Application and comparison of two different intraspecific protoplast fusion methods in Trichoderma harzianum and their effect on -glucosidase activity

Abstract

In an attempt to construct superior Trichoderma harzianum isolates for improving β-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal agents and their productivities of carboxymethylcellulase (CMCase) and β-glucosidase to be introduced into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion). Three crosses were carried out among the selected four isolates. Results showed that, the number of fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG) method. In addition, high productivities of CMCase and β- glucosidase were obtained after electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good and effective method for obtaining T. harzianum fusants with higher productivity of β- glucosidase enzyme.

Full text

African Journal of Biotechnology Vol. 10(52), pp. 10683-10690, 12 September, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.119
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Application and comparison of two different
intraspecific protoplast fusion methods in Trichoderma
harzianum and their effect on -glucosidase activity
Ahmed M. El-Bondkly1*, Aboshosha, A. A. M.2, Radwan, N. H.2 and Dora, S. A.2
1Genetics and Cytology Department, National Research Center, Dokki, Giza, Egypt.
2Genetics Department, Faculty of Agriculture, Kafrelsheikh University, Kaferelsheikh, Egypt.
Accepted 10 May, 2011
In an attempt to construct superior Trichoderma harzianum isolates for improving -glucosidase
productivity, protoplast fusion technique was applied. After application of different mutagenic
treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four
antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal
agents and their productivities of carboxymethylcellulase (CMCase) and -glucosidase to be introduced
into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion).
Three crosses were carried out among the selected four isolates. Results showed that, the number of
fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG)
method. In addition, high productivities of CMCase and - glucosidase were obtained after
electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good
and effective method for obtaining T. harzianum fusants with higher productivity of - glucosidase
enzyme.
Key words: Trichoderma, protoplast fusion, electroporation, -glucosidase.
INTRODUCTION
Cellulose is one of the most abundant substrates
available in nature; the potential importance of cellulose
hydrolysis in the context of conversion of plant biomass
to fuels and chemicals as well as cellulose hydrolysis
also represents one of the largest material flows in the
global carbon cycle (Zhang and Lynd, 2004). The
enzymatic conversion of cellulose is catalyzed by a
multiple enzyme system. Beta-glucosidase (-D-
glucoside glucohydrolase, EC 3.2.1.21) is one of the
essential enzymes in the enzymatic conversion of cellu-
lose. It is an important component of cellulase system
*Corresponding author. E-mail: ahmed_bondkly@yahoo.com.
Abbreviations: CMCase, carboxymethylcellulase; PEG,
polyethylene glycol.
and acts synergistically with endogluconase and cello-
biohydrolase for complete degradation of cellulose
(Harhangi et al., 2002; Szengyel et al., 2000).
Members of the fungal genus Trichoderma are
considered the main producer of extracellular cellulolytic
enzymes. This fungus belongs to the fungi imperfecti and
contains seven chromosomes (Mäntylä et al., 1992) or
sex chromosomes (Herrera-Estrella et al., 1993).
Trichoderma harzianum is well known as producer of
cellulolytic enzymes that are extensively used for the
degradation of cellulose particularly in textile and paper
industries, beside its use in wastewater treatment
(Prabavathy et al., 2006a and b).
Fungal protoplasts are important tools in physiological
and genetic research, as well as genetic manipulation
which can be successfully achieved through fusion of
protoplasts in filamentous fungi that lack the capacity for
sexual reproduction (Hayat and Christias, 2010; Lalithakumari,

10684 Afr. J. Biotechnol.
2000; Mrinalini and LalithaKumari, 1998; Pe,er and Chet,
1990; Stasz et al., 1988).
The aim of the present study is to apply and compare
two different intraspecific protoplast fusion methods
[polyethylene glycol (PEG) and electroporation] in T.
harzianum and to construct the strains of the fungus T.
harzianum having the genetic ability to produce the
highest carboxymethylcellulase (CMCase) and -gluco-
sidase activities.
MATERIALS AND METHODS
Strains of T. harzianum
T. harzianum NRRL 13879 strain and its mutants (EL-Bondkly et
al., 2010; Table 1) were used in the present study and maintained
on YMGA medium slants (Strauss and Kubicek, 1990).
