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First published online March 9, 2007; 10.1104/pp.106.090365 Plant Physiology 144:18-31 (2007) © 2007 American Society of Plant Biologists
Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from TobaccoUnité de Génétique et d'Amélioration des Fruits et Légumes, UR1502, Institut National de la Recherche Agronomique (INRA), F–84143 Montfavet cedex, France (M.M., E.B., F.F., J.-P.B., B.M.); and Laboratoire de Biologie Cellulaire, UR501, INRA, F–78026 Versailles cedex, France (B.C., M.-C.C., Y.C., H.L.)
The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3  -hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.
Continuous advances in whole-genome sequencing have resulted in important breakthroughs in plant science. Current data obtained on a few model species (Arabidopsis [Arabidopsis thaliana] and rice [Oryza sativa]) demonstrate that the degree of collinearity is rather low at the level of the whole genome and ongoing genome projects in other plant species might not be able to rely on the information obtained from already sequenced genomes to the extent that had been expected (Aert et al., 2004  ). Consequently, the importance of individual genome projects in higher organisms should not be underestimated. Unfortunately, traditional sequencing approaches are time consuming and expensive, and the time and cost increase proportionally with the size of the genome, which is a major obstacle in the accumulation of sequences and in the discovery of genes and nongenic functional elements in many agriculturally and industrially important plant genomes. Methods that allow rapid identification of the sequence involved in an interesting character (or a particular alteration of a phenotype) in a plant are promising, especially in plants for which genomic and whole-genome sequencing are not available. In this context, reverse-genetics tools such as insertional mutagenesis play an important role. Transposable elements provide attractive tools for constructing mutant collections and for gene tagging. Among these transposable elements, retrotransposons have unique features that give them several advantages over traditional insertion elements (Hirochika, 1997; Kumar and Bennetzen, 1999; Kumar and Hirochika, 2001). Because they transpose via a replicative mode, the mutations they induce are stable. As they transpose via an RNA intermediate that is reverse transcribed into extrachromosomal DNA, transposition target sites are not linked with the site of the original copy. Their transposition is regulated and most of the retrotransposons used for mutagenesis until now are activated by tissue culture, enabling large-scale generation of mutated populations. Retrotransposons have already been used successfully as mutagens in plants: Tos17, an endogenous retrotransposon of rice, is used for gene tagging in rice (Yamazaki et al., 2001). Moreover, it was shown that Tto1 and Tnt1 transpose in Arabidopsis (Okamoto and Hirochika, 2000; Courtial et al., 2001), and Tnt1 from tobacco (Nicotiana tabacum) is used in Medicago truncatula (d'Erfurth et al., 2003; Tadege et al., 2005). These studies revealed another important advantage of the use of retrotransposons over more traditional DNA-type elements: They can be highly mutagenic because some of them prefer to transpose into gene-rich regions. This last feature could make them ideal genetic tools for gene tagging in plants with a large genome.
Our purpose was to explore the opportunities of developing a retrotransposon mutagenesis strategy in lettuce (Lactuca sativa) using the heterologous element Tnt1. Lettuce is an important crop species belonging to the large Compositae family. The Compositae Genome Initiative (http://cgpdb.ucdavis.edu) has generated an enormous amount of DNA sequence data, especially ESTs, from the two major representatives of this family: lettuce and sunflower (Helianthus annuus). However, lettuce has a genome of 2.65 pg/1C, corresponding to 2,600 Mb per haploid genome, approximately 18, 6, and 5 times larger than Arabidopsis, rice, and Medicago, respectively (Arumuganathan and Earle, 1991
In this article, we show that Tnt1 transposes efficiently in lettuce during in vitro culture and that the insertions are stable and genetically unlinked. We also demonstrate the feasibility of gene tagging in lettuce by identifying and isolating an insertion of Tnt1 into a GA
Transposition of Tnt1 during in Vitro Transformation of Lettuce Occurred Simultaneously with T-DNA Integration
Tnt1 is an active 5.3-kb-long copia-like long-terminal repeat (LTR) retroelement isolated from tobacco (Grandbastien et al., 1989
