U.S. patent application number 13/413131 was filed with the patent office on 2012-06-28 for method for altering the metabolism of plant.
Invention is credited to Mark A. Holland, Joseph C. Polacco.
Application Number | 20120167257 13/413131 |
Document ID | / |
Family ID | 22433691 |
Filed Date | 2012-06-28 |
United States Patent
Application |
20120167257 |
Kind Code |
A1 |
Holland; Mark A. ; et
al. |
June 28, 2012 |
METHOD FOR ALTERING THE METABOLISM OF PLANT
Abstract
The invention is related to a method of altering the metabolism
of a plant by carrying out the steps of treating seeds of the plant
with non-genetically engineered selected mutant pink-pigmented
facultative methylotroph having a plant altering capability, and
growing the plants from the treated seeds, wherein the plants that
grow from such treated seeds exhibit an altered characteristic
relative to non-treated plants.
Inventors: |
Holland; Mark A.;
(Salisbury, MD) ; Polacco; Joseph C.; (Columbia,
MO) |
Family ID: |
22433691 |
Appl. No.: |
13/413131 |
Filed: |
March 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11347579 |
Feb 6, 2006 |
8153118 |
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13413131 |
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09958175 |
May 8, 2002 |
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PCT/US00/09307 |
Apr 7, 2000 |
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11347579 |
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60128111 |
Apr 7, 1999 |
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Current U.S.
Class: |
800/298 ; 435/29;
435/410; 435/415; 435/430 |
Current CPC
Class: |
C12N 15/01 20130101;
A01N 63/00 20130101; C12N 1/20 20130101; C12N 5/04 20130101; C12R
1/01 20130101 |
Class at
Publication: |
800/298 ;
435/430; 435/29; 435/410; 435/415 |
International
Class: |
C12N 5/04 20060101
C12N005/04; A01H 5/10 20060101 A01H005/10; A01H 5/12 20060101
A01H005/12; C12Q 1/02 20060101 C12Q001/02 |
Claims
1-19. (canceled)
20. A method of increasing vitamin B12 content of a plant or tissue
thereof, comprising growing a plant from plant tissue treated with
a mutant pink-pigmented facultative methylotroph (PPFM) selected
for overproduction of vitamin B12, wherein the plant or a tissue
thereof that is grown from the treated plant tissue exhibits an
increase in vitamin B12 content relative to a non-treated plant or
tissue thereof.
21. The method according to claim 20, wherein the mutant
pink-pigmented facultative methylotroph produces at least 7.5 ng
vitamin B12 per ml of culture supernatant.
22. The method according to claim 20, wherein the mutant
pink-pigmented facultative methylotroph produces at least 15 ng
vitamin B12 per ml of culture supernatant.
23. The method according to claim 20, wherein said mutant is
Methylobacterium mesophilicum, Methylobacterium organophilum,
Methylobacterium extorquens, Methylobacterium fujisawaense,
Methylobacterium radiotolerans, Methylobacterium rhodesianum,
Methylobacterium rhodinum, or Methylobacterium zatmanii.
24. The method according to claim 20, wherein said plant or tissue
thereof exhibits a vitamin B12 content of at least about 0.01
micrograms per gram fresh weight of tissue.
25. The method according to claim 20, wherein said plant or tissue
thereof exhibits a vitamin B12 content of at least about 0.1
micrograms per gram fresh weight of tissue.
26. The method according to claim 23, wherein said mutant is
Methylobacterium mesophilicum.
27. The method according to claim 20, wherein said plant is a
soybean plant or a lettuce plant.
28. The method according to claim 20, wherein said tissue is a seed
or a leaf
29. The method of claim 28, wherein said seed is heat-treated
before being treated with mutant pink-pigmented facultative
methylotroph (PPFM).
30. A method for selecting a mutant pink-pigmented facultative
methylotroph that overproduces vitamin B12 for treatment of plants,
comprising selecting a pink-pigmented facultative methylotroph for
stimulation of vitamin B12 auxotroph growth, wherein said selected
pink-pigmented facultative methylotroph provides for an increase in
vitamin B12 content in a treated plant or tissue relative to a
non-treated plant or tissue thereof.
