Modified plant containing a bacterial insculant

A method for the production of a plant of reduced stature is provided comprising the introduction of a bacterial cell into a seed or a plant, the bacterial cell being capable of replicating in the plant and of inducing a reduction in plant stature. A seed and a plant modified by introduction of such a bacterial cell are also provided.

FIELD OF THE INVENTION 
The present invention relates to a modified seed and a modified plant, 
respectively, into each of which a bacterial cell is affirmatively 
introduced, the modified seed and modified plant being capable of 
developing into a modified plant of reduced stature. A method of producing 
a plant of reduced stature is also provided as well as a method of 
introducing a bacterial cell into a seed. 
BACKGROUND OF THE INVENTION 
One of the goals of commercial agriculture is to increase the yield of 
crops. Yield increases have been obtained, for example, by reducing plant 
stature. By reducing the stature of plants, relatively less of the plant's 
biomass becomes involved in the production of stem and leaves and 
relatively more of the plant's biomass becomes involved in grain 
production. Furthermore, by reducing the stature of plants, the resistance 
of plants to lodging, i.e., falling over, is increased. Lodging is 
undesirable because the resultant flattened crop and secondary growth make 
harvesting difficult. Moreover, when plants lodge, the grain becomes dirty 
and wet and rots, thereby necessitating grain drying and causing delay in 
harvesting and loss of yield. 
Throughout the history of agronomy, plant breeders have produced plants of 
reduced stature by selection and breeding, or by induction of mutation 
through irradiation or chemical means, as discussed in Dalrymple, D. G., 
DEVELOPMENT AND SPREAD OF HIGH-YIELDING RICE VARIETIES IN DEVELOPING 
COUNTRIES, Bureau for Science and Technology, Agency for International 
Development, Washington, D.C. (1986). The production of such plants of 
reduced stature, in accordance with the conventional methods, entails 
selection of appropriate parent stock, manipulation of the parent stock by 
hybridization or mutation, propagation of progenies, and further selection 
and breeding. Such processes are time-consuming and expensive. 
Plant growth and development are also known to be regulated by the external 
application of plant growth regulators, such as hormones and other 
chemicals. Responses to such chemical applications, however, are variable, 
as discussed in Herbert, C. C., Growth Regulation in Cereals--Chance or 
Design?, in CHEMICAL MANIPULATION OF CROP GROWTH AND DEVELOPMENT, J. S. 
McLaren, ed., Butterworth Scientific (London 1982), pp. 315-327. Moreover, 
application of chemicals for purposes of regulating plant growth has to be 
performed in accordance with a precise schedule, without much allowance 
for flexibility. 
The plants of reduced stature produced by conventional breeding means, such 
as those described above, are normally adapted to growth and cultivation 
under a specific set of environmental conditions. Because of the elaborate 
processes involved in breeding and adaptation, however, only a limited 
number of dwarf varieties of a particular crop are customarily adapted for 
cultivation in a given region. This often leads to planting of either a 
monoculture or a limited variety of crops by a given farmer. It is a 
concern among commercial farmers, therefore, that should disease strike 
the one or few varieties cultivated, the entire crop will be decimated. It 
would be desirable, therefore, to provide an easier, less expensive and 
less time-consuming method of producing a plant of reduced stature so that 
more varieties of a crop can be planted at any given time. 
Microorganisms have been known to affect plant growth and development. 
Certain types of microorganisms, such as hybrid 
agricultural-chemical-producing endosymbiotic microorganisms, colonize the 
interior of plants and provide useful agricultural chemicals, such as 
pesticides, to the plants. Certain microbial endophytes are capable of 
inducing enhanced resistance in a host to phytopathogens. 
Furthermore, pathogenic strains of Clavibacter xyli that inhabit sugar cane 
and bermudagrass and cause stunting disease in these plants have been 
reported. In Davis, M. J. et al. [J. System. Bacteriol. 34:107-117 
(1984)], ratoon stunting disease of sugarcane is attributed to infection 
by Clavibacter xyli subsp. xyli. The stunting effect of this infection was 
associated with significant yield losses. In Davis, M. J. and Augustin, B. 
J. [Plant Disease 68:1095-1097 (1984)], a bermudagrass stunting disease is 
attributed to Clavibacter xyli subsp. cynodontis. The diseased grass 
reportedly appeared as unsightly circular patches of chlorotic and dying 
grass. The bacterial species causing ratoon stunting disease and that 
causing bermudagrass stunting disease were determined to be distinct from 
each other because each was able to induce a disease condition in its 
natural host but not in the natural host of the other. Yet, on the other 
hand, both bacteria could be found growing in the xylem vessels of both 
hosts, as stated in Davis et al. (1984). Subspecies of Clavibacter xyli, 
therefore, have been known to cause disease only in their natural hosts. 
In addition to bacterial cells, fungal endophytes have also been known to 
cause stunting in infected plants, particularly, in infected grasses as 
discussed in Clay, K., Mycol. Res. 92:1-12 (1989). Fungal endophytes, 
however, often produce toxic alkaloids that are not appropriate for 
agricultural crops. In particular, for purposes of producing plants that 
yield edible seeds such as rice, infection by fungal endophytes are 
associated with certain disadvantages. For example, plants that are 
infected with fungal endophytes can become sterile and not produce seeds 
at all, or the fungal spores and toxic materials produced by the fungal 
endophytes, such as alkaloids, may find their way into the seeds, thus 
making the seeds unsatisfactory as food sources. Hence, the use of fungal 
endophytes to produce a reduction in plant stature is not desirable. 
