Plant transformation process with early identification of germ line transformation events

A method is disclosed for making more efficient the particle-mediated germ line genetic transformation of bean species such as soybean. After a particle-mediated transformation event, in the absence of a selectable marker gene, relatively large numbers of plants must be regenerated to find the relatively low likelihood germ line transformation events which have occurred. It has been discovered that using in the transformation process a marker gene linked to the gene of interest, and by excising a segment of the stem of the shoot during the regeneration process and assaying the segment for the marker gene, certain patterns or phenotypes can be identified in the stem segment which are associated with an increased frequency of germ line transformation events. As the plants are regenerated, other indices of gene expression, at the first trifoliate leaf stage and at the third or fourth trifoliate leaf stage, also serve as markers of the likelihood of germ line transformation. By using these markers in the relatively early stages of plant regeneration to assay for likelihood of germ line events, it is possible to concentrate regeneration efforts on plants most likely to yield germ line events, and to discard the others, so as to lower the burden and effort in achieving a desired number of transformation events.

FIELD OF THE INVENTION 
The present invention relates to the genetic transformation of plants in 
general, and relates, in particular, to the efficient germ line 
transformation of soybean plants utilizing particle-mediated techniques 
for plant transformation. 
BACKGROUND OF THE INVENTION 
It is now becoming possible to introduce into some of the major crop plants 
foreign genes of potential interest in those plants. This process of 
genetic engineering has been accomplished with certain model species, such 
as tobacco, petunia, and carrot, and has now been accomplished in such 
major crops species soybean and cotton. In procedures for the genetic 
engineering of plants, it is desired that the genetic transformation of 
the plant tissues be of the germ line of the plant tissues. Germ line 
refers to the inheritable genetic material of the plant which is 
permanently altered by the transformation process in such a fashion that 
the plant will pass to its progeny by normal genetic inheritance the 
inserted foreign gene. While the transformation of somatic, or non-germ 
line cells, may be desired in some instances, in general in the genetic 
engineering of crop plants it is desired that germ line transformation of 
plant lines be achieved as quickly and efficiently as possible. 
The most common previously utilized technique for the genetic engineering 
of plants involves the use of the soil-dwelling plant pathogenic bacterium 
Agrobacterium tumefaciens. A. tumefaciens has the natural ability to 
transfer a portion of its DNA, referred to as T-DNA, into the genome of 
susceptible plant cells. By changing the native T-DNA in an Agrobacterium 
strain, it is possible to use this unique trait of Agrobacterium to 
transfer desired genes into single plant cells. If the introduced gene 
includes a selectable marker, such as a herbicide or antibiotic resistance 
trait, it is possible thereafter to select for transformed cells in a 
tissue culture by imposing the putative transformant tissues to the 
selection pressure of the appropriate antibiotic or herbicide. 
Unfortunately, in soybean almost all cultivars are resistant to 
Agrobacterium infection and are thus very resistant to transformation with 
Agrobacterium. In addition, antibiotic resistant markers, such as 
kanamycin resistance commonly used in Agrobacterium plant transformation 
procedures with other plant species, have been found to be of limited 
utility in soybean transformation experiments. Accordingly, while it may 
be possible to utilize Agrobacterium-mediated transformation techniques in 
soybean, it is a difficult endeavor because of the lack of effective 
selectable markers. 
Other techniques for transforming plants do exist, however. In particular, 
there exists a general approach to the transformation of plant cells which 
is based on delivering the transforming DNA into the plant cells by 
coating the DNA onto small inert carrier particles which are physically 
hurled into the target plant tissues. The technique of particle-mediated 
plant cell transformation was first demonstrated with somatic cells in 
such tissues as the epidermal tissue of onion and other such model cell 
cultures. Klein et al., Nature, 327:70-73 (1987). Later the originators of 
the particle-mediated transformation technique were able to achieve 
genetically engineered tobacco plants by the transformation of tobacco in 
tissue culture, using a selectable marker, which was then subsequently 
regenerated into whole plants. Klein et al., Proc. Natl. Acad. Sci. USA, 
85:8502-8505 (1988). Rather than attempting to use particle-mediated 
transformation techniques on plant cells in culture, another approach was 
developed in which the growing meristems of plants were subjected to a 
particle-mediated transformation event. From such a technique, stable 
transformation of the germ line of soybean plants was achieved. McCabe et 
al., Bio/Technology, 6:923-926 (1988). This technique is not dependent on 
the availability of a selectable marker for the plant species. 
In developing the technique for the germ line transformation of soybean 
plants using a particle-mediated technique based on meristem 
transformation, it was discovered that the transformation events often 
resulted in chimeric plants, which are plants in which some, but not all, 
of the tissues had been genetically transformed with the introduced DNA. 
