Ethylene in relation to protein, peroxidase and polyphenol oxidase activities during rooting in hazelnut cotyledons

During formation of adventitious roots, the effects of 2-chlorethylphosphonic acid (CEPA), 1-aminocyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG) added to a Cheng basal medium, supplemented with indole-3-butyricacid (IBA) and kinetin were determined on peroxidase (PO; EC 1.11.1.7) and poiyphenol oxidase (PPO; EC 1.10.3.1) activities in cotyledon explants of hazelnut ( Corylus avellana L. cv. Casina). CEPA stimulated PO and PPO activities while AVG inhibited. SDS-polyacrylamide gel electrophoresis of preparations from hazelnut cotyledons showed a correlation between proteins and rooting. Ethylene seems to modify total protein content and the activities of PO and PPO. As compared to the control extracts, AVG inhibited the anodic (53, 30.7 and 27 kDa) and cathodic (66.2 and 53.4 kDa) isoperoxidases and anodic (27.5 and 21 kDa) and cathodic (67.5 kDa) isopolyphenol oxidases, whereas CEPA promoted the anodic (30.7, 28.8 and 27 kDa) and cathodic (53.4 and 27.2 kDa) isozymes with PO activity. The increased PO activity during rooting in hazelnut cotyledons could enhance isozymes with lignin biosynthetic activity.     

Identification of hazelnut (Corylus avellana) cultivars by RAPD analysis

The random amplified polymorphic DNA (RAPD) technique offers a useful tool to detect DNA polymorphisms. It can also be used to distinguish different clones and cultivars. We have developed a comprehensive RAPD-based procedure for the routine molecular typing of various plants. Here we report the application of this technique for the correct identification of six hazelnut cultivars (Corylus avellana) widespread in the Campania region (south Italy). The analysed hazelnut cultivars were successfully distinguished by their RAPD fingerprints using the DNA primers U2, U3, U4, U11 and U14. However, in each cultivar we observed very low genetic heterogeneity among the clonal variants. Since this technique is among the simplest and easiest methods used to fingerprint DNA, it could be easily transferred to less sophisticated laboratory infrastructures (e.g. outstations of crop regulatory agencies). Received: 20 December 1997 / Revision received: 6 August 1998 / Accepted: 13 November 1998     

Exogenous sugars overcome incompatibility in hazelnut (Corylus avellana L.)

Summary Petreatment of hazelnut (Corylus avellana L.) stigmas with L M or 2 M sugar solutions before pollination with incompatible pollen, or pollination with 11 mixtures of incompatible pollen and finely ground sugars, prevented rejection of incompatible pollen by the stigma surface. Lower sugar concentrations or lower ratios of sugar to pollen were less effective. No specificity for overcoming incompatibility was observed among 18 simple sugars and related sugar compounds.Oregon Agricultural Experiment Station Technical Paper No. 7613     

欧洲榛子贮藏及萌发生理特性研究

对欧洲榛子(Corylus avellanaL.)在沙藏、萌发、幼苗生长阶段的种仁养分和保护酶活性动态变化特点进行了研究,结果表明:种子萌发后107 d营养耗尽;沙藏过程中种仁的干重、粗脂肪含量略下降,萌发后下降迅速且与时间呈线性负相关;可溶性糖在沙藏至萌发后12 d内下降而后上升,萌发24 d后上升迅速,72 d后进入高峰期;淀粉含量在沙藏过程中下降,萌发后上升并在萌发48 d取得11.12%的峰值后下降;游离氨基酸总量在沙藏期间下降,萌发前12 d开始升高,在萌发36~48 d达155.31 mg/100 g的高峰后降低;可溶性蛋白50.87 mg/g的峰值出现在萌发前12 d;SOD在萌发前12 d至萌发后12 d、CAT由萌发起24 d内、POD在萌发36 d后分别出现活性高峰,对种仁营养按测定时间进行的聚类分析结果与依据幼苗干重划分的幼苗生长阶段相吻合。     

Identification of a caleosin associated with hazelnut (Corylus avellana L.) oil bodies

• Caleosins are involved in several cellular and biological processes that are closely associated with the synthesis, degradation and stability of oil bodies (OB).
• Because of the importance and the multiple roles of these OB‐associated proteins, in silico identification of sequences corresponding to putative caleosins in the hazelnut genome has been performed, and the association with seed OB was verified using a proteomic approach.
• Five full‐length sequences (CavCLO‐H1, CavCLO‐H2, CavCLO‐H3, CavCLO‐L1, CavCLO‐L2), belonging to the two groups of caleosins (H and L), have been identified in the hazelnut genome. The number of identified caleosins is in agreement with that previously observed in other plant species, confirming that caleosins comprise small gene families in plants.
• A proteomic approach allowed us to verify only the presence of CavCLO‐H1 in hazelnut OB, suggesting that several members inside this family could have different roles during plant growth and development. In silico analysis also suggests that CavCLO‐H1 may act as a peroxygenase.

