Aquatic Botany 84 (2006) 79–84

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Anatomical and nutritional requirements for induction and sustained

growth in vitro of Cymodocea nodosa (Ucria) Ascherson
Pilar Garcı́a-Jiménez, Eva P. Navarro, Cristo H. Santana,
Ángel Luque, Rafael R. Robaina *
Departamento de Biologı́a, Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria,
Gran Canaria, The Canary Islands, Spain
Received 13 July 2004; received in revised form 20 June 2005; accepted 6 July 2005

Abstract
In vitro methods of plant micro-propagation are being considered as a possible solution for the decline in seagrass communities registered
worldwide. To achieve successful plant micro-propagation, culture conditions are commonly adjusted empirically within almost species-specific
conditions, to comply to the following three conditions: (i) culture establishment (ii) shoot production and (iii) rooting and hardening for planting
in soil. Cultures of Cymodocea nodosa were established from axenic explants of the apical meristem (approx. 0.5 cm) which regenerated new leaf
or produced leaf regenerating calli (5% of cultivated explants) in media containing 10
6 M of the cytokinin analogue TDZ. Longer ramet
explants, not fully axenic, containing internode with leaf and roots were also affected by 10
6 M cytokinins and auxin type of regulators, as they
promoted leaf extension (in cm), particularly GA. None of the explants progressed further to massive shoot propagation and new plantlet
production. Instead, experiments made with ramet explants which simulated potential produced plantlet revealed that there seems to be a strong
interaction within leaf, rhizome and root, since the carbon fixed in the leaf was rapidly translocated to the rest of the tissue (50% in the roots in a
FW basis). The explants preferred ammonium and dihydrogen inorganic phosphate as a nutrient source, efficiently assimilating the former
regardless of whether such were added to the underground or surface tissue. However, underground tissue was required to maintain P status in the
cultivated explants.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Cymodocea nodosa; Explant anatomy; Growth substance; In vitro; Nitrogen; Phosphorus

1. Introduction Micropropagation is the propagation of plant species in vitro

starting from its cells, tissues or, organs. This can be carried out
The seagrass Cymodocea nodosa (Ucria) Ascherson forms in a shorter time than conventional vegetative propagation, and
dense submerged, ecologically relevant communities as stable produces high biomass from one single plant. In the 1970s
and protected habitats, where other vegetation, animals and biota Murashige stated that three developmental stages of propaga-
live, spawn and feed. Anthropogenic activities have influenced tion seems to be mandatory: (i) culture establishment, (ii) shoot
coastal marine ecosystems to the extent that they are considered production and (iii) rooting and hardening for planting in soil.
to be responsible for the worldwide decline in seagrass Using these ground rules, micropropagation of freshwater
communities. Several strategies such as transplantation and aquatic plants was successfully carried out concluding in
micropropagation may help to avoid complete depletion. normal and healthy plants (Huang et al., 1994; Agrawal and
Transplantation depends on a source (threatened) population Mohan Ram, 1995; Kane et al., 1999).
and its rates of reproductive or vegetative propagation, which are Interesting studies have been produced, aimed at in vitro
commonly slow (West et al., 1990; Molenaar et al., 1993; culture and/or propagation of ecologically relevant marine
Molenaar and Meinesz, 1992, 1995). species, such as Posidonia oceanica, Ruppia maritima or
C. nodosa. Whilst it is clear that it is possible to apply the
essentials of in vitro methodology to establish explant cultures
* Corresponding author. Tel.: +34 928 452904; fax: +34 928 452922. (i.e. stage I), massive shoot induction, rooting control and plant
E-mail address: rrobaina@dbio.ulpgc.es (R.R. Robaina). production (stages II and III) comparable to that of other
0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquabot.2005.07.006
80 P. Garcı́a-Jiménez et al. / Aquatic Botany 84 (2006) 79–84

