Plant Cell Rep (2008) 27:1423–1440
DOI 10.1007/s00299-008-0571-4
REVIEW
A history of plant biotechnology: from the Cell Theory
of Schleiden and Schwann to biotech crops
Indra K. Vasil
Received: 31 March 2008 / Revised: 28 May 2008 / Accepted: 10 June 2008 / Published online: 9 July 2008
Springer-Verlag 2008
Abstract Plant biotechnology is founded on the principles
of cellular totipotency and genetic transformation, which can
be traced back to the Cell Theory of Matthias Jakob Schleiden and Theodor Schwann, and the discovery of genetic
transformation in bacteria by Frederick Griffith, respectively. On the 25th anniversary of the genetic transformation
of plants, this review provides a historical account of the
evolution of the theoretical concepts and experimental
strategies that led to the production and commercialization of
biotech (transformed or transgenic) plants expressing many
useful genes, and emphasizes the beneficial effects of plant
biotechnology on food security, human health, the environment, and conservation of biodiversity. In so doing, it
celebrates and pays tribute to the contributions of scores of
scientists who laid the foundation of modern plant biotechnology by their bold and unconventional thinking and
experimentation. It highlights also the many important lessons to be learnt from the fascinating history of plant
biotechnology, the significance of history in science teaching
and research, and warns against the danger of the growing
trends of ignoring history and historical illiteracy.
Keywords Biotech crops Cell Theory Genetic transformation History of science Adapted from the Opening Plenary Address to the international
conference on ‘‘Plants for Human Health in the Post-Genome Era’’,
held 26–29 August 2007, at Helsinki, Finland.
Communicated by P. Kumar.
I. K. Vasil (&)
University of Florida, Box 110690,
Gainesville, FL 32611-0690, USA
e-mail: [email protected]
Plant biotechnology Plant regeneration Somatic embryogenesis Totipotency Transgenic crops
The further backward you look, the further forward
you can see (Winston Churchill).
Know where you come from, to get where you are
going (Jhumpa Lahiri).
Introduction
Eighteenth January 1983, is arguably one of the most
important dates in the history of plant biotechnology.
Indeed, history was made that day 25 years ago, at the
Miami Winter Symposium, where three independent
groups described Agrobacterium tumefaciens-mediated
genetic transformation, leading to the production of normal, fertile transgenic plants. Although each group had
introduced a bacterial antibiotic resistance gene into
tobacco, a model plant for studies on in vitro regeneration,
there was widespread belief that the new technology would
allow introduction of agronomically important genes into
crop species that are susceptible to agrobacterial infection.
Soon, scores of academic as well as corporate research
groups took up this challenge, along with the difficult task
of developing the necessary technologies for the genetic
transformation of the economically important cereal crops
that at the time were considered to be outside the host
range of Agrobacterium. It is a testament to the ingenuity
of the plant biotechnology community that within the next
decade all major crop species had been transformed with
genes that conferred resistance to non-selective herbicides,
and some pests and pathogens. In the mean time, in
response to concerns about the safety and environmental
impact of biotech (transgenic) crops, elaborate and
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stringent mandatory regulations were developed in the
United States and some other countries to monitor and
regulate the cultivation and use of biotech crops and
products. But plant biotechnology came of age only in
1996, with the planting of nearly five million acres of
biotech crops, mostly in the United States. Thereafter, the
global acreage of biotech crops increased rapidly—at an
annual rate in excess of 10%—so that in 2007 more than
282 million acres (nearly 8% of total world crop acreage)
were planted in 23 countries, for a cumulative (1996–2007)
total acreage of over 1.7 billion acres (James 2007).
For the purposes of this review, plant biotechnology is
defined rather narrowly to include only biotech plants. It
provides a historical perspective of the evolution of a
variety of novel ideas and technologies that are now routinely used in the regeneration and genetic transformation
of plants, and celebrates the contributions of many men and
women whose seminal contributions laid the foundation of
modern plant biotechnology. It was my good fortune to
have known most of the founding figures of plant biotechnology in the twentieth century—from Philip R. White,
Roger J. Gautheret, Kenneth V. Thimann, Armin C. Braun,
George Morel, Frederick C. Steward, Jacob Reinert, Albert
C. Hildebrandt, and Folke K. Skoog, to Jeff Schell and
many others—whose work is chronicled in these pages
(some of these associations are described in an earlier
autobiographical article; Vasil 2002). I am indebted
particularly to Ken Thimann, Phillip White and Al Hildebrandt for their wise counsel, encouragement and support
early in my career, and to Jeff Schell for being one of the
first to believe in, and publicly speak in support of, our
work on embryogenic cultures which continue to be
essential for the production of biotech grasses and cereal
crops. In providing this historical account of the science as
well as the personalities of plant biotechnology, and the
many debates and controversies, the review highlights the
value of historical studies in science education and
research.
The Cell Theory
The theoretical framework and experimental basis of
modern plant biotechnology derive from the concepts of
cellular totipotency (the ability of a single cell to divide
and produce a whole plant) and genetic transformation
(genetic alteration caused by the uptake, stable integration
and expression of foreign genetic material). The concept of
totipotency is inherent in the Cell Theory of Schleiden
(1838) and Schwann (1839), which recognized the cell as
the primary unit—elementary part—of all living organisms. Genetic transformation was first reported by Griffith
(1928) in bacteria, but the genetic basis of the transforming
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factor (DNA) was not elucidated until much later (Avery
et al. 1944).
Matthias Jakob Schleiden was born in 1804 in Hamburg
(Germany). He was trained as a lawyer, but gave up the
practice of law to study medicine at Göttingen. There he
became interested in the study of plants. He continued his
botanical studies in Berlin before assuming the post of
professor of botany at the University of Jena. He was the
first prominent botanist to shift the focus from taxonomic
to structural studies of plants. Theodor Schwann was born
in 1810 in Neuss (Germany). After spending 4 years under
the influence of J.P. Müller in Berlin, he became professor
of physiology at the University of Louvain (Belgium). In
addition to many other notable studies, Schleiden and
Schwann made extensive use of the microscope to study
plants and animals. They had known each other while
studying in Berlin and were friends. In 1838, while they
were enjoying after-dinner coffee, Schleiden talked excitedly about the universality of plant cells. All of the species
that he had examined seemed to be made up of recognizable units or cells. He expressed his belief that the growth
of plants depended on the production of new cells. Schwann was immediately struck by the similarity of
Schleiden’s observations to his own studies of animals.
After dinner they retreated to Schwann’s laboratory to look
at his microscopic preparations. They published their
results a year apart—Schleiden (1838), and Schwann
(1839)—without any reference or acknowledgement of
each other’s contribution (an unfortunate and unacceptable
practice that is still a part of scientific publishing). Their
respective observations on plants and animals, when taken
together, formed the basis for a unified Cell Theory which
applied universally to all living organisms. One can get a
sense of their boldness and originality, and that of Virchow
(1858), from their unambiguous and insightful observations (Box 1).
The Cell Theory went unnoticed and unappreciated for
nearly two decades. During this time Schleiden, who was a
powerful and domineering figure, became embroiled in a
contentious scientific controversy and debate. Some years
earlier, Giovanni Battista Amici (1824), a young Italian
mathematician and astronomer, had made the chance discovery of the pollen tube while examining the stigmas of
Portulaca oleracea with a crudely constructed microscope.
He later noted that the pollen tubes grow/elongate through
the style and eventually enter the ovary and the ovule
(Amici 1830). Schleiden (1837) also studied the subject
extensively and in general confirmed Amici’s observations.