Figure 1B shows the results of the Southern hybridization on T0 genotypes (primary transformants) derived from six bushes. Buds from the same bush are designated by the same number and differ by the letter placed just after. Each blot was successively hybridized with two probes, namely, R-U5 (fragment of Tnt1 localized in the LTR) and Kana (fragment of the nptII gene). When T-DNA inserts were complete, hybridization with the R-U5 probe on HincII-digested genomic DNA revealed two internal fragments of 630 and 1,500 bp (Fig. 1A). Of the 21 buds analyzed, three (MarTnt21a and two buds from the same bush, JesTnt7) did not reveal the two internal fragments and thus probably contained a truncated version of the T-DNA without Tnt1. Two buds, MarTnt27a and JesTnt2a, revealed only the 630-bp fragment, indicating a longer truncated version of the T-DNA containing only the 3'-LTR from Tnt1. Thus, although the Kana probe revealed that all the buds analyzed contained the right part of the T-DNA, four bushes out of the 14 analyzed (30%) contained only a truncated version of the T-DNA in which at least the 5'-part of Tnt1 was missing. Two buds (MarTnt14b and MarTnt10a) of the 21 analyzed displayed only the two expected fragments when the blots were hybridized with the R-U5 probe. The 14 remaining buds (corresponding to eight different bushes of the 14 analyzed) revealed the presence of multiple bands in addition to the 630- and 1,500-bp expected fragments. The high degree of intensity observed specifically at 1,500 bp corresponded to the internal fragment localized inside Tnt1, whereas no such intensity was visible for the 630-bp fragment that corresponded to an internal fragment specific to the T-DNA. The other fragments probably corresponded to transposed copies of Tnt1, although some of them could be due to methylation of some Tnt1 HincII sites (J. Perez, F. Potet, B. Courtial, and H. Lucas, unpublished data). Multiple transposed copies of Tnt1 were thus inserted in the genome of 14 buds originating from the two varieties of lettuce. Hybridization with the Kana probe confirmed the differences between the number of T-DNA inserts and the number of Tnt1 copies. Whereas the number of T-DNA inserts varied between one and three, the number of Tnt1 insertions was very high and difficult to estimate accurately on the blots. Hybridization with the Kana probe confirmed previous observations that buds from the same bush possess the same T-DNA inserts and derive from the same transformation event (Fig. 1C). The multiple-band patterns obtained with the R-U5 probe appeared to be identical for buds from the same bush, suggesting that transposition of Tnt1 occurred very early during the in vitro culture and transformation process, concomitant with the T-DNA transfer process.
When using Tnt1 for insertion mutagenesis in lettuce, it is important to be able to isolate plants carrying only one or a few insertions of Tnt1 to identify the insertions responsible for the useful mutant phenotypes. Transposed copies of the element should also be genetically unlinked to cover the whole genome. In addition, it is important that new rounds of transposition do not happen in subsequent generations. To investigate the genetic independence of the Tnt1 insertions, their segregation was studied in progenies of the MarTnt53a transformant. T1 plants obtained by self-pollination of the MarTnt53a primary transformant were backcrossed to the wild-type genotype ‘Mariska.’ Seventy-seven different BC1 genotypes, originating from 51 different T1 plants, were analyzed by Southern blot. Twenty-eight BC1 plants each containing between nine and 16 different Tnt1 insertions, but together including all the insertions present in the MarTnt53a primary transformant genotype, were selected and again backcrossed to ‘Mariska.’ Southern-blot analysis was performed on 104 different BC2 plants. Twenty-one of 104 BC2 plants analyzed are presented in Figure 2B . At least 28 different Tnt1 insertions were clearly identified. Provided that, among the different T0 genotypes showing transposed insertions, the MarTnt53a genotype was the one containing the lowest number of Tnt1 copies (Fig. 1), it can be assumed that the number of Tnt1 insertions exceeded 28 for the other genotypes obtained in the transformation experiment. For all 28 insertions, it was possible to detect segregation events by Southern-blot analysis. Four BC2 genotypes, each containing two to five Tnt1 insertions and together representing 11 different Tnt1 transposition events, were self-pollinated and 39 to 44 progenies per BC2 line were analyzed by Southern blot. The 12 different progenies presented in Figure 3 show these 11 Tnt1 insertions in different combinations of two or isolated (lanes 2, 3, 4, and 7). We looked for new fragments indicative of germinal transposition events of Tnt1 in the different progenies analyzed, but did not find any, indicating that Tnt1 does not transpose at a high frequency in the germ cells of lettuce. Taken together, the results showed no evidence of linkage between any of the Tnt1 inserts studied, but rather showed Mendelian segregation of the insertions.
