Optimization of Protoplast Isolation From The Gametophytes of Brown Alga Undaria Pinnatifida Using Response Surface Methodology
Optimization of Protoplast Isolation From The Gametophytes of Brown Alga Undaria Pinnatifida Using Response Surface Methodology
Optimization of Protoplast Isolation From The Gametophytes of Brown Alga Undaria Pinnatifida Using Response Surface Methodology
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Abstract
The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications,
including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good
candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study
reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of
commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme
combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast
isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subse-
quently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS,
2 U mL−1 alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L−1 H2O and adjusted to pH 5.8–
6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 106 protoplasts g−1 fresh weight for male gametophyte and 2.11 ±
0.08 × 106 for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase,
respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effec-
tiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful
method for rapidly producing the higher yields of protoplasts from brown alga.
Keywords Phaeophyceae . Commercial enzymes . Undaria pinnatifida . Gametophytes . Protoplast isolation . Response surface
methodology
et al. 2005). These features make gametophytes suitable for boxes (5–8 °C) within 48 h after collection. Cleaning of well-
cellular biotechnology techniques, and many of them rely on matured sori and sporulation were performed according to
protoplasts (Reddy et al. 2008). Redmond et al. (2014). After spore attachment, spores were
Currently, several studies on protoplast isolation and cul- cultured in PES at low density (102–101 spores mL−1) in 12-
ture from sporophytes and gametophytes of U. pinnatifida well plates under 14:10-h light/dark photoperiod with light
have been limited in using non-commercial enzymes (Wu intensity 50–80 μmol photons m−2 s−1 at 13 °C. Male and
1988, Zha and Kloareg 1996, Benet et al. 1997, Matsumura female gametophyte clones were isolated after 2 weeks culture
et al., 2001, Xiaoke et al. 2003). Zha and Kloareg (1996) and thereafter cultured separately in 100 × 40 mm Petri dishes
obtained high protoplast yields from U. pinnatifida gameto- filled with 50 mL modified PES medium (m-PES), which did
phyte using non-commercial alginate lyases from marine not contain Fe3+ supplementation because removing Fe3+ can
herbivores and cellulase from filamentous fungi. Also, Benet effectively inhibit gametogenesis (Suzuki et al. 1995).
et al. (1997) reported inconsistent yields using a mixture of Cultures were maintained in vegetative stage at 20 °C under
commercial cellulase and non-commercial alginate lyases 40 μmol photons m−2 s−1 of the blue LED (DyneBio Co.,
from Haliotis tuberculata and Pseudomonas alginovora. Korea) and the same photoperiod (Morita et al. 2003; Xu
However, the use of non-commercial enzymes makes the iso- et al. 2005). Medium was renewed every two weeks. After
lation process expensive and time-consuming because they 7 months in culture, gametophyte clones accumulated much
have to be produced. In addition, contaminating enzymes biomass and were transferred into 1-L flat-bottomed round
(e.g. protease, lipase, nuclease and carbohydrolase) in crude flasks filled with 1 L m-PES medium under aeration. Air
preparations and variation of the enzymatic activity from was sterilized with 0.22-μm SFCA syringe filters (Corning,
batch to batch compromise the reproducibility of the results Germany). Medium was renewed every two weeks. Light in-
(Gupta et al. 2011; Inoue et al. 2011). Thus, an optimal pro- tensity was 40–60 μmol photons m−2 s−1. Clone spheres were
tocol for protoplast isolation using commercial enzymes is fragmented monthly using an Ultra-Turrax homogenizer
fundamental for the development of a protoplast system for (T25, Ika Works Inc., USA) in order to maintain homogenous
this species. cell-filament suspension cultures.
