| Belonging to Ascomycetes in biological classification,morels are world-famous edible and medicinal mushrooms with unique flavor and many biological activities such as antioxidant,anti-inflammatory,anti-bacterial,immune stimulation and anti-tumor properties.In 2012,field cultivation of Morchella mushrooms was successfully carried out for the first time in Sichuan Province,China,and radiated rapidly to the whole country.However,contrary to their rapid development of artificial cultivation,the fundamental study of morels is still relatively lagged,which limits the sustainable healthy and stable development of morel industry.Thus,the mechanismal study of sclerotial development,strain quality evaluation and application of new technology in morel breeding will be conducive to the healthy development of morel artificial cultivation.In the presented paper,the morphological and physiological changes during the sclerotial development of Morchella importuna were studied by electron microscopy and confocal laser microscopy.The ruseluts suggested that autophagy,apoptosis and necrosis were involved in sclerotial development.The energy storage material in sclerotia was fat.The technology of assay of the identity,mating type and vitality（IMV）of cultivated strains of Morchella mushrooms were established,which can be used to test the adaptability of the strains in large-scale artificial cultivation.The technologies of protoplast release,regeneration and rusion of M.importuna and M.sextelata were optimized,and some fusion strains were obtained.The study was of practical significance for promoting the development of protoplast fusion in breeding of Morchella fungi.The main results are as follows:（1）Sclerotial primordia were formed by the expansion of repeated branches of mycelia and terminal or sub-terminal branches of mycelia in the cultivated strain of M.importuna No.1.The development of sclerotium was characterized by both terminal and lateral forms,and was complex one.Transmission electron microscopy（TEM）and confocal laser imaging of living cells suggested that autophagy,apoptosis and necrosis were involved in sclerotial development of M.importuna.In addition,the study also revealed that the energy storage substance in sclerotia was lipid.Qualitative determination of fat showed that more fats were accumulated in sclerotia than those in vegetative mycelia.（2）The genomic DNA of six Morchella strains was amplified using primers of ITS-1/ITS-4,RPB1B-F/RPB1B-R and RPB2B-F/RPB2B-R,respectively.The sequences of ITS,RPB1B and RPB2B genes were determined and compared.The results showed that taxonomy status of strains 4 and Xuchang were M.importuna,strains 201,13 and SZL-3 were M.sextelata,and the homology between CuBing and M.kaibabensis was 98.9%and the reliability was 100%.The specific primers to amplify the mating types of Morchella fungi were designed and screened.It was found that the primers of Mat1-1/Mat1-1-1 could specifically identify the mating type of M.sextelata,and Mat1-1-1/Mat1-2-1 could identify the mating type of M.importuna.The results of PCR with selected primers found that each pair of primers resulted in one band,and the mixed primers could amplify two bands.These results indicated that the selected primers could be used to analyze the mating type of the strains of both morels.The study showed that no mating type deletion was found in the tested strains of both morels,which demonstrated that M.importuna and M.sextelata were both typical heterozygous fungi.The lipid peroxidation levels of tested strains were determined to evaluate the aging of the strains.The results suggested that strains SZL-3 and CuBing were seriously aged,strains 201 and 13 had the strongest activity,and strains 4 and XuChang falled in between them.（3）The conditions of protoplasts release for M.importuna and M.sextelata were optimized.The results showed that the optimum conditions for protoplast preparation of M.importuna were as follows:osmotic stabilizer was 0.6 M mannitol,enzyme liquid system was 2%lysozyme+1%snail enzyme+1%cellulase,and the culture period was 3 days.Under the optimum conditions,the protoplast yield of M.importuna was 1×106/mL,and the protoplast regeneration rate of the prepared protoplasts under optimized conditions could reach 1.8%.The optimum conditions for protoplast preparation of M.sextelata were as follows:osmotic stabilizer was 0.6 M mannitol,enzyme liquid system was 2%lysozyme+1%snail enzyme+1%cellulase,and culture time of mycelia was 3 days.Under the optimum conditions,the protoplast yield of M.sextelata was 6.5×105/mL,and the protoplast regeneration rate of such protoplasts could reach 1.5%.（4）The fusion conditions of double inactivated protoplasts between M.importuna and M.sextelata were optimized.The results showed that the optimum conditions for protoplast fusion between the two species of Morchella were as follows:protoplast fusion temperature 25℃,solution pH 8.0,final concentration of CaCl2 50mmol/L,fusion agent PEG 25%.Under the optimum conditions,the protoplast fusion rate could reach 0.8%.（5）The specific RAPD（random amplified polymorphism DNA）molecular markers were screened for identification of the Morchella parents and protoplast fusants.Cluster analysis showed that the parents and fusants could be divided into three groups:the first group consisted of parental M.importuna（P1）,the second group consisted of all fusants,and the third group consisted of parental M.sextelata（P2）.Sixteen Fusants can be divided into three groups:the first group included fusants1,5,6,8,9,10,11,12,13,14,15 and 16,the second group contained fusants 4 and 7and the last group were fusants 2 and 3.These results indicate that the protoplast fusants obtained by technology of double inactivation fusion were real ones.（6）Fusant 1（R1）,fusant 2（R2）,fusant 4（R4）and their parents were selected to cultivate on plates,and their colony morphology were observed.It was found that cultures M.importuna（P1）,M.sextelata（P2）,R1,R2 and R4 all produced dotted sclerotia.The order of mycelial growth rate was R4>P1>P2>R2>R1,however the growth rates of five strains were not significantly different.The recovery time after inoculation of five strains was also different,among which the recovery time of P2was the longest and that of P1 was the shortest,and fusants R1,R2 and R4 were between them.The hyphae of P1,R1,R2 and R 4 were intense and P2 was sparse,and the colony edges of all strains were neat.These results indicated that the fusants were real ones.（7）Artificial cultivation showed that the ascocarp morphology of Fusants R1 and R4 was close to that of parents 2（P2,Morchella sextelata）,R5 and R8 were close to that of parents 1（P1,Morchella importuna）,while R2 and R3 were between parents.（8）Artificial cultivation showed that there were significant differences between the yield of 8 strains of 2 parents and 6 fusants.No fusant strain produced more than P1 parent strain,while the yield of R2 fusant was lower than P2 parent strain,suggesting that the selection of parents was very important in protoplast fusion breeding（P2 parents had degenerated and aged）.The limitation of strains is great,but this technology has great potential in the breeding of fusion strains which synthesize other good traits of parents. |
