PlantCell Reports
Plant Cetl Reports (1996) 16:58-62
© Sprioger-Verlag1996
Relationship between endogenous auxin and cytokinin levels and morphogenic responses in Actinidia deliciosa tissue cultures M.L. Centeno, A. Rodriguez, I. Feito, and B. Fermindez * Lab. Fisiologia Vegetal, Dpto. KO.S., Fac. Biologia, Univ. Oviedo, C/Catedrfitico Rodrigo Urla, E-33071 Oviedo, Spain Received 30 June 1995/Revised version received 12 December 1995 - Communicated by E. W. Weiler
Summary. The in vitro culture of A ctinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxirdcytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot
formation when they were transferred to proliferation medium. Most important differences fotmd in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed, Abbreviations: BM, basal medium, BA, 6-benzyladenine, CIM, callus induction medium, CPM, callus proliferation medium, (dill)Z, dihydrozeatin, DW, dry weight, ELISA, enzymelinked immunoassay, FW, freshweight, ttPLC, high performanceliquid chromatography,IAA, indole-3-aeetic acid, iP, N6-(A2=isopentenyl)adenine, NAA, 1naphthaleneacetic acid, PBS, phosphate buffer, PGR, plant growth regulator, (diH)[9R]Z, 9-13-D-ribofuranosyl-(diH), [9R]iP, 9-13-Dribofuranosyl-iP, [9R]Z, 9-13-D-ribofuranosylzeatin, TBS, trishydroxymethyl-aminomethanebuffer, Z, zeatin. Inlroduetion
culture offers the possibility of studing differentiation on pieces of tissues detached from the whole plant (van der Krieken et al. 1990). Morphogenesis of in vitro cultured tissues, as well as plant development, are regulated by plant hormones, specially auxins and cytokinins (Skoog and Miller 1957). This hormonal control has been studied mainly by exogenous application of PGRs and only in few cases in relation to endogenous levels (Letham and Palni 1983).
In
vitro
Correspondence to: B. Fernfindez
The regeneration of shoots from callus is a differentiation process whose physiology has been a poorly developed area of research (Aitchison et al. 1977). This route for plant multiplication includes callus induction and formation from an initial explant, and shoot stimulation and development from the induced callus. Each of these two steps may not be discrete or synchronous and the levels of the exogenous hormones could be different (Krikorian 1995). In some species, callus induction was associated with high auxin/cytokinin ratios exogenously applied (Smulders et al. 1988; Branca et al. 1991) or with high auxin endogenous contents (Besse at al. 1992; Michalczuk et al. 1992). Nevertheless, the presence of cytokinin, is generally required for callus formation too (Aitchison et al. 1977). Moreover, cytokinins have been regularly incorporated into tissue culture for shoot regeneration (George and Sherrington 1984). Our attempt is to establish a relationship between exogenous applied regulators, endogenous hormonal contents, and in vitro morphogenic responses. In this way, the analysis of the same problem in morphogenic areas, explants or genotypes, as well as in non-morphogenic ones is a good way to study the control of morphogenesis in in vitro cultures (Tran Thanh Van 1981). Therefore, we compare the endogenous auxin and cytokinin levels in A ctinidia deliciosa tissues with different callogenic and caulogenic abilities. Material and methods Plant Material and culture conditions. Petioles (2-2.5 era) from four
kiwiplants (Actinidia deliciosa (A.Chev) Liang and Ferguson cv. Hayward) were used for callus induction. These plants (1, 2, 3, and 4), originated from cuttings, were grown 4-5 years in the greenhouse. The
59 petioles of the five most apical leaves were explanted, so some of them
to achieve its quantification. Radioactivity was measured after addition
were still developing. Explants were surface sterilized by washing 1 rain
of 2 ml scintillation liquid (Pico-Aqua, Packard) in a Packard 2500TR
in
70% (v/v) ethanol and 20 min (v/v) in 9% commercial NaC10
scintillation analyzer. The dried HPLC cytokinin fractions were
(Domestos), The petioles were rinsed 4 times in sterile distilled water.
resuspended in tris-hydroxymethyl aminornethane buffer (TBS) (25 raM,
The basal medium (BM) used was MS (Murashige and Skoog 1962)
pH 7.5) and quantified by ELISA, using three different polyclonal rabbit
supplemented with sucrose (2.5% w/v) and solidified with agar (0.7%
antibodies (Fermindez et al. 1995): i) anti[9R]Z antibodies to mesure Z
w/v). The pH was adjusted to 5.6 before autoclaving. Each culture vessel
and [9R]Z; ii) anti (diH)[9R]Z antibodies to mesure (diH)Z and
(200 ml) contained 30 ml of medium.
(diH)[9R]Z; iii) anti[9R]iP antibodies to mesure iP, [9R]iP, and also BA,
Petioles were preconditioned by culture in BM for 7 days. Afterwards
since it has high cross-reactivity with the [9R]iP antibodies.
the petiole ends were eliminated by excision and longitudinal incisions
Cytokinin conjugatesof alkalinephosphatase. The cytokinin-alkaline
were made on both extremes. These explants were placed on callus
phosphatase tracer conjugates were prepared following Ebefle et al.
induction medium (CIM), consisting of BM supplemented with 2.2 gM
(1986), except that 3 mg of protein were used and therefore the volumes
BA and 0.27 gM NAA. The petioles were always cultured in horizontal
of other reagents were proportionally scaled up. The dialysed conjugates
position. After 30 days of culture, induced callus was transferred to
were stored in 50% (v/v) glycerol at -80°C.
proliferation and maintenance medium (CPM = BM + 4.4 gM BA + 2.7
ELISA forcytokinin.~ ~luantification. Assays ofcytokinin were carried
gM NAA). Two pieces of callus per flask were cultured, and subculture
out in flat bottom, Immunolon M129A Micro ELISA plates (Nunc) using
took place every 5 weeks.
a modification of the ELISA method described by Eberle et al. (1986).