In recent years, response surface methodology (RSM) has
been successfully used for optimizing the protoplast yield
from plants (Rezazadeh and Niedz 2015), fungi (Wei et al. Protoplast isolation
2016) and red seaweeds (Gupta et al. 2011). RSM is a collec-
tion of statistical and mathematical techniques useful for de- The commercially available cell wall lytic enzymes used for
veloping, improving and optimizing processes. It is particu- this study were cellulase Onozuka RS and R-10, macerozyme
larly applied when dealing with products or processes that are R-10 (Yakult Co. Ltd., Japan), alginate lyase, pectinase and
potentially influenced by several input variables. It can iden- driselase from Basidiomycetes sp. (Sigma-Aldrich, USA).
tify significant factors and their interactions in a faster and Different enzyme combinations were evaluated for male (from
more economical way than the classical one-variable at a time Geoje island) and female gametophyte (from Jindo island)
or full factorial experimentation (Reddy et al. 2008; Myers clones and the optimal enzyme mixture was selected for the
et al. 2009; Krishnaiah et al. 2015). To date, RSM has not highest protoplast yield (Table 1). Various concentrations for
been used for protoplast production in brown seaweeds. each enzyme within the optimal mixture were also tested.
In this study, we assessed key parameters influencing pro- Protoplast isolation was performed following the protocols
toplast isolation such as enzyme composition, chelation pre- described by Benet et al. (1997) and Coelho et al. (2012) with
treatment, growth, temperature, incubation time, pH and os- some modifications. Approximately 10–20 mg of Undaria
molarity for male and female gametophytes of U. pinnatifida
Table 1 Combinations and concentrations of enzyme mixtures for
using commercial lytic enzymes. RSM via Box-Behnken de- protoplast isolation from Undaria pinnatifida gametophytes
sign (BBD) was used to design the experiment, generate a
model and optimize the protoplast isolation conditions. Commercial enzymes Composition of enzyme mixtures
A B C D E F G H I
pinnatifida gametophytes from 1-L round flasks was incubat- Table 2 Level of factors used in split-plot design for protoplast produc-
tion from Undaria pinnatifida gametophytes. HEPES was used for
ed in a 0.22-μm filter-sterilized enzymatic solution (400 mM
adjusting pH at 7.5. Osmolarity was calculated as 1570 (initial osmolar-
NaCl, 130 mM MgCl2·6H2O, 22 mM MgSO4, 160 mM KCl, ity) and 3140 (2 times the initial osmolarity) mOsm L−1 H2O for enzy-
2 mM CaCl2, 10 mM MES, pH 6.3, 1570 mOsm L−1 H2O) matic solution at 1× and 2×, respectively
containing different combinations of enzymes at 20 °C with
Factor Levels
shaking at 70 rpm for 6 h in the dark. Protoplasts were filtered
using a 25-μm nylon mesh to remove undigested filaments Low (− 1) High (+ 1)
and concentrated by centrifugation at 100×g for 10 min.
Protoplast yields were estimated by using a haemocytometer Temperature (C°) 15 25
and expressed as protoplasts g −1 fresh weight (FW). Incubation time (h) 3 15
Protoplast isolation was repeated four times in each treatment. pH 5.8 7.5
Also, we tested the effect of chelation pre-treatment on proto- Osmolarity 1× 2×
plast yield. Chelation pre-treatment was conducted with a
calcium-chelating solution (665 mM NaCl, 30 mM MgCl2·
6H2O, 30 mM MgSO4, 20 mM KCl, 20 mM ethylene gly- by Minitab 17.1 which included 12 base run experiments and
col-bis(β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid triplicates at the center point to estimate experimental errors.
tetrasodium salt (EGTA-Na4) as calcium chelator, pH 5.5) The whole experiment was carried out in duplicate giving a
for 20 min prior to enzymatic digestion (Coelho et al. 2012). total of 30 run experiments. For predicting the optimal condi-
tion, the quadratic polynomial equation was fitted to correlate
Growth experiments the relationship between variables and response (i.e. proto-
plast yield), and estimated with the following equation:
Gametophyte clones were inoculated in 1-L flat-bottomed n n n−1 n
round flasks containing 1 L m-PES at 1 g FW L−1, cultured Y ¼ b0 þ ∑ bi X i þ ∑ bii X ii þ ∑ ∑ bij X i X j
i¼1 i¼1 i¼1 j¼iþ1
at 20 °C, under 40–60 μmol photons m−2 s−1 and 14:10-h
light/dark photoperiod following being cut into short frag- where Y is the predicted response, b0 is a constant coefficient,
ments (200–300 μm in length) with an Ultra-Turrax homoge- bi is a linear coefficient, bii is the quadratic equation, bij is the
nizer. FW of gametophyte clones was measured at 2-day in- interaction coefficient, and Xi and Xj are the input variables.