31. The method of claim 30, wherein said methylotroph produces at
least 7.5 ng vitamin B12 per ml of culture supernatant.
32. The method according to claim 30, wherein said methylotroph
produces at least 15 ng vitamin B12 per ml of culture
supernatant.
33. The method of claim 30, wherein said mutant is Methylobacterium
mesophilicum, Methylobacterium organophilum, Methylobacterium
extorquens, Methylobacterium fujisawaense, Methylobacterium
radiotolerans, Methylobacterium rhodesianum, Methylobacterium
rhodinum or Methylobacterium zatmanii.
34. The method of claim 30, wherein said treated plant or tissue
thereof exhibits a vitamin B12 content of at least about 0.01
micrograms per gram fresh weight of tissue.
35. The method according to claim 30, wherein said treated plant or
tissue thereof exhibits a vitamin B12 content of at least about 0.1
micrograms per gram fresh weight of tissue.
36. The method of claim 30, wherein said mutant was isolated from a
natural sample or was subjected to a mutagenizing amount of
chemicals, irradiation, or stress.
37. A plant tissue comprising a pink-pigmented facultative
methylotroph mutant selected for overproduction of vitamin B12.
38. The isolated plant tissue according to claim 37, wherein said
methylotroph produces at least 7.5 ng vitamin B12 per ml of culture
supernatant.
39. The isolated plant tissue according to claim 37, wherein said
methylotroph produces at least 15 ng vitamin B12 per ml of culture
supernatant.
40. The plant tissue according to claim 37, wherein said mutant is
Methylobacterium mesophilicum, Methylobacterium organophilum,
Methylobacterium extorquens, Methylobacterium fujisawaense,
Methylobacterium radiotolerans, Methylobacterium rhodesianum,
Methylobacterium rhodinum, or Methylobacterium zatmanii.
41. The plant tissue according to claim 37, wherein said plant
tissue exhibits a vitamin B12 content of at least about 0.01
micrograms per gram fresh weight of tissue.
42. The method according to claim 37, wherein said treated plant
tissue exhibits a vitamin B12 content of at least about 0.1
micrograms per gram fresh weight of tissue.
43. The plant tissue according to claim 37, wherein said tissue is
a seed or a leaf
44. The plant tissue according to claim 37, wherein said plant is a
soybean plant or lettuce.
Description
[0001] This application claims the benefit of priority date of U.S.
Provisional Application No. 60/128,111, filed Apr. 7, 1999, the
content of which is incorporated into the present application in
its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to isolating novel naturally-occurring
mutant pink-pigmented facultative methylotrophs and altering a
plant's metabolism with such strains by treating the plant with
said strains.
BACKGROUND OF THE INVENTION
[0003] Pink-pigmented facultative methylotrophs (PPFMs) of the type
Methylobacterium mesophilicum are bacteria normally found on plant
surfaces. It has been demonstrated that these bacteria interact
with plants, by 1) producing a variety of compounds that affect
plant metabolism; and by 2) consuming plant metabolic waste, most
notably methanol. Production compounds include, but are not limited
to vitamins (Basile et al 1985. Bryologist 88 (2):77-81.), enzymes
(Holland and Polacco 1992. Plant Physiol. 98:942-948.); and
cytokinins (Long et al 1996. In: Lidstrom and Tabita eds. Microbial
Growth on Cl Compounds, Kluwer Academic Publishers). Strains of the
bacteria have also been identified that produce amino acids (Izumi,
1985 J. Ferment. Technol. 63:507-513., U.S. Pat. No. 3,663,370;
U.S. Pat. No. 3,907,673; U.S. Pat. No. 3,907,641 all of which are
incorporated herein by reference).
[0004] The inventors have previously described a method for
altering the metabolism of a plant (U.S. Pat. No. 5,268,171 herein
incorporated by reference) that employs PPFMs that have been
genetically engineered. The method includes engineering PPFMs to
produce a product where the presence of such a product would confer
desirable qualities to plants. Once identified and isolated a
population of the genetically altered bacteria are placed on or
near a plant targeted for improvement. Such bacteria are expected
to provide their host plant with the product the bacteria are
engineered to produce. The inventors also described a method that
uses PPFMs to enhance seed germination (U.S. Pat. No. 5,512,069
herein incorporated by reference). The second method does not
specify the genetic makeup or the origin of the PPFMs employed.