Some petiole tissues and calli were cultured in CIM with 1.32 nM
The wells of the microtiter plates were filled with 200 gl of diluted
(0.89 nCi/culture vessel) [3H]NAA (specific activity 22.6 Ci/mmol) or
antibody anti-[9R]Z, (diH)[9R]Z or [9R]iP, dissolved in 50 mM NaHCQ
in CPM with 0.66 nM (0.45 nCi/culture vessel) [3H]NAA respectively
buffer, pH 9.6 (Femfindez et al. 1995). Plates were incubated overnight
in order to quantify NAA levels in the tissues.
at 4°C. The solution was poured-off and the wells washed three times
Growth conditions were 25"2°C under a 16 h photoperiod provided
with TBS buffer. Then, the wells were incubated for 30 additional min
by cool-white fluorescent lamps (Sylvania F40W/154-RS) at a photon
at room temperature with 250 gl of 0.1% (w/v) bovine serum albumin
flux density of 33 gmol m "2 s"~.
in TBS buffer. After rinsing with TBS buffer° the coated wells were
To determine the rate of callus growth during the fifth subculture, two
filled with 100 gl of the antigen standard solution or sample solution,
pieces of callus (1±0.05 g fresh weight) were cultured per vessel. Every
diluted in TBS buffer. To determine the maximum binding of tracer,
6 days, they were removed, frozen in liquid N2, lyophilized and weighed
TB S was used instead of standard. Nonspecific binding was determined
to determine their dry mass.
by adding an excess (100 pmoI) of cytokinin standard. The plates were
Harvest and preparation of plant extracts. Samples from the initial
incubated for 30 min at 4°C. The corresponding diluted enzyme tracer
plant material and explants at the end of the different culture phases
(100 gl) was then added to each well and the plates were incubated for
were harvested. The petioles harvested after their culture on BM were
another 2.5 h at 4°C. Tracer dilutions were done in TBS buffer
separated in three portions of equal length (apical, middle and basal) and
supplemented with 0.1% (w/v) gelatin. The plates were decanted and
those cultured in CIM were divided in two parts (apical and basal), in
washed three times with TBS buffer. Phosphatase activity was measured
order to know the distribution of PGRs in the explants. Sampled tissues
by incubating each well for 1 h at 37°C with 200 gl of a freshly
were deep-frozen in liquid nitrogen, powdered and lyophilized.
prepared solution of 4.24 mM p-nitrophenylphosphate in 0.9 M
Extraction, purification and separation of plant growth regulators. Extraction and purification were performed according to Femfindez et al.
diethanolamine buffer pH 9.8. The absorbance was measured at 405 nm in a microplate reader (Lambda Reader, Perkin-Elmer).
(1995). Fractions containing NAA were dissolved in 50 gl methanol and applied on 0.25 mm silica-gel thin layer chromatography plates with a
Results
fluorescence indicator (Merck 60F254).
Levels o f PGRs and their distribution in the explanted petioles.
Plates were run with
cloroform:methanol:acetic acid, 75:20:4 (v/v/v) (Caboche et al. 1984) and 0.1 gmol of pure NAA and [H3]NAA were cochromatographed.
Cytokinins.
Several
cytokinins
were
identified
by
After separation, the plates were visualized under ultravioletqight at 254
comparison of the retention times of pure compounds with
nm.
liquid
the immunoreactive fractions. Immunoreactivity was found
chromatography (HPLC) and the automatic equipement used have been
in f r a c t i o n s c o r r e s p o n d i n g to t h e r e t e n t i o n t i m e s o f Z ,
previously described (Fermindez et al. 1995).
( d i l l ) Z , [ 9 R ] Z , ( d i H ) [ 9 R ] Z , iP, [ 9 R ] i P , a n d B A .
Separation
of
cytokinins
by
high
performance
Quantification of plant growth regulators. IAA samples were
T a b l e 1 s h o w s t h e a m o u n t o f e n d o g e n o u s c y t o k i n i n s in
methylated according to Neill and Horgan (1987) prior to their analysis
t h e e x p l a n t e d p e t i o l e s a n d a f t e r t h e i r c u l t u r e in B M (initial
by enzyme linked immunoassay (ELISA) (Phytoscience test kits). After
conditions
determination of NAA position on plates by cochromatography with
h i g h e s t l e v e l s o f Z a n d its r i b o s i d e , b u t at t h e e n d o f t h e
authentic standards, the silica was scraped off into scintillation vessels
preconditioning phase, these cytokinins declined by three
for
callus
induction).
Petioles
showed
the
60 Table 1. Cytokininconcentrationin petioles of kiwiplants,and in petiole portions (apical, middle and basal) and whole petioles cultured in vitro. The values are mean of 3 biological and 3 analyticalreplications(n= 9), :sstandarerrors, p= petiolar portion. Culture
Plant
conditions
tissue
Concentrationof cytokinins,nmol g-I Z
in vivo
whole p e t i o l e
BM in vitro p. apical
(diH)Z
[9R]Z
(diH)[9R]Z
3.81±0.22 0.01:50.00 11.20:51.34 0.024-0.01
iP
[9R]iP
BA
0.524-0.05 0.054-0.02
Total 15.61:51.35
0.97:50.61
0.34:50.05 1.094-0.13
0.23:t:0.03 1.00:50.03 1.28:50.11
4.9l:50.64
cultures
p. middle
1.11:50.55
0,024-0.01
0.06:50.01 0.23:50.01 0.06:50.00
2.40:50.56
(7 days)
p. basal
1.214-0.46
0.02:50.01 0.69:50.21 0.03:50.00 0.22±0.01 0.03:50.02
0.924-0.11
2.20:50.51
petiole (mean value) 1.09:50.24
0.12:50.05 0.914-0.09 0.11:50.04 0.49:50.14 0.55:50.19
3.274-0.35
CIM in vitro p. apical
0.17:50.01
0.36:50.06 0.13:50.01 0.06:50.02 0.99:50.04 0.66:50.02 0.81:50.04 3.18:50.09
cultures
0.16:50.02
0.06:50.02 0.18:50.01 0.04:50.01 0.69:50.04 0.59:50.03 0.36:50.03 2.084-0.07
(30 days)
p. basal
petiole (mean value) 0.17:50.01
0.21+0.05 0.164-0.01
0.05:50.01 0.84:50.05 0.63:50.02 0.59:50.06 2.65:50.09
BM: basal medium without PGRs, preconditioningmedium. CIM: BM + 2.2 gM BA + 0.27 gM NAA, callus induction medium. and twelve times respectively. On the contrary, (dill)Z,
and
(diH)[9R]Z, and [9R]iP increased thirteen, four and ten
cytokinins, its levels being higher in the basal end. No
this
auxin
showed
a
distribution opposed
to
fold respectively. The analysis in the different portions in
differences were found for NAA content between apical
which the petioles were sectioned, showed an unequal
and basal portions (Tab. 2).
distribution of (dill)Z, (diH)[9R]Z, iP, an [9R]iP; the highest content being observed in the apical portion. Auxins.
After culture on BM, the IAA content of
petioles increased about three times (Tab. 2). The middle portion of explants had lower IAA content than both apical and basal sections.
