In vitro organogenesis of two Sansevieria trifasciata cultivars on different concentrations of benzyladenine

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AGRIVITA VOLUME 33 No. 2 JUNE-2011 ISSN : 0126-0537
IN VITRO ORGANOGENESIS OF TWO SANSEVIERIA CULTIVARS
ON DIFFERENT CONCENTRATIONS OF BENZYLADENINE (BA)
Yusnita*), Wiry Pungkastiani2) and Dwi Hapsoro3)
Plant Science Laboratory, Department of Agronomy, College of Agriculture, The University of Lampung.
Jl. Sumantri Brojonegoro no. 1 Bandar Lampung 35145
*) Corresponding author Phone: +62-8128145990 E-mail: yusnita.said@yahoo.com
Received: June 23, 2010/ Accepted: March 2, 2011
ABSTRACT
In vitro regeneration of buds and shoots via
organogenesis in two genotypes of Sansevieria
trifasciata was established. Leaf segments (1cm
x 1cm) of cv. Hahnii and cv. Lorentii were
cultured on Murashige and Skoog (MS) basal
medium containing 2.4-dichloro-phenoxyacetic
acid (2.4-D) for 1 week, trans-ferred into MS
medium without plant growth regulator (MS0) for
1 week, and then cultured to MS medium
containing different concentrations of
benzyladenine (BA:0; 0.1; 0.25; 0.5; 1 and 2
mg/l) for 3 consecutive passages with 4 week
intervals. The results showed that BA proved to
be an effective cytokinin to induce the formation
of adventitious buds and shoots in two cultivars
of Sansevieria trifasciata. The maximum of 12
propagules per explant of Lorentii and 9.3
propagules of Hahnii were obtained in medium
with 2 mg/l BA after 14 weeks. Furthermore,
regenerative capacity to form shoot buds and
propagules was genotype dependent. The
popagules number formed by cv. Lorentii was
significantly higher than those formed by cv.
Hahnii. The average shoot length formed by cv.
Lorentii was also higher than those of cv. Hahnii.
Shoots of both cultivars were successfully ex
vitro rooted and acclimatized to the greenhouse
with high survival rate (95.9 -100%).
Keywords: organogenesis, Sansevieria trifas-
ciata, benzyladenine, genotypes
INTRODUCTION
Sansevieria (Sansevieria trifasciata) or
snake plant is a genus of ornamental foliage
suitable for either outdoor or indoor plants.
Plants from this genus are widely used for their
beautiful and various shapes, colors, and
patterns of their leaves. The color of its leaves
varies from dark green, pale green, grayish
green or a combination of green and white or
yellow with a various snake skin pattern of the
lamina. In addition to ornamental foliage,
sansevieria is used as a plant that absorbs
hazardous pollutant from the atmosphere. A
research conducted by NASA found that snake
plants were capable of absorbing carbon
monoxide and lead from atmosphere. In Japan,
it was reported that sansevieria could absorb
many hazardous gases from atmospheres such
as chloroform, formaldehyde, trichloroethylene,
benzene and xylene in a significant amount
(Suara Pembaharuan, 2005). People in Thailand
believe that snake plants could be used as
traditional medicinal plants for several diseases
such as diabetes and cancer. Korean people
believe that this genus can be used to overcome
effect of various radiation, and some people of
China think that planting sansevieria brings
some good luck.
Since the year 2000, demand of sansevie-
ria in Indonesia has dramatically increased and
reached the highest in 2004, and remains high
up to now. The cultivars of sansevieria mostly
needed are cv. Lorentii or sword type and cv.
Hahnii or the short type. Some of the local
markets of sansevieria increase significantly due
to the high demand for export. During the last
five years, demand of sansevieria, especially
from Korea and Japan, has been considerably
high. For instance, since April 2005, 3500 plants
of cv.Hahnii had been delivered for Malaysian
buyers and one container of sansevieria was
ordered by each of the buyers from Korea,
Japan and Europe (Kontan, 2005).
The increase in sansevieria demand leads
to an increase in both local and export market
which in turn makes farmers grow this foliage
extensively. However, extensive culture of

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
sansevieria could not fulfill the demand yet due
to slow growing manner of plant propagation.
