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Sweet orange (C. sinensisL. Osbeck) protoplasts were isolated from nucellar-derived embryogenic callus, cultured in alginate beads for 5–30 days, and the resulting p-calli released by liquefaction and cultured on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-day-old p-calli. There were no significant differences in the numbers of embryos produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies were observed. The minimum size of p-calli to form embryos was 77.84 μm in diameter. Embryos were smaller from p-calli than those produced from embryogenic callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos derived from embryogenic callus ranged between 1 and 2 mm.Sweet orange (C. sinensisL. Osbeck) protoplasts were isolated from nucellar-derived embryogenic callus, cultured in alginate beads for 5–30 days, and the resulting p-calli released by liquefaction and cultured on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-day-old p-calli. There were no significant differences in the numbers of embryos produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies were observed. The minimum size of p-calli to form embryos was 77.84 μm in diameter. Embryos were smaller from p-calli than those produced from embryogenic callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos derived from embryogenic callus ranged between 1 and 2 mm.

Summary: Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm−1) vector DNA concentration (100 μgml−1), carrier DNA concentration (100 μgml−1), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg−1 (protein) min−1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.

Summary: Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphogenesis of somatic embryos. To normalize development, glycerol-induced globular-stage somatic embryos of sweet orange [C. sinensis (L.) Osbeck cv. ‘Hamlin’] were cultured on 6000–8000 MW cutoff cellulose acetate, >400 000 MW cutoff cellulose acetate, nitrocellulose, polyvinylidene fluoride (PVDF), cellulose filter paper, or positive or neutral charged nylon membranes. Only the two cellulose acetate membranes resulted in the development of normal, two-cotyledon, bipolar, heart-shaped embryos, and no aberrant teratoma-like structures. Heart-shaped embryos developed and germinated normally on Murashige and Tucker basal medium with 0.5% sucrose and 1 μM gibberellic acid. Culture of embryogenic callus directly onto cellulose membranes also resulted in the development of normal heart-shaped embryos, indicating that glycerol induction of globular-stage embryos is not necessary. Heart-shaped embryos were not observed when the osmotic potential of the medium was increased by the addition of 2.5–15% polyethylene glycol; neither were they observed when the matric potential of the medium was increased by increasing the gelling agent concentrations of agar and Gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively.

Summary: Controlling fungal and bacterial contamination of woody plant material can be extremely difficult. Isothiazolone biocides and sodium dichloroisocyanurate have been used singly and in combination to reduce microbial contamination in bud explants derived from greenhouse- and field-grown citrus trees. Explants from greenhouse-grown trees were effectively disinfested (<5% vs. 85% contamination) using a ‘standard’ disinfestation (SD) procedure followed by culture on medium containing 5 ml l−1 Plant Preservative Mixture, or by the SD procedure but substituting 300 ppm sodium dichloroisocyanurate for 48 h for the treatment with 1.05% NaOCl in the SD procedure. Disinfestation of explants from field-grown trees was less effective than explants from the greenhouse, but was improved (10% vs. 47% contamination) by using a combination of the SD procedure with 1.05% NaOCl, or 100 or 300 ppm sodium dichloroisocyanurate followed by culture onto medium containing 5 ml l−1 Plant Preservative Mixture.

We have purified a β-1,3-endoglucanase (EC 3.2.1.39) from nonembryogenic Citrus sinensis(L.) Osbeck cv. Valencia callus to electrophoretic homogeneity by means of pH precipitation and ion exchange chromatography. The protein has an apparent Mr of 32,000, a pl pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestansinfection. The enzyme hydrolyzes laminarin (Lam- inaria digitata) optimally at pH 5 and 50 °C. The enzyme will hydrolyze pachyman and laminarin extensively and yeast glucan slightly, but does not hydrolyze lichenin, barley glucan, cellulose, or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. The protein is N-terminal blocked, however, partial internal amino acid sequence analysis revealed that the peptide shared homology with a number of β-1,3-endoglucanases. Antibody to the purified protein was raised in a rabbit and used to screen an amplified cDNA library prepared from Citrus sinensis(L.) Osbeck cv. Valen- cia callus. A positive clone (pBGVC-1) containing a 1,249 by insert was isolated. A full length sequence of the clone was obtained and it contained a 1,229 by open reading frame starting at nucleotide 20. Sequence analysis indicated that the clone is homologous to other 0-1,3-endoglucanase genes. The predicted amino acid sequence was homologous with other 0-1,3-glucanases, contained both N- and C-terminal signal sequences, the glycosyl hydrolase family 17 signature sequence, and the sequence identical to the peptide that was sequenced from the purified protein.