Isolation of antifungal resistant mutants
For the isolation of antifungal resistant mutants, hypertonic
regeneration medium (EL-Bondkly, 2006) and antifungal agents
were used separately; concentrations of antifungal agents added
were as follows: 0.5 and 1.0 µg/ml Benomyl; 10 and 25 µg/ml
miconzole; 75 and 100 µg/ml cycloheximide and 250 µg/ml
griseofulvin. A part of the mycelium of each isolate was inoculated
on the surface of the antifungal medium plates; the plates were
incubated at 28°C for six days. Colonies that exhibited resistance or
sensitivity to a specific antifungal were retested on the same
antifungal dose to be sure of their stability concerning resistance or
sensitivity and used as markers to select the fusants.
Protoplast formation
Protoplasts were prepared through enzymatic hydrolysis of
mycelium suspension using the procedure of EL-Bondkly (2002).
Cultures were grown in liquid protoplast medium, containing (g/l in
distilled water): glucose, 80; NH4NO3, 2; KH2PO4, 10; MgSO4.7H2O,
0.25; FeCl3.6H2O, 0.02; MnSO4, 0.14 and the initial pH of the
medium was adjusted to 4.5. Fifty milliliters (50 ml) of medium were
dispensed into 250 ml Erlenmeyer flask for the development of
mycelium. The flasks were incubated at 30°C for 20 h on a shaker
maintained at 160 rpm. After incubation, the mycelium was
collected by centrifugation, washed twice with 0.7 M KCl in 25 mM
phosphate buffer, pH 5.8 and then resuspended in 50 mg/ml
phosphate buffer containing 0.7 M KCl and 10 mg/ml Novozyme
234 (Sigma Co.). The lytic mixtures were incubated at 30°C with
gentle shaking for 3 h. Incubated mixtures were filtered and
protoplasts were counted in the filtered lysate.
Protoplast fusion techniques
PEG method
According to the CMCase and -glucosidase activities and
resistance or sensitivity to one or more antifungal agents, equal
numbers of protoplasts from the two mutants were mixed and
centrifuged at 3000 rpm for 5 min, the residue (mixture of
protoplast) was suspended in 2 ml of prewarmed (30°C) solution of
PEG 6000 at 30 % (w/v). The PEG containing 0.05 M CaCl2 and
0.05 M glycine-NaOH buffer (pH 7.5). After incubation at 30°C for
10 min, the suspension was centrifuged at 3000 rpm for 5 min.
Electroporation process
The process of electrofusion of protoplasts was conducted in the
gene pulser Bio-Rad CO., (USA) with an electrofusion chamber of 1
ml working volume. Process parameters: 1 or 2 impulses
immediately following one another with a field intensity of 200 v/cm
and an exposition time of 1000 min at the stage of
dielectrophoresis, 1 impulse with a field intensity of 500 v/cm and
an exposition time of 20 min at the stage of fusion, regulated
temperature of 4°C before and after the process, rounding time of
ca 20 min (ukowska et al., 2004).
Isolation of recombinant fusants
Through the present study, PEG or electroporation methods,
treated protoplast suspensions were plated onto an antifungal
selective medium. It contains the same components of the
protoplast medium with addition of cellulose, as a carbon source
instead of glucose, 0.7 M KCl and one or more of the antifungal
agents. Treated protoplast pellets were resuspended in 1 ml of
asomatically balanced phosphate buffer, diluted appropriately and
were plated on the hypertonic selective and nonselective
regeneration media. The plates were incubated at 28°C until the
colonies were grown on plate's surface. The grown colonies were
considered as complementary fusants. They were transplanted and
subcultured several times onto selective and nonselective media
before further studies. Fusion frequency was expressed as the ratio
of the number of colonies formed on selective and nonselective
media.
Fermentation and determination of CMCase and -glucosidase
activities
The wild type strain, mutants and fusants were grown in
fermentation medium (Haapala et al., 1995), which is optimal for
CMCase and -glucosidase productivities. The medium was
inoculated with 10% spore suspension from 8-day old slants and
flasks were incubated with shaking (200 rpm) at 28°C for ten days.
CMCase and -glucosidase activities were assayed in the culture
supernatant according to Vaheri et al. (1979).
