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Orchid micropropagation: the path from laboratory to commercialization and an account of several unappreciated investigators

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A commonly held view is that the ideas and basis for the practice of orchid micropropagation arose de novo in 1960 from the work of Georges Morel in France. In this paper we argue that the crucial developments in micropropagation were made by Gavino Rotor in 1949 in the USA and Hans Thomale in 1957 in Germany, and that Morel's work needs to be seen in the context of a long line of research achievements in the in vitro culture of a wide range of explanted tissues and organs from plants of many species. A critical, historical, analysis of the events as they relate to clonal orchid multiplication is offered here. Two important technical innovations for orchid micropropagation — the use of activated charcoal to darken nutrient media and the adoption of liquid culture environments for part of the process — are examined in detail. In addition, an unusual US patent claiming invention of ‘a method for rapidly reproducing orchids’, especially cattleyas, is analysed. The origin of the micropropagation process claimed in this patent, said by the nominal inventor to go back as far as 1950, is discussed, but the claim remains unsubstantiated. Finally, consideration is given to the problems of adjudicating unequivocal priority for ‘discovery’ of a process as complicated and as broad as micropropagation.
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Orchid micropropagation: the path from
laboratory to commercialization and an account
of several unappreciated investigators
JOSEPH ARDITTI F.L.S.
Department of Developmental and Cell Biology, University of California, Irvine,
CA92697-2300, U.S.A.
AND
ABRAHAM D. KRIKORIAN
Department of Biochemistry and Cell Biology, and Institute for Cell and Developmental Biology,
State University of New York, Stony Brook, NY11794-5215, U.S.A.
Received August 1995, accepted for publication June 1996
A commonly held view is that the ideas and basis for the practice of orchid micropropagation arose de
novo in 1960 from the work of Georges Morel in France. In this paper we argue that the crucial
developments in micropropagation were made by Gavino Rotor in 1949 in the USA and Hans Thomale
in 1957 in Germany, and that Morel’s work needs to be seen in the context of a long line of research
achievements in the in vitro culture of a wide range of explanted tissues and organs from plants of many
species. A critical, historical, analysis of the events as they relate to clonal orchid multiplication is offered
here. Two important technical innovations for orchid micropropagation —the use of activated charcoal
to darken nutrient media and the adoption of liquid culture environments for part of the process— are
examined in detail. In addition, an unusual US patent claiming invention of ‘a method for rapidly
reproducing orchids’, especially cattleyas, is analysed. The origin of the micropropagation process
claimed in this patent, said by the nominal inventor to go back as far as 1950, is discussed, but the claim
remains unsubstantiated. Finally, consideration is given to the problems of adjudicating unequivocal
priority for ‘discovery’ of a process as complicated and as broad as micropropagation.
©1996 The Linnean Society of London
ADDITIONAL KEY WORDS: activated charcoal – ‘mericloning’ – propagation in vitro – stem tip
culture – tissue culture.
CONTENTS
Introduction .......................... 184
Orchid seed germination and the first in vitro culture of orchids ......... 184
Foundations of stem tip culture .................... 187
Plant hormones ....................... 187
Auxins .......................... 188
Coconut water and cytokinins ................... 188
Culture of stem tips ........................ 189
Botanical Journal of the Linnean Society (1996), 122: 183–241. With 56 figures
183
0024–4074/96/011183+ 59 $25.00/0 ©1996 The Linnean Society of London
The second aseptic culture of an orchid explant .............. 194
Plant diseases and meristems ..................... 199
The third aseptic culture of an orchid explant ............... 200
The fourth aseptic culture of an orchid explant ............... 210
A patent ........................... 211
Darkening of media ....................... 212
Terminology and semantics ..................... 212
Subsequent research ....................... 213
Cell and protoplast cultures ..................... 213
Concluding remarks ....................... 220
Acknowledgments ........................ 225
Appendix ........................... 225
References ........................... 228
INTRODUCTION
It is hardly possible to state any truth strongly without apparent injustice to
some other (E. Mach, 1838–1916, quoted in Gaffron, 1969).
Micropropagation, mass rapid clonal propagation of plants in vitro by a technique
commonly referred to as tissue culture
1
(all notes are in the Appendix, pp. 225–228),
is currently used world-wide with numerous species ranging from ferns to trees. Over
the years, a large body of literature dealing with basic science and practical
applications has been generated for this important area of plant biotechnology. Since
a Cymbidium orchid was the first plant to be propagated commercially using this
method, it is appropriate to attempt a reconstruction of the research process which
led to it. Our hope is that by doing so we will convey some of the flavour of the events
associated with the path from laboratory bench to commercialization, and place in
perspective the role played by key participants. This account will also make apparent
the fact that methods of this sort do not emerge suddenly and fully formed in
‘Eureka’ fashion. Rather, they result from the findings of many who may have had
little concern for fame or financial reward. In fact, some may not have appreciated
partially, much less fully, the implication of their work.
ORCHID SEED GERMINATION AND THE FIRST IN VITRO CULTURE OF ORCHIDS
In 1891 a brief notice was published in L’Orchidophile that a grower named
Perrenoud (no first name given) who saw reports in so-called ‘journaux anglais’,
placed sections of Phalaenopsis roots in humid enclosures and obtained a plant
(Anonymous, 1891). This is reminiscent of micropropagation, and while no details
are available, it is known that Phalaenopsis roots can produce buds and plants
Figures 1–11. Historical figures in research on orchid seed germination and micropropagation, plant hormone
studies and tissue culture research. Fig. 1. Professor Lewis Knudson (Arditti, 1990; photograph courtesy Department
of Botany, Cornell University; signature courtesy Cornell University Archives). Fig. 2. Professor Wilhelm Pfeffer
(Wittrock, 1897–1903). Fig. 3. Dr Gavino Rotor (photograph courtesy of Dr Gavino Rotor; signature from a letter
to J.A.). Fig. 4. Phalaenopsis floral-stalk node cultures (Rotor, 1949). Fig. 5. Professor John T. Curtis (Skoog, 1951).
Fig. 6. Professor Gottlieb Haberlandt (White, 1943). Fig. 7. Professor Hans Fitting (photograph from a Kodachrome
slide taken by Ms. Brigitta H. Flick, J.A.’s technician, c. 1970; signature from letter to J.A.). Fig. 8. Professor Kenneth
V. Thimann (photograph from Skoog, 1951; signature from an autographed copy of Went & Thimann, 1937 owned
by J.A.). Fig. 9. Professor Frits W. Went (photograph and signature from Went, 1990). Fig. 10. Professor Johannes
van Overbeek (photograph from Skoog, 1951; signature from letter to J.A.). Fig. 11. Professor Albert F. Blakeslee
(Skoog, 1951).
184 J. ARDITTI AND A. D. KRIKORIAN
185HISTORY OF ORCHID MICROPROPAGATION
(Churchill, Ball & Arditti, 1972); this account, therefore, could perhaps be described
as being part of its pre-history.
Modern orchid micropropagation began in 1949, when “a new [tissue culture or
in vitro], simple and practical method for vegetative [clonal] propagation of
Phalaenopsis [orchids] was developed at Cornell [University]” (Rotor, 1949) five years
before the first published report of orchid stem tip cultures and thus any suggestion
that these methods could be used for micropropagation (Thomale, 1956, 1957). The
nutrient medium used for these cultures was ‘Knudson C’ formulated for the
asymbiotic germination of orchid seeds by Lewis Knudson (1884–1958; Fig. 1),
2
Professor of Plant Physiology at Cornell University (see Arditti, 1990 for a biography
of Knudson).
Knudson’s first solution, a modification of a formulation devised by the German
plant physiologist Wilhelm Pfeffer (1845–1920; Fig. 2) known as ‘Pfeffer’s Solution’,
was his medium B (‘Knudson B’). It was, and still is, a reasonably good medium but
Knudson improved it and published his solution C (‘Knudson C’, KC) in 1946
(Knudson, 1946). This is still used widely for the germination of orchid seeds (Arditti
et al., 1982) and the micropropagation of some orchids (for a review see Arditti &
Ernst, 1993).
Gavino Rotor Jr. (Fig. 3) was born in Manila on 26 March 1917.
3
His mother was
an avid orchid enthusiast and by the time Gavino entered high school he knew the
scientific names of the major Philippines species. He went on to major in agriculture
at the University of the Philippines where he received his B.S. in Agriculture in 1937.
Rotor was waiting to go abroad for further study when World War II broke out. This
caused him to delay but not cancel his plans. He “chose Cornell University for
several reasons, the most important ones being Dr Knudson’s presence there and its
impressive reputation in the horticultural sciences”. After receiving his M.S. degree
in 1947 and “hearing Dr Kenneth Post’s lectures on the effects of day length and
temperature on the growth and flowering of various florist crops … [Rotor] …
decided to focus on the responses of orchids to temperature and day length” for his
doctorate at Cornell University. His major professor was the floriculture crop
physiologist Kenneth Post (1904–1955), whereas Knudson was a member of his
doctoral thesis committee.
Rotor conceived the idea of propagating orchids while attending a lecture by
Knudson on the role of sugars in plant growth. He cut Phalaenopsis inflorescences into
segments and placed nodal sections, each with a bud, on KC in the hope that they
would produce plants. The buds became swollen and leaves appeared after 14–60
days. Roots were produced after 2–3 leaves were formed (Fig. 4). Only seven of 65
buds failed to develop (Rotor, 1949). Knudson’s eyes brightened when Rotor showed
him the first successful propagation and told him how he got the idea from
Knudson’s lecture (Arditti, 1990:48).
There is no question that Gavino Rotor invented micropropagation of orchids and
was the first to publish a scientific report on clonal multiplication of a higher plant
in vitro.
4
Rotor’s method involved a nutrient culture medium, aseptic techniques and
explants. Some might argue that his procedure was not ‘true micropropagation’ for
it only produced one shoot from a given explant with a pre-existing bud. Certainly
his procedure did not involve callus formation or proliferation (for a more extensive
discussion see Arditti & Ernst, 1993). However, neither multiple plantlet production
nor callus proliferation are part of the definition or requirements for
‘micropropagation’.
1
186 J. ARDITTI AND A. D. KRIKORIAN
Rotor’s method was not widely noticed or appreciated at the time.
5
One reason
for this may have been its publication in the American Orchid Society Bulletin, a hobbyist
publication. Orchid growers who read it may have failed to grasp its importance and
probably found the procedure difficult and complicated.
6
Scientists who might have
appreciated Rotor’s method probably did not read the American Orchid Society Bulletin.
When Rotor’s discovery was finally noticed, other claims of priority had become
widely accepted. However, it seems clear that in vitro clonal propagation (or whatever
other term is used to describe the process for any higher plant in aseptic culture) was
first achieved by Gavino Rotor Jr. in 1949 at Cornell University. The number of
plants which can be produced by Rotor’s method is not large, but it has practical
significance.
During the same period, Professor John T. Curtis (1913–1961; Fig. 5) and co-
workers in the Department of Botany at the University of Wisconsin described in
detail the formation of many growing points on proliferating callus of Cymbidium and
Vanda (Curtis & Nichol, 1948). They used the word ‘calloid’ to describe
protuberances which developed from young asymbiotically germinated protocorm
stage seedings after treatment with barbiturates. They observed that these tissue
masses often had a capacity for continued growth into complete plants (Curtis &
Nichol, 1948). Their appreciation of the potential for clonal multiplication is apparent
in the statement “the practical ability to produce clonal lines of plants of potentially
unlimited numbers would be of obvious value in many types of genetic and plant
production work”. Nevertheless, there is a big difference between the drawing of
attention, almost as an aside, to potential by Curtis and Nichol and the achievement of
a well-conceived goal by Rotor.
FOUNDATIONS OF STEM TIP CULTURE
Three separate lines of research led to the development of micropropagation
methods for orchids: (1) in vitro culture of stem tips of other plants and regeneration
of plantlets from them; (2) production of disease (mainly virus)-free clones of
important crops; and (3) clonal propagation. The background of these will be
explored separately and brought to the point where they converge. A short history
of plant hormones will also be presented because these substances are often critical
to the in vitro culture of plant cells, tissues and organs (see Krikorian, 1995 for a
review).
Plant hormones
Gottlieb Haberlandt (1854–1945; Fig. 6), Professor of Plant Physiology in Berlin,
was the first to suggest that hormones might play an important role in plant cell
culture media. This is clear from his suggestion that “the effect (also reported by
[Hans] Winkler) of the pollen tube on the development of the ovule in orchids, the
swelling of ovaries etc., [occurs] probably [because] substances (Wuchsenzyme), are
involved here which, released from the pollen tube, [act] as a chemical stimulus to
the growth and division of the cells concerned … it would be worthwhile to culture
together vegetative cells and pollen tubes; perhaps the latter would induce the former
to divide” (Haberlandt, 1902; English translation by Krikorian & Berquam, 1969:
187HISTORY OF ORCHID MICROPROPAGATION
83). It does not seem that Haberlandt followed his own suggestion.
7
Had he done so,
it is possible but not probable that his culture experiments would have been
successful. Nevertheless, Haberlandt was the first to suggest clearly that exogenous
hormones are part of the requirements for culturing cells and it is interesting to note
that the first successes with animal cells by Ross G. Harrison (1870–1959) are said to
have derived their inspiration from Haberlandt’s researches (Oppenheimer, 1966:
533 et seq.).
Auxins
Pollen tubes release at least one substance which brings about post-pollination
phenomena and ovule development in orchids (Avadhani et al., 1994). Hans Fitting
(1877–1970; Fig. 7) was the first to show that it exists as a result of his work with
Phalaenopsis pollinia and pollination at the Buitenzorg (now Bogor) Botanical Gardens
in the then Netherlands Indies (now Indonesia) (Fitting, 1909a, b, 1910, 1911, 1921;
several personal communications with J.A. shortly before his death; for reviews see
Arditti, 1971, 1979, 1984, 1992; Avadhani et al., 1994). Fitting named the substance
Pollenhormon and by doing so became the first to do more than speculate that plants
have hormones. He was also “the first investigator to work with hormones and active
extracts in plants” (Went & Thimann, 1937). Fitting claimed until his death that
Pollenhormon was a specific substance, different from auxin. Current evidence suggests
that his extracts contained several substances including auxin (for a review see
Avadhani et al., 1994). In 1932 Friedrich Laibach reported that the active substance
in Fitting’s Pollenhormon could be extracted with diethylether (Laibach, 1932) and
several years later Kenneth V. Thimann (b. 1904; Fig. 8) showed that the ether
extract contained auxin. The story ends here as far as Fitting’s Pollenhormon is
concerned. Frits W. Went (1904–1990; Fig. 9) suggested once (in a conversation with
J.A. at Irvine during the early 1970s) that Fitting could have discovered auxin, but
did not because his interest shifted to other problems in plant physiology including
Reizung (which is best translated as ‘irritability’ or to use the modern term, ‘sensory
physiology’).
Went’s discovery of auxin (Went 1926, 1928) and its subsequent identification as
indole-3-acetic acid (IAA) in 1934 (Went & Thimann, 1937; Haagen-Smit, 1951)
opened the road to early successes in tissue culture (Gautheret, 1983, 1985).
Lyophilized leaf extract from leaves of dodder host plants (a preparation which
probably contained auxin) and pure IAA were incorporated in culture media with
mixed results starting about a decade after the discovery of this hormone (Fielder,
1936; Geiger-Huber & Burlet, 1936; for reviews see Gautheret, 1935, 1937; Loo,
1945a).
Coconut water and cytokinins
Inspiration and insight both seem to have played a part in discoveries associated
with tissue culture. One such instance was responsible for the incorporation of
coconut water (at that time commonly called coconut milk) by Johannes van
Overbeek
8
(1908–1988; Fig. 10) into culture media for immature or recalcitrant
embryos of Datura stramonium produced by Albert F. Blakeslee (1874–1954; Fig. 11).
188 J. ARDITTI AND A. D. KRIKORIAN
When it was shown that the embryos grew (van Overbeek, Conklin & Blakeslee,
1941) a new and complex additive (see Arditti & Ernst, 1993 for composition)
9
became available for supplementing culture media.
10
Also, the growth of explants
with requirements which could not then and still may not be satisfied with defined
compounds can be fostered by addition of coconut water to the medium. Moreover,
coconut water was shown to induce cell division in quiescent cells in carrot root
phloem explants (Caplin & Steward, 1948; Krikorian, 1975; Gautheret, 1985) and to
work synergistically with 2,4-dichlorophenoxyacetic acid (2,4-D) in potato tuber
explants where neither coconut milk nor 2,4-D would alone bring about cell division
(Steward & Caplin, 1951).
Tissue culture research broadened and accelerated in the late 1940s and early
1950s and investigators faced a number of problems, one of them being the
recalcitrance of tobacco pith tissues (Gautheret, 1985; Skoog, 1994). In his attempts
to culture these tissues Folke Skoog (b. 1908; Fig. 12) and his students and associates
at the University of Wisconsin formulated media (for details see Skoog, 1944, 1951;
Skoog & Tsui, 1948; Skoog & Miller, 1957) and evaluated the growth stimulating
effects of a number of substances. These included a preparation of herring sperm
DNA which had been stored for a long time (the time frame suggested that Skoog
may have used nucleic acid preparations left over from research on orchid seed
germination by Prof. John T. Curtis; however, Prof. Carlos O. Miller
12
(b. 1923; Fig.
13) recalls that the relations between Skoog and Curtis would have precluded this
from happening). This research resulted in the discovery of plant kinins (later re-
named cytokinins
11
) by Skoog, Miller and others in the Wisconsin Botany
Department group (for historical details see Strong, 1958; Miller, 1961, 1977;
Leopold, 1964; Gautheret, 1985; Skoog, 1994). With the need for certain vitamins
(Gautheret, 1945) and auxins in culture media already established, Toshio
Murashige (b. 1930; Fig. 14) refined an existing nutrient culture solution and
formulated the now widely used Murashige and Skoog (MS) medium (Murashige &
Skoog, 1962; for historical coverage of the development of MS medium see Smith &
Gould, 1989; Skoog, 1994). Since then (as now) an appropriate medium (along with
the explant source) was a very important factor in the establishment of a tissue
culture (Krikorian, 1982, 1995), a large number of plants earlier viewed as
recalcitrant became amenable to culture.
CULTURE OF STEM TIPS
The idea of using stem tips or buds for mass rapid clonal propagation is over 100
years old. In 1893 Carl Rechinger in Vienna attempted to culture stem sections and
excised buds of Populus nigra and Fraxinus ornus as well as portions of roots on sand
moistened with tap water (Rechinger, 1893; Krikorian, 1982; Gautheret, 1983). His
attempts failed, but he concluded that to develop properly, sections must be thicker
than 1.5mm. Rechinger’s procedures would not be called ‘tissue culture’ by today’s
standards, but they foreshadowed it because he used a medium (tap water), a support
(sand) and explants. Orthodox tissue culture procedures now include: (1) a nutrient
medium which contains several components, including organics like sucrose, that
make it advisable to use aseptic techniques; (2) an explant; and (3) in some instances
agar (or a related substance) as a solidifier or support. However, except for the
explant, some of these factors are often not an absolute requirement and a matter of
189HISTORY OF ORCHID MICROPROPAGATION
190 J. ARDITTI AND A. D. KRIKORIAN
convenience or sometimes invoked for pedantic rather than functional reasons.
Many media, especially those used for orchids (for a review see Arditti & Ernst, 1993)
are very dilute and liquid, i.e. ‘unsolidified’. Even sterility, which is certainly to be
preferred, is not an absolute requirement as long as microbial contaminants can be
prevented from over-running the explants (Thurston, Spencer & Arditti, 1978, 1979;
Spencer et al., 1979/1980; Brown et al., 1982, 1984; Johnson, Perera & Arditti, 1982;
Cvitanic & Arditti, 1984).
