Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis. L. Scaramelli, A. Balestrazzi and M. Confalonieri have contributed equally to this work.     

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

Transformation of kiwifruit using the <Emphasis Type="Italic">ipt</Emphasis> gene alters tree architecture

To manipulate the architecture of woody plants by controlling endogenous cytokinin levels, the isopentenyl transferase gene (ipt) from Agrobacterim tumefaciens was introduced to kiwifruit using stable transformation. Consequently, eight transgenic lines were obtained. Transgenic shoots harboring the ipt gene were recalcitrant to rooting under tissue-culture conditions; thus, their in vitro-cultivated shoots were directly grafted onto potted wild-type kiwifruit seedlings to evaluate their morphological features, and three lines (tmr2-4, tmr2-G, tmr3-C) were successfully grafted. The grafted transgenic plants had dwarfing and branching phenotypes, both of which are typical features of cytokinin overproduction. In addition, the number of buds increased and internode length was shorter in the grafted transgenic plants. The content of a precursor, trans-zeatin riboside, and an active cytokinin, trans-zeatin, increased in one transgenic line, in which the level of ipt gene expression was high, indicating that morphological changes were related to expression levels of the ipt gene and cytokinin content. Possibilities for potential utilization of the ipt gene in manipulating tree shape are discussed.     

Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis>

The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt ⁻, gus ⁻ but lacZ ⁺ as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene.     

Expression of the PsMT A1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8-hydroxy-2′-deoxyguanosine mediated-DNA damage

Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT _A1 gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT _A1 -NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT _A1 -NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl₂, a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMT_A1 plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMT_A1 lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2′-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.     

Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation

Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.Abbreviations BA benzyladenine - CW coconut water - Cef cefotaxime - P peppermint - SS scotch spearmint - NS native spearmint - OM orange mint - BM basal medium - MS Murashige and Skoog (1962) - PAR photosynthetically active radiation - CTAB hexadecylatrimethylammonium bromide - ipt isopentenyl transferase Received for publication 1994. Published as Miscellaneous Paper No. 1482 of the Delaware Agricultural Experiment Station. Contribution No. 317 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.     

Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium,but not nitrogen‐fixing rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR‐Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.     

Effective selection system for generating marker-free transgenic plants independent of sexual crossing

 In a previous report, a novel selection protocol termed "the MAT-vector system" for generating marker-free transgenic plants (MFTPs) was presented. The first stage of the system is visual selection of morphologically abnormal transgenic shoots, ipt-shooty, that have lost apical dominance and rooting ability. The second stage involves elimination of the ipt gene and the appearance of MFTPs free of ipt gene influence. The present report describes a practical MAT-vector in which removal of the ipt gene is efficiently mediated by the site-specific recombination system R/RS from Zygosaccharomyces rouxii, in place of the maize transposable element Ac, used previously. This improved MAT-vector produced MFTPs from 39% of moderate ipt-shooty and 70% of extreme ipt-shooty lines. These results are superior to the previous MAT-vector which produced MFTPs from only 5% of ipt-shooty lines. The present novel system also induced direct development of MFTPs from adventitious buds without production of ipt-shooty intermediates. The presence of β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) genes of interest, and the absence of the ipt gene were verified by a GUS histochemical assay, NPTII assay, and molecular analysis. Received: 19 June 1998 / Revision received: 4 December 1998 / Accepted: 18 December 1998     

Production of marker-free transgenic Nierembergia caerulea using MAT vector system

Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt ⁻, GUS⁺ and excision⁺ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.     

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR limited host range - WHR wide host range - onc oncogenicity genes - iaaH indoleacetamide hydrolase gene - iaaM tryptophan monooxygenase gene - ipt isopentenyl transferase gene - tzs transzeatin secretion gene - NAA -naphthalene acetic acid - BAP 6-benzylaminopurine - Km kanamycin - Neo neomycin - Cm chloramphenicol     

Expression of isopentenyl transferase gene (<Emphasis Type="Italic">ipt</Emphasis>) in leaf and stem delayed leaf senescence without affecting root growth

A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T₂ progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T₂ progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root growth and morphology of the plant were not affected in the transgenic tobacco.     

Combining a regeneration-promoting <Emphasis Type="Italic">ipt</Emphasis> gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted in the apricot cultivar ‘Helena’. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants by site-specific recombination.