The isolation and molecular characterisation of genes expressed during somatic embryogenesis in Pinus radiata

Abstract

Pinus radiata somatic embryogenic tissue offers many advantages over seed material for plantation forestry in New Zealand. Although much progress has been made in the past few years, research is needed to increase both the number of responsive genotypes and the conversion of immature to mature embryos. An improved basic understanding of the physiological and molecular processes that underlie the development of somatic embryos is needed. The aim of this study was to conduct an initial molecular characterisation of early stage somatic embryogenesis with the objective of understanding some of the developmental events that are involved in the formation of somatic embryos in P. radiata. A small P. radiata cDNA library was constructed from mRNA isolated from somatic embryogenic tissue that was forming stage 1 embryos. This library was differentially screened against non-embryogenic tissue (roots, shoots and needles) so that genes that were preferentially expressed during conifer embryogenesis were isolated. Altogether, 24 of the 28 cDNA clones that were isolated showed high mRNA transcript levels in embryogenic tissue with little or no expression in non-embryogenic or callus tissue. The 24 cDNA clones, representing seven separate gene families, encoded putative extracellular proteins (i.e. germin, β-expansin,2l kDa protein precursor and a cellulase), cytochrome P450s, α-glucosidase and PRE87, a gene of unknown function. Representative full-length clones from five of these families (i.e. germin, β-expansin, 2l kDa protein precursor, cytochrome P450 and PRE87) were fully sequenced and their sequences compared with genes in the database. Southern blot analyses show that all of the gene families were multicopy within the genome, indicating that they were members of complex gene families. Northern analyses showed that the five clones were 'embryo-abundant' in that their transcripts were detected at high levels throughout embryo development and maturation and were not detected in Picea abies somatic embryogenic tissue (apart from cytochrome P450). They were also detected in the 'embryogenic' lines that had lost their ability to develop into plants. To provide information about the role of germin during somatic embryogenesis, transgenic lines were made containing germin in sense and antisense orientation, with the aim of reducing physiological levels of germin. Using biolistic transformation, l6% of the bombarded plates gave rise to stable transformants for P. abies, although for p. radiata, the transformation rate was only 0.95% and only four transgenic lines were obtained. However, in both P. radiata and P. abies, a co-transformation strategy was successfully used to co-bombard four genes on two plasmids (nptII and uidA in pCWl22, bar and PRGerl in either pSeGerl or pAsGerl). Transcripts from all four genes were expressed in the majority of the transformed lines containing germin in sense orientation; however, in the antisense lines germin was not expressed. The mRNA transcripts for germin were detected in both P. radiata and P. abies transgenic lines when germin was over-expressed, but were not detected in the antisense lines. In all the lines the morphology of the somatic embryogenic tissue was similar and in p. abies, the number of cotyledonary-stage embryos produced were similar between lines. In p. radiata, there was a reduction in the number of cotyledonary-stage embryos produced in the one 'sense' line that was examined; however, these results are not sufficient to conclude anything because the sample size was too small. Bar mRNA transcripts were also detected in the transgenic lines that were transformed with pSeGerl and pAsGerl, two plasmids harbouring the bar gene under the control of the Ubi-1 promoter. Subsequent work to that reported in this thesis showed that p. radiata and P. abies plantlets regenerated from these transgenic lines were tolerant to operational levels of 'Basta', a herbicide containing phosphinothricin.