Nearly 20 years later the German experimental morphologist Karl [later von]
Goebel
13
(1855–1932; Fig. 15) tried to grow excised buds of the water fern Ceratopteris
thalictroides in peat moss, but obtained only abnormal plants (Goebel, 1902;
Krikorian, 1982). This was not ‘tissue culture’ either as the term has been used
during the last 30–40 years, but Goebel did use ‘explants’ and a medium. Studies on
the effects of polarity, water and presence or absence of cork on root formation in
Salix by the German botanist Herman V¨ochting (1847–1917; Fig. 16) were more
tangential (V¨ochting, 1906), but nevertheless important steps on the road to tissue
culture (Krikorian, 1982: 162).
The pace of research in the area was accelerating but some 15 years passed before
the first stem- and root-tip cultures were attempted (Krikorian, 1982; Gautheret,
1983). William J. Robbins (1890–1978; Fig. 17) working at the University of Missouri
germinated seeds of peas, maize and cotton under aseptic conditions, excised root
and stem tips and attempted to grow them in the dark on sterile ‘Pfeffer’s Solution’
with and without glucose or fructose (see Knop, 1884; Pfeffer, 1900; White, 1943,
1945; Krikorian, 1975, 1982; Murashige, 1978; Arditti, 1977, 1992; Arditti et al.,
1982; Arditti & Ernst, 1993 for composition of media). The cotton explants did not
grow normally, but those of maize and peas did (Robbins, 1922). They produced
roots but were chlorotic and showed characteristics which were “typical of plants
grown in the dark” (Robbins, 1922). The results obtained by Robbins are easy to
explain from our present vantage point on culture requirements. He did not have
plant hormones or vitamins at his disposal. Indeed, he did not then know that the
latter may be required by explants.
14
Neither did he realize that the cultures would
have benefited from illumination. Despite this, Robbins and his associates managed
to maintain the root tip cultures for nearly 4.5 months (Robbins & Maneval, 1923,
1924).
Independently of Robbins, but at the same time, one of Haberlandt’s students in
the Pflanzenphysiologische Institut in Berlin-Dahlem, Walter Kotte (Fig. 18),
cultured pea roots. He used a solution of Knop’s salts (Knop, 1884) as his basic salt
medium and added to them glucose, peptone, asparagine, alanine, glycine, a meat
extract and a digest of pea seeds. Kotte’s medium was more sophisticated than the
Figures 12–25. Botanists who played major roles in the history of plant hormone, tissue culture and orchid research.
Fig. 12. Professor Folke Skoog (Janick, 1989). Fig. 13. Professor Carlos O. Miller (photograph courtesy C. O. Miller;
signature from a note to J.A.). Fig. 14. Professor Toshio Murashige (Janick, 1989). Fig. 15. Professor Karl von Goebel
(Wittrock, 1897–1903). Fig. 16. Professor Hermann V¨ochting (photograph and signature from portrait in Fitting,
1919). Fig. 17. Professor William J. Robbins (Gautheret, 1985). Fig. 18. Dr Walter Kotte (photograph and signature
from White, 1943). Fig. 19. Dr Philip R. White (photograph from Gautheret, 1985; signature from White, 1943). Fig.
20. Professor Loo Shih-wei (from a transparency by Dr Franz Hoffmann taken in Beijing c. 1985; English and
Chinese character signatures from a letter to J.A.). Fig. 21. Professor Roger J. Gautheret. Fig. 22. Professor Pierre
Noub´ecort (Gautheret, 1985). Fig. 23. Professor Ernest A. Ball (photograph from a Kodachrome transparency by
J.A.; signature from Ph. D. Dissertation by Dr Michael S. Strauss). Fig. 24. Mr Hans Thomale (photograph courtesy
Hans Thomale; signature from a letter to J.A. both obtained with the help of E. Lucke and Dr N. Haas-von
Schmude). Fig. 25. Professor Hans Burgeff (photograph from Haber, 1963; signature from a letter to Professor
Robert Ernst).
191HISTORY OF ORCHID MICROPROPAGATION
one used by Robbins and probably contained vitamins, some plant hormones and
inositol, all of which were probably components of the complex additives. The roots
grew, but could not be subcultured (Kotte, 1922a, b; White, 1943).
Philip R. White (1901–1968; Fig. 19) of The Rockefeller Institute for Medical
Research at Princeton, New Jersey reached the conclusion that apical and
intercalary meristems “would be best to choose [as] materials for our first
experiments” (White, 1931, 1933b). While on a visit to Haberlandt’s plant
physiology institute at the University of Berlin in the winter of 1930 and spring and
summer of 1931 White attempted to culture root tips (White 1932a, 1933a) and
“some 400 stem tips” of the “common weed” Stellaria media in hanging drops of the
medium of Uspenski & Uspenkaja (1925), a formulation designed for pure cultures
of Volvox minor and V. globator. White used this nutrient solution earlier for the culture
of root tips, embryos and other explants (White, 1933b). He managed to keep the tips
alive “for periods up to three weeks … [and] during this time there … occurred
active cell division … growth … differentiation … into leaves, stems and floral
organs” (White, 1933b). But his results were disappointing by today’s standards.
Accumulation of “excretory products, and the exhaustion of nutrient materials” were
given as reasons for the limited success (White, 1933b). A more plausible explanation
is the composition of the medium. It had no ammonium ion, and contained no
vitamins or hormones because they were not yet discovered, new to science at the
time, or not yet known to be required by organ and tissue explants.
15
Still, White’s medium was one of the best available at the time and maize shoot
tips cultured on it produced plants (Segelitz, 1938). If the tips were shorter than 2cm
in length they had to be cultured under illumination. Longer shoots (2–4cm) could
grow in darkness (Segelitz, 1938). This is one of the earliest successes in culturing a
monocotyledon in vitro. It was reported long before what has sometimes been claimed
to have been the first real success with this group (Morel & Wetmore, 1951a). To be
precise, however, it should be noted that Morel and Wetmore dealt with callus
production in their cultures. That success was appropriately viewed as particularly
significant since monocotyledons do not normally make wound tissue and hence
many monocot cultures even today grow only with difficulty (see also Swamy &
Sivaramakrishna, 1975; Hunault, 1979 on monocotyledonous recalcitrance).
The second monocotyledon to be propagated by what can in retrospect be
described as a crude or ‘prehistoric form’ of tissue or explant culture was taro
(Colocasia esculenta), an important and ancient crop in Hawaii and the Pacific region.
16
An attempt was made to accelerate taro propagation, though by culturing normally
dormant buds “ borne in the axils of the leaves on the surface of the taro corm”
(Kikuta & Parris, 1941). Tuber slices, 2–5cm thick and buds “together with
approximately 1 cubic centimeter of corm tissue”, planted in sterilized soil produced
plants. Thus, excised buds and corm explants were cultured in a sterile medium, soil
in this case, and produced plants. There is no valid reason why only a solution (semi-
solid or liquid) should be defined as a culture medium. This method of taro
multiplication (Kikuta & Parris, 1941) is analogous to present day tissue culture
propagation even if the techniques are somewhat crude and the cultures are not in
vitro. Unfortunately, this procedure and related ones are mentioned only in a few
instances (Arditti & Ernst, 1993; Arditti & Strauss, 1979; Krikorian, 1994a) and are
generally missing from historical reviews (Gautheret, 1980, 1982, 1983, 1985). Taro
was cultured in vitro for the first time 30 years later (by Mapes & Cable in 1972; see
also Krikorian, 1994a).
192 J. ARDITTI AND A. D. KRIKORIAN
Rye was cultured early (de Ropp, 1945). Stem tips (the plumules) of excised
embryos were cultured on White’s medium containing 2% (w/v) sucrose. When “any
isolated stem tip developed a root, the entire growing point was stimulated to
meristematic activity, and leaves normal in form and size developed” (de Ropp,
1945). These explants were embryonic in nature and it may be suggested that they
were not equivalent to shoot tips of mature plants. However, current evidence (at
least that obtained from orchids, see Arditti & Ernst, 1993 for a review) suggests that
embryonic stem tips from seedlings and mature plants do not differ greatly with
respect to their requirements in vitro.
From the mid-1930s to the 1950s the California Institute of Technology in
Pasadena was arguably the world centre for research in plant physiology. Its faculty
(which included such major figures in plant physiology as Kenneth V. Thimann,
James Bonner, Frits W. Went, Herman Dolk, Arie J. Haagen Smit, Johannes van
Overbeek and others) attracted excellent graduate and post-doctoral students from
all continents (Thimann, 1980). One of these was Shih-Wei Loo
17
(b. first decade of
the 1900s; Fig. 20). For his doctoral dissertation Loo cultured excised stem tips,
5–10mm long, of Asparagus officinalis on a medium utilized by James Bonner for the
culture of tomato roots (Loo, 1945a). Some of Loo’s explants developed buds, but
none formed roots. He concluded that growth of the excised stem tips was
“potentially unlimited” (Loo, 1945b). It seems reasonable to assume that the tips
would have produced roots had IAA been added to the medium. Loo moved to
Columbia University after his first paper on the subject (Loo, 1945b) and published
yet another report on asparagus shoot tips (Loo, 1946a). He showed that a solution
rendered semi-solid with agar was “as good, if not better, than liquid medium”. In
the process of ascertaining this fact, he devised a simple method for supporting stem
tips (Loo, 1946a). Growth of the explants was normal and they were still alive after
22 months and 35 transfers (Loo, 1946a).
Loo also cultured stem tips of the parasite dodder (Cuscuta campestris). His cultures
failed to produce leaves and roots but nevertheless fortuitously flowered in vitro (Loo,
1946b). This is probably the first case where “floral organs … developed on excised
stem tips in vitro” (Loo, 1946b). Again, it is reasonable to speculate that dodder
explants would have formed leaves and roots with hormones in the medium
(Galston, 1948). Unfortunately Loo did not add any, but he did conclude that the
explants required sugar for growth in vitro, a conclusion which was relatively new at
the time [although, in some cases orchid explants develop in a more desirable fashion
only on sugar-free medium (see Arditti & Ernst, 1993 for a review)]. Another
contribution by Loo was the cultivation and flowering in vitro of the composite Baeria
chrysostoma, a small annual sometimes grown in gardens and which belongs to a
California genus of c. 20 species (Loo, 1946c).
Loo’s papers suggest that tissue culture of angiosperms and micropropagation
would have advanced more rapidly had he remained in the U.S.A. and/or if
conditions in China had been different. His important contributions to stem tip
culture and ultimately to micropropagation have thus far received only passing credit
in a few reviews (Krikorian, 1982; Gautheret, 1983) and a few research papers
(Steward & Mapes, 1971b; Koda & Okazawa, 1980). Loo’s work is certainly not as
well-known as it should be. It is worth mentioning here that Segelitz, de Ropp and
Loo (independently of each other), and not subsequent workers (Morel & Wetmore,
1951a; Gautheret, 1983, 1985) were the first to have significant success in the
culturing of monocotyledons in vitro.
193HISTORY OF ORCHID MICROPROPAGATION
Frits Went was also indirectly associated (through a gift of auxin) with the first
successful culture by Carl D. LaRue (1888–1955) of an axillary bud meristem, that
of water cress on White’s mineral nutrients supplemented with 20g (w/v) sucrose l
–1
and “1 hetero-auxin added, 1 part to 20 millions” (LaRue, 1936).
That plant tissues can be cultured “for unlimited periods of time was announced
independently” and almost at the same time but not “simultaneously” (for a review
see Gautheret, 1985) by Phillip R. White (c. 31 Dec 1938), Roger J. Gautheret (b.
1910, Fig. 21; 9 January 1939) and Pierre Nob´ecourt (1895–1961, Fig. 22; on 20
February 1939). These findings on the potentially unlimited growth of callus cultures
set the stage for the first successful culture of a stem tip.
Ernest A. Ball (b. 1909; Fig. 23) was interested in shoot tips and apical meristems
(Ball & Boell, 1944), “the capacity for growth and development of vegetative plant
cells”, “polarity of the buds and subjacent cells, “the relation between respiration and
development, independence of the tip from the rest of the plant, production of
subjacent tissues by the apex”, and the “totipotentiality of all living plant cells” (Ball,
1946). He excised shoot apices of nasturtium, Tropaeolum majus L. (“55µhigh and
140µthick”) and lupin, Lupinus albus L. (“81µhigh and 250µthick”); the sections
were 400–430µ
3
in volume
18
(Ball, 1946). Ball made “no provisions to achieve and
maintain asepsis”, “inoculations were performed in the laboratory”. He placed
explants on Robbins’ modification of ‘Pfeffer’s Solution’ plus micro-elements and in
some cases “unautoclaved coconut milk”.
19
The medium was made semi-solid with
agar which changed in colour from brown to white after being washed with thirty
24-hour changes of distilled water. His explants grew well (Ball, 1946; personal
communication to J.A. in many conversations) and any insinuations to the contrary
(Morel, 1974:177) are without foundation.
20
THE SECOND ASEPTIC CULTURE OF AN ORCHID EXPLANT
Even before the availability of cytokinins and the formulation of MS, several
culture media (Skoog, 1944; Skoog & Tsui, 1948, 1951; Miller & Skoog, 1955;
White, 1951) were adapted for less demanding plants, especially with the addition of
auxins, vitamins and coconut water. Four such media were used to culture geranium,
Pelargonium zonale and cyclamen, Cyclamen persicum (Mayer, 1956), and this led a
German horticulturist to the first reported culture of sections (Teilst¨ucken or
Pflanzenteile) and tissues (Gewebe) of orchids (Thomale, 1956, 1957:89–90, fig. 39).
Hans Thomale (Fig. 24) was born in Herne, Westphalia, Germany on 16 October
1919, raised in Cologne and now lives in Lemgo where he still grows orchids.
21
He
started to study chemistry and medicine just before World War II broke out. When
he “was half ready” Thomale was drafted and had to interrupt his studies. After
World War II he “was forced to learn potato [cultivation] in a well-known nursery
which had more orchids … than vegetables”. The owner of the nursery, Mr H.
Kuhlman, also had a “daughter [Lieselotte] who [earned] the title ‘Doctor of Botany’
[while] I was forced to be a soldier”. They later married.
Thomale became interested in orchid seed germination and asked Prof. [Hans]
Burgeff [1883–1976; Fig. 25] for his book Samenkeimung der Orchideen and used it to
teach himself both symbiotic and asymbiotic seed germination. In 1946 he
established a laboratory and utilized it to produce hybrids between the “many fine
orchids [Mr Kuhlman] bought [in] England and Belgium before the war … after
194 J. ARDITTI AND A. D. KRIKORIAN
that I tried to raise … orchids [via] clonal propagation”. The laboratory work
brought him offers from Dorset Orchids Ltd., Plush, Dorset, U.K. in 1949 and
Sanders Orchids, St Albans, Herts., U.K. in 1950 (neither exists any longer) to
establish laboratories for them. Thomale wanted to propagate both tropical orchids
and those which were native to Germany. It is clear from his writings that Thomale
read widely and was familiar with the work of Gautheret, Mayer, Rotor, Skoog, Tsui
and others.
Thomale based his own work on orchids on a paper by Dr Lucie Mayer (Mayer,
1956; Fig. 26) and on 23 September 1956 he was able to report to a meeting of the
Deutsche Orchideen Gesellschaft [German Orchid Society] that explants of
Dactylorhiza (Orchis) maculata (Fig. 27) and some tropical orchids in vitro produced
shoots (Figs 28, 29) and subsequently plants. Thomale recollects, albeit with some
uncertainty, that Mr Lecoufle of the French orchid firm Vacherot and Lecoufle (see
below) was present at that meeting. A photograph of the Orchid maculata culture (Fig.
28) was published in the second edition of Die Orchideen (Thomale, 1957). The caption
reads: “Section of Orchis maculata on agar medium (Mayer’s method), which was
induced to form roots and shoots” (Figs 28, 29). Thomale appreciated immediately
the potential of his discovery. He wrote (Fig. 30, the translation below is from Arditti
& Ernst, 1993):
It should be noted that efforts to find a propagation method for European
terrestrial orchids, based on the work by Dr L. Mayer [Mayer, 1956], through
the culture of sterile explants on an agar medium were successful. It is well
known that vegetative parts of orchids, for example, sterile sections of
Phalaenopsis flower stalks [Rotor, 1949], which bear at least one adventitious
bud [Note by J.A. and A.D.K.: these buds are lateral on the scape and not
necessarily adventitious, at least not in the strict sense of the word], can
produce shoots when cultured on an agar medium. Recently it has become
possible to culture undifferentiated tissues on certain nutrient media to
produce roots and shoots from them. Since sufficient details were not available
by the time this book went to press [i.e., the second edition which appeared in
1957; the first edition was published in 1954], it is only possible to mention
that whole plants can be produced from tissue explants one cubic centimeter
in size. This is a form of vegetative multiplication whose potential cannot be overlooked
[emphasis added]!
Thomale’s work and his conviction about the value of explant culture as a means
of mass rapid propagation was published (Thomale, 1957) before the first reports of
Cymbidium ‘meristem’ cultures (Morel, 1960; Wimber, 1963), but it was overlooked.
In this respect Thomale’s work was similar to Rotor’s. It is of some interest to note
here that people were apparently aware of Rotor’s work on Phalaenopsis propagation
from stem sections, even if not, as Thomale states, “well known”, but drawing
attention to it in German language publications did little to publicize it. Another
important point is that Thomale behaved professionally by calling attention to
Rotor’s work, first by mentioning his name (Thomale, 1956) and later by referring
to Phalaenopsis (Thomale, 1957). Had Thomale not mentioned Rotor and Phalaenopsis
he could have created the impression that he originated the entire idea of clonal
propagation in vitro. Thomale did not describe his techniques in detail but credited
Mayer’s. In fact, Dr Mayer participated in Thomale’s initial attempts (Haas-von
Schmude, Lucke & Arditti, 1995; personal communication to J.A. by E. Lucke and
Dr N. Haas-von Schmude, Wettenberg, Germany). Dr Mayer recalls that they also
195HISTORY OF ORCHID MICROPROPAGATION
196 J. ARDITTI AND A. D. KRIKORIAN
excised and cultured Cymbidium stem tips, but they never published that part of their
work and therefore cannot be credited with it.
The following reasons may be responsible for the fact that Thomale’s work did not
become well known: (1) his findings were first published in German in an orchid
hobbyist publication which at the time was not well-known outside Germany; (2) the
second publication, also in German, was in a relatively obscure book aimed primarily
at hobbyists and commercial orchid growers. As a result, few scientists read about
Thomale’s discovery. Practical growers who read it probably did not appreciate the
technique and/or were bewildered by it (note this parallel between Rotor’s and
Thomale’s publications).
Georges Morel (1916–1973; Fig. 31) is generally given exclusive credit for being
the ‘first’ to culture an orchid explant in vitro. Clearly, he was familiar with Thomale’s
work at least as early as 1965 (Fig. 32). However, Morel cited it for the first time
nearly 10 years later in a chapter written for Carl L. Withner’s The Orchids-Scientific
Studies. This was published some 14 years after his fame in the orchid world had been
firmly established (Morel, 1974; Hass-von Schmude, Lucke & Arditti, 1995). Even
then, Morel only cited Thomale’s 1957 book and although he accurately reported
that “pieces from the bulb of Orchis maculata, aseptically cultivated on nutrient
medium, soon regenerated stems and roots …” He also added the qualifier “that
[cases like this] are very exceptional.” Morel included in his chapter a copy of a
photograph provided by Thomale (Fig. 28) with the caption “Regeneration of roots
and shoots occurring on a piece of tuber of Orchis maculata. (After Thomale.)” The
wording (“stems and roots”) tends to minimize Thomale’s achievement by implying
that what was produced were not ‘whole’ plants, and the context (included in a
section entitled ‘Regeneration from Inner Parenchyma’) would seem to suggest that
the new plants were produced from inner parenchyma rather than from buds,
through bud formation, or via some other process commonly associated with tissue
culture propagation (see Morel, 1974:170 ff.). Moreover, the photograph was not
‘after Thomale’; it was provided by Thomale to Morel in response to his request (Fig.
