Download DIFFERENCES IN PLOIDY AND DEGREE OF INTERCELLULAR

J. Cell Sci. 12, 37-53 (1973)
Printed in Great Britain
37
DIFFERENCES IN PLOIDY AND DEGREE OF
INTERCELLULAR CONTACT IN
DIFFERENTIATING AND NONDIFFERENTIATING SYCAMORE CALLUSES
K. WRIGHT AND D. H. NORTHCOTE
Department of Biochemistry, University of Cambridge, Cambridge CB2 iQW, England
SUMMARY
Comparisons have been made between a sycamore callus (S4) isolated in 1970 and one first
isolated in 1958 (S2). Incubation of S4 on media containing different concentrations of auxin
and kinetin showed that the ratio of the hormones was the factor which controlled the growth
pattern within the tissue. High ratios of auxin: cytokinin favoured a white friable tissue,
whereas a compact callus with a much harder texture was formed at low ratios. Roots were
differentiated at intermediate values.
Callus (S2) has not been induced to undergo cellular differentiation. No soluble factors which
were capable of stimulating differentiation in S2 were produced during the differentiation of S4,
and S2 did not affect the ability of S4 to form roots.
Divisions seen in suspension cultures of S4 callus were mainly diploid; the cells of S2 were
predominantly tetraploid. When S2 suspensions were grown in the cytokinin-free medium used
for S4 a considerable number of diploid cells were seen. A number of S2 clones have been
isolated and all these developed chloroplasts on exposure to continuous light and, when grown
on the medium used to induce differentiation in S4, five of them showed a growth pattern in
which the cells were more closely packed than is usually the case in S2 callus.
A greater degree of cell contact has also been induced by including polyethylene glycol in the
medium and so restricting the availability of water to the callus. Under these conditions the
cells must have a greater capacity for interaction with one another. The effect of tissue texture
on the extent of cell contact and adhesion between the cells is discussed in relation to the
composition of the plant cell wall and ideas on the totipotency of plant tissue cultures.
INTRODUCTION
Many callus tissues have been found to have the ability to undergo cellular differentiation. In some cases this occurs spontaneously, whereas in others the differentiation can be experimentally induced, usually by an appropriate supply of auxins and
cytokinins. In this paper we examine some of the factors which control the differentiation and organization of sycamore callus.
Cytokinins are known to exert an effect on cell division (Das, Patau & Skoog, 1956;
Miller, Skoog, Von Saltza & Strong, 1958; Roberts, 1969) and differentiation is
usually considered to be possible only immediately following cell division (Sinnott &
Bloch, 1945; Fosket, 1968). In some instances, however, differentiation can be induced
in parenchyma cells without immediate prior cytokinesis (Sachs, 1969). Fosket &
Torrey (1969) have used soybean tissue to show that kinetin above a certain concentra-
38
K. Wright and D. H. Northcote
tion is required for xylem element formation to occur, but that this is not a consequence
of its effect on cell division.
Kinetin can also affect the ploidy of a tissue (Torrey, 1959, 1961, 1967) and this is
presumably connected with its role in cell division. Torrey (1959, 1961, 1967) found
that when kinetin or yeast extract was present in the medium on which a pea-root
callus was initiated the callus became tetraploid and lost its ability to differentiate roots.
When kinetin was omitted from the medium however, the callus remained diploid and
was still capable of organ formation. The degree of polyploidy of particular tissues
within an intact plant is characteristic both of the tissue and of the plant (D'Amato,
1952) and the role of polyploidy as a factor in plant development has been reviewed by
D'Amato (1964).
A sycamore callus (S2) has not been induced to undergo cellular differentiation since
its isolation in 1958. This tissue has been cultured on a medium containing coconutmilk, a component of which has been shown to have cytokinin activity (Zwar & Bruce,
1970). We have reported the induction of root formation from a new sycamore callus
(S4) grown on a defined medium (Wright & Northcote, 1972) and the stimulus for
this differentiation was the supply of kinetin in the growth medium. In the present
investigations we examine the role of kinetin in the growth and differentiation of sycamore tissue cultures.
A great many theoretical and practical consequences of tissue culture studies depend
upon the ideas of totipotency and the ability of the cells to differentiate. It would be of
great value, therefore, if a general method were available for the induction of differentiation within a callus. With this in mind we have examined some differences between
the S2 and S4 tissues and describe some experiments in which we have induced each
of the cultures to assume some of the properties of the other.
MATERIALS AND METHODS
Tissue culture
The culture and isolation, of S4 callus have been described by Wright & Northcote (1972).
