Institut für Pflanzenernährung, Abteilung Gewebekultur, der Justus-Liebig-Universität

Gießen, Federal Republic of Germany

Investigations on the Influence of Pre-culture in IAA- and

Kinetin-containing Culture Media on Subsequent Growth of
Cultured Carrot Explants
L. BENDER and K.-H. NEUMANN

Received 31 October 1977 . Accepted 16 February 1978

Summary
Positive interactions among the effects of IAA, kinetin and m-inositol on fresh weight and
cell division activity of carrot tissue cultures are reported. For the manifestation of the effects
of these positive interactions on growth, neither the presence of IAA nor kinetin in the
nutrient solution throughout a culture period of several weeks was required. Whereas a
supplement of IAA for up to 6 days is sufficient, the corresponding period for kinetin
amounts to about 12 days.

Key words: Tissue culture, Daucus carota, IAA, kinetin.

Introduction
Phytohormones are presently regarded as the most powerful tools for regulating
the development of the whole higher plant as well as of cell and tissue cultures
derived thereof. Furthermore, it is thought that some environmental factors, in
addition to genetic factors, exert their influence on the ontogenetic .development of
higher plants by altering the internal hormonal system. In earlier publications a
strong genetic influence on the reaction pattern of tissue cultures of carrot and of
some Brassicaceae to exogenously applied phytohormones such as IAA or gibberellic
acid was reported (NEUMANN and GARCIA, 1974; BUIATTI et al., 1974; ELMSHEUSER et
al., 1978 a and b). Furthermore, although distinct differences in the contents of
soluble IAA, GA 3 and abscisic acid between genetic'ally different sources of explants
for tissue culture experiments have been demonstrated, no correlation between the
content of the free forms of these phytohormones in the original explants and the
developmental reaction of these explants to exogenously applied phytohormones of
the same kind has been found (ELMSHEUSER et al., 1978 C; ELMSHEUSER, 1977). One of
the possible conc1usions drawn from these experiments was the assumption that
exogenous phytohormones merely stimulate an autonomous phytohormone synthesis
in the cells of explants, as a consequence of which a hormonal system typical of the

Z. PJlanzenphysiol. Bd. 88. S. 201-208. 1978.

202 L. BENDER and K.-H. NEUMANN

particular species and variety in tissue culture c'onditions will be established. In

another paper it was in fact shown that carrot tissue cultures at least are able to
synthesize their own IAA (BENDER and NEUMANN, 1978). To provide more
information concerning the role of exogenous phytohormones in growth initiation
and the development of tissue and ceIl cultures, the present paper reports on
experiments in which carrot explants were pre-cultured in IAA- or
kinetin-containing nutrient media for a certain length of time and then transferred to
nutrient media free of these growth substances. The growth patterns of these explants
treated in this fashion were foIlowed and compared to treatments in which the
explants remained In the original nutrient solution throughout the entire
experimental period.

Material and Methods

a) T issue culture
Carrot root tissue cultures (origin: sec. phloem) were obtained according to a method
described previously (NEUMANN, 1965, 1968). Cultures were continuously illuminated with
about 5000 lux (Osram-L 15 W /25 universal white). All tissue culture experiments were
carried out aseptically. The medium used (NEUMANN, 1965) was either hormone-free or
supplemented with IAA (2 ppm), m-inositol (50 ppm) or kinetin (0.1 ppm) as well as with
various combinations of these hormones as is indicated in the tables. In order to check the
influence of light in the tissue culture system on the IAA conte nt of the medium, culture tubes
with nutrient solution + hormones but without explants were also maintained according to
this system for three days, after which the IAA content was determined. Control tubes were
shielded from light by covering them with aluminum foi!. In some of the experiments,
cultures were started in IAA- or kinetin-containing nutrient solutions for a preculture period
of various duration followed by transfer of the explants to freshly prepared nutrient solutions
without IAA or kinetin, respectively, and then grown in this nutrient solution until the end of
the experimental period (28th day). Control cultures were maintained in the original growth
medium without transfer until the 28th day. In all experiments control and test explants were
taken from the same carrot root.

b) Cell counting
As described by NEUMANN and STEWARD (1968) in detail, explants were macerated in a
mixture of 0.1 n HCI and 10 % chromic acid (1 : 1, v/v) for 24 hours, using 10 ,ul per mg of
tissue. Aliquots of the resulting cells suspension were transferred to a haemocytometer plate
and counted microscopically. 6 parallel countings were carried out on each sampie.

c) Extraction and pre-puri/ication 0/ IAA

IAA-containing fractions from nutrient solutions, as well as from tissue, were obtained
according to the modified method of POWELL (1964). The nutrient solution (60 ml per sampie)
in which explants had been grown was freed from cells and tissue particles by passage
through a gl ass filter. The volume was then reduced to about 25 ml in a vacuum rotary
evaporator at 40°C and the sampies were adjusted to pH 3 with HCl and extracted 6 times
with dichloromethane (CH2 CI 2 , analytical grade) which had previously been further purified
by an additional distillation step in a Liebig distillation apparatus. This step appeared to be
of some importance, because at least some of the batches of dichloromethane (analytical

z. P/lanzenphysiol. Bd. 88. S. 201-208. 1978.

