As
vividly described in the review by Vasil (2008), the alleged friendship between
the botanist Matthias Jakob Schleiden (1804–1881) and the animal physiologist Theodor
Schwann (1810–1882) stimulated, among others, the formation of the “cell
theory”. Schleiden (1838) was the first to formulate the hypothesis that all plant
or animal structures are composed of cells (or their derivatives) that preserve
the complete functional potential of the organism.
Half a century after the cell theory had
been formulated, both plant and animal biologists started to verify it
experimentally. Initially, attempts were made to demonstrate the “immortality”
of animal and plant somatic cells by means of their in vitro culture. The next
challenge – only successful for plants – was to regenerate functionally
complete organisms from these cultured tissues.
Against the preconceived expectation
that the high functional autonomy of autotrophic plant cells would make it
easier to cultivate them in vitro, it was an “animal” researcher who won the
first round of this race when Ross Granville Harrison, working at Johns Hopkins
Medical School and later at Yale University, published his results of
experiments from 1907 to 1910, which established the methodology of animal
tissue culture (Harrison 1907). It took a further quarter of a century before
comparable results were achieved for plants, by the independent work of White
(1939), No ´becourt (1939) and Gautheret (1939).
The possible reasons for Haberlandt’s
persistent failure to persuade isolated plant cells to divide in vitro have
been extensively discussed in various critical reviews. Maybe the most
objective conclusion is that reached by Gautheret (1985): Unfortunately, he
(Haberlandt) was influenced by Schleiden and Schwann’s cell theory rather than
by experimental expectation. And he neglected both Duhamel’s results as well as
Vo ¨chting’s and Rechinger’s experiments (Note: experiments with plant parts
containing various meristematic tissues, thus generating calli or regenerating
buds; cf. Gautheret). His dogmatic attitude and the ignorance of the past
explain the failure of his own attempts. But he appreciated very clearly that,
when the technical difficulties were removed, the method of cultivating
isolated cells in nutrient solution should make possible the experimental study
of many outstanding problems from a new point of view. He, therefore, chose to
work with single cells. Appreciating the importance of photosynthesis he
presumed that green cells would be the best material. However, he neglected the
fact that green cells of phanerogams are relatively differentiated and cannot
recover meristematic competence without stimulating substances which were
unknown at the time. He worked with palisade cells of Lamium purpureum, pith
cells from petioles of Eichhornia crassipes, glandular hairs of Pulmonaria and
Urtica, stamen hairs of Tradescantia, stomatal guard cells of Ornithogalum, and
many other materials.
At
this time it was recognized that asepsis was absolutely necessary when culture
media were enriched in organic substances metabolized by microorganisms.
Haberlandt’s media contained glucose and peptone, he carefully avoided
contamination and his cultures remained free of microorganisms. The results,
however, were disappointing. The cells survived for several weeks. They were
capable of synthesizing starch and enlarging, but they were never dividing.
Fifty six years passed before the realization of Haberlandt’s dogmatic
dream....
Undoubtedly, it was mainly thanks to the
use of these phytohormones that Muir et al. (1954, 1958) succeeded in obtaining
new cell colonies from isolated cells of Nicotiana tabacum and Tagetes erecta.
Application of a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), in
the culture medium allowed Steward et al. (1966) not only to obtain a
well-growing suspension culture of carrot callus cells and their aggregates but
also to regenerate from them somatic embryos of potentially unicellular origin.
Eventually, as a result of the long-term competition between the Skoog and
Steward teams to isolate and identify the active compound in the plant “fetal
serum”, i.e. coconut milk (for a review, see Amasino 2005), cytokinins were
first isolated and became a universal component of the plant tissue culture
media. Vimla Vasil and Hildebrandt (1965a, b) convincingly documented the whole
procedure “from a somatic cell to a regenerated plant” by means of time-lapse
photography of individual cells of the hybrid Nicotiana glutinosa ? Nicotiana tabacum.
From a recovered single-cell-based colony, they regenerated the entire flowering
plants, exploiting the results by Skoog and Miller (1957) on the influence of
auxin to cytokinin ratio on regeneration.
These experiments, however, failed to
prove the validity of Haberlandt’s vision “from individual somatic cells to
complete plants”; the problem was that the primary explants used were multicellular
and thus the original callus colonies from which the so-called single-cell
clones were derived. Later technology, how-ever, finally confirmed Haberlandt’s
original dream in all its details. It was shown that when single cells were
stripped of their cell walls to produce the so-called protoplasts (Cocking
1960, 1972, 2000; Takebe et al. 1971), these protoplasts were able to
regenerate not only into walled cells (Nagata and Takebe 1970; Opatrny ´et al.
1975, 1980) but also into complete plants (Takebe et al. 1971; Schumann et al.
1980). Isolated protoplasts, cultured separately in microchambers, could regenerate
real “protoclonal plants”.
Through the alternative methodology of
pollen culture, developed in the late 1970s, new (even haploid) plant organisms
can be generated directly from plant spores.
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