The method of isolation and culture of S2 callus has been described by Stoddart & Northcote
(1967) and except where stated it was grown on a medium containing coconut-milk, sucrose,
2,4-dichlorophenoxyacetic acid (2,4-D) and the same mineral salts and vitamins as were used
by Heller (1953) (Table 1). S4 was grown on a medium based on Gamborg's (1966) PRL4
medium (Wright & Northcote, 1972) whose composition is shown in Table 1. Two per cent
sucrose was supplied except where otherwise stated. All callus cultures were maintained in the
dark at 26 ± 2 °C.
Suspensions of cells in liquid media were grown in conicalflasksin an orbital shaker operating
at 97 rev/min and maintained in the dark at 26 ± 1 °C. Suspension cultures (100 ml) in a 500-ml
conical flask were subcultured every week by transferring 10 ml into 90 ml of fresh medium;
sterile precautions were observed throughout (Wright & Northcote, 1972).
Microscopy
The techniques used have been described by Jones & Northcote (1972) and Wright &
Northcote (1972).
Differences between sycamore calluses
39
Table 1. Composition of media used for sycamore callus
Medium for S2
Inorganic components, mg/1.
NaH,PO 4 .H 2 O
Na2HPO4
KC1
NaNO3
MgSO 4 .7H,O
(NH4)2SO4
KNO 3
CaCl2.6H2O
FeCl3
Fe2+EDTA
ZnSO 4 .7H,O
H3BO3
MnSO 4 .4H 2 O
AICI3
NiCl 2 .6H 2 O
KI
CuSO 4 .5H 2 O
Na 2 MoO 4 .2H,O
CoCl 2 .6H 2 O
Organic components, mg/1.
w-Inositol
Pyridoxine HC1
Nicotinic acid
Calcium D-pantothenate
Thiamine
2,4-Dichlorophenoxyacetic acid
i-Naphthylacetic acid
N-Z-amine type 'A', g/1.
(Sheffield Chemical, N.Y., U.S.A
Sucrose, g/1.
Coconut milk, ml/1.
125
—
75°
600
250
—
—
75
1
—
1
1
o-oi
0-03
0-03
o-oi
0-03
•—
—
Medium (PRL4)
forS4
90
30
300
250
200
1000
220
—
28
3
3
13
—
—
o-75
025
0-25
0-25
—
—
—
100
I
—
I
10
—
6
—
—
1
1
1
2
20
20
200
—
Chromosome counting
After 3 days a sample of the subculture was placed on a slide, excess moisture was removed,
and the tissue was flooded with aceto-orcein and squashed between slide and coverslip (Roberts
& Northcote, 1970). The preparations were examined using a Zeiss Ultraphot model II and
the chromosomes were counted in squashes of 80-100 dividing cells for each culture. Occasional
squashes were impossible to count and if cells of a particular ploidy level did not squash so well
as the others the results will be unavoidably biased against these.
Cloning procedure
Samples were removed from liquid suspension cultures and plated thinly on to solidified
medium in a Petri dish. The positions of single cells were noted with the aid of a dissecting
microscope. After 5 weeks clones arising from these single cells were removed and grown
alongside nurse tissue (Muir, Hildebrandt & Riker, 1954) on solidified medium contained in
sterile plastic jars (Wright & Northcote, 1972) until the colony was large enough to be cultured
alone.
K. Wright and D. H. Northcote
4°
005
0-2
W
NAA, mg/l.
0-5
W
w
W
0 01
20
08
10
10
W
w
W
w
W
W
50
0-1
80
100
100
w
005
20
w
01
05
0-5
1 0 ;2
10
Wxp
02
20
0-25
W
0-4
05
1 25
0 25
0 63
0-125
Cxp
08
0063
Cxp
20
0025
Cxp
Cxp
0-1
0-25
Cxp
0-4
Fig. i. The effect of different NAA and kinetin concentrations on the growth and
differentiation of S4 callus. The results were recorded 8 weeks after subculture.
# , Death; ^ , root formation; texture of the callus, W = white; C = compact (see
text). Calluses in which xylem and phloem were found are marked (xp). The value in
the bottom right-hand corner of each square is the ratio NAA:kinetin (w/w) in the
incubation.
