Method for producing new varieties of plants

A method for increasing the proportion of mutants in a generation in a first plant species having a recognized and established phenotype involves the simultaneous somatic exposure of germinal plants of the species to contact with whole cells and associated material of a second species of plants, and to electrophoretic conditions. The plants of the first species are preferably in a germinal state, such as seeds or seedlings, while the whole cells and associated materials of the second species can be a seedling root tip, a seedling, a tissue macerate (suspended in either water or agar) root nodules, fruit tissue or root tissue. When the cells of the first and second species have different membrane potentials, the step of electrophoretic exposure can be carried out by simply placing the cells in contact with one another. Preferably, however, an electropotential difference such as a constant DC voltage is disposed across the somatic cells of the first species of the plant and the whole cells and associated materials of the second species of plant, for example, by attaching one of a cathode and anode to the first species of plant, and the other of anode or cathode to the second species of plant.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for inducing mutations in plants, 
and more particularly to a method for increasing the number of plants of a 
first species which exhibit a phenotype or characteristic normally 
associated with a different species. 
2. Description of the Prior Art 
The members of a given species of plant typically share a number of 
well-established physical characteristics associated with the genetic 
materials of their cells; these characteristics are known as phenotypes. 
However, it is well known that plants of a given species having one or 
more new and distinctive characteristics, generally referred to as sports 
or mutations, occur naturally as a small fraction of any plant population. 
For centuries, mutants have been selectively bred to produce new varieties 
or modifications of existing plants. In natural populations of plants, 
however, the frequency of mutations is generally considered to be less 
than 1 in 500,000, so that the selection of desirable mutants after such 
breeding is a slow and laborious process, particularly since it is well 
recognized that mutants exhibiting a desirable phenotype are rare, and 
progeny outputs are often low. 
Several methods for increasing the occurrence of mutants in a population of 
a given species are well known; for example, the exposure of such a 
population to ionizing radiation. Such techniques, however, are typically 
subject to the drawbacks that the individually resulting mutants are 
generally weak, and must still be subjected to the time-consuming and 
labor-intensive techniques of isolation and selective breeding for a large 
number of generations, before a sufficient number of mutants possessing 
the new phenotype are obtained for use in outcrossing or agricultural 
growth. 
Recombinant DNA and protoplast fusion techniques are potentially useful for 
producing new varieties of plants without isolation of mutants or 
selective breeding. The use of these techniques is subject to several 
drawbacks, however. First, these techniques are tedious and slow, 
requiring elaborate instrumentation involving a large number of chemical 
processes, and a substantial investment in the education and training of 
the personnel conducting the procedures. Presently, these techniques are 
very expensive and time consuming. Indeed, Applicant is aware of no 
reported instance of the inducement of a functional expression of a novel 
gene (phenotype) from one species of plant to a population of another 
species of plant, employing these genetic engineering techniques. 
SUMMARY OF THE PRESENT INVENTION 
The present invention overcomes these and other difficulties encountered in 
prior methods of inducing mutations in a population of a first species of 
plant by providing a method for increasing the number of mutants 
exhibiting altered phenotypic characteristics, characteristics which are 
stable in successive generations, where such phenotypic characteristics 
are an established trait of a second different species of plant. The 
method of the present invention allows for the production of large numbers 
of plants having substantial modifications from the parent generation, 
without the delay of several generations for selective breeding and 
establishment of characteristics as stable by outcrossing, and which does 
not require the complex instrumentation or large numbers of chemical 
reactants and steps inherent in present recombinant DNA or protoplast 
fusion techniques. 
The method according to the present invention involves placing a plurality 
of germinal plants of a first or recipient species, this first species 
exhibiting at least one established phenotype, in contact with the whole 
cells and associated materials of a second species of plant, while 
exposing the germinal plants of the first species to electrophoretic 
conditions, such as an ionophoretic current. The germinal plants are grown 
to adult plants, or to a stage sufficient to observe any changes from the 
established phenotype. The exposure of the germinal plants of the first 
species to electrophoretic conditions can be carried out by simply 
abutting a portion of seedlings of the first species with seedlings of a 
second plant species, when the cells of the first and second species have 
differing membrane potentials. This can be carried out by excising 
complimentary sections from the root of seedlings of the first and second 
plant species, and abutting the cut surfaces of the roots. Preferably, 
however, an external DC current is applied across the germinal first 
species plants and whole cells and associated materials of the second 
plant species by attaching an anode to the plants or materials of one 
species, and a cathode to the plants or cells of the other species. 
Typically the plants and materials are exposed to a constant DC voltage 
having a current density in the range of 10 to 100 microamps per 
centimeters applied at a potential difference of from 1 to 50 volts for 
periods of five minutes to 24 hours. In effect, the donor material of the 
second species acts as an electrode substrate or base contactable with the 
seedlings of the first species. The donor material is prepared as either a 
tissue macerate or as whole tissue. The donor material can be placed on 
sterile cotton or a filter paper which in turn rest on a stainless steel 
plate electrode. Most preferably, the acceptor tissue or plants of the 
first species are exposed at the seed or early seedling stage, typically 
24 to 96 hours after germination by placing the root apex in contact with 
the donor-coated electrode, and the shoot apex, cotyledons or coleoptile 
in contact with the electrode of opposite polarity. 
The method of the present invention is preferably carried out with 
genetically pure, stable and homozygous inbred varieties of lines as the 
host or acceptor first species. Such well-established lines were used in 
all of the examples described below, and are commercial varieties which 
have been released from university or USDA breeding programs for public 
use. 
After exposure, the test seedlings or germinal plants of the first species, 
along with untreated controls, are developed to maturity under field 
conditions or in a greenhouse, depending upon expediency. Typically, 
alterations are observed in the growth rates and yields of the germinal 
plants actually treated, depending upon the type of donor and the exposure 
parameters; however, a stable expression of an altered phenotype is 
typically not seen until at least the second generation bred from the 
treated plants. The frequency of inherited, varietal alterations resulting 
from the present method ranges from 5% to 95% of the test population, 
typically, depending upon the specific procedure and plant species 
involved. This is a substantial improvement over the proportion of one in 
a few thousands or several thousands of cells or plants treated by 
recombinant DNA and protoplast fusion methods. 
Not only does the present method yield a significantly increased proportion 
of mutants in the treated plants, but a significant proportion of the 
resulting mutants exhibit an altered phenotypic characteristic which was, 
in fact, an established phenotypic characteristic of the second or donor 
species of plant. It is believed that this transferred phenotype results 
from the transduction of genetically associated cell tissue components and 
macromolecular complexes from the second or donor species into the intact, 
somatic cells of the first or acceptor species, in such a manner as to 
alter the genotype and/or phenotype of the plants of the first species. 
For this reason, plants treated in accordance with the method of the 
present invention, or grown from plants treated in accordance with the 
present invention, are designated by generation with the letter "T". For 
example, the first treated generation of the first species of plant is 
described as the T-1 generation, while a second inbred generation grown 
from the adult plants of the T-1 generation are referred to as the T-2 
generation. This designation of generations is intended to avoid confusion 
with the system of F-1, F-2 and so on, normally employed in conventional 
plant breeding, when crossing for hybrid vigor. 
It is thus an object of this invention to provide a method, by means of 
electrophoresis techniques, for the production of new plant mutations 
consisting of types and varieties having altered genotypic and/or 
phenotypic characteristics, that is simple when compared with the 
recombinant DNA and protoplast fusion methods known in the art. The 
methods of the present invention do not require complex instrumentation, 
nor drastic alterations in cell wall-membrane contiguity, particularly the 
removal of the cell wall as required by prior techniques, or detailed 
elucidation of chromosome maps. 
Another object of the present invention is to provide a method for the 
production of new varieties of plants that can quickly yield large numbers 
of healthy plants having substantial modifications from the parent plants, 
thus eliminating the delay of several generations and large test 
populations required in prior selective breeding programs, which have been 
conventionally necessary before the plants can be used in out-crossing. 
Both conventional breeding programs and the recombinant DNA and protoplast 
fusion methods generally produce a low yield of mutants which must be 
selectively grown and bred for a large number of generations, before a 
sufficient number of stable plants are available for use in programs for 
developing plant varieties; in contrast, the production of such stable 
plant varieties is remarkably more rapid in the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENT INVENTION 
The method of the present invention both increases the proportion of 
mutants in a generation of a first species of plant (the species having at 
least one established phenotype) while simultaneously causing at least 
some of the resultant mutant plants to exhibit a phenotype, characteristic 
or trait of a second species of plant. Several illustrative techniques and 
specific examples of the present invention are described hereinafter. It 
should be understood that the technique of the present invention is 
generally intended to be used on a substantial number of plants sought to 
be modified, so as to provide a ready supply of mutant plants for 
subsequent varietal development. The electrophoretic techniques described 
however, can also clearly be conducted on a single plant cell as the 
acceptor, employing micromanipulative techniques in order to apply an 
ionophoretic current across the single acceptor cell and donor material. 
Such a technique is, of course, within the scope of the present invention. 
In such a case, the acceptor electrode can comprise a thin needle or wire 
inserted into or in contact with the acceptor cell. In all cases, however, 
the cell wall and plasmalemma are preferably not breached by the present 
method. 
It is believed that germinal plants, such as seeds or seedlings about one 
to five days after germinations, are most susceptible to successful 
treatment by the method of the present invention. While some variation may 
occur in the percentage of mutant plants grown from the treated seeds or 
seedlings, such as percentage varying with the species of plants used and 
the particular technique employed, the method of the present invention 
will generally result in a substantially greater percentage of mutations 
than results when radiation or the like are employed to create mutant 
plants. Moreover, whereas radiation and the like cause random mutations 
having widely varying characteristics, in general a substantial percentage 
of mutants formed by this invention tend to exhibit substantially similar 
characteristics. 
With reference first to FIG. 1, a first embodiment of the method of the 
present invention is thereshown involving a joined pair of seedlings 10 of 
two different species of plants. One or more seedlings 12 of first species 
of plant are prepared by longitudinally excising a section consisting of 
about one-half of the root's side, to expose a fresh cut surface 16. 
Preferably, the germinated seedlings include radicles in the range of 1 to 
6 centimeters in length, and the radicle tip is left intact when the 
surface is cut, exposing the procambium, protophleom and protoxylem cells. 
