Adventive plants from ovules and nucelli in Citrus

Summary 1- to 8-week-old ovules and nucelli from three Citrus cultivars—Shamouti and Valencia (Citrus sinensis) oranges and Marsh Seedless (C. paradisi) grapefruit—were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/l malt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GA₃). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GA₃ to the basal medium stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development.     

In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant

The nature of the explant, seedling age, medium type, plant growth regulators, complex extracts (casein hydrolysate, coconut milk, malt extract and yeast extract) and antioxidants (activated charcoal, ascorbic acid, citric acid and polyvinylpyrrolidone) markedly influenced in vitro propagation of Gymnema sylvestre. A maximum number of shoots (57.2) were induced from 30 day old seedling axillary node explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (1 mg l⁻¹), kinetin (0.5 mg l⁻¹), 1-napthalene acetic acid (0.1 mg l⁻¹), malt extract (100 mg l⁻¹) and citric acid (100 mg l⁻¹). High frequency of rooting was obtained in axillary node explant derived shoots (50%) on half strength MS medium supplemented with IBA (3 mg l⁻¹). The plantlets, thus developed, were hardened and successfully established in natural soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

A Method for the Removal of Mineral Salts from Coconut Milk and a Comparison of Some of the Growth-Promoting Properties of Whole and Desalted Coconut Milk in Carrot Root Tissue Cultures

Various methods of separating the growth-promoting activityof coconut milk from its mineral salts constituents are examined.It is shown that coconut milk can be freed from mineral saltselectrolytically without serious impairment of its ability tostimulate growth in carrot root tissue cultures. Fresh weightand dry weight data, cell counts, and anatomical observationsshow that the growth of cultures in electrolytically desaltedcoconut milk is substantially the same as in whole coconut milk. The anatomical heterogeneity of carrot tissue cultures grownin both desalted and whole coconut milk is demonstrated.     

Experimental Studies of Young Ginkgo Embryos—the Effect of Coconut Milk on the Embryos Cultured in Vitro

The present investigation deals with the effect of coconut milk upon the growth of in vitro culture of young Ginkgo embryos. The basic medium consists of White mineral salts in which Fe-citrate was used instead of Fe2(SO4)3 and vitamins (B1, 0.5 ppm, B6, 0.5 ppm, Ca-pantothenate, 0.5 ppm, niacin, 1 ppm and glycine, 2.5 ppm). 8% sucrose was used for younger embryos as the carbon source and 5% for the larger ones. The pH value of the medium was adjusted to about 6. 0.7% agar was used. The cultures were kept in an incubator (about 20–23 ℃) and no artificial light was used. The coconut milk, both filtered and autoclaved, was tested for its effect on the growth and structure of the young embryos. The important results obtained are summarized as follows: 1. The coconut milk, used both filtered and autoclaved, greatly promoted the growth and differentiation of the Ginkgo embryos cultured in vitro. 2. The optimum concentration of the coconut milk is 10%–20% for the younger embryos (900–1,600μ), while the higher concentrations (30% and over) may induce callus formation. 3. For the larger embryos the coconut milk was less effective, no callus formation occurred for the embryos over 2,800μ at isolation and for them 40% coconut milk was found more effective than 10% coconut milk. 4. There was no significant difference of the effect between the filtered and the autoclaved coconut milk. 5. From the experimental data obtained the authors conclude that the coconut milk at adequate concentration greatly promotes the rate of cell division and may initiate the meristematic regions around the shoot apex and over the whole surface of the cotyledons of the treated embryos.     

Enabling mycelial application of Hirsutella thompsonii for managing the coconut mite

Laboratory and field studies were conducted to examine the prospect of mycelial application of Hirsutella thompsonii as an alternative to the use of mycelial-conidial formulations of the fungus in the suppression of the coconut mite, Aceria guerreronis. In a series of laboratory experiments, glycerol, yeast extract powder and dehydrated malt extract broth were found to be the best among nine substances investigated as possible adjuvants for use on coconut palms in the field along with H. thompsonii mycelia. H. thompsonii biomass in the presence of adjuvants not only produced more colonies but also yielded more conidia per pellet. In terms of the density of conidia generated on a mycelial mat the treatments varied highly significantly in two methods, with glycerol showing an average of 106% increase over control. Though irradiance with simulated sunlight resulted in reduced conidiogenesis, in general, adjuvant-treated pellets, both exposed and unexposed to simulated sunlight, produced substantial conidiation compared with control, irrespective of the two incubation conditions. Better conidiation was observed under alternating light-dark regime than under total darkness in all the treatments. Glycerol boosted the pathogenicity of H. thompsonii by 16.5% over control. In the field, a newly developed mycelial formulation of H. thompsonii applied after tank-mixing separately with the three selected adjuvants brought down the post-treatment population of the coconut mite by 85.6-97.1%. Application of the fungus in combination with glycerol resulted in a tolerable mean nut damage grade of 2.0 during the pre-harvest stage, compared with an acute score of 4.0 in control palms.     