However, he went beyond the scope of his observations
and carelessly proposed that the tip of the pollen tube—
after entering the embryo sac—directly becomes the
embryonal vesicle which undergoes a number of divisions
to form the embryo. According to him, therefore, the
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Box 1
Schleiden (1838): every plant and animal is ‘‘an aggregate of fully individualized, independent, separate beings’’, that is, cells.
Schwann (1839): ‘‘Some of these elementary parts (cells) which do not differ from others, are capable of being separated from the organism and
continuing to grow independently. Hence we can conclude that each of the other elementary parts, each cell, must possess the capacity …to
grow, and that therefore each cell possesses …an independent life, as a result of which it too would be capable of developing independently, if
only there be provided the external conditions under which it exists in the organism. We must therefore ascribe an independent life to the cell.
That not every cell grows, when separated from the organism, is no more an argument against this theory than is the fact that a bee soon dies
when separated from its swarm a valid argument against the individual life of the bee’’
Virchow (1858): ‘‘Where a cell arises, there must have been a cell before, even as an animal can come from nothing but an animal, a plant from
nothing but a plant…nor can any developed tissue be traced back to anything but a cell’’
embryo sac was nothing more than an incubator for the
embryo that developed from the pollen tube. The debate
between Amici (1844) and Schleiden (1845), and their
respective supporters, was quite acrimonious and continued
for many years. In the mean time, in response to the controversy, the Imperial Institute of the Netherlands had
announced that it will award a prize to anyone who can
conclusively resolve the question of the origin of the
embryo. After examining all of the relevant evidence, it
awarded the prize to H. Schacht (1850), one of Schleiden’s
most ardent supporters, for his thesis that was in agreement
with Schleiden’s view of the embryo arising directly from
the pollen tube. Fortunately, the definitive studies of Amici
(1847), as well as those of Wilhelm Hofmeister (1849),
soon showed the fallacy of Schleiden’s arguments and
Schacht’s observations, by providing unequivocal evidence
that indeed the embryo originated from a preexisting cell in
the embryo sac and not from the pollen tube. Ultimately,
the mounting evidence against Schleiden’s views provided
by many investigators became so overwhelming that he
was forced to retract his opinions. He became depressed,
soon gave up all botanical work, and retreated to Dresden
as a private teacher of history and philosophy. The final
blow to Schleiden’s ideas was the discovery of syngamy
(fertilization) by Edward Strasberger (1884), and double
fertilization by Nawaschin (1898). The vigorous debate
about the origin of the embryo, which involved many
prominent individuals for many years, is a powerful illustration of the bedrock principle of science that what matters
in the end is not who you are or how powerful you are, or
how many prizes you have received or supporters you
have, but rather how good and reproducible your science is.
Interest in the Cell Theory was revived in 1858 by the
famous aphorism of Ludwig Karl Virchow: ‘‘Omnis cellula
a cellula’’ (all cells arise from cells). Virchow was a
pathologist at the Charité Hospital in Berlin (still considered one of the leading teaching hospitals in Europe). He
determined that diseased cells always arose from healthy
cells, and rejected the prevailing view of spontaneous
generation. He is rightfully acknowledged as the founder of
the modern science of pathology. Virchow was a manyfaceted individual. He is recognized for his many important
contributions to medicine, social medicine, anthropology
and politics, and his service as a member of the Bundestag
(parliament) in Germany.
Although the concept of totipotency is evident in the
writings of Schleiden, Schwann and Virchow (Box 1), it
was Herman Vöchting (1878), who attempted to demonstrate totipotency experimentally by dissecting and
growing smaller and smaller fragments of plant tissues. He
concluded that ‘‘In every fragment, be it ever so small, of
the organs of the plant body, rest the elements from which,
by isolating the fragment, under proper external conditions,
the whole body can be built up’’ (Vöchting 1878).
Totipotency of plant cells
No sustained or organized attempts were made to test the
validity of the concept of totipotency that is inherent in
the Cell Theory (see Box 1), until the beginning of the
twentieth century. The Austro-German botanist Gottlieb
Haberlandt was the first to try to obtain experimental
evidence of totipotency by culturing plant cells in nutrient solutions in the hope of regenerating whole plants.
He was born in 1854 in Ungarisch-Altenburg, Hungary.
After completing his studies at the University of Vienna
(Austria), he held professorships at universities in Graz
(Austria) and Berlin (Germany). He presented the results
of his pioneering experiments, in a prophetic and now
classic paper, entitled ‘‘Experiments on the culture of
isolated plant cells’’, before the Mathematics and Natural
Sciences Section of the Vienna Academy of Sciences in
Berlin, on 6 February 1902 (Haberlandt 1902; see
Krikorian and Berquam 1969; Laimer and Rücker 2002,
for English translation). He cultured chloroplast-containing differentiated cells from leaves of Lamium
purpureum, cells from the petioles of Eichhornia crassipes, glandular hairs of Pulmonaria and Urtica, and
stamen hairs of Tradescantia, in Knop’s (1865) salt
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solution in hanging drop cultures. The cells grew in size,
but did not undergo any cell divisions. Eventually, the
cultures were lost to infection. In hindsight, his results
are not surprising. He failed largely because of his
unfortunate choice of experimental material, and because
of the inadequacy of nutrition provided by Knop’s salt
solution. Even now, more than a century later, the culture
of isolated leaf cells, and other materials used by
Haberlandt, is either extremely difficult or impossible in
most species. Nevertheless, based on his experiences,
Haberlandt made some bold predictions. He advocated
the use of embryo sac fluids (coenocytic liquid endosperm, such as coconut milk that was later widely and
successfully used in tissue culture studies) for inducing
cell divisions in vegetative cells, and pointed to the
possibility of successfully cultivating artificial embryos
(i.e., somatic embryos, which are now the predominant
means of plant regeneration in a wide range of species;
see Thorpe 1995; Vasil 1999) from vegetative cells in
nutrient solutions (see Box 2). It is for these reasons that
Haberlandt is rightfully credited with being the founder
of the science of plant cell culture.
Improved nutrient solutions and unlimited growth
of plant tissues
The lack of availability of a nutrient solution that could
support the growth of isolated plant cells and tissues hindered further progress for many years. Molliard (1921) in
France, Kotte (1922) in Germany, and Robbins (1922) in
the United States, cultured fragments of embryos, and
excised roots, on Knop’s (1865) mineral solution. Some
growth was observed on this minimal medium, but none of
the cultures could be maintained for more than a few
weeks.
Philip Rodney White in the United States was the first
to obtain indefinite growth of cultured plant tissues. He
was working with the famed biochemist and virologist
Wendel Stanely at Rockefeller Institute in Princeton, NJ
(now the Rockefeller University, New York), and was
trying to cultivate viruses in plant tissues. He was able to
maintain excised root tips of tomato and other plants in
culture for indefinite periods of time, while also demonstrating multiplication of tobacco mosaic virus in the
cultured roots (White 1934a, b). He noted that in some
instances no virus could be detected in subcultured roots.
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This led to the discovery that root meristems were often
virus-free, and the application of this information many
years later by George Morel to obtain virus-free plants
from cultured shoot meristems (Morel and Martin 1952,
1955; Kartha 1984).
