Scientia Horticulturae, 46 ( 1 9 9 1 ) 2 8 3 - 2 9 4

283

Elsevier Science Publishers B.V., A m s t e r d a m

Factors affecting areole activation in vitro in the

cactus Sulcorebutia alba Rausch
M.A.A. Dabekaussen, R.L.M. Pierik, J.D. van der Laken and J. Hoek Spaans
Department of Horticulture, Agricultural University, P.O. Box 30, 6700 AA Wageningen
(The Netherlands)
(Accepted for publication 16 August 1990)

ABSTRACT
Dabekaussen, M.A.A., Pierik, R.L.M., van der Laken, J.D. and Hoek Spaans, J., 1991. Factors affecting areole activation in vitro in the cactus Sulcorebutia alba Rausch. Scientia Hortic., 46: 283-294.
A cultivation system was developed to study areole activation (AA) in explants of the cactus Sulcorebutia alba Rausch. Explants containing three areoles were cut from the central piece of the cladodes, which were grown in vitro. The optimal culture medium for AA consisted of macro-salts according to Murashige and Skoog at a strength of 1.5, MS micro-salts at full strength except iron,
NaFeEDTA 25 mg 1-~, sucrose 2.5%, BA 0.25-1 mg 1 ~, Daichin agar 0.5% and pH 5.5. Cytokinin
was an essential prerequisite for AA; BA yielded best results, followed by kinetin. The cytokinins PBA,
2iP and IPA were not suitable as they gave rise to heavy callus formation. Optimal physical growth
conditions for AA were a temperature of 27 C and an irradiance of 2.3-5 W m - 2 (photosynthetically
active radiation).
Keywords: activation; areole; cactus; in vitro; micropropagation; Sulcorebutia alba Rausch.
Abbreviations: AA=areole
activation; ABA=abscissic acid; BA=6-benzylaminopurine;
GA := gibberellic
acid;
IAA = indole-3-acetic
acid;
2iP = N 6- ( 2 - i s o p e n t e n y l ) a d e n i n e ;
IPA := isopentenyladenoside; MS = Murashige and Skoog ( 1962 ); NAA = 1-naphthaleneacetic acid;
nac--=number of abnormal cacti; n n c = n u m b e r of normal cacti; PBA=6-(benzylamino)-9-(2tetrahydropyranyl )-9H-purine; TIBA = 2,3,5-triiodobenzoic acid; wat = fresh weight of abnormal
tissu e; wnc = fresh weight of normal cacti.

INTRODUCTION

Relatively little is known about the propagation of Cactaceae in vitro. The

application of tissue culture techniques could eliminate the required maintenance of large numbers of stock plants and could result in more uniform material, eliminating seedling variability. Tissue culture propagation might also
prove useful for maintaining populations of rare and endangered species
(Johnson and Emino, 1979a,b). Johnson and Emino (1979a) propagated
Mammillaria elongata by shoot initiation from callus cultures. However,
0304-4:238/91/$03.50

1991 - - Elsevier Science Publishers B.V.

284

M.A.A. DABEKAUSSENET AL.

Minocha and Mehra ( 1974 ) could not induce differentiation in callus of Neomammillaria prolifera, although they had sporadic root initiation in some
cultures. Vyskot and Jara (1984) propagated Mammillaria carmenae, Mammillaria prolifera, Astrophytum myriostigma and Trichocereus spachionus by
AA in vitro. Ault and Blackmon (1987) also described the use of AA in the
propagation of Ferocactus acathodes and Mauseth ( 1976, 1979) and Mauseth and Halperin (1975) described its use in the propagation of Opuntia
polyacantha. Starling ( 1985 ) described the AA of Leuchtenbergia principis
and Escobar et al. ( 1986 ) described that of Opuntia amyclaea.
The present paper summarizes the effects of plant, nutritional, hormonal
and physical factors on AA in the cactus species Sulcorebutia alba Rausch in
vitro. This species was chosen as the most suitable from preliminary experiments (Pierik et al., 1987a,b).
MATERIALS AND METHODS