Table 2, Auxin concentrationin petioles of kiwiplants and in petiolar portions (apical, middle and basal) and whole petioles cultured in vitro. The values are mean of 3 biologicaland 3 analyticalreplications(n=9), ±standar errors, n= netiolar portion. Culture Plant conditions tissue
Concentrationof auxins, nmol g-i IAA
Callus i n d u c t i o n
NAA
Total
in vivo
whole petiole 21.75:53.46
21.754-3.46
BM in
p. apical p. middle p. b a s a l
84.45±20.41 13.06:51.05 91.2715.70
84.45:520.41 13.06:51.05 91.274-15.70
petiole (mean 62.92:513.69 value)
62.924-13.69
When petiole explants were transferred from BM to CIM, callus formation took place between 10 and 20 days after inoculation. This occurred mainly at the basal end of the
vitro
explants (88% callogenic response compared to the 4%
cultures (7 days)
produced at the apical end of the explants). However, shoots and/or leaves regenerated at the apical side (1-5 per explant) at a percentage of 32%. P G R s in p e t i o l e s a f t e r callus induction. There was a
change in the content of PGRs when petiole explants were
C1M m vitro
cultures (30 days)
p. a p i c a l p. b a s a l
13.024-4.00 2.334-0.03 15.354-4.00 29.31:t:6.38 2.424-0.02 31.734-6.38
petiole(mean 21.17:55.27 2.36:50.01 23.53:55.27 value)
cultured in CIM (Tab. 1 and 2). C y t o k i n i n s . The amounts of all zeatin-type cytokinins
decreased significantly with the exception of (dill)Z,
BM: basal medium without PGRs, preconditioningmedium. CIM: BM + 2.2 gM BA + 0.27 gM NAA, callus induction medium.
which increased (Tab. 1), iP was enhanced approximately
Calli p r o l i f e r a t i o n a n d their o r g a n o g e n i c r e s p o n s e
twice, and no changes were observed for [9R]iP. Natural
After 30 days of culture in CIM, the calluses induced at
cytokinins were homogeneously distributed between the
the basal end of the petioles reached a diameter between
apical and basal portions with the exception of iP and
0.5-1 cm and at this stage they were transferred to CPM
(dill)Z, whose level was about six fold higher at the
for callus proliferation. In this medium, calluses from
apical end. Interestingly, the amount of BA was two times
kiwiplant no. 3 showed a high organogenic capacity (75-
higher at the apical than in the basal end.
100% of calli with response along 10 subcultures) giving
A u x i n s . IAA level decreased to the initial level (Tab. 2)
rise to leaves and/or shoots (1-10 per callus). At the same
61 Table 3. Cytokinin concentration in calluses of line A and line B of kiwiplant at the end of the fifth subculture on CPM. The values are mean of 3
biological and 3 analytical replications (n=9), ± standar errors. Callus line cultured in CPM (35 days)
Concentration of cytokinins, nmol g-XDW Z
(diH)Z
[9R]Z
(diH)[9R]Z
iP
[gR]iP
BA
Total
(A) canlogenic
0.19±0.01
0.10±0.02
0.54±0.03
0.16±0.01
0.68±0.06
1.19±0.03
1.09±0.09
3.95±0.12
(B) non-caulogenic
0.16±0.02
0.06±0.01
0.66±0.04
0.19±0.02
0.86±0.04
1.18±0.07
0.47±0.07
3.57±0.13
CPM: BM + 4.4 gM BA + 2.7 M gNAA. Callus proliferation and maintenance medium. time, oniy 4-23% o f calluses from plants 1, 2 and 4
content in callus lines A and B were measured at the end
showed this caulogenic response and it disappeared after
o f the fifth subculture (Tab. 3 and Tab. 4).
4 subcultures. Therefore, we can establish two callus
Cytokinins. Slight differences were observed in natural
lines: line A, callus with high caulogenic capacity and line
cytokinin levels between both callus lines. The difference
B, callus with low or null capacity.
in BA content was the most noticeable, being double in line A (Tab. 3).
4-
Auxins. No significant differences were observed
3,5-
between the auxin content in both lines (Tab. 4). Discussion
The original physiological status o f explants and their
x~ 2,5-
hormonal content are very important factors in directing o a.
in vitro responses (Almnirato 1986). The large reduction o f total cytokinin content, and specially of Z and [9R]Z,
1,5
taking place in kiwi petioles cultured in vinv, could be associated with glycosylation or oxidative metabolism ( M c G a w 1995). The I A A increment, detected only at the 0
6
12
18 24 Time (days)
30
36
42
Fig. 1. The growth of caulogenic (ll) and non-caulogenic ([5) callus lines (line A and B) during the fifth subculture on proliferation medium. Each point was derived from an average of nine flasks (two pieces of callus in each flask). Vertical bars indicate standard errors (n=9).
apical and basal portions o f petioles, could be a consequence o f the subtances liberation taking place in the tissues when they are wounded (Aitchison et al. 1977). Thereby, the stress conditions accompanying in vitro culture (Debergh and Read 1991) induced a great change in hormonal content in kiwi tissues.
Twelve days after initiation o f the fifth subculture on
When petioles were cultured on CIM, Z and [9R]Z
CPM, the growth (in fresh weight) o f callus line B was
levels showed a great decline, which could be due to the
slightly higher than line A (Fig. 1). However, the
exogenous applications o f N A A (Palni et al. 1988) and
accumulation o f dry matter (measured as the ratio D W / F W ) was always higher in line A; being 1 g FW,
also to B A application trought the increment in cyt0kinin oxidase activity (Chatfield and Armstrong 1986; Kaminek
0.102:t:0.0025 and 0.0724-0.005 g D W at the beginning o f
and Armstrong 1990). The decline o f I A A levels, is in
subculture in lines A and B respectively, and this ratio
agreement with the results reported by Ludwing-Mfiller et
was practically unchanged during subculture.
al. (1993), who observed that the addition o f N A A in the
PGRs in caulogenic and non-caulogenic callus'. PGRs Table 4. Auxin concentration in calluses of line A and B of kiwiplant at the end of the fifth subculture on CPM. The values are mean of 3 biological and 3 analytical replications (n=9). :kstandarerrors. Callus line cultured in Concentrationof auxins, nmol g-LDW CPM (35 days) IAA NAA Total (A) caulogenic
30.75±3.18
30.81±5.08 61.56±5.94
(B) non-caulogenic
31.05±6.85 31.72±3.54 62.22±7.71
CPM: BM + 4.4 gM BA + 2.7 gM NAA, callus proliferation and maintenance medium.
early stages o f growth o f A rabidopsis thaliana seedlings caused a decrease in the amount o f free I A A and indole 3-butyric acid. Therefore, both PGRs exogenously applied ( N A A and BA) could be related to modifications in natural auxin and cytokinin levels in kiwi tissues. In the same way that ocurred in petioles o f kiwi (present results; Pedrieri et al. 1988), the response of callus development increased from the apical to the basal sections of leaf explants (Ammirato 1986: W e m i c k e and Milkovits 1987). Therefore, it seemed interesting to study PGRs distribution in the explants since sometimes there
62 is a relationship between this differential response and the
Refe~nces
hormonal content o f t issue sections (Okubo et al. 1991).
