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Plant-viroid interactions result in systemic infections that are often accompanied by developmental aberration indicative of a general perturbation in phytohormone metabolism and signaling. This chapter provides an overview of the effects of viroid infection, particularly potato spindle viroid disease, on the content of endogenous phytohormones and the expression of phytohormone-related genes in host plants, with a specific focus on salicylic acid, jasmonic acid, brassinosteroids and auxins. The effect of exogenous phytohormones on the development of viroid infections is also described. A summary of recent studies that propose mechanisms of phytohormone signaling involved in plant responses against pathogens and regulatory crosstalk between different signaling pathways is included. A better understanding of the role of phytohormones in plant-viroid interactions could improve our knowledge about host strategies to survive viroid attack and help the development of hormone-based breeding programs aiming to improve plant resistance/tolerance to viroids.

Herbra Epimedii is a common and widely used traditional Chinese medicine for thousands of years. Epimedium alpinum L. is one of the representative herb of Epimedium species in Europe, which is used as a diaphoretic and applied in breast illness in folk medicine. In this paper, an efficient UHPLC-Q-TOF-MS method was established for rapid discovery and identification of prenylated flavonoid glucosides in E. alpinum from Croatia. Based on the MS2 fragment pathways, rules and intensity of 12 reference compounds in negative and positive ion mode, the kinds of substituents and site of saccharides linkage of 79 prenylated flavonoid glucosides were tentatively characterized. Different substituents at C-3 and C-4 in the ring B formed four skeletons, including skeleton A with hydrogen and hydroxyl, skeleton B with hydrogen and methoxyl, skeleton C with hydroxyl and methoxyl and skeleton D with two hydroxyls, were identified in E. alpinum. Based on our knowledge, the skeleton D was first identified in herb medicine and 23 new compounds with skeleton D were characterized. According to these findings, prenylated flavonoids glucosides of 40 leaves, rhizomes, buds and tissue cultural products of E. alpinum were further systematically investigated. Chemicals in E. alpinum from Croatia were obviously different from those in E. koreanum Nakai and E. brevicomu Maxim from China via principal component analysis (PCA). This study provide an effective strategy for new compounds discovery in herbal extract, which is helpful to quality control and pharmacological evaluation of E. alpinum from Croatia.

Although structurally simple, viroids can trigger numerous changes in host plants and cause loss of yield in agronomically important crops. This study investigated changes in the endogenous status of phytohormones and antioxidant enzyme activity in Solanum tuberosum cv. Désirée in response to Potato spindle tuber viroid (PSTVd) infection. Phytohormone analysis showed that the content of endogenous jasmonic acid (JA) and its precursor cis-OPDA significantly increased in leaves, while the content of castasterone (CS) increased in both leaves and tubers of systemically infected plants compared to mock- inoculated control plants at 8 weeks post- inoculation. The indole-3-acetic acid content moderately increased only in tubers, while no differences in salicylic acid and abscisic acid content were observed between infected and control plants. Changes in endogenous phytohormone content were associated with upregulated expression of genes involved in the biosynthesis of JA and brassinosteroids, and the metabolism of auxins. Additionally, PSTVd infection provoked overproduction of hydrogen peroxide, which coincided with increased activity of guaiacol peroxidase in leaves and ascorbate peroxidase in potato tubers. The activity of catalase decreased in leaves, while superoxide dismutase activity remained steady regardless of the treatment and organ type. Total ascorbate and glutathione did not change significantly, although a shift towards oxidized forms was observed. Results suggest the existence of organ-specific differences in phytohormone and antioxidative responses in potato upon PSTVd infection. Possible effects of the observed changes on symptom development are discussed.

