| Walnut?Juglans regia L.?has the ability of apomixis,using of non-fusion embryo to establish walnut in vitro regeneration system,which has a very important theoretical and practical significance in cultivation and breeding.In this study,the non-fusion reproductive rate of different walnut cultivars?lines?was observed.The methods of appropriate explant sterilization and controlling browning were studied,The culture medium and culture conditions of walnut embryo germination and walnut stem culture were screened.The main results are as follows:1.The results of bagging isolation showed that all the tested walnut cultivars had a certain degree of non-fusion reproductive capacity,the average non-fused reproductive rate was 52.47%,up to 56.67%.The germination seedling rate of each cultivar was54.67% in Xiang Ling,52.33% in Yuan Lin,56.67% in Rui Jia,49.67% in Rui Xiu and49.00% in Y17.The results of variance analysis showed that there was no significant difference in the fusion ability between the cultivars.2.The optimum medium for the germination of walnut in vitro was DKW + 6-BA2.The optimum medium for the germination of walnut in vitro was DKW + 6-BA2.0mg·L-1 + PVP 200mg·L-1 + sucrose 30g·L-1 + agar 7g·L-1.Optimal sterilization conditions and selection of medium.The explant was used as the explant embryo of five tested cultivars.The walnut embryo had the best sterilization effect when the 70% alcohol was sterilized for 30 s and the 0.1% sodium chloride was disinfected for 5min.The pollution rate was only 1.11%.The germination rate of walnut embryo in MS,DKW,1/2MS,WPM basic medium was significant,and the highest germination rate in DKW was 73.50%.When the concentration of sucrose was 30g·L-1,the highest germination rate of walnut embryo was 72.39%,and the germination rate of walnut embryo induced by different medium of sucrose was significant.When the p H value of the culture medium was5.8,the higest germination rate of walnut embryo was 73.04%,and the germination rate of walnut embryo induced by different p H was significant.The determination of the best extraction time and the germination rate of differentThe determination of the best extraction time and the germination rate of different varieties?lines?.The germination rate and survival rate of walnut embryo were high when the drawing materials time on July 15,and the rate of germination to seedling was 78.10%.There was a significant difference in seed germination rate between different extraction time.The highest germination seedling rate of Y17 was 79.29% in 5 varieties of walnut,and the germination seedling rate of walnut embryo between the five cultivars was significant.Thegermination seedling rate of imature walnut embryos was higher than that of mature walnut embryos.The optimum conditions of culture were temperature,humidity and light intensity.When the incubator temperature was 26?,humidity was 40%,and light intensity was3000 lx,the germination rate of walnut embryo was the highest,Respectively,71.35%,71.37%,71.33%,and the difference between the different temperature conditions,the humidity conditions,and the light intensity is significant.When the concentration of PVP was 200mg·L-1,walnut embryo browning rate was 2.26%,and different concentrations of PVP on the walnut embryo browning rate was significant.And the transfer time interval of4 days could reduce the browning rate of walnut embryo.Screening of Plant Growth Regulators.The effect of different plant growth regulators on the germination and development of walnut embryos showed that when the concentration of 6-BA was 2.0mg·L-1,the highest germination seedling rate of walnut embryo was73.58%.When the concentration of KT was 1.0mg·L-1,the highest germination seedling rate of walnut embryo was 70.46%.When the 2,4-D concentration was 2.0mg·L-1,the highest germination seedling rate of walnut embryo was 60.68%.The effects of 6-BA,KT and 2,4-D on the germination seedling of walnut embryos were significant,but the effect of6-BA was the best.Picloram can induce adventitious buds of walnut embryos,and when Picloram concentration is 0.01mg·L-1,the adventitious buds are the most,and the adventitious buds are the best.Different concentrations of Picloram are different from the adventitious buds of walnut embryos.The effect of different concentrations of IBA on the germination and development of walnut embryos was not significant,and the induction rate was very low.3.The best medium combination of germination and differentiation of adventitious buds of walnut stem was DKW + 6-BA 1.5mg·L-1 + IBA 0.01mg·L-1 +PVP 300mg·L-1+sucrose 30g·L-1 + agar 7g·L-1.In the stem culture of walnut,when the combination of plant growth regulators were 6-In the stem culture of walnut,when the combination of plant growth regulators were 6-BA 1.5mg·L-1 and IBA 0.01mg·L-1,KT 1.0mg·L-1and IBA 0.01mg·L-1,the number of the walnut stem buds were many,grew well,and the former was stronger than the latter,the latter induced stem segments grew many ineffective callus.When the concentration of PVP was 300mg·L-1,the browning rate of walnut stem could be reduced to 9.79%.The varieties of walnut stem were different,and the number of buds were different.Among the five cultivars,the highest of the multiplication coefficient was Y17 was 3.63. |
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