BIBLIOGRAPHY IBIS, JEZREEL ZYRA D. OCTOBER...
BIBLIOGRAPHY
IBIS, JEZREEL ZYRA D. OCTOBER 2008. Protocols for Optimizing Hybrid
Production Between Two Pleurotus spp. Benguet State University, La Trindad, Benguet.
Adviser: Bernard S. Tad-awan, PhD.
ABSTRACT
A study determining culture medium appropriate for spore germination,
comparing spore punch and agar block method to obtain single spore, determining effect
of length of time of exposure of germinated spore to ultra violet light on dikaryotization
and determining for incidence of dikaryotic mycelium production was conducted at the
Plant Pathology Laboratory, Benguet State University from October 2007 to October
2008.
Results revealed that malt extract agar is the most suitable culture medium for
spore germination and growth after two days of incubation as compared to potato
dextrose agar which yielded shorter mycelial length at the same period of incubation and
water agar which yielded a germinated spore after six to ten days of incubation.
For obtaining a well isolated single spore, spore punch method proved to be more
reliable than that of the agar block method. Spore punch method gives higher chance of
obtaining a single spore and is less laborious than that of the agar block method.
Monokaryons exposed to ultra violet light at different lengths of time from 10 to
60 minutes did not produce a dikaryon. Rather, an inhibition zone was very evident in all
treated plates except that exposed to UV light for 60 minutes. This was subjected to
mycelial diameter measurement. Mycelium from both parents and the supposed dikaryon
was measured and compared but no difference was observed.
It is recommended that studies be conducted testing other mutagenic agents using
the given protocols on culture media used and technique on single sporing.
ii
TABLE OF CONTENTS
Page
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iii
INTRODUCTION . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
The Mushrooms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Importance of Hybrid Mushroom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
MATERIALS AND METHODS
Collection of Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Collection of spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Optimizing Protocols for Spore Germination . . . . . . . . . . . . . . . . . . . . . . .
9
Single Sporing Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Optimizing Protocols for Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Presence of Dikaryotic Mycelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Data Gathered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
RESULTS AND DISCUSSION
Physical Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Microscopic Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Effect of the Kind of Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
Appropriate Technique of Single Sporing . . . . . . . . . . . . . . . . . . . . . . . . .
14
iii
Exposure Time to Ultra Violet (UV) Light . . . . . . . . . . . . . . . . . . . . . . . . .
16
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
iv
INTRODUCTION
Background of the Study
Mushrooms are fleshy spore-bearing fruiting bodies of fungi. Most are
saprophytic, wood-decomposing fungi. Thus, many are seen growing on twigs, chips of
wood, stump of logs and soil. Kaul (1997) mentioned that mushrooms appear almost
everywhere – deserts, open fields, gardens, farmyards, marshes, forests, alpine-areas in
almost all the biomes on this planet. However, the majority are confined to wooded areas.
From the earliest days, mycologists recognized the fact that both large and small fungi
are ecologically connected to herbaceous plants and tress amongst which they grow.
Because of their medicinal properties, nutritional content and potential for the
global market, mushroom cultivation is growing and gaining popularity. A lot have cited
of their effect on the immune system. Ganoderma, for example is considered a natural
regulator of the immune system. Oyster mushrooms are best known medically for their
cardiovascular and cholesterol-controlling benefits. Shiitake on the other hand is
recognized for its antitumor action. Mushrooms are also good sources of protein,
carbohydrates and fiber.
The demand for good quality of mushrooms is high especially in the urban areas
due to the emergence of mushroom producers that compete for market. Mushroom
standards are then raised to that which can produce more and that with better quality than
of the existing criteria.
It should be noted that degeneration occurs after frequent subculturing from the
original strain. Cultivars used for commercial spawn production that were maintained on
various agars or cereal grains with periodic subculturing of growing mycelium to a fresh
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
2
medium were reported cases of culture degeneration periodically
(http://pubs.cas.psu.edu). It is also commonly observed that tissue cultures often give
lower yields than the original cultures (www.krishiworld.com). Tissue culture is in a
sense cloning. It gives rise to mushrooms with similar characteristics with that of the
original, thus, no room for strain improvement. Improving mushroom strain is necessary
to likewise improve their yield and quality, enabling them to compete in the market.
Hybridization calls an important role in this intent. According to Eger (1978) as cited by
Arias et al. (2000), large number of hybrids, obtained by pairing monosporic cultures
need to be cultivated to evaluate their production characteristics. This would bring about
a trial and error process during the selection of promising hybrids which would take quite
sometime.
Importance of the Study
Breeding is the only controllable means by which desired traits of different stocks
or strains can be combined. The quality of the crop depends as much upon the inherited
potential of the strain used as does upon conditions for growth and development (Raper,
1978). Fritsche (1978) said that by continued selection of mushrooms, bad characteristics
are eliminated.
Hybridization offers greater possibility for genetic improvement because of the
exchange of genetic information. This results from the fusion of two haploid nuclei.
Regardless of this, most spawn is made with mycelium from a stored culture,
rather than mycelium whose parent was a spore. This is because spores are likely to yield
a new strain and performance would be unpredictable (http://pubs.cas.psu.edu). The issue
of unpredictability might be reduced by testing each hybrid. Arias et al. (2000) obtained
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
3
high yielding Pleurotus ostreatus by cultivating hybrids of this species resistant to 2
Deoxyglucose (2DG).
This study aimed to:
1. determine culture medium appropriate for spore germination;
2. compare techniques for single sporing;
3. determine the effect of length of time of exposure of germinated single spore
to Ultra Violet (UV) light on dikaryotization; and
4. determine for incidence of dikaryotic mycelium production.
Time and Place of the Study
This study was conducted at the Plant Pathology Service Laboratory from
October 2007 to October 2008.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
4
REVIEW OF LITERATURE
The Mushrooms
Mushrooms are good cash crops; they are rather easy to grow and are brimming
with protein, B vitamins and minerals. They also have medicinal properties like anti-
cancer (Oei, 2005).
Because of their medicinal properties, nutritional content and potential for the
global market, mushroom cultivation is growing and gaining popularity. A lot have cited
of their effect on the immune system. Ganoderma, for example is considered a natural
regulator of the immune system. Oyster mushrooms are best known medically for their
cardiovascular and cholesterol-controlling benefits. Shiitake on the other hand is
recognized for its antitumor action. Mushrooms are also good sources of protein,
carbohydrates and fiber.
Importance of Hybrid Mushrooms
The purpose of breeding is to combine desired traits present in separate
individuals using controlled crosses and selection of offspring (Sonnenberg, 2007).
Several studies showed the significance of breeding mushrooms. A study
conducted by Arias, et al. (2000) resulted to six hybrids that showed improved fruiting
characteristics and had maximum productivity. Anderson, et al. (2001) found out that
hybrid off-white strains of Agaricus bisporus exhibited intermediate susceptibility to
Trichoderma harzianum, with mean yield losses of 56% to 73%. According to Miller
(2005), hybridization of Cordyceps amplified target medicinal compounds to five times
its potency. Hybridization concentrates the dosage, lowering volume ingested
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
5
substantially over the wild varieties. With hybrid cordyceps, we obtain cordyceps for
target medicinal compounds, vigorous or fast growing, and resistance to infection with
other organisms. Hybridization also may modify the substrate used in cultivating some
mushrooms. Bak et al. (2005) states that FRI 169, a hybrid shiitake mushroom turned out
to be a superior strain for sawdust-based cultivation.
Hybridization
Unlike plant and animal breeding, mushroom breeding is a relatively new applied
science. This is not surprising since only recently have mushrooms been produced
commercially on a large scale (Sonnenberg, 2007).