The present inventors have recognized that there exists a need for an easy, 
inexpensive and efficient method of producing a plant of reduced stature, 
particularly, that of an agricultural crop. They have recognized that it 
would be desirable if a microorganism can be found that can be introduced 
into a nonnatural host to produce a plant of reduced stature and yet, 
would not produce any harmful or undesirable side effects associated with 
stunting due to a natural infection of such a microorganism in its natural 
host. Such a microorganism should not produce any toxic compounds such as 
alkaloids and should not sporulate or grow in the seeds of the host so as 
not to contaminate, for example, the seeds of cereal crops. Infection of 
plants by a microorganism that can produce the above-described effect 
would be an easy, efficient and inexpensive method of producing plants of 
reduced stature. 
SUMMARY OF THE INVENTION 
It is, therefore, an object of the present invention to provide a method of 
producing a plant of reduced stature in a manner that overcomes the 
disadvantages of conventional techniques. 
It is a further object of the present invention to provide a method of 
producing a plant of reduced stature in a manner that is easy, inexpensive 
and efficient. 
It is another object of the present invention to provide a plant of reduced 
stature by use of a microorganism. 
It is another object of the present invention to provide a plant of reduced 
stature by use of a microorganism that will not affect the yield of the 
plant, that will not transmit the microorganism to the seed of the plant 
and that will not result in any toxic chemicals in the seed. 
It is yet another object of the present invention to provide a modified 
seed containing a microorganism, the modified seed being capable of 
developing into a plant of reduced stature, and the microorganism having 
the characteristics described above. 
It is still another object of the present invention to provide a modified 
plant containing a microorganism, the modified plant being capable of 
developing into a plant of reduced stature, the microorganism also having 
the characteristics described above. 
In accomplishing these objects, there has been provided, in accordance with 
one aspect of the present invention, a method for the production of a 
plant of reduced stature comprising introducing a bacterial cell into a 
seed of the plant to produce a modified seed, and developing the modified 
seed into a modified plant, the bacterial cell being capable of 
replicating in the modified plant to produce a reduction in stature of 
said modified plant. 
In accordance with a further aspect of the present invention, there has 
been provided a method for the production of a plant of reduced stature 
comprising introducing a bacterial cell into a plant to produce a modified 
plant, and developing said modified plant, the bacterial cell having the 
characteristics described above, and the plant is a dicotyledonous plant 
or a monocotyledonous plant such as a cereal crop. 
In accordance with another aspect of the present invention, there has been 
provided a method for introducing bacterial cells into a seed by adding 
the bacterial cells to a biologically compatible liquid carrier to form a 
suspension, impregnating the seeds with the suspension, and removing the 
excess carrier from the seeds, to form a viable seed comprising a viable 
bacterial cell. 
In accordance with a another aspect of the present invention, there has 
been provided a modified seed comprising a seed of a plant and a bacterial 
cell introduced into the seed, the modified seed being capable of growing 
into a modified plant comprising the bacterial cell, and the bacterial 
cell being capable of replicating in the modified plant to produce a 
reduction in stature of the modified plant. 
In accordance with another aspect of the present invention, there has been 
provided a modified plant comprising a plant and bacterial cell introduced 
into the plant, the bacterial cell having the characteristics as described 
above, and the plant is a dicotyledonous plant or a monocotyledonous plant 
such as a cereal crop. 
Further objects, features and advantages of the present invention will 
become apparent from the following detailed description. It should be 
understood, however, the detailed description and specific examples, while 
indicating preferred embodiments of the invention, are given by way of 
illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Reference will now be made in detail to the presently preferred embodiments 
of the invention, which, together with the following examples, serve to 
explain the principles of the invention. 
It has been discovered that a seed of a plant can be modified by 
introduction of a bacterial cell into the seed with limited or no injury 
to the seed. In the context of the present invention, the seed modified in 
this manner is capable of growing into a modified plant in which the 
bacterial cell can replicate and produce a reduction in stature of the 
modified plant. In addition, applicants have discovered that a plant 
itself can also be modified by introduction of a bacterial cell into the 
plant with limited or no injury to the plant, the bacterial cell is 
capable of replicating in the plant and producing a modified plant of 
reduced stature, the plant being a dicotyledonous plant or a 
monocotyledonous plant such as a cereal crop. 
Furthermore, applicants have discovered a method of producing a plant of 
reduced stature by introduction of a bacterial cell into a seed to produce 
a modified seed, or into a plant to produce a modified plant, the modified 
seed, the modified plant and the bacterial cell each having the 
characteristics described above. The process of introduction of a 
bacterial cell into a seed or plant can be accomplished by any acceptable 
technique that will preserve the viability of the seed or the plant and of 
the bacterial cell. 