To better understand the role of phytohormones and antioxidative responses in plant-viroid interaction, changes in endogenous phytohormone contents and activities of key antioxidant enzymes during latent infection of Solanum laxum by potato spindle tuber viroid (PSTVd) were examined. The results showed that endogenous jasmonic acid (JA) and castasterone (CS) content significantly increased, whereas abscisic acid content significantly decreased in symptomless leaves of systemically infected plants compared to that of mock-inoculated control plants. Regarding endogenous salicylic acid and indole-3-acetic acid content, there were no differences between infected and control plants. PSTVd infection also caused excessive production of hydrogen peroxide and enhanced the activity of ascorbate peroxidase (APX) and superoxide dismutase but not the activity of peroxidase and catalase (CAT). When expressed on a protein-weight base, only APX activity increased while CAT activity decreased almost by half in systemic leaves of infected plants. Furthermore, there was a fall in total glutathione content but also a rise in the content of oxidized forms of both ascorbate and glutathione, indicating that the regeneration of glutathione is insufficient. Results of this study strongly suggest the involvement of JA, CS and enzymatic and non-enzymatic antioxidants in the physiological response of S. laxum during latent infection by PSTVd.

Herbra Epimedii is a common and widely used traditional Chinese medicine for thousands years. Modern pharmacology reported that Epimedium have various efforts, such as anti- nociceptive, anti-osteoporosis, antirheumatic and aphrodisiac. These years, the flavonoid glucosides of Epimedium species from China has a wildly research. Epimedium alpinum L. is one of the representatives of Epimedium species in Croatia. In folk medicine, the leaf is used as a diaphoretic and applied in breast illness. In this paper, an efficient method was established for rapid discovery and identification of flavonoid glucosides in Epimedium alpinum L. from Croatia. Based on the MS/MS fragment pathways, rules and intensity, 10 reference compounds in negative ion mode was systematically characterized by UHPLC-Q-TOF-MS. Different substituents at C-3 and C-4 in the B ring formed four skeletons, including hydrogen and hydroxyl for skeleton A, hydrogen and methoxyl for skeleton B and hydroxyl and methoxyl for skeleton C and two hydroxyls for skeleton D, which had been identified in Epimedium alpinum L. from Croatia. Based on these findings, the flavonoids glucosides of leaves, rhizomes, buds and cultural products of 45 Epimedium alpinum L. samples had been a clearly identification. Through the principal component analysis, sample from Croatia has an obviously difference in compounds of Epimedium koreanum Nakai and Epimedium brevicomu Maxim from China. This study is helpful for the quality control and extended the pharmacological application of Epimedium alpinum L. from Croatia.

The Harmonization of Seismic Hazard Maps in the Western Balkan Countries Project (BSHAP) was funded for 7 years by NATO-Science for Peace Program to support the preparation of new seismic hazard maps of the Western Balkan Region using modern scientific tools. One of the most important outputs of the BSHAP is an updated and unified BSHAP earthquake catalogue that is compiled directly from the datasets of earthquake data providers of the region. In the framework of BSHAP, the regional free field strong motion network capacity was increased significantly by the purchased and installed recorders and the BSHAP strong motion database that includes both pre- BSHAP (mostly analog) and post-BSHAP (all digital) recordings was compiled. The BSHAP strong motion database is used for proper selection of the ground motion prediction equations (GMPEs) for the probabilistic seismic hazard assessment (PSHA) by comparing the compiled strong ground motions with the predictions of candidate global, European, and Euro- Med GMPEs in a systematic manner. BSHAP collected relevant knowledge about the geological structure of southwestern Balkans provided a better understanding of the prevailing stress regime in the region. The main output of BSHAP is the new probabilistic seismic hazard maps for Western Balkans, obtained by implementation of the smoothed- gridded seismicity approach. The results are expressed in terms of peak horizontal acceleration (PGA) for 95 and 475 years return periods aligned with Eurocode 8 requirements. The seismic hazard maps derived in this project are a good basis to characterize the seismic hazard of Western Balkans. They will help the national authorities, public and private institutions, civil emergencies agencies, etc., for urban planning, disaster preparedness, and seismic hazard mitigation.