32).
By the time Thomale was given any recognition (Arditti & Ernst, 1993; Haas-von
Schmude, Lucke & Arditti, 1995), total credit for priority of discovery had essentially
been established for and by Morel. This occurred, it may be argued, not only by
virtue of the fact that Morel was already a well-known and established senior scientist
in the world of plant physiology and plant pathology, but also due to his extensive
travels and lectures. Orchid scientists who were unfamiliar with the historical details
presented here, admiring hobbyists and grateful commercial growers, played a major
role in elevating Morel to the position of being virtually the sole participant in the
‘invention’. There was also, we submit, resistance to new knowledge (Gaffron, 1969)
about which more will be said under our ‘Concluding remarks’.
A note marking Thomale’s 75th birthday (Lucke, 1994) makes no mention of his
discovery because a statement to that effect was edited out by the editors of Die
Figures 26–30. The culture of Orchis maculata and Dr Lucie Mayer. Fig. 26. Dr Lucie Mayer (courtesy E. Lucke and
Dr Norbert Haas-von Schmude). Fig. 27. Flowers and inflorescences of Orchis maculata (Landwehr, 1977). Fig 28.
Explant of Orchis maculata in culture (courtesy of Mr Hans Thornale obtained with the help of E. Lucke and Dr N.
Haas-von Schmude). Fig. 29. Plants of Orchis maculata produced from in vitro explants like the one in Fig. 28 (courtesy
Dr Lucie Mayer obtained with the help of E. Lucke and Dr N. Haas-von Schmude). Fig. 30. The first description
of the Orchis maculata cultures (from pages 89–90 in Thornale, 1957).
197HISTORY OF ORCHID MICROPROPAGATION
198 J. ARDITTI AND A. D. KRIKORIAN
Orchidee (Dr Norbert Haas-von Schmude, Wettenberg, Germany, personal commu-
nication to J.A.). However his important contribution was eventually recognized in
an American publication (Haas-von Schmude, Lucke & Arditti, 1995).
PLANT DISEASES AND MERISTEMS
The idea that healthy clones of horticultural plants can be obtained from stem tips,
root cuttings and even leaves is at least half a century old (see Krikorian, 1982;
North, 1953 for literature citations). A method for establishing Verticillium-free clones
of chrysanthemums by making tip cuttings from 4–6 inch long shoots which were
shown to be disease-free was reported by Arthur W. Dimock (1908–1972) during
World War II (Dimock, 1943a, b) and subsequently refined and extended to other
diseases (Brierly, 1952; Dimock, 1956). Similar methods were used for carnations
(Dimock, 1943a, b, 1951; McFarland, 1948; Forsberg, 1950; Andreasen, 1951;
Guba, 1952; Hellmers, 1955; Thammen, Baker & Foley, 1956).
That tips of virus-infected roots could be free of infection was reported over 60
years ago (White 1934a, b, 1943). Before that, viruses or ‘abnormalities’ could not be
seen in stem tips of tobacco, tomato and Solanum nodiflorum (Clinch, 1932; Sheffield,
1933, 1942) but clearly virus infections were obtainable from them. For example,
aucuba (Aucuba, Cornaceae is a genus of ornamental shrubs known as Japanese,
greenleaf or sulphur leaf aucuba) and tobacco mosaic virus infections were obtained
from isolated shoot- and root-tips (Sheffield, 1942). Thus, supposed absence of virus
could have been due to the manner in which the tissues were excised, or to very low
virus content (Samuel, 1934). Nevertheless, by 1948 stem tip cuttings could be used
to eliminate the spotted wilt virus from Dahlia (Holmes, 1948, 1955). This method
was extended to leaf spots associated with the internal-cork disease in sweet potato,
Ipomea batatas (Holmes, 1956a) as well as aspermy virus (Holmes, 1956b) and other
viruses (Brierley & Olson, 1956) in Chrysanthemum.
The use of stem tip cuttings to eliminate spotted wilt of Dahlia (Holmes, 1948) very
clearly suggested that apical meristems might be virus-free. This was confirmed a
year later in studies with tobacco mosaic infection of Nicotiana tabacum var. Samsun
(Limasset & Cornuet, 1949). These findings were fortuitous. It is well-known today
that apical meristems are not necessarily free of virus infection and this has led to
considerable difficulties in freeing many clones and cultivars of viruses (Kassanis,
1967).
A problem facing French horticulture c. 1950 was viral infection of certain potato
and Dahlia cultivars which would have caused them to be abandoned (Lecoufle,
1974a, b). Given the previous findings with Dahlia (Holmes, 1948) and tobacco
(Limasset & Cornuet, 1949) the culture of stem tips provided a means of freeing these
plants of viruses. And, indeed, Pierre Limasset and Pierre Cornuet “suggested to
their colleagues Georges Morel and Claude Martin to cultivate shoot meristems of
Figures 31–35. Georges Morel: in vitro cultures, correspondence and folklore. Fig. 31. Dr Georges Morel (Orchids
Orlando, n.d.). Fig. 32. Letter from Dr Morel requesting permission to use Thomale’s photograph of Orchis maculata
(copy of letter provided by Hans Thomale; photograph courtesy Hans Thomale obtained with the help of E. Lucke
and Dr N. Haas-von Schmude). Fig. 33. Protocorm-like body (PLB) of Cymbiium (Morel, 1960). Fig. 34. Cymbidium
plantlet produced from a PLB like the one in Fig. 33 (Morel, 1960). Fig. 35. Part of the cover of the Orchids Orlando
catalog which listed orchids that were propagated clonally by the French firm of Vacherot and Lecoufle (Orchids
Orlando, n.d.).
199HISTORY OF ORCHID MICROPROPAGATION
infected plants” (Gautheret, 1983, 1985). The suggestion was excellent, the attempts
were successful and virus-free Dahlia (Morel & Martin, 1952) and potato (Morel &
Martin, 1955a, b; Morel & Muller, 1964; Gautheret, 1983, 1985) plants were
obtained from infected ones. The Dahlia and potato shoot obtained from stem tips in
vitro by Georges Morel and his co-workers did not produce roots. Shoots produced
in vitro by previous workers also failed to form roots. Therefore, following established
laboratory practice, the shoots produced by Morel and his associates were grafted
onto healthy seedlings (Gautheret, 1983). Later, other investigators were able to get
rooting (Quak, 1961; Hollings & Stone, 1983). Attempts to free potatoes of virus
through the culture of shoot tips were also undertaken by a number of others
(Kassanis, 1957; Pirie, 1973; see Hirst & Harrison, 1988 for historical
perspectives).
The success with Dahlia, potatoes and other plants (Morel & Martin, 1955b;
Morel, 1964a) led Morel and his associates to the in vitro culture of Cymbidium shoot
tips (Morel, 1960; Figs 33, 34). As already mentioned, this achievement has been
heralded in a wide array of publications. A particularly adoring account in an
advertisement-catalogue makes the claim on its cover that “a funny thing happened
to the orchid when they operated on a sick potato“ (Fig. 35) and in the text that “a
beautiful thing happened to the orchids when they operated on a sick potato
[because] Dr Georges Morel, distinguished French botanist, discovered the orchid
meristem process while he was trying to figure out a way to prevent virus in potatoes”
(Orchids Orlando, n.d; Fig. 36). Less maudlin but equally inaccurate statements
asserting the same abound in the scientific and horticultural literature as well (for
examples see Bertsch, 1966, 1967; Marston & Voraurai, 1967; Vacherot, 1967,
1977; Borriss & H¨ubel, 1968; Vanseveren & Freson, 1969; Hahn, 1970;
Kukulczanka & Sarosiek, 1971; Lecoufle, 1971; Lucke, 1974; Allenberg, 1976;
Champagnat, 1977; Rao, 1977; Loo, 1978; Murashige, 1978; Goh, 1983; Bouriquet,
1986; Griesbach, 1986; Hetherington, 1992). Much less frequently does one
encounter attempts to be more precise about orchid micropropagation history
(Arditti, 1977; Stewart, 1989; Arditti & Ernst, 1993).
Horticulture and plant agriculture are the major beneficiaries of stem tip culture
in terms of the generation of plants free from specific pathogens as well as massive
and rapid clonal propagation. The fact that both objectives can sometimes be
accomplished simultaneously with one and the same explant has created “an
apparent conception among horticulturists that tissue culturing and diseases-freedom
(sic!) are synonymous. The same misconception was true of the so-called meristem
cultured plants… A classic example of this misconception can be seen in the orchid
industry… Before ‘mericloning’ orchid viruses were a minor problem … However
[they] are now common, wide-spread and costly” (Langhans, Horst & Earle, 1977)
because careless culturing spread rather than contained or eliminated viruses (see
also Toussaint, Dekegel & Vanheule, 1984).
THE THIRD ASEPTIC CULTURE OF AN ORCHID EXPLANT
Most accounts and reviews of orchid micropropagation seem to start with a
citation or at least a mention of Morel’s 1960 paper on Cymbidium shoot tip culture.
Figures 36 & 37. Two accounts of Dr Georges Morel’s work by firms which sold plants produced through his
methods. Fig. 36. Page from the catalogue of Orchids Orlando (Orchids Orlando, n.d.). Fig. 37. Part of a letter to
J.A. from the late Maurice Lecoufle, owner of the French orchid firm, Vacherot and Lecoufle.
200 J. ARDITTI AND A. D. KRIKORIAN
201HISTORY OF ORCHID MICROPROPAGATION
A few examples are: “the potential of propagating orchids through tissue culture was
observed first by Morel” (Murashige, 1974). Similarly, “… credit for the initiation of
meristem culture technique goes to the late Dr G. Morel of INRA [Institut National
de la Recherche Agronomique], Versailles, France” (Rao, 1977). Assertions that “the
first application [of micropropagation] concerned the clonal propagation of orchids
(Morel, 1960)” can be found in historical accounts by a ‘founding father’ of plant
tissue culture (Gautheret, 1983, 1985). Since such reviews are often re-stated or
quoted in other papers [for example, “the potentials of tissue culturing for plant
propagation … have been … reviewed by Murashige …” (Langhans et al., 1977)] a
historical ‘factoid’ has been elevated to truth and dogma. Once such a
transformation happens, the forces which usually resist knowledge tend to maintain
the status quo and thus strive to support dogma (Gaffron, 1969).
These factors seem to have come to bear on the history of orchid micro-
propagation. Attempts to question the accepted views have led to polemical
exchanges in the literature (Arditti, 1985; Torrey, 1985a, b). Editorial demands for
changes in manuscripts have also had to be agreed to (see e.g. Arditti & Arditti,
1985). The accepted history will be examined here for the sole purpose of placing
historical facts in the most accurate perspective possible. Unfortunately, it may not
be possible to do that “without apparent injustice to some other” (see opening quote).
Indeed, this historical outline may appear, to some at least, to be somewhat ‘unjust’
only because many previous accounts have been imprecise enough to have done
considerable violence to the truth.
Georges Morel (1916–1973) was born on 16 April 1916 in B´ethune, France
(Gautheret, 1977). In 1934 he entered l’Institut de Chemie in Paris where his
interests led him to agriculture, plant pathology and I.N.R.A. (Gautheret, 1977).
Drafted into military service in 1939, Morel served with an artillery unit and was
taken prisoner at the Belgian front in 1940. He escaped in 1941 (Gautheret, 1977).
On returning to I.N.R.A. Morel was soon appointed “chef de travaux”. In 1943 he
joined Professor Gautheret’s laboratory (Lecoufle, 1974a, b) and worked there
towards his doctorate, which he received in 1948.
He went to the U.S.A. during the same year and worked until 1951 with Professor
Ralph W. Wetmore (1892–1989) in the Biological Laboratories at Harvard
University. They worked on tissue culture of monocotyledonous plants (Morel &
Wetmore, 1951a) and ferns (Morel & Wetmore, 1951b).
22
Morel became friends and
collaborated with Armin C. Braun (1912–1986) of the Rockefeller Institute in New
York City on studies dealing with habituation and hormone autonomy in various
plant tumours (Braun & Morel, 1950). Braun, a distinguished researcher on plant
tumorigenesis, especially those induced by the crown-gall bacterium Agrobacterium
tumefaciens, is appropriately regarded as one of the founding fathers of modern-day
plant genetic engineering (Braun, 1982). Some gene transfer or transformation
techniques rely heavily on the use of the Ti plasmid from that bacterium as a vector
for inserting new genetic information (Bevan & Chilton, 1982). On Morel’s return to
France, he was appointed Maˆıtre de recherches (in 1951 or 1952) and in 1956 Directeur
de recherches of the Station Centrale de Physiologie V´eg´etale du Centre National des
Recherches Agronomiques, Minist`ere de l’Agriculture (Lecoufle, 1974a, b).
It may be argued that Morel’s first paper on shoot tip culture of Cymbidium (Morel,
1960) more closely resembled a news release or notice than a scientific paper. It
sketchily reported what was done, minimally described the excision process and
culture conditions, and referred to a nutrient medium, “Knudson III”, which does
202 J. ARDITTI AND A. D. KRIKORIAN
not last and may have been ‘Knudson C’. The report concluded by stating “that it
is relatively easy to free a Cymbidium from the mosaic virus … each bud will give
several plants so the stock of a rare or expensive variety can be increased … [and
that] experiments of the same kind are now being conducted with … Cattleya,
Odontoglossum, and Miltonia, contaminated with different viruses” (Morel, 1960).
Morel’s paper, did however, introduce a new phrase into orchid terminology and
the English language. He used the term “protocorm-like body”, (generally
abbreviated as PLB)
23
to describe the “small flat bulblet looking exactly like [a]
protocorm” (Fig. 33) which preceded the orchid plantlet formation (Fig. 34).
Significantly, Morel’s paper includes only two literature citations. One pertains to the
mosaic disease (Jensen, 1951), the other deals with freeing plants from viruses
through stem tip culture (Morel & Martin, 1955b). It would have been very difficult
for anyone to repeat Morel’s work because his article did not present sufficient details
(competent plant scientists who took the trouble to study all of his previous work
might have been able to reconstruct the procedures and medium or media; hobbyists
or commercial growers would have had much more serious problems in doing that
since many of them would have been looking for a detailed recipe, even a ready-
made magic ‘formula’). However, as the record shows, the orchid firm of Vacherot
and Lecoufle ‘La Tuilerie’, Boissy-Saint Leget (Seine-et-Oise) had enough informa-
tion to start commercial micropropagation of ‘rare or expensive’ orchids before any
other establishment. They moved quickly enough to have a clonally propagated
plant of Vuylstekeara Rutiland ‘Colombia’ bloom in December 1965 (Vacherot, 1966;
Lecoufle, 1967).
24
This was 24 years before the publication of a specific method for
this hybrid genus (Kukulczanka, Kromer & Roginska, 1989) and only two years after
the reported excision date of the stem tips (Vacherot, 1966), and the development of
culture methods (which were not published in detail at the time) for stem tips of the
parent genera (Morel, 1963).
Two years from protocorm-like body to flowering appears to be very fast growth
and development (but perhaps not impossibly so) for hybrids available at that time.
In fact, according to one view “it will take just as long to grow the plants produced
from meristem tissue as it takes to grow a new hybrid from seed” (Scully, 1964). As
a rule, orchid plants grown from seed require at least 3 years to flower (excluding
some recent Phalaenopsis hybrids which can be considerably faster) but there are also
reports of hybrids which flowered only after 10 or more years (Goh, Strauss &
Arditti, 1984; Goh & Arditti, 1985). However some “meristem-cultured plants may
mature more quickly than plants raised from seeds” (Lecoufle, 1967). Plantlets of
Odontonia Boussole ‘Blanche’ and Odontonia Moliere ‘Lanni’ removed from their flasks
on 30 April 1965 “flowered ten to eleven months later and in blocks of hundreds, less
than two years after being deflasked”
25
(Lecoufle, 1967). If the Odontonia plantlets
were ‘deflasked’ on 30 April 1965, the cultures were probably started in 1964 or 1963
which is before the publication of culture procedures for this hybrid genus and its
parent genera (Odontoglossum ×Miltonia), but after Morel seems to have developed
appropriate methods for them without publishing them (for a review see Arditti &
Ernst, 1993).
In a subsequent paper, published in French, Morel added anatomical details
regarding the protocorm-like body mentioned earlier and referred to attempts to
extend the Cymbidium method to Odontoglossum, Miltonia and Phajus (Morel, 1963).
However this paper did not provide additional details about excision or culture
conditions.
26
It also tended to add to the confusion about a medium which those
203HISTORY OF ORCHID MICROPROPAGATION
seeking to duplicate his results might employ by reporting the use of ‘Knop’s
Solution’ supplemented with 2% glucose (Morel, 1963). The exact composition of
the medium was not given. Morel used a modification of ‘Knop’s Solution’ for potato
stem tips but that paper (Morel & Martin, 1955a) was published in a journal not
widely read outside France and is not cited in the orchid article. Therefore, it is safe
to conclude that it would not have been easy for orchid scientists, and even more
difficult for horticulturists, to gain access to the paper or the recipe.
27
Even if orchid
scientists could find the composition of the potato stem tip medium there were no
indications that it would be suitable for orchids. In fact, the potato stem tip medium
is quite different from that subsequently used for orchids by Morel. It is also
interesting to note that Georges Morel was very familiar with ‘Knop’s Solution’ and
the modified Berthelot trace elements formulation because he used them routinely in
his doctoral dissertation work (Morel, 1948:137 et seq.). Those trying to learn more
about the media used by Morel for orchids could have learned much from this paper
(Morel, 1948), but the connection was not obvious, the published thesis was not well
known, the journal was relatively obscure, and it was published in French.
Modifications of ‘Knop’s Solution’ have been used for the culture of vegetative
axis nodes of Dendrobium and Bletilla (Yam, 1989), and floral stem or scape nodes of
Phalaenopsis (Ball, Reisinger & Arditti, 1974–1975; for a review see Arditti & Ernst,
1993) but there is no indication that these, or any, modifications of this solution
would be suitable for shoot tips of other orchids. This is not surprising because the
available evidence suggests that at present there is no single solution which is suitable
for all orchids (see Arditti & Ernst, 1993 for a review).
A third paper (the second published in English) appeared a year later (Morel,
1964b). It was longer, had more illustrations, added the results of more work with
three genera (Cattleya, Miltonia, Phajus) to those that were being cultured, and
described the culture conditions. It has the potential to lead to confusion rather than
clarification regarding the culture medium because it was listed as “Knudson III”
again. This paper left no doubt that the culture of shoot tips could be used for mass
rapid clonal multiplication but it still did not provide enough information for others
to duplicate the technique. In retrospect, it is clear that even those who were familiar
with all three papers (Morel, 1960, 1963, 1964b) would have had to guess which
medium to use and how to modify it.
28
Guessing would not be conducive to success
especially for commercial and hobby growers. Development of another suitable
medium would have required time (i.e. caused delays for other investigators) and
delayed knowledge of the ‘right’ formulation would have decisively secured for
Vacherot and Lecoufle the lead they already enjoyed. This is an important point
since the only published procedures in the literature even now for Miltonia and Phajus
are the ones published (albeit unclearly) by Morel (see Arditti & Ernst, 1993 for
details). It is not known, however, whether the medium is really pivotal, that is
whether it is crucial to success. Several procedures and media are currently available
for Cattleya and other orchids (Arditti & Ernst, 1993). The same may be true for
Miltonia and Phajus.