RESULTS
The effect of kinetin at different l-naphthylacetic acid (NAA) concentrations
Kinetin has been identified as a factor necessary for differentiation to occur in S4
callus (Wright & Northcote, 1972). In order to investigate this further S4 callus was
grown on media containing kinetin at concentrations of between o and 2 mg/1. and for
each medium NAA was included over the concentration range 0-10 mg/1. (Fig. 1). The
results after 8 weeks are shown in Fig. 1. Each experiment was performed at least in
duplicate. The numbers in the bottom right-hand corner of each square show the
ratios of NAA: kinetin (w/w) in the incubation. Calluses are classified as ' white' or
'compact'. Apart from their colour, white calluses were characterized by extensive
growth, little if any differentiation and a much greater friability than the compact
calluses which were very hard, had a red-brown pigmentation and usually contained
Differences between sycamore calluses
41
some differentiated tissue that could be identified microscopically. The difference in
the external appearances of these 2 types of tissue is illustrated in Fig. 3. The results
summarized in Fig. 1 show that white calluses were produced on high NAA: kinetin
ratios while low ratios resulted in compact tissues. At ratios varying between 0-5 and
20 the calluses had some external features of both the white and the compact tissues
and roots often grew from the surface. Selected calluses grown on media containing
extreme ratios of hormones were processed for microscopical examination and those
incubations in which xylem and phloem were identified are appropriately marked in
Fig. 1. Since it is difficult to be certain about negative results these are not marked on
the diagram. It can be seen that xylem and phloem were formed even at the lowest
NAA: kinetin ratios tested and some well developed areas were observed (Figs. 6 and
10). However, although it was qualitatively the same, differentiation seen in sections
of tissues grown in the presence of these low hormone ratios was less extensive than
that found in calluses where roots were formed (Wright & Northcote, 1972). Differentiation was found in only one case where the NAA concentration was 10 mg/1. and we
have previously shown that differentiation did not occur in the absence of kinetin
(Wright & Northcote, 1972).
Kinetin has a striking effect on the size of the cells as well as on the extent of
differentiation in the tissue. Figs. 9 and 10 show cross-sections through the surface
areas of white and compact calluses. With a low NAA: kinetin ratio the cells were much
smaller than in the absence of kinetin and there were localized areas of high mitotic
activity. When no kinetin was supplied the cells were larger and much more uniform
in size than those grown in the presence of kinetin.
Some S4 callus has been subcultured on a kinetin-containing medium (PRL4 with
1 mg/1. NAA and o-i mg/1. kinetin) throughout 6 subcultures (approximately 12
months). No roots have been formed since the second subculture and sections of the
callus show that only a few isolated areas contain differentiated tissue. The callus still
retains its hard texture.
Interaction between S2 and 1S4 calluses on kinetin-containing media
From the above experiment and also from previous work (Wright & Northcote,
1972) a medium based on PRL4, that contains 1 mg/1. NAA and 2 % sucrose together
with o-i mg/1. kinetin was shown to be suitable for testing the ability of sycamore
callus to differentiate. Simpkins, Collin & Street (1970) did not report any differentiation when kinetin was added to the medium in which suspensions of the original
isolate (S2) were growing. Tissues (S2 and S4) were cultured together on the medium
described above in order to test whether any extracellular water-soluble substances
produced by one tissue could affect the other. This technique is analogous to the' nurse
tissue' procedure of Muir et al. (1954). Pieces of one tissue were arranged around one
piece of the other on solidified medium in a 500-ml conical flask. In controls, tissue
from only one callus was used and inocula came from the medium on which these
calluses are normally maintained in this laboratory. The experimental arrangement
is shown in Fig. 4. Roots were frequently produced by S4 when it surrounded S2,
although no differentiation was found in S2. Callus S4 was still able to differentiate
42
K. Wright and D. H. Northcote
when surrounded by S2 and controls containing only S4 formed xylem, phloem and
roots while those containing only S2 did not. A sample of S2 from theflaskin which this
was surrounded by S4 callus was removed to a further flask and surrounded by fresh
S4 inocula. Again no differentiation was found in the S2 callus after growth had
occurred and this result was also obtained when the S2 was subcultured for a third
time. Finally, a sample of S2 from the third subculture was transferred to a medium
containing all the nutrients and hormones described above and to which had been
added a homogenate of approximately 20 g fresh weight of S4 callus which had been
incubated for 3 weeks on a medium causing differentiation. Thus the aqueous factors
present in differentiating S4 callus were made available to S2 tissue. After a lag
period of 2 weeks the callus began to grow very rapidly but after growth for a further
4 weeks, sections of the callus revealed no differentiation within the S2 callus.
The effect of kinetin on the ploidy of the calluses
Roberts & Northcote (1970) established that S2 callus grown in suspension is
predominantly tetraploid and their method of chromosome counting has been employed to study the chromosome number of S2 and S4 calluses grown on different
media. After squashing and staining with aceto-orcein chromosome counts were made
on between 80 and 100 dividing cells from suspension cultures. The chromosome
number distribution of S2 tissue grown on its usual medium is shown in Fig. 2 A. The
diploid number of sycamore is 52 (Foster, 1933) and while it is difficult to specify the
accuracy of counts with high numbers of small chromosomes, the clustering about the
tetraploid number is unmistakable. The corresponding distribution for S4 grown as
a suspension in PRL4 with 1 mg/1. NAA is shown in Fig. 2 D and here the chromosome
count is distributed about the diploid number although occasional tetraploid mitoses
were seen. Thus there was a fundamental difference between the sycamore callus
prepared in 1958 and the one isolated in May 1970.
