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Seasonal growth is characteristic for many tree species including almond. Varying conditions during the season are responsible for growth cessation, bud set, dormancy entry, cold hardening, and bud burst. Here, we report the characterization of an almond homologue of the Arabidopsis GIGANTEA (AtGI) gene (designated as PdGI, GenBank accession No. KJ502316). We propose a role for this gene in the transition to dormancy and cold acclimation. The complementary DNA (cDNA) sequence of PdGI was 4 322 bp long and contained an open reading frame of 3 512 bp. The deduced amino acid sequence of PdGI shared 76 % identity with AtGI. The expression of PdGI at ambient day/night temperatures of 22/20 oC was differentially regulated under a 16-h or 12-h photoperiod, increasing during the day and decreasing after dusk. However, this diurnal regulation was disrupted when plants were transferred to cold (12 oC) conditions. In addition, we have assessed the expression of PdGI and putative almond homologues of the downstream target genes CONSTANS (PdCO-like) and FLOWERING LOCUS T (PdFT-like) in flower buds and shoots from adult trees during the bud break period in autumn and early winter. Our results show a clear increase in transcript abundance towards anthesis, suggesting a role of these genes in flower development.

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

Background Cynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities. Results In this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3′-hydroxylase (C3′H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3′H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3′H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms. Conclusion In this work we identified allelic variants with putative impact on C3′H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3534-8) contains supplementary material, which is available to authorized users.

17 pags., 8 Figs., High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice., This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through national funds allocated to research projects [POCI/BIA-BCM/56063/2004 and PTDC/BIA-BCM/099836/2008] and PhD scholarships [SFRH/BD/31011/2006 to TS, SFRH/BD/29258/2006 to DF, SFRH/BD/74946/2010 to AC, SFRH/BD/65229/2009 to DA, SFRH/BPD/34943/2007 to TL]. NS and IA were supported by Programa Ciência 2007, financed by POPH (QREN). AS and BCM work was supported by funding from Programa Euroinvestigación 2008 [EUI2008-03612].

Soil salinity is a major abiotic stress that results in considerable crop yield losses worldwide. However, some plant genotypes show a high tolerance to soil salinity, as they manage to maintain a high K+/Na+ ratio in the cytosol, in contrast to salt stress susceptible genotypes. Although, different plant genotypes show different salt tolerance mechanisms, they all rely on the regulation and function of K+ and Na+ transporters and H+ pumps, which generate the driving force for K+ and Na+ transport. In this review we will introduce salt stress responses in plants and summarize the current knowledge about the most important ion transporters that facilitate intra- and intercellular K+ and Na+ homeostasis in these organisms. We will describe and discuss the regulation and function of the H+-ATPases, H+-PPases, SOS1, HKTs, and NHXs, including the specific tissues where they work and their response to salt stress.

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

Plant development depends on the perception of external cues, such as light, gravity, touch, wind or nutrients, among others. Nevertheless, little is known regarding signal transduction pathways integrating these stimuli. Recently, we have reported the involvement of a rice E3-ubiquitin ligase (OsHOS1, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1), previously associated with abiotic stress response, in root responses to mechanical stimuli. We showed that OsHOS1 is involved in the regulation of root curling after mechanosensing and that RNAi::OsHOS1 plants failed to exhibit the root curling phenotype observed in WT. Interestingly, the straight root phenotype of these transgenics correlated with the up-regulation of rice ROOT MEANDER CURLING (OsRMC, a negative regulator of rice root curling) and was reverted by the exogenous application of jasmonic acid. Altogether, our results highlight the role of the proteasome modulating plant responses to mechanical stimuli and suggest that OsHOS1 is a hub integrating environmental and hormonal signaling into plant growth and development.

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.

Low temperature plays a crucial role in seasonal development of woody plants and may directly impact crop production, more particularly in temperate fruit trees. Given its high genetic variability and adaptability to different climatic conditions, almond (Prunus dulcis Mill.) is an interesting model to understand the mechanisms regulating low temperature sensing in fruit trees. In this paper, we report the cloning and characterization of two genes (PdCBF1 and PdCBF2) belonging to the C-repeat-binding factor (CBF) family of transcription factors. Southern blotting analysis showed that this family is composed of at least five members. In almond shoots propagated in vitro, transcription of these genes was rapidly induced by low temperature, suggesting an involvement in cold acclimation. Transactivation assays showed that PdCBF1 and PdCBF2 could bind to dehydration responsive element/C-repeat containing sequences, as activators of gene expression. In addition, induction of both PdCBFs by cold was higher towards the end of the day, which agreed with the expression pattern of PdDehydrin1, a predicted CBF target gene. Furthermore, PdCBF1 and PdCBF2 were also transiently induced by abscisic acid and drought treatments. Considering the bin mapping analysis that correlated PdCBFs and PdDHN1 (respectively in linkage groups 5 and 7) with two different quantitative trait locicontrolling blooming time, it is relevant to perform further association studies that may validate their effect on this trait.

