J. CellSci. 17, 11-26(1975)
Printed in Great Britain
„
QUANTITATIVE MEASUREMENT OF THE
COURSE OF BEAN CALLUS DIFFERENTIATION
LINDSAY E. HADDON AND D. H. NORTHCOTE
Department of Biochemistry, University of Cambridge,
Tennis Court Road, Cambridge, CBz iQW, England
SUMMARY
Two strains of callus have been isolated from bean hypocotyl and grown on a defined maintenance medium supplemented with 2 mg/1. 2:4-dichlorophenoxyacetic acid (2:4D) and 2 %
sucrose. Root initiation was observed in one strain and formation of nodules containing xylem
and phloem in both strains after transfer to an induction medium supplemented with 1 mg/1.
naphthyleneacetic acid, 0-2 mg/1. kinetin and 3 % sucrose, after 3 transfers to maintenance
medium.
The number of nodules per gramme increased 10-fold between 6 and 12 days after transfer,
and thereafter remained constant. Phenylalanine ammonia lyase (PAL) activity rose to a
maximum value when the rate of nodule formation was greatest, and decreased after the maximum nodule concentration was reached. The final constant value for PAL activity was above
that of callus grown on maintenance medium. /?i -»• 3 glucan synthetase activity rose to a
maximum 15 days after transfer, and then fell gradually to a level above that measured in callus
on maintenance medium.
Callus was transferred from maintenance medium after 3, 4, 5 and 6 transfers. The concentration of nodules after 21 days on induction medium decreased as the callus was kept in
culture. No further differentiation could be induced after 6 transfers. The fall in nodule
formation was paralleled by a decrease in PAL and /?i -»• 3 glucan synthetase activities measured
21 days after transfer.
INTRODUCTION
Plant tissue cultures have been used to study the differentiation of plant cells, since
they provide a system in which known growth conditions can be imposed upon a
fixed population of cells.
Having established the conditions necessary for the induction of differentiation in a
callus, it should be possible to develop quantitative methods for the estimation of
differentiation, using biochemical rather than histological techniques. This would
then allow the time course of the induction to be followed as well as a comparison of
the ability to differentiate, both of the same callus after it had been kept for varying
times in culture and between different callus lines of the same species but initiated
from different tissues.
Histological detection of differentiation generally depends upon the specific staining
of a component localized in the cell type under consideration, and quantitative biochemical estimations can examine either the actual amount of these components or the
activity of the enzymes believed to control their biosynthesis. The latter approach has
the advantage that enzyme activity is a more sensitive indicator of a change in the cell's
development than the total amount of end product accumulated throughout the
12
L. E. Haddon and D. H. Northcote
growth of the cell. The pattern of variation of activity could also suggest possible
mechanisms for the molecular basis of differentiation.
The French bean Phaseolus vulgaris L. was chosen for this study since sterile bean
tissue is readily obtained and a bean stem callus originally isolated in this laboratory
in 1964 (Jeffs & Northcote, 1966) retained for several years an ability to form vascular
nodules containing both xylem and phloem. New strains of bean stem callus were
isolated and grown on defined medium in the hope that organogenesis could be
induced by a known change in the medium.
General metabolic changes have been found to occur in differentiating callus and
some, such as increased activity of enzymes concerned with sugar utilization (Thorpe
& Laishley, 1973), have been estimated. These methods, however, indicate a change
in the growth pattern of the whole callus rather than the production of a specific cell
typeSince vascular tissue is fairly readily formed by bean callus and vascularization is
necessary for organogenesis, the 2 differentiated cell types chosen for study in this
work were the xylem vessel and the phloem sieve tube.
Xylem vessels are generally detected by their heavily lignified walls, although the
walls of other cells, such as extraxylary fibres, are lignified to some extent. Chemical
analysis has shown that the lignin content of callus does rise when xylem is formed
(Jeffs & Northcote, 1966). The activity of the enzyme phenylalanine ammonia lyase
(PAL) has been correlated with xylogenesis of sycamore cambium (Rubery & Northcote, 1968), as well as with induced xylogenesis in culture (Rubery & Fosket, 1969).