Tnt1 Is Transcriptionally Active in the Early Stage of Regeneration
To analyze Tnt1 transcriptional regulation in lettuce, we introduced the pHLV5501 construct, a translational fusion between Tnt1 LTR and the GUS reporter gene (Courtial et al., 2001
Tnt1 Transposition Can Be Reactivated in Lettuce Activation of Tnt1 transposition by in vitro regeneration of lettuce genotypes containing one Tnt1 element could be an easy way to increase the size of a tagged population. To test the ability of several Tnt1 copies present in the lettuce genome to be activated for transposition, we used the 12 genotypes possessing one or two Tnt1 insertions issued from the MarTnt53a genotype by several backcrosses with the wild-type ‘Mariska,’ as presented previously (Fig. 3). Leaf explants taken from the 12 genotypes were submitted to in vitro regeneration with or without preliminary Agrobacterium coculture. As verified by Southern blot with a kanamycin probe (data not shown), all 11 Tnt1 inserts covered by the 13 studied genotypes were previously transposed copies without T-DNA. One to 15 independently regenerated shoots from each genotype were used for DNA extraction and Southern analysis. Figure 5 shows a Southern blot for the 14 regenerated shoots originating from MariskaTnt53a-51-2-4-6, obtained after cocultivation with an empty Agrobacterium followed by regeneration. Each new fragment indicates a transposition event of Tnt1, and seven of the 14 segregated shoots contained new transposition events. As summarized in Table I , all genotypes, except one (MarTnt53a-51-2-4-24), gave at least one regenerated plantlet with new Tnt1 transposed copies. It was not possible to find an obvious correlation between the frequency of transposition in the regenerated shoots and Tnt1 copy number or a specific insertion locus in the starting material. When Tnt1 transposition occurred, it generally gave rise to a large number of new insertion events (data not shown). Cocultivation of explants with an empty Agrobacterium strain prior to regeneration did not result in a significant increase in the frequency of Tnt1 transposition (Table I). Overall, around 40% of the regenerated plantlets contained new transposition events of Tnt1.
Transgenic Lettuces Containing Tnt1 Insertions: A Small Highly Mutagenic Population No phenotypic variation was observed in the T0 plants acclimated in the greenhouse from transformed bushes. However, phenotypic variants were found in four of eight T1 families studied. Three different genotypes (JesTnt1, JesTnt6, and JesTnt8) showed abnormal phenotype at the seedling stage in their T1 progenies. They were blocked at the cotyledon stage or the cotyledons showed a pigmentation defect (white for JesTnt1a-7 and yellow for JesTnt6a and JesTnt8a), and the roots were shorter than those of the wild type (Fig. 6A ). Segregation analysis of the mutant phenotypes observed in JesTnt6a and JesTnt8a T1 and T2 families indicated that they corresponded to a single recessive mutation (Table II ). Segregation analysis of the mutant phenotype in the JesTnt1a-7 T2 family suggested that this phenotype could be due to recessive mutations in two unlinked genes (the ratio of white to green seedlings was between those expected for one and two recessive mutations).
Other phenotypic variations were observed in the greenhouse at later stages of development in the T1 progeny of three genotypes. Dwarf plants appeared in JesTnt6 progeny with a very short internodal stem (Fig. 6C). Marked modification in the structure of the flower (absence of ligules and stamens) was observed in JesTnt1 progeny (Fig. 6B) and in the color of the ligule (light yellow) in MarTnt53 progeny. Segregation analysis of these mutant phenotypes in T1, T2, and T3 families suggested that all three corresponded to single recessive mutations (Table II). The high number of Tnt1 insertions in the genotypes presenting phenotypic alterations associated with the recessive character of those mutations did not allow identification of the inserts involved, except for the dwarf mutation for which several rounds of backcrosses were performed. Consequently, we cannot exclude the involvement of some endogenous transposable elements or other mutations in addition to Tnt1 to generate these phenotypic alterations.
