By Ian M. Sussex

One of the stellar achievements of twentieth century plant biologywas the
genetic transformation of somatic cells enabling the regeneration of whole
plants that were stably transformed and capable of transmitting the inserted
genetic material to subsequent generations.
This achievement grew out of three independent lines of research initiated
early in the twentieth century: plant tissue culture, regeneration of plants
from single somatic cells, and the study of crown gall disease. The early
discoveries made in these areas represent a combination of basic scientific
research and technological innovations and led to the development of
genetically transformed crop species expressing traits unobtainable by
conventional breeding. Each of these fields can be traced back to a single
research publication (Haberlandt, 1902; Smith and Townsend, 1907; White,
1939a) that later came to be considered as the foundation of the field. It
is instructive to follow how these three fields were established,
progressed, converged, and finally coalesced. Early workers, of course,
could not know the ultimate way in which their discoveries would be applied
to modern plant biotechnology (Figure 1). Instead, they posed questions that
were of interest to them and to the scientific communities of which they
were a part, and some of their discoveries led in directions that would
later prove to be productive in the progression to biotechnology. This essay
is intended to provide a review of the crucial discoveries that ultimately
led to modern plant biotechnology and show how they contributed to this
progression.

PLANT TISSUE CULTURE

Philip White worked at the Rockefeller Institute for Medical Research in
Princeton, New Jersey in the 1930s to develop an experimental system with
which to study metabolism in a completely undifferentiated tissue where all
cells are identical and hence exert similar influences on one another. White
(1939a) defined a plant tissue culture as a system in which cells satisfied
two main requirements of remaining "undifferentiated yet capable of
unlimited growth" (White, 1939a). Earlier attempts, starting in the late
nineteenth century, to grow plant parts in isolation from the organism did
not satisfy these requirements and failed for a variety of reasons,
including microbial contamination, inadequate nutrient media, and poor
selection of tissues to culture (White, 1931; Gautheret, 1983; Hoxtermann,
1997).

The most successful of the early attempts involved the culture of maize,
pea, and cotton root tips (Kotte, 1922; Robbins, 1922). These could be
excised from the plant with minimal trauma and grown aseptically for a few
weeks in nutrient media, but ultimately growth ceased. White (1934)
succeeded in growing excised tomato root tips for potentially unlimited
periods of time in a liquid medium containing inorganic salts, 2% sucrose,
and 0.01% yeast extract. Tips were excised from seedling roots and
subcultured at regular intervals. After 52 subcultures over .400 d, the
cultured roots showed no diminution of growth rate. White calculated that by
this time any nutrients and regulatory substances in the original root tip
would have been diluted to ;10240, and he concluded that the nutrient medium
had supported indefinite growth of the root tips. Thus, while he had
demonstrated "potentially unlimited growth," the second part of his
definition of a tissue culture had not been met because the roots clearly
were not undifferentiated.

White (1939a) addressed this second question using a hybrid between
Nicotiana glauca and N. langsdorffii. The hybrid plants produced tumor-like
calluses and galls on the stem and leaves. Tissue removed aseptically from
young stems was cultured on the same nutrient medium used for tomato roots.
Proliferated masses from the original explants were divided and subcultured
at weekly intervals through 40 subcultures. White calculated that any
material from the original explant would have been diluted to at least 10217
by this time and that the cultures therefore satisfied the criterion of a
capacity for unlimited growth. The question remained as to whether the cells
in the culture were undifferentiated. Histological examination revealed only
mature parenchyma cells, regions of dividing meristematic cells, and
occasional isolated xylem cells. Despite this level of cellular
heterogeneity, White concluded that these tissues approximated an
undifferentiated condition and grew for potentially unlimited periods and
therefore were true tissue cultures.

Scientific results are greatly strengthened when other workers with
appropriate expertise replicate the original findings. Remarkably, this
occurred within 6 weeks of the publication of White's results. Two French
workers, Roger Gautheret in Paris and Pierre Nobecourt in Grenoble, both of
whom had been attempting to culture plant tissues for several years,
reported potentially unlimited growth of cultures derived from carrot tap
root tissue when the growth substance indole-3-acetic acid (IAA) was
incorporated in the culture medium (Gautheret, 1939; Nobecourt, 1939).
Neither cited White (1939a), and presumably they were unaware of his
results. Neither study was as detailed as White's. Gautheret cultured tissue
for 13 months making bimonthly subcultures, and Nobecourt cultured tissue
for 20 months making seven subcultures.