aquatic plants, such as the above mentioned species, have not penicillin, nystatin, ampicillin (150 mg 1
1 each) and germa-
been achieved (Loquès et al., 1990; Koch and Durako, 1991; nium dioxide (5 mg 1
1). This method produced not axenic
Terrados-Muñoz, 1995; Bird et al., 1993, 1996, 1998). cultures of such a centimeter long explants, but together with
In spite of the relevance of these physiological traits to the sterilization through autoclaved seawater, nutrient solutions,
success of in vitro propagation of seagrasses, nothing is known sand and the culture vessel, ensured they were clean enough to
about the actual or potential nutritional situation of the explants avoid contaminant overgrowth and interference during the
and plantlets produced in culture. The ecophysiological experimental time (up to 15 days). Axenic explants were
literature relating to these problems commonly addresses the obtained from cylindrical fragments (0.5  0.1 cm) excised
question in the light of the respective roles of root versus leaf as from the meristematic apex of the plagiotropic rhizome
the organ involved in nutrient absorption (commonly N and P). (henceforth, apical explants).
This is the same in the case of the acquisition of carbon, its
fixation and allocation during photosynthesis, although it is 2.2. Culture conditions
clear that seagrasses translocate oxygen and carbon from leaf to
non-photosynthetic tissues (Pérez et al., 1994; Terrados and To check the effects of plant growth regulator (PGRs)
Williams, 1997; Kraemer and Mazzella, 1999; Touchette and auxins, indole acetic acid (IAA), indole butyric acid (IBA), 2,4-
Burkholder, 2000a,b). In fact, clone identity experiments dichlorophenoxyacetic acid (2,4-D); cytokinins, kinetin (KIN),
revealed that the subsistence of an apical meristem in C. nodosa benzyladenine (BA), N-phenyl-N0 -1,2,3-thidiazol-5-ylurea
seems to depend on the rhizome (up to 50 cm Terrados et al., (thidiazuron or TDZ), and gibberellic acid (GA) were added
1997). individually as filter-sterilised stock solutions at 10
6 M final
In this work, explants of C. nodosa, containing only the concentration ramets were cultured in 30 ml Provasoli’s
apical meristem, or a ramet, containing the internode, leaf, and medium (PES, Provasoli, 1968) in tubes, supplemented with
associated root (i.e. simulating plantlet), were cultivated under the respective growth regulator. Axenic apical explants were
different nutritional conditions, which included plant growth cultivated in solidified (agar 0.8%) PES medium (prepared
regulators, and nitrogen and phosphorus sources to determine: with diluted seawater to adjust the osmolarity to that of the
(i) the effect of growth regulators, (ii) the preferred chemical seawater; Robaina et al., 1990) in Petri dishes, supplemented
form of the nutrients and (iii) the relative importance of the with 10
6 M of the same regulators and sucrose 60 g 1
1.
underground and overground parts of the explant in N, P and C Preliminary experiments carried out with most of the
assimilation. This was an attempt to clarify whether growth regulators tested did not reveal the existence of a clear dose
regulators affect growth, to optimise the type and form of response, thus effective concentration was directly adjusted to
addition to the culture vessel of the limitant nutrients, and to 10
6 M as a standard hormonal concentration ramets and
evaluate to what extent both parts are necessary to avoid poor apical explants were kept for 15 days in their respective media.
nutritional status during the propagation of the regenerated Experiments were repeated three times with five replicates of
plantlet during stages II and III of propagation. each regulator (i.e. 15 ramets and 15 apical explants per growth
regulator).
2. Materials and methods Enriched seawater Provasoli medium in Magenta1-G7
(Sigma Co.) culture vessels was used as a culture medium in the
2.1. Plant material experiments with nutrients. The vessels were filled with 40 ml
autoclaved sand and 200 ml of liquid culture medium as shown
Samples of C. nodosa (Ucria) Ascherson were collected at in Fig. 1. Enrichment of the medium was achieved by adding
Castillo del Romeral (on the southeast coast of Gran Canaria, nutrients from sterilized stock solutions to the seawater.
the Canary Islands, 278480 0000 N; 158250 4000 W) from shore Nutrient enriched sand was obtained by incubating autoclaved
pools of 1–2 m depth. To avoid any damage to the populations, sand during 4 days in the appropriate nutrient solution. To
the experiments were regularly carried out with material check nutritional preferences for different chemical forms of N
collected in the Winter and Spring of the years from 2001 to and P, a simplified factorial experimental design of Box–
2003. Within 2 h after collection, the youngest and cleanest Behnken was used (Tox and Behnken, 1969) for three factors
plant material was selected at the laboratory, where explants of (nitrate, glutamic, ammonium, for N, and inorganic KH2PO4
approx. 3 cm consisting of internode rhizome, leaf, and and organic glyceraldehyde-3-phosphate for P) with three
associated root were excised from the rhizome (henceforth concentration levels based on regular PES enrichment of sand
ramets, Fig. 1). Leaves from ramets were further cut and water (0, 0.05, 0.1 mM, and 0, 0.5, 1 mM phosphorus and
transversely in order to analyse the effect of regulators on nitrogen, respectively) and three central points. A semiquanti-
new leaf tissue generation (meaning both the growth (cm) of the tative response variable (GH) was used to measure greenness
young leaf and new leaf tissue produced by those that were cut and youthful vigor of leaves during the experimental time (15
off). Explants were surface sterilized previous to cultivation, by days). One ramet per vessel (Fig. 1) was used together with five
dipping in distilled water and immersion in 1% sodium replicates per treatment (GH = 1 no evident change or even
hypochloride in autoclaved seawater for 5 min. After rinsing degeneration symptoms, GH = 2 healthy as pigmented, and
three times in sterile seawater, explants were incubated for 48 h GH = 3 healthy and regenerating new leaves. Fig. 1C and D).
with a filter-sterilized antibiotic mixture containing rifampicin, The entire experimental design was performed twice with five
 P. Garcı́a-Jiménez et al. / Aquatic Botany 84 (2006) 79–84 81

