Font Size: a A A

Regeneration And Microarray Examination Of Functional Genomic Expression Of Transgenic Norway Spruce (Picea Abies) For Low-content Lignin

Posted on:2003-10-05Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Z ChenFull Text:PDF
GTID:1100360092993015Subject:Forest cultivation
Abstract/Summary:PDF Full Text Request
Microarray Analysis and Plantlets Regeneration of the Transgenic Norway Spruce (Picea abies) for Low-Content LigninNorway spruce is one of the two most important forest tree species in Europe and the preferred species for pulp manufacture. The three main constituents of plant cell walls are lignin, cellulose and hemicellulose. Lignin composes about 15~36% by dry weight in Norway spruce wood and has to be removed in chemical pulping for the manufacture of high quality paper. A slight decrease of lignin in wood's is estimated to significantly reduce the consumption of environmentally harmful chemicals during chemical pulping. An attractive objective in tree breeding is therefore to reduce the content of lignin. The decrease of lignin has to be achieved without interfering the stability of the tree, the efficiency of water and nutrient transport, and its resistance to pathogen. Recent developments in molecular biotechnology are making this possible.In the work described here, three transgenic sublines of embryogenic Norway spruce calli: A78-3, A78-4 and A78-5 were studied. They contained an antisense constructed for the spruce gene encoding CCR (cinnamoyl alcohol dehydrogenase), an enzyme of monolignol synthesis. The antisense RNA method is a technique for reducing the expression of a resident target gene. The transgenic sublines were produced by particle bombardment at the Dept of Forest Genetics, Swedish University of Agricultural Sciences. Plantlets have been regenerated from the embryogenic calli. The expressions of the target gene were confirmed by RT-PCR. Furthermore the expressions of all the genes in a representative sample were examed by the recently developed method of hybridization to cDNA arrays. This was intended to strengthen the theoretical background for the screening ofNorway spruce genotypes with low lignin content.The calli of the transformed sublines A78-3, A78-4, A78-5 and the untransformed control A95:88:22 were successfully induced to form mature embryos from which plantlets were established. The results shows that germination with 1/4 strength SH medium after maturation for 5 weeks with DKM or LP-M maturing medium(containing ABA) is a suitable combination for these sublines since the viability reached 80%.Total RNA was isolated from the calli and plantlets of each transgenic subline and respective controls. RT-PCR results showed that the presence of the CCR antisense band in sublines A78-3 and A78-4 but not in A78-5 or the untransformed control A95:88:22. These confirmed the successful transformation of the A78-3 and A78-4 on selection medium containing Basta.Microarray on membranes were fabricated from a set of 384 Pinus taeda genes (cDNAs) related to lignin synthesis, adaptation or primary metabolism for examination of gene expression in the sublines. The results showed: (1) the correlation coefficients between the transgenic sublinesA78-3 and A78-4 and the untransformed control A95:88:22 were 0.8028-0.9028, while those of A78-5 are 0.8897-0.9302. These indicated that the expressed difference of A78-3 and A78-4 were much higher than that of A78-5; (2) In comparison to the control line, 0.26%-2.60% of the total genes in the array expressed significant differences in sublines A78-3 and A78-4, but not in A78-5. These also confirmed the RT-PCR result once again and revealed some related genes have been affected by the expression of the antisense CCR. (3) Differences in gene expression between plantlets and calli were considerable. In calli subcultured for one week, 18.75% of the genes expressed differently from plantlets; and in calli subcultured for two weeks, the proportion was 15.6%. In contrast, there was very little difference in gene expression between calli subcultured for one week and two weeks with only 0.78% of the genes showing significant different expression. (4) The 14 genes with different expression level between the control and the transformed sublines A78-3 and A78-4 were classified by their function. It turned out that the most represented group was Group...
Keywords/Search Tags:Norway spruce (Picea abies), Lignin, transformation, Embryogenesis, Plantlet Regeneration, CCR(Coumaroyl Co-A reductase), Antisense CCR, CAD (Cinnamyl-Alcohol Dchydrogenase), Microarray
PDF Full Text Request
Related items