tervals after m-PES was squeezed out by hand using a 25-μm Statistical analysis of the resulted models for the optimum
aperture nylon mesh. After FW measurement, protoplast iso- conditions of variables was evaluated by ANOVA at
lation was performed using the optimal mixture. Culture du- p < 0.01. The adequacy of the developed models was tested
ration was maintained over 18 days. The duration of the ex- by performing coefficient of determination (R2), adequate pre-
ponential phase was determined using the semi-logarithmic cision, the Mallow’s Cp statistics and coefficient of variance
plot of FW (x) as a function of time (t). Four replicate flasks (CV %) (Dawson and Martinez-Dawson 1998; Myers et al.
were used per treatment. 2009; Yetilmezsoy et al. 2009). The response surface plot was
drawn to visualize the input-output relationships.
Multivariate optimization
microscope fitted with a Leica EL6000 external light source g −1 FW), followed by mixture A with chelation pre-
for fluorescence excitation and equipped with a 470/40 nm treatment (2.41 ± 0.70 × 105 protoplasts g−1 FW), mixture A
emission filter and a 515 nm suppression filter. The removal without chelation pre-treatment (1.50 ± 0.73 × 105 protoplasts
(true protoplasts) of cell walls was confirmed by staining the g−1 FW) and mixture H (cellulase RS, alginate lyase, driselase
protoplasts with 0.01% calcofluor white M2R (Sigma- and pectinase) with chelating pre-treatment (1.45 ± 0.47 × 105
Aldrich, USA) and by examining under a Leica DMi8 protoplasts g−1 FW).
inverted microscope equipped with a 360/40 nm emission Significant variation in protoplast yields was recorded for
filter and a 425 nm suppression filter. both gametophytes under different mixtures and chelation pre-
treatment. A significant interaction between the effects of both
Statistical analysis factors was observed only for male gametophyte (Table 3).
Chelation pre-treatment showed positive effects with mixture
Normality and homoscedasticity were checked with Shapiro- G (1.7-fold increase) for male gametophyte, while mixture G
Wilk and Levene tests, respectively, prior to conducting two- (1.5-fold increase) and H (5.6-fold increase) for female game-
way ANOVA for the comparison of protoplast yield under tophyte (Table 4). Removing cellulase RS or alginate lyase
different enzyme mixtures and chelation pre-treatment. One- from the optimal mixture (mixture G) reduced significantly
way ANOVA was performed to examine the effects of differ- the protoplast production or gave inconsistent yields.
ent enzyme concentrations in the optimal mixture. Only treat- Increasing or decreasing the enzyme concentrations did not
ments with at least three non-zero values were considered in affect the protoplast yield (data not shown). For the next ex-
these analyses. The effect of days in culture on protoplast yield periments, enzyme concentrations in the optimal mixture were
was assessed using repeated-measures ANOVA. The Huynh– set at 1% cellulase RS, 2 U mL−1 alginate lyase and 1%
Feldt correction was used to adjust for sphericity violations driselase with chelation pre-treatment.
when necessary. Tukey’s post hoc test was used when the
results of ANOVA were significant. In the case of repeated-
measures ANOVA, the Bonferroni method was chosen for Effect of growth on protoplast yield
multiple comparisons (Park et al. 2009). Effect sizes
(Sullivan and Feinn 2012) were presented for ANOVA anal- Male gametophyte presented a fast biomass increase during
ysis as ω2 or η2p (Field 2009, Lakens 2013). The significance the first 10 days of culture (exponential phase), followed by a
threshold was set at p = 0.01 in order to reduce the true type I gradual decrease in the next 8 days (deceleration phase).