RESULTS
Response of the original strain and the selected
mutants to some antifungal agents
To induce new fungal recombinants through protoplast
fusion, the original strain in addition to the 20 selected
mutants were exposed to four antifungal agents. Table 1
summarizes the response of the original strain and the 20
selected mutants to four antifungal agents (benomyle; 0.5
and 1.0 g/ml, miconzole; 10 and 25 g/ml, cyclo-
heximide; 75 and 100 g/ml and griseofulvin; 250 g/ml)
as well as their CMCase and -glucosidase activities.

El-Bondkly et al. 10685
Table 1. Sources, CMCase and -glucosidase productivities and response of the selected T.harzianum mutants and their original strain to four antifungal agents.
Mutant number Source of mutant
CMCase and -glucosidase productivities Antifungal agents (µg / ml)
CMCase -Glucosidase Benomyle Miconzole Cycloheximide Griseofulvin
U/ml % from W.T. U/ml % from W.T. 0.5 1.0 10 25 75 100 250
W.T.
Original strain 2.5 100.0 6.0 100.0 -
-
+ + - - +
W1/9 Original strain with 0.1% colchicines 3.0 120.0 7.5 125.0 - - + - - - +
W2/9 Original strain with 0.2% colchicines 3.2 128.0 7.8 130.0 - - + - - - +
L1/1 (9/8) mutant with 0.1% colchicines 4.3 172.0 10.0 166.7 - - + - - - +
L1/9 (9/8) mutant with 0.1% colchicines 4.3 172.0 10.5 175.0 - - + - - - +
L1/15* (9/8) mutant with 0.1% colchicines 5.0 200.0 10.5 175.0 - - + + - - +
L2/11 (9/8) mutant with 0.2% colchicines 5.0 200.0 11.5 191.7 - - + - - - +
L2/16 (9/8) mutant with 0.2% colchicines 5.2 208.0 11.5 191.7 - - + - - - +
P1/8 (15/4) mutant with 0.1% colchicines 4.3 172.0 10.5 175.0 - - + - - - +
P2/9 (15/4) mutant with 0.2% colchicines 5.0 200.0 11.0 183.3 - - + - - - +
E1/9 (50/30/17) mutant with 0.1% colchicines 5.0 200.0 10.0 166.7 - - + - + - +
E2/3* (50/30/17) mutant with 0.2% colchicines 4.7 188.0 10.0 166.7 - - + - + - +
R2/10 (100/30/44 n) mutant with 0.2%
colchicines 4.8 192.0 14.0 233.3 - - + - - - +
D1/1 (125/30/12) mutant with 0.1% colchicine 6.2 248.0 15.5 258.3 - - + - - - +
D1/4* (125/30/12) mutant with 0.1% colchicine 6.5 260.0 17.2 286.7 - - + - + - +
D1/8 (125/30/12) mutant with 0.1% colchicine 5.8 232.0 17.0 283.3 - - + - + - +
D1/14* (125/30/12) mutant with 0.1% colchicine 6.0 240.0 17.2 286.7 - - + + - - +
D2/1 (125/30/12) mutant with 0.2% colchicine 5.8 232.0 16.5 275.0 - - + - - - +
D2/2 (125/30/12) mutant with 0.2% colchicine 5.8 232.0 16.5 275.0 - - + + - - +
D2/11 (125/30/12) mutant with 0.2% colchicine 5.8 232.0 16.5 275.0 - - + + - - +
D2/14 (125/30/12) mutant with 0.2% colchicine 5.8 232.0 16.8 280.0 - - + + - - +
L1 and L2 mutants were obtained from original strain after treatment with 9 min UV exposure time; P1 and P2 mutants were obtained from original strain after treatment with 15 min UV exposure time;
E1 and E2 mutants were obtained from original strain after treatment with concentration of 50 µg/ml NTG for 30 min; R2 mutant was obtained from original strain after treatment with concentration of
100 µg/ml NTG for 30 min; D1 and D2 mutants were obtained from original strain after treatment with concentration of 125 µg/ml NTG for 30 min (EL-Bondkly el al., 2010). *Four mutants (L1/15, E2/3,
D1/4 and D1/14) were selected to be used in the intraspecific protoplast fusion.
