering transforming
DNA segments to plant cells is microprojectile bombardment. In this
method, particles may be coated with nucleic acids and delivered
into cells by a propelling force. Exemplary particles include those
comprised of tungsten, gold, platinum, and the like.
[0152] It is contemplated that in some instances DNA precipitation
onto metal particles would not be necessary for DNA delivery to a
recipient cell using microprojectile bombardment. In an
illustrative embodiment, non-embryogenic BMS cells were bombarded
with intact cells of the bacteria E. coli or Agrobacterium
tumefaciens containing plasmids with either the
.beta.-glucoronidase or bar gene engineered for expression in
maize. Bacteria were inactivated by ethanol dehydration prior to
bombardment. A low level of transient expression of the
.beta.-glucoronidase gene was observed 24-48 hours following DNA
delivery. In addition, stable transformants containing the bar gene
were recovered following bombardment with either E. coli or
Agrobacterium tumefaciens cells. It is contemplated that particles
may contain DNA rather than be coated with DNA. Hence it is
proposed that DNA-coated particles may increase the level of DNA
delivery via particle bombardment but are not, in and of
themselves, necessary.
[0153] An advantage of microprojectile bombardment, in addition to
it being an effective means of reproducibly stably transforming
monocots, is that neither the isolation of protoplasts (Cristou et
al., Plant Physiol., 87, 671 (1988)) nor the susceptibility to
Agrobacterium infection is required. An illustrative embodiment of
a method for delivering DNA into maize cells by acceleration is a
Biolistics Particle Delivery System, which can be used to propel
particles coated with DNA or cells through a screen, such as a
stainless steel or Nytex screen, onto a filter surface covered with
maize cells cultured in suspension (Gordon-Kamm et al., The Plant
Cell, 2, 603 (1990)). The screen disperses the particles so that
they are not delivered to the recipient cells in large aggregates.
It is believed that a screen intervening between the projectile
apparatus and the cells to be bombarded reduces the size of
projectiles aggregate and may contribute to a higher frequency of
transformation by reducing damage inflicted on the recipient cells
by projectiles that are too large.
[0154] For the bombardment, cells in suspension are preferably
concentrated on filters or solid culture medium. Alternatively,
immature embryos or other target cells may be arranged on solid
culture medium. The cells to be bombarded are positioned at an
appropriate distance below the macroprojectile stopping plate. If
desired, one or more screens are also positioned between the
acceleration device and the cells to be bombarded. Through the use
of techniques set forth herein one may obtain up to 1000 or more
foci of cells transiently expressing a marker gene. The number of
cells in a focus which express the exogenous gene product 48 hours
post-bombardment often range from about 1 to 10 and average about 1
to 3.
[0155] In bombardment transformation, one may optimize the
prebombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment, and also the nature of the
transforming DNA, such as linearized DNA or intact supercoiled
plasmids. It is believed that prebombardment manipulations are
especially important for successful transformation of immature
embryos.
[0156] Accordingly, it is contemplated that one may wish to adjust
various of the bombardment parameters in small scale studies to
fully optimize the conditions. One may particularly wish to adjust
physical parameters such as gap distance, flight distance, tissue
distance, and helium pressure. One may also minimize the trauma
reduction factors (TRFs) by modifying conditions which influence
the physiological state of the recipient cells and which may
therefore influence transformation and integration efficiencies.
For example, the osmotic state, tissue hydration and the subculture
stage or cell cycle of the recipient cells may be adjusted for
optimum transformation. Results from such small scale optimization
studies are disclosed herein and the execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0157] IV. Production and Characterization of Stable Transgenic
Maize
[0158] After effecting delivery of a preselected DNA segment to
recipient cells by any of the methods discussed above, the next
steps of the invention generally concern identifying the
transformed cells for further culturing and plant regeneration. As
mentioned above, in order to improve the ability to identify
transformants, one may desire to employ a selectable or screenable
marker gene as, or in addition to, the expressible preselected DNA
segment. In this case, one would then generally assay the
potentially transformed cell population by exposing the cells to a
selective agent or agents, or one would screen the cells for the
desired marker gene trait.
[0159] A. Selection
[0160] An exemplary embodiment of methods for identifying
transformed cells involves exposing the bombarded cultures to a
selective agent, such as a metabolic inhibitor, an antibiotic,
herbicide or the like. Cells which have been transformed and have
stably integrated a marker gene conferring resistance to the
selective agent used, will grow and divide in culture. Sensitive
cells will not be amenable to further culturing.
[0161] To use the bar-bialaphos or the EPSPS-glyphosate selective
system, bombarded tissue is cultured for about 0-28 days on
nonselective medium and subsequently transferred to medium
containing from about 1-3 mg/l bialaphos or about 1-3 mM
glyphosate, as appropriate. While ranges of about 1-3 mg/l
bialaphos or about 1-3 mM glyphosate will typically be preferred,
it is proposed that ranges of at least about 0.1-50 mg/l bialaphos
or at least about 0.1-50 mM glyphosate will find utility in the
practice of the invention. Tissue can be placed on any porous,
inert, solid or semi-solid support for bombardment, including but
not limited to filters and solid culture medium. Bialaphos and
glyphosate are provided as examples of agents suitable for
selection of transformants, but the technique of this invention is
not limited to them.
[0162] An example of a screenable marker trait is the red pigment
produced under the control of the R-locus in maize. This pigment
may be detected by culturing cells on a solid support containing
nutrient media capable of supporting growth at this stage and
selecting cells from colonies (visible aggregates of cells) that
are pigmented. These cells may be cultured further, either in
suspension or on solid media. The R-locus is useful for selection
of transformants from bombarded immature embryos. In a similar
fashion, the introduction of the C1 and B genes will result in
pigmented cells and/or tissues.
[0163] The enzyme luciferase is also useful as a screenable marker
in the context of the present invention. In the presence of the
substrate luciferin, cells expressing luciferase emit light which
can be detected on photographic or x-ray film, in a luminometer (or
liquid scintillation counter), by devices that enhance night
vision, or by a highly light sensitive video camera, such as a
photon counting camera. All of these assays are nondestructive and
transformed cells may be cultured further following identification.
The photon counting camera is especially valuable as it allows one
to identify specific cells or groups of cells which are expressing
luciferase and manipulate those in real time.
[0164] It is further contemplated that combinations of screenable
and selectable markers will be useful for identification of
transformed cells. In some cell or tissue types a selection agent,
such as bialaphos or glyphosate, may either not provide enough
killing activity to clearly recognize transformed cells or may
cause substantial nonselective inhibition of transformants and
nontransformants alike, thus causing the selection technique to not
be effective. It is proposed that selection with a growth
inhibiting compound, such as bialaphos or glyphosate at
concentrations below those that cause 100% inhibition followed by
screening of growing tissue for expression of a screenable marker
gene such as luciferase would allow one to recover transformants
from cell or tissue types that are not amenable to selection alone.
In an illustrative embodiment embryogenic type II callus of Zea
mays L. was selected with sub-lethal levels of bialaphos. Slowly
growing tissue was subsequently screened for expression of the
luciferase gene and transformants were identified. In this example,
neither selection nor screening conditions employed were sufficient
in and of themselves to identify transformants. Therefore it is
proposed that combinations of selection and screening will enable
one to identify transformants in a wider variety of cell and tissue
types.
[0165] B. Regeneration and Seed Production
[0166] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In an
exemplary embodiment, MS and N6 media have been modified (see Table
1) by including further substances such as growth regulators. A
preferred growth regulator for such purposes is dicamba or 2,4-D.
However, other growth regulators may be employed, including NAA,
NAA+2,4-D or perhaps even picloram. Media improvement in these and
like ways was found to facilitate the growth of cells at specific
developmental stages. Tissue is preferably maintained on a basic
media with growth regulators until sufficient tissue is available
to begin plant regeneration efforts, or following repeated rounds
of manual selection, until the morphology of the tissue is suitable
for regeneration, at least two weeks, then transferred to media
conducive to maturation of embryoids. Cultures are transferred
every two weeks on this medium. Shoot development will signal the
time to transfer to medium lacking growth regulators.
[0167] The transformed cells, identified by selection or screening
and cultured in an appropriate medium that supports regeneration,
will then be allowed to mature into plants. Developing plantlets
are transferred to soilless plant growth mix, and hardened, e.g.,
in an environmentally controlled chamber at about 85% relative
humidity, about 600 ppm CO.sub.2, and at about 25-250
microeinsteins m.sup.-2-s.sup.-1 of light. Plants are preferably
matured either in a growth chamber or greenhouse. Plants are
regenerated from about 6 weeks to 10 months after a transformant is
identified, depending on the initial tissue. During regeneration,
cells are grown on solid media in tissue culture vessels.
Illustrative embodiments of such vessels are petri dishes and Plant
Con.RTM.s. Regenerating plants are preferably grown at about 19 to
28.degree. C. After the regenerating plants have reached the stage
of shoot and root development, they may be transferred to a
greenhouse for further growth and testing.
[0168] Mature plants are then obtained from cell lines that are
known to express the trait. If possible, the regenerated plants are
self pollinated. In addition, pollen obtained from the regenerated
plants is crossed to seed grown plants of agronomically important
inbred lines. In some cases, pollen from plants of these inbred
lines is used to pollinate regenerated plants. The trait is
genetically characterized by evaluating the segregation of the
trait in first and later generation progeny. The heritability and
expression in plants of traits selected in tissue culture are of
particular importance if the traits are to be commercially
useful.
[0169] Regenerated plants can be repeatedly crossed to inbred maize
plants in order to introgress the preselected DNA segment into the
genome of the inbred maize plants. This process is referred to as
backcross conversion. When a sufficient number of crosses to the
recurrent inbred parent have been completed in order to produce a
product of the backcross conversion process that is substantially
isogenic with the recurrent inbred parent except for the presence
of the introduced preselected DNA segment, the plant is
self-pollinated at least once in order to produce a homozygous
backcross converted inbred containing the preselected DNA segment.
Progeny of these plants are true breeding and the level of an
osmoprotectant, or the degree of resistance or tolerance to a
reduction in water availability, in these progeny are compared to
the level of the osmoprotectant, or the degree of resistance or
tolerance to a reduction in water availability, in the recurrent
parent inbred, in the field under a range of environmental
conditions (see below). The determination of the level of tolerance
or resistance to a reduction in water availability are well known
in the art.
[0170] Alternatively, seed from transformed monocot plants
regenerated from transformed tissue cultures is grown in the field
and self-pollinated to generate true breeding plants. Progenies
from these plants become true breeding lines which are evaluated
for resistance or tolerance to reduced water availability, or
production of an osmoprotectant, in the field under a range of
environmental conditions.
[0171] Progeny and subsequent generations are grown in the field
and assayed for their performance under a range of water
availability conditions. Both qualitative and quantitative measures
of the plant's ability to withstand water stress are made. Seeds
are germinated in the greenhouses, growth chambers and field
conditions under ample water supply. At one or more times during
the plant's life cycle, water availability is reduced in order to
identify plants that exhibit tolerance or resistance to a reduction
in water availability. In addition to the visual signs of wilting,
which may only be observed under more pronounced drought stress,
measures of plant water status are made. These measures include,
but are not limited to, total water potential, osmotic potential
and turgor potential are quantitatively measured and detection of
differences in turgor or the ability of the plants not to wilt.
These measurements can be made even when no signs of plant stress
are visible to the eye. Plants expressing the most favorable water
status result in superior growth under water stress. Different
measures of growth are used to document this superior performance
including, but not limited to, measures of cell and leaf area
expansion.
[0172] The physiological and biochemical activity of the
transformed plant tissue is indicative of its improved stress
tolerance. Such screening of plants with the measurement of
photosynthetic activity or transpirational activity are only two
examples of the types of measurement that can be done to identify
the superiority of the transgenic plants compared to
non-transformed plants. Measurements of reproductive capacity
including, but not limited to, the synchrony of pollen shed and
silk emergence are indicators of improved stress tolerance when the
preselected DNA segment is expressed. It is contemplated that
barrenness will not be a problem.
[0173] Once the initial breeding lines are selected by criteria,
which may include the criteria described above, test crosses are
made and hybrid seed is produced. The testcross hybrids and
breeding populations are planted in several different arrays in the
field. One scheme of evaluation is to grow populations of hybrid
plants containing the preselected DNA segment in many different
locations and measure the performance of the plants at these
different locations. Given the variability of rainfall
distribution, the different locations receive different quantities
of rainfall and in some locations, the plants will receive stress.
Yield information as well as measures which quantify plant response
to stress as described earlier, are made. The information regarding
the performance of these hybrids along with that of the performance
of non-transformed hybrids is compared. It is anticipated that the
hybrids expressing the preselected DNA segment will be higher in
yield performance and stability at a given level of water
availability than the controls.
[0174] Where irrigation is available, more controlled comparisons
are made through the establishment of differential irrigation
treatments. The same entries of hybrids or lines are grown under
contrasting irrigation treatments. Such an approach limits the
number of variables at work in the evaluation. Aside from the same
types of measurements as defined above, differential responses are
calculated because of the contrast in the data. It is anticipated
that preselected DNA segment expressing hybrids will have less
yield reduction when grown under irrigated versus non-irrigated
conditions when compared to hybrids without the gene.
[0175] Upon the identification of the superior performance of
transgenic plants, the parent selections are advanced and inbred
lines are produced through conventional breeding techniques. Hybrid
plants having one or more parents containing the preselected DNA
segment are tested in commercial testing and evaluation programs
and performance documented. This testing includes performance
trials over a wide geographical area as well as dedicated trials
where water availability is varied to reveal performance advantage
and hence value.
