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Nutrient requirements of in vitro cultured
Halophila ovalis and Posidonia coriacea:
nitrogen source
INTRODUCTION
Halophila ovalis
Contd.
Posidonia ostenfeldii (den Hartog, 1970)
OBJECTIVES
• to establish the in vitro culture of P. coriacea
and H. ovalis
• examine the response of these two species to
varying nutrient availability
• to report the effect of increasing nitrogen
concentration on growth of both species
METHODOLOGY
H. ovalis seeds collected
from Swan
River sediment, in
metropolitan Perth
Two apical rhizome
sections of
H. ovalis, consisting of
1–2 nodes each, were
subcultured into fresh
BMH from stock
cultures
surface sterilized in
ultra pure water
for 20 min
five replicates
were used for each
treatment.
Stock cultures were
subcultured to fresh
BMH supplemented
with 5.0 lM 6furfurylaminopurine
(kinetin)
every 4 weeks for
approximately 2 years
prior to the
start of the experiment
2% (v/v) benzalkonium
chloride (in
10% (v/v) ethanol) for a
further 20 min followed
by
three rinses in sterile
distilled water.
Seeds were
germinated in 10 ml
Basal Medium for H.
ovalis
(BMH) (control
medium)
Seedlings was
subcultured into 250 ml
polycarbonate tubs
containing 100 ml BMH
to produce stock
cultures.
Fruit of P. coriacea were
collected from the Perth
metropolitan area, on
Parmelia Bank, Western
Australia
Each seedling
culture was considered
one replicate and ten
replicates
were used per
treatment
five replicates
were used for each
treatment.
Spathes containing 20–
30 fruit removed from
parent plants,
transported to
laboratory and
maintained in aerated
tanks containing
seawater at 21C for a
maximum of 48 h.
Seedlings with no visual
contamination were
transferred
to experimental media.
BMP was used as the
control
medium
Fruit were
surface sterilised
Seedling cultures
were established by
dissecting the seed
from the fruit
then transferring to 10
ml of basal medium
After 3 weeks, the
seedling cultures had
two leaves, one root
and were visually
assessed for
contamination
each component
for each case for
both species with
five concentrations
(0, ¼, ½, 1 and 2
MS)
The first
experimental media
tested three broad
medium components
of MS medium , MS
macronutrients, MS
micronutrients and
MS vitamins
• The second
experiment
involved varying
the concentrations
of NH4NO3 and
the source of
nitrogen, either as
NH4+ or NO3
All cultures
were
maintained at
21 ± 1C and
were
subcultured
every 4 weeks
Total nitrogen (mM) ranged from
0 to 60 mM and was supplied as
NO3 (KNO3 or Ca(NO3)2) alone or
with NH4NO3.Halophila experiments ran for 4
weeks, Posidonia 8 weeks and
were subcultured to fresh medium
after 4 week
Result & Discussion
Halophila ovalis
• Effect of MS macronutrients
Parameter
Effect
Fresh weight
• Sig. affect growth of H.ovalis
• Fresh weight reduces with increasing
conc. MS macro
Total no. leaves
No sig effect
Total no. roots
No sig. effect
Chlorophyll
½ MS macro= highest chlorophyll level
DO
1/2 MS macro= highest DO level
• Same effect observed when MS micronutrients is used.
• MS Vitamins no effect observed
• Nitrogen treatment have sig. affect. Increases leaf no, no.
roots, chlorophyll and DO level. But reduces length of root
Contd.
• All media have effect except MS vitamins due
to:
– Opportunistic in nutrient uptake
– Have rapid turnover of rhizomes
– Do not store carbohydrates to the extent
Posidonia coriacea do.
– Adaptable to changes or stresses
Posidonia coriacea
Fresh weight
• Sig affect growth
• Highest conc. MS macro= decrease
growth
Total no of leaves
• Increases with increasing conc. of MS
macronutrients.
• At highest conc. MS macro= reduced
no. leaves.
• No sig. effect
Total no of roots
• No sig. effect
• No effect of MS micronutrients and MS vitamins
observed
• No sig effect of Nitrogen on P.coriacea
Contd.
• MS macro have little effect on growth due to:
– Slow growing nature
Effect of nitrogen
• pH in both cultures decreases at the end of
experiment due to:
– Plants actively absorbs NH4+,lowering the pH due
to an exchange with H+ ion.
– Plants favors NH4+ over NO-3 due to different
oxidation state and ease of exchange
Conclusion
• For H.ovalis, chlorophyll content and DO level
is optimum when ½ MS macronutrients and
MS micronutrients are used.
• ½ MS is the most suitable for both seagrass.
• Highest nitrogen content=highest leaf and
root number for H.ovalis.However root is
short and no root hair when NH4NO3 is
present.
LABORATORY CULTURE OF THE SEAGRASS,
Halophila ovalis (R.Br.) HOOKER f
•
9 out of 12 genera of seagrasses; ThaZassia, HaZoduZe, HalophiZa,
Posidonia, Zostera, Cymodocea, Syringodium, EnhaZus and
ThaZassodendron have been successfully cultured in synthetic seawater
and
under
controlled
environmental
conditions
by
McMillan (1976,1978, 1980a, 1980b).
•
facilitated studies involving the biological, ecological and phenological
aspects of seagrasses (McMillan, 1980a; McMillan 1980b; McMillan et aI.,
1981).
•
14 species of seagrasses of which 5 species belong to Halophila and
Halophila ovalis, being the most common occurred in lagoons, along the
shallow inter-tidal coast and sub-tidal areas. Although common, its
biology and phenology have not been examined (Japar Sidik et aI., 2006).