White's objective, of studying metabolism in an undifferentiated tissue
wherein all the cells are identical and presumably exert similar influences
on one another, appears not to have been followed up by him or his
contemporaries. It was not until many years later that other scientists
began using methods developed by White to study cellular metabolism.
Notably, H.E. Street at Manchester University used cultured excised tomato
roots in an extensive series of studies to examine the metabolism of
inorganic ions, carbohydrates, amino acids, and hormones (Street, 1957). A
more direct approach to White's original intention was followed by F.C.
Steward and colleagues at Cornell University. They examined metabolic states
in three contrasting carrot tap root cultures: secondary phloem cells as
they existed in the intact root, excised tissue maintained on a minimal
medium where cells grew predominantly by enlargement, and actively
metabolizing cells stimulated by coconut milk in the medium to divide as
rapidly as possible. They documented significant differences in rates of
accumulation of RNA and DNA under the different culture conditions (Steward
et al., 1952, 1964).

With White's original goal for plant tissue culture having been achieved and
confirmed, attention turned in other directions. Gautheret and Nobecourt
both reported the occurrence of roots that developed on their carrot tissue
cultures. White (1939b) found that shoots were produced from Nicotiana tumor
tissues submerged in a liquid medium, and Michael Levine (Levine, 1947)
reported the spontaneous formation of shoots and roots on cultured carrot
tissue. These observations turned attention to the question of totipotency.
That is, did tissue cultured cells retain the full genetic competence of the
zygote to form a complete plant?

This question was addressed by Folke Skoog and his collaborators at the
University of Wisconsin. Starting from White's discovery of shoot formation
in submerged Nicotiana tumor cultures, they found that auxin (IAA) was a
potent inhibitor of shoot formation in tobacco tissue cultures (cv Wisconsin
38), but high concentrations of auxin stimulated the formation of roots. The
nucleic acid base adenine supplied with low auxin concentrations stimulated
shoot formation in these cultured tissues. The amount of shoot and root
formation depended on the proportions of auxin and adenine supplied in the
culture medium (Skoog and Tsui, 1948). In further work, they isolated from
autoclaved DNA an adenine derivative, 6- furfurylaminopurine, that they
named kinetin. Using tobacco callus cultures they found that by adjusting
the relative concentrations of auxin and kinetin in the culture medium it
was possible to induce the formation of shoots and roots or the growth of
undifferentiated callus (Skoog and Miller, 1957). Thus, they demonstrated
that tobacco callus tissues retained the potentialities of the zygote to
form both shoots and roots, but their research did not prove that a single
cell had these potentialities.

Studies that approached this question were initiated in the laboratory of
F.C. Steward at Cornell University. Previously this group had compared
metabolism of carrot secondary phloem explants in basal medium or medium
supplemented with coconut milk, a source of cell division factors. Explants
in the coconut milk medium produced new cells that became detached and
proliferated freely in the culture medium, resulting in large numbers of
single cells and cell aggregates. Among these aggregates they noted some
that formed roots and subsequently shoots to produce whole plants that could
later be transferred to agar media and then to soil where they flowered and
completed the life cycle (Steward, 1958; Steward et al., 1958a, 1958b).

From these experiments they concluded that "no single parenchyma cell can
directly recapitulate the familiar facts of embryology, but, through the
formation first of an unorganized tissue culture, which is in fact a colony
of dividing cells, the necessary degree of organization is recaptured, first
to form roots and then to form shoots" (Steward et al., 1958a). The
implication of this statement was that the origin of the regenerated plants
was from single cells. If so, cellular totipotency would have been proven
for carrot cells, at least. However, other workers (cited in Sussex, 1972)
pointed out that there was no incontrovertible proof that single cells
rather than cell aggregates that may have contained cells with different
genetic potentialities were the source of the new plants that they obtained.
These tissue culture studies that demonstrated the potentially unlimited
growth of undifferentiated cells and the production from them of roots,
shoots, and entire plants did not contribute further to the questions that
we are examining because of their failure to assure the single cell clonal
origin of regenerated plants and thus the genetic totipotency of single
cells. However, the study of plant tissue and suspension cultures was
continued in different directions, including the commercial production of
secondary products (Ramawat and Merillon, 2007) and commercial production of
trees, crops, and horticultural plants, most notably species of orchids
(Arditti and Krikorian, 1996).

REGENERATION OF WHOLE PLANTS FROM SINGLE SOMATIC CELLS

Gottlieb Haberlandt, working in Graz, Austria, was the first to culture
isolated somatic cells of higher plants in vitro. He began these
investigations in 1898 and published the results in 1902 (Haberlandt, 1902).
His intention was to study "the properties and potentialities which the cell
as an elementary organism possesses" (Krikorian and Berquam, 1969;
translation from Haberlandt's text). Although Haberlandt failed to induce
divisions in any of the cells that he cultured, he is recognized as the
founder of plant cell culture because of the novelty of the methods he
proposed and the concluding paragraph in his article ".I believe, in
conclusion, that I am not making too bold a prediction if I point to the
possibility that, in this way, one could successfully cultivate artificial
embryos from vegetative cells."