Extracellular proteins and glycoproteins secreted by ammonium- or auxin-induced somatic embryogenic cultures of pumpkin were analyzed. Despite an overall similarity in developmental characteristics between these embryogenic cultures, distinct expression patterns of extracellular proteins and glycoproteins were observed. Ammonium, when supplied as the sole source of nitrogen, caused acidification of the culture medium and significantly reduced protein secretion. Buffering pH in the ammonium-containing medium restored extracellular protein secretion and glycosylation and an enhanced cell aggregation but not the development of later embryo stages. As revealed by Concavalin A (Con A) immunodetection, extracellular glycoproteins containing α-D-mannose and α-D-glucose were most abundant in proembryogenic cultures grown in a buffered ammonium-containing medium and in a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). We assume that extracellular proteins (Mr 28, 31, and 44 kDa) and Con Abinding glycoproteins (Mr 26, 30, 40, 53, and 100 kDa) found in both proembryogenic cultures may have a role during somatic embryogenesis induction. The glycan components of proteins were further characterized by affinity blotting with different lectins. Binding patterns of mannose-specific lectin from Galanthus nivalis partially correlated with those detected with Con A, whereas no signal was observed with lectins from Datura stramonium and Arachis hypogea regardless of the treatment applied. Results indicate that complex N- or O-glycans are not typical for early phases of pumpkin embryo development. The accumulation of extracellular glycoproteins with high-mannose-type glycans from 30 to 34 kDa, observed after the transfer from the ammonium- or 2,4-D-containing media into a maturation medium, appeared to be associated with development of later embryo stages. This study also revealed the presence of EP-3-like endochitinases in pumpkin embryogenic cultures, particularly in cultures grown in the buffered ammonium-containing medium, however, these proteins should be examined further.

Viroids of the genus Pospiviroid are able to induce diseases in a wide range of host plants including important crop species. Although occasional disease outbreaks of Potato spindle tuber viroid (PSTVd) and closely related pospiviroids have been reported in potato and tomato, recent studies found an increase in number of latent infections in ornamental solanaceous species. In order to verify the presence of PSTVd and other pospiviroids in Croatia, a survey was conducted between 2009 and 2012. A total of 182 samples belonging to five ornamental species and two solanaceous crops were analyzed. Eight plants belonging to two different species (Solanum jasminodes and Lycianthes rantonnetii) were found infected by PSTVd and, in addition, one S. jasminoides plant infected by Tomato apical stunt viroid (TASVd). Viroid infection was confirmed by mechanical inoculation on tomato plants to observe symptom expression and molecular tests. Molecular characterization of the isolates was done and complete viroid sequences were submitted to the GenBank. This is the first evidence of the presence of PSTVd and TASVd and their variability in Croatia.

Habituated embryogenic line of pumpkin contained more CKs and IAA, but less ABA than the non-habituated line. Pronounced hypomethylation correlated with the absence of 2,4-D, addition of 5-azaC, and the process of habituation. A comparative analysis between habituated and non-habituated embryogenic cultures of pumpkin (Cucurbita pepo L.) in relation to endogenous phytohormones, global DNA methylation, and developmental and regeneration capacities of the cultures was conducted. The analysis revealed more cytokinins (CKs) and indole-3-acetic acid (IAA), but less abscisic acid (ABA) in the habituated HEC line than in the non-habituated DEC line. Ribosides and ribotides were the most abundant CK forms in both HEC and DEC lines (75.9 and 57.6 %, respectively). HEC contained more free-base CKs (5.8 vs. 3.2 %), whereas DEC contained considerably more O-glycosides (39.1 vs. 18.3 %). Although prevalence of IAA was common for both lines, relative ratio of CKs and ABA differed between DEC and HEC lines. ABA was prevailing over CKs in DEC, while CKs prevailed over ABA in HEC line. Taking into account the importance of ABA for embryo maturation, the reduced endogenous ABA content in HEC line might be the reason for a 5-fold reduction in regeneration capacity compared to DEC. Both habituated and non-habituated embryogenic lines were highly methylated in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). Pronounced hypomethylation correlated with the absence of 2,4-D, addition of 5-azacytidine (5-azaC), but also with the process of habituation. The habituated line was resistant to the effect of hypomethylation drug 5-azaC and remained highly methylated even after the addition of 5-azaC. Also, 5-azaC did not change the developmental pattern in the habituated line, indicating the existence of separate mechanisms by which 2,4-D influences global DNA methylation in comparison to habituation-related global DNA methylation.

