We report the use of TILLING (targeting induced local lesions in genomes), a reverse genetic, nontransgenic method, to improve a quality trait in a polyploid crop plant. Waxy starches, composed mostly of amylopectin, have unique physiochemical properties. Wheat with only one or two functional waxy genes (granule-bound starch synthase I, or GBSSI) produces starch with intermediate levels of amylopectin. We have identified 246 alleles of the waxy genes by TILLING each homoeolog in 1,920 allohexaploid and allotetraploid wheat individuals. These alleles encode waxy enzymes ranging in activity from near wild type to null, and they represent more genetic diversity than had been described in the preceding 25 years. A line of bread wheat containing homozygous mutations in two waxy homoeologs created through TILLING and a preexisting deletion of the third waxy homoeolog displays a near-null waxy phenotype. This approach to creating and identifying genetic variation shows potential as a tool for crop improvement.

A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING

Soil salinity is a major environmental constraint to crop productivity worldwide. The “biological” approach to this problem focuses on the management, exploitation, or development of plants able to thrive on salt‐affected soils. This chapter reviews strategies by which plants can be enabled to grow on saline soils. The first strategy is to prime seeds before planting by treating them with inorganic or organic chemicals and/or with high or low temperatures. The second strategy involves exogenous application of organic chemicals, such as glycine betaine, proline, or plant growth regulators, or inorganic chemicals to plants under salinity stress. Considerable improvements in growth and yield have been reported in a number of crops using these approaches. The third strategy is to employ selection and breeding. Major efforts have been made to develop salt‐tolerant lines or cultivars of crops using conventional plant breeding. However, the complexity of the tolerance mechanisms, lack of selection criteria, and variation in responses of plants at different developmental stages have resulted in only limited success. The emphases for developing salt‐tolerant lines/cultivars are now on marker‐assisted breeding and genetic transformation. The development of salt‐tolerant transgenic plants is still at an early stage but may become increasingly more effective as better knowledge of the complex mechanisms involved in plant salt tolerance is acquired. Furthermore, the rapid expansion in knowledge on genomics and proteomics will undoubtedly accelerate the transgenic and molecular breeding approaches However, to date, there are few conclusive reports indicating successful performance of transgenic cultivars under natural stressful environments.

Some Prospective Strategies for Improving Crop Salt Tolerance

Drought stress is a major constraint to the production and yield stability of soybean [Glycine max (L.) Merr.]. For developing high yielding varieties under drought conditions, the most widely employed criterion has traditionally been direct selection for yield stability over multiple locations. However, this approach is time consuming and labor intensive, because yield is a highly quantitative trait with low heritability, and influenced by differences arising from soil heterogeneity and environmental factors. The alternative strategy of indirect selection using secondary traits has succeeded only in a few crops, due to problems with repeatability and lack of phenotyping strategies, especially for root-related traits. Considerable efforts have been directed towards identifying traits associated with drought resistance in soybean. With the availability of the whole genome sequence, physical maps, genetics and functional genomics tools, integrated approaches using molecular breeding and genetic engineering offer new opportunities for improving drought resistance in soybean. Genetic engineering for drought resistance with candidate genes has been reported in the major food crops, and efforts for developing drought-resistant soybean lines are in progress. The objective of this review is to consolidate the current knowledge of physiology, molecular breeding and functional genomics which may be influential in integrating breeding and genetic engineering approaches for drought resistance in soybean.

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Physiological and molecular approaches to improve drought resistance in soybean.

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The testing and release of genetically modified organisms (GMOs)—in particular GM plants—is tightly regulated internationally to prevent any negative effects on the environment or human health. However, these regulations are based on transgenic organisms and do not discriminate between transgenic plants and cisgenic plants, although we believe that they are fundamentally different (see sidebarNow, cisgenic plants fall under regulations designed for transgenic organisms, possibly because there have not yet been any applications for the approval of the deliberate release of cisgenic plants into the environment.

Definitions of key terms in relation to plants

Cisgenesis is the genetic modification of a recipient plant with a natural gene from a crossable—sexually compatible—plant. Such a gene includes its introns and is flanked by its native promoter and terminator in the normalsense orientation.Cisgenic plants can harbour one or more cisgenes, but they do not contain any transgenes.