The development of improved nutrient solutions, use of
the newly discovered plant growth regulator indole-3-acetic acid (IAA), informed choice of plant material, and
appreciation of the importance of aseptic cultures, led to
sustained growth of root tips, and carrot root and tobacco
stem tissues, for indefinite periods of time (Gautheret 1934,
1939, 1985; Nobécourt 1939; White 1939). This important
milestone was achieved by Roger Gautheret and Pierre
Nobécourt in France, and Philip White in the United States,
within weeks of each other. However, with the beginning
of World War II, the European and American scientists
remained unaware of each other’s results for many years.
White’s original cultures were maintained for nearly four
decades, with regular subcultures, but without any evidence of changes in growth habits or nutritional
requirements. He often carried a flask in his jacket pocket
to meetings, and proudly showed it around to impress the
fact that his original root cultures were still going strong
after hundreds of subcultures.
White (1932) found Knop’s nutrient solution, as well as
the formulation used by Robbins (1922), to be unsatisfactory. This prompted an effort by many individuals to
develop nutrient solutions that could adequately support
the growth of isolated plant tissues. White developed a new
nutrient solution—the White’s medium (White 1943,
1963)—that was based on the formulation of Uspenski and
Uspenskaia (1925) for algae, and the microelements of
Trelease and Trelease (1933), and included glycine, nicotinic acid, thiamine and pyridoxine. It was the most widely
used nutrient solution for plant tissue cultures until the
1960s. Knop’s solution was used in Roger Gautheret’s
laboratory in France for a long time, until it was replaced
by a new formulation developed by one of his students,
René Heller (1953). Many scientists in Europe considered
it to be superior to White’s medium.
As the only son of a well-to-do businessman, Gautheret
could have led a very comfortable life without ever having
to work for a living. Yet, he chose a scientific career under
the guidance of the famed French cytologist Guillermond
(Gautheret 1985). After three frustrating years, he was
among the first, along with White and Nobécourt, to obtain
Box 2
‘‘To my knowledge, no systematically organized attempts to culture vegetative cells from higher plants in simple nutrient solutions have been
made. Yet the results of such culture experiments should give some interesting insight into the properties and potentialities which the cell as an
elementary organism possesses …I am not making too bold a prediction if I point to the possibility that, in this way, one should successfully
cultivate artificial embryos from vegetative cells’’ (Haberlandt 1902)
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unlimited growth of cultured plant tissues. His manuscripts
describing the historic results were rejected for publication
in the official journal of the French Academy of Sciences.
Ironically, he was later elected to the Academy, and for a
long time was its pre-eminent Academician in plant biology. For nearly four decades, his students and colleagues
dominated the field of plant cell culture in France. Unfortunately, all of his work was published in French, and
remained unknown for a long time in much of the Englishspeaking world. It was White—his competitor—who
introduced his work to a wider audience through citations
in his papers and books.
Philip Rodney White was born in Chicago, on 25 July
1901, one of twin sons. He obtained his Ph.D. from Johns
Hopkins University. He studied also in France, and worked
in Germany, apart from holding positions in Jamaica and
Panama. He spent much of his professional career at the
famed Roscoe B. Jackson Memorial Laboratory in remote
Bar Harbor, Maine, which was also a major center for
animal cell culture and genetics. It was during his stay in
Berlin in 1930–1931, that he met Haberlandt who challenged White to pursue plant tissue culture research, but
also warned him of the high possibility of failure and
disappointment. White had begun his work on plant tissue
culture as a means to grow viruses, but had quickly
expanded it to include the culture of roots, and stem and
other tissues. Forty years after, he died prematurely of
hepatitis, on 25 March 1968, in Bombay, while on an
extensive lecture tour in India, White is still a much revered figure in plant biotechnology. He is remembered and
honored for his significant and seminal scientific contributions to the science of plant tissue culture, for the
development of a widely used nutrient solution, and for his
influential books—the first of their kind—on the culture of
plant cells that have inspired and enabled a generation of
scientists to use cell culture techniques for physiological
and developmental studies of plants. He was an unassuming but outspoken individual with strong opinions, which
did not endear him to some of his contemporary colleagues. He was invariably kind to young scientists, always
encouraging and supporting them, and telling them that his
generation had done what it could, and now it was up to the
younger generation to take up the new challenges. White
was an internationalist in the true sense. He was convinced
that great advances in science can come only through
international understanding and cooperation. It was in this
spirit that he organized the highly successful Decennial
Review Conference in 1956, for the American Cancer
Society, while he was President of the Tissue Culture
Association (now the Society for In Vitro Biology); about
one-fourth of the program was devoted to plant tissue
cultures. He followed it by hosting the first truly international conference on plant cell and tissue culture, in 1963,
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at Pennsylvania State University, where he was a Visiting
Professor (White and Grove 1965). It was there, with his
encouragement and guidance, that the International Council of Plant Tissue Culture, now the International
Association for Plant Biotechnology (IAPB), was founded.
The IAPB is the largest international professional organization representing the interests of the plant biotechnology
community. It continues to meet every 4 years in different
parts of the world. It too is a part of his legacy. Published
proceedings of these conferences provide a continuous
historical record of trends and key developments in plant
biotechnology (see Vasil 2003b; Xu et al. 2007).
Arguably, the most detailed and systematic studies on
the nutrient requirements of cultured plant tissues were
carried out by Albert C. Hildebrandt at the University of
Wisconsin, in an attempt to develop an ideal nutrient
solution (Hildebrandt and Riker 1949, 1953; Hildebrandt
et al. 1946; Vasil and Hildebrandt 1966c; Ozias-Akins and
Vasil 1985). This resulted in the development of solutions
that contained greatly elevated levels of mineral salts, such
as the tobacco high salts medium (Vasil and Hildebrandt
1966c). In the mean time, Toshio Murashige, a graduate
student in the laboratory of Folke Skoog, also at the University of Wisconsin, was trying to obtain optimum and
predictable growth of cultured tobacco pith tissues which
Skoog needed for performing reliable bioassays of cytokinin activity. He found that the addition of an aqueous
extract of tobacco leaves to White’s medium resulted in
greater than fourfold increase in growth. This was determined to be caused largely by the inorganic constituents of
the leaf extract. Similar results were obtained when the ash
of tobacco leaf extracts, or large amounts of ammonium,
nitrate, phosphate and potassium salts were added to the
White’s medium. These results led to the formulation
of a new and completely defined nutrient solution, the
Murashige and Skoog (1962) or MS medium, which also
included chelated iron in order to make it more stable and
available during the life of the cultures, myo-inositol, and a
mixture of four vitamins. In a sense then, the MS medium
is reconstituted tobacco leaf extract. It is the most widely
used formulation for culture of plant tissues, and the publication describing the MS medium (Murashige and Skoog
1962) remains one of the most highly cited publications in
plant biology.
Discovery of plant growth substances
Charles Darwin (1880) was one of the first to observe and
describe the curvature of seedlings toward light. He discovered that this effect could be prevented by covering the
tip of the coleoptile. Boysen-Jensen (1910) found that the
complete removal of the tip abolished the effect of light,
and pointed to the possibility of a ‘‘material substance’’
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being responsible for the curvature. Frits Went (1928), at
Leiden in The Netherlands, successfully isolated and
quantified the amount of ‘‘material substance’’, which he
called ‘‘wuchstoff’’ (hormone), and coined the phrase
‘‘Ohne Wuchstoff, kein Wachstum’’ (without hormone, no
growth). The hormone, later to be identified as the naturally
occurring auxin, IAA, was first isolated in crystalline form
from the urine of pregnant women by Kögl (Kögl et al.