A series of experiments was carried out in order to grow S2 on a medium containing
no kinetin and to facilitate future comparative studies PRL4 with 1 mg/1. NAA was
chosen. A suspension in medium containing coconut milk was subcultured into one
based on PRL4 that included 1 mg/1. NAA and o-i mg/1. kinetin; it was then transferred to a medium with a lower kinetin concentration (o-oi mg/1.) and finally into
one with 1 mg/1. NAA but no kinetin. The chromosome number distributions of S2
that had been grown in PRL4 with 1 mg/1. NAA after 5 and 8 subcultures are shown in
Fig. 2B, c. It can be seen that the chromosome number distribution changed from
predominantly tetraploid to predominantly diploid. A number of intermediate values
were also found.
The isolation of S2 clones
Samples from the seventh subculture of S2 in PRL4 with 1 mg/1. NAA were used
to isolate clones of single-cell origin in an attempt to obtain clones of cells with different
ploidies. Twenty such clones have been maintained and tested for (1) their ability to
differentiate into a green tissue by development of chloroplasts on exposure to continuous light (500-1500 lux) and (2) their response to the medium which induced
Differences between sycamore calluses
Diploid (52)
Tetraploid (104)
•
T
_r
40
30
_
20
-
10
-
43
1
'—i
1
l
1 i
l
i
i
i i l
1
1
1 1
B
30
o
DO
«
20
S 10
c
wit
1
1 |
I
i
1—1
! !
11
C
11
£° 30
1 20 -
I io 1
1 |
| ^
1 1
1
J
, , |
1—<—1
1 1
D
30 -
r-.
20 -
uS
10
1
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t^\
1—1
to \o ^o r^. r*^ co co o^ o^ t
L^l
imm3
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t, ,>
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tmm3
Ify
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Chromosome number range
Fig. 2. The chromosome number distributions of sycamore tissues on different media
(Table i ) ; A, S2 in its usual medium; B, after 5 subcultures in PRL4 with 1 mg/1.
NAA; c, after 8 subcultures in PRL4 with 1 mg/1. NAA; and D, S4 in PRL4 with
1 mg/1. NAA.
xylem and phloem formation in S4. Simpkins et al. (1970) have already reported that
uncloned S2 callus can become green when exposed to light.
All the clones showed a certain amount of greening after 8 weeks in the light but
histological examination of the calluses grown for 6 weeks on PRL4 with 1 mg/1. NAA
and 0-1 mg/1. kinetin did reveal differences in the type of growth which occurred. In
most cases the cells of the callus showed little cell contact and many of the cells had
broken up (Fig. 7), but 5 clones contained areas where the cells were in close contact
with one another giving the callus an appearance more characteristic of S4 than S2
(Figs. 11, 12). These clones are being maintained on PRL4 with 1 mg/1. NAA.
44
K. Wright and D. H. Northcote
The effect of polyethylene glycol on S2 callus
The effect of the hormone ratios on S4 callus led us to consider a method of
changing the texture of S2 callus. Thus we included polyethylene glycol
(mol.wt.iooo) and sucrose in various combinations in a solidified medium based
on PRL4 which also contained 1 mg/1. NAA and o-i mg/1. kinetin. The appearance of
the callus after 6 weeks is shown in Fig. 5. Although 16 % polyethylene glycol appeared
to inhibit the growth of the callus, other concentrations did not substantially affect the
amount of growth which occurred. Histological examination of the tissue showed that
it was the concentration of polyethylene glycol which affected the pattern of growth
within the callus; over the concentration range used no visible effect of sucrose was
noted. Calluses grown on media containing no polyethylene glycol were watery and
did not stand up well to processing for cytological examination, and only occasional
areas maintained their integrity to show well separated cells (Fig. 8). The inclusion
of polyethylene glycol in the medium caused the calluses to be much harder, comparable with compact S4, and the cells were more closely packed together. This was
most apparent with 16 % polyethylene glycol (Fig. 13), but many of the cells had been
killed. At 4 and 8% polyethylene glycol the cells were also closely packed although
they seemed less constrained to grow in rows than in the presence of 16 % polyethylene
glycol (Fig. 14). Some areas of high mitotic activity occurred although no cellular
differentiation was found in the calluses incubated on any of the media containing
polyethylene glycol.
