ACTA
PHYSIOLOGIAE
Effect of medium composition and type of vessel closure on axillary shoot
production of magnolia in vitro
A urelia Kamenicka, Maria Lanakova
Institute of Forest Ecology, Slovak Academy of Sciences, Vieska nad Zitavou, 951 52 Slepcany, Slovak Republic
Key words: Magnolia x soulangiana Soul.-Bod.,
axillary shoots, media composition, media concen-
tration, type of vessel closure
Abstract
Proliferationof axillaryshoots from nodal segmentsof saucer
magnolia (Magnolia x soulangiana Soul.-Bod.)was achieved
on modifiedStandardiand Catalano(S medium)and Lloydand
McCown (WPM) medxa containing 1.33 gmol.dm- BA and
0.54 ~mol.dm " NAA. The greatestnumber of axlllaryshoots
was producedon S-mediumwith full strengthmacronutrients.
Statisticallysignificantwere the differencesin biomassof axil-
lary shoots cultured in vessels sealed with plastic closures.
Rootingof the shoots was achievedon half strengthS medium
supplemented with 4.9, 9.8, 14.7 and 19.6 gmol-drn- IBA.
Rootedplantletswere able to resume independentgrowthafter
a short period of acclimatization.
List of abbreviations: 6-benzylaminopurine (BA),
(x-naphtaleneacetic acid (NAA), 4-(3-indolyl) bu-
tyric acid (IBA)
Introduction
Environmental factors are very important for the
growth, development and morphogenesis of tissue
cultures. Plant growth in vitro is affected by many
factors, such as photoperiod, temperature, exoge-
nous growth regulators, relative humidity in the
culture vessels and medium composition (Kozai et
PLANTARUM
Vol. 22. No. 2. 2000:129-134
al. 1995). Shoot induction, proliferation and bio-
mass production directly and indirectly are influ-
enced by the type of culture vessels and their clo-
sures as well, (Monette 1986, Mackay, Kitto 1988,
McClelland, Smith 1990). The type of culture ves-
sel closures stimulated growth of the explant cul-
ture (Tricoli 1983, Kamenicka 1996). The culture
closures can have not only a stimulative effect on
the growth of explant culture (Fitter, Krikorian
1985), but they can induce necroses of shoot tips
and branching of shoots (Sha et al. 1985).
Tissues of magnolias are difficult to disinfect prior
to attempting to place them in sterile culture
(Tubesing 1998). For the establishment of magno-
lia culture juvenile shoots, axillary buts, shoot tips
(direct regeneration) or unripe embryos (somatic
embryogenesis) are used as primary explant in mi-
cropropagation. Biederman (1987) concluded that
the best time for primary explants is the period after
dormancy. Success in regeneration depends on the
choice of the culture media and growth conditions.
Some species and cultivars of magnolia have differ-
ent culture media requirements, modification of
culture medium is often inevitable for magnolia re-
generation in vitro (Biedennan 1987, Kamenicka,
Valka 1997).
129
A. KAMENICKA & M. LANAKOVA
The objective of this research was to study the in-
fluence of media composition, media concentration
and type of vessel closure on propagation of mag-
nolia in vitro.
Materials a n d M e t h o d s
Apical shoots (7-10 cm long) of saucer magnolia
(Magnolia x soulangiana Soul.-Bod.) consisting of
3 to 4 internodes were taken in spring (April) from
100-year old trees growing at the Arboretum
Mlyfiany. After removal of leaves the shoots were
sterilized in solution of HgC12 (0.1-0.3 %) with
three drops of Tween 20 (0.03-0.05 %) for 5-7 min.
Then the shoots were soaked in 70 % ethanole (20 s)
and rinsed (three times) with sterile water. Shoots
were cut for nodal segments (3-5 mm long) and cul-
tured on S medium. The primary culture was trans-
ferred after 6-8 weeks on fresh medium (Fig. 1).
Shoots were cultured on S medium o r S1/2 concen-
tration medium (Standardi, Catalano 1985) or
WPM or WPM1/2 m e d i u m (Lloyd, M c C o w n
1980). Shoots were cultured in 100 ml culture ves-
sels containing 25 ml of medium. The culture ves-
sels were closed with metal (SMet) or plastic (Spvc)
light permeable closures with vent membranes
(FORING-LOCK M 82.5 x 24/6). The culture me-
dia were supplemented with 20 g-dm -3 of sucrose, 7
g.dm -3 of agar, 1.33 gmol.dm -3 BA and 0.54
~tmol.dm-3 NAA. All cultures were kept at a 16 h
light (35-40 ~tmol.m-2.s -1) and 8 h dark photocycle
at 20-24 °C.
Table 1. In vitro establishmentof magnoliaculture on differentculture media
Media ContaminationCulturesshowing Shoots perexplant±SE Length ofshoot
(%) new growth (%)
WPM 11 62.4 4.92±0.135
WPM1/2 14 68.6 4.42±0.134
S 12 89.4 8.30±0.440
Sin 10 74.2 6.17_+0.277
Table 2. Biomassof magnoliaculture
Media Fresh weight(g)±SE
WPM 0.678_+0.020
WPMln 0.528±0.020
S 1.693±0.066
Sin 1.123±0.049
130
After 8 weeks number, length, fresh and dry weight
of axillary shoots were recorded. In each experi-
ment, 70 explants were used in three repetition.
Shoots 3.0 to 4.0 cm long were separated from the
culture and rooted on S1/2 medium suplemented
with 4.9, 9.8, 14.7 and 19.6 ~amol.dm-3 IBA, 0.3 %
activated charcoal, 20.0 g.dm -3 sucrose and 7.0
g.dm -3 agar (Phyto Agar from Duchefa Biochemie
B.V. Haarlem, The Netherlands). Rooting medium
without IBA was marked as control. All chemicals
were purchased from SIGMA (St. Louis, MO,
USA). Five shoots were placed into 250 ml culture
jar. Growth conditions were the same as above.
After 5 weeks, number of rooted shoots, number of
roots, total root length, fresh and dry weight of roots
were recorded. Results were evaluated using
SAS/Statistical program package for personal com-
puters (SAS Institute Inc., USA, 1995). Signifi-
cance of the treatment effects was determined by
analysis of variance (Statgraphics, Version 6, SAS
Institute Inc.USA).
Results and Discussion
Culture e s t a b l i s h m e n t a n d multiplication
The effect of different culture media on the forma-
tion of axillary shoots was examined (Table 1).
Contamination (caused by fungi) of primary cul-
ture was generally low. A comparison of nodal ex-
plants cultured on WPM medium with WPM1/2 in-
dicated that differences were similar. Shoots pro-
Totalcapacity
(mm)±SE ofproli~ration
14.94±0.255 73.5
17.04_+0.446 75.3
16.24±0.066 134.8
16.67±0.335 102.9
Dry weight(g)_+SE FM/DM
0.070+0.002 9.69
0.065-+0.003 8.12
0.131_+0.004 12.92
0.096_+0.003 11.70

