STRAIN IMPROVEMENT OF Lentinus edodes (Berk.) Sing. BY PROTOPLAST FUSION TECHNIQUE
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Date
2013
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Punjab Agricultural University
Abstract
Protoplast fusion is one of the techniques to combine genetic characters across species barriers. In the
present studies, dikaryotic mycelium of Lentinus edodes strains was used for protoplast fusion using
polyethylene glycol (PEG) to develop hybrids with improved desirable traits like total yield, temperature
tolerance, sporelessness/less spore producing and shorter cropping cycle. Optimized protocol was developed for
protoplast isolation of using parameters like enzyme: combinations and concentrations, age of mycelium,
duration of incubation period, osmotic stabilizers type. It was found that for both LeS and LeC, 3 days old
mycelial culture, 0.6M NaCl prepared in 0.01 M phosphate buffer gave the maximum protoplast yield (2.40 x 107
and 1.80 x 107 protoplasts/mL) after 3 hours of incubation when enzyme combinations from group I of chitinase
3, β-1, 3-glucanase 4, driselase 7 and lyticase 5 concentrations (mg/mL) were used. The protoplasts subjected to
polyethylene glycol (PEG) mediated fusion was regenerated on three regeneration media with four osmotic
stabilizers. Maximum regeneration efficiency of putative hybrid fusants was obtained on SMY (starch-malt-yeast
extract) medium (3.0%) where 0.6M sucrose was used. All together 124 intraspecific putative hybrid fusants
were obtained and five hybrid fusants (L4, L13, L31, L58 and L76) were selected on the basis of radial growth.
All the hybrid fusants shared almost similar colony morphology with some variation in growth pattern. Some
unique characteristics like production of red exudate, softening of agar in hybrid fusants were also seen. Selected
hybrid fusants were further characterized for genetic variability by high enzyme production, isozyme analysis
and RAPD-PCR.Qualitative estimation of endoglucanase activity of parents and hybrid fusants indicated highest
enzyme production by hybrid fusant L58 with 1.27 z/c value. Quantitative endoglucanase activity indicated
highest enzyme production by hybrid fusant L58 (8.85 μmol/min/mL) on saw dust supplementation.Highest
xylanase activity showed by hybrid fusant L58 (18.85 μmol/min/mL) on wheat straw supplementation. Highest
laccase activity was observed in L4 (8.33 μmol/mL of culture filtrate) on saw dust supplementation. Isozyme
analysis was done using Native-PAGE. Protein content found maximum in L58 hybrid fusant (182.5µg/mL).
Isozyme analysis of parents and hybrid fusants revealed a maximum of 4 malate dehydrogenase isozyme, with
maximum by hybrid fusant L58 at the Rm value of 0.33, 0.43 and 0.56. Peroxidase produced two isozyme at Rm
value of 0.62 and 0.70 while superoxide dismutase produced only one isoform. A set of 10 primers were used for
molecular characterization of hybrids along with the parents using RAPD-PCR. Out of these 4 primers gave
distinct amplification products. These 4 primers yielded 69 scorable bands ranging from less than 100 bp to more
than 1000 bp for all the genotypes. Maximum similarity coefficient of value 0.952 was obtained between hybrid
fusants L58 and L13. Similarity coefficient of parent LeC with parent LeS was 0.416 which could be compared
with hybrid fusants L13 and L58. A combined dendrogram was obtained from the RAPD data of all the four
primers together and it was found that three hybrid fusants i.e. L58, L13 and L31 were closer to LeS parent
strain. Cultivation of all the five hybrid fusants developed by protoplast fusion along with the parent strains LeS
and LeC was carried out on combination of sawdust with wood chips and wheat straw as substrate. Spawn run
period varied from 47 days (L31) to 65 days (LeS). Hybrid fusant L58 was found to give significantly higher
yield (57.2 % and 56.9 % with wheat straw and sawdust and wood chips of red meranti tree respectively) than
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Keywords
sawdust, wood chips