The root side and tip of a corresponding number of seedlings 14 of a 
second, different species of plant are excised, the radicle tip as well as 
the longitudinal portion of the side being removed, to form a cut surface 
18. The seedlings 14 of the second species are preferably of similar 
radicle development as the seedlings 12 of the first species, and the 
procambium, protophloem and protoxylem of the seedlings 14 of the second 
species similarly form the cut surface 18. 
The cut surfaces 16 and 18 of the two species of plants are then 
immediately abutted and a thin cord is wrapped or tied about the abutted 
roots in order to insure good contact between them and maintain them in 
abutment. The excisions on each of the seedlings 12 and 14 should be 
complementary in order to maximize contact between the cut surfaces 16 and 
18. The joined seedlings 10 are planted and nurtured to adult plants, at 
which time either seeds from the plants are harvested for growth of a T-2 
generation from which plants having desired traits are selected; or the 
T-1 adult plants are directly selected for desired traits. The former is 
the particularly preferred procedure in this invention. 
In the embodiment disclosed in FIG. 1, the seedlings 14 of the acceptor 
species are exposed to electrophoretic conditions through the existence of 
a difference between the natural membrane potentials known to exist about 
both plant and animal cells, Jaffe, Nature, 256: 600-602 (1977). Although 
natural membrane potentials are known to be of low magnitudes, generally 
on the order of 1 to 100 millivolts, the adjacent disposition of cells of 
different species will result in a mutual electrophoretic process. Because 
each plant species has its own distinctly characteristic metabolic cycle 
and timing of activity, the biochemical cycles in plants 12 of one species 
will likely be at a phase different from that of plants 14 of the second 
species. Consequently, since at one growth stage the mutual potentials may 
be complementary and at another stage of development they may be opposed, 
this can provide a potential gradient quite different from that which the 
cells of the plants 12 of the first species would experience under normal 
conditions of development. 
Because the plant radicle or root tip is responsible for the production of 
vitamins and other important enzymes used in the development of germinal 
plants, the plant 14 having the root tip excised will be acceptor plant, 
while the plant 12 having the root tip retained will be the donor plant. 
Applicant has measured the current density in the region where the cut 
tissues contact, when abutted as disclosed in FIG. 1. For example, when 
four day old seedlings from different species such as corn and soybean are 
paired, the current density reaches a maximum value of about 0.7 
microamperes per square centimeter at about 40 minutes after initial 
abutment, with a very gradual decline over the next 10 hours. In contrast, 
when seedlings of the same species are paired in a similar fashion, such 
as soybeam-soybean pairings, the current density is only around 0.01 
microamperes per square centimeter, again showing a very gradual decline 
with time. Typically, even at this low electric potential difference 
between the seedlings of disparate species, new traits appear in the 
acceptor plants at about a 5% mutuation level and are often in the nature 
of phenotypical alterations such as plant shape, size and foliage color. 
The T-1 generation is then selfed to yield a T2 generation, and the 
altered phenotypes exhibited by the mutated members of the new generations 
do not segregate out in succeeding generations. 
With reference now to FIG. 2, a second preferred embodiment of the 
invention is thereshown in which the natural membrane potential difference 
between seedlings of two different species is augmented or reversed, as 
desired, by the application of an ionophoretic current across the joined 
seedlings. More particularly, the root tips of seedlings 12 and 14 of two 
different species are excised, and the cut portions of the seedlings 
abutted together. A pair of electrodes 22 are then afixed to the seedlings 
12 and 14 generally opposite the abutted root portions, for example, in 
the shoots or cotyledons. An electrical potential such as provided by a 
constant direct electrical current is then applied through the electrodes 
22 across the pair of joined seedlings 20. The voltage applied to the 
seedlings will generally range between 1 to 45 volts, and preferably on 
the order of 1.5 to 22.5 volts, for times of about 5 minutes to 24 hours, 
and preferably about 5 minutes to about 3 hours. This yields a current 
density across the region where the cut seedlings abut one another in a 
range of about 10 to 100 microamperes per square centimeter. Preferably, 
the direction of current applied is chosen to augment the difference in 
membrane potential of the cells between the different species of plants. 
Once subjected to such a potential, the seedling pairs 20 are then 
separated into individual seedlings 12 and 14, which are separately 
nurtured to adult plants. The selfed T2 generation from the treated T1 
generation plants are then selected for desired traits. 
Preferably, the electrodes 22 are constructed from iron, since iron 
electrodes can be inserted into the seedlings without causing detrimental 
effects to the seedlings. Other electrodes which are not deleterious to 
plants can also be used, and stainless steel electrodes are particularly 
preferred for this purpose. 
The donor material employed in the present invention need not be a whole 
plant or seedling. Instead, as shown in FIG. 3, tissues of a donor species 
of plant can be macerated, such as by blending in water, in order to 
produce an aqueous donor liquor. The aqueous liquor is collected and added 
to a support medium such as agar or gelatin, to produce a treated medium 
26. The treated medium 26 is disposed in a test tube or vial 28, or other 
convenient container, and the root tip of the seedlings 14 of the second 
species of plant are placed in contact with or immersed in the medium 26. 
Most preferably, the radicle of the acceptor plant seedling 14 is placed 
in the medium. Once electrode is contacted with the shoot of the seedling, 
while another electrode is disposed in contact with the support medium 26. 
An electrical potential is then applied across the seedling 14 and medium 
26, of the type, time and intensity described in the preceding embodiment. 
Following the application of this electrical potential, the seedlings 14 
are removed from the treated medium 26 and grown to adult plants, which 
are then either selected for desired traits or are selfed in order to 
determine which traits in a T2 generation are inheritable and stable. 
The use of agar or gelatin as a medium 26 for suspending the aqueous liquor 
is desirable, but not essential to use of an aqueous liquor of the donor 
plant cells. In the embodiment shown in FIG. 3, the medium can be replaced 
by the aqueous liquor itself. Moreover, the aqueous liquor can itself be 
subjected to an electrical potential prior to its contact with the 
acceptor seedling 14, as shown in FIGS. 4 through 6. As above, the tissue 
of the donor species of plant is macerated in distilled water, and the 
resulting liquor 30 collected. The aqueous liquor 30 is then deposited in 
a petri dish 32, and a positive electrode 34 and a negative electrode 36 
are placed in the aqueous liquid 30. An electrical potential 38 is then 
applied to the aqueous liquor 30 across the positive electrode 34 and the 
negative electrode 36. While the electrodes can be constructed from 
silver, it is preferred that the electrodes are constructed from platinum 
in order to reduce oxidation of the electrodes, and minimize the effect of 
the electrode material upon the aqueous liquor 30. Generally a potential 
of about 5 to 20 volts is applied for a time of about 10 to 30 minutes. 
Consitutents of the aqueous liquor will migrate towards or away from one 
or the other of the electrodes 34 and 36, depending upon the charge 
possessed by the various tissue constituents. As shown in FIG. 5, the 
portion of the aqueous liquor 30 which is located about the anode or 
positive electrode 34 (the anode solution) is removed from the remainder 
of the aqueous liquor 30 by withdrawal into a hypodermic syringe 40. The 
portion of the liquor 30 surrounding the negative electrode or cathode 36 
(the cathode solution) is removed by drawing into a syringe 42. 
The syringes 40 and 42 containing the anode and cathode solutions are then 
inserted into opposite ends of seedlings 14 of the acceptor species, as 
shown in FIG. 6, and pressure is applied to the syringes 40 and 42 to 
inject a portion of the anode and cathode solutions to the tissue of the 
seedlings. For example, the anode solution contained in the syringe 40 can 
be inserted into the shoot 44 of the seedling 14, while the cathode 
solution contained in the syringe 42 can be injected into the root of the 
seedling 14, preferably into the radicle 46. The positive electrode 34 and 
negative electrode 36 are then connected to the syringes 42 and 40, 
respectively (opposite to the electrodes from which the syringes collected 
a portion of the aqueous liquor), the syringes preferably having metal 
tips to facilitate electrical contact with the seedling 14. A potential 
difference of about 1 to 50 volts and preferably of about 1.5 to 22.5 
volts is applied to the seedling through the syringes for a time of about 
5 minutes to 24 hours, and preferably for about 5 minutes to about 3 
hours. Subsequent to the application of the potential difference, the 
needles are removed from the seedling 14, and the seedling 14 grown to an 
adult plant. A plurality of seedlings are selected for the desired traits 
in either the T1 or T2 generation, as described earlier. 
In another preferred embodiment of the present invention, only one of the 
electrode solutions needs to be applied to the acceptor species of plant 
in order to obtain the high proportion of mutations encountered in the 
present invention. With particular reference to FIG. 7, either of the 
anode or cathode solutions collected by the syringes 40 and 42 can be 
applied to a porous medium, such as a filter paper 48. A seedling 14 of 
the acceptor species of plant is positioned on the filter paper 48 with 
both its radical 46 and its shoot 44 in contact with the filter paper 48 
containing the donor electrode solution. The donor-containing filter paper 
48 is placed in contact with a first electrode 50 while a second electrode 
52 of opposite polarity is inserted into the shoot 44 of the seedling 14. 
As above, the polarity of the electrode 50 in contact with the filter 
paper 48 is opposite to the sign of the electrode 40 or 42 from which the 
anode or cathode solution was collected. Because at least some of the 
constituents of the anode or cathode solution will be of the type to 
migrate towards the electrode opposite in sign so that of the electrode 50 
in contact with the filter paper, these constituents will tend to migrate 
towards the second electrode 52 upon the application of the potential 
difference across the electrodes 50 and 52, and thereby across the 
seedling 14. The length of time and type and strength of potential 
difference applied across the seedling 14 are as disclosed above. 
Subsequent to the application of the potential difference, the electrodes 
50 and 52 are removed from the seedling, and the seedling 14 grown to 
either the T-1 or T-2 generation, and selected for any desired traits. The 
electrodes 50 and 52 are preferably constructed of iron or stainless 
steel, because of their minimal effects on biological systems. 