[0005] One of the inventors also described a method of treating
crop plants with a spray treatment of PPFM bacteria to enhance
yield (U.S. Pat. No. 5,961,687 herein incorporated by reference).
By this method, the application of PPFM bacteria to flowering
soybean plants in the field resulted in as great as a 70% increase
in yield compared to control plants. This method does not specify
the genetic makeup or origin of the PPFMs employed.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to a method of altering
the metabolism of a plant comprising treating plant tissues,
especially seeds of the plant, with a non-genetically engineered
selected mutant microorganism having a plant altering capability;
and growing the plants from the treated tissue, said growing plants
from the treated tissue being treated plants, said treated plants
exhibiting an altered characteristic relative to non-treated
plants. As a preferred embodiment of the invention, the
microorganism is a pink-pigmented facultative methylotroph, the
preferred tissue is a seed and the preferred plant is soybean.
[0007] The present invention is also directed to a non-genetically
engineered mutant microorganism having the ability to alter plant
metabolism.
[0008] The invention is further directed to plants or plant tissues
that are treated with the non-genetically engineered selected
microorganism.
DETAILED DESCRIPTION OF THE INVENTION
[0009] A method of the invention includes selecting variants from
untreated or mutagenized PPFM populations and using them to
establish "elite lines" of the bacterium. Selection, for example,
could be for enhanced production of amino acids, vitamins, enzymes
or cytokinins or for their efficient removal of plant products such
as methanol. Variants or mutants can be selected and/or isolated by
unusual physical characteristics, chemical characteristics, or by,
for instance, resistance to a particular chemical or metabolite not
possessed by the related or surrounding microorganism populations.
Other examples of such physical or chemical characteristics
include, but not limited to, the overproduction of a flavor or
fragrance-enhancing compound, as well as the production of
compounds that cause plant tissue regeneration in culture
conditions. Other examples include the ability of the bacteria to
fix atmospheric nitrogen and provide useable nitrogen to the host
plant.
[0010] As used herein, "plant metabolism" is a combination of the
biochemical activities of the plant and its PPFM symbiont. Thus, a
plant with "altered metabolism" means a plant whose physical,
biochemical or nutritional attributes have been changed due to the
activities of PPFM bacteria living on it.
[0011] As used herein, with respect to a mutant that overproduces a
certain compound, such as "methionine mutant", "vitamin mutant",
and so on, it refers to any mutant microorganism that overproduces
the product. Thusly, as used herein, "methionine mutant" refers to
a mutant microorganism that overproduces methionine as compared
with a normal microorganism. It can be seen that any mutant that
produces a metabolite or substance that enhances the growth or
otherwise affects the growth or characteristics of the associated
plant is within the purview of the invention. For example, a
cytokinin mutant overproduces cytokinin (e.g., zeatin or zeatin
riboside) to alter the growth and regeneration of the associated
plant.
[0012] As used herein, "vitamin mutant" refers to a microorganism
that overproduces vitamins. Preferably, the vitamin is vitamin
B-12.
[0013] As used herein, "vigor mutant" refers to a microorganism
that grows well under conditions detrimental to plant growth and
development. Such conditions, for example, might be low soil
temperatures during spring planting.
[0014] As used herein, "triterpenoid mutant" refers to a
microorganism that overproduces terpenoid compounds normally found
in PPFMs or which produces novel terpenoid compounds.
[0015] As used herein, "threonine mutant " refers to a
microorganism that overproduces the amino acid threonine.
[0016] As used herein, "L-glutamic acid mutant" refers to a
microorganism that overproduces the amino acid L-glutamic acid.
[0017] As used herein, "L-lysine mutant" refers to a microorganism
that overproduces the amino acid L-lysine.