Transgenesis is the genetic modification of a recipient plant with one or more genes from any non‐plant organism, or from a donor plant that is sexually incompatible with the recipient plant. This includes gene sequences of any origin in the anti‐sense orientation, any artificial combination of a coding sequence and a regulatory sequence, such as a promoter from another gene, or a synthetic gene.

Traditional breeding encompasses all plant breeding methods that do not fall under current GMO regulations.As the European legal framework defines GMOs and specifies various breeding techniques that are excluded from the GMO regulations,we use this framework as a starting point, particularly the European Directive 2001/18/EC on the deliberate release of GMOs into the environment (European Parliament, 2001). Excluded from this GMO Directive are longstanding cross breeding, in vitro fertilization, polyploidy induction,mutagenesis and fusion of protoplasts from sexually compatible plants (European Parliament, 2001).

Although transgenesis and cisgenesis both use the same genetic …

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/pdf/cisgenic-plants-are-similar-to-traditionally-bred-plants-2yhqsjfnic.pdf

Cisgenic plants are similar to traditionally bred plants: International regulations for genetically modified organisms should be altered to exempt cisgenesis

Section A: Interaction of Bacterial Pathogens with Plants. Section B: Rhizobium--Plant Symbiotic Interactions. Section C: Interaction of Fungi with Plants. Section D: Isolation of Plant Disease Resistance Genes. Section E: Plant Responses to Pathogens and Resistance Mechanisms. Section F: Engineered Resistance to Plant Pathogens. Index.

Advances in molecular genetics of plant-microbe interactions : proceedings of the 7th International Symposium on the Molecular Plant-Microbe Interactions, Edinburgh, U.K., June 1994

During the past seventy years, worldwide more than 2250 varieties have been released that have been derived either as direct mutants or from their progenies Induction of mutations with radiation has been the most frequently used method for directly developed mutant varieties The prime strategy in mutation-based breeding has been to upgrade the well-adapted plant varieties by altering one or two major traits, which limit their productivity or enhance their quality value In this paper, the global impact of mutation-derived varieties on food production and quality enhancement is presented In addition, the economic contribution of the selected mutant varieties of rice, barley, cotton, groundnut, pulses, sunflower, rapeseed and Japanese pear is discussed In several mutation-derived varieties, the changed traits have resulted in synergistic effect on increasing the yield and quality of the crop, improving agronomic inputs, crop rotation, and consumer acceptance In contrast to the currently protected plant varieties or germplasm and increasing restrictions on their use, the induced mutants have been freely available for plant breeding Many mutants have made transnational impact on increasing yield and quality of several seed-propagated crops Induced mutations will continue to have an increasing role in creating crop varieties with traits such as modified oil, protein and starch quality, enhanced uptake of specific metals, deeper rooting system, and resistance to drought, diseases and salinity as a major component of the environmentally sustainable agriculture Future research on induced mutations would also be important in the functional genomics of many food crops

Global impact of mutation-derived varieties

Many spontaneous and a large number of induced mutants that show altered nodulation pattern have been isolated in pea, soybean, common bean, faba bean, chickpea, groundnut and pigeonpea. Available information on nodulation mutants in these crops is summarised. The importance of nodulation mutants in basic studies on plant-microbe symbiotic interactions, nitrogen fixation and breeding of cultivars with higher yield and nitrogen fixation rate are examined. The nodulation mutants, after inoculation with specific bacterial strains or a number of different strains, show either: no nodulation (nod-), few nodules (nod+/-), ineffective nodulation (fix-), hyper nodulation (nod++) or hypernodulation even in the presence of otherwise inhibitory nitrate levels (nts). No spontaneous hypernodulation or nts mutants have been found, all have been induced in independent experiments using different cultivars of pea, soybean and common bean after mutagenising seeds. Most nodulation mutants show monogenic recessive inheritance, though semi-dominant and dominant inheritance is also reported. Nodule number is controlled by a process known as autoregulation; hypernodulating mutants show relaxed autoregulation. By grafting shoots of hypernodulating soybean mutant on normal nodulating soybean, mungbean and hyacinth bean, presence of a common, translocatable signal has been shown. Nodulation mutants have contributed to the understanding of the genetic regulation of host-symbiont interactions, nodule development and N fixation. Initially, the hypernodulating mutants were found to be poor in yield. Using the induced hypernodulating mutant, a new soybean cultivar ‘Nitrobean 60’, has been released in Australia. This cultivar is reported to have given 15% higher yield over cv. ‘Bragg,’ and contributed a higher amount of fixed N to the following cereal crop in rotation. Prospects of using the nodulation mutants in developing grain legume cultivars that combine high yield with high residual N, within the bioenergetic constraints, for developing sustainable cropping systems are examined.