The transcription factor encoded by the gene Knotted1 is a nuclear homeodomain protein, regulating meristematic cells at the shoot apical meristem. It has been demonstrated that Knotted1 (KN1) expression specifies stem cell fate in adventitious shoot induction in herbaceous plants. This gene may thus potentially identify the initiation of meristem development in adventitious shoot induction in difficult-to-regenerate plants such as Prunoideae. We isolated an almond (Prunus dulcis Mill.) KN1-type gene using degenerate primers targeting the most conserved regions of Knotted1 gene. The 5′ and 3′ ends of the isolated sequence were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), and the gene was named P. dulcis Knotted1-like (PdKn1). PdKn1 transcripts were detected by reverse transcription (RT)-PCR mainly in shoot apical and axillary meristems. The RT-PCR and RT-quantitative PCR PdKn1 expression in almond leaf explants was found to anticipate the organization of adventitious shoot meristems. Apricot RNA isolated from induced leaf explants cross-hybridized with the almond probe PdKn1 in Northern blotting. We suggest that almond PdKn1 may be a useful marker to study the adventitious regeneration system by revealing the potential organogenic conditions, not only in almond but also in other Prunoideae.

Family Azollaceae has seven species with a controversial taxonomy. The identification of species without reproductive structures relies on vegetative characters but some are variable, leading to misinterpretations. The molecular methods may be helpful, but until now, they did not provide a conclusive Azolla taxonomy. Therefore, we studied the family Azollaceae at vegetative and molecular levels. Analysis of vegetative, random amplified polymorphic DNA (RAPD) and combined data showed a comparable grouping of the Azolla species in two main clusters: cluster I, referred to as section Rhizosperma (A. pinnata and A. nilotica) and cluster II, referred to as section Azolla (A. filiculoides, A. microphylla, A. caroliniana and A. mexicana), with the exception of A. rubra, which clustered differently depending on the method. All the Azolla species were distinguished by the 13 polymorphic vegetative characters, the 211 RAPD markers or the combined data, with the latest showing the highest discrimination. The Shannon Index diversity was greater with RAPD (2.276) than with vegetative characters (0.054), highlighting the higher discriminating power of the molecular data. The partitioning of diversity was, as expected, high among species for all the types of data and low within species, with the lowest diversity obtained for morphological data. Both data sets (vegetative and RAPD) allowed the distinction of all the species and their clustering into sections Rhizosperma and Azolla, suggesting this as the most correct for this family. The dendrogram from the combined data was the most accurate, highlighting the benefit of integrating different types of data to study the family Azollaceae.

Global climate change, i.e. higher and more variable temperatures, and a gain in soil salinity are increasing plant stress with direct consequences on crop yield and quality levels. Rice productivity is strongly affected by abiotic stress conditions. The regulation of chromatin structure in response to environmental stress is poorly understood. We investigated the interphase chromatin organization from rice plants in non-stress versus stress conditions. We have used a cytogenetic approach, based on fluorescence in situ hybridization (FISH) with 45S, 5S rDNA and centromeric probes on rice tissue sections. The abiotic stress conditions included cold, heat and mild salinity and were applied during seed germination. In contrast to cold, saline and heat stresses caused extensive decondensation of 45S rDNA chromatin and also an increase in the distance between the 2 homologous 5S rDNA loci. 5-Azacytidine (5-AC), a DNA hypomethylating drug, greatly increased 45S rDNA chromatin decondensation and interestingly was able to induce polarization of centromeres in rice interphase nuclei. The abiotic stresses tested did not perturb the spatial position of centromeres, typically with circular arrangement around the nucleolus. The results suggest a role for chromatin plasticity in a world of climate changes.