One significant advantage of using retrotransposons for gene tagging in plants with large genomes is the preference some display to transpose into gene-rich regions. To test Tnt1 mutagenic activity in lettuce, 25 different lettuce Tnt1 insertion sites longer than 100 nucleotides were isolated by sequence-specific amplification polymorphism (S-SAP) and then sequenced (Table III
). Sequences were processed to obtain lettuce DNA fragments corresponding to the 5'-Tnt1 flanking sequence. The cleaned sequenced DNA fragments varied in size from 114 to 557 nucleotides. Sequences were compared with four of The Information for Genomic Research (TIGR) databases (lettuce, sunflower, tomato [Solanum lycopersicon], and potato [Solanum tuberosum]) using the BLASTN program. We also compared the translated sequences of the sequenced DNA fragments with these translated databases and with two protein databases (Arabidopsis and Swiss-Prot TrEmbl) using the BLASTX program to see whether we could detect any homology with known or hypothetical proteins. Of the 25 sequences analyzed by BLASTN, eight sequences (32%) showed no homology. Fourteen sequences (56%) showed some similarities with ESTs or known mRNAs, with E values between 7.8e-103 (perfect match, i.e. certain) and 5.7e-05 (probable but not certain). E values above 1e-05 were regarded as not significant and not included in Table III. Apart from sequence HEL185 (which perfectly matched lettuce Ls3h1 mRNA coding for the GA 3
Tnt1 Insertion into a GA -Hydroxylase Gene Induces a Dwarf Phenotype in LettuceTo demonstrate that Tnt1 was responsible for the mutant phenotype observed, we decided to study the dwarf mutant in more detail. The mutant phenotype was detectable at an early stage of development, and, despite the absence of a flower stem, the plants were partially fertile and generated 100% mutant progenies after self-pollination. Southern-blot analysis of 60 T3 JesTnt6a plants showed that the Tnt1 copy number was too high to allow correlation with the presence of a specific fragment with the dwarf mutation (data not shown). One T3 mutant plant was backcrossed with the wild type. Five BC1 plants selected to posses the lowest Tnt1 copy number (between 25 and 30) among the 100 analyzed were grown in the greenhouse and self-pollinated. Dwarf plants were selected from among these BC1 progenies and the Tnt1 copy number was assessed by Southern blot for 100 of them. The plant that contained the lowest copy number of Tnt1 (between eight and 10) was backcrossed to the wild type and five BC2 plants out of the 50 analyzed by Southern blot, containing four to five Tnt1 inserts, were allowed to grow in the greenhouse for self-pollination. Fifteen to 25 progenies per BC2 plant were allowed to grow in the greenhouse for detection of the mutant phenotype and mutant genotypes were analyzed by Southern blot. The mutant plant containing the smallest copy number (two) was then self-pollinated, giving 100% mutant progenies with some containing only one Tnt1 copy (Fig. 6D, 1).
The S-SAP fragment corresponding to a Tnt1 insertion site in the lettuce GA
In this article, we have shown that the tobacco retrotransposon Tnt1 transposes in lettuce. Molecular analysis of several individuals isolated from a common regenerated ancestor bush demonstrated that the transposition of Tnt1 occurred very early during the lettuce transformation process, concomitantly to T-DNA transfer. Of the 10 different bushes containing a complete version of the T-DNA analyzed, two bushes contained only the T-DNA insert, whereas the remaining eight contained multiple transposed copies of Tnt1. The number of transposed copies per plant was particularly high and difficult to estimate accurately on the T0 Southern blots. Whereas in Arabidopsis the number of transposed Tnt1 copies fluctuated between 0 and 26 (Courtial et al., 2001 ) and in Medicago between four and more than 30 (d'Erfurth et al., 2003), the number of Tnt1 insertions obtained in lettuce was estimated by molecular analysis performed on successive backcrosses to be 28 for one genotype and much higher for the seven remaining T0 genotypes, revealing a highly efficient rate of transposition. This high copy number per plant will be an advantage for lettuce mutagenesis because it reduces the number of transgenic lines that have to be produced to saturate the genome, although the time needed to segregate these elements afterward to show that the mutant phenotype is tagged could be a limiting factor. Based on the equation of Krysan et al. (1999) and assuming an average gene length of 3.5 kb and that Tnt1 insertions are random, we estimate that 2,225,400 insertions would be required to have a 95% probability of tagging any given gene in the 2,600 Mb lettuce genome. With a postulate that a Tnt1 average copy number of 50 insertions per line inserted into unlinked loci in lettuce, the same level of saturation could be obtained in 44,508 lines. Contrary to results obtained with Medicago showing plants containing transposed Tnt1 copies without T-DNA insertions, the four different regenerated lettuce bushes containing only truncated versions of the T-DNA did not show any transposed copies of Tnt1.
