Since their introduction in 1996, the area grown to transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has grown rapidly. In 2004, Bt plants were grown on over 13 million ha in the U.S. and 22.4 million ha worldwide^1,2. Bt corn and Bt cotton comprised about one-third and one-half, respectively, of the U.S. corn and cotton markets in 2004. Worldwide, approximately 15 million ha of Bt corn and 7.4 million ha of Bt cotton were grown in 2004. The economic and environmental benefits of Bt crops, compared to other technologies, have been well-documented³. Reports from China have also shown that fewer insecticide poisonings occur when Bt cotton is used⁴.

Despite the extensive use of Bt crops over an 8-year period, there have been no reports of product failure or increased resistance in insect pests⁵. To put this in perspective, Bt crops have already exceeded the length of time that typically passes in the field before resistance is first documented with most conventional neurotoxic pesticides, despite undergoing what has been hailed as one of the world’s largest selection for resistance⁶. However, because of the demonstrated ability of two insect species (the diamondback moth and the cabbage looper) to evolve resistance to sprays of Bt proteins in commercial settings, there is concern that some insects may also evolve resistance in the field to Bt plants. To prevent or delay resistance to Bt crops, various insecticide resistance management (IRM) strategies have been proposed. These strategies include manipulation of the transgenic elements of the plants, such as the genes and promoters, and the manner in which plants are deployed in the landscape. The most widely employed tactic to delay resistance is the use of plants expressing a high dose of a single Bt protein throughout the life of the plant, combined with a "refuge" of non-Bt plants nearby that can maintain an adequate supply of susceptible alleles within the insect population. We believe this "high dose/refuge" strategy has played a major role in the lack of resistance to date for Bt plants, but recognize that other strategies must be developed as the use of Bt plants continues to grow and selection for resistance intensifies.

Over the past 15 years, our cooperative program has tested various IRM strategies using a unique system. The system is composed of different populations of diamondback moth that have evolved resistance to one or two Bt proteins (Cry1A and/or Cry1C) and broccoli plants that express either one or both of these proteins. Our greenhouse and field tests are described in a recent paper⁶ in a historical perspective of IRM and Bt plants. In that paper, we describe the use of this unique system to demonstrate the effectiveness of the "high dose/refuge" strategy and the use of an inducible promoter as "proof of concept" by using the inducible promoter so that the Bt protein is expressed only at a specific period of time, thus reducing the selection pressure for resistance. Such inducible promoters allow Bt proteins to be expressed only during specific periods of time, and could be used in situations in which plants could withstand some insect defoliation early in their growth, but the marketable part of the plant must be kept clean during the later stages of growth (e.g., tomatoes).

An IRM strategy outlined in a 1998 paper by Roush⁷ suggested that pyramiding two dissimilar Bt proteins in the same plant could delay resistance development compared to plants that expressed only one Bt protein. The models contained in the paper were evaluated using our diamondback moth/Bt broccoli system, and we found that such Bt pyramided plants significantly delayed the evolution of resistance⁸.

Pyramided cotton plants ("Bollgard II") with two genes derived from Bt (Cry1Ac and Cry2Ab2) were approved for commercial use in Australia and the U.S. in 2002, and several companies are developing new cotton and corn varieties with pyramided Bt genes. However, there is concern that the optimal benefits of pyramided Bt genes for resistance management may be lost if one-gene plants sharing similar Bt toxins continue to be deployed. Newly developed pyramided varieties of Bt cotton and corn currently contain the same or similar genes as one-gene (Cry1Ac for Bt cotton, Cry1Ab for Bt corn) Bt plants already marketed. If market forces result in a complicated landscape mix of one- and two-gene Bt plants, the benefits of pyramided Bt plants for slowing resistance evolution could be undermined. For example, a modeling study⁹ suggested that Cry2A resistance evolution in a cotton pest was maximized when Bt cotton varieties expressing one- and two-genes were both available, and that the overall durability of two-gene plants would be greater if they were deployed alone, compared to a sequential or mosaic deployment with Bollgard (Cry1Ac alone). However, the risk of pest adaptation to pyramided Bt plants used in conjunction with one-gene plants had not been quantified empirically.