1934; Kögl and Kostermans 1934) in Copenhagen, Denmark, and from large scale cultures of the fungus, Rhizopus
suinus, by Thimann (1935) at the California Institute of
Technology (Caltech) in the United States. Kenneth Vivian
Thimann was born in 1904 in Ashford (Kent, England), and
studied for his graduate degree at Imperial College, University of London. Soon after, he accepted a position at
Caltech (he later worked at Harvard, and University of
California, Santa Cruz). Thimann, and his many students,
including James Bonner and Folke Skoog, continued their
studies on auxins and contributed greatly to our understanding of the structure/function of auxins, and the
development of a wide variety of synthetic auxins—such as
2,4-dichlorophenoxyacetic acid (2,4-D)—which are used
widely in the culture of plant tissues, and in agriculture.
In addition to auxins, the other naturally occurring plant
growth substances that have had a profound impact on the
culture of isolated plant cells are the cytokinins. They were
discovered by Folke Skoog and his colleagues at the University of Wisconsin. Skoog was born in 1908 in Halland
(Sweden), and immigrated to the United States during a
visit as a high school student to California, in 1925. He was
an avid sportsman, and had competed—finishing fourth—
in the 1,500-m race at the 1932 Olympics. He carried out
his graduate work with Thimann at Caltech, where he also
came under the influence of Frits Went and Johannes van
Overbeek, all distinguished pioneers in the discovery and
study of auxins.
Haberlandt (1913) had continued his attempts to grow
plant tissues in culture and had shown that phloem diffusates induced cell divisions in parenchyma tissue of potato
tubers. Crushed cells yielded a material that induced cell
division activity, but this effect was lost when the crushed
tissue was rinsed with water (Haberlandt 1921). Many years
later, Skoog was continuing his own studies on auxins, first
at Harvard with Thimann, and then at Wisconsin. During a
visit to White’s laboratory he had seen bud formation in
cultured tobacco tissues, and started his work with hybrid
tobacco tissue cultures that had been obtained from White
(1939) by Thimann. They were lost during the war and so he
started using tissues from Nicotiana tabacum cv Wisconsin
38. The initial success in obtaining unlimited growth of
plant tissues in culture by Gautheret, Nobécourt and White,
had been limited to the use of explants that contained
meristematic cells. Continued cell divisions and bud
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formation were soon obtained by Skoog and Tsui (1948),
when tobacco pith tissues were cultured on nutrient solutions containing IAA, or adenine and high levels of
phosphate (Skoog and Tsui 1951). Interestingly, they
noticed that the cells failed to divide and grow unless the
explant included some vascular tissue, or its extract was
added to the medium (Jablonski and Skoog 1954). This led
to the addition of a variety of plant extracts, including
coconut milk (the beneficial effect of coconut milk, the
liquid endosperm from immature fruits, on plant tissue
cultures was first shown by Van Overbeek et al. 1941), to
the nutrient medium in an attempt to replace the effect of
vascular tissues, and to identify the active factors responsible for their stimulatory effect. Among these, yeast extract
was found to be most effective, and its active component
was shown to have purine-like properties. This prompted
the addition of DNA to the medium which greatly enhanced
cell division activity in cultured pith tissues (see also Vasil
1959). These findings eventually led to the isolation of kinetin from aged autoclaved herring sperm DNA by Carlos
Miller, then a post-doc in Skoog’s laboratory (Miller et al.
1955). Kinetin, which was active in inducing cell divisions
at astonishingly great dilutions (as low as 1 part per billion)
in the presence of auxin, was soon identified as N6-furfurylaminopurine, and synthesized (Miller et al. 1956).
Kinetin, as well as several other similar molecules, were
initially given the generic name kinin, which was later
changed to cytokinin in order to avoid confusion with a
similar name used in animal systems for a very different
class of substances. Only a few naturally occurring cytokinins have been identified, but thanks to Skoog and his
colleagues’ studies on their structure/function relationships,
a number of cytokinins with varying levels of activity were
synthesized. Soon after the discovery of cytokinins, Skoog
and Miller (1957) made another significant contribution, by
demonstrating the hormonal (auxin–cytokinin) regulation
of morphogenesis in plants, which allowed the controlled
formation of shoots and/or roots in cultured callus tissues,
and is rightly considered an important milestone in understanding plant morphogenesis, and in micropropagation and
regeneration of plants from cultured tissues.
Somatic embryogenesis
Haberlandt (1902), in his famous publication, had predicted the cultivation of artificial embryos (today’s somatic
embryos) from vegetative cells (see Vasil and Vasil 1972).
Experimental production of somatic embryos was first
attained by Jakob Reinert (1958a, b, 1959) in callus, and by
Frederick C. Steward (Steward and Pollard 1958; Steward
et al. 1958a, b), in cell suspension cultures of carrot root
tissues. Reinert, who worked initially at the University of
Tübingen (Germany) and later occupied the position (and
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the laboratory) at the Free University of Berlin which was
once held by Gottlieb Haberlandt, published all of his
original papers in German. With his slicked black hair and
heavy wool suits, he looked like a prosperous European
banker. He was reserved, not well traveled and was
uncomfortable speaking English. Steward was just the
opposite of Reinert. He had a self-confident and forceful
personality. He was born and educated in England, had a
great command over the English language, and was a
dramatic and spell-binding speaker. He traveled extensively. It would be a rare individual who would forget the
experience of listening to his famous ‘‘carrots and coconuts’’ lectures, which were well-produced and rehearsed
performances. He published many review articles, books
and book chapters. It is for these reasons that Steward’s
work is better known and widely cited. The basic thrust of
his argument was that carrot root tissues cultured in the
presence of 2,4-D and coconut milk gave rise to embryogenic cultures, and that somatic embryos formed in such
cultures germinated to form carrot plants. However, he also
insisted that the dissociation of cells into cell suspension
cultures, the presence of 2,4-D, and addition of coconut
milk to the medium, were all essential, and that somatic
embryogenesis occurred almost exclusively in members of
the Umbelliferae (the carrot family). He further dramatized
this by saying that the dissociated single cells floating in a
medium containing the liquid endosperm of coconut, ‘‘felt’’
and behaved like zygotes which in nature are surrounded
by the endosperm. Steward also maintained that carrot
embryogenic cultures grew best in ‘‘nipple flasks’’, especially designed and made by attaching culture tubes along
the periphery of the basal part of an Erlenmeyer flask,
which rotated on a horizontal axis. This resulted in the
tissue pieces being intermittently immersed in liquid
medium and sloughing off of cells from the surface of the
explant. Without making an explicit statement, and without
providing any convincing evidence, he implied in his many
publications and lectures that the somatic embryos were
derived directly from single cells. None of these assertions
was found to be correct. Indeed, Halperin (1966, 1970)
showed that although 2,4-D is essential for the induction of
embryogenic cultures in carrot, it actually inhibits the
formation of somatic embryos, which are formed only
when it is either removed or its level in the medium is
greatly reduced. At the same time, Vasil and Hildebrandt
(1966a, b) induced somatic embryogenesis in cultures of
Cichorium endivia (Compositae) and Petroselinum hortense (Umbelliferae), without 2,4-D or coconut milk. Much
of the accumulated evidence shows that somatic embryos
in culture rarely, if ever, arise directly from isolated single
cells. Single cells in plated emrbyogenic cultures of carrot
first form a proembryonal mass of cells, followed by the
formation of somatic embryos (Backs-Hüsemann and
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Reinert 1970). Steward, who did not particularly appreciate
challenges to his work or to be shown that he was wrong,
was not amused. There is clear evidence from studies in
grasses and other species, that somatic embryos in cultured
tissues (not isolated single cells) arise from single cells
(Konar and Nataraja 1965a, b; Lu and Vasil 1982; Vasil
and Vasil 1982; Conger et al. 1983; Ho and Vasil 1983;
Botti and Vasil 1983; Vasil et al. 1985; Jones and Rost
1989).