Results in Table 1 showed that, the original
strain NRRL13879 exhibited resistance to both
miconzole and griseofulvin antifungal agents,
while it was sensitive to the other two antifungal
agents. In addition, different responses appeared
after exposure of the 20 selected mutants to these
antifungal agents. All isolates were sensitive to
the two benomyle concentrations and the high
concentration (100 g/ml) of cycloheximide,
whereas they exhibited complete lethality. On the
other hand, complete resistance was observed
after exposure of all isolates to both miconzole (10
g/ml) and griseofulvin (250 g/ml). Meanwhile,
different responses were noticed after exposing
the 20 mutants to high concentration of miconzole

10686 Afr. J. Biotechnol.
(25 g/ml) and low concentration of cycloheximide (75
g/ml). Five isolates (L1/15, D1/14, D2/2, D2/11 and
D2/14) were resistant to miconzole in concentration of 25
g/ml, while the rest isolates exhibited complete lethality.
In addition, the results showed also that, four isolates
(E1/9, E2/3, D1/4 and D1/8) were resistant to the low
concentration of cycloheximide, whereas, the rest
isolates were sensitive to the same antifungal concentra-
tion.
Protoplasts formation and fusion
On the basis of the CMCase and -glucosidase
activities shown in Table 1 and resistance or sensitivity to
one or more of the four used antifungal agents, only four
mutants (L1/15, E2/3, D1/4 and D1/14) were selected
and used in the intraspecific protoplast fusion. Data in
Table 1 clearly showed that, two out of the four selected
isolates (L1/15 and E2/3) showed low productivity of
CMCase and -glucosidase, while the other two isolates
showed high productivity. In addition, these isolates
exhibited different response to the two antifungal agents
that is, miconzole and cycloheximide. According to the
obtained results, these four isolates were used to carry
out three intraspecific crosses using two different methods,
classical (PEG) and electropration method. The first
cross was applied between the low CMCase and -
glucosidase producer isolates (L1/15 and E2/3), the
second cross was performed between the low and high
producer isolates (E2/3 and D1/14), while the third cross
was carried out between the two higher isolates (D1/4
and D1/14) .
According to the conditions described under materials
and methods, enzymatic treatments and subsequent
examination of the treated mycelia from the selected
parental strains with a phase-contrast microscope
showed that, gradual degradation of fungal mycelia
started after the addition of 10 mg/ml novozyme 234
enzyme. The whole cell wall digestion was achieved
following incubation at 30°C with gentle shaking for 3 h.
Maximum release of protoplasts differed from one mutant
to the other. From the mycelium of mutant L1/15, the
highest yield of protoplasts (2.8 x 107/ml) was obtained
after the 3 h incubation period. The maximum release of
protoplasts was obtained with mutant E2/3 that yields 1.9
x 107 protoplasts per ml. On the other hand, 3.0 x 107 and
3.5 x 107/ml protoplasts were released from mutant D1/4
and D1/14 mycelium after the 3 h incubation time,
respectively. Two different protoplast fusion techniques
were the main subject to be evaluated in this study. They
are tools for inducing genetic recombinants especially in
fungi like, T. harzianum, where the sexual cycle is
unknown, in order to isolate higher CMCase and -gluco-
sidase producing recombinants (Hayat and Christias,
2010). However, the use of these techniques requires
labeling the parental strains before protoplasting and
fusion (EL-Bondkly, 2006). Some of the highest and
lowest CMCase and -glucosidase producer isolates
were used for intraspecific protoplast fusion through this
study.
Fusion frequency, estimated as the ratio of the number
of colonies regenerating on the nonselective medium to
the number of colonies formed on the selective medium,
was found to be different from one cross to another. The
fusion frequencies were 1.8 x 10-3 and 2.5 x 10-3 in the
case of fusion between isolates L1/15 and E2/3 when
PEG and electrofusion methods were applied, respect-
tively. On the other hand, the frequencies of intraspecific
protoplast fusion between E2/3 and D1/14 were
increased to 2.0 x 10-3 (PEG method) and 2.6 x 10-3
(electrofusion method). The highest frequencies were
between mutants D1/4 and D1/14 yielding 2.5 x 10-3 for
PEG method and 2.8 x 10-3 for electrofusion method.
Other investigators mentioned that, the formation,
regeneration and fusion of protoplasts are affected by
different factors, for example, enzymes, time of treat-
ments, mycelial age, regeneration medium, protoplast
fusion method (EL- Bondkly and Talkhan, 2007;
Lalithakumari, 2000; Prabavathy et al., 2006b).