Within the content of the present invention, a "reduction in stature of a 
plant" refers to an effect caused by the affirmative introduction of a 
bacterial cell into a seed or a plant, whereby the height of the mature 
plant measured from the ground to the highest visible ligule ("hvl") is 
reduced from the average height of a similar plant in the field which is 
not so infected. 
The bacterial cell that is suitable for use in the present invention 
belongs to a species of bacteria that is capable of replicating in the 
interior tissues of a plant and of effecting, either directly or 
indirectly, a reduction in plant stature. Under normal field conditions, 
the bacterial cell within the present invention does not ordinarily 
inhabit the seed or the plant into which the bacterial cell is introduced. 
Such a bacterial cell can be a gram-positive bacterium, a gram-negative 
bacterium or a species of actinomycetes. The bacterial cell can be 
unmodified or modified, either by genetic engineering techniques to 
incorporate foreign genes or by mutagenesis. In a preferred embodiment, 
the bacterial cell is an unmodified species of either Corynebacteria, 
Clavibacter, Pseudomonas, Xanthomonas or Erwinia, the corynebacteria and 
clavibacters being as defined in Davis M. J. et al. (1984), loc. cit. In a 
particularly preferred embodiment, the bacterial cell is an unmodified 
strain of Clavibacter xyli. In a most preferred embodiment, the bacterial 
cell is an unmodified Clavibacter xyli subspecies cynodontis (hereafter 
"Cxc"). A preferred strain of Clavibacter xyli subsp. cynodontis is one 
that is on deposit at American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. 20852, U.S.A., under Accession No. 33973. In an 
alternative embodiment, Cxc can be isolated from infected bermudagrass, a 
natural host species, from areas where Cxc is known to be found, e.g., in 
Louisiana. 
As used herein, the term "genetically engineered" or similar terms refer to 
bacterial cells that have been manipulated by human intervention to delete 
or rearrange DNA from those cells, or to add to those cells DNA or RNA, 
including DNA or RNA from a different organism, by conventional techniques 
including, but not limited to, recombinant DNA, recombinant RNA, cell 
fusion, protoplast fusion, conjugation, plasmid transfer, transformation, 
transfection and transduction. 
Known protoplast and spheroplast fusion techniques are described in D. A. 
Hopwood, "Genetic Studies With Bacterial Protoplasts," Ann. Rev. 
Microbiol., 35:237-72 (1981), and in R. L. Weiss, J. Bacteriol., 
128:668-70 (1976), both of which are specifically incorporated herein by 
reference. Selection of a technique by which the fusion hybrids of the 
present invention may be formed will generally be within the capabilities 
of one of ordinary skill in the art based on the above-referenced 
scientific literature. 
In general, the fusion procedure involves the removal of the cell wall from 
the bacteria to be fused, fusion of the bacterial cells in a 
fusion-inducing medium, such as polyethylene glycol, and regeneration of 
the cell wall about the fusion hybrids. Fusion and regeneration of the 
cell wall are conducted at low temperatures so that rates of expressible 
genetic recombination are favored in relation to rates of enzymatic 
destruction of genetic material in the newly formed hybrids. 
Following initial formation of fusion hybrids, the genetic make-up of the 
bacteria population is relatively unstable for a period of 2 or 3 days, 
during which much genetic recombination apparently occurs. During this 
time period, those stable fusion hybrids capable of manifesting the one or 
more selectable traits are selected. 
In a preferred embodiment of the present invention, the bacterial cell that 
is suitable for use in the present invention is capable of growing in the 
vascular tissues of a plant, e.g., the xylem. It is preferable that the 
bacterial cell be nonsporulating and it is most preferable that the 
bacterial cell is not transmittable to the seed of the modified plant. 
Virtually any type of seeds may be used in the present invention, provided 
they are seeds and they produce plants both of which the bacterial cell of 
the present invention does not ordinarily inhabit or infect under normal 
field conditions. These include seeds of both monocotyledonous and 
dicotyledonous plants that may be useful for agronomic, horticultural, or 
ornamental purposes. A preferred group of plants that provide seeds that 
are suitable for the present invention is either dicotyledonous plants or 
monocotyledonous plants such as cereal crops. The cereal crops include, 
but are not limited to, wheat, barley, rye, rice, and oats as well as 
corn, millet, and sorghum. Particularly preferred seeds are those for 
rice. Among the monocotyledonous plants, the seeds of which are suitable 
for use in the present invention, a cereal crop or plant that is capable 
of utilizing either the C3 (Calvin) or the C4 (Hatch-Slack) photosynthetic 
pathway is preferred. 
In another embodiment of the present invention, the plant itself is 
modified by introduction of a bacterial cell. In such an instance, the 
plant is one that the bacterial cell of the present invention does not 
ordinarily inhabit or infect under normal field conditions. Preferably, 
the plant is either a monocotyledonous plant such as a cereal crop, or a 
dicotyledonous plant. Among the cereal crops that are suitable for use 
herein, wheat, barley, rye, oats, as well as sorghum, corn, millet are 
preferred, and rice is particularly preferred. 
In carrying out the method of producing a plant of reduced stature of the 
present invention, a bacterial cell is affirmatively introduced into a 
seed to produce a modified seed that can develop into a modified plant of 
reduced stature, or into a plant to produce a modified plant of reduced 
stature. Further, in carrying out the method of the present invention, the 
bacterial cells and the seeds or plants that are suitable for use are 
bacterial cells that are not found in the seeds or plants sought to be 
modified, absent affirmative inoculation of such bacterial cells. 