Breeding is the only controllable means by which desired genetic traits of
different stocks or strains can be combined. In the higher fungi, its minimal requirements
are: a recognizable sexual interaction between mated strains, fruiting competence,
meiosis and viability of spores (Raper, 1978). She further says that the quality of the crop
depends as much upon the inherited potential of the strain used as does upon conditions
for growth and development. By continued selection, bad characteristics are eliminated
(Fritsche, 1978).
The true success of the hybrid would be if it produces fruits that has spores. Only
at this stage would the true hybrid strain be accomplished. No genetic information is
exchanged between two strains until their separate haploid nuclei have fused and then
undergone meiosis. Recombination would then occur between the two separate strains
forming a third strain. Its offspring (spores) would be new combinations of the two donor
strains. Simply cloning the original mating that fruited will be a hybrid as well, but not a
true hybrid, because there has been no recombination between the two strains, no mixing
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
6
of genes. There has simply been a successful coexistence of two haploid nuclei, one from
each strain, acting independently, but together to create fruits (www.shroomery.org).
Thus, the importance of single sporing.
Elliott and Langton (1981) as cited my Yadav et al. (1999) emphasized that the
hybridization methods based on the combination of non-fertile, homokaryotic single
spore cultures offer better prospects for genetic improvement than the traditional
selection methods. Yadav made use of single sporing technique by serially diluting an
obtained spore mass and isolating the germinated single spore cultures from the serial
dilution. Similarly, Arias et al. (2000) produced dikaryons by pairing monokaryotic
strains, either neohaplons or single spore isolates. Sexual compatibility was assessed by
the positive clamp connection seen under the microscope.
In the process, not all sub-strains within a strain will fuse, some are completely
incompatible. There is a zone of zero growth between the two strains on the Petri plate
(www.shroomery.org); (www.nwbotanicals.org). Holliday (2004) reported that when
different strains were inoculated together onto one Petri dish, the strains grew towards
each other until they formed a zone of inhibition. But through the addition of snake
venom to the agar, the zone of inhibition becomes short lived until the mycelial strands
fuse and exchange nuclear material through their venom-weakened cell wall. Miller
(2005) also observed that snake venom changes the form of the Cordyceps by breaking
down its cell walls, rather than being added into the final product and indicated that snake
venom is an essential catalyst for hybridization allowing the fusion of the two differing
Deoxyribonucleic acids (DNA’s).
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
7
Ultra Violet (UV) is a strong physical mutagen and it is directly absorbed by the
DNA bases (Fincham et al., 1975). Perera et al. (2005) observed the conversion of
ergosterol in mushrooms to vitamin D2 when exoposed to UV.
Santos et al. (1989) were able to obtain thermotolerant and high alcohol yielding
mutant of Saccharomyces cerevisae when treated with acridine mustard (AM) and nitrous
acid (NA). Accordingly, acradine mustard is a frameshift mutagen and nitrous acid is a
demeaning agent. Santiago et al, (1991) claimed that nitrous acid is an effective mutagen
for the isolation of mutant in Agaricus bisporus and Lentinula edodes. He added that the
nitous acid dose depends upon the type of species and nature of the desired mutants.
With the occurrence of mutation, performance of the new strain is unpredictable.
Hybrids must be cultivated to evaluate their production characteristics. Arias et al. (2000)
reported that the use 2-deoxyglucose was used to select high yielding resistant hybrids.
2-Deoxy-D-glucose has a molecular formula of C6H12O5. It is a molecule which
has the 2-hydroxyl group replaced by hydrogen, so that it cannot undergo further
glycolysis (http://en.wikipedia.org). 2-Deoxyglucose is a rare and natural
monosaccharide. It is the basic structure of the anticancer drugs such as daunomycin,
adriamycin, carminomycins and antibiotics.
2-DG is a white crystalline hydroscopic powder. It is odorless, tastes sweet and
very soluble in water, partially soluble in hot methanol, ethanol, acetone, and butanol. It
is not soluble in ether, chloroform, petroleum ether and toluene. 2-DG is a polyhydroxy
aldehyde with reducing ability can react with Fehling or Benedict reagent and produce
red or yellow precipitation of copper oxide. 2-DG acts to inhibit the phosphorylation of a
glucose molecule that produces glucose-6-phosphate in the glycolysis cycle, therefore
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
8
inhibiting the production of ATP and can also decrease the temperature in muscle cells. It
can restrain viral infections and fermentation, microbes and cancer cell growth. It also
controls metabolism and possesses physiological and therapeutically effects.
(http://www.2dgpro.com)
The effect of several concentrations of the toxic analogue of glucose, 2-deoxy-D-
glucose (2-DG), was observed on the rate of apical growth of strains of Pleurotus
ostreatus using different carbon sources. The growth of the strains under these conditions
distinguished between tolerant and sensitive strains. It was observed that the phenotype
which was tolerant to 2-DG was correlated with high productivity of the strains in pilot
production farms (Sanchez et al., 1996).
To eliminate the trial and error process during hybridization, Arias et al. (2000)
used 2DG in the selection of recovered Pleurotus ostreatus. Resistance to this toxic
analogue of glucose had been used to select overproducing microorganisms. In their
study, six hybrids showed resistance to 2-DG out of the original thirteen hybrids. The 2-
DG resistant hybrids showed required 62 days to fruit and achieved maximum
productivity in a fruiting period of 28 days, with fruit bodies of 18.5g average weight.
There were similar results of that of Kirimura et al. (1992) with Aspergillus niger
mutants resistant to 2-DG that produced citric acid earlier than sensible individuals and
that of Sanchez and Viniegra – Gonzales (1996) with Pleurotus ostreatus resistant
strains gave higher yield as reported by Arias et al. (2000)
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
9
MATERIALS AND METHODS
Collection of Specimens
Ten mushrooms were collected, five each from both white (Pleurotus
cornucopiae) and gray (Pleurotus ostreatus) oyster species. These were characterized as
to 1.) size, shape, and color of the cap; 2.) size, shape, color of spores and; 3.) form of
hymenophore.
Collection of Spores
Collection of spores was done by removing the cap and laying it faced down. Half
of the cap was laid over white paper and the other half on black paper. After hours, the
deposited spores were collected for single sporing and for microscopic characterization.
Calibrating the Microscope
Calibration of the microscope was done by inserting the ocular micrometer into
the eyepiece and the stage micrometer on the stage. Using the low high power objective
(40x), zero (0) point of both stage and ocular micrometers sere set to coincide with each
other. The ocular divisions that cover the space between zero and the coincident line were
counted. Calibration Factor (CF) or Calibration Constant was calculated using this
formula:
CF= n divisions of stage micrometer x 10 units/division
n divisions of ocular micrometer
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
10
Protocols for Optimizing Spore Germination
Effect of the Kind of Media and Species Used
Spore germination of each mushroom species was tested on differential
media namely. Each treatment was replicated four times and arranged in a
factorial completely randomized design (CRD) with mushroom species as factor
A and different media used as factor B. The treatments were as follows:
Factor A
A1 – P. ostreatus
A2 – P. cornucopiae
Factor B
B1 – Water agar
B2 – Potato Dextrose Agar (PDA)
B3 – Malt Extract Agar (MEA)
Commercial sterilized distilled water was poured into the Petri dish
containing the spores after which 0.1 mm was obtained and spread over the
differential media. Plates were incubated at 23°C and presence of germinated
spores was observed after 48 hours. Treatments were replicated four times.
