A Method For Microclonal Propagation of Staurogyne Repens in Tissue Culture

A Method for Microclonal Propagation of
Staurogyne repens in Tissue Culture

M.M. Sereda, E.V. Lutsenko, V.A. Chokheli, A.V. Vereschagina,
K.Y. Rachkovskaya, V.S. Lysenko and T.V. Varduny
ABSTRACT
Objective: Staurogyn (Staurogyne repens (Nees) Kuntze) is a Services

perennial herb, widely used in aquarium landscape design.
Representatives of the Staurogyne species are sources of unique Related Articles in ASCI
glycosides in their chemical composition, which are taste
stimulators. However, a method of the tissue culture of S. repens Similar Articles in this Journal
was not described in the literature until now. In this study, such a
method is proposed. Materials and Methods: Node bines with Search in Google Scholar
axillary buds were taken as explants. Surface sterilization with View Citation
mercuric chloride (HgCl2) was found to be the most effective (60%
survivors of sterile explants). Solid and liquid media contained 3%Report Citation
sucrose and various combinations of plant hormones:
Benzyladenine (BA), thidiazuron (TDZ) and indole-3-acetic acid
(IAA) were applied. Results: It was shown that the intensive
multiplication of shoots can be induced using a liquid Murashige-
Skoog (MS) medium with the following combination of
phytohormones: Benzyl adenine (2 mg L–1) and indole-3-acetic acid
(0.5 mg L–1). The MS concentration of 0.2 mg L–1 was the most
effective for the rhyzogenesis. The adaptation of plants was
successful enough under conditions of regular liquid media
changeout and under maintaining overwater air humidity of 90%
when the plants were grown in emergent conditions. Conclusion:
The developed tissue culture method for micropropagation of
aquatic plant Staurogyne repens can used be for commercial
purposes.
How to cite this article:
M.M. Sereda, E.V. Lutsenko, V.A. Chokheli, A.V. Vereschagina, K.Y. Rachkovskaya, V.S.
Lysenko and T.V. Varduny, 2017. A Method for Microclonal Propagation of Staurogyne
repens in Tissue Culture. Journal of Plant Sciences, 12: 17-21.
DOI: 10.3923/jps.2017.17.21
URL: https://scialert.net/abstract/?doi=jps.2017.17.21
Received: October 26, 2016; Accepted: November 25, 2016; Published: December 15, 2016
INTRODUCTION
Staurogyn (Staurogyne repens (Nees) Kuntze) belongs to the family acanthus

(Acanthaceae), order lamiales, class dicotyledonous. Taxonomically, this species are close to
its parent type-Hygrophila. In nature these perennial ground-covering plants are found in
the countries of the South American continent with a hot climate as a part of coastal aquatic
communities. Ecologically, they are typical hydrophytes 1. Having a number of decorative
properties, staurogyn was being cultivated recent years and now it is of a commercial
interest in the market of decorative plants designated for landscaped aquariums. Traditional
breeding techniques can be used to keep up staurogyn in collection. However, the
accompanying flora of lower plants, such as different types of filamentous green algae could
endanger the development of the entire artificial aquarium ecosystem. Therefore, the
development of technologies of mass reproduction of this plant represents a profound
interest. The composition of biologically active compounds of staurogyn is an object of
recent studies. Thus, Hiura et al.2 have identified five new glycosides in Staurogyne
merguensis Wall.
Staurogyn also have a food value. After adding of milled leaves of S. merguensis the water
becomes a pleasant taste. This plant grows wild and native people often cook rice with its
leaves to give a pleasant taste to the rice.
It should be noted here, that the relatively close parentage between S. repens and S.
merguensis allowed to hope that the S. repens propagation protocol, would it be developed
can be applied for a more valuable S. merguensis. In general, the very fast speed of
micropropagation, high rate of multiplication of bines would make S. repens a successful
model object for experiments in plant physiology, cellular engineering purposes, etc.
Reproduction of staurogyn in vitro tissue culture seems to be the most effective way of
its mass propagation, in which the plants are devoid of any detrimental effects of
concomitant biota.
In spite of that some microclonal propagation protocols for aquatic and semi-aquatic plants3-
7
were developed, the tissue culture of S. repens was not described in the study until now.
In this study it described a method for the microclonal propagation of S. repens as well as
the results of the studies on the adaptation of S. repens grafts (obtained in tissue culture)
to the aquarium conditions.
MATERIALS AND METHODS
The S. repens grafts were obtained using the plants (Fig. 1a) from the collection of
Botanical garden of Southern Federal University. They were dissected so that each cutting
has one node and small fragments of clipped laminas (Fig. 1b). A several-step sterilization
of grafts was performed in accordance with our previous experience and the known
methodological principles of aquatic tissue culture8-10.
Before sterilization, the grafts were washed with 0.01% tween-80 for 15 min with constant
shaking and then running water. The subsequent steps were carried out in a laminar box.
The grafts were immerged in water for 30 sec. Then they were treated with 70% ethanol
followed by washing with gnotobiotic water. Further, the following reagents were attempted
in the process of sterilization-5% solution of chloramine B, 1% sodium hypochlorite
solution, 0.1% mercuric chloride solution. Surfaces of the sterilized grafts were washed
three times in gnotobiotic water for 15 min.
Grafts (explants) were placed on a solid agar (0.6% agar) with the Murashige-Skoog (MS)
medium11 or on a liquid MS. Solid and liquid media contained 3% sucrose and various
compositions of plant hormones: Benzyladenine (BA), thidiazuron (TDZ) and indole-3-acetic
acid (IAA) and were adjusted to pH 5.7. IAA was applied in the following concentrations in
attempts to induce rhizogenesis; 0.1, 0.2 and 0.5 mg L –1. In the cases of both solid and
liquid media, the cultivation was carried out at 25±1°C, 16 h photoperiod under illuminance
of 50 mmol photons m2 sec–1. If the explants were placed into a liquid medium, they were
cultured on a shaker with a speed of 50 rpm min–1.
The regenerants obtained in the course of cultivation and having an extensive root system
were transferred to ex vitro conditions to the bottom of a glass reservoir of 10 L with
different levels of water. A solid mixture containing clay, quartz sand and mineral wool were
used as a substrate.
RESULTS AND DISCUSSION
As a result of the present study, the staurogyn (Staurogyne repens) plants were
micropropagated for the first time using tissue culture method which was optimized for
this purpose.
Surface sterilization of explants gave the best results with 0.1% mercuric chloride (5 min).
In this case, about 60% of the explants appeared to be not contaminated. The usage of a
sterilizing reagents: 5% chloramine B, 1% sodium, hypochlorite brought to damage the
plants (Table 1) or didn’t prevent the development of micro-organisms.
On the 2nd week of cultivation the explants which were placed on a solid agar medium
demonstrated indications of growth; elongation of bine and leaf development. About 60% of
the bines were rooted.
Fig. 1(a-d): Micropropagation of S. repens, (a) Mother plants of S. repens, (b) Explant with axillary
buds, (c) Multiple shoot formation on MS medium treated with 2 mg L–1 BAP and 0.5
mg L–1 IAA and (d) Multiple shoot formation on liquid MS medium
Table 1: Effectiveness of influence of the sterilizing

agents on staurogyn explants, percentage of
survived plants
*Mean±SD, n = 20
Cultivation of explants in a liquid medium gave better results. There were observed the
emergence of new bines on the third week, under the influence of a particular combination
of plant hormones (Fig. 1c).
The mix of BA (2 mg L–1) and IAA (0.5 mg L–1) gave the largest multiplication factor. In this
case, one bine formed 7-9 adventitious bines (Table 2). The alone BA usage showed the
worst results. About 1 mg L–1 BA induced formation of 2-3 bines from one explants in a
longer period of time. The higher concentrations of BA caused the vitrification of staurogyn
tissues.
Table 2: Effect of different concentrations and