To assess the risk of resistance evolution to pyramided plants when they were simultaneously deployed with single gene plants containing one of the Bt genes in the pyramided Bt plants, we used broccoli plants transformed to express different Cry toxins (Cry1Ac, Cry1C, or both) and populations of diamondback moth carrying resistance to each of the Bt Cry toxins⁸. Three treatments were tested in separate large greenhouse cages: (1) 45% Cry1Ac and 45% two-gene plants plus 10% refuge; (2) 45% Cry1C and 45% two-gene plants plus 10% refuge; (3) 90% two-gene plants plus 10% refuge. Diamondback moths with a known frequency of resistance alleles to Cry1Ac and Cry1C were then introduced into each cage. We allowed the insect populations in each cage to reproduce normally over time. Every few generations, we counted the number of insects produced in each cage and determined the frequency of resistance alleles in the insect population. The results were rather dramatic. After 24 – 26 generations of selection in the greenhouse, the concurrent use of one- and two-gene plants resulted in control failure of both types of Bt plants. When only two-gene plants were used in the cage, no or few insects survived in subsequent tests on one- or two-gene Bt plants. Overall, this clearly indicated that the concurrent use of transgenic plants expressing a single and two Bt genes will select for resistance to two-gene plants more rapidly than the use of two-gene plants alone. The results of this experiment agree with the predictions of a Mendelian deterministic simulation model.

What does this mean for the commercial use of Bt plants? Simply put, the concurrent use of single and two-gene Bt plants can offer exposed insect populations a "stepping stone" to develop resistance to both toxins. Thus, from a resistance management perspective, it appears that using pyramided Bt plants simultaneously with single-gene plants, if they share similar Bt toxins, will negate some of the benefits of the two-gene plants. In Australia, pyramided Bt cotton (Bollgard II) has been commercially available since 2002. The use of both one- and two-gene varieties was permitted for the first two years following the introduction of Bollgard II, but now only two-gene varieties are allowed. The rapid phaseout of one-gene varieties was intended to minimize pest exposure to the single Bt toxin and thus to reduce the risk of resistance to pyramided plants. In the U.S., plants with a single Bt gene remain the dominant Bt varieties. Our results indicate that the introduction of pyramided plants with currently deployed single gene plants should be examined carefully by regulatory agencies. Our data are consistent with models and suggest that, from an IRM standpoint, it could be advantageous for regulatory agencies to consider deregulating single gene plants as soon as pyramided plants are available.

We recognize that economic considerations must be factored into decisions regarding the deployment of single and pyramided gene plants. From an industry standpoint, however, there could be distinct economic and marketing advantages for promoting pyramided plants rather than single gene plants. In addition to providing superior resistance management, pyramided plants may provide improved control of some harder-to-kill insects, and require a smaller area set aside for the refuge. The smaller refuge size has particular relevance for IRM; models have indicated that a 30–40% refuge for single gene plants is equivalent to a 5–10% refuge for pyramided plants⁷. Thus, the two-gene strategy is especially suitable for developing countries such as China, where farms on average are only 0.5 ha, and the practice of setting aside land for a refuge is highly impractical.

Naturally-occurring cytoplasmic male sterility (CMS) has been known for over 100 years. CMS systems are used to produce commercial F1 hybrid lines. Ruiz and Daniell (2005) recently reported the first engineered cytoplasmic male sterility system in plants. They studied the effect of light regulation of the phaA gene coding for β-ketothiolase engineered via the chloroplast genome. The phaA gene was efficiently expressed in all tissue types examined, including leaves, flowers, and anthers. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation decreased the inner space of the locules, affected pollen grain, and resulted in the irregular shape or collapsed phenotype. Reversibility of the male sterile phenotype was observed under continuous illumination, resulting in viable pollen and a copious amount of seeds. This study offers a new tool for transgene containment for both nuclear and organelle genomes and provides an expedient mechanism for F1 hybrid seed production.

History of Cytoplasmic Male sterility
Male-sterility-inducing cytoplasms have been known for over 100 years. In 1921, Bateson and Gairdner reported that male sterility in flax was inherited from the female parent. Chittenden and Pellow observed in 1927 that male sterility in flax was due to an interaction between the cytoplasm and nucleus. In 1943, Jones and Clarke established that male sterility in onion is conditioned by the interaction of the male-sterile (S) cytoplasm with the homozygous recessive genotype at a single male-fertility restoration locus in the nucleus. The authors also described the technique used today to exploit cytoplasmic-genic male sterility (CMS) for the production of hybrid seeds. CMS inbred lines have been widely used for hybrid production of many crops. The first application of organelle biotechnology was the role played by cytoplasmic male sterility in hybrid seed production, a major contribution towards the "Green Revolution." The use of cytoplasmic male sterility in hybrid seed production has been recently reviewed by Havey (2004).