McCabe et al., supra. Although the technique was thus useful to create 
genetically engineered plants, it was somewhat burdensome in the sense 
that large numbers of tissues had to be subject to transformation events, 
and large numbers of plants had to be cultivated from the putative 
transformed tissues in order to discover those particular shoots and 
plants which properly expressed the introduced DNA. Therefore, the ability 
to identify transformation events early in the process giving rise to 
heritable germ line transformation of tissues creates the ability to 
effectuate dramatic savings in the practical and cost-effective genetic 
transformation of plants resulting in reduced labor costs and reduced time 
and energy expended in cultivating the non-transformed tissues which were 
subject to the transformation events. 
In seeking a germ line transformation of a plant species, such as soybean, 
it would be helpful if the progenitor tissue of the germ cells of the 
plant were identified in the growing meristem or shoot. Unfortunately, the 
science of developmental morphology of plant cells has not developed to 
the point that the ancestor cells of the soybean germ cells is known. 
Accordingly, if it is a plant meristem or embryo that is being 
transformed, no present knowledge exists as to which precise cells in that 
meristem or embryo must be transformed to achieve germ line 
transformation. Therefore, any correlation between categories of cells 
transformed in a growing soybean plant and a germ line transformation 
event would have to be determined empirically. 
SUMMARY OF THE INVENTION 
The present invention is summarized in that a process for creating and 
identifying germ line transformed soybean plants is provided in which a 
number of growing meristems of soybean plants are subject to a 
particle-mediated transformation procedure and that the resulting tissues 
are subject to a series of early-stage tests of phenotypic markers in 
primary transformant tissues to search for phenotypic markers which have a 
high correlation to germ line transformation events, so that only the 
marked tissues will be subject to the investment of energy in regenerating 
whole plants therefrom. 
It is an object of the present invention to provide a method of 
particle-mediated transformation of plants which is inherently more 
efficient and cost-effective than previously available techniques. 
It is an object of the present invention to make more cost effective the 
genetic engineering of plants, and soybean plants in particular, by 
identifying, at the earliest stage possible in the process, those plants 
which are most likely to yield germ line transformants so that only those 
plants need to be cultivated into mature plants. 
It is another object of the present invention to optimize the use of 
reporter or marker genes in plant transformation experiments even in the 
absence of selectable markers to make possible the genetic engineering of 
plants in those plants for which reliable dominant selectable markers are 
not available. 
Other objects, advantages, and features of the present invention will 
become apparent from the following specification when taken in conjunction 
with the accompanying drawings.

DESCRIPTION OF THE INVENTION 
In accordance with the method of the present invention, a series of indices 
are described which are used to identify high likelihood germ line 
transformation events among the shoots and plants produced from a 
particle-mediated plant transformation process. These indices are based on 
a classification of certain phenotypes of expression of a marker gene in 
tissues of a regenerating plant at certain defined stages. 
To fully understand the advantages of the present method, it is helpful to 
appreciate certain considerations about the nature of accelerated 
particle-mediated transformation of plants. Since the transforming DNA is 
carried into the plant tissues on particles, and since the number of 
particles must be limited to avoid destroying the plant tissues 
transformed, only a percentage of the cells in a treated plant tissue will 
receive transforming DNA, and only a percentage of those cells will be 
transformed. Therefore, in the absence of a selection agent to kill 
non-transformed cells preferentially, the result of regenerating plants 
from such treated tissues will be plants most of the tissues of which are 
not transformed. It has been found that even of the plants which are at 
least in part transformed, most of the transformations do not result in 
germ line transformations. This invention is therefore directed to 
identifying those desired germ line transformation events at an early 
stage so that regeneration of the non-germ line plants can be minimized. 
Thus it is not necessary to use the early germ line identification process 
described here to achieve a germ line transformation of soybean. It is 
possible to regenerate all plants recovered from the treated tissue, 
sexually propagate all the plants and assay all the progeny. The drawback 
to this approach is that most of the effort in the regeneration and 
propagation process will be wasted on non-germ line transformation events. 
The present invention helps to avoid that waste and thereby assist in the 
efficient creation of lines of genetically transformed soybean. 
The present invention is thus based on a technique involving the 
particle-mediated transformation of plant cells. Therefore, to better 
understand the context of the present invention, it is necessary to 
understand the general technique of particle-mediated transformation of 
plant cells and the apparatus which may be used therefore. 
