Intercellular protuberances in the ground tissue ofCocos nucifera L.

Summary Prolific filamentous intercellular protuberances have been observed in the intercellular spaces of the ground parenchyma tissue in the stems ofCocos nucifera. They are visually similar to some intercellular material reported in several other plant tissues but their chemical composition is unknown. Tests for lignin, cellulose, callose, suberin and waxes have proved negative and those for pectin inconclusive. The amount of intercellular material is closely related to the thickness of the parenchyma cell wall and the protuberances appear to be produced continuously by an active cytoplasm.     

A comparative biochemical study of conifer pollen allergens

In the order Coniferales, only the family Cupressaceae is regarded as being a significant source of airborne allergens, withJuniperus ashei characterized as the most significat aeroallergen. Pollen of the closely related speciesJ. virginiana has been shown to cross-react withJ. ashei pollen, however,J. virginiana pollen is not considered an important aeroallergen. Although there have been several reports of allergies toPinus pollen, the pollen of this genus is regarded as hypoallergenic. Our previous studies have shown that pollen extracts ofJ. ashei, J. virginiana, J. pinchotii, Cupressus macrocarpa, Pinus echinata andP. taeda all contained several proteins with the same molecular weights including the reported allergen ofJ. ashei. The present study compared the biochemistry ofJ. ashei, J. virginiana andP. echinata pollen. A time course experiment ofJ. ashei, J. virginiana andP. echinata showed thatJ. ashei released a greater quantity of protein within the first minute of moistening. SDS-PAGE analyses showed that the reported allergen ofJ. ashei pollen extracts was released in large quantities within the first minute of extraction. It was also determined that individual pollen grains ofP. echinata contained a greater quantity of protein than the pollen ofJ. ashei andJ. virginiana, but due to the large size of pine pollen there was less protein per gram of pollen. Lipid analysis of these three taxa showed that the pollen ofP. echinata contained more lipid per grain and per gram of pollen. Results indicate that the rapid release of the reported allergen fromJ. ashei pollen contributes to the allergenicity of this species compared to bothJ. virginiana andP. echinata.     

Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)

The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5-monophosphate (ZR5P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Embryo development in Cocos nucifera L.: A critical contribution to a general understanding of palm embryogenesis

InCocos (and probably in all other palms) the embryogenesis shows a number of primitive characters, such as differentiation of the embryo proper from one cell of the pluricellular proembryo, origin of the single cotyledon from a position lateral to the terminal stem tip, and a tendency to cleavage polyembryony.     

Studies on potassium-magnesium interaction in coconut (Cocos nucifera)

The widespread deficiency of magnesium in coconut acts as a limiting factor to increased production. Two ongoing experiments on potassium-magnesium (K-Mg) interaction in coconut conducted in lateritic gravels (ultisols), in the wet and intermediate agroclimatic zones of Sri Lanka showed significant yield responses (P=0.01) to differential K treatments, in the wet zone, but not in the intermediate zone. Differential Mg treatments, however, did not give rise to yield responses. Leaf and nut water analysis showed significant changes in the concentrations of Na, K and Cl (P=0.001), with a distinct inverse relationship between Na and K when K was applied. Differential Mg applications showed a significant effect only for leaf Mg (P=0.001), in the fourth year of the experiment, in the wet zone. Results indicate the usefulness of nut water analysis as an additional diagnostic tool, for Na, K and Cl.     

Ultrastructural features of megasporogenesis inTorreya nucifera (Taxaceae)

In megasporogenesis ofTorreya nucifera (Taxaceae) more than one product of meiosis can start to germinate, a process previously observed inTaxus. Ultrastructural analysis ofT. nucifera revealed that this behaviour is cytologically determined by the presence of a transfer cell type wall-membrane apparatus and by the presence of uncommonly complex plasmalemmasomes in the megasporocyte. These features may determine a uniform supply of nutrients to the entire megasporocyte cytoplasm, so that after meiosis the germination potential is not restricted to the chalazal megaspore. Even if more than one product of meiosis can originate the female gametophyte in bothTaxus andTorreya, this process involves different ultrastructural aspects in the two species. In conclusion, the extension of the germination potential is a common characteristic to both species but it is most probably the result of some evolutionary convergence.     

Morphological and histological changes during somatic embryo formation from coconut plumule explants

Summary Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing., At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic componenets. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103 000 daltons. Xan VIa was a glycoprotein of molecular weight 17 000 daltons. The carbohydrate moiety of Xan Vla was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

Enhanced aerobic respiration improves <Emphasis Type="Italic">in vitro</Emphasis> coconut embryo germination and culture

Summary Germination of coconut (Cocos nucifera L.) zygotic embryos was tested in liquid or solid medium. Percent germination was greater when embryos were cultured in solid medium, particularly when embryos were placed with their micropyle end facing upwards in relation to the vial orientation, independently of orientation in relation to gravity. This occurred because solid medium allowed embryos to be positioned with their micropyle end exposed to the ambient atmosphere of the vial. Germination of embryos facing upwards was suppressed when the ambient atmosphere was replaced with N₂ or when aerobic respiration inhibitors were added to the medium, whereas it was not suppressed when the atmosphere was replaced with a N₂/O₂ (50∶50, v/v) mixture, thus indicating that aerobic respiration is required for embryos to germinate. Germination in facing upwards mode also resulted in improved conversion and plantlet development. These improvements in recovery are important since they will help to avoid unintentional selection due to low in vitro germination and corresponding conversion of a population subset. These results set the basis for the improvement of current protocols for in vitro coconut embryo culture, allowing safe movement of germoplasm both in terms of phytosanitation and conservation of genetic diversity in a population of interest.     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^–1 min^–1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with subunits complex, the enzyme activity completely disappeared, whereas when subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity

The endo--1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.     