 Influence of pre-culture 203

grade) were found to contain some impurlues which interfered with the subsequent
GLC-analysis and which could be removed by the additional distillation.
The dichloromethane fraction containing the acid indoles including IAA (POWELL, 1964)
was reduced to about 1 ml in a vacuum rotary evaporator at 40 oe. To avoid loss of IAA due
to oxidation or sublimation during all evaporation steps under reduced pressure (MANN and
]AWORSKI, 1972), care was taken to prevent the sampies from evaporating to dryness. The
remaining volume of about 1 ml was quantitatively transferred to the reaction vials and
evaporated to dryness at 50°C at normal pressure under a continuous stream of nitrogen,
which was followed by the silylation procedure.
In order to check the IAA content of explants freshly excised from the sec. phloem of the
carrot root, about 50 gr of tissue were ground in an ice-cold mortar with insoluble polyvinyl
pyrrolidone (PVP), which was included to remove phenols. The homogenate was then
extracted under occasional stirring for one hour in the dark with 250 ml of ethanol (80 Ofo) to
which 0.02 Ofo (w/v) sodiumdiethyldithiocarbamate had been added (MANN and ]AWORSKI,
1970).
After one hour the extracted suspension was centrifuged at OOC and 10,000 rpm for 30
min, the supernatant was recovered and the residue was resuspended in ethanol (80 °/0) and
centrifuged again twice. The resulting supernatants were combined and the volume was
reduced to about 25 ml in a vacuum rotary evaporator, acidified to pH 3 with HCI and
extracted 6 times with an equal volume of dichloromethane. The dichloromethane fraction
containing the acid indoles was further treated as described above for the analysis of the IAA
content in the nutrient solution.
d) Silylation procedure and GLC-analysis
The dry dichloromethane fraction was dissolved in 0.2 ml of acetone (analytical grade)
which contained 5-bromoindoxylacetate (Fluka) as an internal standard (500 ,ug/ml). 0.2 ml
of bis-(trimethylsilyl-)trifluoroacetamide (BSTFA) were added and the reaction vial was
sealed with a teflon-coated rubber disc and a screw cap. Derivatization was carried out for
30 min at 50 oe.
2 ,ul of the solution were injected into a Packard-Becker Mod 417 gas chromatograph
equipped with a flame ionisation detector (FID) and a 185 cm (3 mm i.d.) glass column
packed with 3 Ofo SE 30 on Chromosorb W (HP) 80/100 mesh (pierce chemical company).
Temperature of the injection port and FID: 260 oe.
Temperature of the column: initially for 3 min at 140°C, followed then by a linearly
programmed temperature increase (10 °C/min) to 250 oe.
Carrier gas: N 2 , purity 99.995 Ofo, flow rate 30 ml/min.
Synthetic air: (80 Ofo N 2 , 20 °/0 O 2 ), flow rate 170 ml/min.
Hydrogen: purity 99.995 Ofo, flow rate 170 mllmin.
Chart speed: 10 mm/min.

Results and Discussion

In table 1 the influence of the various growth substances (IAA, kinetin, inositol)
separately and in combination on fresh weight are summarized. As can be seen, the
separate addition of IAA, kinetin or inositol to carrot tissue cultures increases fresh
weight yield only very slightly in comparison to the hormone-free control treatment.
However, the addition of two hormones together resulted in fresh weight increments
which indicate positive interactions between these 3 growth substances. A
particularly strong positive inter action apparently takes place on the joint

z. Pjlanzenphysiol. Bd. 88. S. 201-208. 1978.

 204 L. BENDER and K.-H. NEUMANN

application of kinetin and inositol. A further increase in fresh weight was achieved
by adding all three substances together. Apparently in addition to the positive
interactions between IAA and kinetin, IAA and inositol, and kinetin and inositol, a
multiple interaction between IAA, inositol and kinetin appears to be effective in
bringing about an inc'rease in fresh weight as great as that usually observed upon the
addition of coconut milk, by some still regarded as the most powerful growth
stimulant in this tissue culture system.

Table 1: The influence of various phytohormones (IAA, kinetin, inositol) and coconut milk
(CM) on the fresh weight of carrot tissues cultured for 3 weeks (sec. phloem of the carrot
root).

