The GENERAL OBJETIVE of the project was the identification of morphological, physiological and molecular markers for the characterization of tissues in the juvenile and mature phase and for rapid change from vegetative into reproductive phase.

The main objetives of this project were:

The tree species selected for this project consisted of gymnosperms Pinus radiata (radiata pine), Pinus pinaster (maritime pine), Pinus pinea (umbrella stone pine), Pinus nigra (black pine) and angiosperms Prunus avium (wild cherry), Corylus avellana (hazelnut), Prunus persica (peach) and Malus domestica (apple).

The plant material available comprehended parental trees, seeds and seedlings from different progenies, rejuvenated shoots by in vitro or ex vitro manipulations, graftings, re-graftings and cuttings from selected mature trees in different developmental stages. Different types of tissues and vegetative structures were analysed within this plant material.

During this project different types of markers for phase change from juvenile to the adult vegetative stage, to the adult reproductive stage and for rejuvenation were identified in model species and developed for the forest species mentioned before. These markers included molecular markers based on tissue specific mRNAs, isozymes, plant growth regulators and other physiological, histological and morphological markers. In order to test their general applicability, molecular markers developed by individual participants were interchanged and tested by other groups on different plant species and using different juvenile, mature and rejuvenated tissues. On the other hand plant material of different species were made available to the participants studying isozymes, plant growth regulators, physiological and histo-morphological markers in order to extent their analyses to other species and tissues. Promising markers for propagation and flowering behaviour were validated at larger scale through field trials. All results were made available through an electronic database.

To accomplish the main objectives mentioned above, the following detailed objectives were fulfilled:

This objective included on one hand the development of differentially expressed cDNA markers in certain model species. On the other hand the applicability of known, available genes with differential expression was tested on this plant material.

In this objective enzyme activities and specific isoforms of catalase (CAT) and superoxide dismutase (SOD), with prooven differential expression in the course of plant development were determined in different tissues of adult and juvenile plant material. Genes coding for these enzymes were identified and their expression patterns were analyzed in the plant material and tissues under study.

This objective comprehended the analyis of the variation on meristem characteristics (morphology, zonation) and the in situ localization of biochemical and molecular markers linked to phase change in meristems and shoot apěces of juvenile, adult vegetative and rejuvenated tissues and during the transition to flowering.

In this objective the importance of intracellular pH, membrane potential and Ca2+ for the regulation of cell cycle and development was determined in juvenile and adult plant material.

This objective included the optimization of physico-chemical and inmunological techniques for PGR quantification and the determination of PGR levels and ratios in different juvenile and adult plant material. The analysis of phytohormones (auxins, cytokinins, abscisic acid and ethylene), polyamines, phenolic compounds and ionic content allowed to establish a relationship between levels of these compounds and the physiological state, in order to determine specific PGR ratios for the characterization of maturation and rejuvenation.

Identified markers on the specific material of each group were tested first intensively in the same species. e.g. different crossings, other accessions and tissues, and then, if appropiate, on other species used by the different participants in order to test their general applicability.

For this purpose two working tools, a Molecular Marker Set (MMS) containing available and developed useful cDNA clones and a Plant Material Package (PMP) containing different juvenile and mature plant material of different species was made available to all participants in order to extent their analyses to other species or to test the suitability of cDNAs from other participants.

In this objective the applicability of the most promising markers - in terms of simplicity, economical feasability and reproducibility - which had been identified on a reduced set of plant material in objectives 1 and 2, were evaluated at a larger, commercial scale through field trials. For this purpose either the expression of molecular and non molecular markers on material with known propagation and flowering characteristics was tested, or the predictive power of these markers on material analyzed previously for marker expression was validated.

The results on the different phase change markers were published in an electronic database which is available on Internet.

Initially partner P01 adapted successfully all basic techniques which were applied to P. radiata.Two different strategies (DDRT-PCR, cDNA-AFLP) were used for the identification of differentially expressed cDNAs. After confirmation by Northern analysis and discarding ribosomal bands a total of 5 DDRT-PCR fragments and 5 cDNA-AFLP fragments were obtained with the model system, which consisted of parental trees and progeny genotypes of the controlled cross P68xP40.

Partner P01 obtained in total 10 reproduceable, differentially expressed cDNAs. Four of them appeared only in the mature tissues of the parental trees of our cross, while the others were only present in pools of juvenile tissues from the progeny seedlings. Most of the detected fragments were of similar size, around 300 bp; with cDNA-AFLP also some larger fragments between 500 and 600 bp were obtained. Promising bands were characterized further by cloning and sequencing. Sequence analysis showed that the PR5J fragment shows homology (63%) with a chromosomal gene from Arabidopsis thaliana of unknown function. No significant homologies with any other known sequences were found for the other fragments.

Partner P01 obtained eight potentially useful candidate genes involved in plant development and flowering. These were analyzed by Northern analysis and liquid hybridizations using a magnetic bead technique in order to identify homolgous genes in P. radiata. In two constructs no inserts werer found, three genes gave no amplification products and in the other three cases amplification products were detected. However, these were identical both in juvenile and adult material of radiata pine, so that they were not useful for our purposes. Results could be confirmed by Northern analyses.

Partner P01 constructed in total four cDNA libraries from adult material (P68+P40, P34) and from the corresponding pools of progeny genotypes. Libraries were further enriched for phase specific genes by PCR based substraction using magnetic beads techniques. They were used to derive the cDNA-AFLP markers, to evaluate the candidate genes and the markers obtained from other partners (MMS).

Partner P01 analyzed markers MET, prpS28 and a peach SF gene (obtained from P03) and MADS box genes (obtained from P07) in Pinus radiata. Amplification products were obtained always in genomic DNA as well as cDNA. However, no differential expression could be detected in the model system.