A trio of additional papers appeared within the next three years (Morel, 1965a, b.
1966) and at this point the recipes were finally given. Some did but others did not
resemble ‘Knudson C’ medium (more than likely Morel’s ‘Knudson III’), Knop’s
solution or the potato stem tip substrate (Morel & Martin, 1955a) enough to be called
a modification of any of them. One medium for Cymbidium was actually described as
“potato meristem medium” (Morel, 1966). Therefore, one is left wondering about
204 J. ARDITTI AND A. D. KRIKORIAN
the listing of media (Morel, 1960, 1963, 1964b, 1965a, b) especially since the
Knudson and Knop solutions were given as suitable for Miltonia and Cymbidium in a
subsequent paper (Morel, 1970). That paper and an earlier one (Morel, 1966) also
contained additional information about the micropropagation of Cattleya. Informa-
tion about Vandaceous and European orchids and Dendrobium was published
between 1966 and 1970 (Morel, 1966, 1970; see Arditti & Ernst, 1993 for details).
Two reviews (Morel, 1971a, 1974) were Morel’s final contributions in English on
the micropropagation of orchids. Both are excellent and contain a considerable
amount of basic information. His last review (Morel, 1974), like some of the previous
papers (Morel, 1965a, 1966), covers culture media and their components in some
detail. The discussion is both interesting and enlightening. Media recipes and details
about culture conditions are unambiguous. However, by that time the information
was much less important and useful than it would have been in 1960. This is so
because by 1965 a single firm in France (Vacherot and Lecoufle) had established a
monopoly; also, as a result of research carried out throughout the world, several
culture media and procedures for the micropropagation of orchids had been
formulated and published. Publication of a suitable medium in 1960 would have
made the technique available to all who wanted to use it even if (a) the medium used
by Morel was not pivotal, and (b) several media were later shown to be suitable for
some orchids (see Arditti, 1977 and Arditti & Ernst, 1993 for lists and media
recipes).
With one exception (Morel, 1963), the initial orchid papers and several subsequent
ones were published in periodicals aimed at hobbyists and commercial growers
(Morel, 1960, 1964b, 1965a, b, 1966, 1970) and in proceedings of meetings (Morel
& Champagnat, 1969; Morel, 1971a, b, c) rather than peer-reviewed scientific
journals. One reason for this could have been the laudable intent to make the
procedures available to growers. But if this was so, important information (e.g.
culture media recipes, details about techniques etc.) would have had to have been
included in each of them. It was not. Another conspicuous deficiency in these papers
is the lack of literature citations. Previous papers by others which may have been the
source of ideas, media and methods were not cited. This is not in line with the
accepted standards of scientific publication. Lack of citations creates the erroneous
impression that the ideas are original. Peer-reviewed scientific journals would have
probably rejected most of these papers due to insufficient information about methods
and media, and lack of citation of previous work. Yet there is no question whatever
that the calibre of the early research was high enough to justify papers which could
have been published in peer-reviewed scientific journals.
A key problem which cannot currently be solved is why there was only a single
early, incomplete, paper in English, on Cymbidium (Morel, 1960) in a non-reviewed
hobbyist journal. Subsequent papers on Cymbidium (Champagnat, 1965; Champag-
nat, Morel & Gambade, 1966; Champagnat et al., 1968), Cattleya (Champagnat &
Morel, 1969; Champagnat, Morel & Mounetou, 1970), Neottia nidus-avis (Champag-
nat, 1971), and Oprys (Champagnat & Morel, 1972) were published in reviewed
French journals. French is a language which has long since lost its scientific
importance and is one with which most orchid growers in the world were, and are,
not familiar. These papers did contain more details than the first one, but by this
time the importance of the information was much reduced because a detailed
procedure, complete with a medium recipe, had already been published by Wimber
(1963).
205HISTORY OF ORCHID MICROPROPAGATION
“In 1955 Dr Morel performed the meristematic tissue culture of Cymbidium,
Cattleya, Miltonia and Phajus” (Lecoufle, 1971). He “discovered in 1956 that it was
possible to cultivate shoot apices” (Morel, 1964b). Also, “in 1956 [Morel] started to
apply the techniques of meristem culture … previously developed to free potatoes,
dahlias and carnations from viruses, to various Orchids” (Morel, 1965a). Addition-
ally, in 1956, the year “meristem culture was achieved by Dr Morel”, “Dr C. Martin
[one of Morel’s first students] … was received at Vacherot & Lecoufle in 1956
explaining especially … the great achievement made by Dr Morel” (Fig. 37; letter
dated April 1 1985 to J.A. from the late Maurice Lecoufle). These statements raise
yet another question about the first paper on Cymbidium which showed an 18-month
old explant and included the statement that “some plants that are…10cm high”
(Morel, 1960). A Cymbidium plant in vitro or in a pot would certainly grow more than
10cm in 4–5 years (from 1955 or 1956 to 1959 or 1960 when the time the paper was
submitted and published). Therefore it is by no means clear whether the statements
in the paper are accurate (Morel, 1960) or if the report is about plants which were
produced specifically for that article.
“The possibility of producing unlimited numbers of plants from any single orchid
clone” drew the attention of Dr Walter Bertsch, then living in Paris, who was
involved with the breeding programme at Vacherot and Lecoufle (Bertsch, 1966).
Bertsch suggested that Vacherot and Lecoufle enter the field. They did and were
successful immediately (Bertsch, 1966, 1972). As a result “Vacherot and Lecoufle
became the first nursery to develop, on an industrial basis, the meristemming of
orchids. For ten years they held the monopoly” (Lecoufle, 1995). This monopoly
started in the early 1960s or late 1950s (Orchid Digest Staff, 1995). Vacherot and
Lecoufle published a full-page advertisement in the American Orchid Society
Bulletin for June 1964 which included a photograph of a flask containing plantlets of
Laeliocattleya Chine ‘Bouton D’Or’ and stated “we do it” (Fig. 38). The fact that this
cross was registered in 1962 (Royal Horticultural Society, 1961–1963) and the size
of the plantlets suggest that the cultures were started before publication of culture
media for this hybrid genus or its parent genera (Cattleya and Laelia).
Vacherot and Lecoufle’s “we do it” advertisement was followed in December of
that year with a photograph of technicians performing aseptic manipulations in what
appears to be a sophisticated laboratory (Fig. 39). The two advertisements appeared
approximately one year before Morel first published extensive details about his
procedure and the composition of some of his culture media. Georges Morel, the
Vacherots and the Lecoufles were friends. One might speculate that this friendship
prompted him and/or one of his associates to teach Vacherot and Lecoufle the
technique and then to delay publication and withhold information for a while
(Arditti, 1985). Morel was certainly willing to oblige an American orchid
establishment which represented Vacherot and Lecoufle (Fig. 40a, b) with an
endorsement (Fig. 41; Anonymous, c. 1965). At the time, this was not a common
action for a research scientist. Another possibility, of course, is that the delay in
divulging procedural details openly and generally was brought about by an unselfish
and patriotic intent to allow a French firm to capture the market.
Figures 38–41. Commercialization of orchid ‘meristem’ culture. Fig. 38. The first advertisement by the French
orchid firm, Vacherot and Lecoufle announcing the availability of in vitro-produced clonal divisions (American Orchid
Society Bulletin, June 1964, p. 535). Fig. 39. Partial view of the Vacherot and Lecoufle laboratories c. 1964 (American
Orchid Society Bulletin, page 1097). Fig. 40. Text (a) and photograph (b) in an advertisement by Orchids Orlando
(American Orchid Society Bulletin, October 1964, pp. 898–899). Fig. 41. Commercial endorsement by Dr Georges Morel
(Orchids Orlando, n.d.).
206 J. ARDITTI AND A. D. KRIKORIAN
207HISTORY OF ORCHID MICROPROPAGATION
The questions posed here may be evaluated in connection with the facts presented
above, and the rapid (approx. 2 years from the start of research to publication date)
publication of the potato and Dahlia papers. Following that line of reasoning, it may
be argued that the orchid papers might well have been published even more
promptly because the stem tip cultures were well established by then.
Many consider Morel’s orchid work to be highly original and innovative.
However, a somewhat different picture emerges from our critical evaluation of the
facts. None of what Morel did with potatoes, Dahlia and orchids was original. Media
for plant tissue culture in general and stem tips of orchids in particular existed
(Knudson, 1946; Rotor, 1949; Mayer, 1956, Thomale 1956, 1957) before Morel
formulated his own by modifying existing ones. Several explant types (shoot tips,
buds, nodes) from monocotyledonous plants in general and orchids in particular
(Rotor, 1949; Thomale, 1956, 1957) were cultured before Morel did so. Further, a
number of procedures were published following established scientific protocol prior
to his. Shoot tips had been used to free plants of virus infection before Morel’s work
with dahlias, potatoes and orchids (see above). Even Morel’s work on potatoes and
dahlias was suggested by others, namely P. Limasset and P. Cornuet (Gautheret,
1983:402, 1985:42).
Morel’s most significant achievement was to produce protocorm-like bodies (PLBs)
which were sustainable via subculture and this made true mass and rapid clonal
propagation possible. He did that by cleverly combining existing procedures and
culture techniques into a very useful new application. Having done that, he was also
able to generate publicity for an advance whose time had come. Clearly, he should
be credited with imaginatively applying existing knowledge and technology to a new
application. Indeed, in this he played a decisive role. However, he should not be
given the accolades normally reserved for those who originate novel ideas, make
basic discoveries and formulate new principles.
In the course of more recent reminiscences relating to the history of plant tissue
culture, it has been claimed that “Ball is really the father of the so called
micropropagation method” (Gautheret, 1985:16–17). Perhaps Gautheret felt
justified in crediting Ernest Ball because he showed that stem tips can be cultured in
vitro. But Ball does not seem to have appreciated and certainly did not express in
print
20
the practical potential of his work. He was interested in the basic aspects of
growth and development from meristems.
29
Therefore, he is perhaps better viewed
as more of an ‘uncle’ than a ‘father’. If we accept that Ball is not the father, then
Morel could have been, except that (1) Gavino Rotor Jr. first thought of and
implemented in vitro clonal propagation; (2) Hans Thomale was the first to culture
orchid tuber explants; he also drew special attention to the mass propagation
potential of his work, and (3) Donald Wimber was the first to publish a detailed shoot
‘meristem’ culture procedure.
Figures 42–48. Orchid tissue culture: people, plants, companies and a medium component (charcoal). Fig. 42.
Samuel Mosher (Anonymous, n.d.). Fig. 43. Professor Donald E. Wimber (photograph from an Ektachrome
transparency taken by J.A. in Hiroshima in 1987, signature from a letter to J.A.). Fig. 44. Protocorm-like bodies in
liquid culture (Wimber, 1963). Fig. 45. Plantlets in vitro (Wimber, 1963). Fig. 46. Mr Everest McDade (photograph
courtesy Everest McDade, signature from a letter to J.A.). Fig. 48. Rivermont Orchids from the air (back cover,
American Orchid Society Bulletin, January 1952; this advertisement appeared over a number of years, several times and
in more than one orchid magazine).
208 J. ARDITTI AND A. D. KRIKORIAN
209HISTORY OF ORCHID MICROPROPAGATION
THE FOURTH ASEPTIC CULTURE OF AN ORCHID EXPLANT
Samuel Mosher (1893–1970; Fig. 42) grew orchids and eventually established the
Dos Pueblos Orchid Company in Goleta, California. Mosher’s enterprise included
what was described as “the world’s largest establishment for the breeding and
growing of Cymbidium orchids” (Anonymous, n.d.). Mr Mosher was an enlightened
and earnest grower and student of orchids, in many ways a throwback to the great
British firms of yesteryear like Sanders, Veitch, Black and Flory McBean,
Charlesworth and others (Arditti, 1990). He established a modern and well-equipped
laboratory and hired a cytogeneticist, Dr Donald E. Wimber (b.1930; Fig. 43), to
study orchid chromosomes and to manage a large, modern laboratory.
Wimber received his B.S. from San Diego State College in 1952 and M.S. and
doctorate from Claremont College in 1954 and 1956 respectively. He became
associated with the Dos Pueblos Orchid Company and worked there until 1957. In
1963 Wimber accepted an appointment at the Biology Department, University of
Oregon, where he has remained and where he became a distinguished and honored
(American Orchid Society Gold Medal) scientist (Ernst, 1992).
While associated with the Dos Pueblos Orchid Company, Wimber studied orchid
cytology and engaged in seed germination. He had been introduced to the technique
by Emil Vacin, co-formulator of the Vacin and Went medium (Ernst, 1992).
Observing young plants and seedlings led Wimber to tissue culture of orchids. His
first attempt was never published, but it pre-dated both Thomale’s and Morel’s work.
The following account is based on a letter to J.A. from Dr Wimber dated 13
December 1976.
Research with embryonic leaves was carried out in the summer of 1955 while
Wimber was still a graduate student. It involved several immature shoots from a
Cymbidium lowianum clone. The shoots were 4–5cm long. They were surface sterilized
with a 10% dilution of the laundry bleach Clorox after a few of the outside scale
leaves were removed. Several additional leaves were removed before the last 4–6
embryonic leaves were broken off and placed on semi-solid Vacin and Went nutrient
medium. In addition, Wimber made several thin transverse sections through the
shoot axis after removing many of the covering leaves. PLBs developed at the bases
of the embryonic leaves and along the thin sections.
When some of the PLBs were quartered and spread on agar, the sections produced
plantlets. Wimber showed his results to Sam Mosher and Kermit Hernlund,
manager of Dos Pueblos at the time. They were not impressed because the tissues
grew slowly. By Christmas of that year the plantlets were only 2–3mm tall. In 1957
Wimber had a dozen plants in 10–15cm (4–6 inch) pots. Wimber concluded his
letter by stating “I knew I had something, but was rather fearful that some sort of
chromosomal change might have occurred so that a faithful reproduction of the
parent might not occur.” If the cytogeneticist in Wimber had been less persuasive
than the propagator he could have been the one credited with the discovery of mass
rapid clonal propagation of orchids (see below under ‘Concluding remarks’).
In 1963, Wimber published his first paper on clonal propagation of Cymbidium
(Wimber, 1963). Like Morel’s first paper on shoot tip culture of Cymbidium, Wimber’s
report was published in the American Orchid Society Bulletin (Figs 44, 45), but the
similarity ends there. Wimber followed standard scientific practice and provided full
procedural details, gave the recipe of the medium (modified Tsuchiya) he used and
carefully described the culture conditions (continuous illumination of 100 foot-
210 J. ARDITTI AND A. D. KRIKORIAN
candles or less, constant temperature of 22°C, rotary shaker, 125ml Erlenmeyer
flasks sealed with rubber stoppers). Also, Wimber was very clear in calling attention
to the propagation potential of shoot tip cultures. This wealth and clarity of details
is especially remarkable in view of the fact that the procedure was developed while
Wimber was employed by a commercial concern which had every right to keep the
details secret. Morel, on the other hand, worked in a government laboratory and at
one point received funding from the American Orchid Society. Anyone with the
appropriate training or experience with orchid seed germination and the needed
facilities could repeat Wimber’s work immediately. (Incidentally, when Lewis
Knudson established how orchid seedlings could be established asymbiotically, he
too promptly published full details, see Arditti, 1990). A subsequent paper elaborated
on the initial procedures (Wimber, 1965). Indeed, it could even be argued that
Wimber was the first to publish on clonal propagation of orchids through stem tip
culture because his was a scientific (albeit non-reviewed) paper (Wimber, 1963),
rather than what can be called an announcement or news release (Morel, 1960).
30
A PATENT
Following publication of the ‘mericlone’ process by Morel a claim was made by
Mr Everest McDade (b. c. 1916; Fig. 46) that he developed the ‘mericloning’ process
secretly as early as 1950 and kept it secret or at least did not publicize it at the time.
This information (Bergman, 1972) as well as publications by Morel, Professor Harry
C. Kohl (Kohl, 1962) and Wimber were used (Torrey, 1985b) to obtain eventually
a patent (U.S. 3,514,900) for the process. According to McDade, an electronic
engineer, science teacher and co-owner of Rivermont Orchids, Signal Mountain,
Tennessee (Fig. 47) until c. 1949 (the firm no longer exists), the idea originated from
a “photo of a Cymbidium bulb, with a cluster of buds at its base” (letter to J.A. from
Everest McDade, Asheville, North Carolina). An article from c. 1946 or 1947 (which
McDade does not have and claims he is still trying to find) that accompanied the
photograph “was a very sudden clear message to us [McDade]. Just what we had
been looking for: a renewable source of ‘Ramets’…. We wanted to use the process
for Cattleya types … I adapted the Cymbidium idea to cattleyas”.
According to McDade, his secretary Dorothy Smith “made the first meristem
cultures in 1950”. He “wrote scores of letters … to authors … botany and genetics
journals. [but] Only a few people took them seriously, or even guessed that we had
discovered cloning and patents in [the] year 1950.” McDade also claims that “in
October, 1952 [he] actually gave the cloning process paper to the [American Orchid
Society Bulletin] … for publication and demonstrated a growth developing from a Cattleya
flower stem, a flask, and community pot of a clone” (emphasis by McDade).
However, McDade’s claims are not borne out by the paper he published in the
American Orchid Society Bulletin (McDade, 1952). Rivermont advertisements from that
period did not offer for sale any orchids described as being clonally propagated. The
granted patent itself ‘relied on’ work by Morel, Kohl and Wimber (U.S. patent
3,514,900 filed August 11, 1967 and awarded June 2, 1970; Torrey, 1985a, b). The
chronology itself is suspect. It involves an idea which supposedly originated in 1946
or 1947, cultures that were presumably first made in 1950, work which was
published between 1955 and 1963, and a patent not issued until 1970. An equally
211HISTORY OF ORCHID MICROPROPAGATION
good case, perhaps a better one, can be made for a suggestion that the patent was
an attempt to benefit from the ‘mericlone’ process.
DARKENING OF MEDIA
Orchid seed germination and micropropagation media are often darkened with
activated charcoal because the plants grow better in the presence of this additive.
John T. Curtis was the first to darken a nutrient medium for orchid seedlings in vitro.
He did it in an unsuccessful effort to simulate natural conditions and thereby bring
about the germination of Cypripedium reginae, C. pubescens, C. parviflorum, C. candidum and
C. acaule seeds (Curtis, 1943). Curtis used lampblack which has very little in common
with activated charcoal except colour. Lampblack is soot produced by the burning of
petroleum hydrocarbons. It does not have the large internal surface area, adsorptive
properties and pore structure of charcoal. Lampblack has long been used in the
production of black inks and paints.
Vegetable charcoal (Fig. 47) is made from wood, sawdust, peat and organic
residues which are recovered during the production of pulp (West Virginia Pulp and
Paper, n.d.), carbonized and activated to produce a large surface area. As a result for
instance, one gram of Nuchar may contain up to 120 billion particles and have a
total surface area of 500 to 2000m
2
. Pore distribution can range from <10µm to
>500µm (Yam et al., 1990). The pore to volume ratio is 0.9 cc g
–1
. Charcoals can
contain many elements, some in very small amounts (Yam et al., 1990). Activation is
carried out through treatment of the carbonized pyrolysis product with steam or
carbon dioxide (Yam et al., 1990).
Charcoal was first used to darken an orchid culture medium by Prof. Peter
Werkmeister in Germany (Werkmeister, 1970a, b, 1971). At the same time it was
employed to germinate moss spores and grow filamentous algae (Proskauer &
Berman, 1970; Krikorian, 1988). Werkmeister darkened the medium to study the
growth of roots, gravitropism and proliferation of clonally propagated plantlets. He
died not long after publishing the last of his orchid papers.