error rate (at least 7% but typically close to 15%, Sellke et al. Cultures did not reach the stationary phase during the exper-
2001). All the statistical tests were performed using Minitab iments. Female gametophyte presented a lag phase during the
17.1, with the exception of Mauchly’s sphericity test and first 4 days of culture, followed by a fast biomass increase for
Huynh–Feldt correction which were conducted with R pro- 8 days (exponential phase) and then by a stationary phase.
gramming package within the graphical interface R-Studio (R Protoplast yield was affected by days in culture (male game-
Core Team 2016, https://www.R-project.org/). tophyte: p < 0.001, η2p = 0.88; female gametophyte: p =
0.006, η2p = 0.85), reaching its maximum values in the mid-
exponential phase (day 6), for male gametophyte, and at the
Results end of the lag phase (day 4), for female gametophyte (Fig. 1).
SS, sum of squares; df, degrees of freedom; MS, mean square; F, F statistic; p, significance level; ω2 , omega
squared (effect size); NS, not significant
IY, inconsistent yield (at least two zero values per treatment); NP, no protoplasts
J Appl Phycol
Fig. 1 Growth curve (black triangles) and protoplast yield (white bars) protoplast yield (n = 4, p < 0.01). Only mean values are presented for
from Undaria pinnatifida gametophytes. a Male gametophyte. b Female FW (n = 4). The duration of each growth phase is indicated
gametophyte. Different letters indicate significant differences for
gametophyte; and an incubation time of 3.8 h, pH of 5.8 and 6 days in culture. Cell division and regeneration as clear proof
1.65× osmolarity (2591 mOsm L−1 H2O) give 2.15 × 106 pro- of protoplast viability have been observed in both male and
toplasts g−1 FW for female gametophytes. Under these condi- female gametophytes and will be reported elsewhere.
tions, experimental values were found to be 3.12 ± 0.51 and
2.11 ± 0.08 × 106 protoplasts g−1 FW for male and female ga-
metophytes, respectively. These mean values were compared
Discussion
with the predicted values and indicated the suitability of the
developed quadratic models. The percentage deviation of the
The present study demonstrates a successful method for iso-
experimental and theoretical results was found as − 4.29%
lating a large number of viable protoplasts from male and
(male) and − 1.86% (female). Protoplasts were released
female gametophytes of the economically important brown
through apical or one-sided holes in the cell wall (incomplete
seaweed U. pinnatifida using commercial enzymes with che-
cell wall digestion). They were spherical in shape with an
lation pre-treatment. Protoplast isolation conditions were op-
average diameter of 9.50 ± 1.64 μm in male gametophyte
timized using RSM via BBD, representing the first report of
and 12.38 ± 2.68 μm in female gametophyte, and with periph-
this technique in the production of protoplasts from brown
erally arranged chloroplasts. Calcofluor white staining con-
seaweeds.
