Molecular Basis of Ionic Homeostasis and Salt Tolerance in Plants
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Juan March Foundation Workshop (October 22-24, 2001, Madrid, Spain)
“Molecular Basis of Ionic Homeostasis and Salt Tolerance in Plants”.
Blumwald E & Rodriguez-Navarro A, organizers.
(see: Serrano R, Rodriguez PL EMBO Reports 3: 116-119, 2002)
It describes what the audience considered essential and sufficient experimental process for the analysis and description of the effect of transgenes on engineering or altering what are generally described as complex genetic and physiological traits, such as tolerance to salinity stress, drought, low temperature, or freezing.
Evaluating Transgene Impact
Genetic engineering of salt tolerance is becoming a mature field, so it is appropriate to define, for the purposes of publication, a minimum set of criteria for establishing unambiguously that transgenic plants do indeed show tolerance that is attributable to the transgene (somewhat analogous to Koch’s postulates for infectious disease). The following guidelines (a series of “do’s” and “do nots”) were agreed:
DO:
- Establish that the stress-tolerant phenotype of interest is shown by a fair-sized population of primary transformants (e.g., ten independent transgenics) that have been shown to express the transgene. This avoids being misled by insertional mutagenesis, positional effects, and somaclonal variation. A range of expression levels may be more informative than selecting the highest expressers, especially in studies involving metabolic traits.
- Likewise, show that a comparably-sized population of control plants harboring the empty vector does not show the stress-tolerant phenotype. Another desirable type of control (so far not used) is using an inactivated transgene (e.g., lacking its catalytically essential residues). This type of control is unsuitable for oligomeric proteins due to dominant negative effects.
- For progeny of primary transformants, work with single-insert lines. Either backcross the primary transformants to the wild type and compare the hemizygotes (S of progeny) to the azygotes, or self the primary transformants and compare the homozygotes and azygotes (each S of progeny). This controls for somaclonal effects.
- Carry out stress tests using the established, statistically sound procedures that are mandatory in the agronomy literature. Blind designs (in which the experimenter is unaware of the plants’ identities) are advisable.
- For Arabidopsis, deposit viable seeds of transgenic lines in a stock center.
- Discourage institutional publicity officers or the press from extrapolating from small advances in basic research to major benefits to agriculture.
- Use only wild type as control.
- Use the stress-tolerant phenotype of interest as the first criterion for screening transgenic lines to be studied (and discard the others).