The S-allele characterisation of ‘Alzina’ and ‘Garondes’, two local almond cultivars from the island of Majorca, by a multidimensional approach has allowed the confirmation of the presence of the Sf-allele and the identification of a new allele not previously described in almond, S36. When these cultivars were phenotypically evaluated, both showed a self-incompatible phenotype and were cross-incompatible, as assessed by artificial self- and cross-pollinations and fruit sets after field pollinations, confirming that their Sf-allele is in its active form, Sfa. Thus a new CGI group in almond is proposed and named XXVIII. These results confirm the wide diversity of S-alleles in almond both at genotypic and phenotypic levels, as well as their similarity with the S-alleles from other close Prunus species. This similarity suggests the possibility of allele introgression between species or allele identity by descent from a common ancestor.

The pistil (S-RNase) and the pollen [S-haplotype-specific F-box protein (SFB)] components of the Sf allele, presumably conferring self-compatibility in almond {Prunus amygdalus Batsch [syn. P. dulcis (Mill.) D.A. Webb]}, were identified and sequenced in ‘Ponç’, a local Spanish almond cultivar, confirming their identity with the published sequences of these components. Despite the presence of the Sf allele, the ‘Ponç’ phenotype was self-incompatible as confirmed by different pollination tests, including self pollen tube growth, fruit set after self-pollination, and fruit set in bagged branches. However, the pistil and the pollen of ‘Ponç’ were fully viable when pollinated by a cross-compatible pollen or used on a cross-compatible pistil. The fact that ‘Ponç’ presents two different S-proteins with RNase activity may indicate an active function of its Sf -RNase, whereas in the self-compatible almond cultivars thus far studied, the Sf -RNase has been inactive. This activation indicates that the presence of the Sf allele may not be the exclusive source of self-compatibility in almond, and other factors may also be involved in the expression of almond self-compatibility.

SummaryAlmond [Prunus amygdalus Batsch syn. Prunus dulcis Mill. (D.A. Webb)] has a gametophytic self-incompatibility system (GSI). The GSI is haplotype-specific and controlled by a multi-allelic S-locus that codes for RNases involved in pollen rejection in a specific manner. The different S-alleles can be used for molecular identification and characterisation of the S-haplotypes. PCR analyses with molecular markers, including primers designed from conserved sequences and for the second intron of almond S-RNases, were used to characterise the S-genotype of 39 almond accessions, including 29 Spanish cultivars, eight releases from the CITA almond breeding programme, and two foreign cultivars. The S-allele compositions of 34 of the cultivars have been characterised for the first time, and two cultivars were revised. The use of conserved primers permitted amplification of two putative S-alleles that had not been amplified previously by PCR, for which the genotypes S31 and S32 are proposed. Moreover, the Sf all...

An improved almond transformation system was developed with a 100× increase in efficiency (12.3%) as compared to the existing transformation method (0.1%) in spite of the lower regeneration ability of the explants used. Leaf transformation was performed with Agrobacterium EHA105/p35 SGUSINT. Main modifications introduced in the transformation protocol were the use of 150 μM acetosyringone (AS) during the 21-days induction period, and a different selection strategy. Transformed shoots were assayed using PCR, GUS analyses and Southern blotting. The improved methodology is being applied for transformation of in vitro propagated cultivars and opens the possibility of using almond as model for functional studies in Prunoideae.

Transformed root cultures of Levisticum officinale (lovage) were established by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes strain A4 carrying plasmid pRiA4::70GUS. Hairy roots growth in four different types of liquid culture media was determined by the dissimilation method and by measuring the fresh and dry weight of the roots. The composition of the essential oils from the hairy roots and from lovage plant roots was analysed by GC and GC–MS. The main components of the oil samples from the hairy roots were falcarinol, ( Z )-ligustilide, ( Z )-3-butylidenephthalide, trans -β-farnesene, β-phellandrene, n -octanal, γ-elemene and n -heptanal, in varying amounts depending on the culture media tested. The hairy roots essential oil yields ranged from 0.006 to 0.018% (v/fr. wt.). The main components of the oil from the lovage plant roots were ( Z )-ligustilide, β-pinene, pentylcyclohexadiene and α-pinene. The yield of the oil from the lovage plant roots was 0.16% (v/fr. wt.).

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l−1 polyethylene glycol (PEG) and 10 g l−1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.

