【作者】 刘璐；

【导师】 杜双田；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2017， 硕士

【摘要】 香菇产业是我国食用菌产业经济的重要支柱之一,在调整农村经济结构与产业结构,促进农村经济发展中发挥着重要作用。我国是香菇生产大国,但不是香菇生产强国,育种工作不能满足产业发展的需求。因此,育种工作迫在眉睫。本文利用YJ-01等23个香菇品种,通过原生质体诱变与单孢杂交进行选育,以期得到高产优质的香菇菌株。主要研究结果如下:1.确定了香菇菌株YJ-01原生质体制备的最适条件:以0.5 mol/L Mg SO4作为稳渗剂,在p H=5.0的条件下,利用混合酶(1%蜗牛酶+1%纤维素酶),在27.9℃条件下酶解5 d龄菌丝体2 h,原生质体产量最高。2.确定了香菇菌株YJ-01原生质体再生的最适条件:以0.5 mol/L蔗糖作为稳渗剂,在p H=5.5的条件下,利用混合酶(1%蜗牛酶+1%纤维素酶),在27.8℃条件下酶解6.5 d龄菌丝体2.9 h,原生质体再生率最高。3.通过原生质体紫外诱变获得547株再生菌株。(1)利用不同栽培料进行筛选,得到17株优良再生菌株。其中,菌株Y209最适于苹果木屑上生长,菌丝生长指数(79.88)是YJ-01的3.56倍;菌株Y72最适生长于沙柳木屑,生长指数(70.63)是YJ-01的3.67倍;菌株Y369最适宜生长于杨桐木屑,培养43d后的连接料量(338.24 g),是YJ-01的2.76倍。(2)对17株优良再生菌株进行连续传代培养与液体出菇检测。结果表明:菌株Y150的菌丝生长性能最稳定;菌株Y440,Y365,Y314,Y243和Y283能够在液体培养基中形成原基。(3)对17株优良再生菌株进行酯酶同工酶与RAPD鉴定。结果表明:菌株Y263,Y440,Y314,Y454,Y381,Y317,Y211,Y150和Y463为变异菌株。其中,菌株Y440和Y314表现最为突出。4.收集香菇品种23个,对18个品种进行单孢分离得到1166株单核菌株。通过7个杂交方案,经单孢杂交得到杂交菌株1367株。(1)对9608-1和808-1的杂交的238株杂交经菌丝生长性能筛选,得到18株优良菌株(B80D111,B80D20,B46D111,B10D175,B6D178,B80D51,B70D21,B6D125,B60D111,B81D111,B60D99,B10D167,B9D176,B76D20,B6D149,B27D21,B81D99和B46D20)。其中,B60D111在杨桐木屑上表现最优,培养43 d后的连接料量为261.54 g,分别是9608-1的1.29倍、808-1的1.60倍。B70D184在PDA平板上表现最优,生长指数(32.73)分别是808-1和9608-1的2.42倍和3.32倍。(2)对18株优良菌株进行连续传代培养与液体出菇性能鉴定。结果显示:菌株B6D178的菌丝生长性能最稳定。菌株B10D167和B10D175能够在液体培养基中形成原基。(3)对18株优良菌株进行酯酶同工酶与RAPD鉴定。结果显示:菌株B60D99,B6D149,B60D111,B27D21,B10D175,B10D167,B80D111,B80D51和B70D20与亲本条带具有明显差异。通过综合比较,确定菌株B10D175为优良杂交菌株(4)对其余1129株杂交菌株进行耐高温筛选。结果表明:高温处理后,有133株杂交菌株能恢复生长,其中F149B92,F`47B90和A59D97等20株杂交菌株能在35℃下生长;26株杂交菌株在高温处理后,经过6 d 25℃恢复,长势依旧较好,其中F149B92长势最优。更多还原

