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 Plant biotechnology Á Plant regeneration Á of cellular totipotency and genetic transformation, which can Somatic embryogenesis Á Totipotency Á Transgenic crops be traced back to the Cell Theory of Matthias Jakob Schle- iden and Theodor Schwann, and the discovery of genetic The further backward you look, the further forward transformation in bacteria by Frederick Griffith, respec- you can see (Winston Churchill). tively. On the 25th anniversary of the genetic transformation Know where you come from, to get where you are of plants, this review provides a historical account of the going (Jhumpa Lahiri). evolution of the theoretical concepts and experimental strategies that led to the production and commercialization of biotech (transformed or transgenic) plants expressing many Introduction useful genes, and emphasizes the beneficial effects of plant biotechnology on food security, human health, the environ- Eighteenth January 1983, is arguably one of the most ment, and conservation of biodiversity. In so doing, it important dates in the history of plant biotechnology. celebrates and pays tribute to the contributions of scores of Indeed, history was made that day 25 years ago, at the scientists who laid the foundation of modern plant biotech- Miami Winter Symposium, where three independent nology by their bold and unconventional thinking and groups described Agrobacterium tumefaciens-mediated experimentation. It highlights also the many important les- genetic transformation, leading to the production of nor- sons to be learnt from the fascinating history of plant mal, fertile transgenic plants. Although each group had biotechnology, the significance of history in science teaching introduced a bacterial antibiotic resistance gene into and research, and warns against the danger of the growing tobacco, a model plant for studies on in vitro regeneration, trends of ignoring history and historical illiteracy. there was widespread belief that the new technology would allow introduction of agronomically important genes into Keywords Biotech crops Á Cell Theory Á crop species that are susceptible to agrobacterial infection. Genetic transformation Á History of science Á 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 Adapted from the Opening Plenary Address to the international that at the time were considered to be outside the host conference on ‘‘Plants for Human Health in the Post-Genome Era’’, held 26–29 August 2007, at Helsinki, Finland. range of Agrobacterium. It is a testament to the ingenuity of the plant biotechnology community that within the next Communicated by P. Kumar. decade all major crop species had been transformed with genes that conferred resistance to non-selective herbicides, & I. K. Vasil ( ) and some pests and pathogens. In the mean time, in University of Florida, Box 110690, Gainesville, FL 32611-0690, USA response to concerns about the safety and environmental e-mail: ivasil@ufl.edu impact of biotech (transgenic) crops, elaborate and 123 1424 Plant Cell Rep (2008) 27:1423–1440 stringent mandatory regulations were developed in the factor (DNA) was not elucidated until much later (Avery United States and some other countries to monitor and et al. 1944). regulate the cultivation and use of biotech crops and Matthias Jakob Schleiden was born in 1804 in Hamburg products. But plant biotechnology came of age only in (Germany). He was trained as a lawyer, but gave up the 1996, with the planting of nearly five million acres of practice of law to study medicine at Go¨ttingen. There he biotech crops, mostly in the United States. Thereafter, the became interested in the study of plants. He continued his global acreage of biotech crops increased rapidly—at an botanical studies in Berlin before assuming the post of annual rate in excess of 10%—so that in 2007 more than professor of botany at the University of Jena. He was the 282 million acres (nearly 8% of total world crop acreage) first prominent botanist to shift the focus from taxonomic were planted in 23 countries, for a cumulative (1996–2007) to structural studies of plants. Theodor Schwann was born total acreage of over 1.7 billion acres (James 2007). in 1810 in Neuss (Germany). After spending 4 years under For the purposes of this review, plant biotechnology is the influence of J.P. Mu¨ller in Berlin, he became professor defined rather narrowly to include only biotech plants. It of physiology at the University of Louvain (Belgium). In provides a historical perspective of the evolution of a addition to many other notable studies, Schleiden and variety of novel ideas and technologies that are now rou- Schwann made extensive use of the microscope to study tinely used in the regeneration and genetic transformation plants and animals. They had known each other while of plants, and celebrates the contributions of many men and studying in Berlin and were friends. In 1838, while they women whose seminal contributions laid the foundation of were enjoying after-dinner coffee, Schleiden talked excit- modern plant biotechnology. It was my good fortune to edly about the universality of plant cells. All of the species have known most of the founding figures of plant bio- that he had examined seemed to be made up of recogniz- technology in the twentieth century—from Philip R. White, able units or cells. He expressed his belief that the growth Roger J. Gautheret, Kenneth V. Thimann, Armin C. Braun, of plants depended on the production of new cells. Schw- George Morel, Frederick C. Steward, Jacob Reinert, Albert ann was immediately struck by the similarity of C. Hildebrandt, and Folke K. Skoog, to Jeff Schell and Schleiden’s observations to his own studies of animals. many others—whose work is chronicled in these pages After dinner they retreated to Schwann’s laboratory to look (some of these associations are described in an earlier at his microscopic preparations. They published their autobiographical article; Vasil 2002). I am indebted results a year apart—Schleiden (1838), and Schwann particularly to Ken Thimann, Phillip White and Al Hilde- (1839)—without any reference or acknowledgement of brandt for their wise counsel, encouragement and support each other’s contribution (an unfortunate and unacceptable early in my career, and to Jeff Schell for being one of the practice that is still a part of scientific publishing). Their first to believe in, and publicly speak in support of, our respective observations on plants and animals, when taken work on embryogenic cultures which continue to be together, formed the basis for a unified Cell Theory which essential for the production of biotech grasses and cereal applied universally to all living organisms. One can get a crops. In providing this historical account of the science as sense of their boldness and originality, and that of Virchow well as the personalities of plant biotechnology, and the (1858), from their unambiguous and insightful observa- many debates and controversies, the review highlights the tions (Box 1). value of historical studies in science education and The Cell Theory went unnoticed and unappreciated for research. 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 The Cell Theory earlier, Giovanni Battista Amici (1824), a young Italian mathematician and astronomer, had made the chance dis- The theoretical framework and experimental basis of covery of the pollen tube while examining the stigmas of modern plant biotechnology derive from the concepts of Portulaca oleracea with a crudely constructed microscope. cellular totipotency (the ability of a single cell to divide He later noted that the pollen tubes grow/elongate through and produce a whole plant) and genetic transformation the style and eventually enter the ovary and the ovule (genetic alteration caused by the uptake, stable integration (Amici 1830). Schleiden (1837) also studied the subject and expression of foreign genetic material). The concept of extensively and in general confirmed Amici’s observations. totipotency is inherent in the Cell Theory of Schleiden However, he went beyond the scope of his observations (1838) and Schwann (1839), which recognized the cell as and carelessly proposed that the tip of the pollen tube— the primary unit—elementary part—of all living organ- after entering the embryo sac—directly becomes the isms. Genetic transformation was first reported by Griffith embryonal vesicle which undergoes a number of divisions (1928) in bacteria, but the genetic basis of the transforming to form the embryo. According to him, therefore, the 123 Plant Cell Rep (2008) 27:1423–1440 1425