The terms "affirmative introduction of the bacterial cells into the seed" 
or "impregnation of seeds," within the context of the present invention, 
mean to actively or positively introduce the bacterial cells to the inside 
of the outer, protective seed coat, i.e., the pericarp or the testa. 
Moreover, in certain instances, for example, in the case of corn seeds, at 
least for Clavibacter xyli subsp. cynodontis, the microorganisms must be 
in the embryo itself for the resulting plant to be colonized. 
For purposes of affirmatively introducing bacterial cells into the seeds, 
the bacterial cell of the present invention may be micro-encapsulated with 
a suitable nutrient source. Such nutrient sources include, for example, 
dehydrated culture medium; components of a culture medium such as cysteine 
and bovine serum albumin; and complex carbohydrate or amino acid sources, 
such as milk, starch or yeast extract. The nutrient source and the 
bacterial cell can be both encapsulated by freeze-drying, spray-drying, 
liposome entrapment or other conventional techniques. 
In one embodiment of the present invention, the bacterial cell is 
introduced into the seed by wounding the seed and contacting the bacterial 
cell with the wound. In another embodiment of the present invention, the 
bacterial cell is introduced into the seed by (1) placing a bacterial cell 
in a biologically compatible liquid carrier to form a suspension, and (2) 
impregnating the seeds with the suspension. Preferably, excess suspension 
or carrier is removed from the seeds, such as by evaporation. This process 
of introducing bacterial cells into seeds is accomplished in such a way 
that the viability of both the bacterial cell and the seeds is maintained, 
so that the seed is capable of germinating and the bacterial cell is 
capable of replicating in the resulting plant. 
The bacterial cell to be introduced into both plants and seeds can be 
cultured or maintained in any medium that permits growth or sustains 
viability, respectively. The cell cultures can be concentrated or diluted 
to obtain the desired density for inoculation into seeds or plants. 
Preferably, the bacterial cells used herein are derived from cultures that 
are substantially pure, that is, substantially free of unwanted 
microorganisms. 
The biologically compatible liquid carrier for the bacterial cell, within 
the present invention, is any liquid in which the bacterial cell can form 
a suspension and which is not lethal to either the bacterial cell or the 
seeds. Such a liquid carrier includes water and a water-based buffer, such 
as phosphate buffered saline. In a preferred embodiment, the biologically 
compatible liquid carrier comprises a solution of water and a 
water-soluble gum, which may be natural or synthetic. Such gums include 
gelatin, sodium alginate, gum ghatti, xanthan gum, karaya gum, Dow-Corning 
1944/B polymer, which is a silicone oil from Dow-Corning, polyethylene 
oxide, Natrolsol.TM., which is a hydroxyethyl cellulose from Hercules 
Chemical Co., tragacanth gum, guar, gum, gum arabic, locust bean gum, 
methylcelluose, carboxmethycellulose, starch, and Rhoplex.TM. B-15, which 
is an aqueous acrylic emulsion from Rohm & Haas. Particularly preferred 
gums are methylcellulose, carboxymethycellulose, xanthan gum, and 
Rhoplex.TM.. The gums are preferably used in a concentration of from about 
0.1% to about 10% weight per volume of water. 
The liquid carrier may also contain a buffer, which assists in maintaining 
the viability of the microorganism, and a surfactant. Suitable surfactants 
for use herein are, e.g., Tween 20 (polyoxyethylenesorbitan monolaurate), 
Tween 40 (polyoxyethylenesorbitan monopalmitate), Tween 60 
(polyoxyethylenesorbitan monopalmitate), Tween 80 (polyoxyethylenesorbitan 
monoleate), Tween 85 (polyoxyethylenesorbitan trioleate), Regulaid 
(polyoxyethylenepolypropoxypropanol and alkyl-2-ethoxy ethanol dihydroxy 
propane), and Surfel (83% paraffin-based petroleum oil; 15% polyol fatty 
acid esters and polyethoxylated derivatives and 2% unidentified 
components). Organic solvents and penetrants such as dimethyl sulfoxide 
(DMSO) or N,N-dimethyl formamide (DMF), also serve as liquid carriers and 
promote colonization. Particularly preferred concentrations are 1% DMSO or 
3% DMF. 
In addition, chemicals such as fungicides can be added to the liquid 
carrier and used at a concentration that is not lethal to the bacterial 
cell. For example, Clavibacter xyli subsp. cynodontis has been found to 
tolerate significant levels of certain fungicides. 
In an alternative embodiment, the biologically compatible liquid carrier is 
an organic solvent. In such instances, particularly, when it is desirable 
that the bacterial cell be dormant, the organic solvent should be 
substantially anhydrous. Otherwise, the presence of water in the organic 
solvent can cause the bacterial cell to reenter into an active metabolic 
state. Dormancy can be induced in the bacterial cell by conventional 
techniques, e.g., by lyophilization. 
