IN VITRO

Vol. 8, No. 3, 1972

TOTIPOTENCY A N D E M B R Y O G E N E S I S IN P L A N T CELL A N D
TISSUE CULTURES ~

INDRA K. VASIL AND VIMLA VASIL

Department o] Bota~y and Department o] Agronomy, University o] Florida,
GainesviUe, Flordia 32601

SUMMARY
An important development in the field of plant cell and tissue culture has been the
demonstration in the past decade of the totipotency of higher plant cells. Isolated single
cells were first successfully grown on a nurse tissue separated by a filter paper and gave
rise to a callus tissue. Later, completely isolated single cells of tobacco were grown in
microchambers to form small clumps of cells which then could be differentiated to form
adult tobacco plants. Indirect evidence of the totipotency of higher plant cells has
also been provided in a number of other plants. Embryo-like structures (or embryoids) or
whole plants, or both, have been obtained from such highly differentiated cells as the
pollen grains (gametic and haploid), photosynthetic palisade cells in leaves, epidermal
cells from the hypocotyl, and the triploid endosperm cells; all of these cell types
perform very highly specialized functions in the plant. Plant protoplasts (cell wall is
digested with enzymes) have also been cultured to give rise to normal adult plants. In
many instances embryoids have been produced in vitro from several species of flowering
plants which do not show such asexual activity in nature. These embryoids are normally
indistinguishable morphologically from embryos produced by gametic fusion, often fol-
low the same pattern of cell divisions and differentiation as the developing zygote, and
are economically important as they provide clonal populations. Early work in this area
emphasized the necessity of dissociating tissues into single cells and providing a nutri-
tional environment identical to that of tim zygote in the embryo sac (usually by
supplementing the medium with liquid endosperm from coconuts), before the cells could
be released morphogenetically to express their totipotency by forming embryoids. Much
of the recent work, however, has shown that perfect development of embryoids can be
obtained in completely synthetic media in callus tissues as well as in suspension cultures.

Plant cell and tissue cultures have two impor- zygote after normal gametic fusion. In this re-
tant characteristics; the ability to regenerate spect, higher plant cell and tissue cultures differ
normal adult plants from single cells or groups of from similar animal cell and tissue cultures and
highly specialized cells, and in many cases to offer an important experimental system for the
recapitulate various stages of cell division, differ- study of cell function and differentiation. This
entiation, and morphogenesis undergone by the paper reviews and summarizes some of the
outstanding demonstrations of totipotency and
* This paper is dedicated to the memory of the embryogenesis in plant cell and tissue cultures.
late Professor Philip R. White, a dear friend who
provided much counsel and inspiration to us both, TOTIPOTENCY IN PLANT C E L L
for his pioneering work, valuable contributions, and AND TrSSUE CULTURES
untiring efforts in developing the science of plant
cell, tissue, and organ culture. The concept of cellular totipotency is inherent
Florida Agricultural Experiment. Stations Jour- in the Cell Theory of Sehleiden (1) and
nal Series No. 4699. Schwann (2), proposed during 1838-1839, and
Presented in the Symposium on Functional Dif-
ferentiated Systems at the 23rd Annual Meeting popularized by Virchow (3) in 1858 with his
of the Tissue Culture Association, Los Angeles, famous aphorism "omnis cellula e cellula"
California, June 5-8, 1972. (every cell from a cell). However, no attempts
117
118 VASIL AND VASIL
were made to obtain any experimental evidence
to support the idea of totipotency until 1878,
when VSchting (4) succeeded in dissecting
plants into smaller and smaller fragments and
keeping the fragments viable and growing. The
ultimate and unequivocal proof of cellular totip-
otency could come only from the cultivation
and growth of isolated cells into whole orga-
nisms. This problem was set forth clearly and
boldly more than half a century after the for-
mulation of the cell theory, by the famous bota-
nist Gottlieb Haberlandt (5), in a classically
prophetic paper entitled "Experiments on the
Culture of Isolated Plant Cells" (a complete
English translation of the original paper has
been published by Krikorian and Berquam (6),
along with an analysis of Haberlandt's work),
presented before a meeting of the Mathematics
and Natural Sciences Section of the Vienna
Academy of Sciences in Berlin on February 6,
1902. He made a number of important and far-
reaching predictions in his paper, based on pre-
liminary and largely unsuccessful laboratory ex-
periments, which nonetheless were to be experi-
mentally tested and proved correct half a cen-
tury later. For example, he suggested using em-
bryo sac fluids (coenocytic liquid endosperm)
for inducing cell divisions in vegetative cells, the
possibility of successfully cultivating artificial
embryos from vegetative cells, using the tech-
nique of the culture of isolated plant cells in
nutrient solutions for the investigation of the
properties and potentialities of cells, and using
such experimental techniques to understand the
"interrelationships and complementary influ-
ences to which cells within a multicellular whole
organism are exposed."