In many vascular plants, zygotic reproduction regularly alternates with different types of asexual reproduction, so embryos can develop not only from fertilized egg cells, but also from induced somatic cells through the process of somatic embryogenesis. Although somatic and zygotic embryogenesis are not directly correlated, their common features are presented, demonstrating that the origin and development of the somatic embryo morphologically and physiologically resemble zygotic embryogenesis at certain points. To initiate embryogenesis, both competent egg and somatic cells require activation either by fertilization or specific environmental signals, respectively. During induction of somatic and zygotic embryogenesis, modulation of DNA methylation, activation of particular hormonal and stress-related mechanisms and changes in cell wall properties are triggered. Here, we give an overview and discuss the most recent research in the field of plant somatic and zygotic embryogenesis, with special attention given to the onset of embryogenesis and early embryo development as well as to embryogenesis-related interconnections between plant hormones, stress responses, DNA methylation and regulatory gene expression.

When supplied as the sole nitrogen source, ammonium can induce somatic embryogenesis in some plant species, including pumpkin. To get an insight into the involvement of phytohormones in this adaptive developmental response to nitrogen availability, the status of endogenous abscisic acid (ABA), indole-3-acetic acid (IAA), and their conjugates was analyzed in an ammonium-induced embryogenic pumpkin culture. Results were compared with endogenous hormone status in an embryogenic culture induced by exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D). In the NH4 +-induced somatic embryogenic culture, the maximum concentration of ABA was measured after 21 days of cultivation, while in the 2,4-D-induced culture ABA peaked after 7 days of cultivation, which suggested a possible activation of different ABA-regulated stress responses. The NH4 +-induced embryogenic culture was also characterized by increased accumulation of endogenous IAA to the level similar or higher than the one induced by 2,4-D. A considerable decrease (threefold) in endogenous IAA content was observed after buffering the NH4 +-medium with MES or after nitrate re-supply. In contrast, buffering the NH4 +-medium increased endogenous ABA by five times. Significant ammonium- and pH-related changes were also detected in ascorbate and glutathione redox status, and discussed regarding the corresponding changes in cell size and mitotic activity in the cultures. Furthermore, a negative correlation between free IAA content and the activity of ascorbate peroxidase (EC 1.11.1.11) and auxin-sensitive peroxidase CpAPRX (EC 1.11.1.7) was observed. The results strongly suggest a mediating role of external pH in the regulation of endogenous phytohormones and the ascorbate–glutathione cycle during ammonium-induced acquisition of embryogenic competence in pumpkin.

Reproductive development in the Christmas rose (Helleborus niger L.) differs from that in commonly investigated model plants in two important aspects: (i) the perianth develops a photosynthetic system, after fertilization, and persists until seed ripening; and (ii) the ripe seed contains an immature embryo which continues to mature off the mother plant. The possible roles of cytokinins in these processes are investigated here by analysing extracts of the perianth and the carpels/maturing fruit prepared during anthesis and four stages of post-floral development. trans-Zeatin, dihydrozeatin, N6-(Delta2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5'-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins were mostly localized in the seeds. Their overall concentrations increased dramatically during early seed development and remained high for 6-8 weeks, until shortly before seed ripening (the last time point covered in this work). Overall cytokinin levels in the perianth did not change markedly in the period covered, but the level of N6-(Delta2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.

In an attempt to develop a transformation procedure for somatic embryos of Picea omorika (Panc.) Purk. using Agrobacterium, the sensitivity of non-transformed embryogenic tissues of P. omorika to antibiotics was measured. Two groups of antibiotics were tested: antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin and cefotaxime), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin, neomycin and hygromycin). Carbenicillin (500 mg l−1) reduced proliferation of P. omorika embryogenic tissue by 50% as compared to the control. Cefotaxime (500 mg l−1) was less toxic to embryogenic tissue growth (93% of the control) and is thus more suitable for the elimination of A. tumefaciens from embryogenic tissue of P. omorika. Embryogenic tissue showed severe susceptibility to kanamycin and hygromycin. Induction of secondary embryogenesis was blocked by 10 mg l−1 of kanamycin or 2 mg l−1 of hygromycin. The aminoglycoside analogs of kanamycin, paromomycin and neomycin inhibited embryogenic tissue induction at concentrations of 5 and 100 mg l−1, respectively. The higher tolerance of embryogenic tissue to neomycin indicated that of the antibiotics tested neomycin may be most useful for selection of nptII-transformed embryogenic tissue of Picea omorika.