The use of CMS for hybrid seed production received a "black eye" after the epidemic of Bipolaris maydis on T-cytoplasmic maize. This epidemic is often cited as a classic example of genetic vulnerability of our major crop plants. In addition to Southern corn blight (CMS-T), cold susceptibility (CMS Ogura) and Sorghum Ergot infection in the unfertilized stigma have been reported. However, these disease linkages were successfully broken by somatic cell genetics and conventional plant breeding. Hybrids of other crop plants may be produced using nuclear male sterility. A natural source of nuclear male sterility was identified in leek. Engineered sources of nuclear male sterility have been developed in model systems. A problem with these approaches is that they segregate for male fertility or sterility and must be over-planted and rogued by hand or sprayed with herbicides to remove male-fertile plants. Male-sterility systems have been created by several different mechanisms; most of these affect tapetum and pollen development. Unfortunately, additional severe phenotypic alterations that were due to interference with general metabolism and development had precluded their use in agriculture.

Advantages of the chloroplast transformation system
A chloroplast genetic engineering approach offers a number of attractive advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position effect due to site-specific transgene integration, and lack of pleiotropic effects due to sub-cellular compartmentalization of transgene products. Genetically engineered cytoplasmic male sterility via the chloroplast genome may be used for the safe integration of foreign genes via the nuclear genome and in those rare cases in which plastid genomes are paternally or biparentally transmitted. Recently, plastid transformation was demonstrated in carrot with their ability to grow in very high saline conditions (up to 400 mM sodium chloride). Additionally, plastid transformation of recalcitrant crops such as cotton and soybean allows the application of the cytoplasmic male sterile system to commercially important crops. For a recent review of developments in this field, see Daniell et al. (2005) and Grevich & Daniell (2005).

Novel mechanism of Cytoplasmic Male Sterility
PHB synthesis takes place by the consecutive metabolic action of β-ketothiolase (phaA gene), acetoacetyl-CoA reductase (phaB) and PHB synthase (phaC). Nawrath et al. (1994) introduced the phbA, phbB, and phbC genes in individual nuclear Arabidopsis transgenic lines and reconstructed the entire pathway, targeting all enzymes to the plastids. This approach resulted in PHB expression up to 14% leaf dry weight. This study suggests that the depletion of metabolites from essential metabolic pathways in the cytoplasm might have caused the pleiotropic effects, and that by targeting the enzymes to the chloroplast, which is a compartment with high flux through acetyl-CoA, the adverse effects were overcome. With expression of optimized gene constructs, PHB yield increased up to 40% of leaf dry weight, but this was accompanied by severe growth reduction and chlorosis (Bohmert et al., 2000), indicating that targeting the PHB pathway to the chloroplast can result in pleiotropic effects, at higher concentrations of polymer synthesis. In an attempt to address the role of phaA expression in the pleiotropic effects observed in transgenic plants expressing PHB, Bohmert et al. (2002) expressed the phbA gene via the nuclear genome. Continuous expression of the phbA gene led to a significant decrease in transformation efficiency, inhibiting the recovery of transgenic lines, and prevented analysis of plants expressing the β-ketothiolase gene. Such a toxic effect exerted by phbA expression was speculated to be the result of PHB biosynthesis intermediates or its derivatives, the depletion of the acetyl-CoA pool, or of the interaction of β-ketothiolase with other proteins or substrates.

While investigating the phb operon, Ruiz & Daniell (2005) proposed to address the specific role of β-ketothiolase (phaA gene) that results in pleiotropic effects in transgenic plants expressing the polyhydroxybutyrate pathway. Therefore, they hyper-expressed the β-ketothiolase gene by inserting the transgene into the chloroplast genome under light regulation and assessed the effects of phaA expression in the chloroplast transgenic plants at molecular and physiological levels. In this study, the phaA gene was constitutively expressed, and fully regenerated transgenic plants were recovered. Therefore, evaluation of the specific effect of β-ketothiolase in the transgenic lines was possible for the first time. Detailed characterization revealed that severe pleiotropic effects such as stunted phenotype and chlorosis observed during polyhydroxybutyrate expression were not observed during phaA expression via chloroplast transformation, with the exception of complete (100%) male sterility. Such elimination of pleiotropic effects was not surprising because the chloroplast genetic engineering system has been able to overcome the toxic effects (stunted growth, chlorosis, and infertility) associated with nuclear expression of the trehalose, xylanase, and cholera toxin B subunit genes, producing totally normal plants that were healthy and fertile.