Although Reinert and Steward are generally credited
with being the first to experimentally produce somatic
embryos, there is sufficient evidence in the writings of
Harry Waris, a professor of botany at the University of
Helsinki (Finland), showing that he too was among the first
to observe the formation of embryos, in cultures of
Oenanthe aquatica, also a member of the carrot family,
Umbelliferae (Waris 1957, 1959; see also Krikorian and
Simola 1999).
Embryogenic cultures are now routinely produced in a
wide variety of species, including most of the economically
important crops and woody tree species (see Thorpe 1995;
Vasil 1999), and have become the preferred method for the
regeneration of most of the commercially cultivated biotech crops.
Experimental demonstration of totipotency
The formulation of chemically defined nutrient solutions
that supported vigorous and sustained growth of plant tissues in culture, the discovery of plant growth substances,
the hormonal regulation of morphogenesis, and the
experimental induction of embryogenic cultures and formation of somatic embryos, were all important milestones
that together led to the development of efficient technologies for the regeneration of plants from a wide variety of
cultured cells and tissues (Vasil and Vasil 1972; Vasil
1984; Vasil and Thorpe 1994). Yet, the demonstration of
totipotency of plant cells, that is, the regeneration of
complete plants from single cells, so eloquently and
explicitly stated by Schleiden and Schwann in the Cell
Theory (see Boxes 1, 2), and the central requirement for
genetic transformation, remained elusive.
In the 1950s and 1960s, a number of groups were
attempting to culture single cells. A well-organized and
sustained effort to demonstrate the totipotency of plant
cells was made at the University of Wisconsin in the
laboratory of Albert C. Hildebrandt. He was a plant
pathologist by training, and, among other things, was
interested in studying the growth of viruses in plant cells.
His group was one of the first to establish cell suspension
cultures of several species. Cells isolated from such
cultures were used in much of the early work on single
cell culture, which was technically very challenging. To
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overcome this problem, an ingenuous technique was
developed in Hildebrandt’s laboratory. Cells from suspension cultures of grape, marigold and tobacco were isolated
and placed on 8-mm2 pieces of filter paper, which had
previously rested for two or more days on the surface of an
actively growing callus of the same species, and had
absorbed ‘‘secretions’’ from the underlying callus tissue
(Muir et al. 1954, 1958). The filter paper square with the
single cells was then replaced on the surface of the callus
tissue which now acted as a ‘‘nurse tissue’’. Nutrients as
well as growth substances evidently passed through the
filter paper to support sustained cell divisions leading to
the formation of a callus, which could be removed from the
filter paper in 6–10 weeks, and cultured directly on agar
medium. No cell divisions were obtained without the nurse
tissue, emphasizing its critical role in the nourishment of
the isolated cells. Placement of the isolated cells on filter
paper was an obvious disadvantage as it did not allow
continuous monitoring of the cultures and documentation
of cell divisions. Plating of large numbers of single cells in
agar medium in Petri dishes allowed some degree of
monitoring, and the isolation of single cell clones (Bergmann 1959, 1960). The ‘‘nurse effect’’ was obvious even in
plated cultures as cells in close proximity to each other, or
to cell groups, grew better (Reinert 1963).
In the mean time, Hildebrandt’s group developed the
microculture chamber for the culture of completely isolated
cells, which were totally removed from the influence of
other cells. A single cell was placed in a microdrop of
nutrient solution on a glass microscope slide. A cover glass
was placed on either side of the cell on droplets of heavy
mineral oil. A third cover glass was placed to bridge the
two cover glasses to form a ‘‘microculture chamber’’. The
culture was then sealed all around with mineral oil. Such
preparations were ideal for monitoring under the microscope, and were used by Jones et al. (1960) to obtain many
single cell clones. However, they still found it necessary to
use a ‘‘conditioned’’ nutrient solution (medium from an
actively growing culture), and generally have more than
one cell per culture in order to obtain continued cell divisions and growth.
Direct and unequivocal evidence of the totipotency of
plant cells was finally provided by Vasil and Hildebrandt
(1965a, b, 1967), who regenerated plants from a completely isolated single cell of a hybrid tobacco grown in a
fresh (as opposed to conditioned) nutrient solution in a
microculture chamber. These results not only validated the
concept of totipotency, which was one of the most
important tenets of the Cell Theory of Schleiden and
Schwann, but also created the theoretical framework for the
genetic transformation of plants in the 1980s. Steward, who
had been openly skeptical of the possibility of regenerating
plants from isolated single cells during a visit to
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Wisconsin, graciously congratulated the authors on their
achievement and its wider implications.
Work with single cells was technically and physically
very demanding and difficult. In the 1960s, nutrient solutions were prepared by individually weighing each of the
numerous components. There were no laminar flow hoods.
Experiments were carried out on an open bench in a small
room without heating or air-conditioning. The bench top,
and the dissecting and compound microscopes, were disinfected with paper towels soaked in ethanol. Micropipettes
to pick up single cells were made by heating and pulling
the long narrow tips of Pasteur pipettes on a flame, and
then fitting them with a rubber bulb. Entry to the room was
highly restricted. Physical movement was kept to a minimum. Breathing was controlled and measured. All of this
was done in order to minimize air movement and avoid
infection of the cultures. The fact that most of the cultures
remained free of infection showed that the precautions had
the desired effect.
Genetic transformation
Like so many scientific discoveries and inventions, genetic
transformation was discovered by chance, and not by
design, by Frederick Griffith, a British medical officer, in
1928. He was trying to develop a vaccine for prevention of
pneumonia epidemics after the Spanish flu pandemic had
killed nearly 50 million people at the end of World War I.
He used two strains of Streptococcus pneumoniae: the nonvirulent rough strain (R strain) which lacked a polysaccharide capsule, and the deadly smooth strain (S strain)
which had a polysaccharide capsule (Griffith 1928). Mice
injected with S strain bacteria developed pneumonia and
died after a few days, while those injected with the R strain
remained healthy. The infectious nature of the S strain
could be abolished by heat inactivation. However, mice
injected with a mixture of inactivated S strain and normal
R strain developed pneumonia. In his attempt to understand
this curious and unexpected finding, Griffith discovered
that all of the bacteria isolated from mice that had been
injected with a mixture of inactivated S and normal R
strain, were of the S strain, were infectious, and maintained
their phenotype over many generations. He postulated that
some unknown ‘‘transforming principle’’ from the inactivated S strain had converted the non-capsulated and nonvirulent R strain into the encapsulated and virulent S strain.
Griffith was killed in 1941 during an air raid in London,
and the genetic nature of his ‘‘transforming principle’’,
DNA, was not determined until 1944, by Oswald T. Avery,
a Canadian physician and bacteriologist, and his colleagues
Colin M. MacLeod and Maclyn McCarty (Avery et al.
1944). They found that extracts of heat-inactivated S strain
Plant Cell Rep (2008) 27:1423–1440
containing almost pure DNA were capable of transforming
R strain bacteria, but lost this ability when treated with
deoxyribonulcease. In so doing, they established the role of
DNA as the basis of heredity.