Single Sporing Technique
Two modified techniques of single sporing according to Manzanares et al.
(1994) and Webster et al. (1981) were tested. Treatments used were:
S1
Agar block method
S2
Spore Punch Method
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
11
For the agar block method, the media was cut into cubes at approximately
1 mm x 1 mm then observed under the microscope for well isolated single spores.
The second method is the use of spore punch. An agar disc bearing a
germinated spore was obtained using a spore punch mounted in the microscope.
Agar blocks and spore punches containing a single spore were transferred
to the media that gave the best result on initial test of media. Each plate contained
a spore each of white and gray oyster mushroom. Treatments were replicated four
times.
Hybridization between species was also tested by inoculating two spores
from the same kind of mushroom (i.e. P. ostreatus with P. ostreatus and P.
cornucopiae with P. cornucopiae) but from different fruiting bodies into a Petri
dish.
Isolates that produced mycelia were cut and transferred to plates and were
paired with other isolates that also produced mycelia.
Protocols for Optimizing Hybridization
Exposure Time to Ultra Violet (UV) Light
Petri plates containing the single spores were exposed to Ultra Violet
(UV) light with a distance from the lamp of 15 cm (Santiago, Jr., et al.). The
treatments were:
T0 – Control
T1 – 10 minutes
T2 – 20 minutes
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
12
T3 – 30 minutes
T4 – 40 minutes
T5 – 50 minutes
T6 – 60 minutes
Presence of Dikaryotic Mycelia
Treated isolates were observed under the microscope for the presence of clamp
connection. Additionally, mycelia were taken from both parent spores and from meeting
points of both mycelia which is treated as a possible hybrid candidate and were measured
as to mycelial diameter.
Data Gathered
The data gathered were the following:
1.
Physical description of parent mushroom. Mushroom fruiting bodies were
described as to (a) size of cap, (b) shape of cap, (c) color of cap and (d) form of
hymenophore.
2. Microscopic description. The size, shape and color of spores were described.
3.
Effect of media on germination. Presence of germination was noted as
well as length of mycelium.
4. Presence of clamp connection.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
13
RESULTS AND DISCUSSIONS
Physical Description
Mushroom characterization is presented in Table 1. The mushrooms used were
harvested after 36 hours from pinhead formation. Results reveal that white oyster
mushroom (Pleurotus cornucopiae) is bigger in size than gray oyster mushroom
(Pleurotus ostreatus). Both have a convex, fan-shaped cap and are gilled.
Table 1. Physical description of parent mushroom
Specimen
Size of cap
Shape of cap
Color of cap
Form of
(cm)
hymenophore
P. cornucopiae
5.2
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.0
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.4
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.2
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.0
Convex, fan-shaped
White
Gilled
P. ostreatus
5.0
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.8
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.0
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.7
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.8
Convex, fan-shaped
Gray
Gilled
Microscopic description
Spore shape for both mushrooms is kidney-shaped with white to pinkish spore
print. At 500x magnification, spore size for both mushrooms measures 10.71 x 3.57.
a.
b.
Figure 1. (a) White to pinkish spore print and (b) a spore of P. cornucopiae.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
14
Effect of the Kind of Media and Species Used
Spore germination was observed after two days from inoculation. Table 2 shows
that there are no significant differences on the length of the mushrooms as affected by the
species. These signifies that the P. cornucopiae and white P. ostreatus are not
significantly different from each other in terms of there mycelium length. Moreover, the
effect of the media on mycelia length is highly significant. Table 2 shows that MEA gave
significantly higher mean length of 47.77µ. followed by PDA and water agar with mean
mycelia length of 29.018 µ and 0.000 µ, respectively.
Analysis further shows that there are no significant effects of species and medium
interaction on the length of the mycelium. This means that the species and the medium
are independent from each other. That the differences on the length of the mycelium is
not a result of the interaction of the species and the medium, rather, it is either a result of
the effect of the species or of the medium alone. In this case, the significant effect ion the
kind of media used.
Table 2. Mean mycelium length as affected by species and media used
Mean Length (µ)
Species
P. ostreatus
27.83a
P. cornucopiae
23.36a
Medium
MEA
47.77a
PDA
29.018b
Water Agar
0.000c
Species x Medium
ns
CV (%)
26.34
Means of the same letter are not significantly different from at each other at 5% level of
significance by DMRT.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
15
a.
b.
c.
a.
b.
Figure 2. Geminated spores of Pleurotus ostreatus on (a.) MEA, (b.) PDA 2 days after
inoculation and (c.) Water Agar 10 days after inoculation at 500x.
a.
b.
c.
a.
b.
Figure 3. Germinated spores of P. cornucopiae on (a) MEA, (b) PDA 2 days after
inoculation and (c) Water Agar 10 days after inoculation at 500x.
Appropriate Technique of Single Sporing
Single sporing methods were tested on MEA as it is a promising medium for
spore germination and growth. Spore punch method generated higher possibility of
obtaining a well isolated, germinated, single spore as compared to the agar block method.
Moreover, spore punch method is a more rapid way to obtain a single spore than that of
the agar block method where a lot of agar blocks should be made to increase the chance
of obtaining a single spore.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
16
a.
b.
Figure 4. Comparison of single spore methods (a) spore punch and (b) agar block method
at 100x for P. cornucopiae.
Mycelia emerging from the single spore was observed after 6 to 10 days from
incubation at 23ºC only from spores of Pleurotus ostreatus.
a.
a.
b.
Figure 5. Plates showing growth of spore after 6 days from inoculation. (a) mycelium is
observed from spore punch of P.ostreatus (Gray) while (b) no growth is
observed from spore punch of P. cornucopiae.
A second set of single sporing was done this time pairing P. ostreatus spores but
from different fruiting bodies. After 15 days from inoculation, mycelia from both spores
were seen growing towards each other but only up to a certain point where one became
inhibitory to the other thus the formation of an inhibition zone. This result is supported by
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
17
Holliday’s (2004) findings on Cordyseps sinensis. He stressed that not all sub-strains
within a strain will fuse, some are completely incompatible.
Figure 6. Formation of inhibition zone after 15 days from isolation.
Exposure Time to Ultra Violet (UV) Light
To increase the chance of hybridization, before subjecting it to UV light, five
isolates from five single spores were transferred into a Petri dish. Results show that
regardless of the length of time exposed to UV light, no clamp connections were
observed in all plates. Fig. 8.g reveals no evident inhibition zone. Subjecting it though to
mycelium diameter measurement also indicates no dikaryotization that took place.
a.
b.
a.
b.
Figure 7. Monokaryons exposed to UV Light for 40 minutes (a) Inhibition zone between
two different mycelia. (b) Zone of inhibition as seen under the microscope at
100x magnification.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
18
a.
a.
b.
c.
a.
c.
d.
e.
f.
f.
g.
Figure 8. One week old culture of monokaryons exposed to Ultra Violet (UV) Light at
different lengths of time (a) Control, (b) 10 minutes, (c) 20 minutes, (d) 30
minutes (e) 40 minutes (f) 50 minutes and (g) 60 minutes.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
19
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
This study was conducted to determine the kind of culture medium appropriate for
spore germination, compare techniques for single sporing, determine length of time of
exposure of germinated single spore to Ultra Violet light on dikaryotization and to
determine for incidence of dikaryotic mycelium production.
Based on the results, malt extract agar is the most suitable medium for spore
germination compared to potato dextrose agar and water agar. Although PDA and WA
also sustain spore germination and growth, PDA produces shorter mycelium after two
days from inoculation compared to MEA while water agar takes a longer time to be able
to produce a germinated spore.