OBJECTIVES:
(i) the sustain growth and development of the H. ovalis
population
(ii) the reproductive biology (flowering and fruiting)
(iii) the pattern of seedling development from seeds to
mature plants
METHODOLOGY
•
•
•
•
0.42 m x 0.42 m x 1.22 m glass
aquarium
provided with 3-4 em thick of
substrate
flooded with 45 liters of 30 psu
ofInstant Ocean synthetic seawater.
The substrate was flooded with
distilled water for a
month to reduce the nutrients in the
H. ovalis
•
•
•
•
natural
Plugs of H. ovalis explants collected
two types of substrate;
H. ovalis native substrate of calcareous
sandy
mud from Merambong shoal and
commercial
artificial sand
under separate light regimes
of -90 ~lmollm2/sec
six months, 25% by volume of the
water was replaced to replenish the
nutrients and to improve the water
quality of the aquarium.
•
•
•
RESULTS
Plugs of explants were successfully grown in
natural substrate and under both light regimes
of -90 lmol m2s' and -200 lmol m2sl
The sequence of events on the growth and
development, and reproductive biology of H.
ova lis plug (Fig.2) to the formation of
population
Better growth performance of
plugs was observed when grown in natural
substrate and under the light regime of 200lmol m-2 S·I.
INTRODUCTION
• In the Mediterranean Sea, the most important
seagrass
• In this, Isolation of protoplasts = combination of
cellulase Onozuka R-10, hemicellulase and
pectinase
Posidonia Oceanica
Cymodocea nodosa
Collect
-At Ligurian
Sea
Exercised &
Washed
-Youngest
leaves
Cut
tranversally
-1-2mm²
Rotatory
shaker
-Release
protoplast
Culture of
Protoplast
-petri dish
-0.5x105
Sterelized Enzyme
Mixture
-10ml of 5g tissue
Monitoring by
fluorescence
microscope
-isolation
-cell wall regeneration
-7-10h
Icubatiom
-30 min
METHODS
Mesophyll
protoplasts
released from
the cut end of a
P. oceanica leaf
Viable
protoplasts of P.
oceanica
stained with
fluorescein
diacetate
Callus-like
structure in P.
oceanica after
20 days in
culture
Mesophyll
protoplasts
from
Cymodocea
nodosa
 Protoplasts of P. oceanica proved.
 Cymodocea nodosa requires
optimization.
 Affected by the developmental state
and environmental conditions
 Electrophysiology and biophysics.
 Characterization of membrane
properties and ion transport
processes.
 Adaptations and stress resistance in
these plants.
RESTORATION OF CYMODOCEA NODOSA
(UCHRIA)
ASCHERSON SEAGRASS MEADOWS
THROUGH SEED
PROPAGATION. GERMINATION IN VITRO,
SEEDLINGS
CULTURE AND FIELD TRANSPLANTS
Nor Suhada Zakaria 174578
INTRODUCTION
•
Concern has arisen over the decline of seagrass ecosystems
worldwide as a result of adverse human activities in coastal
areas (Walker and McComb 1992, Duarte et al. 2004, Orth
et al. 2006, Duarte and Gattuso 2008, Hughes et al. 2009).
•
The use of seeds and seedlings germinated in vitro is widely
considered to be a more cost-effective alternative (Thorhaug
1985, Orth et al. 1994, 2000, 2009, Balestri et al. 1998, Kirkman 1998, Harwell and
Orth 1999, Granger et al. 2002, Pickerell et al. 2005, Ailstock and Shafer 2006).
OBJECTIVE
•
To enhance biomass development, and outplanting
in the wild under prevailing conditions ensuring
enhanced survival rates
•
To improve the conservation of established
meadows by introducing genetic variation.
MATERIALS & METHODS
Seed collection
& culture
methods
Induction of
germination
Acclimation of
seedlings
Transplants of
developed
seedling to the
field
Results & discussion
Figure 1 Cymodocea nodosa.
(A–C) Three different stages of
development: (A) Stage I,
germinating seeds (arrow),
bars0.5 cm; (B) Stage II,
elongation of cotyledon,
bars1 cm; (C) Stage III,
development of first new leaves
(arrow), bars1 cm; (D–G)
Acclimation: (D–E) Seedlings
acclimating in aquaria or tanks;
(D) bars0.5 m; (E) bars0.4 m;
(F–G) Seedlings with leaves
attached to a young incipient
rhizome with up to two leaves
and root development after 30
days of acclimation; (H–J) Field
transplant. Persistent patch
formed from clustered seedlings
transferred directly
into the sediment: (H) Plants at
day zero, bars5 cm; (I) Plants at
day 30. Note plagiotropic
Figure 2 Cymodocea nodosa: plant development during acclimation.
(A–B) Leaf and root lengths over 45 days. (C–D) Shoot and root regeneration over 45
days seedling acclimation (values are means"SD,
ns30).
Figure 3 Cymodocea nodosa: field trials.
(A) Survival (%). (B) Average leaf length (cm) of acclimated plantlets transferred to
a natural meadow (27859920.440 N; 15822912.450 W,Gran Canaria, Spain) using
three different transplant methodologies: a)seedlings scattered on nylon nets (rs3,
ns60), b) seedlings clustered in biodegradable trays (rs3, ns108), and c) seedlings
planted directly into the sediment (ns76). Seedling monitoring was performed over
nine months (values are means"SE).
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