Cross-breeding and Selection of Flammulina velutipes Hybrid Strains for Industrial Cultivation

Cross-breeding and Selection of Flammulina velutipes HybridStrains for Industrial CultivationAbstract:White Flammulina velutipes strains FM, F21, F10 and F3-W were selected as the parental strains in a single-spore cross-breeding experiment. Thirty basidiospore-derived monokaryons were selected randomly from each parental strain, and mating was carried out according to the mating groups: FM×F21, FM×F3-W and F3-W×F10. Integrative analysis revealed that hybrid progenies from the FM×F21 mating group exhibited the highest mating rate (79%). However, only 59.2% of these hybrids produced measurable fruit body yields compared with 82.6% and 84.4% of the hybrids generated from FM×F3-W and F10×F3-W crosses respectively, perhaps indicating that certain hybrid progeny were better adapted to the standard set of cultivation conditions adopted in the study. Mating rates for FM×F3-W and F10×F3-W crosses were 28% and 42%, respectively indicating that the number of mating type genes may vary among the monokaryons constituting each of the three mating groups. Mycelial growth rates of most FM×F21 hybrid progeny ranged between 4.0~7.0 mm/d. Corresponding values for progeny from FM×F3-W and F10×F3-W crosses were 3.0~6.0 mm/d and 2.0~6.0 mm/d, respectively although a small number of dikaryons from these two mating groups exhibited much higher/lower growth rates. No correlation between mycelial growth rate and fruit body yield was evident for any of the three sets of hybrid progeny. Fruit bodies produced by all the hybrid progeny were white in color.Key words:Flammulina velutipes;single-spore cross-breeding;mycelium growth rate;fruit body yieldIn recent years, among all the edible fungi produced in China, industrial production of Flammulina velutipes has undergone the most rapid development. Fruit body color has become an important commercial characteristic and most F. velutipes strains that are currently cultivated on an industrial scale have a white fruit body［1］.F. velutipes breeding programs have two main objectives: the development of new high-yielding strains with shorter cultivation periods in order to reduce energy costs associated with the maintenance of artificial cultivation envi-ronments, and the protection of intellectual property rights.Cross-breeding among edible fungi involves genetic recombination and the selection of strains possessing the target traits, and crosses between single spore-derived monokaryons has been most widely used in mushroom breeding programs due to the precision of this method［2］. In this study, spore-derived monokaryons from four parental strains of F. velutipes, constituting three mating groups, have been crossed, and the mycelial growth rates, fruiting periods and fruitbody yields of the hybrid progenies have been compared.1 Materials and Methods1.1Materials1.1.1StrainsTest strains FM, F21, F10 and F3-W (white inbred progeny of F3) were provided by the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences.XU Zhen, SHANG Xiaodong, TAN Qi1.1.2Growth media and cultivation substratePotato dextrose agar (PDA): potato 200 g, glucose 20 g, agar 20 g, water 1000 mL, pH 7.Spawn substrate formula (w/w): sawdust 80%, wheat bran 20%, moisture content 50%, pH 6.5.Cultivation substrate formula (w/w): sawdust 30%, corn-cob meal 20%, cotton seed hulls 20%, wheat bran 15%, rice bran 15%, moisture content 65%, pH 6.5.1.2Methods1.2.1Single spore isolationCaps of fresh, mature and good quality F. velutipes basidiocarps were placed in sterile Petri dishes overnight in order to obtain basidiospore prints［3］. Spores were suspended in sterile water and diluted to approximately 2×10.4 spores/mL (using a haemocytometer). Aliquots of the spore suspensions (50 μL) were spread over the surface of PDA plates (9 cm dia. with 20 mL PDA) and incubated at 23 ℃for 3~5 days until visible colonies appeared. Small colonies were transferred to fresh PDA plates and, after further incubation for 3~5 days, hyphae were examined under the microscope (400× magni-fication). Hyphae without clamp connections were retained and stored at 4 ℃as spore monokaryons for further study［4］.1.2.2Crossing between monokaryonsThirty monokaryons were selected randomly from each parental strain and matings carried out between all the possible pairings defined by the crossing groups: FM×F21, FM×F3-W and F3-W×F10. Crosses were undertaken by transferring small pieces of agar (0.5×0.5 cm.2), cut from the growing edges of the two testmonokaryotic colonies, to a fresh PDA plate. Mycelium from the intersection was examined microscopically (×400) for the presence of clamp connections, and hybrid dikaryons were retained and stored at 4 ℃for further study.1.2.3Mycelial growth ratesAverage mycelial growth rates on the cultivation substrate after 7 d incubation were calculated from determinations of the growing edge of triplicate samples.1.2.4Fruit body cultivationMushroom cultivation was carried out in 1100 mL bottles containing 800 g wet substrate. Bottles were sterilized at 121 ℃for 2 h and, after the temperature at the centre of the bottle had dropped to below 25 ℃, were inoculated with 3% (w/w) of spawn using automatic inoculation equipment. Bottles were incubated at 20~22 ℃(air temperature), 60%～65％relative humidity (RH) and 2000～2500 ppm