Reversibility of male sterility
To test whether depletion of the acetyl-CoA pool destined for de novo fatty acid biosynthesis in the chloroplast by β-ketothiolase is the cause of the male sterility phenotype, the authors explored if a continuous light could revert male fertility of the chloroplast transgenic lines. Recent reports have shown light-dependent regulation of the key enzyme in fatty acid biosynthesis. Therefore, acetyl-CoA carboxylase (ACCase) male sterile transgenic lines were exposed to continuous light for a period of 10 days. The authors observed that from 20 flowers produced during the 10 days of illumination by the two transgenic plants, four flowers produced pollen, normal length anther filaments, fruit capsules, and seeds. The seeds recovered from the reverted male fertility study were able to germinate in the selection medium, confirming that these seedlings were transgenic and contained the transgene; wild type tobacco seedlings were bleached. These findings support the hypothesis that an increase in ACCase activity outcompetes the removal of acetyl-CoA by β-ketothiolase.

Conclusions
Havey (2004) documents the worldwide use of CMS to produce competitive hybrid cultivars. Major investments of time and resources are required to backcross a male-sterility-inducing cytoplasm into elite lines. These generations of backcrossing could be avoided by transformation of an organellar genome of the elite male-fertile inbred line to produce female inbred lines for hybrid seed production. Because the male-fertile parental and male-sterile transformed lines would be developed from the same inbred line, they should be highly uniform and possess the same nuclear genotype, excluding mutations and residual heterozygosity. Therefore, the male-fertile parental line becomes the maintainer line to seed-propagate the newly transformed male-sterile line. A few generations of seed increases would produce a CMS-maintainer pair for hybrid seed production. An additional advantage of organellar transformation would be the diversification of CMS sources used in commercial hybrid-seed production. Transformation of the chloroplast genome would allow breeders to introduce different male-sterility-inducing factors into superior inbred lines. Introduction of a male-sterility inducing transgene into one of the organellar genomes of a higher plant would be a major breakthrough in the production of male-sterile inbred lines. This technique would be of great potential importance in the production of hybrid crops by avoiding generations of backcrossing, an approach especially advantageous for crop plants with longer generation times. Moreover, transgenes that are engineered into annual crops could be introgressed into wild crops, persist in the environment, and thereby have negative ecological consequences. Therefore, it may be necessary to engineer a male sterility system that is 100% effective.

For vegetable, fruit, or forage crops, restoration of male fertility in the hybrid is not necessary. This simplifies the production of hybrids because effort can be concentrated on maintainer line development, without concern over whether the pollinator restores male fertility in the hybrid. For crops with seeds as the economically important product, such as canola, sunflower, or maize, one or both of the hybrid’s parents must bring in male-fertility restoration factors or the male-sterile hybrid seed must be blended with male-fertile hybrid seed. In currently available cytoplasmic male sterile lines, the nuclear genome controls various restoration factors and such factors are often located at multiple loci and are poorly understood. However, the authors report restoration of male fertility by changing conditions of illumination. Therefore, this is a novel approach for creating male sterile transgenic plants, which may help advance the field of plant biotechnology through effective transgene containment.

Bohmert K et al. (2002) Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants. Plant Physiol 128, 1282-1290

Bohmert K et al. (2000) Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight. Planta 211, 841-845

H. Daniell, S. Kumar & Duformantel N. (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology 23, 238-245

Grevich J & Daniell H (2005) Chloroplast genetic engineering: Recent advances and perspectives. Critical Reviews in Plant Sciences 24, 1-25

Havey MJ (2004) The use of cytoplasmic male sterility for hybrid seed production. In Daniell H, Chase C, eds, Molecular Biology and Biotechnology of Plant Organelles, Springer Publisher: The Netherlands, pp 617-628

Nawrath C, Porier Y, & Somerville C (1994) Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana result in high levels of polymer accumulation. PNAS 91,12760-12764