Virtually any organic solvent that is not toxic to the seed or to the 
dormant bacterial cell, and which can maintain the dormancy of the 
bacterial cell may be used. These include acetone, dichloromethane, 
trichloromethane (i.e., chloroform), carbon tetrachloride, DMSO, DMF, 
methanol, ethanol, benzene, n-hexane, cyclohexane, ortho-xylene, 
meta-xylene, and para-xylene, isopropanol, and n-butanol. Volatile organic 
solvents are particularly preferred because they have the advantage of 
being easily evaporated at the end of the process. 
Various oils have also been found to be useful as organic solvents. These 
include oils that are relatively inert and nontoxic to the bacterial cell 
and the seeds such as vegetable oil, mineral oil, linseed oil, and 
silicone oil. 
The parameters of the impregnation process, including the type of liquid 
carried, the presence and concentration of surfactants, solvents and 
penetrants, may be varied and optimized on a case-by-case basis by 
conventional means. The process of impregnation of seeds with bacterial 
cells, in the context of the present invention, can be accomplished by any 
conventional techniques as long as the viability of the seeds and of the 
bacterial cells is maintained. In an embodiment of the present invention, 
seeds can be impregnated in a manner described 
(1) adding beneficial microorganisms to a biologically compatible liquid 
carrier to form a suspension of the microorganism in the carrier; and 
(2) impregnating the seeds with the suspension or otherwise introducing the 
suspension into the seeds. 
Preferably, the excess suspension or carrier is removed from the seeds, 
such as by evaporation. This process is accomplished in such a way that 
the viability of the microorganisms and the seeds is maintained, so that 
the seeds are capable of germinating and the microorganisms are capable of 
colonizing the resulting plant. 
The microorganisms can be in any culture medium that permits growth or 
sustains viability. Preferably, the cultures are substantially pure, that 
is, substantially free of unwanted microorganisms. 
The biologically compatible liquid carrier is any liquid in which the 
microorganisms can form a suspension and which is not lethal to either the 
microorganisms or the seeds. This includes water and a water-based buffer, 
such as phosphate buffered saline. The suspensions are formed by adding 
cultures of the microorganisms to the liquid carrier and mixing 
microorganisms with the carrier and by related techniques known to persons 
skilled in the art. 
In a preferred embodiment, the biologically compatible liquid carrier 
comprises a solution of water and a water-soluble gum. Such gums may be 
natural or synthetic. They include gelatin, sodium alginate, gum ghatti, 
xanthan gum, karaya gum, Dow-Corning 1944A/B polymer (a silicone oil made 
by Dow-Corning), polyethylene oxide, Natrosol.TM. (a hydroxyethyl 
cellulose made by Hercules Chemical Co.,), tragacanth gum, guar gum, gum 
arabic, locust bean gum, methylcellulose, carboxymethylcellulose, starch, 
and Rhoplex.TM. B-15 (an aqueous acrylic emulsion made by Rohm & Haas). 
Methylcellulose, carboxymethylcellulose, xanthan gum, and Rhoplex.TM. are 
particularly preferred. These gums appear to act as a drying and 
preserving agent for the microorganisms as well as binding them to 
microscopic cracks within the seed or to intracellular spaces. They are 
preferably used in a concentration from about 0.1% to about 10% weight per 
volume of water. 
After the microorganisms have been impregnated into the seeds, it is 
preferable that the excess suspension and carrier be removed. When the 
liquid carrier is an anhydrous organic solvent, drying may be accomplished 
rapidly at ambient temperature. When the liquid carrier contains water, 
the seeds are dried and careful selection of the dry-down temperature by 
means known to those of ordinary skill in the art is beneficial. The 
drying causes the microorganisms to become dormant. When the gum is used, 
it appears to act as a matrix in which enough moisture is removed so that 
the microorganisms become dormant yet enough moisture is maintained so 
that they remain visible. 
The liquid carrier may also contain a buffer, which assists in maintaining 
the viability of the microorganisms, and surfactants, such as Tween 20 
(polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan 
monopalmitate), Tween 60 (polyoxyethylenesorbitan monostearate), Tween 80 
(polyoxyethylenesorbitan monooleate), Tween 85 (polyoxyethylenesorbitan 
trioleate), Regulaid (polyoxyethylenepolypropoxypropanol and 
alkyl-2-ethoxy ethanoldihydroxy propane), and Surfel (83% paraffin-based 
petroleum oil; 15% polyol fatty acid esters and polyethoxylate derivatives 
and 2% unidentified components). The surfactants aid in permitting the 
suspension of the microorganisms and the carrier to penetrate microscopic 
cracks and fissures in the hard, outer seed coat so that the 
microorganisms end up within the coat. Organic solvents and penetrants 
such as dimethyl sulfoxide (DMSO) or N,N-dimethyl formamide (DMF), also 
promote colonization, probably by nature of their unique properties as 
universal solvents and their ability to reduce surface tension. 
Particularly preferred concentrations are 1% DMSO or 3% DMF. In addition, 
chemicals such as fungicides can be added to the liquid carrier, if they 
are not lethal to the microorganisms. The toxicity of the fungicides can 
be determined by the person skilled in the art on a case-by-case basis. 
For example, Clavibacter xyli subsp. cynodontis has been found to tolerate 
significant levels of certain fungicides. 