During the past four decades, a wide variety of experimental approaches have been proposed, and used, as
possible means to improve the quality as well as productivity of crops in order to meet increasing demands for food
(Vasil 2003a). Extensive world-wide efforts to create novel
genetic variability in plants by means of embryo rescue
(Raghavan 1986), androgenetic haploids (Guha and
Maheshwari 1966; Nitsch and Nitsch 1969; Guha-Mukherjee 1999), somatic hybrids (Giles 1983; Gleba and
Sytnik 1984), etc., have proven to be useful only in a few
species, and have generally not lived up to their purported
promise. Others, such as the much-hyped somaclonal variation (Larkin and Scowcroft 1981), have been found to be
of dubious value. Arguably, much of the success in the
production of plants with novel and useful agronomic traits
has come from the infinitely more precise and predictable
methods of genetic transformation, in which well characterized single or multiple genes introduced into single cells
are stably integrated into the nuclear genome, and are
transmitted to progeny as dominant Mendelian genes.
The following three methods are widely used for the
introduction of foreign genes into plants: (1) Indirect
or vector-based gene transfer, such as A. tumefaciensmediated transformation. (2) Direct DNA delivery into
protoplasts by osmotic or electric shock. (3) Direct DNA
delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles.
Agrobacterium-mediated transformation
The tumor-forming neoplastic crown-gall disease of plants,
which causes considerable damage to perennial crops, has
been known for a long time. However, it was not until
100 years ago that Smith and Townsend (1907) identified
A. tumefaciens as its causative agent (see Braun 1982, for
the colorful and fascinating history of the early work on
crown gall). The prevailing view, until the 1990s, was that
only dicotyledonous species were susceptible to infection
by Agrobacterium, whereas monocotyledons and gymnosperms were considered to be outside its natural host range
as there were no reports of tumor formation in these
species.
Armin C. Braun, working at the Rockefeller Institute
(now Rockefeller University in New York), and a pioneer
in crown gall research, had demonstrated the transformed
nature of crown gall tumor cells by showing that tumor
cells which were free of bacteria could be grown indefinitely on hormone-free nutrient solution, while normal,
non-transformed cells required media supplemented with
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plant growth regulators for continued cell divisions (White
and Braun 1942; Braun 1958, 1982). He proposed that a
tumor-inducing principle (TIP), present in the bacteria, was
responsible for hormone-independent autonomous growth,
and transformation (Braun 1947). A number of substances,
such as toxins, plant growth regulators, bacteriophages,
chromosomal DNA, small RNAs, etc., were at various
times considered to be the TIP.
A key discovery in the development of Agrobacterium
as a vector for plant transformation was made by Georges
Morel, a former student of Roger Gautheret, in Versailles
(France). He found that bacteria-free tumor cells made and
excreted large amounts of opines (octopine, nopaline, etc.),
a new class of metabolites (amino acid and sugar derivatives) that are not formed by normal plant cells (Menagé
and Morel 1964; Morel et al. 1969; Petit et al. 1970). The
opines were used by the bacteria as a sole source of
nitrogen and carbon. The type of opine—octopine or
nopaline—formed by crown gall cells was a function of the
bacterial strain used for transformation, rather than the
plant (Goldman et al. 1968; Petit et al. 1970; Bomhoff et al.
1976). This finding strongly indicated that a functional
bacterial gene had become a part of the plant cell, but this
revolutionary idea was initially met with considerable
skepticism. Nevertheless, it stimulated attempts to identify
the TIP, to search for the presence of agrobacterial DNA in
crown gall cells, and to elucidate the genetic basis of tumor
formation. Several research groups in Europe and the
United States—led by Jeff Schell at Ghent in Belgium, Rob
Schilperoort at Leiden in The Netherlands, and Eugene
Nester at Seattle in the United States—embarked on the
difficult task of resolving this challenging problem (see
Kahl and Schell 1982; Tzfira and Citovsky 2008).
It was first thought that tumor induction might be
caused by an extrachromosomal element (plasmid) carrying tumor-inducing genes, or by a tumorigenic virus
similar to oncogenic viruses that had been found in
animals (Kerr 1969, 1971; Hamilton and Fall 1971;
Hamilton and Chopan 1975). These efforts were greatly
aided by the timely discovery of restriction enzymes and
the development of various recombinant DNA technologies that for the first time made it possible to dissect the
genome into discrete fragments, which could then be
separated by gel electrophoresis to detect specific, short
gene sequences in large genomes (Southern 1975). The
timely availability of the new experimental tools played
an important role in the discovery by Ivo Zaenen in
Schell’s group at Ghent that the virulent strains of
Agrobacterium contained a large extrachromosomal element that harbored the genes responsible for the induction
of crown gall (Zaenen et al. 1974). The extrachromosomal
element was shown to be an exceptionally large plasmid—megaplasmid—and aptly named the tumor-inducing
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or Ti plasmid. The loss of virulence by virulent strains of
bacteria grown at high temperatures was accompanied by
the loss of the Ti plasmid (Van Larebeke et al. 1974;
Zaenen et al. 1974). The transfer of the Ti plasmid to a
non-virulent strain converted it into a tumor-inducing
virulent strain (Van Larebeke et al. 1975). Only a small
part of the Ti plasmid, christened T-DNA, was shown to
be responsible for tumor formation (Chilton et al. 1977).
It was present only in the nuclear DNA fraction of tumor
cells (Chilton et al. 1980; Willmitzer et al. 1980), and was
found to integrate at random sites rather than at any
specific locus. These investigations also made it clear that
the capacity of crown gall tumor cells to undergo
autonomous growth was related to their capacity, acquired
as a result of transformation, to synthesize plant growth
substances and metabolites required for sustained growth.
In order to appreciate the many technical difficulties and
challenges faced by the early workers, one needs only to
read the fascinating account provided by Chilton (2001):
‘‘The catalog of restriction endonucleases was unrecognizably thin. What few enzymes were available often were
tainted. Kits were unknown. Procedures often did not work.
We sized DNA and determined its percentage of G and C
in the model E ultracentrifuge. We measured small volumes with 5-, 20-, 50-, or 100 lL glass capillaries. We
cultured our plant calli in jelly jars and fleakers. Instead of
laminar flow hoods we worked in still air hoods. …we
taught ourselves how to do gel electrophoresis, and we
designed and built our own gel rigs. …We made combs
from square aluminum rod, using double-stick tape to
mount teeth that were pieces of glass cut with great difficulty from microscope slides’’.
Detailed examination of T-DNA isolated from a number
of independently transformed lines showed that it was a
well-defined segment of the Ti plasmid, and that it was
transferred to the plant cell without any changes as a linear
stretch of DNA. The fact that a readily identifiable part (TDNA) of the Ti plasmid was transferred and integrated into
the nuclear genome of the host plant during tumor formation (transformation), suggested that the plasmid could be
used as a vector to transfer other genes.
This was indeed a fortunate coincidence. Agrobacterium
is the only prokaryotic organism that has the natural ability
to transfer DNA to eukaryotic cells (Ream 1989; Tzfira and
Citovsky 2008). This unique characteristic of Agrobacterium for inter-kingdom transfer of genes, evolved millions
of years ago in nature in the form of a highly effective
transformation vector (Ti plasmid), was soon to be
exploited widely for the genetic transformation of plants. It
is humbling to realize that humans were to use an approach
that a ‘‘lowly’’ bacterium had evolved millions of years ago
in order to engineer plants to custom produce its ‘‘food’’
(opines).