Fig. 1. In vitro culture of Cymodocea nodosa. (A) Leaf regenerating callus growing from an axenic apical explant, obtained after 15 days in media supplemented with
10
6 M of the synthetic cytokinin N-phenyl-N0 -1,2,3-thidiazol-5-ylurea (thidiazuron or TDZ). (B) Ramet with node, leaves and associated roots, over the grid used to
homogenize size. (C and D) Growth of healthy explant after 15 days in culture vessels (i.e. GH = 3) with sandy ground and overlying water layer used in this study
(note the extension of the leaves towards the surface, arrow). Scale bar 0.2 cm in part (A), and 1 cm in the rest.

replicates (i.e. 10 ramets per treatment). Here, we present the dilute assimilated nutrients. The water and/or the sand part of
pooled data. the cultivation system in the culture vessel was enriched with
l mM ammonium or 0.1 mM KH2PO4, and the content of each
2.3. Leaf versus root nutrient assimilation nutrient in the ramet was analysed. A series of experiments
were performed to check whether kinetin addition (10
6 M)
Once reduced N and inorganic form of P were determined as might also contribute to N and P assimilation by the explants.
the preferred chemical form, experiments were performed to The content of nitrogen (%N) and phosphorus (%P) were
check whether these nutrients were being absorbed by the determined in dried powdered plant material cultivated in each
ramets preferably from the enriched water (i.e. overground of the conditions tested (n = 6–9 samples per treatment).
tissue) or from the enriched sand (i.e. underground tissue) Nutrients were extracted and determined colorimetrically
prepared as described above. Short-term design (48 h) was following standard methods for plant analysis (Walinga
performed in order to avoid any increase of biomass that may et al., 1995). As a reference of nutrient contents, some samples
82 P. Garcı́a-Jiménez et al. / Aquatic Botany 84 (2006) 79–84

of C. nodosa (the whole plant powder) were send to Estación

Experimental of Zaidin (CSIC, Granada, Spain), a reputed
reference laboratory for nutrient content in plants. There, N and
P were determined colorimetrically after microwave-assisted
digestion of vegetal material.

