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In Vitro CeII. Dev. Biol.-PIant 34:244-248, JuIy-September 1998
@ 1998 Society for In Vitro BioIogy
1054-5476198 $05.00+0.00
CONSERVATION OF HYPERICUM FOUOSUM AITON, AN ENDEMIC AZOREAN SPECIES,
BY MICROPROPAGATION
MONICA MOURA
Departamento de Biologia, Universidade dos Açores, Apartado 1422, 9502 Ponta Delgada (Açores) Codex, Portugal
(Received 15 ApriI1997; accepted 20 March 1998; editor M. R. Becwar)
SUMMARY
One of lhe first Azorean endemic vascular taxa chosen for lhe development of in vitro multiplication techniques was
Hypericumfoliosum Aiton, due to its colonizing ability (Sjogren, 1984), a loss of seed germination capacity after only 1 yr
of storage (Maciel, 1994), and lhe populations' generally low number of individuais. The following culture media were
tested using Hypericumfoliosum's single node cuttings: Murashige and Skoog (1962), Roest and Bockelmman (1973), Uoyd
and McCown (1980), Côrte and Mendonça (1985), and Cellárová et aI. (1992). Further experiments were performed on CM
medium supplemented with four different growth regulators: a-naphthaleneacetic acid (NAA), N6-benzyladenine (BA), y, y-
(dimethylallyl) aminopurine (2iP), and kinetin (KIN). The acclimatization stage was carried out in Jiffy 7@ pois and in a
2:1 or 1:1 peat/perlite mixture. We found that micropropagation of Hypericumfoliosum is possible on CM medium and that
lhe best results with growth regulators were achieved with lhe following supplements: 0.1 mg/l (0.4 11M) BA and 0.5 mg/l
(2.6 11M> NAA + 1.0 mg/l (4.4 11M) BA (in lhe initiation stage), anq 0.1 mg/l (0.4 11M> BA (in lhe elongation stage). As
for culture multiplication, 0.1 mg/l (0.4 11M) BA (in lhe initiation stage) and 0.5 mg/l (2.6 11M> NAA + 1.0mg/l (4.4 11M)
BA (both in lhe initiation and elongation stages), proved to be lhe most efficient concentrations. The acclimatization stage
was successfully performed in Jiffy 7@ pellets.
Key words: micropropagation; Hypericumfoliosum Aiton; conservation; endemic; nodal explants.
INTRODUCTION altered vegetation patches, located in lhe feeding areas of an Azorean
endemic bird known as "Priôlo" (Pyrrhula murina).
As to previous in vitro cultures of Hypericum spp., lhe only avail-
able information referred to lhe shoot production and rooting of Hy-
pericum peiforatum (Roest and Bockelmann, 1973; Cellárová et al.,
1992) and Hypericum canarieme (Mederos, 1991). Roest and Bock-
elman obtained adventitious shoots Eram capitula, in RB medium
supplemented with 1 mg/l of N6-benzyladenine (BA) and 5 mg/l su-
crase. The media was solidified with 6 g/l agar. Rooting was accom-
plished ex vitro, after previously dipping lhe shoots in 1% indole-3-
acetic acid (IAA) talco Mederos (1991) initiated lhe culture of Hy-
pericum canarieme from apical and axillary buds in an Almacigo
medium (Mederos and Rodríguez, 1990). The author considers lhe
MS macronutrient solution, [Murashige and Skoog 1962], with BA
and a-naphthaleneacetic acid (NAA) added, as lhe most effective to
propagate lhe species. Rooting was obtained in a 1/2 diluted MS
macronutrient solution to which was added indole-3-butyric acid
(IBA) or NAA and lhe other remaining regular Almacigo additives.
The author also refers to lhe importance of adding rosmanol to pre-
vent explant and media darkening during lhe culture initiation stage,
as lhe only condition to allow in vitro production of shoots. Cellárova
et alo (1992) obtained several adventitious shoots from whole Hyper-
icum peiforatum plantlets, excised leaves, shoots, and roots, using
MS mineral salts, PRL-4-C Gamborg vitamin solution (Gamborg et
al., 1968), and Skoog amino acids. The medium was supplemented
with 87.6 X 10-3 M of sucrose, 0.5 or 0.91 mg/l BA, and solidified
with 0.6% agar. The pH was set to 5.6 before autoclaving. The shoots
The Azores, with its archipelago status and an increasing human
pressure to which the natural vegetation is subjected, has become a
priority region for the development of conservation methodologies.