DISCUSSION
As in the experiments of Skoog & Miller (1957) the ratio of auxin to cytokinin was
found to be the factor which controlled differentiation in S4. Roots were most often
induced when the ratio was between 0-5 and 20 and usually at a stage when the calluses could not be classified as appearing white or compact. At low ratios of hormones
the tissues were pigmented and very hard; xylem and phloem formation was observed
although root formation was infrequent. At high ratios the calluses had a characteristic
white colour and differentiation was found only once. It may be that the ratio of auxin
to cytokinin was not the only controlling factor since ratios of 4 and 5 which usually
induced root formation failed to do so when associated with 10 mg/1. NAA. The calluses
on these media were white in appearance and in general showed much less differentiation.
The increased pigmentation in the presence of kinetin may have been the result of
the production of a secondary product from the tissue. Deposition of secondary material
in the vacuole of S4 has been observed (Wright & Northcote, 1972) and this has recently been reported in S2 by Carceller, Davey, Fowler & Street (1971).
Since no differentiation was found in S2 when it was cultured alongside S4 that was
differentiating in the same flask S4 did not produce any diffusible extracellular watersoluble substance capable of inducing differentiation in S2 callus. Equally S2 did not
produce any factors which were capable of inhibiting differentiation in S4 tissue
and the reasons for the inability of S2 to differentiate must be sought elsewhere.
Differences between sycamore calluses
One obvious difference between the tissues S2 and S4 was the ploidy. The cells of
S2 were mainly tetraploid while the new tissue S4 was predominantly diploid.
Torrey (1958, 1961, 1967) found a similar variation with pea-root callus and as with
the pea tissue the tetraploidy of S2 may have been induced by the cytokinins that were
present in the coconut-milk. By transferring the S2 suspensions through a series of
media with decreasing kinetin concentrations and finally to the one used to culture the
diploid S4 (PRL4 with 1 mg/1. NAA) it has been possible to induce a population of
predominantly diploid S2 cells. Although there are a number of differences between
the media used for S2 and S4 (Table 1, p. 39) it is most probable that it was the
difference in kinetin concentration that was responsible for this change in ploidy. It
might have arisen by some kind of reduction division of the tetraploid cells or by mitosis
and preferential selection of diploid cells that were present in the original culture in
small numbers or that had reverted to diploid in the absence of kinetin. Clones have
been isolated from such suspension cultures in order to try and isolate a pure diploid
callus strain of S2 which will be tested for its ability to differentiate. So far 5 clones
have been identified which have a growth pattern such that a greater number of cells
are in contact with one another. If a tetraploid clone can also be isolated it will be
possible to define more closely the mechanism of the reduction from tetraploid to
diploid. In a reciprocal way tissues of S4 have been cultured on kinetin-containing
media and have ceased to produce roots after 2 subcultures.
It can be seen that the relationships of kinetin to polyploidy and differentiation are
complex. Kinetin stimulates polyploid mitoses in pea root segments (Torrey &
Fosket, 1970) and even in salamander tissue (Kevin, Witkus & Berger, 1966) and it is
also known that many cells in the intact plant are polyploid (D'Amato, 1964). We
have shown that kinetin acts on the predominantly diploid callus S4 to induce roots,
whereas prolonged culturing in the presence of kinetin yields a tetraploid tissue that
will not differentiate. A possible explanation is that the cells must undergo the transition from diploid to tetraploid (or higher degrees of polyploidy) under the correct
conditions for differentiation to be induced as a result of the environmental factors
controlling the transcription of the DNA. The requirement that cell division should
immediately precede differentiation has been noted in plants (Fosket, 1968) and animals (Stockdale & Topper, 1966) and these authors suggest that it is the milieu in
which cell division occurs that determines the pathway of development of the daughter
cells. Thus polyploid cell formation may be a way of making the DNA susceptible to
environmental control in the same way that this occurs at a normal cell division
(Kauffman, 1967).
Partanen (1965) has considered the function of polyploidy in the intact plant and
argued that cells may become polyploid when the nutrient supply falls so low that the
amount of energy available does not reach an envisaged predetermined level necessary
for the discrete process of cytokinesis. DNA replication, being a more prolonged
process, might continue slowly in the presence of this low amount of energy with the
result that the cell would become polyploid. This theory neither indicates how the
normal precise control mechanisms involved in correlating DNA replication and cytokinesis (Mather, 1965) could be overcome, nor does it explain the inducement of
45
46
K. Wright and D. H. Northfote
polyploidy by kinetin. Although kinetin causes the cells to become smaller and more
active it seems unlikely that this occurs to such an extent that the carbon supply
becomes limiting. Cell division and differentiation are usually thought to be controlled
by the ratio of the various hormones in the environment of the cell and possibly by the
gradient of these substances across the cell. This may also be true of the control of
polyploid cell formation. Auxins are synthesized by leaves (Jacobs, 1952) and cytokinins are produced by roots (Kende, 1965) and both of these hormones may be formed
during the controlled autolytic processes of xylem and phloem formation (Wooding &
Northcote, 1964; Northcote & Wooding, 1966; Sheldrake & Northcote, 1968). The
ratio of these hormones at a particular point within a plant would depend on the
physical dimensions of the plant as well as on other factors and thus such a ratio may
differ from plant to plant. Conditions in some plants but not in others might favour
polyploid cell formation during differentiation of certain tissues and in both cases
there would appear to be the same potential for allowing re-programming of the DNA.