Ruiz O & Daniell H (2005) Engineering cytoplasmic male sterility via the chloroplast genome. Plant Physiology 138, 1232-1246

HOMOLOGOUS PROMOTER USE IN GENETIC MODIFICATION
Keerti S. Rathore & Ganesan Sunilkumar

Genetic modification generally requires stable transgene expression at a desired level in the transgenic organism, without adversely affecting native gene activities. The concept of gene stacking to introduce multiple agronomic traits requires coordinated expression of several genes. In addition, second and third generation biotechnology products require controlled expression of several transgenes. These applications necessitate a heterologous and/or a homologous promoter to drive the expression of one or more transgenes. For certain applications, a homologous promoter may be a better choice since it is likely to provide a more precise level of developmental and spatial control and its activity may be stronger in the native environment.

The benefits of homologous promoter use were demonstrated recently in a study on the characterization of a cotton α-globulin promoter, which showed that this promoter resulted in a significantly higher level of gusA gene expression in cotton, compared to that of two other heterologous systems, Arabidopsis and tobacco¹. However, it is generally believed that homologous promoters should be avoided, as they can lead to transgene and/or the resident gene/transgene silencing^2-5. This notion mainly stems from studies conducted by two independent groups who demonstrated that, in some tobacco lines, reintroduction of a heterologous promoter into a transgenic plant that contains a previously introduced copy of that promoter silenced transgenes driven by each promoter^6-8. Methylation of the promoters was found to be the basis of their inactivation.

If homology-dependent promoter inactivation is based on a general mechanism, it should apply not only to the multiple uses of a heterologous promoter but also to the single use of a homologous promoter. There are several reports on the successful use of homologous promoters for transgene expression (see references in ref. 9). However, to our knowledge, no systematic study has been conducted to specifically address the negative impact of a transgenic, homologous promoter on the activity of the endogenous promoter. Inactivation of a transgenic and/or resident promoter is a serious concern in agricultural biotechnology that requires a thorough investigation.

We have directly addressed this important issue in transgenic lines that were created to enhance the levels of oleic acid in cottonseed⁹. The promoter region isolated from a cotton α-globulin B gene was used to drive an antisense construct of a ∆-12 desaturase gene in cotton. The seeds from several antisense lines exhibited increased levels of oleic acid and a concomitant decrease in the levels of linoleic acid. These lines exhibiting the antisense-mediated phenotype, as well as some transgenic lines that did not exhibit the high-oleate phenotype, provided a suitable resource to study the impact of the use of a homologous promoter on the activity of an endogenous promoter.

The level of the α-globulin B protein in the seed is expected to accurately reflect the activity of its promoter. Therefore, we examined the quantity of this protein by estimating the intensity of the 52 kDa band on a Coomassie Brilliant Blue-stained SDS-PAGE gel. The levels of α-globulin B protein in the seeds from four selected, highest-oleate lines were compared with control seeds. The controls consisted of non-transgenic cottonseeds as well as seeds from two different null segregant plants derived from one of the high-oleate lines. Total proteins were extracted from a pooled sample of 10 seeds from each plant and the quantitation was performed on three replicate protein extracts that were fractionated on three different gels. The α-globulin B protein band was quantified from the digital photographs of the gels using AlphaEase^TM software. The levels of α-globulin B protein in the high-oleate lines were not significantly different from control seeds. Therefore, the endogenous promoter as well as the transgenic promoter in high-oleic acid lines were functioning normally.

As with any transformation experiment, we obtained a few lines that did not exhibit the transgenic phenotype, i.e., higher levels of oleic acid in the seeds. One reason for the absence of the transgenic phenotype may be that the transgene was not expressed due to inactivation of the promoter. In this scenario, if the promoter homology-based mutual silencing mechanism was in operation, the possibility exists that the corresponding endogenous promoter was also silenced. To test this possibility, the levels of α-globulin B protein in the seeds from eight transgenic lines, which showed little or no increase in seed oleic acid, were compared with control seeds. The levels of α-globulin B protein were not significantly different between the seeds of control and transgenic lines that exhibited wild-type levels of oleic acid. These results, taken together, show that the seed-specific, α-globulin promoter can be used effectively for genetic modification of cottonseed without interfering with the activity of the corresponding endogenous promoter⁹.