The starting material was adult cacti of Sulcorebutia alba Rausch and two
other species ( Sulcorebutia mentosa and Sulcorebutia flavissima ), originating from Bolivia and collected at a height of 2900 m. All tested species of
Sulcorebutia could be induced to form cacti in vitro, although they all reacted
differently during callus formation and cactus growth. Sterile cacti, cultivated
in vitro, were obtained by AA on explants isolated from adult plants grown in
the greenhouse. Cactus segments were surface sterilized as follows: a few seconds in ethanol (70%), 20 min in 1.5% NaOC1 with 0.25 ml 1-~ Tween 20,
followed by three rinses in sterile tap water for 3, 10 and 20 min. Areole activation took place as described below. After about 16 weeks, large cacti were
formed in vitro, from which explants were again taken to obtain AA. This
reproduction system was continuously used to produce new starting material
for the experiments.
For the AA experiments, in vitro-grown cacti were chosen which, after activation, were further grown on a m e d i u m without regulators. Explants containing three areoles and 1.5 m m of the underlying tissue, were cut from the
centre piece of the cladode (cactus body). Explants from the terminal or basal
part of the cactus showed irregular activation. The explants were placed with
their wounded surface on the culture m e d i u m and divided at random over
the various treatments.
The basic culture medium, which was used as a control in all experiments,
consisted of: macro- and micro-salts according to Murashige and Skoog
( 1962 ) at full strength except iron; 25 mg 1-~ NaFeEDTA; 3.5% sucrose; 0.5
mg 1-~ BA; 0.5% Daichin agar (Brunschwig Chemie, Amsterdam, The Netherlands). The pH was adjusted to 5.5 with KOH before autoclaving. Daichin
agar was used as it resulted in a better AA and growth than Gelrite or Difco
Bacto agar. About 12 ml of the m e d i u m were dispensed into each of the Pyrex

AREOLE ACTIVATION IN CACTUS

285

test tubes, which were then sealed with cotton plugs and autoclaved at 121 C
for 20 min. After inoculation, the tubes were closed with Vitafilm (Good
Year). Cultures were incubated in a growth chamber at 27C with a 16 h
photoperiod provided by fluorescent light (Philips TL 57/40W). The irradiance at plant level; was reduced to 2.3 W m -2 (photosynthetically active
radiation) by covering the tubes with one layer of cheesecloth.
All experiments had, in principle, one variable factor (nutritional, hormonal or physical). Each treatment consisted of 24 test tubes, each containing one explant. The experiments were repeated once.
Eight weeks after inoculation the number of activated normal cacti (nnc),
the fresh weight of normal cacti (wnc), the number of abnormal cacti (nac)
and the fresh weight of abnormal tissue (wat) were determined per explant.
Mean values were calculated per replication and per treatment. Mean values
were calculated per replication and per treatment. If in a two-way analysis of
variance (ANOVA) a significant ( P < 0.05 ) effect of the treatment was found,
treatment means were separated by Tukey's honest significant difference
(HSD) test ( P = 0 . 0 5 ) . This is indicated by letter codes in the figures. Treatment means (two replications) indicated by different letters were significantly different.
RESULTS

Histological and morphological studies showed that the areoles of Sulcorebutia alba Rausch are situated on the tubercle, in the furrow on the apical side
of the spines. Within 3 days of inoculation in vitro, the areole meristem was
activated, increased in size and started to form a new cladode. These results
showed clearly that the newly formed cacti were not adventitious but axillary
in origin.
Explants with three areoles were chosen as these showed an increase in
number and weight of activated cacti as compared with explants with one or
two areoles.
There was a low percentage of irregular root formation in vitro; in general,
adventitious roots originated at the basal part of the newly formed cacti. Adventitious root formation was not influenced by the factors described below.

Agar..- Daichin agar was used as it resulted in a better AA and growth than
Gelrite or Difco Bacto agar. The role of the Daichin agar concentration is
shown in Figs. 1 and 2. The weight of normal cacti decreased significantly
when 1:he agar concentration was raised from 0.5 to 1.0%. There was no significant effect on the numbers of activated cacti or the weight of abnormal
tissue. An agar concentration of 0.5% proved to be optimal, a lower concentration did not solidify the culture medium sufficiently.

286

M.A.A. DABEKAUSSEN ET AL.