In the process of particle-mediated transformation of plant cells, a 
carrier particle consisting of a small inert relatively dense particle of 
material is coated with a transforming genetic construction of DNA and 
then is physically accelerated so that it is delivered into the interior 
of the growing meristematic or embryonic cells to be transformed. The 
transforming DNA is thus carried into individual cells, but the carrier 
particles are small enough such that the individual cells are neither 
destroyed nor seriously incapacitated. It has been found that by 
delivering DNA on such carrier particles in such a fashion into the cells 
of plants, such as soybeans, whole transformed germ line plants can be 
obtained to result in transgenic plants in plant lines. 
There are several factors which are necessary to be considered in the 
creation of germ line plant transformations in these fashions. The genetic 
construction must be one properly constructed to be expressed in plant 
tissues. The apparatus utilized must be of a type capable of delivering 
the carrier particles with the coated DNA on them into plant cells in such 
a fashion that a suitable number of cells are transformed. There are many 
types of mechanical systems which can be envisioned to accelerate 
biologically inert small carrier particles. Possible mechanisms include 
ballistic explosive acceleration of particles, centrifugal acceleration of 
particles, electrostatic acceleration of particles, or any other analogous 
system capable of providing momentum and velocity to small inert 
particles. The mechanism used herein to achieve particle-mediated plant 
transformation is based on an adjustable electric voltage spark discharge 
device. The apparatus is illustrated in a schematic fashion in FIG. 1. 
The particle-acceleration apparatus is generally indicated at 10 in FIG. 1. 
The apparatus consists of a spark discharge chamber 12 into which are 
inserted two electrodes 14 which are spaced apart by a distance of 
approximately one to two millimeters. The spark discharge chamber 12 is a 
horizontally extending rectangle having two openings 16 and 18 extending 
out its upward end. The opening 16 is covered by an access plate 20. The 
opening 18, located on the side of the rectangle of the spark discharge 
chamber 12 opposite from the electrodes 14, is intended to be covered by a 
carrier sheet 22. The electrodes 14 are connected to a suitable adjustable 
source of electric discharge voltage. Such a source of electric discharge 
voltage would preferably include suitable electric switching connected to 
a capacitor of the one to two microfarad size range, with the amount of 
the voltage of the charge introduced onto the capacitor being adjustable, 
such as through the use of an autotransformer, through a range of perhaps 
1 to 50,000 volts. Suitable high voltage electric switching (not shown) is 
provided so that the capacitor can safely be discharged through the 
electrodes 14 so that the apparatus can be used conveniently by a user. 
The carrier sheet 22 intended to be placed upon the opening 18 in the spark 
discharge chamber 12 is a planar sheet of relatively stiff material, such 
as a sheet of aluminized saran coated mylar. Above the opening 18 in the 
discharge chamber 12, positioned approximately 15 millimeters above it, is 
a retaining screen 24. Placed above the retaining screen 24 at a distance 
of approximately 5 to 25 millimeters above the retaining screen, is a 
target surface 26. The target surface 26 can be any suitable culture 
surface onto which the material to be transformed 28 may readily be placed 
such as, most conveniently, an overturned petri dish into which the plant 
tissues have been positioned for culture. Copies of the exogenous foreign 
genetic construction intended to be transformed into the plant tissues are 
prepared by suitable DNA preparation techniques well known to those of 
ordinary skill in the art, and multiple copies of the genetic construction 
are made. The copies of the foreign genetic construction, in aqueous 
solution, are then coated onto small particles of a durable dense 
biologically inert carrier material, such as gold, the carrier particles 
typically being in a size range of 1 to 3 microns. The carrier particles 
with the exogenous genetic construction thereon are then placed upon the 
carrier sheet 22, which is inserted at the proper opening on the top of 
the spark discharge chamber 12. The target surface 26, including the 
living plant material thereon, is then placed in position above the 
retaining screen 24. A small droplet of water, preferably about 10 
microliters in size, is then placed bridging between the ends of the 
electrodes 14. The access cover 20 is placed in position on top of the 
spark discharge chamber 12. At this point the entire apparatus is enclosed 
in a vacuum chamber and a vacuum is drawn until it is in the range of 
approximately 500 millimeters of mercury. As the vacuum is being drawn, a 
supply of helium is bled into the vacuum chamber, replacing the remaining 
atmosphere in the chamber with helium. The lower relative density of 
helium, combined with the reduced pressure, helps to lower the drag on the 
gold particles. 