firmed the absence of cell walls and red chlorophyll autoflu-
The highest protoplast yields of 3.12 and 2.11 × 106 proto-
orescence showed they were viable (Fig. 4). Other gameto-
plasts g−1 FW obtained in this study for male and female
phyte strains from Korea showed protoplast yields ranging
gametophytes, respectively, were superior to the number of
from 2.57 to 8.17 × 106 protoplasts g−1 FW in male gameto-
protoplasts reported by Benet et al. (1997). However, Zha
phyte and 2.74 to 7.91 × 106 protoplasts g−1 FW in female
and Kloareg (1996) reported higher protoplast values than us
gametophyte (Table 7). Protoplasts regenerated cell walls with
(15 to 25 × 106 protoplasts g−1 FW) with a mix of non-
Fig. 2 Normal probability plot of the standardized effects for protoplast yield from Undaria pinnatifida gametophytes. a Male gametophyte. b Female
gametophyte
J Appl Phycol
Table 5 Box-Behnken
experimental design with three Run Incubation time (h) pH Osmolarity Protoplast yield, Y (× 105 protoplasts g−1 FW)
independent variables for
protoplast production from X1 Code X2 Code X3 Code Male Female
Undaria pinnatifida
gametophytes. HEPES was used 1 9 0 5.8 −1 1× −1 4.63 1.73
for adjusting pH at 7.5. 2 9 0 5.8 −1 2× +1 18.00 16.07
Osmolarity was calculated as
3 9 0 7.5 +1 1× −1 3.91 0.96
1570 (initial osmolarity), 2355
(1.5 times the initial osmolarity) 4 9 0 7.5 +1 2× +1 3.42 5.29
and 3140 (2 times the initial 5 3 −1 6.65 0 1× −1 4.83 3.18
osmolarity) mOsm L−1 H2O for 6 3 −1 6.65 0 2× +1 11.46 8.91
enzymatic solution at 1×, 1.5×
7 15 +1 6.65 0 1× −1 3.60 1.21
and 2×, respectively. Each run
was carried out in duplicate. 8 15 +1 6.65 0 2× +1 11.00 8.50
Values are mean of duplicate runs 9 3 −1 5.80 −1 1.5× 0 33.57 20.89
10 3 −1 7.50 +1 1.5× 0 14.67 7.81
11 15 +1 5.80 −1 1.5× 0 21.37 9.08
12 15 +1 7.5 +1 1.5× 0 11.83 12.40
13a 9 0 6.65 0 1.5× 0 32.30 18.05
14a 9 0 6.65 0 1.5× 0 25.82 17.04
15a 9 0 6.65 0 1.5× 0 32.10 19.45
a
The left point was replicated three times
commercial enzymes which might have had additional active Kloareg and Quatrano 1988). Our results suggest that
ingredients digesting the cell walls of gametophytes. macerozyme R-10 and pectinase can be excluded when iso-
A simple mix of commercial cellulase RS (1%), alginate lating protoplasts from Phaeophyceae.
lyase (2 U mL−1) and driselase (1%) was found to be the best The addition of cation chelators has improved protoplast
enzymatic combination. Cellulase RS was more effective than production in Ectocarpales (Mejjad et al. 1992; Coelho et al.
cellulase R-10, especially in female gametophyte, mainly due 2012) and Laminariales (Butler et al. 1989; Kloareg et al.
to the higher xylanase and cellulase activities in the RS prep- 1989). However, its effect has not been tested in gametophytes
aration (Thayer 1985). Alginate lyase (a mannuronate lyase from Laminariales. In our study, the effect of chelation pre-
according to the manufacturer) was also critical in the enzy- treatment was dependent on the specific type of enzymatic
matic mixture, although the incomplete cell wall digestion can mixture. Only the best enzymatic combination (cellulase, al-
be attributed to its inefficient degradation of alginate (Formo ginate lyase and driselase) showed significant increases in
et al. 2014). both gametophytes following incubation in the chelating
Driselase increased significantly the number of protoplasts solution.
in male gametophyte while marginally improved their release Our results showed that protoplast yield was affected by the
in the female gametophyte. Driselase is a natural mixture of growth phase of culture, reaching maximum values in the
enzymes (e.g. cellulase, hemicellulase and pectinase) that can mid-exponential phase (male gametophyte) or just before the
cleave, among other polysaccharides, mixed-linked glucan beginning of this one (female gametophyte). Differences in
(MLG) also known in fungi as lichenan (Thibault and growth patterns can be attributed to distinct reproductive strat-
Rouau 1990). Recently, Salmeán et al. (2017) demonstrated egies in gametophytes (Destombe and Oppliger 2011).