--~Ir- wnc:

1.50

1.00

a
A.

a
\ \ b

A~ b
~A
0.50

0,00

0.4

i
0.6

Agar

0.8

1.0

concentration

12

(%)

Fig. 1. The influence of the Daichin agar concentration on the weight of normal cacti (wnc) per
explant, 8 weeks after culture.

0.5

0.7

0.6

0,8

1.0

Daichin, %
Fig. 2. The influence of the Daichin agar concentration on areole activation, 8 weeks after culture.

- Figure 3 shows that the weight of normal cacti reached

an optimum on the medium with MS-macroelements at a strength of 1.5. The
numbers of activated cacti and the weight of abnormal tissue were not influenced significantly by the strength of MS macroelements (data not shown).
MS-macroelements.

287

A R E O L E A C T I V A T I O N IN C A C T U S

--11~- WnC
1.50

/
a b /~"

Ik

/!
1.00

/
/I
!

0.50

0.00
0.0

,
0.5

E
1.0

MS-macroelements

,
1.5

,
2.0

2.5

(strength)

Fig. 3. The influence of MS macroelements on the weight of normal cacti (wnc) per explant, 8
weeks al~ter culture.
Sucrose.
- Sucrose was preferred to glucose which caused more callus formation. The influence of the sucrose concentration is shown in Figs. 4 and 5.
The number of normal cacti was relatively low at a sucrose concentration of
1.5%, and the cacti showed an elongated shape and were coloured light green
at their basal parts. The number of normal cacti was the same in the sucrose
range :2.5-5.5%. However, increasing the sucrose concentration to 5.5% resulted in an increasing weight of abnormal tissue (maximum at 4.5% sucrose). Therefore, a sucrose concentration of 2.5% was considered to be
optimal.
Osmotic
potential
- Lowering the osmotic potential of the culture medium
by adding mannitol, showed results comparable with a high concentration of
sucrose, MS-macroelements or agar. Mannitol was added to the medium in
the fol]Lowingconcentrations: 0 g 1-1 (0 atm ); 8.1 g 1- i ( _ 1 atm ); 16.3 g 1- l
( - 2 atm); 24.4 g 1-1 ( - 3 atm); 32.5 g 1-1 ( - 4 atm); the corresponding
calculated change of the osmotic potential of the medium is given in parentheses; 1 a t m = 10 -5 Pa. The number of abnormal cacti was reduced significantly when mannitol was used (Fig. 6). However, weight of normal cacti
and abnormal tissue also decreased when the osmotic potential of the medium was lowered (Fig. 6 ), making it undesirable to use mannitol in the culture medium.

pH.

The pH of the culture medium strongly influenced AA. The number of

288

M.A.A. DABEKAUSSEN ET AL.

~nc

~4a{

nnc

2.50
b

/7
'

b
. - - --A/.,

a c
..

"~ .: '. ab
~.w.--..:,_.~

,/

..b

..

2.00

.5o

,A...,,/ ab
./ .."" "~

1
ab, /

." a b

%00

ab
N "A,

,.'

~,

b
0.50

a.
ti

~
1.0

2.0

3.0

Suc, r c ~ e

t
4.0

i
5.0

0.00
6.0

(%)

Fig. 4. The influence of the sucrose concentration on the number of normal cacti (nnc), the
weight of normal cacti (wnc) and the weight of abnormal tissue (wat) per explant, 8 weeks
after culture.

Fig. 5. The influence of the sucrose concentration on the areole activation, after 8 weeks of
culture.

289

AREOLE ACTIVATION IN CACTUS

~;~C

Wa~

nnc

2.50

a
a
~--~

ab

""-~.~

2.00

"~'~
1.5o

1.00

-,[
[a

........ ......
o

!
0 L

0.50

"J,~
~

10

20

b
~

30

4
i

b 0.00
40

MamitoF cone. (g I-L)

Fig. 6. The influence of the mannitol concentration on the number of normal cacti (nnc), the
weight of normal cacti (wnc) and the weight of abnormal tissue (war) per explant, after 8 weeks
of cultcxe.