At this point, the spark discharge between the electrodes 14 may be 
initiated by the user. This is done by means of the appropriate electric 
switching which applies the voltage stored in the capacitor across the 
terminals of the electrodes 14. The force of this electric discharge 
bridges the spark discharge gap between the electrodes 14 instantly 
vaporizing the small droplet of water previously placed therebetween. The 
force of the vaporization of that water creates a shockwave within the 
spark discharge chamber 12 which radiates outward in all directions. The 
impact of the radiating shockwave upon the carrier sheet 22 propels the 
carrier sheet 22 upward with great velocity. The upwardly traveling 
carrier sheet 22 accelerates until it contacts the retaining screen 24. 
The use of the helium within the vacuum containment for the apparatus 
provides less drag on the flight of the carrier sheet 22 as well as the 
carrier particles. At the retaining screen 24, the carrier sheet 22 is 
retained, and the carrier particles coated with the exogenous genetic 
construction previously coated thereon fly off of the carrier sheet and 
travel freely onward toward the target tissues 28. The small carrier 
particles then proceed into the cells of the target tissues 28 placed on 
the target surface 26, and pass freely into the cytosol of the cells 
placed thereon. The actual momentum of the carrier particles as they 
impact the surface of the target tissues is adjustable, based upon the 
voltage of the initial electric discharge applied to the electrodes 14. 
Thus, by varying the amount of the electric discharge applied across the 
electrodes 14, the velocity by which the particles impact the target can 
be adjusted, and thus the depth of penetration of the carrier particles 
into the tissue of the target tissues can be adjusted continuously 
throughout the range of adjustment provided for the electric voltage 
applied across the electrodes 14. 
To be useful in a particle-mediated transformation technique, the 
transforming exogenous genetic construction must be capable of performing 
some useful function in the cells of target plant tissues. The 
transforming genetic construction, which will normally be a chimeric 
construction in the sense that its DNA originates from more than one 
organism, should be capable of expressing in the target tissues gene 
product such as a foreign protein of interest or an antisense RNA strand. 
Such foreign genetic constructions, usefully embodied in expression 
cassette vectors for use in plant cells, are known to those of ordinary 
skill in the art. Typically such plant expression cassette vectors 
includes, besides the coding sequence of the desired exogenous or foreign 
gene, appropriate flanking regulatory sequences suitable for expression of 
the foreign gene in the plant cells, such as a promoter sequence capable 
of initiating transcription and a translational terminator to terminate 
translation of a message if protein synthesis is desired. It has been 
previously demonstrated that typical promoters and transcription 
terminators found to be effective in other plant tissues are effective in 
soybean as well. 
The transforming genetic construction may also include a marker gene. Such 
a marker gene need not be a selectable marker. It need only be a marker 
the presence of which can be assayed using a minimal amount of plant 
tissue, by appropriate biochemical or phenotypic trait which may be 
observed in transgenic plant tissues. Such a marker gene may be the 
transforming DNA itself, if a completely biochemical assay for the 
presence of the transforming DNA is used, such as a polymerase chain 
reaction type of assay for the DNA itself. One convenient type of marker 
gene capable of detection by a phenotypic assay is the GUS gene described 
by Jefferson et al., Embo J., 6:3901-3907 (1987). The GUS gene, coding for 
the enzyme beta-glucuronidase which can be expressed in plant cells and 
the expression of which, in a tissue-destructive assay, will turn a 
convenient substraight, indigo-glucuronide, or 5-bromo-4-chloro-3-indolyl 
glucuronide blue in color in an in situ assay in plant tissues. Thus, the 
use of the GUS gene provides a convenient colorimetric assay for the 
expression of introduced DNA by phenotypic analysis in transformant plant 
tissues. Thus, in a typical transformation procedure, the desired gene of 
interest would be coupled in tandem in a single genetic construction, a 
DNA strand, with a GUS gene, and then the detection of the transforming 
DNA in plant tissues would be done by phenotypic analysis for the 
expression of the GUS enzyme in the target plant tissues. 
Several plant tissues of soybean may be genetically transformed by such a 
particle-mediated transformation technique using the apparatus of FIG. 1. 
It has been found most conveniently that the excised embryonic axes from 
immature or mature soybean seeds may be readily transformed utilizing the 
procedure. The embryonic axes are excised from the soybean seeds and the 
primary leaves are removed to expose the meristem of the embryo. The axes 
are then plated on target plates containing 1% water-agar. The plates are 
then used as a target surface in the apparatus of FIG. 1 for a 
particle-mediated transformation event. The axes may then be plated in the 
dark on MS basal medium as modified by Barwale et al., Planta, 167:473-481 
(1986) for regenerating zygotic embryos. This particular medium contains a 
high level of benzylaminopurine which induces multiple shoot formation in 
the plated embryonic tissues. Following incubation for one to two weeks in 
the dark, the tissues are transferred to the same basal medium with lower 
concentrations of benzylaminopurine, and then are cultivated in the light 
to promote shoot elongation. Using this technique, multiple shoots will be 
derived from the primary and axillary meristems on the embryonic tissues. 