[0018] As used herein, "a single-cell protein mutant" refers to a
microorganism that contributes to the nutritional quality of its
plant host by overproducing a high quality, nutritionally complete
protein. Single cell protein is a high quality protein produced by
a microorganism, and is used directly as a food supplement in
animal feeds or by people. In certain instances, the microorganism
is used directly for food because of its nutritional qualities.
[0019] As used herein, "a mutant that over-produces the amino acids
L-lysine, L-aspartic acid, L-alanine, L-valine, L-leucine, and
L-arginine" has the characteristic of overproducing all of the
above-named amino acids.
[0020] The invention is not limited to Methylobacterium
mesophilicum. For example, microorganisms that can be used in the
invention include any other pink-pigmented facultative
methylotrophs (PPFMs). Preferably, PPFM includes, but is not
limited to, Methylobacterium mesophilicum, Methylobacterium
organophilum, Methylobacterium extorquens, Methylobacterium
fujisawaense, Methylobacterium radiotolerans, Methylobacterium
rhodesianum, Methylobacterium rhodinum, or Methylobacterium
zatmanii. In the most preferred embodiment, the microorganism is
Methylobacterium mesophilicum.
[0021] The type of plants that can be altered by the method of the
invention include, but not limited to, nonvascular plants, vascular
plants, gymnosperms, angiosperms, dicots, or monocots. Preferably,
the plant belongs to the vascular plants. More preferably, the
plant is an angiosperm. Most preferably, the plant is soybean.
[0022] In the seed treatment method of the invention, the treatment
method can be any method so long as the microorganism is able to
bind to the seed. Such a method may include taking dry plant seeds
and soaking them in suspensions of washed cells of the mutants,
preferably for about 6 hours, and allowing the seeds to imbibe the
mutant bacteria. Following this treatment, the seeds can be planted
as normal to produce treated plants. Control seeds also can be
planted to produce control plants. Harvested tissue from the
treated plants is assayed for increased amounts of Vitamin B-12 as
compared with control plants. Another treatment method may utilize
electricity to cause the microorganism to adhere to the plant seed.
Another method may use the PPFMs as a seed coating or powdered
inoculum.
[0023] The introduction and transfer of the non-genetically altered
or engineered strains can be accomplished by several methods. For
example, cured or uncured seed can be imbibed in bacterial
suspensions. Alternatively, cured or uncured scions can be grafted
onto bacteria associated stocks pursuant to well known methods
practiced in agriculture. Bacterial-associated embryos can be
propagated under cell culture isolation that induces plantlet
formation. Plant tissue cultures can be co-cultivated with, or
inoculated with the bacteria. Another method capable of introducing
and transferring the communal bacterium to the host plant is to
vacuum infiltrate bacteria into seedlings or somatic embryos. Plant
cuttings could be rooted in water or nutrient solutions containing
bacteria. Finally, plants could be sprayed with a suspension of
bacterial cells.
[0024] As used herein, "non-genetically engineered" PPFM mutant
means any mutant that is isolated from a natural sample, and thus
called "naturally occurring". Alternatively, the mutant may be
subject to a mutagenizing amount of chemicals, irradiation, or
stress such that the microorganism exhibits an altered metabolism
phenotype. The phenotype may be, but not limited to, overexpression
of various nutritional compounds, such as vitamins or amino
acids.
[0025] Various prior art methods of genetically altering bacteria
can be used in accordance with the present invention. For example,
spontaneous and induced mutants can be recovered and selected for
resistance to a series of antibiotics, such as pipericillin,
rifampicin, etc. If desired, multiple resistances can be
assembled.
[0026] An alternate approach for obtaining the bacteria is to
recover bacteria that have acquired promiscuous plasmids bearing
drug resistance (gene conferring drug inactivation) by
non-genetically engineered methods.
[0027] Another method of altering the bacteria is to recover
bacteria resistant to, or able to inactivate herbicides such as
glyphosate and sulfonyl urease which inhibit the
5-enolpyruvylshikimic acid-3-phosphate synthase and acetolactate
dehydrogenase, respectively.