Aitchinson PA, Macleod AJ, Yeoman MM (1977). In: Street HE (ed) Plant Tissue and Cell Culture. University California Press, Berkeley, pp 267-306 Ammirato PV (1986). In: Withers LA, Alderson PG (eds) Plant Tissue Culture and Its Agricultural Applications. University Press, Cambridge, pp 267-306 Barbieri C, Morini S (1988). Acta Hortie 227:470-472 Besse I, Verdeil JL, Duval Y, Sotta B, Maldiney R, Miginiac E (1992). J Exp Bot 43:983-989 Branea C, Bucei G, Domiano P, Rieei A, Torelli A, Bassi M. (1991). Plant Cell Tiss Org Cult 24:105-114 Caboche M, Aranda G, Poll AM, Huet JC, Leguay JJ (1984). Plant Physiol 75:54-59 Chatfield JM, Armstrong D (1986). Plant Physiol 80:493-499 Debergh PC, Read PE (1991). In: Debergh PC, Zimmerman, RH (eds) Micropropagation: Technology and Application. Kluwer Academic Publishers, London, pp 1-13 Eberle J, Arnscheidt A, Klix D, Weiler EW (1986). Plant Physiol 81:516-521 Fem~indez B, Centeno ML, Feito I, Sfinchez-Tamts R, Rodriguez A (1995). Phytochem Anal 6:49-54. George EF, Sherrington PD (1984) In: Plant Propagation by Tissue Culture. Handbook and Directory of Commercial Laboratories. Exegetics Limited, Eversley Heylen Ch, Vendrig JC, van Onekelen H (199l). Physiol Plant 83:578584 Horgan R, Scott IM, (1987). In: Rivier L, Crozier (eds). The Principles and Practice of Plant Hormone Analysis. Academic Press, London, pp 303-365 Kaminek M, Armstrong D (1990). Plant Physiol 93:1530-1538 Krikorian D (1995). In: Davies PJ (ed) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, London, pp 774-798 Leva AR, Bertocei F (1988). Acta Hortic 227:447-449 Letham DS, Palni MS (1983). Ann Rev Plant Physiol 34:163-197 Ludwing-Mtiller J, Sass S, Sutter EG, Wodner M, Epstein E (1993). Plant Growth Regul 13:179-187 McGaw BA (1995). In: Davies PJ (ed) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, London, pp 87-117 Michalezuk L, Cooke TJ, Cohen JD (1992). Phytochem 31(4):10971103 Murashige T, Skoog F (1962). Physiol Plant 15:473-497 Neill B, Horgan R (1987). In: Rivier L, Crozier A (eds) The Principles and Practice of Plant Hormone Analysis. Academic Press, London, pp 111-168 Okubo H, Wada K, Uemoto S (1991). Plant Cell Rep 10:501-504 Palni LMS, Burch L, Horgan R (1988). Planta 14:231-234 Predieri S, Mezzetti B, Ranieri R (1988). Revista di Frutticoltura 11: 69-72 Skoog F, Miller CO (1957). Symp Soc Exp Biol 11:118-131 Smulders MJM, Croes AF, Wullems GJ (1988). Plant Physiol 88:752756 Tran Thanh Van KM (1981). Ann Rev Plant Physiol 32:291-357. van der Krieken WM, Croes AF, Smulders MJM, Wullems GJ (1990). Plant Physiol 92:565-569 Wernicke W, Milkovits L (1987). Physiol Plant 69:23-28
The auxin/cytokinin ratio in A ctinidia petioles was always advantageous to auxin, and higher in the basal end o f explants than in the apical one, due to unequal distribution o f I A A and cytokinins, mainly B A and (diH)Z. This could explain the basal callus formation, because tissue dedifferentiation and disorganized growth have been closely associated with abnormally high auxin/cytokinin ratios or high endogenous auxin content in other species (Branca et al. 1991; Besse et al. 1992; Michalczuk et al. 1992). The incapacity o f apical cells to form callus, in spite o f the high I A A levels, could be related to their developmental status, being more advanced than in basal cells (Wernicke and Milcovits 1987). On the other hand, the greater content o f cytokinins in the apical end could be the cause o f occasional shoot regeneration. In relation to growth o f kiwi callus, the differences observed between both lines (caulogenic and noncaulogenic), are in agreement with the results o f Barbieri and Morini (1988) and Leva and Bertocci (1988) that related the higher organogenic potential with lower growth,
lower water uptake and higher dry matter
accumulation in A ctinidia calluses. Caulogenic expression in callus line A can not be associated with a different auxirdcytokinin balance from line B, but it can be so with the B A content, which is double in caulogenic callus. The importance o f cytokinins for the initiation o f organogenic centres was earlier reported by Heylen et al. (1991) in Nicotiana. On the other hand, the role o f I A A and N A A could be associated to proliferation and maintenance o f callus, since their concentration was the same in both callus lines. Similar results were found in embryogenic and non-embryogenic callus o f carrot (Michalczuk et al. 1992). In conclusion, in this work we attributed a major role to auxins in callus formation and maintenance, and to cytokinins in the caulogenic responses o f kiwi tissues, appearing BA in callus, and BA and (diH)Z in petioles, the key compouds. However, this does not imply that both PGRs
groups,
auxins and cytokinins, can have
an
individual action. Studies o f endogenous content o f PGRs, together with uptake and metabolism o f N A A and BA at early periods o f culture, are necessary to get a better understanding o f this point. Acknowledgments. We are very grateful to Dr. J. R. Toyos for his assistence in the immunization experiments. This research was supported by the Direcci6n General de Investigaci6n Cientifica y Ttcnica (PB 920999) and M.L.C. and I.F. were supported by predoctoral fellowships from the Ministerio de Educacitn y Ciencia (Spain).