[0176] An additional advantage of the expression of the preselected
DNA segment is the superior performance of the parental inbred
lines in production of hybrids. Less stress related parent yield
loss is associated with higher green seed yield and thereby higher
economic margins.
[0177] It is anticipated that the performance advantage will not
only be present under stress conditions. Given the overall role of
water in determining yield, it is contemplated that maize plants
expressing the preselected DNA segment may utilize water more
efficiently. This will improve overall performance even when soil
water availability is not limiting. Through the introduction of the
preselected DNA segment(s) and the improved ability of maize to
maximize water usage across a full range of conditions relating to
water availability (i.e., including normal and stressed
conditions), yield stability or consistency of yield performance
will be achieved. These studies are conducted in maize and other
monocots.
[0178] C. Characterization
[0179] To confirm the presence of the preselected DNA segment(s) or
"transgene(s)" in the regenerating plants, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays well known to those of skill in the art, such as
Southern and Northern blotting and PCR; "biochemical" assays, such
as detecting the presence of a protein product, e.g., by
immunological means (ELISAs and Western blots) or by enzymatic
function; plant part assays, such as leaf or root assays; and also,
by analyzing the phenotype of the whole regenerated plant.
[0180] 1. DNA Integration, RNA Expression and Inheritance
[0181] Genomic DNA may be isolated from callus cell lines or any
plant parts to determine the presence of the preselected DNA
segment through the use of techniques well known to those skilled
in the art. Note that intact sequences will not always be present,
presumably due to rearrangement or deletion of sequences in the
cell.
[0182] The presence of DNA elements introduced through the methods
of this invention may be determined by polymerase chain reaction
(PCR). Using this technique discreet fragments of DNA are amplified
and detected by gel electrophoresis. This type of analysis permits
one to determine whether a preselected DNA segment is present in a
stable transformant, but does not prove integration of the
introduced preselected DNA segment into the host cell genome. In
addition, it is not possible using PCR techniques to determine
whether transformants have exogenous genes introduced into
different sites in the genome, i.e., whether transformants are of
independent origin. It is contemplated that using PCR techniques it
would be possible to clone fragments of the host genomic DNA
adjacent to an introduced preselected DNA segment.
[0183] Positive proof of DNA integration into the host genome and
the independent identities of transformants may be determined using
the technique of Southern hybridization. Using this technique
specific DNA sequences that were introduced into the host genome
and flanking host DNA sequences can be identified. Hence the
Southern hybridization pattern of a given transformant serves as an
identifying characteristic of that transformant. In addition it is
possible through Southern hybridization to demonstrate the presence
of introduced preselected DNA segments in high molecular weight
DNA, i.e., confirm that the introduced preselected DNA segment has
been integrated into the host cell genome. The technique of
Southern hybridization provides information that is obtained using
PCR, e.g., the presence of a preselected DNA segment, but also
demonstrates integration into the genome and characterizes each
individual transformant.
[0184] It is contemplated that using the techniques of dot or slot
blot hybridization which are modifications of Southern
hybridization techniques one could obtain the same information that
is derived from PCR, e.g., the presence of a preselected DNA
segment.
[0185] Both PCR and Southern hybridization techniques can be used
to demonstrate transmission of a preselected DNA segment to
progeny. In most instances the characteristic Southern
hybridization pattern for a given transformant will segregate in
progeny as one or more Mendelian genes (Spencer et al., Plant Mol.
Biol., 18, 201 (1992); Laursen et al., Plant Mo. Biol. 24, 51
(1994)) indicating stable inheritance of the gene. For example, in
one study, of 28 progeny (Ra) plants tested, 50% (N=14) contained
bar, confirming transmission through the germline of the marker
gene. The nonchimeric nature of the callus and the parental
transformants (R.sub.0) was suggested by germline transmission and
the identical Southern blot hybridization patterns and intensities
of the transforming DNA in callus, R.sub.0 plants and R.sub.1
progeny that segregated for the transformed gene.
[0186] Whereas DNA analysis techniques may be conducted using DNA
isolated from any part of a plant, RNA may only be expressed in
particular cells or tissue types and hence it will be necessary to
prepare RNA for analysis from these tissues. PCR techniques may
also be used for detection and quantitation of RNA produced from
introduced preselected DNA segments. In this application of PCR it
is first necessary to reverse transcribe RNA into DNA, using
enzymes such as reverse transcriptase, and then through the use of
conventional PCR techniques amplify the DNA. In most instances PCR
techniques, while useful, will not demonstrate integrity of the RNA
product. Further information about the nature of the RNA product
may be obtained by Northern blotting. This technique will
demonstrate the presence of an RNA species and give information
about the integrity of that RNA. The presence or absence of an RNA
species can also be determined using dot or slot blot Northern
hybridizations. These techniques are modifications of Northern
blotting and will only demonstrate the presence or absence of an
RNA species.
[0187] 2. Gene Expression
[0188] While Southern blotting and PCR may be used to detect the
preselected DNA segment in question, they do not provide
information as to whether the preselected DNA segment is being
expressed. Expression may be evaluated by specifically identifying
the protein products of the introduced preselected DNA segments or
evaluating the phenotypic changes brought about by their
expression.
[0189] Assays for the production and identification of specific
proteins may make use of physical-chemical, structural, functional,
or other properties of the proteins. Unique physical-chemical or
structural properties allow the proteins to be separated and
identified by electrophoretic procedures, such as native or
denaturing gel electrophoresis or isoelectric focussing, or by
chromatographic techniques such as ion exchange or gel exclusion
chromatography. The unique structures of individual proteins offer
opportunities for use of specific antibodies to detect their
presence in formats such as an ELISA assay. Combinations of
approaches may be employed with even greater specificity such as
western blotting in which antibodies are used to locate individual
gene products that have been separated by electrophoretic
techniques. Additional techniques may be employed to absolutely
confirm the identity of the product of interest such as evaluation
by amino acid sequencing following purification. Although these are
among the most commonly employed, other procedures may be
additionally used.
[0190] Assay procedures may also be used to identify the expression
of proteins by their functionality, especially the ability of
enzymes to catalyze specific chemical reactions involving specific
substrates and products. These reactions may be followed by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical or chemical procedures.
Examples are as varied as the enzyme to be analyzed and may include
assays for PAT enzymatic activity by following production of
radiolabelled acetylated phosphinothricin from phosphinothricin and
.sup.14C-acetyl CoA or for anthranilate synthase activity by
following loss of fluorescence of anthranilate, to name two.
[0191] Very frequently the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
These assays also may take many forms including but not limited to
analyzing changes in the chemical composition, morphology, or
physiological properties of the plant. Chemical composition may be
altered by expression of preselected DNA segments encoding enzymes
or storage proteins which change amino acid composition and may be
detected by amino acid analysis, or by enzymes which change starch
quantity which may be analyzed by near infrared reflectance
spectrometry. Morphological changes may include greater stature or
thicker stalks. Most often changes in response of plants or plant
parts to imposed treatments are evaluated under carefully
controlled conditions termed bioassays.
[0192] D. Establishment of the Introduced DNA in Other Maize
Varieties
[0193] Fertile, transgenic plants may then be used in a
conventional maize breeding program in order to incorporate the
preselected DNA segment into the desired lines or varieties.
Methods and references for convergent improvement of maize are
given by Hallauer et al. (In: Corn and Corn Improvement, Sprague et
al. (eds.), pp. 463-564 (1988)), incorporated herein by reference.
Among the approaches that conventional breeding programs employ is
a conversion process (backcrossing). Briefly, conversion is
performed by crossing the initial transgenic fertile plant to elite
inbred lines. The progeny from this cross will segregate such that
some of the plants will carry the preselected DNA segment whereas
some will not. The plants that do carry the preselected DNA segment
are then crossed again to the elite inbred lines resulting in
progeny which segregate once more. This backcrossing process is
repeated until the original elite inbred has been converted to a
line containing the preselected DNA segment, yet possessing all
important attributed originally found in the parent. Generally,
this will require about 6-8 generations. A separate backcrossing
program will be generally used for every elite line that is to be
converted to a genetically engineered elite line.
[0194] Generally, the commercial value of the transformed maize
produced herein will be greatest if the preselected DNA segment can
be incorporated into many different hybrid combinations. A farmer
typically grows several hybrids based on differences in maturity,
standability, and other agronomic traits. Also, the farmer must
select a hybrid based upon his or her geographic location since
hybrids adapted to one region are generally not adapted to another
because of differences in such traits as maturity, disease, drought
and insect resistance. As such, it is necessary to incorporate the
gene into a large number of parental lines so that many hybrid
combinations can be produced containing the preselected DNA
segment.
[0195] Maize breeding and the techniques and skills required to
transfer genes from one line or variety to another are well known
to those skilled in the art.
[0196] Thus, introducing a preselected DNA segment, preferably in
the form of recombinant DNA, into any other line or variety can be
accomplished by these breeding procedures.
[0197] E. Uses of Transgenic Plants
[0198] The transgenic plants produced herein are expected to be
useful for a variety of commercial and research purposes.
Transgenic plants can be created for use in traditional agriculture
to possess traits beneficial to the grower (e.g., agronomic traits
such as resistance to water deficit, pest resistance, herbicide
resistance or increased yield), beneficial to the consumer of the
grain harvested from the plant (e.g., improved nutritive content in
human food or animal feed), or beneficial to the food processor
(e.g., improved processing traits). In such uses, the plants are
generally grown for the use of their grain in human or animal
foods. However, other parts of the plants, including stalks, husks,
vegetative parts, and the like, may also have utility, including
use as part of animal silage or for ornamental purposes. Often,
chemical constituents (e.g., oils or starches) of maize and other
crops are extracted for foods or industrial use and transgenic
plants may be created which have enhanced or modified levels of
such components.
[0199] Transgenic plants may also find use in the commercial
manufacture of proteins or other molecules, where the molecule of
interest is extracted or purified from plant parts, seeds, and the
like. Cells or tissue from the plants may also be cultured, grown
in vitro, or fermented to manufacture such molecules.
[0200] The transgenic plants may also bemused in commercial
breeding programs, or may be crossed or bred to plants of related
crop species. Improvements encoded by the preselected DNA segment
may be transferred, e.g., from maize cells to cells of other
species, e.g., by protoplast fusion.
[0201] The transgenic plants may have many uses in research or
breeding, including creation of new mutant plants through
insertional mutagenesis, in order to identify beneficial mutants
that might later be created by traditional mutation and selection.
An example would be the introduction of a recombinant DNA sequence
encoding a transposable element that may be used for generating
genetic variation. The methods of the invention may also be used to
create plants having unique "signature sequences" or other marker
sequences which can be used to identify proprietary lines or
varieties.
[0202] Success in producing fertile transgenic monocot plants
(maize) has now been achieved where others have failed by methods
described herein. Aspects of the methods of the present invention
for producing the fertile, transgenic maize plants comprise, but
are not limited to, isolation of recipient cells using media
conducive to specific growth patterns, choice of selective systems
that permit efficient detection of transformation; modifications of
DNA delivery methods to introduce genetic vectors with exogenous or
recombinant DNA into cells; invention of methods to regenerate
plants from transformed cells at a high frequency; and the
production of fertile transgenic plants capable of surviving and
reproducing.
[0203] F. Preferred Methods of Delivering DNA to Cells
[0204] Preferred DNA delivery systems do not require protoplast
isolation or use of Agrobacterium DNA. There are several potential
cellular targets for DNA delivery to produce fertile transgenic
plants: pollen, microspores, meristems, immature embryos and
cultured embryogenic cells are but a few examples.
[0205] One of the newly emerging techniques for the introduction of
preselected DNA segments into plant cells involves the use of
microprojectile bombardment. The details of this technique and its
use to introduce preselected DNA segment into various plant cells
are discussed in Klein et al. (Plant Physiol., 91, 440 (1989)),
Wang et al. (Plant Mol. Biol., 11, 433 (1988)) and Christou et al.
(Plant Physiol., 87, 671 (1988)). One method of determining the
efficiency of DNA delivery into the cells via microprojectile
bombardment employs detection of transient expression of the enzyme
.beta.-glucuronidase (GUS) in bombarded cells. For this method,
plant cells are bombarded with a DNA construct which directs the
synthesis of the GUS enzyme.
[0206] Apparati are available which perform microprojectile
bombardment. A commercially available source is an apparatus made
by Biolistics, Inc. (now bDuPont), but other microprojectile or
acceleration methods are within the scope of this invention. Of
course, other "gene guns" may be used to introduce DNA into
cells.
[0207] Several modifications of the microprojectile bombardment
method were made. For example, stainless steel mesh screens were
introduced below the stop plate of the bombardment apparatus, i.e.,
between the gun and the cells. Furthermore, modifications to
existing techniques were developed for precipitating DNA onto the
microprojectiles.
[0208] Another newly emerging technique for the introduction of
preselected DNA segment into plant cells is electroporation of
intact cells. The details of this technique are described in
Krzyzek et al. (U.S. Pat. No. 5,324,253, issued Jan. 24, 1995).
Similar to particle bombardment, the efficiency of DNA delivery
into cells by electroporation can be determined by using the
.beta.-glucuronidase gene. The method of electroporation of intact
cells and by extension intact tissues, e.g., immature embryos, were
developed by Krzyzek et al., and represent improvements over
published procedures. Generation of fertile plants using these
techniques were described by Spencer et al. (cited supra (1993))
and Laursen et al. (cited supra (1994)).
[0209] Other methods may also be used for introduction of DNA into
plants cells, e.g., agitation of cells with DNA and silicon carbide
fibers.