These shoots may then be grafted onto soybean roots, or induced to form 
roots, to regenerate whole intact and sexually mature soybean plants. It 
is these shoots, and the soybean plants regenerated therefrom, which are 
subject to the screening techniques described herein so as to result in 
high numbers of germ line transformant plants. 
When the soybean shoots or plantlets resulting from tissues subject to a 
transformation process, such as has been described above, are regenerated 
into whole mature plants, it has been found that only a fraction of the 
resulting soybean plants will prove to be genetically transformed with the 
foreign DNA. The plants which are regenerated from the shoots resulting 
from this tissue are referred to as R0 plants, while their progeny in 
successive generations are referred to as R1 and R2, etc. In addition to 
having a low frequency of genetic transformation within the R0 plant 
generation, of the R0 plants which exhibit some genetic transformation, 
most of the plants will be chimeric. By the use of the term chimeric in 
this sense, it is intended to signify that the plants will be composed of 
tissues which are not genetically identical, i.e., the plants will have 
only a portion or fraction of their tissues transformed, while the 
remainder of the tissues are not genetically transformed. Since it is the 
object of the plant transformation process such as that described herein 
to create stably transformed plants which carry the foreign DNA in their 
germ plasm, and are capable of passing the inserted DNA into their 
progeny, it is necessary then to implement a screening procedure to 
ascertain how to recover the germ line transformation events from a 
number, and perhaps a large number, of putatively transformed R0 plants or 
plant tissues. 
The method disclosed herein is intended to enable the screening of R0 
shoots and plants from the putatively transformed plant culture to isolate 
those plants which will give rise to, or at least be most likely to give 
rise to, progeny plants which have had their germ line transformed. In 
this regard it should be noted that for the progeny grown from the R0 
plants, that is to say from the R1 generation onward, if the progeny 
plants contained the inserted foreign DNA at all, they are clonal, that is 
to say, non-chimeric and have their germ line cells transformed. While 
events are observed in which the inserted DNA appears to be present in a 
clonal fashion in the R0 plant, but then is found completely absent in the 
R1 and R2 plants, if the gene is found to be present in the R1 plant, it 
will prove to be stably inheritable thereafter in those of the progeny of 
the R1 plant which inherit the inserted gene as a trait. 
The present method is a screening technique rather than a selection 
technique. By that it is meant that the plants, or portions of the plants, 
are screened for the presence of certain marker characteristics, but are 
not subjected to a selection criteria such as would be achieved by 
antibiotic or herbicide resistance routines. In part the use of such a 
screening technique, as opposed to a selecting technique, is required due 
to the absence of reliable selectable marker gene effective in soybean 
tissues. The preferred marker gene, as mentioned, to be the object of the 
screening process, is the GUS gene. The GUS assay is, however, destructive 
of the plant tissues and thus cannot be performed in vivo. It must 
therefore be performed on a portion of the tissues recovered from the 
plants to be screened and the portion of the tissue selected must be one 
that balances the need to gain an accurate reflection of the prospects for 
germ line transformation on the particular plant with the need to maintain 
the vigor of the plant tissues involved so that the very events sought are 
not lost during the assay process. 
The method of the present invention is based on the selection of certain 
strategically located portions of plant tissue from the R0 plants during 
the regeneration process. In particular, three indices of the state of 
transformation of the R0 plants are utilized. The first index is a section 
of stem segment from the R0 shoot collected from the shoot prior to 
rooting. The second index is a section of the leaf phenotype at the first 
trifoliate stage of development of the seedling. The third index is a 
petiole/mid-rib section of a trifoliate leaf in a more mature plant, at 
the third or fourth trifoliate leaf stage. By doing histochemical assays 
for the enzyme coded by the GUS gene in each of these three stages, it is 
possible to predict with a high degree of confidence the germ line 
transformation events which have occurred. In fact, it is possible at a 
relatively early stage in the procedure to discard most (as many as 
90-99%) of the regenerating R0 shoots and plantlets to concentrate on the 
remaining shoots or plantlets which have a high probability of yielding 
germ line transformation events. While the discarding of many such shoots 
or plantlets may seem wasteful, and will almost certainly involve the 
discarding of occasional truly transformed plants, the amount of labor and 
time saved by not taking large numbers of non-transformed plants to seed 
is large in comparison to the time involved in creating additional 
transformation events. Thus the laborious part of the process, i.e., 
taking the putatively transformed plants to seed and growing and testing 
the progeny, can be reduced to a minimum by the screening methodology 
disclosed herein. 
