Mutation Detection

The detection of novel induced mutations has long been a bottleneck in plant mutation breeding. However, with recent breakthroughs in high-throughput mutation detection technologies the PBG sub-program is assisting Member States in acquiring and establishing appropriate capacities to increase the efficiency of screening for desired traits and to accelerate the development of mutant lines into commercial varieties for farmers. In doing so, the PBGL develops and disseminates simple and user-friendly protocols for phenotyping (plant based) and genotyping (DNA based) of target traits in a wide range of crops that can be easily validated and adopted by Member States.

Mutation Detection Current priorities are to develop and disseminate screening protocols for important biotic and abiotic stress-related traits, such as pest and disease resistance and tolerance to drought, heat, and salinity. The PBGL recently generated protocols for low-cost DNA extraction and analysis, and for the screening of salt tolerance in rice and other cereals, such as wheat and barley, and is currently developing screens for disease resistance.

The process of selecting mutant plants with improved traits involves two major steps: mutant screening and mutant confirmation (also referred to as mutant verification). Mutant screening involves the evaluation of a large number of mutant plants to identify the rare mutant individuals that meet the selection criterion (e.g. improved resistance to disease, better quality). For example, M2 mutant plants flowering three days before their non-mutated (wild type) parent(s) are screened as potential early flowering mutants and plants without disease symptoms might be screened as potential disease resistant mutants. Early flowering is an important trait to farmers as plants that flower earlier allow food to be harvested before the onset of some insect pest challenges or also adverse climatic conditions such as frosts or flooding that can ruin a crop. Since flowering is dependent on both genotypic and environmental factors they can only be regarded as “putative mutants”, which means they are not necessary “true mutants”. This is the case for many traits including disease resistance as here non-infection may simply be the result of the absence of the pathogen.

Mutation Detection Screening by phenotyping has been the common method in plant breeding to select improved crops since plants were first domesticated. Phenotypic screening can be easily exemplified by the above. The crop breeder evaluates his or her field and finds plants producing flowers much earlier than normal. However phenotypic screening has some disadvantages. The process can be very time consuming and plant phenotypes are often influenced by environmental factors. This is the case for many traits including disease resistance where non-infection may simply be the result of the absence of the pathogen.

Genotypic screening can be employed to address the bottlenecks and limitations of phenotypic evaluation. The process involves evaluating the DNA of plants to identify variation that is linked to and inherited with the trait of interest. Once DNA variation has been identified, molecular assays can be developed so that a population can be rapidly screened. The development of so-called molecular markers is also an important tool for breeders to move the new mutant trait into different cultivars for improvement (genetic introgresson). The PBGL has developed protocols and guidelines and routinely trains counterparts on diverse genotypic screening techniques and their appropriate application in mutant populations.

After successful selection mutant conformation is the process of re-evaluating the putative mutants under replicated and stringent conditions in the field, using larger sample sizes (usually the progenies of selected putative individuals, e.g. M3 or M4 lines of putative selected mutants). Many putative mutants of quantitative traits, e.g. growth duration, yield, quality, disease resistance, might be proven to be false mutants.

References:

Plant Mutation Breeding and Biotechnology. Edited by Q. Y. Shu , B. P. Forster and H. Nakagawa . Wallingford, UK: CABI (2012), pp. 608, ISBN 978-178064-085-3