Plant Cetl Reports (1996) 16:58-62
© Sprioger-Verlag1996
Relationship between endogenous auxin and cytokinin levels and morphogenic responses in Actinidia deliciosa tissue cultures M.L. Centeno, A. Rodriguez, I. Feito, and B. Fermindez * Lab. Fisiologia Vegetal, Dpto. KO.S., Fac. Biologia, Univ. Oviedo, C/Catedrfitico Rodrigo Urla, E-33071 Oviedo, Spain Received 30 June 1995/Revised version received 12 December 1995 - Communicated by E. W. Weiler
Summary. The in vitro culture of A ctinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxirdcytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot
formation when they were transferred to proliferation medium. Most important differences fotmd in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed, Abbreviations: BM, basal medium, BA, 6-benzyladenine, CIM, callus induction medium, CPM, callus proliferation medium, (dill)Z, dihydrozeatin, DW, dry weight, ELISA, enzymelinked immunoassay, FW, freshweight, ttPLC, high performanceliquid chromatography,IAA, indole-3-aeetic acid, iP, N6-(A2=isopentenyl)adenine, NAA, 1naphthaleneacetic acid, PBS, phosphate buffer, PGR, plant growth regulator, (diH)[9R]Z, 9-13-D-ribofuranosyl-(diH), [9R]iP, 9-13-Dribofuranosyl-iP, [9R]Z, 9-13-D-ribofuranosylzeatin, TBS, trishydroxymethyl-aminomethanebuffer, Z, zeatin. Inlroduetion
culture offers the possibility of studing differentiation on pieces of tissues detached from the whole plant (van der Krieken et al. 1990). Morphogenesis of in vitro cultured tissues, as well as plant development, are regulated by plant hormones, specially auxins and cytokinins (Skoog and Miller 1957). This hormonal control has been studied mainly by exogenous application of PGRs and only in few cases in relation to endogenous levels (Letham and Palni 1983).
In
vitro
Correspondence to: B. Fernfindez
The regeneration of shoots from callus is a differentiation process whose physiology has been a poorly developed area of research (Aitchison et al. 1977). This route for plant multiplication includes callus induction and formation from an initial explant, and shoot stimulation and development from the induced callus. Each of these two steps may not be discrete or synchronous and the levels of the exogenous hormones could be different (Krikorian 1995). In some species, callus induction was associated with high auxin/cytokinin ratios exogenously applied (Smulders et al. 1988; Branca et al. 1991) or with high auxin endogenous contents (Besse at al. 1992; Michalczuk et al. 1992). Nevertheless, the presence of cytokinin, is generally required for callus formation too (Aitchison et al. 1977). Moreover, cytokinins have been regularly incorporated into tissue culture for shoot regeneration (George and Sherrington 1984). Our attempt is to establish a relationship between exogenous applied regulators, endogenous hormonal contents, and in vitro morphogenic responses. In this way, the analysis of the same problem in morphogenic areas, explants or genotypes, as well as in non-morphogenic ones is a good way to study the control of morphogenesis in in vitro cultures (Tran Thanh Van 1981). Therefore, we compare the endogenous auxin and cytokinin levels in A ctinidia deliciosa tissues with different callogenic and caulogenic abilities. Material and methods Plant Material and culture conditions. Petioles (2-2.5 era) from four
kiwiplants (Actinidia deliciosa (A.Chev) Liang and Ferguson cv. Hayward) were used for callus induction. These plants (1, 2, 3, and 4), originated from cuttings, were grown 4-5 years in the greenhouse. The
59 petioles of the five most apical leaves were explanted, so some of them
to achieve its quantification. Radioactivity was measured after addition
were still developing. Explants were surface sterilized by washing 1 rain
of 2 ml scintillation liquid (Pico-Aqua, Packard) in a Packard 2500TR
in
70% (v/v) ethanol and 20 min (v/v) in 9% commercial NaC10
scintillation analyzer. The dried HPLC cytokinin fractions were
(Domestos), The petioles were rinsed 4 times in sterile distilled water.
resuspended in tris-hydroxymethyl aminornethane buffer (TBS) (25 raM,
The basal medium (BM) used was MS (Murashige and Skoog 1962)
pH 7.5) and quantified by ELISA, using three different polyclonal rabbit
supplemented with sucrose (2.5% w/v) and solidified with agar (0.7%
antibodies (Fermindez et al. 1995): i) anti[9R]Z antibodies to mesure Z
w/v). The pH was adjusted to 5.6 before autoclaving. Each culture vessel
and [9R]Z; ii) anti (diH)[9R]Z antibodies to mesure (diH)Z and
(200 ml) contained 30 ml of medium.
(diH)[9R]Z; iii) anti[9R]iP antibodies to mesure iP, [9R]iP, and also BA,
Petioles were preconditioned by culture in BM for 7 days. Afterwards
since it has high cross-reactivity with the [9R]iP antibodies.
the petiole ends were eliminated by excision and longitudinal incisions
Cytokinin conjugatesof alkalinephosphatase. The cytokinin-alkaline
were made on both extremes. These explants were placed on callus
phosphatase tracer conjugates were prepared following Ebefle et al.
induction medium (CIM), consisting of BM supplemented with 2.2 gM
(1986), except that 3 mg of protein were used and therefore the volumes
BA and 0.27 gM NAA. The petioles were always cultured in horizontal
of other reagents were proportionally scaled up. The dialysed conjugates
position. After 30 days of culture, induced callus was transferred to
were stored in 50% (v/v) glycerol at -80°C.
proliferation and maintenance medium (CPM = BM + 4.4 gM BA + 2.7
ELISA forcytokinin.~ ~luantification. Assays ofcytokinin were carried
gM NAA). Two pieces of callus per flask were cultured, and subculture
out in flat bottom, Immunolon M129A Micro ELISA plates (Nunc) using
took place every 5 weeks.
a modification of the ELISA method described by Eberle et al. (1986).