[0210] Histological analysis of stressed and unstressed tissue from
transformed and untransformed plants are performed (Sylvester et
al., Light Microscopy I: Dissection and Microtechnique, In: The
Maize Handbook, pp. 83-95, Springer-Verlag, NY (1994)). Cross
sections through the appropriate tissues (e.g. leaves or roots)
reveal any structural aberrations. Transmission and scanning
electron microscopy are used to characterize transformed and
untransformed cell structure and epidermal surfaces. Leaf surfaces
are also examined for normal stomate structure and density using
epidermal peels (Ristic et al., Bot. Gaz., 152, 173 (1991)).
[0211] The invention has been described with reference to various
specific and preferred embodiments and will be further described by
reference to the following detailed examples. It is understood,
however, that there are many extensions, variations, and
modifications on the basic theme of the present invention beyond
that shown in the examples and description, which are within the
spirit and scope of the present invention.
EXAMPLE I
[0212] Because a link was observed between (1) the maintenance of
turgor level via shifts in osmotic potential and yield of hybrids
under stress conditions, and (2) more negative osmotic potentials
and increased yield levels in hybrids under irrigated conditions,
monocot cells were transformed with a preselected DNA segment
encoding an enzyme which catalyzes the synthesis of an
osmoprotectant so as to result in a transformed monocot plant with
improved cellular osmotic relations. The expression of the
preselected DNA segment includes expression in the cytosol or the
chloroplast, or both. In addition to constitutive gene expression,
differential expression in shoots, roots and reproductive tissues,
developmental, temporal, as well as inducible expression of a
preselected DNA segment, is within the scope of the invention.
[0213] Monocot plant cells can be transformed with more than one
preselected DNA segment, so as to result in a synergistic effect
for plant performance, under either, or both, water-stress and non
water-stress conditions. Thus, it is also contemplated that
expression of a preselected DNA segment in plants, when those
plants are grown under relatively non-stress conditions or typical
conditions, can result in a yield performance over plants which do
not express the preselected DNA segment, or do not express the DNA
at altered, increased or elevated levels.
[0214] Construction of mtlD Vectors
[0215] One embodiment of the invention is a vector constructed to
direct constitutive expression of the preselected DNA segment. For
example, a preferred embodiment of the invention is an expression
cassette comprising the Cauliflower Mosaic Virus 35S promoter
(Odell et al., Nature (1985)) 5' to the mtlD gene. Alternatively
the rice actin gene promoter (Wang et al., Mol. Cell. Biol., 12,
3399 (1992)) is placed 5' of the mtlD gene. It is anticipated that
all promoters which direct constitutive gene expression in maize
will be useful when operably linked to a mtlD gene. Sequences which
direct polyadenylation are preferably linked 3' to the mtlD gene.
These sequences include, but are not limited to, DNA sequences
isolated from the 3' region of Agrobacterium tumefaciens nopaline
synthase, octopine synthase or transcript 7, or potato proteinase
inhibitor II genes. It is anticipated that constitutive expression
of the mtlD gene in all tissues of a monocot plant, such as maize,
will enhance the ability of the plant to maintain water turgor
under conditions of decreased water availability.
[0216] It is further contemplated that tissue specific expression
of a preselected DNA segment, e.g., mtlD, will enhance the
agronomic performance of a monocot plant, such as maize. Vectors
for use in tissue-specific targeting of mtlD genes in transgenic
plants will typically include tissue-specific promoters and may
also include other tissue-specific control elements such as
enhancer sequences. Promoters which direct specific or enhanced
expression in certain plant tissues will be known to those of skill
in the art in light of the present disclosure. These include, for
example, the rbcS promoter, specific for green tissue; the ocs, nos
and mas promoters which have higher activity in roots or wounded
leaf tissue; a truncated (-90 to +8) 35S promoter which directs
enhanced expression in roots, an a-tubulin gene that directs
expression in roots and promoters derived from zein storage protein
genes which direct expression in endosperm. It is particularly
contemplated that one may advantageously use the 16 bp ocs enhancer
element from the octopine synthase (ocs) gene (Ellis et al., EMBO
J. 6, 3203 (1987)); Bouchez et al., EMBO J., a, 4197 (1989)),
especially when present in multiple copies, to achieve enhanced
expression in roots.
[0217] Expression of mtlD in transgenic plants may be desired under
specified conditions. For example, the expression of mtlD genes may
be desired only under actual stress conditions. It is known that a
large number of genes exist that respond to the environment. For
example, expression of some genes such as rbcS, encoding the small
subunit of ribulose bisphosphate carboxylase, is regulated by light
as mediated through phytochrome. Other genes are induced-by
secondary stimuli. For example, synthesis of abscisic acid (ABA) is
induced by certain environmental factors, including but not limited
to water stress. A number of genes have been shown to be induced by
ABA (Skriver et al., Plant Cell, 2, 503 (1990)). Promoter regions
that regulate expression of these genes will be useful when
operably linked to mtlD.
[0218] It is proposed that in some embodiments of the present
invention expression of mtlD in a transgenic plant will be desired
only in a certain time period during the development of the plant.
Developmental timing is frequently correlated with tissue specific
gene expression. For example, expression of zein storage proteins
is initiated in the endosperm about 15 days after pollination.
[0219] To provide a transgenic monocot plant that is substantially
resistant or tolerant to a reduction in water availability, several
vectors were constructed containing a gene that encodes an enzyme
which catalyzes the synthesis of an osmoprotectant. Such genes
include, but are not limited to, the mtlD gene from E. coli and the
HVA-1 gene from barley. The mannitol operon was originally cloned
and characterized by Lee et al. (J. Bacteriol., 153, 685 (1983)).
The mtlD gene has been shown to confer water stress resistance on
transgenic tobacco plants (Tarczynski et al., Science, 259, 508
(1993)).
[0220] Construction of Vector pDPG451.
[0221] The mannitol operon (mtlC, mtla, mtlD) was obtained as a
plasmid in C600 E. coli from Malthius Muller, Univ. of Freiburg
(pDPG409). To isolate the plasmid DNA, the plasmid was first
amplified using chloramphenicol and then isolated using Qiagen
large-scale plasmid preparation. The mtlD gene was excised from the
pDPG409 plasmid by digesting the DNA with restriction enzymes NsiI
and PstI. The digested DNA was run on a 1.1% SeaKem agarose gel in
TAE buffer (see Sambrook et al., Molecular Cloning: A Laboratory
Manual (1989)) to separate the fragments by size and the
appropriate fragment was isolated from the gel using S&S NA45
membrane (Schleicher & Schuell, Keene, N.H.).
[0222] The mtlD gene fragment was next cloned into the maize
expression vector pDPG431 (35S promoter-adh1 Intron1-Tr7 3' end).
Vector pDPG431 DNA was digested with restriction enzymes NsiI and
PstI to open up the backbone and the mtlD fragment inserted by
ligation. The ligated DNA was transformed into DH5.alpha. cells and
the resulting colonies screened by mini-preps to identified those
containing the correct gene construct. The new vector was
designated pDPG451. A map of the plasmid is shown in FIG. 1.
[0223] Construction of Vector pDPG480.
[0224] The NsiI-PstI fragment from vector pDPG409 containing the
mtlD gene used to construct pDPG451 was cut with restriction
enzymes AvaI and HindIII. This removed about 122 bp of untranslated
sequence from the 5' end of the mtlD fragment and about 69 bp of
untranslated sequence from the 3' end of the fragment. The
AvaI-HindIII fragment was ligated into pUC19 DNA that had
previously been digested with AvaI and HindIII to open up the
plasmid backbone in the region of the multiple cloning sites. The
pUC19/mtlD construct was then digested with restriction enzymes
Sacd and HindIII to release a fragment containing the mtlD gene.
This fragment was isolated by running the digestion reaction on an
agarose gel and the appropriately-sized fragment extracted from the
gel using a S&S Elu-Quik DNA purification kit, per the
manufacturer's instructions.
[0225] The DNA fragment was next ligated into pcDNAII DNA that had
previously been digested with SacI and HindIII to open up the
plasmid backbone in the region of the multiple cloning sites. The
pcDNAII/mtlD vector was then digested with restriction enzymes
BamHI and PstI to release a fragment containing the mtlD gene. This
fragment was isolated by running the digestion reaction on an
agarose gel and the appropriately-sized fragment extracted from the
gel using a S&S Elu-Quik DNA purification kit, per the
manufacturer's instructions. The DNA fragment was next ligated into
pDPG431 DNA that had previously been digested with the restriction
enzymes BamHI and PstI and the backbone fragment containing the 35S
promoter-adhI IntronI and Tr7 3' end isolated by gel purification.
The resulting maize expression vector was designated pDPG480. A map
of the plasmid is shown in FIG. 3.
[0226] Construction of Vector pDPG493.
[0227] DNA from vector pDPG480 was modified to remove approximately
120 bp of untranslated DNA from the 3' end of the mtlD gene
fragment. To modify the 3' region, two oligonucleotides were made
(DNA International, Inc.) to anneal together and then used to
replace about 150 bp of the 3' end of the mtlD gene fragment. The
first oligonucleotide (mtlD-B1) had a sequence of: 5' GTA ACC GCT
TAT AAA GCA ATG CAA TAA TGA GTA CTC TGC AG 3' (SEQ ID NO: 1). The
second oligonucleotide (mtlD-B2) had a sequence of: 5' GAG TAC TCA
TTA TTG CAT TGC TTT ATA AGC G 3' (SEQ ID NO: 2). The annealed
oligos duplicated the last twenty base pairs of the mtlD gene
starting at the BstEII restriction site and running up to and
including the stop codon and created a new sequence after the stop
codon. This new sequence created new ScaI and PstI sites.
[0228] The new vector was constructed in the following manner.
Vector pDPG480 plasmid DNA was digested with restriction enzymes
BstEII and NsiI to remove the 3' end of the gene fragment. The
digested DNA was run on an agarose gel to size separate the
fragments and the appropriately-sized vector fragment was extracted
from the gel using a S&S Elu-Quik DNA purification kit, per the
manufacturer's instructions. Oligonucleotides mtlD-B1 and mtlD-B2
were annealed together and ligated into the digested pDPG480 DNA
fragment. The resulting vector was designated pDPG493. A map of the
plasmid is shown in FIG. 4.
[0229] Construction of Vector pDPG586.
[0230] A DNA fragment containing the mtlD gene was removed from
vector pDPG480 by digesting the plasmid DNA with restriction
enzymes BamHI and PstI. The DNA fragment containing the gene was
isolated by gel purification and extraction from the gel using a
S&S Elu-Quik DNA purification kit per the manufacturer's
instructions. A DNA fragment containing the G1b1 promoter and G1b1
terminator was isolated by digesting vector pDPG423 DNA with
restriction enzymes BamHI and PstI to open up the backbone in the
polylinker region. The two fragments were then ligated together to
create vector pDPG586. A map of the plasmid is shown in FIG. 5.
[0231] Construction of Vector pDPG587.
[0232] Vector pDPG411 was digested with restriction enzymes XhoI
and SacI to release a DNA fragment containing the 35S promoter and
a maize transit peptide sequence (MZTP). This DNA fragment was
isolated. by gel purification and extraction from the gel using a
S&S Elu-Quik DNA purification kit per the manufacturer's
instructions. A DNA backbone fragment containing the mtlD gene was
generated by digesting the pcDNAII/mtlD vector described above with
restriction enzymes XhoI and NsiI to open up the vector in the
polylinker region. These two fragments along with a Nsi-StuI-SacI
linker (Keystone Laboratories, Inc.) were ligated together to
create a vector designated MZTP/mtlD. Plasmid DNA of this vector
was digested with restriction enzyme PstI to open up the vector at
the 3' end of the mtlD gene sequence.
[0233] A DNA fragment containing the Tr7 terminator was isolated
from plasmid DNA of vector pDPG527 by digesting with restriction
enzyme PstI. This DNA fragment was isolated by gel purification and
extraction from the gel using a S&S Elu-Quik DNA purification
kit per the manufacturer's instructions. The MZTPImtlD and Tr7
terminator DNA fragments were ligated together to create the maize
expression vector pDPG587. The region from the end of the 35S
promoter, through the MZTP sequence and into the mtlD gene was
sequenced by dideoxy DNA sequencing to confirm the correct
composition of this region and to ensure that the MZTP and mtlD
gene are in frame with one another. A map of the plasmid is shown
in FIG. 6.
[0234] An additional expression vector for the mtlD gene was
created by removing the bar gene from pDPG182 using SmaI. After
blunting the ends of the mtlD gene, it was ligated into the
pUC-based vector; between the maize AdhII promoter/AdhI.sub.1
intron and the transcript 7 3' end from Agrobacterium lumefaciens
(provided in pCEV5 from Calgene, Inc., Davis, Calif.). This plasmid
vector was designated pDPG469.
EXAMPLE II
Preparation of Type II Callus for Transformation
[0235] Initiation and Maintenance of Cell Line AT824
[0236] Immature embryos (0.5-1.0 mm) were excised from the
B73-derived inbred line AT and cultured on N6 medium with 100 .mu.M
silver nitrate, 3.3 mg/L dicamba, 3% sucrose and 12 mM proline
(Medium 2004, see Table 1). Six months after initiation, type I
callus was transferred to Medium 2008. Two months later type I
callus was transferred to a medium with a lower concentration of
sucrose (Medium 279). A sector of type II callus was identified 17
months later and was transferred to Medium 279. This cell line is
uniform in nature, unorganized, rapid growing, and embryogenic.
This culture is easily adaptable to culture in liquid or on solid
medium.
