Strain improvement of Calocybe SPP. through interspecific hybridization

Abstract

The present study entitled “Strain improvement of Calocybe spp. through interspecific hybridization” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021, for the development and evaluation of improved strain(s) of Calocybe spp. with desirable traits by interspecific hybridization. Two Calocybe spp. viz., Calocybe indica, Calocybe gambosa were obtained from AICRP on Mushroom and Instructional Farm, College of Agriculture, Vellayani. The pure culturing and morphological studies of cultures revealed that the mycelia was septate with clamp connections. The mycelial growth was white fluffy and with slow growth in C. indica, whereas it was off white cottony mycelia with fast growth in C. gambosa. The mycelial width was larger (26.55 μm) in C. gambosa. Basidia and cheilocystidia were comparatively larger in C. indica. The basidiospores were hyaline, broadly ellipsoid, thin walled and produced on clavate basidia on the hymenial layer of the sporocarps in both the Calocybe spp. The size of the basidiospores were comparatively larger in C. gambosa. Mycelial run was faster in C. gambosa than C. indica in paddy grains when used as spawn substrate. Paddy straw and amended cocopeat were used as substrate for mushroom bed preparation. C. indica took minimum number of days for complete spawn run (25), pinhead formation (37), first harvest (44), total crop period (60) with comparatively more number of sporophores (16) with lesser sporophore weight than C. gambosa. C. gambosa took 31 days for spawn run, 45 days for pin head formation, 52 days for first harvest with larger sporophores and biological efficiency of 84.78 % whereas in C. indica it was 61.59%. The colour of sporophores were milky white in C. indica while creamish white in C. gambosa. The pileus shape in both the species were convex with varied diameter and thickness of the pileus. Length and diameter of stipe was higher in C. gambosa. C. indica showed more number of gills cm-2 (24), while in case of C. gambosa it was 19. The development of improved strains by hybridization was initiated with the isolation of single spores. The single spores of Calocybe spp. were isolated by serial dilution technique. Pure culturing of single spores was done in potato dextrose agar medium. Nature of their mycelial growth, colour, growth rate and days taken for complete growth in Petri dishes were recorded. The experiment was laid out in CRD with five replication. Hybridization was carried out between single spore cultures of C. indica and C. gambosa by dual culture technique. 121 crosses were done with 42 positive crosses (compatible) and 79 inhibitions (incompatible). The hybrids were confirmed by the presence of clamp connection in the mycelium from the barrage zone. Out of the 42 crosses 22 compatible crosses were identified based on their parental mycelial characters, nature of barrage formation (clamp connection) and mycelial run in spawn. In vitro and in vivo studies were conducted to evaluate the parents and their interspecific hybrids. In vitro studies were conducted to observe nature of mycelial growth, colour, growth rate and days taken for complete growth in Petri dish for 22 interspecific hybrids and their parents. four were selected for further studies based on the prior experiment. In vivo studies were conducted by preparing the beds of the selected 4 interspecific crosses with five replication in CRD. The days taken for spawn run and incidence of disease were recorded for all the four crosses. Fruiting body production was observed only in A2B4. The parents A2 and B4 failed to produce any fruiting body indicating the monokaryotic condition. Days taken for pin head initiation, first harvest, number of sporophores emerged, average weight of sporophore and incidence of pest were recorded for this cross. The microscopic observations of A2B4 sporophore revealed basidia of 35.64 x 5.21 μm with thin ellipsoidal basidiospores of size 10.25 x 7.56 μm. Comparative studies on the biomass of the inter specific hybrids (A1B5, A2B4, A4B2 and A4B6) and their parents revealed that A2B4 hybrid had higher biomass (0.42 g 100ml-1) when compared to other crosses. The crude fibre estimation studies for the parents (C. indica and C. gambosa) and hybrid revealed that A2B4 hybrid had higher fibre content of 24.43 % crude fibre when compared to the parents C. indica and C. gambosa 11.79 and 16.47 % respectively. Molecular characterization of improved strain along with parents were analysed by RAPD (Random Amplified Polymorphic DNA). Pairwise comparisons of the strains, based on the presence or absence of unique and shared amplicons, were used to generate similarity coefficient of Jaccard. The results were analysed using the unweighted pair-group method with arithmetic average (UPGMA). The analysis revealed that the hybrid A2B4 had 69.6 per cent similarity with C. gambosa and 54.5 per cent similarity with C. indica. The present study revealed the integration of traits of parents into the hybrid. Enhancement in characters of sporophore viz., like colour, shape, earlier initiation, increased sporophore weight were observed in hybrid when compared to parents. Also faster completion of crop cycle, reduced incidence of pest and disease were noticed. There was an improvement in biomass, crude fibre content in hybrid when compared to the parents. The hybrid A2B4 can be recommended for the production after yield stabilization studies.