Introduction

The formation of embryos within the seeds of pine trees differs from seed formation in traditional crop and vegetable plants. While in both cases an embryo forms inside a seed, develops and finally germinates to form a plant, the morphology, biochemistry and genetics of the processes in gymnosperms (eg. pine) and angiosperms (e.g. carrot) are distinctly different. Embryogenesis is the result of a regulated program of gene activity and the elucidation of the program for loblolly pine is the goal of this project. The multiplication of pine embryos in a laboratory setting and the production of plantlets, has great implications for reforestation.

After corn, forest products are the second most valuable US agricultural product at $22.5 billion. In the southern United States, one of nine manufacturing jobs can be linked to southern pine forests. Loblolly pine (Pinus taeda L.) is the primary commercial species in southern conifer forests covering 13.4 million ha. Over 1.5 billion loblolly pine seedlings are planted annually. Somatic embryogenesis (SE), a tissue culture technique for embryo multiplication, is an excellent experimental tool for investigating embryogenesis and has great potential for supplying fiber to the US Forest Products industry. SE, however, is very genotype-dependent and remains inefficient. Knowledge of the program of gene expression in natural and laboratory-cultivated embryos will provide insight into varying metabolism of these embryos and a basis for protocol improvement.

We will create a cDNA library from loblolly pine zygotic (natural) and somatic (lab cultivated) embryos, representing a combined set of transcripts expressed during all stages of embryogenesis thus gaining a 'snapshot' of genes participating in the formation of the pine embryo. Randomly selected cDNA clones will be 5' end sequenced for gene discovery to generate 35,000 Expressed Sequence Tags (ESTs). These ESTs will be clustered and assembled to construct a non-redundant pine EST database (TIGR Gene Index) for identification of unique transcripts. A non-redundant cDNA clone set will be identified and used to develop a 10,000 pine cDNA clone microarray, which will be utilized for characterization of gene expression profiles during stages of zygotic and somatic loblolly pine embryogenesis.

Objectives of the NSF Genomics of Loblolly Pine Embryogenesis Project

• Sequence ESTs from loblolly pine embryonic tissues
• Develop a Pine Gene Index
• Develop robust methods for pine cDNA arrays using micro-levels of RNA
• Develop a ~10,000 clone loblolly pine cDNA array enriched in sequences expressed in embryogenesis
• Profile gene expression patterns at various stages of somatic and zygotic embryo development in pine and other conifers
• Outreach to undergraduates and high school teachers

Current Status of the NSF Genomics of Loblolly Pine Embryogenesis Project

We have been performing quality assessment tests on various pine cDNA libraries constructed from a series of embryonic RNA populations. We have identified a library constructed from pooled mRNA for further sequencing. We have released to Genbank the first 5' sequences from this library. Click here for the current statistics on EST sequencing. The sequences can also be retrieved from the TIGR Pine FTP site.

We have constructed a Pine Gene Index. The index provides a representation of pine EST and mRNA sequences obtained from Genbank. These have been clustered and assembled into tentative consensus sequences and singleton ESTs. The sequences and their annotation can be accessed here.

We have developed robust methods for the fabrication of loblolly pine cDNA arrays and hybridization with total RNA isolated from loblolly pine. We have optimized amplification conditions for loblolly pine mRNA to enable robust hybridization with as little as 2 ug of total RNA. Click here for our protocols on pine microarrays.

Participants of the NSF Genomics of Loblolly Pine Embryogenesis Project

The NSF Genomics of Loblolly Pine Embryogenesis is a joint effort by The Institute of Paper Science and Technology (IPST) and The Institute for Genomic Research (TIGR).

Investigators:

• John Cairney, Principal Investigator, IPST, john.cairney@ipst.gatech.edu
• C. Robin Buell, co-Principal Investigator, TIGR, rbuell@tigr.org

The NSF Genomics of Loblolly Pine Embryogenesis Project at IPST and TIGR is funded by the National Science Foundation.