Somatic Embryogenesis And Cryopreservation For Embryogenic Callus Of Nematode-resistant Pinus Massoniana Lamb.

As an evergreen tree,Pinus massoniana Lamb.is widely distributed in China and one of the most important timber species in China.However,in recent years,the death of Pinus massoniana in a large-scale has decimated forest resources and forest ecosystem in China,due to the invasion of Bursaphelenchus xylophilus.A seed orchard of nematode-resistant Pinus massoniana has been established in Anhui,and the seeds have been harvested,but the number of seeds is limited.Therefore,it is urgent to establish somatic embryogenesis for nematode-resistant Pinus massoniana to obtain highly efficient regeneration plants.Embryonic callus was induced by immature zygotic embryo of nematode-resistant Pinus massoniana in the early stage of our laboratory.In a previous study,the emmbryogenic system of nematode-resistant Pinus massoniana had been established and the regenerated plants had been obtained.However,there still have some problems in the system,such as low induction rate of embryogenic callus,low synchronization of suspension cell,weak rooting ability of somatic embryo seedlings,and low survival rate of transplantation.The embryogenicity of callus gradually degenerated with time.Therefore,the somatic embryogenesis of nematode-resistant Pinus massoniana was further optimized based on the previous research.And the cryopreservation of nematode-resistant Pinus massoniana callus was preliminarily established in order to provide technical support for the long-term maintenance of embryogenic callus of nematode-resistant Pinus massoniana.The main results are as follows:1.Optimization of induction and proliferation for embryogenic callus of resistant P.massonianaThe response between different genotypes of resistant Pinus massoniana and induction medium were discussed,and the effects of Brassinolide and Phytosulfonin on the induction of embryogenic callus of resistant Pinus massoniana were also discussed.At the same time,the differences of stable proliferation of embryogenic callus among different genotypes of resistant Pinus massoniana were compared.It was found that 2,4-D and 6-BA were necessary for induction of embryogenic callus of resistant Pinus massoniana,and the concentration of two hormones should be controlled.Some families,such as‘he14-2’and‘Guang26-3’needed KT and NAA to initiate response.Minimal medium for different families were also not the same.Adding Brassinolide(10^-4-10^-3mg/L)and phytosulfonopeptide（0.5 mg/L）in induction medium could effectively improve the induction rate.In addition,the proliferation rate of embryogenic callus from different genotypes of resistant Pinus massoniana was also significantly different under the same culture conditions.In this experiment,the proliferation rate of genotype‘quan 172-1-1’was higher than that of genotype‘quan 172-1-2’.2.Synchronization of suspension cells of resistant Pinus massonianaThe effects of screening,starvation and thymidine blocking on synchronization of embryonic suspension cells of resistant Pinus massoniana were preliminarily discussed.The results showed that the degree of cell mass differentiation between 0.4 mm and 0.6 mm was more concentrated and could be used for differentiation culture;the aggregation of embryonic head of cells mass<0.4 mm was too low,and the differentiation was lower,which could be used for proliferation;while the cells>0.6 mm were mostly malformed cells.The results of starvation treatment also showed that the lower tconcentration of carbon source supplied was,the higher the synchronization of cells.When 1 mmol/L thymidine nucleoside（Td R）was added,most of the embryonic heads and suspension cells accumulated,while the cell activity decreased with the increase of Td R concentration,indicating that the appropriate concentration of thymidine nucleoside could effectively control the synchronization of resistant Pinus massoniana embryonic suspension cells.3.Cultivation of Regenerated Plants of Resistant Pinus massonianaThere were great differences in the growth and development of somatic embryo seedlings in different germination stages.The results showed that among the four types of somatic embryo tested seedlings,the somatic embryo seedlings with normal root and cotyledon development could be used for strong seedling rooting.Somatic embryo seedlings with normal cotyledon and hypocotyl development but without root system could be also used to developing roots,while the somatic embryo seedlings with normal cotyledon development but malformation of hypocotyl or cotyledon hypocotyl development should be eliminated.Different matrix ratios had great influence on root development.The results showed that different ratios of perlite&vermiculite could affect the root development of somatic plants,and culture in a matrix of perlite:vermiculite=2:1 would promote root development of somatic plants.In addition,adding 0.4 mg/L of paclobutrazol could increase root length and area,while paclobutrazol at too high concentration could also inhibit root growth.Treating by Boletus edulis could increase the plant height and ground diameter of resistant Pinus massoniana,but there was no significant difference compared with the control,and the survival rate of the plants decreased.This may be due to the fact that at the beginning of transplantation,the somatic embryo seedlings need to adapt to the environment with microbes.The treatment of Boletus edulis is an invasive process for somatic embryo seedlings,and the growth of Boletus edulis mycelium is faster,which may affect the growth of plant root system at the early stage of root development.Therefore,it is necessary to further screen the species of mycorrhizal fungi and the appropriate time for application.4.The cryopreservation of nematode-resistant Pinus massoniana embryogenic callusThe cryopreservation of embryogenic callus of resistant Pinus massoniana was screened.The results showed that pretreatment for 36-48 hours effectively maintained the viability of cryopreserved cells.Although the ratio of cryoprotectants between the two tested cell lines was different,the best cryoprotectants were mixed（DMSO,sucrose and PEG4000）.The optimum thawing temperature for resistant Pinus massoniana was 30-35oC.The subculture time and genotypes of embryonic callus had a great influence on the survival rate of callus.The embryogenic callus induced in 2017（quan 172-1-1）was stronger than that induced in 2014（ma7-6）in the adaptability to ultra-low temperature environment and the ability of callus recovery and growth.There was no significant difference in morphology between restored embryogenic callus and subcultured callus,and there was no significant difference in microstructures of callus before and after cryopreservation.The recovered callus still has the ability to differentiate into somatic embryos after mature culture.Therefore,cryopreservation preserves the potential activity of embryogenic callus and has the ability to maintain its embryogenicity for a long period of time.