Fig. 1. Primary magnolia culture planted on S mediumcontain-
ing 1.33 pmol.dm~' BA and 0.54 pmol.dm-3 NAA
duced on WPM medium were shorter but had more
nodes. The type of culture medium significantly in-
fluenced the mean number of shoots formed per ex-
plant. The capacity of proliferation was different
ranging from 134.8 to 73.5. A small quantity of cal-
lus was associated with the base of culture on WPM
media. The highest number of shoots per expant
was obtained on S medium and the greatest shoot
length was obtained on WPM1/2 medium. The
number of axillary shoots produced on WPM media
was approximately half in comparison with S me-
dia (Table 1). The shoots grown on S medium were
characterized by great leaf size and succulence.
These results indicated the importance of medium
composition (ionic strength and proportion) for the
growth and development of magnolia culture. In
the present study, as in those of other forest tree spe-
cies (Bonga, Aderkas 1992, Iliev, Trifonov 1996),
the success of in vitro shoot proliferation depends
on culture media.
T h e mean fresh and dry weight were higher on S
media in comparison with W P M media (Table 2).
Variation in FW and D W of explant were related to
water and minerals absorption of explant during
culturing (Valova et al. 1996). Explants grown on S
EFFECT OF MEDIUM COMPOSITION ...
/ U .......... :" ....... ........................ : .......... ............. ::;" °:i
Dcor~rot
Jcu~e
0 10 20 30 56
days
Fig. 2. Changes ofpH medium during growth of magnolia cul-
ture on S medium.
media had FW and D W values higher than WPM
media (Table 2). Culture media (S) FW/DW ratio
reached high values. Difference between W P M and
S media was measured in total biomass production,
we suspected that the level macroelements of the
media may be responsible for these values (as
demostrated by the fresh weight/dry weight ratios,
Table 2).
T h e presence of explant on culture medium in-
duced change of pH. A level of pH medium with
culture increased during 56 days of culturing, pH
media without culture remained stable (Fig. 2).
Singha et al. (1987) reported that pH monitoring is
important, especially if the cultures are growing
fast. In accordance with literature to date, we found
that pH medium from 5.6 to 6.7 was optimal for cul-
turing because magnolia prefer fertile soil with al-
kalinine reaction (Hunt 1998).
Effect of vessel closures on multiplication
The effect of different vessel closures on formation
of axillary shoots is shown in Table 3. The results
have confirmed that the differences in the number
of axillary shoots between cultures grown in culture
vessels closed with metal or plastic caps are not sig-
nificant. A higher number of axillary shoots was
produced on medium with plastic closures. The
mean length of shoots statistically was not signifi-
cant.
Stimulative effect of the plastic closures was found
on production of fresh weight (Table 3). Mackay
and Kitto (1988) found that various types of clo-
sures used to seal vessels influenced the fresh
weight, although dry weight of shoots statistically
131
A. KAMENICKA & M. LANAKOVA