Currently, various endemic Azorean taxa appear only in small dis-
persed populations or are restricted to a single locality in one or more
islands. As part of an autoecological conservationist plan, a micro"
propagation unit was established in the Biology Department of the
University of Azores, and is fully working since late 1993.
One of the first Azorean endemic vascular taxa chosen for the
development of in vitro multiplication techniques was Hypericumfo-
liosum Aiton (Guttiferae family). This plant (Fig. 1 A), commonly
known as "Furalha" or "Malfurada," is an Azorean endemic nano-
phanerophyte (Franco, 1971), occurring on alI islands of the archi-
pelago (Dias, 1989). It is a woody plant that, according to Sjogren
(1984), grows preferentially above 400 m and occasionally below 100
m, on both sheltered and exposed places. On São Miguel, the most
developed populations were found on the northeast part of the island
(at altitudes ranging from 570 to 887 m), in open, treeless sites. The
plants colonizing ability (Sjogren, 1984) was verified by our study,
as several individuaIs were observed in recent landslide areas and
man-made glades.
It is algO known that the seeds of this species lose the ability to
germinate afteronly 1 yrof storage (Maciel, 1994) and its populations
are usually formed by a small number of individuaIs. Micropropa-
gation will overcome such problems and was made a component of
the LIFE project, whose major objective is the recuperation of several
:l44
MICROPROPAGATION OF HYPERlCUM FOUOSUM 245
% 100
80
60
40
20
o
DABCDE rGHI JKLM
SURF ACE..STERlUZA TION PROCEDURES
B
E
c
F
flG. 1. Stages in lhe micropropagation of Hypericumfoliosum Aiton from single nade explants. A, Field-established planto B, Ger-
minated seedling. C, Single nade stem cuttings (each pair of leaves will be excised before placing lhe explant in lhe culture vessel). D,
Visible contamination percentages for lhe surface-sterilization procedures tested. The values were taken after 4 wk in culture. O Without
visible contamination. .Bacteria + yeasts. m Fungi. E, Single nade explant in CM medium (CÔrte and Mendonça, 1985), with differ-
entiated shoots and roots. F, Acclimatized young planto
246 MOURA
TABLE 1
;URFACE-STERILIZAll0N PROCEDURES TESTED FOR
HYPERICUM FOUOSUM
Alcohol
(%) Time
(min.) Bleach
(%) Time
(min.) HgCl2
(%) Time
(min.)
Plant Material Pr.
Whole
Plants 10
15
20
10
10
10
10
10
10
15
15
15
10
15
15
15
10
10
10
15
20
20
10
15
20
10
A
B
C
D
E
F
G
H
I
70
70
70
70
70
70
70
70
70
70
96
-
0.1
0.2
0.3
0.1
0.1
0.2
0.1
0.1
0.1
0.1
-
5
5
5
5
5
5
5
5
5
5
4--ó cm Stem
Portions
Without
Leaves
K
L
M
The culture vessels used were 125 X 25 mm Pyrex@ test tubes with Ka-
put@ translucent closures. AlI cultures were placed in a growing charnber at
21 :!: 10 C, with a light intensity ofapproximately 56j!mol'm-2's-1 and a
photoperiod of 16 h.
The acclimatization stage was carried out in Jiffy 7@ pois and in a 2:1 or
1:1 peat/perlite mixture. Single plants were kept inside 330 ml plastic green-
houses and, REter 6 to 8 wk, were transplanted to pois with humus-enriched
soil (Fig. 1 E).
The experiments conducted with seed-produced explants were carried out
in 22 to 24 test tubes, with a single explant each and repeated twice. For lhe
growth regulator tests and due to lhe plants reduced number of individuais,
each test tube with one single explant, was considered lhe experimental unit
and was replicated 48 times. The values used in lhe statistical analysis were
taken REter 4 wk in culture.
The multiplication rales (i.e., lhe number of single node cuttings produced
in vitro), were determined in lhe initiation stage by lhe product between lhe
number of nodes per normal shoot and lhe number of normal shoots per
explant. In lhe elongation stage, lhe multiplication rales were determined by
lhe number of nodes obtained per normal shoot.
When evaluating lhe contarnination rales, in lhe few cases where both fungi
and bacteria/yeast contaminants were visible, lhe contamination was consid-
ered fungal in lhe statistical analysis, because fungi development inside lhe
test tubes overcame those of lhe other contaminants.