     

Chloroplast DNA variation,postglacial recolonization and hybridization in hazel,Corylus avellana

To unravel the postglacial migration history of hazel, Corylus avellana, the genetic variation at two types of chloroplast DNA markers, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellites, was assessed in 26 natural hazel populations distributed across the range of C. avellana. In addition a sequence of 2468 base pairs, which contains the matK gene, was analysed in seven individuals. Very little variation was detected overall [hT:PCR-RFLP= 0.091, hT:microsatellite= 0.423, pi (nucleotide diversity) = 0.00093] but the microsatellite markers, which have the highest levels of variation, show a clear geographical structure that divides Europe into two areas: (i) Italy and the Balkans, on one hand and (ii) the rest of Europe, on the other hand. These data exclude Italy and the Balkans as possible origins of the postglacial recolonization but cannot unambiguously show which other area is the origin, since the genetic data does not indicate the direction of spread. If we take the pollen record into account, the most likely scenario would be an expansion from southwestern France into most of Europe except Italy and the Balkans, and then a local expansion in the latter area. The two main haplotypes identified with both PCR-RFLP and sequencing, A and B, were found not only in C. avellana but also in other European Corylus species and cultivars. Haplotype A, which is dominating all investigated natural populations of C. avellana, is also found in the European tree hazel (C. colurna) and haplotype B, which is rare in C. avellana, has been identified in the filbert (C. maxima) and C. avellana cultivars. This pattern seems to indicate a history of past hybridization among the European Corylus species and cultivars.     

CaMADS1, a MADS box gene expressed in the carpel of hazelnut

Hazelnut (Corylus avellana L.) is a species of economic interest that shows a peculiar floral biology. Unlike most of the angiosperms, which produce ovules during floral development such that they are ready for pollen at anthesis, hazelnut ovary development is delayed and triggered by compatible pollination. In order to elucidate the mechanisms regulating this unusual process and the role of the MADS box genes in ovary development, a cDNA library from pollinated styles of hazelnut was screened with a mixture of MADS box genes from different plant species. CaMADS1 (Corylus avellana MADS box), a floral-specific MADS box gene, was isolated, and characterized as belonging to the sub-family of the AGAMOUS genes. Northern blot, RT-PCR analyses and in situ hybridization experiments show a precise correlation between ovary development and CaMADS1 expression, indicating a role of this MADS box gene in the processes of floral organogenesis.     

Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.) Carr.] and high frequency in vitro rooting of shoots

Shoot formation from cotyledons of Sitka spruce was induced by a 2 h incubation in an aqueous solution of 400 μM benzyladenine, followed by culture on medium lacking plant growth regulators. Several basal media were assessed. Murashige and Skoog (1962) based medium was found to be optimal on which, after 25 weeks, 62.5% of cotyledons produced shoots with a mean of 10.7 shoots per cotyledon. Shoots, detached from cotyledons and propagated individually by axillary bud proliferation, rooted with a frequency of 84% following culture in half-strength MS-based medium lacking growth regulators under low illumination (1.5 μmol m⁻²s⁻¹). Rooted plants were successfully transferred to ex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection and Molecular Characterization of ‘Candidatus Phytoplasma asteris’ in European Hazel (Corylus avellana) in Poland

Stunted European hazel (Corylus avellana L.) plants showing leaf yellowing were observed in south‐eastern Poland. Phytoplasma‐specific primers P1/P7 and R16F2n/R16R2, as well as primers specific for aster yellows (16SrI), X‐disease (16SrIII) and apple proliferation (16SrX) groups were singly used in nested polymerase chain reaction (PCR) to amplify the 16S rDNA from 22 symptomatic and asymptomatic hazel plants. Restriction fragment length polymorphism with MseI, HhaI, RsaI and BfaI enzymes of the 16S rRNA gene fragments amplified with the primers R16F2n/R16R2 from three symptomatic hazel plants of cvs Katalonski, Webba and Halle revealed patterns identical to those from the AY1 strain related to ‘Candidatus Phytoplasma asteris’. The nucleotide sequence analysis confirmed this result. This is the first report of the natural occurrence of ‘Ca. P. asteris’ in European hazel in Poland.     

Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases.

Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone containing a complete LOX gene was isolated and fully characterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed.     

Shoot development and bud mite infestation in hazelnut (Corylus avellana)*

The interaction of environmental and genetic variation in hazelnut (Corylus avellana) shoot development and the behaviour, survival, and colonisation of eriophyid bud mites (Phytoptus avellanae and Cecidophyopsis vermiformis) were studied. The distribution of galled buds on shoots indicated that mites colonised only those buds formed during the mite migration period. The point of entry is probably the growing shoot tip. Once within this structure, as the shoot develops the mites have access to a succession of newly-formed, bud primordia that are unprotected by bud scales. The relative accessibility of the apical meristem and bud primordia may affect host susceptibility.     

Micropropagation of Sicilian cultivars with an aim to preserve genetic diversity in hazelnut (Corylus avellana L.)

The use of a small number of cultivars in agriculture can lead to a loss of agrobiodiversity. Since in vitro techniques are valuable tools for conserving plant biodiversity, an efficient micropropagation protocol for four Italian hazelnut cultivars, ‘Carrello’, ‘Ghirara’, ‘Minnulara’, and ‘Panottara’, was developed. The highest axillary bud survival was obtained after decontamination with 40?min 1% sodium hypochlorite followed by 40?min 0.1% sodium merthiolate in ‘Minnulara’ and ‘Ghirara’, while the 35?+?35?min treatment was the best for ‘Carrello’ and ‘Panottara’. Shoot multiplication was higher in ‘Minnulara’ and ‘Ghirara’ when 6.6?μM N⁶-benzyladenine was used, even if some hyperhydric shoots were observed, while metatopolin was more effective in the other two cultivars. In vitro rooting, performed in ‘Carrello’ and ‘Panottara’, was higher with 17.6 than with 9.8 µM indole-3-butyric. Following in vitro root induction with 17.6 µM indole-3-butyric acid for 7 days, rooting and acclimatisation in greenhouse exceeded 85% for all four cultivars.     

Desiccation sensitivity and successful cryopreservation of oil seeds of European hazelnut (Corylus avellana)

The sensitivity of a Polish provenance of dormant Corylus avellana seeds to extreme desiccation and cryopreservation in liquid nitrogen (LN, ?196°C) was investigated. No sensitivity to desiccation was observed as seeds readily germinated and exhibited seedling emergence, even when the critical water content (WC) of seeds (nuts devoid of pericarp) was reduced to 0.027 g H₂O/g dry mass, g g^?1 by drying. Results of germination and seedling emergence tests indicated that seeds tolerated cooling in LN when desiccated to a WC in the range of 0.05–0.10 and 0.08–0.10 g g^?1, respectively. The results of this study demonstrate the feasibility of long‐term cryopreservation of European hazelnut seeds. As the seeds of this species have been classified in several different categories (orthodox, suborthodox and recalcitrant), based on their response to desiccation and low temperature, the assignment of the seeds of hazelnut to a specific category is provided and discussed.     

Phytohormone contents in Corylus avellana and their relationship to age and other developmental processes

Endogenous levels of indole-3-acetic acid, abscisic acid, and cytokinins (Z-type: dihydrozeatin, dihydrozeatin riboside, zeatin, and zeatin riboside; iP-type: N ⁶-isopentenyl adenine and N ⁶-isopentenyl adenosine), were determined in leaves of hazelnut (Corylus avellana L.) (adult material from spring, autumn and forced outgrowth, and juvenile material). Our results showed high levels of indole-3-acetic acid, abscisic acid and total cytokinins in spring samples and low levels of the same hormones in autumn and forced outgrowth materials. The ratios of iP-type/Z-type cytokinins were low in autumn and spring leaves, while they were high in the juvenile and forced outgrowth samples. Both juvenile and forced-outgrowth hazel tissues also showed a high morphogenetic potential, suggesting that the ratio of iP-type/Z-type cytokinins may be a good index of in vitro potential of hazelnut materials.     

Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source

Shoot cultures established from mature trees of hazelnut (Corylus avellana L.) cvs. Nonpareil and Tonda Gentile Romana were used to determine the effects of basal media, carbon sources and concentrations, pH and cytokinins on shoot multiplication. All factors except pH affected the multiplication rate. Shoot multiplication was the best on a modified Driver and Kuniyuki medium for Paradox walnut (DKW) supplemented with 6-benzylaminopurine (BA) (1.5–3 mg/l). Plants grown on 3% glucose or fructose medium produced more and longer shoots than those on sucrose. The general appearance and growth habit of shoots were better on medium with glucose than fructose. Nonpareil shoots elongated better than those of Tonda Gentile Romana. Changes in medium pH from 4.7 to 5.7 did not significantly affect the multiplication rate. More than 10 genotypes propagated well on modified DKW medium with glucose. This is the first report of the effect of carbon sources on shoot multiplication of hazelnut and provides a basis for further research in the improvement of hazelnut micropropagation.Abbreviations MWPMC modified woody plant medium for chestnut (Yang et al. 1986) - DKW Driver and Kuniyuki (1984) medium for Paradox walnut - BA 6-benzylaminopurine - Z zeatin - 2iP N6-(2-isopentenyl) adenine - K kinetin - IBA indole-3-butyric acid     

Hyper-expression of small nucleolar RNAs (snoRNAs) in female inflorescences of hazelnut (Corylus avellana L.) supports rRNA aggregation in vitro

Under certain in vitro (salt and temperature) conditions rRNA aggregation occurs in female inflorescences but not in leaves or pollen RNA preparations from hazelnut (Corylus avellana L.), a species of economic interest. This paper describes experiments addressing an explanation of this phenomenon. The experiments demonstrate that: (i) trans-acting factors induce rRNA aggregate formation in female inflorescences RNA preparations; (ii) these factors support aggregation also of heterologous rRNA; (iii) aggregation is a function of temperature pre-treatment of rRNA and not of source 18S rRNA; (iv) the factors inducing rRNA aggregates are sensitive to RNase; (v) antisense small nucleolar RNAs (snoRNAs) participate in rRNA aggregate formation. snoRNAs are involved in pre-rRNA spacer cleavages, and are required for the two most common types of rRNA modifications: 2'-O-ribose methylation and pseudouridylation. Even though it is questionable whether rRNA aggregation really happens in female inflorescence in vivo, the phenomenon observed in vitro may reflect the abundance of snoRNAs in these reproductive structures. In fact the level of accumulation of three tested snoRNAs, R1, U14 and U3, is much higher in female inflorescence than in leaves or pollen of hazelnut. This finding opens the possibility of studying the role of snoRNAs in tissue development in plants.     

Characterization and evaluation of microsatellite loci in European hazelnut (Corylus avellana L.) and their transferability to other Corylus species

In this work, 18 microsatellite loci were developed in the European hazelnut (Corylus avellana L.) using three enriched genomic libraries. They were evaluated on a set of 20 accessions of this species on the basis of number of alleles (mean: 7.1), expected heterozygosity (mean: 0.67), power of discrimination (mean: 0.77) and polymorphism information content (mean: 0.64). Cross‐species transferability was evaluated using seven other Corylus species. All primer pairs amplified in all species, except for CaT‐C505 in Corylus ferox and CaT‐A114 in Corylus californica.     

硼、锌对平欧杂交榛光合生理、结实和果仁品质的影响

通过对黑龙江省东宁地区的4年生平欧杂交榛（Corylus heterophylla×Corylus avellana）联合施用不同浓度的硼（0.2%、0.3%、0.4%）、锌（0.3%、0.4%、0.5%）肥处理,研究两种微量元素对平欧杂交榛光合生理、结实、果仁和叶片营养状态的影响。研究结果表明：（1）硼、锌对净光合速率影响显著,其交互作用对净光合速率影响极显著。（2）硼对出仁率影响极显著,对果仁重影响显著,对单果重和座果率影响不显著。锌对单果重和果仁重影响极显著,对出仁率影响显著,对座果率影响不显著。硼、锌交互作用对出仁率影响显著,对单果重、果仁重和座果率影响不显著。（3）硼、锌和其交互作用对果仁脂肪酸含量、果仁蛋白质含量影响不显著。硼、锌和其交互作用对4年生平欧杂交榛的生理和结实影响明显,提高了平欧杂交榛叶片的净光合速率,同时对果仁重、出仁率和单果重有不同程度的促进作用。本地区4年生平欧杂交榛硼、锌叶面施肥的浓度分别以0.3%和0.4%为宜。     

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997