2.4. Carbon fixation

Inorganic carbon fixation was monitored by incubating the

ramets (n = 3) for 90 min in 20 ml PES with 0.5 mCi [14C]-
NaHCO3 (42.6 mCi mmol
1, 1.6 GBq mmol
1, NEN, UK) and
70 mmol photons m
2 s
1, provided by daylight fluorescent at
the level of the incubation vessel. After incubation, HCl4N was
added to remove the remaining [14C]-NaHCO3, and the ramets
were split into leaf, rhizome and roots to be extracted and the
fixed dpm counted separately. Carbon fixation rates were
calculated using the equation: Fig. 2. Effect of plant regulators on the length of the leaf tissue and number of
not fully axenic ramet explants showing leaf extension after 15 days in culture
dpm S DIC 1:05 (mean  S.E., n = 5 in three replicates). All regulators were supplemented at
fixed carbon ðmg C h
1 Þ ¼
dpm T t 10
6 M. IAA = indole acetic acid, IBA = indole butyric acid, 2,4-D = 2,4-
dichlorophenoxyacetic acid, KIN = kinetin, BA = benzyladenine, TDZ = N-
where dpm S is the dpm in the DMSO (10 ml) soluble fraction phenyl-N0 -l,2,3-thidiazol-5-ylurea or thidiazuron, GA = gibberellic acid. (*)
of each leaf, rhizome and root sample; DIC is the carbon (mg C) denotes significant difference with control (P < 0.05).
in 20 ml of media with 1.86 mM dissolved inorganic carbon;
1.05 is a correction factor; dpm T is the total dpm added to the Short-term experiments of N and P enrichment, revealed
incubation vessel; and t is the time in hours. Data were normal- that, as compared to control plants, N status was kept in all
ized to g FW and mg Chl a in the leaf, by extracting 90% cultivated explants, regardless of whether it was added to the
acetone and quantified spectrophotometrically using the equa- water or to the sand. Only explants cultivated in sand enriched
tions of Jeffrey and Humphrey (1975).
Table 1
2.5. Statistics Analysis of the Box–Behnken simplified factorial design for N and P (including
ammonium as the best N source)
Analysis of the Box–Behnken simplified factorial design for Source d.f. Mean squares F-ratio P-value
N and P Statgraphics Plus 5.1 statistical software (Statistical
A: nitrate 1 2.0 5.71 0.062
Graphics Co, MA, USA). Tukey HSD post hoc test (SPSS 13.0.
B: glutamic acid 1 1.125 3.21 0.133
SPSS Inc., IL, USA) was used for multiple comparison of data C: ammonium 1 10.125 28.93 0.003
from the experiments with N, P, and PGR’s enrichment. AA 1 0.923 2.64 0.165
AB 1 2.25 6.43 0.052
3. Results AC 1 0.25 0.71 0.436
BB 1 0.230 0.66 0.453
BC 1 0 0 1.000
None of the plant growth regulators tested promoted growth CC 1 2.076 5.93 0.058
or regeneration in apical explants, but outstanding results were
Total error 5 0.35 – –
obtained with N-phenyl-N0 -1,2,3-thidiazol-5-ylurea (TDZ) r2 = 75.98%
since leaf regeneration was observed in 80% of the explants,
A: ammonium 1 2.076 41.54 0.001
and leaf regenerating callus were to be observed sprouting from
B: KH2PO4 1 1.125 22.5 0.005
the edge of the explants (Fig. 1A) in a frequency of up to 5% of C: Glyceraldehyde-3P 1 0.932 18.46 0.007
the cultivated explants. Neither the calli nor the leaflet AA 1 1 0.125 2.5
progressed further as they browned and died off after a few AB 1 4 80 0.003
weeks. In the larger not fully axenic explants, plant growth AC 1 0.25 5 0.075
BB 1 2.076 41.54 0.001
regulators influenced both new leaf regeneration and extension
BC 1 0.25 5 0.075
in ramet explants (Fig. 2), with the highest records for CC 1 0.5 10 0.250
gibberellic acid. Indole butyric acid (IBA) was apparently
Total error 5 0.05 – –
inhibitory as compared to the rest. r2 = 94.03%
The results on nutrient preferences are presented in Table 1
and Fig. 3. Block design revealed that, in terms of the GH The ANOVA table partitions the variability in GH (growth and health variable)
for each of the effects, and test the statistical significance of each factor by
variable, ammonium and inorganic phosphate sustained better comparing the mean squares against an estimate of the experimental error.
the ramet as they were the most significant factors explaining Significant effects are in bold letter (P < 0.050). r2 statistic indicates the
up to 94% of the variance of the GH observed (Table 1). percentage of the variability of GH explained by the model.
 P. Garcı́a-Jiménez et al. / Aquatic Botany 84 (2006) 79–84 83