Using degenerated primers for a KNAT gene obtained from partner P03, two amplification products were obtained by partner P01. One PCR product of 800 bp was only present in the adult parental genotypes, the other of 400 bp did not show differential expression, but showed large difference in abundance between the two stages in Northern analysis. Sequence analysis revealed that the 400 bp product represented a homologous gene in Pinus radiata of a KNAT of Arabidopsis thaliana. The 800 bp product showed homology of 95% with a chloroplast gene from Pinus tumbergii.

Gene expression from structures (buds, leaves and stems) of basal shoots (juvenile material) and apical shoots (adult material) were compared by DDDRT-PCR experiments by partner P03. Only 5% of the cDNA markers confirmed their differential expression after Northern analyses. Four markers were selected, based on their differential expression, base pair length and possible relation to developmental stages. Several other cDNA clones were collected when data bank sequence analysis confirmed homology to gene of interest.

Partner P03 characterized the following genes:

RPS28 gene. RPS28 encodes the cytoplasmic ribosomal protein S28. The gene was characterised structural and expression level (Giannino et al., 2000) during juvenile, juvenile-like and adult phases (both vegetative and reproductive). RPS28 expression appeared to mark with a more abundant message leaves borne on juvenile and juvenile-like rather than apical shoots. Although RPS28 expression is not an constant effective phase specific marker it is a good indicator of tissue mitotic/metabolic stage with its mature/precursor expression pattern. In fact, precursor accumulation marks tissues at late developmental stage or senescence.

PYGLO marker was found to be homologous to a membrane protein of Picea abies and yeast (YGLO). Partial characterisation at structural and expression level of the gene was carried out. Gene expression signalling appeared to exclusively mark leaves of basal shoots rather than apical shoots and shoots borne on seed derived juvenile plants

PAR marker is single copy gene encoding a message homologous to a Monohydro Acorbate Reductase from Cucumis sativus. It was more expressed in leaves of juvenile, juvenile-like shoots and in vitro juvenile clones although faint signal is observed at low level in leaves of adult shoots. PAR appeared to mark leaves of juvenile shoots although the difference in message abundance is modulated and not clearly distinguishable in all the case tested.

PORF shared no homology with any gene and deduced protein in databank although its sequence harboured an open reading frame. Until now Northern analyses have only revealed a higher gene expression in buds born on apical shoots. No stage specific expression was observed in leaves of different types of shoots. Gene expression appeared to mark with more abundant messages the buds of apical rather than basal shoots.

Other partial cDNA clones were identified as putatively differentially expressed: PGgh, geranylgeranyl hydrogenase, PRuBP, 1-5 biphosphate carboxylase/oxygenase activase, Lhcb-Pp2, Prunus persica light harvesting chlorophyll binding A/B protein. The differential expression of these markers was not tested by Northern analyses and they may still turn to be effective markers.

Partner P03 evaluated the following known genes:

DNA Methyltransferase is a regulatory gene and involved in several developmental processes in plants and animals. METPE structure, deduced products and expression were characterised. RT-PCR technique was performed to detect an on/off type expression in leaves of juvenile and adult plants but no signifying differential expression was observed. Comparison between adult vegetative and reproductive phases was carried out by in situ experiments on triple buds at different developmental stages. Distinct patterns of message localisation were observed between central buds (vegetative fate) and side buds (reproductive fate) during their development. A dynamic pattern of METPE expression was outlined hypothesising that this gene is involved in the apex programming for a) the cell competence in the signal perception for flower induction and b) the activation of phase change genes.

A leading hypothesis proposes that knotted-like genes control cell vegetative identity and that the reduction, if not a turn off, in gene expression activate the signals to determine cell fate. Two peach clones KNAPE1 and KNAPE2 were amplified by RT-PCR. They both fell into the knotted 1 class, but diverged one from the other (50% homology). Two distinct patterns in Southern analysis suggested that each gene is present in one or two copies in peach genome. Signals of KNAPE1 were only detected at a low level in differentiated buds, whereas KNAPE2 messages were equally signalled in vegetative tissue of juvenile and adult plants. No expression was detected in the stamens and petals of adult plants and in the roots of all the individuals examined. KNAPE1 messages were also localised by in situ hybridisation to compare buds with vegetative and reproductive destiny. A distinct localisation pattern characterised the two bud types. KNAPE1 zonation pattern may mark buds with distinct destiny.

Partner P03 obtained a genomic library of Prunus persica cv Stark Earlyglo and screened this library. Double strand cDNA from leaves of in vitro juvenile clones of Prunus persica cv Chiripa was synthesised.

Partner P07 used two approaches. First, a Differential Display to identify markers for the juvenile-to-adult transition in apple. The limited number of differentially expressed bands in the Differential Display could not be confirmed by Northern blots. Second, in an approach targeted at transcription factors, degenerate primers were used for the conserved MADS box region in RT-PCR to isolate apple orthologues of MADS box genes that may be involved in the juvenile-to-adult transition or serve as markers for the reproductive period. Five different genes (named MdMADS12-16) were isolated, and their expression pattern was determined with Northern blot analyses and RT-PCR. MdMADS16 is most promising as a reproductive phase/maturation stage marker: no PCR product was detected in juvenile leaves, but a clear PCR product was present in RT-PCR with RNA from leaves of trees that flowered, and also with RNA from trees that did not flower at the time of sampling, but would flower the next year.

Partner P04 achieved by means of RT-PCR experiments and using the 5’/3’ RACE methodology from juvenile tissues of peach the isolation of the full length cDNAs of Mn-SOD and two catalases, Cat-1 and Cat-2, of peach. The same strategy allowed the isolation of a partial fragment of Mn-Sod from umbrella stone pine. Fragments of Mn-Sod and Cat cDNAs were cloned and used as probes for hybridisation experiments. A non radioactive DIG system was used for Northern blot hybridisation and detection.