Previous studies^6,8 that led to the notion of promoter homology-mediated silencing were based on observations on a few, isolated transgenic events containing multiple T-DNA inserts or rearranged T-DNA structures. These events were subjected to detailed characterizations in subsequent investigations by each group. It is possible that the extensive nature of these series of studies may have helped create a perception that promoter homology causes silencing problems. In our study, we have directly addressed this issue and found that introduction of a homologous promoter, per se, does not cause silencing of either the transgenic or resident promoter.

1. Sunilkumar G et al. (2002) Transgenic Res. 11, 347-359

2. Finnegan J & McElroy D. (1994) Biotechnol. 12, 883-888

3. De Wilde C et al. (2000) Plant Mol. Biol. 43, 347-359

4. Halpin C et al. (2001) Plant Mol. Biol., 47, 295-310

5. Potenza C, Aleman L & Sengupta-Gopalan C (2004) In Vitro Cell. Dev. Biol.-Plant 40, 1-22

6. Matzke MA et al. (1989) EMBO J., 8, 643-649.

7. Matzke MA, Neuhuber F & Matzke AJM. (1993) Mol. Gen. Genet. 236, 379-386

8. Vaucheret H (1993) C. R. Acad. Sci. Paris 316, 1471-1483

9. Sunilkumar G et al. (2005) Plant Biotechnology Journal 3, 319-330

QTL DETECTION AND APPLICATION TO PLANT BREEDING
Motoyuki Ashikari and Makoto Matsuoka

QTL cloning for grain productivity and plant height
Rice (Oryza sativa L.) is a staple food and approximately 50% of the human population depends on rice as their main source of nutrition. In particular, it is the most important crop for people living in the monsoonal areas of Asia where rice has a long history of cultivation; it is deeply ingrained in the daily lives of Asian people.

Rice is a model monocot because it has the smallest genome size (390 Mb) among the major cereals, its genome is syntenic with the genomes of other cereals, and it can be transformed easily. As a result, The International Rice Genome Sequencing Project (IRGSP) was launched in 1998 to sequence the rice genome; the task was completed in 2004. These accomplishments and recent technological innovations have greatly facilitated gene cloning and provided a new breeding strategy for rice as well as for other major cereals.

In contrast to monogenic characteristics, such as disease and insect resistance, many important agronomic traits including yield, heading date, culm length, grain quality, and stress tolerance show continuous phenotypic variation. These complex traits usually are governed by a number of genes known as quantitative trait loci (QTLs) derived from natural variations. Although these polygenic characteristics, including QTLs, were previously very difficult to analyze using traditional plant breeding methods, recent progress in rice genomics has made it possible to search QTLs.

QTL analysis is a powerful approach to discover agronomically useful genes. Grain number and plant height are important traits that directly contribute to grain productivity. Why is plant height important for grain production? Dwarf rice and wheat varieties were developed by classical plant breeding methods, contributing to the green revolution in the 1960’s. Higher yields were obtained from these dwarf crops because their short stature reduced lodging from wind or rain^1,2. Our lab has aimed to identify genes of QTLs for grain number, Gn1, and plant height, Ph1, not only to elucidate molecular mechanisms for grain productivity, but also to utilize these genes for breeding³.

A choice of parental lines that show wide phenotypic variation in the targeted traits is necessary for QTL analysis because QTL detection is based on natural allelic differences between parental lines. An indica rice variety, Habataki,

and a japonica variety, Koshihikari, were chosen in QTL analysis since not only do they exhibit large differences in grain number and plant height, but also many molecular markers are available. QTL analysis using progenies from the cross with Habataki and Koshihikari revealed the presence of five QTLs for increasing grain number (Gn1-5) and four QTLs for plant height (Ph1-4). The most effective QTLs for grain number, Gn1, and plant height, Ph1, were chosen as targets for cloning.

QTL cloning is facilitated by using nearly isogenic lines (NILs) carrying only one target QTL, because NIL can eliminate the effects of other QTLs. By using a suitable NIL, the QTL of interest in the NIL can be treated as a single Mendelian factor. We produced the NIL-Gn1 and NIL-Ph1 lines carrying the Gn1 or Ph1 region from Habataki in the Koshihikari background and used these lines for cloning. Base on fine mapping analysis of QTL-Gn1, we found that Gn1 could be divided as two linked QTLs (QTL-Gn1a and QTL-Gn1b). We focused on the Gn1a, since it could be mapped between two molecular markers.