Mutations affecting nodulation in grain legumes and their potential in sustainable cropping systems

One hundred and sixty-three cultivars of annual oilseed crops, developedusing induced mutations, have been officially approved and released forcultivation in 26 countries. The maximum number of cultivars have beenreleased in soybean (58), followed by groundnut (44), sesame (16), linseed(15), rapeseed (14), Indian mustard (8), castorbean (4), white mustard(3) and sunflower (1). The majority (118 of 163) of the cultivars havebeen developed as direct mutants and 45 of 163 by using the inducedmutants in a crossing programme. While in soybean 53 out of 58 cultivarswere selected as direct mutants, in groundnut 22 from 44 were developedafter hybridization. Eighty-three cultivars were developed directly byexposing seeds to gamma or X-rays. Attempts have been made to inferthe successful dose range, defined as the range which led to thedevelopment, registration and release of the maximum number of mutantcultivars for gamma and X-rays. The successful dose ranges in Gy forthe main oilseed crops are: soybean 100-200, groundnut 150-250, rapeseed600-800, Indian mustard 700 and sesame 100-200. The maincharacteristics of the new cultivars, besides higher yield, are altered planttype, early flowering and maturity and oil content. Mutants altering fattyacid composition have been isolated in soybean, rapeseed, sunflower,linseed and minor oil crops. New cultivars having altered fatty acidcomposition have been released in rapeseed, sunflower and linseed. Thelatter, previously grown for non-edible oil, has been converted to a newedible oil crop.

Oilseed cultivars developed from induced mutations and mutations affecting fatty acid composition

Methodologies for generating variability. Part 4: Mutation techniques.

Two hundred and sixty-five grain legume cultivars developed using induced mutations have been released in 32 countries. A maximum number of cultivars have been released in soybean (58), followed by common bean (50), groundnut (44), pea (32) and mungbean (14). Gamma or x-ray exposures of seeds led to the direct development of 111 cultivars, while neutron and chemical mutagen treatments resulted in 8 and 36 cultivars respectively. One hundred and three cultivars have been developed using mutants in cross breeding. Attempts have been made to estimate the successful dose range for gamma and x-rays, defined as the dose range, which led to the development, registration and release of a maximum number of mutant cultivars. Exposures to seeds ranging between 100-200 Gy in all grain legumes, except faba bean, resulted in 49 out of 111 cultivars being developed as direct mutants. Successful doses reported for faba bean are lower than 100 Gy. Modified crop plant characters are listed. Besides the development of new cultivars, a large number of induced mutants that show altered nodulation pattern have been isolated in grain legumes. Such mutants have made a significant contribution in basic studies on host-symbiont interactions and towards cloning of plant genes related to symbiosis and nitrogen fixation. Their exploitation in breeding programs for enhancing nitrogen fixation is just beginning. Available information on nodulation mutants in grain legume crops is summarised. Mainly, four types of nodulation mutants have been isolated. They show either: no nodulation (nod -), few nodules (nod +/-), ineffective nodulation (Fix-), hypernodulation (nod ++) or hypernodulation even in the presence of otherwise inhibitory nitrate levels (nts). Hypernodulating and nts mutants are of great interest. A soybean cultivar incorporating nts trait has been released in Australia. (author)

K. Nichterlein

Papers

Global impact of mutation-derived varieties

Mutations affecting nodulation in grain legumes and their potential in sustainable cropping systems

Oilseed cultivars developed from induced mutations and mutations affecting fatty acid composition

Methodologies for generating variability. Part 4: Mutation techniques.

Grain legume cultivars derived from induced mutations, and mutations affecting nodulation