The patterns of development which occur in the intact plant depend on cell-to-cell
interactions. Cells in the plant make direct contact by means of plasmodesmata and
there are common cell walls between adjacent cells. It is probable that the walls can
act to carry material between cells and this function will vary according to the composition and developmental stage of the wall. The properties of the cell wall also determine the degree of mutual adhesion of the cells and this is especially important for the
cell-to-cell interaction of callus tissues.
Sycamore callus tissue cell walls contain relatively large amounts of pectic materials
similar to cambial pectic substances (Stoddart, Barrett & Northcote, 1967; Stoddart
& Northcote, 1967). A physiological low auxin concentration is usually thought to be
required for differentiation to occur (Fosket & Roberts, 1964), and Rubery & Northcote (1970) have shown that under these conditions less arabinose is inserted in the
neutral blocks and side chains of the pectin giving less branching and resulting in a
tighter cell wall. Kinetin induces a harder texture in S4 and a certain amount of kinetin
is required before differentiation can occur (Wright & Northcote, 1972). If the callus
becomes too hard however root formation is uncommon; Torrey & Shigemura (1957)
also found this using pea-root callus.
The inclusion of polyethylene glycol in the medium causes the cells of S2 to come
into greater contact and recently divided cells give the appearance of being physically
constrained by their close neighbours. When Doley & Leyton (1970) applied polyethylene glycol to Fraxinus twigs a greater amount of xylem was formed in the wound
callus which was also harder than in the absence of polyethylene glycol. Water can be
considered a structural component of the plant cell wall and the amount of water
available to pectin when it forms a gel can markedly affect the texture of the cell wall
(Northcote, 1972). Thus it may be possible to increase the extent of cell interaction
by either altering the chemical nature of the wall constituents, which occurs naturally
during development, or by artificially regulating the amount of the water component
which can be incorporated into the cell wall.
Kochhar, Bhalla & Sabharwal (1971) have reported the induction of vegetative buds
in tobacco callus by chelating agents and there is evidence to suggest that chelating
Differences between sycamore calluses
47
agents cause a loosening of the wall by removing the calcium ions associated with the
pectin (Xorthcraft, 1951). Thus the texture of the cell wall can be altered physically,
chemically and hormonally and in all cases the pattern of differentiation in the tissue
changes. It may be therefore that in order to induce differentiation of a callus tissue it is
necessary to have not only genetically competent cells and the correct hormonal
environment, but tissues of a texture compatible with allowing the appropriate cell-tocell interaction to take place.
K. W. is grateful to the Science Research Council for a grant during the tenure of which this
work was carried out. We would like to thank Mrs M. Wilson for typing the manuscript and
Mr L. Jewitt and Mr R. Pilcher for assistance with photography.
REFERENCES
CARCELLER, M., DAVEY, M. R., FOWLER, M. W. & STREET, H . E. (1971). The influence of
sucrose, 2,4-D, and kinetin on the growth, fine structure and lignin content of cultured sycamore cells. Protoplasma 73, 367-385.
D'AMATO, F. (1952). Polyploidy in the differentiation and function of tissues and cells in plants.
A critical examination of the literature. Caryologia 4, 311—358.
D'AMATO, F. (1964). Endopolyploidy as a factor in plant tissue development. Caryologia 17,
41-52.
DAS, N. K., PATAU, K. & SKOOG, F. (1956). Initiation of mitosis and cell division by kinetin
and indoleacetic acid in excised tobacco pith tissue. Physiologia PL 9, 640-51.
DOLEY, D. & LEYTON, L. (1970). Effects of growth regulating substances and water potential on
the development of wound callus in Fraxinus. New Phytol. 69, 87-102.
FOSKET, D. E. (1968). Cell division and the differentiation of wound-vessel members in cultured
stem segments of Coleus. Proc. natn. Acad. Sci. U.S.A. 59, 1089-1096.
FOSKET, D. E. & ROBERTS, L. W. (1964). Induction of wound-vessel differentiation in isolated
Coleus stem segments in vitro. Am. J. Bot. 51, 19-25.