Robert Ernst (b. 1916; Fig. 49), a surfactant chemist and manufacturer, and
Professor of Biology at the University of California, Irvine, was the first to add
charcoal to practical seedling culture media and found that Paphiopedilum and
Phalaenopsis seedlings grew well on it (Ernst, 1974, 1975, 1976). Ernst’s findings
resulted in the formulation and widespread use of charcoal-containing media for
orchid seed germination, seedling culture and micropropagation (Ernst, 1974, 1975,
1976; Yam et al., 1990). The reasons for the beneficial effects of charcoal are still
under discussion even if not the subject of intensive research (for reviews see Yam et
al., 1990; Mohammed-Yasseen et al., 1995) and beyond the scope of this account.
TERMINOLOGY AND SEMANTICS
By 1964 clonal multiplication of orchids by means of aseptic culture had become
an important part of orchid horticulture (Dillon, 1964; Scully, 1964). Discussions
centered on techniques, growth, mutations during culture, costs and nomenclature.
The question was what to call plants produced by this method which was
inappropriately called “meristem culture” (Dillon, 1964; morphologically what was
212 J. ARDITTI AND A. D. KRIKORIAN
being cultured were stem tips). Terms such as ‘mass rapid clonal propagation’,
‘micropropagation’, or ‘tissue culture propagation’ had not yet been invented. One
suggestion by Dr Robert D. Patton of Galena, Ohio, namely, ‘meristem division’ was
deemed “a good term” (Dillon, 1964), but ‘mericlone’, a term proposed by then Lt.
and now orchid grower for Carter & Holmes Orchids in Newberry, South Carolina,
Gene Crocker (Fig. 50) was judged to be better (Dillon, 1964). This term seems to
have excellent marketing appeal. However, it is scientifically inaccurate and
uninformative. Moreover, it is a linguistic abomination (Arditti & Ernst, 1993),
whether used as a verb (“to mericlone a plant”), a noun (“this plant is a mericlone”),
a description (“the mericlone industry”, or “he/she is a mericloner”) or part of the
language (“this book is about mericloning”). The only potentially charitable
statement that may be made for the term is that it upholds a long-standing ‘tradition’
in tissue culture in that it perpetuates the propensity to adopt misleading
terminologies. I.W. Bailey (1884–1967), a pioneer and influential American plant
anatomist and morphologist at Harvard University early complained that the term
‘tissue culture’ was a misnomer because only rarely are cultures derived from or are
comprised of specific tissues (Bailey, 1943). His exhortations had little effect on the
then relatively new field and the term ‘tissue culture’ seems to be with us forever.
SUBSEQUENT RESEARCH
The early history of orchid micropropagation ends around 1965. Many workers
entered the field at that time. Scientists, hobbyists, commercial growers and
laboratory operators attempted to culture stem tips and other explants from many
genera. As a result, the number of papers on orchid micropropagation is now
enormous (for reviews see Morel, 1974; Arditti, 1977; Arditti & Ernst, 1993).
Numerous orchids have been cultured (Table 1; only the first publication regarding
the aseptic culture or micropropagation of a specific genus or explant type is listed).
Significantly, some genera of hobby or commercial interest like Paphiopedilum have
not yet yielded to micropropagation despite being cultured experimentally in the
laboratory (see Arditti & Ernst, 1993 for details). Many north temperate species
including another ‘slipper’ genus, Cypripedium, have also been recalcitrant.
CELL AND PROTOPLAST CULTURE
The first attempts to culture free plant cells utilized mechanically isolated ones.
Although fairly intensive efforts were made by Gottlieb Haberlandt in 1898 and
1902 (Krikorian & Berquam, 1969; Krikorian, 1975, 1982; Steward & Krikorian,
1975), he did not succeed. A suggestion that Haberlandt failed because he neglected
the findings of a French naval architect and agronomist, Henri-Louis Duhamel du
Monceau (1700–1782) who also studied wound healing in trees (Gautheret, 1985)
probably has its roots in Gallic chauvinism rather than scientific reality. Haberlandt
failed for the following reasons: (1) his ideas were more advanced than the plant
science ‘biotechnology’ of his day; (2) his selection of cells to culture (mature,
differentiated, specialized non-meristematic) was inappropriate; (3) his culture media
did not include all necessary componentshe used “tap water, one to five percent
sucrose solutions, and Knop’s solution with or without sucrose, dextrose, glycerine,
213HISTORY OF ORCHID MICROPROPAGATION
TABLE 1. Chronology of formulation of protoplast, cell, tissue and organ culture procedure
s
for orchidsa,b
Genus Explant Reference
Acampe praemorsa Protoplasts Seeni & Abraham, 1986
Acampe rigida Leaf tips Yam, 1989; Yam & Weatherhead,
1991a
Aëridachnis Apical and axillary buds Lim-Ho, 1981
Aerides Protoplasts Seeni & Abraham, 1986
Anacamptis pyramidalis Shoot tips Morel, 1970
Angraecum eburneum as Protoplasts Price & Earle, 1984
Angraecum giryamae
Anoectochilus elatus Protoplasts Gopalakrishnan & Seeni, 1987
Anoectochilus formosanus Lateral bud explants, Chow, Hsie & Chang, 1982
(Anoectochilus roxburghii) cuttings, layering
Arachnis hookeriana Terminal and lateral buds Vajrabhaya & Vajrabhaya,
1976a, b
Arachnostylis Apical and axillary buds Lim-Ho, 1981
Aranda Shoot tips Goh, 1973
Protoplasts Loh & Rao, 1985
Aranthera Apical and axillary buds Irawati et al., 1977
Leaf bases Lim-Ho, 1981
Arundina Shoot tips Mitra, 1971
Ascocenda Young leaves Fu, 1978
Bud explants Lim-Ho, 1981
(as Schlechterara) Shoot meristems Ichihashi, 1979
Ascocentrum Protoplasts from flowers, leaves Oshiro & Steinhart, 1991
and roots
Ascofinetia Inflorescences Intuwong & Sagawa, 1973
Barlia Protoplasts Pais et al., 1982
Bletilla striata Root tips, stem nodes Yam, 1989
Brassavola Protoplasts Seeni & Abraham, 1986
Brassia Protoplasts Capesius & Meyer, 1977
Brassia brachiata Protoplasts from root tip Yasugi, 1989a, b
Brassocattleya Buds Scully, 1967
Bulbophyllum Protoplasts Seeni & Abraham, 1986
Burkillara Apical and axillary buds Lim-Ho, 1981
Calanthe Protoplasts Seeni & Abraham, 1986;
Yasugi et al., 1986
Catasetum Root tips Kerbauy, 1984
Cattleya Shoot tips Morel, 1964a, 1970
Lateral buds Reinert & Mohr, 1967
Dormant backbulb buds Vajrabhaya, 1978
Leaf bases Champagnat et al., 1970
Leaf tips Arditti, Ball & Churchill, 1971
Leaf tissues Fu, 1978
Protoplasts Capesius & Meyer, 1977
Protoplasts from flowers Oshiro & Steinhart, 1991
Cattleya aurantiaca Protoplasts from leaves and Yasugi, 1989a, b
roots
Cattleya skinneri Protoplasts Yasugi, 1900
Cymbidium Cells, sloughed off from shoot Steward & Mapes, 1971a
tip cultures
Cymbidium Protoplasts Capesius & Meyer, 1977
Cymbidium Floral organs Kim & Kako, 1982
Protoplasts Oshiro & Steinhart, 1991
Cymbidium ensifolium Shoot tips some cultures Wang et al., 1984, 1988a, b
flowered in vitro
Cymbidium faberi Rhizomes Hasegawa, Ohashi & Goi, 1985
Various Hasegawa, 1987
Cymbidium forestii Various Hasegawa, 1987
214 J. ARDITTI AND A. D. KRIKORIAN
TABLE 1. (continued)
Genus Explant Reference
Cymbidium goeringii Shoot tips Wang et al., 1981
Various Hasegawa, 1987
Cymbidium hakuran Various Hasegawa, 1987
Cymbidium hybrids Shoot tips Morel, 1960; Wimber 1963
Cymbidium insigne Shoots Ueda & Torikata, 1972
Various Hasegawa, 1987
Cymbidium kanran Various Hasegawa, 1987
Cymbidium Kenny
‘Wine Color’ Protoplasts Yasugi, 1990
Cymbidium sinense Various Hasegawa, 1987
Cypripedium Various Morel, 1971b
Cyrtopodium Root tips Sanchez, 1988
Dactylorchis Dormant shoot (not details) Stokes, 1974
Darwinara Embryonic callus S. Ichihashib
Protoplasts S. Ichihashib
Plants regenerated from
protoplasts S. Ichihashib
Dendrobium No details of any kind Morel, 1965a, b
Dendrobium Flower stalks Singh & Sagawa, 1972
Protoplasts Price & Earle, 1984
Protoplasts Yasugi, 1990
Protoplasts and cells were
used to regenerate plants Sajise et al., 1990
Dendrobium aggregatum Protoplasts Yasugi et al., 1986
Dendrobium antenatum Axillary buds Kukulczanka & Wojciechowska,
1983
Dendrobium aduncum Stem nodes Yam, 1989
Dendrobium crumenatum Leaves Manorama et al., 1986
Dendrobium chrysanthum Pseudobulb segments Vij & Pathak, 1989
Dendrobium hybrids Axillary buds Sagawa & Soji, 1967
Nodes Arditti, Mosich & Ball, 1973
Protoplasts Yasugi et al., 1986; Yasugi,
1989a, b
Dendrobium loddigesii Stem nodes Yam, 1989
Dendrobium nobile Ovary Ito, 1966
Dendrobium phalaenopsis Shoot tips Gandawijaja, 1980
Dendrobium transparens Shoot tips Yam, 1989
Disa Shoot apices Haas & Lückel, 1977
Doritaenopsis Lateral buds from
flower stalks Lim-Ho, 1981
Embryogenic callus S. Ichihashib
Doritis Leaves Sagawa & Kunisaki, 1982
Epidendrum Node sections Stewart & Button, 1976
Root tips Churchill, Ball & Arditti, 1972
Leaf tips Churchill, Ball & Arditti, 1973
Protoplasts Yasugi et al., 1986
Protoplasts from flowers Oshiro and Steinhart, 1991
Epidendrum radicans Protoplasts Yasugi, 1990
Epiphronitis Shoot tips Kusumoto, 1981
Eulophia hormusjii Rhizome segments Vij, Sood & Pathak, 1989
Geodorum densiflorum Protoplasts Seeni & Abraham, 1986
Grammatophyllum elegans Protoplasts Seeni & Abraham, 1986
Hetaeria Creeping rhizome sections Yam, 1989
Holttumara Apical and axillary buds Lim-Ho, 1981
Kagawara Apical and axillary bds Lim-Ho, 1981
Laelia Shoots Kako, 1973
Laeliocattleya Shoot tips Ishii, 1974
Leaf tips Arditti et al., 1971
215HISTORY OF ORCHID MICROPROPAGATION
TABLE 1. (continued)
Genus Explant Reference
Liparis nervosa Stem sections Yam, 1989
Liparis rigida Leaf tips Yam, 1989
Ludisia (Haemaria) Single node sections Teo, 1978
Luisia trichorhiza Leaf segments Vij & Pathak, 1988a
Luisia zeylanica Protoplasts Seeni & Abraham, 1986
Lycaste No details of any kind Morel, 1965a, b
Malaxis acuminata Stem sections Yam, 1989
Maxillaria tenuifolia Protoplasts Price & Earle, 1984
Miltonia No details of any kind Morel, 1965a, b
Shoot tips Kusumoto, 1981
Mokara Flower buds Lim-Ho, Teo-Lee & Phua, 1984
Axillary buds Lim-Ho, 1981
Inflorescence tips Abdul Ghani c.1988 as reported
in Arditti & Ernst, 1983 (the
following papers were just being
written then: Abdul Ghani,
Haris & bin Haji Ujang, 1992b;
Abdul Ghani & Haris, 1992)
Young leaves Abdul Ghani c. 1988 as reported
in Arditti & Ernst, 1993 (the
following papers were just being
written then: Abdul Ghani et al.,
1992b; Abdul & Ghani Haris,
1992
Mormodes Pseudobulb sections Hölters & Zimmer, 1990a
Mormodes histrio Root explants Hölters & Zimmer, 1990b
Neofinetia falcata Embryogenic callus S. Ichihashib
Neostylis Various Sagawa & Kunisaki, 1982
Neottia Root Champagnat, 1971
Nigritella Tubers Haas, 1977a, 1977b
Oberonia Protoplasts Seeni & Abraham, 1986
Odontoglossum No details of any kind Morel, 1965a, b
Apical and axillary buds Khaw, Ong & Nair, 1978a, b
Protoplasts Price & Earle, 1984
Oncidium Flower stalks Lim-Ho & Lee, 1987
Apical shoot explants Khaw et al., 1978a, b
Apical buds Lim-Ho, 1981
Oncidium papilio Flower stalk tips Fast, 1973
Ophrys apifera Tuber sections Champagnat & Morel, 1972
Tuber and shoot base Hoppe & Hoppe, 1987a, b, 1988
Ophrys bombylifera Protoplasts Pais et al., 1983
Ophrys fuciflora Tuber sections Champagnat & Morel, 1972
Ophrys lutea Protoplasts Pais et al., 1983
Orchis coriophora Bud meristems Allenberg, 1976
Orchis maculata Tuber sections Thomale, 1956 (first suggestion
that mass rapid clonal
propagation of orchids in vitro
is possible), 1957 (first
photograph and report of
a regenerated culture in vitro)
Pachystoma senile Tubers Vij et al., 1983
Paphiopedilum Protoplasts Yasugi, 1989a, b
Paphiopedilum insigne Protoplasts Yasugi, 1990
Paphiopedilum species and hybrids Shoot tips Bubeck, 1973
Leaf tips Allenberg, 1976
Protoplasts Price & Earle, 1984
Phajus (misspelled as Phaius) No details of any kind Morel, 1965a, b
216 J. ARDITTI AND A. D. KRIKORIAN
TABLE 1. (continued)
Genus Explant Reference
Phalaenopsis Inflorescence nodes Rotor, 1949; first time an orchid,
or any plant, was propagated
vegetatively in vitro
Shoot tips Intuwong & Sagawa, 1974
Leaf tissue Koch, 1974
Root tips Tanaka, Senda & Hasegawa, 1976
Inflorescence internodes Lin, 1986
Protoplasts Teo & Neumann, 1978a, b;
Chen et al., 1990
Sajise et al., 1990
Plants from protoplasts Kobayashi et al., 1993
Embryogenic callus S. Ichihashib
Electrofusion of protoplasts Chen et al., 1990, 1991, 1995
Phalaenopsis amabilis Protoplasts Yasugi, 1990
Pholidota cantonensis Root tips Yam, 1989
Pholidota chinensis Leaf tips Yam, 1989
Phragmipedium Axillary bud explants Stokes, Thomas & Holdgate,
1975; paper did not present any
details and should be questioned
No details of any kind Morel, 1971b, c
Flower buds Fast, 1973
Pleione Shoot tips Weatherhead & Harberd, 1980
Renantanda Leaf explants Goh & Tan, 1982
Shoot tips Abdul Ghani as reported in
Arditti & Ernst, 1993
(Abdul Ghani, Haris & bin Haji
Ujang, 1992a was just being
written)
Protoplasts Teo & Neumann, 1978b, c
Rhynchostylis gigantea Buds and shoot tips Vajrabhaya & Vajrabhaya, 1970
Rhynchostylis retusa Leaf segments Vij, Sood & Plaha, 1984
Root segments Sood & Vij, 1986
Protoplasts Seeni & Abraham, 1986
Rhynchostylis retusa Root- and leaf-tips Sharma & Chaturvedi, 1988
Schomburgkia superbiens Shoot tips Scully, 1967
Sophrolaeliocattleya Shoot tips Kako, 1969
Spathoglottis Flower buds Intuwong & Sagawa, 1974
Spathoglottis plicata Rhizomes Bapat & Narayanaswami, 1977
Protoplasts Seeni & Abraham, 1986
Thunia alba Flower stalks Singh & Prakash, 1984
Vanda Leaf tips Chaturvedi & Sharma, 1986
Inflorescences Valmayor, Pimentel & Martinez,
1986
Leaf explants Tanaka, Hasegawa & Goi, 1974
Protoplasts from flowers Oshiro & Steinhart, 1991
Embryogenic callus S. Ichihashib
Vanda coerulea Leaf bases Anonymous, 1987, Seeni, 1988
Vanda Miss Joaquim Shoot tips Kunisaki, Kim & Sagawa, 1972
Stems Sagawa & Sehgal, 1967
Axillary buds and root-tips Goh, 1970
Vanda praemorsa Protoplasts Seeni & Abraham, 1986
Vanda sp. Protoplasts Sajise et al., 1990
Vanda testacea Root tips Vij & Pathak, 1988b
Vanda TMA X V. teres Root- and leaf-tips Sharma & Chaturvedi, 1988
Vandofinetia Not mentioned Intuwong & Sagawa, 1974
Vanilla planifolia Nodal stem sections Kononowicz & Janick, 1984
Lateral buds Gu, Arditti & Nyman, 1987
Aerial root-tips Philip & Nainar, 1986
Axillary buds Cervera & Madrigal, 1980
217HISTORY OF ORCHID MICROPROPAGATION
TABLE 1. (continued)
Genus Explant Reference
Protoplasts Seeni & Abraham, 1986
Vascostylis Inflorescences Intuwong & Sagawa, 1973
Vuylstekeara Shoots Kukulczanka et al., 1989
aGenera are listed alphabetically; the reference given is the first known for each genus and/or type of explant.
Some of the genera listed here are hybrids. Their parentage and year of registration are listed below.
Year of
Hybrid genus Parentage registration
Aëridachnis Aerides X Arachnis 1954
Arachnostylis Arachnis X Rhynchostylis 1966
Aranda Arachnis X Vanda 1937
Aranthera Arachnis X Renanthera 1936
Ascocenda Ascocentrum X Vanda 1949
(some Vanda species were transferred to
the genus Euanthe, see also Schlechterara)
Ascofinetia Ascocentrum X Neofinetia 1961
Brassocattleya Brassavola X Cattleya 1889
Burkillara Aerides X Arachnis X Vanda 1967
Darwinara Ascocentrum X Neofinetia X
Rhynchostylis X Vanda 1980
Doritaenopsis Doritis X Phalaenopsis 1935
Epiphronitis Epidendrum X Sophronitis 1890
Holttumara Arachnis X Renanthera X Vanda 1958
Kagawara Ascocentrum X Renanthera X Vanda 1968
Laeliocattleya Cattleya X Laelia 1887
Mokara Arachnis X Ascocentrum X Vanda 1969
Neostylis Neofinetia X Rhynchostylis 1965
Renantanda Renantherra X Vanda 1935
Schlechterara Ascocentrum X Euanthe X Vanda 1966
(some Vanda species were transferred to the genus
Euanthe, see also Ascocenda)
Sophrolaeliocattleya Cattleya X Laelia X Sophronitis 1897
Vandofinetia Neofinetia X Vanda 1960
Vascostylis Ascocentrum X Rhynchostylis X Vanda 1964
Vuylstekeara Cochlioda X Miltonia X Odontoglossum 1911
bPersonal communication from Dr. Syoichi Ichihashi, Department of Life Science, Aichi University of Education,
Aichi, Japan.