Recognizing the lack of knowledge of the nutrient requirements of higher
plant cells, he used as a culture medium the seven inorganic elements that
had been identified by Knop (1865) as sufficient for the water culture of
higher plants, with additions of sucrose, dextrose, glycerine, asparagine,
and peptone in various concentrations and combinations.

Haberlandt first attempted to culture green, photosynthetic cells from leaf
bract mesophyll of Lamium purpureum. Bracts were teased apart in liquid
until microscopy examination revealed numerous isolated palisade and spongy
mesophyll parenchyma cells. These were then transferred by finely drawnout
pipettes to hanging drops or dishes of culture medium. Microbial sterility
was attempted by flaming instruments and glassware but usually failed to
eliminate bacterial and fungal contamination completely. Cultures were
maintained in lighted rooms at ambient temperature or in darkness. Some
cells remained alive for a month in lighted cultures but died soon in
darkness. Haberlandt noted several changes in cell structure during the
culture period. Cells expanded in length and girth. and cell walls
thickened. Plastids remained green in light, photosynthesized, and
accumulated starch. However, no cells were observed to divide. He then
attempted to culture cells from other species: photosynthetic cells from
Eichhorina crassipes, glandular hairs of Pulmonaria mollissima, stinging
hairs of Urtica dioica, staminal filament hairs of Tradescantia virginica,
and stomatal cells of Ornithogalum umbellatum, Erythronium denscanis, and
Fuschia magellanica with equal lack of success (reviewed in Krikorian and
Berquam, 1969).

In retrospect, Haberlandt's failure to obtain dividing cells can be
attributed to lack of microbial sterility, culture media that lacked
hormones and growth factors that were unknown at that time, and his
selection of highly differentiated mature cells. However, he made immense
contributions to plant and animal cell culture studies by his technical
innovations, including the use of hanging drop culture methods and use of
micropipettes to manipulate single cells. Similarly his prediction that
cocultivation of vegetative cells with pollen tubes, that were then known to
produce chemical stimuli that induced growth of orchid ovules, foreshadowed
nurse culture technology, and his prediction that embryo sac fluids might be
used as components of the culture medium to induce divisions in isolated
vegetative cells foreshadowed the use of coconut milk. Each of these
predictions has led to advances in cell culture technology.

Despite continued efforts by Haberlandt's collaborators and others, no
significant progress on cultures derived from single cells was made for 56
years when W.H. Muir, Albert Hildebrandt, and Albert Riker at the University
of Wisconsin investigated this question (Muir et al., 1958). They used
tissue cultures of tobacco and carrot and crown gall cultures of grape,
marigold, periwinkle, and sunflower. By testing the capacity for growth in
liquid culture media, they identified several that produced large numbers of
single cells. Single cells were then transferred by micropipette to filter
paper placed on nurse cultures of the same or other species growing on an
agar medium. Those of tobacco, marigold, sunflower, and grape divided and
produced macroscopic cultures, some of which were transferred through 25 or
more agar subcultures without diminution of growth rate. Marigold clones
consistently produced roots, and hybrid tobacco clones produced shoots on
media containing adenine and kinetin.

Subsequently, Vimla Vasil and Hildebrandt (Vasil and Hildebrandt, 1965a,
1965b) transferred single tobacco hybrid cells to a drop of culture medium
on a microscope slide that could be observed and photographed repeatedly
under phase contrast microscopy. Cells were observed to divide to form a
filament and subsequently a microcallus mass that was transferred to an agar
medium for further growth, where roots and leafy shoots were differentiated.
Rooted shoots were transferred to soil where they produced buds and flowers.
Thus, these studies demonstrated that plantlets derived from single cultured
cells had the capacity to produce whole plants. However, they did not prove
that the whole plants were the direct product of a single cell, rather than
the product of a tissue mass within which somaclonal or other genetic
changes might have taken place during growth to produce a chimeric tissue
mass.

The final step proving Haberlandt's prediction that "one could successfully
cultivate artificial embryos from vegetative cells" came from the research
of Dietlinde Backs-Husemann and Jakob Reinert in Berlin. They cultured
single carrot cells from suspension cultures on microscope slides where they
could be observed and photographed repeatedly. Isolated cells divided to
form a mass of embryogenic and parenchyma cells, and the embryogenic cells
developed into heart-shaped and torpedoshaped embryos with recognizable
cotyledons, hypocotyls, and radicles (Backs-Husemann and Reinert, 1970).