normal cacti remained constant when the pH was raised from 5.0 to 6.0, but
decreased when the pH was raised from 6.0 to 7.0 (Fig. 7). Weight of normal
cacti and abnormal tissue decreased significantly when the pH was raised over
5.0 (Fig. 7 ). The number of abnormal cacti was not influenced significantly
by the: pH values tested. In conclusion, a pH of 5.5-6.0 can be considered as
optimal.
The influence of BA, kinetin, PBA, 2iP and IPA on AA was
compared by adding equimolar quantities (2.22 X 10 -6 M) of growth regulators to the medium. Figure 8 shows that BA resulted in a significantly lower
number of abnormal cacti. There were not significant differences in number
and weight of normal cacti between the cytokinins used. Although IPA and
2iP seemed to result in more abnormal tissue than BA, kinetin and PBA, these
differences were not significant.
The influence of the BA concentration is presented in Figs. 9 and 10. On
the culture medium without BA there was hardly any AA as shown in Fig. 10.
The weight of normal cacti increased when the BA concentration was raised
(Figs. 9 and 10). There was no significant difference between 0.1, 0.25, 0.5
and 1 mg 1- ' BA. The BA concentration had no significant effect on the number of activated cacti or the weight of abnormal tissue. A BA concentration of
between 0.25 and 1 mg 1- ' can be considered as optimal.
Cytokinins.

290

M.A.A. DABEKAUSSEN ET AL.

",~'a '~

wnc

iqnc

2,50
a

2.00

"~ C
%5o

1.00
'

\\b

..&
b-.

AX
\

\ C

bc
0
4.5

~
5.5

0.50

.&.

-\ C

bg

~
0.00
7.5

6.5
pH

Fig. 7. The influence of the pH on the number of normal cacti (nnc), the weight of normal cacti
(wnc) and the weight of abnormal tissue (wat) per explant, after 8 weeks of culture.
r~ac
1.00

0.80

0.60
c

bc
///,

bc

0.40

, / / ,

, / / ,

///~

....

///.

K/IA

0.20

V I I A
~,'//A
v /

....

K//A
v / / ~

~ / / I

"///

,///

0.00
BA

Kin

PBA
Cytokinin

2iP

IPA

Fig. 8. The influence of the kind of cytokinin at a concentration of 2.22 X 10 6 M on the number
of abnormal cacti (nac), obtained after 8 weeks of culture.
N A A was a d d e d ( a l w a y s in c o m b i n a t i o n with 0.5 m g 1-~ B A ) to the
culture m e d i u m in five concentrations: 0, 0 . 0 0 1 , 0.01, 0.1 and 1 m g 1-~. A
c o n c e n t r a t i o n o f 0.1 m g 1-~ c a u s e d a significantly higher weight o f n o r m a l

Auxin.

AREOLE

ACTIVATION

291

IN CACTUS

- & - wi'lc
1.5C

b
b

~k

1.0C,[ ~ / //A

1:/
0.5C i/t

0.0(3

'
0.0

t .

03

0.6

BA c w ~ e n l r a t i o n

0.9
(rr~l I-')

.
1.2

Fig. 9. The influence of the BA concentration on the weight of normal cacti (wnc) per explant,
obtained after 8 weeks of culture.

,,z :

10-7

i! ~ i ? i ~ ~ ~ ~ ~
~ii ~i ! ~ ~ i ~

Fig. 10. The influence of the BA concentration on the areole activation, obtained after 8 weeks
of culture.

cacti but also more abnormal tissue than the other concentrations tested (results not shown). The numbers o f cacti per explant were not significantly influenced by the NAA concentration used. The addition of NAA is therefore
thought to be of little importance for AA.

292

M.A.A. DABEKAUSSEN ET AL.

Other growth regulators. - The use of TIBA, ABA, GA4+7 and ethrel had a
negative effect on both AA and growth.
Irradiance. - Irradiances of 1.3, 2.3, 4, 5 and 8 W m -~ were tested. The irradiance in the growth chamber did not influence the number or weight of activated cacti. However, the shape of the cacti was clearly influenced by the
irradiance. Low irradiance (1.3 W m -2) caused elongated, light-green cacti
and high irradiance ( 8 W m - 2 ) resulted in small, compact cacti with a darkgreen colour. The optimal irradiance for areole activation is situated between
2.3 and 5 W m-2 (results not shown).
Temperature. - An incubation temperature of 27 C was chosen as it was shown
that increasing the temperature from 21 to 27 C increased the weight of normal cacti considerably, but an increase to 29C caused an increase in callus
formation.
DISCUSSION