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

The interaction of genotype and culture medium on the tissue culture responses of wheat (Triticum aestivum L. em. thell) callus

The effect of the interaction of genotype and culture medium on the initiation of callus from immature embryos and subsequent plant regeneration was investigated in eight hexaploid wheat lines. Intervarietal differences in culture response and interaction of the genotype with coconut milk are reported. The relative contributions of media and genotype effects to culture performance are assessed. The observation that primordia and shoot development was promoted by coconut milk in some lines and inhibited in others is particularly significant given that coconut milk is widely used to try to improve culture response. This report shows that this effect is dependent on the genotype of the tissues in culture.     

Astaxanthin hyperproduction by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> (now <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>) with raw coconut milk as sole source of energy

Natural carbon sources, such as those present in cane sugar molasses and grape juice, promote the synthesis of astaxanthin in different Phaffia rhodozyma yeasts. One of these, coconut milk, has a very rich nutrient composition. The aim of this work was to investigate the utility of coconut milk as sole source of energy for astaxanthin pigment production by P. rhodozyma strains. Currently, coconut pulp is widely used in industrial processes in Mexico for the production of shampoos, candies, food, etc. However, coconut milk is a waste product. We show that coconut milk enhances astaxanthin production. The fermentation yielded 850 g/g yeast with the NRRL-10921 wild-type strain and 1,850 g/g yeast with the mutated R1 strain. Production was better than reported results employing other natural carbon sources.     

Enzymic Maceration of Citrus Callus and the Regeneration of Plants from Single Cells

The maceration medium comprised a basal nutrient medium (BM)containing an optimum concentration of 3% (w/v) sucrose. Mannitoland sorbitol were inferior osmotica. Addition of potassium dextransulphate adversely affected maceration. ‘Macerozyme’was not as effective as ‘Macerase’ in the productionof single cells. The optimal concentration of ‘Macerase’was found to be 2–3% (w/v). Single cells obtained by filtering the macerate were rinsedwith BM and cultured in, and on, agar media comprising: BM;BM + 500 mg 1^–1 malt extract (ME); and BM + 10% (v/v)coconut milk (CM). No growth or organization was observed incultures where cells were mixed in with warm medium prior togelling. When spread on the surface of gelled media supplementedwith ME and CM, proliferation and organization occurred. Manymicroscopic globular proembryoids developed within 3 weeks onthe supplemented media. Microscopic torpedo-shaped embryoidswere frequently observed on BM + CM, rarely on BM + ME, andnot at all on unsupplemented BM. The high frequency of microscopic globular proembryoids, andlater of macroscopic pseudo-bulbils, formed on BM + ME leadsus to postulate that pseudobulbils are derived from globularproembryoids in which polarity is not established by the 16to 32-cell stage. Microscopic torpedo-shaped embryoids probablygive rise to macroscopic heart-shaped embryoids which developinto plantlets. The technique reported in this article provides an ideal systemfor examining embryogenesis per se and for studying the effectsof various treatments on embryogenesis and organ differentiationin vitro. It also affords excellent opportunities for the breedingof solid mutant plants.     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

Coconut and Salmonella Infection

Raw, unprocessed coconut supports the growth of salmonellae as well as that of other enteric bacteria, salmonellae being particularly resistant to subsequent desiccation. Original contamination is not due to carriers or to polluted water supplies, but to contact with bacteria-containing soils followed by dispersion via infected coconut milk and shells. Pasteurization of raw coconut meat in a water bath at 80 C for 8 to 10 min effectively killed such bacteria, did not injure the product, and provided a prophylactic method now widely used by the coconut industry.     

Morphogenesis in Tissue Cultures of Different Organs of Nigella sativa

Calli were isolated from root, stem and leaf segments of Nigella sativa (Fam. Ranunculaceae) on White's medium containing napthaleneacetic acid and coconut milk. From all the three types of calli, roots were differentiated. Shoot development occurred in stem and leaf calli only after the omission of first coconut milk and then of auxin from the basal medium and also in IAA (2.0 mg/ml) and coconut milk (15 % v/v) in the medium. Frequency of organ formation and the maintenance of this capacity depend upon the nature as well as the age of the callus tissue.     