This early research in plant tissue culture demonstrated that tissues
isolated from plants can be grown in culture for indefinite periods of time,
they can produce shoots and roots, and finally, single isolated cells in
culture can produce embryos. These studies provided the platform for genetic
transformation of plants, as described below.

CROWN GALL DISEASE

In 1907, Erwin Smith, working at the USDA Bureau of Plant Industry on
diseases of plants, reported that the cause of crown gall disease of Paris
Daisy (Chrysanthemum frutescens) was a bacterium that he named Bacterium
tumefaciens (Smith and Townsend, 1907). This was subsequently reclassified
as Phytomonas tumefaciens and then as Agrobacterium tumefaciens (Conn,
1942). Smith established that this bacterium was the cause of the disease by
plating bacteria from galls onto an agar medium, inoculating uninfected
plants with subcultures of the bacteria, reisolating bacteria from the galls
produced, and inducing galls on new plants (Koch's postulates). Tumors were
also produced on stems of tobacco, tomato, and potato and on roots of sugar
beet and peach trees that were inoculated with B. tumefaciens (Smith and
Townsend, 1907). The latter galls closely resembled peach crown gall disease
on which Smith had been working for several years without identifying the
cause. In a subsequent publication, Smith et al. (1911) reported that the
bacterium isolated from Paris Daisy caused tumors on 24 dicot species but
not on nine other dicots or the single monocot (Allium cepa) that they
tested.

Smith also observed secondary tumors that developed on stems or leaves of
infected plants of some species at some distance from the primary gall.
Based on histological examination, he concluded that secondary tumors
developed from tumor strands that were root-like outgrowths from the primary
gall (Smith et al., 1912). Smith believed that tumor strands might be
comparable to certain types of metastases that occurred in malignant tumors
of animals and humans (Smith, 1916).

In addition to establishing the cause of crown gall disease, Smith and
Townsend (1907) suggested that their results might shed light on the origin
of cancerous growths in animals. Smith frequently alluded to the
similarities between plant and animal tumors (Smith, 1916) for which he
received a Certificate of Honor from the American Medical Association in
1913, and in 1925, he was elected to the presidency of the American
Association for Cancer Research.

However, little progress was made on the nature of crown gall disease until
Armin Braun at the Rockefeller Institute for Medical Research, began an
investigation of crown gall disease in the 1940s that lasted for 40 years
and that laid the foundation for the molecular studies that were to come. He
began this investigation by examining the question of tumor strand
connections between primary and secondary tumors in sunflower. By examining
tissue sections cut between the primary and secondary tumors, he found no
histological support for a tumor strand connection and concluded that the
mechanism of formation of secondary tumors may not be identical to that
concerned in the formation of the primary tumor (Braun, 1941). Although
crown gall tissues did not always yield cultures of the inducing bacterium,
it had been assumed that the bacteria had been present at some stage in the
development of the tumor. Thus, the discovery that secondary tumors lacked
direct cellular continuity with the primary tumor raised questions as to
their origin and development, some of which are still unresolved. Braun
collaborated with Philip White of the same institution to investigate these
questions. Specifically, they set out to investigate whether host cells
under the influence of the bacteria acquire the capacity for autonomous
growth. First, they induced secondary tumors in sunflower and showed by
culturing tissue from these tumors that those separated by more than one
internode from the primary gall were free of crown gall bacteria. Next, they
cultured tissue from secondary tumors for 30 subcultures on a basal medium
known to support growth of Phytomonas (Agrobacterium). Secondary
tumor-derived tissue grew rapidly, whereas tissue from noninfected plants
grew very slowly. They did not observe growth of Phytomonas in any of these
subcultures. To test for the presence of Phytomonas contained within the
cultured secondary tumor tissues, they tested these serologically with
negative results. Secondary tumor tissue when grafted onto healthy sunflower
or artichoke (Helianthus tuberosus) plants produced typical tumors. From
these experiments they concluded that the secondary tumor tissue had
acquired the capacity for autonomous growth both in vivo and in vitro and
that this permanent change had been induced by the bacteria (White and
Braun, 1941, 1942).