The first index analysis of the transformed plants occurs when regeneration 
of shoots commences from the originally transformed plant explant. As the 
regenerating soybean shoots reach a finite and defined size, typically 
approximately 2 centimeters in size, the shoots may be isolated from the 
original explant and prepared for propagation, either by grafting or by 
hormone treatment to induce root formation. However, at the time the 
shoots are separated from the original explant, it is convenient at that 
point to take a relatively small segment of the stem from the basal 
portion of the separated shoot, i.e., a segment size of perhaps 2 
millimeters. The stem segment can then be fixed, and subjected to the GUS 
histochemical assay. The result is a cross-sectional view of the stem of 
the soybean plant which will exhibit a color, i.e., blue, in those 
portions of the tissues of the plant which have been transformed. Based on 
an analysis of those tissues, it is then possible to predict for those 
stems which exhibit some enzyme activity, those plants which are most 
likely to yield a germ line transformation event. 
A classification scheme has been devised to categorize the results of the 
stem segment assay. The transgenic primary regenerates were designated "B" 
when the GUS enzyme assay revealed that the gene was expressing in all of 
the epidermis, the cortex, and the pith of the stem segments assayed. The 
regenerates were designated "C*" or "P*" when the expression of the GUS 
gene was confined to 100% of either the cortex or the pith respectively of 
the shoot segment which was assayed. The regenerating shoots were 
classified as "C" when at least half of the cortex expressed the enzyme, 
and "c" when less than 50% of the cortex showed activity. Similarly a 
classification of P or p indicates GUS activity in more or less than 50% 
of the pith of the stem. The shoots were scored as "e" when the activity 
of the GUS gene could only be seen in the epidermis of the shoot. Examples 
were also observed in which the codes were used to indicate localized 
activity in multiple areas, for example, a score of c/P* indicates that 
the shoot expressed the GUS marker gene in 100% of its pith tissue, but in 
less than 50% of its cortex tissue. A classification of E*/p indicates the 
expression of the enzyme in 100% of the epidermis but in less than 50% of 
the pith and none of the cortex of the transformant plant. 
FIGS. 3-8 illustrate the classification scheme of the stem segments. FIG. 3 
is a highly stylized drawing of an ideal soybean stem cross-section in 
which 30 indicates the whole stem, 32 indicates the epidermis, which is 
greatly exaggerated in width, 34 indicates the cortex and 36 indicates the 
pith. Shading in FIGS. 4 to 8 is intended to represent blue color in the 
GUS assay. FIG. 4 illustrates a stem 30 classified as "E" since only the 
epidermis is expressing the GUS gene. FIG. 5 illustrates a stem 30 which 
would be classified as "c/p" since less than 50% of cortex or pith are 
expressing. The stem 30 of FIG. 6 would be classified as "c/P*" since all 
the pith but less than 50% of the cortex was transformed. The stem 44 of 
FIG. 7 would be "C*". As should be apparent, the stem 30 of FIG. 8 
represents a "B" stem segment. All of the results of FIGS. 4-8 represent 
actual events. 
For most of the shoots recovered for the plant transformation process using 
the apparatus of FIGS. 1 and 2, no expression of the GUS gene in the stem 
section was found. Of those shoots in which expression in the stem segment 
was found, and classified in one of the categories as described above, a 
number of shoots of each category were either induced to root or were 
grafted onto healthy root stock and cultivated further into plantlets. 
When the plantlets developed either two or three trifoliate leaves, one or 
more leaflets from the first trifoliate leaf was harvested from each of 
the plants and the trifoliate leaflet itself was assayed for activity for 
the GUS enzyme. Thus new phenotypes could be identified which could be 
correlated to the phenotypes from the classification on the original stem 
of the primary regenerate shoot. A number of the plants were found only to 
express the GUS enzyme in epidermal trichomes and/or hairs either 
throughout the leaflet or only in certain areas within the leaflet. Other 
plants were found to express GUS activity only in mesophyll cells or 
stomata, and the activity in these tissues could extend throughout the 
leaflets or be confined to one or more specific areas within the leaflet. 
In other cases, the enzyme activity would be found to be localized in the 
mid-rib or to extend in varying degrees to one or both sides of the 
mid-rib in sectors of the leaflet. Examples were even found in which the 
GUS activity extended from the mid-rib to the perimeter only on one-half 
of the leaf. Again, the most optimal phenotype observed was that in which 
the blue color was not either localized into a sector or confined to the 
epidermis. 