In an alternative embodiment, the biologically compatible liquid carrier is 
a substantially anhydrous organic solvent. Most organic solvents are toxic 
to metabolically active microorganisms. In such cases, the microorganisms 
should be dormant, and the organic solvent should be substantially 
anhydrous because water could cause microorganisms to reenter the active 
state. Microorganisms can be made dormant before being added to the 
solvent by conventional techniques. Lyophilization is preferred. 
Virtually any organic solvent, which is nontoxic to the seed or the dormant 
microorganisms and which maintains the dormancy of the microorganisms, may 
be used. These include acetone, dichloromethane, trichloromethane 
(chloroform), carbon tetrachloride, DMSO, DMF, methanol, ethanol, benzene, 
n-hexane, cyclohexane, ortho-, meta-, and para-xylene, isopropanol, and 
n-butanol. Volatile organic solvents are particularly preferred because 
they have the advantage of being easily evaporated at the end of the 
process. 
Various oils have also been found to be useful as organic solvents. These 
include vegetable, mineral, linseed, and silicone oil. The oils are 
relatively inert and nontoxic to the microorganisms and the seeds. 
The parameters of the impregnation process, including type of liquid 
carrier, presence and concentration of surfactants, solvents and 
penetrants, may in light of the specification, be varied on a case-by-case 
basis depending on the variety of seed or plant, by means known to one of 
ordinary skill in the art to optimize the impregnation process. 
One method of impregnation of seeds that is suitable for use herein 
comprises mixing a suspension of bacterial cells with the seeds to be 
impregnated and applying a vacuum, as described, e.g., in Goth, Plant 
Disease Reporter, 50:110-111 (1966), the contents of which are 
incorporated herein by reference. At the end of the evacuation process, 
the system is allowed to rapidly repressurize, thereby allowing the 
suspension to be drawn into the seeds. The degree of the vacuum and the 
length of time it is applied can be determined by a person skilled in the 
art in view of the teachings herein. A preferred time period for 
evacuation is about 40 minutes or less. When this technique is used with a 
water-based system, the seeds may be presoaked to enhance the ease of 
penetration by the bacterial cell suspension. It is preferable that some 
seeds, for example, corn, are presoaked or imbibed overnight for 
approximately 17 hours or for one day. 
A second method of impregnating the seeds in the present invention involves 
applying the bacterial cell suspension to the seeds or otherwise mixing 
the seeds and the suspension and applying pressure. The degree of pressure 
and the length of time it is applied can be determined by a person skilled 
in the art in view of the teachings herein. In the pressure treatment, any 
gas may be used but, preferably, O.sub.2, N.sub.2 or compressed air. 
A third technique for impregnation of seeds within the present invention 
involves forceful injection of a bacterial cell suspension into the seeds. 
This can be done by any effective technique, e.g., with a needle and 
syringe, a medical needleless jet injector, or by coating the suspension 
onto high velocity microprojectiles and propelling projectiles into the 
seeds with sufficient force to cause them to penetrate the coat, as 
described, for example, in Klein et al., Nature, 327:70-74 (1987), the 
contents of which are incorporated herein by reference. Forceful injection 
can also be done by wounding the seed, e.g., by puncturing the seed coat 
with a solid needle and contacting the wound with the bacterial cell 
suspension, or by vacuum or pressure infiltration. 
Needleless injection is accomplished by propelling a small amount of from 
about 10 microliters to about 100 microliters of bacterial cells suspended 
in a liquid carrier through a small jet or orifice under very high 
pressure. The stream of liquid remains coherent over a short distance and 
"punctures" a hole for itself in a solid substrate. Any number of these 
injectors that have been developed for the medical products industry can 
be used to propel the bacterial cells into the seed or plant tissue 
according to the methods of the present invention. With the 
microprojectile technique, it is possible to coat a culture of the 
bacterial cells directly onto, or place the bacterial cells into the 
microprojectiles without first suspending them in the biologically 
compatible liquid carrier. 
The carrier need only be applied to the seeds and allowed to remain in 
contact with the seeds for a period of time sufficient for the carrier to 
naturally penetrate the seed coat and bring the microorganisms into the 
seed. For certain embodiments of the invention, it is preferable, but not 
necessary, to add a finely divided inorganic solid, such as diatomaceous 
earth, microparticulate glass, or carborundum, to the suspension to cause 
"cracks" which enhance penetration of the bacterial cell. 
After the seeds have been impregnated with the bacterial cell suspension, 
preferably, the excess suspension and/or carrier is removed. When the 
liquid carrier contains water or a volatile organic solvent, the water or 
solvent is allowed to evaporate at ambient temperature before the seeds 
are stored. The evaporation may, however, be assisted by drying of the 
seeds in accordance with conventional techniques, the only requirement 
being that viability of the seeds and of the bacterial cells be 
maintained. The seeds impregnated with bacterial cells may be planted or 
otherwise germinated immediately or stored until germination is desirable. 
If the seeds are stored, it is preferable that they be stored at a 
controlled level of temperature and humidity. It is within the skill of 
one of ordinary skill in the art to select the appropriate temperature and 
humidity for storage to maintain optimum viability. 