[0237] The first suspension cultures of AT824 were initiated 31
months after culture initiation. Suspension cultures were initiated
in a variety of culture media including media containing 2,4-D as
well as dicamba as the auxin source, e.g., media designated 210,
401, 409, 279. Cultures were maintained by transfer of
approximately 2 ml packed cell volume (PCV) to 20 ml fresh culture
medium at 3.5 day intervals. AT824 was routinely transferred
between liquid and solid culture media with no effect on growth or
morphology.
[0238] Suspension cultures of AT824 were initially cryopreserved
33-37 months after culture initiation. The survival rate of this
culture was improved when it was cryopreserved following three
months in suspension culture. AT824 suspension cultures have been
cryopreserved and reinitiated from cryopreservation at regular
intervals since the initial date of freezing. Repeated cycles of
freezing have not affected the growth or transformability of this
culture.
1TABLE 1 Illustrative Embodiments of Tissue Culture Media Which are
Used for Type II Callus Development, Development of Suspension
Cultures and Regeneration of Plant Cells (Specifically Maize Cells)
OTHER BASAL COMPONENTS** MEDIA NO. MEDIUM SUCROSE pH (Amount/L) 101
MS 3% 6.0 MS vitamins 100 mg myo-inositol Bactoagar 189 MS -- 5.8 3
mg BAP .04 mg NAA .5 mg niacin 800 mg L-asparagine 100 mg
casaminoacids 20 g sorbitol 1.4 g L-proline 100 mg myo-inositol
Gelgro 201 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D 100 mg
casein hydrolysate 2.9 g L-proline Gelgro 210 N6 3% 5.5 N6 vitamins
2 mg 2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 790 mg
L-asparagine 100 mg casein hydrolysate 1.4 g L-proline 2 mg glycine
Hazelton agar 223 N6 2% 5.8 3.3 mg dicamba 1 mg thiamine 0.5 mg
niacin 800 mg L-asparagine 100 mg casein hydrolysate 100 mg
myo-inositol 1.4 g proline Gelgro 3 mg bialaphos 227 N6 2% 5.8 2 mg
L-glycine 100 mg casein hydrolysate 2.9 g L-proline Gelgro 279 N6
2% 5.8 3.3 mg dicamba 1 mg thiamine 0.5 mg niacin 800 mg
L-asparagine 100 mg casein hydrolysate 100 mg myo-inositol 1.4 g
proline Gelgro 401 MS 3% 6.0 3.73 mg Na.sub.2EDTA 0.25 mg thiamine
1 mg 2,4-D 2 mg NAA 200 mg casein hydrolysate 500 mg
K.sub.2SO.sub.4 400 mg KH.sub.2PO.sub.4 100 mg myo-inositol 409 MS
3% 6.0 3.73 mg Na.sub.2EDTA 0.25 mg thiamine 9.9 mg dicamba 200 mg
casein hydrolysate 500 mg K.sub.2SO.sub.4 400 mg KH.sub.2PO.sub.4
100 mg myo-inositol 425 MS 3% 6.0 3.73 mg Na.sub.2EDTA 0.25 mg
thiamine 9.9 mg dicamba 200 mg casein hydrolysate 500 mg
K.sub.2SO.sub.4 400 mg KH.sub.2PO.sub.4 100 mg myo-inositol 3 mg
bialaphos 501 Clark's 2% 5.7 Medium* 607 0.5 .times. MS 3% 5.8 0.5
mg thiamine 0.5 mg niacin Gelrite 734 N6 2% 5.8 N6 vitamins 2 mg
L-glycine 1.5 mg 2,4-D 14 g Fe sequestrene 200 mg casein
hydrolysate 0.69 g L-proline Gelrite 735 N6 2% 5.8 1 mg 2,4-D 0.5
mg niacin 0.91 g L-asparagine 100 mg myo-inositol 1 mg thiamine 0.5
g MES 0.75 g MgCl.sub.2 100 mg casein hydrolysate 0.69 g L-proline
Gelgro 739 N6 2% 5.8 1 mg 2,4-D 0.5 mg niacin 0.91 g L-asparagine
100 mg myo-inositol 1 mg thiamine 0.5 g MES 0.75 g MgCl.sub.2 100
mg casein hydrolysate 0.69 g L-proline Gelgro 1 mg bialaphos 750 N6
2% 5.8 1 mg 2,4-D 0.5 mg niacin 0.91 g L-asparagine 100 mg
myo-inositol 1 mg thiamine 0.5 g MES 0.75 g MgCl.sub.2 100 mg
casein hydrolysate 0.69 g L-proline Gelgro 0.2 M mannitol 1 mg
bialaphos 758 N6 2% 5.8 1 mg 2,4-D 0.5 mg niacin 0.91 g
L-asparagine 100 mg myo-inositol 1 mg thiamine 0.5 g MES 0.75 g
MgCl.sub.2 100 mg casein hydrolysate 0.69 g L-proline Gelgro 3 mg
bialaphos 2004 N6 3% 5.8 1 mg thiamine 0.5 mg niacin 3.3 mg dicamba
17 mg AgNO3 1.4 g L-proline 0.8 g L-asparagine 100 mg casein
hydrolysate 100 mg myo-inositol Gelrite 2008 N6 3% 5.8 1 mg
thiamine 0.5 mg niacin 3.3 mg dicamba 1.4 g L-proline 0.8 g
L-asparagine Basic MS medium described in Murashige et al., (cited
supra (1962)). This medium is typically # modified by decreasing
the NH.sub.4NO.sub.3 from 1.64 g/l to 1.55 g/l, and omitting the #
pyridoxine HCl, nicotinic acid, myo-inositol and glycine. N6 medium
described in Chu et al., Scientia Sinica 18 659 (1975). NAA =
Napthol Acetic Acid IAA = Indole Acetic Acid 2-IP = 2, isopentyl
adenine 2,4-D = 2,4-Dichlorophenoxyacetic Acid BAP = 6-benzyl
aminopurine ABA = abscisic acid *Basic medium described in Clark,
J. Plant Nutrition, 5, 1039 (1982)
[0239] Initiation and Maintenance of Type IT callus of the genotype
Hi-II.
[0240] The Hi-II genotype of corn was developed from an
A188.times.B73 cross. This genotype was developed specifically for
a high frequency of initiation of type II cultures (100% response
rate, Armstrong et al., Maize Genetics Coop Newsletter, 65, 92
(1991)). Immature embryos (8-12 days post-pollination, 1 to 1.2 mm)
were excised and cultured embryonic axis down on N6 medium
containing 1 mg/L 2,4-D, 25 mM L-proline (Medium 201) or N6 medium
containing 1.5 mg/L 2,4-D, 6 mM L-proline (Medium 734). Type II
callus was initiated either with or without the presence of 100
.mu.M AgNO.sub.3. Cultures initiated in the presence of AgNO.sub.3
were transferred to medium lacking this compound about 14-28 days
after culture initiation. Callus cultures were incubated in the
dark at about 23-28.degree. C. and transferred to fresh culture
medium at about 14-21 day intervals.
[0241] Hi-II type II callus was maintained by manual selection of
callus at each transfer. Alternatively, callus was resuspended in
liquid culture medium, passed through a 1.9 mm sieve and replated
on solid culture medium at the time of transfer. This sequence of
manipulations enriches for recipient cell types. Regenerable Type
II callus that is suitable for transformation was routinely
developed from the Hi-II genotype and hence new cultures were
developed every 6-9 months. Routine generation of new cultures
reduces the period of time over which each culture is maintained
and hence insures reproducible, highly regenerable, cultures that
routinely produce fertile plants.
[0242] Initiation of embryos of the genotype Hi-II.
[0243] Immature embryos of the Hi-II genotype (8-12 days post
pollination, 1.0-2.5 mm) were excised and cultured embryonic axis
down on Medium 201, or other equivalent or similar medias, with or
without the addition of 100CM AgNO.sub.3. Immature embryos were
cultured in the dark at about 23-28.degree. C. for about 0-14,
preferably about 2-4, days prior to transformation.
EXAMPLE III
[0244] Transformation of Cell Cultures
[0245] Microprojectile Bombardment: AT824.
[0246] AT824 suspension culture cells were subcultured to fresh
Medium 401, at about 0-3, preferably at about 2, days prior to
particle bombardment. Cells were plated on to solid Medium 279, or
other similar medias, at about 0-24, preferably about 4, hours
before bombardment of about 0.5-1.0 ml packed cell volume per
filter. Tissue can be treated with or without the addition of about
200 mOsm sorbitol or mannitol for about 0-5, preferably about 3,
hours prior to bombardment.
[0247] DNA was precipitated on to gold particles as follows. A
stock solution of gold particles was prepared by adding 60 mg of 1
.mu.m gold particles to 1000 .mu.l absolute ethanol and incubating
for at least 3 hours at room temperature followed by storage at
about -20.degree. C. Twenty to thirty-five .mu.l sterile gold
particles are centrifuged in a microcentrifuge for 1 minute. The
supernatant is removed and one ml sterile water is added to the
tube, followed by centrifugation at 2000 rpm for 5 minutes.
Microprojectile particles are resuspended in 30 .mu.g total DNA
containing a selectable marker, such as bar, EPSPS, or deh, and the
mtlD gene which is operably linked to a promoter. Approximately 220
.mu.l sterile water, 250 .mu.l 2.5 M CaCl.sub.2, and 50 .mu.l
spermidine stock are then added. The mixture is thoroughly mixed
and placed on ice, followed by vortexing at 4.degree. C. for 10
minutes and centrifugation at 500 rpm for 5 minutes. The
supernatant is removed and the pellet resuspended in 600 .mu.l
absolute ethanol. Following centrifugation at 500 rpm for 5 minutes
the pellet is resuspended in 36 .mu.l of absolute ethanol.
[0248] Approximately 5-10 .mu.l of the particle preparation was
dispensed on the surface of the flyer disk and the ethanol was
allowed to dry completely. DNA was introduced into cells using the
DuPont Biolistics PDS1OOOHe particle bombardment device. Particles
were accelerated by a helium blast of approximately 1100 psi. Zero
to seven, preferably about 1-4, days following bombardment, cells
were transferred to 10-20 mls liquid Medium 401, or other similar
medias. Tissue was subcultured twice per week. In most cases,
during the first week there was no selection pressure applied.
Microprojectile Bombardment: Type II callus from the genotype
Hi-II.
[0249] Hi-II callus cultures are bombarded similarly to AT824
suspension cultures. Approximately 0.5-1.0 ml packed cell volume
was plated on to Whatman filters after a brief liquid phase. Cells
were either plated on to solid media or left on a bed of wet
filters prior to bombardment. Cells can be bombarded with or
without the addition of an osmoticum before bombardment (liquid or
solid) in a manner similar to that described above for AT824.
Following particle bombardment cells remained on solid Medium 201,
or other similar medias, in the absence of selection for about 0-2
weeks, preferably for about 1 week. At this time cells were removed
from solid medium, resuspended in liquid Medium 201, or other
similar medias, replated on Whatman filters at about 0.1-1.0 ml PCV
per filter, and transferred to Medium 201, or other similar medias,
containing about 0.5-3.0 mg/L bialophos.
[0250] Bombardment of Immature Embryos.
[0251] Immature embryos (1.0-2.5 mm in length) were excised from
surface-sterilized, greenhouse-grown ears of Hi-II about 10-12 days
post-pollination. Approximately 30 embryos per petri dish were
plated axis side down on Medium 201, or other similar medias.
Embryos were cultured in the dark for about 1-14 days at about
23-28.degree. C.
[0252] Approximately four hours prior to bombardment, embryos were
transferred to Medium 201 with the sucrose concentration increased
from about 3% to 12%. When embryos were transferred to the high
osmoticum medium they were arranged in concentric circles on the
plate, starting 2 cm from the center of the dish, positioned such
that their coleorhizal end was orientated toward the center of the
dish. Usually two concentric circles were formed with about 25-35
embryos per plate.
[0253] Preparation of gold particles carrying plasmid DNA was
performed as described above. The plates containing embryos were
then placed on the third shelf from the bottom, at about 5 cm below
the stopping screen. The 1100 psi rupture discs were used. Each
plate of embryos was bombarded once. Embryos were allowed to
recover about 0-7, preferably about 1, days on high osmotic
strength medium prior to initiation of selection.
[0254] Stable Transformation of SC716 and AT824 Cells Using pDPGI65
and pDPG208 by Electroporation
[0255] Maize suspension culture cells were enzyme treated and
electroporated using conditions described in Krzyzek-et al. (PCT
Publication WO 92/12250, incorporated by reference herein). SC716
or AT824 suspension culture cells, three days post subculture, were
sieved through 1000 .mu.m stainless steel mesh and washed, 1.5 ml
packed cells per 10 ml, in incubation buffer (0.2 M mannitol, 0.1%
bovine serum albumin, 80 mM calcium chloride, and 20 mM
2-(N-morpholino)-ethane sulfonic acid (MES), pH 5.6). Cells were
then treated for 90 minutes in incubation buffer containing 0.5%
pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan) at a density
of 1.5 ml packed cells per 5 ml of enzyme solution. During the
enzyme treatment, cells were incubated in the dark at approximately
25.degree. C. on a rotary shaker at 60 rpm. Following pectolyase
treatment, cells were washed once with 10 ml of incubation buffer
followed by three washes with electroporation buffer (10 mM
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.4 mM
mannitol). Cells were resuspended in electroporation buffer at a
density of 1.5 ml packed cells in a total volume of 3 ml.
[0256] Linearized plasmid DNA, 100 .mu.g of EcoRI digested pDPG 165
and 100 .mu.g of EcoRI digested pDPG208, was added to 1 ml aliquots
of electroporation buffer. The DNA/electroporation buffer was
incubated at room temperature for approximately 10 minutes. To
these aliquots, 1 ml of suspension culture cells/electroporation
buffer (containing approximately 0.5 ml packed cells) were added.