[0028] Once bacterial strains are selected, seeds of plants can be
soaked in suspensions containing these strains and allowed to
imbibe bacterial cells. Such treated seeds can be planted. Mature
plant and seed products can be harvested and tested for increases
in such compounds as certain desired amino acids, vitamins, enzymes
or cytokinins. Isolated PPFMs growing under extreme conditions
might also impart superior hardiness or vigor to plants grown from
seed imbibed or inoculated with such PPFMs.
Other Mutants
[0029] The application of selected strains of PPFM bacteria to
plants for the purpose of altering their metabolism will be useful
as a technology only to the extent that PPFMs produce a variety of
products of interest or which may be manipulated. In fact, a number
of useful strains have already been described for other purposes
and are included here to serve as examples of the type of mutant
PPFM cell lines that can be made, such as: Methylobacterium
rhodinum (ATCC# 43282) over-produces the amino acid threonine;
Methylobacterium sp. (ATCC# 21371) over-produces the amino acid
L-glutamic acid; Methylobacterium sp. (ATCC# 21372) over-produces
the amino acid L-glutamic acid; Methylobacterium sp. (ATCC# 21926)
over-produces the amino acid L-lysine; Methylobacterium sp. (ATCC#
21969) over-produces the amino acid L-glutamic acid;
Methylobacterium sp. (ATCC# 21927) over-produces the amino acids
L-lysine, L-aspartic acid, L-alanine, L-valine, L-leucine, and
L-arginine; and Methylobacterium sp. (ATCC# 21438) produces
single-cell protein.
[0030] PPFM bacteria are also documented to produce triterpenoid
compounds, which is significant because these might be implicated
in development of novel flavors and fragrances or condition pest
resistance (Bisseret, P., Zundel, M., Rohmer, M. 1985. Eur. J.
Biochem. 150:29.; Renoux, J. M., Rohmer, M. 1985. Eur. J. Biochem.
151:405.; Zundel, M., Rohmer, M. 1985. Eur. J. Bioch. 150:35.)
EXAMPLES
Example 1
Boosting Methionine Content In Soybeans
[0031] Mutant PPFMs (methionine mutant 1) were isolated from the
general population of PPFMs by their resistance to the methionine
analog ethionine. Phenotypes of putative variants or mutants were
confirmed as methionine-excreting and overproducing by their
ability to support the growth of an E. coli methionine auxotroph.
This organism was deposited with the American Type Culture
Collection, (ATCC), 10801 University Boulevard, Manassas, Va.,
20110, USA, under provisions of the Budapest treaty and assigned
ATCC 202213. The deposit will be maintained for the life of the
patent as required by Treaty. All restrictions imposed by the
depositor on the availability to the public of ATCC 202213 will be
irrevocably removed upon granting the patent. Dry soybean seeds
were soaked for 6 hours in suspensions (10.sup.9 cells/ml) of
mutant cells washed in 0.5% saline, allowing the seeds to imbibe
the mutant bacteria. A duplicate set of dry soybean seeds were
soaked in non-mutant isolates of PPFMs (control) designated as ATCC
202211.
[0032] Following this treatment, all seeds were planted as normal
to produce treated plants and control plants. Beans harvested from
the treated plants and control plants were sent to an independent
laboratory for analysis. It was determined that seeds harvested
from the plants treated with the mutant strain exhibited a 12%
increase in methionine content over control levels.
Example 2
Boosting Methionine Content In Soybeans
[0033] The experiment above was duplicated using a second strain of
methionine secreting and overproducing PPFMs (methionine mutant 2).
This organism was deposited with the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va.,
20110, USA, under provisions of the Budapest treaty and assigned
ATCC 202212. The deposit will be maintained for the life of the
patent as required by Treaty. All restrictions imposed by the
depositor on the availability to the public of ATCC 202212 will be
irrevocably removed upon granting the patent. Dry soybean seeds
were soaked in suspensions of washed cells of the mutant for 6
hours, allowing the seeds to imbibe the mutant bacteria. A
duplicate set of dry soybean seeds were soaked in non-mutant
isolates of PPFMs. Plants from seeds thus treated produced seeds
exhibiting an 8% increase in methionine content over control
seeds.