that MLG is common in brown algal cell walls, including Actively dividing cells or fast-growing plants have produced
U. pinnatifida sporophyte. This can explain the effectiveness higher amounts of protoplasts in yeasts (Shahin 1972), land
of driselase on protoplast isolation, especially in male plants (Strauss and Potrykus 1980; Nakagawa et al. 1985) and
gametophyte. in green and red seaweeds (Björk et al. 1990, 1992; Gómez
The addition of macerozyme R-10 and pectinase to the best Pinchetti et al. 1993). During cell division and growth, cell
enzymatic mixture did not improve the protoplast release. wall elasticity is necessary and, consequently, its composition
Benet et al. (1997) reported that pectin-degrading enzymes changes (Burns et al. 1982a, 1982b, 1984). Elasticity and
were inefficient at increasing protoplast yields in gameto- rigidity of alginate in the cell wall are due to mannuronate
phytes from Laminariales. Reddy et al. (2006) demonstrated (M) and guluronate (G) blocks, respectively. M-block content
that macerozyme R-10 is unnecessary in enzyme mixtures if increase is expected during the exponential phase due to a
the algal cell walls do not contain pectin or its derivatives, high cell division (Kloareg and Quatrano 1988; Lee et al.
which is the case for brown algae (Cronshaw et al. 1958; 2012). The alginate lyase used in this study mainly cleaves
J Appl Phycol
Table 6 ANOVA table of the Box-Behnken design model for optimized parameters
Factors Statistics
SS df MS F p
Male gametophyte
Model 3329.21 9 369.91 7.12 < 0.001
X1 69.90 1 69.90 1.35 0.260
X2 477.94 1 477.94 9.20 0.007
X3 181.08 1 181.08 3.49 0.077
X 21 166.11 1 166.11 3.20 0.089
X 22 182.63 1 182.63 3.52 0.075
X 23 2289.56 1 2289.56 44.09 < 0.001
X1X2 43.79 1 43.79 0.84 0.369
X1X3 0.30 1 0.30 0.01 0.941
X2X3 96.24 1 96.24 1.85 0.189
Residual 1038.68 20 51.93
Lack of fit 88.07 3 29.36 0.53 0.671
Pure error 950.61 17 55.92
Female gametophyte
Model 1306.38 9 145.153 15.09 < 0.001
X1 23.04 1 23.04 2.39 0.137
X2 113.59 1 113.59 11.81 0.003
X3 250.82 1 250.82 26.07 < 0.001
X 21 67.83 1 67.83 7.05 0.015
X 22 45.15 1 45.15 4.69 0.043
X 23 675.61 1 675.61 70.22 < 0.001
X1X2 134.43 1 134.43 13.97 0.001
X1X3 1.22 1 1.22 0.13 0.725
X2X3 50.07 1 50.07 5.20 0.034
Residual 192.43 20 9.62
Lack of fit 14.62 3 4.88 0.47 0.710
Pure error 177.81 17 10.46
SS, sum of squares; df, degrees of freedom; MS, mean square; F, F statistic; p, significance level
M blocks, which can explain the better digestibility of the cell and the physiological state of the explant can explain the var-
wall during the exponential phase. iations of the time periods (Reddy et al. 2008). Interestingly,
The range of temperatures tested (15–25 °C) did not affect female gametophyte showed a shorter incubation time com-
protoplast yield in U. pinnatifida gametophytes. The optimal pared with male gametophyte, suggesting that cell wall com-
temperature employed for isolating protoplasts is more closely position may vary between them.
related to that what species of macroalgae naturally grow Optimal enzyme pH values were found to be 5.8–6.1.
(Huddy et al. 2013). Morita et al. (2003) showed that game- Previous studies on protoplast from gametophytes of
tophytes from U. pinnatifida can grow in the range of 10 to Laminariales have used a pH 6.5 (Benet et al. 1997;
25 °C with an optimal of 20 °C. This wide range of tempera- Varvarigos et al. 2004). This difference could be due to the
tures might explain the lack of effect of incubation tempera- type of enzyme mixtures used because the activity of the en-
ture on protoplast yield. zymes is pH dependent (Bhojwani and Razdan 1996).