Evaluation and comparison of PEG and
electroporation methods
Cross 1
This cross was carried out between the two low CMCase
and -glucosidase producer isolates (L1/15 and E2/3) as
shown in Table 2 with the application of two fusion
methods. Eleven and 15 recombinants were obtained
from this cross on the basis of resistance or sensitivity to
both antifungal agents; miconzole and cycloheximide
were marked from F1/1 to F1/11 (PEG method) and from
F4/1 to F4/15 (electoporation method). The CMCase
productivity of the parental isolates (E2/3 and L1/15) was
4.7 and 5.0 U/ml, while their productivities of -
glucosidase were 10.0 and 10.5 U/ml, respectively. The
highest productivity of both enzymes among the 11
fusants obtained from PEG method was recorded by
fusants number F1/6 and F1/10, where they gave 20 and
14% of CMCase and -glucosidase more than the higher
parent (L1/15), respectively.
Concerning CMCase and -glucosidase activities deter-
mined for the 15 fusants obtained from electroporation
method, ten fusants (F4/1, F4/2, F4/3, F4/4, F4/7, F4/8,
F4/9, F4/13, F4/14 and F4/15) out of them, exhibited
higher productivity of both enzymes when compared

El-Bondkly et al. 10687
Table 2. CMCase and -glucosidase productivities for the intraspecific fusants resulted from cross 1.
PEG Electroporation
Parent and
fusant
CMCase -glucosidase Parent and
fusant CMCase -glucosidase
U/ml % from the
higher parent U/ml % from the
higher parent U/ml % from the
higher parent U/ml % from the
higher parent
W.T. 2.5 50.0 6.0 75.14 W.T. 2.5 50.0 6.0 57.14
E2/3 4.7 94.0 10.0 95.23 E2/3 4.7 94.00 10.0 95.23
L1/15 5.0 100.0 10.5 100.0 L1/15 5.0 100.0 10.5 100.0
F1/1 5.0 100.0 10.5 100.0 F4/1 6.0 120.0 12.5 119.04
F1/2 5.0 100.0 10.5 100.0 F4/2 6.5 130.0 12.5 119.04
F1/3 4.9 98.0 11.0 104.66 F4/3 6.5 130.0 12.5 119.04
F1/4 5.5 110.0 11.5 109.52 F4/4 6.0 120.0 12.0 114.28
F1/5 3.7 74.0 10.0 95.23 F4/5 4.7 94.0 10.0 95.23
F1/6 6.0 120.0 12.0 114.28 F4/6 4.7 94.0 10.0 95.23
F1/7 4.7 49.0 10.0 95.23 F4/7 6.0 120.0 12.0 114.28
F1/8 4.9 98.0 11.0 104.66 F4/8 6.0 120.0 12.0 114.28
F1/9 5.0 100.0 10.5 100.00 F4/9 6.0 120.0 12.0 114.28
F1/10 6.0 120.0 12.0 114.28 F4/10 4.7 94.0 10.0 95.23
F1/11 5.5 110.0 11.5 109.52 F4/11 5.0 100.0 10.5 100.0
F4/12 4.3 86.0 9.5 90.47
F4/13 6.5 130.0 12.0 114.28
F4/14 6.5 130.0 12.0 114.28
F4/15 6.5 130.0 12.5 119.04
with the parental isolates. CMCase productivity of these
fusants ranged from 20% produced by fusants (F4/1,
F4/4, F4/7, F4/8 and F4/9) to 30% produced by fusants
(F4/2, F4/3, F4/13, F4/14 and F4/15) more than the
higher parent (L1/15). In the meantime, fusants (F4/4,
F4/7, F4/8, F4/9, F4/13 and F4/14) produced 14.28%,
whereas fusants (F4/1, F4/2, F4/3 and F4/15) gave
19.04% -glucosidase more than the higher parent.
Cross 2
The second cross was achieved between the highly
efficient CMCase and -glucosidase producer isolate
(D1/14) and the lower efficient one (E2/3). Results in
Table 3 revealed that, only 11 fusants (F2/1 to F2/11)
resulted from the PEG method, in comparison with 15
fusants (F5/1 to F5/15) which were obtained through
electroporation method. Concerning the 11 recombinants
obtained from PEG method, it was noticed that five
fusants (F2/1, F2/5, F2/6, F2/10 and F2/11) showed
higher activity in CMCase and -glucosidase production,
since they gave 6.8, 6.8, 6.6, 6.8 and 6.6 U/ml of
CMCase, respectively, and also produced 18.5, 18.5,
18.0, 18.5 and 18.0 U/ml of -glucosidase, respectively.