Some petiole tissues and calli were cultured in CIM with 1.32 nM
The wells of the microtiter plates were filled with 200 gl of diluted
(0.89 nCi/culture vessel) [3H]NAA (specific activity 22.6 Ci/mmol) or
antibody anti-[9R]Z, (diH)[9R]Z or [9R]iP, dissolved in 50 mM NaHCQ
in CPM with 0.66 nM (0.45 nCi/culture vessel) [3H]NAA respectively
buffer, pH 9.6 (Femfindez et al. 1995). Plates were incubated overnight
in order to quantify NAA levels in the tissues.
at 4°C. The solution was poured-off and the wells washed three times
Growth conditions were 25"2°C under a 16 h photoperiod provided
with TBS buffer. Then, the wells were incubated for 30 additional min
by cool-white fluorescent lamps (Sylvania F40W/154-RS) at a photon
at room temperature with 250 gl of 0.1% (w/v) bovine serum albumin
flux density of 33 gmol m "2 s"~.
in TBS buffer. After rinsing with TBS buffer° the coated wells were
To determine the rate of callus growth during the fifth subculture, two
filled with 100 gl of the antigen standard solution or sample solution,
pieces of callus (1±0.05 g fresh weight) were cultured per vessel. Every
diluted in TBS buffer. To determine the maximum binding of tracer,
6 days, they were removed, frozen in liquid N2, lyophilized and weighed
TB S was used instead of standard. Nonspecific binding was determined
to determine their dry mass.
by adding an excess (100 pmoI) of cytokinin standard. The plates were
Harvest and preparation of plant extracts. Samples from the initial
incubated for 30 min at 4°C. The corresponding diluted enzyme tracer
plant material and explants at the end of the different culture phases
(100 gl) was then added to each well and the plates were incubated for
were harvested. The petioles harvested after their culture on BM were
another 2.5 h at 4°C. Tracer dilutions were done in TBS buffer
separated in three portions of equal length (apical, middle and basal) and
supplemented with 0.1% (w/v) gelatin. The plates were decanted and
those cultured in CIM were divided in two parts (apical and basal), in
washed three times with TBS buffer. Phosphatase activity was measured
order to know the distribution of PGRs in the explants. Sampled tissues
by incubating each well for 1 h at 37°C with 200 gl of a freshly
were deep-frozen in liquid nitrogen, powdered and lyophilized.
prepared solution of 4.24 mM p-nitrophenylphosphate in 0.9 M
Extraction, purification and separation of plant growth regulators. Extraction and purification were performed according to Femfindez et al.
diethanolamine buffer pH 9.8. The absorbance was measured at 405 nm in a microplate reader (Lambda Reader, Perkin-Elmer).
(1995). Fractions containing NAA were dissolved in 50 gl methanol and applied on 0.25 mm silica-gel thin layer chromatography plates with a
Results
fluorescence indicator (Merck 60F254).
Levels o f PGRs and their distribution in the explanted petioles.
Plates were run with
cloroform:methanol:acetic acid, 75:20:4 (v/v/v) (Caboche et al. 1984) and 0.1 gmol of pure NAA and [H3]NAA were cochromatographed.
Cytokinins.
Several
cytokinins
were
identified
by
After separation, the plates were visualized under ultravioletqight at 254
comparison of the retention times of pure compounds with
nm.
liquid
the immunoreactive fractions. Immunoreactivity was found
chromatography (HPLC) and the automatic equipement used have been
in f r a c t i o n s c o r r e s p o n d i n g to t h e r e t e n t i o n t i m e s o f Z ,
previously described (Fermindez et al. 1995).
( d i l l ) Z , [ 9 R ] Z , ( d i H ) [ 9 R ] Z , iP, [ 9 R ] i P , a n d B A .
Separation
of
cytokinins
by
high
performance
Quantification of plant growth regulators. IAA samples were
T a b l e 1 s h o w s t h e a m o u n t o f e n d o g e n o u s c y t o k i n i n s in
methylated according to Neill and Horgan (1987) prior to their analysis
t h e e x p l a n t e d p e t i o l e s a n d a f t e r t h e i r c u l t u r e in B M (initial
by enzyme linked immunoassay (ELISA) (Phytoscience test kits). After
conditions
determination of NAA position on plates by cochromatography with
h i g h e s t l e v e l s o f Z a n d its r i b o s i d e , b u t at t h e e n d o f t h e
authentic standards, the silica was scraped off into scintillation vessels
preconditioning phase, these cytokinins declined by three
for
callus
induction).
Petioles
showed
the
60 Table 1. Cytokininconcentrationin petioles of kiwiplants,and in petiole portions (apical, middle and basal) and whole petioles cultured in vitro. The values are mean of 3 biological and 3 analyticalreplications(n= 9), :sstandarerrors, p= petiolar portion. Culture
Plant
conditions
tissue
Concentrationof cytokinins,nmol g-I Z
in vivo
whole p e t i o l e
BM in vitro p. apical
(diH)Z
[9R]Z
(diH)[9R]Z
3.81±0.22 0.01:50.00 11.20:51.34 0.024-0.01
iP
[9R]iP
BA
0.524-0.05 0.054-0.02
Total 15.61:51.35
0.97:50.61
0.34:50.05 1.094-0.13
0.23:t:0.03 1.00:50.03 1.28:50.11
4.9l:50.64
cultures
p. middle
1.11:50.55
0,024-0.01
0.06:50.01 0.23:50.01 0.06:50.00
2.40:50.56
(7 days)
p. basal
1.214-0.46
0.02:50.01 0.69:50.21 0.03:50.00 0.22±0.01 0.03:50.02
0.924-0.11
2.20:50.51
petiole (mean value) 1.09:50.24
0.12:50.05 0.914-0.09 0.11:50.04 0.49:50.14 0.55:50.19
3.274-0.35
CIM in vitro p. apical
0.17:50.01
0.36:50.06 0.13:50.01 0.06:50.02 0.99:50.04 0.66:50.02 0.81:50.04 3.18:50.09
cultures
0.16:50.02
0.06:50.02 0.18:50.01 0.04:50.01 0.69:50.04 0.59:50.03 0.36:50.03 2.084-0.07
(30 days)
p. basal
petiole (mean value) 0.17:50.01
0.21+0.05 0.164-0.01
0.05:50.01 0.84:50.05 0.63:50.02 0.59:50.06 2.65:50.09
BM: basal medium without PGRs, preconditioningmedium. CIM: BM + 2.2 gM BA + 0.27 gM NAA, callus induction medium. and twelve times respectively. On the contrary, (dill)Z,
and
(diH)[9R]Z, and [9R]iP increased thirteen, four and ten
cytokinins, its levels being higher in the basal end. No
this
auxin
showed
a
distribution opposed
to
fold respectively. The analysis in the different portions in
differences were found for NAA content between apical
which the petioles were sectioned, showed an unequal
and basal portions (Tab. 2).
distribution of (dill)Z, (diH)[9R]Z, iP, an [9R]iP; the highest content being observed in the apical portion. Auxins.
After culture on BM, the IAA content of
petioles increased about three times (Tab. 2). The middle portion of explants had lower IAA content than both apical and basal sections.