Haberlandt attempted the culture of palisade
and mesophyll cells from leaves in Knop's sim-
ple salt solution in hanging drop cultures. Ex-
tension growth of cells continued up to 3 weeks
in culture and resulted in a 2-fold increase in
length as well as width. No nuclear or cell divi-
sion, however, took place. The absence of much
growth or any cell divisions in his cultures can
be attributed to many factors. He noted that
"the cultured plant cells were impaired only
slightly in their progress by the presence of nu-
merous bacteria in the culture solutions," and
therefore, did not try to achieve complete steril-
ity in his cultures. His choice of experimental
material was also unfortunate and perhaps the
most important factor in the failure of his ex-
periments. Further, he used the mature and
highly differentiated mesophyll and palisade
cells from leaves, ill the belief that the natural
photosynthetic ability of these cells will prove
advantageous for their survival and growth. He
also used tissues from many monocotyledonous
species. We know that even now, 70 years later,
and with modern techniques and access to highly
improved nutrient media and plant growth sub-
stances, it is generally very difficult, if not im-
possible, to culture successfully mesophyll and
palisade cells, or tissues from many monocotyle-
donous species.
Haberlandt thus introduced the idea of plant
cell and tissue and organ culture, but it was left
to Molliard (7), Kotte (8, 9), and Robbins (10,
11) to cultivate successfully fragments of plant
embryos and excised plant roots for brief peri-
ods of time. The first successful culture of ex-
cised roots for indefinite periods of time was
achieved by White (12) in 1934. A few years
later, in 1939, Gautheret (13) in Paris,
Nob~court (14) in Grenoble, and White (15) in
Princeton, independently succeeded in cultivat-
ing cambial tissues of carrot and tobacco for
prolonged periods. The original cultures of ex-
cised tomato roots started by White in 1934, and
of carrot phloem tissue started by Gautheret in
1937, are still being routinely grown and main-
tained.
Extensive cell proliferation, and the formation
of a relatively undifferentiated and homogeneous
mass of cells called callus, can be obtained in a
variety of higher plants. One of the common
methods used to obtain such calli, which are
capable of indefinite growth with periodic sub-
culturing, is to grow explants from various parts
of a plant in a nutrient agar medium supple-
mented with an auxin (2,4-dichlorophen-
oxyacetic acid, indoleaeetic acid, naphthalene-
acetic acid, etc.) and a eytokinin (kinetin or
6-furfurylaminopurine), or very often an auxin
like 2,4-dichlorophenoxyacetic acid alone. Cells
which proliferate under the influence of the
above plant growth substances are rather large,
thin-walled, vaeuolated, differentiated, and ma-
ture parenchyma cells and must undergo dedif-
ferentiation in order to become meristematic and
mitotically active. During this process, particu-
larly in the presence of auxins, many changes
are noticeable in cells of the explant. These in-
 PLANT TISSUE CULTURES
dude enlargement of nuclei and nucleoli, appear-
ance of vacuoles in nucleoli, an increase in the
density of nucleohistones around the nucleoli,
and increased synthesis of proteins, DNA, and
particularly ribosomal RNA (16-23). Increased
differentiation of the granular zone in the nu-
cleolus and the formation of ribosomal precursor
particles or the ribonucleoprotein particles takes
place during cell proliferation induced by the
au.'dn herbicide, 2,4-dichlorophenoxyaeetie acid
(22).
Once a continuing callus culture has been ob-
tained, segments of it are transferred into a liq-
uid nutrient medium and incubated on a gyra-
tory shaker. Viable and growing cell suspensions
can be maintained in liquid media by transfer-
ring small aliquots of the cell suspension to fresh
media every 2 to 4 weeks. Such suspension cul-
tures yield large populations of single cells, as
well as groups of cells, and can be used as con-
venient sources for obtaining single cells.
It is with such suspension cultures that most
of the work on single cell culture and totipo-
tency in higher plants has been done. Muir, Hil-
debrandt, and Riker (24, 25) devised an inge-
nious technique to culture single plant ceils.
Cells from suspension cultures of grape, mari-
gold, and tobacco were isolated and placed on
8-ram = pieces of sterile filter paper which had
rested for 2 or more days on the surface of an
actively growing callus from the same species.
The filter paper squares were then replaced on
the surface of the callus tissue which now acted
as a nurse tissue for the isolated single cell. Nu-
trients as well as growth substances required for
the growth and division of the isolated cell evi-
dently passed through the filter paper and stim-
ulated the cell to divide repeatedly to form a
callus. Within 6 to 10 weeks a callus about 4 mm
in diameter was formed from the single cell and
could be transferred directly onto nutrient agar
media for further growth. These calli can be
cultured indefinitely by subculturing and, given
our present knowledge about the control of or-
ganogenesis, can be used for differentiating
shoots or roots, or whole plants.
The results from the nurse tissue culture ex-
periments dearly demonstrated for the first time
that single isolated cells of higher plants could
give rise to masses of tissue which retained the
capacity to differentiate and undergo morphogen-
esis (24, 25). An important fact brought out by
119
these experiments was the essential role of the
nurse tissue in the survival and growth of the
isolated single cell. However, it was not possible
by this technique to monitor continuously and
record the growth and divisions of the isolated
cell. Bergmann (26, 27), therefore, introduced
the plating technique which made it possible to
grow a large number of single cell clones in a
relatively easy manner, and also permitted some
degree of freedom in observing the cells in cul-
ture under an inverted microscope. Suspension
cultures are filtered through sterile gauze of var-
iable porosity to obtain a nearly uniform popu-
lation of single cells; almost always these prepa-
rations are "contaminated" by the presence of
small clmnps containing two or more cells. Sin-
gle cell suspensions are then mixed in nutrient
agar medium and plated over agar media in
Petri dishes. In Bergmann's experiments, about
20% of the plated single cells grew to form col-
onies which could be subcultured. Essentially
the same procedure with a higher plating
efficiency was later used by Gibbs and Dougall
(28), Blakely (29), Blakely and Steward (30),
and Earle and Torrey (31, 32) to obtain colonies
from single cells of a number of different plants.