The accompaniment of xylogenesis by an increase in PAL activity therefore seems
fairly well established in those systems where xylem initiation is not accompanied by
pigment formation. Cinnamic acid, the product of PAL activity, is also used in the
biosynthesis of polyphenol pigments, and PAL activity can be correlated with
anthocyanin formation in a callus which does not show xylogenesis (Heinzmann &
Seitz, 1974). Since the formation of anthocyanins has not been shown in bean callus,
and vascular nodules were the only portion of the callus stained by phloroglucinol,
PAL activity was used as a biochemical marker for xylogenesis.
Phloem vessels can be identified by the presence of paired pads of callose (a /?i -*• 3
glucan.) on the sieve plates. In contrast to monocotyledonous plants whose hemicellulose and storage polymers contain considerable proportions of /?i -»• 3 linked glucose
(Northcote, 1969), callose is the major polysaccharide containing /?i -> 3 linked
glucose in dicotyledons such as Phaseolus. The precise mechanism of polysaccharide
synthesis is unknown but it seems likely that one control step is the transfer of the
sugar moiety from the activated form of nucleotide-diphosphate-sugar to the growing
chain. Glucose transferase preparations from the related species Phaseolus aureus have
been extensively studied and it seems agreed that UDP-glucose is the activated
precursor of /?i -> 3 glucosyl bonds (Chambers & Elbein 1970), although the glucose
moiety may also contribute to /?i -> 4 linkages (Villemez, Franz & Hassid, 1967). The
ratio of fli -> 3 to fti ->• 4 links varies considerably with growth conditions (Clark &
Villemez, 1972). The activity of UDP-glucose :/?i -> 3 glucan glucosyl transferase
(callose synthetase) was therefore used as a marker for phloem formation.
Measurement of bean callus differentiation
13
MATERIALS AND METHODS
Materials
Analytical grade chemicals and glass-distilled water were used wherever possible. UDPD-[U-14C]glucose was obtained from the Radiochemical Centre, Amersham, Bucks; sterile
containers for media from Sterilin Ltd and Richardsons of Leicester Ltd; Difco Special Agar
Noble from Difco Laboratories, Detroit, Michigan, U.S.A.
Media
The media used in these experiments were chemically defined and contained the salts and
vitamins of the B5 medium of Gamborg, Miller & Ojima (1968), supplemented with sucrose and
plant growth hormones as required and solidified with 1 % agar, added before sterilizing by
autoclaving at 103-4 kNm~* (120 °C) for 30 min. Molten sterile medium was dispensed in
15-20 ml portions into sterile screw-top containers. The medium used routinely for isolation
and subculture, referred to as maintenance medium, contained in addition to salts and vitamins,
2 mg/1. 2:4-dichlorophenoxyacetic acid (2:4D) and 2 % sucrose. All transfers were performed
with sterile instruments in a sterile air bench.
Isolation of callus
Seeds of Phaseolus vulgaris L. var. Canadian Wonder were surface-sterilized in a buffered
solution of sodium hypochlorite ('Milton' (Richardson-Merrill Ltd)), washed and placed on
sterile water-agar to germinate. When the hypocotyls were 60—100 mm long, 5-mm sections
were excised and placed on maintenance medium.
The callus which formed after 3-4 weeks growth at 25 °C in the dark was removed from the
explant and transferred to fresh maintenance medium and thereafter portions (approx. 1 g)
were transferred to fresh medium every 4-6 weeks. All callus samples were grown at 25 °C in
the dark.
Three transfers to fresh maintenance medium were performed before use in differentiation
experiments in order to ensure complete removal of parent tissue. Three callus lines have been
isolated in the past 12 months. Experiments on strain I and strain II are reported here. Strain
III has been transferred only twice to fresh maintenance medium.
Tissue cultures were also obtained from root, epicotyl, leaf and other tissues of the bean
seedlings.
Induction of differentiation
Portions of callus (approx. 1 g) were transferred from maintenance medium to various
modified media and incubated for 3 weeks before examination for vascular differentiation. All
the modified media contained 3 % sucrose, the concentration found by Wetmore & Rier (1963)
to induce xylem and phloem formation. The various hormone levels of these modified media
are described separately. The medium adopted for routine use, which is referred to as induction
medium contained salts, sucrose, vitamins and 1 mg/1. naphthyleneacetic acid (NAA) and
C2 mg/1. kinetin (furfuryl aminopurine).