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 the modern science of pathology. Virchow was a many- embryo that developed from the pollen tube. The debate faceted individual. He is recognized for his many important between Amici (1844) and Schleiden (1845), and their contributions to medicine, social medicine, anthropology respective supporters, was quite acrimonious and continued and politics, and his service as a member of the Bundestag for many years. In the mean time, in response to the con- (parliament) in Germany. troversy, the Imperial Institute of the Netherlands had Although the concept of totipotency is evident in the announced that it will award a prize to anyone who can writings of Schleiden, Schwann and Virchow (Box 1), it conclusively resolve the question of the origin of the was Herman Vo¨chting (1878), who attempted to demon- embryo. After examining all of the relevant evidence, it strate totipotency experimentally by dissecting and awarded the prize to H. Schacht (1850), one of Schleiden’s growing smaller and smaller fragments of plant tissues. He most ardent supporters, for his thesis that was in agreement concluded that ‘‘In every fragment, be it ever so small, of with Schleiden’s view of the embryo arising directly from the organs of the plant body, rest the elements from which, the pollen tube. Fortunately, the definitive studies of Amici by isolating the fragment, under proper external conditions, (1847), as well as those of Wilhelm Hofmeister (1849), the whole body can be built up’’ (Vo¨chting 1878). 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 Totipotency of plant cells the embryo sac and not from the pollen tube. Ultimately, the mounting evidence against Schleiden’s views provided No sustained or organized attempts were made to test the by many investigators became so overwhelming that he validity of the concept of totipotency that is inherent in was forced to retract his opinions. He became depressed, the Cell Theory (see Box 1), until the beginning of the soon gave up all botanical work, and retreated to Dresden twentieth century. The Austro-German botanist Gottlieb as a private teacher of history and philosophy. The final Haberlandt was the first to try to obtain experimental blow to Schleiden’s ideas was the discovery of syngamy evidence of totipotency by culturing plant cells in nutri- (fertilization) by Edward Strasberger (1884), and double ent solutions in the hope of regenerating whole plants. fertilization by Nawaschin (1898). The vigorous debate He was born in 1854 in Ungarisch-Altenburg, Hungary. about the origin of the embryo, which involved many After completing his studies at the University of Vienna prominent individuals for many years, is a powerful illus- (Austria), he held professorships at universities in Graz tration of the bedrock principle of science that what matters (Austria) and Berlin (Germany). He presented the results in the end is not who you are or how powerful you are, or of his pioneering experiments, in a prophetic and now how many prizes you have received or supporters you classic paper, entitled ‘‘Experiments on the culture of have, but rather how good and reproducible your science is. isolated plant cells’’, before the Mathematics and Natural Interest in the Cell Theory was revived in 1858 by the Sciences Section of the Vienna Academy of Sciences in famous aphorism of Ludwig Karl Virchow: ‘‘Omnis cellula Berlin, on 6 February 1902 (Haberlandt 1902; see a cellula’’ (all cells arise from cells). Virchow was a Krikorian and Berquam 1969; Laimer and Ru¨cker 2002, pathologist at the Charite´ Hospital in Berlin (still consid- for English translation). He cultured chloroplast-contain- ered one of the leading teaching hospitals in Europe). He ing differentiated cells from leaves of Lamium determined that diseased cells always arose from healthy purpureum, cells from the petioles of Eichhornia crass- cells, and rejected the prevailing view of spontaneous ipes, glandular hairs of Pulmonaria and Urtica, and generation. He is rightfully acknowledged as the founder of stamen hairs of Tradescantia, in Knop’s (1865) salt

123 1426 Plant Cell Rep (2008) 27:1423–1440 solution in hanging drop cultures. The cells grew in size, This led to the discovery that root meristems were often but did not undergo any cell divisions. Eventually, the virus-free, and the application of this information many cultures were lost to infection. In hindsight, his results years later by George Morel to obtain virus-free plants are not surprising. He failed largely because of his from cultured shoot meristems (Morel and Martin 1952, unfortunate choice of experimental material, and because 1955; Kartha 1984). of the inadequacy of nutrition provided by Knop’s salt The development of improved nutrient solutions, use of solution. Even now, more than a century later, the culture the newly discovered plant growth regulator indole-3-ace- of isolated leaf cells, and other materials used by tic acid (IAA), informed choice of plant material, and Haberlandt, is either extremely difficult or impossible in appreciation of the importance of aseptic cultures, led to most species. Nevertheless, based on his experiences, sustained growth of root tips, and carrot root and tobacco Haberlandt made some bold predictions. He advocated stem tissues, for indefinite periods of time (Gautheret 1934, the use of embryo sac fluids (coenocytic liquid endo- 1939, 1985; Nobe´court 1939; White 1939). This important sperm, such as coconut milk that was later widely and milestone was achieved by Roger Gautheret and Pierre successfully used in tissue culture studies) for inducing Nobe´court in France, and Philip White in the United States, cell divisions in vegetative cells, and pointed to the within weeks of each other. However, with the beginning possibility of successfully cultivating artificial embryos of World War II, the European and American scientists (i.e., somatic embryos, which are now the predominant remained unaware of each other’s results for many years. means of plant regeneration in a wide range of species; White’s original cultures were maintained for nearly four see Thorpe 1995; Vasil 1999) from vegetative cells in decades, with regular subcultures, but without any evi- nutrient solutions (see Box 2). It is for these reasons that dence of changes in growth habits or nutritional Haberlandt is rightfully credited with being the founder requirements. He often carried a flask in his jacket pocket of the science of plant cell culture. to meetings, and proudly showed it around to impress the fact that his original root cultures were still going strong Improved nutrient solutions and unlimited growth after hundreds of subcultures. of plant tissues White (1932) found Knop’s nutrient solution, as well as the formulation used by Robbins (1922), to be unsatisfac- The lack of availability of a nutrient solution that could tory. This prompted an effort by many individuals to support the growth of isolated plant cells and tissues hin- develop nutrient solutions that could adequately support dered further progress for many years. Molliard (1921)in the growth of isolated plant tissues. White developed a new France, Kotte (1922) in Germany, and Robbins (1922)in nutrient solution—the White’s medium (White 1943, the United States, cultured fragments of embryos, and 1963)—that was based on the formulation of Uspenski and excised roots, on Knop’s (1865) mineral solution. Some Uspenskaia (1925) for algae, and the microelements of growth was observed on this minimal medium, but