DEVELOPMENT OF HYBRID(S) OF Agaricus bisporus (Lange) IMBACH AND THEIR EVALUATION FOR HIGHER YIELD

Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan 173 230 (HP) India Department of Mycology and Plant Pathology Title of Thesis : “Development of hybrid(s) of Agaricus bisporus (Lange) Imbach and their evaluation for higher yield”. Name of the Student : Manju Sharma Admission Number : H-2008-15-D Major Advisor : Dr. B. C. Suman Major Field : Plant Pathology Minor Field : Microbiology Biotechnology Degree Awarded : Ph. D. (Mycology and Plant Pathology) Year of Award of Degree : 2012 No. of Pages in thesis : 181+V No. of words in Abstract : 603 ABSTRACT The present investigations entitled “Development of hybrid(s) of Agaricus bisporus (Lange) Imbach and their evaluation for higher yield” were undertaken to isolates the homokaryons from different parental strains, their identification by fruiting trials and by ISSR markers, matings compatible homokaryons to produce hybrids, spawn preparation and production efficiency including other aspects like physiological studies and molecular characterization of developed hybrids. one thousand six hundred forty two single spore isolates were raised from parental strains namely A-15, S 11, S140 and U3 of Agaricus bisporus. These 1642, single spore isolates showed fluffy, appressed and strand types of growth. Out of those only 36 single spore isolates (nine from each parent strains to maintain uniform pattern) which were supposed to be homokaryons on the basis of slow mycelial growth rate and appressed types of colony morphology were selected for future studies. All single spore isolates with appressed types of colony morphology produced no fruit bodies in any replication. However the strandy slow growing single spore isolates produced fruit bodies in all replication. For the molecular characterization of selected monokaryons from each parents, six inter simple sequence repeats (ISSR) marker were used. In the ISSR fingerprints of putative single spore isolates from A-15, S11, S140 and U3 strains of Agaricus bisporus, homokaryons lacked amplification products at multiple loci; these grows slowly and all of them had appressed colony morphology. Primers P3 in A-15, P39 in S11, P30 in S-140 and P8 in U3 strains of Agaricus bisporus were proved to be the best due to the sharpness of banding patterns. Out of the two hundred fifty three (253) crosses, only seven crosses between the parents were compatible because compatible colonies got fused with each other and at the point of their contact some raised growth was observed whereas in case of incompatible colonies in Petri plate, growth usually stoped as colonies approached near each other. Anastomosis or intermingling of compatible myceliums were also observed under the microscope. The yield studies showed that that hybrid Hb4, produced highest average yield (26.94kg/100kg compost) and also exhibited better qualities as compared to other isolates. Four hybrids with higher production than parent strains were taken up for studying physiological requirement and molecular characterization in detail. Among various solid media studied malt extract agar media supported maximum average mycelial growth and in case of liquid media, glucose asparagine medium supported maximum average mycelia growth and dry weight and in case and was used as a basal medium for further physiological studies. The optimum temperature and pH for the growth of different hybrids and parent strains were found to be 25C and 6.5 respectively. Among the carbon and nitrogen sources, best growth of different hybrids including the parent strains was recorded on maltose and proline respectively on liquid as well as on solid media. In case of vitamins and growth regulators thiamine and gibberelic acid supported maximum growth of all the hybrids including the parent strains both on liquid and solid medium. RAPD studies shown that the percentage of polymorphism detected was 60.00 per cent. Maximum number of bands were produced by OPP-07 primer, while primer OPO-02 yielded maximum number of unique bands in hybrid Hb4 as compared to other primers. ITS studies showed that all the isolates exhibited identical ITS length of approximately near about 650 bp. The newly developed best hybrids namely Hb1 and Hb4 showed 33.00 per cent and 31.00 per cent molecular variation respectively as compared to the commercially used strain (U3). The ITS region showed single nucleotide polymorphisms (SNPs) at six nucleotide position of 281, 290, 291,292, 293, 295 and 300 base pair in the ITS region of the best developed hybrids (Hb1, Hb4) and parent strains namely A-15, U3 of A. bisporus Signature of Major Advisor Signature of the Student