combinations of auxins and cytokinins on
multiple shoot formation and rooting shoots of
S. repens (liquid media)
*Mean±SD, n = 20
The usage of thidiazuron was not effective. Its influence led to plant growth arrest.
There were significant differences in characteristic of S. repens bines developed on solid and
a liquid media. Basically they relate with the shape and dimensions of the leaf. In the first
case, the leaves have a relatively well-developed blade in the form close to natural. In the
second case the leaves appear to have 7-9 pieces per explants on average, with a relatively
small and rounded leaf blade.
Duration of a single subcultivation was 2 weeks. About 30% of the explants spontaneously
formed roots at the end of each subcultivation-5 pieces/explant on average. The remaining
(non-rooted) explants were transferred on an IAA-contained medium where they rooted on
the 10-12th day, while the most effective concentration of IAA for rooting was 0.2 mg L –1.
In general our results support the observation3,4 that the micropropagation of plants in the
liquid media (without agar) gives better results than on the solid media. Thus, in our study,
the coefficient of propagation of S. repens was increased from 1:5 on the solid media to
1:15 on the liquid media.
In addition, the time period between subcultivations was found to be increased when using
liquid media compared with solid media. The entire period of S. repens development lasted
longer than 8 weeks.
The results of this study have also showed that the application of the alone IAA
phytohormone for aquatic plants gives a higher proportion of rooted bines than the
application of its mix with BA (Table 2) or than the alone BA (Table 2) which most
frequently was used for the aquatic plants cultivation without other phytohormones 12-15.
Under ex vitro conditions the regenerants were successfully developed on a quartz sand
substrate. There were some difficulties to optimize submersion depth of in aquarium. In a
fully submerged condition the regenerants continued to grow and but then turned into a
resting phase. Most of these plants remain viable for several weeks. In attempt to cultivate
them on a solid substrate, the regenerantes quickly died. It was found that the regenerants
should be submerged so that to remain leaves on the surface. Furthermore, the adaptation
of plants was effective at a regular changeout of MS and maintaining the overwater air
humidity in the aquarium about 90-100%. After the appearance of well-developed leaves,
the plants were being able to successfully adapt to both subwater and overwater conditions.
CONCLUSION
A method for tissue culture of micropropagation of aquatic plant Staurogyne repens has
been developed. The most effective mode sterilization of explants was found to be applied
using 0.1% mercuric chloride. Various phytohormone compositions were tested to optimize
growth of explants. The maximum multiplication factor of bines (7-9 shoots explant–1) was
achieved on a liquid Murashige-Skoog medium with addition of benzyladenine (2 mg L–1)
and indole-3-acetic acid (0.5 mg L–1). Massive formation of roots was observed with use of a
liquid MS media with addition of indole-3-acetic acid at concentration of 0.2 mg L–1.
ACKNOWLEDGMENT
The project is supported by grant from the Southern Federal University No. 213.01-07-
2014/06PChVG and performed with the equipment of Laboratory of plant physiology and
ecology, Laboratory of cellular and genomic plant’s technologies of Botanical Garden of
Southern Federal University. The research was solely supported by a single grant from
Southern Federal University (No. 213.01-07-2014/06PChVG).
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The biology and in vitro propagation of the
ornamental aquatic plant, Aponogeton
ulvaceus
Melissa Yit Yee Kam, Li Chin Chai, and Chiew Foan Chin
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Abstract
Aponogeton ulvaceus Baker (Aponogetonaceae) is a commercially important ornamental aquatic
plant species with traditional medicinal uses. Due to the low survival rate of seedlings,
propagation by conventional means has been met with many difficulties. In this study, botanical
aspects of A. ulvaceus were examined with regards to the morphology, anatomy and physiology
of the plant and an efficient protocol for its in vitro propagation using immature tuber explants
has been established. The existence of glandular trichomes on the leaves was discovered and the
occurrence of circumnutation in A. ulvaceus has been demonstrated. Immature tuber segments
with meristems were cultured on MS medium supplemented with various combinations (0, 1, 2,
and 3 mg/L) of BAP and NAA for callus induction. The highest percentage of callus production
(100 %) was obtained in two different treatments: 1 mg/L BAP and 3 mg/L NAA, and 2 mg/L
BAP and 3 mg/L NAA. For shoot and root organogenesis, the combination of 1 mg/L BAP and
1 mg/L NAA was shown to be significant for A. ulvaceus regeneration when compared to
control, which yields a mean shoot and root number of 22.50 and 29.50 respectively. The current
protocol is the first reported successful establishment of in vitro clonal propagation of A.
ulvaceus.
Electronic supplementary material
The online version of this article (doi:10.1186/s40064-016-3041-4) contains supplementary

material, which is available to authorized users.
Keywords: Aponogeton ulvaceus, Circumnutation, Glandular trichomes, Callus induction, Plant

growth regulators, Immature tuber
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Background
Aponogeton ulvaceus Baker (Madagascar water lettuce), is a submerged aquatic monocot native
to the rivers of central and northern Madagascar (James 1986; Les et al. 2005; Azan 2011). This
herbaceous perennial belongs to the family Aponogetonaceae (order Alismatales). Along with
other 51 Aponogeton species of perennial aquatics, this plant predominantly distributes in the
subtropical and tropical areas of Africa, Madagascar, India, Sri Lanka, Southeast Asia, Australia
and New Guinea (Robinson 2011; Grímsson et al. 2014). It thrives well in both stagnant and
flowing waters with varying light conditions (extreme shade to intense sunlight) and fluctuating
water temperature (22–28 °C) (James 1986). The leaves, arranged in a rosette, may reach up to
60 cm in length and 10 cm in width (Fig. 1a) (Thabrew 2014). They are borne on petioles of
equal length arising from a slightly hairy, cone-shaped tuber (Fig. 1b). This Madagascan species
survives seasonal dry periods by entering dormancy, whereby the dormant tuber functions as
food storage (Ingrouille and Eddie 2006). Inflorescences with a yellow colour grow on long
peduncles of varying length according to the water depth (Fig. 1c). The self-sterile inflorescences
are composed of 2 spikes and may attain a maximum length of 15 cm (Azan 2011). Flowers are
hypogynous, small and sessile (Fig. 1d). They are arranged more or less spirally along the rachis.
The ripe fruit of A. ulvaceus is a follicle and it is schizocarpic with a mostly distinct terminal,
often curved beak (Fig. 1e) (Lye 1989; Grímsson et al. 2014).
Fig. 1
Morphology of A. ulvaceus. a Aquarium grown A. ulvaceus plant; scale bar 5 cm. b A mature
tuber with shoots arising from the shoot apical meristem; scale bar 1 cm. c An immature
inflorescence borne on rachis (arrow); scale bar 1 cm. d Individual flowers along spikes of
mature inflorescence; scale bar 0.5 cm. e Ripe follicles of A. ulvaceus; scale bar 0.5 cm. Note
the distinct, curved beak and shrivelled stamens
The genus Aponogeton has been regarded as ideal for use as aquarium plants or water garden
ornamentals (Les et al. 2005; Azan 2011). It comprises some of the commercially important
aquatic species utilized in the aquatic plant trade. Among the species in the family of
Aponogetonaceae, A. ulvaceus has stood out as the popular ornamental aquarium plant that is
large and imposing. In addition to its commercial importance and aesthetic appeal, Aponogeton
is reported to have medicinal properties such as antidiabetic activity (Munasinghe et al. 2010;
Dash et al. 2014). It is also used for treating stomach disorder as well as reviving the digestive
system (Shankar and Mishra 2012). Likewise, Aponogeton forms part of the diet of the
Anuradhapura District of Sri Lanka (Munasinghe et al. 2010) and some regions of Africa
(Pemberton 2000) whereby the consumption of flowers, flower stalks and tubers has been
reported. In Cape South Africa, for instance, inflorescence of A. distachyos (waterblommetjie) is
a domesticated food crop and a unique South African delicacy.
The unique contortion of the lamina under high light irradiance, resulting in the formation of
twisted leaves in a cork-screw manner gave A. ulvaceus its intricate morphology (Czaja 1930). It
appears that there are some form of associations between such behaviour and light-induced
stress. This could possibly be a strategy of A. ulvaceus to survive excess illumination through
leaf movement by orientating and exposing the abaxial surface to light instead of the adaxial
side. Intriguingly, the leaves may develop a rufescent colouration under the same conditions
(James 1986; Thabrew 2014).
Due to the low survival rate of seedlings in the early stages and thus, difficult to propagate
conventionally (Sweeney 2008), A. ulvaceus is in short supply. To meet the high demands for A.
ulvaceus, most plants offered for sale in the aquarium trade are harvested from native
populations and overharvesting exert pressures on wild sources, resulting in diminutions in
density and abundance.
In vitro culture is an excellent alternative method for the large-scale production of this valuable
Malagasy species, mitigating both supply and quality predicaments. To date, numerous
experimental studies on aquatic plant tissue culture have been reported. Aquatic plant species
that have been successfully propagated in vitro include A. madacasgariensis (Carter and
Gunawardena 2011), Cryptocoryne wendtii (Stanly et al. 2011), Hygrophila polysperma (Roxb.)
T. Anderson (Çinar et al. 2013), Myriophyllum spicatum L. (Zhou et al. 2006), Nymphoides
indica (Jenks et al. 2000), Pogostemon helferi (Wangwibulkit and Vajrodaya 2016) and
Potamogeton crispus L. (Zhou et al. 2006). To the best of our knowledge, this is the first report
describing the micropropagation of A. ulvaceus. In this study, specific aspects of A. ulvaceus
morphology, anatomy and physiology were investigated in order to supplement the earlier
literature and an efficient clonal micropropagation system via indirect organogenesis was
established.
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Methods
Plant materials and culture conditions
A. ulvaceus plants were supplied by local aquarium plant trader. The plants were maintained in
water-filled tanks until flowering. Mature flowers were cross-pollinated with fresh pollens from
plants of the same species. Spikes with seed-bearing follicles were disinfected in 70 % (v/v)
ethanol for 30 s, followed by 0.4 % (v/v) sodium hypochlorite for 20 min. Seeds were
subsequently removed from follicles and germinated on basal MS medium (Murashige and
Skoog 1962) (Duchefa Biochemie B. V.) containing 3 % (w/v) sucrose (Fisher Scientific) and
0.35 % (w/v) Phytagel (Sigma-Aldrich). To prevent desiccation, seeds were submerged in liquid
MS medium. All media were adjusted to pH 5.8 and autoclaved at 121 °C for 15 min. Cultures
were maintained at 25 ± 1 °C under a 16 h photoperiod (40 μmol m−2 s−1) provided by cool-white
fluorescent lamps (Akari TLD36W/54). In vitro-raised plantlets were subcultured at 21-day
intervals into fresh MS basal medium.
Morphological, anatomical and physiological studies
Developing seedling and mature plant samples were used for morphological analysis. Plant
samples were subjected to imaging with an AZ100 dissecting microscope equipped with a
Digital Sight DS-5Mc camera (Nikon) to characterize the distinct developmental features of A.
ulvaceus. To identify the anatomical structures, transverse sections of fresh leaf midrib and
petiole samples were prepared and mounted in sterile distilled water to prevent desiccation for
microscopic examination.
An interesting phenomena with respect to the physiology of A. ulvaceus known as