Reinert (33) used a highly friable strain of the
crown gall tissue cultures of Vitis vinifera, which
could be easily spread with a spatula on the
surface of chemically defined media in Petri
dishes. He reported that, "Usually division of
the isolated cells starts during the first 12 days
after spreading. However, others remain quies-
cent for between 30 and 50 days. Most of these
quiescent cells were in regions of the agar free
from other cells and cell groups and their divi-
sion was found to occur as such regions of the
agar became invaded with growing cells. It is
therefore concluded that the initiation of divi-
sion in such cells is dependent on their coming
under the sphere of influence of actively growing
cells." Thus, even in the plating experiments, the
neighboring cells and cell groups perform the
function of the nurse tissue of Muir et aI. (24,
25).
Jones et al. (34) used the microculture cham-
ber to grow single cells of hybrid tobacco. Mi-
crochambers are prepared by placing two 22-
mm 2, no. 1, cover glasses about 18 mm apart on
droplets of sterile U.S.P. heavy mineral oil on a
glass slide. A drop of the conditioned medium
(medium in which cells have already grown for
120 VASIL AND VASIL
some time) with one or more cells is placed in
between the two cover glasses and immediately
covered by another cover glass and sealed
around the edges with mineral oil. Many single
cell clones were obtained by this method, which
also allowed continuous observation of cells dur-
ing their growth, enlargement, division, matura-
tion, and senescence. It was still necessary to use
a conditioned medium, and generally have more
than one cell per culture, to obtain continued
growth and cell divisions.
The microculture chamber was used later by
Vasil and Hildebrandt (35) to obtain cell colo-
nies from completely isolated (only one cell in
each microeulture) single cells of hybrid tobacco
growing in fresh and previously unconditioned
nutrient media. The same authors soon provided
an unequivocal evidence of the totipoteney of
the higher plant cell by successfully growing a
single and isolated tobacco cell to a mature and
flowering plant (36, 37). The concept of cellular
totipoteney was proved beyond doubt by these
experiments, and was further strengthened by
indirect evidence of totipotency demonstrated
by several other workers using diploid tissues of
many other angiosperms (38-42).
Recent work has shown that it is not only the
diploid somatic cells of higher plants which re-
tain their totipoteney through countless mitotic
divisions and cellular differentiation, but even
haploid gametic tissues and triploid tissues
formed after meiosis and gametic fusion are ca-
pable of proliferating and giving rise to haploid
and triploid plants, respectively. Anther cultures
of Datura (43-45), tobacco (46-48), rice (49),
Brassica (50), and some other plants 1 have been
used to obtain large numbers of haploid plants.
In most eases it is the vegetative cell within the
young pollen grain which divides repeatedly and
gives rise either to a callus or directly to an
embryoid and a plantlet. Clonal populations of
haploid plants can be obtained by this tech-
nique; monozygous diploids can be obtained in
tobacco by culturing stem segments of haploids
which undergo duplication of chromosome num-
ber during cell proliferation. The generally tri-
ploid endosperm tissue of angiosperms, formed
by the fusion of two haploid female nuclei with
a haploid sperm nucleus, has also been shown to
proliferate in culture and undergo morphogene-
sis to give rise to triploid plants (51-53).
1See note 1, p. 127.
In the last few years improved techniques
have been developed to remove tile cell wall
from plant cells by enzyme treatments to pro-
duce large populations of viable plant proto-
plasts (54). Nagata and Takebe (55) and Tak-
ebe, Labib, and Melehers (56) have recently
succeeded in developing mature and fertile to-
bacco plants from protoplasts of tobacco meso-
phyll cells? We have recently used the mieroeul-
ture chamber technique to grow protoplasts
from mesophyll cells of tobacco, and mesophyll
and petal protoplasts of Petunia (57). Detailed
observations on cell wall regeneration, move-
ment of cell organelles, cytoplasmic streaming,
and cell division in the protoplasts can be made
by this technique. There are several advantages
in using protoplasts as a source of single cell
clones: protoplasts can generally be obtained in
pure single cell populations as compared to sin-
gle cells and cell groups grown in suspension
cultures, plating efficiency of protoplasts is
higher than that of plated cell suspensions, and
mesophyll cells appear to be uniformly diploid
in comparison to variations in the ploidy of cells
in callus or suspension cultures.
Highly differentiated and mature haploid, dip-
loid, and triploid cells of higher plants, normally
destined for diverse and highly specialized func-
tions in the plant body, have thus been shown to
retain their full genetic potential which can be
unmasked under appropriate conditions in vitro
to produce normal and mature plants.
EMBRYOGENESIS IN PLANT CELL
AND TISSUE CULTURES
Regeneration of whole plants from cells, tis-
sues, or segments from different parts of the
plant body is a relatively common phenomenon in
most plant groups and is an important means of
vegetative propagation in nature as well as in
horticulture and agriculture. Buds formed from
cells or groups of cells in roots of Cichorium and
Convolvulus, leaves of Bryophyllum and Kalan-
ehoe, inflorescences and flowers of Agave and
Liriope, and from epidermal cells in the leaves
of Begonia or the hypocotyl of Linum develop
into normal adult plants. Buds, shoots, and roots
also develop readily in tissue cultures derived
from various tissues and organs of a wide vari-
ety of flowering plants. There is good experi-
mental evidence to indicate that such differen-
2See note 2, p. 127.