An enzymatic digestion period of 4–7 h was optimum ac- According to the manufacturer, the optimum pH for cellulase
cording to the RSM. Benet et al. (1997) reported 15 h incuba- RS and alginate lyase is 5–6 and 6.3, respectively. The optimal
tion time for gametophytes from Laminariales, while values obtained in this work were within this range. Optimal
Varvarigos et al. (2004) obtained high amounts of protoplasts pH for the commercial enzymes used also explain the decrease
from female gametophytes of Macrocystis pyrifera with a pe- of protoplast yield by increasing the pH.
riod of 4–5 h. The type of enzymes used, cell wall complexity
J Appl Phycol
The highest protoplast yields were obtained at 2481– the holes in the cell wall, or protection of the protoplast mem-
2591 mOsm L−1 H2O (around 1.6× enzymatic solution). brane (Xiaoke et al. 2003).
Although our optimal osmolarity values were higher than The models developed using RSM were evaluated using
those ones used previously (Benet et al. 1997; Xiaoke et al. several parameters. The R2 coefficient for both models was
2003; Varvarigos et al. 2004; Mussio and Rusig 2006), proto- superior to that one reported by Gupta et al. (2011) for proto-
plasts did not show damages. Instead, high osmolarity plasts from Gracilaria dura and G. verrucosa. Male and fe-
favoured the isolation process probably due to the stimulation male gametophytes reported adequate precision of 6.88 and
of alginate lyase activity by increasing salt concentrations 11.63, respectively. Values greater than 4 indicate an adequate
(Huang et al. 2013), promotion of protoplast release through signal and confirm that all predicted models can be used to
J Appl Phycol
navigate the design space (Anderson and Whitcomb 2017). for both gametophytes, validation of the model showed less
The Mallow’s Cp statistics were 10.00 for both gametophytes. than 5% deviation between experimental and theoretical
Cp can be used to determine how many terms can be omitted values indicating the suitability of the developed quadratic
from the response surface model. When Cp ≤ p, where p is the models. Moreover, high numbers of protoplasts were isolated
number of parameters or variables in the regression model from other gametophyte strains using the optimum conditions.
including the intercept term (p = 10 in our study), the predic- In conclusion, high amounts of viable and true protoplasts
tion model is very good (Dawson and Martinez-Dawson could be obtained from U. pinnatifida gametophytes cultures
1998). Although the CV% values were somewhat elevated in exponential phase using RSM and a simple mixture of
Table 7 Protoplast yields from different gametophyte strains of Undaria pinnatifida isolated in Korea. Values are presented as mean ± SD (n = 4)
1 Geomun island, Yeosu, South Jeolla Province 25 March 2016 Male 2.57 ± 0.27
Female 4.93 ± 0.82
2 Gampo, Gyeongju, North Gyeongsang Province 1 April 2016 Male 4.67 ± 1.36
Female 7.91 ± 1.97
3 Jindo island, Jindo, South Jeolla Province 5 May 2016 Male 5.16 ± 0.75
4 Heuksan island, Sinan, South Jeolla Province 19 May 2016 Male 8.17 ± 1.74
Female 2.74 ± 0.95
5 Hondgo island, Sinan, South Jeolla Province 20 May 2016 Male 5.45 ± 1.02
Female 4.55 ± 1.15
J Appl Phycol
commercial enzymes (cellulase RS, alginate lyase and Destombe C, Oppliger V (2011) Male gametophyte fragmentation in
Laminaria digitata: a life history strategy to enhance reproductive
driselase) with chelation pre-treatment. RSM via BBD can
success. Cah Biol Mar 52:1–9
be applied as a useful method for increasing the protoplast Dwiranti F, Hiraoka M, Taguchi T, Konishi Y, Tominaga M, Tominaga A
production in brown algae. (2012) Effects of gametophytes of Ecklonia kurome on the levels of
glucose and triacylglycerol in db/db, prediabetic C57BL/6J and
Funding information This study was supported by a research grant from IFN-γ KO mice. Int J Biomed Sci 8:64–75
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