A New Method to Estimate the Population Size of Coconut Mite, Aceria Guerreronis, on a Coconut

Studies of the coconut mite, Aceria guerreronis, often require accurate assessment of the population density on individual infested coconuts. Here an efficient and accurate method was developed to estimate its population density on an infested coconut. The coconut mites were removed by washing the bracts and surface of an infested coconut with 30 ml of a detergent solution. Shaking the wash for 5 s allowed the mites to distribute uniformly. The number of mites in the first 1 ml of the first wash (X) yielded a very accurate predictor of the total number of mites on a coconut (Y): Y = 30.1X (R² = 0.99; p <0.0001), also confirming that the wash was indeed homogeneous. The advantages and disadvantages of this method are discussed.     

Transgenic ryegrasses (Lolium spp.) with down-regulation of main pollen allergens

Ryegrass pollen (Lolium species) is a widespread source of air-borne allergens and is a major cause of hayfever and seasonal allergic asthma, which affect approximately 25% of the population in cool temperate climates. The main allergens of ryegrass pollen are the proteins Lol p 1 and Lol p 2. These proteins belong to two major classes of grass pollen allergens to which over 90% of pollen-allergic patients are sensitive. The functional role in planta of these pollen allergen proteins remains largely unknown. Here we describe the generation and analysis of transgenic plants with reduced levels of the main ryegrass pollen allergens, Lol p 1 and Lol p 2 in the most important worldwide cultivated ryegrass species, L. perenne and L. multiflorum. These transgenic plants will allow the study of the functional role in planta of these pollen proteins and the determination of potential for development of hypo-allergenic ryegrass cultivars.     

The making of the kato alu—A traditional tongan basket

We investigated the weaving of the kato alu, a traditional Tongan basket. Unlike most ordinary baskets which are woven from coconut or pandanus leaves, kato alu are woven from tuaniu (Cocos nucifera, Palmae, midrib of leaf) and alu [Epipremnum pinnatum, Araceae, aerial roots]. Anciently the common people wove and decorated the kato alu and gave them to chiefs, nobles and kings as gifts at wedding and funeral ceremonies. Today, however, the weaving techniques are known to only a few people. It appears that these techniques will likely disappear from Tonga within a generation.

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Résumé  Ko e lipooti ko eni na’e vakai’i ’ae ngaahi founga a’ia ’oku kau ki he lalanga ’o e kato faka-Tonga, koe kato alu, pea na’a ku vakai’i foki ’a e tuaniu (Cocos nucifera) mo e akau, ko e alu (Epipremnum pinnatum) a ’ia ’oku na kau i he lalanga ’o e kato ia. ’I he ngaahi kuonga kuo hili, na’e lalanga ’a e kato alu ’e he kakai, ’o nau foaki me’a’ofa ’a kato ko ia ki he kau hou’eiki pea mo e Tu’i ’i he taimi na’a nau fai mali mo e fai putu. Ko e ’uhinga ’a e lipooti ko eni, ke fakamatala pe fakailonga ’a e ngaahi founga faka-Tonga ki hono lalanga ’o e kato mahu’inga ko eni, ’a’ai koe kato alu.

     

Detection of a <Emphasis Type="Italic">SERK</Emphasis>-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.     

Rearing of coconut mite Aceria guerreronis and the predatory mite Neoseiulus baraki in the laboratory

A method was developed for the rearing of coconut mite, Aceria guerreronis Keifer (Acari: Eriophyidae), and its predatory mite Neoseiulus baraki (Athias-Henriot) (Acari: Phytoseiidae) on embryo culture seedlings of coconut (Cocos nucifera) in the laboratory. Seedlings in the ages of <2, 2–4 and 4–6 months were infested with 75 field-collected coconut mites and the population growth was determined up to six weeks after introduction. The populations of coconut mites increased exponentially up to five weeks after introduction and declined thereafter on seedlings of all ages with significant differences among the three groups of seedlings occurring over time. At week 5, a significantly higher mean number (±SE) of coconut mites (20,098 ± 3,465) was bred on 4–6-month-old seedlings than on smaller seedlings, and on the largest seedlings the numbers were highest at all time intervals, except at week 2. Neoseiulus baraki was reared on embryo culture seedlings of the three age groups infested with coconut mites, by introduction of five female deutonymphs and one male, three weeks after introducing coconut mites. Predator numbers progressed significantly over time, but the size of seedlings did not significantly influence the numbers. On all groups of seedlings, the mean number of N. baraki increased up to two weeks after introduction on to seedlings and then declined. Many coconut mites were successfully reared in the laboratory for a longer period by this method and it could also be used as an alternative method to rear N. baraki. Development of this method may contribute to the progress of studies on the biology and ecology of coconut mite and its interactions with natural enemies.