A cDNA library from juvenile needles of Pinus pinea, was constructed. The library screening using specific primers designed on the basis of the partial sequence of peach catalase already isolated allowed the isolation of a 890 bp catalase fragment of umbrella stone pine.

Isoenzyme analysis of CAT and SOD was performed by partner P04 in umbrella stone pine, Pinus pinea and peach Prunus persica. The results obtained, both in peach and in umbrella stone pine, allowed the identification of Mn-SOD as the isoform which is specifically expressed in the juvenile tissues and in vitro micropropagated shoots. By contrast SOD isoenzymes were not correlated to rooting ability, since trials performed with peach clones showing different rooting abilities did not show differences among the isoform activity. The results obtained on the catalase isoenzymes demonstrated that the different isoforms, are active during leaf development in the stem and in buds. However, the native PAGE results suggested that catalases were more specific for the tissue than for the phase. In umbrella stone pine, out of the isoforms detected, CAT-1, the fast migrating isoform, resulted specifically correlated to seedling and microshoot tissues. The bands which we referred as CAT-2 and CAT-3 resulted specific for the needles, irrespective from the age of the tree.

Morphometric analysis of shoot-tips realized by partners P06+P09 showed that the meristem size exhibits age-dependent seasonal variations. The meristem size increases from a minimum observed in winter to a maximum reached earlier in juvenile (spring) than in adult material (summer). In october, in april and during summer, all parameters describing the meristem shape allow to distinguish juvenile from adult material. Furthermore, in april and, to a certain extent, at the end of the growth season, the meristem width exhibits significant differences between the adult vegetative and the adult reproductive material. Pratically, simple meristem width measurement at appropriate periods allow the discrimination of juvenile, adult vegetative and adult reproductive material.

The internal cytophysiological zonation of meristem revealed by in situ detection of the RNA/DNA- rich regions also exhibits age-dependent seasonal variations and could be used as supplementary marker.

The polysaccharides accumulation and within shoot-tip distribution show age-dependent seasonal variations. As the meristem width, examination of the polysaccharidic status of the shoot-tips can be used as a diagnostic test for juvenile versus mature phase characterization. Tannins content in shoot-tips follow parallel evolution with polysaccharides and could be also considered as a potential phase specific histocytological marker.

Lipid reserves exhibited seasonal variations similar in juvenile and in adult material. They were not appropriate for phase change characterization.

The comparison between somatic and zygotic embryos in the first stages of germination revealed important differences in term of meristem shape and content of stored food. The lack of megagametophyte in somatic embryos is involved in these initial differences. At further developmental stages however both types of embryos become similar. This suggests that compared to zygotic embryos, somatic embryos had not reached complete maturation.

In situ localization tests made on seedlings revealed that the within shoot-tips antioxidative activity decreased as fast as the stored lipids disappeared and the polysaccharides accumulation arose, thus confirming the oxidative conversion of fats to carbohydrates during germination. In shoot-tips of plants in their second growth season, this spatio-temporal relationship between the antioxidative activity and lipid reserves consumption was not observed. This could be due to the activation of additional phenomenons producing oxidative radicals in meristematic tissues.

The sequential histological study realised in Prunus Persica by Partner P02 showed a very simple structure of in vitro buds. The apical bud is constituted by small and isodiametric cells with big nucleus and thin walls and the vascular system is formed by slightly diferentiated cells. By contrast, embrionary apical buds presented a higher level of organisation with a more developed vascular system with secondary wall thickening and phenolic deposits. Juvenile and mature apical zones developed a great number of lateral buds rounding the apical meristem. In juvenile and mature buds two types of carbohydrate deposits were observed. In general, lipids were located, principally, in active cells of apical buds and in leave primordia. In juvenile and mature tissues, the more developed primordia presented a low intensity of staining and lipids were located in the zones near cell wall. No differences in protein location between the four developmental stages of Prunus persica were observed.

Partner P05 found that PH and calcium maps show similar features in P.radiata and P. pinaster. Both calcium and pH show typical patterns in seedlings and adult trees, the differences are clearer pronounced for pH. Average pH at the apex is higher in seedlings and decreases during development, with a treshold value of 6.0 between seedlings and adult trees. Responsivity to environmental signals measured as electro physiogrammes is higher in seedlings than in adult trees of both, P.radiata and P. pinaster.

The analysis followed by Partner P02 in terminal and axillary buds of different ages of P. radiata trees, leaves from C.avellana sprouts submited to various severe pruning treatements and terminal buds of P. pinea trees showed that polyamine(PAs)-S-fraction decreased along maturation and permited to characterize tree physiological ageing. PAs-SH fraction reached superior levels after flowering, maintaining inferior levels on juvenile and mature-vegetative phases. This marker allowed to discriminate plant tissues in relation to their flower ability. The ratio S/SH presented specific values for juvenile-vegetative and mature-reproductive states. Percentage of 5-mC in genomic DNA showed specific values for juvenile and mature states and mature reinvigorated trees: 5mC 60% characterizes mature-reproductive phase, about 35% 5mC is proper for mature-vegetative phase. Lower percentages of 5mC characterize juvenile phase.

With respect to hormonal analyses, in both pine species partner P02 found an increase in Z-type Cks (cytokinins) along tree maturation. No differences were obtained neither for ABA, nor for IAA. A decreasing ratio of iP-type/Z-type Cks was found in C. avellana cotyledons and in P. radiata apical and axillary buds, and was validated in P. radiata one, 11 and 13-month-old buds during their maturation and in reinvigorated material. ABA analysis showed similar values between juvenile and adult material in P. radiata. IAA showed the same increasing pattern until P4 in both kinds of buds and afterwards amounts of this hormone were maintained. In organ maturation studies, a negative correlation between IAA and iP-type Cks levels in the basal portions of C1 needles and their developmental state was found. In the case of C3, similar results were found in [9R]iP as well as a high decrease in iP levels between one and 11-month-old needles, followed by a reduction in the IAA amounts between 11 and 13-month-old needles. IAA in 1 and 11-month-old needles was accumulated in the apical and basal fragments, whereas in 13-month-old needles this hormone was found mainly in the apical part. Cks were accumulated in the basal portion, but at the end of the growth period (13-month-old needles) they were concentrated in the apical zone.