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There was strong competition to develop the Ti plasmid as a vector for plant transformation. To this end,
genes were introduced into the T-DNA region in the
laboratories of Schell, Schilperoort, and Chilton, who had
earlier worked in Nester’s laboratory (Van Haute et al.
1983; De Framond et al.1983; Hoekema et al. 1983). A
problem that had been noticed early was that crown gall
tumor tissues growing on hormone-free media formed
only highly aberrant shoots. The autonomous nature of
crown gall tissues, and the formation of aberrant shoots,
was found to be related to the presence of large amounts
of auxin and cytokinin in tumor cells, which were formed
by the auxin and cytokinin synthesis genes located in
T-DNA (Barry et al. 1984; Schröder et al. 1984;
Thomashow et al. 1986).
Deletion of the auxin and cytokinin synthesis genes
from T-DNA produced transformed tissues that required
auxin and cytokinin for continued growth in culture.
Normal plants were regenerated from such cultures by
careful manipulation of the level of auxin and cytokinin
added to nutrient solutions (Bevan et al. 1983; Fraley
et al. 1983; Herrera-Estrella et al. 1983; De Block et al.
1984; Horsch et al. 1984, 1985). These historic results,
ushering in the era of plant biotechnology, were first
presented at the Miami Winter Symposium, on 18 January
1983, by the groups led by Mary-Dell Chilton, Robb T.
Fraley, and Jeff Schell.
At that time, there were no reports of Agrobacteriuminduced tumor formation in monocotyledonous species,
which include the economically important cereals that
account for 66% of the world food supply. This led to the
widely held view that the new technology could not be
used to transform cereals. Elaborate arguments and schematics were presented as to how and why Agrobacteriummediated transformation would not work with cereals
(Potrykus 1990). However, continued efforts by the Schell
and Schilperoort groups soon demonstrated T-DNA transformation of a few monocot species (Hernalsteens et al.
1984; Hooykaas-Van Slogteren et al. 1984). Additional
evidence came from the development of agro-infection, a
technique by which Agrobacterium is used to transfer a
virus into a higher plant (Grimsley et al. 1986). One of the
major barriers to the use of Agrobacterium to transform
cereals was the absence of wound response, and the associated activation of virulence genes. These problems were
overcome by Toshihiko Komari and his colleagues in the
laboratories of Japan Tobacco Inc. The co-cultivation of
actively dividing embryogenic cells with super-virulent
strains of A. tumefaciens in the presence of acetosyringone,
a potent inducer of virulence genes, led to Agrobacteriummediated transformation of rice first, and then all of the
cereals (Hiei et al. 1994; see also Komari and Kubo 1999;
Vasil 1999, 2005, 2007).
Plant Cell Rep (2008) 27:1423–1440
Direct DNA delivery into protoplasts
As a newly appointed lecturer in plant physiology at the
University of Nottingham (UK), Edward C. Cocking was
interested in producing ‘‘isolated cells from plants with the
aspiration of developing a plant cell ‘‘bacterial’’ type of
culture system in which plant cells would divide, separate
and produce a culture of single cells’’ (Cocking 2000). He
successfully isolated protoplasts by incubating segments of
roots and other tissues in a cellulose preparation from
Myrothecium verrucaria, or in filtrates of fungal cultures
containing crude mixtures of cell wall hydrolyzing
enzymes (Cocking 1960, 2000; see also Vasil 1976; Giles
1983). The small numbers of isolated protoplasts obtained
were used mostly for physiological studies. It was the
availability of commercial preparations of Trichoderma
viride cellulase and other cell wall degrading enzyme
preparations from Japan, which made it possible to isolate
large numbers of protoplasts from leaf and other tissues.
Two key developments in 1970 and 1971, pointed to the
potential use of protoplasts for plant improvement.
Tobacco mesophyll protoplasts cultured by Itaru Takebe, a
distinguished and soft-spoken Japanese plant virologist,
regenerated cell walls, underwent sustained divisions
(Takebe et al. 1968; Nagata and Takebe 1970), and
regenerated whole plants (Takebe et al. 1971). At the same
time, Cocking’s group showed that protoplasts of diverse
species—irrespective of taxonomic relationships—could
be fused by chemical treatment (Power et al. 1970). Taken
together, the reports from Takebe’s and Cocking’s laboratories pointed to the possibility of producing novel and
useful hybrids by protoplast fusion. This created a whole
new field of research that attracted the interest of hundreds
of scientists around the world. During the next 20 years,
plants were regenerated from protoplasts (mostly leaf
protoplasts) of scores of species. Once again, regeneration
of protoplasts of cereals and other monocots proved to be
difficult. There was a long and vigorous debate about the
best strategies to regenerate plants from cereal protoplasts
(Vasil 2005), until it was shown decisively that protoplasts
isolated from embryogenic cell suspension cultures (Vasil
and Vasil 1980, 1992) were the best—and possibly the
only—source of totipotent cereal protoplasts (see Cocking
2000; Potrykus 2001; Vasil 2005). Over the years a variety
of somatic hybrids of related as well as unrelated species
have been obtained. Unfortunately, few of these—except
those of Brassica, Citrus, Solanum spp.—have proven to be
of any practical use.
Indeed, the principal use of protoplasts has been in the
genetic transformation of plants. The absence of the cell
wall allows DNA to be easily delivered into protoplasts by
osmotic or electric shock, which temporarily perturbs the
plasma membrane. Methods for the direct delivery of DNA
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into protoplasts developed during the early 1980s (Marton
et al. 1979; Davey et al. 1980; Paszkowski et al. 1984;
Shillito 1999), proved to be especially useful for cereals
which were considered at the time to be outside the host
range of Agrobacterium, and therefore, not amenable to
Agrobacterium-mediated transformation. Indeed, grasses
and cereals were first transformed using protoplasts isolated from cell suspension cultures (Potrykus et al. 1985;
Fromm et al. 1986; Hauptmann et al. 1988; Rhodes et al.
1988; see Vasil and Vasil 1992, Vasil 1999).
Use of protoplasts for genetic transformation became
less attractive after it was shown that monocotyledons,
including the cereals, could be transformed by co-cultivation of embryogenic cell cultures and super-virulent strains
of A. tumefaciens, in the presence of acetosyringone.
Nevertheless, it is important to note that transformation of
cereals was first achieved by the use of embryogenic
protoplasts.
Direct DNA delivery into intact cells
Technical difficulties faced with the isolation and longterm maintenance of embryogenic cultures (the only source
of totipotent cereal protoplasts), as well as perceived
limitations of Agrobacterium-mediated transformation,
encouraged further search for universal methods of transformation. The most notable success in these efforts was
achieved by John Sanford, at Cornell University (US).