The study conducted on two consecutive backcrosses and one self-pollination performed with a given T0 transformant demonstrated the genetic independence of the different Tnt1 copies and the Mendelian segregation of the insertions. Furthermore, several different single-copy insert plants were easily obtained after only three backcrosses and one self-pollination, starting with a plant containing 28 copies of Tnt1, demonstrating the feasibility of separating a given Tnt1 insertion from the others even from a genotype containing large numbers of transposed copies. No new transposition events were observed in any of the progenies studied, as already observed in Arabidopsis and Medicago (Lucas et al., 1995
Using LTR-GUS fusion, we demonstrated that transcription of the element, driven by its LTR promoter, is highly regulated. LTR-GUS products are poorly expressed in most intact lettuce leaf tissues of transgenic plants, except roots. However, Tnt1 transcription is strongly induced in lettuce by in vitro culture regeneration experiments. Transcription activation occurs very early, 2 d after the beginning of the experiments, and then decreases to no transcription at all in newly regenerated plantlets. Thus, the absence of germinal transposition events in the progeny of transgenic plants carrying Tnt1 and the early occurrence of transposed copies obtained in transgenic lettuce after in vitro regeneration and transformation are in keeping with the element's transcription pattern. Tnt1 transcription has previously been shown to be strongly regulated and induced by different biotic or abiotic stress factors, such as treatment with herbicides, microbial elicitors, wounding, and 2,4-dichlorophenoxyacetic acid (Pouteau et al., 1991
We also showed that transposition of integrated copies of Tnt1 in the lettuce genome can be reactivated by tissue culture. As already observed for T0 genotypes obtained after transformation, when transposition occurred, the number of Tnt1 copies was particularly high. Whereas transposition was obtained for all Tnt1 insertions studied, showing their functional integrity, only 38% of the regenerated plantlets studied showed transposition events. Cocultivation of explants with an empty Agrobacterium strain prior to regeneration did not result in a significant increase in the frequency of Tnt1 transposition compared to simple regeneration experiments. Thus, Agrobacterium does not seem to particularly induce Tnt1 transposition, contrary to the in vitro culture regeneration. The absence of transposed copies observed in some of the regenerated plantlets, as well as in some of the regenerated bushes obtained after the transformation experiment, might be due to the lettuce regeneration protocol used. Indeed, lettuce is a plant that is easily regenerated in in vitro culture and thus does not need large quantities of growth regulators in the regeneration medium (0.15 mg/L benzyladenine acid and 0.3 mg/L indole acetic acid) to obtain 100% regeneration of the leaf explants placed on the medium. Modification of this regeneration protocol by using higher amounts of growth regulators or 2,4-dichlorophenoxyacetic acid, previously shown to induce Tnt1 transposition (Pauls et al., 1994
To obtain efficient mutagenesis in plants with large genomes, it is important to use a transposable element that prefers to insert in genes rather than in noncoding regions. The genome of most crop plants is large compared to the plant model Arabidopsis (145 Mb). The size of the lettuce genome is 2,639 Mb (i.e. similar to that of higher plants like maize or sunflower; Arumuganathan and Earle, 1991 In addition, we demonstrated the feasibility of using Tnt1 as a gene-tagging tool in lettuce by isolating the Tnt1 insertion responsible for dwarf mutation. Even if the involvement of Tnt1 insertions was not demonstrated for the other mutant phenotypes observed in the progeny of the T0 plants, their surprising number with respect to the small starting T0 population generated provides indirect additional proof of Tnt1 preference for insertion in transcribed regions and its potentially highly mutagenic characteristics.