A single factor analysis of variance (ANOVA) was performed whenever
there were sufficient amounts of data to justify it. Veri/ication of lhe homo-
geneity of variances, for each pararneter analyzed, was accomplished through
a graphical representation of residuais. When homoscedasticity was not ob-
served, lhe data was normalized with one lhe following transformations: x'
= arcsenv'x, x' = logx or x' = log (x + 1). For a multiple comparison of
meROS, Tukey's test was used at lhe 5% signi/icance leveI. The statistical
analysis and graphics were done with SYSTAT 5.2,running in a Macintosh
LCIII.
were rooted in the same medi um, without growth regulators. Though
there were no references to the use of Uoyd and McCown (1980) and
Côrte and Mendonça (CM, 1985) media for micropropagating this
specific genus, it was decided to also include them in the experiment,
because these media are known to have produced good results with
other woody plants (Côrte and Mendonça, 1985; George et aI., 1987;
Matos, 1992). RESULTS AND DISCUSSION
MATERIAIS AND METHODS
Plane material. Ali plant material carne from São Miguel island. The ex-
plant sources were young seedlings (Fig. 1 B and C), by in viera culture and
this year's growth from field established plants. The whole plants, produced
by seed germination on petri dishes with moist Whatman~ No. 1 papel, or
4--6 cm stem sections taken from field-established specimens, without leaves
nor apical portions (including lhe last three to falir nades), were previously
washed in running tap water for 3 h. The material was then surface-sterilized
and several procedures were tested (Table 1). 0.01% ofTween 20 was added
to ali treatments. The last stage was also identical in ali lhe experiments and
consisted of three rinses with sterile distilled water.
ln lhe initiation stage, lhe explants used were singie nade cuttings without
leaves and in lhe elongation and rooting stage, axillary shoots produced in
viera. The explants obtained from seed-produced plants were solely used in
lhe media experiments, while lhe field-collected material was uniquely used
in lhe growth regulator experiments.
Culture conditíons. The following basal media were tested: MS (Murashige
and Skoog, 1962), RB (Roest and Bockelmman, 1973), WPM (Lloyd and
McCown, 1980), CM (Côrte and Mendonça, 1985), and CK (Cellárová et ai.,
1992).
CM medium consists of a 2/5 dilution of lhe MS macronutrient solution,
lhe MS micronutrient solution, 15 mg/i (40.9 I!M) of NaFeEDTA, lhe Rose
Galzy (1964) vitamin formulation, and 1 mg/i (2.61!M) of vitamin D3. ln lhe
CM medium, vitamin D3 was omitted. Ali lhe media were supplemented with
20 g of sucrose, solidified with 7 g of Bacto Agar Difco~, and autoclaved at
pH 5.8. Furtber experiments were performed using CM medium supple-
mented with falir different growth regulators: NAA, BA, y,y-(dimethylallyl)
aminopurine (2iP), and kinetin (KlN).
ln lhe initiation stage, lhe effect of lhe falir growth regulators, in lhe fol-
lowing concentrations, was tested: 0.1, 0.5, and 1.0 mg/i. The matrix had ali
possible combinations between auxin and cytokinins. ln lhe elongation and
rooting stage, lhe medium was supplemented with 0.1 mg/i (0.41!M) BA, 0.1
mg/i (0.51!M) NAA + 1.0 mg/i (4.41!M) BA, 0.5 mg/i (2.61!M) NAA + 1.0
mg/i (4.41!M) BA, and 1.0 mg/i (4.91!M) 2iP. To specifically induce rooting,
two auxins were added to lhe basal medium: 0.1 (0.5 I!M) and 1.0 mg/i (5.3
!lM) NAA, 0.1 (0.41!M) and 0.5 mg/i (2.41!M) lBA.
Surface sterilization. The contamination (Fig. 1 D) in lhe tests
performed with laboratory seed-germinated plants (Table 1, proce-
dures A and B) was significantly different from those observed in
field-obtained stem cuttings (by Tukey's test, at lhe 5% leveI). In B,
no visible fungal contamination was detected. The best results using
field material was with procedures F; I, and M, with I being lhe most
efficient one, relative to necrosis (data not shown).
In vitro culture. CM medium gave lhe best results (Table 2, Fig. 1
E) in lhe initiation stage, with a multiplication rale of 2.8 nodes per
explant, against 2.2 for WPM, 1.8 for MS, 0.9 for CK, and 0.8 for
RB. In lhe elongation stage, CM algO produced lhe longest shoots,
lhe highest number of rooted explants, and lhe longest roots, though
these values were not significantly different by Tukey's testo
Although CM and CK produced lhe highest number of nodes per
normal shoot in lhe elongation stage (respectively, 2.8 and 3.0 nodes),
CK algO produced lhe highest percentages of shoot necrosis (approx-
imately 15% in lhe initiation and 23% in lhe elongation stages).