Cells and DNA in electroporation buffer were incubated at room
temperature for approximately 10 minutes. One half ml aliquots of
this mixture were transferred to the electroporation chamber
(Puite, Plant Cell Rep., 4, 274 (1985)) which was placed in a
sterile 60.times.15 mm petri dish. Cells were electroporated with a
70, 100, or 140 volt (V) pulse discharged from a 140 microfarad
(.mu.f) capacitor.
[0257] Approximately 10 minutes post-electroporation, cells were
diluted with 2.5 ml Medium 409 containing 0.3 M mannitol. Cells
were then separated from most of the liquid medium by drawing the
suspension up in a pipet, and expelling the medium with the tip of
the pipet placed against the petri dish to retain the cells. The
cells, and a small amount of medium (approximately 0.2 ml) were
dispensed onto a filter (Whatman #1, 4.25 cm) overlaying solid
Medium 227 (Table 1) containing 0.3 M mannitol. After five days,
the tissue and the supporting filters were transferred to Medium
227 containing 0.2 M mannitol. After seven days, tissue and
supporting filters were transferred to Medium 227 without
mannitol.
[0258] Electroporation of Immature Embryos
[0259] Immature embryos (0.4-1.8 mm in length) were excised from a
surface-sterilized, greenhouse-grown ear of the genotype H99 11
days post-pollination. Embryos were plated axis side down on a
modified N6 medium containing 3.3 mg/l dicamba, 100 mg/l casein
hydrolysate, 12 mM L-proline, and 3% sucrose solidified with 2 g/l
Gelgro.RTM., pH 5.8 (Medium 726), with about 30 embryos per dish.
Embryos were cultured in the dark for two days at about 24.degree.
C.
[0260] Immediately prior to electroporation, embryos were
enzymatically treated with 0.5% Pectolyase Y-23 (Seishin
Pharmaceutical Co.) in a buffer containing 0.2 M mannitol, 0.2%
bovine serum albumin, 80 mM calcium chloride and 20 mM
2-(N-morpholino)-ethane sulfonic acid (MES) at pH 5.6. Enzymatic
digestion was carried out for 5 minutes at room temperature.
Approximately 140 embryos were treated in batch in 2 ml of enzyme
and buffer. The embryos were washed two times with 1 ml of 0.2 M
mannitol, 0.2% bovine serum albumin, 80 mM calcium chloride and 20
mM MES at pH 5.6 followed by three rinses with electroporation
buffer consisting of 10 mM HEPES and 0.4 M mannitol at pH 7.5. For
the electroporations, the final rinse of electroporation buffer was
removed and the embryos were incubated with 0.33 mg/ml linearized
pDPG165, 0.33 mg/ml supercoiled pDPG215, or 0.33 mg/ml linearized
pDPG344 in electroporation buffer. One half ml aliquots of DNA in
electroporation buffer and twenty embryos were transferred to the
electroporation chamber that was placed in a sterile 60.times.15 mm
petri dish. An electrical pulse was passed through the cells from a
500 .mu.f capacitor that was charged to 100 volts (400 V/cm field
strength, 160 ms pulse decay time; exponential pulse).
[0261] Immediately following electroporation, embryos were diluted
1:10 with Medium 726 containing 0.3 M mannitol. Embryos were then
transferred to GelgroA solidified Medium 726 containing 0.3 M
mannitol. Embryos were incubated in the dark at about 24.degree. C.
After five days embryos were transferred to Gelgro solidified
Medium 726 containing 0.2 M mannitol. Two days later embryos were
transferred to selection medium.
EXAMPLE IV
Identification of Transformed Cells Using Selectable Markers
[0262] In order to provide a more efficient system for
identification of those cells receiving DNA and integrating it into
their genomes, it is desirable to employ a means for selecting
those cells that are stably transformed. One exemplary embodiment
of such a method is to introduce into the host cell a marker gene
which confers resistance to some normally inhibitory agent, e.g.,
an antibiotic or herbicide. The potentially transformed cells are
then exposed to the agent. In the population of surviving cells are
those cells wherein generally the resistance-conferring gene has
been integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA. Using embryogenic suspension cultures, stable
transformants are recovered at a frequency of approximately 1 per
1000 transiently expressing foci.
[0263] One of the difficulties in cereal transformation, e.g.,
corn, has been the lack of an effective selective agent for
transformed cells, from totipotent cultures (Potrykus, Trends
Biotech, 7, 269 (1989)). Stable transformants were recovered from
bombarded nonembryogenic Black Mexican Sweet (BMS) maize suspension
culture cells, using the neo gene and selection with the
aminoglycoside, kanamycin (Klein et al., Plant Physiol., 91 440
(1989). This approach, while applicable to the present invention,
is not preferred because many monocots are insensitive to high
concentrations of aminoglycosides (Dekeyser et al., Plant Physiol.,
X, 21-7 (1989); Hauptmann et al., Plant Physiol., 86, 602 (1988)).
The stage of cell growth, duration of exposure and concentration of
the antibiotic, may be critical to the successful use of
aminoglycosides as selective agents to identify transformants
(Lyznik et al., Plant Mol. Biol., L3, 151 (1989)); Yang et al.,
Plant Cell Rep., 7, 421 (1988); Zhang et al., Plant Cell Rep., 2,
379 (1988)). For example, D'Halluin et al. (The Plant Cell, 4, 1495
(1992)) demonstrated that using the neo gene and selecting with
kanamycin transformants could be isolated following electroporation
of immature embryos of the genotype H99 or type I callus of the
genotype PA91. In addition, use of the aminoglycosides, kanamycin
or G418, to select stable transformants from embryogenic maize
cultures can result in the isolation of resistant calli that do not
contain the neo gene.
[0264] One herbicide which has been suggested as a desirable
selection agent is the broad spectrum herbicide bialaphos.
Bialaphos is a tripeptide antibiotic produced by Streptomyces
hygroscopicus and is composed of phosphinothricin (PPT), an
analogue of L-glutamic acid, and two L-alanine residues. Upon
removal of the L-alanine residues by intracellular peptidases, the
PPT is released and is a potent inhibitor of glutamine synthetase
(GS), a pivotal enzyme involved in ammonia assimilation and
nitrogen metabolism (Ogawa et al., Sci. Rep., Meija Seika, 13, 42
(1973)). Synthetic PPT, also known as Glufosinate.RTM., the active
ingredient in the herbicides Basta.RTM. or Libert.RTM. is also
effective as a selection agent. Inhibition of GS in plants by PPT
causes the rapid accumulation of ammonia and death of the plant
cells.
[0265] The organism producing bialaphos and other species of the
genus Streptomyces also synthesizes an enzyme phosphinothricin
acetyl transferase (PAT) which is encoded by the bar gene in
Streptomyces hygroscopicus and the pat gene in Streptomyces
viridochromogenes. The use of the herbicide resistance gene
encoding phosphinothricin acetyl transferase (PAT) is referred to
in DE 3642 829 A wherein the gene is isolated from Streptomyces
viridochromogenes. In the bacterial source organism, this enzyme
acetylates the free amino group of PPT preventing auto-toxicity
(Thompson et al., EMBO J. 6, 2519 (1987)). The bar gene has been
cloned (Murakami et al., Mol. Gen. Genetics, 205, 42 (1986);
Thompson et al., supra) and expressed in transgenic tobacco, tomato
and potato plants (De Block, EMBO J. 6, 2513 (1987)) and Brassica
(De Block et al., Plant Physiol., 91, 694 (1989)). In previous
reports, some transgenic plants which expressed the resistance gene
were completely resistant to commercial formulations of PPT and
bialaphos in greenhouses.
[0266] EP patent 0 242 236 refers to the use of a process for
protecting plant cells and plants against the action of glutamine
synthetase inhibitors. This application also refers to the use of
such of a process to develop herbicide resistance in determined
plants. The gene encoding resistance to the herbicide LIBERTY
(Hoechst, phosphinothricin or Glufosinate.RTM.) or Herbiace (Meiji
Seika, bialaphos) was said to be introduced by Agrobacterium
infection into tobacco (Nicotiana tabacum cv Petit Havan SRI),
potato (Solanum tuberosum cv Benolima) and tomato (Lycopersicum
esculentum), and conferred on these plants resistance to
application of herbicides.
[0267] Another herbicide which is useful for selection of
transformed cell lines in the practice of this invention is the
broad spectrum herbicide glyphosate. Glyphosate inhibits the action
of the enzyme EPSPS which is active in the aromatic amino acid
biosynthetic pathway. Inhibition of this enzyme leads to starvation
for the amino acids phenylalanine, tyrosine, and tryptophan and
secondary metabolites derived thereof Comai et al., U.S. Pat. No.
4,535,060, issued Aug. 13, 1985 describe the isolation of EPSPS
mutations which infer glyphosate resistance on the Salmonella
typhimurium gene for EPSPS, aroA. The EPSPS gene was cloned from
Zea mays and mutations similar to those found in a glyphosate
resistant aroA gene were introduced in vitro. The mutant gene
encodes a protein with amino acid changes at residues 102 and 106.
Although these mutations confer resistance to glyphosate on the
enzyme EPSPS, it is anticipated that other mutations confer the
same phenotype.
[0268] An exemplary embodiment of vectors capable of delivering DNA
to plant host cells is the plasmid, pDPGI65 and the vectors
pDPG433, pDPG434, pDPG435, and pDPG436. The plasmid pDPG165 is
illustrated in FIG. 2. A very important component of this plasmid
for purposes of genetic transformation is the bar gene which
encodes a marker for selection of transformed cells exposed to
bialaphos or PPT. Plasmids pDPG434 and pDPG436 contain a maize
EPSPS gene with mutations at amino acid residues 102 and 106 driven
by the actin promoter and 35S promoter-Adh1 intron I, respectively.
The mutated EPSPS gene encodes a marker for selection of
transformed cells.
[0269] Transformation of Cell Line AT824 Using Bialaphos Selection
Following Particle Bombardment--Selection in Liquid Medium
[0270] A suspension culture of AT824 was maintained in Medium 401.
The bombardment was done as described above, with a few variations.
Four filters of AT824 suspension cultures were plated out at
approximately 0.75 ml PCV on to Medium 279. There were 4 filters
bombarded with pDPG165 (FIG. 2, 35S-bar-Tr7) and pDPG480 (FIG. 3,
35S-mtlD-Tr7). The cells were left on the solid Medium 279 for 4
days and then put into liquid Medium 401. Liquid selection was
started after one passage (3.5 days) using 1 mg/L bialaphos. Cells
were thin plated one week later at 0.1 ml PCV (2 weeks after
bombardment) on to Medium 279+3mg/L bialaphos. Putative
transformants were observed about 8 weeks later. A total of 46
bialaphos-resistant lines and 25 lines containing mtlD DNA, as
determined by a polymerase chain reaction, were obtained.
[0271] Transformation of Cell Line AT824 Using Bialaphos Selection
Following Particle Bombardment--Solid Medium Selection
[0272] Cells were bombarded as described above, except the gold
particle-DNA preparation was made using 25 .mu.l pDPG319 DNA (bar
gene and aroa expression cassette containing the .alpha.-tubulin
promoter). Following particle bombardment cells remained on solid
Medium 279 in the absence of selection for one week. At this time
cells were removed from solid medium, resuspended in liquid Medium
279, replated on Whatman filters at 0.5 ml PCV per filter, and
transferred to Medium 279 containing 1 mg/L bialaphos. Following
one week, filters were transferred to Medium 279 containing 3 mg/L
bialaphos. One week later, cells were resuspended in liquid Medium
279 and plated at 0.1 ml PCV on Medium 279 containing 3 mg/L
bialaphos. Nine transformants were identified 7 weeks following
bombardment.
[0273] Transformation of Hi-II callus using Bialaphos Selection
Following Particle Bombardment.
[0274] Hi-II callus was initiated and bombarded as described above.
Four filters were bombarded with pDPGI65 FIG. 2, (35S-bar-Tr7) and
pDPG493 (FIG. 4, 35S-mtlD-Tr7). After bombardment, cells were
allowed to recover on solid media for 3 days. The four original
bombarded filters were transferred to Medium 201 containing 1 mg/L
bialaphos for 2 weeks. After this time, cells were removed from
solid medium, resuspended in liquid medium, replated on Whatman
filters at 0.5 ml PCV per filter, and transferred to Medium 201
containing 1 mg/L bialaphos. Following 2-3 weeks, cells were
resuspended in liquid medium and plated at 0.1 ml PCV on Medium 201
containing 3 mg/L bialaphos. Putative transformants were visible
about 5-6 weeks after thin plating. There were 8
bialaphos-resistant lines, and out of these 4 transformants
contained mtlD DNA, as determined by PCR.
[0275] Another consideration is that plants may need to have very
high levels of osmoprotectant to show a significant change in
stress resistance. Thus, a combination of mtlD constructs with
different promoters was transformed into Hi-II callus, and mtlD
PCR.sup.+ transformants were obtained. Southern and PCR analysis
can determine which mtlD constructs have been incorporated into
which transformants.
[0276] Transformation of Immature Embryos of the Genotype Hi-II
Using Bialaphos as a Selective Agent Following Particle
Bombardment.
[0277] Immature embryos of the genotype Hi-II were bombarded as
described above using pDPG670 (H3C4-adh1-bar-Tr7) and pDPG598
(Act1-mtlD-Tr7). Embryos were allowed to recover on high osmoticum
medium (Medium 201+12% sucrose+100 .mu.M AgNO.sub.3) for about 1-3
days, preferably at least overnight, i.e., for about 16-24 hours,
and were then transferred to selection medium containing 1 mg/l
bialaphos (Medium 201+1 mg/l bialaphos+100 .mu.M AgNO.sub.3).
