Apple Malus x domestica Borkh. has a long period of juvenility during which plants are unable to produce flowers and fruits. Flowering is only possible on trees which have passed the transition from juvenility to the adult stage. Several genes have been identified which control this switch and promote or delay flowering in different plant species. FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) are two flowering genes of Arabidopsis which are similar, but act antagonistically. FT and TFL1 homologous genes are present in the apple genome, but little is known about their function. Therefore we produced transgenic apple lines which over-expressed the MdFT2 gene under the control of the constitutive CaMV 35S promoter. Transgenic shoots expressing the MdFT2 gene driven by the CaMV 35S promoter flowered in vitro. To study the function of MdTFL1 we silenced the gene by expressing an MdTFL1 specific hairpin under the control of the CaMV 35S promoter. Transgenic plants of different apple cultivars flowered in vitro and later in the greenhouse. Transgenic flowers were evaluated for phenotypic differences and tested on their usability for crossbred breeding programs.

We used an RNAi based approach to induce post-transcriptional gene silencing of the MdTFL1 gene in apple in order to reduce the juvenile phase. The MdTFL1 protein is homologous to TFL1 of A. thaliana which suppresses the floral meristem identity genes LFY and AP1 and maintains the inflorescence meristem. A binary vector was constructed which contains a constitutively expressed nptII gene and a chimeric gene construct encoding for a hairpin RNA homologous to the coding sequence of MdTFL1. The vector was used to transform the apple (Malus domestica Borkh.) cvs. ‘Holsteiner Cox’ and ‘Gala’ via Agrobacterium tumefaciens-mediated transformation. Regenerated shoots were proven for transgenity by Southern blot and RT-PCR. Quantitative real time PCR analysis showed that the expression of MdTFL1 was markedly reduced in transgenic lines compared to non-transformed control plants. The regenerated transgenic plants started to flower six month after the transformation under in vitro conditions. The plants were recently transferred to the greenhouse where they continued to flower.

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants including pathogen defence. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Modifying flavonoid biosynthesis in fruit crops, such as apple, offers the opportunity to increase plant resistance against pathogens and the health benefit potential of the fruit. Both overexpression and RNAi-based suppression strategies were used to modify flavonoid biosynthesis in apple. Introducing the maize Lc transcription factor gene, responsible for controlling the expression of structural genes of the flavonoid biosynthetic pathway in maize, into Malus domestica Borkh. cv. 'Holsteiner Cox' resulted in increased mRNA levels for most of the structural genes of the flavonoid pathway. Lc transgenic plants accumulated higher levels of the anthocyanin idaein, the flavan-3-ols epicatechin, catechin, and some distinct dimeric proanthocyanidins. In a second approach, the consequences of RNAi silencing of the genes for anthocyanidin synthase (ANS) and UDPgalactose: flavonoid 3-O-galactosyltransferase (FGT) gene to induce a shift towards flavan-3-ols were examined. Preliminary results obtained from transgenic plants of the cv. 'Holsteiner Cox' and of the red-leaved type TNR 31-35 (a descendent from Malus sieversii var. sieversii f. niedzwetzkyana (Dieck)) revealed that suppression of ANS or FGT resulted in increased amounts of flavan-3-ols and flavonols. In the red leaved type TNR 31-35, anthocyanin accumulation decreased after ANS silencing.

RNA silencing is based on the processing of double stranded RNA into small RNAs by DICER-like proteins. These small RNAs guide ARGONAUTE-like proteins to complementary sequences resulting in posttranscriptional gene silencing. Silencing is a non-cell autonomous event. We used the binary vector pHELLSGATE8::gus containing nptII and a gene construct encoding for a hpRNA homolog to the gene gusA (sigus) for transformation of a ‘Pinova’ descendant, to evaluate the mobility of silencing for the first time on a woody plant like apple. 13 sigus transgenic apple lines were obtained. Following, gusA transgenic apple shoots were grafted onto sigus transgenic rootstocks in the greenhouse and in vitro. GUS staining assays revealed that the gusA expression in the gusA transgenic scion was clearly decreased in leaves and shoots compared to control shoots grafted onto non-transgenic shoots. Real-time PCR analyses showed that the reduction of gusA expression occurs on the RNA level.

Preliminary results are presented on a screening of resistance to Verticillium on Asian Fragaria species after inoculation under greenhouse conditions. Different accessions of the same species exhibit different degrees of resistance. Three clones of Fragaria nubicola resulting from one seed source showed differences in resistance reaction from susceptible to tolerant.

