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Preliminary Studies on Cryopreservation

of Vegetative Thalli of Some
Economically Important Seaweeds of
India
a a a
P. L. Lalrinsanga , Geetanjali Deshmukhe , S. K. Chakraborty ,
a a a
Alkesh Dwivedi , Nimai Barman & Niraj Kumar
a
Central Institute of Fisheries Education , Versova, Mumbai, India
Published online: 09 Dec 2009.

To cite this article: P. L. Lalrinsanga , Geetanjali Deshmukhe , S. K. Chakraborty , Alkesh Dwivedi ,

Nimai Barman & Niraj Kumar (2009) Preliminary Studies on Cryopreservation of Vegetative Thalli of
Some Economically Important Seaweeds of India, Journal of Applied Aquaculture, 21:4, 250-262, DOI:
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 Journal of Applied Aquaculture, 21:250262, 2009
Copyright Taylor & Francis Group, LLC
ISSN: 1045-4438 print/1545-0805 online
DOI: 10.1080/10454430903317096

Preliminary Studies on Cryopreservation of

1545-0805
1045-4438
Journal
WJAA of Applied Aquaculture,
Aquaculture Vol. 21, No. 4, October 2009: pp. 00

Vegetative Thalli of Some Economically

Important Seaweeds of India

P. L. LALRINSANGA*, GEETANJALI DESHMUKHE*,

Cryopreservation
P. L. Lalrinsanga etofal.
Vegetative Thalli

S. K. CHAKRABORTY, ALKESH DWIVEDI,

NIMAI BARMAN, and NIRAJ KUMAR
Central Institute of Fisheries Education, Versova, Mumbai, India
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The effects of cryoprotectants and their concentrations, prefreezing

temperature, equilibration time, thawing temperature, and the
regenerative efficacy of cryopreserved vegetative thalli (apical tips)
of Gracilaria corticata J. Agardh., Ulva lobata Duetzing, and
Hypnea musiformis (Wulfar) Lamouroux were evaluated. The api-
cal tips were suspended in various cryoprotective solutions and
slowly cooled to 40C over a period of 4 h. After the slow cooling
step, the suspensions were immediately immersed in liquid
nitrogen (LN), which were thawed after 2 days. Both U. lobata and
H. musiformis survived maximally in 10% DMSO, whereas 10%
glycerol was found most suitable for G. corticata. A slow cooling
temperature of 40C and thawing temperature of 40C were
found most suitable irrespective of the species. The equilibration
time of 60 min was found most suitable for G. corticata, while 45
min was sufficient for both H. musiformi and U. lobata. Cryopre-
served vegetative thalli were then successfully regenerated, though
the survival was considerably reduced up to a period of 28 days
after which it was stabilized.

KEYWORDS cryopreservation, seaweeds, Hypnea, Ulva, Gracilaria,

survival rate

*Present address: Central Institute of Freshwater Aquaculture, P.O. Kausalyaganga,

Bhubaneswar 751002, Orrisa, India.
Address correspondence to Geetanjali Deshmukhe, Central Institute of Fisheries
Education, Fisheries University Road, Seven Bunglow, Versova, Mumbai-400061, India.
E-mail: dgeetanjali@gmail.com

250
 Cryopreservation of Vegetative Thalli 251

INTRODUCTION

Macroalgae are important resources of the marine ecosystem. In India,

seaweeds are mostly used as raw material for phycocolloids production
such as agar, alginates, and carrageenan. Gracilaria, Gellidiella, Gelidium,
Sargassum, Turbinaria, and Hypnea sp. are some of the commercially
important seaweeds of the country. Since the farming of these species is still
under trial, natural seaweeds are heavily exploited for industrial and human
consumption. Gracilaria corticata, Hypnea musiformis, and Ulva lobata are
some of the important marine macroalgae found along the coast. Recent
studies revealed that there is a decreasing trend in the number of these
species along the coast (Dhargalkar, Untawale, & Jagtap 2001). Cryopreser-
vation offers an opportunity for long-term storage and preservation of
biological specimens, which can be revived again at any desired time and
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thus is helpful in germplasm preservation in an effective, fast and safe