Sansevieria is conventionally propagated by
suckers and leaf cuttings. Both methods need
plenty amount of plant materials and a long
period of time to produce significant number of
propagules. Generally, only 1-3 propagules are
obtained from 1 leaf cutting for 2 months, and
until 5 months the number of propagules
remains constant without proliferarion. Plant
tissue culture could be an alternative to the
conventional methods to produce a large
number of propagules in a relatively short time.
One pattern of in vitro regeneration of
sansevieria is via organogenesis.
Blazich and Novitzky (1984) previously
reported in vitro organogenesis of Sansevieria
tifasciata using leaf segments as explants. In
their report, explants were cultured on Murashige
and Skoog (MS) (1962) medium supplemented
with 0.25 mg/l 2,4-dichlorophenoxy acetic acid
(2,4-D) for 2 weeks followed by cultures for 2
more weeks on MS without growth regulator
(MS0) to induce callus formation. To induce
adventitious shoot formation, explants were then
transferred to MS + 0.3 mg/l kinetin.
Wahyuningsih (2006) found that when 1x1 cm2
leaf segments of Sansevieria trifasciata var.
Lorentii were cultured on callus induction
medium, followed by explant transfer to MS
medium containing benzyladenine or kinetin,
formation of adventitious shoots was observed.
Responses of different cultivars to a certain
growth regulator could be different as reported
by Veltcheva and Svetleva (2005) in in vitro
organogenesis of Phaseolus vulgaris L. This
research was aimed to study the formation of
adventitious shoots of two varieties of
Sansevieria trifasciata (cv. Hahnii and cv.
Lorentii) as affected by different concentrations
of benzyladenine (BA).
MATERIALS AND METHODS
Plant Materials and Explant Preparation
This research was conducted in Plant
Tissue Culture Laboratory, Department of Agro-
nomy, College of Agriculture, The University of
Lampung from March to August 2006. The plant
materials were obtained from the local market in
Lampung. The source plants of Sansevieria
trifasciata cv. Hahnii and cv. Lorentii were
maintained at green house with routine culture
procedures including watering, cleaning of the
leaves and spraying with pesticide. Explants
were taken from fully expanded young leaves
cut at ¾ of the upper part for Hahnii and ½ of the
upper part for Lorentii. Explant surface-
sterilization was started by washing the leaf
cuttings using detergent under tap water for
approximately 20 minutes. After that, leaf
segments of 3 cm in size were rinsed in 5% of
commercial bleach solution for 15 minutes
followed by rinsing with tap water. The second
sterilization was done in laminar air flow cabinet
(LAFC, Bassaire, Hampshire, England). The leaf
segments were soaked in 10% of Bayclin
commercial bleach (Johnson, Jakarta, Indonesia,
5.25% NaOCl) solution for 15 minutes followed by
three-time rinsing with sterile aquadest. One
leaf blade of sansevieria cv. Hahnii was cut into
± 20 explants, whereas that of cv. Lorentii was
cut into 40-50 explants.
All disecting tools, ie., Petri dishes (Iwaki,
Asahi Glass Co. Ltd. Thailand), forceps, scalpels
(Maiden Stainless, Japan), bottles (Jakarta,
Indonesia) and aquadest were sterilized by
autoclaving (Tommy®, Tokyo, Japan) at 1210C
and 1.5 kg/cm2 for 30 min. After the surface
was sterilized, explants were cut aseptically to
approximately 1 cm x 1 cm and cultured on
callus inducing medium which was MS basal
medium supplemented with 30 g/l sucrose,
vitamine and 0.25 mg/l 2,4-D (Merck KgaA,
Darmstadt, Germany) for one week, followed by
transferring to MS medium defoid of growth
regulators (MS0). After being incubated for one
week in MS0, all explants were transferred to
shoot-inducing medium with different con-
centrations of benzyladenine (BA) (Merck KgaA,
Darmstadt, Germany). Subcultures to the fresh
media with the same treatments were conducted
twice in 4 weeks intervals. All cultures were
incubated in a culture room at 26±20C provided
by cool-white fluorescent light (Phillip, Indonesia)
at 2000 lux continuously.