It should be evident that the embodiments disclosed in FIGS. 3 and 7 are 
readily adaptable to use in exposing a single cell or isolated protoplast 
cell of an acceptor species to the aqueous liquor or cathode or anode 
solutions from the donor species. More particularly, in FIG. 9 there is 
disclosed another preferred embodiment of the present invention in which a 
pair of non-reactive electrodes 70 and 72 (preferably platinum electrodes) 
are used to place an electrical potential across a single plant cell or 
isolated protoplast 74. The cell 74 is carried on the end of a glass tube 
76, the tube 76 being filled with water 78 or another conductive liquid so 
as to permit manipulation of the cell 74 within the tube 76. The use of a 
water-filled tube to carry a single plant cell is, of course, a known 
micromanipulative technique. One of the electrodes, for example, the 
cathode 72, is electrically connected through the tube 76 and disposed in 
contact with the liquid 78 in the tube 76. The other of the electrodes, 
for example, the anode, is electrically connected through the wall of 
another glass tube 80 and disposed in contact with a donor medium 82 
contained in the tube 80. The donor medium 82 is the same as the media 
prepared in accordance with the preceeding embodiment of the invention. 
Pressure is applied to the medium 82 to express a small droplet 84 of the 
medium 82 out of the end of the glass tube 80. The tubes 76 and 80 are 
mounted to a micromanipulator (not shown), which aligns the tubes 76 nd 80 
and permits the droplet 84 to be brought into contact with the plant cell 
74. Alignment and contact can be visually monitored through a microscope 
86. The plant cell 74 is then subjected to electrophoretic conditions by 
the application of a DC voltage across the electrodes 70 and 72. The 
applied voltage should be sufficient to produce a current density in the 
range of 1.0 to 100 microamps per square centimeter, for a time of about 
minutes to three hours. 
A final preferred embodiment of the general method of present invention is 
shown in FIG. 8 in which a seed 62 of an acceptor species of plants id 
disposed between two pieces of porous material or filter paper 54 and 56. 
Cathode and anode solutions of a tissue macerate of a donor species are 
prepared as described above. The filter papers 54 and 56 are placed on 
electrodes 58 and 60, and infused with the anode or cathode solution 
collected from the electrode 34 or 36 of potential opposite to the 
electrodes 58 and 60. One of the filter papers 54 or 56 is placed in 
contact with the hilum or embryo end of the seed 62. The electrodes 58 and 
60 can be constructed of various materials, preferably stainless steel or 
other iron material. It is preferred that the electrodes do not contact 
the seed 62 directly. An electric potential is the applied to the 
electrodes 58 and 60, and thus applied across the seed 62. The potential 
can be applied to the dry seed 62, or the seed can be allowed to be 
partially or completely imbibed with water or the anode and cathode 
solutions, before the potential difference is applied. A constant direct 
current of 20 to 90 volts is applied to the dry seeds, or a potential of 1 
to 40 volts is applied to the partially imbibed seeds, for about 5 minutes 
to 1 hour. After such treatment, the seed may be returned to the quiescent 
state and stored until it is convenient to plant them. Alternatively, the 
seeds may be germinated immediately, sprouted and grown to adult plants. 
Adults in the T2 generation, selfed from the T1 plants, are selected for 
desired traits. 
The methods of the present invention are further illustrated by several 
following examples. Some of the examples have been followed through the T5 
generation in extensive agricultural testing. In general, it has been 
found in the invention that the induced mutations recognizably segregate 
in the T2 or subsequent generations, so that selections for further 
crossings or further development can accordingly be made in the T2 
generation. For the most careful screening of the types of mutations, it 
has been fund advantageous to examine plant row tests in the T2 
generations, that is, to use seeds from the individual treated plants of 
the T1 generation for inbred or selfed plant row replications, in the T2 
generation testing. This allows a more efficient screening and 
categorization of the induced mutations from the T1 generation since 
traits or characteristics which are not reproduced in a selfed or inbred 
generation are neither stable nor of particular commercial value. 
The high percentage of mutants obtained in the method of the present 
invention allows a relatively small number of seeds or seedlings to be 
treated in the T1 generation (which is also referred to as the transduced 
series), on the order of 15 to 20 seedlings of each transduction polarity 
being examined for differences in growth and for phenotypic variations, 
along with a control group of untreated seedlings or seeds of equal 
number. Thus, about one-third of the plants field tested at the time of 
the T1 generation are control plants. After the T1 generation, each of the 
treated lines and controls are grown in three replicated rows of 40 to 50 
seeds each within statistically randomized test plots. Unless otherwise 
indicated, the Latin Square method of randomization was employed. 
Subsequent to the T2 generation the lines are selected and expanded 
according to the apparent importance of the new characteristics of the 
mutant plants. 
The acreage necessary to adequately insure that the new characteristics are 
stabilized in the particular treated lines will vary according to the 
percentage of mutants obtained in the T1 generation and the number of 
lines that appear desirable to investigate. For example, in 1984, 
applicant produced T1 transduced series of acceptor species including 
corn, tomato, soy beans and navy beans. Less than a one acre test plot was 
required for 124 transduction series and controls. These particular tests 
were made in lower Michigan. By 1985, the subsequent T3 generation testing 
involved an area of 10 acres, while the 1986 T5 generation required over 
70 acres of primary growth, in addition to replicated tests at several 
locations and in several states. The T2 and T4 generations of these 
transduced series were seed expansion grow-outs in Hawaii, in order to 
shorten the time necessary to achieve the T5 generation. 
The following examples serve to further illustrate the present invention: 
EXAMPLE ONE 
Longitudinal sections from soy bean (Glycine max) seedling roots (the donor 
species) were excised in a plurality of seedlings, and longitudinal 
sections including the root tip were excised from a plurality of bush bean 
(Phaseolus vulgaris) seedlings (the acceptor species). Each seedling has a 
radicle in the range of 1 to 6 centimeters in length, and the excised 
portions were of complementary shape, such as to expose the procambium, 
protophloem and protoxylem cells of each root tip. The cut portions of 
pairs of seedlings of the different species were abutted and bound with 
thread, as shown and described in conjunction with FIG. 1. The pairs of 
joined seedlings were grown to adult plants. 
One in twenty bush bean seedlings so treated resulted in an adult plant 
that was shorter than the control plants and which has more compact 
foliage than the control plants, characteristics which are of commercial 
importance in the harvesting of bush beans. Tall plants tend to lodge and 
intertwine, and are thus less efficiently harvested. These plants also had 
leaves of a deeper green color than the control plants, the fruit of these 
plants and these plants exhibited greater drought resistance than the 
control plants. Yields under field condition, however, were found to abe 
about the same as those of the control plants. The seeds of these plants 
were observed to be intermediate in shape between the soy bean and bush 
bean progenator seeds. 
These new characteristics were stable; they were observed without change 
through seven inbred or selfed generations with no reversion back to the 
height, bushiness, color, sweetness, and drought resistance of the 
original and control bush bean plants. Six generations of the mutated 
plants, along with an equal number of controls, were grown under field 
test conditions as described earlier. The maintenance of these 
characteristics for seven generations demonstrates that the changes were 
inheritable. The fact that the inbred, transduced plants do not segregate 
or revert, that is, return to the characteristics of the control plants, 
demonstrates that the method can provide new varieties of plants which 
breed true. As will be subsequently discussed, this non-segregating, 
stable nature of the growth alterations suggests a non-Mendelian or 
cytoplasmic type of inheritance. 
EXAMPLE TWO 
Longitudinal sections from a plurality of bush bean seedlings (Phaseolus 
vulgaris) roots, the donor species, and longitudinal sections including 
the root tips from soy bean (Glycine max) seedlings, the acceptor species, 
were excised to expose procambim, protophlem and protoxylum cells on each 
seedling. Each seedling was germinated and possessed radicles in the range 
of 1 to 6 centimeters in length. The excised portions of pairs of 
seedlings of different species were cut in complementary shapes, and the 
exposed cut portions of the seedlings were joined together to form pairs 
of joined seedlings in the fashion shown in and described in conjunction 
with FIG. 1. Each pair of joined seedlings contained a bush bean seedling 
and a soy bean seedling. The pairs of seedlings were then grown to adult 
soy bean plants, and one in ten of the soy bean plants so grown exhibited 
seeds that were intermediate in shape and color between the seeds of the 
bush bean and the soy bean progenators. The leaves of the one in ten 
altered soy bean plants were less lobed in shape than the leaves of the 
control plants, the stem node lengths were reduced as compared to those of 
the soy bean control plants, and the number of stem nodes was increased as 
compared to the controls as well. This resulted in a line of altered soy 
bean plants which had more compact foliage than the control plants and was 
thus more resistant to lodging under field conditions. These changed 
characteristics were maintained in inbred or selfed plants grown through 
four generations. Three of these generations were grown along with an 
equal number of controls under field conditions. 
This example is, of course, the reciprocal or reverse of the transduction 
which occured in Example 1, that is, the donor and acceptor species are 
reversed. Significantly, the percentage of altered or mutated plants 
obtained is of the same order of magnitude in each example, demonstrating 
that the method allows modifications to be made to plants in two 
directions. Typically, attempts to induct positive and viable mutations in 
plants by conventional methods such as by chemical or ionizing radiation 
treatments yields an expected frequency of useful, viable mutations or 
phenotypic alterations of one in five hundred thousand test plants (a 
frequency equal to 0.000002). Examples 1 and 2 demonstrate that the method 
of the invention can produce plants having new, inheritable 
characteristics at a rate of 25,000 times that expected under conditions 
of conventional chemical or radiation treatment. This increase in the rate 
of mutation is highly significant and commercially valuable in terms of 
time, space, and the volume of plants needed to be treated or exposed in 
order to produce positive mutations. 
EXAMPLE THREE 
Tissue from the immature fruit of tomato (Lycopersicon esculentum) was 
macerated in distilled water and the resulting aqueous liquor placed in a 
perti dish. A pair of spaced silver electrodes were inserted in the 
macerate liquor and a constant direct current electrical potential of 9 
volts was applied for 20 minutes. A portion of the liquor surrounding each 
electrode was drawn into a hypodermic syringe having a conductive needle 
tip. The conductive syringe tips were inserted into the root and shoot of 
a plurality of soy bean (Glycine max) seedlings in the fashion shown in 
and described in conjunction with FIG. 5, 6 and 6, above. A negative 
electrode was then connected to the syringe containing the solution which 
has surrounded the positive electrode in the petri dish, while a positive 
electrode was connected to the syringe containing the other electrode 
solution. A constant DC electrical potential of 22.5 volts was then 
applied for five minutes, so that a current of approximately 100 microamps 
was passed through the seedlings. 