manner. As the culture maintained in laboratories increased, the technique
of cryopreservation is becoming increasingly important for conservation and
management of algal resources.
Successful cryopreservation of marine algae has been previously
reported around the world (Taylar & Fletcher, 1999; Kuwano & Saga, 2000).
However, information on regenerative efficacy of cryopreserved thalli under
environmental condition and factors affecting the survival are still limited.
Further, no information on cryopreservation of marine algae from Indian
waters is yet available. Therefore the present investigation was conducted
with the aim of standardizing the cryopreservation protocol for economi-
cally important species like Gracilaria, Hypnea, and Ulva to assess factors
affecting survival, while evaluating the regenerative efficacy of the cryopre-
served thalli under natural environmental conditions.

MATERIALS AND METHODS

Test Organisms and Culture
Vegetative thalli (apical tips) of Gracilaria corticata (J. Agardh.) J. Agardh.,
Hypnea musiformis (Wulfar) Lamouroux, and Ulva lobata Duetzing were
used in the present study. The samples were collected from Maharashtra
coast of India (Malvan 16 04 N 73 28 E, 18 54 N; Colaba, Mumbai 72
47 E and Dahanu 19 58 N 72 44 E) and thoroughly washed with filtered
seawater three to four times and maintained in Provosoli-enriched seawater
(PES) (Provasoli 1968) media. Germanium oxide (GeO2 10 mg.l-1) and
antibiotics mixture (1 ml.l-1) were added initially to control the diatom and
bacterial growth. The cultures were then maintained at 22C25C and 15 mol.
photon. m-2s-1 provided by cool white fluorescent lamp with a
photoperiod of 14:10 hr light:dark cycle. The apical tips of thalli were cut
 252 P. L. Lalrinsanga et al.

into small 12 mm fragments using sterile surgical blade. The resulting frag-
ments were cultured under the same condition as mentioned above, for one
week prior to cryopreservation.

Addition of Cryoprotectants
Three different cryoprotectantsDimethylsulfoxide (DMSO), Ethylene glycol,
and glycerolwere tested at three different concentrations of 5%, 10%, and
15%, each with HEPES (0.01M) buffer. They were dissolved in seawater
(34) at twice the intended final concentration and buffered with 0.01M
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) at pH 8. The
cryoprotectants solutions were chilled in ice bath and added slowly to an
equal volume of ice-chilled apical tips suspension; the addition of cryopro-
tectant solution was performed with stirring for a period of 15 min to avoid
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sudden change in osmotic pressure (Kuwano, Aruga, & Saga 1993). After
adding the cryoprotectants in the sample suspension, aliquots of 4.0 ml
were dispensed in cryogenic vials (5-ml capacity). The vials were then
mounted in an aluminum holder and allowed to stand for equilibration at
room temperature as pretreatment is generally thought to be most successful
at room temperature (20C23C) (Taylor & Fletcher 1999).
Experiments were also made to assess the suitability of the different
equilibration time by using 30, 45, and 60 min equilibration periods.

Cooling
Five different prefreezing temperature of 20C, 30C, 40C, 50C, and
60C with a non-linear cooling rate of <1C min-1 for 4 h were examined.
After the slow cooling step, the vials were immediately immersed in LN and
stored for 24 h. The effect of the prefreezing temperature was examined by
directly thawing the suspension after prefreezing treatment without
immersion in the LN and cultured. The survival percentage was compared
with the survival percentage of the thalli, which were thawed after LN
immersion.

Thawing and Removal of Cryoprotectants

Five different thawing temperatures at 20C, 30C, 40C, 50C, and 60C
were tested by agitating the vials in a water bath and transferred to an ice
bath just before the ice melted completely. The melted suspensions were
then serial diluted to six-fold volume of ice-chilled seawater for 30 min to
remove the cryoprotectants, and diluted cryoprotectants were discarded.
The thalli were rinsed twice and maintained in the PES culture media at
room temperature under the condition mentioned earlier.
 Cryopreservation of Vegetative Thalli 253

Viability Assay
Viability of the vegetative thalli (apical tips) was assessed by pigmenta-
tion index as described by van der Meer and Simpson (1984) and growth
measurements in terms of increase in length. The thalli were incubated
in a fresh PES culture medium and after incubation viability was
assessed for one month. Pigmentation of the thalli was determined every
two days interval, under microscopic observation of pigment retention,
phycoerythrin for both G. corticata and Hypnea musiformis and chloro-
phyll for U. lobata. Observation on pigmentation was taken for up to 15
days of the incubation period when the pigment loss was reduced and
stabilized. The percentage of thalli retaining the pigments on the final
day of observation was approximated as the real survival. Simulta-
neously, the growths were also recorded by length measurements at
two-week intervals. Viability was examined immediately after thawing
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and after post-thawing incubation.