In addition, ten fusants (F5/2, F5/3, F5/4, F5/8, F5/9,
F5/10, F5/11, F5/12, F5/14 and F5/15) out of the15
obtained from electroporation method showed increase in
both CMCase and -glucosidase than the higher parental
isolate (D1/14). The CMCase activity recorded by these
fusants yielded between 13.33 and 20% more than the
higher parent (D1/14). Productivity of -glucosidase
ranged from 5.81% for F5/11 and F5/12 fusants to 7.55%
for the rest eight isolates over the higher parent.
Cross 3
This cross was done between the highest CMCase and
-glucosidase producer isolates (D1/4 and D1/14).
Fourteen fusants were obtained from this cross using
PEG method and 15 immediately after the application of
electroporation method on the basis of antifungal test as
shown in Table 4. These tested fusants showed variable
levels of CMCase and -glucosidase activities. Out of the
14 tested fusants obtained, six (F3/4, F3/5, F3/8, F3/9,
F3/10 and F3/14) proved to have higher productivity of
CMCase and -glucosidase when compared with their
parents. CMCase productivity of these fusants ranged
from 7.5 U/ml (produced by the fusants F3/4, F3/9, F3/10
and F3/14) to 8.0 U/ml (produced by the fusants F3/5 and
F3/8). While, -glucosidase productivity of these fusants
ranged from 19.0 U/ml (produced by the fusants F3/4,
F3/5, F3/9, F3/10 and F3/14) to 19.5 U/ml (produced by

10688 Afr. J. Biotechnol.
the fusant F3/8). In addition, nine fusants out of the 15
obtained through electroporation technique proved to
have higher productivity of CMCase, in which four fusants
(F6/4, F6/9, F6/10 and F6/14) produced 33.33% more
than the higher parent (D1/4). While five fusants (F6/5,
F6/6, F6/11, F6/13 and F6/15) exhibited about 41.66%
CMCase more than D1/4. Furthermore, ten fusants (F6/2,
F6/4, F6/5, F6/6, F6/9, F6/10, F6/11, F6/13, F6/14 and
F6/15) proved to have high productivity of -glucosidase,
their products giving from 4.65 to 16.27% more than their
parents (D1/4 and D1/14), respectively.
DISCUSSION
Protoplast fusion is an effective tool for inducing genetic
recombinations and developing superior hybrid strains in
filamentous fungi (Mrinalini and LalithaKumari, 1998;
Pe,er and Chet, 1990; Stasz et al., 1988). Genetic recom-
bination is a powerful method for developing superior
industrial strains. Comparing both methods of protoplast
fusion used in this study (PEG and electroporation), the
obtained results clearly showed that, the number of
recombinant fusants obtained after application of
electrofusion was more than that obtained after
application of PEG method. On the other hand, higher
productivity of CMCase and -glucosidase was recorded
after electrofusion compared with the PEG method in the
three crosses carried out through of this study.
Regarding the first cross (Table 2) carried out between
the two low producer isolates (L1/15 and E2/3), ten
fusants obtained after electrofusion method produced
from 20 to 30% CMCase more than the higher parent
(L1/15). Also, they produced from 14.28 to 19.04% -
glucosidase more than the higher parent. On the other
hand, four fusants (F1/4, F1/6, F1/10 and F1/11) obtained
after PEG method produced from 10 to 20% CMCase,
while other five fusants (F1/3, F1/4, F1/6, F1/8, F1/10 and
F1/11) produced -glucosidase ranged between 4.66 and
14.28% more than the higher parent (L1/15) as shown in
the same table.
The second cross was carried out between the lower
producer isolate (E2/3) and the higher producer (D1/14);
results in Table 3 showed that, ten fusants obtained after
electrofusion method showed higher productivity of the
two enzymes. The CMCase productivity of these fusants
was ranged between 13.33 and 20% over the higher
parent (D1/14). While, -glucosidase productivities was
ranged from 5.81 to 7.55% over the higher parent. On the
other hand, five fusants (F2/1, F2/5, F2/6, F2/10 and
F2/11) obtained after PEG method showed from 10 to
13.33% CMCase productivity more than the higher parent
(D1/14), as well as, produced from 4.65 to 7.55% -
glucosidase more than the higher parent (Table 3).