Sanford was a plant breeder, with neither any interest nor
training in molecular biology or plant tissue culture. Following on the earlier studies of Kamla K. Pandey in New
Zealand, and Dieter Hess in Germany (see Box 3), he was
‘‘looking for ways to utilize pollen to transmit foreign
DNA into natural zygotes and embryos, thereby producing
transgenic seed directly’’ (Sanford 2000). These included
the use of irradiated pollen, direct uptake of DNA into
pollen, electroporation of pollen, agro-infection of pollen,
and cutting holes in pollen by microlaser beams to facilitate DNA entry. These efforts were soon abandoned as
none of the experiments yielded convincing evidence of
transformation. He and Ed Wolf, who was an electrical
engineer at Cornell, then developed the idea of shooting
DNA-coated tungsten particles into epidermal cells of
onion with the blast of a BB-gun (Klein et al. 1987; Sanford et al. 1987). These attempts were initially met with
laughter and ridicule by their colleagues at Cornell, but it
was the crude BB-gun-based ‘‘Macroparticle Accelerator’’
that later evolved into the hugely successful biolistic
delivery system or the gene gun (Sanford 2000). Early
versions of the instrument actually used blank 0.22 caliber
bullets to accelerate the DNA-coated microparticles. They
were eventually replaced with a safer version which uses
air pressure to achieve the same results. Within a few years
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Box 3
Unconfirmed claims of transformation
Soaking (imbibition) of seeds in solutions containing DNA (Ledoux and Huart 1969; Hess 1969a, b)
Transgenosis—the use of phages as vectors for the transfer and expression of bacterial genes in plant cells (Doy et al. 1973; Hess and Dressler
1984)
Pollen-mediated transformation (Hess et al. 1976; Hess 1978, 1979, 1980; De Wet et al. 1986; Ohta 1986)
Use of irradiated pollen: pollen transformation (Pandey 1975, 1977, 1983; Grant et al. 1980; Jinks et al. 1981)
Application of Agrobacterium tumefaciens to seedlings (Graves and Goldman 1986; Zhao et al. 2006) or spikelets (Hess et al. 1990)
Injection of DNA into bases of flowering tillers (De la Pena et al. 1987)
Pollen tube pathway: application of DNA to cut post-pollination styles (Luo and Wu 1988; Zilberstein et al. 1994)
the high-velocity bombardment of DNA-coated microprojectiles was used to transform a wide variety of plant
species, including the economically important cereals and
legumes, and many woody species. Along with Agrobacterium, it is now the most commonly used method for the
genetic transformation of plants.
Tales of unconfirmed results
The history of plant biotechnology is replete with claims of
genetic transformation of plants achieved with novel and
often simple technologies, including those that do not require
the use of tissue cultures (Box 3). Many of these studies were
authored by well-known and respected scientists, and published in reputable journals. Initially, they generated much
enthusiasm, but never gained the confidence of the broader
scientific community as they failed to provide unambiguous
molecular and genetic evidence of transformation, and could
not be independently confirmed. Consequently, they are
seldom used and have largely been abandoned.
Twenty-five years of plant biotechnology
Keeping in mind the historical perspective of plant biotechnology provided in the preceding pages, it is useful to
look at the achievements of the past 25 years, from the
production of the first biotech plants in 1983, followed by
the first commercial cultivation in 1996, to its present
status (see James 2007; Box 4).
Canola, cotton, maize and soybean are the only major
biotech crops that have been grown commercially on a large
scale during the past decade, and have become an integral
part of international trade and agriculture. At the same time, a
wide variety of useful genes have been transformed into a
large number of economically important plants, including
most of the food crops, scores of varieties of fruits and
vegetables, and many tree species. Biotech crops are also
being increasingly used for the production of vaccines and
many pharmaceuticals (Routier and Nickell 1956; Tulecke
and Nickell 1959; Staba 1980; Constabel and Vasil 1987,
1988; Ma et al. 2005; Fox 2006; Murphy 2007). Admittedly,
there is an urgent need to broaden the diversity of biotech
crops by introducing a wider variety of genes into a larger
number of species. Nevertheless, these are, by any standard
of measurement, truly remarkable achievements for a relatively new technology. Mathhias Jakob Schleiden, Theodor
Schwann, Gottlied Haberlandt, Frederick Griffith, and all
others who came after them, would be justifiably proud.
Plant biotechnology has been also advanced considerably by the sequencing of plant genomes, such as those of
Arabidopsis, rice and others. During the past two decades,
Box 4
Plant biotechnology 1996–2008
From 1996 to 2007, biotech crops were cultivated cumulatively on 1.7 billion acres in 12 developing and 11 developed countries, at[10% annual
rate of growth. In 2007, biotech crops were cultivated on [282 million acres, representing 8% of the world crop land
World acreage for biotech crops in 2007: soybean 57%, maize 25%, cotton 13%, canola 5%. Comparable 2007 acreage for biotech crops in the
United States: soybean 90%, cotton 85% and maize 50%. Developing countries accounted for 43% of the world acreage of biotech crops
Biotech foods have been consumed by [1 billion humans
More than 90% or 11 million of the 12 million farmers planting biotech crops are small, resource-poor farmers in developing countries
Cumulatively, biotech crops have thus far contributed US$16.5 billion and US$17.5 billion, to the economies of developing and developed
countries, respectively
Cultivation of biotech crops has reduced the use of agro-chemicals, has increased productivity and economic development, is improving human
health, and is aiding efforts directed at conservation of the environment and biodiversity
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Box 5
Lessons to learn from the history of plant biotechnology
To see what everyone else has seen but think what no one else has thought before (Albert Szent-Györgyi, Nobel Prize, 1937)
Think big, think bold, and think different
Think outside the box
Challenge conventional wisdom
Challenge dogmas and ‘experts’
Be creative, not clannish
Be critical of published work
Controversies and debates are useful and the essence of elegant and inspiring science
a wealth of new information about useful genes, and gene
regulation, expression and function, has become available,
and has improved our understanding of the molecular basis
of plant development. These insights, combined with the
availability of molecular maps and markers, has helped
plant breeders produce new and useful varieties. Molecular
breeding is increasingly becoming an important and powerful tool for plant improvement and variety development.
Fears and exaggerated claims of possible adverse effects
of biotech foods and crops on humans and the environment
are unfounded and have no basis in fact. Indeed, it is
remarkable that no such ill effects have been documented
after 12 years of extensive cultivation in diverse environments, and the consumption of biotech foods by more than
a billion humans and even a larger number of animals.
Many practical applications of plant biotechnology,
including the introduction into the market place of other
biotech crops with useful and highly desirable characteristics, have been prevented by political and ideological
opposition from vested interests—including the European
Union, a few developing countries, and some self-serving
environmental groups—and by the many redundant, overly
stringent, time-consuming and exorbitantly expensive
regulatory requirements (Vasil 2003a, b). It is imperative
now to take a fresh look at the entire regulatory framework
for biotech crops, and gradually phase out and eliminate
many of the unnecessary restrictions imposed by overzealous regulators, and politically motivated decisions by
some countries in Europe and elsewhere.
Lessons from the history of plant biotechnology
It is said that history teaches us many valuable lessons,
and that those who ignore it do it at their own peril. Yet,
history is not given much importance in modern science
education and research. The unfortunate result is that we
are producing scientists who are historically illiterate.
Like the rest of modern society, many scientists today are
interested more in the ‘‘here and now’’, and show little
interest in the past or concern for the future. Most contemporary publications lack a historical perspective, and
few scientists bother to consult or cite literature that is
older than 10–15 years. Literature searches are generally
confined to what is readily available on the internet. Such
historical ignorance and neglect present a highly distorted
and biased view of science, deny credit to earlier workers,
and unjustifiably inflate the importance of modern
research. Authors, reviewers and editors share equal
responsibility for this very undesirable practice, which
is unhealthy for science and for the training of young
scientists. There is also an increasing tendency among
scientists to be clannish, and to follow only the most
popular areas of investigation. This habit is fueled by the
unwillingness of funding agencies to support and
encourage truly novel and high risk proposals. A study of
the history of plant biotechnology clearly shows that there
are many important and meaningful lessons to be learnt
from the wisdom and cumulative experience of all those
who came before us (Box 5). It is up to us now, to benefit
from the opportunities presented by the history of our
science, and reverse the growing trends of historical
ignorance and illiteracy.
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