CM's performance can most likely be explained by its two-fifth's
dilution of lhe MS macronutrient solution, for it has already been
previously stated that a high ionic concentration (which is lhe case
with MS and CK media) has growth-inhibitory effects in several
woody species (McCown and Sellmer, 1987).
The complete lack of vitamins in RB's formulation can be pointed
as a possible explanation for its low efficiency, being referred by
several authors lhe importance of these substances, particularly thi-
amine, for in vitro cultures (Gamborg and Shyluck, 1981; Bhojwani
and Razdan, 1983; Boccon-Gibod, 1989);
The positive results obtained in lhe initiation stage in CM media
with added growth regulators are shown in Table 3. From these vari-
ous treatments tested in lhe elongation stage, only two produced posi-
247
MICROPROPAGATION or HYPERICUM FOUOSUM
TABLE 2
EFFECT OF BASAL MEDIUM ON SHOOT AND ROOT GROWTH OF HYPERlCUM FOUOSUM EXPLANTS
NOrnlal Shoots/Expl. Nodes/Nornlal Shoot Nodes/NornIaI Sh(K)! Shoot Length (mm) Rooted Shoots (%)
Elongation and Rooting Longes! Roo! Length (mm)
Media Inilialion
3.0a
2.3 a
2.5 a
1.4 b
2.4 a
3.0 a
2.8 a
2.2 ab
1.1 b
2.0 ab
5.5 a
6.9 a
4.8 a
4.5 a
4.4 a
8a
39a
Oa
26 a
18 a
2.5
5.6
CK
CM
MS
RB
WPM
0.3 a**
1.2 b
0.7 ab
0.6 a
0.9ab 2.2 a
3.8 a
*CK = Cellárová et al., 1992; CM = Côrte and Mendonça, 1985; MS = Murashige and Skoog, 1962; RB = Roest and Bockelmann, 1973; WPM = Uoyd
and McCown, 1981.
**Significantly different from b, by Tukey test, at lhe 5% levei.
TABLE 3
EFFECT OF DIFFERENT AUXIN AND CYTOKININ
CONCENTRATIONS ON CULTURE INffiATION OF
HYPERlCUM FOUOSUM
Growth Regula!ors
(mg/i)*
Nonnal Shoots/Explant Nodes/Nomlai Shoot
!nitiation
1.0
0.1
0.2
0.1
0.4
0.2
0.5
0.1
0.1
0.0
0.7
0.1
2.0
3.0
1.5
4.0
2.1
2.6
2.4
1.0
1.7
1.0
3.1
1.5
0.1 BA
0.5BA
1.0 BA
0.12iP
1.02iP
O.lNAA + 0.5BA
O.lNAA + 1.0BA
O.lNAA + 0.52iP
0.5NAA + O.lBA
0.5NAA + 0.5BA
0.5NAA + 1.0BA
0.5NAA + 1.02iP
*BA = N6-benzyladenine; 2iP = Y,Y-(dimethylallyl) aminopurine; NAA
= a-naphthaleneacetic acid.
tive results: 2.9 nodes per nonnal shoot with a mean shoot length of
7.0 mm in the media supplemented with 0.1 mg/l BA, and 4.4 nodes
per nonnal shoot with a mean shoot length of 4.7 mm in 0.5 mg/l
NAA + 1.0 mg/l BA. Neither one of the treatments promoted rooting.
Considering the various growth regulators, when added singly to
the medium, BA was the most efficient in promoting shoot differen-
tiation and proliferation, as stated by Cellárová et alo (1992) and
Roest and Bockelmann (1973) for Hypencum peiforatum. The best
results using an auxin and a cytokinin simultaneously happened in
an NAA + BA supplemented media. This was algo stated by Med-
eros (1991) for Hypencum canarien.se. In contrast to what was said
by Cellárová et aI. (1992) for Hypericum peiforatum, high BA con-
centrations did not improve results. The absence of rooting in the
presence of NAA and IBA, as mentioned by the same authors, was
algO confinned for Hypencum foliosum.
An increase in NAA, generally proved to be inversely proportional
to shoot differentiation. Its inhibitory effect was only counterbalanced
by BA's concentrations of 1 mg/l. It was observed that even with the
highest concentrations of BA, the results were very poor when in the
presence of 1.0 mg/l NAA. The inhibitory effect of NAA was further
confinned by a totallack of shoot differentiation on the modalities
supplemented only with this auxin.