FOSKET, D. E. & TORREY, J. G. (1969). Hormonal control of cell proliferation and xylem differentiation in cultured tissues of Glycine max var. Biloxi. PL PhysioL, Lancaster 44, 871-880.
FOSTER, R. C. (1933). Chromosome number in Acer and Staphylea. J. Arnold Arbor. 14,
386-393.
GAMBORG, O. L. (1966). A/omatic metabolism in plants. II. Enzymes of the shikimate pathway in suspension cultures of plant cells. Can. J. Biochem. 44, 791-799.
HELLER, R. (1953). Recherches su la nutrition minerale des tissus vegetaux cultives in vitro.
Annh Sci. nat. Bot. Biol. Ve'g. 14, 1-223.
JACOBS, W. P. (1952). The role of auxin in differentiation of xylem around a wound. Am. jf.
Bot. 39, 301-309.
JONES, M. G. K. & NORTHCOTE, D. H. (1972). Nematode induced syncytium - a multinucleate
transfer cell. J. Cell Sci. 10, 789-809.
KAUFFMAN, S. (1967). Sequential DNA replication and the control of differences in gene
activity between sister chromatids - a possible factor in cell differentiation. J. theor. Biol.
17, 483-497KENDE, H. (1965). Kinetin-like factors in the root exudate of sun-flowers. Proc. natn. Acad. Sci.
U.S.A. 53, 1302-1307.
KEVIN, S. P., WITKUS, E. R. & BERGER, C. A. (1966). Effects of kinetin on cell division in
Triturus viridescens. Expl Cell Res. 41, 259-264.
KOCHHAR, T . S., BHALLA, P. R. & SABHARWAL, P. S. (1971). In vitro induction of vegetative
buds in tobacco callus by chelating agents. Can. jf. Bot. 49, 391-394.
MATHER, K. (1965). Genes and cytoplasm in development. In Encyclopedia of Plant Physiology,
vol. 15 (ed. W. Ruhland), pp. 41-67. Heidelberg: Springer-Verlag.
MILLER, C. O., SKOOG, F., V O N SALTZA, M. H . & STRONG, F. M. (1958). Kinetin, a cell
division factor from deoxyribonucleic acid. J. Am. chetn. Soc. 77, 1392.
48
K. Wright and D. H. Northcote
Mum, W. H., HILDEBRANDT, A. C. & RIKER, A. J. (1954). Plant tissue cultures produced from
single isolated cells. Science, N.Y. 119, 877-878.
NORTHCOTE, D. H. (1972). Chemistry of the plant cell wall. A. Rev. PI. Physiol., Lancaster
23. 113-132NORTHCOTE, D. H. & WOODING, F. B. P. (1966). Development of sieve tubes in Acer pseudoplatanus. Proc. R. Soc. B 163, 524-537.
NORTHCRAFT, R. D. (1951). The use of oxalate to produce free-living cells from carrot tissue
cultures. Science, N.Y. 113, 407-408.
PARTANEN, C. R. (1965). On the chromosomal basis for cellular differentiation. Am. J. Bot. 52,
204—209.
ROBERTS, K. & NORTHCOTE, D. H. (1970). The structure of sycamore callus cells during division
in a partially synchronized suspension culture. J. Cell Sci. 6, 299-321.
ROBERTS, L. W. (1969). The initiation of xylem differentiation. Bot. Rev. 35, 201-250.
RUBERY, P. H. & NORTHCOTE, D. H. (1970). The effect of auxin (2,4-dichlorophenoxyacetic
acid) on the synthesis of cell wall polysaccharides in cultured sycamore cells. Biochim.
biophys. Ada 222, 95-108.
SACHS, T . (1969). Polarity and the induction of organised vascular tissues. Ann. Bot. 33,
263-275.
SHELDRAKE, A. R. & NORTHCOTE, D. H. (1968). The production of auxin by autolysing tissues.
Planta 80, 227-236.
SIMPKINS, K., COLLIN, H. A. & STREET, H. E. (1970). The growth of Acer pseudoplatanus cells
in a synthetic liquid medium: response to the carbohydrate, nitrogenous and growth hormone
constituents. Physiologia PI. 23, 385-396.
SINNOTT, E. W. & BLOCH, R. (1945). The cytoplasmic basis of inter-cellular patterns in vascular
differentiation. Am. J. Bot. 32, 151-156.
SKOOG, F. & MILLER, C. O. (1957). Chemical regulation of growth and organ formation in plant
tissues cultured in vitro. Symp. Soc. exp. Biol. 11, 118-131.