The finding that the plant cells had been permanently altered after
interaction with the bacteria led Braun to begin experiments to determine
the time when this interaction took place. These experiments depended on
observations that Phytomonas was killed at a temperature of 46[degrees]C,
whereas periwinkle plants (Vinca rosea) tolerated this temperature for
several days. Plants were inoculated with bacteria via needle punctures to
the stem, placed at 25[degrees]C for varying periods, then transferred to 46
to 47[degrees]C for 5 d and returned to 25[degrees]C for continued growth.
Plants transferred to the heat treatment 1 d after inoculation failed to
form tumors when returned to 25[degrees]C. Those transferred after 1.5 to 3
d produced small tumors, and those transferred after 4 to 5 d produced
tumors comparable to those on control plants that had not been subjected to
the heat treatment. These large tumors were bacteria free. Thus, the
plant-bacteria interaction must have occurred between 1 and 4 d, after which
time tumor growth became autonomous and independent of the continued
presence of bacteria (Braun, 1943). Additional temperature shift experiments
narrowed the time of maximal tumor formation to 24 to 48 h after wounding in
plants maintained at 25[degrees]C. These experiments suggested that an
active principle resulting from the plant-bacterium interaction was
responsible for the transformation, and Braun suggested four possible
categories for it: (1) a metabolic product of the crown-gall bacterium; (2)
a host constituent converted by the bacterium to a tumor-inducing substance;
(3) a chemical fraction of the bacterial cell that is capable of initiating
in the host cell a permanent developmental alteration; and (4) a viral or
other agent present in the crown-gall organism (Braun, 1947). This active
principle whose chemical nature was still unknown was named the tumor
inducing principle (TIP) (Braun and Mandle, 1948).

Initial understanding of the biochemical nature of the TIP came from
research conducted by Georges Morel at the Centre National de Recherches
Agronomiques, Versailles, France, who was studying amino acid changes during
tuber development in Jerusalem artichoke (Solanum tuberosum), paying
particular attention to arginine metabolism (Duranton and Morel, 1958). When
they compared tuber tissue cultures with crown gall cultures of this
species, they found that the latter contained a conjugate of arginine with
pyruvate that had previously been identified from octopus muscle and named
octopine (Menage and Morel, 1964). Subsequently, when investigating crown
gall cultures of the cactus Opuntia vulgaris, they discovered another
arginine derivative, a conjugate with a-ketoglutarate, which they named
nopaline, from Nopal, the French common name for Opuntia (Goldmann et al.,
1969). They performed a systematic study of these arginine derivatives,
named guanidines or opines, in crown gall cultures induced by 43 different
strains of A. tumefaciens and showed that with few exceptions they contained
either nopaline or octopine and that the opine produced was specific to the
inducing bacterial strain (Petit et al., 1970). Opines could not be isolated
from normal, habituated, or genetic tumor tissue cultures of the same
species. Since the opinespecifying information was proposed to move with the
TIP from the bacterial cell to the plant cell where it was maintained in a
functional state in bacteria-free crown gall tissue cultures, it represented
a useful marker for the transformed state.

Further information on the nature of the transforming agent TIP came from
two studies. First, Alan Kerr working at the Waite Agricultural Research
Institute in South Australia found that tomato plants inoculated with
Agrobacterium sp, a virulent strain, became contaminated with water-splashed
soil containing A. radiobacter, an avirulent strain. Approximately 50% of
the A. radiobacter contaminants were then found to be virulent and
indistinguishable from A. tumefaciens. Kerr (1969) concluded that this was
"the first unequivocal evidence for transfer of virulence from a plant
pathogenic to a saprophytic species of bacterium" and suggested that this
may have resulted from DNA transfer. Second, Hamilton and Fall (1971)
working at Pennsylvania State University found that the tumor- initiating
ability of A. tumefaciens was lost after incubation at high temperatures.
They incubated cultures of A. tumefaciens C-58, an extremely virulent
strain, at 36[degrees]C for different lengths of time and found a
progressive decline in the number of virulent colonies, so that after 120 h
at 36[degrees]C no virulent colonies could be recovered. They concluded that
this result could be explained by a loss of the virulence factor (Hamilton
and Fall, 1971).

Suggestions that crown gall transformation resulted from transfer of genetic
material from the bacterium to plant cells generated several studies to
investigate this possibility. However, these studies, based on filter or
solution hybridization using the whole Agrobacterium genome, yielded either
negative results or results that could not be substantiated (Chilton, 2001).

The resolution of these diverse conflicting results came from a study by Ivo
Zaenen working with Marc Van Montagu and Jeff Schell at the University of
Ghent, Belgium. Alkaline or neutral lysis of Agrobacterium B6-S3 cells
followed by sucrose density gradient centrifugation or dye-buoyant density
centrifugation and electron microscopy examination revealed the presence of
a large supercoiled circular plasmid in this crown gall-inducing bacterial
strain. The plasmid was present as one or a few copies per cell. Examination
of other crown gall-inducing Agrobacterium strains belonging to seven
different groups revealed the presence of plasmids of lengths comparable to
those in B6-S3 in all of them, whereas plasmids were not detected in eight
different nonpathogenic strains. They proposed the hypothesis that "the
tumor-inducing principle (Braun, 1947) in crown-gall inducing Agrobacterium
strains is carried by one or several large plasmids of various lengths"
(Zaenen et al., 1974). Confirmation of this hypothesis came within a year
when three reports of the essentiality of the A. tumefaciens plasmid for
crown gall induction were published (Van Larebeke et al., 1974, 1975; Watson
et al., 1975).