Self-incompatibility (SI)-associated S-RNase genes represent an extreme example of allele polymorphism. We have cloned and analysed eight novel S-RNase cDNA sequences from almond (Prunus dulcis) cultivars of European origin. Insertions/deletions of 10 to 20 amino acid residues are present between RC4 and C5 domains in Amygdaloideae S-RNases. Synonymous and non-synonymous substitution analysis of the codon data identified positive selection regions. S-RNase genes of almond and sweet cherry and of apple and pear show little heterogeneity in substitution rates, however, significant difference was detected between the two subfamilies Amygdaloideae and Maloideae. Gene sequences of almond S-RNases were amplified by PCR with degenerate primers. The introns located between C2 and C3 are very different in sizes, ranging from 80 to 1100 bp. However, these introns display conserved features in primary sequences. S-allele specific PCR, coupled with RFLP analysis of the PCR products was performed to identify S-genotypes of almond cultivars mainly from Portugal. Up to now, twenty-four almond cultivars were identified genotypically. Some S-allele combinations were further confirmed by comparing the data of S-allele analysis with those of breeding tests.

A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S8) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.

Transformed root cultures of Anethum graveolens were induced by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes carrying plasmid pRi 1855. The main component of the essential oils from the fruits and from the roots of the parent plant was carvone, whereas α-phellandrene and apiole were dominant in the oil from, respectively, the aerial parts and the hairy roots. The essential oils from the fruits, aerial parts and roots of the parent plant were at 2%, 0.3% and 0.06% (v/w), respectively, but only 0.02% (v/w) in the hairy root cultures. Growth of the hairy root cultures reached 600 mg dry wt/50 ml medium after 50 days. The essential oil composition did not change significantly during their growth.

In Portugal, almond (Prunus dulcis Mill.) is a traditional crop with good possibilities in terms of production and fruit quality, especially in the Southern (Algarve and Baixo Alentejo) and Northern (Tras-os-Montes) regions. A study of the variability and relationship among our cultivars is important for the development of a breeding program and for the organisation and conservation of the collections maintained in Portugal. The genetic relatedness among 35 almond cultivars from Portugal, France and Spain, and 5 putative wild almond plants was estimated using RAPD, msp-PCR and AFLP markers. The analysis of the data obtained was performed using Dice coefficient and UPGMA clustering. Three different molecular techniques revealed high levels of polymorphism and were capable of resolving intervarietal differences. In the case of almond, msp-PCR seems to be a good option for varietal discrimination, due to its low costs, simplicity and reliability.

The essential oils isolated from roots of two Achillea millefolium populations (BGL and CGA) and from two hairy root cultures (A4 and LBA) derived from one of these were analysed by GC and GC–mass spectrometry. The essential oils from the plant roots were obtained in a yield of 0.10% (BGL) and 0.05% (CGA) (v/w), whereas that of both hairy root cultures attained 0.05% (v/w). Compared on a dry weight basis, the yield from the hairy root cultures was similar to or higher than that from the plant roots. The oxygen-containing sesquiterpene fraction of the root oils from both plant populations was the most characteristic one, epi-cubenol being the main component of these oils (18% for BGL and 26% for CGA). Oxygen-containing monoterpenes constituted the most important fraction of the essential oils from both hairy root cultures, neryl isovalerate being the main component (27% and 43% for A4 and LBA, respectively). No major differences were detected in the root essential oil profiles of the two A. millefolium plant populations, but qualitative and quantitative differences were found between the essential oils from the plant roots and those from the hairy roots, and also between the oils from the two hairy root cultures.

Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (β-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed to selection 20 days after cocultivation. From 1419 original leaves, four shoots (A, B, C and D) were obtained that showed amplification of the predicted DNA fragments by polymerase chain reaction (PCR). After micropropagation of these shoots, only those cloned from shoot D gave consistently positive results in histochemical GUS detection and PCR amplification. Southern blot hybridisation confirmed stable transgene integration in clone D, which was also negative in PCR amplification of an Agrobacterium gene. Additional molecular analysis suggested that the remaining three shoots (A, B and C) were chimeric.

Hairy root cultures of Pimpinella anisum were established following inoculation of aseptically grown plantlets with an A 4 pRiA 4 70 GUS strain of Agrobacterium rhizogenes . The essential oils from the hairy roots, maintained in four different media, and from the fruits and roots of the parent plant were analysed and their compositions compared by GC and GC-mass spectrometry. The major components of the essential oils from the hairy root cultures were trans -epoxypseudoisoeugenyl 2-methylbutyrate, geijerene, pregeijerene, zingiberene and β-bisabolene, in varying amounts depending on the light or dark growth conditions and on the culture media tested. trans -Epoxypseudoisoeugenyl 2-methylbutyrate, β-bisabolene and pregeijerene were the major components of the essential oil from the roots of the parent plant, whereas the main component of the fruit oil was trans -anethole. Geijerenes were not detected in the fruit oil. The essential oil yield of the transformed roots grown in one of the media was comparable with that obtained for the roots of the parent plant and, calculated on a dry weight basis, the oil yield of these hairy roots was comparable with that of the fruits.

