The Scientific Roots of Modern Plant Biotechnology
Year: 2008
Type: article
Abstract: Chronology of Research Leading to Modern Plant Biotechnology. Some of the major steps and publications are listed. Further details, definitions, and additional citations are provided in the main text. Philip White worked at the Rockefeller Institute for Medical Research in Princeton, New Jersey in the 1930s to develop an experimental system with which to study metabolism in a completely undifferentiated tissue where all cells are identical and hence exert similar influences on one another. White (1939a) defined a plant tissue culture as a system in which cells satisfied two main requirements of remaining “undifferentiated yet capable of unlimited growth” (White, 1939a). Earlier attempts, starting in the late nineteenth century, to grow plant parts in isolation from the organism did not satisfy these requirements and failed for a variety of reasons, including microbial contamination, inadequate nutrient media, and poor selection of tissues to culture (White, 1931; Gautheret, 1983; Höxtermann, 1997). The most successful of the early attempts involved the culture of maize, pea, and cotton root tips (Kotte, 1922; Robbins, 1922). These could be excised from the plant with minimal trauma and grown aseptically for a few weeks in nutrient media, but ultimately growth ceased. White (1934) succeeded in growing excised tomato root tips for potentially unlimited periods of time in a liquid medium containing inorganic salts, 2% sucrose, and 0.01% yeast extract. Tips were excised from seedling roots and subcultured at regular intervals. After 52 subcultures over >400 d, the cultured roots showed no diminution of growth rate. White calculated that by this time any nutrients and regulatory substances in the original root tip would have been diluted to ∼10−40, and he concluded that the nutrient medium had supported indefinite growth of the root tips. Thus, while he had demonstrated “potentially unlimited growth,” the second part of his definition of a tissue culture had not been met because the roots clearly were not undifferentiated. White (1939a) addressed this second question using a hybrid between Nicotiana glauca and N. langsdorffii. The hybrid plants produced tumor-like calluses and galls on the stem and leaves. Tissue removed aseptically from young stems was cultured on the same nutrient medium used for tomato roots. Proliferated masses from the original explants were divided and subcultured at weekly intervals through 40 subcultures. White calculated that any material from the original explant would have been diluted to at least 10−17 by this time and that the cultures therefore satisfied the criterion of a capacity for unlimited growth. The question remained as to whether the cells in the culture were undifferentiated. Histological examination revealed only mature parenchyma cells, regions of dividing meristematic cells, and occasional isolated xylem cells. Despite this level of cellular heterogeneity, White concluded that these tissues approximated an undifferentiated condition and grew for potentially unlimited periods and therefore were true tissue cultures. Scientific results are greatly strengthened when other workers with appropriate expertise replicate the original findings. Remarkably, this occurred within 6 weeks of the publication of White's results. Two French workers, Roger Gautheret in Paris and Pierre Nobécourt in Grenoble, both of whom had been attempting to culture plant tissues for several years, reported potentially unlimited growth of cultures derived from carrot tap root tissue when the growth substance indole-3-acetic acid (IAA) was incorporated in the culture medium (Gautheret, 1939; Nobécourt, 1939). Neither cited White (1939a), and presumably they were unaware of his results. Neither study was as detailed as White's. Gautheret cultured tissue for 13 months making bimonthly subcultures, and Nobécourt cultured tissue for 20 months making seven subcultures. White's objective, of studying metabolism in an undifferentiated tissue wherein all the cells are identical and presumably exert similar influences on one another, appears not to have been followed up by him or his contemporaries. It was not until many years later that other scientists began using methods developed by White to study cellular metabolism. Notably, H.E. Street at Manchester University used cultured excised tomato roots in an extensive series of studies to examine the metabolism of inorganic ions, carbohydrates, amino acids, and hormones (Street, 1957). A more direct approach to White's original intention was followed by F.C. Steward and colleagues at Cornell University. They examined metabolic states in three contrasting carrot tap root cultures: secondary phloem cells as they existed in the intact root, excised tissue maintained on a minimal medium where cells grew predominantly by enlargement, and actively metabolizing cells stimulated by coconut milk in the medium to divide as rapidly as possible. They documented significant differences in rates of accumulation of RNA and DNA under the different culture conditions (Steward et al., 1952, 1964). With White's original goal for plant tissue culture having been achieved and confirmed, attention turned in other directions. Gautheret and Nobécourt both reported the occurrence of roots that developed on their carrot tissue cultures. White (1939b) found that shoots were produced from Nicotiana tumor tissues submerged in a liquid medium, and Michael Levine (Levine, 1947) reported the spontaneous formation of shoots and roots on cultured carrot tissue. These observations turned attention to the question of totipotency. That is, did tissue cultured cells retain the full genetic competence of the zygote to form a complete plant? This question was addressed by Folke Skoog and his collaborators at the University