These results stimulated further attempts to demonstrate the presence of
whole plasmid genomes in plant tumor cells, but these were not successful
and raised the possibility that only part of the plasmid genome was
transferred to the plant. This was demonstrated soon after by Mary-Dell
Chilton and her colleagues Milton Gordon and Eugene Nester at the University
of Washington, Seattle. They digested radioactively labeled plasmid DNA from
A. tumefaciens strain A277 with the restriction endonuclease SmaI and
electrophoresed the digest. Nineteen bands were resolved, the 17 largest of
which were then hybridized to tobacco tumor DNA or to control DNAs.
Renaturation kinetics indicated that bands 3B and 10 showed homology to
tumor DNA (Chilton et al., 1977), indicating that only part of the plasmid
DNA was present in the tumor DNA. By 1978, terminology for the plasmid,
called Ti plasmid, and the transferred DNA, called T-DNA, had become
established (Chilton et al., 1978; Depicker et al., 1978). The experiments
of Chilton et al. (1977) did not address questions of the location of the
plasmid DNA in the plant cells, whether covalently integrated, or located in
the nuclear or plastid genomes.

Three years later, research groups in Washington and Europe (Belgium and
Germany) reported that Ti plasmid DNA was present in the crown gall cell
nucleus and not in plastids or mitochondria (Chilton et al., 1980;
Willmitzer et al., 1980). Whether the DNA was integrated into plant
chromosomes or functioned as an independent replicon was not yet
established. However, almost simultaneously with these reports two studies
showed by molecular cloning and sequencing of a border fragment of T-DNA and
flanking plant DNA that the T-DNA was covalently integrated into the plant
nuclear genome in tobacco teratoma cell lines (Yadav et al., 1980; Zambryski
et al., 1980). The next step in elucidating the role of T-DNA in plant
tumorigenesis was to determine the function of the genes that were presumed
to be located in it. This was done by disrupting T-DNA genes by insertion of
foreign DNA or by deletion of T-DNA sequences. The first studies using this
approach were those of Peter Klapwijk and Gert Ooms in the lab of Robbert
Schilperoort in Leiden (Klapwijk et al., 1980; Ooms et al., 1981). A Ti
plasmid insertion mutant containing a Tn904 transposon in the center of the
T-DNA region induced tumors on tobacco plants that gave rise to roots.
Insertion of an IS element, IS60, encoding streptomycin resistance into the
left arm of the T region of an octopine Ti plasmid gave rise to tumors that
produced shoots. Shoots were also produced on in vitro- cultured tissue of
the IS60 mutant tumors, and Ooms concluded that "the tumor phenotype should
depend on relative concentrations of various phytohormones present in a
tumor" (Ooms et al., 1981).

At about the same time, David Garfinkel and collaborators working with Milt
Gordon and Gene Nester made a detailed genetic analysis of the T-DNA of the
octopine plasmid pTiA6NC using Tn3 and Tn5 transposon inserts. Twenty-five
insertions defined three distinct loci affecting tumor morphology: tms, tmr,
and tml, mutations in which caused shooty tumors, rooty tumors, and
abnormally large tumors, respectively. These three loci were all located in
the core T-DNA segment present in all octopine-type tumors and were found to
encode enzymes involved in auxin or cytokinin biosynthesis. tms1 codes for
tryptophan monooxygenase, and tms2 codes for indoleacetamide hydrolase, both
of which specify steps in auxin (IAA) biosynthesis (Klee et al.,1984), and
tmr codes for isopentenyl transferase, which catalyzes the first step in
cytokinin biosynthesis (Akiyoshi et al., 1984; Barry et al., 1984). A fourth
locus, ocs, was located outside this core region and codes for octopine
synthase (Garfinkel et al., 1981).

Using a similar approach of DNA deletions or transposon insertions, genes
that were present in a common core of both octopine and nopaline tumor T-DNA
were identified by Willmitzer et al. (1983) and Joos et al. (1983), working
with Marc Van Montagu and Jeff Schell. Two of these genes inhibited shoot
formation and ensured tumorous growth. The third gene inhibited root
formation. Mutants missing all three genes did not induce tumors, nor shoot
or root formation, although the mutant T-DNA was transferred to plant cells.
This last fact supported the earlier conjecture of Garfinkel et al. (1981)
for the "eventual use of the Ti plasmid as a vehicle for introducing genes
of choice into the genomes of higher plants." Plasmids lacking all oncogenic
genes would be of use for such introductions and were said to be "disarmed"
(Binns, 2002).