218 J. ARDITTI AND A. D. KRIKORIAN
asparagine and peptone in various combinations and concentrations” (Krikorian &
Berquam, 1969);
31
(4) his choice of plants included monocotyledonous species which
can be recalcitrant; (5) he had no previous information to guide him; and (6) his
cultures, although sanitary, were not aseptic (Haberlandt, 1902 translated by
Krikorian & Berquam, 1969).
Not as well known as his work with orchids, but well ahead of its time was Lewis
Knudson’s attempt to culture sloughed-off root cap cells of maize and Canada field-
pea (Knudson, 1919). As a culture medium he employed water and, foreshadowing
his work with orchids (for a review see Arditti, 1990), ‘Pfeffer’s Solution’ modified
through the use of monobasic potassium phosphate instead of the dibasic salt and
with or without 0.5% sucrose. Some of the Canada pea cells survived for 50 days
when roots were present in the culture medium. They lived for another 21 days after
the roots were removed despite becoming contaminated. These experiments
suggested the diffusion from roots of growth substances which the cells needed, but
it needs to be borne in mind that this work was done: (1) 8 years before the discovery
of auxins (Went, 1928, 1990; Thimann, 1980); (2) 20 years before it was shown that
vitamin B
1
, niacin and other factors enhance the growth of stem sections in general,
and excised roots in vitro in particular (Bonner, 1937, 1938, 1940a, b; Addicott &
Bonner, 1938; Bonner & Devirian, 1939); (3) 35 years before the discovery of
cytokinins (Miller et al., 1955a, b; Miller, 1961, 1977; Skoog, 1994). Even if he
surmised that his cells needed growth substances, few if any were available at the
time and Knudson’s attempts failed. However, it is surprising that he did not use
aseptic techniques since the procedures were available in his laboratory by that time,
even though this research was carried out 1–2 years before he started work on non-
symbiotic germination of orchid seeds (for a review see Arditti, 1990).
The first isolated cells to be cultured successfully were those of tobacco, Nicotiana
tabacum and marigold, Tagetes erecta. They were grown on filter paper platforms placed
on top of proliferating callus masses (for reviews see Muir, Hildebrandt & Riker,
1954, 1958; Steward & Krikorian, 1975; Krikorian, 1975, 1982; Gautheret, 1983,
1985). Proof that the colonies on the platforms did not form from cells of callus origin
that grew through the paper was obtained by culturing a single cell of Tagetes erecta
on a platform placed on top of a sunflower callus (Muir et al., 1958). Other research
followed (Bergmann, 1960) and convincing proof that a single isolated cell can divide
and eventually yield a large callus mass was provided by tobacco cells which divided
in drops of medium in microculture (Vasil & Hildebrandt, 1965a, b). Shortly after
that, isolated mesophyll cells of Arachis hypogea were prompted to divide in culture and
produced what can be described as protocorm-like bodies or structures which look
like them (Joshi & Ball, 1968a, b).
Using an apparatus that slowly (1 r.p.m.) rotates ‘nipple’ culture flasks around a
horizontal axis, F[rederick] C[ampion] Steward (1904–1994; Fig. 51) and his
associates at Cornell University, Marion O. Mapes (1913–1981; Fig. 52) and
Kathryn Mears (Fig. 53) obtained suspension cultures of carrot cells and eventually
regenerated plants from them (for reviews see Krikorian, 1975, 1982, 1989b;
Steward & Krikorian, 1975; Gautheret, 1983, 1985; Arditti & Ernst, 1993). The
same technique was used to obtain the first orchid cell suspension cultures from
which plants, those of Cymbidium, were regenerated (Steward & Mapes, 1971a). More
recently Phalaenopsis plants have been regenerated from embryoids derived from a
loose-celled callus (Sajise, Valmayor & Sagawa, 1990). See Krikorian (1996) for a
discussion of ‘free’ cell culture and related terminology.
219HISTORY OF ORCHID MICROPROPAGATION
Protoplast preparations were first obtained in 1892 by surgically releasing them
from plasmolysed cells of the water aloe Stratiotes aloides. Digestion of cell walls
became the eventual method of choice and (for historical surveys see Steward &
Krikorian, 1975; Krikorian, 1982). What is probably the first preparation of orchid
protoplasts resulted from work with leaves (i.e. mesophyll cells) of Cymbidium Ceres
and virus free protocorms of Cymbidium pumilum, Brassia maculata and Cattleya
schmobocattleya (Capesius & Meyer, 1977).
32
The protoplasts were used for the
isolation of nuclei but apparently no effort was made to produce callus masses or
regenerate plants from them.
Production of orchid protoplasts and subsequent fusion between and within
genera was first reported in 1978, but the ultimate fate of the fusion products has not
been described in the literature (Teo & Neumann, 1978a, b, c). Subsequent isolations
of orchid protoplasts have been reported from Portugal (Pais, Anjos, & Rangel de
Lima, 1982, 1983); the U.S.A. (Price & Earle, 1984; Sajise et al., 1990), Singapore
(Loh & Rao, 1985; Hew & Yip, 1986; Hew, 1987; Koh, Goh & Loh, 1988), Japan
(Yasugi, Kagimiya & Katsura, 1986; Yasugi, 1986, 1989 a, b, 1990; Kobayashi,
Kameya & Ichihashi, 1993), India (Seeni & Abraham, 1986; Gopalakrishnan &
Seeni, 1987), and Taiwan (Chen et al., 1990). More recently, 4 310
6
protoplasts
were obtained per gram of young leaf tissue of Phalaenopsis (Chen et al., 1995). Their
average diameters were 31.2µm. The diameters of protoplasts from root tips and
petals were 36.4µm and 31.1µm respectively. Approximately 90% of the protoplasts
were viable. Some of the protoplasts divided after 5 days. Only a few divided twice
after 10 days. Very few clusters were formed after 21 days, and they eventually died
(Chen et al., 1995). However, Yoneo Sagawa (b. 11 October 1926; Fig. 54) and his
co-workers at the University of Hawaii have reported regeneration of Phalaenopsis
plants from protoplasts (Sajise et al., 1990).
A direct electrical current pulse of 2500Vcm
–1
of 2 milliseconds duration was
sufficient to cause fusion in 10% of Phalaenopsis protoplasts to form hybrid cells. The
fate of these fusion products was not described, but the context of the paper (Chen
et al., 1995) suggests that they did not survive. Studies of transgenic Phalaenopsis are
also in progress (Chen et al., 1995).
CONCLUDING REMARKS
Orchids were the first plants to be propagated in vitro, from seeds (symbiotically by
No¨el Bernard in France c. 1900 and asymbiotically by Lewis Knudson in the U.S.A.
in 1921), or clonally (for reviews see Arditti, 1967, 1977, 1979, 1990, 1992; Arditti
et al., 1982; Arditti & Arditti, 1985; Arditti & Ernst, 1993). The first papers on in vitro
seed propagation were published in well-known scientific journals with complete
details on all aspects of the procedures in line with accepted scientific standards. This
was not the case with all of the early, and even several later papers on in vitro clonal
propagation of orchids. Most of the papers were not peer-reviewed and were
published in journals intended for orchid hobbyists. As a result, early publications
often either remained unnoticed or did not meet all accepted scientific standards.
They frequently lacked sufficient detail to enable duplication of results.
A person who could have gained substantial credit for her work in the early use
of aseptic techniques to propagate plants but did not do so because none was
published is Clara S. Hires (1897–1984; Fig. 55). She was encouraged by her family
220 J. ARDITTI AND A. D. KRIKORIAN
Figures 49–56. Persons and equipment associated with in vitro culture of orchids and other plants. Fig. 49.
Professor Robert Ernst (photograph by J.A., signature from J.A.’s files). Fig. 50. Mr Gene Crocker
(photograph taken by J.A. in Brisbane, Australia in 1984). Fig. 51. Professor F.C. Steward, alone (a) and
with Mr Russell C. Mott (b) admiring a Cymbidium plant derived from a cell suspension cultured by Mrs
Marion O. Mapes (Fig. 52) under his direction. Mott was in charge of the Conservatory of the Liberty
Hyde Bailey Hortorium at Cornell University and reared the ‘free’ cell-derived orchids to maturity. Fig.
53. Miss Kathryn Mears (now Mrs Trupin) placing ‘nipple’ culture flask of carrot cells on a rotating wheel
or ‘auxophyton’ in F.C. Steward’s laboratory in the Plant Sciences Building (Figs 51–53 from
photographs and documents in the possession of A.D.K.; see also Krikorian, 1989). Fig. 54. Professor
Yoneo Sagawa lecturing in Nagoya, Japan in 1990). Fig. 55. Miss Clara S. Hires examining a flask near
a window (photograph supplied to A.D.K. by Clara Hires). Fig. 56. A rotating drum shaker for cultures
(Morel, 1966).
221HISTORY OF ORCHID MICROPROPAGATION
to spend time in Knudson’s laboratory after she had gotten her undergraduate
degree at Cornell in the late 1920s so as to learn about asymbiotic orchid seed
germination. The aim was to keep up supplies of vanilla for the family owned Hires
Root Beer Company. Later Miss Hires operated a biological supply house under the
title of ‘Mistaire Laboratories’ in Milburn, New Jersey. It provided various and
sundry items like ferns in different stages of development to schools and colleges etc.
These were aseptically propagated and/or multiplied in vitro. Pictures of the
laboratory when it was in full operation are impressive and in many respects it was
state-of-the-art. However, virtually none of the aseptic culture work was published
(Hires, 1940). No doubt she was a pioneer in the industrialization of aseptic methods
for propagating plants in the U.S.A. She not only grew lower plants (some of which
increased vegetatively ‘spontaneously’ in vitro) but she was concerned with cinchona
and, of course, orchids (Krikorian, 1979, 1982:171). Had Clara Hires published, one
would perforce credit her work with lower vascular and non-vascular plants as pre-
dating the higher plant ‘micropropagation’ work of Gavino Rotor Jr. with
Phalaeonopsis.
We have already discussed the situation where workers suggest, peripherally or
even directly, a process or a use but do not follow through on it (Curtis & Nichol,
1948 and p. 187 of this paper). In a similar vein, one may mention the carrying out
of a process by an investigator not fully appreciating the potential of what he or she
has accomplished. Finally, there are situations where valuable discoveries are not
appreciated for a number of reasons.
From our more narrow perspective on orchid micropropagation, the problem
caused by not strictly adhering to the rules of scientific publication was that the
procedures did not become promptly available to all scientists and practical growers.
As it turned out, only a single French commercial orchid establishment managed to
obtain the information needed to produce plants from stem tips on a commercial
scale. They held a monopoly for a long time. Growers wishing to have increased
stocks of plants were obliged to send materials to France for ‘mericloning’. These
activities pre-dated substantially the development of ‘new’ technologies for
germplasm exchange of in vitro materials (Roca, 1984; Krikorian, 1994a, b and
references therein). Partial publication of orchid micropropagation research has
made it difficult to unravel fully the ‘muddied’ historical track and adjudicate or
resolve some largely unprovable or incorrect claims of priority in discovery. It is
interesting that once perceptions of priority of discovery become established, they are
indeed maintained by ‘resistance to knowledge’.
‘Resistance to Knowledge’ is the title of a prefatory chapter by Hans Gaffron
(1902–1979), a plant physiologist of note who worked on certain aspects of
photosynthesis (Gaffron, 1969). While the chapter does not have much to do directly
with photosynthesis or plant physiology, it points out eloquently that established
institutions and dogma do not take kindly toward new ideas or challenges to ‘proven’
or generally accepted ‘truths’. Two points in the chapter are of particular relevance
to the question of how micropropagation came about. The first concerns
‘Heisenberg’s formulation’, which states that ‘science clears the fields on which
technology can build’. In turn, technology delivers to science bigger and better
bulldozers to clear the fields ever faster (Gaffron, 1969). This is relevant to orchid
micropropagation history because most discussions of the subject by-pass early
research and many pioneers. They ignore the clearing of the fields and start with, or
222 J. ARDITTI AND A. D. KRIKORIAN
even showcase secondary developments and personalities that have attracted the
most attention (i.e. louder and bigger bulldozers).
A second point made by Gaffron is that “those establishments whose power rests
with God-given ignorance and thoughtlessness of their subjects rightly recognize
free-science as an ever-present threat”. He continues, “in the twentieth century …
the refined modern way of minimizing the subversive power of research is to distract
the minds of scientists from certain problems by offering them unheard-of-
opportunities to run after … others …”. In the context of orchid micropropagation
the ‘establishments’ held generally accepted and widely held views which were, in
large measure, derived from flawed information, unnoticed or ignored truths and/or
constant repetition. The resistance to knowledge is indeed a fierce protector of these
‘establishments’. Gaffron’s “free-science” and the “subversive power of research” can
be equated with attempts to seek and present facts which shake the foundations of the
‘establishments’ as well as the science and technology that preceded secondary
discoveries. This, in turn, may eliminate the pedestals which support established but
not always deserving idols. There is much in Gaffron’s scholarly and entertaining
essay to appeal to iconoclasts.
There is an extensive literature on what makes scientists ‘tick’, revealing how egos
come into play in the scientific process (Schilling, 1958; Snow, 1961; Merton, 1973;
uller-Hill, 1993). Increasingly, attention has been given to how some scientists even
come to ‘betray the truth’ (Broad & Wade, 1982), or go so far as to re-write
history.
33,34
In addition to recognizing the human aspects of the problem, it is instructive to
consider the orchid micropropagation story in the context of the ‘revolution’ in plant
biotechnology. From the outset, there was substantial confusion even among so-
called experts about what was really achieved. F.C. Steward and A.D. Krikorian felt
the need to denounce the increasingly common and disturbing trend in the reporting
of plant ‘biotechnology’ research (Steward & Krikorian, 1975, 1979): “research,
instead of an avocation quietly conducted by the few with limited means and little
thought of priority or publicity [as in the so-called good old days], must now face the
continued realities of funding. The temptation is to exploit ever-narrower objectives
and, in the struggle for survival, minutiae are exaggerated, possibilities seen soon
masquerade as realities, and, often aided by television and radio, the public or
industry becomes involved so that patents and propaganda soon confuse the trail”
(Steward & Krikorian, 1979:222; Krikorian, 1982, 1988, 1989a).
Plant scientists learned very early that it is virtually impossible to patent the
processes of growth. It is easier to patent the products of plant breeding efforts and
there are now many plant patents to protect ‘breeders’ rights’. It may be argued that
perhaps the maximum commercial advantage for those interested in utilizing a new
biotechnology process is derived from obtaining early access to information. This can
be far more advantageous than attempting to defend a so-called process patent
because it is, in the final analysis, relatively easy to achieve a given end in plant tissue
culture using a variety of strategies. Sooner or later the competitors will have figured
it all out. The ‘sooner’ is, however, what really matters from the commercialization
perspective!
The technology of meristem culture was fairly simple to implement in a great
many genera. However, it is very often a simple point of procedure that can make
a major difference in the ease or efficiency with which a technique may be
implemented. A nominally ‘minor’ procedural detail, overlooked or unappreciated
223HISTORY OF ORCHID MICROPROPAGATION
by one investigator but reported by another, can allow a person who has reached a
‘bottle-neck’ to proceed.
Morel’s first orchid ‘meristem’ paper reports the use of a semi-solid nutrient
medium. The use of such a medium limits the responsiveness of meristem explant,
and a fair amount of time, ranging from days to weeks, is required to get the system
going. A major procedural innovation was the adoption of a liquid nutrient medium
and gentle shaking on a rotary ‘shaker’ (Fig. 44) which significantly speeded up the
establishment of cultures. “Morel by 1960, was able, however, to grow Cymbidium
meristems, and in 1963 Wimber grew clonal populations from rotating liquid
meristem cultures of Cymbidium apices, using a mineral medium with sucrose and
tryptone supplements” (Morel, 1974:177–178). This seems to indicate that Morel’s
group, at the outset at least, would have been severely limited in terms of the
efficiency of the process for serious production purposes. Efficiency would have
improved dramatically, however, upon adoption of liquid media and culture
rotation.
35
As late as 1964 the indication was that 1.2% agar was being used in
‘Knudson C’ medium for Cymbidium etc. (Morel, 1964c:735). However, a photograph
(Fig. 56) of a rotation aparatus for Cattleya was included by Morel in a 1965 article
published in German (Morel, 1965b).
Again, it is significant to recognize that use of liquid medium for various tissue
cultures was not ‘invented out of the blue’ and in fact, early in its history it
encountered considerable criticism from the small, tightly ‘networked’ and
sometimes arrogant ‘tissue culture establishment’.
36
Caplin and Steward developed an apparatus, the ‘auxophyton’, that rotated
T-shaped tubes around a horizontal axis (Caplin & Steward, 1949). The tumbling
action causes explants and cells to be alternately bathed in liquid nutrient and
exposed to air (Steward, Caplin & Millar, 1952). Animal culture workers used roller
tubers which bathed cells in liquid in their work whenever they could (the tubes
apparently date back to a suggestion by Alexis Carrel (1873–1944) as early as 1913
(Gey, 1933)). Variations of the Steward & Caplin ‘auxophyton’ were adopted in
several laboratories because liquid and appropriate aeration improved growth of
carrot root plug cultures over what could be obtained on semi-solid media. For their
early cell culture work, Albert J. Riker (1894–1982) and his co-workers at Wisconsin
used a drum aerator that was a modification of the ‘auxophyton’ described by Caplin
and Steward (Muir et al., 1958: fig. 2).
Phillip R. White (1953), mentioned above as one of the nominal ‘inventors’
37
of
plant tissue culture, took issue with the claims for increased growth. Roger J.
Gautheret in France was no less displeased with the liquid culture work of Caplin
and Steward, for his laboratory had concluded that liquid media presented nearly
insurmountable problems. Gautheret and his student Heller devised a means of
supporting cultures on a filter paper raft which drew nutrients by capillarity (Heller
& Gautheret, 1949).
Further examination from a historical perspective of the particulars associated
with the incorporation of liquid culture methods into the cloning protocols and the
advantages that derived from this may well confirm one day that this was the pivotal
development in orchid mericloning. Be that as it may, these points serve to
underscore that it will not be possible to resolve all the issues relating to who made
the most important contributions to the development of orchid micropropagation,
and when they did it.
It is clear that Georges Morel was only one of the major participants in the
224 J. ARDITTI AND A. D. KRIKORIAN
process. The work of many others, which received far less publicity, paved the way
and provided seminal information. With most of the key participants now deceased,
and crucial laboratory and business records not available, it will perhaps never be
possible to fully re-construct the events. Nevertheless, if this account serves to provide
a more historically precise record of the role of the many participants who were
involved than has hitherto been attempted, then our efforts will have been
justified.
ACKNOWLEDGMENTS
I dedicate my contribution to Professor Emeritus John L(uther, not Lucifer) Mohr,
University of Southern California in Los Angeles for introducing me during my days
as a graduate student to the pleasures and rewards of tracing the history of biology
and to Professor Emeritus Arnold Samuel Dunn, also of U.S.C. for making it
possible for me to study it 30 years later.J.A.
I dedicate my contribution to the memory of the late George H. Pride. For many
years he taught the only botany course at high school level in the U.S.A. His high
academic standards and demand for precision and sustained critical analysis instilled
a love for botanical and horticultural inquiry in several generations of students at
South High School, Worcester, Massachusetts.A.D.K.
APPENDIX
All comments and notes (superscript numerals) listed in the body of the paper can be found in this Appendix.
1. Micropropagation was originally defined as any aseptic procedure involving the manipulation of plant organs,
tissues or cells that produces a population of plantlets thereby making it possible to by-pass conventional sexual or
vegetative propagation. In some areas the term has largely been restricted to the use of stem tips and lateral buds
for clonal multiplication in vitro (Krikorian, 1982:170). For orchids it describes all in vitro clonal propagation (Arditti
& Ernst, 1993).