circumnutation was investigated. Plant movements are often considered as a result of an
environmental stimulus reaction. Circumnutations, however, are autonomous helical movements
of plant organs whereby the tips outline a circular, full-ellipsis, pendulum-like shape or irregular
zigzags over a sufficiently long period (using time-lapse video method) (Stolarz 2009). For
circumnutation observations and measurements, a mature plant was cultivated in a shallow-water
acrylic tank measuring 26 cm in depth from the water surface to the base of the plant at room
temperature (25 ± 1 °C) under continuous illumination. Time-lapse video of the oscillating
growth patterns of a 4-day-old shoot apex was recorded by lapse it (interactive universe).
Camera parameters remained constant throughout the experiment and time-lapse images were
recorded one frame per 5 min intervals for 9 h. The plant was filmed from the top-view. Time-
lapse images were digitized using Circumnutation Tracker (CT) (Stolarz et al. 2014) and
Microsoft Excel programs.
Callus induction and organogenesis
Immature tubers with meristems obtained from in vitro-raised plantlets were used as explant
materials. Tuber explants were longitudinally dissected into four equal sections and cultured on
MS medium supplemented with different concentrations of BAP (0, 1, 2, and 3 mg/L) in
combination with NAA (0, 1, 2 and 3 mg/L) for callus induction and subsequent regeneration
(Carter and Gunawardena 2011). To prevent desiccation, liquid MS was poured over the explants
to submerge the entire tuber segment. Plant growth regulators (PGRs) were filter-sterilized
(0.2 μm pore size, Minisart, Sartorius Stedim Biotech) before being added to molten autoclaved
MS. The experimental design was completely randomized with four culture samples per
treatment and each treatment was replicated thrice under the same conditions as above. The
frequency of explants producing callus was determined after 28 days of culture. Subsequently,
the evaluation was repeated 56 days later on the shooting and rooting responses. Likewise, the
number of shoots and roots per explant was determined.
Statistical analysis
Statistical tests were performed with Genstat 10 for Windows. Percentage data of initial callusing
was analysed using Kruskal–Wallis test. Prior to statistical analysis, the mean number of both
shoots and roots at day 28 was subjected to logarithm transformation. The square root was used
for the mean number of shoots and roots analysis at day 56 and arcsine transformation for the
percentage of organogenic explants experiment in day 28 and 56. Data were tested for normality
and analysed using one-way ANOVA. The mean data were compared using Duncan’s multiple
range test with a 0.05 confidence interval.
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Results
Morphological features
Owing to the lack of literature on this economically important aquatic macrophyte, little is
known about its morphology and almost nothing in regards to the anatomical structures of A.
ulvaceus. Therefore, the present study examined several characteristics of this plant species and
provided a detailed description on these aspects. Observational data showed that A. ulvaceus is
monoecious, with perfect flowers, where both male and female organs occur together (Fig. 2a).
The stamens generally appear in 4–7, but most often six surrounding the female structures in the
centre. Anthers are of 2-thecate and pollens are always yellow. Likewise, the darker green
carpels exist in 3–5 (usually three free carpels), unilocular and are pear-shaped. Each carpel will
give rise to individual fruit. The isobilateral bright green (sometimes pale green) leaves are
translucent and wavy; blades are often undulated and sometimes contorted (Fig. 1a). The blades
usually have a distinct midrib with three or four pairs of parallel, confluent nerves connected by
numerous cross-veins.
Fig. 2
Morphological and anatomical features of A. ulvaceus. a Close up image of individual flowers of

A. ulvaceus; scale bar 0.2 cm. b Glandular trichomes (arrows) on leaf surface; scale bar 100 μm.
c Cross section of a petiole; scale bar 500 μm. d A transverse section of the leaf midrib; scale
bar 1000 μm. e A closer view of the honeycomb aerenchyma (HA), extensive lacunae and
diaphragms; scale bar 200 μm
Trichomes discovered fortuitously and may contribute to plant resistance against

herbivory
Remarkably, glandular trichomes were observed on A. ulvaceus in the present study (Fig. 2b).
No findings related to the development of trichome have thus far been reported. Located on both
the adaxial and abaxial surfaces of the leaves and along the petioles, trichomes formed sparingly
and were scattered over the entire surface. Of note, young leaves remained glabrous and during
the third week of growth, trichome formation initiated. In the case of in vitro-raised plantlets,
however, trichome development was not detected. Interestingly, a single type of detritivore was
observed coexisting in the water in which the analysed plants were maintained. Therefore, we
hypothesized that a resistance mechanism could have been triggered in response to damage
caused by the detritivores. In an effort to test the possibility of induced trichome production, an
observational analysis was conducted ex vitro on in vitro-raised plants whereby these were
transferred to a water-filled tank containing sterile aquarium substrate. Again, trichome
formation was not observed (0 out of 5). Thus, these results imply that A. ulvaceus is capable of
induced trichome formation in response to herbivory.
Anatomical adaptations of A. ulvaceus to an aquatic environment
Dissection and microscopic examination exhibited that honeycomb type aerenchyma,

characterized by the irregular and large air spaces, was observed in the shoots of A. ulvaceus
(Fig. 2c, d). Freshly cut transverse sections of leaf midrib revealed that a large proportion of the
cellular volume is occupied by an air-filled lacunae system (Fig. 2d). Parenchyma cells that
surround the lacunae and at least seven vascular bundles occupied the remaining cellular volume.
The same midrib cross section showed the presence of small hexagonal diaphragm cells in some
of the lacunae (Fig. 2e). Together, these results demonstrate the successful adaptation of A.
ulvaceus to a submerged aquatic environment, as anatomical modifications of the shoot such as
the existence of air-filled aerenchyma, the extensive lacunae system and cellular diaphragms are
evident.
Circumnutation behaviour of A. ulvaceus shoots
The movements of a growing 4-day-old shoot were tracked using the time-lapse video method
(Additional file 1: Video S1). Circumnutation parameters and trajectory of shoot apex were
subsequently generated using CT software. The oscillation pattern of the shoot depicts a wide
ellipse with a shape coefficient of 0.84 and it circumnutated in a counter-clockwise (ccw)
direction (left-handed) (Fig. 3a). It was found that the tip organ assumes the irregularly zigzag
trajectory of circumnutation, although previously it was demonstrated that rosette-like trajectory
also occurs in A. ulvaceus (Fig. 3b). The circumnutation period of the shoot typically reaches
110 min with amplitude (length) of 77.15 mm. Taken together, these qualitative observations
demonstrate the occurrence of circumnutation in the aquatic plant A. ulvaceus, especially in the
actively growing parts of plant organs.
Fig. 3
Top-view of the circumnutation trajectories of A. ulvaceus shoots. a Irregular zigzag

circumnutation. Shoot movement marked by colour gradients beginning from red to blue line.
Trajectory was recorded at 5 min intervals and extracted from CT. b Rosette-like circumnutation;
scale bar 2 cm. Each point indicates tip position at 5 min intervals. Direction of movement as
shown by arrowhead
In vitro regeneration of shoots and roots
Indirect organogenesis of A. ulvaceus under the influence of different concentrations of BAP and
NAA was investigated. Results on callus induction and organogenesis using immature tuber
tissues with meristems are shown in Table 1. Compact, yellowish green globular calli were
formed after 4 weeks of incubation (Fig. 4a–c). Among the PGR combinations tested, T5 has
yielded the highest number of shoots per explant (3.67) in day 28 (Table 1). However, after
8 weeks of incubation, shoot number of T3 culture increased up to 14.7-fold as compared to
28 days of growth with an overall highest mean shoot number (50.44). It is worth noting that
longer exposure time of explants to BAP and NAA is critical for shoot regeneration and
subsequent mass propagation as can be seen in the significant increase in % shoots and mean
number of shoots in all the treatments compared to the control (Table 1).
Table 1
Effects of BAP and NAA in combination on shoot regeneration from callus of A. ulvaceus after
day 28 and 56 of culture
Plant growth regulators Mean number of shoots per

% shooting
(mg/L) explant
Treatment
Day Day
BAP NAA Day 28 Day 56
28 56
T0 0 0 25.0ab 33.3a 0.67 ± 1.15a 6.92 ± 11.98a
Plant growth regulators Mean number of shoots per
% shooting
(mg/L) explant
Treatment
Day Day
28 56
T1 1 1 33.3ab 100.0b 1.75 ± 1.25a 22.50 ± 5.24b
T2 1 2 50.0ab 91.7b 2.67 ± 3.78a 37.17 ± 38.11b
T3 1 3 50.0b 83.3b 3.42 ± 1.42a 50.44 ± 30.63b
T4 2 1 25.0ab 100.0b 2.92 ± 3.17a 40.34 ± 12.57b
T5 2 2 41.7ab 75.0b 3.67 ± 1.28a 39.33 ± 1.91b
T6 2 3 66.7b 91.7b 2.17 ± 1.28a 31.65 ± 16.40b
T7 3 1 30.0ab 91.7b 0.75 ± 0.25a 31.33 ± 11.75b
T8 3 2 8.3a 66.7b 1.50 ± 2.60a 15.00 ± 9.69b
T9 3 3 50.0b 91.7b 2.25 ± 1.52a 29.41 ± 19.30b
Results represent mean ± standard deviation (SD) of three replicated experiments. Different
letters in each column indicate significant difference of mean values (P < 0.05) using Duncan’s
multiple range test
Fig. 4
Indirect organogenesis from tuber-induced callus. a–c Developing yellowish green globular
callus after 4 weeks. Callus formed on the edges of excised surface and proliferate on the entire
surface of tubers; scale bars 0.5 cm. Note the purple colouration of callus in c. d Multiple shoot
and root development from a single callus piece. Long shoots are trimmed to ease handling;
scale bar 2 cm. e Multiple shoot apical meristems developing on callus; scale bar 0.8 cm. f An
individual immature tuber with two shoots growing from the callus tissue; scale bar 0.6 cm. g–h
Root development from callus tissue; scale bars 0.6 and 0.8 cm respectively
The morphogenetic response of callus to form roots on different BAP and NAA combinations is
presented in Table 2. It was observed that root development commonly initiates following the
development of shoots from the callus (Fig. 4d). T1 showed a similar positive effect on root
regeneration that yielded an average root number of 29.50, which is significantly higher than
control (i.e. 7.9-fold). The best response was observed in T4 after 56 days with the highest root
number of 44.58. At 9 weeks of culture, multiple shoot apical meristems with proliferating green
shoots and roots are clearly visible on the globular calli (Fig. 4e–h). Thus, these results
corroborate the feasibility of A. ulvaceus micropropagation from callus induction using immature
tuber tissues.
Table 2
Effects of BAP and NAA in combination on root regeneration from callus of A. ulvaceus after
day 28 and 56 of culture
Plant growth regulators Mean number of roots per