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response., This project was funded by "Fundacao para a Ciencia e a Tecnologia" (FCT) within a National Consortium (COEC - Cork Oak ESTs Consortium) that supported 12 sub-projects (SOBREIRO/033, 035, 014, 034, 015, 017, 038, 019, 029, 039, 030, 036/2009). The authors further wish to acknowledge FCT for ten doctoral (BD) and post doctoral (BPD) fellowships (Tania Almeida: SFRH/BD/44410/2008, Tiago Capote: SFRH/BD/69785/2010, Ines Chaves: SFRH/BPD/20833/2004, Ana S. Fortunato: SFRH/BPD/47563/2008, Marilia Horta: SFRH/BPD/63213/2009, Liliana Marum: "SFRH/BPD/47679/2008, Andreia Miguel: SFRH/BD/44474/2008, Margarida Rocheta: SFRH/BPD/64905/2009, Tatiana E. Santo: SFRH/BD/47450/2008, Monica Sebastiana: SFRH/BPD/25661/2005). Andreas Bohn, Nelson J.M. Saibo, Rita Teixeira were supported by the Programa Ciencia 2007, financed by POPH (QREN) and Isabel A. Abreu, Susana Araujo, Dora Batista, A. Margarida Fortes, Jorge A.P. Paiva, Sonia Goncalves by Programa Ciencia 2008, also funded by POPH (QREN). A Margarida Santos was funded through iBET (PEst-OE/EQB/LA0004/2011). Maintenance of the CorkOakDB is supported by the Instituto Gulbenkian de Ciencia.

Adventitious shoot regeneration was achieved from almond leaves, cv. Boa Casta, excised fromin vitro cultures of juvenile and adult material. Murashige and Skoog (1962) medium (MS) was found to be more efficient for adventitious shoot induction than a modified medium of Quoirin et al. (1977) when using identical growth regulator supplements. Thidiazuron (TDZ) at 4.54, 5.90, 6.81, and 9.08 μM was used in all induction media, together with indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or a combination of IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). When N6-benzyladenine (BA) was used instead of TDZ, no adventitious shoots were induced. Leaf explants of juvenile origin yielded the highest regeneration rates (40.0 and 38.2%) and required higher concentrations of TDZ for shoot induction than leaves of adult origin. An increase from 15.0 to 35.3% in the regeneration ability of adult leaf explants, tested on one of the induction media, modified medium of Quoirin et al. (1977) supplemented with 5.90 μM TDZ and 2.85 μM IAA], was achieved when donor shoots were subcultured twice on a medium with a low BA concentration of 1.33 μM.

Plants can cope with adverse environmental conditions through the activation of stress response signalling pathways, in which the proteasome seems to play an important role. However, the mechanisms underlying the proteasome-mediated stress response in rice are still not fully understood. To address this issue, we have identified a rice E3-ubiquitin ligase, OsHOS1, and characterized its role in the modulation of the cold stress response. Using a RNA interference (RNAi) transgenic approach we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1A. This was correlated with an increased amount of OsICE1, a master transcription factor of the cold stress signalling. However, the up-regulation of OsDREB1A was transient and the transgenic plants did not show increased cold tolerance. Nevertheless, we could confirm the interaction of OsHOS1 with OsICE1 by Yeast-Two hybrid and bi-molecular fluorescence complementation in Arabidopsis protoplasts. Moreover, we could also determine through an in vitro degradation assay that the higher amount of OsICE1 in the transgenic plants was correlated with a lower amount of OsHOS1. Hence, we could confirm the involvement of the proteasome in this response mechanism. Taken together our results confirm the importance of OsHOS1, and thus of the proteasome, in the modulation of the cold stress signalling in rice.

In temperate fruit trees, seasonal dormancy and cold acclimation have a major impact on blooming time and, consequently, fruit production. To gain insight into the still unclear molecular processes underlying blooming, expression of genes putatively involved in the cold response was studied in almond (Prunus dulcis Mill.), which is the earliest fruit tree in the family Rosaceae to bloom. The transcript levels of two C-repeat binding factor (PdCBF) genes and one of their putative targets, PdDehydrin1 (PdDHN1), were analysed in flower buds and shoot internodes during seasonal dormancy up to bud break. In parallel, expression of candidate genes related to flower development was also followed. In a 2-year study, PdCBF2 showed a progressive increase in transcript abundance during the autumn in close correlation with cold acclimation, while high transcript levels of PdCBF1 and PdDHN1 were already found by summer. After bud break, with temperatures still within the chilling range, both PdCBF genes and PdDHN1 were found to sharply reduce transcription in flower buds and internodes, suggesting damping of CBF-mediated cold signalling during growth resumption, in correlation with cold hardiness decline. Flower bud break was also followed by a decrease in the expression of PdGA20OX, a candidate gene involved in gibberellin biosynthesis, and an increase in the expression of two homeotic genes related to floral organ development, PdMADS1 and -3. These genes may also be relevant players in the regulation of anthesis in this model Rosaceae species.