Two series of 20 seedlings were treated and grown along with 20 non-treated 
controls. In one treated series, the electrode solution from the positive 
electrode in the petri dish was applied to the seedling roots. In the 
other series, the electrode solution from the negative electrode in the 
petri dish was applied to the roots of the seedlings. After such 
treatments, all of the seedlings were grown under field test conditions as 
described above, and the results obtained are given in Table I below. The 
asterisk indicates data which is statistically significant at about a 95% 
confidence level (P less than 0.05). The observed increases in pod and 
seed yields continued in two subsequent generations of selfed or inbred 
plants, grown under field test growth conditions. 
TABLE I 
__________________________________________________________________________ 
(N = 20 plants per series) 
ELECTRODE SOLUTION 
PODS PER PLANT 
AVERAGE SEED YIELD 
APPLIED TO ROOT 
AVERAGE 
S.D. 
(GRAMS PER PLANT) 
__________________________________________________________________________ 
ANODE (+) 59.1* 43.6 
16.76 
CATHODE (-) 49.6* 36.7 
14.06 
CONTROLS 32.5 13.7 
9.50 
__________________________________________________________________________ 
EXAMPLE FOUR 
A portion of root tissue from Eastern Marsh Cabbage Plant (Symplocarpus 
foetidus) was excised in early Spring (mid-March), macerated in distilled 
water and admixed with a sufficient quantity of agar to create a donor 
macerate of moderate viscosity. A portion of this donor macerate was 
placed in a test tube. The radicles of a plurality of tomato seedling 
(Lycopersicon esculentum) were immersed in the donor medium. One electrode 
was inserted into each of the seedlings, while another was positioned in 
contact with the donor medium. A direct current 9 volt potential 
difference was applied across the electrodes, and thus across the 
seedlings and macerate, for five minutes. 
Tomato is well known to be one of the agronomic crops which can be 
commercially grown both under greenhouse and field conditions, while it 
has been noted that the Marsh Cabbage possesses a high metabolic output in 
its early stages of growth, R. M. Knutson, Science 186: 746-747 (1974). In 
view of the hypothetical model set forth in the discussion following the 
examples herein, and in light of the fact that certain characteristics of 
the transduced plants in Examples 1 and 2 were intermediate the 
characteristics of the donor and acceptor species, it was thought there 
was a significant chance that the high metabolic output of Marsh Cabbage 
could be imparted to tomato seedlings to increase their fruit yields, and 
thereby increase the commercial value of the crop. 
A number of tomato seedlings so treated were grown in a greenhouse, and the 
number of plants resulting from treatment, and the number of fruit borne 
by those plants at the time of fruit ripening, are shown in Table II. Both 
the positive and negative electrode orientation data were combined in the 
data reported in Table II, since in this case there were no apparent 
polarity differences. Again, the asterisk indicates data which is 
significant at a 95% confidence level (P less than 0.05). 
TABLE II 
______________________________________ 
FRUIT/PLANT 
DONOR AVERAGE AND s.d. N-PLANTS 
______________________________________ 
Macerate 
*4.33 (2.64) 15 
Controls 
2.43 (2.42) 21 
______________________________________ 
This same donor/host transduction was repeated for the purpose of examining 
yield levels under field conditions. Using three different varieties, a 
total of 24 test series were prepared with 30 transduced seedlings in 
each series (15 per electrode polarity) plus 15 control, non-transduced 
plants. Exposure was again conducted with the apparatus shown in, and the 
method described in conjunction with, FIG. 3, at a direct current 
potential of 9 volts and an exposure time of five minutes. All plants were 
handles and reared under similar conditions of field environment. Yields 
from individual plants were recorded at the time of optimum fruit harvest 
(approximately two-thirds mature fruit per plant). 
Of the 24 test series, 7 of them, or 29.2%, disclosed a statistically 
significant yield advantage (based on mean weight of fruit per plant) over 
the control or non-transduced groups, at a confidence level of 95% (P less 
than 0.05). Within the groups showing yield increases, there were also 
concomitant, statistically significant increases in growth rates and in 
plant size. The yield data possessing this significance ranged from +35% 
to +70% fruit weight increases over the controls. An experienced plant 
geneticist and breeder observing the transduced series selected one with a 
+50% (P less than 0.05) yield increase as having improved phenotypic 
characteristics for traits desirable for commercial harvesting, 
specifically, upright plants having good clustering of fruit. 
Individual plants were selected and grown in T2 generation field 
replications as plant rows (30 plants per row) from this particular 
exceptional plant series as well as from several of the T1 generation 
series, including some that showed no yield advantages. Yields were again 
recorded in the T2 generation. These plants row data disclosed that those 
plants showing growth and yield advantages in the T1 generation also gave 
high growth rates and yields in the T2 generation, whereas those showing 
no growth or yield advantages in the T1 generation gave no growth or yield 
increases in the T2 generation. From the T1 generation exceptional 
progenitor plant, a total of 10 plant rows gave statistically significant 
yield advantages ranging from +40.7% to +55.6%, compared with the T2 
generation control plant yields. T3 inbred generations of these high 
yielding plants are currently being compared in several large scale field 
tests (approximately five acres at four different locations) with two high 
yield commercial varieties, as well as with the non-transduced F3 
generation controls. In all locations the transduced line is still showing 
significant growth and development advantages over the non-transduced 
varieties. 
This example illustrates the consistency of the induced phenotypic effects 
and practical increases in the rates of fruit production in a commercially 
valuable crop, when the method of the present invention is practiced. 
EXAMPLE FIVE 
The Symplocarpus foetidus root tissue used as donor material in Example 
Four was prepared in the early spring (mid-March) growth period, when the 
metabolic activity in the root was at a high rate. Donor tissue prepared 
from different tissue regions of the donor plant and taken at a later 
stage of maturity can have significantly different effects on the growth 
rate in the acceptor plant Lycopersicon esculentum. Tissues from the root, 
the development spadix in the lower stem of the plant, and the leaf 
foliage of the Marsh Cabbage were collected in mid-April, and donor 
macerates were prepared as described in Example Four and shown in FIG. 3. 
Seedlings from four different commercial and established varieties of 
tomato plants were treated with these macerates and with a direct current 
9 volt potential and five minute exposure. The tomato seedlings were grown 
under greenhouse conditions and periodic growth data was obtained. Table 
III presents data obtained at six weeks of growth which shows the 
percentage of the total series which possessed growth statistically 
significantly higher (P less than 0.05) than the corresponding control 
series. 
TABLE III 
______________________________________ 
DONOR TESTS WITH SIGNIFICANT 
TISSUE GROWTH N-TEST SERIES 
______________________________________ 
Root 2.8% 36 
Spadix 19.4% 36 
Foliage 
30.0% 20 
______________________________________ 
The foliage employed as a donor, with its high rate of protein synthesis, 
yielded the highest percentage of tests showing significant growth 
increases in the acceptor series, when compared with the controls. It is 
noteworthy that the root macerate used in the above test produced 
significant growth increases in only 2.8% of the series, whereas in 
Example Four the root tissue obtained about one month earlier (when at its 
high level of metabolic activity) induced high growth in 29.2% of the test 
series. 
This example serves to illustrate the importance of the selection of tissue 
for the transduction donor, as well as considering its state of maturity. 
EXAMPLE SIX 
Many varieties of plants in the pea and bean family (legumes) have the 
ability to more efficiently utilize or fix nitrogen from the atmosphere 
than other plants. This diazotrophy occurs through bacteria which live 
symbiotically on the plant roots and form outgrowths or root nodules. The 
results of this example suggest that an acceptor species in the cereal 
family such as corn, which does not fix nitrogen, could have mutations and 
growth stimulation induced therein from a donor bean species which has 
these root nodules. 
A donor extract was prepared from soy bean (Glycine max) root nodules 
excised from plants grown from seeds which were initially inoculated with 
the bacterium Rhizobium japonicum, which is known to produce diazatrophy 
in soy beans. The macerated nodule liquor was mixed uniformly with agar as 
a base, and corn (Zeas mays) and sunflower (Helianthus annuus) seedlings 
were both treated with this donor extract in fashion shown in and 
described in conjunction with FIG. 3. 
EXAMPLE SIX (A) 
Both corn and sunflower seedlings were placed in the base medium and 
exposed to a potential giving an initial current of about 30 microamps 
through the seedlings. After exposure the seedlings (along with equal 
numbers of controls) were planted in a field test plot, with no fertilizer 
added. Growth and development studies were conducted on three separate 
test series of corn and two separate test series of sunflower seedlings. 
The growth and development enhancement produced by the root nodule extract 
treatment was consistently observed in all five test series. Examples of 
growth and development data are presented in Table IV for a field test 
series of corn and in Table V for a field test series of sunflower plants. 
The corn seedlings were exposed to the current for one hour, with the 
cathode inserted into the donor medium; the data of Table IV were obtained 
from twelve plants in each series. The sunflower seedlings were exposed to 
the current for 30 minutes, with the cathode inserted into the donor 
medium; the data of Table V were obtained from twenty plants in each 
series. The differences in growth shown in the last column of each table 
were significant at the 99% confidence level (P less than 0.01). 
TABLE IV 
__________________________________________________________________________ 
DAYS AFTER 
ROOT NODULE EXTRACT 
CONTROLS GROWTH 
PLANTING AVE. S.E. AVE. S.D. DIFF. 
__________________________________________________________________________ 
7 9.25 cm 
2.14 cm 
5.09 cm 
1.64 cm 
+81.7% 
19 47.00 6.41 31.45 
8.89 
+49.4% 
31 83.58 9.99 64.64 
16.83 
+29.3% 
46 104.92 13.14 80.45 
19.44 
+30.4% 
62 109.00 25.04 74.64 
37.37 
+46.0% 
__________________________________________________________________________ 
After 73 days of field growth, the root nodule group disclosed an 84% near 
development and the control group only a 16% ear development. After 90 
days field growth, the average ear weight of the root module series was 
55.0 g and the average ear weight of the control series 28.7 g. The 
kernals on the treated series were also more fully developed than were 
those of the controls. 
TABLE V 
__________________________________________________________________________ 
DAYS AFTER 
ROOT NODULE EXTRACT 
CONTROLS GROWTH 
PLANTING AVE. S.D. AVE. S.D. DIFF. 