% rooting
(mg/L) explant
Treatment
Day Day
28 56
T0 0 0 8.3a 33.3a 0.33 ± 0.58a 3.75 ± 6.50a
T1 1 1 16.7a 75.0b 0.67 ± 0.63ab 29.50 ± 3.68b
T2 1 2 16.7a 58.3b 0.33 ± 0.38ab 30.00 ± 19.64b
T3 1 3 41.7a 50.0ab 4.25 ± 4.26b 31.58 ± 12.35b
T4 2 1 16.7a 91.7b 2.42 ± 2.27ab 44.58 ± 17.89b
T5 2 2 8.3a 66.7b 0.08 ± 0.14a 24.17 ± 9.15b
T6 2 3 8.3a 75.0b 0.42 ± 0.72ab 27.08 ± 7.37b
T7 3 1 25.0a 91.7b 1.00 ± 0.87ab 32.00 ± 20.22b
T8 3 2 8.3a 66.7b 0.75 ± 1.30ab 20.17 ± 8.14b
T9 3 3 16.7a 83.3b 1.00 ± 1.73ab 22.25 ± 20.88b
Results represent mean ± SD of three replicated experiments. Different letters in each column
indicate significant differences of mean values (P < 0.05) using Duncan’s multiple range test
Go to:
Discussion
As aforementioned, scientific literatures of A. ulvaceus are lacking, especially the morphology
and anatomical structures, about which little is known. Consequently, discrimination of true
species from hybrids is difficult and this will affect the marketable quality of the plant. In the
present study several important characteristics of this Malagasy species were identified by
observational analysis. The discovery that A. ulvaceus is monoecious, as corroborated by the
presence of both male and female reproductive organs in a single flower, is consistent with the
premise that Aponogetonaceae is usually monoecious and seldom dioecious (van Bruggen 1998).
Other features such as the occurrence of stamens in six and carpels in three as well as blade
structures were described in details in the current study. The unique foliage shape of A. ulvaceus,
with long, wavy and often contorted leaves indicates its adaptability to rapidly flowing water
similar to other Aponogeton species (Hellquist and Jacobs 1998). In addition, the undulated leaf
margins of A. ulvaceus is suggested to help minimize the boundary layer thickness in low water
velocities by creating turbulence, which in turn increases the uptake of dissolved inorganic
carbon (Raven et al. 1985).
In this study, we report, for the first time, the presence of glandular trichomes on leaf surfaces
and petioles of A. ulvaceus. However, we found that none of the in vitro-cultured plants have
trichomes. Likewise, leaf surfaces of in vitro-raised plants grown ex vitro remained glabrous. A
marked difference observed was there were no detritivores (organisms feeding on dead plant
materials) inhabiting the water in the ex vitro study, thus in vitro-raised plants were not damaged.
The detritivores could have inflicted damage to live cells while feeding on dead plant parts. It is,
therefore, reasonable to speculate that the leaf trichome formation in A. ulvaceus is a response
following damage caused by herbivory. In this regard, A. ulvaceus, that was previously glabrous,
produces new leaves with trichomes that start developing in the third week of growth which may
be favourable, as it increases resistance against herbivores (Dalin et al. 2008). This is similar to
the resistance mechanism of several plant species whereby new leaves with increased trichome
number or density are produced (Traw and Bergelson 2003; Björkman et al. 2008). Our finding
provides the first evidence that such a response occurred in A. ulvaceus and further field studies
addressing functional and adaptive significance of induced trichome development should be
evaluated in the context of effects on tritrophic interactions and plant fitness under varying
abiotic conditions.
Submerged aquatic macrophytes are highly advanced as they have evolved specialized
anatomical structures, enabling them to flourish in aquatic environments while growing in anoxic
rooting substrate. It has been suggested that aerenchyma is responsible for the oxygen supply to
aquatic plant roots, thereby facilitating internal oxygen transport and enhancing metabolic
efficiency (Armstrong et al. 1991). The aerenchyma lacunae form a relatively continuous
pipeline system through plants which generally provide a pathway that allows adequate aeration
of roots, supporting respiration of submerged tissues (Maricle and Lee 2002). However, Rascio
(2002) pointed out that while emergent and floating-leaf plants are dependent on the internal
oxygen transport from leaves to roots via the air lacunae system, submerged species acquire
these gases through diffusion. Thus, leaves of the latter species are usually thin, translucent and
do not possess developed aerenchyma, if leaves are entire. Interestingly, in A. ulvaceus, the
presence of honeycomb-like aerenchyma and an extensive lacunae system were observed in the
midrib. On the basis of such evidence, A. ulvaceus plants could have evolved to adapt in both
lotic and lentic environments. It is possible that during seasonal dry periods, leaves are exposed
to the oxygen-rich atmosphere, thus A. ulvaceus will rely on the aerenchyma for oxygen supply
since the diffusion coefficient of oxygen transport in water is much lower than in air (Aachib et
al. 2004). Similarly, respiration of A. ulvaceus when fully submerged underwater will be
facilitated by diffusion via its leaves, as oxygenation is higher due to turbulence in the lotic
environment (Lusa et al. 2011). The existence of cellular diaphragms within the lacunae,
exhibiting a marked cell dimorphism, is yet another anatomical adaptive strategy found in A.
ulvacues. Apart from providing lateral support to stem and cross bundles, other functions of
diaphragms include tannin storage (Snow 1914), carrying laticifers (Stant 1964), photosynthetic
capability (Kaul 1973) and preventing internal flooding caused by injury (Soukup et al. 2000).
The twisting and rotating movements of rapidly elongating plant organs (hypocotyl, tendril,
shoot or root) have long became a fascination to biologists and have spawned a variety of studies
on terrestrial plants. In the present investigation, the occurrence of circumnutation has been
demonstrated in the aquatic plant A. ulvaceus using the novel software CT. The mechanisms and
functional importance underlying circumnutation has long been debated since Darwin’s time. At
present, it is presumed that circumnutation is dependent upon the internal oscillatory movement
and gravity sensing. In the hydrophyte Vallisneria, however, female flowers are capable of
circumnutating in the absence of gravitropism. The paper by Kosuge et al. (2013) indicated that
the helical intercalary growth of the peduncle, the internal oscillator, is the driving force of
circumnutation. As buoyancy minimizes the gravity effect, which renders it difficult to study
circumnutation in land plants due to the innate nature of gravitropic response, shoot movements
underwater are interesting. Then again, mutant analyses on agravitropic mutants of morning
glory that lack starch-filled amyloplasts in endodermal cells were defective in shoot
circumnutation, indicating the importance of gravitropic response (Kitazawa et al. 2005).
Additionally, there is increasing evidence that circumnutations possess distinct ecological
functions and are not simply a way of growth. In Oryza sativa, circumnutating roots have a
significant role in seedling establishment on waterlogged and soft soil (Inoue et al. 1999).
Likewise, circumnutation in Vallisneria is crucial for hydrophilous pollination on the water
surface (Kosuge et al. 2013). In this regard, circumnutation proves to be a complex phenomenon
and dependent on plant species, the organs involved and the developmental stage (Kitazawa et al.
2005; Stolarz 2009). Clearly, further analysis is warranted to elucidate the molecular mechanism
of circumnutation in A. ulvaceus as well as the implicated ecological functions of shoot
movements.
The procedure described here is the first successful in vitro micropropagation system for A.
ulvaceus via indirect organogenesis. Based on the evidence, immature tubers with meristems
were the most successful explant in callus induction and subsequent organogenesis. Due to their
physiological state of being young, healthy and nutrient-rich, immature tubers are ideal explant
sources. Younger explants exhibiting greater morphogenic potential has already been established
(Yepes and Aldwinekle 1994) owing to their meristematic properties and increased totipotency
in vitro (Razdan 2003). However, in other sources such as leaves, petioles and root tips of A.
ulvaceus, lethal browning of tissue was one of the impediments to callus induction. Our findings
are in conformity with the results of Carter and Gunawardena (2011), who observed no callus
formation in petioles and root tips of A. madagascariens due to browning. Such phenomenon can
be attributed to the exudation and oxidation of phenolic compounds in the culture medium as a
defence response following tissue wounding or stress (Jones and Saxena 2013).
In A. ulvaceus, the BAP and NAA combination was found to be suitable for callus induction and
organogenesis. This hormonal combination has been extensively utilized for indirect
organogenesis in various protocols developed for other plant species of Aponogeton (Carter and
Gunawardena 2011), Artemisia (Tariq et al. 2014) and Saussurea (Zhao et al. 2001; Dhar and
Joshi 2005). Taking into consideration that indirect morphogenic routes can generate somaclonal
variation in micropropagated progenies causing both genetic and epigenetic alterations (Ramírez-
Mosqueda and Iglesias-Andreu 2015), these genetic variabilities can be exploited for genetic
improvement of ornamental species with growing economic importance such as A. ulvaceus (Sun
et al. 2013; Krishna et al. 2016).
In the present investigation, regeneration capacity of A. ulvaceus shoots and roots was
significantly enhanced with the addition of BAP and NAA to MS medium. Maximum root
numbers (45 roots per explant) were produced in response to 2 mg/L BAP and 1 mg/L NAA
(T4). These results are in agreement with Oh et al. (2008), who reported that a lower
concentration of auxin is optimal for rooting. Under the same treatment condition, however,
shoot proliferation response was onefold (40 shoots per explant) lower than that obtained with
1 mg/L BAP and 3 mg/L NAA (T3) (50 shoots per explant). This suggests the importance of the
ratio of auxin (NAA) to cytokinin (BAP) in the manipulation of plant regeneration in A.
ulvaceus. The significance of the auxin to cytokinin ratio in culture medium has been highlighted
by several reports. It was indicated that auxins at lower concentrations along with cytokinins are
critically important in plant regeneration in a number of systems such as Coffea arabica
(Zoriniants et al. 2003), Eleusine indica (Yemets et al. 2003) Juncus effusus (Xu et al. 2009) and
Senecio candicans (Hariprasath et al. 2015). Though findings in relation to the effectiveness of
culture media supplemented with BAP (2 mg/L) and NAA (1 mg/L) on shoot regeneration have
been reported in S. candicans (Hariprasath et al. 2015), the differential response may be
attributed to the inhibitory effect of higher BAP concentrations on shoot multiplication. Similar
observations on the inhibitory effect of BAP were also reported in Tinospora cordifolia (Raghu
et al. 2006) and Sida cordifolia (Sivanesan and Jeong 2007).
With no statistical difference between PGR treatments on shoot regeneration, the best responses
were found in MS media supplemented with 1 mg/L BAP and 3 mg/L NAA, and 2 mg/L BAP
and 1 mg/L NAA. However, in the latter treatment, it was shown to be more effective on root
regeneration. Despite the increased regeneration capacity induced by both treatments, it was
found that suboptimal shoot and root numbers of 22.50 and 29.50, respectively, were
significantly produced with 1 mg/L BAP and 1 mg/L NAA (T1) when compared to control
explants. Based on these observations, it appears that A. ulvaceus is rather responsive to
phytohormones, as the addition of low concentrations of PGR is sufficient to induce regenerative
responses in tuber explants. Our results are in agreement with recent reports on Humulus lupulus
whereby explants exhibited high sensitivity to changes in auxin concentration and low IAA
concentration was beneficial for shoot initiation from callus tissues (Trojak-Goluch et al. 2015).
As highlighted by Hill and Schaller (2013), plants capable of accumulating higher levels of
bioactive cytokinin are generally more receptive to methods employed for the induction of de
novo shoot organogenesis and in vitro plant regeneration. It is possible that the presence of the
mechanisms necessary for plant regeneration may positively impact A. ulvaceus regenerative
capability, allowing it to readily undergo callus induction and organogenesis. Another possible
explanation is that tuber or bulb explants having larger nutrient reserves tend to readily
regenerate in vitro and are more independent of the hormones in growth medium (Yildiz 2012).
In consideration of the production cost of in vitro mass propagation of A. ulvaceus, the use of
lower concentrations of plant hormones is more cost effective. Also, low hormone concentrations
are often associated with lower incidence of or no somaclonal variation (Swartz 1991). Thus, it
can be concluded that MS medium containing 1 mg/L BAP with 1 mg/L NAA was the optimal
combination for callus induction and subsequent organogenesis of A. ulvaceus. This protocol
could potentially be used for the commercial propagation of this economically valuable aquatic
plant. In addition, with the present regeneration system, further research should focus on
evaluating: (1) the functional and adaptive significance of induced trichome formation; (2) the
underlying molecular mechanism of circumnutation in A. ulvaceus and its implicated ecological
functions of shoot movements; and (3) the mutational possibilities during regeneration which
could affect plant behaviour including circumnutation.
Go to:
Conclusion
This study provides useful information on the biology of the ornamental plant A. ulvaceus.
Through close examination of A. ulvaceus, glandular trichomes were found to be present on the
leaves. By recording time-lapse videos to track the movement of shoots, it was found that the
shoots moved in a specific trajectory known as circumnutation. The study on the morphology,
anatomy and physiology of A. ulvaceus will provide a more in depth understanding of the plant
as well as its functions and applications.
Since A. ulvaceus is a much sought after ornamental aquatic plant, mass multiplication of the
plants is essential for preserving this rare species in their natural habitat. We have successfully
developed an in vitro regeneration protocol in this study through an intermediate callus phase
using MS medium supplemented with 1 mg/L BAP and 1 mg/L NAA. This protocol will provide
an alternative method with the potential to mass propagate A. ulvaceus.
Go to:
Authors’ contributions
MYYK conducted the experiment and prepared the manuscript. LCC did the data analysis and
samples preparations. CFC initiated the project and prepared the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
The authors would like to thank the Ministry of Education for the FRGS grant and the School of
Biosciences, Faculty of Science of the University of Nottingham Malaysia Campus for the
internal fund provided to support this project. Also, the authors would like to thank Dr. Ahimsa
Campos-Arceiz for kindly allowing us to use his time-lapse video camera.
Competing interests
The authors declare that they have no competing interests.
Additional file
10.1186/s40064-016-3041-4 Time-lapse video of shoot circumnutation in Aponogeton

