Search

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

Forests are vital to the world's ecological, social, cultural and economic well-being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial-scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter-lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.

This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.

It is commonly held that confined field trials (CFTs) used to evaluate the potential adverse environmental impacts of a genetically engineered (GE) plant should be conducted in each country where cultivation is intended, even when relevant and potentially sufficient data are already available from studies conducted elsewhere. The acceptance of data generated in CFTs “out of country” can only be realized in practice if the agro-climatic zone where a CFT is conducted is demonstrably representative of the agro-climatic zones in those geographies to which the data will be transported. In an attempt to elaborate this idea, a multi-disciplinary Working Group of scientists collaborated to develop a conceptual framework and associated process that can be used by the regulated and regulatory communities to support transportability of CFT data for environmental risk assessment (ERA). As proposed here, application of the conceptual framework provides a scientifically defensible process for evaluating if existing CFT data from remote sites are relevant and/or sufficient for local ERAs. Additionally, it promotes a strategic approach to identifying CFT site locations so that field data will be transportable from one regulatory jurisdiction to another. Application of the framework and process should be particularly beneficial to public sector product developers and small enterprises that develop innovative GE events but cannot afford to replicate redundant CFTs, and to regulatory authorities seeking to improve the deployment of limited institutional resources.

A series of frameshift, deletion, and inversion mutations were made in the coat protein (CP) gene of the icosahedralcucumber necrosis tombusvirus (CNV) to investigate the role of the CP protruding (P) domain in the production of virus particles and, also, to investigate the basis for the accumulation of CP deletion derivatives previously reported in plants inoculated with PD(−), a P-domainless CNV CP mutant. P-domainless coat protein subunit could be detected in extracts of CP mutant-infected plants; however, virus-like particles could not, suggesting that the P domain is essential for tombusvirus particle assembly and/or stability. In addition, each of the P-domain mutants analyzed invariably produced coat protein deletion derivatives in infected plants whereas shell domain mutants rarely produced deletion derivatives. Finally, P-domain inversion and deletion mutants accumulated deletion derivatives very rapidly in comparison to P-domain frameshift mutants. Protoplast studies show that PD(−) RNA inoculum does not undergo further deletion in infected protoplasts, suggesting that PD(−) CP deletion derivatives preferentially accumulate in plants because they have a greater capacity for cell-to-cell movement.

A cucumber necrosis virus (CNV) mutant which lacks the coding sequence for the coat protein protruding domain, PD(-), was constructed by site-directed mutagenesis of an infectious CNV cDNA clone, pK2/M5 (wild-type, 4701 nt). Transcripts of PD(-) were infectious on Nicotiana clevelandii; however, local lesions produced were significantly smaller than those on the corresponding leaves of plants inoculated with wild-type transcript. In addition, systemic symptoms took 8 to 12 days longer to develop than in wild-type-inoculated plants. The distinctive PD(-) phenotype was lost when N. clevelandii was inoculated with sap from systemically infected leaves of PD(-) transcript-inoculated plants and was replaced by symptoms that were the same as those with wild-type infections. High-molecular-weight RNA from mutant- and wild-type-infected plants was extracted and analyzed by Northern blotting. Full-length PD(-) RNA could be detected only rarely in RNA preparations from transcript-inoculated leaves; a further deleted, stable RNA species of approximately 3800 nucleotides was found in preparations from systemically infected leaves of PD(-) transcript- and sap-inoculated plants. CNV coat protein could not be detected by ELISA or ISEM in PD(-)infected leaf material. The ca. 3.8-kb RNA, when cloned and sequenced, was found to have lost all but 74 of the 1140 nucleotides of the CNV coat protein open reading frame. Transcripts from this coat proteinless CNV cDNA clone produced wild-type symptoms on N. clevelandii . It would appear that CNV is able to replicate and move systemically, in both transcript-inoculated and sap-inoculated N. clevelandii , in the absence of a functional coat protein. Additionally, mechanical transmission of this virus occurs in the absence of the coat protein; however, such transmission is less efficient when compared with wild-type infections.