Thallus Regeneration
The regenerative efficacies of the cryopreserved thalli were estimated by
maintaining the thalli in seawater (34) in non-axenic outdoor tanks in the
condition simulated to the natural environment by giving daily immersion
and exposing. After incubation in the culture media for the viability test for
a period of one month, the thalli were directly transferred to the outdoor
tank. Observation on the growth was taken by length measurement at an
interval period of two weeks for a period of two months. The effect of
freezing on the thalli was studied by taking a section of both the dead and
live cryopreserved thalli and observing under microscope for any disruption
or damage in the tissue.
Each of the above experiment setups were kept in triplicate along with
control and data were analyzed through one-way ANOVA.

RESULTS

Among the three cryoprotectants used singularly, 10% glycerol gave the
highest survival rate of 85.06 2.1% for G. corticata (Table 1). However,
15% glycerol was found to be detrimental for the plant, and the survival at
5% was also found to be low (Table 1). Ethylene glycol at 5% gave the low-
est survival rate of 51.04 2.8 (Table 1). In the case of H. musiformis and U.
lobata, 10% DMSO was found to be the most suitable, which gave a survival
of 28.92 2.1% and 51.03 2.2%, respectively (Table 1). However, DMSO
at 15% was found to reduce the survival for both H. musiformis and U.
lobata to 16.24 2.7% and 22.85 2.3%, respectively (Table 1).
 254 P. L. Lalrinsanga et al.

TABLE 1 Effects of Various Cryoprotectants on the Survival of Vegetative Thalli After the
First Slow Cooling to -40C and Immersion in Liquid Nitrogen (N=50)

Survival (%)*
Cryoprotectant
concentration Gracilaria corticata Hypnea musiformis Ulva lobata

5% DMSO 56.1 2.3 13.8 0.9 29.4 2.3

10% DMSO 73.3 1.8 28.9 2.1 51.0 2.2
15% DMSO 58.4t 3.6 16.2 2.7 22.8 3.3
5% Ethylene Glycol 51.0 2.8 6.8 2.1 23.7 2.3
10% Ethylene Glycol 74.9 3.5 6.1 2.8 28.2 2.9
15% Ethylene Glycol 72.4 2.5 6.2 2.8 46.5 3.0
5% Glycerol 66.4 2.9 17.4 1.3 39.0 2.2
10% Glycerol 85.0 2.1 17.6 1.5 46.5 2.5
15% Glycerol 68.1 3.3 5.9 2.5 45.2 2.9
Control (Seawater) 0.0 0.0 0.0 0.0 0.0 0.0
*The thalli were immersed in liquid nitrogen after the first slow cooling to 40C. Survival rate was
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estimated after post-thawing incubation for 15 d.

The viability estimation by pigmentation index method showed that the

cryopreserved thalli thatsurvived the freezing treatment retained their pig-
ments even after a long period of incubation in the culture media, whereas
the dead thalli were found to lose their pigments within a short period of
time.
When the thalli was immersed in liquid nitrogen, the survival of thalli
prefrozen to 20C to 30C reduced considerably (Figures 1, 2 & 3). How-
ever, prefreezing to 40C and below decreased the reduction in survival
progressively. The optimum prefreezing temperature was 40C regardless
of cryoprotectant concentration (Figures 1, 2 & 3).
The equilibration time had significant effect on the survival of the cryo-
preserved thalli, with an equilibration time period of 60 min being the most
suitable for G. corticata, which were treated with 10% glycerol (Figure 4).

100

80
Survival (%)

60

40

20

0
20 30 40 50 60
Prefreezing Temperature (C)

Survival (%) after liquid nitrogen treatment

Survival (%) without liquid nitrogen treatment

FIGURE 1 Survival of the vegetative thalli of H. musiformis suspended in 10% DMSO.