ulvaceus.(3.4M, wmv)
Go to:
Contributor Information
Melissa Yit Yee Kam, Email: ym.ude.mahgnitton.liamxe@ykm5xyhk.
Li Chin Chai, Email: ym.ude.mahgnitton@hlc1xyhk.
Chiew Foan Chin, Phone: +60-3-8924 8216, Email: ym.ude.mahgnitton@nihc.naof-weihc.
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Micropropagation of three species of aquatic

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Article (PDF Available) · January 2006 with 2,119 Reads
 Maurizio Micheli
 22.87
 Università degli Studi di Perugia
  De Gasperis A
  Prosperi F
 
Alvaro Standardi
o 26.24
Abstract
An efficient protocol for in vitro propagation of three aquatic plant species (Cryptocoryne
becketti, Cryptocoryne lutea and Rotala rotundifolia) was established. For the sterilization of the
initial explants, 1.0 and 1.5% sodium hypochlorite was successfully employed for Rotala and for
Cryptocoryne spp. respectively. Satisfactory shoot proliferation was obtained in Linsmaier and
Skoog medium (1965) enriched with 0.5 mg l-1 naphtalene acetic acid (NAA) and two different
concentrations of 6- benzylaminopurine (BAP) (1 or 4 mg l-1). Both BAP concentrations
induced also adventitious root emission in all species. In the medium supplemented with 4 mg l-
1 BAP the multiplication rate values were higher, but root number and length were lower than in
the medium containing 1 mg l-1 BAP. For acclimatization in aquarium of the rooted clumps a
natural condition-like soil (peat:clay:sand at 1:1:10/v:v:v ratio) offered better results in terms of
shoot and root growth compared to Compo Cactea© commercial mix. Moreover, during this step
better root systems developed in plantlets obtained in presence of 1 mg l-1 BAP during the
proliferation-rooting-phase. Rotala showed the highest adaptability to in vitro propagation, since
proliferation activity was very high; between the two Cryptocoryne species, no differences in
proliferation and rooting were observed.
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1
MICROPROPAGATION OF THREE SPECIES OF AQUATIC PLANTS
M. Micheli, A. De Gasperis, F. Prosperi, A. Standardi*
Dipartimento di Scienze Agrarie e Ambientali, Università di Perugia
SUMMARY - An efficient protocol for in vitro propagation of three aquatic plant species
(Cryptocoryne
initial ex-
plants, 1.0 and 1.5% sodium hypochlorite was successfully employed for Rotala and for
Cryptocoryne spp.
respectively. Satisfactory shoot proliferation was obtained in Linsmaier and Skoog medium
(1965) en-
riched with 0.5 mg l-1 naphtalene acetic acid (NAA) and two different concentrations of 6-
benzy-
laminopurine (BAP) (1 or 4 mg l-1). Both BAP concentrations induced also adventitious root
emission in
all species. In the medium supplemented with 4 mg l-1 BAP the multiplication rate values were
higher,
but root number and length were lower than in the medium containing 1 mg l-1 BAP. For
acclimatization
in aquarium of the rooted clumps a natural condition-like soil (peat:clay:sand at 1:1:10/v:v:v
ratio) of-
fered better results in terms of shoot and root growth compared to Compo Cactea© commercial
mix.
Moreover, during this step better root systems developed in plantlets obtained in presence of 1
mg l-1 BAP
during the proliferation-rooting-phase. Rotala showed the highest adaptability to in vitro
propagation,
since proliferation activity was very high; between the two Cryptocoryne species, no differences
in prolif-
eration and rooting were observed.
Key words: Cryptocoryne becketti, Cryptocoryne lutea, in vitro culture, Rotala rotundifolia,
vegetative multi-
plication.
INTRODUCTION
Aquatic plants are ornamental species:
they are planted in aquaria for their beauty,
but also to maintain water quality. They are
selected for shape, plant colour and size
(Stodola, 1980). The demand is limited to
specific markets and in five years (1990-
1995) the production in Holland increased
from 417,000 to 1,204,000 plants, some of
which propagated in vitro(Pierik and Ruib-
ing, 1997).
The most common aquatic plants are rep-
resented by Cryptocoryne and Rotala gen. na-
tive in vast areas of southeast Asia and In-
donesia. These plants are submerged or
emerged (Kasselmann, 1999). Propagation
by seed and by division of rhizomes are not
productive and require a long time. Conse-
quently, these methods are not used (Winde-
low, 1987). Most plants commercialized in
the USA are collected in their natural habitat.
The clumps are divided into single plants
and acclimatized and grown in baths before
sale. The information about in vitro propaga-
tion of aquatic plants is very limited and
fragmentary, perhaps because the experi-
mental activities are conducted within pri-
vate companies (Kane et al., 1988, 1990; Star-
itski, 1977).
The aim of this study was to develop a mi-
cropropagation protocol for three aquatic
species (two in the gen. Cryptocoryne and one
of the Rotala), focusing the attention to the ef-
fect on the proliferation activity of cy-
tokinins added to the medium.
Agr. Med. Vol. xxx, xxx-xxx (2006)
* Corresponding Author: borgo XX Giugno 74, 06121 Perugia, Italy - E-mail: asep@unipg.it
2
AGRICOLTURA MEDITERRANEA
Three species were studied: Cryptocoryne
becketti Trimen and Cryptocoryne lutea (Shott)
(Araceae) and Rotala rotundifolia (Buch.-
Ham. ex Roxb.) Koehne (Lythraceae). The
initial explants were excised from mother
plants (Fig. 1) which were bought from a
speciality store and were grouped by habit
and shape types. The plants were transferred
into aquaria in order to limit the incidence of
the pathogens.
In vitro stabilization
Vigorous plants free from seaweed infesta-
tion were washed in warm water; one cen-
timetre-long pieces of rhizome were then
taken from C. becketti and C. Lutea, and unin-
odal segments (0.5 cm) were used in R. ro-
tundifolia. The portions of rhizomes were
washed in ethanol solution (50% v/v) for 5-
10 seconds and then dipped in sodium
hypochlorite solution (1.5% w/v) for 20 min.
All explants were rinsed in sterile distilled
water. Sodium hypochlorite solution at 1.0%
(w/v) concentration was used for the steril-
ization of the uninodal segments of R. rotun-
difolia. Each explant was vertically posi-
tioned into a glass micro-jar (100 ml) con-
taining 15 ml of Linsmaier and Skoog (LS)
medium (1965), supplemented with naph-
thalene acetic acid (NAA) (0.5 mg l-1) and 6-
benzylaminopurine (BAP) (2.0 mg l-1). The
cultures were transferred to a growth cham-
ber and maintained at 22 ± 1 °C and 16-h
light photoperiod, under cool white fluores-
cent light (40 µE m-2s-1). After 6 weeks of sta-
bilization, shoots 10-15 mm in length which
had proliferated from the initial explants
were transferred into 500 ml-glass jars (10
explants/jar) containing 100 ml of LS medi-
um, enriched with growth regulators as
above. Every four weeks the proliferated
shoots were subcultured in the same nutri-
tive and environmental conditions and after
4 months enough plant material was pro-
duced for experiments.
Fig. 1 - Mother plants of Cryptocoryne becketti.
3
M. MICHELI, A. DE GASPERIS, F. PROSPERI ET AL.
Proliferation: Effect of cytokinins
Four different concentrations of BAP were
added to the LS medium (0, 1, 4 and 20 mg l-
1) and tested during three subsequent subcul-
tures. In all cases LS medium was supple-
mented with 0.5 mg l-1 NAA; one hundred
millilitres of medium were transferred into
each 500 ml-glass jar before sterilization in
autoclave at 115 °C for 20 minutes. In each jar
5 single shoots were aseptically transferred.
After 4 weeks in the growth chamber, the
proliferation was evaluated by assessing the
number of shoots obtained from each initial
explant, the length of proliferated shoots and
the fresh weight of the proliferated masses.
The rooting activity, recorded during the
proliferation, was evaluated measuring the
number of rooted clumps, the amount and
the length of roots.
The experiments followed a randomised
block design in which 20 explants per treat-
ment were employed; each jar represented
one replication.
Acclimation: Effect of basal substrate
The rooted clumps (plantlets) were direct-
ly acclimated in aquarium (110 x 40 x 55 cm),
made of PVC and glass, containing total 160