Table 2, Auxin concentrationin petioles of kiwiplants and in petiolar portions (apical, middle and basal) and whole petioles cultured in vitro. The values are mean of 3 biologicaland 3 analyticalreplications(n=9), ±standar errors, n= netiolar portion. Culture Plant conditions tissue
Concentrationof auxins, nmol g-i IAA
Callus i n d u c t i o n
NAA
Total
in vivo
whole petiole 21.75:53.46
21.754-3.46
BM in
p. apical p. middle p. b a s a l
84.45±20.41 13.06:51.05 91.2715.70
84.45:520.41 13.06:51.05 91.274-15.70
petiole (mean 62.92:513.69 value)
62.924-13.69
When petiole explants were transferred from BM to CIM, callus formation took place between 10 and 20 days after inoculation. This occurred mainly at the basal end of the
vitro
explants (88% callogenic response compared to the 4%
cultures (7 days)
produced at the apical end of the explants). However, shoots and/or leaves regenerated at the apical side (1-5 per explant) at a percentage of 32%. P G R s in p e t i o l e s a f t e r callus induction. There was a
change in the content of PGRs when petiole explants were
C1M m vitro
cultures (30 days)
p. a p i c a l p. b a s a l
13.024-4.00 2.334-0.03 15.354-4.00 29.31:t:6.38 2.424-0.02 31.734-6.38
petiole(mean 21.17:55.27 2.36:50.01 23.53:55.27 value)
cultured in CIM (Tab. 1 and 2). C y t o k i n i n s . The amounts of all zeatin-type cytokinins
decreased significantly with the exception of (dill)Z,
BM: basal medium without PGRs, preconditioningmedium. CIM: BM + 2.2 gM BA + 0.27 gM NAA, callus induction medium.
which increased (Tab. 1), iP was enhanced approximately
Calli p r o l i f e r a t i o n a n d their o r g a n o g e n i c r e s p o n s e
twice, and no changes were observed for [9R]iP. Natural
After 30 days of culture in CIM, the calluses induced at
cytokinins were homogeneously distributed between the
the basal end of the petioles reached a diameter between
apical and basal portions with the exception of iP and
0.5-1 cm and at this stage they were transferred to CPM
(dill)Z, whose level was about six fold higher at the
for callus proliferation. In this medium, calluses from
apical end. Interestingly, the amount of BA was two times
kiwiplant no. 3 showed a high organogenic capacity (75-
higher at the apical than in the basal end.
100% of calli with response along 10 subcultures) giving
A u x i n s . IAA level decreased to the initial level (Tab. 2)
rise to leaves and/or shoots (1-10 per callus). At the same
61 Table 3. Cytokinin concentration in calluses of line A and line B of kiwiplant at the end of the fifth subculture on CPM. The values are mean of 3
biological and 3 analytical replications (n=9), ± standar errors. Callus line cultured in CPM (35 days)
Concentration of cytokinins, nmol g-XDW Z
(diH)Z
[9R]Z
(diH)[9R]Z
iP
[gR]iP
BA
Total
(A) canlogenic
0.19±0.01
0.10±0.02
0.54±0.03
0.16±0.01
0.68±0.06
1.19±0.03
1.09±0.09
3.95±0.12
(B) non-caulogenic
0.16±0.02
0.06±0.01
0.66±0.04
0.19±0.02
0.86±0.04
1.18±0.07
0.47±0.07
3.57±0.13
CPM: BM + 4.4 gM BA + 2.7 M gNAA. Callus proliferation and maintenance medium. time, oniy 4-23% o f calluses from plants 1, 2 and 4
content in callus lines A and B were measured at the end
showed this caulogenic response and it disappeared after
o f the fifth subculture (Tab. 3 and Tab. 4).
4 subcultures. Therefore, we can establish two callus
Cytokinins. Slight differences were observed in natural
lines: line A, callus with high caulogenic capacity and line
cytokinin levels between both callus lines. The difference
B, callus with low or null capacity.
in BA content was the most noticeable, being double in line A (Tab. 3).
4-
Auxins. No significant differences were observed
3,5-
between the auxin content in both lines (Tab. 4). Discussion
The original physiological status o f explants and their
x~ 2,5-
hormonal content are very important factors in directing o a.
in vitro responses (Almnirato 1986). The large reduction o f total cytokinin content, and specially of Z and [9R]Z,
1,5
taking place in kiwi petioles cultured in vinv, could be associated with glycosylation or oxidative metabolism ( M c G a w 1995). The I A A increment, detected only at the 0
6
12
18 24 Time (days)
30
36
42
Fig. 1. The growth of caulogenic (ll) and non-caulogenic ([5) callus lines (line A and B) during the fifth subculture on proliferation medium. Each point was derived from an average of nine flasks (two pieces of callus in each flask). Vertical bars indicate standard errors (n=9).
apical and basal portions o f petioles, could be a consequence o f the subtances liberation taking place in the tissues when they are wounded (Aitchison et al. 1977). Thereby, the stress conditions accompanying in vitro culture (Debergh and Read 1991) induced a great change in hormonal content in kiwi tissues.
Twelve days after initiation o f the fifth subculture on
When petioles were cultured on CIM, Z and [9R]Z
CPM, the growth (in fresh weight) o f callus line B was
levels showed a great decline, which could be due to the
slightly higher than line A (Fig. 1). However, the
exogenous applications o f N A A (Palni et al. 1988) and
accumulation o f dry matter (measured as the ratio D W / F W ) was always higher in line A; being 1 g FW,
also to B A application trought the increment in cyt0kinin oxidase activity (Chatfield and Armstrong 1986; Kaminek
0.102:t:0.0025 and 0.0724-0.005 g D W at the beginning o f
and Armstrong 1990). The decline o f I A A levels, is in
subculture in lines A and B respectively, and this ratio
agreement with the results reported by Ludwing-Mfiller et
was practically unchanged during subculture.
al. (1993), who observed that the addition o f N A A in the
PGRs in caulogenic and non-caulogenic callus'. PGRs Table 4. Auxin concentration in calluses of line A and B of kiwiplant at the end of the fifth subculture on CPM. The values are mean of 3 biological and 3 analytical replications (n=9). :kstandarerrors. Callus line cultured in Concentrationof auxins, nmol g-LDW CPM (35 days) IAA NAA Total (A) caulogenic
30.75±3.18
30.81±5.08 61.56±5.94
(B) non-caulogenic
31.05±6.85 31.72±3.54 62.22±7.71
CPM: BM + 4.4 gM BA + 2.7 gM NAA, callus proliferation and maintenance medium.
early stages o f growth o f A rabidopsis thaliana seedlings caused a decrease in the amount o f free I A A and indole 3-butyric acid. Therefore, both PGRs exogenously applied ( N A A and BA) could be related to modifications in natural auxin and cytokinin levels in kiwi tissues. In the same way that ocurred in petioles o f kiwi (present results; Pedrieri et al. 1988), the response of callus development increased from the apical to the basal sections of leaf explants (Ammirato 1986: W e m i c k e and Milkovits 1987). Therefore, it seemed interesting to study PGRs distribution in the explants since sometimes there
62 is a relationship between this differential response and the
Refe~nces
hormonal content o f t issue sections (Okubo et al. 1991).