Improved rooting was demonstrated in coast redwood (Sequoia sempervirens (D. Don.) Endl.) using an Agrobacterium-mediated gene transfer system. Microcloned shoot-tips of redwood (clone CA3) were inoculated with Agrobacterium rhizogenes wild strain 8196 or with the pRi A4 transconjugant strain of Agrobacterium tumefaciens (C58 chromosomal background) at the basal-cut end. Roots were formed in 58–69% of explants, but without a hairy-root phenotype. Rooting frequency and the number of adventitious roots on inoculated shoot-tips were slightly moderated by the bacterial strain, duration of inoculation and explant support system. Growth of excised roots was very slow on hormone-free medium and opine production could not be confirmed. Callus cultures derived from putative transformed roots were established on medium supplemented with 1.13 μM 2,4-D and 2.22 μM BA. PCR analysis of callus tissues DNA confirmed that integrative transformation had occurred in Sequoia adventitious roots generated after infection with Agrobacterium strains 8196 or A4. This is the first report of transgenic Sequoia tissue using A. rhizogenes-mediated gene transfer.

KEY MESSAGE : Stress hormones, particularly jasmonic acid, influenced root growth, auxin levels, and transcription of auxin amidohydrolase BrIAR3 in Brassica rapa seedlings, while auxin conjugate synthetases BrGH3.1 and BrGH3.9 were down-regulated by all treatments. The influence of stress hormones: jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) on 1-day-old seedlings of Chinese cabbage (Brassica rapa L. ssp. pekinensis) was investigated with particular focus on auxin levels and the regulation of reversible auxin conjugation as a mechanism of auxin homeostasis. At the physiological level, stress hormones inhibited root growth, where JA was the most prominent inhibitor with an IC50 value 3.1 μM, which is one and two orders of magnitude lower than that found for ABA and SA, respectively. JA treatment significantly increased the total auxin content, by induction of free and conjugated forms. Also, the stress hormones affected the transcription of genes involved in the process of the reversible auxin conjugation: auxin amidohydrolases BrIAR3 and BrILL2, and auxin conjugate synthetases BrGH3.1 and BrGH3.9. JA treatment increased the transcript level of BrIAR3 two-fold, while it did not affect the transcription of BrILL2. SA and ABA down-regulated the transcription of both auxin amidohydrolase genes by 30 %. Transcription of both auxin conjugate synthetases was significantly down-regulated by all treatments by 30-70 %. Among the investigated biochemical stress markers, glutathione along with protein carbonylation appeared the most affected upon treatments. The redox status of the seedlings was shifted to the more oxidized state upon JA and ABA treatments, whereas SA caused more reduced redox state in comparison to the control. The principal component analysis visualized relationship among auxin and stress parameters upon treatments. Accordingly, the role of auxin in stress response of Brassica seedlings was discussed.

Wounding of explanted Pinus nigra primary explants followed by infection with Agrobacterium rhizogenes wild strains 8196, 15834, or with the pRiA4abc transconjugant strain of A. tumefaciens (C58 chromosomal background) resulted in adventitious root induction. Roots were formed in 60-97% of explants (1-3 roots/explant) but without a hairy root phenotype. The presence of T-DNA of pRi8196 or pRiA4abc in regenerated roots was confirmed by the opine (mannopinic acid) content. Transformation response was influenced by the bacterial strain, age of explants and period of co-cultivation. Both the aggregate state (liquid) of medium and the season of the year (spring) had a positive effect on the root induction and their development. Histological analysis of the transformed roots showed that complete elements of primary and secondary root structures were present but roots were always triarch or tetrarch in the central cylinder as opposed to the primary roots of the untransformed seedling which are diarch.

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.