of Wisconsin. Starting from White's discovery of shoot formation in submerged Nicotiana tumor cultures, they found that auxin (IAA) was a potent inhibitor of shoot formation in tobacco tissue cultures (cv Wisconsin 38), but high concentrations of auxin stimulated the formation of roots. The nucleic acid base adenine supplied with low auxin concentrations stimulated shoot formation in these cultured tissues. The amount of shoot and root formation depended on the proportions of auxin and adenine supplied in the culture medium (Skoog and Tsui, 1948). In further work, they isolated from autoclaved DNA an adenine derivative, 6-furfurylaminopurine, that they named kinetin. Using tobacco callus cultures they found that by adjusting the relative concentrations of auxin and kinetin in the culture medium it was possible to induce the formation of shoots and roots or the growth of undifferentiated callus (Skoog and Miller, 1957). Thus, they demonstrated that tobacco callus tissues retained the potentialities of the zygote to form both shoots and roots, but their research did not prove that a single cell had these potentialities. Studies that approached this question were initiated in the laboratory of F.C. Steward at Cornell University. Previously this group had compared metabolism of carrot secondary phloem explants in basal medium or medium supplemented with coconut milk, a source of cell division factors. Explants in the coconut milk medium produced new cells that became detached and proliferated freely in the culture medium, resulting in large numbers of single cells and cell aggregates. Among these aggregates they noted some that formed roots and subsequently shoots to produce whole plants that could later be transferred to agar media and then to soil where they flowered and completed the life cycle (Steward, 1958; Steward et al., 1958a, 1958b). From these experiments they concluded that “no single parenchyma cell can directly recapitulate the familiar facts of embryology, but, through the formation first of an unorganized tissue culture, which is in fact a colony of dividing cells, the necessary degree of organization is recaptured, first to form roots and then to form shoots” (Steward et al., 1958a). The implication of this statement was that the origin of the regenerated plants was from single cells. If so, cellular totipotency would have been proven for carrot cells, at least. However, other workers (cited in Sussex, 1972) pointed out that there was no incontrovertible proof that single cells rather than cell aggregates that may have contained cells with different genetic potentialities were the source of the new plants that they obtained. These tissue culture studies that demonstrated the potentially unlimited growth of undifferentiated cells and the production from them of roots, shoots, and entire plants did not contribute further to the questions that we are examining because of their failure to assure the single cell clonal origin of regenerated plants and thus the genetic totipotency of single cells. However, the study of plant tissue and suspension cultures was continued in different directions, including the commercial production of secondary products (Ramawat and Merillon, 2007) and commercial production of trees, crops, and horticultural plants, most notably species of orchids (Arditti and Krikorian, 1996). Gottlieb Haberlandt, working in Graz, Austria, was the first to culture isolated somatic cells of higher plants in vitro. He began these investigations in 1898 and published the results in 1902 (Haberlandt, 1902). His intention was to study “the properties and potentialities which the cell as an elementary organism possesses” (Krikorian and Berquam, 1969; translation from Haberlandt's text). Although Haberlandt failed to induce divisions in any of the cells that he cultured, he is recognized as the founder of plant cell culture because of the novelty of the methods he proposed and the concluding paragraph in his article “…I believe, in conclusion, that I am not making too bold a prediction if I point to the possibility that, in this way, one could successfully cultivate artificial embryos from vegetative cells.” Recognizing the lack of knowledge of the nutrient requirements of higher plant cells, he used as a culture medium the seven inorganic elements that had been identified by Knop (1865) as sufficient for the water culture of higher plants, with additions of sucrose, dextrose, glycerine, asparagine, and peptone in various concentrations and combinations. Haberlandt first attempted to culture green, photosynthetic cells from leaf bract of were in liquid until examination revealed isolated and parenchyma cells. These were then transferred by to or of culture was attempted by and but failed to and were maintained in at or in Some cells remained for a in cultures but in Haberlandt noted several in cell the culture in and and cell remained in and However, no cells were to He then attempted to culture cells from other photosynthetic cells from of of of and cells of and with lack of in and Berquam, In Haberlandt's failure to dividing cells can be to lack of microbial culture media that hormones and growth that were at that and his selection of mature cells. However, he to plant and cell culture studies by his including the of culture methods and of to single cells. his prediction that of vegetative cells with that were then to produce that growth of culture and his prediction that be used as of the culture medium to induce divisions in isolated vegetative cells the of coconut of these to in cell culture Despite continued by Haberlandt's collaborators and no significant on cultures derived from single cells was for years when and at the University of Wisconsin this question et al., They used tissue cultures of tobacco and carrot and cultures of and the capacity for growth in liquid culture media, they