Spore punch method offers higher chances of obtaining single spores and is less
laborious than that of the agar block method where the chances of obtaining a well
isolated spore is slim.
Single spores obtained were paired together and were exposed to Ultra Violet
light at different length of time. Regardless of this, no clamp connection was observed
from these paired isolates.
Mycelia from both parents were compared to that of the supposed dikaryotic
mycelium. These were measured but no difference in mycelial diameter was observed
among the parents and the supposed hybrid.
Conclusions
Based on the findings of the study, it could be concluded that:
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
20
1. Among the media used, Malt Extract Agar is the most suitable medium for
Pleurotus spp. spore germination and growth.
2. Spore punch method is the best technique to be able to obtain a well isolated
single spore. It is much less laborious and requires shorter time of obtaining a single
spore compared to the agar block method.
3. Regardless of Ultra Violet exposure at different lengths of time from 10
minutes to 60 minutes, dikaryotization did not take place, as observed in the plates and
under the microscope.
Recommendations
Based on the findings, the following are recommended:
1. Further research need to be undertaken regarding growth of monokaryons in
controlled setting among white oyster mushrooms.
2. Hybridization study on other mushrooms can be done with the given
protocols on the kind of medium and single sporing technique from this study as a guide.
3. Other hybridization techniques including the use of mutagenic agents may
be tested to improve the study.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
21
LITERATURE CITED
ANDERSON, M. G., D. M. BEYER and P. J. WEST. 2001. Yield Comparison of
Hybrid Agaricus Mushroom Strains as a Measure of Resistance to Trichoderma
Green Mold. Retrieved at http://www.apsnet.org/pd/pdfs/2001/0511-02R.pdf
ANONYMOUS. 2007. Is it Possible to Cross Two Strains? Shroomery. Magic
Mushrooms Demystified. Retrieved at http://www.shroomery.org/8492/Is-it-
possible-to-cross-2-strains
______. 2008. 2-Deoxy-D-glucose. Retrieved from http://en.wikipedia.org/wiki/2-
Deoxy-D-glucose
______. Undated. College of Agricultural Sciences. Retrieved from
http://pubs.cas.psu.edu
______. Undated. Mushroom. Krishiworld. The Pulse of Indian Agriculture. Retrieved at
http://www.krishiworld.com/html/mushroom.html
______. Undated. P-2DG® 2-Deoxy-D-Glucose. Retrieved from
http://www.2dgpro.com/2dginfo.htm
ARIAS, A., R. RAMIREZ and H. LEAL. 2000. Cultivation of Pleurotus ostreatus
hybrids resistant to 2DG obtained by pairings of neohaplonts from selected
dikaryons. Rotterdam: Van Griensven. pp. 305-309.
BAK, W. C., B. H. LEE, M. K. Kim, K. H. Yoon, K. H. Ka AND H. Park. Breeding of
Shiitake strain for sawdust-cultivation by di-mon mating method. Abst. Retrieved
at http://www.worldmushroomsociety.com/
FRITSCHE, A. 1978. The biology and cultivation of edible mushrooms. New York:
Academic Press, Inc. pp. 139-143.
HOLLIDAY, J. C., P. CLEAVER, and M. LOOMIS-POWERS. 2004. The hybridization
of Cordyceps synensis strains and the modifications of their culture parameters,
in order to optimize the production of target medicinal compounds. Retrieved at
www.nwbotanicals.org/nwb/lexicon/hybridcordyceps.htm
KAUL, T. N. 1997. Introduction to mushroom science (Systematics). U.S.A.: Science
Publishers, Inc. p. 61
MILLER, R. 2005. Lab-grown cordyceps sinensis hybrtid: A Nano-processed Medicinal
Mushroom that Really Delivers. Retrieved at
http://pharmaceuticalmushrooms.nwbotanicals.org/cordycepssinensisheaa.pdf
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
22
OEI, P. 2005. Small-scale mushroom cultivation. Retrieved at
http://www.anancy.net/uploads/file_en/40-e-2005-mushrom_screen.pdf
PERERA, C. O., V.J. JASINGHE. 2005. Distribution of ergosterol in different tissues of
mushrooms and its effect on the conversion of ergosterol to vitamin D2 by UV
irradiation. Food Chemistry. Abst., 92:3. Retrieved at
www.cababstractsplus.org/google/abstract.asp?AcNo=20053082147
RAPER, C. A. 1978. The biology and cultivation of edible mushrooms. New York:
Academic Press, Inc. p. 110
SANCHEZ, C., G. VINIEGRA-GONZALES, and D. MOORE. 1996. Correlation
between Tolerance to 2-deoxy-D-glucose and Productivity of Strains of Pleurotus
ostreatus. Abst. Retrieved at http://www.worldmushroomsociety.com/
SANTIAGO, C. M., JR., B.B. MERCADO, E.D. DE LEON, R.P. FLORES, R.P.
GARCIA, M.B. BIGOL. 1991. Strain improvement of selected species of edible
fungi. Phil. J. Sci. 120 (2): 159.
SANTOS, M. E., D. A. RAMIREZ, P. C. SANCHEZ, W. L. FERNANDEZ. 1989. The
production of thermotolerant and high alcohol yielding yeasts by chemical
mutagenesis and hybridization. The Phil. J. Sci. 118 (13) : 239.
SONNENBERG, A.S.M. 2007. Breeding Mushrooms: State of the Art. WSMBM.
Retrieved at http://www.mushworld.com/tech/view.asp?cata_id=1130&vid=7742
YADAV, M. C., R.N. VERMA and B. L. DHAR. 1999. Studies on development of
improved strains and hybrids of white button mushroom. Retrieved at
www.worldmushroomsociety.com/upload/f2003610144435_3rdICMBMP_011.p
df
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
23
APPENDICES
Appendix Table 1. Effect of media and species on mycelial length
REPLICATION
TREATMENT
I II III IV
TOTAL
MEAN
Media
MEA
46.42 58.91 75.00 48.22
228.55
57.14
PDA
33.04 25.90 28.57 28.60
116.11
29.03
WA
0
0
0
0
0
0
Species
P. ostreatus
25.00 32.74 22.62 30.95
111.31
27.82
P. cornucopiae
27.98 23.81 21.43 20.24
99.43
24.86
ANALYSIS OF VARIANCE
SOURCE DEGREE
COMPUTE
TABULAR
OF
OF
SUM OF
MEAN
OF
D
Significanc
F
VARIATIO FREEDO SQUARE SQUARE
e
0.0
N
M
S
S
F
0.0
5
1
Model
5
9447.077 1889.415
41.591**
.000 2.7 4.2
7
5
Species
1
119.483
119.483
2.630ns
.122 4.4 8.2
1
9
Media
2
9267.055 4633.527 101.997**
.000 3.5 6.0
5
1
Species x
2
60.538
30.269
0.666ns
.526 3.5 6.0
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
24
Media
5
1
Intercept
1
15721.98
15721.98 346.086**
8.2
5
5
.000 4.41 9
Error
18
817.704
45.428
Total
24
25986.766
ns = not significant
CV = 26.34%
** = highly significant
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
IBIS, JEZREEL ZYRA D. OCTOBER 2008. Protocols for Optimizing Hybrid
Production Between Two Pleurotus spp. Benguet State University, La Trindad, Benguet.
Adviser: Bernard S. Tad-awan, PhD.
ABSTRACT
A study determining culture medium appropriate for spore germination,
comparing spore punch and agar block method to obtain single spore, determining effect
of length of time of exposure of germinated spore to ultra violet light on dikaryotization
and determining for incidence of dikaryotic mycelium production was conducted at the
Plant Pathology Laboratory, Benguet State University from October 2007 to October
2008.