Partner P01 defined three different adult-juvenile systems in radiata pine for the evaluations of markers obtained in the model system: System Progeny (SP): Seedlings and parental trees from controlled crosses or open pollinations; System Rejuvenation (SR): cascade graftings and cuttings of adult material;

System Flowering (SF): genotypes showing precocious flowering and non flowering individuals.

Plant material, RNA or membranes were distributed to other partners or these came for sampling. Graftings were performed to distribute adult material. Markers were evaluated via RT-PCR using original primers or specific primers based on sequence information and by Northern analysis.

From the DDRT-PCR primers only PR4J and PR6J showed consistently diiferential expression in all SP subsystems applying RT-PCR. Results could be confirmed by Northern. In cascade graftings they behave as adults and in cuttings as juveniles (SR). However, no differentiation in the SF system neither in the field trial material was observed.

With respect to the cDNA-AFLP markers, only cPR8J and cPR9A were successfully in all SP subsystems, which could be confirmed by marked intensity polymorphisms in Northern analysis. In addition, cPR8J showed adult expression in graftings and juvenile expression in cuttings. In the field trials, only cPR8J was useful in the rooting assay (FT I). All non-rooted genotypes showed one extra band which was absent in the rooted genotypes. Instead, no differential behaviour was observed in the flowering assay (FT II).

Degenerated as well as specific KNAT primers showed differential expression in all SP subsystems, behaved in cascade graftings as adults and with cuttings as juveniles. Furthermore, specific KNAT primers showed differential expression in the rooting assay of Field trial I and in family nş 79 of FT II. Results could be confirmed also by Northern analyses.

Using cPR8J and KNAT-P600 products as probes in Northern analysis also differential expression between juvenile and adult material of Pinus pinea (FTIII) was observed.

Partner P03 obtained the following results:

Cherry and peach are phylo-genetically related, so peach probes were used to test gene expression in fully expanded leaves of one, two, three year old and adult cherry plants. As for RPS28, a single copy gene in cherry genome, no relevant difference in message abundance was found among the fully expanded leaves from plants of different ages. Similarly to what occurs in peach fully expanded leaves, two signals were observed: a mature message of c.a. 0.6 kb and a 1.2 kb product, the latter being alleged to be a precursor. Precursor accumulation was observed in relation to leaf ageing. Regarding PYGLO, the peach probe cross hybridised with cherry genomic DNA and suggested that a homologous small family gene is also occurring. No clear differential expression occurred among the leaf tissues of juvenile and adult plants. PAR putative gene was signalled as a single band by RT-PCR in leaves borne on juvenile and adult, but no differential signal discriminated the distinct samples. A METPE probe indicated a homologous single copy in cherry genome. RT-PCR experiments were performed on the standard material and single products of equal abundance were amplified in both leaves borne on adult and juvenile plants. As for KNAPE1, the peach probe, suggested the occurrence of one or two copies in cherry. The expression was not tested since the gene is only lowly expressed in peach buds. When KNAPE2 probe was used, a single band pattern appeared in cherry DNA gel blot analyses. Even in this case no differential expression was observed in leaves of juvenile and adult plants.

The creation of species-specific probes was a mandatory step for the functional characterisation of genes derived from other plant genera. Consequently, primers were designed on possible conserved amino-acidic domains and delivered to the partners. Peach markers testing by RT-PCR was first performed on P. radiata system of P01 then extended to Pinus pinea if positive results occurred. RPS28 was tested by partners P01 and P04, PAR primers were effective to amplify a single product but no quantitative differences in message abundance were revealed among the samples of different ages. PYGLO primers lead to a multiple band amplification with no clear and significative difference in signal abudance, whereas PORF primers were not effective to amplify any gene product METPE primers were tested on Pinus radiata leaves, but no differential expression was observed. Knotted-like genes primers were tested by P01 and P03 collaborated in sequence analysis and characterisation of gene structure and tissue expression.

P07 has used plant material (several tissues) of controlled crosses of Malus domestica for the evaluation of the isolated apple MADS box genes. Several tissues of crosses that were seeded in 1992 (for juvenile/adult material, sampled in 1997, 1998 and 1999, during which period most seedlings started to flower for the first time) , in 1995 (sampled in 1999) and in 1996 (genuine juvenile material sampled in 1998 and in 1999) were used. The marker MdMADS16, that was not expressed when flowering was more than one year away, was also not expressed in the juvenile tissues of the 1996 cross. This is consistent with the gene being an expression marker for the mature phase in apple trees.

Expression analysis of catalase cDNAs was also performed by partner P04 in apple and radiata pine. In apple two transcripts were evidenced in both the leaf of microshoot and adult tree, by contrast in leaf of juvenile tree the expression was barely detectable. A high level of transcription was observed in the radiata pine seed, but no specific differences were evidenced in the needles from trees with different age.

Determination of CAT and SOD activities and electrophoretic pattern analysis was performed by partner P04 in Pinus radiata, P. pinaster and Malus domestica. In pines native PAGE results revealed the specific presence of Mn-SOD in cotyledons, needles in the early stages of seedling development and in vitro propagated material. Its potential as phase specific marker is however confined only to early stage of development and cotyledon germination. On the contrary, the lack of CAT-1 in the mature adult tissues makes worthy to investigate further at molecular level the potential of this protein as juvenile marker. Electrophoretic analysis on Malus domestica revealed the specific presence of Mn-SOD in juvenile phase and in vitro propagated tissues evidencing its potential as juvenile specific marker in apple as well.