Aitchinson PA, Macleod AJ, Yeoman MM (1977). In: Street HE (ed) Plant Tissue and Cell Culture. University California Press, Berkeley, pp 267-306 Ammirato PV (1986). In: Withers LA, Alderson PG (eds) Plant Tissue Culture and Its Agricultural Applications. University Press, Cambridge, pp 267-306 Barbieri C, Morini S (1988). Acta Hortie 227:470-472 Besse I, Verdeil JL, Duval Y, Sotta B, Maldiney R, Miginiac E (1992). J Exp Bot 43:983-989 Branea C, Bucei G, Domiano P, Rieei A, Torelli A, Bassi M. (1991). Plant Cell Tiss Org Cult 24:105-114 Caboche M, Aranda G, Poll AM, Huet JC, Leguay JJ (1984). Plant Physiol 75:54-59 Chatfield JM, Armstrong D (1986). Plant Physiol 80:493-499 Debergh PC, Read PE (1991). In: Debergh PC, Zimmerman, RH (eds) Micropropagation: Technology and Application. Kluwer Academic Publishers, London, pp 1-13 Eberle J, Arnscheidt A, Klix D, Weiler EW (1986). Plant Physiol 81:516-521 Fem~indez B, Centeno ML, Feito I, Sfinchez-Tamts R, Rodriguez A (1995). Phytochem Anal 6:49-54. George EF, Sherrington PD (1984) In: Plant Propagation by Tissue Culture. Handbook and Directory of Commercial Laboratories. Exegetics Limited, Eversley Heylen Ch, Vendrig JC, van Onekelen H (199l). Physiol Plant 83:578584 Horgan R, Scott IM, (1987). In: Rivier L, Crozier (eds). The Principles and Practice of Plant Hormone Analysis. Academic Press, London, pp 303-365 Kaminek M, Armstrong D (1990). Plant Physiol 93:1530-1538 Krikorian D (1995). In: Davies PJ (ed) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, London, pp 774-798 Leva AR, Bertocei F (1988). Acta Hortic 227:447-449 Letham DS, Palni MS (1983). Ann Rev Plant Physiol 34:163-197 Ludwing-Mtiller J, Sass S, Sutter EG, Wodner M, Epstein E (1993). Plant Growth Regul 13:179-187 McGaw BA (1995). In: Davies PJ (ed) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, London, pp 87-117 Michalezuk L, Cooke TJ, Cohen JD (1992). Phytochem 31(4):10971103 Murashige T, Skoog F (1962). Physiol Plant 15:473-497 Neill B, Horgan R (1987). In: Rivier L, Crozier A (eds) The Principles and Practice of Plant Hormone Analysis. Academic Press, London, pp 111-168 Okubo H, Wada K, Uemoto S (1991). Plant Cell Rep 10:501-504 Palni LMS, Burch L, Horgan R (1988). Planta 14:231-234 Predieri S, Mezzetti B, Ranieri R (1988). Revista di Frutticoltura 11: 69-72 Skoog F, Miller CO (1957). Symp Soc Exp Biol 11:118-131 Smulders MJM, Croes AF, Wullems GJ (1988). Plant Physiol 88:752756 Tran Thanh Van KM (1981). Ann Rev Plant Physiol 32:291-357. van der Krieken WM, Croes AF, Smulders MJM, Wullems GJ (1990). Plant Physiol 92:565-569 Wernicke W, Milkovits L (1987). Physiol Plant 69:23-28
The auxin/cytokinin ratio in A ctinidia petioles was always advantageous to auxin, and higher in the basal end o f explants than in the apical one, due to unequal distribution o f I A A and cytokinins, mainly B A and (diH)Z. This could explain the basal callus formation, because tissue dedifferentiation and disorganized growth have been closely associated with abnormally high auxin/cytokinin ratios or high endogenous auxin content in other species (Branca et al. 1991; Besse et al. 1992; Michalczuk et al. 1992). The incapacity o f apical cells to form callus, in spite o f the high I A A levels, could be related to their developmental status, being more advanced than in basal cells (Wernicke and Milcovits 1987). On the other hand, the greater content o f cytokinins in the apical end could be the cause o f occasional shoot regeneration. In relation to growth o f kiwi callus, the differences observed between both lines (caulogenic and noncaulogenic), are in agreement with the results o f Barbieri and Morini (1988) and Leva and Bertocci (1988) that related the higher organogenic potential with lower growth,
lower water uptake and higher dry matter
accumulation in A ctinidia calluses. Caulogenic expression in callus line A can not be associated with a different auxirdcytokinin balance from line B, but it can be so with the B A content, which is double in caulogenic callus. The importance o f cytokinins for the initiation o f organogenic centres was earlier reported by Heylen et al. (1991) in Nicotiana. On the other hand, the role o f I A A and N A A could be associated to proliferation and maintenance o f callus, since their concentration was the same in both callus lines. Similar results were found in embryogenic and non-embryogenic callus o f carrot (Michalczuk et al. 1992). In conclusion, in this work we attributed a major role to auxins in callus formation and maintenance, and to cytokinins in the caulogenic responses o f kiwi tissues, appearing BA in callus, and BA and (diH)Z in petioles, the key compouds. However, this does not imply that both PGRs
groups,
auxins and cytokinins, can have
an
individual action. Studies o f endogenous content o f PGRs, together with uptake and metabolism o f N A A and BA at early periods o f culture, are necessary to get a better understanding o f this point. Acknowledgments. We are very grateful to Dr. J. R. Toyos for his assistence in the immunization experiments. This research was supported by the Direcci6n General de Investigaci6n Cientifica y Ttcnica (PB 920999) and M.L.C. and I.F. were supported by predoctoral fellowships from the Ministerio de Educacitn y Ciencia (Spain).