CO 2concentration for approximately 35 days, and then transferred to the cultivation house for fruiting after the surface of the substrate had been scraped off. Environmental conditions within the cultivation house were controlled by an “ULTRA UMC GR (Computer for Mushroom Cultivation) ” system and set at 8～12 ℃, 85％～95％RH and 2000～3000 ppm CO 2 concen-tration［5］. Harvesting was carried outwhen the fruit bodies in each bottle culture had reached approximately 80% maturation, and color, yield and fruiting periods were recorded.1.2.5Data analysisThe u test was employed to determine significance level when comparing hybrid progenies with their parental strains, and the t test was employed to identify relationships between mycelium growth rate and average fruit body yield［6］.2 Results and Analysis2.1Mating testsCrossing groups and progeny characte- ristics are summarized in Table 1 (see the Chinese version).2.2Mycelial growth rates of hybrid progenyThe distribution of different mycelial growth rates among the various hybrid progeny is shown in Fig. 1 (see the Chinese version). Mycelial growth rates of most FM×F21 hybrid progeny ranged between 4.0~7.0 mm/d. Corresponding values forprogeny from FM×F3-W and F10×F3-W crosses were 3.0~6.0 mm/d and 2.0~6.0 mm/d, respectively although a small number of dikaryons from these two mating groups exhibited much higher/lower growth rates. Lower contami-nation rates (e.g. mite infestation, green mold infections) during cultivation were recorded in the case of hybrid progeny derived from FM×F3-W and F10×F3-W crosses compared with the FM×F21 mating group.2.3Comparison of hybrid progeny with parentalstrainsHybrid progeny and their parental strains were subjected to integrative analysis with respect to average mycelial growth rates and fruiting periods (period between scratching treatment and fruit body harvesting) (see Table 2 in the Chinese version). Average mycelial growth rates of hybrid progeny derived from FM×F21 crosses were higher compared with the two parental strains, but were lower than the parental strains in the case of F10×F3-W progeny. Average fruiting periods of all the hybrid progeny were intermediate between the values recorded for the respective parental strains.2.4Fruit body yields from hybrid progenyThe distribution of different average fruit body yields among the various hybrid progeny is shown in Fig. 2 (see the Chinese version). Yields obtained from FM×F21 hybrids were highly diverse and most ranged between 50 and 230 g per bottle, whereas yields produced by FM×F3-W and F10×F3-W progeny varied between 20 and 70 g per bottle.Hybrid progeny and their parental strains were subjected to integrative analysis with respect to average fruit body yields (see Table 3 in the Chinese version). Average yields produced by hybrid progeny derived from FM×F21 crosses were intermediate between the yields obtained from the two parental strains, but were lower than the yields produced by both parental strains in the case of hybrids derived from FM×F3-W and F10×F3-W crosses.2.5Relationship between mycelial growth rate and average yield of hybrid progenyNo correlation between mycelial growth rate and fruit body yield was evident for any of the three sets of hybrid progeny (see Fig. 3 in the Chinese version).3 DiscussionAmong the three mating groups, mono-karyons used in FM×F21 crosses exhibited the highest mating rate (79%) compared with the relatively low mating rates recorded for the monokaryons used in FM×F3-W (27.8%) and F10×F3-W (41.8%) crosses. Variations in the number of mating type genes present in the three matinggroups may explain these differences, but confirmation or otherwise would require analysis of the mating type factors existing in each of the basidiospore-derived monokaryons. Our data suggest a relatively distant genetic relationship between parental strains FM and F21.Considerable variation was also observed in terms of the relative proportion of hybrid progeny derived from each mating group that were suitable for cultivation as a result of differences in the incidence of mite infestation and fungal (e.g. green mold) contamination during mycelium incubation. Marked differences were also recorded in the number of cultivated hybrid progeny derived from each of the three mating groups in terms of the production of measurable yields of fruit bodies. In both cases, lowest values were recorded for hybrid progeny derived from FM×F21 crosses, indicating that these hybrids were perhaps more effete and required more stringent cultivation conditions. Since the cultivation experiments were carried out using identical protocols and environmental parameters for all the hybrids tested, our data suggest that some adjustment in these conditions may be necessary for individual strains in order to establish the most effective cultivation model.In summary, our data indicated that hybrid progenies derived from FM×F21 crosses may, in view of their relatively high mycelial growth rates and wider range of fruit body yields, provide a better source of strains suitable for industrial cultivation.References[1]XIE BG，JIANG YJ，WU WL. Fruit body color inheritance of Flammulina velutipes［J］. Mycosystema，2004，23（1）：79-84. (in Chinese with English abstract)［2］WANG LZ，YE JD，YOU ZX，et al. Edible fungi cultivation［M］. 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