Partner P05 found that pH and calcium patterns are not species specific. Electro physiogrammes show specific pattern for specific tree features: developmental stage, rooting capacity and flowering capacity, depending also on triggering environmental signal.

In Prunus Persica, Partner P02 observed that [9R]iP, iP and (diH)Z levels decreased in apical buds parallel to increasing developmental stages. The other Cks analysed did not show any clear tendency. Total Cks decreased along tree maturation. The percentage of iP-type Cks with respect to total Cks decreased parallel to the increase of the trees developmental state, which involved the subsequent decrease in the iP-type/Z-type Cks balance. IAA levels decreased from embryonic to juvenile buds, but the higher amounts were found in mature ones. The Cks/IAA ratio and ABA levels decreased parallel to the developmental state. P. pinea showed an increase in Z-type Cks along tree maturation followed by a drop in more than 30-year-old trees. IAA did not show differences in its levels, and ABA did not show any pattern along tree maturation. Prunus Persica and Pinus pinea, such as Pinus radiata, presented the same specific PA evolution during organ and individual maturation. Organ maturation was characterised by a decrease in total endogenous PA content. Individual maturation in all the species tested was characterised by a descent in S/SH ratio.

Investigations made by partners P06+P09 in Pinus pinea and P. radiata seedlings revealed strong similarity with P. pinaster in the variations of the meristems size and shape as well as in the reserve accumulation and consumption within shoot-tips during the first growth season.

In contrast, in juvenile, adult vegetative and adult reproductive materials between-species differences could be observed in the timing of the seasonal variations of the meristem characteristics. Indeed, compared to P. pinaster, the observed sequence of morphometric variation was late in P. pinea and more precocious in P. radiata. This resulted in modified optimal periods for the application of age-discriminating markers for these species.

In P. radiata, discrimination of rooting from non rooting ortets can only be done using the examination of the polysaccharidic status of the shoot-tips.

All the Pinus radiata genotypes provided by Partner P01 were established in vitro by Partner P02. Trees younger than 5 years could be directly successfully established. Conversely older P. radiata selections must be introduced in vitro after monophasic homo-micrografting. Quantification of shoot proliferation and rooting permits to rank the plant material response as follows: P1,P4, NF, C3, NR, AA, C1 (see Tables 2.i in the annex).

Prunus persica: several selected genotypes of peach (including "GF677", a peach x almond hybrid used as a peach rootstock) were micro-propagated, maintained in-vitro and, where necessary, rooted and acclimatized in greenhouse. Results show that the aptitude to micro-propagation depends on the variety.

Pinus pinea: three different Pinus pinea selected genotypes (YO1, YO2 and O2) were put in culture starting by seeds and maintained in vitro. In-vitro rooting experiments gave negative results for all the tested genotypes.

In-vitro cultures of Pinus radiata, Pinus caribaea, Pinus nigra and additional selected genotypes of Pinus pinea were also successfully maintained in-vitro.

Prunus persica: Seeds gathered from three selected peach cultivars ("Regina di Venver", "Falcon Crest" and "Cardinale") and from a wild genotype "Slav" were sown in greenhouse at the beginning of January 1999. The sowing gave positive results only for the wild genotype. Peach variety "Duchessa D’Este", were succesfully grafted in September 1998 onto a "Slav" genotype and cultivated in greenhouse. Cuttings of the same selected peach cultivars previously mentioned were performed in December 1998 and repeated in June 1999. The cuttings were cultivated in a heated greenhouse, under optimal conditions, but the results were negative in both cases.

Pinus pinea: seedlings of several selected Pinus pinea genotypes were successfully acclimatized in greenhouse, as well as 1-year-old Pinus halepensis plantlets selected as rootstock for Pinus radiata. Cuttings and homologous graftings performed in October 1998 on Pinus pinea genotypes OY1, OY2 and O2 were grown in greenhouse during winter 1998-99. The results show that propagation by grafting is more suitable than cuttings for Pinus pinea.

Pinus radiata: graftings of Pinus radiata selected genotypes onto Pinus halepensis (performed in February 1998) failed on account of an exceptionally early seasonal trend. Nevertheless, a set of homologous graftings (P. radiata onto P. radiata), previously realised by Partner 01, were successfully grown in greenhouse, with little differences depending on genotypes. The same is true for Pinus radiata cuttings and seedlings.

Prunus persica clones N° 2-8 and 10, as well as"GF677" show a quite good multiplication rate (between 1,5 and 2), and the quality of their shoots is, as a whole, satisfying. Neither clones of Prunus persica, neither "GF 677" showed any difficulty in rooting. On the contrary, selected genotypes for field trials (P1RV, P2FC, P3C) gave a no good response to micro-propagation.

All in-vitro cultures of Pinus pinea, Pinus radiata and other pine species (Pinus nigra and Pinus caribaea) showed a relatively low multiplication rate (except for Pinus caribaea which multiplies more quickly); with regard to Pinus pinea, it was possible to start an in-vitro culture only by seeds (juvenile material); all in-vitro rooting tests performed on Pinus pinea gave negative results.

Prunus persica: woody cuttings performed in December 1998 (on the genotypes P1RV, P2FC, P3C), showed an almost void surviving, so that a new set of green cuttings was performed on June 1999. Nevertheless, the results were negative once again, confirming propagation by cutting as a very unsuitable propagation system for our Prunus persica varieties.

Pinus pinea: results about cuttings performed in October 1998 on genotypes YO1, YO2 and O2 indicated a high mortality. Seedlings born in 1998 from genotypes YO1,YO2, O1 and O2 showed a good growth rate and a survival rate over 70% for each genotype. Graftings performed in October 1998 on genotypes YO1, YO2 and O2, showed a survival rate of about 30% for each genotype.

Pinus radiata: seedlings born from genotypes P56 and P11, cultivated in greenhouse, show a good survival rate. With respect to graftings only genotype P11 showed a very high rate of survival; other genotypes (P 45, P32 and P56) showed a survival rate between 40% and 60%. Cuttings (performed in July 1998) showed a very low survival rate (less than 15% for all the genotypes).