If the Ti plasmid was to be the vehicle for introduction of genes of choice
into plant genomes, three molecular requirements had to be satisfied. First,
a promoter that functioned in plant cells had to be identified; second, a
dominant selectable marker that indicated the functioning presence of the
introduced DNA and that would replace the opine synthesis locus (which was a
screenable, but not selectable, marker); and finally, polyadenylation
termination signals that would function in plant cells. Surprisingly, at the
Miami Winter Symposium, January 1983, three research groups (Jeff Schell,
Rob Horsch, and Mary-Dell Chilton) all reported success in producing
chimeric genes that satisfied these criteria and that functioned in
transformed plant cells (Downey et al., 1983). All three groups used the NOS
(nopaline synthetase) promoter spliced to the bacterial NPT II (neomycin
phosphotransferase) coding sequence as a dominant selectable marker and NOS
polyadenylation signals or a variation of this strategy. The first
peer-reviewed publication reporting similar results was that of
Herrera-Estrella et al. (1983a). The technological innovation that underlay
these reports was the development of binary plant vector systems in which
the vir region (virulence region) and the T-region of the A. tumefaciens Ti
plasmid were located on different plasmids. With this approach, the T-DNA,
located on a wide host-range replicon, could be easily genetically
manipulated and modified in Escherichia coli then reintroduced into A.
tumefaciens cells that harbored the vir- containing plasmid (Hoekema et al.,
1983).

Using this binary vector approach, Herrera-Estrella et al. (1983b), working
with Marc Van Montagu and Jeff Schell, used the NOS promoter sequence and
the dominant selectable markers APH(3#)II of Tn5 or DHFR Mtx of the R67
plasmid. APH(3#)II inactivates a number of aminoglycoside antibiotics, such
as kanamycin, neomycin, and G418. Kanamycin, G418, and methotrexate are very
toxic to plant cells and thus function as selectable markers for transformed
cells. The chimeric genes transferred to tobacco cells via the Ti plasmid as
a vector were expressed and conferred resistance to the antibiotics. Fraley
et al. (1983) at Monsanto produced chimeric genes containing the NOS
promoter and regulatory regions linked to NPT type I or II from bacterial
transposons Tn5 or Tn601. Cocultivation of A. tumefaciens containing these
chimeric genes with cells derived from protoplasts of petunia, tobacco,
sunflower, and carrot resulted in transformed cells that expressed the
chimeric genes and were resistant to inhibitory levels of the antibiotic.
Mike Bevan, Richard Flavell, and Mary-Dell Chilton (Bevan et al., 1983)
combined the NOS promoter and regulatory sequences with NPTII from
transposon Tn5 to transform sterile stem explants of tobacco. The successes
of these three research groups heralded the way for the genetic
transformation of crop plants.

However, it remained to be shown that such transformed cells could be
regenerated into intact normal plants and that the inserted DNA would be
inherited in subsequent generations in a stable manner. Proof of these
requirements came quickly in several reports. Ken Barton and colleagues,
working with Mary-Dell Chilton, regenerated tobacco plants that contained
fulllength copies of genetically engineered T-DNA that were transmitted to
the R1 progeny (Barton et al., 1983). Patty Zambryski, working with Marc Van
Montagu and Jeff Schell, inoculated decapitated tobacco plants and
regenerated transformed plants from callus that developed at the inoculation
sites (Zambryski et al., 1983). Finally, Marc De Block, working with Luis
Herrera-Estrella, Marc Van Montagu, Jeff Schell, and Patty Zambryski,
infected single protoplasts of tobacco with Agrobacterium- containing
chimeric genes and regenerated plants from the resulting calli (De Block et
al., 1984). These plants developed normally, flowered, and set seed. F1
seedlings when grown on an antibiotic- containing medium segregated in a
Mendelian manner. These three studies all reported successful production of
transformed tobacco plants from transformed cells.

The broader application of gene transfer into plants was reported by Rob
Horsch and colleagues of Monsanto, who transformed tobacco, petunia, and
tomato (several cultivars) with genetically engineered T-DNA (Horsch et al.,
1985). They cocultivated A.tumefaciens containing a plasmid-encoded
NOS/NPTII/NOS chimeric gene with leaf discs of these species. The leaf discs
were subsequently transferred to callus-inducing medium containing
carbenicillin and kanamycin. Shoots that developed from the callus were
rooted and transferred to soil for further growth. F1 generation plants of
all species expressed kanamycin resistance in a simple Mendelian fashion.
This fact suggests that the regenerated shoots had originated from single
transformed cells and were not chimeric in origin.