Upon further development, a third assay was developed based on petiole and 
mid-rib sections on a trifoliate leaf higher up the regenerating plant. It 
was found that if this last assay were not performed, certain non-germ 
line transformation events would be allowed to go to seed, since certain 
transformation events seem to give rise to plants in which the transformed 
tissues only extended a finite length up the stem of the regenerating 
plant. It became a not uncommon observation to observe GUS activity only 
in the first trifoliate leaf and not in certain other trifoliates further 
up the plant. It was, however, the general observation that the majority 
of plants which had GUS activity in the third or fourth trifoliate leaves 
would exhibit the same activity in all or most of the leaves of the 
plants. 
The use of these three indicia led to the observation that the 
transformation process could be optimized to the total overall labor 
involved if only the plants which were characterized as "B", "P*" or some 
combination of P and c or C, during the assay of the stem segment, were 
fully regenerated and allowed to propagate into R1 plants. Even plants 
which are characterized as B, however, would not prove to have germ line 
transformation events if the expression of the GUS gene was not also found 
in the first trifoliate leaf and also in the petiole mid-rib assay in the 
mature leaf. The best use of the second and third indicia of 
transformation events were used as negatives, that is to say, plants which 
failed to properly exhibit the GUS activity at these stages could also be 
discarded, since the likelihood that such plants would yield germ line 
transformation events was found to be relatively minimal. Of plants which 
had been classified as B during the stem segment, and which showed major 
GUS activity in the pith or pith and cortex during the assay of the 
petiole and mid-rib sections of the third or fourth trifoliate stage, all 
plants had activity which was later determined to involve germ line 
transformation events as evidenced by the recovery of progeny plants which 
were clonal and expressing the GUS gene. All plants which were classified 
in any other of the categories described above exhibited a dramatically 
lower frequency of germ line transformation events, although such events 
did occur with other classes of tissues. For example, two plants out of 
363 which showed activity only in their epidermis at the stem segment 
stage also gave rise to transformed progeny. However, the likelihood of 
any transformation events from any other categories of plants was thus so 
low as to justify, on a practical basis, discarding all but the plants 
designated above during the stem segment and further to sexually propagate 
those plants categorized likely to lead to germ line transformation which 
both exhibited GUS activity at the first trifoliate stage and also showed 
major pith and cortex involvement during the assay of the petiole and 
mid-rib of the mature leaf of the plant. 
An excess of one-third of the plants classified as "B" would give rise to 
germ line transformation events. By contrast, plants categorized in other 
categories during the stem segment as they were found to have a 
dramatically lower likelihood of germ line transformation event. For 
example, a plant that is classified as c/p*, the likelihood of a germ line 
transformation event was less than 10% while for plants showing activity 
in greater than half their cortex, and classified C, the ratio of 
transformation events was on the order of 2%. Of plants which had major 
cortex and pith activity, designated C/P, of which only eight plants were 
tested, two yielded germ line transformation events. Of six plants which 
showed activity in less than 50% of the pith, one germ line transformation 
event was recovered. Thus the exact classifications that are regenerated 
may depend on a cost analysis of the cost of regeneration of shoots into 
plants and seeds as compared to the cost of generating more putatively 
transformed shoots. 
EXAMPLES 
The target tissues used in the soybean transformation procedures described 
herein were the excised embryonic axes from immature and mature soybean 
seeds of elite lines of soybean. The axes were excised from the seeds and 
plated on target plates on agar. The methodology for excising the seeds 
and performing the transformation experiment is described in detail in 
McCabe et al., Bi0/Technology, 6:923-926 (1988). The apparatus used in the 
transformation experiments was the apparatus of FIGS. 1 and 2. 
After the transformation event was conducted, in excess of 5,000 shoots 
were recovered, and subject to a stem segment assay. In performing the 
stem segment assay, the shoots were allowed to reach a length of 
approximately 2 centimeters before they were severed from the underlying 
tissue. At the time of severing, a 2 to 4 millimeter section of the base 
of the shoot was cut off, and assayed for GUS activity. Of the 757 stem 
segments assayed which showed GUS activity, the plants were categorized on 
the basis of the kind of activity which was observed in the stem by the 
categorization system described above, i.e., either B, C/P*, C, C/P, p, c 
or e. The distribution of the plants as classified can be seen in Table I. 
TABLE I 
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Germ Line Event Frequency 
Stem Segment Number Number 
Assay Number of expressing expressing 
Classification 
Plants in first leaf 
in mature leaf 
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B 30 27 13 
C/P* 13 5 1 
C 45 20 1 
C/P 8 4 2 
p 6 1 1 
c 292 9 1 
e 363 7 1 
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All of the plants for whom the stem segment assay was positive to any 
degree were then either grafted or rooted to create plantlets. The first 
trifoliate leaf from each of the plantlets was assayed for GUS activity. 