Two types of field trials have been set up by partner P1:

A) Rooting assay(FT I; 2 years) – consisting of 50 cuttings from each progeny genotypes of controlled cross (P42 x P45) which showed marked differences in rooting ability. For marker evaluation maternal trees of three stable rooting and non-rooting genotypes were selected.

B) Flowering assay(FT II) – consisting of 50 precocious and late flowering halfsib or fullsib families.

For marker evaluation two flowering and non flowering genotypes in an abundant flowering and in a scarce flowering family were selected. Results of the marker evaluation in the field trial material are described under Task 2.

Prunus persica: final results on Prunus persica field trials performed by Partner P08 (cuttings, grafting, seedlings, micro-propagation) showed a high degree of homogeneity between the genotypes P1RV, P2FC and P3C. No important difference for flowering behaviour between these peach genotypes was found.

Therefore, it was necessary to introduce differences in the plant systems for validation of marker prediction. For this purpose one-hundred seedlings germinated from a very productive "Slav" peach variety (as positive response to seed propagation), and clones N. 2-8-10 (as positive micro-propagation behaviour) were added to previously selected plant material for field trials.

Pinus pinea: selected genotypes "YO1", "YO2" and "O2" did not show any important differences with respect to their propagation behaviour (seeds, micro-propagation, graftings and cuttings), neither with respect to flowering. Therefore, only differences between the age of the selected pine trees (very young, young, adult -just after the first flowering, mature adult; very old) could be considered for the evaluation of marker prediction.

Morphological traits evaluated on Prunus persica and Pinus pinea genotypes selected for field trials are listed below:

Furthermore, general evaluations about the success of ther used propagation systems were realised.

A database has been established by partner P01 on the World Wide Web of Internet. The database contains general information about the project and the participants (including links) as well as the results of the research activities. Furthermore as part of the deliverables, a list of phase change markers and their description as well as a technical brochure with the specific protocols for Phase Change Marker Methodology and methods for propagation of plant material is included. The address of the database is:

Partner P01 found, that the efficiency of DDRT-PCR in generating differentially displayed bands in Pinus radiata is low and in many cases these putative bands are not truely differential fragments based on the corresponding Northern analyses or belong to ribosomal RNA. Similar results have been obtained also by partners P03 and P07. Instead, using the substracted and enriched cDNA libraries together with cDNA-AFLP, new promising markers were obtained.

Partner P03 found that DDDRT-PCR methodology allowed to identify differentially expressed genes in structures borne on basal (juvenile material) and apical shoots (adult material). However, the efficiency of this technique was very low, that is 5% of the putative differentially expressed cDNA markers. Moreover, several experiments of confirmation are required to establish a tight correlation, if any, between gene expression and tissue/organ developmental stage and age. In peach the expression of gene-markers selected are partially effective to specifically identify, mark and/or correlate to tissue and organ developmental stage. RPS28 gene expression appeared to mark with a more abundant message leaves borne on juvenile and juvenile-like rather than apical shoots. This is observed during the vegetative period of peach plants. Moreover, the increase and accumulation of gene precursor is a distinctive indicator of ageing tissues. As for PYGLO, PAR and PORF there are still few data in the scientific literature to support an articulate discussion about these DDRT-PCR markers which also need more general characterisation. Their differential expression partially confirmed by Northern analyses may be not specifically due to the plant age, though a differentiated behaviour during organ/tissue developmental may be suggested. Repeated tests should lead to solve this dilemma and establish the right correlation. Since METPE, KNAPE1 and KNAPE2 belong to regulatory gene classes though they have quite distinct mechanism of regulation. The former regulates gene expression by cytosine methylation in gene active sequences, the latter two by encoding signals that trigger tissue differentiation and developmental processes. Their cell specific expression pattern is more effective in marking cell layers with distinct destiny rather than the measurement of gene message abundance in the tissues. Specifically, the METPE peculiar zonation pattern in the SAMs appeared to mark the differences between buds (and their cell layers) with a vegetative and reproductive destiny at very early stages of development, that is likely to be before the occurrence of induction period (when exogenous and endogenous destiny induction signals are thought to occur). A genomic library of Prunus persica cv Stark Earlyglo has been made available and successfully screened. Double strand cDNA from leaves of in vitro juvenile clones of Prunus persica cv Chiripa was synthesised and it is ready to be cloned in to appropriate phage vectors.

Partner P07 used specific primers in RT-PCR to test known apple MADS box genes (isolated from flowers and fruit RNA) and five MADS box genes isolated in this project from vegetative shoot tips. MdMADS16 is interesting as a potentially useful marker in apple for the reproductive phase, or at least a marker for the maturation stage. The gene is expressed in vegetative shoot tips and leaves of flowering plant and plants that do not flower but will start to flower the next year. The gene was not expressed in those plants that would not flower the next year, and also not in 2 year old juvenile leaves.

Northern analysis of Mn-Sod realised by partner P04 in leaves at various stages of development in P. persica clearly indicates that Mn-Sod has a differential expression in the tissues in the growth phases considered in this research, thus confirming the data obtained with native PAGE. Data on juvenile tissues and the buds confirmed high level of transcription for this gene and suggest its potentiality as marker. Nevertheless, because of the influence of stress factors on the expression of Sods, it is worthy to test the efficacy of Mn-Sod as marker also in stressing environmental conditions.

As far as catalase expression analysis in peach is concerned, results evidenced an intense transcription activity of Cat-2 in adult and in vitro leaves and in buds, whilst Cat-1 transcripts were equally expressed in adult and juvenile leaves. The high level of expression of Cat-2 observed in the buds and in vitro material makes this gene a potential marker for active growing tissues. However as above reported for Mn-Sod, its potential use as phase specific marker has been so far tested in standard environmental conditions. In the case of stone pine, adult tree needles showed level of CAT-1 transcript similar to that observed in young tree therefore excluding its potential use as marker.