Organogenesis of Two Sansevieria Trivasciata
Cultivars on Different Concentrations of BA
Treatments were arranged in factorial with
2 factors, i.e., first, Sansevieria trifasciata
cultivars (Hahnii and Lorentii), and the second
were concentrations of BA (0, 0.1, 0.25, 0,5, 1
and 2 mg/l). The experiment in this research
was done in randomized block design with 5
replicates and each replicate consisted of 4

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
explants. Number of shoot buds (meristemoid
structures < 0.5 cm), shoots ( ≥ 0.5 cm in length)
and propagules (i.e., buds + shoots) and length
of shoots were recorded after 14 weeks; in some
treatments, it was after 18 weeks of cultures. In
addition, cultures of sansevieria cv Lorentii with
BA 0.5, 1.0 and 2.0 mg/l were subcultured three
times, and the number of shoot buds and shoots
were counted after 18 weeks. To measure all
observed parameter, the plants were removed
aseptically to a sterile Petri dish with millimeter
block placed under the dish. Data was subjected
to analysis of variance (ANOVA) using Minitab
Statistical Software version 13.20. If there was
any significant results of ANOVA, Least Signi-
ficant Difference (LSD) method (Lentner and
Bishop, 1986) at P≤ 0.05 was used to detect
differences among treatments.
The basal medium formulation used for
callus, meristemoid and shoot induction was
macro and micro salts of Murashige and Skoog
(1962) medium supplemented with 30 g/l
sucrose, 0.1 mg/l thiamine–HCl, 0.5 mg/l nico-
tinic acid, 0.5 mg/l pyridoxine-HCl, 2 mg/l glycine
and 100 mg/l myo-inositol (Merck KgaA,
Darmstadt, Germany). The pH of medium was
adjusted to 5.8 with 1N KOH or 1N HCl prior to
addition of 7 g/l agar powder. The mixture was
then boiled to make the agar soluble and a 30
ml-aliquots of medium were dispensed to 250
ml- culture bottles before autoclaving at 1210C
and 1.5 kg/cm2 for 20 min.
Ex vitro rooting of shoots (4-5 cm in
length) was done by treating the base of the
shoots with 0.2% (w/w) indolebutyric acid (IBA)
(Merck KgaA, Darmstadt, Germany) and
planting them in a 10 cm pot in diameter
containing (1:1) mixture of sand and compost,
then placed on benches in a shaded
greenhouse. Irrigation with tap water was given
once a day. After two months, survival of
acclimatized plantlets was recorded.
RESULTS AND DISCUSSION
Results of this experiment showed that in
vitro indirect organogenesis of two varieties of
Sansevieria trifasciata can be done successfully
from leaf segments as explants. Organogenesis
of Sansevieria trifasciata leaf explants was
initiated by the formation of callus at the cutting
edge of explants. All explants from the two
cultivars tested swelled, and formed callus and
greenish meristemoid during the first two weeks
of cultures, and when explants were transferred
to shoot-inducing medium containing various
concentrations of BA, shoot bud and shoot
initiation occured.
In general, both BA concentrations and
sansevieria genotypes affected organogenesis
capacity of the explants tested. However, there
was no interaction between BA concentrations
and cultivars which affected variables recorded.
The highest number of shoot buds and
propagules in both cultivars was formed at
medium with 2 mg/l BA. In addition, Sansevieria
trifasciata cv. Lorentii showed higher
organogenic capacity than that of cv. Hahnii
(Table 1). The culture appearance of Sanse-
vieria trifasciata cv. Hahnii is presented in Figure
1, while that of cv. Lorentii is in Figure 2. Those
figures showed that regenerated shoots of cv.
Hahnii were shorter than those of cv. Lorentii.
The mode of regeneration of two cultivars
of Sansevieria trifasciata reported in this
experiment was similar to the ones reported by
Shahzad et al (2009) in Sanseviera cylindrica,
i.e through indirect organogenesis. In these
experiments, leaf explants were induced to form
callus on IBA-containing medium or to form
nodules on 2,4-D or 2,4,5-T containing medium,
then the explants were induced to form shoots
on BA+NAA containing medium. Their results
showed that a maximum number of 17.6 shoots
per culture was obtained from callus culture on
medium with 5µM (1.1 mg/l) BA and 2 µM (0.4
mg/l) NAA, whereas from nodule culture, a
maximum of 25 shoots was obtained on medium
with 5µM (1.1 mg/l) BA and 1µM (0.2 mg/l) NAA.