Effect of plant extracts on development ofCapsella embryos in ovules cultured invitro

When distantly related plants are crossed, embryos abort on the mother plant. One of the methods used to rescue the embryo is to cultivate the ovulein vitro. When the ovule is precociously detached from the mother plant, survival rate of the embryo is very low. To increase the chance of survival as well as the growth of the embryo, the medium was supplemented by different extracts of plants after either autoclaving or filtering. Autoclaved tomato extract seemed to have no simulating effect in spite of reports from different authors. Filtered coconut milk increased growth and filtered cucumber extract improved survival. Other new extracts had a stimulating action on both growth and survival and are of interest for further study.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biosynthesis of flavour-active furanones by Saccharomyces cerevisiae during fermentation depends on the malt type used in medium preparation

Extracts of a coloured malt contained 4-hydroxy-5-monomethyl-3(2H)-furanone (HMMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) but not 4-hydroxy-5(or 2)-ethyl-2(or 5)-methyl-3(2H)-furanone (HEMF). Extracts of a pale malt did not contain any of the furanones. HMMF and HDMF were produced by Saccharomyces cerevisiae during fermen-tation of both types of malt extract. About 0.09 mg HEMF l ⁻¹ was synthesised during fermentation of the coloured malt extract but none was produced with the pale malt extract. Final concentrations of HDMF (2.0 mgl ⁻¹) and HEMF (0.09 mgl ⁻¹) were in excess of their aroma threshold values in water (0.16 and 0.02 mgl ⁻¹ respectively) after fermen-tation of the coloured malt extract. This revised version was published online in November 2006 with corrections to the Cover Date.     

GROWTH OF EXCISED PINE EMBRYOS AND THE ROLE OF THE COTYLEDONS DURING GERMINATION IN VITRO

A study was made of the role of the cotyledons, embryo orientation, surgical treatment, darkness, light, and autoclaved coconut milk on the growth of Pinus lambertiana Dougl. embryos in vitro. The embryos did not require an haustorial function of the cotyledons in vitro. Removal of the shoot meristem drastically altered the developmental physiology of the embryo and the function of the root meristem was severly inhibited. Positional effects on embryo growth were reversed by darkness. In the light vertical-inverted tube cultures displayed better growth than horizontal-inverted tube cultures, whereas in the dark the horizontal-inverted tube cultures displayed better growth than the ver ical-inverted tube cultures. Autoclaved coconut milk had no statistically demonstrable effect on embryo growth as measun d by the analysis of variance and Student-Newman-Keul's range test; however, the graphical analysis suggests that, in conjunction with the presence of the shoot meristem, there may be a slight beneficial response to autoclaved coconut milk.     

Harry Belafonte and the secret proteome of coconut milk

The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk.     

Production of Chlamydospores and Conidia in Submerged Culture by Rhynchosporium alismatis, a Mycoherbicide of Alismataceae in Rice Crops

The objective of this work was to evaluate the production of chlamydospores and conidia of Rhynchosporium alismatis in a liquid Czapex-Dox based medium supplemented with increasing concentrations of sodium nitrate and malt extract. In addition, the germination of chlamydospores was evaluated. A high concentration of malt extract (4.4 g L^-1) as the sole carbon source and a high level of sodium nitrate as the sole nitrogen source (3.3 g L^-1) were shown to increase chlamydospore production while agitation (150 rpm) enhanced conidial yields. Maximum chlamydospore production (2.03×10⁵±0.7 total chlamydospores mg DW^-1) was achieved in cultures grown in a medium supplemented with 8.8 g L^-1 malt extract and 5.74 g L^-1 sodium nitrate. Two days growth was required for maximum chlamydospore and conidial production, while 6 days was necessary to obtain maximum dry weight accumulation (350 mg per flask). Germination of chlamydospores (90%) was significantly higher than germination of conidia (47%) after 2 days growth.     

Induction of laccase production inCyathus bulleri under shaking and static culture conditions

Laccase production byCyathus bulleri was lower in lignins and phenolic compounds as compared to malt extract medium (8 U/mL) which increased significantly on supplementing these compounds with malt extract. Of the different lignins and phenolic compounds, Reax, lignin and orcinol exhibited maximum laccase formation (12 and 68 U/mL, respectively) under static culture conditions, while sugars repressed it. Laccase activity inC. bulleri was higher under static than under shaking cultivation conditions. Moreover, agitation repressed laccase formation even in the presence of inducers.