none of Trelease and Trelease (1933), and included glycine, nico- the cultures could be maintained for more than a few tinic acid, thiamine and pyridoxine. It was the most widely weeks. used nutrient solution for plant tissue cultures until the Philip Rodney White in the United States was the first 1960s. Knop’s solution was used in Roger Gautheret’s to obtain indefinite growth of cultured plant tissues. He laboratory in France for a long time, until it was replaced was working with the famed biochemist and virologist by a new formulation developed by one of his students, Wendel Stanely at Rockefeller Institute in Princeton, NJ Rene´ Heller (1953). Many scientists in Europe considered (now the Rockefeller University, New York), and was it to be superior to White’s medium. trying to cultivate viruses in plant tissues. He was able to As the only son of a well-to-do businessman, Gautheret maintain excised root tips of tomato and other plants in could have led a very comfortable life without ever having culture for indefinite periods of time, while also demon- to work for a living. Yet, he chose a scientific career under strating multiplication of tobacco mosaic virus in the the guidance of the famed French cytologist Guillermond cultured roots (White 1934a, b). He noted that in some (Gautheret 1985). After three frustrating years, he was instances no virus could be detected in subcultured roots. among the first, along with White and Nobe´court, 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)

123 Plant Cell Rep (2008) 27:1423–1440 1427 unlimited growth of cultured plant tissues. His manuscripts at Pennsylvania State University, where he was a Visiting describing the historic results were rejected for publication Professor (White and Grove 1965). It was there, with his in the official journal of the French Academy of Sciences. encouragement and guidance, that the International Coun- Ironically, he was later elected to the Academy, and for a cil of Plant Tissue Culture, now the International long time was its pre-eminent Academician in plant biol- Association for Plant Biotechnology (IAPB), was founded. ogy. For nearly four decades, his students and colleagues The IAPB is the largest international professional organi- dominated the field of plant cell culture in France. Unfor- zation representing the interests of the plant biotechnology tunately, all of his work was published in French, and community. It continues to meet every 4 years in different remained unknown for a long time in much of the English- parts of the world. It too is a part of his legacy. Published speaking world. It was White—his competitor—who proceedings of these conferences provide a continuous introduced his work to a wider audience through citations historical record of trends and key developments in plant in his papers and books. biotechnology (see Vasil 2003b; Xu et al. 2007). Philip Rodney White was born in Chicago, on 25 July Arguably, the most detailed and systematic studies on 1901, one of twin sons. He obtained his Ph.D. from Johns the nutrient requirements of cultured plant tissues were Hopkins University. He studied also in France, and worked carried out by Albert C. Hildebrandt at the University of in Germany, apart from holding positions in Jamaica and Wisconsin, in an attempt to develop an ideal nutrient Panama. He spent much of his professional career at the solution (Hildebrandt and Riker 1949, 1953; Hildebrandt famed Roscoe B. Jackson Memorial Laboratory in remote et al. 1946; Vasil and Hildebrandt 1966c; Ozias-Akins and Bar Harbor, Maine, which was also a major center for Vasil 1985). This resulted in the development of solutions animal cell culture and genetics. It was during his stay in that contained greatly elevated levels of mineral salts, such Berlin in 1930–1931, that he met Haberlandt who chal- as the tobacco high salts medium (Vasil and Hildebrandt lenged White to pursue plant tissue culture research, but 1966c). In the mean time, Toshio Murashige, a graduate also warned him of the high possibility of failure and student in the laboratory of Folke Skoog, also at the Uni- disappointment. White had begun his work on plant tissue versity of Wisconsin, was trying to obtain optimum and culture as a means to grow viruses, but had quickly predictable growth of cultured tobacco pith tissues which expanded it to include the culture of roots, and stem and Skoog needed for performing reliable bioassays of cyto- other tissues. Forty years after, he died prematurely of kinin activity. He found that the addition of an aqueous hepatitis, on 25 March 1968, in Bombay, while on an extract of tobacco leaves to White’s medium resulted in extensive lecture tour in India, White is still a much rev- greater than fourfold increase in growth. This was deter- ered figure in plant biotechnology. He is remembered and mined to be caused largely by the inorganic constituents of honored for his significant and seminal scientific contri- the leaf extract. Similar results were obtained when the ash butions to the science of plant tissue culture, for the of tobacco leaf extracts, or large amounts of ammonium, development of a widely used nutrient solution, and for his nitrate, phosphate and potassium salts were added to the influential books—the first of their kind—on the culture of White’s medium. These results led to the formulation plant cells that have inspired and enabled a generation of of a new and completely defined nutrient solution, the scientists to use cell culture techniques for physiological Murashige and Skoog (1962) or MS medium, which also and developmental studies of plants. He was an unassum- included chelated iron in order to make it more stable and ing but outspoken individual with strong opinions, which available during the life of the cultures, myo-inositol, and a did not endear him to some of his contemporary col- mixture of four vitamins. In a sense then, the MS medium leagues. He was invariably kind to young scientists, always is reconstituted tobacco leaf extract. It is the most widely encouraging and supporting them, and telling them that his used formulation for culture of plant tissues, and the pub- generation had done what it could, and now it was up to the lication describing the MS medium (Murashige and Skoog younger generation to take up the new challenges. White 1962) remains one of the most highly cited publications in was an internationalist in the true sense. He was convinced plant biology. that great advances in science can come only through international understanding and cooperation. It was in this Discovery of plant growth substances spirit that he organized the highly successful Decennial Review Conference in 1956, for the American Cancer Charles Darwin (1880) was one of the first to observe and Society, while he was President of the Tissue Culture describe the curvature of seedlings toward light. He dis- Association (now the Society for In Vitro Biology); about covered that this effect could be prevented by covering the one-fourth of the program was devoted to plant tissue tip of the coleoptile. Boysen-Jensen (1910) found that the cultures. He followed it by hosting the first truly interna- complete removal of the tip abolished the effect of light, tional conference on plant cell and tissue culture, in 1963, and pointed to the possibility of a ‘‘material substance’’ 123 1428 Plant Cell Rep (2008) 27:1423–1440 being responsible for the curvature. Frits Went (1928), at formation were soon obtained by Skoog and Tsui (1948), Leiden in The Netherlands, successfully isolated and when tobacco pith tissues were cultured on nutrient solu- quantified the amount of ‘‘material substance’’, which he tions containing IAA, or adenine and high levels of called ‘‘wuchstoff’’ (hormone), and coined the phrase phosphate (Skoog and Tsui 1951). Interestingly, they ‘‘Ohne Wuchstoff, kein Wachstum’’ (without hormone, no noticed that the cells failed to divide and grow unless the growth). The hormone, later to be identified as the naturally explant included some vascular tissue, or its extract was occurring