The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L.) cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos., Analizirani su izvanstanični proteini tri ustaljene embriogene linije bundeva (Cucurbita pepo L.) koje su uzgajane na četiri različite podloge MS s obzirom na udjel dušika i prisutnost/odsutnost auksina. Izvanstanični proteini što sadrže α-D-manozu specifično su dokazani lektinom konkavalinom A. Tijekom uzgoja embriogenoga tkiva na podlozi s dodatkom reduciranog dušika, razvitak većine embrija bio je zakočen u predglobularnom i globularnom razvojnom stadiju, što se podudaralo s nedostatkom izlučivanja proteina. Izlučivanje proteina od 76, 68, 37 i 34 kDa dokazano je u sve tri linije tijekom uzgoja na podlozi koja pospješuje razvoj embrija, neovisno o dodatku 2,4-diklorofenoksioctene kiseline ili tunikamicina. Glikoprotein od 64 kDa utvrđen je u sve tri linije uzgajane na podlozi MS bez dodatka hormona i s nepromijenjenim izvorima dušika, te se mogao povezati sa sazrijevanjem embrija na ovoj podlozi. Izlaganje tunikamicinu nije utjecalo na embriogenezu, iako je imalo specifično djelovanje na glikozilaciju proteina ispitanih linija. Rezultati pokazuju da osim auksina i izvori dušika imaju važnu ulogu u pravilnoj glikozilaciji proteina, njihovom izlučivanju i dinamici razvitka somatskih embrija bundeve.

Gibberellins (GAs) were identified and quantified during flower and fruit development in the Christmas rose (Helleborus niger L.), a native of south-eastern Europe with a long international horticultural tradition. Physiologically, the plant differs from popular model species in two major aspects: (i) following anthesis the, initially white or rose, perianth (formed, in this species, by the sepals) turns green and persists until fruit ripening ; and (ii) the seed is shed with an immature embryo, a miniature endosperm, and a prominent perisperm as the main storage tissue. GA1 and GA4 were identified by full-scan mass spectra as the major bioactive GAs in sepals and fruit. LC-MS/MS-selected ion monitoring in accord with previously verified protocols also afforded analytical data on 12 precursors and metabolites of GAs. In the fruit, GA4 peaked during rapid pericarp growth and embryo development and GA1 during the subsequent period of rapid nutrient accumulation in the seeds and continued pericarp enlargement. In the sepals, the flux through the gibberellin biosynthetic pathway was highest prior to the light green stage when the photosynthetic system was induced. Unfertilized, depistillated, and deseeded flowers became less green than the seed-bearing controls ; chlorophyll accumulation could be restored by applying GA1, GA4 and, less efficiently, GA3, to the deseeded fruit. The sepals of unfertilized and depistillated flowers indeed contained very low levels of GA4 and gradually decreasing levels of GA1. However, the concentrations of their precursors and metabolites were less affected. These data suggest that a signal(s) from the fruit stimulates GA biosynthesis in the sepals resulting in greening. The fruit derived GAs appear to be mainly involved in pericarp growth and seed development.

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.

This book contains 46 chapters, divided into 4 sections : A) 12 chapters on conifers, B) 14 chapters on fruits, C) 14 chapters on angiosperms, and D) 6 chapters on histology, bioencapsulation, protoplasts, cryopreservation, double staining and thin cell layer sectioning. The book provides stepwise protocols for somatic embryogenesis of a range of selected woody plants in order to assist researchers to initiate somatic embryogenic cultures without too much alteration in protocols. Each chapter provides information on initiation and maintenance of embryogenic cultures ; somatic embryo development, maturation, and germination ; acclimatization and field transfer of somatic seedlings. Some chapters include applications of somatic embryogenic cultures, e.g. protoplasts, encapsulation, cryopreservation, genetic transformation, genetic fidelity with molecular markers, and bioreactor.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 m M NH(4)Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 micro M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.