+IAA
0 + IAA + Inositol + Inositol CM

-Kin 4 8 6 22"")
100
+ Kin 8 20 43 91
.~) root formation

Differences between the treatment not including a hormonal supplement and the IAA,
kinetin and inositol treatments respectively, as well as the difference due to interactions,
are highly significant at the 0.01 010 level, with the exception of the interaction between IAA
and inositol (0.05010).
Conc. of kinetin (Kin) = 0.1 ppm
Conc. of IAA = 2 ppm
Conc. of Inositol (I) = 50 ppm
CM = 10010

The experiments documented in table 1 were carried out in growth chambers with
continuous illumination. It is known from other systems that IAA is very rapidly
degraded in light, which in fact can be also concluded from the results presented in
table 2. Although free rAA could not be detected in the original explants (BENDER,
1976), it is interesting to note that the IAA content in sampies supplemented with
explants was somewhat higher after illumination than in vessels containing only the
nutrient solution.

Table 2: The influence of autoclaving, of illumination and of the explants on the rAA
content of the nutrient solution.

freshly prepared nutrient solution 200 ,ag/100 ml

immediately after autoclaving 182.6 ,ag/lOO ml
after 3 days in darkness, without explants 179.2 pg/100 ml
after 3 days of illumination, without explants 2.7 ,ag/100 ml
after 3 days of illumination, with explants*) 15.7 ,ag/lOO ml
*) average of 6 treatments (± kin) after 3 days of culture.

z. Pflanzenphysiol. Bd. 88. S. 201-208. 1978.

 Influence of pre-culture 205

Table 3: The influence of kinetin and a pre-culture period of various duration (6 h, 24 h,

144 h) in an IAA-containing medium on the fresh weight and the number of cells per
explant at two stages of a culture period of 28 days (controls were cultured in the original
nutrient solution throughout the experimental period). For conc. of growth substances s.
table 1.

Explants transferred from: IAA + I to I IAA + I + KintoI + Kin

Time of cellsl cellsl
pre-culture mg/Expl. Expl. X 103 mg/Expl. Expl. X 103

original explants 4
harvest at 15 th day
5 h pre-culture 19.9 133 73.4 1830
24 h pre-culture 11.2 84 71.1 1830
144 h pre-culture 12.1 105 138.3 3370
control 12.9 101 118.8 3570
harvest at 28 th day
5 h pre-culture 27.6 200 276.3 4860
24 h pre-culture 31.4 257 304.0 6480
144 h pre-culture 30.6 245 550.4 9140
control 25.0 183 573.0 9830

Table 3 summarizes the results of experiments in which freshly excised carrot

explants were pre-cultured for various periods of time in an IAA-containing medium
and then transferred for subsequent culture to a nutrient solution free of IAA.
Apparently a pre-culture period of 5 h in an IAA containing medium free of kinetin
is sufficient to induce an ~ncrease in fresh weight and cell division activity
comparable to those of control explants which remained in the original
IAA-supplemented nutrient solution throughout the experiment. Treatments with
kinetin led to comparable results with respect to fresh and dry weight after a pre-
culture period of up to 6 days.
As was shown by the experiments summarized in table 1, a strong interaction
between kinetin and inositol was shown, which resulted in fresh weight gains about
half as great as those induced by the combined addition of IAA, inositol and kinetin.
To test the influence of the kinecin/inositol interaction on the growth of the explants
after transfer to the IAA-free medium, the experiments documented in table 4 were
carried out. As can be recognized from the data, the transfer of explants from an
IAA-supplemented nutrient solution resulted in an increase of fresh weight. This
fresh weight increment was only very small in the treatments without inositol,
whereas a considerable increase was noted in the presence of this compound. At
present no explanation can be made respective of this observation. Since nutrient
uptake by the explants during their 6-day pre-culture period is very small (NEUMANN
et al., 1978), it is unlikely that the increase in growth due to the transfer of the

z. Pjlanzenphysiol. Bd. 88. S. 201-208. 1978.

206 L. BENDER and K.-H. NEUMANN

Table 4: The influence of kinetin, m-inositol and the transfer from an IAA-containing
nutrient medium to an IAA-free medium on the fresh weight of carrot tissue cultures during
a 28-day growth period. For conc. of growth substances s. table 1.

-Kinetin + Kinetin
- Inositol + Inositol - Inositol + Inositol
transferred *) 47 70 118 273
not transferred"*) 42 48 100 179
(control treatment)
"-) after 6 days in an IAA-containing medium (2 ppm), the explants were transferred to an
IAA-free medium supplemented with inositol and/or kinetin according to the treatment
as during the pre-culture period.
**) the explants remained in the original nutrient medium supplemented with IAA through-
out the experiment.

explants to a freshly prepared medium was a result of nutritional factors.