 PLANT TISSUE CULTURES
tiation and organogenesis is under hormonal
control. In many flowering plants, particularly
several varieties of Citrus and mango, vegetative
embryos are produced within the seed without
any fusion of gametes. These apomietie embryos
arise by the development of diploid somatic cells
of the nueellus, which surrounds the embryo sac,
and generally follow the same sequence and pat-
tern of cell divisions as undergone by the zygote
during normal embryogenesis. In many cases
perfect embryo-like structures or embryoids,
which closely simulate the development of the
zygotic embryo, develop in vitro from cell and
tissue cultures derived from virtually any part
of the plant body.
Sequential development of root and shoot pri-
mordia and whole carrot plantlets in callus cul-
tures was reported more than 20 years ago (58,
59). Formation of embryoids from higher plant
~issues in vitro was, however, first reported by
Reinert (60-62), who showed that cells in callus
tissue derived from the root of carrot form em-
bryo-like structures. Tissue cultures obtained
from the secondary phloem of carrot root have
since been used extensively and have provided
an important system for the study of embryo-
genesis in vitro. Steward et al. (38) have cul-
tured cells and cell groups from segments of
carrot root in suspension cultures. Cells and cell
clusters withdrawn periodically from such cul-
tures show a variety of forms, some of which
simulate various stages of embryogenesis in car-
rot. Many unorganized masses of tissue are also
formed in the same cultures, which later give
rise to nodules with roots. The rooted nodules,
when transferred to nutrient agar media, form
shoots and ultimately normal and adult carrot
plants. Cell suspensions obtained from shaking
carrot embryos in liquid media proliferate vigor-
ously and contain some large, vacuolated, undif-
ferentiated cells which occur singly or in clus-
ters, and large numbers of embryo-like forms,
which recapitulate in a surprisingly faithful
fashion the various stages of normal embryogen-
esis before forming viable carrot plants (38, 39,
63). The cells which give rise to the embryoids
are small, spherical, densely packed with starch,
and usually form globular colonies. The liquid
endosperm of coconut (coconut milk or coconut
water), a highly complex and incompletely un-
derstood mixture of various organic and inor-
ganic chemical substances, is said to be very
effective in inducing cell divisions and embryo-
121
genesis in normally quiescent carrot tissues and
cell suspensions, respectively. A 2- to 2.5-mg ex-
plant of carrot secondary phloem, containing
25,000 to 28,000 cells, grew to a weight of 200 to
300 mg in 3 weeks and contained about
2,500,000 cells, whereas an aliquot of cells ob-
tained from a single embryo of carrot in suspen-
sion culture, formed up to 100,000 young em-
bryoids when plated in a single Petri dish. Co-
conut milk is the principal nutritive substance
which bathes the young coconut embryo during
its development in nature. Endosperms of most
flowering plants, whether free nuclear or cellular
in development, perform a similar function; like
the coconut they can be extracted with relative
ease in species like horse chestnut and corn and
are advantageous in plant tissue culture media.
On the basis of their extensive investigations of
the carrot tissue culture system, Steward et al.
(63) concluded that the tendency of diploid cells
in culture to behave as zygotes is a result of the
fulfillment, in such cultures, of certain conditions
normally found in the angiosperm embryo sac,
and that in order to "release the totipotency of
any diploid carrot cell it is necessary (a) to
obtain it, like the zygote, as a free cell, and (b)
to cause it to divide under the influence of the
special nutrients drawn from the environment of
natural, immature, zygotic embryos."
These conclusions have been vigorously chal-
lenged by many workers who have duplicated
Steward's work on carrot tissues in relatively
compact callus tissues grown on completely de-
fined nutrient media. As is shown by several
examples in the following discussion, it is not
essential to dissociate plant cells into single cells
to induce them to behave like zygotes and form
embryoids. However, it is not yet entirely clear
whether the embryoids formed in callus tissues,
or from epidermal cells or nueellar cells sur-
rounding the embryo sac in the ovule, originate
from cells which are isolated from neighboring
cells by the severance of all plasmodesmatal
connections and the thickening of cell walls? Iso-
lation of mierospore and megaspore mother cells,
during the critical stages of meiosis, from the
surrounding diploid somatic cells is well docu-
mented in flowering plants (64). "The zygote
and primary endosperm nucleus are formed si-
multaneously in the embryo sac by a process of
double fertilization, and, although the embryo
8 See n o t e 3, p. 127.
122 VASIL AND VASIL
usually depends on the endosperm for its nutri-
tion, there is no reason to suppose that the en-
dosperm confers embryological competence on
the zygote or directs it into its unique develop-
mental pathway. Moreover, there are no experi-
mental data showing that coconut milk or other
endosperms specifically induce or even have a
qualitative positive effect on embryogenesis in
cell cultures (65)." Callus or suspension cul-
tures, or both, of many flowering plants have
been shown to form typical embryoids in syn-
thetic media (39, 66-72). Some of these are dis-
cussed in greater detail in the following pages.
Single cells dissociating from the surface of
friable callus tissue of carrot tap root, and float-
ing on to the wet surface of the agar medium,
have been shown to follow a course of develop-
ment "quite similar to that observed in the ordi-
nary embryogenesis from zygote to plantlet
(73)." In most cases a conditioned medium was
required for such development, although some
times the same effect could be obtained by
transplanting a mass of single cells onto fresh
nutrient media.