Histological detection of differentiation
Samples of callus were examined to determine the amount of growth and root initiation.
Weighed portions were then macerated in a solution of phloroglucinol in concentrated HC1,
and the number of stained, lignified nodules counted under a low-power ( x 10) microscope.
Portions were also fixed, embedded and sectioned for optical microscopy according to Wright
& Northcote (1972). Sections were stained with safranin and fast green to detect xylem, using
polarized light to detect young vessels which were not sufficiently lignified to stain with
safranin. Phloem was detected with u.v. fluorescence optics after staining with dilute aniline
blue (Currier & Strugger, 1956).
14
L.E. Haddon and D. H. Northcote
Estimation of phenylalanine ammonia lyase
PAL was extracted and assayed according to the method of Rubery & Northcote (1968),
except that in the extraction buffer 8 mM ethanethiol was replaced by 8 mM sodium diethyl
dithiocarbamate, and PAL was extracted from the acetone powder with o-i M borate buffer,
pH 8-8, overnight at 4 °C. Assays were carried out in a double-beam specrrophotometer
(Beckmann DB-GT) measuring the absorbance at 290 ran, the absorbance peak of the reaction
product, iranj-cinnamic acid.
Callose synthetase estimation
The assay for /?i -> 3 glucan synthetase was based on the method of Chambers & Elbein
(1970). Callus was homogenized in the presence of Ballotini beads (no. 12) in 0 1 M-tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7-0. Particles sedimenting between 3000 g
and 20000 g were resuspended in o-i ml of Tris buffer for each gramme of fresh callus used.
The protein content of this suspension was calculated from the absorbance at 260 and 280 nm.
Tubes containing 0 1 ml Tris buffer, o-i ml of enzyme suspension, 10 fil of 005 M MgCl,,
o-i fid of UDP-D-[U- 14 C]glucose (260 mCi/mmol) and 10 /il of a 12-2 mg/ml solution of cold
U D P G to make the final U D P G concentration 1 mM, were incubated at 37 °C for 30 min. The
reaction was stopped by heating to 100 °C and the precipitate isolated by centrifugation was
washed well with water. The nature of the linkage formed was determined by periodate
oxidation and borohydride reduction (Smith degradation) of the precipitate, following the
method of Elbein (1969). The degraded polysaccharide was hydrolysed with 4 % (w/w) H S SO 4
for 1 h at 120 °C, neutralized with BaCO, and deionized by passage through Zeokarb 225 resin
in the H+ form and Amberlite IRA 400 resin in the CO, 1 " form. The products were separated
by descending paper chromatography in ethyl acetate:pyridine:water (8:2:1 by vol.). In this
solvent glucose (from 1 ->• 3 linked chains), erythritol (from 1 ->• 4 linked chains) and glycerol
(from the ends of ohains) were well separated (R0io value for erythritol 3; for glycerol, 6).
Radioactivity was determined in a Nuclear Chicago Unilux scintillation counter after adding
toluene/PPO/POPOP scintillant to 40 x 10 mm strips of the chromatogram (Harris & Northcote, 1970). Doubling the time of exposure to periodate did not increase the amount of erythritol
detected.
In earlier experiments 3 N HC1 was used at 100 °C for 3 h for hydrolysis. The recovery was
similar to that after H,SO 4 hydrolysis. -vThe latter was preferred since it gave complete hydrolysis without further degradation of the products.
RESULTS
Isolation of callus
All the callus lines isolated from the hypocotyl were at first similar in appearance.
The cultures were cream or white and had a friable texture. A few samples became
harder after several subcultures while retaining their white colour. When transferred
to fresh medium, some samples of this hard callus remained hard, while others
became friable again (Figs. 3, 4). All the cultures required subculture every 4-6 weeks,
and strains I and II were maintained through 8 subcultures without change in appearance or growth rate. Unless otherwise stated only friable white calluses obtained from
the hypocotyl were used for the work described below.
Induction of differentiation
Microscopic examination of callus grown on maintenance medium showed that
most of the cells were large and vacuolate, like parenchyma, although a few nodules
containing differentiated tissue could be detected by phloroglucinol staining. There
Measurement of bean callus differentiation
15
was no difference between the number of nodules in friable and hard callus samples.