l of deionised water and pre-conditioned at
16-h photoperiod and artificial light (25
Watt). Apump allowed to introduce filtered
water, maintained between 20 and 25 °C in-
to the aquarium. In a 150 mm layer at the
bottom of the aquarium two different basal
substrates were placed: the first was very
similar to the natural soil conditions of these
species and was composed of peat, fine clay
and sand (1:1:10/v:v:v); the other was repre-
sented by Compo-cactea®, a commercial
substrate used for the cultivation of Cac-
taceae. All substrates were sterilized by auto-
claving. The two species of Cryptocoryne
were transferred both in the natural soil and
in Compo-cactea®; in Rotala rotundifolia the
aclimation phase was carried out on the lay-
er of Compo-cactea® (Fig. 2). For each
aquarium condition, fifty plantlets per geno-
type were maintained completely sub-
merged. At the end of acclimation (15 days)
the following parameters were recorded:
number of living plants, total number of
roots in each clump and root length.
All collected data were analysed by the
SAS (Statistical Analysis System); a GLM
(General Linear Models) procedure was
used with a Duncan’s test (p ≤0.05).
RESULTS AND DISCUSSIONS
Sterilization resulted in a high percentage
of explants’ survival: 70.0% in Rotala rotundi-
folia, 55.0% in Cryptocoryne becketti and 65.0%
in C. lutea, which after 14 days of establish-
ing culture developed shoots that exceeded
10 mm in length. After another four weeks
the shoots generally had 4-5 leaves.
The absence of growth regulators induced
necrosis on the shoot apex, while the highest
Fig. 2 - Cryptocoryne sp. clumps in the natural sub-
strate (left) and in Compo-Cactea© together Rotala ro-
tundifolia (right) during the acclimatation phase.
4
concentration of BAP caused vitrification as
reported by George (1996), Jenks et al. (2000)
and Roger (2003) who described the effect of
BAP on the shoot proliferation in aquatic
plants. For this reason, after the first subcul-
ture, media without cytokinins as well as
media containing 20 mg l-1 of BAP were
eliminated. In all species, the medium con-
taining 4 mg l-1 of BAP resulted in higher
multiplication rates as clearly shown in
Table 1. Under these growth conditions the
multiplication rate observed in Rotala rotun-
difolia, reached 67.5% and the quality of pro-
liferated shoots was satisfactory as shown in
Figure 3; this demonstrates the adaptability
of this genotype to in vitro conditions. How-
ever, the values of shoot length were higher
when the lowest concentration of BAP (1 mg
l-1) was employed (Tab. 1). It may be due to
the well-known inverse relation between the
Tab. 1 - Mean effect of BAP concentration on the parameters connected with the proliferation
activity after three
subcultures.
Species BAP Multiplication Shoot Fresh weight
Rate length of clumps
(mg l-1) (n) (mm) (g)
C. becketti 1 3.8 b 28.3 a 1.77 a
4 5.5 a 19.5 b 1.20 a
C. lutea 1 3.9 b 22.4 a 1.44 a
4 5.7 a 17.3 b 1.12 a
R. rotundifolia 1 37.2 b 26.4 a 11.22 a
4 67.5 a 19.5 b 5.49 b
In each column and for each species the values within different letters indicate significant
differences (p ≤0.05).
Fig. 3 - Proliferated clumps of Rotala rotundifolia after
4 weeks in LS medium containing 4 mg l-1 BAP.
Tab. 2 - Mean effect of BAP concentration on the parameters connected with the rooting activity
after three sub-
cultures.
Species BAP Roots
(mg l-1)Number/clumps (mm)
C. becketti 1 4.0 a 12.0 a
4 2.0 b 6.0 b
C. lutea 1 4.0 a 12.0 a
42.0 b 6.0 b
R. rotundifolia 1 40.0 a 6.0 a
430.0 b 3.0 b
5
multiplication rate values and shoots length
(Pierik, 1987). Proliferated masses of Crypto-
coryne (Tab. 1) showed no significant differ-
ences in fresh weight values between 1 and 4
mg l-1 of BAP, while in Rotala rotundifolia the
higher value was observed in the presence of
1 mg l-1 of BAP (11.2 mm). BAP concentra-
tions (1 and 4 mg l-1) produced no callus, no
necrosis and low vitrification. The colour of
the proliferated shoots was always green-red
in Cryptocoryne becketti, light green in Crypto-
coryne lutea and dark green in Rotala rotundi-
folia; these colorors were similar to the nor-
mal and typical of the three species.
In all cases the proliferation was accompa-
nied by root formation. As consequence, it is
possible to avoid a separate stage dedicated
to rooting. We were able to transfer the
plantlets directly to an aquarium with all
their proliferated shoots; moreover, the low-
er concentration of BAP (1 mg l-1) induced a
higher number and length of roots (Tab. 2).
In Cryptocoryne the roots appeared fleshy,
while in Rotala they were fibrous. In general,
considering the comparison between the two
substrates, the conclusion was that the use of
the substrate containing 4 mg l-1 of BAP was
more productive because it induced a higher
number of shoots, while the substrate poor
in cytokinins could be employed to obtain a
better quality of rooting (Fig. 4) and to in-
duce shorter periods for acclimation, con-
firming the observation of Pierik (1987).
Therefore, the use of 1 mg l-1 BAP should be
reccommended for subculture when the at-
tention is focused to obtain plantlets suitable
for acclimation.
At the end of the acclimation period of 15
days in the aquarium, in all species 100.0%
of survived plantlets were observed.
In Cryptocoryne the comparison of the two
substrates showed that the natural matrix re-
sulted in a higher number of roots, which,
moreover, were longer in both concentration
of BAP (Tab. 3).
Fig. 4 - Plantlets (rooted clumps) of Cryptocoryne lutea
before the acclimatation phase.
Tab. 3 - Mean values of number and length of total roots observed in Cryptocoryne species after
15 days of accli-
mation on two substrates.
BAP Substrate Roots
1Natural 7.4 a 35.0 a
1 Compo-Cactea© 4.2 b 25.0 b
4 Compo-Cactea© 3.4 a 18.0 b
Within BAP treatment, in each column the values within different letters indicate significant
differences (p ≤
0.05).
Received: Accepted:
6
Moreover, the plantlets produced in pres-
ence of 1 mg l-1 BAP developed a better root
system, both in terms of number and length
of roots.
In Rotala rotundifolia no differences were
observed between plants obtained in medi-
um containing 1 or 4 mg l-1 BAP: during ac-
climation the plantlets produced twenty
new roots which reached 100 mm in length
(data not shown).
CONCLUSIONS
This study shows that micropropagation is
an efficient method to produce plants of
Cryptocoryne becketti, Cryptocoryne lutea and
Rotala rotundifolia. This is in agreement with
the results reported by Staritsky (1977) for
other aquatic plants. Rotala rotundifolia was
more productive than Cryptocoryne species
which did not show significant differences
among species during the proliferation and
rooting phases.
All species rooted easily during prolifera-
tion which mase possible to transfer all
plantlets directly to the acclimation phase,
eliminating the cost for the rooting phase. Fi-
nally, the acclimation was without difficul-
ties in all species: the use of a natural sub-
strate matrix resulted in good acclimation in
terms of plant survived and time of acclima-
tion.
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and Organ Culture, 63: 1-8.
KANE M.E., MCCONNEL D.B., SHEEHAN T.J., DE-
HGAN B. (1988) - A laboratory exercise to
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velopment biology plant, 39: 1-5.
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ryne. Aqua-Planta, 1: 3-6.
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(Italy): Olimpia Ed.
WINDELOW H. (1987) - Aquarium Plants. Neptune
City (New Jersey, USA): T.F.H. Editions, 54-96.
 ... aquarium plants namely Aponogeton sp. (Kukulczanka et al., 1980), Cryptocoryne
lucens (Kane et al., 1990), Anubias barteri (Huang et al., 1994), Cryptocorine wendtii
(Kane et al., 1999; Mo and Jiang, 2003), Ludwigia repens (Ozturk et al., 2004),
Cryptocorine becketti, Cryptocorin lutea and Rotala rotundifolia (Micheli et al., 2006).
However, there has been no report on the effect of medium on the shoot proliferation of
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Micropropagation of three species of aquatic