Normal dicotyledonous embryos develop
within 6 to 10 days from two- to five-celled
embryogenic structures in suspension cultures of
carrot tissue grown in a hormone-free liquid me-
dium containing only salts, sucrose, and thiamin
(74). Auxins are, therefore, not essential for the
production of embryoids. In fact, it has been
demonstrated that the removal of auxins (indole-
acetic acid or 2,4-dichlorophenoxyacetic acid)
from the nutrient medium results in the forma-
tion of embryoids in carrot tissue cultures (62).
Auxins are, however, essential for proliferation
of the initial explant, although cytokinins can be
two to three times more effective. However, Hal-
perin (65) has clearly shown that embryogenesis
is a vigorous process only iu those suspension
cultures which are derived from explants in-
duced to proliferate in a medium containing an
auxin as the only hormone. Cytokinins and gib-
berellins cause partial or complete inhibition of
the appearance of embryogenic cells, but have
little effect on the ability of embryogenic cells,
once formed, to produce embryoids. Nutrient
media used for the proliferation of the initial
explants, therefore, are most critical in the abil-
ity of the cells to undergo embryogenesis.
Another important nutritive factor affecting
embryogenesis in the carrot system in vitro is
the form in which nitrogen is present in the
nutrient medium. Ammonium ions and casein
hydrolysate are strongly stimulatory to embryo-
genesis in comparison to nitrate and glutamine
(65, 75). Tazawa and Reinert (72) have shown
that embryoids are readily produced in media
containing relatively large amounts of ammo-
nium ions and nitrate, but embryoids are never
formed on a medium containing only nitrate in
a low concentration. "Although the occurrence of
NH, + in the medium is not necessary for embryo
formation in vitro it appears that a certain level
of intracellular NH, § is a prerequisite for this
process. Since there is a positive correlation be-
tween embryo formation and the contents in the
cultures of both soluble and insoluble organic ni-
trogen, it is probable that NHd§ is important for
embryo formation because it is an essential sub-
strate for the synthesis of organic nitrogen com-
pounds such as amino acids and proteins (72)."
Tissues which are cultured for long periods of
time lose their ability to synthesize organic nitro-
gen from ammonium ions and their capacity to
form embryoids. Halperin (65) believes that it is
unlikely that the nearly identical effects of ammo-
nium and casein hydrolysate in his experiments
are related to a general stimulation of protein
synthesis, since glutamine is clearly much more
stimulatory to growth than ammonium but still
is a poor nitrogen source for the induction of
embryogenesis in carrot tissue cultures.
Callus tissue derived from the leaf petiole of
parsley and maintained for several years on a
medium containing coconut milk and the auxin
herbicide, 2,4-dichlorophenoxyacetic acid, still
retains the ability to form embryoids when
grown on a completely defined nutrient agar me-
dium containing adenine sulphate, indoleacetic
acid, and kinetin (66). Adenine sulphate is es-
sential for the induction of embryogenesis in
parsley, since no embryoids are formed in media
containing only indolcacetic acid and kinetin.
Embryoids are formed from the cortical cells of
roots which differentiate during the first few
days of culture, and also from the epidermal
cells of embryoids themselves. Embryogenesis in
parsley very closely simulates the stages of nor-
mal development and the germination of a dicot-
yledonous embryo. Embryoid formation has
also been reported in cell suspensions of endive
embryo callus tissue in defined media containing
indoleacetic acid and kinetin (67).
Formation of embryoids from epidermal cells
of cultured stem segments of Ranunculus scelera-
 PLANT TISSUE CULTURES
tus (68) and carrot (76) and from hypoeotyle-
donary segments of tobacco (77) has also been
reported. Differentiation of embryoids from epi-
dermal cells is often associated with extensive
disorganization of surrounding cells and may in-
dicate severance of plasmodesmatal connections
and comparative isolation of embryoid-initiating
cells?
Tissue cultures obtained from diploid somatic
cells of the stem, root, leaf, floral organs, and
embryo have often been induced to form em-
bryoids when grown in defined nutrient media
on agar or in suspension cultures. Production of
embryoids has also been achieved recently from
haploid gametic cells in several species of flower-
ing plants (43-50, 78). Microspores and young
pollen grains of tobacco have proved to be the
most suitable material for such studies. The
minimal medium on which pollen grains of to-
bacco proliferate consists of 2% sucrose solidified
with agar. On such a medium, however, develop-
ment of the embryoids stops at the globular
stage. Lack of iron in the medium also arrests
development at about the same stage. A com-
plete nutrient agar medium, without any auxins
or other plant growth substances, supports the
development of haploid embryoids through char-
acteristic globular, heart-shaped, torpedo-
shaped, and cotyledonary stages resembling the
development of zygotic embryos. Auxins im-
prove somewhat the yield of anthers producing
haploid embryoids.
The marked change in the developmental pat-
tern of pollen described above can be achieved
only during specific stages of their ontogeny.
Contrary to some of the earlier reports, young
pollen grains in which vegetative and generative
cells have been formed are still capable of pro-
ducing embryoids. However, proliferation and
embryoid formation can not be induced after the
central vacuole disappears and storage of starch
begins in the pollen cytoplasm. It is the vegeta-
rive cell which proliferates in culture to form em-
bryoids; in vivo protein and RNA synthesis oc-
cur rapidly in this cell, filling the central vacuole,
but there is no DNA synthesis in its nucleus. In
the generative cell in vivo, which generally re-
mains undivided in vitro, DNA synthesis takes
place in preparation for the formation of the two
male gametes, but there is no protein or RNA
synthesis (78).