Isoenzymes native PAGE analysis in peach realised by partner P04 revealed the specific presence of Mn-SOD in juvenile phase and in vitro propagated tissues evidencing its potential as phase specific marker; on the contrary SOD isoenzymes cannot be considered marker for rooting ability. Furthermore catalase isoforms can not be regarded as a phase specific proteins being probably, much more specific, for the tissue than for the phase. In umbrella stone pine isoenzymes native PAGE analysis revealed the specific presence of Mn-SOD in cotyledons, needles in the early stages of seedling development and in vitro propagated material. Its potential as phase specific marker is confined only to early stages of development and cotyledon germination. Furthermore catalases do not seem to represent a phase related protein.

From the analysis of the seasonal variations observed by partners P06+P09 in shoot-tips and meristems characteristics, 3 diagnostic tests using a combination of 2 morphometric and/or histological markers can be proposed for routine characterization of juvenile, adult vegetative and adult reproductive state in Pinus pinaster:

The sequential histological study realised in Prunus Persica by Partner P02 showed different structures of buds in buds of different developmental stages. Also distribution patterns of phenolic compounds ans lipids were different.

According to partner P05 all tested markers are developmental stage specific in both P.radiata and P. pinaster. However, calcium patterning is less useful as marker because the method is time consuming and cost intensive and the differences between developmental stages are less pronounced.

The increase in the Z-type Cks and final decrease in more than 30-year old trees along maturation, found by Partner P02, in P. radiata as well as in P. pinea trees of similar developmental states, offers the possibility to establish for this kind of phytohormones maturation indexes in Pinus sp. The decreasing iP-type/Z-type Cks ratio found in P.radiata tree maturation, could be considered as a maturation index in woody species, which was validated in several plant materials (apical and axillary buds as well as in organ maturation and in reinvigorated material) and several species (P. persica and C. avellana). This allows Partner P02 to propose a negative correlation between the maturity degree of a tree or organ, loss of their morphogenic competence and a decrease in the previous cited ratio. Neither ABA nor IAA results could be considered as maturation indexes. Changes in PAs and DNA methyladion during maturation and between juvenile and mature phases allowed P02 to define specific markers. Maturation: PAs-S, Put (S), Put/Spd+Spm (S) ratio, PAs-PH. Vegetative vs reproductive growth, PAs-SH, S/SH ratio, 5-mC. Reinvigoration, PAs-S, Put (S), 5-mC.

Phase change markers are only of commercial use if they are universal, e.g. if they can be applied in all genetic backgrounds and for different kinds of plant material.

Therefore Partner P01 had defined the different types of plant material in radiata pine mentioned above and realised in addition two field trials for rooting and flowering behaviour. Marker evaluation included validation by RT-PCR and Northern hybridizations. The differential expression of some DDRT-PCR markers could be verified in several SP subsystems However, they failed for confirming the results of the field trials. Therefore and for the reasons mentioned above, P01 does not consider them as useful for the obtainment of phase change markers.

On the contrary cDNA AFLP markers cPR9A and particularly cPR8J are promising, since they appear in different genetic backgrounds of juvenile and adult material in RT-PCR experiments. Furthermore, they show marked intensity polymorphisms in Northern analyses. The marker cPR8J also showed differential expression in field trial I and can be considered as a real phase change marker for rooting capacity at least in P. radiata.

The KNAT marker has been proven to be useful for the different SP systems and in rejuvenated material of Pinus radiata. Furthermore differential expression was observed in field trial I and in part also in field trial II (Flowering behaviour). Therefore, this marker could be not only a phase change marker for rooting ability but also a marker for precocious flowering in more juvenile material, at least in P. radiata. Identification of homologous probes and specific primer design may lead to useful markers also in other pine species.

Peach RPS28, PYGLO, PAR, METPE, KNAPE2 were tested by partner P03 in cherry tissues. None of these gene exhibited an expression pattern that was age specific. Only cherry RPS28 expression marked the leaf developmental phases by precursor accumulation during ageing, similarly to the peach case. PYGLO, PAR and PORF expression failed to mark tissue, organ and plant developmental stages by RT-PCR despite primers were mostly functional from peach to these gymnosperms.

To further test the putative marker MdMADS16, partner P07 included material from other crosses, and tested, next to pools of material, also individual trees. The expression of MdMADS16 in leaves follows generally the concept of a maturation marker, since all trees that flower display expression of MdMADS16, and most of the trees that will not flower the next year, do not.

The hybridisation results obtained by partner P04 using CAT-1 and CAT-2 fragments as heterologous probes in apple do not allow to draw conclusions about the differential spatial and temporal expression of the two genes. As far as radiata pine is concerned, results obtained are quite similar to those obtained in umbrella stone pine, therefore similar conclusions can be drawn for this and more in general for the other Pinus species.

Isoenzyme results obtained by partner P04 in Malus domestica revealed the specific presence of Mn-SOD in juvenile phase and in vitro propagated tissues evidencing its potential as phase specific marker. The results of the analyses performed in apple confirmed the data obtained in peach.

According to partner P05 all markers gave reliable results in the studied species. Electro physiogrammes show the highest specificity, not just for developmental stages, but also for other physiological characteristics such as rooting and flowering capacities. Therefore, they are proposed to be the most useful marker. The method is non invasive, fast and simple to use. Reliability of the method was checked also in field trials. However, it has to be improved for routine use and triggering signals giving the most specific responses for different characteristics have to be selected.