identified several that produced large numbers of single cells. cells were then transferred by to on cultures of the same or other species growing on an agar of and divided and produced cultures, some of which were transferred through or more agar subcultures diminution of growth rate. produced roots, and hybrid tobacco produced shoots on media containing adenine and kinetin. and and transferred single tobacco hybrid cells to a of culture medium on a that could be and under were to divide to form a and subsequently a that was transferred to an agar medium for further where roots and shoots were shoots were transferred to soil where they produced and Thus, these studies demonstrated that derived from single cultured cells had the capacity to produce whole However, they did not prove that the whole plants were the direct of a single rather than the of a tissue within which or other genetic have growth to produce a tissue The Haberlandt's prediction that could successfully cultivate artificial embryos from vegetative from the research of and in They cultured single carrot cells from suspension cultures on where they could be and cells divided to form a of and parenchyma cells, and the cells developed and embryos with and and This early research in plant tissue culture demonstrated that tissues isolated from plants can be grown in culture for indefinite periods of they can produce shoots and roots, and single isolated cells in culture can produce These studies provided the for genetic of plants, as In working at the of Plant on of plants, reported that the of of Paris was a that he named and This was subsequently as and then as that this was the of the by from galls an agar medium, plants with subcultures of the from the galls and galls on new plants were produced on stems of and and on roots of and that were with and The galls on which had been working for several years the In a et reported that the isolated from Paris on species but not on other or the single that they secondary that developed on stems or of plants of some species at some from the on he concluded that secondary developed from tumor that were from the et al., that tumor be to of that occurred in of and In to the of and that their results on the origin of in to the between plant and for which he a of from the Medical in and in he was to the of the for However, was on the of until at the Rockefeller Institute for Medical began an of in the that for 40 years and that the for the studies that were to He began this by examining the question of tumor between and secondary in examining tissue between the and secondary he found no for a tumor and concluded that the of formation of secondary may not be identical to that in the formation of the tumor Although tissues did not cultures of the it had been that the had been at some in the of the Thus, the discovery that secondary direct cellular with the tumor questions as to their origin and some of which are with Philip White of the same to these they out to whether cells under the of the the capacity for growth. they secondary in and showed by tissue from these that by more than one from the were of they cultured tissue from secondary for subcultures on a basal medium to growth of tissue grew tissue from plants grew They did not growth of in any of these subcultures. for the of contained within the cultured secondary tumor they these with results. tumor tissue when or plants produced From these experiments they concluded that the secondary tumor tissue had the capacity for growth both in and in and that this had been by the and The that the plant cells had been with the to experiments to the time when this These experiments depended on observations that was at a of plants this for several were with to the at for then transferred to to for and to for continued growth. transferred to the failed to form when to transferred to produced and transferred to produced to on plants that had not been to the These large were Thus, the have occurred between and d, which time tumor growth became and of the continued of experiments the time of tumor formation to to in plants maintained at These experiments that an resulting from the was for the and possible for a metabolic of the a by the to a a of the cell that is capable of in the cell a and a or other in the organism This was was named the tumor and 1948). of the of the from research by at the was studying amino acid in attention to metabolism and they compared tissue cultures with cultures of this they found that the contained a of with that had been identified from and named and 1964). when cultures of the they derivative, a with which they named from the French for et al., They a study of these named or in cultures by different of and showed that with few they contained or and that the produced was to the et al., could not be isolated from or genetic tumor tissue cultures of the same the was proposed to with the from the cell to the plant cell where it was maintained in a in tissue cultures, it a for the Further on the of the from two working at the Research Institute in found that tomato plants with a became with soil containing an of the were then found to be and from concluded that this was “the first for of from a plant to a species of and that this may have from DNA and working at University found that the of was at high They cultures of an at for different of time and found a in the of that at no could be They concluded that this could be by a of the and that from of genetic material from the to plant cells several studies to this However, these on or using the whole results or results that could not be The of these results from a study by working with and at the University of or of cells followed by or and examination revealed the of a large in this The was as one or a few of other to seven different revealed the of of to in in all of were not in different They