auxin, IAA, was first isolated in crystalline form added to the medium (Jablonski and Skoog 1954). This led from the urine of pregnant women by Ko¨gl (Ko¨gl et al. to the addition of a variety of plant extracts, including 1934;Ko¨gl and Kostermans 1934) in Copenhagen, Den- coconut milk (the beneficial effect of coconut milk, the mark, and from large scale cultures of the fungus, Rhizopus liquid endosperm from immature fruits, on plant tissue suinus, by Thimann (1935) at the California Institute of cultures was first shown by Van Overbeek et al. 1941), to Technology (Caltech) in the United States. Kenneth Vivian the nutrient medium in an attempt to replace the effect of Thimann was born in 1904 in Ashford (Kent, England), and vascular tissues, and to identify the active factors respon- studied for his graduate degree at Imperial College, Uni- sible for their stimulatory effect. Among these, yeast extract versity of London. Soon after, he accepted a position at was found to be most effective, and its active component Caltech (he later worked at Harvard, and University of was shown to have purine-like properties. This prompted California, Santa Cruz). Thimann, and his many students, the addition of DNA to the medium which greatly enhanced including James Bonner and Folke Skoog, continued their cell division activity in cultured pith tissues (see also Vasil studies on auxins and contributed greatly to our under- 1959). These findings eventually led to the isolation of ki- standing of the structure/function of auxins, and the netin from aged autoclaved herring sperm DNA by Carlos development of a wide variety of synthetic auxins—such as Miller, then a post-doc in Skoog’s laboratory (Miller et al. 2,4-dichlorophenoxyacetic acid (2,4-D)—which are used 1955). Kinetin, which was active in inducing cell divisions widely in the culture of plant tissues, and in agriculture. at astonishingly great dilutions (as low as 1 part per billion) In addition to auxins, the other naturally occurring plant in the presence of auxin, was soon identified as N6-furfu- growth substances that have had a profound impact on the rylaminopurine, and synthesized (Miller et al. 1956). culture of isolated plant cells are the cytokinins. They were Kinetin, as well as several other similar molecules, were discovered by Folke Skoog and his colleagues at the Uni- initially given the generic name kinin, which was later versity of Wisconsin. Skoog was born in 1908 in Halland changed to cytokinin in order to avoid confusion with a (Sweden), and immigrated to the United States during a similar name used in animal systems for a very different visit as a high school student to California, in 1925. He was class of substances. Only a few naturally occurring cyto- an avid sportsman, and had competed—finishing fourth— kinins have been identified, but thanks to Skoog and his in the 1,500-m race at the 1932 Olympics. He carried out colleagues’ studies on their structure/function relationships, his graduate work with Thimann at Caltech, where he also a number of cytokinins with varying levels of activity were came under the influence of Frits Went and Johannes van synthesized. Soon after the discovery of cytokinins, Skoog Overbeek, all distinguished pioneers in the discovery and and Miller (1957) made another significant contribution, by study of auxins. demonstrating the hormonal (auxin–cytokinin) regulation Haberlandt (1913) had continued his attempts to grow of morphogenesis in plants, which allowed the controlled plant tissues in culture and had shown that phloem diffu- formation of shoots and/or roots in cultured callus tissues, sates induced cell divisions in parenchyma tissue of potato and is rightly considered an important milestone in under- tubers. Crushed cells yielded a material that induced cell standing plant morphogenesis, and in micropropagation and division activity, but this effect was lost when the crushed regeneration of plants from cultured tissues. tissue was rinsed with water (Haberlandt 1921). Many years later, Skoog was continuing his own studies on auxins, first Somatic embryogenesis at Harvard with Thimann, and then at Wisconsin. During a visit to White’s laboratory he had seen bud formation in Haberlandt (1902), in his famous publication, had pre- cultured tobacco tissues, and started his work with hybrid dicted the cultivation of artificial embryos (today’s somatic tobacco tissue cultures that had been obtained from White embryos) from vegetative cells (see Vasil and Vasil 1972). (1939) by Thimann. They were lost during the war and so he Experimental production of somatic embryos was first started using tissues from Nicotiana tabacum cv Wisconsin attained by Jakob Reinert (1958a, b, 1959) in callus, and by 38. The initial success in obtaining unlimited growth of Frederick C. Steward (Steward and Pollard 1958; Steward plant tissues in culture by Gautheret, Nobe´court and White, et al. 1958a, b), in cell suspension cultures of carrot root had been limited to the use of explants that contained tissues. Reinert, who worked initially at the University of meristematic cells. Continued cell divisions and bud Tu¨bingen (Germany) and later occupied the position (and 123 Plant Cell Rep (2008) 27:1423–1440 1429 the laboratory) at the Free University of Berlin which was Reinert 1970). Steward, who did not particularly appreciate once held by Gottlieb Haberlandt, published all of his challenges to his work or to be shown that he was wrong, original papers in German. With his slicked black hair and was not amused. There is clear evidence from studies in heavy wool suits, he looked like a prosperous European grasses and other species, that somatic embryos in cultured banker. He was reserved, not well traveled and was tissues (not isolated single cells) arise from single cells uncomfortable speaking English. Steward was just the (Konar and Nataraja 1965a, b; Lu and Vasil 1982; Vasil opposite of Reinert. He had a self-confident and forceful and Vasil 1982; Conger et al. 1983; Ho and Vasil 1983; personality. He was born and educated in England, had a Botti and Vasil 1983; Vasil et al. 1985; Jones and Rost great command over the English language, and was a 1989). dramatic and spell-binding speaker. He traveled exten- Although Reinert and Steward are generally credited sively. It would be a rare individual who would forget the with being the first to experimentally produce somatic experience of listening to his famous ‘‘carrots and coco- embryos, there is sufficient evidence in the writings of nuts’’ lectures, which were well-produced and rehearsed Harry Waris, a professor of botany at the University of performances. He published many review articles, books Helsinki (Finland), showing that he too was among the first and book chapters. It is for these reasons that Steward’s to observe the formation of embryos, in cultures of work is better known and widely cited. The basic thrust of Oenanthe aquatica, also a member of the carrot family, his argument was that carrot root tissues cultured in the Umbelliferae (Waris 1957, 1959; see also Krikorian and presence of 2,4-D and coconut milk gave rise to embryo- Simola 1999). genic cultures, and that somatic embryos formed in such Embryogenic cultures are now routinely produced in a cultures germinated to form carrot plants. However, he also wide variety of species, including most of the economically insisted that the dissociation of cells into cell suspension important crops and woody tree species (see Thorpe 1995; cultures, the presence of 2,4-D, and addition of coconut Vasil 1999), and have become the preferred method for the milk to the medium, were all essential, and that somatic regeneration of most of the commercially cultivated bio- embryogenesis occurred almost exclusively in members of tech crops. the Umbelliferae (the carrot family). He further dramatized this by saying that the dissociated single cells floating in a Experimental demonstration of totipotency medium containing the liquid endosperm of coconut, ‘‘felt’’ and behaved like zygotes which in nature are surrounded The formulation of