 Cryopreservation of Vegetative Thalli 255

90
80
70

Survival (%)
60
50
40
30
20
10
0
20 30 40 50 60
Prefreezing Temperature (C)

Survival (%) after Liquid nitrogen treatment

Survival (%) without Liquid nitrogen treatment

FIGURE 2 Survival of the vegetative thalli of U. lobata suspended in 10% DMSO.

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100

80
Survival (%)

60

40

20

0
20 30 40 50 60
Prefreezing Temperature (C)
Survival (%) after liquid nitrogen treatment
Survival (%) without liquid nitrogen treatment

FIGURE 3 Survival of the vegetative thalli of G. corticata suspended in 10% glycerol.

An equilibration period of 45 min was sufficient for both H. musiformis and

U. lobata, while a period of more than 45 min showed no significant differ-
ence in survival. However, periods less than 45 min showed reduction in
survival (Figure. 4).
It was also found that thawing temperature was influential on the sur-
vival of thalli during post-thawing incubation. The survival of thalli was
highest at 40C thawing temperature irrespective of the species (Figures 5 & 6).
However, there was less significant difference in the survival when thawed
at a temperature above 40C. The frozen thalli thawed at a temperature
lower than 40C showed a decrease in survival (Figure 5); thalli thawed
directly after prefreezing incubation at a different temperature without
immersion in the LN showed a gradual decrease in survival with the
decreasing prefreezing temperature (Figures 5 & 6).
 256 P. L. Lalrinsanga et al.

100
80

Survival (%)
60
40
20
0
30 45 60
Equilibration time

FIGURE 4 Effects of equilibration time on the survival (%) of H. musiformis, U. lobata,

and G. corticata.
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90
80
70
Survival (%)

60
50
40
30
20
10
0 I
20 30 40 50 60

Thawing Temperature (C)

FIGURE 5 Effects of different thawing temperature on the survival (%) of vegetative thalli of
H. musiformis and U. lobata suspended in 10% DMSO and G. corticata suspended in 10%
glycerol.

90
80
70
Survival (%)

60
50
40
30
20
10
0
1 14 28 42 56 70
Days of Culture

FIGURE 6 Survival (%) of cryopreserved thalli cultured and regenerated in outdoor tanks.
 Cryopreservation of Vegetative Thalli 257

TABLE 2 Growth Performances of Vegetative Thalli After Different Periods of

Suspension in Outdoor Tanks

Average length of thalli (in mm)

Days of culture Gracilaria corticata Hypnea musiformis Ulva lobata

1 1.56 0.06 1.93 0.04 1.95 0.04

14 2.68 0.04 2.56 0.06 2.24 0.06
28 3.61 0.12 3.16 0.04 2.57 0.02
42 5.05 0.04 3.87 0.05 3.07 0.07
56 6.34 0.08 4.23 0.05 3.40 0.06
70 7.19 0.04 4.62 0.08 3.74 0.03

While culturing the cryopreserved thalli in an outdoor tank and

with condition simulated to the natural environment, the survival was
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considerably reduced. The survival percentage of G. corticata was

reduced considerably from 85.06 2.1% to 58.97% (Figure 6). The sur-
vival rate was decreased until a 28 days period of culture after which the
survival was stabilized. The survival of H. musiformis and U. lobata,
which were maintained under the same condition, was also reduced
from 28.92 2.1% to 08.16% and 51.03 2.2% to 12.50%, respectively
(Figure 6). The growth performance of cryopreserved thalli is presented
in Table 2.