STOCKDALE, F. E. & TOPPER, Y. J. (1966). The role of DNA synthesis and mitosis in hormonedependent differentiation. Proc. natn. Acad. Sci. U.S.A. 56, 1283-1289.
STODDART, R. W., BARRETT, A. J. & NORTHCOTE, D. H. (1967). Pectic polysaccharides of grow-
ing plant tissues. Biochem. J. 102, 194-204.
STODDART, R. W. & NORTHCOTE, D. H. (1967). Metabolic relationships of the isolated fractions
of the pectic substances of actively growing sycamore cells. Biochem. J. 105, 45-59.
TORREY, J. G. (1959). Experimental modification of development in the root. In Cell, Organism
and Milieu, 17th Symp. Soc. Study Dev. Growth (ed D. Rudnick), pp. 189-222. New York:
Ronald Press.
TORREY, J. G. (1961). Kinetin as a trigger for mitosis in mature endomitotic plant cells. Expl
Cell Res. 23, 281-299.
TORREY, J. G. (1967). Morphogenesis in relation to chromosomal constitution in long-term
plant tissue cultures. Physiologia PI. 20, 265-275.
TORREY, J. G. & FOSKET, D. E. (1970). Cell division in relation to cytodifferentiation in cultured
pea root segments. Am. J. Bot. 57, 1072-1080.
TORREY, J. G. & SHIGEMURA, Y. (1957). Growth and controlled morphogenesis in pea root
callus tissue grown in liquid media. Am. J. Bot. 44, 334-344.
WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The development of the secondary wall of
the xylem in Acer pseudoplatanus. J. Cell Biol. 23, 327-337.
WRIGHT, K. & NORTHCOTE, D. H. (1972). Induced root differentiation in sycamore callus.
J. Cell Sci. 11, 319-337.
ZVVAR, J. A. & BRUCE, M. I. (1970). Cytokinins from apple extract and coconut milk. Aust. J.
biol. Sci. 23, 289-297.
{Received 11 April 1972)
Differences between sycamore calluses
Polyethylene glycol, %
Figs. 3-5. For legend see p. 50.
49
K. Wright and D. H. Northcote
Fig. 3. The difference in external appearance of white (top) and compact (bottom)
S4 calluses. The white callus was friable and the compact one hard and pigmented.
Callus diameter was about 2-5 cm.
Fig. 4. Pieces of S4 callus surrounding a piece of S2 after 6 weeks growth together on
solidified PRL4 with 1 mg/1. NAA and o-i mg/1. kinetin. Roots (arrows) are visible on
some of the S4 calluses. Actual size.
Fig. 5. The effect of sucrose and polyethylene glycol on S2 callus. Calluses grown in
the presence of polyethylene glycol were similar in texture to the compact S4 shown
in Fig. 3. Diameter of calluses grown in the presence of o, 4 and 8 % polyethylene glycol
was about 2-5 cm.
Fig. 6. A well developed area of xylem and phloem in a compact callus (S4). (Medium
containing 0 8 mg/1. NAA, 2 mg/1. kinetin). The section was stained with aniline-blue
and observed with ultraviolet fluorescence optics, x 440.
Figs. 7—14 are sections of material embedded in wax and stained with safranin and
picric aniline blue.
Fig. 7. A cross-section through an area of an S2 clone in which the cells showed little
intercellular contact. This was the normal growth pattern of S2 calluses, x 100.
Fig. 8. Callus (S2) grown o n P R L 4 with img/1. NAA and 5-2 % sucrose, but no polyethylene glycol. The cells are well separated, x 100.
Fig. 9. A cross-section through the surface area of a typical white callus (S4). The
cells are in contact with one another but they are large and there is no organization,
x 145.
Fig. 10. A cross-section through a compact callus (S4). (Medium containing
0-5 mg/1. NAA, o-8 mg/1. kinetin.) There is more organization and the cells are very
much smaller than in a white callus (Fig. 9). One nodular area containing xylem is
encircled, x 140.
Differences between sycamore calluses
4-?
52
K. Wright and D. H. Northcote
Figs, i i , 12. Two different S2 clones isolated onPRL4with 1 mg/1. NAA in which
the degree of cell contact is similar to that in S4 (Fig. 9) rather than to that in typical
S2 (Fig. 7). Fig. 11, x 135; Fig. 12, x 200.
Fig. 13. The surface region of an S2 callus grown in the presence of 1 6 % polyethylene glycol. Some of the cells appear to have been constrained to divide in
radial columns (arrows), x 140.
Fig. 14. S2 callus grown in the presence of 4 % polyethylene glycol. There is little
intercellular space. Contrast with Fig. 8 where no polyethylene glycol was added,
x 160.
Differences between sycamore calluses
53