Example 3
Increasing the Vitamin Content of Plants
[0034] Vitamin B12 is an essential nutrient for human health.
Normally, it is made by intestinal bacteria and absorbed through
the gut. Alternatively, it is found in meat. Plants are not a
source of the vitamin and it has been questioned whether plants
require it themselves. However, several enzymes that include a B12
co-factor have been reported in plants (Poston, J M 1977. Science
195:301.; Poston, J M 1978. Phytochemistry 17:401.) and B12
produced by Methylobacterium (PPFM bacteria) was reported to
stimulate the growth of liverworts in culture (Basile, D. V.,
Basile, M. R., Li, Q. Y., Corpe, W. A. 1985. Bryologist 88 (2):77.)
Since B12 is extremely light labile, it seems likely that plants do
not accumulate large quantities of it even though it is normally
present in their tissues (Nelson, D. L., Cox, M. M. 2000. Lehninger
Principles of Biochemistry. 3.sup.rd edition, Worth.).
[0035] We selected naturally-occurring mutants of PPFM bacteria
that overproduce vitamin B12 based on their ability to stimulate
the growth of Arthrobacter (ATCC#12834), a B12 auxotroph.
Thirty-six putative mutants were initially selected; that number
was reduced to 17 upon re-screening. Culture supernatants from
these isolates were assayed for vitamin B12 content by the
microbiological method of Capps et al. (Capps, Hobbs, Fox. 1949 J.
Biol. Chem. 178:517.) using a commercial B12 assay medium (Difco
#0360) and Lactobacillus delbrueckii subsp. Lactis ATCC# 4797. The
results of this assay are shown in Table 1 for 17 selected strains
of PPFM.
TABLE-US-00001 TABLE 1 Vitamin B12 content of PPFM culture
supernatants (ng vit. B12/ml culture medium) Strain # ng B12/ml 1
5.4 2 4.2 4 5.8 5 5.7 6 5.9 7 6.2 8 6.9 9 6.5 10 7.5 11 15.0 13 5.5
15 7.5 21 8.0 26 4.2 29 5.0 32 6.0 34 4.9
[0036] Among the B12 over-producing mutants isolated, #11, as shown
in Table 1, was a standout, producing three times the amount of B12
produced by the other isolates. This organism, designated
Methylobacterium mutant B12-11, was deposited with the American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va., 20110, USA, on Apr. 4, 2000, under provisions of the
Budapest treaty. The deposit will be maintained for the life of the
patent as required by Treaty. All restrictions imposed by the
depositor on the availability to the public of this strain will be
irrevocably removed upon granting the patent. We tested mutant
B12-11 on plant tissue to see whether it could increase the B12
content of the plant.
[0037] Seeds of leaf lettuce, Lactuca sativa cv. Green Ice (Burpee
Seed Co.) were heated in a dry oven at 50.degree. C. for 48 hours
to reduce the resident PPFM bacteria population. Following this
treatment, they were imbibed in a suspension of mutant B12-11 cells
for six hours (the method is as described for the methionine
over-producing mutants described above), then planted in the
greenhouse. A second set of seeds was imbibed in mutant B12-11
cells, but was not heated before imbibition. Control plants were
not heated, nor were they imbibed in cells of the mutant. After one
month of growth, leaf tissue was harvested from the plants and
aqueous extracts were prepared from them. These extracts were
assayed for Vitamin B12 content as described above. B12 was not
detected in control plants, but measured about 0.1 .mu.g/gram fresh
weight lettuce in the heat-treated seeds and about 0.01 .mu.g/gram
fresh weight lettuce in the unheated seeds. This is a very small
amount of the vitamin to be sure, but given the fact that the
recommended daily allowance of B12 is only 2 .mu.g for adults, it
is significant.
[0038] These effects are not limited to leaf lettuce. Soybean is
also expected to accumulate B12 in bean tissue to even higher
levels than in leaf lettuce.
Example 4
Plants of Induced Vigor
[0039] Seeds of crop plants frequently germinate and grow poorly
under cold, wet field conditions early in the spring. Because PPFM
bacteria influence both the rate of germination and growth, PPFMs
that grow under lower than normal temperatures are selected.
