Books & Proceedings

Mutation Techniques for Gene Discovery and Crop Improvement.
Shu, Q.Y. and Lagoda, P.J.L. (2007) Molecular Plant Breeding, Vol. 5, pp. 193-195

If you wish more information about this publication, then please E-mail to Q.Y. Shu

Summary

One of the most important breakthroughs in the history of genetics was the discovery that mutations can be artificially induced in organisms (van Harten, 1998). Artificially induced mutations, by physical and chemical mutagens, have greatly advanced the understanding of genetics of higher organisms. Starting in the late 1960's, the International Atomic Energy Agency (IAEA) and the Food and Agriculture Organization (FAO) of the United Nations sponsored extensive research on mutation induction and their application to breeding of food and industrial crops that resulted in the introduction of new varieties of rice, wheat, barley, apples, citrus, sugar cane, banana, and others (more than 2500 officially released new varieties are to be found in the FAO/IAEA Mutant Varieties Database) (http://mvgs.iaea.org/). However, the usefulness of mutation techniques has been underappreciated in research communities, particularly during the last decade, when more and more researchers and breeders were rushing into molecular marker techniques and transgenic plants. In this paper, after a brief review of the past accomplishments of mutation induction and its application, we discuss the uniqueness of induced mutations in gene discovery and how to integrate induced mutants into functional genomics programs; we also explore the possibilities of increasing the efficiency and efficacy of mutation techniques in crop breeding and research, by integrating up-to-date knowledge and state-of-the-art technologies.

Mutation techniques have been used almost exclusively for plant breeding from the 1960's to 1990's. The outcome was indeed remarkable: about 2000 new varieties were developed in almost all continents except Africa, where progress was limited (Ahloowalia et al., 2004). This fact is even more spectacular, taking into consideration that: (1) Only a very limited number of breeders, compared to the large number of cross breeders, had been using mutation techniques in their programs, due to both under-appreciation of the technology, limited resources or capacities; (2) Many mutant varieties became the national leading variety, e.g. the rice variety Yuanfengzao in the 1970's and early 1980's, and Zhefu 802 in the late 1980's and early 1990's in China for the early season rice production (Ahloowalia et al., 2004; Liu et al., 2004); (3) Several mutated genes have been integrated into most modern varieties, e.g. the two independent sd1 mutant alleles first induced in Reimei in Japan and in Calrose 76 in the US are now fully integrated into new rice varieties in these two countries and beyond (Yamaguchi, 2001; Rutger, 2006) ; most spring barley varieties contain various mutant alleles of the disease resistant gene mlo in Europe and Australia (http://www.crpmb.org/mlo/#Pifanelli); (4) Mutation techniques have been used to improve almost all important traits, from tolerance to abiotic stress (i.e. salinity, cold, acidity) to disease resistances, from food and nutritional quality to market preference, and from plant structure to yield potential; (5) Mutation techniques sometimes are the only way for improving overall performance while keeping the particular trait unchanged, e.g., traditional varieties like Basmati rice varieties in India and Pakistan (Patnaik et al., 2006). These results unequivocally prove that mutation techniques are useful and unique as a tool in plant breeding.

Besides the mutations that have been successfully integrated into new varieties, a large amount of mutants were induced as by-products in many mutation projects. Most of these mutants are not useful for breeding new varieties and thus were often discarded. However, there have also been some collections preserved by various groups; for example, the Carlsberg Collection of flavonoid mutants comprises 724 induced barley mutants (http://grain.jouy.inra.fr/ggpages/bgn/18/c18-07.html), and the collection of Antirrhinum majus stocks has 300-400 mutants at the John Innes Centre (http://www.jic.bbsrc.ac.uk/staff/enrico-coen/Rosemary/Mutants.html). Most breeders using mutation techniques may have their own mutant collections based on their personal curiosity for the material. Systematic development, characterization and collection of chemically or physically induced mutants came rather recently, when researchers started realizing the potential of such mutants in functional genomic studies (Li et al., 2001). For example, the IR64 mutant collection in the International Rice Research Institute comprises more than 38,000 M4 lines (http://www.iris.irri.org) (Wu et al., 2004).

Compared with the T-DNA mutant lines, induced mutants are commonly more limited in number, because they are considered less straightforward in gene identification. Nevertheless, induced mutations are unique and to some extent irreplaceable in gene discovery. For example, most T-DNA insertions cause knock-outs of genes, and no mutant can be recovered if such genes are essential for plant survival, while induced mutations are more knock-down mutations and thus could be useful for gene discovery and gene function analyses of these types of genes. Besides, more T-DNA insertions are in the inter-genic region or in introns than in the exons, and thus are less useful for gene discovery - e.g. in rice, there are far more T-DNA insertion lines than rice genes, however, the number of lines with T-DNA insertions in exons are much fewer than the number of potential genes (http://signal.salk.edu/cgi-bin/RiceGE). In many other crops, it is not always possible to generate large numbers of such insertion mutants, because of the resilience to transformation or inefficient regeneration protocols, but it is possible almost for all crops to generate induced mutants through physical or chemical mutagenesis. In addition, recent developments in biotechnology - especially in understanding the structure and function of plant genomes - confirms mutation induction as one of the most efficient and cost-effective tools for functional genomics projects dealing with both forward and reverse genetics strategies. Thus, induced mutants are bridging the so-called phenotype gap (PhG) - the gulf between the available mutant resource and the full range of phenotypes.

The rapid advance in molecular biology and DNA technologies has provided never before dreamed of possibilities for increasing both the efficiency and efficacy of mutation techniques in crop breeding and research. Firstly, for traits of which the controlling gene(s) is already known, the high throughput selection of mutations has become possible at the DNA level, e.g. using the TILLING (Targeting Induced Local Lesions IN Genomes) technique (Henikoff et al., 2004), particularly, when simplified methods are applicable, i.e. detection of SNPs on agarose gels (Raghavan et al. 2007) and in ployploid crops (Slade et al., 2005). Secondly, for some important mutations, linked DNA tags are greatly facilitating its use in cross breeding programs, e.g., using techniques such as marker-assisted selection for pyramiding various promising alleles into a breeding line. Thirdly, DNA technologies are also important for the set-up of an efficient mutation induction protocol, for example, identifying proper doses by assaying DNA damage for different crops and systems, which is indeed the very basis of any mutation induction project. Last but not least, the accumulating knowledge of DNA damage, repair and mutagenesis will lead to knowledge-based design of mutation researches, which, together with the manipulation of these critical genes using transgenic tools, can greatly increase the efficiency of mutation techniques.