Embryos were maintained in the dark at 24.degree. C. After two to
four weeks on the initial selection plates about 50% of the embryos
had formed Type II callus and were transferred to selective medium
containing 3 mg/l bialaphos (Medium 201+3 mg/L bialaphos).
Responding tissue was subcultured about every two weeks onto fresh
selection medium (Medium 201+3 mg/L bialaphos). Six
bialaphos-resistant lines were recovered from this experiment.
[0278] If cells are producing too much mannitol at the callus
level, there may be possible cell death due to swelling or
bursting. Immature embryo transformation experiments have been
conducted using a-low level of mannitol during selection. It is
possible that osmoticum in the medium may counteract mannitol
producing cells to make a more isotonic environment. It may be
possible to obtain high mtlD expressing transformants by doing
so.
EXAMPLE V
Plants From Transformed Cells
[0279] For use in agriculture, transformation of cells in vitro is
only one step toward commercial utilization of these genotypically
new plant cells. Plants must be regenerated from the transformed
cells, and the regenerated plants must be developed into full
plants capable of growing crops in open fields. For this purpose,
fertile corn plants are required. The following protocol describes
a method for regenerating plants, but one of skill in the art will
be familiar with other equally efficient protocols.
[0280] During suspension culture development, small cell aggregates
(10-100 cells) are formed, apparently from larger cell clusters,
giving the culture a dispersed appearance. Upon plating these cells
to solid media, somatic embryo development can be induced, and
these embryos can be matured, germinated and grown into fertile
seed-bearing plants. The characteristics of embryogenicity,
regenerability, and plant fertility are gradually lost as a
function of time in suspension culture. Cryopreservation of
suspension cells arrests development of the culture and prevents
loss of these characteristics during the cryopreservation
period.
[0281] Regeneration of AT824 Transfor-mants and HiII callus
[0282] Transformants were produced as described above. For
regeneration tissue was first transferred to solid Medium 223 or
Medium 201+1 mg/L bialaphos and incubated for two weeks.
Transformants can be initially subcultured on any solid culture
that supports callus growth, e.g., Medias 223, 425, 409, and the
like. Subsequently transformants were subcultured one to three
times, but usually twice on Medium 189 (first passage in the dark
and second passage in low light) and once or twice on Medium 101 in
petri dishes before being transferred to Medium 607 in Plant
Cons.COPYRGT.. Variations in the regeneration protocol are normal
based on the progress of plant regeneration. Hence some of the
transformants were first subcultured once on Medium 425, twice on
Medium 189, once or twice on Medium 101 followed by transfer to
Medium 501 in Plant Cons.COPYRGT.. As shoots developed on Medium
101, the light intensity was increased by slowly adjusting the
distance of the plates from the light source located overhead. All
subculture intervals were for about 2 weeks at about 24.degree. C.
Transformants that developed 3 shoots and 2-3 roots were
transferred to soil.
[0283] Plantlets in soil were incubated in an illuminated growth
chamber and conditions were slowly adjusted to adapt or condition
the plantlets to the drier and more illuminated conditions of the
greenhouse. After adaptation/conditioning in the growth chamber,
plants were transplanted individually to 5 gallon pots of soil in
the greenhouse.
EXAMPLE VI
Determination of MDH Activity
[0284] Mannitol-1-P Dehydrogenase (MDH) Spectrophotometric
Assay
[0285] The MDH assay has been used to determine if there is
expression of the mtlD gene in transformed callus or leaf tissue.
The spectrophotometer measures differences at the 340 nm
wavelength, looking for a change from NAD+to NADH, a result of
expression of the mtlD gene changing mannitol-l-phosphate to
fructose-6-phosphate.
[0286] Bacterial extracts are used as controls. An aliquot of the
glycerol stocks of bacteria containing the bar gene (p 165) or
containing the mtlD gene (p480) was put into LB media (100 mg/L
ampicillin). These cultures are grown overnight at 37.degree. C.
The next day cultures are spun down at 5,000 rpm for 5 minutes. The
pellet is rinsed with either Tris-citrate (0.1 M Tris-citrate, pH
8.5) or PAT buffer (50 mM Tris-HCl, pH. 7.5, 2 mM EDTA, 0.15 mg/ml
leupeptin, 0.15 mg/ml PMSF, 0.3 mg/ml BSA, 0.3 mg/ml DTT)and spun
down again. Then the pellet, about 200 .mu.l of glass beads, and
500 .mu.l of buffer are put into a 1.5 ml eppendorf tube and shaken
twice for 20 seconds on "high" (MINI-BEADBEATER.TM., Biospec
Products). The tubes are then spun down and the supernatant is used
for the assay. All tubes are kept on ice.
[0287] For callus or plant extracts, about 0.5 g of tissue is used.
Tissue is homogenized with approximately 250 .mu.l of Tris-citrate
or PAT buffer. Extracts are spun down in the microfuge at 14,000
rpm for 5 minutes. Protein is quantified using the BioRad
assay.
[0288] For the MDH assay, a master assay mix is made to be used for
all the samples. The mix includes: 2.5 ml 0.1 M Tris-citrate, pH
8.5, 0.1 ml of 4 mM NAD.sup.+ (dissolve one 20 mg vial of SIGMA,
P-nicotinamide adenine dinucleotide, in 7.15 ml ddH.sub.2O), and
0.1 ml of 6 mM mannitol-1-phosphate (SIGMA)).
[0289] The spectrophotometric readings were done as follows: 1 ml
of assay mix was put into a cuvette. Then 2-100 .mu.g of protein
was added. The cuvette was inverted about 3 times and then the
reading was initiated. Measurements were taken for up to 5 minutes
at 340 nm.
[0290] Bioassays for Mannitol
[0291] Callus assays were conducted on transformants derived from
AT824 (S80HO-52) and Hi-II callus (HCO5II-55), as well as on
controls. Callus growth assays were started by plating 0.1-0.5 g
callus fresh weight on to Whatman filters. Filters were then put on
to media with additional concentrations of osmoticum. The osmoticum
includes mannitol (0, 0.3, 0.6, 0.9 M) and NaCl (0, 50, 150, 250
mM). Fresh weight gains were taken after 2-3 weeks in culture.
[0292] To determine if there is a significant amount of mannitol
being produced at the callus level, osmotic potential readings can
be conducted on 0.1 g callus samples using the Psychrometer (Wescor
Inc. C-52 sample chambers) by methods well known to the art.
EXAMPLE VII
Transformant Plants into the Greenhouse and Characterization of
R.sub.0 Plants
[0293] Once plants are regenerated, hardened off in the growth
chamber, plants are transferred to the greenhouse to obtain seed.
Leaf samples are taken of the Ro plants as well as subsequent
generations and crosses and endogenous mannitol levels are
determined. Phenotypic changes in the plants possessing the
transgene were documented.
[0294] To determine the mannitol content of these plants,
approximately 30 grams (fresh weight) of the tip of mature, healthy
leaves are sampled. The leaf samples are placed in 50 ml
polypropylene test tubes in a -70.degree. C. freezer. Frozen leaf
samples are then dried in a freeze drier and stored until analysis.
In separate 50 ml polypropylene test tubes, 1.0 to 1.5 gram
quantities of dried leaf sample are weighed. The samples are then
homogenized in 40 mls of 80% ethanol (v/v) using a Polytron. The
resulting solutions are incubated in a 72.degree. C. water bath for
30 minutes, with a brief vortexing step at approximately 15
minutes. Following the incubation, the solutions are heated in a
boiling water bath for 2 minutes. The samples are then centrifuged
at 3000.times.g for 15 minutes. The resulting supernatants are then
recovered and taken to dryness overnight in a 40.degree. C.
nitrogen evaporator. The remaining paste is frozen then
freeze-dried for approximately 2 hours. The dried material is
dissolved in 0.5 mls of distilled, deionized water to form the
aqueous simple carbohydrate extract. The extract is purified prior
to HPLC separation techniques by passing it through a C-18 solid
phase extraction column (Varian Bond Elut.RTM.) and a 1.2 micron
acrodisc filter.
[0295] Mannitol content of the simple carbohydrate extracts are
determined using HPLC separation techniques. An RCM monosaccharide
column (Phenomenex.RTM.) is used, with water as the mobile phase.
The separated simple sugars are detected with an Erma.RTM. ERC-7512
refractive index detector. The resulting sample chromatograms are
analyzed using Maxima(D peak integration software and compared to
chromatograms of mannitol standards.
[0296] The above procedure for mannitol extraction and
quantification from corn leaf material was tested using a plant
species which was known to possess naturally occurring endogenous
levels of mannitol. Extracts were prepared from leaves, roots,
small stems, and large stalks of the celery plant. All four
extracts were found to possess detectable levels of mannitol. Based
on chromatograms obtained from standards, the amount of mannitol in
the tissue was estimated to be between 20 mg (roots) to 112 mg
(stems) per gram of dry weight.
[0297] During the mid vegetative stage of development, greenhouse
grown R.sub.0 maize plants were sampled for leaf mannitol content,
according to the above described procedure. Over a 10 month period,
leaf samples from one hundred four R.sub.0 plant clones from
seventeen callus cell lines were assayed. Carbohydrate extracts of
R.sub.0 clones from several cell lines were found to exhibit HPLC
chromatograms which contained peaks with retention times similar to
mannitol standards. Although leaf samples from most of the cell
lines expressed relatively small amounts of leaf tissue mannitol,
those derived from two cell lines were found to express putative
levels of mannitol which were over 3.0 milligrams per gram of dry
weight (mg/g dry wt.). Addition of mannitol to the extracts
resulted in an increase in the area of the "mannitol" peak without
the production of any new peaks. Levels of leaf tissue mannitol in
R.sub.0 clones ranged from 19.31 mg/g dry wt. for the cell line
HCO5II-55 (derived from Hi-II callus) to 3.63 mg/g dry wt. for the
cell line S80HO-52 (derived from AT824).
[0298] Transformation Using the G1b1 promoter
[0299] Transformed plant cell lines derived from AT824 suspension
(S87KM) and immature embryos (H168KM) which were PCR.sup.+ for the
pDPG586 construct have been in regeneration. The pDPG586 vector is
potentially sensitive to ABA induction at the callus level due to
the presence of the G1b1 promoter. Moreover, levels of ABA are
increased in drought sensitive plants during a period of drought
(Landi et al., Maydica, 40 (1995)), indicating that an ABA
inducible promoter is also drought inducible.
[0300] Droughted pDPG586-containing transgenic plants are tested
for the production of ABA and for increased levels of mannitol.
HPLC analyses showed low levels of mannitol in leaf tissue from
these plants. Young transgenic seedlings are exposed to ABA and
differences in mannitol expression determined at later plant
stages. MDH assays are conducted on ABA treated callus from tissue
transformed with this construct. Seed viability after drought is
also tested to determine whether mannitol is expressed in the
embryo.
[0301] Transformation Using the Maize Transit Peptide (MZTP)
[0302] The MZTP was used to express mtlD in the chloroplast.
Increased mtlD expression in the chloroplast can give protection to
the chloroplastic photosynthetic system under reduced water
availability conditions. The expression of mtlD thus allows the
chloroplast to osmotically adjust to the cellular conditions that
change as a result of changes in the water relations in the plant.
In addition, if mannitol is expressed exclusively in the cytosol,
some disruption of chloroplast function could occur due to the
imbalance of osmotic relations between the compartments of the
cell. Moreover, increased mtlD expression in the chloroplast may
also provide anti-oxidant activity.
[0303] One construct, pDPG587 (35S-MZTP-mtlD-Tr7 3'), has been
tested using AT824 suspension (S85KN, S87KN, S88LG), Hi-II callus
(HS06LG, HZ04LG), and immature embryos (HI88LG, H189LG, HI9OLG,
IH07LW, DL04LW, IH16LW, CS12LW, DTOILW). PCR.sup.+ transformants
with the construct were obtained. Furthermore, the presence of
mannitol was detected in transformants containing the
(35S-MZTP-mtlD-Tr7 3') expression cassette.
[0304] Chloroplast viability assays, magnetic isolation of
chloroplasts, and greenhouse and field studies of the resultant
transformed plants under a range of water stress or non-stress
conditions are performed, by methods described herein or by other
methods well known to the art.
EXAMPLE VIII
Characterization of R.sub.1 Transformants
[0305] Seed were recovered from several outcrosses of S80HO-52 and
HC05II-55 R.sub.0 plants. The first R.sub.1 seed became available
from the outcrosses involving S80HO-52 X AW. The R.sub.1 seed was
evaluated in three separate greenhouse plantings.
[0306] The first planting of twenty-two R.sub.1 seeds resulting
from the cross of S80HO-5207 X AW were planted in the greenhouse to
compare results from HPLC determined leaf tissue mannitol levels to
PCR-derived data. The carbohydrate profiles obtained from the
twenty-two plants revealed twelve as expressing levels of mannitol
comparable to the R plant. The results were found to agree with the
PCR data developed from the same set of plants.
[0307] A second, larger, planting of R.sub.1 populations was made
in the greenhouse after additional seed became available. The
planting included eight populations from various outcrosses of
S80HO-52 R.sub.0 plants to AW. Twenty seeds were planted per
population among ten 15-gallon pots, two seeds from the transgenic
population per pot plus the common tester, AW. Therefore, a total
of three plants per pot were planted. During the mid-late
vegetative stage of development a drought episode was imposed on
the plants for 33 days. During midday and predawn sampling periods,
several whole plant physiological measurements were collected when
appropriate, including the following: 1. water relations parameters
(under water stress and rewatered conditions), 2. gas exchange
measurements, 3. leaf temperature, 4. leaf mamiitol samples, 5.
plant height, 6. flowering synchrony, and 7. Glufosinate.RTM.
sensitivity test.