chemically defined nutrient solutions by the endosperm. Steward also maintained that carrot that supported vigorous and sustained growth of plant tis- embryogenic cultures grew best in ‘‘nipple flasks’’, espe- sues in culture, the discovery of plant growth substances, cially designed and made by attaching culture tubes along the hormonal regulation of morphogenesis, and the the periphery of the basal part of an Erlenmeyer flask, experimental induction of embryogenic cultures and for- which rotated on a horizontal axis. This resulted in the mation of somatic embryos, were all important milestones tissue pieces being intermittently immersed in liquid that together led to the development of efficient technolo- medium and sloughing off of cells from the surface of the gies for the regeneration of plants from a wide variety of explant. Without making an explicit statement, and without cultured cells and tissues (Vasil and Vasil 1972; Vasil providing any convincing evidence, he implied in his many 1984; Vasil and Thorpe 1994). Yet, the demonstration of publications and lectures that the somatic embryos were totipotency of plant cells, that is, the regeneration of derived directly from single cells. None of these assertions complete plants from single cells, so eloquently and was found to be correct. Indeed, Halperin (1966, 1970) explicitly stated by Schleiden and Schwann in the Cell showed that although 2,4-D is essential for the induction of Theory (see Boxes 1, 2), and the central requirement for embryogenic cultures in carrot, it actually inhibits the genetic transformation, remained elusive. formation of somatic embryos, which are formed only In the 1950s and 1960s, a number of groups were when it is either removed or its level in the medium is attempting to culture single cells. A well-organized and greatly reduced. At the same time, Vasil and Hildebrandt sustained effort to demonstrate the totipotency of plant (1966a, b) induced somatic embryogenesis in cultures of cells was made at the University of Wisconsin in the Cichorium endivia (Compositae) and Petroselinum hor- laboratory of Albert C. Hildebrandt. He was a plant tense (Umbelliferae), without 2,4-D or coconut milk. Much pathologist by training, and, among other things, was of the accumulated evidence shows that somatic embryos interested in studying the growth of viruses in plant cells. in culture rarely, if ever, arise directly from isolated single His group was one of the first to establish cell suspension cells. Single cells in plated emrbyogenic cultures of carrot cultures of several species. Cells isolated from such first form a proembryonal mass of cells, followed by the cultures were used in much of the early work on single formation of somatic embryos (Backs-Hu¨semann and cell culture, which was technically very challenging. To 123 1430 Plant Cell Rep (2008) 27:1423–1440 overcome this problem, an ingenuous technique was Wisconsin, graciously congratulated the authors on their developed in Hildebrandt’s laboratory. Cells from suspen- achievement and its wider implications. sion cultures of grape, marigold and tobacco were isolated Work with single cells was technically and physically and placed on 8-mm2 pieces of filter paper, which had very demanding and difficult. In the 1960s, nutrient solu- previously rested for two or more days on the surface of an tions were prepared by individually weighing each of the actively growing callus of the same species, and had numerous components. There were no laminar flow hoods. absorbed ‘‘secretions’’ from the underlying callus tissue Experiments were carried out on an open bench in a small (Muir et al. 1954, 1958). The filter paper square with the room without heating or air-conditioning. The bench top, single cells was then replaced on the surface of the callus and the dissecting and compound microscopes, were dis- tissue which now acted as a ‘‘nurse tissue’’. Nutrients as infected with paper towels soaked in ethanol. Micropipettes well as growth substances evidently passed through the to pick up single cells were made by heating and pulling filter paper to support sustained cell divisions leading to the long narrow tips of Pasteur pipettes on a flame, and the formation of a callus, which could be removed from the then fitting them with a rubber bulb. Entry to the room was filter paper in 6–10 weeks, and cultured directly on agar highly restricted. Physical movement was kept to a mini- medium. No cell divisions were obtained without the nurse mum. Breathing was controlled and measured. All of this tissue, emphasizing its critical role in the nourishment of was done in order to minimize air movement and avoid the isolated cells. Placement of the isolated cells on filter infection of the cultures. The fact that most of the cultures paper was an obvious disadvantage as it did not allow remained free of infection showed that the precautions had continuous monitoring of the cultures and documentation the desired effect. 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 (Berg- Genetic transformation mann 1959, 1960). The ‘‘nurse effect’’ was obvious even in plated cultures as cells in close proximity to each other, or Like so many scientific discoveries and inventions, genetic to cell groups, grew better (Reinert 1963). transformation was discovered by chance, and not by In the mean time, Hildebrandt’s group developed the design, by Frederick Griffith, a British medical officer, in microculture chamber for the culture of completely isolated 1928. He was trying to develop a vaccine for prevention of cells, which were totally removed from the influence of pneumonia epidemics after the Spanish flu pandemic had other cells. A single cell was placed in a microdrop of killed nearly 50 million people at the end of World War I. nutrient solution on a glass microscope slide. A cover glass He used two strains of Streptococcus pneumoniae: the non- was placed on either side of the cell on droplets of heavy virulent rough strain (R strain) which lacked a polysac- mineral oil. A third cover glass was placed to bridge the charide capsule, and the deadly smooth strain (S strain) two cover glasses to form a ‘‘microculture chamber’’. The which had a polysaccharide capsule (Griffith 1928). Mice culture was then sealed all around with mineral oil. Such injected with S strain bacteria developed pneumonia and preparations were ideal for monitoring under the micro- died after a few days, while those injected with the R strain scope, and were used by Jones et al. (1960) to obtain many remained healthy. The infectious nature of the S strain single cell clones. However, they still found it necessary to could be abolished by heat inactivation. However, mice use a ‘‘conditioned’’ nutrient solution (medium from an injected with a mixture of inactivated S strain and normal actively growing culture), and generally have more than R strain developed pneumonia. In his attempt to understand one cell per culture in order to obtain continued cell divi- this curious and unexpected finding, Griffith discovered sions and growth. that all of the bacteria isolated from mice that had been Direct and unequivocal evidence of the totipotency of injected with a mixture of inactivated S and normal R plant cells was finally provided by Vasil and Hildebrandt strain, were of the S strain, were infectious, and maintained (1965a, b, 1967), who regenerated plants from a com- their phenotype over many generations. He postulated that pletely isolated single cell of a hybrid tobacco grown in a some unknown ‘‘transforming principle’’ from the inacti- fresh (as opposed to conditioned) nutrient solution in a vated S strain had converted the non-capsulated and non- microculture chamber. These results not only validated the virulent R strain into the encapsulated and virulent S strain. concept of totipotency, which was one of the most Griffith was killed in 1941 during an air raid in London, important tenets of the Cell Theory of Schleiden and and the genetic nature of his ‘‘transforming principle’’, Schwann, but also created the theoretical framework for the