The recalcitrance of some species, especially monocotyledons, to
transformation by Agrobacterium led to searches for other methods for
introducing DNA into plant cells. These included electroporation,
microinjection, floral dipping, and particle bombardment/biolistics (Pen a,
2005). The most successful and widely used of such methods is particle
bombardment by the gene gun, developed by John Sanford and colleagues at
Cornell University in 1984 (Klein et al., 1987; Sanford, 2000). This was the
method used in the first successful transformation of maize cells and the
regeneration of fertile transgenic plants from them that transmitted the
introduced genes to the R1 generation (Gordon-Kamm et al., 1990).

These workers at DEKALB Plant Genetics (now Monsanto) bombarded cells from
maize embryogenic suspension cultures with tungsten particles coated with
plasmids containing the selectable marker gene bar. This gene confers
resistance to the herbicide bialaphos, which was used to select transformed
callus cells. Transformed calli were shown to contain the integrated bar
gene and to express the enzyme phosphinothricin acetyltransferase encoded by
bar. Fertile transformed plants were produced from the calli, and of 53
progeny tested, 29 had phosphinothricin acetyltransferase activity. In other
experiments, they cotransformed embryogenic suspension culture cells with a
mixture of two plasmids, one containing the bar gene and the other
containing the gene encoding b-glucuronidase. Regenerated plants expressed
both genes. The authors concluded that "this system provides a new, powerful
tool for both the study of basic plant biology and the introduction of
important agronomic traits into one of the world's major crops" (Gordon-Kamm
et al., 1990).

CONCLUDING REMARKS

Modern plant biotechnology, defined as the genetic modification of plants,
resulted from a century-long combination of basic research findings and
technological innovations. The basic scientific findings that underlay this
include in vitro tissue culture, auxin/cytokinin regulation of
organogenesis, single cell culture, discovery of cellular totipotency, the
bacterial cause of crown gall disease, the TIP, opines as markers of
transformed cells, transfer of virulence between Agrobacterium strains,
T-DNA, the genes that determine tumor morphology (tms1, tms2, and tmr),
disarmed plasmids, and regeneration of transformed cells. The technological
innovations include aseptic tissue/cell culture, hanging drop culture,
micropipettes, nurse cultures, binary plant vectors, and gene gun
transformation. Of course, some basic discoveries and technological
innovations were adopted from other disciplines, such as plant culture
medium requirements and plasmid genetic manipulation in E. coli, but many
originated as the research that led to the genetic transformation of plants.
A remarkable feature of this research is the changing aspect of publication
frequency and author contribution. From 1900 to 1949, 20 articles that are
here regarded as crucial were published by 29 authors. This is a frequency
of 0.4 articles per year and 1.5 authors per article. From 1950 to 1969, 13
articles were published by 30 authors with a frequency of 0.7 articles per
year and 2.3 authors per article. From 1970 to 1990, 34 articles were
published by 185 authors with a frequency of 1.7 articles per year and 5.4
authors per article. These numbers reflect the changing pace of plant
biology and the increasing attractiveness of it as a scientific career where
large laboratory groups have come to characterize the field.

This story emphasizes the relationship between basic scientific research and
technological developments and the necessity for both. In retrospect, we can
trace the sequence of research that ultimately led to the genetic
modification of plants, but could we have predicted it? Obviously not in
1902, 1907, or 1939, when the three founding articles in crown gall disease
causation, single plant cell culture, and plant tissue culture,
respectively, were published. It was not until the early 1980s that research
articles were predicting the use of the Ti plasmid as a vehicle for transfer
of genes of choice into plants.

POSTSCRIPT

The first transgenic food crop to be commercialized was Flavr Savr, a
delayed ripening tomato, in 1994 (Martineau, 2001). In 2006, transgenic
crops were planted on 102 million hectares (252 million acres) in 22
countries (11 industrial countries and 11 developing countries) by 10.3
million farmers: 9.3 million of these farmers were resource-poor with small
farms in developing countries. Soybean was the principal transgenic crop in
2006, occupying 58.6 million hectares, followed by maize (25.2 million
hectares), cotton (13.4 million hectares), and canola (4.8 million
hectares).

The first field trials of transgenic crops were conducted in 1986 to test
herbicide tolerance in tobacco. By 2005, 3647 field trials had been
conducted at .15,000 sites in 34 countries on 56 crop species. The eight
most frequently tested species were maize, canola, potato, tomato, tobacco,
soybean, cotton, and melon (James, 2006). In 2007, it was estimated that
;140 species of angiosperms had been genetically transformed (James, 2007).

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