3 See note 3, p. 127.
123
The formation of shoots, roots, embryoids,
and plantlets from such highly differentiated,
determined, and often quiescent cells, with such
specialized functions in the life of the plant as
the storage parenchyma, pollen grains, meso-
phyll cells, epidermal cells, endosperm cells, etc.
--with variable ploidy levels, clearly demon-
strates that the determined pattern of differen-
tiation can be altered under suitable conditions.
It is no longer true that tissue cultures derived
from normal zygotic embryos form embryoids
more easily than those derived from roots or
other parts of the plant. In fact, depending on
the tissue being used, it may not even be neces-
sary to provide any special growth substances to
induce a cell to undergo embryogenesis in vitro.
Thus, epidermal cells in the hypocotyl of R.
sceleratus seedlings form embryoids in simple nu-
trient media containing mineral salts, vitamins,
and sucrose only, whereas the pollen grains of
tobacco form embryoids in sucrose alone, jelled
with agar.
In conclusion, although it has been possible in
the last 10 to 15 years to demonstrate the totip-
otency of a variety of specialized higher plant
cells and to cultivate artificial embryos from so-
matie and gametic cells in defined nutrient
media under controlled experimental conditions,
we are still far from understanding the precise
internal physiological and biochemical changes
which must take place before a cell can exhibit
its totipotency or acquire embryological compe-
tence like a zygote. Hopefully, the next decade or
so will be devoted to bridging this gap in our
understanding of totipotency and embryogenesis
in higher plant cells.
REFERENCES
1. Schleiden, M. J. 1838. Beitdige zur Phytogene-
sis. Arch. Anat. Physiol. Wiss. Med. (J. Miil-
ler) 1838: 137-176.
2. Schwann, T. 1839. Mikroskopische Untersu-
chungen i~ber die Ubereinstimmung in der
Struktur und dem Wachstume der Tiere und
Pflanzen. Leipzig: W. Engelmann, Nr. 176,
Ostwalds Klassiker der exakten Wissen-
schaften, 1910.
3. Virehow, R. 1858. Die Cellularpathologie in
ihrer Begri~ndung au] physiologische und
pathologische Gewebelehre. A. Hirschwald,
Berlin.
4. VSchting, I-I. 1878, 1884. ~]ber Organbildung im
Pflanzenreich. Part I, 1878, Part II, 1884. Max
Cohen, Bonn.
5. Haberlandt, G. 1902. Kulturversuche mit iso-
lierten Pflanzenzellen. Sitzungser. Mat. Nat.
K. Kais. Akad. Wiss. (Wien) 111 : 69-92.
124 VASIL A N D VASIL
6. Krikorian, A. D., and D. L. Berquam. 1969.
P l a n t cell and tissue culture: the role of
Haberlandt. Bot. Rev. 35 : 59-88.
7. Molliard, M. 1921. Sur le d~veloppement des
plantules fragment~es. C. R. Soc. Biol. (Paris)
84 : 770-772.
8. Kotte, W. 1922. Wurzelmeristem in Gewebe-
kultur. Ber. Deut. Bot. Ges. 40 : 269-272.
9. Kotte, W. 1922. Kulturversuche mit isolierten
Wurzelspitzen. Beitr. Allg. Bot. 2 : 413-434.
10. Robbins, W. J. 1922. Cultivation of excised root
tips and stem tips under sterile conditions.
Bot. Gaz. 73 : 376-390.
11. Robbins, W. J. 1922. Effect of autolyzed yeast
and peptone on growth of excised corn root
tips in the dark. Bot. Gaz. 74 : 59-79.
12. White, P. R. 1934. Potentially unlimited growth
of excised tomato root tips in a liquid me-
dium. P l a n t Physiol. 9: 585-600.
13. Gautheret, R. J. 1939. Sur la possibilit6 de
rgaliser la culture ind~finie des tissus de tu-
bercules de carotte. C. R. Acad. Sci. (Paris)
208 : 118-120.
14. Nob6court, P. 1939. Sur la perennit~ et l'aug-
m e n t a t i o n de volume des cultures de tissus
v~g~taux. C. R. Soc. Biol. (Paris) 130: 1270.
15. White, P. R. 1939. Potentially unlimited growth
of excised plant callus in an artificial nutrient.
Am. J. Bot. 26 : 59-64.
16. Chrispeels, M. J., and J. B. Hanson. 1962.
C o n t e n t of cytoplasmic particulates of soy-
bean hypocotyl induced by 2,4-dichlorophe-
noxyaeetic acid. Weeds 10: 123-125.
17. Key, J. L., and J. C. Shannon. 1964. Enhance-
m e n t by auxin of ribonucleic acid synthesis
in excised soybean hypocotyl tissue. P l a n t
Physiol. 39 : 360-364.
18. Key, J. L., C. Y. Lin, E. M. Gifford. Jr., and
R. Dengler. 1966. Relation of 2,4-D-induced
growth aberrations to changes in nucleic acid
metabolism in soybean seedlings. Bot. Gaz.
127 : 87-94.
19. Gifford, E. M., Jr., and J. P. Nitsch. 1969. Re-
sponses of tobacco pith nuclei to growth sub-
stances. Planta (Berlin) 85: 1-10.
20. Johnson, J. M. 1969. A study of nucleolar vacu-
oles in cultured tobacco ('ells using radioau-
tography, actinomycin D, and electron mi-
croscopy. J. Cell Biol. 43 : 197-206.