The supplements added to the four modified media are shown in Table 1 and transfer
of callus to any one of these media resulted in a slight browning of the callus without
affecting the growth rate. Phloroglucinol squashes of the cultures after 21 days indicated that all the modified media were capable of inducing differentiation, and microscopic examination indicated both xylem and phloem formation within nodules in all
cases (Figs. 6, 7) (Jeffs & Northcote, 1967). A quantitative comparison of nodule
Table 1. Modified media used for the induction of differentiation
Sucrose
%
2:40,
mg/1.
NAA,
mg/1.
IAA
mg/1.
Kinetin,
mg/1.
Nodules/g
(strain I,
21 d)
(1)
(2)
2
3
2
—
—
1
—
—
—
0-2
6
45
C (3)
-. (4)
I (5)
3
3
3
2
—
—
—
—
—
—
o-i
o-i
0-2
—
°'2
30
50
18
Medium*
Maintenance medium
Induction medium
Other media
All media contain salts and vitamins of Gamborg et al. (1968)
formation (Table 1) showed that a medium supplemented with o-i mg/1. of indolylacetic acid (IAA) induced the greatest concentration, but since this medium supported
the growth of only 3 of 5 cultures which had been isolated from the various bean
tissues, a medium supplemented with 1 mg/1. of NAA and 0-2 mg/1. of kinetin which
allowed growth and induced vascular differentiation in all 5 cultures was chosen as the
routine induction medium, since it would be more useful for future comparative
studies. This induction medium supported the growth of a callus friable in texture,
but containing small hard portions which were generally found to stain with phloroglucinol (Figs. 3, 4).
Comparison of hypocotyl callus strains I and II
Portions of strain I callus were transferred to induction medium at the fourth and
seventh subcultures. PAL and glucan synthetase activities were measured after both
subcultures and the callus was examined for differentiation after 3 weeks of growth.
No organ initiation was observed after either transfer and nodule formation was
found when the transfer to induction medium was made at the fourth but not at the
seventh subculture.
Portions of strain II callus were transferred to induction medium at the fourth
subculture and the time course of the appearance of PAL and callose synthetase
activities and nodule formation measured over a period of 6 weeks. Root initiation was
observed in about 50% of the samples after 15-18 days (Fig. 5). After 6 weeks some
of the roots were 10—20 mm long but no other organs were initiated. Portions of
strain II callus were also transferred to induction medium for the first time at the
fifth, sixth and seventh subcultures. PAL and glucan synthetase activities and nodule
i6
L. E. Haddon and D. H. Northcote
concentration were estimated 21 days after transfer to maintenance or induction
medium. No root initiation was observed after these transfers.
Time course of nodule formation in strain II
Three i-g samples of callus were taken, from at least 2 containers, and stained with
phloroglucinol at 3-day intervals after transfer either to induction or maintenance
medium. Fig. 1 shows that cultures immediately before the fourth subculture contained 5 nodules/g and that in callus subsequently transferred to maintenance medium
10
15
20
35
Days after transfer
Fig. 1. The time course of differentiation in strain II callus after 3 transfers to maintenance medium and then either to maintenance medium (—#—) or to induction
medium (—A—). A, nodule concentration; B, PAL activity; c, callose synthetase
activity.
the number of nodules/g remained fairly constant, although it rose to 10 after 12 days
(actual values 10, 12, 9) (Fig. 1). However, when callus was transferred to induction
medium the number of nodules began to rise after 9 days and by 12 days had reached a
level of 40/g which was maintained until at least 6 weeks after transfer.
Measurement of bean callus differentiation
Time course of the appearance of PAL
17
activity
Duplicate assays were performed on each acetone powder and gave values for PAL
activity within 10 % of each other. The PAL activities of 2 acetone powders prepared
from duplicate callus samples agreed within 10% but insufficient callus was available
to prepare duplicate acetone powders throughout the experiment.