The results of that assay are indicated by the third column in Table I. 
All plants which continued to have GUS activity in the first trifoliate 
leaf were then propagated into plants containing multiple leaves, and 
mature third or fourth trifoliate level leaves were harvested from the 
plants and the petiole and mid-rib sections were taken through that leaf 
and were fixed and assayed for GUS activity. The results of those assays 
are indicated in the fourth column in Table I. All plants indicated in the 
fourth column were found to have experienced a germ line transformation 
event. 
The fourth column in Table I indicates those plants which had activity in 
the pith or pith and cortex of the third or fourth stage trifoliate leaf. 
All of the plants which had GUS activity were allowed to self-pollinate 
and reproduce progeny. Analysis of the progeny was then done for GUS 
activity. It was determined that all of the plants which were indicated in 
column 4 of this chart to have activity in the pith or pith and cortex of 
the third or fourth trifoliate leaf stage were found to involve germ line 
transformation events as indicated by the recovery of progeny plants which 
expressed the GUS gene. 
Of plants which failed to have pith or pith and cortex activity in the 
third or fourth stage trifoliate leaf, the results were more mixed. Of 
plants which had been designated B by the stem segment assay, during the 
assay of the third or fourth trifoliate leaf, 8 of the plants only showed 
activity in the epidermis while 6 were found to be completely negative in 
GUS assay. Two of the plants which showed only activity in their epidermis 
gave rise to transformed progeny. Thus the indication of classification as 
B is not a predictor alone and of itself of a germ line transformation 
event. The classification of B thus indicates a likelihood of a high level 
of recovery of germ line transformation, but cannot be considered alone a 
highly confident predictor of germ line transformation event in the 
absence of pith or pith and cortex involvement in the third or fourth 
trifoliate leaf of the soybean plant. 
For the plants for which the stem segment assay caused categorization in 
another category, the results also gave rise to germ line transformation 
events, although at a lower frequency. Of the 13 plants characterized as 
C/P*, 5 plants were recovered which seemed to express GUS throughout their 
tissues, but only 1 of the plants proved to be a germ line transformation 
event. Of the 45 plants showing activity in excess of 50% of their cortex, 
and designated C, 20 GUS expressing plants were found only 1 of which was 
found to be a germ line transformation event. Of the 8 plants categorized 
as C/P by virtue of activity in excess of 50% of their cortex, 4 plants 
were recovered which expressed GUS in their leaves which yielded only 2 
plants in which a germ line transformation event was found. Of the 6 
plants which showed activity in less than 50% of their pith, a single germ 
line transformation event was found. Of the 292 R0 plants which showed 
minor activity in the cortex, 9 plants were recovered which expressed GUS 
in their leaves and 1 germ line transformation event was recovered. Of 363 
R0 plants which showed activity in the epidermal layer only, 7 plants were 
recovered which seemed to express the GUS in their leaves and only 1 event 
proved to be a germ line transformation related event. 
Based on the data presented in Table I, and related experiments, it was 
determined that of the soybean plants subjected to the particle-mediated 
transformation process described herein, between 10 and 15% of the 
recovered shoots exhibit some form of expression of the GUS gene during 
the stem segment assay. Of all of the shoots which are the subject of the 
procedure the percentage which were found to be germ line transformed will 
ultimately be in the range of 0.2 to 0.5% of the regenerated shoots. Thus 
by performing the stem segment assay, the number of plants which need to 
be carried through the regeneration process can be reduced by a factor of 
85 to 90%. Then, since it has been found that the highest concentration of 
germ line transformation events occurs for segments which are categorized 
as B, C/P*, C, or C/P, all other plants falling in all other categories 
during the stem segment assay could be discarded on the grounds of low 
likelihood of finding a germ line transformation event. Since these plants 
categorize approximately 15% of the total stem segments which exhibit GUS 
activity at all, again this allows a discard of approximately 85% of the 
recovered plants so that the efforts for regeneration can be concentrated 
on the remaining 15% which will show a high likelihood of germ line 
transformation events. In this way approximately 2 to 2.5% of the shoots 
recovered from the transformation process need ultimately be propagated, 
thus saving 97 to 98% of the labor and effort involved in the plant 
propagation and progeny testing process. Of the 2% which are regenerated 
through the transformation process, between 10 and 25% of the plants have 
been found, by assay of the progeny to GUS activity, to have undergone a 
germ line transformation yielding progeny which are stably transformed and 
inheritably possess the inserted foreign DNA.