DNA, was not determined until 1944, by Oswald T. Avery, genetic transformation of plants in the 1980s. Steward, who a Canadian physician and bacteriologist, and his colleagues had been openly skeptical of the possibility of regenerating Colin M. MacLeod and Maclyn McCarty (Avery et al. plants from isolated single cells during a visit to 1944). They found that extracts of heat-inactivated S strain 123 Plant Cell Rep (2008) 27:1423–1440 1431 containing almost pure DNA were capable of transforming plant growth regulators for continued cell divisions (White R strain bacteria, but lost this ability when treated with and Braun 1942; Braun 1958, 1982). He proposed that a deoxyribonulcease. In so doing, they established the role of tumor-inducing principle (TIP), present in the bacteria, was DNA as the basis of heredity. responsible for hormone-independent autonomous growth, During the past four decades, a wide variety of experi- and transformation (Braun 1947). A number of substances, mental approaches have been proposed, and used, as such as toxins, plant growth regulators, bacteriophages, possible means to improve the quality as well as produc- chromosomal DNA, small RNAs, etc., were at various tivity of crops in order to meet increasing demands for food times considered to be the TIP. (Vasil 2003a). Extensive world-wide efforts to create novel A key discovery in the development of Agrobacterium genetic variability in plants by means of embryo rescue as a vector for plant transformation was made by Georges (Raghavan 1986), androgenetic haploids (Guha and Morel, a former student of Roger Gautheret, in Versailles Maheshwari 1966; Nitsch and Nitsch 1969; Guha-Muk- (France). He found that bacteria-free tumor cells made and herjee 1999), somatic hybrids (Giles 1983; Gleba and excreted large amounts of opines (octopine, nopaline, etc.), Sytnik 1984), etc., have proven to be useful only in a few a new class of metabolites (amino acid and sugar deriva- species, and have generally not lived up to their purported tives) that are not formed by normal plant cells (Menage´ promise. Others, such as the much-hyped somaclonal var- and Morel 1964; Morel et al. 1969; Petit et al. 1970). The iation (Larkin and Scowcroft 1981), have been found to be opines were used by the bacteria as a sole source of of dubious value. Arguably, much of the success in the nitrogen and carbon. The type of opine—octopine or production of plants with novel and useful agronomic traits nopaline—formed by crown gall cells was a function of the has come from the infinitely more precise and predictable bacterial strain used for transformation, rather than the methods of genetic transformation, in which well charac- plant (Goldman et al. 1968; Petit et al. 1970; Bomhoff et al. terized single or multiple genes introduced into single cells 1976). This finding strongly indicated that a functional are stably integrated into the nuclear genome, and are bacterial gene had become a part of the plant cell, but this transmitted to progeny as dominant Mendelian genes. revolutionary idea was initially met with considerable The following three methods are widely used for the skepticism. Nevertheless, it stimulated attempts to identify introduction of foreign genes into plants: (1) Indirect the TIP, to search for the presence of agrobacterial DNA in or vector-based gene transfer, such as A. tumefaciens- crown gall cells, and to elucidate the genetic basis of tumor mediated transformation. (2) Direct DNA delivery into formation. Several research groups in Europe and the protoplasts by osmotic or electric shock. (3) Direct DNA United States—led by Jeff Schell at Ghent in Belgium, Rob delivery into intact cells or tissues by high velocity bom- Schilperoort at Leiden in The Netherlands, and Eugene bardment of DNA-coated microprojectiles. Nester at Seattle in the United States—embarked on the difficult task of resolving this challenging problem (see Agrobacterium-mediated transformation Kahl and Schell 1982; Tzfira and Citovsky 2008). It was first thought that tumor induction might be The tumor-forming neoplastic crown-gall disease of plants, caused by an extrachromosomal element (plasmid) car- which causes considerable damage to perennial crops, has rying tumor-inducing genes, or by a tumorigenic virus been known for a long time. However, it was not until similar to oncogenic viruses that had been found in 100 years ago that Smith and Townsend (1907) identified animals (Kerr 1969, 1971; Hamilton and Fall 1971; A. tumefaciens as its causative agent (see Braun 1982, for Hamilton and Chopan 1975). These efforts were greatly the colorful and fascinating history of the early work on aided by the timely discovery of restriction enzymes and crown gall). The prevailing view, until the 1990s, was that the development of various recombinant DNA technolo- only dicotyledonous species were susceptible to infection gies that for the first time made it possible to dissect the by Agrobacterium, whereas monocotyledons and gymno- genome into discrete fragments, which could then be sperms were considered to be outside its natural host range separated by gel electrophoresis to detect specific, short as there were no reports of tumor formation in these gene sequences in large genomes (Southern 1975). The species. timely availability of the new experimental tools played Armin C. Braun, working at the Rockefeller Institute an important role in the discovery by Ivo Zaenen in (now Rockefeller University in New York), and a pioneer Schell’s group at Ghent that the virulent strains of in crown gall research, had demonstrated the transformed Agrobacterium contained a large extrachromosomal ele- nature of crown gall tumor cells by showing that tumor ment that harbored the genes responsible for the induction cells which were free of bacteria could be grown indefi- of crown gall (Zaenen et al. 1974). The extrachromosomal nitely on hormone-free nutrient solution, while normal, element was shown to be an exceptionally large plas- non-transformed cells required media supplemented with mid—megaplasmid—and aptly named the tumor-inducing 123 1432 Plant Cell Rep (2008) 27:1423–1440 or Ti plasmid. The loss of virulence by virulent strains of There was strong competition to develop the Ti plas- bacteria grown at high temperatures was accompanied by mid as a vector for plant transformation. To this end, the loss of the Ti plasmid (Van Larebeke et al. 1974; genes were introduced into the T-DNA region in the Zaenen et al. 1974). The transfer of the Ti plasmid to a laboratories of Schell, Schilperoort, and Chilton, who had non-virulent strain converted it into a tumor-inducing earlier worked in Nester’s laboratory (Van Haute et al. virulent strain (Van Larebeke et al. 1975). Only a small 1983; De Framond et al.1983; Hoekema et al. 1983). A part of the Ti plasmid, christened T-DNA, was shown to problem that had been noticed early was that crown gall be responsible for tumor formation (Chilton et al. 1977). tumor tissues growing on hormone-free media formed It was present only in the nuclear DNA fraction of tumor only highly aberrant shoots. The autonomous nature of cells (Chilton et al. 1980; Willmitzer et al. 1980), and was crown gall tissues, and the formation of aberrant shoots, found to integrate at random sites rather than at any was found to be related to the presence of large amounts specific locus. These investigations also made it clear that of auxin and cytokinin in tumor cells, which were formed the capacity of crown gall tumor cells to undergo by the auxin and cytokinin synthesis genes located in autonomous growth was related to their capacity, acquired T-DNA (Barry et al. 1984; Schro¨der et al. 1984; as a result of transformation, to synthesize plant growth Thomashow et al. 1986). substances and metabolites required for sustained growth. Deletion of the auxin and cytokinin synthesis genes In order to appreciate the many technical difficulties and from T-DNA produced transformed tissues that required challenges faced by the early workers, one needs only to auxin and cytokinin for continued growth in