PAL activity in strain II callus after the fourth transfer is shown in Fig. 1. On
maintainance medium PAL activity remained constant up to 6 weeks after transfer
except for a slight peak between 9 and 12 days. On induction medium PAL activity
rose rapidly between 6 and 9 days after transfer and reached a value 5 times that of the
control after 12 days. After this, PAL activity declined to about half the maximum
value after 21 days and remained at this level for a further 3 weeks.
A similar time course had been obtained with strain I callus after the fourth
transfer. A constant level of PAL activity was reached by 21 days after the transfer
and this level was 80 % of the constant value obtained with strain II. PAL activity on
transfer to maintainance medium again remained constantly low.
Time course of appearance callose synthetase activity
Three incubations were carried out with each enzyme preparation: 2 contained
active enzyme and a control contained boiled enzyme. Control samples incorporated
20-30 cpm total radioactivity which was mainly located at the origin of the chromatogram of the products of the Smith degradation and was not therefore a product of
polysaccharide oxidation and hydrolysis. The chromatograms from the active enzyme
preparations showed a small amount of radioactivity located at the origin, a peak of
radioactivity corresponding to glucose and a smaller peak corresponding to glycerol.
No erythritol was detected either by counting radioactivity or by staining the paper in
alkaline silver nitrate (Trevelyan, Procter & Harrison, 1950). Results are expressed as
cpm of radioactive glucose per gramme of fresh callus detected after Smith degradation.
The values are corrected for the incorporation by the boiled preparation and represent
the mean of the 2 replicates which agreed within 20%. The incorporation of radioactivity by samples from duplicate enzyme preparations also agreed within 20%.
Where significant differences are claimed there was no overlap between values for
callus on induction medium and callus on maintenance medium. The protein content
of the enzyme preparation varied from 19 to 23 mg/mJ and there was no consistent
difference between samples from callus grown on the 2 different media.
The incorporation of radioactive glucose into /?i ->• 3 links by strain II callus was
about 100 cpm/g when it was growing on maintenance medium, although there was
considerable fluctuation (Fig. 1). On induction medium incorporation began to rise
after 9 days, rising steadily to a peak of 920 cpm/g at 15 days and thereafter declining
to a level of 720 cpm/g at 21 days and 600 cpm/g at 35 days.
A very similar time course was obtained with strain I callus after the fourth transfer.
Although incorporation of radioactive glucose from UDPG by strain I callus on
maintenance medium was about double that of strain II, and was rather less variable,
2
CEL
17
i8
L. E. Haddon and D. H. Northcote
No. of subcultures
Fig. 2. Differentiation of bean callus (AI, BI, CI, strain i; AZ, B2, C2, strain II) 21 days
after transfer from maintenance medium to maintenance medium ( • ) or induction
medium ( 0 ) . Ai, A2, nodule concentration; Bi, B2, PAL activity; c i , C2, callose
synthetase activity.
Measurement of bean callus differentiation
19
callus transferred to induction medium showed a maximum value of incorporation of
960 cpm/g at 14 days falling to 700 cpm/g after 21 days.
Effect of continued subculture on maintenance medium
Strain I callus was transferred either to maintenance or induction medium after 6
subcultures on maintenance medium and PAL and glucan synthetase activities were
measured at 3-day intervals.
The level of PAL activity on both media followed a similar time course to strains I
and II on maintenance medium after the fourth transfer, the value remaining constant
up to 21 days in culture but with a slight peak after 9 days. Callose synthetase activity
was also the same on both media and its value was similar to that measured in callus
grown on maintenance medium 3 subcultures earlier. Even after 21 days there was no
significant difference between callus on the 2 media, both activities having risen to
30 % above the initial value.
Examination of the callus for nodule formation showed that this strain was no
longer capable of forming more vascular nodules when it was transferred from
maintenance to induction medium (Fig. 2).
Fig. 2 shows that continued subculture of strain II callus to fresh maintenance
medium resulted in a gradual loss of its ability to form vascular nodules when transferred to induction medium. Most nodules were formed when callus was transferred
at the fourth subculture. Fewer nodules were formed on induction medium after the
fifth and sixth subcultures and at the seventh subculture transfer to induction
medium did not increase the concentration of nodules.