Several transposon systems have already been constructed for the functional analysis of genes (reverse-genetics analysis) and gene tagging in plants (for review, see Parinov and Sundaresan, 2000 In this article, we showed that insertional mutagenesis and gene tagging using the retrotransposon Tnt1 is also feasible in lettuce, despite its large genome, 5 to 6 times bigger than that of rice or Medicago. The results obtained show that retrotransposons should not be underestimated as alternative tools especially in plants amenable to tissue culture but in which classical genomics and whole-genome sequencing are not easy to achieve. In the future, this could help to identify the similarities or differences in coding sequences or coded functions existing between well-studied model species and a crop plant with agronomical useful traits or between plants belonging to distant families.
Plant Material and Plant Growth Conditions Two butterhead lettuce plants (Lactuca sativa) were used: ‘Mariska’ from Numhems and ‘Jessy’ from Caillard. For germination, seeds were sown in petri dishes containing filter paper moistened with water and exposed to 4°C in the dark for 48 h before being placed in a growth room (24°C, 16-h photoperiod, 60 µE m–2 s–1) for 5 to 10 d. Seedlings were first grown on compost in 7-cm pots in a greenhouse for 3 weeks before being transplanted in 3-L compost pots and watered with nutritive solution. Mutant plants whose stems failed to elongate in the greenhouse were treated by applying 5 mL of a solution containing 0.02% (w/v) of GA3 (C19H2206; catalog no. G 0907; Duchefa) to the top of the plants in the greenhouse at the heading stage.
The Tntk23 vector (Fig. 1A) is a derivative of the pBin19 vector carrying the autonomous Tnt1 retroelement (Lucas et al., 1995
Plasmids were introduced in Agrobacterium tumefaciens strain C58 pGV2260 (Deblaere et al., 1985
Lettuce genetic transformation procedures are described by Dinant et al. (1997)
Newly in vitro-regenerated plantlets (without transformation) were produced from true leaves taken from sterile seedlings grown in vitro or from selected plants growing in the greenhouse, surface sterilized by immersing in a disinfecting solution for 30 min (Mazier et al., 2004
Genomic DNA was extracted from leaf tissues using a modified cetyltrimethylammonium bromide method (Bernatzky and Tanksley, 1986 Hybridization with 32P-labeled probes was performed at 65°C in hybridization buffer (750 mM NaCl, 125 mM sodium citrate, 0.6% SDS, 50 mM Na2HPO4/NaH2PO4, pH 7.5, 5x Denhart, 2.5 mM EDTA, 5% dextran sulfate, 2.5 mg DNA salmon sperm) for 16 to 24 h. The filters were then washed one time in 2x SSC; 0.1% SDS at 65°C for 20 min followed by one wash in 1x SSC; 0.05% SDS at 65°C for 20 min and a final wash in 0.5x SSC; 0.05% SDS at 65°C for 10 to 20 min. Filters were exposed and analyzed by autoradiography.
PCR analysis of the Tnt1 insertion responsible for the dwarf mutation was performed using primers LTR1R, 5'-GGCTACCAATCCAACAAGGA-3' (complementary to Tnt1); Gi1F, 5'-CAAACGCCATGAAGCTTGT-3' (GA
The sequences flanking Tnt1 integration sites were isolated by S-SAP and sequenced as described by Courtial et al. (2001)
Sequence chromatograms were analyzed using phred software (Ewing et al., 1998
Histochemical staining for GUS activity was carried out according to Jefferson (1987) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF397531 to EF397555.
We are grateful to V. Sarnette for excellent plant care and backcrossings. We thank Y. Bellec and E. Martin for technical assistance during cultivation of T0 and T1 plants and their progenies. We wish to thank M. Pitrat, P. Ratet, and S. Muños for their advice and helpful comments, and J. Chadoeuf for help in calculations and probability formulas. Received September 26, 2006; accepted January 30, 2007; published March 9, 2007.
1 Present address: Laboratoire National de la Protection des Végétaux, Unité de Détection des OGM, 93 Rue de Curembourg, 45404 Fleury-les-Aubrais cedex, France.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Marianne Mazier (mazier{at}avignon.inra.fr). www.plantphysiol.org/cgi/doi/10.1104/pp.106.090365 * Corresponding author; e-mail mazier{at}avignon.inra.fr; fax 33–4–32–72–27–02.
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-amylase expression in aleurone and flower development. Plant Cell 16: 33–44