The introduction of the bacterial cells into seeds may be performed without 
taking special precautions to prevent seed contamination by undesirable or 
harmful microorganisms. However, it is preferred that contamination be 
controlled by treatment of the seeds with fungicides or other conventional 
seed treatments. Such treatments may be applied, for example, via coating, 
pelleting, or film-coating, in order to prevent seed or plant damage. 
A bacterial cell can also be introduced directly into a plant itself, 
instead of via the seed, to produce a modified plant in which the 
bacterial cell can replicate. In a preferred embodiment of the present 
invention, the bacterial cell does not, under normal field conditions, 
ordinarily infect or colonize the plant into which the introduction of 
bacterial cells is desired. The term "affirmative introduction of 
bacterial cells into a plant" as used herein means to actively or 
positively introduce the bacterial cells into the interior tissues of a 
plant, such as the vascular tissues. In practicing the present invention, 
the bacterial cells can be added to a biologically compatible liquid 
carrier, as previously described to form a suspension. This bacterial cell 
suspension can then be used for affirmative introduction into plants, 
using any conventional techniques that are capable of preserving the 
viability of the plants and of the bacterial cells. Vacuum and pressure 
infiltration are most conveniently used with seedlings. Injection with 
needleless medical injectors, or with microprojectiles is more 
conveniently done with more mature plants. The technique of needleless 
injection is described, for example, in Wastie, Plant Pathology, 33:61-64 
(1984), the contents of which is incorporated herein by reference. 
In a further embodiment of the present invention, the bacterial cells are 
affirmatively introduced into a plant by wounding a plant and contacting 
the bacterial cells with the wound. A plant can be wounded, for example, 
by stem stabbing, that is, wounding the plant by means of a sharp 
instrument. A preferred method of stem stabbing involves the use of a 
scalpel or other sharp instrument that is first coated with bacterial 
cells to simultaneously wound and deliver the bacteria into the interior 
of the plant. In another embodiment of the present invention, the 
bacterial cells are introduced into the plants by stem injection. "Stem 
injection" refers to the process of puncturing the stem of a plant by a 
sharp instrument, e.g., a needle, such as a turberculin intradermal 
syringe, and gently delivering the bacterial cell in the syringe into the 
stem. 
In a further embodiment of the present invention, the bacterial cells are 
introduced into plants either by injection into the petiole of a 
dicotyledonous plant or by deposition onto a previously broken petiole. In 
still another embodiment, the bacterial cells can be introduced by 
intercellular infiltration, in which a suspension of bacterial cells is 
injected into the intercellular spaces of a leaf. 
In a particularly preferred embodiment, the plant to be inoculated can be 
trimmed with clipper blades, for example, either at a low cutting height 
at about the highest visible ligule ("hvl") or at a high cutting height of 
about 3 cm above the hvl. Simultaneously, the clipper blades and rice 
plants can be sprayed with an inoculum of the bacteria, e.g., Clavibacter 
xyli subsp. cynodontis, at a high concentration, e.g., about 
1.4.times.10.sup.11 cfu/ml in phosphate buffered saline ("PBS"). 
In as yet another embodiment of the present invention, the bacterial cells 
are introduced into the part of a plant where they are most likely to grow 
and replicate, such as the vascular tissues. In the case of rice, for 
example, Clavibacter xyli subsp. cynodontis is affirmatively introduced 
into the xylem of the plant. 
A seed can be modified, in the context of the present invention, by 
impregnating it with bacterial cells in accordance with the methods 
described above. The modified seed can be allowed to germinate in 
greenhouse flats and later transplanted to field conditions for 
development into a modified plant. Similarly, a plant, preferably, a young 
seedling, can be modified by impregnating it with bacterial cells, under 
greenhouse conditions, and later transplanted to field conditions. 
Colonization of the modified plant by the introduced bacterial cells can be 
monitored by examination of the stem tissues or by examination of the sap 
expressed from the stem tissues of such plants by conventional techniques. 
For example, sap can be expressed from a stem section of the modified 
plant and examined under phase-contrast microscopy, to determine the 
presence of the bacterial cells. Alternatively, sap can be expressed and 
cultured on an agar medium or a in a liquid medium that allows for growth 
of the bacterial organism. The latter process is generally referred to as 
"culture indexing." Moreover, a radioimmunoassay or a fluorescent antibody 
assay can be used to determine colonization. For example, polyclonal 
antibodies to the bacteria can be induced in laboratory animals such as 
rabbits. The polyclonal antibodies from the laboratory animals can be 
labelled with a fluorescein dye for fluorescent antibody assays or can be 
used with, e.g. goat anti-rabbit antibodies that are radioactively 
labelled, for radioimmunoassays. 
It is to be understood that the application of the teachings of the present 
invention to a specific problem or environment will be within the 
capabilities of one having ordinary skill in the art in light of the 
teachings contained herein. An example of the products of the present 
invention and processes for their production appears as follows which is 
illustrative only and is in no sense limiting. 
EXAMPLE 1 
Introduction of Clavibacter xyli subsp. cynodontis into rice plants for 
production of a modified rice plant of reduced stature. 
A trial was established at a commercial rice farm in Leroy, La., in a small 
leveed field planted to the variety Mercury. A central section of the 
field large enough to accommodate the trial was cleared of rice and plowed 
to produce a good seedbed for receiving transplants. 