Two different selection systems which do not require the use of antibiotics or herbicides were tested in apple transformation: the mannose/phosphomannose isomerase (pmi) and the galactose/UDP-glucose:galactose-1-phosphate uridyl-transferase (galT) system. Pmi transgenic plants are capable of converting the non-metabolisable sugar mannose into a usable source of carbon, fructose. The toxicity of galactose is attributed to accumulation of galactose-1-phosphate. GalT converts galactose-1-phosphate to UDP-galactose and glucose-1-phosphate, that might be sufficient to provide a reduction of galactose toxicity suitable to allow transgenic plants to survive on galactose media. Sensitivity of apple to the selective agents mannose and galactose was tested by exposing leaf explants to various combinations of mannose and sorbitol or galactose and sorbitol supplemented media. Results indicated that apple is not able to utilize these sugars as a carbon source. Young leaves of Malus domestica Borkh. 'Holsteiner Cox' were transformed via Agrobacterium tumefaciens with the binary vectors pNOV2819-GUS (pmi and GUS gene) or pBI-galT (galT, nptll and GUS gene) and selected using mannose, galactose or kanamycin. Several transgenic lines were regenerated using the pmi/mannose system. Transgene integration was confirmed by PCR and southern blot analysis. Explants transformed with pBI-galT developed only callus when galactose was used as selective agent. Thus no transgenic plants were recovered using the galT/galactose selection system.

A positive selection system based on the use of the phosphomannose isomerase (pmi) gene as a selectable marker gene and mannose as the selective agent was used for the transformation of apple (Malus domestica Borkh.). pmi converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells are capable of utilizing mannose as a carbon source. Explant sensitivity to mannose was tested by transferring leaf explants on regeneration media supplemented with various combinations of mannose and sorbitol. Results indicated that apple is not able to utilize mannose as a carbon source. Young leaves of the apple cv. 'Holsteiner Cox' (Malus domestica Borkh.) were transformed via Agrobacterium tumefaciens. In order to develop and to optimize selection and transformation conditions, the histochemical marker gene for beta-glucuronidase (GUS) was used to monitor transformation events. Transgenic plants regenerated on media containing various concentrations mannose and sorbitol. Integration of the phosphomannose isomerase gene was proven by PCR. The results indicated that the mannose selection system could be used for apple transformation, although selection conditions need to be optimised.

Independently transformed Malus domestica 'Elstar' and 'Holsteiner Cox' lines, each harbouring different copy numbers of the stilbene synthase gene from grapevine (Vitis vinifera L.) and the bar gene from Streptomyces hygroscopicus, were used to assess transgene expression stability in apple plants and their fruit. The stilbene synthase is responsible for the synthesis of the phytoalexin resveratrol in grapevine and was introduced to improve fungal resistance. The bar gene confers resistance against the herbicide glufosinate ammonium and was cotransferred as a selectable marker. The investigation included analysis of leaf material from long-term in vitro cultures, from plants growing on their own roots as well as grafted on a rootstock for three years under greenhouse conditions, and from young and mature transgenic apple fruit. RT-PCR analysis for detecting specific mRNAs, HPLC for proving resveratrol synthesis and leaf paint assays with glufosinate ammonium to examine herbicide detoxification revealed stable expression of the transgenes in all lines except one 3.5 years after the initial transformations. Preliminary results using fruit material revealed that the genes are also active in apple fruit tissue.

Resveratrol is a phytoalexin produced in different unrelated plant species such as grapevine and peanut. The effect of resveratrol and its glucoside trans-resveratrol-3-O-beta-D-glycopyranosid (piceid) against Venturia inaequalis, the causal agent of apple scab, was evaluated using a newly established test based on enzymatically isolated cuticular membranes (CMs) from apple (Malus domestica Borkh.) leaves. The test substances resveratrol and piceid were either sorbed to CMs before inoculation with spores or were applied simultaneously with the Venturia inaequalis spores to the CMs, and their effect on germination, appressoria formation, and penetration was examined. Resveratrol had no influence on spore germination but a significant inhibiting effect on penetration when applied simultaneously as well as before. A percentage inhibition of 89.7 +/- 11.5 and 61.8 +/- 35.1 was observed for simultaneous and preapplication, respectively. The resveratrol glucoside piceid had a significant inhibitory effect on germination and completely inhibited penetration of the fungus at concentrations between 200 and 400 microg mL(-1) when applied simultaneously with the spores to the CMs. On piceid-enriched CM (preapplication), spores germinated but penetration was inhibited nearly completely (96.1 +/- 5.1%). Thus, in vitro experiments showed that resveratrol and its glucoside in fact could contribute to improving the pathogen resistance of apple leaves.

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstarand Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase(Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi (Actinidia deliciosa) were transferred into Holsteiner Coxand Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.

Based on the observation that most of the shoot regeneration from apple leaf explants is associated with cutting edges and wounds caused by forceps and that Agrobacterium tumefaciens-mediated transformation depends on wounding (Norelli et al., 1996) we tested the influence of sonication on regeneration and transformation efficiencies by using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transfer, Trick and Finer, 1997). Leaf explants from three apple varieties, Holsteiner Cox, Gloster and Elstar, were treated in Agrobacterium (EHA101, pIBGUS) suspension with sonication for various timed intervals (10, 20, 40 and 80s). First results indicate an increased stable beta-glucuronidase expression in 'Holsteiner Cox' leaf tissue. Improvement of transformation efficiencies seems to be genotype dependent, because the transformation efficiencies in 'Elstar' and 'Gloster' were not influenced. Regeneration rates of the three varieties, however, are drastically reduced through sonication, depending on the duration of the treatment.