__________________________________________________________________________ 
10 8.00 cm 
1.56 cm 
6.00 cm 
1.81 cm 
+33.3% 
22 20.30 3.15 16.00 
3.89 
+26.9% 
37 67.00 10.87 56.25 
11.18 
+19.1% 
53 103.10 16.41 86.90 
18.17 
+18.6% 
__________________________________________________________________________ 
At maturity the mean seed pod weight (before seed removal) of the 
nodule-treated group was 21.7% higher than the mean seed pod weight of the 
control group. 
In these field test series, the polarity conditions were limited to the 
donor medium electrode being the cathode. The reason for examining only 
the one polarity condition was the fact that preliminary studies with the 
soybean root-nodule extract disclosed a greater growth response with the 
donor medium media electrode being negative than with the medium electrode 
being positive. 
This example shows the induction of more efficient growth, development and 
yield from the root-nodule extracts, as compared to the control plants. 
EXAMPLE SIX (B) 
Corn seedlings were placed in the donor medium and exposed to a direct 
current 15 volt potential giving an initial current in the range of 30 
microamps through the seedlings. After 10 minute exposures, groups of 15 
test seedlings along with equal number of controls were planted in a field 
test plot, no fertilizer added, and growth data taken periodically during 
the growth cycle. The test was conducted with five pure and commercially 
available inbred varieties, Mo17-Ht, A634-Ht, A632-Ht, B73-Ht and W117-Ht, 
and thus provided range of different lines of stable but homozygous test 
material. The use of different inbred lines also provided a germ plasm for 
subsequent hybrid crossing studies. 
A total of 18 test series were prepared and examined under field conditions 
in accord with this protocol. The observed development alterations in the 
T1 generation were primarily in the rates of maturity or tassel 
development, in growth rates and in changes in root structure and 
morphology. The roots in several of the treated series disclosed a much 
more branching or dendritic patterning, with thickening at the terminus of 
the root. The roots of the control plants had less branching with no 
thickening at their termini. This formation of inchoate nodules and 
alterations in the root morphology of the corn plant is indicative of the 
initial stages of diazotrophy induction in this cereal plant. Plants from 
those groups disclosing significant increases in the rate of tasseling or 
growth were then selected on an individual plant basis for T2 generation 
self pollination. These T2 general plants were then used in T3, T4 and T5 
generations, for both inbred and hybrid crosses. 
The advantage of the use of genetically pure, homozygous inbred varieties 
or lines as the acceptor materials is that mutations in corn can be keyed 
to alterations in particular chromosomes from known listings. Specifically 
in the case of corn, new genotypic and phenotypic expressions can be 
compared with those listed in The Mutants of Maize, N. G. Neuffer, et al 
Crop Science Society of America (Madison, Wisc.), 1968; and Maize for 
Biological Research, W. F. Sheridan, Ed. Plant Molecular Biology 
Association, (University Press, N. Dak.), 1982. It is well known to those 
skilled in the art that if a particular characteristic appears in a 
subsequent generation of a plant line where this characteristic was not 
previously present, a point mutation has occurred on a particular 
chromosome. Indeed, these point mutations are cataloged in this fashion. 
The treatment of corn seedlings by the present invention with the soybean 
root module donor material produced a number of changes in characteristics 
in the T2 generation which are known to be associated with particular 
point mutations. In particular, a number of these mutations are known to 
be located on chromosome which occurred in the T2 generation plants 
obtained from some of the treated series. A summary of the frequency of 
mutations found in two of the inbred treated series (derived from the 
A632-Ht acceptor) are listed below in Table VI. There were no mutations or 
phenotypic alterations (a zero percent level) observed in several thousand 
control or non-transduced plants from this same inbred line. 
TABLE VI 
______________________________________ 
CHROMOSOME-3 POINT MUTUATIONS 
(T2 GENERATION FROM TRANDUCED INBRED A632-Ht) 
MUTATION SERIES M33-1-18 
SERIES M33-1-7 
______________________________________ 
Dwarf 7 (20%) 0 
Short 7 (20%) 0 
Dwarf-Crinkly Leaf 
5 (14%) 13 (30%) 
rinkly Leaf 0 8 (19%) 
Short-Romosa 0 8 (19%) 
Dwarf-Crinkly- 
0 1 (2%) 
Romosa 
Normal 16 (46%) 13 (30%) 
TOTAL PLANTS 35 43 
______________________________________ 
The probability of any one of these mutations occuring in one plant by 
change alone is about 1 in 500,000 whereas in Table VI there are shown 
several cases in which a number of plants expressed two mutations and in 
one case, a single plant expressed three mutuations. Now, from the laws of 
strict probability, the odds that these percentages occurred by random 
chance are, in the case of two mutuations on the same plant, one in 
2.5.times.10.sup.11 and, in the case of three mutuations on the same 
plant, one in 1.25.times.10.sup.17. In addition to point mutuations, other 
transduced series were observed to express large increases in point 
mutuations which are known to involve several gene alleles. Examples of 
these multiple allele mutuations are listed below in Table VII. 
TABLE VII 
______________________________________ 
MUTATION 
TRANSDUCED 
SERIES VAR- 
PLANTS ALBINO LUTEUS IEGATED TOTAL 
______________________________________ 
MED. 27 26% 27% 0 144 
(A632-Ht) 
MED. 25 0 0 10.7% 28 
(A632-Ht) 
CONTROLS 0.024% 0.037% 0% 8179 
(A632-Ht) 
______________________________________ 
The data of Table VII show a mutuation increase for both albino and luteus 
of about a thousand times the level observed in the control population. 
Many of these mutations are not of commercial interest. For example, 
albino plants do not produce chlorophyll and expire before maturity. 
However, there were other mutations which have importance in plant 
breeding. The dwarf plants listed in Table VI are an example of a useful 
mutation. These plants are about one half the height of the control 
plants, but the ear size and production were comparable to those of the 
controls. This normal ear size on the mutuant dwarf plants is an important 
and commercially beneficial distinction from dwarf corn plants derived 
from conventional breeding programs, the difference being that the ears on 
the conventionally bred dwarf plants are small when compared with the 
normal hybrid ears, and have large areas on the ears which do not develop 
kernels at all. 
Field studies of dwarf plants obtained from corn seedlings treated in 
accordance with the method of the present invention establish the 
existence of a number of commercially important characteristics. The 
following has been shown to be true from five generations of field trials: 
The inbred, dwarf mutants have held their recessive characteristics through 
the T5 generation and exhibit a 50%-60% reduction in plant height, when 
compared with untreated parent inbred control corn plants, yet produce 
full ears of normal size, as compared to the controls. 
Using this same method and the same soybean root-nodule macerate as the 
donor material, the dwarf traits have been produced in treated series from 
four of the five original inbred varieties. 
When T5 generation dwarf plants originating from two different treated 
inbred lines are crossed in a normal manner to produce a hybrid, the dwarf 
characteristics are transferred to the hybrid. The resulting hybrid is 
uniformly about 40% of the height of the hybrid resulting from a cross 
between two untreated, inbred parent lines. 
The ear size and kernel formation in the dwarf hybrids are about the same 
as in the untreated hybrid controls. The commercial significance of this 
is that a smaller plant size in the dwarf hybrid allows a higher plant 
density under field planting conditions, which in turn results in a higher 
yield per acre. 
Lastly, the ears on the dwarf plants are located much lower on the plant 
than on the normal or control hybrids, and thus are more efficiently 
harvested than those on taller control plants. 
Additionally, a male sterile, cytoplasmic mutation (Cms) was observed in 
100% of the plants in one of the transduced, Mo17-Ht inbred lines. This 
mutation is commercially important in the development of inbred lines 
which do not require the laborious task of de-tasseling in the normal 
production of hybrids. 
In the T3 generation, a number of plants selected for phenotypic growth and 
yield advantages were used for hybrid crossing studies. In general, the 
early development and high yield traits present in the T3 generation 
plants were transmitted into the hybrids when the treated progeny were 
expressed through the female line of the hybrid. An example of this is a 
soybean root-nodule donor series expressing the mutuation "prolific", 
which relates to the percentage of plants with multiple ears. A normal 
hybrid line has about a 10% level of prolific plants. In hybrid crosses, 
using female parents from T2 generation inbreds, a direct correlation was 
observed between the percentage of plants with prolific mutations and the 
resulting yields. The yields from three field replications were compared 
with a good producing commercial hybrid. The yield from one of these high 
producing treated lines is compared with the control hybrid in Table VIII 
below. 
TABLE VIII 
______________________________________ 
PRO- YIELDS 
HYBRID SERIES 
LIFIC (g/plant) 
YIELD INCREASE 
______________________________________ 
Control (HL2454) 
8% 200.09 -- 
Female Transduced 
47% 257.87 +28.9% (P &lt; 0.05) 
______________________________________ 
This example illustrates the number and type of mutations which can be 
induced by the methods of the present invention. Many of the mutuations 
have utility in the production of new varieties and in the hybridization 
of plants. The useful mutant characteristics are selected from the test 
populations by conventional segregation testing methods commonly employed 
by plant breeders. The useful mutations are also expressed when employed 
in hybrid crosses. 
EXAMPLE SEVEN 
As noted in Example Six, the Eastern Marsh Cabbage (Symplocarpus foetidus) 
has a high metabolic output during early spring growth, the result of 
which is development of the plant during a period of temperatures too low 
for growth to proceed in most plant species. This metabolic response can 
be imparted to corn (Zea mays) by the method of the present invention, 
when a donor extract from Marsh Cabbage is applied to the corn seedlings. 
Potential benefits of such a characteristic might be expressed as higher 
yields, faster development rates or other useful mutations. A new variety 
with some or all of these attributes could be grown in regions of the 
world where the growing season is conventionally believed to be too brief 
for corn development. 
EXAMPLE SEVEN(A) 
A donor medium was prepared from the macerated roots of the Easter Marsh 
Cabbage, and corn seeds were exposed to a direct current during initial 
inbibition with the medium with the apparatus shown in, and by the method 
described in conjunction with, FIG. 8. After treatment, the 
extract-exposed and control series were examined under field growth 
conditions. Table IX discloses growth data taken just before mid-maturity 
(36 days after exposure). Each series contained 16 plants. Only the series 
having a positive base plate polarity during exposure of the seeds 
exhibited a statistically significant increase (P less than 0.05) in 
growth, as compared to the controls. 