In Pinus pinea and Prunus persica plant material, Partner P02 observed that iP-type/Z-type Cks, iP-type/total Cks and iP levels were the same during tree maturation, which is indicative of the same physiological state of the tissues, pointing out their importance in phase change of trees. These balance could be used as physiological marker for the developmental stage. Although IAA levels and the Cks/IAA balance did not show similar tendeces in both species, Cks/IAA could be used in P. Persica as maturation index. Results allowed Partner P02 to validate S/SH as a maturation and vegetative-reproductive marker across species, both for gymnosperms and angiosperms.

Similar results as in P. pinaster were obtained by partners P06 + 09 in P.radiata and P. pinea. However the test proposed for mature from juvenile state discrimination must be adapted specifically for these species.

Partner P02 confirmed that the chronological age and the physiological state are decisive for the in vitro establishment and multiplication of field material. All these factors depend on the specific genic regulation state of the individual. Also the loose of morphogenic potential of mature individuals is a genic ageing-related process that may be reverted by reinvigoration techniques.

Prunus persica: micro-propagation can be a suitable way to propagate some peach genotypes such as clones N°2,8 and 10. On the contrary, other peach varieties ("Regina di Venver", Flavour Crest", "Cardinale") resulted recalcitrant to micro-propagation, starting from adult plant material, even after repeated attempts.

Pinus pinea: after several attempts, it was finally possible to put in culture Pinus pinea adult plants starting from seeds (both embryos and cotyledons) but, even in this case, their multiplication rate was very low, and the in-vitro-rooting was unsuccessful. Therefore, it is impossible to get a clone of the adult mother plant.

Prunus persica: graftings performed on commercial variety "Duchessa d’Este" (onto a "Slav" variety rootstock) grew well in greenhouse, as well as seedlings of varieties usually propagated by seeds ("Slav"). Cuttings performed and cultivated in a heated greenhouse on three different varieties of peach ("Regina di Venver", Flavour Crest" and "Cardinale"), showed a very low survival rate, both in December and in June.

Pinus pinea seedlings (of different age, genotype or origin) seem to respond well to acclimatisation and cultivation in greenhouse. Pinus pinea was also successfully propagated by graftings in October, in a cold greenhouse, regardless of the genotype. On the contrary, cuttings performed in autumn, and cultivated in a heated greenhouse, showed a very low survival rate, although they were performed under optimal conditions.

Pinus radiata: cuttings of Pinus radiata, performed in summer, showed a very low percentage of rooting. A possible explanation for this may consist in the stress sustained by fresh material during the journey from the forest nursery in Spain to Italy. Anyway, Pinus radiata seems to be more suitable for cutting propagation than Pinus pinea. As well as the homologous graftings and the seedlings performed by Partner 01 in 1998, the survived plants propagated by cuttings grew well in greenhouse.

Prunus persica: clones 2 ,8 and 10 showed an appreciable aptitude to in-vitro multiplication and rooting, even if they don’t tolerate well cold storage periods longer than three months (at 2°C). On the contrary, Prunus persica genotypes selected for field trials (P1RV, P2FC and P3C) are scantly suitable for micro-propagation. Propagation by seeds is a good system only for a few selected varieties (conserving some "wild" characteristics), usually utilised as rootstock. Also propagation by cuttings is inefficient with peach. On the contrary, propagation by graftings seemed to be the better way to propagate some commercial varieties, such "Regina di Venver", "Flavour Crest" and "Cardinale".

Pinus pinea adult plants could be put in culture only starting from seeds (embryos or cotyledons) and it is impossible to obtain a clone of a selected adult mother plant. Moreover, once the in-vitro cultures were established, their multiplication rate is very low, and the rooting phase is often unsuccessful. A possible alternative, for a commercial exploitation of pine micro-propagation, can be represented by micro-grafting technique, which is time consuming, and requires a specific experience of handling. Propagation by seed results very suitable for a large scale production, probably being the quickest and the cheapest way to obtain a great number of plants. Both seedlings and graftings can be successfully acclimatised and cultivated in greenhouse, even though, for a long-term cultivation, the young plants grow better outside (under Mediterranean climate conditions). On the contrary, propagation by cutting seems not to be suitable for Pinus pinea, whereas for Pinus radiata the results are more encouraging.

The final purpose of this project about phase change markers was to validate the most promising markers for phase change in FIELD TRIALS, including also an economical evaluation.

Two field trials have been set up by partner P01. One considers rooting capacities of progeny genotypes from a controlled cross P42xP45, the other flowering capacities of different genotypes in a seed orchard consisting of many half sib families. The observed variability of the different clones of radiata pine in these field trial was useful to test these markers. Results are discussed under Task 2.

Prunus persica genotypes selected by partner P08 for field trials (P1RV, P2FC and P3C) showed a homogeneous, negative response to micro-propagation, as well as to seeds propagation. Therefore, in order to introduce differences for marker test validation, Prunus persica clones 2-8-10 (which showed a positive micro-propagation behaviour) and a "Slav" peach variety (with a remarkable seed production) has been proposed as additional plant material to our field trials.

All the Pinus pinea available genotypes selected for field trials (YO1, YO2 and O2) showed insignificant differences with regard to their propagation and flowering behaviour. Therefore, only the difference between the age of the selected pine trees can be taken into account for marker prediction.

Prunus persica: propagation by seeds can be considered a good system only for a few selected varieties, usually utilised as rootstock. Also propagation by cuttings is inefficient with commercial varieties selected for field trials. On the contrary, propagation by graftings seems to be the a good system, whereas the response to micro-propagation differs depending on the varieties.

Pinus pinea seedlings and graftings seem to respond well to acclimatisation and cultivation in greenhouse, even though, for a long-term cultivation, the young plants grow better outside (under Mediterranean climate conditions). Propagation by cuttings and micro-propagation seems not to be a suitable propagation system for Pinus pinea.

The project database established by partner P01 provides detailed information about the project and the results including protocols for phase marker methodology. In this way detailed information is available for the scientific community and for other companies or institutions of the forest or related sectors, which are interested to exploit the results, to advance further in the research or to initiate related projects.