One of the main concerns about genetically modified foods and their potential impacts on human health is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. We intended to evaluate, and to compare, using quantitative real time RT-PCR technique, the expression of 5 already known maize allergens (Zea m14, Zea m25, Zea m27kD, 50kD Zein and trypsin inhibitor) in MON 810 vs. its non-transgenic counterpart, throughout seed development (10, 16 and 23 days after pollination). We have shown that none of the tested allergen genes presented differential expression, with statistic significance, along all tested seed development stages, in MON810 vs. its conventional counterpart. We have also used bidimensional gel electrophoresis followed by Western blotting with plasma from two maize allergic subjects to characterize their immunologic responses against MON 810 vs. its non-transgenic control. Immunoreactive spots were characterized by MS. We have identified fourteen new IgE-binding proteins present in both transgenic and non-transgenic maize.

The effects of the ectopic expression of a barley transcription factor (HvCBF4) under the control of a constitutive (maize Ubi1) or a stress-inducible (Arabidopsis RD29A) promoter in the abiotic stress response in rice (Oryza sativa L.) was investigated. The transformed plants were analyzed both at molecular and physiological level and the AtRD29A::HvCBF4 plants were further analyzed using the GeneChip® rice genome array under control conditions. Only the plants constitutively expressing HvCBF4 have shown increased survival to drought stress, but not to cold or high-salinity. These plants have also shown better photosynthetic capacity, as determined by chlorophyll fluorescence. Plants expressing AtRD29A::HvCBF4 did not show increased survival to any of the stresses applied. However in the GeneChip® microarray, these plants have shown up-regulation of many stress-responsive genes (> 400) as compared to non-transformed plants. Interestingly, RT-PCR analysis revealed not only differential gene expression between roots and shoots, but also between transgenic lines with the different promoters. Our results indicate that different HvCBF4 expression levels resulted in different transcriptomes and drought tolerance. Given that AtRD29A::HvCBF4 plants did not show increased tolerance to any of the imposed stresses, we may conclude that this promoter may be inappropriate for rice transformation aiming for enhanced abiotic stress tolerance. Fundação para a Ciência e Tecnologia e Fundo Social Europeu

Plant transformation is an important tool with many applications in modern plant biology. Although this technique is primarily used to produce superior crop varieties, it is also being utilized to answer basic questions concerning gene function and regulation in contemporary functional genomics research. In our laboratory, we have established a transformation system for Hypericum perforatum . This protocol involves the transfer of foreign DNA into H. perforatum organogenic nodule explants via particlebombardment and the regeneration of shoots from the explants under selection pressure. We have successfully used this method to express ß-glucuronidase and hygromycin phosphotransferase genes in H. perforatum . Molecular analyses of putative phenotypically normal transgenic plants show stable integration of the transgenes into the plant nuclear genome. Here we describe the procedure for the transformation of H. perforatum ., Fundação para a Ciência e a Tecnologia (FCT)