【Abstract】 Lentinula Edodes industry,one of the most important pillar in Chinese edible fungus,plays an significant role in adjusting rural economic and industry stucture and in promoting rural economy development.Although China has produced a large number of the Lentinus Edodes,but commercial process is very low.Lagging of breeding work has a certain restriction on the development of Lentinula Edodes industry.Therefore,breeding work is imminent.This paper,researching work starts on the YJ-01 and thestrains which from laboratory.In order to obtain high yield and quality Lentinula Edodes new strains,using protoplast mutagenesis and single spore hybridization technology.The main findings are as follows:1.The optimum conditions for the protoplast preparation of Lentinus Edodes strains YJ-01 were settled: pH=5.0,0.5 mol L-1 MgSO4 as stabilizing agent,mixed enzyme(1% snail enzyme +1% cellulase)to enzyme hydrolysis of 5th-day mycelium for 2 h at 27.9℃,obtain the highest protoplast production.2.The optimum conditions for protoplast regeneration of Lentinus edodes strains YJ-01 were settled: pH=5.5,0.5 mol L-1 sucorse as stabilizing agent,using mixed enzyme(1% snail enzyme +1% cellulase)to enzyme hydrolysis of 6.5th-day mycelium for 2.9 h at 27.8℃,obtain the highest protoplast regeneration rate.3.547 regenerated strains were obtained by UV mutagenesis of protoplast.(1)17 strains of fine regenerated strains were obtained by screening with different cultivated materials.Strain Y209 is most suitable for the apple sawdust,the growth index(79.88)is 3.56 times higher than that of YJ-01;Strain Y72 is most suitable for willow wood sawdust,the growth index(70.63)is 3.67 times higher than that of YJ-01;Strain Y369 is most suitable for cleyera sawdust,the material connecting quantity(338.24 g)with daily average connecting quantity 7.87 g d-1,2.76 times higher than that of YJ-01.(2)Continuous subculture of 17 excellent regenerated strains and test if fruitbodies could carry out in liquid medium.The results showed that strain Y150 has the most stable mycelial physiological properties,and strains Y440,Y365,Y314,Y243 and Y283 could form the fruitbodies in the liquid medium.(3)Using isozyme and RAPD to identify these 17 of excellent regenerated strains,results showed that strains Y263,Y440,Y314,Y454,Y381,Y317,Y211,Y150 and Y463 were variant strains.By comprehensive comparison,the mutant strains Y440 and Y314 were identified as excellent mutants.4.23 species were collected and 1166 strains were isolated from 18 strains and 1367 strains of hybrid strain were obtained according to hybridization(1)The physiological properties of 238 hybrid strains of 9608 and 808 strain were screened.18 strains of dominant strains were obtained(B80D111,B80D20,B46D111,B10D175,B6D178,B80D51,B70D21,B6D125,B60D111,B81D111,B60D99,B10D167,B9D176,B76D20,B6D149,B27D21,B81D99 and B46D20).Among which,strain B60D111 is the best in cleyera sawdust,the material connection quantity(261.54 g),with daily average connecting quantity 6.08 g d-1,is 1.29 times of the parents 9608-1,1.6 times of 808-1.B70D184 performed optimally on PDA tablets,the growth index(32.73)was 2.42 times and 3.32 times than that of parents 808-1 and 9608-1.(2)Continuous subculture of 18 excellent regenerated strains and test if fruitbodies could carry out in liquid medium.The results showed that strain B6D178 has the most stable mycelial physiological properties,and strains B10D167 and B10D175 could form the fruitbodies in the liquid medium.(3)Using isozyme and RAPD to identify these 18 of excellent regenerated strains,results showed that strains B60D99,B6D149,B60D111,B27D21,B10D175,B10D167,B80D111,B80D51 and B70D20 were variant strains.By comprehensive comparison,B10D175 was identified as excellent hybrid strain.(4)Screening of the remaining 1129 strains for high temperature resistance.The results showed that after high temperature treatment,133 strains of hybrid strains were able to restore growth,of which F149B92、F`47B90、A59D97 and other 20 strains can grow under 35℃.After high temperature treatment and 6 d 25℃ recovery,the growth of 26 hybrid strains is still good,of which F149B92 shows best,the growing rate relative to the processing before the decrese 65.99%,after the F203B92(38.42%),F98B92(64.19%)and F133B92(49.08%).F203B92、F149B92、F133B92、F98B92 and F128B66 were determined to be high temperature resistant strains.更多还原