Results revealed that malt extract agar is the most suitable culture medium for
spore germination and growth after two days of incubation as compared to potato
dextrose agar which yielded shorter mycelial length at the same period of incubation and
water agar which yielded a germinated spore after six to ten days of incubation.
For obtaining a well isolated single spore, spore punch method proved to be more
reliable than that of the agar block method. Spore punch method gives higher chance of
obtaining a single spore and is less laborious than that of the agar block method.
Monokaryons exposed to ultra violet light at different lengths of time from 10 to
60 minutes did not produce a dikaryon. Rather, an inhibition zone was very evident in all
treated plates except that exposed to UV light for 60 minutes. This was subjected to
mycelial diameter measurement. Mycelium from both parents and the supposed dikaryon
was measured and compared but no difference was observed.
It is recommended that studies be conducted testing other mutagenic agents using
the given protocols on culture media used and technique on single sporing.
ii
TABLE OF CONTENTS
Page
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iii
INTRODUCTION . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
The Mushrooms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Importance of Hybrid Mushroom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
MATERIALS AND METHODS
Collection of Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Collection of spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Optimizing Protocols for Spore Germination . . . . . . . . . . . . . . . . . . . . . . .
9
Single Sporing Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Optimizing Protocols for Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Presence of Dikaryotic Mycelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Data Gathered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
RESULTS AND DISCUSSION
Physical Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Microscopic Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Effect of the Kind of Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
Appropriate Technique of Single Sporing . . . . . . . . . . . . . . . . . . . . . . . . .
14
iii
Exposure Time to Ultra Violet (UV) Light . . . . . . . . . . . . . . . . . . . . . . . . .
16
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
iv
INTRODUCTION
Background of the Study
Mushrooms are fleshy spore-bearing fruiting bodies of fungi. Most are
saprophytic, wood-decomposing fungi. Thus, many are seen growing on twigs, chips of
wood, stump of logs and soil. Kaul (1997) mentioned that mushrooms appear almost
everywhere – deserts, open fields, gardens, farmyards, marshes, forests, alpine-areas in
almost all the biomes on this planet. However, the majority are confined to wooded areas.
From the earliest days, mycologists recognized the fact that both large and small fungi
are ecologically connected to herbaceous plants and tress amongst which they grow.
Because of their medicinal properties, nutritional content and potential for the
global market, mushroom cultivation is growing and gaining popularity. A lot have cited
of their effect on the immune system. Ganoderma, for example is considered a natural
regulator of the immune system. Oyster mushrooms are best known medically for their
cardiovascular and cholesterol-controlling benefits. Shiitake on the other hand is
recognized for its antitumor action. Mushrooms are also good sources of protein,
carbohydrates and fiber.
The demand for good quality of mushrooms is high especially in the urban areas
due to the emergence of mushroom producers that compete for market. Mushroom
standards are then raised to that which can produce more and that with better quality than
of the existing criteria.
It should be noted that degeneration occurs after frequent subculturing from the
original strain. Cultivars used for commercial spawn production that were maintained on
various agars or cereal grains with periodic subculturing of growing mycelium to a fresh
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
2
medium were reported cases of culture degeneration periodically
(http://pubs.cas.psu.edu). It is also commonly observed that tissue cultures often give
lower yields than the original cultures (www.krishiworld.com). Tissue culture is in a
sense cloning. It gives rise to mushrooms with similar characteristics with that of the
original, thus, no room for strain improvement. Improving mushroom strain is necessary
to likewise improve their yield and quality, enabling them to compete in the market.
Hybridization calls an important role in this intent. According to Eger (1978) as cited by
Arias et al. (2000), large number of hybrids, obtained by pairing monosporic cultures
need to be cultivated to evaluate their production characteristics. This would bring about
a trial and error process during the selection of promising hybrids which would take quite
sometime.
Importance of the Study
Breeding is the only controllable means by which desired traits of different stocks
or strains can be combined. The quality of the crop depends as much upon the inherited
potential of the strain used as does upon conditions for growth and development (Raper,
1978). Fritsche (1978) said that by continued selection of mushrooms, bad characteristics
are eliminated.
Hybridization offers greater possibility for genetic improvement because of the
exchange of genetic information. This results from the fusion of two haploid nuclei.
Regardless of this, most spawn is made with mycelium from a stored culture,
rather than mycelium whose parent was a spore. This is because spores are likely to yield
a new strain and performance would be unpredictable (http://pubs.cas.psu.edu). The issue
of unpredictability might be reduced by testing each hybrid. Arias et al. (2000) obtained
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
3
high yielding Pleurotus ostreatus by cultivating hybrids of this species resistant to 2
Deoxyglucose (2DG).
This study aimed to:
1. determine culture medium appropriate for spore germination;
2. compare techniques for single sporing;
3. determine the effect of length of time of exposure of germinated single spore
to Ultra Violet (UV) light on dikaryotization; and
4. determine for incidence of dikaryotic mycelium production.
Time and Place of the Study
This study was conducted at the Plant Pathology Service Laboratory from
October 2007 to October 2008.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
4
REVIEW OF LITERATURE
The Mushrooms
Mushrooms are good cash crops; they are rather easy to grow and are brimming
with protein, B vitamins and minerals. They also have medicinal properties like anti-
cancer (Oei, 2005).
Because of their medicinal properties, nutritional content and potential for the
global market, mushroom cultivation is growing and gaining popularity. A lot have cited
of their effect on the immune system. Ganoderma, for example is considered a natural
regulator of the immune system. Oyster mushrooms are best known medically for their
cardiovascular and cholesterol-controlling benefits. Shiitake on the other hand is
recognized for its antitumor action. Mushrooms are also good sources of protein,
carbohydrates and fiber.
Importance of Hybrid Mushrooms
The purpose of breeding is to combine desired traits present in separate
individuals using controlled crosses and selection of offspring (Sonnenberg, 2007).
Several studies showed the significance of breeding mushrooms. A study
conducted by Arias, et al. (2000) resulted to six hybrids that showed improved fruiting
characteristics and had maximum productivity. Anderson, et al. (2001) found out that
hybrid off-white strains of Agaricus bisporus exhibited intermediate susceptibility to
Trichoderma harzianum, with mean yield losses of 56% to 73%. According to Miller
(2005), hybridization of Cordyceps amplified target medicinal compounds to five times
its potency. Hybridization concentrates the dosage, lowering volume ingested
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
5
substantially over the wild varieties. With hybrid cordyceps, we obtain cordyceps for
target medicinal compounds, vigorous or fast growing, and resistance to infection with
other organisms. Hybridization also may modify the substrate used in cultivating some
mushrooms. Bak et al. (2005) states that FRI 169, a hybrid shiitake mushroom turned out
to be a superior strain for sawdust-based cultivation.
Hybridization
Unlike plant and animal breeding, mushroom breeding is a relatively new applied
science. This is not surprising since only recently have mushrooms been produced
commercially on a large scale (Sonnenberg, 2007).
Breeding is the only controllable means by which desired genetic traits of
different stocks or strains can be combined. In the higher fungi, its minimal requirements
are: a recognizable sexual interaction between mated strains, fruiting competence,
meiosis and viability of spores (Raper, 1978). She further says that the quality of the crop
depends as much upon the inherited potential of the strain used as does upon conditions
for growth and development. By continued selection, bad characteristics are eliminated
(Fritsche, 1978).