Positional cloning and molecular characterization revealed that Gn1a encodes a cytokinin oxidase/dehydrogenase, OsCKX2, an enzyme that degrades a phytohormone cytokinin. The OsCKX2 gene of Koshihikari and Habataki consists of four exons and three introns and encodes proteins of 565 or 563 amino acids, respectively. A comparison of the DNA sequences between the cultivars revealed several nucleotide changes, including a 16-bp deletion in the 5’-untranslated region, a 6-bp deletion in the first exon, and three nucleotide changes, resulting in amino acid variation in the first and fourth exon of the Habataki allele. Both OsCKX2 alleles of Habataki and Koshihikari encode an enzyme capable of degrading cytokinin. However, the expression level of OsCKX2 in Habataki was lower than in Koshihikari, resulting in less cytokinin accumulation in the inflorescence meristems of Habataki than Koshihikari. Cytokinin (CK) is known to influence various aspects of plant growth and development, including seed germination, apical dominance, leaf expansion, reproductive development, and delay of senescence. The reduced expression of OsCKX2 can explain the increased cytokinin accumulation, and hence, increased grain number. The semi-dominant inheritance of Gn1a is also consistent with the function of the OsCKX2 enzyme that degrades cytokinin.

On the other hand, Ph1 was located near the sd1 gene that encodes gibberellin 20-oxidase. Comparing the sequence of sd1 in the Habataki and Koshihikari alleles revealed that Habataki had a 383-bp deletion in the SD1 gene, which is the same mutation found in ‘IR8’, a variety that led to the green revolution in rice. This observation demonstrated that the short stature of Habataki mainly depends on the sd1 locus³.

QTL pyramiding breeding
QTL pyramiding is an efficient strategy for crop improvement. This strategy is based on the combination of desirable QTLs through conventional crossing using molecular markers. Once desirable QTLs are detected, a strategy for QTL pyramiding employs the use of NILs harboring only one target QTL. The NIL-QTLs are produced by backcrossing and marker selection. A parent line with a positive QTL is backcrossed with a recurrent parent lacking the QTL. Subsequently, a line that carries only a positive QTL region from the mother line in the recurrent parent genome background is selected by molecular markers. The NILs can be used to accurately evaluate the effect of each QTL individually. Once QTLs with important effects are identified in this manner, the appropriate NIL-QTLs are crossed to pyramid two or more QTLs in the same background. The two NILs, the NIL-Gn1 and NIL-sd1, carrying the Gn1 region or sd1 region from Habataki in the Koshihikari genome background, were produced by backcrossing with Koshihikari and using marker selection. We then derived a NIL-sd1+Gn1 plant with the two desirable QTL alleles derived from the cross NIL-Gn1 and NIL-sd1. The NIL-sd1+Gn1 has a high yielding, semi-dwarf phenotype (Fig.1)³.

Future outlook
Food shortage is one of the most serious global problems in this century. The world population is expanding rapidly due to a significant decline in mortality rates resulting from advancements in modern medicine and human health care, while the availability of land for cultivation has dramatically declined as the result of desertification caused by reckless deforestation and construction. The global population, now at 6.4 billion, is still growing rapidly and is projected to reach 8.9 billion people by 2050. Cereals are an important source of calories for humans, both by direct intake and as the main feed for livestock. Approximately 50% of the calories consumed by the world population originate from three cereals, rice (23%), wheat (17%), and maize (10%). To meet the expanding food demands of the rapidly growing world population, crop grain production will need to increase by 50% by 2025.

Now that genomic information and tools are available for rice, we have to apply these for human benefits. Today the cloning of genes and production of transgenic plants are common technologies utilized in plant science. Such technologies are very powerful and efficient strategies for producing ‘ideal’ crop plants. Actually, some transgenic crop plants with traits such as insect resistance and herbicide tolerance have already been commercialized. Despite concerns about the impact of GMOs on the environment and their safety to humans, we think transgenic crops are necessary to meet the demand for food in the near future. However, we should not ignore the conventional breeding approach—the QTL pyramiding approach results from a combination of recent crop genomics and conventional breeding, and it is efficient for crop breeding. We believe both strategies, GMO strategies and QTL pyramiding, are necessary and the cooperation of molecular geneticists and breeders is required to accomplish this goal.