Previously, Anis and Shahzad (2005) have
reported direct shoot regeneration from leaf disc
explants of Sansevieria cylindrica. In their
experiment, the highest number of shoots per
explants were obtained in MS medium
supplemented with 10 µM (2.25 mg/l) BA and
0.1 µM NAA.

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
Table 1. Effects of cultivars and benzyladenine (BA) concentrations on number of shoot buds, number of
shoots, number of propagules per explant and shoot length of two sansevieria cultivars after 14
weeks of cultures
Sansevieria
cultivars
BA
concentrations
Number of
Shoot buds
per explants
Number of
Shoots per
explants
Number of
Propagules per
explants
Shoot length (cm)
cv. Hahnii 0.00
0.10
0.25
0.50
1.00
2.00
2.0 f
4.0 e
4.1 e
4.8 de
5.4 d
7.0 b
1.5 c
2.5 ab
3.3 a
2.0 bc
1.8 bc
2.3 bc
3.5 d
6.5 c
7.1 c
6.8 c
7.2 c
9.3 b
0.7 cd
0.9 abc
0.7 cd
0.6 cd
0.5 d
0.6 cd
cv. Lorentii 0.00
0.10
0.25
0.50
1.00
2.00
2.0 f
4.5 de
5.0 d
6.1 c
7.6 b
8.6 a
1.6 c
3.5 a
2.6 ab
2.4 bc
2.6 ab
3.4 a
3.6 d
8.0 c
7.6 c
8.5 c
9.2 b
12.0 a
0.6 cd
1.4 a
1.1 bcd
0.8 bcd
0.8 bcd
0.9 bc
Significancy based on ANOVA (analysis of variance)
ns = non-
significant
* P ≤ 0.05
** ≤ 00.01
Main Effects :
Cultivars (C)
BA (B)
C x B
**
**
ns
ns
**
ns
**
**
Ns
**
*
ns
Remarks= Mean values at the same column followed by the same letters indicate that there was no significant
difference among treatment based on LSD at P≤ 0.05.
\\
Figure 1. Appearance of Sansevieria cv. Hahnii cultures on MS medium with various concentrations of BA
after 14 weeks

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
Figure 2. Appearance of cultures of Sansevieria cv. Lorentii on MS medium with various concentrations of
BA after 14 weeks.
In this experiment, callus and meristemoid
induction occured when explants were cultured
on MS medium supplemented with 0.25 mg/l
2.4-D for 1 week followed by transferring to MS
medium without growth regulator (MS0) for 1
week. In previous reports (Blazich and Novitsky,
1984; Wahyuningsih, 2006), callus and
meristemoid induction was done by culturing
explants on MS+2.4-D for 2 weeks, followed by
transferring to MS0 for 2 weeks, so merestemoid
formation required 4 weeks.
Results of this experiment also revealed
that genotypes significantly affected regeneration
capacity of explant to form shoot buds or shoots.
Sansevieria trifasciata cv. Lorentii showed
higher organogenic capacity than cv. Hahnii,
which was indicated by the higher number of
shoot buds (8.6).The propagules (12.0) obtained
in cv. Lorentii compared to those of cv. Hahnii
(7.0 and 9.3, repectively). The mean length of
regenerated shoots of cv. Lorentii was also
higher than that of cv. Hahnii. The optimum
concentration of cytokinin which resulted in the
best multiplication rate of any micropropagation
could be species or even cultivar specific.
Hodson de Jaramilo et al. (2008) found that
cultivars affected shoot regeneration capasity in
chrysanthemum (Dendranthema grandiflora)
organogenesis. When subjected to the same
BA+NAA containing medium, explants from the
three chrysanthemum cultivars tested showed
different responses. Genotype specificity for in
vitro shoot regeneration was also reported by
Hirimburegama and Gamage (1997) in tissue
culture of banana and plantain. In in vitro
organogenesis of sugar cane, Gandonou et al.