The true success of the hybrid would be if it produces fruits that has spores. Only
at this stage would the true hybrid strain be accomplished. No genetic information is
exchanged between two strains until their separate haploid nuclei have fused and then
undergone meiosis. Recombination would then occur between the two separate strains
forming a third strain. Its offspring (spores) would be new combinations of the two donor
strains. Simply cloning the original mating that fruited will be a hybrid as well, but not a
true hybrid, because there has been no recombination between the two strains, no mixing
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
6
of genes. There has simply been a successful coexistence of two haploid nuclei, one from
each strain, acting independently, but together to create fruits (www.shroomery.org).
Thus, the importance of single sporing.
Elliott and Langton (1981) as cited my Yadav et al. (1999) emphasized that the
hybridization methods based on the combination of non-fertile, homokaryotic single
spore cultures offer better prospects for genetic improvement than the traditional
selection methods. Yadav made use of single sporing technique by serially diluting an
obtained spore mass and isolating the germinated single spore cultures from the serial
dilution. Similarly, Arias et al. (2000) produced dikaryons by pairing monokaryotic
strains, either neohaplons or single spore isolates. Sexual compatibility was assessed by
the positive clamp connection seen under the microscope.
In the process, not all sub-strains within a strain will fuse, some are completely
incompatible. There is a zone of zero growth between the two strains on the Petri plate
(www.shroomery.org); (www.nwbotanicals.org). Holliday (2004) reported that when
different strains were inoculated together onto one Petri dish, the strains grew towards
each other until they formed a zone of inhibition. But through the addition of snake
venom to the agar, the zone of inhibition becomes short lived until the mycelial strands
fuse and exchange nuclear material through their venom-weakened cell wall. Miller
(2005) also observed that snake venom changes the form of the Cordyceps by breaking
down its cell walls, rather than being added into the final product and indicated that snake
venom is an essential catalyst for hybridization allowing the fusion of the two differing
Deoxyribonucleic acids (DNA’s).
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
7
Ultra Violet (UV) is a strong physical mutagen and it is directly absorbed by the
DNA bases (Fincham et al., 1975). Perera et al. (2005) observed the conversion of
ergosterol in mushrooms to vitamin D2 when exoposed to UV.
Santos et al. (1989) were able to obtain thermotolerant and high alcohol yielding
mutant of Saccharomyces cerevisae when treated with acridine mustard (AM) and nitrous
acid (NA). Accordingly, acradine mustard is a frameshift mutagen and nitrous acid is a
demeaning agent. Santiago et al, (1991) claimed that nitrous acid is an effective mutagen
for the isolation of mutant in Agaricus bisporus and Lentinula edodes. He added that the
nitous acid dose depends upon the type of species and nature of the desired mutants.
With the occurrence of mutation, performance of the new strain is unpredictable.
Hybrids must be cultivated to evaluate their production characteristics. Arias et al. (2000)
reported that the use 2-deoxyglucose was used to select high yielding resistant hybrids.
2-Deoxy-D-glucose has a molecular formula of C6H12O5. It is a molecule which
has the 2-hydroxyl group replaced by hydrogen, so that it cannot undergo further
glycolysis (http://en.wikipedia.org). 2-Deoxyglucose is a rare and natural
monosaccharide. It is the basic structure of the anticancer drugs such as daunomycin,
adriamycin, carminomycins and antibiotics.
2-DG is a white crystalline hydroscopic powder. It is odorless, tastes sweet and
very soluble in water, partially soluble in hot methanol, ethanol, acetone, and butanol. It
is not soluble in ether, chloroform, petroleum ether and toluene. 2-DG is a polyhydroxy
aldehyde with reducing ability can react with Fehling or Benedict reagent and produce
red or yellow precipitation of copper oxide. 2-DG acts to inhibit the phosphorylation of a
glucose molecule that produces glucose-6-phosphate in the glycolysis cycle, therefore
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
8
inhibiting the production of ATP and can also decrease the temperature in muscle cells. It
can restrain viral infections and fermentation, microbes and cancer cell growth. It also
controls metabolism and possesses physiological and therapeutically effects.
(http://www.2dgpro.com)
The effect of several concentrations of the toxic analogue of glucose, 2-deoxy-D-
glucose (2-DG), was observed on the rate of apical growth of strains of Pleurotus
ostreatus using different carbon sources. The growth of the strains under these conditions
distinguished between tolerant and sensitive strains. It was observed that the phenotype
which was tolerant to 2-DG was correlated with high productivity of the strains in pilot
production farms (Sanchez et al., 1996).
To eliminate the trial and error process during hybridization, Arias et al. (2000)
used 2DG in the selection of recovered Pleurotus ostreatus. Resistance to this toxic
analogue of glucose had been used to select overproducing microorganisms. In their
study, six hybrids showed resistance to 2-DG out of the original thirteen hybrids. The 2-
DG resistant hybrids showed required 62 days to fruit and achieved maximum
productivity in a fruiting period of 28 days, with fruit bodies of 18.5g average weight.
There were similar results of that of Kirimura et al. (1992) with Aspergillus niger
mutants resistant to 2-DG that produced citric acid earlier than sensible individuals and
that of Sanchez and Viniegra – Gonzales (1996) with Pleurotus ostreatus resistant
strains gave higher yield as reported by Arias et al. (2000)
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
9
MATERIALS AND METHODS
Collection of Specimens
Ten mushrooms were collected, five each from both white (Pleurotus
cornucopiae) and gray (Pleurotus ostreatus) oyster species. These were characterized as
to 1.) size, shape, and color of the cap; 2.) size, shape, color of spores and; 3.) form of
hymenophore.
Collection of Spores
Collection of spores was done by removing the cap and laying it faced down. Half
of the cap was laid over white paper and the other half on black paper. After hours, the
deposited spores were collected for single sporing and for microscopic characterization.
Calibrating the Microscope
Calibration of the microscope was done by inserting the ocular micrometer into
the eyepiece and the stage micrometer on the stage. Using the low high power objective
(40x), zero (0) point of both stage and ocular micrometers sere set to coincide with each
other. The ocular divisions that cover the space between zero and the coincident line were
counted. Calibration Factor (CF) or Calibration Constant was calculated using this
formula:
CF= n divisions of stage micrometer x 10 units/division
n divisions of ocular micrometer
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
10
Protocols for Optimizing Spore Germination
Effect of the Kind of Media and Species Used
Spore germination of each mushroom species was tested on differential
media namely. Each treatment was replicated four times and arranged in a
factorial completely randomized design (CRD) with mushroom species as factor
A and different media used as factor B. The treatments were as follows:
Factor A
A1 – P. ostreatus
A2 – P. cornucopiae
Factor B
B1 – Water agar
B2 – Potato Dextrose Agar (PDA)
B3 – Malt Extract Agar (MEA)
Commercial sterilized distilled water was poured into the Petri dish
containing the spores after which 0.1 mm was obtained and spread over the
differential media. Plates were incubated at 23°C and presence of germinated
spores was observed after 48 hours. Treatments were replicated four times.
Single Sporing Technique
Two modified techniques of single sporing according to Manzanares et al.
(1994) and Webster et al. (1981) were tested. Treatments used were:
S1
Agar block method
S2
Spore Punch Method
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
11
For the agar block method, the media was cut into cubes at approximately
1 mm x 1 mm then observed under the microscope for well isolated single spores.
The second method is the use of spore punch. An agar disc bearing a
germinated spore was obtained using a spore punch mounted in the microscope.
Agar blocks and spore punches containing a single spore were transferred
to the media that gave the best result on initial test of media. Each plate contained
a spore each of white and gray oyster mushroom. Treatments were replicated four
times.