21. Jordan, E. G., and J. M. Chapman. 1971. Ultra-
structural changes in the nucleoli of Jerusa-
lem artichoke (Helianthus tuberos~s) tuber
discs. J. Exp. Bot. 22 : 627-634.
22. Vasil, I. K. 1971. Physiology and ultrastructure
of callus formation. In: Les Cultures de Tis-
sus de Plantes. Colloq. Int. C.N.R.S. (Paris)
193 : 103-112.
23. Yeoman, M. M. 1970. Early development in
callus cultures. Int. Rev. Cytol. 29: 383-409.
24. Muir, W. H., A. C. Hildebrandt, and A. J.
Riker. 1954. Plant tissue cultures produeed
from single isolated cells. Science 119: 877-
878.
25. Muir, W. H., A. C. Hildebrandt, and A. J.
Riker. 1958. The preparation, isolation, and
growth in culture of single cells from higher
plants. Am. J. Bot. 45 : 589-597.
26. Bergmann, L. 1959. A new technique for isolat-
ing and cloning cells of higher plants. N a t u r e
(London) 184: 648-649.
27. Bergmann, L. 1960. Growth and division of
single cells of higher plants in vitro. J. Gem
Physiol. 43 : 841-851.
28. Gibbs, J. L., and D. K. Dougall. 1963. Growth
of single plant cells. Science 141 : 1059.
29. Blakely, L. M. 1964. Growth and organized de-
velopment of cultured cells. VI. The behav-
iour of individual cells on n u t r i e n t agar. Am.
J. Bot. 51 : 792-807.
30. Blakely, L. M., and F. C. Steward. 1964. Growth
and organized development of cultured cells.
V. The growth of colonies from free cells on
n u t r i e n t agar. Am. J. Bot. 51 : 780-791.
31. Earle, E. D., and J. G. Torrey. 1965. Colony
formation by isolated Convolvulus cells
plated oi1 defined media. P l a n t Physiol. 40:
520-528.
32. Earle, E. D., and J. G. Torrey. 1965. Morpho-
genesis in cell colonies grown from Convolvu-
lus cell suspensions plated on synthetic me-
dia. Am. J. Bot. 52 : 891-899.
33. Reinert, J. 1963. Growth of single cells from
higher plants on synthetic media. Nature
(London) 200 : 90-91.
34. Jones, L. E., A. C. Hildebrandt, A. J. Riker,
and J. H. Wu. 1960. Growth of somatic to-
bacco cells in microculture. Am. J. Bot. 47:
468-475.
35. Vasil, V., and A. C. Hildebrandt. 1965. Growth
and tissue formation from single, isolated to-
bacco cells in microculture. Science 147:
1454-1455.
36. Vasil, V., and A. C. Hildebrandt. 1965. Differ-
entiation of tobacco plants from single, iso-
lated cells in microcultures. Science 150: 889-
892.
37. Vasil, V., and A. C. Hildebrandt. 1967. Further
studies on the growth and differentiation of
single, isolated cells of tobacco in vitro.
P l a n t a (Berlin) 75 : 139-151.
38. Steward, F. C., M. O. Mapes, and K. Meats.
1958. Growth and organized development of
cultured cells. II. Organization in cultures
from freely suspended cells. Am. J. Bot. 45:
705-708.
39. Steward, F. C., M. O. Mapes, A. E. Kent, and
R. D. Holsten. 1964. Growth and develop-
ment of cultured plant cells. Science 143:
20-27.
40. Vasil. I. K., A. C. Hildebrandt, and A. J. Riker.
1964. Endive plantlets from freely suspended
cells and cell groups grown in vitro. Science
146 : 76-77.
41. Halperin, W.. and D. F. Wetherell. 1964. Ad-
ventive embryony in tissue cultures of the
wild carrot, Daucus carota. Am. J. Bot. 51:
274-283.
42. Backs-Husemann, D., and J. Reinert. 1970. E m -
bryobildung durch isolierte Einzelzellen aus
 PLANT TISSUE CULTURES 125

Gewebekulturen yon Daucus carota. Proto- trolle an Gewebekulturen aus Karotten. Na-
plasma 70 : 49-60. turwissenschaften (Berlin) 45: 244-245.
43. Guha, S., and S. C. Maheshwari. 1964. In vitro 62. Reinert, J. 1959. Uber die Kontrolle der Mor-
production of embryos from anthers of Da- phogenese und die Induktion yon Adven-
tufa. Nature (London) 204: 497. tivembryonen an Gewebekulturen aus Karot-
44. Guha, S., and S. C. Maheshwari. 1966. Cell di- ten. Planta (Berlin) 53 : 318-338.
vision and differentiation of embryos in the 63. Steward, F. C., L. M. Blakely, A. E. Kent, and
pollen grains of Datura in vitro. Nature M. O. Mapes. 1963. Growth and organization
(London) 212: 97. in free cell cultures. Brookhaven Symp. Biol.
45. Narayanaswamy, S., and L. P. Chandy. 1971. 16 : 73-88.
In vitro production of haploid, diploid, and 64. Vasil, I. K., and H. C. Aldrich. 1970. A histo-
triploid androgenic embryoids and plantlets chemical and ultrastructural study of the on-
ill Datura metel L. Ann. Bot. 35 : 535-542. togeny and differentiation of pollen in Podo-
46. Nitsch, J. P., and C. Nitsch. 1969. Haploid carpus macrophyllus D. Don. Protoplasma
plants from pollen grains. Science 163: 85-87. 71 : 1-37.