culture. read the fascinating account provided by Chilton (2001): Normal plants were regenerated from such cultures by ‘‘The catalog of restriction endonucleases was unrecog- careful manipulation of the level of auxin and cytokinin nizably thin. What few enzymes were available often were added to nutrient solutions (Bevan et al. 1983; Fraley tainted. Kits were unknown. Procedures often did not work. et al. 1983; Herrera-Estrella et al. 1983; De Block et al. We sized DNA and determined its percentage of G and C 1984; Horsch et al. 1984, 1985). These historic results, in the model E ultracentrifuge. We measured small vol- ushering in the era of plant biotechnology, were first umes with 5-, 20-, 50-, or 100 lL glass capillaries. We presented at the Miami Winter Symposium, on 18 January cultured our plant calli in jelly jars and fleakers. Instead of 1983, by the groups led by Mary-Dell Chilton, Robb T. laminar flow hoods we worked in still air hoods. …we Fraley, and Jeff Schell. taught ourselves how to do gel electrophoresis, and we At that time, there were no reports of Agrobacterium- designed and built our own gel rigs. …We made combs induced tumor formation in monocotyledonous species, from square aluminum rod, using double-stick tape to which include the economically important cereals that mount teeth that were pieces of glass cut with great diffi- account for 66% of the world food supply. This led to the culty from microscope slides’’. widely held view that the new technology could not be Detailed examination of T-DNA isolated from a number used to transform cereals. Elaborate arguments and sche- of independently transformed lines showed that it was a matics were presented as to how and why Agrobacterium- well-defined segment of the Ti plasmid, and that it was mediated transformation would not work with cereals transferred to the plant cell without any changes as a linear (Potrykus 1990). However, continued efforts by the Schell stretch of DNA. The fact that a readily identifiable part (T- and Schilperoort groups soon demonstrated T-DNA trans- DNA) of the Ti plasmid was transferred and integrated into formation of a few monocot species (Hernalsteens et al. the nuclear genome of the host plant during tumor forma- 1984; Hooykaas-Van Slogteren et al. 1984). Additional tion (transformation), suggested that the plasmid could be evidence came from the development of agro-infection, a used as a vector to transfer other genes. technique by which Agrobacterium is used to transfer a This was indeed a fortunate coincidence. Agrobacterium virus into a higher plant (Grimsley et al. 1986). One of the is the only prokaryotic organism that has the natural ability major barriers to the use of Agrobacterium to transform to transfer DNA to eukaryotic cells (Ream 1989; Tzfira and cereals was the absence of wound response, and the asso- Citovsky 2008). This unique characteristic of Agrobacte- ciated activation of virulence genes. These problems were rium for inter-kingdom transfer of genes, evolved millions overcome by Toshihiko Komari and his colleagues in the of years ago in nature in the form of a highly effective laboratories of Japan Tobacco Inc. The co-cultivation of transformation vector (Ti plasmid), was soon to be actively dividing embryogenic cells with super-virulent exploited widely for the genetic transformation of plants. It strains of A. tumefaciens in the presence of acetosyringone, is humbling to realize that humans were to use an approach a potent inducer of virulence genes, led to Agrobacterium- that a ‘‘lowly’’ bacterium had evolved millions of years ago mediated transformation of rice first, and then all of the in order to engineer plants to custom produce its ‘‘food’’ cereals (Hiei et al. 1994; see also Komari and Kubo 1999; (opines). Vasil 1999, 2005, 2007). 123 Plant Cell Rep (2008) 27:1423–1440 1433

Direct DNA delivery into protoplasts into protoplasts developed during the early 1980s (Marton et al. 1979; Davey et al. 1980; Paszkowski et al. 1984; As a newly appointed lecturer in plant physiology at the Shillito 1999), proved to be especially useful for cereals University of Nottingham (UK), Edward C. Cocking was which were considered at the time to be outside the host interested in producing ‘‘isolated cells from plants with the range of Agrobacterium, and therefore, not amenable to aspiration of developing a plant cell ‘‘bacterial’’ type of Agrobacterium-mediated transformation. Indeed, grasses culture system in which plant cells would divide, separate and cereals were first transformed using protoplasts iso- and produce a culture of single cells’’ (Cocking 2000). He lated from cell suspension cultures (Potrykus et al. 1985; successfully isolated protoplasts by incubating segments of Fromm et al. 1986; Hauptmann et al. 1988; Rhodes et al. roots and other tissues in a cellulose preparation from 1988; see Vasil and Vasil 1992, Vasil 1999). Myrothecium verrucaria, or in filtrates of fungal cultures Use of protoplasts for genetic transformation became containing crude mixtures of cell wall hydrolyzing less attractive after it was shown that monocotyledons, enzymes (Cocking 1960, 2000; see also Vasil 1976; Giles including the cereals, could be transformed by co-cultiva- 1983). The small numbers of isolated protoplasts obtained tion of embryogenic cell cultures and super-virulent strains were used mostly for physiological studies. It was the of A. tumefaciens, in the presence of acetosyringone. availability of commercial preparations of Trichoderma Nevertheless, it is important to note that transformation of viride cellulase and other cell wall degrading enzyme cereals was first achieved by the use of embryogenic preparations from Japan, which made it possible to isolate protoplasts. large numbers of protoplasts from leaf and other tissues. Two key developments in 1970 and 1971, pointed to the Direct DNA delivery into intact cells potential use of protoplasts for plant improvement. Tobacco mesophyll protoplasts cultured by Itaru Takebe, a Technical difficulties faced with the isolation and long- distinguished and soft-spoken Japanese plant virologist, term maintenance of embryogenic cultures (the only source regenerated cell walls, underwent sustained divisions of totipotent cereal protoplasts), as well as perceived (Takebe et al. 1968; Nagata and Takebe 1970), and limitations of Agrobacterium-mediated transformation, regenerated whole plants (Takebe et al. 1971). At the same encouraged further search for universal methods of trans- time, Cocking’s group showed that protoplasts of diverse formation. The most notable success in these efforts was species—irrespective of taxonomic relationships—could achieved by John Sanford, at Cornell University (US). be fused by chemical treatment (Power et al. 1970). Taken Sanford was a plant breeder, with neither any interest nor together, the reports from Takebe’s and Cocking’s labo- training in molecular biology or plant tissue culture. Fol- ratories pointed to the possibility of producing novel and lowing on the earlier studies of Kamla K. Pandey in New useful hybrids by protoplast fusion. This created a whole Zealand, and Dieter Hess in Germany (see Box 3), he was new field of research that attracted the interest of hundreds ‘‘looking for ways to utilize pollen to transmit foreign of scientists around the world. During the next 20 years, DNA into natural zygotes and embryos, thereby producing plants were regenerated from protoplasts (mostly leaf transgenic seed directly’’ (Sanford 2000). These included protoplasts) of scores of species. Once again, regeneration the use of irradiated pollen, direct uptake of DNA into of protoplasts of cereals and other monocots proved to be pollen, electroporation of pollen, agro-infection of pollen, difficult. There was a long and vigorous debate about the and cutting holes in pollen by microlaser beams to facili- best strategies to regenerate plants from cereal protoplasts tate DNA entry. These efforts were soon abandoned as (Vasil 2005), until it was shown decisively that protoplasts none of the experiments yielded convincing evidence of isolated from embryogenic cell suspension cultures (Vasil transformation. He and Ed Wolf, who was an electrical and Vasil 1980, 1992) were the best—and possibly the engineer at Cornell, then developed the idea of shooting only—source of totipotent cereal protoplasts (see Cocking DNA-coated tungsten particles into epidermal cells of 2000; Potrykus 2001; Vasil 2005). Over the years a variety onion with the blast of a BB-gun (Klein et al. 1987; San- of somatic hybrids of related as well as unrelated species ford et al. 1987). These attempts were initially met with have been obtained. Unfortunately, few of these—except laughter and ridicule by their colleagues at Cornell, but it those of Brassica, Citrus, Solanum spp.