Nevertheless the data in table 4 also indicate that independent of the inositol
supplement pre-cuIture in an IAA-containing medium is sufficient to produce a fresh
weight increment comparable to that evidenced by explants remaining in a nutrient
solution supplemented with IAA throughout the entire experimental period of 28
days.
In table 5, the results of experimenJts concerning the influence of pre-culture in a
kinetin-containing nu trient medium for various length of time followed by
cultivation in a kinetin-free medium are summarized. Here again, cultivation of
explants in a kinetin-supplemented nutrient solution throughout the whole of the

Table 5: The influence of pre-culture in a kinetin-containing medium for various periods of

time on the growth of cultured carrot root explants in a kinetin-free medium. The data
represent the average values from 3 experiments using explants of 3 different carrot roots.
The nutrient solutions were supplemented with IAA and inositol in all treatments. The
entire experiment ran for 28 days. After this period the fresh weight and the number of
cells per explant were determined for all treatments. For conc. of growth substances s.
table 1.

duration of pre-culture mg fresh weightl expl. ceIlsl explants X 103

(days) at transfer at harvest at transfer at harvest

3 2 32 6.5 206.8
6 2 102 6.7 431.2
9 5 154 11.1 652.8
12 19 242 152.5 1468.7
Explants cultured in
kinetin-containing medium 168 1385.8
throughout the experiment

z. Pflanzenphysiol. Bd. 88. S. 201-208. 1978.

 Influence of pre-culture 207

culture period of 28 days seems not to be necessary. However, the pre-culture period
in a nutrient medium with added kinetin required to produce a fresh weight
increment comparable to that of the c'ontrol treatment, i.e. whereby explants
remained in the original nutrient medium (+ kinetin), amounts up to 12 days
(NEUMANN, 1968). This period of time coincides approximately with that required
for the transition of the explants from a heterotrophie to a mixotrophic nutritional
status (NEUMANN et al., 1978). Differences in fresh weight and in the number of cells
of explants transferred after 12 days of pre-culture to that of explants not
tansferred are not statistically significant.

Conclusions
Whereas the separate addition of kinetin, IAA or m-inositol to thenutrient
solution influenced the growth of carrot tissue cultures only negligible, pOSItive
interactions could be observed between IAA and inositol, IAA and kinetin and in a
especially pronounced fashion between kinetin and inositol. Furthermore, in addition
to these individual interactions, a positive multiple interaction between all three
substanc'es was observed, which led to a fresh weight increment per explant
comparable to that obtained by the addition of coconut milk, a supplement which
induces maximal growth in this tissue culture system. This multiple interaction
between IAA, kinetin and inositol was also observed in the development of the axis
of cherry embryos (ABOU ZEID et al., 1977). Therefore this pattern of growth
response apparently is not limited to the carrot tissue culture system.
To evolve the manifestation of this multiple interaction within a culture period of
28 days, the presence of neither IAA nor kinetin is required throughout the entire
experimental period. Whereas the time requirement for IAA to produce the same
amount of fresh weight as does the control kept in nutrient solution supplemented
with IAA amoun:ts to 6 days, 12 days are necessary in the case of kinetin. Since the
log-phase of cell division in this c'arrot tissue culture system ranges. up to 24 days
(and more), during the pre-culture period in IAA- and kinetin-containing media
respectively, either sufficient amounts of these growth substances are taken up and
stored to maintain growth in the media free of these substances or the substances are
required only during the induction period of cell division activity at earlier stages of
the experiment. A further alternative would be the assumption that, subsequent to the
induction of cell division in freshly cut explan:ts, sufficient de novo synthesis of
auxins and cytokinins takes place to maintain high cell division activity as usually
observed du ring the log-phase. In this respect it is of particular interest that the time
requirement for kinetin is longer than that determined for IAA. Although no reliable
explanation for the results reported can be given at present, it has at least been shown
that cultured carrot explants are able to synthesize their own IAA (BENDER, 1976;
BENDER and NEUMANN, 1978) and that the occurrence of a native cytokinin in carrot
tissue cultures has been reported (LINSTEDT and REINERT, 1975).

z. Pflanzenphysiol. Bd. 88. S. 201-208. 1978.

208 L. BENDER and K.-H. NEUMANN

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Dr. L. BENDER, Prof. Dr. K.-H. NEUMANN, Institut für Pflanzenernährung, Abteilung Ge-
webekultur der Justus-Liebig-Universität, Eichgärtenallee 3, D-6300 Gießen/Lahn.

Z. Pflanzenphysiol. Bd. 88. S. 201-208. 1978.