Hybridization between species was also tested by inoculating two spores
from the same kind of mushroom (i.e. P. ostreatus with P. ostreatus and P.
cornucopiae with P. cornucopiae) but from different fruiting bodies into a Petri
dish.
Isolates that produced mycelia were cut and transferred to plates and were
paired with other isolates that also produced mycelia.
Protocols for Optimizing Hybridization
Exposure Time to Ultra Violet (UV) Light
Petri plates containing the single spores were exposed to Ultra Violet
(UV) light with a distance from the lamp of 15 cm (Santiago, Jr., et al.). The
treatments were:
T0 – Control
T1 – 10 minutes
T2 – 20 minutes
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
12
T3 – 30 minutes
T4 – 40 minutes
T5 – 50 minutes
T6 – 60 minutes
Presence of Dikaryotic Mycelia
Treated isolates were observed under the microscope for the presence of clamp
connection. Additionally, mycelia were taken from both parent spores and from meeting
points of both mycelia which is treated as a possible hybrid candidate and were measured
as to mycelial diameter.
Data Gathered
The data gathered were the following:
1.
Physical description of parent mushroom. Mushroom fruiting bodies were
described as to (a) size of cap, (b) shape of cap, (c) color of cap and (d) form of
hymenophore.
2. Microscopic description. The size, shape and color of spores were described.
3.
Effect of media on germination. Presence of germination was noted as
well as length of mycelium.
4. Presence of clamp connection.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
13
RESULTS AND DISCUSSIONS
Physical Description
Mushroom characterization is presented in Table 1. The mushrooms used were
harvested after 36 hours from pinhead formation. Results reveal that white oyster
mushroom (Pleurotus cornucopiae) is bigger in size than gray oyster mushroom
(Pleurotus ostreatus). Both have a convex, fan-shaped cap and are gilled.
Table 1. Physical description of parent mushroom
Specimen
Size of cap
Shape of cap
Color of cap
Form of
(cm)
hymenophore
P. cornucopiae
5.2
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.0
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.4
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.2
Convex, fan-shaped
White
Gilled
P. cornucopiae
5.0
Convex, fan-shaped
White
Gilled
P. ostreatus
5.0
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.8
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.0
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.7
Convex, fan-shaped
Gray
Gilled
P. ostreatus
4.8
Convex, fan-shaped
Gray
Gilled
Microscopic description
Spore shape for both mushrooms is kidney-shaped with white to pinkish spore
print. At 500x magnification, spore size for both mushrooms measures 10.71 x 3.57.
a.
b.
Figure 1. (a) White to pinkish spore print and (b) a spore of P. cornucopiae.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
14
Effect of the Kind of Media and Species Used
Spore germination was observed after two days from inoculation. Table 2 shows
that there are no significant differences on the length of the mushrooms as affected by the
species. These signifies that the P. cornucopiae and white P. ostreatus are not
significantly different from each other in terms of there mycelium length. Moreover, the
effect of the media on mycelia length is highly significant. Table 2 shows that MEA gave
significantly higher mean length of 47.77µ. followed by PDA and water agar with mean
mycelia length of 29.018 µ and 0.000 µ, respectively.
Analysis further shows that there are no significant effects of species and medium
interaction on the length of the mycelium. This means that the species and the medium
are independent from each other. That the differences on the length of the mycelium is
not a result of the interaction of the species and the medium, rather, it is either a result of
the effect of the species or of the medium alone. In this case, the significant effect ion the
kind of media used.
Table 2. Mean mycelium length as affected by species and media used
Mean Length (µ)
Species
P. ostreatus
27.83a
P. cornucopiae
23.36a
Medium
MEA
47.77a
PDA
29.018b
Water Agar
0.000c
Species x Medium
ns
CV (%)
26.34
Means of the same letter are not significantly different from at each other at 5% level of
significance by DMRT.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
15
a.
b.
c.
a.
b.
Figure 2. Geminated spores of Pleurotus ostreatus on (a.) MEA, (b.) PDA 2 days after
inoculation and (c.) Water Agar 10 days after inoculation at 500x.
a.
b.
c.
a.
b.
Figure 3. Germinated spores of P. cornucopiae on (a) MEA, (b) PDA 2 days after
inoculation and (c) Water Agar 10 days after inoculation at 500x.
Appropriate Technique of Single Sporing
Single sporing methods were tested on MEA as it is a promising medium for
spore germination and growth. Spore punch method generated higher possibility of
obtaining a well isolated, germinated, single spore as compared to the agar block method.
Moreover, spore punch method is a more rapid way to obtain a single spore than that of
the agar block method where a lot of agar blocks should be made to increase the chance
of obtaining a single spore.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
16
a.
b.
Figure 4. Comparison of single spore methods (a) spore punch and (b) agar block method
at 100x for P. cornucopiae.
Mycelia emerging from the single spore was observed after 6 to 10 days from
incubation at 23ºC only from spores of Pleurotus ostreatus.
a.
a.
b.
Figure 5. Plates showing growth of spore after 6 days from inoculation. (a) mycelium is
observed from spore punch of P.ostreatus (Gray) while (b) no growth is
observed from spore punch of P. cornucopiae.
A second set of single sporing was done this time pairing P. ostreatus spores but
from different fruiting bodies. After 15 days from inoculation, mycelia from both spores
were seen growing towards each other but only up to a certain point where one became
inhibitory to the other thus the formation of an inhibition zone. This result is supported by
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
17
Holliday’s (2004) findings on Cordyseps sinensis. He stressed that not all sub-strains
within a strain will fuse, some are completely incompatible.
Figure 6. Formation of inhibition zone after 15 days from isolation.
Exposure Time to Ultra Violet (UV) Light
To increase the chance of hybridization, before subjecting it to UV light, five
isolates from five single spores were transferred into a Petri dish. Results show that
regardless of the length of time exposed to UV light, no clamp connections were
observed in all plates. Fig. 8.g reveals no evident inhibition zone. Subjecting it though to
mycelium diameter measurement also indicates no dikaryotization that took place.
a.
b.
a.
b.
Figure 7. Monokaryons exposed to UV Light for 40 minutes (a) Inhibition zone between
two different mycelia. (b) Zone of inhibition as seen under the microscope at
100x magnification.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
18
a.
a.
b.
c.
a.
c.
d.
e.
f.
f.
g.
Figure 8. One week old culture of monokaryons exposed to Ultra Violet (UV) Light at
different lengths of time (a) Control, (b) 10 minutes, (c) 20 minutes, (d) 30
minutes (e) 40 minutes (f) 50 minutes and (g) 60 minutes.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
19
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
This study was conducted to determine the kind of culture medium appropriate for
spore germination, compare techniques for single sporing, determine length of time of
exposure of germinated single spore to Ultra Violet light on dikaryotization and to
determine for incidence of dikaryotic mycelium production.
Based on the results, malt extract agar is the most suitable medium for spore
germination compared to potato dextrose agar and water agar. Although PDA and WA
also sustain spore germination and growth, PDA produces shorter mycelium after two
days from inoculation compared to MEA while water agar takes a longer time to be able
to produce a germinated spore.
Spore punch method offers higher chances of obtaining single spores and is less
laborious than that of the agar block method where the chances of obtaining a well
isolated spore is slim.
Single spores obtained were paired together and were exposed to Ultra Violet
light at different length of time. Regardless of this, no clamp connection was observed
from these paired isolates.
Mycelia from both parents were compared to that of the supposed dikaryotic
mycelium. These were measured but no difference in mycelial diameter was observed
among the parents and the supposed hybrid.