—have proven to be was the crude BB-gun-based ‘‘Macroparticle Accelerator’’ of any practical use. that later evolved into the hugely successful biolistic Indeed, the principal use of protoplasts has been in the delivery system or the gene gun (Sanford 2000). Early genetic transformation of plants. The absence of the cell versions of the instrument actually used blank 0.22 caliber wall allows DNA to be easily delivered into protoplasts by bullets to accelerate the DNA-coated microparticles. They osmotic or electric shock, which temporarily perturbs the were eventually replaced with a safer version which uses plasma membrane. Methods for the direct delivery of DNA air pressure to achieve the same results. Within a few years 123 1434 Plant Cell Rep (2008) 27:1423–1440

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 micropro- look at the achievements of the past 25 years, from the jectiles was used to transform a wide variety of plant production of the first biotech plants in 1983, followed by species, including the economically important cereals and the first commercial cultivation in 1996, to its present legumes, and many woody species. Along with Agrobac- status (see James 2007; Box 4). terium, it is now the most commonly used method for the Canola, cotton, maize and soybean are the only major genetic transformation of plants. 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 Tales of unconfirmed results wide variety of useful genes have been transformed into a large number of economically important plants, including The history of plant biotechnology is replete with claims of most of the food crops, scores of varieties of fruits and genetic transformation of plants achieved with novel and vegetables, and many tree species. Biotech crops are also often simple technologies, including those that do not require being increasingly used for the production of vaccines and the use of tissue cultures (Box 3). Many of these studies were many pharmaceuticals (Routier and Nickell 1956; Tulecke authored by well-known and respected scientists, and pub- and Nickell 1959; Staba 1980; Constabel and Vasil 1987, lished in reputable journals. Initially, they generated much 1988; Ma et al. 2005; Fox 2006; Murphy 2007). Admittedly, enthusiasm, but never gained the confidence of the broader there is an urgent need to broaden the diversity of biotech scientific community as they failed to provide unambiguous crops by introducing a wider variety of genes into a larger molecular and genetic evidence of transformation, and could number of species. Nevertheless, these are, by any standard not be independently confirmed. Consequently, they are of measurement, truly remarkable achievements for a rela- seldom used and have largely been abandoned. tively new technology. Mathhias Jakob Schleiden, Theodor Schwann, Gottlied Haberlandt, Frederick Griffith, and all Twenty-five years of plant biotechnology others who came after them, would be justifiably proud. Plant biotechnology has been also advanced consider- Keeping in mind the historical perspective of plant bio- ably by the sequencing of plant genomes, such as those of technology provided in the preceding pages, it is useful to 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-Gyo¨rgyi, 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 interest in the past or concern for the future. Most con- regulation, expression and function, has become available, temporary publications lack a historical perspective, and and has improved our understanding of the molecular basis few scientists bother to consult or cite literature that is of plant development. These insights, combined with the older than 10–15 years. Literature searches are generally availability of molecular maps and markers, has helped confined to what is readily available on the internet. Such plant breeders produce new and useful varieties. Molecular historical ignorance and neglect present a highly distorted breeding is increasingly becoming an important and pow- and biased view of science, deny credit to earlier workers, erful tool for plant improvement and variety development. and unjustifiably inflate the importance of modern Fears and exaggerated claims of possible adverse effects research. Authors, reviewers and editors share equal of biotech foods and crops on humans and the environment responsibility for this very undesirable practice, which are unfounded and have no basis in fact. Indeed, it is is unhealthy for science and for the training of young remarkable that no such ill effects have been documented scientists. There is also an increasing tendency among after 12 years of extensive cultivation in diverse environ- scientists to be clannish, and to follow only the most ments, and the consumption of biotech foods by more than popular areas of investigation. This habit is fueled by the a billion humans and even a larger number of animals. unwillingness of funding agencies to support and Many practical applications of plant biotechnology, encourage truly novel and high risk proposals. A study of including the introduction into the market place of other the history of plant biotechnology clearly shows that there biotech crops with useful and highly desirable character- are many important and meaningful lessons to be learnt istics, have been prevented by political and ideological from the wisdom and cumulative experience of all those opposition from vested interests—including the European who came before us (Box 5). It is up to us now, to benefit Union, a few developing countries, and some self-serving from the opportunities presented by the history of our environmental groups—and by the many redundant, overly science, and reverse the growing trends of historical stringent, time-consuming and exorbitantly expensive ignorance and illiteracy. 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 References many of the unnecessary restrictions imposed by over- zealous regulators, and politically motivated decisions by Amici GB (1824) Observations microscopiques sur diverses espe`ces de plantes. Ann Sci Nat Bot 2:41–70, 211–248 some countries in Europe and elsewhere. Amici GB (1830) Note sur le mode d’action du pollen sur le stigmate. Extrait d’une lettre d’Amici a` Mirbel. Ann Sci Nat Bot 21:329– 332 Amici GB (1844) Quatrieme reunion des naturalists italiens. Flora Lessons from the history of plant biotechnology 1:359 Amici GB (1847) Sur la fe´condation des Orchide´es. Ann Sci Nat Bot It is said that history teaches us many valuable lessons, 7(8):193–205 Avery OT, MacLeod CM, McCarty M (1944) Studies on the chemical and that those who ignore it do it at their own peril. Yet, nature of the substance inducing transformation of pneumococ- history is not given much importance in modern science cal types. Induction of transformation by a desoxyribonucleic education and research. The unfortunate result is that we acid fraction isolated from Pneumococcus Type III. J Exp Med are producing scientists who are historically illiterate. 79:137–158 Backs-Hu¨semann D, Reinert J (1970) Embryobildung durch isolierte Like the rest of modern society, many scientists today are Einzellen aus Gewebekulturen von Daucus carota. Protoplasma interested more in the ‘‘here and now’’, and show little 70:49–60 123 1436 Plant Cell Rep (2008) 27:1423–1440

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