In the case of the third cross between the two high
producer isolates (D1/4 and D1/14), nine fusants
obtained from electroporation method (F6/4, F6/5, F6/6,
F6/9, F6/10, F6/11, F6/13, F6/14 and F6/15) recorded
33.33 to 41.66% CMCase more than the higher parent
(D1/4 ) and ten fusants (F6/2, F6/4, F6/5, F6/6, F6/9,
F6/10, F6/11, F6/13, F6/14 and F6/15 ) showed from 4.65
to 16.27% -glucosidase more than both parents. On the
other hand, six fusants (F3/4, F3/5, F3/8, F3/9, F3/10 and
F3/14) produced 15.38 to 23.07% CMCase more than the
higher parent (D1/4), as well as showed from 10.46 to
13.37% -glucosidase productivity more than the two
parents when PEG method was applied.
Fifteen (15) self-fusant strains using PEG in STC buffer
from T. harzianum PTh18 strain were isolated
(Prabavathy et al., 2006a). Among them, the strain
SFTh8 produced maximum chitinase with two-fold
increase when compared with the parental strains.
Furthermore, all the self-fusants exhibited increasing of
antagonistic activity against Rhizocotonia solani than the
parents. On the other hand, EL-Bondkly and Talkhan
(2007) applied the intraspecific protoplast fusion in T.
harzianum and they selected eighteen self fusants, four
of them (ATh1/9, ATh1/12, ATh1/14 and ATh1/17)
produced high chitinase activity, while fusant (ATh1/7)
produced 94.3% more chitinase activity than the original
strain. Moreover, Prabavathy et al. (2006b) used
intraspecific protoplast fusion to enhance carboxymethyl-
cellulase activity and they found that, most of the fusants
exhibited fast growth and abundant sporulation compared
to non-fusant and parental strains. Furthermore, two
fusants (SFTr2 and SFT3) recorded more than two-fold
increase in enzyme activity. They suggested that,
protoplast fusion can be used to develop superior hybrid
strains of filamentous fungi that lack inherent sexual
reproduction.
In conclusion, there are two main advantages of
electro-poration method over the traditional PEG method,
the first one is its simplicity and the second advantage;
this method is more reproducible than the classical
method (PEG). The improvement of microbial strains was
conducted in many research centers and most commonly
involve the introduction of additional genes into the cell
genome or an increase in the number of existing genes.
The applied protoplast electrofusion method proved to be
a good and effective method for obtaining T. harzianum
fusants with higher productivity of -glucosidase enzyme.
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El-Bondkly et al. 10689
Table 3. CMCase and -glucosidase productivities for the intraspecific fusants resulted from cross 2.
PEG Electroporation
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CMCase -glucosidase
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F3/3 5.3 81.53 15.2 88.37 F6/3 6.3 96.92 17.2 100.0

10690 Afr. J. Biotechnol.
Table 4 Cont.
F3/4 7.5 115.38 19.0 110.46 F6/4 8.0 133.33 19.5 113.37
F3/5 8.0 123.07 19.0 110.46 F6/5 8.5 141.66 20.0 116.27
F3/6 6.2 95.38 17.2 100.0 F6/6 8.5 141.66 20.0 116.27
F3/7 6.0 92.30 17.2 100.0 F6/7 6.0 92.30 17.2 100.0
F3/8 8.0 123.07 19.5 113.37 F6/8 6.0 100.0 17.2 100.0
F3/9 7.5 115.38 19.0 110.46 F6/9 8.0 133.33 19.5 113.34
F3/10 7.5 115.38 19.0 110.46 F6/10 8.0 133.33 19.5 113.34
F3/11 6.0 92.30 17.2 100.0 F6/11 8.5 141.66 20.0 116.27
F3/12 6.5 100.0 17.2 100.0 F6/12 5.4 90.0 15.3 88.95
F3/13 6.5 100.0 17.2 100.0 F6/13 8.5 141.66 20.0 116.27
F3/14 7.5 115.38 19.0 110.46 F6/14 8.0 133.33 19.5 113.34
F6/15 8.5 141.66 20.0 116.27
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