Relatedness among parents, variation in clonal fertility and background pollination deviate the realized genetic gain and the gene diversity of open pollinated seed orchard from expectation, in particular in wind pollinated species such as Pinus pinaster Aiton. This work investigates the genetic variation, the mating system and the pollen contamination in a P. pinaster clonal seed orchard (CSO), by screening the 60 clones from the CSO and the seeds collected from 21 mother-trees with three nuclear microsatellites. The expected diversity was similar, but the observed heterozygosity decreased 20% in the progenies compared with the parental trees. The outcrossing rate was 90.1%, the biparental inbreeding 21.7% computed through a multilocus approach, and the observed selfing 3.9%. The observed gene flow from outside the CSO was 52.4%. From the results we concluded that the observed gene flow and the biparental inbreeding were high, and care should be taken in the implementation and management of future CSO, in particular clones should be checked for relatedness and the ramet number could be directly proportional to their breeding value., L'apparentement entre parents, la variation de fertilité entre clones et la pollution pollinique font dévier le gain génétique réalisé et la diversité génétique des valeurs attendues en verger à graines de clones à pollinisation libre, en particulier pour une espèce à pollinisation anémophile comme Pinus pinaster Aiton. Ce travail étudie la variation génétique, le système de reproduction et la contamination du pollen d'un verger à graines de clones (VGC) de P. pinaster, en génotypant les 60 clones du VGC et les graines récoltées sur 21 arbres-mères pour trois marqueurs microsatellites nucléaires. La diversité attendue s'est révélée similaire mais l'hétérozygotie observée a diminué de 20 % chez les descendants par rapport aux parents. Le taux d'allofécondation était de 90,1 %, la consanguinité biparentale calculée grâce à une approche multilocus était de 21,7 % et le taux d'autofécondation observé était de 52,4 %. D'après ces résultats, nous avons conclu que les flux de gènes observés et la consanguinité biparentale étaient élevés et que des précautions devaient être prises dans la mise en place et la gestion de futurs VGC, l'apparentement entre clones devrait en particulier être vérifié et le nombre de copies d'un individu pourrait être directement proportionnel à sa valeur génétique.

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47 - 100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm -3 gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.

Mature and immature seeds of Ophrys lutea Cav. and Ophrys fusca Link were germinated in a modified Curtis medium. The protocorms obtained 2 months after sowing, when transferred to orchid culture medium 4 (OCM4) could form protocorm-like tissue with many small protocorms developing many plantlets within 2 months. The tissue could be used for propagation after sectioning and transfer to fresh OCM4. Two months later, rooted plantlets approximately 5 cm in height were obtained. These plantlets could be successfully transferred to soil. Minitubers from O. lutea and Ophrys speculum Link were produced using a modified OCM. These minitubers present the typical morphology and the anatomical characteristics of tubers formed spontaneously by these terrestrial orchid species. They represent a good possibility for vegetative propagation of terrestrial orchids.

We have developed particle bombardment-mediated transformation procedure for Hypericum perforatum L. (St. John’s wort), an important medicinal species that remains highly recalcitrant towards Agrobacterium-mediated transformation. Among the major transformation techniques evaluated in the present study (Agrobacterium tumefaciens-, A. rhizogenes- and biolistics-mediated), particle-bombardment-mediated gene transfer was found to be the most successful one. GUS positive cells were obtained from organogenic nodules bombarded with the plasmid vector pCAMBIA1301 encoding an intron-containing b-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes. After 3 months of continuous selection of bombarded nodules with 20 mg l 1 hygromycin, transgenic hygromycin-resistant callus cultures and subsequently transgenic plants were produced. PCR analysis of DNA isolated from GUS positive plants showed the presence of both gusA and hpt genes. Southern blot analysis confirmed the transgene integration and revealed diverse copy numbers and insertion sites. The data presented here demonstrate for the first time H. perforatum can be efficiently transformed via particle bombardment of organogenic cell suspension. Our results open the possibility of using particle bombardment-mediated transformation to elucidate biosynthetic pathways and to improve secondary metabolite production in H. perforatum., Fundação para a Ciência e a Tecnologia (FCT) - POCTI/AGR/40283/2001, SFRH/BPD/ 17102/2004

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.

Almond shoots produced by axillary branching from clone VII derived from a seedling of cultivar Boa Casta were evaluated for somaclonal variation using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify genetic stability we compared RAPD and ISSR patterns of plantlets obtained after 4 and 6 years of in vitro multiplication. A total of 64 RAPD and 10 ISSR primers gave 326 distinct and reproducible band classes, monomorphic across all 22 plantlets analysed. Thus, a total of 7,172 bands were generated, exhibiting homogeneous RAPD and ISSR patterns for the plantlets tested. These results suggest that the culture conditions used for axillary branching proliferation are appropriate for clonal propagation of almond clone VII, as they do not seem to interfere with the integrity of the regenerated plantlets. These results allowed us to establish the use of axillary branching plantlets (mother-plants) as internal controls for the analysis of somaclonal variation of shoots regenerated from other in vitro culture processes performed with clone VII (adventitious regeneration, regeneration from meristem culture, virus sanitation programs and genetic engineering).

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Coa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Coa plant; P. webbii; and P. persica (outgroup). The segregating Foz Coa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.