Increasing the agar concentration above 0.5% had a negative effect on the
growth of the activated cacti. It is proposed that the uptake of water and nutrients was hampered by the water binding capacities of the agar. Mauseth
and Halperin ( 1975 ) used Difco Bacto Agar in a concentration of 1% for the
AA of Opunthia polyacantha. Other authors failed to mention the kind ofagar
used; the concentrations described in the literature vary between 0.6 and 1.2%.
A low strength of MS macroelements in the medium resulted in poor growth
of the activated areoles. This was probably caused by a lack of nutrients. A
strength of > 1.5 decreased growth and resulted in cacti with a light-green
colour. This could be caused by a hampered uptake of water and nutrients
because of the low osmotic potential of the medium.
Bad activation at low sucrose concentrations can be explained by a lack of
sugar, as sucrose is required for growth in vitro. Tissue cultures often have a
diminished or total lack of photosynthetic activity (George and Sherrington,
1984). Escobar et al. ( 1986 ) found that explants of Opuntia amyclaea turned
dark and died on a medium without sucrose.
The decrease in weight of normal cacti at high sucrose concentrations is
possibly caused by the low osmotic potential of the medium as described for
a medium with a high strength of MS macroelements and a high agar concentration. Mauseth (1979) found that the most rapid growth of Opuntia polyacantha occurred on a medium with 5% sucrose. When the concentration was
either higher or lower, growth was less vigorous. Similar results were found in
previous work (Mauseth and Halperin, 1975). The increase of weight of abnormal tissue at a high sucrose concentration was unexpected considering the
low osmotic potential of the culture medium.

AREOLE ACTIVATION IN CACTUS

293

The use of mannitol in the culture m e d i u m decreased its osmotic potential,

again :restricting the uptake of water and nutrients by the plant tissue. This
resulted in poor growth of the activated areoles.
The pH influences the uptake of nutrients and plant regulators and the solidity of the agar (George and Sherrington, 1984). These factors are probably
the cause of the differences in activation and growth.
It is quite clear that a cytokinin was necessary for the activation of areoles
ofSulcorebutia alba Rausch: BA in a concentration of between 0.25 and 1 mg
l - 1 appears to be optimal, this corresponding to the results of other authors.
Mauseth and Halperin ( 1975 ) and Mauseth ( 1976 ) found positive effects of
BA ( 10 mg 1- l ) on the AA of Opuntia polyacantha. Mauseth (1979) found
for the activation of several cacti, optimal BA concentrations of 1 and 10 mg
1-I. Escobar et al. (1986) found an optimal BA concentration of 2.3 mg 1-1
for the AA of Opuntia amyclaea.
Sulcorebutia alba Rausch did not require NAA for AA. Vyskot and Jara
(1984) activated areoles of Mammillaria carmenae, M. prolifera, Astrophytum myriostigma and Trichocereus spachianus by using kinetin or BA in concentrations of between 0.5 and 5 mg 1-l in combination with 0.5 to 5 mg 1IAA or NAA. Starling (1985) used 10 mg 1-I BA with 0.1 mg 1-l NAA for
the are,ole activation of Leuchtenbergia principis. They did not examine the
influence of a m e d i u m without an auxin.
The irradiance clearly influenced the colour and shape of the cacti. Low
irradiance may result in the production of etioplasts instead of chloroplasts,
which could explain the light-green colour of the cacti.
Finally, it can be concluded that a culture m e d i u m that consists of MS macroelements at a strength of 1.5, MS microelements at full strength except
iron, 25 mg 1- l NaFeEDTA, 2.5% sucrose, 0.25-1 mg 1-l BA and 0.5% Daichin agar is optimal for the AA in vitro of Sulcorebutia alba Rausch. The
m e d i u m should be adjusted to pH 5.5 before autoclaving, and the material
incubated at 27 C with an irradiance between 2.3 and 5 W m -2.
ACKNOWLEDGEMENTS

We are greatly indebted to L.E. Groen from the Department of Plant Taxonomy of the Agricultural University Wageningen, The Netherlands, for the
supply of plant material ofSulcorebutia spp. The authors also thank C.I. Kendrick fi)r correcting the English text.

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294

M.A.A. DABEKAUSSEN ET AL.

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