Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.     

Efficient Identification of Arabidopsis Knock-Out Mutants Using DNA-Arrays of Transposon Flanking Sequences

Abstract: Mutants obtained by insertional mutagenesis are widely used for determining gene-phenotype relationships. In Arabidopsis thaliana, several populations mutagenized either by T-DNA or transposon insertion are available for screening for knockout mutants in genes of interest. We have so far screened our Arabidopsis population mutagenized with the Zea mays transposon En-1/Spm for insertion mutations in 718 genes, using PCR on DNA pools. Although successful, this common approach is too time consuming for use in systematic screening of all 25 498 predicted genes of the Arabidopsis genome. We therefore investigated the use of DNA arrays for the direct identification of mutants in our population. All transposon-flanking regions from individual plants are amplified by PCR and subsequently spotted at high density onto nylon membranes. A single hybridization experiment with a gene-specific probe then allows one to identify candidate mutant plants. The efficiency of each separate step was determined and optimized. Screening of filters representing 2880 plants for insertions in 144 genes and subsequent investigation of some of the potential insertion mutants suggest that an overall screening efficiency of 50 % is attained.     

Facilitating the recovery of phenotypically normal transgenic lines in clonal crops: a new strategy illustrated in potato

Transgenic plants frequently exhibit altered phenotypes, unrelated to transgene expression, which are attributed to tissue culture-induced variation and/or insertional mutagenesis. Distinguishing between these possibilities has been difficult in clonal crops such as potato, due to their highly heterozygous background and the resulting inherent phenotypic variability associated with segregation. This study reports the use of transgene integration as a molecular marker to trace the clonal origin of single cells in tissue culture. Following transformation, multiple shoots have been regenerated from cell colonies of potato (Solanum tuberosum L.) and Southern analysis used to confirm their derivation from a single transformed cell. Analysis of phenotypic variation in field trials has demonstrated marked differences between these multiple regeneration events, the origin of which must have occurred after T-DNA insertion, and consequently during the tissue culture phase. This result unequivocally demonstrates that somaclonal variation occurs during tissue culture and independent of transgene insertion. Furthermore, the first shoots recovered do not necessarily exhibit less somaclonal variation, since later regeneration events can give rise to plants that are more phenotypically normal. Therefore, when developing transgenic lines for genetic improvement of clonal crops, multiple shoots should be regenerated and evaluated from each transformation event to facilitate the recovery of phenotypically normal transgenic lines.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these second generation callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.Abbreviations SH Schenk and Hildebrandt medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.     

Genetic and physiological variability among cultured cells and regenerated plants of Nicotiana tabacum

Summary Tobacco cell lines selected for resistance to picloram (4-amino-3,5,6-trichloropicolinic acid) and plants regenerated from these cell lines manifest several traits not shown by the parental strains. Genetics analyses of the regenerated plants have permitted the sources of this variability to be identified.Tricotyledenous seedlings appeared at a much higher frequency among the progeny of a heterozygous mutant plant (PmR1/+) regenerated from culture than they did among progeny of normal regenerated plants. In crosses with the regenerated heterozygous mutant plant and with homozygous progeny of this plant (PmR1/PmR1) the frequency of tricotyly was influenced more by the generation than by the genotype of the parent plant. Therefore, it is concluded that tricotyly is a physiological response to passage through cell culture.More than half of the picloram-resistant cell lines isolated were also resistant to hydroxyurea. Segregation of these two resistances was analyzed in progeny of crosses with regenerated plants. In all cases hydroxyurea-resistance was genetically stable and inherited as a single dominant nuclear mutation (designated HuR). In crosses with plants PmR1/+ and PmR7/+ the HuR and PmR mutations assorted independently. In contrast, the HuR mutation recovered from plant PmR6/+ was linked to the PmR6 mutation.     

Analysis of genomic DNA methylation patterns in regenerated and control plants of rye (Secale cereale L.)

Rye (Secale cereale L.) is a species that has shown high rates of somaclonal variation when plants obtained by in vitro culture were analysed using different techniques. In this study, using methylation-sensitive amplified polymorphism (MSAP) markers, we analysed the cytosine methylation status at genomic level of regenerated plants of rye that were obtained by somatic embryogenesis. Such plants were originated from three different cell lines and the results were compared with the data obtained from the control plants grown from seeds of the same cultivar and lot. A similar total number of MSAP markers was observed in the regenerated (937) and control plants (1,022), while the mean number detected per plant was significantly higher in regenerated (554.43) than in control plants (356.00). The analysis indicated conservation of the number of partially-methylated CCGG/GGCC sites for all type of plants. However the mean number of non-methylated sites was near twofold in the regenerated plants (442.48) than in control plants (248.19). Methylation changes have been detected in all the regenerated plants when compared within cell lines, with an average frequency of 9.01 % of the detected markers. We also observed that regenerated plants from one or several cell lines shared methylation changes at the same locus pointing to a non-random behaviour of the changes in genomic methylation.