Eight new cDNA sequences for S-RNases were cloned and analysed from almond (Prunus dulcis) cultivars of European origin, and compared to published sequences from other Rosaceae species. Insertions/deletions of 10–20 amino acid residues were detected in the RC4 and C5 domains of S-RNases from almond and sweet cherry. The S-RNases of the Prunus species and those of the genera Malus and Pyrus formed two distinct groups on phylogenetic analysis. Nucleotide substitutions were analysed in the S-RNase genes of these species. The S-genes of almond and sweet cherry have a lower Ka/Ks value than those of apple, pear and wild apple do. The fact that there is no fixed difference between the S-RNase genes of almond and sweet cherry, or between apple and pear, suggests that nucleotide substitutions only introduce transient polymorphism into the two groups, and rarely became fixed and contribute to divergence. Through the comparative study of 17 S-RNase genes from the genus Prunus and 18 from the genera Malus and Pyrus, some fixed nucleotide differences between the two groups were identified. These differences do not appear to be the result of selection for adaptive mutations, since the number of replacement substitutions is not significantly greater than the number of synonymous substitutions. S-RNase genes of almond and sweet cherry, and of apple and pear, showed little heterogeneity in nucleotide substitution rates. However, heterogeneity was observed between the two groups of S-alleles, with the Prunus alleles exhibiting a lower rate of non-synonymous substitutions than alleles from Malus and Pyrus. The evolutionary relationships between these species are discussed.

Nodal segments and shoot tips of axenic shoot cultures of `Hayward' kiwifruit were inoculated on modified Murashige and Skoog (MS) medium supplemented with zeatin at 1 mg·liter-1 and IAA at 0.05 mg·liter-1 (H1) or on MS medium without growth regulators (H2). Inocula cultured on H2 medium all developed into normal plantlets, while those cultured on H1 medium developed into shoots, 18% of them abnormal. Rooting of H1 shoots was induced by a 24-h immersion in a solution of IRA at 20 mg·liter-1. H2 plantlets were directly transferred to soil. Statistical treatment of the results revealed no significant differences, in terms of plant development, between the two micropropagation methods used. However, the presence of a functional root system on 5-week-old H2 plantlets resulted in 100% plant survival, but only 70% of in vivo-rooted shoots from H1 survived. Nevertheless, H1 still allowed for an important reduction of costs and manipulation. Chemical names used: indole3-acetic acid (IAA).

Research in sexual plant reproduction has been boosted by the concentration of means and minds around central issues, such as incompatibility, microspore development or, to a less extent, the progamic phase of fertilisation. These areas are now pivotal since they involve basic biological questions still unanswered and which became recently accessible due to powerful genetic and molecular techniques. While these combined efforts resulted in the outcome of major findings that projected the field to a place not easy to by-pass even in the general context of Biology, this was only possible because of the existence of a few model species, particularly adapted to the experimental needs of the molecular weaponry. Most notably, Arabidopsis now plays a central role, since it possesses most of the development paths that characterise the majority of Angiosperms (with the exception of secondary growth). While this approach is not questionable, since it permits a faster advance in models and concepts, the reductionism underlying the focus on a few model species may create a void of knowledge on the natural variability and diversity of the cellular and molecular mechanisms. Most likely, the majority of the flowering species will never find their place as models species since they cannot compete as regards easiness to grow, maintain and analyse. Yet, an historical perspective shows beyond doubt that some of these species may bear variations to the basic models which may open new avenues and question established paradigms. Such was the case of Arabidopsis, for instance, often neglected and rejected for funding because of its condition of weed (Goldberg 1996).

The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.

Leaves of Humulus lupulus possess two types of glandular trichomes: - peltate (lupulin) and bulbous. Peltate trichomes are formed from a protodermal cell by two anticlinal divisions in perpendicular planes, followed by two periclinal ones that give rise to the initials of the head cells, the basal and the stalk cells. Head cells divide successively in radial and irregular planes. Fully developed peltate trichomes are built of a glandular head consisting of 30 to 72 cells, four stalk cells and four basal cells. Bulbous trichomes are also formed from a protodermal cell by an anticlinal division followed by two periclinal ones that produce the initials of the glandular head cells, and the basal and stalk cells. Fully developed bulbous trichomes consist of four (occasionally eight) head glandular cells, two stalk cells and two basal cells. The density of peltate trichomes decreases with the expansion of the leaves. Both peltate and bulbous trichomes secrete essential oils. Peltate trichomes are the preferential site for the synthesis of bitter resins. Tannic acids could not be detected histochemically either in peltate or in bulbous trichomes. Both types of trichomes produce secretion that accumulates in the subcuticular space, being released, in the case of bulbous trichomes, by rupture of the cuticle.