TABLE IX 
______________________________________ 
BASE PLATE PLANT GROWTH PERCENT 
POLARITY AVE. S.D. CHANGE 
______________________________________ 
(-) 0.878 0.284 +9.5% 
(+) 1.003 0.234 +25.1% 
Controls 0.802 0.134 -- 
______________________________________ 
The polarity differences shown here are consistent with those mentioned in 
Example Six(A). With the base of the apparatus being the positive 
electrode, the embryo or radicle end of the seed was disposed upwardly, in 
contact with the cathode. This arrangement is the one which exhibited a 
statistically significant increase in plant growth. Cathode-radicle 
exposure was also the optimum situation for the plant series reported in 
Table IV and V. This demonstrates the consistency of the electrode 
orientation in the method of the invention. 
A detailed field examination of the plants listed in Table IX disclosed 
five unique plants out of each group of 16 treated series. Each of these 
five plants had definite growth enhancement, larger and greener foliage, 
the foliage being more pronounced than even the other members in the same 
test series. The growth of these designated "sub-groups" are listed in 
Table X, again at 36 days after exposure. The differences in growth 
between the sub-groups and the controls were statistically significant (P 
less than 0.01). 
TABLE X 
______________________________________ 
SUB- 
GROUP PLANT GROWTH GROWTH 
POLARITY AVE. S.D. N-PLANTS DIFF. 
______________________________________ 
(-) 1.208 m 0.039 m 5 +50.4% 
(+) 1.238 0.070 5 +54.4% 
Controls 0.802 0.134 16 -- 
______________________________________ 
Displayed in Table XI are data showing the differences between the leaf 
blade width in the two sub-groups and the controls. These data were taken 
at nodes 6 and 7 at 106 days of maturity. The differences in leaf width 
between the treated and control series are statistically significant (P 
less than 0.01). 
TABLE XI 
______________________________________ 
LEAF 
SUB-GROUP MAX. WIDTH WIDTH 
POLARITY AVE. S.D. N-LEAVES DIFF 
______________________________________ 
(-) 9.29 cm 0.91 cm 16 +15.6% 
(+) 9.44 0.87 16 +17.4% 
Controls 8.04 0.79 16 -- 
______________________________________ 
Development was also more rapid in these sub-group plants. At 82 days 
development, both sub-groups disclosed 100% tassel formation, whereas in 
the controls only 37% possessed tassels. The positive polarity sub-group 
also disclosed two developing ears, with no ear development at all in the 
controls. 
The final yield results for each entire series of plants is shown in Table 
XII. The ear weights are somewhat lower than normal, especially in the 
control series. This was due to a dry period during early ear development, 
a situation which occurred throughout the Midwest in the 1983 growing 
season. All three series were, however, subjected to the same water stress 
conditions. The data in Table XII show the importance of early ear 
development in the two test series which occurred before the water stress 
interval. 
TABLE XII 
______________________________________ 
BASE PLATE EAR WEIGHT WEIGHT 
POLARITY AVE. S.D. DIFFERENCE 
______________________________________ 
(-) 78.5 g 51.3 g +187.6% 
(+) 131.4 80.2 +381.3% 
Controls 27.3 24.2 -- 
______________________________________ 
The final ear weights from the two sub-groups of special high vigor plants 
gave values of 134 g per ear for five negative base plate polarity plants, 
and 230 g per ear for the five positive base plate polarity plants. The 
controls averaged only 27.3 g per ear. The differences are significant at 
a 99% confidence level (P less than 0.01). 
This example demonstrates the induction of a metabolic response having a 
positive effect on both development and yield in corn, when the corn seeds 
are treated in accordance with the method of the present invention. It 
also shows the practical value of selecting outstanding plants in a given 
test series. 
EXAMPLE SEVEN(B) 
A donor medium was prepared in mid-March from the macerated roots of the 
Easter Marsh Cabbage. Corn seedlings were exposed to the donor macerate 
with the apparatus shown in, and in accordance with the method described 
in conjunction with, FIG. 3, and with the test conditions described in 
Example Six(B). The same five inbred lines, also as described in Example 
Six(B), were utilized. After exposure the treated and control series of 
plants were examined under field test conditions as outlined in the 
previous examples. 
In the T1 generation, the plant alterations in the treated series of plants 
were expressed as increased development rates, plant size and plant shape 
variations. From these treated series of plants, individual plants were 
selected for T2 to T3 generation inbred and hybrid crosses. In the T2 
generation, several point mutations were observed, and their degree of 
expression is listed in Table XIII, along with the associated allele and 
chromosome on which the mutuation is known to occur. None of these 
mutuations was found in several thousand untreated controls. 
TABLE XIII 
______________________________________ 
MUTATION 
CHROMO- RECENT 
NAME ALLELE SOME NO. EXPRESSION 
______________________________________ 
Rust Resistant 
Rp 10 100% 
Zebra Necrotic 
zn 10 10% 
Purple pl 6 50-100% 
Pigmy pv 6 25% 
Male Sterile 
msl 6 90% 
Defective de16 4 25% 
Endosperm 
______________________________________ 
From this list there are three point mutations of utility in the commercial 
production of hybrids, namely, rust resistance, pigmy and male sterile. 
The pigmy plants are of quite different phenotype (narrow leaf and other 
known characteristics) from the dwarf mutants discussed in Example Six(b). 
However, they could be utilized for a similar purpose, to produce smaller 
sized hybrids and provide higher plant densities with higher yields. The 
utility of male sterile plants was discussed in Example Six(B) as well. 
In addition to the mutations listed in Table XIII, two important phenotypic 
alterations were observed which continue to be expressed into a T5 
generation currently under study. One new trait involves a line with a 
maturity which is 12-14 days earlier than the untreated controls. The 
second is a "broad leaf" expression with leaf widths on the treated lines 
over 40% greater than those on the untreated controls. The useful nature 
of the broad leaf characteristic lies in the ability of the plant to 
receive and utilize more radiant energy per unit time during 
photosynthetic activity. The result is plant with a more efficient and 
higher biomass output. 
A number of treated series from both the early and broad leaf lines were 
used in hybrid crossing studies. These plants were selected for either 
enhanced growth or for altered plant size. When the female line was the 
treated series, a number of statistically significant yield increases and 
early maturing lines were observed in the replicated field tests. 
This example and previous examples together demonstrate that different 
donor materials produce significantly different mutations and phenotypic 
growth responses, as may be seen by comparing the point mutations in 
Example Six(B) (resulting from soybean root nodule donor material) with 
those in this example, employing Symplocarpus foetidus as the donor 
material. New germ plasm is constantly of importance in commercial plant 
breeding programs and Examples Six(B) and Seven(B) illustrate that 
although the advantageous expression of a mutation, such as male sterile, 
may produce similar results, the fact that different alleles are involved 
in the two examples means that the characteristics in the germ plasm would 
be expressed quite differently in hybird usage. 
CONTROLS 
In order to insure that the results obtained in these examples resulted 
from the combination of subjecting the acceptor species plants to 
electrophoretic conditions and to whole cells and associated materials of 
a second species of plant, controls were conducted in which the materials 
of the second species of plant were replaced by distilled water or by a 
macerate of plants of the same species as the first species. Additionally, 
seedlings of the same species had root portions excised and joined 
together, as well as being exposed to an electropotential difference only. 
In all cases, no statistically significant difference was seen between any 
of the plants so treated and untreated control plants. Thus, the results 
obtained in the examples described in this application necessarily 
resulted from the inclusion of a donor material from a second, different 
species of plants. 
DISCUSSION 
The data obtained in the above examples leads to the inescapable conclusion 
that the frequency of plant mutations can be increased by exposing plants 
in their germinal phase simultaneously to electrophoretic conditions and 
to the whole cells and associated materials of a second species of plant. 
The fat that some of the mutated plants obtained possess characteristics 
which appear to be characteristics associated with the donor material of 
the second species of plant suggests that some genetically associated cell 
tissue components or macromolecular complexes from the donor species of 
plant are transferred to or transduced into the intact living cells of the 
acceptor species of plant, in such a manner as to alter the genotype 
and/or phenotype of the acceptor, to allowing such altered genetic and 
phenotypic characteristics be transmitted to successive generations as 
point mutations or as cytoplasmic transmitted traits. The subsequent 
discussion and examples supporting such a theory should be taken as 
evidence of the theory; however, the theory of transduction of genetic 
materials is not in and of itself essential to an understanding of or a 
practice of the methods of the present invention. Those methods have been 
demonstrated by the preceeding examples to be useful in producing an 
increased number of mutants in a plant population, without regard to 
whether the instant explanation of how such mutations occur is correct. 
The fact that the mutations occur is sufficient support for the invention. 
The theory as to how the present invention operates is straightforward. It 
is believed that in the present invention the application of 
electrophoretic conditions to the cells of an intact organism or whole 
plant allows the tranduction of genetically associated cell tissue 
components and macromolecular complexes from the donor species material to 
the recipient plant species. Migration of these materials would be induced 
by transmembrane ionophoretic currents, either arising from the natural 
difference in membrane potentials between cells of different species, or 
from an externally applied current. The theoretical feasibility of 
electrophoesis occurring laterally or along, but not through, cell 
membranes, has been discussed by Jaffe, Nature, 265: 600-602 (1977), and 
was demonstrated experimentally within the cell membrane and wall by 
Woodruff and Telfer, Nature, 286: 84-86 (1980). However, as opposed to the 
present invention, this ion migration was observed and performed by the 
injection of fluorescent trace-proteins through the cell membranes of an 
insect ooctye, where they were observed to migrate laterally along 
"intercellular bridges" or openings, but was not transferred through the 
membrane barriers without breach of them. Quite simply, the advantage of 
the present invention is the fact that it is conducted with normal, intact 
cells of the acceptor species, and at worst with tissue macerates of the 
donor species. The need to breach or remove the cell wall encountered in 
all previous techniques is avoided. 