4. Discussion

The effectiveness of plant growth regulating substances

(PGRs) is essential to complete the micropropagation of C.
nodosa since this will depend on the availability of feasible
PGR formulations to induce organized growth from explants
and to propagate the plantlets that were produced. The effect of
plant growth regulators has been previously reported in
seagrasses, such as P. oceanica and R. maritima, in which
cytokinin induced shoot and rhizome growth (Loquès et al.,
1990; Koch and Durako, 1991) whilst auxin increased shoot
growth and branch production in rhizome segments obtained
from seeds in Halophila decipiens (Bird et al., 1998). Recently,
auxin naftal and IBA were reported to increase root emergence
and growth in seedlings of P. oceanica (Balestri and Bertini,
2003). In C. nodosa, Terrados-Muñoz (1995) reported the
effects of GA on leaf growth in explants from the rhizome. In
this work, small apical meristem explant cultures could be
established under axenicity, thus producing new leaflet or leaf
regenerating calli when TDZ, a cytokinin-like substance first
used for recalcitrant woody species (Chevreau et al., 1989), was
added to the medium. On the other hand, non-axenic ramet
explants regenerated new leaf tissue as a result of the treatment
with all the plant growth regulators tested, particularly GA. Our
results and previous reports support that seagrasses, like other
aquatic plants and terrestrial counterparts, may acclimatise well
to in vitro culture conditions to complete a stage I of culture
establishment reactive to PGRs. However, it seems rather
difficult to progress further to stages II and III of the
Fig. 3. Nitrogen (%N) and phosphorus (%P) assimilation by the explants
propagation since even the axenic calli did not progress to
cultivated (2 days) in 1 mM ammonium or 0.1 mM KH2PO4, in the presence develop stage II plantlet.
or absence of kinetin (10
6 M). Nutrient supplementation was carried out in the In this respect, the experiments made with ramet explants
water (SW), in the sand (SND) or in both (SW-SND). Bars are means of six to may contribute to improve the culture requirements of future
nine replicates. Vertical lines are S.E. Same letter over the bar denotes regenerated plantlets since, it seems (Table 2, Fig. 3) that there
homogeneous group of means. (*) denotes significant difference with control
(P < 0.05).
is a close interconnection within the leaf–rhizome–root space,
thus no part of the plant anatomy could be avoided in the new
regenerated plantlet. First evidence came from carbon fixation
with P could kept P status, since ramets in water enriched with P experiments, since the carbon fixed at the leaf is rapidly
showed a significant lost. Kinetin addition to sand and water distributed over the leaf itself, the rhizome and the roots (50%
also caused significant loss of both N or P. Its addition to the on a FW basis). Second, the experiments of addition of N and P
water together with P enrichment could contribute towards support that these appeared to fulfil most of the requirements of
maintaining its status (Fig. 3). the cultivated explants (94% of the variable GH explained). In
Rapid translocation of the fixed carbon from the leaf, as the spite of N and P content of our C. nodosa (Fig. 3. and data
point of fixation, to the rhizome and to the roots (Table 2) was from reference laboratory %N = 1.49  0.009; %P = 0.146 
observed after 90 min in presence of the radioactive inorganic 0.0001; mean  S.E.) may support for its consideration as
carbon source. nutrient depleted plants (Duarte, 1990) the cultivated explants
seem not assimilated well nutrient enrichment. On the contrary,
as seen in Fig. 3, without any increase in biomass that might
Table 2 dilute assimilated nutrients, a rapid (48 h) decrease in P nutrient
Carbon fixation rate and allocation after 90 mm of plant incubation in radio- status might be promoted in the cultivated plant, unless P
active inorganic carbon (n = 3)
addition was provided to the underground tissue or kinetin was
Chl a (mg C h
1 mg
1) FW (mg C h
1 mg
1) present in the water.
Leaf 0.185  0.03 (30%) 0.55  0.044 (14%) The respective role and interaction of the aboveground and
Rhizome 0.26  0.07 (43%) 1.26  0.020 (34%) underground tissue on the physiological performance of
Root 0.16  0.04 (26%) 1.92  0.015 (52%) seagrasses has been a matter for significant ecological concern
Data are mean  S.E. Percentage in each organ of the total carbon fixed are in (Pérez et al., 1994; Terrados and Williams, 1997; Terrados
parentheses. et al., 1997; Kraemer and Mazzella, 1999; Touchette and
84 P. Garcı́a-Jiménez et al. / Aquatic Botany 84 (2006) 79–84

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