Selection includes isolating surviving organisms grown at 4.degree.
C. When these PPFMs and added to seeds, enhanced vigor can be
expected from the plants grown from these seeds.
Example 5
Cytokinin Over-Producing Plants
[0040] PPFMs overproducing cytokinins are selected for example, by
their ability to stimulate growth of plant cells in culture. Once
isolated, the PPFMs are placed in seeds, and plants are grown from
such seeds to verify overproduction of the cytokinins by the
plants. Such plants are expected to display superior growth and
yield characteristics compared to untreated plants.
[0041] Alternatively, PPFMs that overproduce cytokinins are sprayed
on plants during flowering to enhance yield. The effectiveness of
this strategy was demonstrated in U.S. Pat. No. 5,961,687 using
unselected lines of PPFMs. Selected mutant lines of the PPFMs are
expected to be even more effective.
Example 6
Plants Producing Unusual Products
[0042] It is known that PPFMs produce unusual products such as
triterpenoid compounds. Isolating PPFMs producing such compounds
and inoculating plant tissues with them generate plants that
contain the triterpenoid. This practice is used to overproduce
other compounds. For example, Zabetakis (1997. Plant, Cell, Tissue
and Organ Culture 50:179-183.) found that tissue cultures of
strawberry that did not include PPFM bacteria did not synthesize
furanones, molecules that participate in the synthesis of
strawberry flavor and fragrance compounds. When the same cultures
were inoculated with PPFMs, flavor and fragrance compounds were
made. This work did not employ selected lines of PPFM bacteria.
[0043] Selected PPFM mutants are expected to confer on cell
cultures or whole plants the ability to make compounds that they do
not normally make or to make greater quantities of compounds (like
flavor or fragrance components) that they normally make at low
levels.
Example 7
Enhancement of Tissue Culture Performance
[0044] Corpe and Basile (1982. Dev. Indust. Microbiol. 23:483.)
reported that tissue cultures of Streptocarpus gracilis that were
inoculated with PPFMs regenerated plantlets while similar cultures
growing without the bacteria did not regenerate. Their work did not
employ selected lines of PPFM bacteria. The performance of plant
cell cultures with respect to growth and regeneration is expected
to be greatly enhanced by the inclusion of selected PPFM
mutants.
Example 8
Bacterial Contributions To Plant Nitrogen Metabolism
[0045] Many of the PPFM bacteria contain a nitrogenase enzyme with
which they convert atmospheric nitrogen gas into a usable form. To
test whether any of this fixed atmospheric nitrogen contributes to
the nitrogen metabolism of plants inhabited by nitrogen-fixing
PPFMs, we performed the following experiment.
[0046] Seeds of tobacco (Nicotiana tabacum) were heated at
50.degree. C. for 48 hours to reduce their resident PPFM
population. The treated seeds were then sown on 15% agar in
distilled water or on 15% agar in distilled water to which a
nitrogen source was added. Untreated (control) seeds were sown on
the same two media. The seeds were grown under continuous light for
two weeks, during which time they were observed for signs of
nitrogen deficiency. At the end of this time, chlorophyll content
of the seedlings was measured, chlorosis being indicative of
nitrogen deprivation. Seedlings grown on nitrogen containing
medium, whether with or without normal levels of PPFM bacteria
showed normal growth and color development. In contrast, seeds with
normal levels of PPFM bacteria growing on medium without added
nitrogen were smaller and contained only 55% as much chlorophyll as
plants grown on nitrogen. Significantly, seedlings with low numbers
of PPFMs growing on nitrogen-free medium not only grew very slowly,
but contained only 29% as much chlorophyll as seedlings grown on
nitrogen. These results suggest that nitrogen fixation by the PPFMs
can help to support the growth of plants. Selected cell lines of
PPFMs are expected to contribute nitrogen to their host plants to
an even greater extent.
[0047] From the above experiments and working examples, applicant
has demonstrated broad utility of the invention. Thus, the
invention is not limited to the examples and disclosure, which
serve purposes of illustration. Rather, the invention is limited by
the scope of the appended claims.
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