plants
Article (PDF Available) · January 2006 with 2,119 Reads

 22.87
 Università degli Studi di Perugia
  De Gasperis A
  Prosperi F
 
Alvaro Standardi
o 26.24
Abstract
An efficient protocol for in vitro propagation of three aquatic plant species (Cryptocoryne
initial explants, 1.0 and 1.5% sodium hypochlorite was successfully employed for Rotala and for
Cryptocoryne spp. respectively. Satisfactory shoot proliferation was obtained in Linsmaier and
Skoog medium (1965) enriched with 0.5 mg l-1 naphtalene acetic acid (NAA) and two different
concentrations of 6- benzylaminopurine (BAP) (1 or 4 mg l-1). Both BAP concentrations
induced also adventitious root emission in all species. In the medium supplemented with 4 mg l-
1 BAP the multiplication rate values were higher, but root number and length were lower than in
the medium containing 1 mg l-1 BAP. For acclimatization in aquarium of the rooted clumps a
natural condition-like soil (peat:clay:sand at 1:1:10/v:v:v ratio) offered better results in terms of
shoot and root growth compared to Compo Cactea© commercial mix. Moreover, during this step
better root systems developed in plantlets obtained in presence of 1 mg l-1 BAP during the
proliferation-rooting-phase. Rotala showed the highest adaptability to in vitro propagation, since
proliferation activity was very high; between the two Cryptocoryne species, no differences in
proliferation and rooting were observed.
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1
MICROPROPAGATION OF THREE SPECIES OF AQUATIC PLANTS
M. Micheli, A. De Gasperis, F. Prosperi, A. Standardi*
Dipartimento di Scienze Agrarie e Ambientali, Università di Perugia
SUMMARY - An efficient protocol for in vitro propagation of three aquatic plant species
(Cryptocoryne
initial ex-
plants, 1.0 and 1.5% sodium hypochlorite was successfully employed for Rotala and for
Cryptocoryne spp.
respectively. Satisfactory shoot proliferation was obtained in Linsmaier and Skoog medium
(1965) en-
riched with 0.5 mg l-1 naphtalene acetic acid (NAA) and two different concentrations of 6-
benzy-
laminopurine (BAP) (1 or 4 mg l-1). Both BAP concentrations induced also adventitious root
emission in
all species. In the medium supplemented with 4 mg l-1 BAP the multiplication rate values were
higher,
but root number and length were lower than in the medium containing 1 mg l-1 BAP. For
acclimatization
in aquarium of the rooted clumps a natural condition-like soil (peat:clay:sand at 1:1:10/v:v:v
ratio) of-
fered better results in terms of shoot and root growth compared to Compo Cactea© commercial
mix.
Moreover, during this step better root systems developed in plantlets obtained in presence of 1
mg l-1 BAP
during the proliferation-rooting-phase. Rotala showed the highest adaptability to in vitro
propagation,
since proliferation activity was very high; between the two Cryptocoryne species, no differences
in prolif-
eration and rooting were observed.
Key words: Cryptocoryne becketti, Cryptocoryne lutea, in vitro culture, Rotala rotundifolia,
vegetative multi-
plication.
INTRODUCTION
Aquatic plants are ornamental species:
they are planted in aquaria for their beauty,
but also to maintain water quality. They are
selected for shape, plant colour and size
(Stodola, 1980). The demand is limited to
specific markets and in five years (1990-
1995) the production in Holland increased
from 417,000 to 1,204,000 plants, some of
which propagated in vitro(Pierik and Ruib-
ing, 1997).
The most common aquatic plants are rep-
resented by Cryptocoryne and Rotala gen. na-
tive in vast areas of southeast Asia and In-
donesia. These plants are submerged or
emerged (Kasselmann, 1999). Propagation
by seed and by division of rhizomes are not
productive and require a long time. Conse-
quently, these methods are not used (Winde-
low, 1987). Most plants commercialized in
the USA are collected in their natural habitat.
The clumps are divided into single plants
and acclimatized and grown in baths before
sale. The information about in vitro propaga-
tion of aquatic plants is very limited and
fragmentary, perhaps because the experi-
mental activities are conducted within pri-
vate companies (Kane et al., 1988, 1990; Star-
itski, 1977).
The aim of this study was to develop a mi-
cropropagation protocol for three aquatic
species (two in the gen. Cryptocoryne and one
of the Rotala), focusing the attention to the ef-
fect on the proliferation activity of cy-
tokinins added to the medium.
Agr. Med. Vol. xxx, xxx-xxx (2006)
* Corresponding Author: borgo XX Giugno 74, 06121 Perugia, Italy - E-mail: asep@unipg.it
2
Three species were studied: Cryptocoryne
becketti Trimen and Cryptocoryne lutea (Shott)
(Araceae) and Rotala rotundifolia (Buch.-
Ham. ex Roxb.) Koehne (Lythraceae). The
initial explants were excised from mother
plants (Fig. 1) which were bought from a
speciality store and were grouped by habit
and shape types. The plants were transferred
into aquaria in order to limit the incidence of
the pathogens.
In vitro stabilization
Vigorous plants free from seaweed infesta-
tion were washed in warm water; one cen-
timetre-long pieces of rhizome were then
taken from C. becketti and C. Lutea, and unin-
odal segments (0.5 cm) were used in R. ro-
tundifolia. The portions of rhizomes were
washed in ethanol solution (50% v/v) for 5-
10 seconds and then dipped in sodium
hypochlorite solution (1.5% w/v) for 20 min.
All explants were rinsed in sterile distilled
water. Sodium hypochlorite solution at 1.0%
(w/v) concentration was used for the steril-
ization of the uninodal segments of R. rotun-
difolia. Each explant was vertically posi-
tioned into a glass micro-jar (100 ml) con-
taining 15 ml of Linsmaier and Skoog (LS)
medium (1965), supplemented with naph-
thalene acetic acid (NAA) (0.5 mg l-1) and 6-
benzylaminopurine (BAP) (2.0 mg l-1). The
cultures were transferred to a growth cham-
ber and maintained at 22 ± 1 °C and 16-h
light photoperiod, under cool white fluores-
cent light (40 µE m-2s-1). After 6 weeks of sta-
bilization, shoots 10-15 mm in length which
had proliferated from the initial explants
were transferred into 500 ml-glass jars (10
explants/jar) containing 100 ml of LS medi-
um, enriched with growth regulators as
above. Every four weeks the proliferated
shoots were subcultured in the same nutri-
tive and environmental conditions and after
4 months enough plant material was pro-
duced for experiments.
Fig. 1 - Mother plants of Cryptocoryne becketti.
3
Four different concentrations of BAP were
added to the LS medium (0, 1, 4 and 20 mg l-
1) and tested during three subsequent subcul-
tures. In all cases LS medium was supple-
mented with 0.5 mg l-1 NAA; one hundred
millilitres of medium were transferred into
each 500 ml-glass jar before sterilization in
autoclave at 115 °C for 20 minutes. In each jar
5 single shoots were aseptically transferred.
After 4 weeks in the growth chamber, the
proliferation was evaluated by assessing the
number of shoots obtained from each initial
explant, the length of proliferated shoots and
the fresh weight of the proliferated masses.
The rooting activity, recorded during the
proliferation, was evaluated measuring the
number of rooted clumps, the amount and
the length of roots.
The experiments followed a randomised
block design in which 20 explants per treat-
ment were employed; each jar represented
one replication.
The rooted clumps (plantlets) were direct-
ly acclimated in aquarium (110 x 40 x 55 cm),
made of PVC and glass, containing total 160
l of deionised water and pre-conditioned at
16-h photoperiod and artificial light (25
Watt). Apump allowed to introduce filtered
water, maintained between 20 and 25 °C in-
to the aquarium. In a 150 mm layer at the
bottom of the aquarium two different basal
substrates were placed: the first was very
similar to the natural soil conditions of these
species and was composed of peat, fine clay
and sand (1:1:10/v:v:v); the other was repre-
sented by Compo-cactea®, a commercial
substrate used for the cultivation of Cac-
taceae. All substrates were sterilized by auto-
claving. The two species of Cryptocoryne
were transferred both in the natural soil and
in Compo-cactea®; in Rotala rotundifolia the
aclimation phase was carried out on the lay-
er of Compo-cactea® (Fig. 2). For each
aquarium condition, fifty plantlets per geno-
type were maintained completely sub-
merged. At the end of acclimation (15 days)
the following parameters were recorded:
number of living plants, total number of
roots in each clump and root length.
All collected data were analysed by the
SAS (Statistical Analysis System); a GLM
(General Linear Models) procedure was
used with a Duncan’s test (p ≤0.05).
RESULTS AND DISCUSSIONS
Sterilization resulted in a high percentage
of explants’ survival: 70.0% in Rotala rotundi-
folia, 55.0% in Cryptocoryne becketti and 65.0%
in C. lutea, which after 14 days of establish-
ing culture developed shoots that exceeded
10 mm in length. After another four weeks
the shoots generally had 4-5 leaves.
The absence of growth regulators induced
necrosis on the shoot apex, while the highest
Fig. 2 - Cryptocoryne sp. clumps in the natural sub-
strate (left) and in Compo-Cactea© together Rotala ro-
tundifolia (right) during the acclimatation phase.
4
concentration of BAP caused vitrification as
reported by George (1996), Jenks et al. (2000)
and Roger (2003) who described the effect of
BAP on the shoot proliferation in aquatic
plants. For this reason, after the first subcul-
ture, media without cytokinins as well as
media containing 20 mg l-1 of BAP were
eliminated. In all species, the medium con-
taining 4 mg l-1 of BAP resulted in higher
multiplication rates as clearly shown in
Table 1. Under these growth conditions the
multiplication rate observed in Rotala rotun-