proposed the that “the 1947) in is by one or several large of various et al., of this within a when three of the of the for were published et al., et al., These results stimulated further attempts to the of whole in plant tumor cells, but these were not successful and the possibility that only part of the was transferred to the This was demonstrated by and colleagues and at the University of They DNA from with the and the were the of which were then to tobacco tumor DNA or to that and showed to tumor DNA et al., that only part of the DNA was in the tumor for the and the transferred had et al., et al., The experiments of et did not questions of the of the DNA in the plant cells, whether or in the or years research in and and reported that DNA was in the cell and not in or et al., et al., the DNA was plant or as an was not yet However, with these two studies showed by and of a of and plant DNA that the was the plant in tobacco cell et al., et al., The in the of in plant was to the of the that were to be in This was by by of DNA or by of The first studies using this approach were of and in the of in et al., et al., A containing a in the of the on tobacco plants that to roots. of an the of the of an to that produced were produced on in tissue of the and concluded that “the tumor on relative concentrations of various in a et al., the same and collaborators working with and a detailed genetic of the of the using and defined three tumor and in which and large These three were all in the in all and were found to involved in auxin or for and for both of which steps in auxin (IAA) et and for which the first in et al., et al., A was this and for et al., Using a similar approach of DNA or that were in a of both and tumor were identified by et and et working with and Two of these shoot formation and growth. The root all three did not induce shoot or root the was transferred to plant cells. This fact supported the of et for the of the as a for of the of higher all would be of for and were to be If the was to be the for of of plant three requirements had to be a that in plant cells had to be a that the of the DNA and that would the was a but not and that would in plant cells. at the three research and all reported in that satisfied these and that in plant cells et al., three used the to the as a and or a of this The first publication similar results was that of et The that these was the of plant in which the and the of the were on different With this the on a could be and in then cells that the et al., Using this et working with and used the and the of or of the a of as and and are to plant cells and thus as for cells. The transferred to tobacco cells the as a were and to the et at produced containing the and regulatory regions to I or from or of containing these with cells derived from of and carrot in cells that the and were to of the and et al., the and regulatory with from to stem explants of The of these three research the for the genetic of However, it remained to be that cells could be regenerated intact plants and that the DNA would be in in a of these requirements in several and working with regenerated tobacco plants that contained of that were to the et al., working with and tobacco plants and regenerated plants from callus that developed at the et al., working with and single of tobacco with and regenerated plants from the resulting et al., These plants developed and when grown on an medium in a These three studies all reported successful production of tobacco plants from cells. The of plants was reported by and colleagues of and tomato with et al., They containing a with leaf of these The leaf were subsequently transferred to medium containing and that developed from the callus were and transferred to soil for further growth. plants of all species in a This fact that the regenerated shoots had from single cells and were not in The of some to by to for other methods for DNA plant cells. These and The most successful and used of methods is by the developed by and colleagues at Cornell University in et al., This was the used in the first successful of cells and the of plants from them that the to the et al., These workers at Plant cells from suspension cultures with with containing the This to the which was used to callus cells. were to the and to the by plants were produced from the and of had In other they suspension culture cells with a of two one containing the and the other containing the plants both The concluded that system a for both the study of plant and the of one of the major et al., Modern plant defined as the genetic of plants, from a of research and The that this in tissue culture, of single cell culture, discovery of cellular the of the as of cells, of between the that tumor and and of cells. The culture, culture, cultures, plant and some and were from other as plant culture medium requirements and genetic in but many as the research that to the genetic of A of this research is the of publication and From to 20 that are as were published by This is a of and From to 13 were published by with a of and From to were published by with a of and These numbers the of plant and the of it as a where large laboratory have to the This the between research and and the for In we can the of research that ultimately to the genetic of plants, but could we have not in or when the three in single plant cell culture, and plant tissue culture, were It was not until the early that research were the of the as a for of of The first to be was a in In were on in and by of these were with in was the in followed by cotton and The first of were in to in had been at in on The most species were maize, and In it was that species of had been I and for the of this and for
Source: The Plant Cell
Author: Ian M. Sussex
Institution: Yale University
Language: English
Subfield: Plant Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2053210153