Electrophoresis can alter cell plasmalemma permability. This permability is 
changed by altering the size or current of charge carrier proteins and 
micropores in the plasmalemma and nuclear envelope. And, as demonstrated 
by the subsequent examples, it is also clear that the application of 
electrophoretic conditions allows the ready passage of nongenetic 
materials through the normal, intact cell wall. Additionally, routine 
commercial gel electrophoresis techniques demonstrate that some sort of 
genetic alteration is associated with the method of the present invention. 
The transmission of certain enzymes, mRNA or tRNA from the cells of the 
donor plant species to the cytoplasm or nucleoplasm of the cells of the 
acceptor species of plant alter the rate or path of one or more specific 
biosynthetic pathways in the acceptor species, which would then alter the 
phenotype of the cells of the plant. A model of such alteration is shown 
in FIGS. 10A and 10B, and is described further below. 
An examination of electrophoretic technology as a testing procedure was 
conducted in 20 transduced corn lines and five untreated control lines, 
from which the test lines were derived. All lines were from the T4 
generation of field testing. Gel electrophoresis indicated the presence of 
10 transduced lines, or 50% of the total test group, having altered gene 
alleles. All five inbred controls displayed uniform, unchanged 
electrophoretic patterns. A total of 8 enzymes, out of the 37 known loci 
in corn, were examined and provided confirmatory evidence of polymorphism 
or new gene alleles. In a second test series, 12 enzymes were examined. 
The test group consisted of 42 transduced lines, 21 from each; of two 
different in-bred host or control lines. The material from the T4 
generation again possessed a high percentage, about 28 percent, of 
transduced lines having altered alleles, with essentially unaltered or 
homozygous patterns in the untreated control samples. 
Applicant has observed that induced dielectrophoretic properties or long 
range dipole interactions of a donor material can influence the spatial 
configuration of organelles within the acceptor cells located within the 
tissue regions of tranduction. For example, when donors are employed which 
have a strong, positive dipole charge, that is, a dipole moment much 
higher than that of water, or donors are employed that have been oxidized 
and thus receive a net positive charge, those donors migrate from the 
anode region and pass through the plasmalemma, and associated with the 
cell nucleus, forming a non-uniform electric field having a maximum 
intensity at the nuclear membrane. This results in an increase in the 
frequency of the collection of chloroplasts and other cell organelles in 
distinct proximity with the surface of the nucleus. In normal, untreated 
tissue, the nuclear-organelle clustering is observed at a low frequency of 
perhaps 1%-5% of cells, while in transduced tissue, the frequency in 
limited regions around the electrode contract zone is observed to be as 
high as 80%-90% of the cells. Chloroplasts and other organelles are 
clearly attracted to the nuclear membrane by long range dipole 
interactions. 
Applicant has also observed that the chloroplasts and organelles clustering 
around the nucleus is not a unique property of one specific donor 
material. For example, other less dipole substances such as distilled 
water, when used as a donor, do not produce the nuclear-organelle 
clustering. In the case of a donor which enters the free space (apoplast) 
of the host tissue and has a marked dipole moment, but is inert with 
respect to passing through the plasmalemma, the influence on the spatial 
patterns of chloroplasts is quite different. In such a case the 
chloroplast and organelle clustering around the nucleus is not observed, 
but rather the collection of the donor material in the free space of the 
cells causes a mass migration of the organelles to the cell wall, the 
direction depending upon the charge characteristics of the donor material. 
These changes in configural associations caused by electrophoretic 
conditions greatly increases the probability level for the exchange of 
genetic information between the nuclear and cytoplasmic DNA, since the 
organelles are disposed in proximity with the nucleus. The cooperative, 
long range dipolar effects occur inside the cell through the 
microdielectrophoretic interactions between the cell organelles. The 
existence of such dipole interactions has been postulated by Pohl, 
Bioelectrochemistry, Plenem Press, New York (1980). By using a 
ferroelectric material, specifically, barium titanate, Pohl was able to 
demonstrate dipolar attraction on the outside surface of animal cells. 
However, as far as the Applicant is aware, the instant observations are 
the first time that microdielectrophoresis has been observed inside living 
cells. 
Applicant believes the following mechanism may be an appropriate 
explanation for the observed migratory phenomenon. It is well known that 
the plant cell wall contains polysaccharides which act as growth and 
development regulators and chemical messengers. As noted by Albershime and 
Darvill, Scientific American, September, 1985, page 58, these regulatory 
molecules are released from the cell wall by enzymes. Different enzymes 
release different oligosaccharides (small polysaccharides). In a 
transduction from a new donor species, a donor enzyme complex enters the 
cell wall matrix and triggers the release of a quite different array of 
oligosaccharides which, after entering the cytoplasm, redirect patterns of 
development and form different genotypic associations with either the cell 
nucleus or the cytoplasmic organelles. As microdielectrophoresis takes 
place as described above, both nuclear and cytoplasmic interactions occur. 
This redirection of growth regulators from the cell walls could not occur 
in the recombinant DNA or protoplast fusion technologies, since the cell 
wall is necessarily removed in the early stages of the techniques. 
Further, the enzymes of one plant species may act as isoenzymes of the 
second plant species and possibly alter the morphogenic properties of the 
cell. Indeed, there may be enough of a potential difference between the 
cells of a difference species to facilitate the formation of intercellular 
cytoplasmic bridges which may allow certain cytoplasmic extranuclear DNA 
or cell organelles to be transferred from one species of plant to another. 
The transferred cytoplasmic extranuclear DNA and organelle systems would 
also exert some influence over the morphogenic determinative components, 
thereby transforming the phenotype of the tissues. 
Additionally, phygocytosis may occur and invaginate certain cell organelles 
through the cell plasmalemma and into the cytoplasm. Because the cell 
organelles and cytoplasmic extranuclear DNA synthesize at least some 
proteins and other materials, which are vital to cell function, the 
addition of cytoplasmic extranuclear DNA and cell organelles from a 
different species of pant may cause the creation of enzymes and proteins 
which are similar enough to the transformed cells' natural products to be 
utilized by the transformed cell but may, in the process, act as 
isoenzymes and "isoproteins" which cause the plant to exhibit different 
phenotypic characteristics, which may then be transmitted to successive 
generations in a non-Mendelian fashion. For example, in the technique 
described in conjunction with FIG. 3, the maceration of the donor tissue 
in distilled water liberates proteins and enzymes inside the cytoplasm of 
the donor cells, and this can facilitate the transfer of these 
constituents, because such constituents need pass only from the medium 
through the cell wall and plasmalemma of the host into the cytoplasm, 
rather than having to pass through at least two whole cell walls and 
plasmalemma, as would be the case for non-macerated donors. 
The procedures of this invention are believed to involve transductions 
within the somatic tissues of the host material. The complete expression 
of a new mutation or phenotypic alteration is not usually observed until 
at least the T2 generation. For this reason any explanation of what takes 
place in the host plant after the application of any of the described 
procedures cannot be based on the concept of a direct, abrupt uptake of 
donor DNA into the host plant cells during the initial transduction 
process. The establishing of a fixed genetic expression arising from a 
transduction appears to be a very gradual process and is believed to occur 
in a series of stages during the entire cycle of plant development. 
For the gradual incorporation of a new genotypic or phenotypic expression 
into the host plant, the transductions are assumed to be operating within 
specific biofeedback control systems involved in the plant morphogenesis. 
To convey this proposed concept of perturbations induced by the genetic 
transduction process, the least complex of known homeostasis pathways is 
adopted as a model, B. C. Goodwin, Temporal Organization in Cells, 
Academic Press, New York (1963). In this simple pathway the alteration 
takes place at a single active gener locus G.sup.0, which mormally leads 
to the synthesis of a cellular metabolite m.sup.0 (or enzyme according to 
the scheme shown in FIG. 10A. In this model, m.sup.0 acts as a repressor 
or co-repressor at the gene site G.sup.0 through the feedback loop. The 
main concern here is with the control of protein (enyzme) synthesis 
Y.sup.0, which regulates the final production of the cellular metabolite. 
The assumption is made that the level of the metabolite m.sup.0 is 
perturbed by the introduction into the cell of a homologous metabolite 
from a different plant species by means of the transduction process. This 
new metabolite m.sup.x acts at the cellular locus and augments the 
concentration of m.sup.0 so that the new level is at the concentration 
m.sup.1 (FIG. 10B) after the transduction is completed. The rate at which 
the effect of the transduction m.sup.x is annulled is, for a small 
perturbation, proportional to the magnitude of the disturbance. From first 
order chemical kinetics, the level as a function of time t after the 
transduction is 
EQU m.sup.x =a(e.sup.-kt) 
where a and k are constants. A very important point here is that m.sup.0 
and m.sup.x must be homologous proteins and very similar in their 
biosynthetic activity in both the donor and host plant systems. if this 
were not the case, the control loop m.sup.0 and repressor level would be 
unaffected, or in the case of an incompatible metabolite, the entire loop 
could be inactivated. This could readily explain shy some species are 
effective as donors and others are not, and why different tissue regions 
of the same species respond different as donors. 
The perturbation of the normal metabolite concentrations m.sup.0 to a new 
level m.sup.x would, through the feedback control, alter the rate of mRNA 
synthesis at the gene site G.sup.0, and a new rate of metabolite 
production would be established in the tissue of the host plant. As the 
somatic tissues develop, the entire pattern of gene expression during 
plant morphogenesis is operating at a different level of temporal 
organization of nucleotides than would be found in the non-transduced 
system. As this perturbed, transduced tissue differentiates into meristem 
regions and ultimately into germ plasm, the kinetics of these altered 
biosynthetic pathways are transcribed as altered gene alleles, with 
permanent expression being established in the DNA code. During 
transcription, the mRNA would contain altered codon sites, which in turn 
would lead to altered protein synthesis as the polypeptide chains are 
synthesized on the ribosome surface. Thus we have the situation of the 
induction of new enzymes synthesized in the epigenetic cycle or enzymatic 
adaption through the introduction of homologue precursors from another 
plant species (the donor). 