DISCUSSION

The freezing of living cells, a complex biological process, is only partly

understood as yet. Formation of intracellular ice crystals during cooling and
subsequent thawing is the major source of damage, and much of an effort in
cryopreservation is directed towards preventing their formation. The two-step
cooling method used in the present study is a widely applicable technique
for the cryopreservation of marine macroalgae. Cell dehydration during the
first cooling prevents intracellular freezing during the second rapid cooling;
intracellular freezing causes the destruction of the intracellular structures,
resulting in cell death (Kuwano & Saga, 2000). During slow cooling to an
intermediate subzero temperature, cells are dehydrated (Mazur, 1984), and
survival at LN temperature requires adequate dehydration, which prevents
intracellular ice formation during the immersion in LN (Fahy et al. 1984).
The elimination of liquid water below 130C is the main reason why cell
viability is stable at liquid nitrogen temperature (Mazur, 1984).
In the present study, 10% glycerol was found to be the most effective
cryoprotectant for G. corticata (Table 1), which may be due to its low toxicity
as compared to other cryoprotectants. Dimethylsulfoxide (DMSO) and
glycerol are known to be very effective cryoprotectants for various types of
 258 P. L. Lalrinsanga et al.

cells, and ethylene glycol has also been reported to have a good cryoprotec-
tive effects on Eisenia bicyclis (Kono, Kuwano, & Saga 1998).
The present study revealed that glycerol is more superior in protection
than both DMSO and ethylene glycol for G. corticata. However, glycerol at
15% was found to be detrimental in this study. In case of H. musiformis and
U. lobata, 10% DMSO was found to be more suitable than glycerol and
ethylene glycol for both the species (Table 1). DMSO at 15% concentration
was found to be effective for Enteromorpha intestinalis (Kono et al. 1997).
The present observation, however, showed that DMSO at 15% has
detrimental effects on the survival of both the species. At concentration
above 10%, DMSO has often been detrimental (Fenwick & Day 1992) possi-
bly due to an inhibitory effect of DMSO at high concentration.
It has been reported in earlier study that DMSO is more suitable for G.
tikvahiae and that glycerol was not satisfactory as cryoprotectants for non-
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axenic culture (van der Meer & Simpson 1984) and has little cryoprotective
effect (Kuwano, Aruga, & Saga 1992). However, the present study showed
that glycerol was more superior in protection than both DMSO and ethylene
glycol for the G. corticata. Careful attention should be given, however, to
the concentration of glycerol because the appropriate concentration is
dependent on the species. Although ethylene glycol was reported to have a
highest cryoprotective effects in brown algae (Kono, Kuwano, & Saga
1998), the present study demonstrated that ethylene glycol was not efficient
for all three species studied. Indeed, it was ineffective especially for H.
musiformis and was also found to be detrimental when used at 5% for both
G. corticata and U. lobata. Therefore, the results of the present study imply
that a special cryoprotective solution is the key to cryopreserving marine
organisms and that the suitability of cryoprotectants is species specific.
Prefreezing is a critical step in cryopreservation and it is important
especially when the two-step cooling technique of cryopreservation is used.
Successful cryopreservation requires the suppression of intracellular ice
crystal formation during cooling. If the cells are properly prefrozen, ice crys-
tals formed in cells during rapid cooling are small enough to be innocuous
(Sakai & Otsuka 1967). However, by lowering the prefreezing temperature,
damage during prefreezing increases and reduction in the survival also
increases. The present investigation showed that the survival of the vegeta-
tive thalli cooled to prefreezing temperatures of higher than 40C was
considerably low as compared to those cooled to prefreezing temperatures
lower than 40C. Maximum survival rate occurred among cells prefrozen at
40C before immersion in LN. When employing a super-optimal (faster
than optimal) cooling rate, which allows for insufficient time for dehydration
of the cells or tissue, intracellular ice may form (Mazur, 1970), which is lethal
to the tissue or cells. Immersion in LN seriously damaged the cells prefrozen
to 20C to 30C. Prefreezing to 40C and below decreases the damage.
Cooling to 40C was adequate to prevent intracellular ice formation.
 Cryopreservation of Vegetative Thalli 259