Efficiency in transformation of three hybrids of Coleus blumei using wild and mutant strains of Agrobacterium sp. was evaluated. Successful transformation depended on specific bacterial strain-plant genotype combination and co-cultivation treatment. The wild type B6S3 of the bacterial strains employed gave the maximum efficiency. Strains C58C1(pArA4abc), GV3101(pGV2215), 8196 and A281 were also effective. Among several co-cultivation conditions tested, the most efficient treatment was as follows: excised leaf explants were incubated immeadiately in bacterial suspension for 5 min and were co-cultured with Nicotiana tabacum crown gall callus for two days. Plant hormone autotrophy, PCR analyses and hybridization analysis confirmed genetic transformation of the cultures. Sixteen different transgenic callus and cell cultures were maintained for more than four years in the absence of exogenous growth regulators and antibiotics without deterioration in the growth rate., Tri genotipa ukrasne koprive (Coleus blumei Benth.) transformirana su s pomoću bakterija Agrobacterium tumefaciens i A. rhizogenes. Uspješnost transformacije ovisila je o sukultivacijskom postupku lisnih eksplantata i bakterija te o interakciji bakterijskoga soja i biljnoga genotipa. Soj divljeg tipa B6S3 bio je najvirulentniji. Najuspješniji je postupak transformacije bio petminutna inkubacija svježe narezanih lisnih eksplantata s bakterijama te nakon toga dvodnevna sukultivacija s tumorskim tkivom duhana (Nicotiana tabacum). Rast kalusnoga tkiva na hranidbenoj podlozi bez regulatora rasta, PCR analiza te hibridizacijska analiza potvrdili su genetičku transformaciju tkiva. Ustanovljeno je 16 transgeničnih kalusnih linija koje su se međusobno razlikovale po fenotipu i stopi rasta. Fenotipske značajke pojedine linije nisu se mogle povezati s bakterijskim sojem upotrijebljenim u poticanju transformiranih linija niti s biljnim genotipom.

The effects of explant source, medium composition and growth regulators were examined in order to optimize the induction and selection of fast growing callus lines of European yew (Taxus baccata L. Washingtonii). Callus cultures were induced from isolated mature zygotic embryos or from segments of juvenile branches. Following two months of growth on induction medium (MS + 3.0 mg/L NAA, 0.5 mg/L kinetin, 100 mg/L arginine, 2.5 % sucrose and 0.8 % agar), callus proliferation was induced in 86.4 % of embryo explants and 100 % of branch-cutting explants. The growth potential of established callus lines was found to vary in response to genetic potential and culture medium composition. The growth rate of stem-derived callus obtained on induction medium was superior to that obtained using all other tested media modifications (duplication time 9.6 days). However, the growth of embryo-derived callus lines was enhanced by increasing the iron content from 27.8 to 55.6 mg/L FeSO4 ∙7H2O in the maintaining MS medium (duplication time for line E2 was 8.5 days). In two out of three embryo-derived lines, tissue growth was further improved by transferring onto modified B5 medium (duplication time for lines E2 end E5 was 4 and 5.7 days, respectively). HPLC analysis confirmed the presence of the anticancer agent cephalomannine in calli grown on B5 medium and a taxane-like substance in calli grown on MS medium., Ispitan je utjecaj eksplantata i sastav hranidbene podloge na rast kalusnih linija europske tise. Kalusne kulture potaknute su na eksplantatima čitavih zrelih zigotnih embrija i odsječaka jednogodišnjih grančica odrasle biljke. Nakon dva mjeseca uzgoja na indukcijskoj podlozi (MS s dodatkom 3,0 mg/L NAA, 0,5 mg/L kinetina, 100 mg/L arginina, 25 g/L saharoze i 8 g/L agara) proliferacija kalusa postignuta je na 86,4 % embrijskih eksplantata i na 100 % eksplantata grančice. Rast ustaljenih kalusnih linija ovisio je o genetskom potencijalu i sastavu hranidbene podloge. Najbolji prirast svježe mase kalusa dobivenog iz tkiva grančice ostvaren je na indukcijskoj podlozi (vrijeme udvostručenja mase kalusa bilo je 9,6 dana). Kalus induciran iz embrija rastao je brže na podlozi MS s povećanim udjelom željeza (55,6 mg/L umjesto 27,8 mg/L FeSO4 ∙ 7H20) pa je masa tkiva linije E2 udvostručena za 8,5 dana. Prirast mase tkiva u dvije od tri embrijske kalusne linije poboljšan je supkultiviranjem na modificiranu podlogu B5 (tkivna masa linija E2 i E5 udvostručena je nakon 4, odnosno 5,7 dana). Analizom HPLC u kalusnim kulturama uzgajanim na podlozi B5 dokazana je prisutnost antikancerogene tvari cefalomanina, a u kalusnom tkivu raslom na podlozi MS prisutnost neodređene tvari slične taksanu.