PAL activity 21 days after transfer to induction medium also fell gradually as the
callus was maintained for a longer time in culture. No significant difference in activity
between callus on maintenance and induction medium was detected after the sixth and
seventh transfers. Callose synthetase activity 21 days after transfer fell in a similar
manner and was significantly higher in callus transferred to induction medium only
after the fourth and fifth transfers.
DISCUSSION
The Phaseolus vulgaris callus previously isolated in this laboratory (Jeffs & Northcote, 1966) was maintained on an undefined medium (H2) supplemented with 20%
coconut milk and 6 mg/1. 2:^Y). We have isolated strains of bean callus which grew
equally well on a fully defined medium supplemented with only 2 mg/1. 2\^D. Several
active components have been isolated from coconut milk (Shantz & Steward, 1964)
including hexitols, amino acids as a reduced nitrogen source and cytokinins. As with
soybean (Gamborg et al. 1968), these components could be replaced by myoinositol
and ammonium sulphate. Bean callus cells did not require added kinetin, presumably
because they were able to synthesize sufficient cytokinin for growth and division.
While the new strains isolated had the advantage that they were grown on a fully
defined medium, enabling the factors stimulating differentiation to be studied more
directly, they changed their properties more rapidly than the old strain, which
20
L. E. Haddon and D. H. Northcote
retained for several years the ability to form nodules, although this has now been lost.
Strain I, however, lost this ability within 9 months of isolation and strain II began to
change its properties after 5 transfers, when root initiation was no longer observed.
This indicates the variability of callus cultures isolated from similar parent tissue, in
this case bean hypocotyl. The variability may be due to differences in the age of the
hypocotyl explant (Lavee & Galston, 1968), to different cell types being stimulated
to divide and form callus, or to the different environments in which the cultures were
maintained.
Skoog & Miller (1957) found that differentiation in tobacco callus was controlled
by the auxin to cytokinin ratio in the medium, and Wright & Northcote (1973) showed
that root initiation in sycamore was also controlled by this ratio, although different
values of the ratio were found to promote root formation in the 2 cases. Anstis &
Northcote (1973) showed that cytokinin was secreted into the medium within one day
of subculture by a potato callus which did not require added kinetin and estimated the
cytokinin content of this callus to be o-oi /tg/g of live tissue. A similar value for a
soybean callus can be estimated from the data of Miura & Miller (1969). Assuming
cytokinin contents of 0-02 /tg/g for callus and 2 mg/1. for coconut milk (Loeffler & van
Overbeek, 1964) the ratio of auxin to cytokinin in media which induced good differentiation in strain I (Media 2-4, Table 1) was between 5 and 10, while the ratio was
higher in maintenance medium and in H2 medium and lower in the medium (Medium
5, Table 1) supplemented with IAA and kinetin, which induced fewer nodules than
media 2-4. The values inducing differentiation were very similar to those inducing
root formation in sycamore (Wright & Northcote, 1973).
We have not isolated a callus which contained no differentiated cells. The average
diameter of a nodule measured after phloroglucinol staining was 0-5 mm, so that
differentiated tissue comprised less than o-i % of the callus volume in cultures growing
on maintenance medium. Transfer of callus to induction medium resulted in a 10-fold
increase in the amount of vascular tissue. A rise in the number of nodules/g was first
detected between 6 and 9 days after transfer to induction medium at the same time as
growth of the callus began after an initial 6-day lag. The greatest increase occurred
between 9 and 12 days after transfer and although callus growth continued after 12
days there was no further increase in the number of nodules/g.
PAL activity also began to rise after 6 days in callus transferred to induction
medium but reached its maximum value before maximum concentration of nodules
(nodules/g) was detected. PAL activity which is required for xylogenesis therefore had
its maximum value, as would be expected, during the period when the rate of nodule
formation was greatest. Heinzmann & Seitz (1974) found that PAL activity rose
immediately before anthocyanin formation could be detected in carrot callus. They
also detected a subsequent fall in activity which was correlated with a decrease in
anthocyanin content, indicating a metabolic balance between synthesis and degradation. During bean xylogenesis PAL activity fell immediately after the maximum concentration of nodules was detected, but this was not accompanied by a decrease in the
number of nodules/g. This is because plant cells do not normally reverse xylogenesis
nor do they contain enzymes which degrade lignin. The PAL activity did not return
Measurement of bean callus differentiation
to its original level even after 6 weeks, since at this time the callus was still growing and
forming nodules to maintain the level of 40/g.