difolia, reached 67.5% and the quality of pro-
liferated shoots was satisfactory as shown in
Figure 3; this demonstrates the adaptability
of this genotype to in vitro conditions. How-
ever, the values of shoot length were higher
when the lowest concentration of BAP (1 mg
l-1) was employed (Tab. 1). It may be due to
the well-known inverse relation between the
Tab. 1 - Mean effect of BAP concentration on the parameters connected with the proliferation
activity after three
subcultures.
Species BAP Multiplication Shoot Fresh weight
Rate length of clumps
(mg l-1) (n) (mm) (g)
C. becketti 1 3.8 b 28.3 a 1.77 a
4 5.5 a 19.5 b 1.20 a
C. lutea 1 3.9 b 22.4 a 1.44 a
4 5.7 a 17.3 b 1.12 a
R. rotundifolia 1 37.2 b 26.4 a 11.22 a
4 67.5 a 19.5 b 5.49 b
Fig. 3 - Proliferated clumps of Rotala rotundifolia after
4 weeks in LS medium containing 4 mg l-1 BAP.
Tab. 2 - Mean effect of BAP concentration on the parameters connected with the rooting activity
after three sub-
cultures.
Species BAP Roots
C. becketti 1 4.0 a 12.0 a
4 2.0 b 6.0 b
C. lutea 1 4.0 a 12.0 a
42.0 b 6.0 b
R. rotundifolia 1 40.0 a 6.0 a
430.0 b 3.0 b
5
multiplication rate values and shoots length
(Pierik, 1987). Proliferated masses of Crypto-
coryne (Tab. 1) showed no significant differ-
ences in fresh weight values between 1 and 4
mg l-1 of BAP, while in Rotala rotundifolia the
higher value was observed in the presence of
1 mg l-1 of BAP (11.2 mm). BAP concentra-
tions (1 and 4 mg l-1) produced no callus, no
necrosis and low vitrification. The colour of
the proliferated shoots was always green-red
in Cryptocoryne becketti, light green in Crypto-
coryne lutea and dark green in Rotala rotundi-
folia; these colorors were similar to the nor-
mal and typical of the three species.
In all cases the proliferation was accompa-
nied by root formation. As consequence, it is
possible to avoid a separate stage dedicated
to rooting. We were able to transfer the
plantlets directly to an aquarium with all
their proliferated shoots; moreover, the low-
er concentration of BAP (1 mg l-1) induced a
higher number and length of roots (Tab. 2).
In Cryptocoryne the roots appeared fleshy,
while in Rotala they were fibrous. In general,
considering the comparison between the two
substrates, the conclusion was that the use of
the substrate containing 4 mg l-1 of BAP was
more productive because it induced a higher
number of shoots, while the substrate poor
in cytokinins could be employed to obtain a
better quality of rooting (Fig. 4) and to in-
duce shorter periods for acclimation, con-
firming the observation of Pierik (1987).
Therefore, the use of 1 mg l-1 BAP should be
reccommended for subculture when the at-
tention is focused to obtain plantlets suitable
for acclimation.
At the end of the acclimation period of 15
days in the aquarium, in all species 100.0%
of survived plantlets were observed.
In Cryptocoryne the comparison of the two
substrates showed that the natural matrix re-
sulted in a higher number of roots, which,
moreover, were longer in both concentration
of BAP (Tab. 3).
Fig. 4 - Plantlets (rooted clumps) of Cryptocoryne lutea
before the acclimatation phase.
Tab. 3 - Mean values of number and length of total roots observed in Cryptocoryne species after
15 days of accli-
mation on two substrates.
BAP Substrate Roots
1 Compo-Cactea© 4.2 b 25.0 b
4 Compo-Cactea© 3.4 a 18.0 b
Within BAP treatment, in each column the values within different letters indicate significant
differences (p ≤
0.05).
Received: Accepted:
6
Moreover, the plantlets produced in pres-
ence of 1 mg l-1 BAP developed a better root
system, both in terms of number and length
of roots.
In Rotala rotundifolia no differences were
observed between plants obtained in medi-
um containing 1 or 4 mg l-1 BAP: during ac-
climation the plantlets produced twenty
new roots which reached 100 mm in length
(data not shown).
CONCLUSIONS
This study shows that micropropagation is
an efficient method to produce plants of
Cryptocoryne becketti, Cryptocoryne lutea and
Rotala rotundifolia. This is in agreement with
the results reported by Staritsky (1977) for
other aquatic plants. Rotala rotundifolia was
more productive than Cryptocoryne species
which did not show significant differences
among species during the proliferation and
rooting phases.
All species rooted easily during prolifera-
tion which mase possible to transfer all
plantlets directly to the acclimation phase,
eliminating the cost for the rooting phase. Fi-
nally, the acclimation was without difficul-
ties in all species: the use of a natural sub-
strate matrix resulted in good acclimation in
terms of plant survived and time of acclima-
tion.
REFERENCES
GEORGE E.F. (1996) - Problems in initiating and
maintaining cultures. In: Plant Propagation by Tis-
sue Culture.Edington (UK): E.F. George, Exegetics
Limited Ltd.
JENKS M.A., KANE M.E., MCCONNEL D.B. (2000) -
Shoot organogeneis from petiole explants in the
aquatic plant Nymphoides indica. Plant Cell, Tissue
and Organ Culture, 63: 1-8.
KANE M.E., MCCONNEL D.B., SHEEHAN T.J., DE-
HGAN B. (1988) - A laboratory exercise to
demostrate adventitious shoot formation using
stem internodes of parrot-feather. Hort. Science,
23: 408.
KANE M.E., GILMAN E.F., JENKS M.A., SHEEHAN
T.J. (1990) - Micropropagation of the aquatic plant
Cryptocoryne lucens. Hort. Science, 25: 687-689.
KASSELMANN C. (1999) - Piante d’acquario. Roz-
zano (Italy): Primaris Ed.
LINSMAIER E.M., SKOOG F. (1965) - Organic growth
factor requirements of tobacco tissue cultures.
Physiologia Plantarum, 18: 100-127.
PIERIK R.L.M. (1987) - In vitro culture of higher
plants. Dordrecht (NL): Kluwer Academic Pub-
lisher.
PIERIK R.L.M., RUIBING M.A. (1997) - Develop-
ments in the micropropagation industry in The
Netherlands. Plant Tissue Culture and Biotechnol-
ogy Vol. 3.
ROGER S.M.D. (2003) - Tissue culture and wetland
establishment of the freshwater monocots Carex,
Juncus, Scirpus, and Thypa. In vitro Cellular & De-
velopment biology plant, 39: 1-5.
STARITSKI G. (1977) - Die vitrokultur von Cryptoco-
ryne. Aqua-Planta, 1: 3-6.
STODOLA J. (1980) - Le piante d’acquario. Firenze
(Italy): Olimpia Ed.
WINDELOW H. (1987) - Aquarium Plants. Neptune
City (New Jersey, USA): T.F.H. Editions, 54-96.
 ... aquarium plants namely Aponogeton sp. (Kukulczanka et al., 1980), Cryptocoryne
lucens (Kane et al., 1990), Anubias barteri (Huang et al., 1994), Cryptocorine wendtii
(Kane et al., 1999; Mo and Jiang, 2003), Ludwigia repens (Ozturk et al., 2004),
Cryptocorine becketti, Cryptocorin lutea and Rotala rotundifolia (Micheli et al., 2006).
However, there has been no report on the effect of medium on the shoot proliferation of
Lindernea sp. ...
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... Moreover, the collection of plant materials from the wild resources can be reduced or
prevented with the production of clonal propagated plants. Previous studies have reported the in
vitro propagation protocols for some aquarium plants, i.e., Aponogeton species (Kukulczanka et
al. 1980); Cryptocoryne lucens (Kane et al. 1990); Anubias barteri (Huang et al. 1994); C.
wendtii (Kane et al. 1999; Mo and Jiang 2003 ); Ludwigia repens (Ozturk et al. 2004); C.
becketti, C. lutea and Rotala rotundifolia (Micheli et al. 2006). However, there has been no
report on the effect of liquid medium on the shoot proliferation of these Cryptocoryne species. ...
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October 2015
Encapsulation and micropropagation technologies were summarized and discussed as tools to
exchange and to propagate valuable genotypes respectively. Each of these technologies shows
advantages and problems for large and commercial diffusion whereas their integration represents
a considerable innovation for the future nursery. In fact, uninodal microcuttings excised from in
vitro proliferated... [Show full abstract]
Read more
Article
Encapsulation of in vitro-derived explants of olive (cv. Moraiolo). I: Effects of pretreatments,
the...
January 2005
Micropropagation overcome the risks related to the diffusion of pest or diseases when plant
material obtained by traditional vegetative multiplication is introduced in new Countries.
However, the transport of micropropagated plantlets is difficult because temperature and/or
fluctuation in relative humidity can damage plantlets, just after acclimatization or during
transport in glass-container.... [Show full abstract]
Read more
Chapter
Encapsulation of in vitro-derived explants: an innovative tool for nurseries.
January 2013
The encapsulation technology consists of the inclusion of some millimeter-long plant portions in
a nutritive and protective matrix. This technology represents a further and promising tool for
exchange of plant material between private and public plant tissue culture laboratories, for short-
and medium-term storage of valuable plant material and for use of in vitro-derived or
micropropagated... [Show full abstract]
Read more
Discover more
Data provided are for informational purposes only. Although carefully collected, accuracy
cannot be guaranteed. Publisher conditions are provided by RoMEO. Differing provisions from
the publisher's actual policy or licence agreement may be applicable.
This publication is from a journal that may support self archiving.
Learn more
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A Method For Microclonal Propagation of Staurogyne Repens in Tissue Culture

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A Method for Microclonal Propagation of

Staurogyne repens in Tissue Culture

Objective: Staurogyn (Staurogyne repens (Nees) Kuntze) is a Services

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Staurogyn (Staurogyne repens (Nees) Kuntze) belongs to the family acanthus

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