[0308] All populations exhibited approximately a 1:1 segregation
for GlufosinateO sensitivity. No visual, morphological differences
were observed between plants which were resistant to GlufosinateO
(and presumably possessed the preselected DNA segment) and those
which were sensitive. This indicates that no deleterious effect on
plant growth and development occurred with the mtlD gene at this
level of expression.
[0309] Data from the twenty plants evaluated among each population
were sorted by resistance versus sensitivity to Glufosinate.RTM.,
then mean values were generated. In all populations, expression of
leaf tissue mannitol, as determined by HPLC, co-segregated with
expression of Glufosinate.RTM. resistance. Levels of leaf tissue
mannitol were found to approximate levels expressed in the R.sub.0
plants.
[0310] A time course of leaf osmotic potential values collected
from the S80HO-5201,-5205, and -5208 populations was assembled from
the water relations data (FIG. 7). With the exception of 1 sampling
period in the -5205 population, plants which exhibited resistance
to Glufosinate.RTM. applications were found to express more
negative predawn osmotic potential values when compared to plants
which were Glufosinate.RTM. sensitive. However, to fully understand
the influence of increased leaf tissue solutes (such as mannitol)
on osmotic potential, the influence of tissue dehydration due to
drops in total water potential must be examined.
[0311] During the first predawn sampling period, total water
potential values were between -1.0 to -2.0 bars. No differences
were observed between plants with and without the gene. Plants were
rewatered prior to the second and third predawn sampling period,
which brought the total water potential values to -0.2 bars. When
the total water potentials approach zero, the osmotic potentials
are directly comparable since the water content is similar. This
indicates that differences in osmotic potential values were
influenced only by differences in accumulated cell solutes and not
by dehydration. During the midday sampling period, total water
potential values were between -10.0 to -15.0 bars, indicating that
tissue dehydration occurred. As shown in FIG. 7, there were no
differences in leaf osmotic potential among any of the populations
during the midday period.
[0312] Midday gas exchange data, leaf rolling observations, plant
heights, and anthesis to silking intervals were also collected in
all of the eight R, populations. No statistically significant
differences were observed between plants with and without the gene
for any of these measurements. This indicates that at this level of
mannitol expression, no deleterious effect on the transgenic plants
was noted.
[0313] The osmotic potential findings in this experiment
represented the first direct link between gene induced leaf
mannitol expression and a significant whole plant physiological
trait related to drought tolerance.
[0314] EXAMPLE IX
Evaluation of the Two Highest Expressing Mannitol Cell Lines,
S8010-5201XAW and ITCO5II-5503 XAW
[0315] In a third greenhouse planting one R, population was
included from each of the two highest mannitol expressing cell
lines. Twenty seeds from each of the populations S80HO-5201 X AW
and HCO5II-5503 X AW were grown to the mid vegetative stage of
development. Plant production and arrangement in the greenhouse was
similar to that described above in Example VIII. LH132 was used as
the common tester. Therefore a total of three plants per pot were
planted. A drought episode was then imposed on half of the twenty
plants from each population. During midday and predawn sampling
periods, the same whole plant physiological measurements used in
the previous R, experiment were collected. All populations
exhibited approximately a 1:1 segregation for Glufosinate.RTM.
sensitivity. Data from the ten plants evaluated among each
population/treatment combination were sorted by resistance versus
sensitivity to Glufosinate.RTM., then mean values were
generated.
[0316] Leaf tissue mannitol, as determined by HPLC, was measured
once during the predawn sampling period. In both populations,
expression of mannitol co-segregated with expression of
Glufosinate.RTM. resistance. As was found in the Ro plants, R.sub.1
plants from the cell line HCO5II-55 expressed leaf tissue mannitol
levels which were approximately 8 times higher than R. plants from
the cell line S80HO-52 (Table 2). Among both populations, plants
which were exposed to the drought stress conditions expressed
higher levels of leaf tissue mannitol than plants grown under well
watered conditions.
2TABLE 2 Predawn leaf mannitol content (mg/g dry wt.) in two cell
lines. GLUFOSINATE .RTM. GLUFOSINATE .RTM. TREATMENT RESISTANT
SENSITIVE HCO5II-5503 X AW Stressed 39.8 0.0 Watered 8.1 0.0
S80HO-5201 X AW Stressed 5.1 0.0 Watered 1.1 0.0
[0317] Water relations data collected at 2 time periods prior to
the rewatering of plants for the HCO5II-55 and S80HO-52 populations
are shown in Tables 3-6. In the HCO5II-55 population, significant
(P.ltoreq.0.05) differences were observed between plants which were
resistant to Glufosinate(compared to plants which were not for both
predawn and midday osmotic and turgor potential values. Among the
S80HO-52 population, significant (P.ltoreq.0.05) differences were
observed between Glufosinate.RTM. resistant and sensitive plants
for predawn osmotic potential values, but not during the midday
period.
3TABLE 3 Leaf water relations during the predawn period for
HCO5II-5503 X AW Glufosinate .RTM. resistant and susceptible plants
grown under water stress and watered conditions. GLUFOSINATE .RTM.
PREDAWN PERIOD TREATMENT RESISTANCE TOTAL OSMOTIC TURGOR bars
Stress No -12.49 -14.99 2.51 Stress Yes -11.73 -16.93* 5.20*
P(.ltoreq.) ns 0.05 0.05 Watered No -0.20 -11.68 11.48 Watered Yes
-0.20 -12.70 12.50 P(.ltoreq.) ns ns ns
[0318]
4TABLE 4 Leaf water relations during the midday period for
HCO5II-5503 X AW Glufosinate .RTM. resistant and susceptible plants
grown under stress and watered conditions. GLUFOSINATE .RTM. MIDDAY
PERIOD TREATMENT RESISTANCE TOTAL OSMOTIC TURGOR bars Stress No
-16.21 -16.14 -0.07 Stress Yes -14.93 -18.94* 4.01* P(.ltoreq.) ns
0.05 0.05 Watered No -4.90 -13.57 8.67 Watered Yes -5.13 -14.25
9.12 P(.ltoreq.) ns ns ns
[0319]
5TABLE 5 Leaf water relations during the predawn period for
S80HO-5201 X AW Glufosinate .RTM. resistant and susceptible plants
grown under watered stress and watered conditions. GLUFOSINATE
.RTM. PREDAWN PERIOD TREATMENT RESISTANCE TOTAL OSMOTIC TURGOR bars
Stress No -13.00 -14.94 1.94 Stress Yes -15.60* -16.40* 0.80
P(.ltoreq.) 0.05 0.05 ns Watered No -0.20 -12.58 12.38 Watered Yes
-0.20 -13.08 12.88 P(.ltoreq.) ns ns ns
[0320]
6TABLE 6 Leaf water relations during the midday period for
S80HO-5201 X AW Glufosinate .RTM. resistant and susceptible plants
grown under stress and watered period. GLUFOSINATE .RTM. MIDDAY
PERIOD TREATMENT RESISTANCE TOTAL OSMOTIC TURGOR bars Stress No
-15.01 -15.30 0.29 Stress Yes -16.37 -15.62 -0.74 P(.ltoreq.) ns ns
ns Watered No -6.30 -15.87 9.57 Watered Yes -6.45 -16.62 10.17
P(.ltoreq.) ns ns ns
[0321] After rewatering of the drought stressed plants,
Glufosinate.RTM. resistant HCO5II-55 plants continued to maintain
more negative osmotic potential values than Glufosinate.RTM.D
sensitive plants for up to 5 days (Tables 7-8). Osmotic adjustment,
as calculated by the difference between rewatered and watered
plants was over 4 bars for both sample periods. These are
significant changes in osmotic potential levels compared to the
plants not having the mtlD gene or expressing mannitol. For the
lower mannitol expressing line S80HO-5201, no significant
differences were observed for changes in osmotic potential between
the Glufosinate.RTM. resistant and susceptible plants (Tables
9-10). The contrast between the higher and lower mannitol
expressing lines may indicate the range of expression needed to
work with in crop improvement.
7TABLE 7 Differences in osmotic potential of HCO5II-5503 X AW
Glufosinate .RTM. resistant and susceptible plants 12 hours after
rewatering. TIME = 12 hrs Rewatered GLUFOSINATE .RTM. REWATERED
WATERED RESISTANCE OSMOTIC OSMOTIC DIFFERENCE bars No -11.70 -10.80
0.90 Yes -17.30*** -11.70 5.6 P(.ltoreq.) 0.001 ns
[0322]
8TABLE 8 Differences in osmotic potential of HCO5II-5503 X AW
Glufosinate .RTM. resistant and susceptible plants 5 days after
rewatering. TIME = 5 days Rewatered GLUFOSINATE .RTM. REWATERED
WATERED RESISTANCE OSMOTIC OSMOTIC DIFFERENCE bars No -12.90 -11.80
1.1 Yes -16.40** -12.10 4.3 P(.ltoreq.) 0.01 ns
[0323]
9TABLE 9 Differences in osmotic potential of S80HO-5201 X AW
Glufosinate.RTM. resistant and susceptible plants 12 hours after
rewatering. TIME = 12 hrs Rewatered GLUFOSINATE .RTM. REWATERED
WATERED RESISTANCE OSMOTIC OSMOTIC DIFFERENCE bars No -11.73 -13.02
-1.29 Yes -12.20 -12.89 -0.69 P(.ltoreq.) ns ns
[0324]
10TABLE 10 Differences in osmotic potential of S80HO-5201 X AW
Glufosinate .RTM. resistant and susceptible plants 5 days after
rewatering. TIME = 5 days Rewatered GLUFOSINATE .RTM. REWATERED
WATERED RESISTANCE OSMOTIC OSMOTIC DIFFERENCE bars No -12.36 -12.44
-0.08 Yes -13.26 -12.94 0.32 P(.ltoreq.) ns ns
[0325] Among both populations, no statistically significant
differences were observed between plants with and without the gene
for midday gas exchange data, leaf rolling observations, plant
heights, and anthesis to silking intervals.
[0326] On the Glufosinate.RTM. resistant HC05II-55 plants, at
flowering and further developing during the grainfill, a leaf
speckling which developed into a leaf chlorosis followed by
necrosis was observed. This abnormality was observed mainly on
plants grown under the well watered treatment. The droughted plants
did not exhibit this leaf expression in the upper most leaves after
rewatering. Due to drought induced leaf firing and senescence it
was not able to read the lower leaves of the stressed plants for
the chlorosis or speckling. The symptoms first appeared on the
oldest leaves of the plant and progressed to younger leaves prior
to the onset of physiological maturity. Other than the leaf
chlorosis, the plants were morphologically normal and set seed.
This chlorosis may disappear with plants where mannitol
accumulation is targeted to the chloroplast.
[0327] Seed planted from this cell line has confirmed that this
chlorosis first starts in the lower most (oldest) leaves and
progresses up the plant as the leaves become older. To determine
what the pattern of mannitol accumulation is and if there is a
correlation to the occurrence of the chlorosis, leaf samples of the
plants from oldest to newest leaves will be analyzed. Also
ultrastructural studies are being done through transmission
microscopy to see the cellular ultrastructure in the chlorotic
areas compared cellular ultrastructure in leaf samples from plants
without the gene and to green sectors on leaves of plants having
the gene.
EXAMPLE X
Evaluation of Mannitol Expressing Transformants in a Field
Environment Under Water Stress and Irrigated Conditions
[0328] Under field conditions, it is necessary to evaluate the
phenotype of plants having different levels of mannitol expression
under irrigated and water stressed condition.
[0329] Germplasm evaluated were the following: (S80H05201X AW) X BK
R2 generation with the mflD gene; (S80H05201X AW) X BK R2
generation without the gene; (HCO5II5503XAW) X BK R2 generation
with the gene; and BK, a standard inbred line.
[0330] The contribution of different levels of mannitol expression
to stress tolerance among R2 generation plants from two mannitol
expressing cell lines, S80HO-52 and HCO5II-55, was evaluated. The
two segregating populations were derived from crosses of greenhouse
grown R1 generation plants, transformed with constructs containing
the mtlD gene and the bar gene, crossed to the elite stiff stalk
inbred designated BK. Stable transformants were determined by
resistance to the herbicide Glufosinate.RTM.. Leaf tissue of RI
generation plants contained mannitol concentrations from at least
about 5.0 mg/g dry weight, for the low expressing cell line, and up
to about 40.0 mg/g dry weight for the high expressing cell line.
The R2 generation populations were planted in a modified randomized
complete block design with 4 repetitions nested within areas of low
and high water supply.
[0331] A drought stress episode was successfully maintained in the
low water supply plot for a period of 12 days at the mid to late
vegetative growth stage. The two populations were treated with a 2%
Glufosinate.RTM. solution and both populations segregated 1:1 for
Glufosinate.RTM. resistance. Within each plot, data were collected
from both Glufosinate.RTM.D resistant and sensitive plants. On
eight separate dates, during the midday sampling period,
measurements of leaf temperature, and associated environmental
data, were collected. The eight dates ranged from the early stages
of the drought stress to seven days after rewatering. Water
relations data were collected on ten separate dates during both
predawn and midday sampling periods. During several stressed and
rewatered sampling dates, leaf samples were collected for mannitol
analysis.
