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Optimization Of Somatic Embryogenesis In Masson Pine(Pinus Massoniana.Lamb)

Posted on:2021-01-17Degree:MasterType:Thesis
Country:ChinaCandidate:J WangFull Text:PDF
GTID:2393330605958860Subject:Forest science
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Masson pine(Pinus massoniana Lamb.)is a native subtropical coniferous tree species that is widely distributed in southern China.It is resistant to drought and barren,adaptable and fast-growing.It plays an important role on economic and ecological development in forestry.The lack of seedlings,low seed yield in seed orchard and the age effect of asexual propagation of cutting seriously hindered the application of elite materials in Masson pine.With the advantages of high efficiency,stability and short cycle,somatic embryogenesis in forest tree species can provide large-scale of selected seedlings for afforestation.At present,somatic embryogenesis in Masson pine has been initially established,but the application of somatic embryogenesis of Masson pine in practice is restricted by the low induction rate of embryogenic mass,the difficulty of maintaining embryogenic mass in proliferation and the low frequency of somatic embryos in maturation.In view of the above mentioned problems,this study addressed on the optimization of the induction and proliferation of embryogenic mass,and explored the conditions for the somatic embryos development to improve the efficiency of somatic embryogenesis(SE)in Masson pine by collecting the immature zygotic embryos of Masson pine in 2018 and 2019.These findings provide an informative technical support to optimizing the somatic embryogenesis in Masson pine.The results are as following:1.Initiation of somatic embryogenesisTo addressing on the low induction rate of embryogenic mass in Masson pine,we explored the effects of different types of explant materials(genotype,zygote development stage)and the medium components(carbon source,nitrogen source)in the initiation of somatic embryogenic in Masson pine.The results showed that selecting the immature zygotes with 2-4 developmental stages was the most sensitive period in the initiation of SE,named "window stage" for initiation of EM.The frequencies in the initiation of SE in different families at the same collection time were significantly different,which may be affected by genotypse or the different developmental stage of immature embryos.We obtained some sensitive family genotypes in 2018 and 2019,with the average induction rate higher than 15%in two families and 20%in three families,respectively.An improved initiation frequency in SE was achieved through adjusting the concentration and level of carbon source(C)and nitrogen source(N)in the initiation medium.For example,among the four families(k1,k2,k3,k4)studied,k1 family obtained the highest induction rate of 40%in the treatment of 30g/L sucrose,while k3 family obtained the highest induction rate of 22%in the treatment of 30g/L glucose,which showed the interaction of genotype,carbon source and its concentration.Overall,in spite of families,the highest initiation frequency was obtained in the treatment of 30g/L glucose.Increasing the concentration of Nitrate nitrogen(NO3-)in the medium can improve the induction rate of the embryogenic mass with the induction rate of 15.48%in 3/2LP,which is higher than that of in the original LP with 9.19%.At the same time,decreasing the concentration of Ammonium nitrogen(NH4+)is beneficial to the induction of the embryogenic mass.and the range of the ammonium/nitrate molar ratio(NH4+/NO3-)between 0.2?0.4 with suitable hormones in the LP basal media was recommended with higher average induction rate of 14.23%than the other treatments.2.Proliferation and maintenance of embryogenic massIn order to study the stability of embryogenic mass(EM)during proliferation,we discussed the influence of temperature of the culture environment for the EM proliferation.The results showed that adjusting the temperature to a lower temperature will slow down the aging process of EM during proliferation and a stable status of EM can be maintained for a long period in proliferation,which provided an effective way to maintain embryogenic mass in the proliferation.When the culture environment temperature was set to low(5?,10?,15?),medium(23?),and high(28?),we found that the culture temperature of 15? was favorable for maintaining the embryogenic mass stability,while the culture temperature of 28? accelerated the aging process of EM in proliferation.Different proliferation and culture methods such as liquid and solid medium had a significant effect on the proliferation of EM.Compared with the solid medium culture method,both the suspension and liquid-solid medium culture methods obtained a higher proliferation.The proliferation in suspension culture was about twice than that of in solid culture medium;meanwhile,in the liquid-solid culture,the proliferation of EM gradually increased before being transferred into the solid medium with the extension of suspended culture medium with different period of 3d,4d,5d,and 6d,respectively.When the EM were being suspended for 6 days before it was transferred to the solid medium culture,the proliferation times was around 13.53,which was significantly higher than that of in solid culture medium with 5.43 only.3.Maturation of somatic embryosDuring the maturation process,when the EM was white,opaque,sticky,and creamy,and there were obvious protrusions on the surface,it would mean that a large amount of embryonic masses had been developed into early somatic embryos.Once the EM has reached such maturation patterns it should be transferred into the the maturation medium for somatic embryos differentiation,otherwise,the EM would become senescence and browning quickly and lost the potential of maturation in continued proliferation medium.Our results showed that the most suitable EM for maturation were those cultures with consecutive proliferation with three sub-culture times.Thus,we must pay more attention to the development stages of EM during proliferation,and transfer the matured EM into the maturation medium in time.4.Cytological observation of the initiation and proliferation cultures of somatic in Masson pineIt was found that the normal embryogenic mass were white,fluffy,and indicating obvious protrusions,and there was a clear embryogenic suspensor mass(ESM)structure under microscope.The results showed that with the increasing number of subcultures,the embryogenic potential quality of the EM showed a trend of increased firstly and then decreased.The most embryogenic potential of the embryogenic mass were those cultures subcultured for three times,in which there were a large number of early somatic embryos formed with obvious polar structure observed under microscope.However,the embryonic mass cultures would gradually be aged with continued subcultured more than four times,and the appearance of the cultures became a lightly yellowish granular mass soon.Those yellowish cultures would lose its maturation potential with the disappearance of EMS,and the aged EM was not suitable embryogenic mass for somatic embryos maturation.
Keywords/Search Tags:Pinus massoniana Lamb., Somatic embryogenesis, Embryogenic mass, Initiation, Proliferation, Maturation
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