(2005) and Ali et al. (2008) also reported that the
best medium and growth regulators for callus
and shoot induction depended upon the clone or
the variety being cultured. The higher number of
shoots per explant which was obtained in
micropropagation of Sansevieria cylindica
reported by Shahzad et al (2009) compared to
the ones obtained in this experiment might be
due to genotype dependent factor as well as the
mode of regeneration and different growth
regulators applied. Sriskandarajah et al. (2006)
suggested that different regenerative capacity in

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
different species or genotypes was due to
different endogenous phytohormones as well as
different activities of several enzymes related to
auxin and cytokinin metabolism, which in turn
shifted the auxin and cytokinin pool.
Shoots of both cultivars were successfully
ex vitro rooted and acclimatized in the green
house with 95.9-100% survival rate. Figure 3
shows the acclimatized plantlets of Sansevieria
trifasciata cv. Hahnii (a) and cv. Lorentii (b) after
2 months in the green house.
The propagation efficiency of the two
cultivars of Sansevieria trifasciata by this
micropropagation method was much higher than
conventional propagation by leaf cuttings. Using
leaf cuttings, we can get maximum of 2 leaf
cuttings per blade of cv. Hahnii and maximum of
5 leaf cuttings per leaf blade of cv. Lorentii. In
two months, if 2-3 plants are regenerated from
each cutting, a maximum of 6 plants of cv.
Hahnii and 15 plants of cv. Lorentii can be
obtained, and since the shoots obtained by leaf
cutting would not proliferate, the number of
plants would not change until four or five
months. Using this micropropagation method,
we can get an average of 20 explants per leaf
blade from cv. Hahnii and 40 explants per leaf
blade from cv. Lorentii. Considering an average
80 % success of sterilization and subculturing,
we can obtain an average of 16 aseptic cultures
of cv. Hahnii and 32 aseptic cultures of cv.
Lorentii per leaf blade. Based on the data
obtained in this experiment, after 4.5 months (18
weeks) we can get potentially 32 x 11 = 352
shoots of cv. Lorentii per leaf, provided that after
the third subcultures (at 18 months) 11.1 shoots
are obtained from cultures with 2 mg/l BA. The
figures of cv Hahnii, on the other hand, based on
the data available (from 14 weeks of cultures),
we can get potentially 16 x 3 shoots = 48 shoots
per leaf blade in 14 weeks (3.5 months). If one
more subculture was conducted, we might
expect at least doubled shoots per explant, and
the figures with Hahnii might become 96 shoots
obtained per leaf. With 95% survival rate of
acclimatization, 91 plants of cv Hahnii and 334
plants of Lorentii would be obtained per leaf
blade. Compared to propagation by leaf cuttings,
micropropagation would potentially be about 15
times as efficient as leaf cuttings for sansevieria
cv Hahnii, and about 22 times as efficient as leaf
cuttings for cv. Lorentii.
a b
Figure 3. The acclimatized plantlets of Sansevieria trifasciata var Hahnii (a) and var. Lorentii (b) in the
greenhouse (two-moth)

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Yusnita et al.,: In Vitro Organogenesis of Two Sansevieria trifasciata Cultivars...............................................................
CONCLUSSIONS
Successful in vitro organogenesis of
Sansevieria trifasciata var. Hahnii and Lorentii was
performed. Results showed that both BA
concentrations and Sansevieria cultivars affected
organogenesis capacity of the explants. However,
there was no interaction between BA con-
centrations and cultivars which affected variables
recorded.
The highest number of shoot buds and
propagules in both cultivars was formed at
medium with 2 mg/l BA. Sansevieria trifasciata cv.
Lorentii showed higher regeneration capacity than
that of cv. Hahnii. Shoots of both cultivars were
successfully ex vitro rooted and acclimatized in the
green house with 95.9 % - 100% survival rate.
Compared to propagation by leaf cuttings, this
micropropagation method would be approximately
15 times as efficient as that for sansevieria cv
Hahnii, and about 22 times as efficient as that for
cv. Lorentii.
ACKNOWLEDGEMENTS
The authors would like to thank to Dr.
Karyanto and others reviewers chosen by the
editorial boards for reviewing this manuscript and
Triani Wahyuningsih for her technical help.
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