[0332] HPLC determinations of leaf tissue mannitol from samples
collected 6 days after the drought imposition are shown in Table
11. In both populations, expression of mannitol co-segregated with
expression of Glufosinate.RTM. resistance. Glufosinate.RTM.
resistant plants from the cell line HCO5II-55 expressed leaf tissue
mannitol levels which were approximately 6-8 times higher than
plants from the cell line S80HO-52. In previous greenhouse
experiments with RI plants, Glufosinate.RTM. resistant plants which
were exposed to drought stress conditions expressed higher levels
of leaf tissue mannitol than plants grown under well watered
conditions. In this experiment, the drought stress episode had
little effect on mannitol levels. In general, the levels of
mannitol expressed among Glufosinate.RTM. resistant plants in this
experiment were less than 20% of the levels observed among the same
cell lines grown in the greenhouse. This difference may be the
result of the compressed (shortened) growth period associated with
the environment employed in this experiment. Expression of the mtlD
gene in these transformants was under the transcriptional control
of the Cauliflower Mosaic Virus 35S promoter.
[0333] The levels of other plant carbohydrates in these lines was
also determined. Glucose was the only carbohydrate to exhibit
significant (P.ltoreq.0.05) differences between Glufosinate.RTM.
sensitive and resistant plants, i.e., Glufosinate.RTM. resistant
plants contained higher levels of glucose relative to
Glufosinate.RTM. sensitive plants. Because glucose is known to have
pleiotropic effects in plant cells, it is contemplated that the
levels of glucose may need to be moderated in these plants.
11TABLE 11 Midday leaf mannitol content of 2 R2 populations grown
under watered and drought stressed conditions in Kihei, HI.
GLUFOSINATE .RTM. GLUFOSINATE .RTM. RESISTANT SENSITIVE TREATMENT
(mg/g dry wt.) High Expressing Population Stressed 5.71 0.0 Watered
5.06 0.0 Prob (.ltoreq.) ns ns Low Expressing Population Stressed
0.84 0.0 Watered 1.36 0.0 Prob (.ltoreq.) 0.01 ns
[0334] Table 12 shows the water relations results for both
populations grown under water stress conditions. The results
represent the average of six midday periods collected prior to
rewatering and indicated more favorable leaf turgor potential
values among plants comprising the mtlD gene compared to plants
which do not contain the gene. These differences were observed in
both the low and high mannitol expressing populations. The
improvements in turgor levels among Glufosinate.RTM. resistant
plants in both populations were the combined results of
improvements in total water potential (.PSI..sub.w) and osmotic
potential (.PSI..sub.s). Since osmotic potential is influenced by
both cellular dehydration and by the active accumulation of
solutes, the less negative .PSI..sub.w values in the
Glufosinate.RTM. resistant plants prevented the detection of
significant differences for .PSI..sub.s. However, the combined
changes among both .PSI..sub.w and .PSI..sub.s led to highly
significant (P<0.01) improvements in leaf turgor.
12TABLE 12 Midday water relations parameters for plants exhibiting
resistance and sensitivity to Glufosinate .RTM. applications among
low and high mannitol expressing R2 populations grown under water
stress conditions in Kihei, HI. Results are the average of 6 dates.
Low Expressing High Expressing Population Population Glufosinate
.RTM. Resistance Resis. Water Relations Resis. Sens. (bars) Sens.
.PSI..sub.w -9.25 -9.73 -8.55 -9.87*** ns .PSI..sub.s -13.03 -12.73
-12.99 -12.88 ns ns .PSI..sub.p 3.77 3.00** 4.44 3.01*** **Prob.
.ltoreq. 0.05 ***Prob. .ltoreq. 0.01
[0335] Table 13 shows the predawn water relations results for the
plants grown under stress. In the high mannitol expressing
population, plants which were resistant to Glufosinate.RTM.
applications expressed significantly (P.ltoreq.0.01) more favorable
values for all three water relations parameters. Differences among
resistant versus susceptible plants in the low expressing
population were not significant.
13TABLE 13 Predawn water relations parameters for plants exhibiting
resistance and sensitivity to Glufosinate .RTM. applications among
low and high mannitol expressing R2 populations grown under water
stress conditions in Kihei, HI. Results are for one sample date.
Low Expressing High Expressing Population Population Glufosinate
.RTM. Resistance Resis. Water Relations Resis. Sens. (bars) Sens.
.PSI..sub.w -1.2 -1.2 -0.85 -1.05*** ns .PSI..sub.s -10.69 -11.03
-10.57 -9.32** ns .PSI..sub.p 9.83 9.49 9.72 8.27*** ns **Prob.
.ltoreq. 0.05 ***Prob. .ltoreq. 0.01
[0336] Table 14 shows the predawn water relations results for the
same plot collected 24 hours after rewatering. Total water
potential differences were eliminated by the rewatering event,
however significant differences in osmotic potential remain and,
since turgor is calculated from total and osmotic water potentials,
the accumulation of mannitol resulted in higher turgor values in
the high expressing population. Again, differences among plants in
the low expressing population were not significant. The
improvements in water relations parameters associated with the
presence of the mtlD gene in plants were smaller in magnitude than
improvements observed in previous greenhouse studies and may be the
result of lower levels of leaf mannitol expression. Because water
potential and higher turgor pressure under water stress are
correlated with a drought resistant phenotype (Morgan, Aust. J.
Agric. Res., 34, 607 (1983)), changes in water relations associated
with the presence of the mtlD gene in maize can provide plants with
an altered ability to utilize available water.
14TABLE 14 Rewatered predawn water relations parameters for plants
exhibiting resistance and sensitivity to Glufosinate .RTM.
applications among low and high mannitol expressing R2 populations
previously grown under water stress conditions in Kihei, HI.
Results are for one sample date. Low Expressing High Expressing
Population Population Glufosinate .RTM. Resistance Resis. Water
Relations Resis. Sens. (bars) Sens. .PSI..sub.w -0.2 -0.2 -0.2 -0.2
ns ns .PSI..sub.s -10.77 -10.82 -10.61 -9.70** ns .PSI..sub.p 10.57
10.62 10.41 9.50** ns **Prob. .ltoreq. 0.05
[0337] During the collection of midday water relations data,
observations of drought-induced leaf rolling were recorded. In
previous field experiments, more favorable water relations
parameters among hybrids and inbreds grown under drought stress
conditions were correlated with decreases in leaf rolling (flatter
leaves). Additionally, less leaf rolling among hybrids have been
correlated with higher relative yield under stress. In this study,
Glufosinate.RTM. resistant plants in both the high and low mannitol
expressing populations exhibited highly significant (P.ltoreq.0.01)
decreases in leaf rolling (Table 15.).
15TABLE 15 Midday leaf rolling scores for plants exhibiting
resistance and sensitivity to Glufosinate .RTM. applications among
low and high mannitol expressing R2 populations grown under water
stress conditions in Kihei, HI. Results are the average of 5 dates.
Low Expressing High Expressing Population Population Glufosinate
.RTM. Resistance Resis. Sens. Resis. Sens. 3.8.sup.1 3.6*** 4.1
3.6*** .sup.1Score: 1 = Severely rolled leaves to 5 = Flat leaves
***Prob. .ltoreq. 0.01
[0338] More favorable water relations parameters have also been
correlated with higher rates of leaf transpiration and, as a
result, cooler leaf temperatures. In one study, leaf temperature
data was collected on eight dates. Analysis of the results
indicated significantly (P.ltoreq.0.01) cooler leaf temperatures
among plants which possess the mtlD gene compared to plants which
did not (FIG. 8). These temperature differences were observed-in
both the low and high mannitol expressing populations.
[0339] The leaf chlorosis symptoms, which were associated with high
levels of mannitol expression in greenhouse studies, were observed
among both populations in this study. The most severe symptoms were
found among Glufosinate.RTM. resistant plants in the high
expressing population. The degree of leaf chlorosis was more severe
than the chlorosis observed in greenhouse grown plants and may have
been exacerbated by the high light intensities which occur in the
field environment employed in those studies.
[0340] Expression of leaf tissue mannitol among both cell lines,
co-segregated with expression of Glufosinate.RTM. resistance and
was lower than that observed in previous greenhouse experiments
with R1 plants. Glufosinate.RTM. resistant plants from the high
mannitol expressing cell line (HCO5II-55) exhibited more favorable
turgor potential levels during midday and predawn water stress
conditions, and during predawn rewatered conditions. The low
mannitol expressing cell line (S80HO-52) exhibited more favorable
turgor potential levels during midday stress conditions.
Glufosinate.RTM. resistant plants from both populations exhibited
less leaf rolling and maintained cooler leaf temperatures. The
occurrence of mannitol-induced leaf chlorosis was more extensive
than in previous greenhouse experiments, and is suspected to be
light intensity dependant. Thus, several improvements in whole
plant drought tolerance traits were observed in plants
co-segregating for Glufosinate.RTM. resistance and the milD gene.
The improvements in water relations parameters, leaf rolling, and
canopy temperature (transpiration) are all important factors in
drought stress resistance.
EXAMPLE XI
Exposure of Maize Plants Expressing Mannitol to Salt Stress
[0341] R3 generation seeds of the high mannitol expressing line
(HCO5II-5503) were germinated in paper towels (12 seeds per towel)
moistened with water containing 1% Glufosinate.RTM.D and 1.2 ml/L
of DOMAIN (fungicide). Seeds were allowed to germinate at
25.degree. C. for 5 days. The resultant surviving seedlings were
transferred to a hydroponics system for further evaluation.
Alternatively, seeds can be germinated without Glufosinate.RTM. and
the segregating population examined.
[0342] The hydroponic system consist of tanks which individually
hold approximately 4 liters of solution. The individual germinated
plants were placed in sponge like material with slits cut to accept
the plants and were inserted into holes in the lid of the tank. The
planting density was twenty seedlings per tank. The hydroponic
solution was described by Clark (J. Plant Nutrition, 5, 1039
1982)). The solution was aerated for the duration of the
evaluation. For the purposes of a seedling assay generally 1/4 or
1/2 strength Clarks solution is used.
[0343] The plants were grown for 6 days, which corresponds to the
2-3 leaf stage of growth in 1/2 strength Clark's solution in the
absence of added NaCl. At this point, the hydroponic solution was
changed (1/2 strength Clark's) and salt (NaCl) added to the
solution. Plants were assayed for resistance to 0, 50, 100, 150,
200 and 250 mM NaCl in 1/2 strength Clark's solution.
[0344] At concentrations of NaCl less than 150 mM, no differences
in appearance of plant growth (no wilting) were observed after a 24
hour exposure to salt. At 200 mM NaCl, and more particularly at 250
mM NaCl, wilting was observed in the control plants, i.e. same
genotype that was used for transformation and not in the
transformed mannitol-containing plants. Upon harvest, which is 7
days after the start of the salt stress, determination of osmotic
potentials demonstrates that a favorable shift in osmotic potential
is associated with the presence of mannitol, resulting in the
maintenance of turgor. Salt-stressed mannitol-expressing
transformants have significantly more dry matter than controls.
EXAMPLE XII
Exposure of Maize Plants Expressing Mannitol to a Range of
Environmental Stresses.
[0345] Salt or Osmotic Stress.
[0346] Transgenic seeds containing the mtlD gene are germinated in
the presence of various salt or osmotically active solutions to
determine whether transgenic seeds demonstrate increased tolerance
or resistance to salt stress. Alternatively, seedlings can be grown
in hydroponic systems and challenged with salt or agents of
differing osmotic potentials at different, or all, developmental
stages in order to assess the response of mannitol expressing
plants to these stresses. Growth and physiological measurements are
used to document the differences.
[0347] Cold.
[0348] To demonstrate whether mannitol expression can confer
increased germination ability under cool conditions, transgenic
seeds containing the mtlD gene are germinated under conditions
similar to the standard cold germination test used in the corn
industry. Alternatively, transgenic seeds are planted under cool
seed bed conditions made cool by artificial environments or
naturally cool seed beds in the field. Additionally, plants
expressing mannitol are challenged during the grain filling period
for cool night time temperatures in order to demonstrate less
inhibition of leaf or canopy activity as a result of cold stress
during this time of crop development. Young transgenic seedlings
are grown at low temperature, such as about 15.degree. C., during
the light and dark period. The expression of mannitol in these
seedlings allows for increased growth and allows the seedlings to
become photosynthetic under such conditions, as well as to survive
and grow.
[0349] Frost/Freeze.
[0350] Mannitol expressing plants are assayed for increased
freezing tolerance at the seedling stage as well as late season
periods. These assays are done in artificial environments to
simulate frost or freeze events. Alternatively, seeds are planted
outside during times when the naturally occurring environment would
impose the stress.
[0351] High Heat.
[0352] Mannitol expressing plants are assayed in artificial
environments or in the field in order to demonstrate that the
transgene confers resistance or tolerance to heat.
EXAMPLE XIII
Mannitol Expression Causes Yield Increase Under Relatively
Non-Stress or More Typical Environment.
[0353] Seeds of mannitol expressing corn plants are planted out in
test plots and their agronomic performance is compared to standard
corn plants using techniques familiar to those of skill in the art.
Optionally included in this comparison are plants of similar
genetic background without the transgene. A yield benefit is
observed and plants exhibiting the increased yield are advanced for
commercialization.
[0354] Furthermore, transgenic plants with increased levels of
mannitol are field tested for agronomic performance under
conditions, including, but not limited to, limited and/or adequate
water availability. When compared to substantially isogenic
nontransgenic plants, mannitol containing plants exhibit higher
yield than their nontransgenic counterparts under non-optimal
growing conditions.
[0355] All publications and patents are incorporated by reference
herein, as though individually incorporated by reference. The
invention is not limited to the exact details shown and described,
for it should be understood that many variations and modifications
may be made while remaining within the spirit and scope of the
invention defined by the claims.
* * * * *