However, cooling to less than 40C had detrimental effect due to excessive
dehydration (Kono, Kuwano, & Saga 1998), and the possible prolonged
exposure to a potentially hyper-osmotic environment or toxic cryopro-
tectants can result in irreversible damage to the cell membrane and other
structures (Steponkus, Langis, & Fugikawa 1992). This optimum freezing
rate is related to the cell membranes permeability. (van Der Meer &
Simpson 1984).
Despite the protection they afford cells during freezing and thawing,
cryoprotective chemicals can themselves be damaging especially when used
in high concentrations (Fahy, 1986). Additionally, toxicity is generally
increased by long-term exposure of the cells to the protectant, though
different algae demonstrate different tolerance levels (Caavate & Lubian
1994). Although van der Meer and Simpson (1984) and Kuwano, Aruga, and
Saga (1993) reported that the equilibration period is not crucial at the
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optimum cryoprotectant concentration, the present investigation showed a

profound effect of the equilibration time on the survival of the species. The
survival of G. corticata increased and reached its highest level when
exposed to protective compound glycerol (10%) for a period of 60 min
prior to freezing, a period less was found to be insufficient. This lower
survival rate at less pretreatment period is attributed to the slow permeabil-
ity properties of glycerol through plasma membrane of the cell. An equili-
bration period of 45 min was sufficient for both U. lobata and H.
musiformis. The less equilibration time requirement may be attributed to
the ability of DMSO to pass through cell membranes freely (Meryman, 1966)
as the effectiveness of cryoprotectant depends on the permeability of cell
membrane (van der Meer & Simpson, 1984).
It is generally considered that the cells stored in LN should be warmed
rapidly; otherwise the ice crystals formed in the cells during rapid cooling
grow to harmful size, resulting in cell death during thawing (Sugawara &
Sakai 1974). As the temperature rises in thawing process, the viscosity falls,
molecular motion becomes slower, and water molecules may diffuse and
rotate into the configuration required to nucleate or add to existing nuclei
producing the phenomenon called recrystalization. Thus, successful
warming should cross this temperature range quickly (Rall, Reid, & Polge
1984). Sakai and Otsuka (1967) also reported that rapid thawing is essential
for any type of cells because slow thawing allows the glass water to
transform into large ice crystals, which destroy intracellular structures.
However, post-thawed removal of cryoprotectants was a critical step in cry-
opreservation (Chen et al., 1984). Therefore, to minimize the damage by
dilution, thalli suspension containing cryoprotectants was serially diluted to
six-fold with ice-chilled seawater over a period of 30 min. Kuwano, Aruga,
and Saga (1992) reported that the difference in warming rate had no effect
on the survival of conchocelis cells of Porphyra yezoensis, which were
thawed from 40C; whereas the decrease in warming rates reduces the
 260 P. L. Lalrinsanga et al.

survival of conchocelis cells, stored in LN. In the present study, the decrease
in warming rates reduced the survival of thalli stored in LN, but had no
effects on the thalli thawed immediately from 40C. It seems that the cell
contents remained unfrozen at 40C due to cryoprotectant effect, and then
were frozen by immersion in LN.
Viability estimation by regeneration in agar (Morris 1976) or in liquid
(McLellan 1989) was reported and is considered reliable because of its
direct link with cell division (Kuwano et al., 2000). Indirect methods of
estimating viability, such as uptake of a particular stain, cell motility, and
oxygen evolution methods, may significantly overestimate the recovery
potential. The pigmentation index (van der Meer & Simpson, 1984) along
with regrowth was employed in the present study; they are regarded as the
definitive indicators of viability (Leeson, Cann, & Morris 1984). Success in
cryopreservation of any biological materials depends on the regenerative
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capability of the cryopreserved materials. In the present study, the thalli

were regenerated in a non-axenic outdoor tank in the condition simulated
to natural environment. The study showed that the damaged thalli lost their
pigments while incubation in the PES culture media and the rate of reduc-
tion in survival was rapid, up to 28th day of incubation, after which the
survival was stabilized. The survival after the 28th day was therefore consid-
ered as approximates to real survivals, which were also reported earlier.
The viability of cells decreased during post-thawing incubation and the
minimum survival level after post-thawing culture approximates to real
survival (Kono, Kuwano, & Saga 1998).
Although the results of survival of the thalli thawed after prefreezing
without LN immersion were higher than those immersed in LN, storing at
prefreezing temperature may be harmful in the long term and may be useful
only for the purpose of short-term storage since LN gives better long-term
storage survival.
The present study shows that the factors affecting the survival during
cryopreservation of marine macroalgae and is the first report of an attempt
on the cryopreservation and regeneration of seaweeds of Indian coasts. This
study would further facilitate the use of the cryopreservation technique to
other marine algae for their conservation and better management of marine
algal resources.

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