Table 3. Effect of vessel closures on growth of magnolia cul- Table 4. Effects of IBA concentration on rooting of magnolia
ture microcuttings
Media Number of Fresh weight_+SE IBA Number Length of Rooting %
shoots+-SE (lamol.dm3) roots/shoot roots (mm)
SMct 4.42-+0.225 0.79-+0.033 0.0 0.9 12.0 28.7
Spvc 5.78+-0.237 1.21-+0.042 4.9 1.3 30.4 45.0
9.8 3.9 26.8 65.7
Analysis of variance 14.7 5.3 24.5 83.3
19.6 6.8 23.4 96.2
Source of df Mean squares
variation Analysis of variance
No shoots/explant Productionoffmshweight
Vessels 1 0.7 7.1"
closure Source of df Mean squares
variation
Error 98 0.1 0.3 No roots/shoot Length of roots
Auxins 4 54.4* 8.3*
*Significant at P = 0.01 Error 55 7.2 0.4
Culture vessels with metal (SMct)and plastic (Spvc) closure
*Significant at P = 0.01
was not significant. McClelland and Smith (1990)
described that the effect of types vessel closure also
depends on the requirements of individual plant R o o t i n g o f microcuttings
species. Our results confirmed that multiplication
S h o o t s with 2-3 c m length were exiced from cul-
of axillary shoots was more intensive in culture ves-
tures and placed on rooting m e d i u m (Table 4). In
sels sealed with light permeable closures. The sig-
the absence of plant growth regulators, shoots grew
nificance o f differences in fresh weight was meas-
normally, but root production was sporadic (0.9).
ured by the analysis of variance.
Increasing level of I B A stimulated root production,
but length of roots decreased. Vitrification and cal-

Fig. 3. Plantlets rooted in

vitro, from left to right
S1/2 medium containing
19.6 pmol.dm-3 IBA and
control medium without
plant growth regulator.

132

2: @: 7:¸¸¸¸ i £ ¸¸
Fig. 4. Magnolia plants
after transferfrom green-
house conditionsto soil.
lus formation at the shoot base were little or without
callus.
Shoot morphology was normal. Increasing IBA
levels increased rooting of microcuttings without
significant increase of callus formation. The high-
est number of roots per shoot was obtained when
the medium was supplemented with 19.6 ~mol.dm-3
IBA, and the greatest root length was measured
when the medium was supplemented with 4.9
~tmol.dm-3 IBA (Fig. 3). Significant differences in
the number of roots per shoot and length of roots
with various levels of IBA were also calculated by
the analysis of variance (Table 4).
Rooted plants were transferred to the greenhouse
and rooted in sand and peat (2:1) mixture. Because
high atmospheric humidity at this stage is very im-
portant for plant survival, polyethylene covers and
mist were used to maintain a relative humidity of
80 % to 95 %. Ninety percent of rooted plants were
successfully acclimatized and established in the
greenhouse. After acclimatization (6-8 weeks),
pots containing the plants were trasferred to a soil
(Fig. 4).
EFFECT OF MEDIUM COMPOSITION ...
:: /
Acknowledgement
Special thanks are expressed for the support of this
project 2/5057/99 to the Grant Agency of the Slo-
vak Academy of Sciences.
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Received July 01, 1999; accepted March 17, 2000