Johal, P.Pnaresh kumar2017-01-202017-01-202013http://krishikosh.egranth.ac.in/handle/1/97399Protoplast 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) thanensawdustwood chipsThesis