2. Many of the photographs used in this paper are re-photographed copies of old, grainy, faded (with age), poorly
printed (due to the technology of the time) sometimes on acid paper, small and/or occasionally scratched originals.
Readers are asked to overlook the poor quality of some of the illustrations.
3. Dr Gavino Rotor Jr. provided biographical information about himself in a letter to J.A.
4. Clara S. Hires (1897–1984) pioneered in the U.S.A. the commercialization of propagation methods via aseptic
seed and spore germination, especially of ferns but also a number of higher plants. She was able to apply and perfect
a number of available laboratory techniques for producing aseptic plants.
5. Post acknowledged Rotor for his help in making drawings for his book on floriculture. Knudson was a
consultant for the orchid section. However, Rotor’s work is not mentioned in the book. The reason for this may be
that both Post’s book and Rotor’s paper were published in 1949 allowing no lag time for inclusion. Also there were
no new editions (only reprintings) of Post’s book after 1949 (Post, 1959).
6. While working for the late Roy J. Scott in Bel Air, California 1957/1958–1960 J.A. tried Rotor’s method. The
first cultures failed and a second attempt was not made.
7. Recognizing that he had to understand more about factors controlling cell division, Haberlandt diverted his
attention to that and what came to be called ‘wound hormones’ (Krikorian, 1975:71 ff.).
8. Like many other Dutch (German and other European) botanists before World War II, van Overbeek spent time
at Buitenzorg. No doubt coconuts and their liquid endosperm, a popular beverage in the then Netherlands Indies,
came to his attention.
The terminology of coconut ‘milk’ versus ‘water’ often presents a problem to those who live or have spent time
in the tropics and/or are familiar with coconuts other than the mature, brown ones purchased in food stores (i.e.
unripe, fresh green fruits with jelly-like solid endosperm and a clear liquid one). The colourless liquid endosperm of
Cocos nucifera is usually referred to as ‘coconut water’. ‘Coconut milk’, on the other hand, is the white liquid obtained
by squeezing, grating or extracting the solid white ‘meat’ (i.e. the solid endosperm which is dried to make copra) of
the coconut.
9. The story of the introduction of coconut ‘milk’ to aseptic ‘tissue culture’ media formulations is of considerable
historical interest and complexity (for reviews see Steward & Shantz, 1955; Tulecke et al., 1961; Krikorian, 1975).
225HISTORY OF ORCHID MICROPROPAGATION
While much of the detail is beyond the scope of this paper, it is interesting to note that van Overbeek and co-workers
initially concluded that at least three factors or complexes that affected Datura embryo growth were present in
coconut milk: (1) a thermolabile factor that caused both growth and differentiation; (2) a thermostable principle that
leads in some cases to callus-like growth but no differentiation; and (3) a heat-stable substance which inhibited root
growth and was probably related to auxin. Van Overbeek found that non-autoclaved coconut water contained an
embryo growth factor, and that there was a toxicity associated with the raw coconut milk. This toxicity could be
largely overcome by treatment with 80% ethyl alcohol. The so-called embryo factor had to be of relatively low
molecular weight since it was dialysable through a cellophane membrane. They also learned that the auxin content
of coconut water could be increased by autoclaving or by treatment with diethyl ether. Autoclaved coconut milk
added to the basal medium yielded an unorganized, callus-like growth of the Datura embryos but this was for all
practical purposes ignored. They focused on the discovery that undifferentiated proembryos grew and became
differentiated embryos in the presence of ‘embryo factor’. Smaller embryos required a higher concentration than
larger ones. It was suggested that the threshold value depended on the embryo’s ability to synthesize its own growth
factor.
The only attempt to identify the growth-promoting substances of coconut milk at that time (van Overbeek, Siu
& Haagen-Smith, 1944) did not proceed to the point of identifying the compounds responsible for the effects but
some concentration of activity was achieved through ethanol extraction. Eventually the effects of coconut water were
attributed largely to the balance of ordinary and well-known, inorganic and organic nutrients rather than to the
presence of unidentified substances.
10. Considerable chemical work has been carried out on coconut water since its first use in plant culture media
but the task of identifying all the components became recognized as gargantuan. A number of sugar alcohols were
isolated from coconut water more than three decades ago (Pollard, Shantz & Pollard, 1961). Several years later the
cytokinin zeatin was isolated from it (Letham, 1968). Some concluded (Galston, 1969) that the identity ‘problem’ of
the components was solved with the identification of zeatin (Letham, 1968; Skoog, 1994; for an alternative view and
summary of what are believed to be still outstanding problems see Steward & Krikorian, 1971). Attempts have been
made from time to time provide summary compilations of components. One such attempt (Tulecke et al., 1961) lists
levels of sorbitol, meso-inositol and scyllo-inositol as being in the range of 15, 0.01 and 0.05 ppm respectively rather
than 15.0, 0.10 and 0.50gm/l. This error, recognized and acknowledged by Tulecke (gracious letter of apology to
Pollard, Shantz and Steward is in possession of A.D.K.) has unfortunately crept into the literature (Raghavan,
1966:10 and Arditti & Ernst, 1993:47).
11. Since animal physiologists had already adopted the term kinins for unrelated, physiologically active molecules,
cytokinin was selected as a term which meets the needs of plant physiologists (Skoog, Strong & Miller, 1965).
12. Carlos Miller also showed that some mycorrhizal fungi produce cytokinins (Miller, 1967, 1969; Crafts &
Miller, 1974). This could have a bearing on our understanding of the physiology of orchid mycorrhizae and
symbiotic seed germination.
13. Like Went, van Overbeek and many other European botanists, Goebel spent some time at the Buitenzorg
Botanical Gardens (Dammerman, 1945).
14. Robbins eventually made many significant contributions to our understanding of the activity of vitamins in
plant tissue cultures (Kavanaugh & Hervey, 1981).
15. Ascorbic acid (vitamin C), first isolated in 1928, was studied more intensively in 1933. Biotin was identified
in egg yolks in 1936. Folic acid was crystallized from liver in 1947, yeast in 1947 and identified in 1948. Although
not really a vitamin, myo-inositol was isolated from muscles in 1850, but was not used in plant tissue culture media
until much later. Niacin (nicotinic acid) was first made by oxidizing nicotine in 1925. Pantothenic acid was isolated
from liver and its structure was first elucidated c. 1940. The structure of riboflavine, originally isolated from eggs, was
described in 1935. Thiamine was isolated from rice bran in 1910–1911, but its structure was elucidated only in 1926
(for a review of vitamins and orchids see Arditti & Harrison, 1977). Of the plant hormones used in tissue culture,
auxins were discovered in 1928 (Went, 1928, 1990) and [cyto]kinins in 1955 (Miller, 1961). Information that
vitamins and hormones are required by explants in culture started to accumulate in 1936–1938 (for a review see
White, 1943; see also Schopfer, 1949 and ˚
Aberg, 1961).
One of the earliest records on the deliberate inclusion of inositol in a modern plant tissue culture medium is by
Jacquiot (1951). Interestingly, results from use of inositol have generally been inconclusive (˚
Aberg, 1961:423). It was
not until the sugar alcohols sorbitol, meso- or myo-inositol and scyllo-inositol were isolated and identified as components
of coconut water (Pollard et al., 1961) that inositol was rationalized as a potentially useful addition to culture media.
Although from time to time it has been implicated in signal perception as part of the phosphoinositide system, it
remains to be shown that it plays a major positive role in the growth of plant tissues in vitro. Nevertheless, routine
addition of myo-inositol as part of the MS medium allows one to ‘play it safe’. At least the addition seems to do no
harm.
16. Taro corms were used to make poi, a paste which was a very important part of the diet of the Hawaiian people
when Cook arrived in the islands. The leaves of taro, Colocasia esculenta, called luau, were used as a vegetable during
cannibalistic feasts. Nowadays, ‘luau’ refers to a Hawaiian barbeque, more often than not, staged for tourists.
17. Loo Shih-wei (Chinese style; the western form, Shih-wei Loo appears on papers he published in the U.S.A.)
returned to China not long after the completion of his studies. This coincided with the communist takeover. He
became a well-known plant scientist there. During the Cultural Revolution Prof. Loo suffered considerably. He
became active again after the ‘Gang-of-Four’ was overthrown and returned to the Shanghai Institute of Plant
226 J. ARDITTI AND A. D. KRIKORIAN
Physiology. S.W. Loo and J.A. started to correspond in 1983. Despite advanced age and failing health, Loo is still
active scientifically.
18. Ernest A. Ball came from NC State University to UC Irvine in 1968 and left after about 10 years. J.A.
collaborated with him for a while on tissue culture of orchids, taro and redwoods. His manual dexterity, ability to
excise minute tissue sections (see for example Ball, 1972), and capacity for work were phenomenal.
19. While at UCI Ball used coconut water for several culture media. There is no doubt that by ‘coconut milk’ he
means ‘coconut water’. In Britain, continental Europe and the U.S.A. the term was, and still is, normally used for
what ought to be termed ‘coconut water’.
Ball, best described as an experimental plant morphologist, was one of the first to use coconut ‘milk’. He did this
in the attempt to culture stem segments of certain dicotyledons. Ball found that tissues subjacent to the stem tip of
garden nasturtium Tropaeolum and lupin, Lupinus grew best in liquid media to which coconut milk was added. One
of us (A.D.K.), while seated next to Professor Ball at a banquet in 1964 learned that he believed his role in
establishing the value of coconut milk in plant tissue culture was pivotal. It is noteworthy therefore that Ball wrote
in 1946 that [his findings] “… argue against the possibility that coconut milk constituted a causative agent in that
growth. This liquid endosperm appears merely to have brought certain organic substances to the culture medium,
without which these small bits of tissue would not have displayed cell division and growth. Its utilization here may
be compared to that in growth of small Datura embryos in vitro by van Overbeek, et al. …”
20. An examination of Ball’s publications indicates that he appears not to have appreciated (or perhaps cared
about?) the significance of his own observations from an applied or practical perspective. This view is justified
because Ball could have at least mentioned in passing the potential for mass multiplication of plants for horticultural
purposes when he wrote an article on regeneration of seedlings after surgically manipulating the central initials of
the meristems (Ball, 1950). He discussed only the theoretical aspects.
21. Thomale’s letter, his photograph and some information were provided by himself, Mr E. Lucke and Dr
Norbert Haas-von Schmude (Lucke, 1974, 1994; Haas-von Schmude, Lucke & Arditti, 1995). They also provided
information about Dr Lucie Mayer and her photograph. Thomale provided a copy of Dr Morel’s letter to him of
15 December 1965. This letter shows that Morel was familiar with Thomale’s work long before crediting it in print
(Haas-von Schmude, Lucke & Arditti, 1995).
22. In a paper that summarized research during a number of years in his laboratory at Harvard, Wetmore (1954)
stated in a footnote on page 22: “The author wishes to acknowledge his indebtedness to Dr Georges Morel whose
cooperation made the in vitro culture techniques a reality in these investigations” (see also Wetmore & Wardlaw,
1951; Torey & Thimann, 1972). In fact, Morel had been invited to Harvard University by Wetmore to help set up
a plant tissue culture laboratory (Torrey & Thimann, 1972:200).
23. The term protocorm was coined by the noted and long-time director of the Buitenzorg Botanic Gardens,
Melchior Treub (1851–1910; for a photograph see Arditti, 1992) to describe a stage in lycopod development. No¨el
Bernard (1874–1911; for photographs see Arditti, 1990, 1992) applied it to orchids between 1899 and 1910 using
it to describe the early corm-like stage of seed germination. Morel modified the term to describe the corm-like
structures which were formed by the Cymbidium stem tips he cultured. By definition ‘protocorm’ is used only for the
structures formed by seeds; ‘protocorm-like body’ (often abbreviated as PLB or plb) is reserved at present for the
bodies which resemble protocorms but develop from explants (see Arditti, 1990, 1992; Arditti & Ernst, 1993 for more
extensive discussions).
24. Vuylstekeara is a trigeneric hybrid between Odontioda (bigeneric hybrid: Cochlioda X Odontoglossum) and
Odontonia (Miltonia X Odontoglossum). A specific procedure for it was first published in 1989 in Poland (Kukulczanka
et al., 1989). Until then the procedures used for Cymbidium (Morel, 1960, 1963, 1964a, b, 1970, 1974) were described
as being suitable for it (Arditti & Ernst, 1993). Morel did develop procedures for Miltonia and Odontoglossum but did
not provide details until 1974 (Morel, 1974).
25. In orchid parlance, ‘flasking’ (a noun incorrectly made into a verb) was initially used to describe the process
of placing orchid seeds in culture (i.e. germinating them in flasks). In recent years its use has extended to the placing
of explants in culture. ‘Deflasking’ (an even worse linguistic abomination) refers to the removal of seedlings or
plantlets from a flask and planting them in a potting mix. ‘Transflasking’ (another linguistic horror) means
transferring protocorms, protocorm-like bodies, seedlings or plantlets from flask to flask.
26. In 1958 Frederika Quak from the Institute of Plant Virology in Wageningen, Netherlands presented a paper
in Colloque-Symposium 4, Diagnostic et Cure des Maladies `a Virus (diagnosis and control of virus diseases) at the
International Horticultural Congress in Nice organized by P[ierre] Cornuet (one of the plant pathologists who
suggested shoot tip cultures to Morel) and C[laude] Martin (one of Morel’s collaborators), of I.N.R.A., Versailles.
The secretary of that symposium was Jean-Paul Marrou, Ing. Hort., Charg´e de Recherches (I.N.R.A.), Station
Centrale de Pathologie V´eg´etale, Route de Saint-Cyr, Versailles (Seine-et-Oise, France). Georges Morel Dir. de
Recherches `a la Station de Physiologie V´eg´etale (I.N.R.A.), 28 rue du Plateau Saint-Antoine, Le Chesnay (Seine-et-
Oise, France) and M
me
Morel were listed as attendees at the conference. In that presentation, which did not get
published until 1961, Dr Quak focused on her work with potato and the use of White’s medium [White, 1954]
supplemented with “10 p.p.m. thiouracil, 0.1 p.p.m. 2,4-D or 0.1 p.p.m. IAA”. (Quak, 1961:146). Nothing was said
in the 1961 paper about orchids. There is no evidence that Morel presented anything at that meeting.
27. Quak and a colleague (Baruch & Quak, 1966) do not cite Morel’s paper on Cymbidium as an example of an
apical meristem culture that could yield a virus-free plant. In connection with their work on iris meristems they state
that “best results were obtained with media based on that of Morel. Therefore the formula of that medium only is
presented here: 1
2concentration Knop solution 1000ml; Berthelot solution 0.5 ml; cystein 1mg; adenine 5 mg;
227HISTORY OF ORCHID MICROPROPAGATION
hydrolysate of casein 200mg; saccharose [sucrose] 20 g; agar 6g; vitamin solution (containing calcium pantothenate
1mg, inositol 100 mg, biotin 10mg, nicotinic acid 1 mg, pyridoxin 1mg, distilled water 100ml. The media were
adjusted to pH 6” (Baruch & Quak, 1966:271). Baruch and Quak started their experiment in January 1963 and used
about 700 meristems of ‘Wedgewood’ iris. The abstract of the paper (in English) draws attention to the fact that the
“medium of Morel (personal communication) gave the best results”. The Dutch summary repeats the statement. So
at least by January 1963 Morel was willing to divulge his nutrient medium recipe and it was published in full by
Quak. This was three years after Morel’s initial publication on orchids and one year before the French orchid firm
of Vacherot and Lecoufle announced that they could propagate orchids via shoot tip cultures. Whether anyone could
or did make a connection between Quak’s paper and orchids is open to speculation.
28. It would be interesting to know whether a written request for the recipe of the nutrient medium would have
elicited a positive response. We do not know if such requests were made regarding any of Morel’s orchid media. No
such requests would have been necessary for the potato stem tip medium because it was published. In his important
work with potato meristems Kassanis (1957) states that … “the apical meristems were excised as described by Morel
and Martin (1955). The medium in which the meristems were cultured was suggested by Dr G. Morel, but differs
from the one which was described by him (Morel and Martin, 1955). It consists of 1
2concentration of Knop solution,
10 drops of Berthelot solution (Morel, 1948)…” Basil Kassanis spent a few months with Morel at Versailles in 1954
(Hirst & Harrison, 1988). At least one British grower made “arrangements … to visit Prof. Morel’s laboratory in May
of 1964 [and] found Prof. Morel and his staff extremely helpful and they taught [him] the technique, giving [him]
details of the formula used to produce plants from meristematic tissue. Late in 1964 Morel also visited McBean
[McBeans Orchids Ltd., Cooksbridge, Lewes, Sussex, U.K.] and [the grower] was privileged to work with him …”
(Bilton, 1985). It is not clear whether these reciprocal visits with the British growers were made on a voluntary basis
or as a consulting arrangement. Nevertheless, by May 1964 the French orchid firm of Vacherot and Lecoufle had
in effect firmly established its monopoly.
29. An example of having achieved something without having fully recognizing its full significance may be cited
from the commentary by Carl D. LaRue, an early pioneer of tissue culture (Krikorian, 1982). LaRue pointed out
on page 39 of the discussion after Ralph Wetmore’s Brookhaven presentation “that years ago (1936) I did succeed
in getting growth of a meristem of Radicula aquatica, which is I now believe, Nasturtium officinale. I did succeed in
growing just a short typical meristem into a whole plant. Now that seems much more remarkable to me in view of
what Dr Wetmore has said, than it did before” (Wetmore, 1954).
30. Ironically, publications now bear the statement that they are ‘advertisements’ to conform with tax regulations.
For example, in the prestigious Proceedings of the National Academy of Sciences of the U.S.A., one may read footnotes to
the effect that “The publication costs of this article were defrayed in part by page charge payment. This article must
therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. § 1734 solely to indicate this fact.”
31. Haberlandt spent time in Buitenzorg (November 1891 to February 1892, see Dammerman, 1945:64) and
several locations in tropical Asia (Haberlandt, 1910). Krikorian & Berquam (1969) raise the question of what might
have happened “had coconuts been generally available in Berlin”, but coconuts may not have caught his fancy.
32. There is no ‘Cattleya schombocattleya’. It is not clear if this was meant to be ‘Cattleya, Schombocattleya’. It could be
Cattleya or Schombocattleya’, ‘Cattleya and Schombocattleya’, or ‘Cattleya X Schombocattleya’. Cattleya is a naturally
occurring genus. Schomboccatleya is a hybrid genus between Schomburgkia and Cattleya. The first hybrid, Schombocattleya
Spiralis was produced in 1905 from the cross Cattleya mossiae X Schomburgkia tibicinis (Garay & Sweet, 1974).
33. In some institutions, attendance at lectures on ‘ethical issues in science’ are mandatory for graduate students
and post-doctoral fellows. At one extreme is the scientist who unwittingly biases the gathering and interpretation of
data, at the other extreme is outright fraud and data fabrication.
34. In 1923 the Nobel Prize in Physiology or Medicine was awarded to Frederick Banting and James J.R. Macleod
for the discovery of insulin. Banting shared his prize money equally with Charles Best, and Macleod shared his with
James B. Collip. It has been pointed out that after Banting’s death in 1941 Best started to re-write history about the
details of the discovery of insulin and worked diligently to promulgate the view that Banting had discovered insulin
with his help only. It has been suggested that claims were made that “were unprovable, misleading, or inaccurate”
Bliss, 1993:257).
35. This also holds in the case of excised apices of bananas and plantains. The system is much more quickly
established in liquid than on semi-solid (Cronauer & Krikorian, 1984). Some orchid explants are also considerably
easier to start or made to proliferate in liquid media (Arditti & Ernst, 1993).