A slight rise in the number of nodules/g was also detected in callus 12 days after
transfer to maintenance medium and PAL activity rose between the sixth and ninth
day in these cultures. This indicates that some xylogenesis occurred in these cultures,
which may have been due to the synthesis of growth factors by the callus resulting in
an auxin to kinin ratio favouring some differentiation at the time when growth started,
although this condition probably existed for only a short time and the ratio changed
rapidly as growth proceeded.
Callose synthetase activity rose during vascular differentiation as was expected,
since formation of phloem was detected. The rise of activity began at the same time as
the rise of PAL activity but the maximum value was reached 3 days later and both
rise and subsequent decline were more gradual. It is not known whether the different
time courses are due to the formation of xylem and phloem at different rates, since the
detection of nodules was dependent on the staining of xylem. Microscopic examination
showed that after 21 days 2 types of nodules were present, only one of which contained
phloem cells (Jeffs & Northcote, 1967). If callose synthetase activity is an indicator
of phloem formation, the gradual rise and fall of activity could be due to the formation
of phloem in nodules which already contained xylem, the proportion of nodules which
contained phloem gradually rising as growth proceeded. This order of development is
the reverse of that found during vascularization in the intact plant (Jacobs & Morrow,
1957), but it is consistent with the observation that xylogenesis can occur in callus
with or without phloem formation, whereas phloem formation is less frequent and
rarely occurs in the absence of xylem formation (Halperin, 1969), although some cases
have been reported (Wetmore & Rier, 1963).
The formation of callose by callus grown on maintenance medium may have been
due to the insertion of a small amount of a fii -> 3 glucan into the cell wall. No
reproducible rise in callose synthetase activity was observed in callus grown on
maintenance medium, suggesting that the transient conditions prevailing at the
initiation of growth favour xylem but not phloem formation.
PAT, and callose synthetase activities of strain I callus were similar to those of
strain II callus of the same age growing on the same medium, and the activities
decreased as the callus formed fewer nodules when transferred to induction medium.
These 2 enzymes are therefore valid biochemical markers for vascular differentiation.
Maximum differentiation was observed after the earliest transfer to induction
medium, suggesting that the ability to form vascular nodules decreased rapidly after
callus was removed from the parent tissue to a simple defined medium. This may have
been due to cells unable to form vascular tissue growing most rapidly under the conditions used, and diluting out slower growing cells capable of differentiation, or to
changes occurring in the cells when cultured in vitro so that they could no longer form
nodules.
No difference in enzyme activity between callus on maintenance and induction
medium was detected after the sixth subculture, although a slightly higher concentration of nodules was observed in callus on induction medium. The activities were,
21
22
L. E. Haddon and D. H. Northcote
however, estimated 21 days after the transfer, by which time both PAL and callose synthetase activities should have reached their constant value (Fig. 1). It is possible that
differences in maximum activity (12 days after transfer for PAL; 15 days for callose
synthetase, Fig. 1) might still have been detected.
L. E. H. thanks the Science Research Council for a Studentship during the tenure of which
this work was carried out.
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Measurement of bean callus differentiation
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VILLEMEZ,
(Received i$July 1974)
24
L. E. Haddon and D. H. Northcote
Fig. 3. Portions of friable callus (strain II) 21 days after transfer to maintenance
medium (left-hand piece) or induction medium (right-hand piece), x 2 8 .
Fig. 4. Portions of hard callus (strain II) 21 days after transfer to maintenance medium
(left-hand piece) or induction medium (right-hand piece), x 2 8 .
Fig. 5. Root initiation in strain II callus 21 days after transfer to induction medium
after 3 transfers to maintenance medium, x 4.
Measurement of bean callus differentiation
L. E. Haddon and D. H. Northcote
*•»
k *
t •r
Fig. 6. Section through a nodule formed after 21 days on induction medium. Xylem
elements can be seen (arrow). Stained with safranin and fast green, x 230.
Fig. 7. As Fig. 6, but stained with dilute aniline blue and viewed with ultraviolet light.
Both xylem and phloem (arrows) can be seen, x 280.