| Masson pine(Pinus massoniana Lamb.) is a native subtropical coniferous tree that is widely planted in southern China. The plant is also tolerance to drought and arid conditions,growing fast and adaptability. It is an important plantation species for timber plantation, resin production, and pollen pini in China. However, the insufficient supply of seed, long breeding cycle and the difficulty of vegetative propagation which has greatly restricted the efficiency plantation of masson pine. The objective of this thesis was to assess the genetic diversity, structure, association and distinctiveness of117elite clones in Chengbu masson pine seed orchard at Hunan province using ISSR markers and to set up an efficient regeneration system via somatic embryogenesis in masson pine. Several key factors on somatic embryogenesis were also discussed. It is our hope that this effort can provide a set of baseline information for the establishment of short cycle breeding programme of masson pine. The main research contents and results were as follows:1) Large ISSR variation was detected in these assayed117masson pine clones. There was different patterns in genetic diversity in different populations according to the origin and the selection of climate in Hunan on populations. The10ISSR primers generated a total of210polymorphic bands. The frequencies of the210polymorphic alleles ranged from0.0044to0.9744with an average of0.4174. Values of Shannon’s entropy for each marker ranged from6.160to4.657with an average of5.283. Population-specific ISSR variation (P values) ranged from77.98%to90.37%and with a total of96.33%. The expected heterozygosity (He) of6populations was ranged from0.3598to0.4252and averaged0.3887. The population-specific Fst values ranged from0.3176to0.3414and averaged0.3276. The average dissimilarity (AD) of a population ranged from0.3306to0.3414with the mean AD of0.3352and Guangxi population were identified as genetically distinct populations with narrow genetics among this assayed clones.2) Clustering117clones with Structure2.3.4resulted in three major clusters. There was a highter ISSR variation of11.32%resided among these clusters, compared to2.60%among6populations,2.77%among4geographic region and2.72%among3provinces with AMOVA analysis, relatively a lower variation of88.68%was presented within the3clusters simulated with Structure2.3.4. This simulated clusters by Structure2.3.4increased the differentiation on different clusters and it would be beneficial to improve the efficiency of parents selection on the heterosis in masson pine orchard. The result of which there was a highter ISSR variation of2.77%harbored among4groups originated from geographic regions compared to2.60%6harbored among populations meaned that there are more effect of geographic regions on the formation of ecotype in masson pine than populations.3) There was different patterns on genetic differentiations between populations in significant difference evaluation and which provided a set of baseline information for the selection of parents in masson pine hybridization and germplasm utilization. The genetic differentiations at the population level showed that there was an extramarked difference between Guangxi and those from Guizhou or Hunan population (p<0.001),also there was an extramarked difference between Guizhou and those from Guangxi,Chengbu or Linwu population. There was a significant difference between Guizhou and those from Suining or Zixing population(p<0.005). There are no difference between Chengbu and Suining population(p>0.05), and the same as Zixing and Linwu population(p>0.05).There was an extramarked difference among groups from4geographic regions(p<0.001). there was the largest variation harbored within the clones from Guizhou geographic regions and the lowest variation presented from Guangxi. The largest pairwise regions FST value was obtained between clones from Guizhou and Guangxi. The lowest pairwise regions FST value was obtained between clones from Xuefeng and Nanling.4) Clustering117clones revealed that these assayed clones were largely grouped into optimal three clusters according to their originated populations with a few exceptions. Each of the three optimal clusters has a considerable proportion of mixed memberships sharing between clusters. To set up a core subset of masson pine germplasm with more genetic diversity, some clones with high similiarity should be filtered out of the breeding population according to the assessment of the genetic associations of117clones using PAUP4.0, Arlequin3.5, STRUCTURE2.3.4and NTSYS-PC PCA softwares. The most genetically distinct clones and populations were identified according to the AD and mean AD. The variation patterns revealed here should share some general baseline information useful for the evaluation and establishment of specific core subsets by determining the weighting or representation for each polulations5) An efficient regeneration system in masson pine for megagametophytes containing immature zygotic embryos via somatic embryogenesis have been developed. The cone collection date in the middle of July was appropriated with the initiation rates arranged from9.66%to22.59%. The induction medium containing2,4-D,6-BA, NAA and KT, and a scope of different concentration hormone level in experiment was benefit to callus induction and proliferation. The somatic embryos with complete structure were achieved with5.0mg/L ABA in the LP maturation medium; After transplanting the embryos to germination medium with no hormone and followed environmental regulation, the embryos could continue to elongation and then form radicle, hypocotyl and cotyledon. A lot of normal somatic embryo seedlings germinated.6) There was some difficult in the induction of masson pine embryonic callus. Hormone6-BA and KT were the key factors which influenced embryonic callus induction rate and callus quality respectively. In appropriate concentration design level for induction rate, the primary and followed influence factors of the four kinds of hormone was6-BA followed by KT, NAA,and2,4-D in order; The optimal desi-gn proposed on induction medium was:LP+2,4-D2.0mg/L+6-BA1.5mg/L+NAA0.5mg/L+KT1.0mg/L; Also the primary and followed influence factors of four hormones which influenced the quality of embryonic callus induction was KT, followed by NAA,2,4-D and6-BA in order; The optimal design proposed on enhancing the quality of callus induction medium was: LP+2,4-D0.5mg/L+6-BA1.5mg/L+NAA0.3mg/L+KT0.5mg/L; Low concentration of2,4-D is beneficial to qualify embryonic callus.7) There was some difficulty in masson pine embryogenic tissue proliferation with limited subculture times, which had an impact on the efficiency of specific SE stages with non synchronous patterns in the proembryogenic mass development. The ageing phenomenon in embryogenic tissue was accompanied by the increasing times of subculture, and at the same time, the cytology microscopic observation on it showed that the compact clump of densely cytoplasmic cells expanded and the primary vacuolated elongated cell increased, with volume size and numbers. The proembryogenic masses(PEMs) which pass through a series of three characteristic stages of PEM Ⅰ、PEM Ⅱ、PEMⅢ and EM as a whole, but the three stages of PEM Ⅰ、PEM Ⅱ、PEMⅢ and EM would be exited in the same time in embryogenic tissue... |
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