The results obtained with NAA in the initiation stage only partially
agrees with Skoog and Miller's theory (1957), which states that shoot
versus root organogenesis is dependent on the proportion between
the exogenous auxinlcytokinin levels in the culture media. According
to these authors, a relatively high auxin concentration will promote
root differentiation and inhibit shoot development. The fact that the
results with NAA in this stage are so extreme, even when used in
low concentrations, suggest a possible high endogenous auxin leveI
present in the field-collected plant material (Tran Thanh Van and
Trinh, 1990).
During elongation, the inefficiency of 1 mg/l2iP and of 0.1 mg/l
NAA + 1 mg/l BA, was obvious. With 0.1 mg/l of BA, an auxin
source did not seem to be needed, whereas it was required when 1.0
mg/l of BA was added. This suggests the need of a compensatory
effect from an exogenous auxin source, which mar be a sign of a
weak apical dominance in the in vitro produced Hypericumfoliosum
shoots.
Tamas (1987), summarizing various work dane in apical domi-
nance, indicates that the lack of apical dominance mar lead to the
channelling of the available cytokinin mainly to the axillary buds in
detriment of the apex, thus implementing an axillary shoot devel-
opment instead of the main shoot growth. On the other hand, and
according to the same author, the accumulation of cytokinin in the
apical bud, due to a strong apical dominance, increases the produc-
tion of endogenous auxins in this structure, which have a suppressive
effect on the axillary caulogenesis.
NAA did not promote rooting, as neither did any of the cytokinins
tested alone or combined with this auxin. The results obtained with
NAA seem to indicate that the caulogenesis suppression effect at-
tributed to this growth regulator is, in this plant, dissociated from its
rooting effect. In fact, having been observed the first in a remarkable
way during initiation, the later was never verified.
Rooting algo was not promoted by the addition of IBA and, in a
last instance, the absence of rooting in alI the growth regulator tests
mar be explained by the fact that these were conducted with material
obtained from field-established plants. Though the year's growth was
used as the preferred explant source, its degree of differentiation is
higher than the one expected in a seedling or in a plantlet, increasing
thus the difficulty of in vitro rooting (Debergh, 1988).
Considering that the number of nodes per explant obtained in the
initiation stage, in a nonsupplemented CM, was slightly higher than
the one obtained with growth regulators, and that the shoot length
and number of nodes per normal shoot in the elongation stage were
248 MOURA
not very different from lhe values obtained in supplemented media,
it seems that lhe micropropagation of Hypericum foliosum could be
efficiently done in a growth-regulator-free CM medi um. However, be-
cause lhe seed-produced explants used in lhe media tests and lhe
field-established explants used in lhe growth regulators tests were
not interchangeable, it is possible that lhe results obtained in a sim-
pIe CM could be improved using growth regulators with seed-pro-
duced explants.
Mter 4 wk of acclimatization to progressively lower humidity lev-
eIs, there were no differences in lhe shoot length and number of
nodes observed between lhe plantlets potted in lhe three media used.
However, lhe plantlets rooted in Jiffy 7@ pellets originated new
shoots. Mter 8 wk of acclimatization, these new shoots looked normal
and lhe plantlets had a well-developed root system. The plantlets
rooted in peatfperlite mixtures not only did not develop any shoots,
but lhe plantlets showed signs of necrosis.
CONCLUSIONS
Culture initiation from laboratory seed-produced plants was the
preferable technique for Hypericumfoliosum's micropropagation. The
most efficient surface-sterilization technique tested with seed-pro-
duced plants was B(15% sodium hypochlorite + 0.01% Tween 20
for 15 min) and lhe procedure labeled as I was the best for field
material (70% alcohol for 1 min + 10% sodium hypochlorite +
0.01 % of Tween 20 for 20 min + 0.2% mercuric chloride for 5 min).
We also conclude that CM was the most efficient medium for alI
culture stages and that the best results with growth regulators are
achieved in the following supplements: 0.1 mg/l (0.4 ~ BA and
0.5 mg/l (2..6 ~NAA + 1.0 mg/l (4.4 ~ BA, in the initiation
stage, and 0,1 ii1g/l(0.4 ).iM)BA in the elongation stage. For culture
multiplication,.O.lmg/l (0.4~ BA (in the initiation stage) and 0.5
mg/l (2.6 I.LM) NAA + 1.0 mg/l (4.4 ~ BA (in the initiation and
elongation stages) are lhe most efficient supplements.
The acclimatization stage was successfully performed in Jiffy 7@
pellets. Using these conditions, it would be possible to produce two
to four plantlets from single nodal explants in approximately 4 mo.
ACKNOWLEDGMENTS
This study was cofinanced by the European Community "CIf:NCIA" pro-
gram and by the "Associaç;ão de Municípios da Região Aut6noma dos
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