47. Nitsch, J. P. 1969. Experimental androgenesis 65. Halperin, W. 1970. Embryos from somatic plant
in Nicotiana. Phytomorphology 19: 389-404. cells. Symp. Int. Soc. Cell Biol. 9 : 169-191.
48. Nakata, K., and M. Tanaka. 1968. Differentia- 66. Vasil, I. K., and A. C. Hildebrandt. 1966. Varia-
tion of embryoids fl'om developing germ cells tions of morphogenetic behaviour in plant
in anther culture of tobacco. Jap. J. Genet. tissue cultures. II. Petroselinum hortense.
43 : 65-71. Am. J. Bot. 53 : 869-874.
49. Niizeki, tI., and K. Oono. 1971. Rice plants ob- 67. Vasil, I. K., and A. C. ttildebrandt. 1966. Varia-
tained by anther culture. In: Les Cultures de tions of morphogenetic behaviour in plant
Tissus de Plantes. Colloq. Int. C.N.R.S. tissue cultures. I. Cichorium endivia. Am. J.
(Paris) 193: 251-257. Bot. 53 : 860-869.
50. Kameya, T., and K. Hinata. 1970. Induction of 68. Konar, R. N., and K. Nataraja. 1965. Experi-
haploid plants from pollen grains of Brassica. mental studies in Ranunculus sceleratus L.
Jap. J. Breed. 20 : 82-87. Development of embryos from the stem epi-
51. Bhojwani, S. S., and B. M. Johri. 1970. Cyto- dermis. Phytomorphology 15: 132-137.
kinin-induced shoot bud differentiation in 69. Reinert, J., D. Backs, and M. Krosing. 1966.
mature endosperm of Scurrula pulverulenta. Faktoren der Embryogenese in Gewebekul-
Z. Pflanzenphysiol. 63 : 269-275. turen aus Umbelliferen. Planta (Berlin) 68:
52. Bhojwani, S. S., and B. M. Johri. 1971. Morpho- 375-378.
genetic studies on cultured mature endosperm 70. Yamada, R., H. Nakagawa, and Y. Sinoto. 1967.
of Croton bonplandianum. New Phytol. 70: Studies on the differentiation in cultured
761-766. cells. I. Embryogenesis in three strains of
53. Johri, B. M., and K. K. Nag. 1968. Experimen- Solanum callus. Bot. Mag. (Tokyo) 80:
tal induction of triploid shoots in vitro from 68-74.
endosperm of Dentrophthoe ]alcata (L.f.) 71. Gamborg, O. L., F. Constabel, and R. A. Miller.
Ettings. Curr. Sci. 37 : 606-607. 1970. Embryogenesis and production of al-
54. Vasil, I. K., and V. Vasil. 1971. Isolation and bino plants from cell cultures of Bromus
culture of plant protoplasts. Plant Sci. Bull. inermis. Planta (Berlin) 95: 355-358.
17: 14-16. 72. Tazawa, M., and J. Reinert. 1969. Extracellular
55. Nagata, T., and I. Takebe. 1971. Platings of iso- and intracellular chemical environments in
lated tobacco mesophyll protoplasts on agar relation to embryogenesis in vitro. Proto-
medium. Planta (Berlin) 99: 12-20. plasma 68: 157-173.
56. Takebe, I., G. Labib, and G. Melchers. 1971. 73. Kato, H., and M. Takeuchi. 1963. Morphogene-
Regeneration of whole olants from isolated sis in vitro starting from single cells of carrot
mesophyll protoplasts of tobacco. Naturwis- root. Plant Cell Physiol. 4 : 243-245.
senschaften (Berlin) 58: 318-320. 74. Halperin, W. 1967. Population density effects
57. Vasil, V., and I. K. Vasil. 1972. Growth and on embryogenesis in carrot cell cultures. Exp.
cell division in tobacco protoplasts cultured Cell Res. 48: 170-173.
in microchambers, in press. 75. Halperin, W., and D. F. Wetherell. 1965. Am-
58. Levine, M. 1950. The growth of normal plant monium requirement for embryogenesis in
tissue in vitro as affected by chemical carci- vitro. Nature (London) 205: 519-520.
nogens and plant growth substances. I. The 76. Kato, H., and M. Takeuchi. 1966. Embryogene-
culture of the carrot tap-root meristem. Am. sis from the epidermal cells of carrot hypo-
J. Bot. 37: 445-458. cotyl. Sci. Pap. Coll. Gen. Edue. Univ. Tokyo
59. Wiggans, S. C. 1954. Growth and organ forma- 16 : 245-254.
tion in callus tissues derived from Daucus 77. Haccius, B., und K. K. Lakshmanan. 1969. Ad-
carota. Am. J. Bot. 41 : 321-326. ventiv- Embryonen--Embryoide--Adventiv _
60. Reinert, J. 1958. Untersuchungen tiber die Knopsen. Ein Beitrag zur Kl~rung der Be-
Morphogenese an Gewebekulturen. Ber. Deut. griffe. Ost. Bot. Z. 116: 145-158.
Bot. Ges. 71 : 15.
78. Sunderland, N. 1971. Anther culture : a progress
61. Reinert, J. 1958. Morphogenese und ihre Kon- report. Sci. Progr. Oxford 59 : 527-549.