The perturbations of biofeedback control mechanisms within more complex 
co-repressor systems could account for incomplete or partial masking of 
dominant alleles in the somatic tissue. In the situation where cytoplasmic 
mutations arise form the transductions, the inherited alterations may be 
brought about in quite a different manner. In this case the presence of 
foreign polypeptides from the donor leads to the possibility that such 
polypeptides become genetic precursors and may be subsequently imported 
into chloroplasts and mitochondria, A. Cashmore et al., Biotechnology, 3: 
803-808, (1895). The plant genome is unstable and capable of generating 
variability, Science, 224: 1415, due to changes in repeated DNA units 
which are more common in plants than animals (more than 75% of all DNA 
sequences fifty base pairs or longer is repetitive DNA). Repeated 
sequences are especially prone to undergo loss or gain because they can 
promote the incorrect pairing of chromosomes during meiosis. If there are 
multiple copies of a gene, one copy may be mutated and lead to a new 
function, as in the above transduction scheme, while the previous function 
is maintained by the remaining members or copies of the gene. Such copies 
have the characteristics of transposable elements, B. Mc Clintock, 
Science, 226: 946, with the result that some specialized cells undergo 
gene activation and phenotype changes. Only DNA loss is irreversible, 
other DNA alterations such as methylation, chromatin structure, 
protein-DNA interactions and the like being reversible and modifiable. The 
mechanisms for all embodiments of this invention are thought to be similar 
to the above recited model. 
Thus, under the application of an electric current across tissues from two 
different species of plants, transmembrane ion migration occurs, with 
specific enzymes, their precursors mRNA and tRNA, and regulatory 
polysaccharides being transmitted from a donor species into the cytoplasm 
of an acceptor species. Current flow across the tissues also effects the 
electric charges on the cell membranes and greatly alters membrane 
permeability and ion pathways through the intrinsic proteins within the 
cell membrane, which control the transfer of ions and large molecules. 
With in the cell, microdielectrophoresis alters spatial configurations of 
the organells, resulting in increased probabilities for the transfer of 
genetic information between the organells and thereby causing increased 
rates of mutation. The following examples demonstrate the ready degree of 
ion migration occuring in cells and germinal plants upon the application 
of electrophoretic conditions. 
EXAMPLE VIII 
To elucidate the mechanisms occuring at the cellular level, donors were 
utilized with known ionic charge characteristics and with both inert and 
biologically active properties. One type of host tissue consisted of the 
chlorophyll containing stems of Pelargonium maculatum. Stems about 5 
centimeters long and 5 to 8 millimeters in diameter were subjected to two 
to four hours at about 10 to 20 volt potentials and a current density of 
about 30 microamperes per square centimeter. The negatively charged, red 
protein pigment from the Amaranth plant was applied as a donor material in 
the apparatus disclosed in and according to the technique described in 
conjunction with FIG. 8, with each end of the host stem contacting a 
pigment-containing electrode. At the cathode end of the test stem the red 
pigment migrated through the section, leaving a zone of stained tissue 
extending several millimeters into the stem. At the boundary of this zone 
of migration, a microscopic examination revealed the stain collecting of 
the nucleus of the parenchyma cells. At the anode end of the test stem, 
the pigment was oxidized and because positively charged. As it migrated 
from the anode end of the stem it gave the host tissue a dark grey color 
zone extending several millimeters into the stem. At the boundary of this 
zone, a microscopic examination revealed a clustering or proximal grouping 
of chloroplasts in the immediate vicinity of the cell nucleus. The 
oxidized Amaranth was observed to collect on the nucleus, and through long 
range dipole intractions (microdielectrophoresis) formed a positive 
electrical field gradient which then attracted the negatively charged 
chloroplasts to the surface of the nucleus at the locations of maximum 
field strength. 
Confirmation of these dielectrophoretic alterations in spatial 
configurations of cell organelles was observed when using a powdered form 
of carbonyl iron having a particle size of one to 10 microns, with a 
positive electric charge. When transduced into geranium stems, as in the 
preceeding example, an electrophoretic migration of 1 to 2 centimeters 
occured at the anode end of the stem. Carbonyl iron is biochemically 
active and was observed to be transduced into the cell cytoplasm where, as 
in the case with the oxidized Amaranth pigment, it caused a long range 
dipolar attraction of the chloroplasts in the cytoplasm. The chloroplasts 
were found to be more tightly grouped around the cell nucleus than in the 
case with the cell Amaranth donor. This is explained by the fact that 
within the same host tissue and under the same conditions of voltage and 
time, when compared with the Amaranth, the carbonyl iron migrates over two 
times the distance into the host tissue. This indicates that the carbonyl 
iron has a higher ionic mobility than the Amaranth pigment. 
A donor macerate of Phaseolus multiflora leaves containing macromolocules 
and proteins with associated charge groups, when electrophoretically 
transduced into the non-chlorophyll tissue of Zea mays radicles using the 
same method as above, causes a clustering of cellular plastids and other 
cell organelles (too minute to identify microscopically) around the cell 
nuclei in the anode region of the host tissue. At the cathode region the 
cell nuclei had a smooth outline and the chromatin structure was uniform. 
Other less ionic donor substances such as distilled water, when transduced 
in a similar manner, with the host tissue being either the geranium stem 
tissue or the non-chloroplast radicle tissue of corn, did not induce the 
observed spatial readjustments in the cell organelles. 
Barium ferrite of particle size 1.3 microns and having a net negative 
charge was then used as a donor for the purpose of examining a 
biochemically inert substance which enters only the free space (apoplast) 
of the host tissue. Using the geranium stem as the acceptor and employing 
exposures as in the preceeding example, the extent of the migration was 
far less than when using the more biochemically active materials. The dark 
stained tissue region was only two to three millimeters into the cathode 
end of the stem section. At the boundary of the migration, the donor 
particles cause the negatively charged chloroplasts to migrate and cluster 
at the cell wall opposite the location of the cathode and migrating barium 
ferrite. The grouping here was of an entirely different spatial patterning 
then when using donors which enter the cytoplasm of the cell of the host 
tissue. 
This example serves to teach that in the process of electrophoretic 
transduction as described in this invention, the donor complex can migrate 
both through the cell free space of the host tissue, as well as through 
the plasma membrane into the cytoplasm of the cell. Furthermore, the 
nature and ionic strength of the molecular dipole charges of the donor can 
significantly alter the natural, more or less random, spatial distribution 
of cell organelles in the cells of tissues being electrophoretically 
transduced. Such altered spatial patterns can greatly influence the 
probability of the exchange of genetic information between the cell 
nucleus and surrounding organelles, and thus provide one mechanism whereby 
mutation rates can be significantly increased. This example also teaches 
that both organic and inorganic molecular species can enter the plant cell 
and interact with the organelles in a physical and/or biochemical manner. 
Components from a macromolecular donor complex produced from plant tissue, 
also enter the cell and are active in the organelle spatial repatterning. 
EXAMPLE NINE 
Dry seeds of corn (Zea mays) were inserted between the stainless steel 
electrodes of the apparatus illustrated in FIG. 8. The electrodes were 
covered with filter paper pads moistened with distilled water. The embroyo 
end of the seed was placed upward, or opposite the base plate electrode. 
At a 45 volt direct current potential a sharp, well defined uniform line 
of black pigment was observed to develop and migrate up the seed if the 
base plate was anodic or positive, or down the seed if the base plate was 
cathodic or negative. 
Since distilled water has a very low ionic content, charge transport in the 
seed occurred through the oxidation of the pigment materials (polyphenols) 
in the test seed. The migration of these oxidation products, as testing 
indicates, is linear with time. This linear relationship is what would be 
expected under conditions of electrophoretic migration. An ionic mobility 
of about 0.54.times.10.sup.-6 centimeters squared per volt per second was 
observed, a value which is consistent with the rate of movement of large 
molecules. Microscopic examination revealed the layer of oxidation 
products to extend laterally through the tests into the outer layers of 
the endosperm. 
This example provides a graphic demonstration of the movement of large, 
physiologically related molecules through the plant tissues under 
conditions of an electrical potential as applied in the methods of the 
present invention. 
EXAMPLE TEN 
The frequencies of altered enzyme loci producing polymorphism in corn 
plants in which Sympolocarpus feotidus is the donor are quite different 
from the frequencies when using the soybean root-nodule extract as the 
donor. These different allelic responses are exemplified by commercial 
electrophoresis tests. In 62 transduced lines produced in accordance with 
the method described in conjunction with FIG. 3, 15 lines were transduced 
with Symplocarpus feotidus as the donor, and 47 lines with the soybean 
root-nodule as the donor. Table XIV provides a listing of the number of 
transduced lines containing a specific enzyme polymorph, as they occurred 
within the two donor test groups. Only those alleles showing positive 
polymorphism are included in this listing; those observed to have only a 
slight variation are excluded. The enzymes listed are those in which 
polymorphism occurred in at least one transduced line. 
TABLE XIV 
______________________________________ 
Number of transduced corn lines 
showing polymorphism 
Soybean 
Enzyme S. foetidus root-nodule 
______________________________________ 
ACP (acid phosphatse) 
6 11 
PGM (phosphogucomutase) 
4 1 
MDH (malate dehydrogenase) 
1 1 
PGD (6-phosphogluconate 
1 0 
dehydrogenase) 
PHI (phosphohexose 
3 1 
isomerase) 
GLU (B-glucosidase) 
1 0 
______________________________________ 
The data in Table XIV demonstrates that in the soybean root-nodule lines 
the majority of the alterations take place at the ACP alleles. In the 
lines with S. foetidus as the donor, there were fewer lines with altered 
ACP alleles and far more lines involving other enzymes. The fact that the 
two enzymes, PGD and GLU revealed polymorphism in the S. foetidus lines 
(comprising only 24% of the total test series) and not in the root-nodule 
lines (comprising 76% of the test series) again emphasizes the influence 
of the donor type on the final genetic response and range of possible 
polymorphic alterations that might be achieved by using other donor types 
and combinations. 
Whatever the mechanism yielding the mutations observed when the methods of 
the present invention are employed, the present invention clearly provides 
methods for increasing the proportion of mutants in plant generations. The 
method of the present invention are significantly advantageous over the 
known methods of recombinant DNA and plasmid fusion techniques, for the 
reasons that the precise genetic structure of the chromosomes mutated need 
not be elucidated, time and effort need not be wasted in removing the cell 
walls, and time and effort need not be wasted in attempting to grow whole 
plants from isolated tissues. Instead, the acceptor plants are whole 
germinal plants, which after treatment can be grown in any conventional 
fashion. 
Having described my invention, however, many modifications thereto will 
become apparent to those skilled in the area to which it pertains, without 
deviation from the spirit of the present invention, as defined by the 
scope of the appended claims.