To ensure colonization of Clavibacter xyli subsp. cynodontis ("Cxc") in the 
rice plants, transplants of rice were produced in greenhouse flats, 
inoculated with Cxc, and subsequently examined for colonization, prior to 
planting in the field. Seeds of the rice variety Lemont were planted to 
trays of 1.5 in. pots containing sterilized field soil on May 10. Shortly 
after germination, benches of trays containing flats were flooded with 
about 3 in. of water. Cxc organisms were inoculated into the rice plant 
seedlings by stem stabbing on June 2, using Cxc isolate 123b that was 
concentrated to over 10.times.10.sup.11 cfu per ml of phosphate buffered 
saline ("PBS"). 
The Cxc isolate 123b was isolated from bermudagrass in the field in 
Louisiana in the following manner: Stem sections of the bermudagrass were 
first examined for the presence of Cxc by phase contrast microscopy. 
Positive samples were selected and sap was expressed from these samples 
and cultured either on SC agar medium or in SC liquid medium. SC medium 
consists of 1000 ml distilled water; 17 g cornmeal for liquid medium or 17 
g cornmeal agar for agar medium; 8 g papain digest of soy meal; 1 g 
K.sub.2 HPO.sub.4 ; 1 g KH.sub.2 PO.sub.4 ; 0.2 g MgSO.sub.4.7H.sub.2 O; 
15 ml of a 0.1% solution, containing about 15 mg of bovine hemin chloride 
in 0.05 N NaOH; 10 ml of a 20% aqueous solution, containing about 2 g of 
bovine serum albumin fraction 5; 1.0 ml of a 50% aqueous solution 
containing about 1 g of cysteine (free base). The Cxc-inoculated media 
were incubated for 6 days at 28.degree. C. .+-.3.degree. C. 
Twenty days after Cxc inoculation, the Lemont rice plants were randomly 
selected from inoculated flats. Sap was expressed from parts of selected 
plants and was examined under phase contrast microscopy to confirm 
colonization. The following day rice plants were hand-transplanted to 
plots, each consisting of six rows of plants, with 7 in. between centers 
and 12 ft. long. An average of 432 plants were planted to each plot, 
separated by 1.5 ft. alleys from adjacent plots. Cxc-inoculated and 
noninoculated controls were arranged according to a paired t-test design 
and replicated six times. The field received a permanent flood directly 
following transplant and recommended cultural practices for rice 
production in Louisiana were followed. 
Colonization of the rice plants by Cxc was assessed at various intervals 
during the crop development from randomly selected plants from each plot 
by either phase-contrast examination of expressed sap or by culture 
indexing. Stand counts were taken on August 15, from four meter long 
sections over the middle four rows of each plot. Plant height measurements 
were taken on July 26 and August 15, respectively, by measuring randomly 
selected plants per plot from the base to the highest visible ligule 
("hvl"). Counts of plants with exposed panicles (heading) were also 
collected on the latter date. 
Growth differences in rice plants between Cxc-inoculated and non-inoculated 
control plots were noted within a few weeks after transplanting. Rice 
plants in inoculated plots were substantially shorter than those in 
control plots on both dates measured, as shown in Table 1. As plants were 
in late boot to early heading stage of development at the time of the 
second measurement, the latter could be considered a terminal height. The 
Cxc-inoculated rice plots produced a 23.5% greater (p&lt;0.06) plant stand 
population than controls. 
TABLE 1 
______________________________________ 
Comparison of rice plant growth between Cxc-inoculated 
and non-inoculated control plots in a field trial at 
Leroy, Louisiana. 
PLANT HEIGHT (cm) 
TREATMENT July 26 August 15 
______________________________________ 
Cxc-inoc. 23.1 45.5 
Control 28.4 54.4 
C.V. 9.6 4.5 
% DIFF. 18.7* 16.4** 
______________________________________ 
*(p &lt; 0.05) 
**(p &lt; 0.01) 
Plant development recorded as a percent of the plants with visible panicles 
on August 15, indicated that inoculated plots were slower to mature, with 
only 0.3% of plants heading compared to 31.6% in the control. This 
developmental delay was probably reflected in grain moisture levels at 
harvest which were 4.7% greater in the Cxc-inoculated plants. Despite the 
effects on plant height and development, the inoculated plots did not 
suffer any apparent yield loss, as shown in Table 2. 
TABLE 2 
______________________________________ 
Yield comparison between Cxc-inoculated and non- 
inoculated control plots in the field trial at Leroy, 
Louisiana. 
TREATMENT PLOT WT. (g) YIELD (kg/ha.sup.1) 
______________________________________ 
Cxc-inoc. 1,381 4,814 
Control 1,321 4,661 
C.V. 6.9 6.9 
% DIFF +4.5 ns +3.3 ns 
______________________________________ 
.sup.1 Yields corrected for 12% moisture. 
ns = not significant 
It will be apparent to those skilled in the art that various modifications 
and variations can be made in the processes and products of the present 
invention. For example, it is anticipated that the degree of seed and 
plant colonization by the bacterial cells can be optimized by those 
skilled in the art, given the teachings of the instant specification. 
Thus, it is intended that the present invention cover such modifications 
and variations, provided that they come within the scope of the appended 
claims and their equivalents.