36. For many years Phillip R. White and Roger J. Gautheret saw themselves as the ‘arch-priests’ of the subject,
and generally acted publicly and in print as though they were the ultimate arbiters of all ‘real’ progress in the field
(Krikorian, 1975). Gautheret refused to mention Rotor’s work in one of his historical accounts even after he was sent
a copy of the paper by J.A.
37. It has been stated that Phillip White was taught aseptic procedures by W.J. Robbins at the University of
Missouri (Kavanaugh & Hervey, 1981:108).
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241HISTORY OF ORCHID MICROPROPAGATION
... In all illustrations 1 is a whole seed; b is magnification of testa wall (source: these scanning electron microscope photographs were taken in Joseph Arditti's laboratory; both the laboratory and the originals no longer exist). scientific journals but in proceedings of meetings and periodicals aimed at growers, amateur and commercial (for a review see Arditti and Krikorian, 1996). These publications were augmented by extensive travels and lectures. ...
... As stated eloquently (Gaffron, 1969), "those establishments whose power rests with God-given ignorance and thoughtlessness of their subjects rightly recognize free-science as an ever-present threat." And, indeed, editorial changes or requests (even demands) that they made were not uncommon when accepted views regarding the discovery of shoot tip cultures and micropropagation of orchids were questioned (for reviews see Haas-von Schmude, L€ ucke, and Arditti, 1995;Arditti and Krikorian, 1996;Arditti, 2009, 2017). ...
... Fortunately, as time passed it became possible to question the "sole discoverer" dogma. In 1996 Dr. Abraham D. Krikorian (now Professor Emeritus at the State University of New York, Stony Brook) agreed to combine his extensive knowledge and understanding of tissue culture and freeing shoot tips of non-Orchidaceous plants from virus with what I knew about these subjects relative to orchids (Arditti and Krikorian, 1996). Our review turned the tide. ...
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When Dr. Paromik Bhattacharyya first asked me to write for this special issue of the South African Journal of Botany, I refused for several good reasons: Retired for 20 years, not having done research for 21 years, 89 1/2 years old, no longer reading as extensively as before my retirement, recovering from surgeries, trying to manage health problems, which even if not severe are vexing, and being lazy. However, Dr. Bhattacharyya would not take no for an answer. After exchanging several (too many) emails I agreed to think about it. I did think and decided that since this issue deals with orchid biotechnology it may be a good idea to clear the air. By “stealing” from my previous/other writings, using my extensive library, accessing literature on line, and utilizing knowledge accumulated since about 1956, I decided to write an article, which establishes beyond any doubt who were first to make several orchid discoveries, formulate a number of procedures and develop some methods. At my age, I have the necessary perspective and knowledge. And, most importantly, antagonizing people (so, what is new?) and making enemies (again, what is new?) will not be damaging. I decided to write about who: •Was the first to recognize orchid seeds as such and describe and illustrate them? There are no arguments or claims and counterclaims about it, but the information is buried in old, rare and not very well-known books in languages other than English. •Invented mass rapid clonal propagation of orchids and how to free plants from virus infections. Believe or not these two are tied to each other, there are unjustified claims of priority, and a widely admired “sole discoverer” did not discover them. •Used serology (now an outdated method) first to detect an orchid virus. There was an unwarranted claim at one time. •Inserted genes into orchids first. It becomes clear if one reads the literature, but what if another claim was repeated several times in writing and talks? •First studied control of flowering in mature orchid plants. There are no claims and counterclaims, but the information is not easy to find. •Managed to induce plantlets or seedlings in vitro to flower. The truth may have been late to come out due to political upheavals.
... Since Morel first cultivated Cymbidium shoot meristems, modern biotechnology has reshaped orchid research and revolutionized the orchid industry (Arditti and Krikorian, 1996). Studies are also needed to find a way to obtain in vitro multiplication to continue the life cycle of this orchid species and to maintain and recover its wild populations (Li et al., 2008), as well as in vitro conservation of ornamental plants (Bonilla Morales, 2015). ...
... In vitro propagation to produce plants for commercial production and to repopulate decimated populations is an alternative that might help in decreasing pressure on natural populations. In vitro culture is a useful method to propagate endemic or endangered plant species for conservation purposes (Arditti and Krikorian, 1996). Many orchids are propagated through seeds, which require specific mycorrhizal associations (Rubluo et al., 1993;Buyun et al., 2004;Hernández et al., 2005). ...
... The technique of orchid micropropagation has been originated since many years, its origin lies in several lines of research and came from the work of many well-known scientists, (Arditti and Krikorian, 1996). Method for the in vitro culture of isolated plant cells, leaves and organs or seeds are not difficult or complex but they do require some equipment and certain and also for micropropagation. ...
Chapter
Bellamya bengalensis (Lamarck, 1822) in one of the most important fresh water, edible, widely distributed snails found in India. This species is present throughout Asia and Africa, and of great medicinal as well as socioeconomic values. Moreover, it is a cheap source of protein which is even higher than some common fish and red meat groups. Besides human, it is also a beneficial food source of many aquatic fishes and birds; hence, Bellamya bengalensis is one the significant part of aquatic ecosystem. Studies revealed that it has been used in the treatment of different human diseases like chronic gastric disorder, arthritis, joint pain, rheumatism, cardiovascular disease, night blindness, asthma, rickets, diarrhea etc. Although it possesses high protein content, it is being neglected than other proteinaceous resources in terms of food choice. Various bacteria present in the gut and flesh of Bellamya bengalensis act positively in the growth and survival of the snails. As snails are mostly found in the polluted water bodies, toxic elements present in polluted water affects the host-bacteria equilibrium, and hence changes in the metabolism and physiology of snails may have been observed. Acute and chronic toxicity studies with different toxic elements like formic acid, copper sulphate, fluoride, mercury etc. from industrial effluent showed behavioral and respiratory alterations along with decreased oxygen consumption rate in Bellamya bengalensis. This review provides an overview of ecological importance and its application on Bellamya bengalensis. Further studies will be done on physiological alterations upon application of toxic elements.
... The technique of orchid micropropagation has been originated since many years, its origin lies in several lines of research and came from the work of many well-known scientists, (Arditti and Krikorian, 1996). Method for the in vitro culture of isolated plant cells, leaves and organs or seeds are not difficult or complex but they do require some equipment and certain and also for micropropagation. ...
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Subhas Sarobar is a medium-sized artificial lake situated in the northeastern region of Kolkata, West Bengal which is controlled by the Calcutta Improvement Trust. This body of water is home to a variety of organisms, including bacteria, benthos, nekton, phytoplankton, and zooplankton, all of which contribute to a healthy and interdependent ecosystem. Seasonal variations in temperature, precipitation, and humidity have an impact on the physicochemical characteristics of the water, which could lead to a shift in the faunal population. Additionally, residential effluents are used on this aquatic system. Therefore, in addition to seasonal variations, home spills, washing clothes and utensils, taking baths, and disposing of plastic garbage next to the lake water all affect the physico-chemical characteristics of the water. The lake water quality ultimately declines as a result of all these human activities. The combination of high nutrient content and low DO creates an ideal habitat for the growth of bacteria in the lake system, including pathogenic bacteria that might potentially harm public health as well as other aquatic creatures. Pathogenic bacteria species found in water are dangerous and can cause several illnesses to human beings. It is possible to successfully stop the spread of disease(s) if the traits and specifics of pathogenic bacteria are understood.
... Ever since the production of protocorm-like bodies (PLBs) from Cymbidium leaf culture by Wimber (1965), several orchids have been successfully propagated in vitro using leaf explants (Arditti and Krikorian, 1996;Khoddamzadeh et al. 2011;Chookoh et al. 2019;Bhowmik and Rahman, 2020a). Propagation using leaves is highly advantageous, especially to monopodial orchids, as the sacrifice of the mother plant is not required (Hardjo and Savitri, 2017). ...
Chapter
Full-text available
Medicinal plants are precious sources of different products that are crucial component of contemporary medicines used to cure a broad range of illnesses and diseases. Nowadays, many species of medicinal plants are facing high risk of annihilation due to anthropogenic factors such as premature harvesting, deforestation, habitat destruction, rapid industrialization, overgrazing by animal and many more. Monitoring and assessment of genetic diversity of medicinal plants are essential as they provide information about different germplasms prevalent and also help environmentalists in framing a blueprint for conservation. Numerous morphological, cytological, biochemical and molecular markers can be used to evaluate the diversity of these medicinal plants for future conservation as well as for the plant improvements. This chapter describes in brief about diverse uses of medicinal plants, importance of diversity, conservation strategies and different markers employed for assessment of diversity stating with some classical examples.Keywords:Medicinal plantsGenetic diversityMolecular markersConservation strategy
... Conservationists and horticulturists worldwide are struggling with this problem, looking for new strategies involving both conventional and modern biotechnology. Although traditional methods, including symbiotic germination and meristem culture, are commonly preferred for mass seedling production (Knudson 1922;Arditti and Krikorian 1996), reintroduction of seedlings produced from these methods directly into natural habitats could be even more challenging. The difficulty is due to the nature of orchids: Establishing a symbiotic association with appropriate fungi is crucial for orchids, and plant robustness depends on the encounter with the fungal partners. ...
Chapter
Genome sequences and gene expression provide important insights into the evolution and function of gene families. A database of complete genome sequences for many plant species, including orchids, is now available. Additionally, transcriptomics via next-generation sequencing can be used to analyze the regulatory mechanisms of various biological processes at the molecular level in many plant species, even nonmodel and wild plants. Recently, whole-genome sequencing and transcriptomic studies have been conducted on some orchids, unveiling the mechanisms underlying orchid mycorrhizal (OM) symbiosis, one of the most important features of Orchidaceae. Because orchids obtain nutrients from their symbiotic fungi during seed germination or even throughout their whole life cycle (mycoheterotrophy), OM symbiosis differs from mutualism, such as arbuscular mycorrhizal (AM) symbiosis. The genetic information of orchids provides a better understanding of how OM symbiosis has evolved, how orchids maintain a delicate balance of immune control during symbiosis, and how OM and AM symbioses differ. This knowledge will help establish a method for maintaining OM symbiosis, which is essential for orchids, and for conserving threatened orchids. The objectives of this chapter are (i) to review genetic study methodologies because practical guidelines of orchid species’ genome sequence and transcriptome analysis are unavailable and (ii) to summarize studies on OM symbiosis.KeywordsGenomicsOrchid mycorrhizal symbiosisTranscriptomics
... Kultur jaringan tanaman anggrek sudah diperkenalkan sejak tahun 1891 namun secara modern dimulai pada tahun 1949, ketika teknik kultur jaringan baru yang mudah dan praktis diaplikasikan pada propagasi vegetatif anggrek bulan dikembangkan di Universitas Cornell, USA oleh Rotor dengan menggunakan media yang diformulasikan oleh Lewis Knudson (Media Knudson C) [16]. Sampai saat ini metode teknik kultur jaringan pada tanaman anggrek terus dikembangkan yang dibuktikan dengan banyaknya publikasi tentang penelitian dalam hal ini. ...
Article
Phalaenopsis orchids are recognized as the most popular orchid genus in the world, especially in horticultural industry due to their large, colorful, and durable flowers as well as their wider adaptability to room conditions. The characteristics of seedling propagated by vegetative means are not uniform; therefore, propagation through tissue culture is desirable. Although the micro propagation of Phalaenopsis has shown very good development, but the wide spread of micro propagation still limited due some problems such as the exudation of phenolic compounds, the PGR concentration, the media used, somaclonal variation, the chosen explants, etc. This paper endeavor to include some important investigations based on the common explants used; leaf and flower stalk. Keywords: Micropropagation, Phalaenopsis, leaf explant, flower stalk ReferencesAnonymous. Orchid (Orchidaceae). Diakes tanggal 13 Januari 2013 dari http://www.rainforest-alliance.org/kids/species-profiles/orchid. 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S.;Kakuta, S.; Kano, A.; Okabe, M.Efficient propagation of protocorm-like bodies of Phalaenopsis in liquid medium. Plant Cell, Tissue and Organ Culture. 1996, 45, 79–85.Park, S. Y. ; Yeung, E. C.; Chakrabarty, D. ; Paek, K. Y. An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture. Plant Cell Reports. 2002, 21, 46–51.Zahara, M.; Datta, A.; Boonkorkaew, P. Effects of sucrose, carrot juice and culture media on growth and net CO2 exchange rate in Phalaenopsis hybrid ‘Pink’. ScientiaHorticulturae. 2016,205, 17–24.Hee, K. H.; Loh, C. S.; Yeoh, H. H. In vitro flowering and rapid in vitro embryo production in Dendrobium Chao Praya Smile (Orchidaceae). Plant Cell Reports. 2007, 26, 2055–2062.Kannan, N. An in vitro study on micropropagation of Cymbidium orchids. Current Biotica. 2009, 3, 244–250.Steward, Jr. N. C. Plant Biotechnology and Genetics. Willey, A john Willey Sons, INC., Publication. 2008.George, E. F.; Sherington, P. D.Biotechnology by tissue culture. Exegetics Ltd. 1994.Nursyamsi. Teknik kultur jaringan sebagai alternatif perbanyakan tanaman untuk mendukung rehabilitasi lahan. Makalah pada ekspose hasil-hasil penelitian balai penelitian kehutanan makasar. Makasar, 2010.Aditi, J. F. L. S.; Krikorian, A. D. Orchid mircropropagation: the path from laboratory to commercialization and an account of several unappreciated investigators. Botanical Journal of of the Linnean Society. 1996, 122: 183-241.Gunawan, L. W. Teknik Kultur Jaringan Tanaman. Pusat Antar Universitas (PAU) Bioteknologi IPB. 1998. Bogor.Chugh, S. Guha, S.; Rao, I. U. Micropropagation of orchids: A review on the potential of different explants. Scientia Horticulturae. 2009, 122, 507–520.Ramdan. Kultur daun dan pangkal batang in vitro anggrek bulan raksasa (Phalaenopsis gigantea J.J.Smith) pada beberapa media kultur jaringan. Departemen agronomi dan hortikultura, Fakultas pertanian IPB. 2011.Latip, M. A. R.; Murdad, Z. A.; Aziz, L. H.; Ting, L. M.; Govindasamy.; R. Pipin. Effects of N6-Benzyladenine and Thidiazuron on Poliferation of Phalaenopsis gigantea Protocorm. AsPac J. Mol. Biol. Biotechnol. 2010, 18(1): 217-220 p.Niknejad, A.; Kadir, M. A.; Kadzimin, B. S. In vitro plant regeneration from protocorms-like bodies (PLBs) and callus of Phalaenopsis gigantea (Epidendroidaceae: Orchidaceae). African Journal of Biotechnology.2010, 10, 11808–11816.Chen, J. T.; Chang, W. C. Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biologia Plantarum. 2006, 50, 169–173.Zahara, M. Disertasi doktor: The Effects of Plant Growth Regulators and Natural Additives on Direct Shoot Regeneration and Plantlet Growth of Phalaenopsis hybrid ‘Pink’. Asian Institute of Technology, Pathumthani. Thailand. 2016.Xu, C. J.; Li, H.; Zhang, M. G. Preliminary studies on the elements of browning and the changes in cellular texture of leaf explant browning in Phalaenopsis. Acta Horticulturae Sinica. 2005, 32, 1111–1113.Tokuhara, K; Mii, M. Induction of embryonic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae). In Vitro Cellular Developmental Biology–Plant. 2001, 37, 457–461Balilashaki, K.; Naderi, R.; Kalantari, S.; Soorni, A. Mircropropagation of Phalaenopsis amabilis cv Cool ‘Breeze’ with using flower stakl nodes and leaves of sterile obtained from node cultures. IJFAS, 2014.Semiarti, E.; Indrianto, A.; Purwanto, A. Agrobacterium-Mediated transformation of Indonesian orchids for micropropagation, genetic transformation, Prof. MarÃa Alvarez (Ed.), ISBN: 978-953-307-364-4, InTech, 2011. Available from: http://www.intechopen.com/books/ genetic-transformation/agrobacterium-mediated-transformation-ofindonesian-orchids-for-micropropagation.
... Ever since the production of protocorm-like bodies (PLBs) from Cymbidium leaf culture by Wimber (1965), several orchids have been successfully propagated in vitro using leaf explants (Arditti and Krikorian, 1996;Khoddamzadeh et al. 2011;Chookoh et al. 2019;Bhowmik and Rahman, 2020a). Propagation using leaves is highly advantageous, especially to monopodial orchids, as the sacrifice of the mother plant is not required (Hardjo and Savitri, 2017). ...
Chapter
rchids are unique ornamental plants with the most highly specialized floral architecture, pollination pattern, and breeding system. They dominate the international floricultural trade with their exceedingly beautiful flowers of varied coloration, patterns, and forms. The plants also possess high therapeutic properties apart from having excellent ornamental value. However, the populations of these multiutility plants have been reduced tremendously due to excessive unregulated commercial collection and mass habitat destruction. Micropropagation through tissue culture techniques offers an effective strategy for orchid conservation by rapidly generating plants on a large scale. Though in vitro orchid propagation has been performed successfully using different explants, the production of true-to-type plants is obligatory for conserving and commercializing the elite genotypes. Genetically identical plants have been produced by assessing the clonal fidelity of several micropropagated orchids using molecular markers. The present chapter emphasizes orchid micropropagation using different explants and their clonal fidelity evaluation employing diverse DNA markers.
... Activated charcoal (AC) can adsorb excessive hormones, vitamins (Fridborg and Eriksson, 1975), and phenolic and carboxylic compounds (Fridborg et al., 1978) and is also used to stimulate the production of PLBs (Chen et al., 2002). Plant growth regulators (PGRs) such as auxins and cytokinins can stimulate the regulation of tissue proliferation and further development into plantlets (Arditti and Krikorian, 1996). ...
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The potentials of tissue culturing for plant propagation and plant breeding have been described by numerous authors and reviewed by Murashige (30) and Nickell (31).. Unfortunately, the popularity and excitement of this field have caused more words than action and the apparent cookbook approach to tissue culture propagation has given the horticulturist the impression it is rather simple in execution and success. To a degree the execution is simple, but a successful propagation system is not. Tissue culturing is not doing all the things we know it is capable of doing, i.e. mass propagation, storage of germ-plasm, fusion of protoplasts, production of disease-free plants, etc., because the practical details have not yet been worked out.
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‘Root’ crops traditionally include plants with swollen hypocotyls (e.g. radish, beets), rhizomes, corms, cormels (e.g. Aroids) and stem tubers (e.g. true yams) as well as true roots (e.g. carrot) and root tubers (e.g. cassava, sweet potato). Because of its importance, potato is covered separately in Chapter 15, but some of the other major ‘root’ and tuber crops will be covered here. A few practical reasons as to why one might wish to carry out aseptic culture studies on these plants are provided under the specific entries. Two extremely valuable references with considerable insight for both broad and specific research needs that readers should be aware of, but might easily be overlooked, are Kay (1973) and Brücher (1989). For ideas on priorities of plant biotechnology research for developing countries reference may be made to National Research Council (1990) and Sasson and Costarini (1990).
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A ‘plantation’ has traditionally been thought of as a large estate in a tropical or subtropical region that is generally cultivated or tended by semi-skilled or unskilled labor under the general direction of a ‘manager’. Anyone who has seen large plantation operations immediately recognizes that the field workers are far from ‘unskilled’. In some places, the term ‘estate’ crop is used synonymously with the term ‘plantation’ crop. Since both terms are steeped in history, and are often flavored with connotations of colonialism or unsavory exploitation (Brockway, 1979), it is perhaps better and more accurate nowadays to think of plantation crops as those wherein a very large or relatively large group of plants, usually but not always perennials, are maintained under cultivation using highly standardized or strictly controlled practices.