Conclusions
Based on the findings of the study, it could be concluded that:
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
20
1. Among the media used, Malt Extract Agar is the most suitable medium for
Pleurotus spp. spore germination and growth.
2. Spore punch method is the best technique to be able to obtain a well isolated
single spore. It is much less laborious and requires shorter time of obtaining a single
spore compared to the agar block method.
3. Regardless of Ultra Violet exposure at different lengths of time from 10
minutes to 60 minutes, dikaryotization did not take place, as observed in the plates and
under the microscope.
Recommendations
Based on the findings, the following are recommended:
1. Further research need to be undertaken regarding growth of monokaryons in
controlled setting among white oyster mushrooms.
2. Hybridization study on other mushrooms can be done with the given
protocols on the kind of medium and single sporing technique from this study as a guide.
3. Other hybridization techniques including the use of mutagenic agents may
be tested to improve the study.
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
21
LITERATURE CITED
ANDERSON, M. G., D. M. BEYER and P. J. WEST. 2001. Yield Comparison of
Hybrid Agaricus Mushroom Strains as a Measure of Resistance to Trichoderma
Green Mold. Retrieved at http://www.apsnet.org/pd/pdfs/2001/0511-02R.pdf
ANONYMOUS. 2007. Is it Possible to Cross Two Strains? Shroomery. Magic
Mushrooms Demystified. Retrieved at http://www.shroomery.org/8492/Is-it-
possible-to-cross-2-strains
______. 2008. 2-Deoxy-D-glucose. Retrieved from http://en.wikipedia.org/wiki/2-
Deoxy-D-glucose
______. Undated. College of Agricultural Sciences. Retrieved from
http://pubs.cas.psu.edu
______. Undated. Mushroom. Krishiworld. The Pulse of Indian Agriculture. Retrieved at
http://www.krishiworld.com/html/mushroom.html
______. Undated. P-2DG® 2-Deoxy-D-Glucose. Retrieved from
http://www.2dgpro.com/2dginfo.htm
ARIAS, A., R. RAMIREZ and H. LEAL. 2000. Cultivation of Pleurotus ostreatus
hybrids resistant to 2DG obtained by pairings of neohaplonts from selected
dikaryons. Rotterdam: Van Griensven. pp. 305-309.
BAK, W. C., B. H. LEE, M. K. Kim, K. H. Yoon, K. H. Ka AND H. Park. Breeding of
Shiitake strain for sawdust-cultivation by di-mon mating method. Abst. Retrieved
at http://www.worldmushroomsociety.com/
FRITSCHE, A. 1978. The biology and cultivation of edible mushrooms. New York:
Academic Press, Inc. pp. 139-143.
HOLLIDAY, J. C., P. CLEAVER, and M. LOOMIS-POWERS. 2004. The hybridization
of Cordyceps synensis strains and the modifications of their culture parameters,
in order to optimize the production of target medicinal compounds. Retrieved at
www.nwbotanicals.org/nwb/lexicon/hybridcordyceps.htm
KAUL, T. N. 1997. Introduction to mushroom science (Systematics). U.S.A.: Science
Publishers, Inc. p. 61
MILLER, R. 2005. Lab-grown cordyceps sinensis hybrtid: A Nano-processed Medicinal
Mushroom that Really Delivers. Retrieved at
http://pharmaceuticalmushrooms.nwbotanicals.org/cordycepssinensisheaa.pdf
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
22
OEI, P. 2005. Small-scale mushroom cultivation. Retrieved at
http://www.anancy.net/uploads/file_en/40-e-2005-mushrom_screen.pdf
PERERA, C. O., V.J. JASINGHE. 2005. Distribution of ergosterol in different tissues of
mushrooms and its effect on the conversion of ergosterol to vitamin D2 by UV
irradiation. Food Chemistry. Abst., 92:3. Retrieved at
www.cababstractsplus.org/google/abstract.asp?AcNo=20053082147
RAPER, C. A. 1978. The biology and cultivation of edible mushrooms. New York:
Academic Press, Inc. p. 110
SANCHEZ, C., G. VINIEGRA-GONZALES, and D. MOORE. 1996. Correlation
between Tolerance to 2-deoxy-D-glucose and Productivity of Strains of Pleurotus
ostreatus. Abst. Retrieved at http://www.worldmushroomsociety.com/
SANTIAGO, C. M., JR., B.B. MERCADO, E.D. DE LEON, R.P. FLORES, R.P.
GARCIA, M.B. BIGOL. 1991. Strain improvement of selected species of edible
fungi. Phil. J. Sci. 120 (2): 159.
SANTOS, M. E., D. A. RAMIREZ, P. C. SANCHEZ, W. L. FERNANDEZ. 1989. The
production of thermotolerant and high alcohol yielding yeasts by chemical
mutagenesis and hybridization. The Phil. J. Sci. 118 (13) : 239.
SONNENBERG, A.S.M. 2007. Breeding Mushrooms: State of the Art. WSMBM.
Retrieved at http://www.mushworld.com/tech/view.asp?cata_id=1130&vid=7742
YADAV, M. C., R.N. VERMA and B. L. DHAR. 1999. Studies on development of
improved strains and hybrids of white button mushroom. Retrieved at
www.worldmushroomsociety.com/upload/f2003610144435_3rdICMBMP_011.p
df
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
23
APPENDICES
Appendix Table 1. Effect of media and species on mycelial length
REPLICATION
TREATMENT
I II III IV
TOTAL
MEAN
Media
MEA
46.42 58.91 75.00 48.22
228.55
57.14
PDA
33.04 25.90 28.57 28.60
116.11
29.03
WA
0
0
0
0
0
0
Species
P. ostreatus
25.00 32.74 22.62 30.95
111.31
27.82
P. cornucopiae
27.98 23.81 21.43 20.24
99.43
24.86
ANALYSIS OF VARIANCE
SOURCE DEGREE
COMPUTE
TABULAR
OF
OF
SUM OF
MEAN
OF
D
Significanc
F
VARIATIO FREEDO SQUARE SQUARE
e
0.0
N
M
S
S
F
0.0
5
1
Model
5
9447.077 1889.415
41.591**
.000 2.7 4.2
7
5
Species
1
119.483
119.483
2.630ns
.122 4.4 8.2
1
9
Media
2
9267.055 4633.527 101.997**
.000 3.5 6.0
5
1
Species x
2
60.538
30.269
0.666ns
.526 3.5 6.0
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
24
Media
5
1
Intercept
1
15721.98
15721.98 346.086**
8.2
5
5
.000 4.41 9
Error
18
817.704
45.428
Total
24
25986.766
ns = not significant
CV = 26.34%
** = highly significant
Protocols for Optimizing Hybrid Production
Between Two Pleurotus spp / Jezreel Zyra D. Ibis. 2008
Document Outline
- Protocols for Optimizing HybridProduction Between Two Pleurotus spp
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- Background of the Study
- Importance of the Study
- Time and Place of the Study
- REVIEW OF LITERATURE
- The Mushrooms
- Importance of Hybrid Mushrooms
- Hybridization
- MATERIALS AND METHODS
- Collection of Specimens
- Collection of Spores
- Calibrating the Microscope
- Protocols for Optimizing Spore Germination
- Protocols for Optimizing Hybridization
- Presence of Dikaryotic Mycelia
- Data Gathered
- RESULTS AND DISCUSSIONS
- Physical Description
- Effect of the Kind of Media and Species Used
- Appropriate Technique of Single Sporing
- Exposure Time to Ultra Violet (UV) Light
- SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
- Summary
- Conclusions
- Recommendations
- LITERATURE CITED
- APPENDICES