Abstract: Volatile constituents of embryogenic and nonembryogenic sweet orange (Citrus sinensis (L.) Osbeck) callus cultures were analyzed by gas chromatography-mass spectrometry to determine if sweet orange flavor essences were produced. Fifteen compounds were identified from the embryogenic callus methylene chloride extracts, with 10 previously reported as volatile constituents of orange juice or peel essential oil, 3 are known fermentation products, 2 have no reported aroma, and 2 were unknown. No volatile compounds were detected from nonembryogenic callus methylene chloride extracts.

Embryogenic callus from Citrus sinensis (L.) Osbeck cv. Hamlin was cultured for 28 days on 20 media arranged in a 5×2×2 factorial varying in the ratio of nitrate to ammonium nitrogen, total inorganic nitrogen, and benzyladenine. Fresh weight increase of callus and final medium pH were significantly affected by total inorganic nitrogen and the ratio of nitrate to ammonium. The nitrate to ammonium ratio accounted for 55% of the variation in the fresh weight increase of the callus and 93% of the variation in the final medium pH. Varying the NO3-:NH4- ratio provided adequate pH control.

Phosphoenolpyruvate carboxylase (PEPCase) and sucrose phosphate synthase (SPS) were active in nucellar calli of ‘Hamlin’ sweet orange,Citrus sinensis (L.) Osbeck, grown on media containing either 5% sucrose or 2% glycerol as the primary carbon source. Activities of the enzymes, however, were much higher in the glycerol-grown tissues than those grown in sucrose. Glycerol, in addition, stimulated embryogenesis and chlorophyll biosynthesis and initiated the onset of ribulose bisphosphate carboxylase-oxygenase (Rubisco) activity. These stimulatory effects, which were not observed in sucrose-grown calli, became more obvious when calli grown on glycerol-containing, agar-solidified medium were transferred and grown in liquid suspension medium containing 2% glycerol. Starch levels in both sucrose- and glycerol-grown tissues were as high as 30% of tissue dry weight. There were, however, higher concentrations of soluble sugars in sucrose-grown calli than those grown on glycerol. Stimulation of embryogenesis, chlorophyll synthesis, Rubisco onset, and activities of PEPCase and SPS by glycerol offered potential prospects in using this compound in citrus tissue cultures.

Cotyledonary explants of 4-day-oldCucumis melo cv. ‘Hale's Best Jumbo’ in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 µM indole-3-acetic acid and 5 µM benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 µmol m-2 s-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 µM benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.

Summary: Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20–60% of the protoplasts emitted an intense green light when illuminated with blue (450–490 nm) light.

The root weevil Diaprepes abbreviatus L. has been continuously reared since 1992 on an artificial diet (F1675) first reported in 1982. The weight of weevils reared on F1675 was more than twice as great as field-collected individuals. Recently, several ingredients included in the original F1675 diet were shown to have little or no effect on insect performance. We examined the effects of varying three ingredients of a simplified diet on the weight of adult D. abbreviatus. We used a geometric design combined with response surface models to evaluate the effects of the amount and proportion of diet ingredients to identify optimal diet recipes for normative weight gain defined in relation to field-collected D. abbreviatus from locations in Florida. Our results showed that it is possible to produce adults of normative weight or, for that matter, any mean weight within a wide range by varying the proportions of cottonseed meal, wheat germ and cellulose, a non-nutritive filler. Although wheat germ contributed to greater weight gain, survival of larvae to adult was lower on diets containing only wheat germ compared with diets containing only cottonseed meal as the principal nutritive components. The analyses of all the variables measured indicate that cottonseed meal is the only major nutritive component, in addition to standard vitamin and salt mixes, required to produce adult D. abbreviatus of normative weight.

A microsomal flavonoid 3′-hydroxylase (F3′H) catalyzing the metabolism of naringenin to eriodictyol in Citrus sinensis (L.) Osbeck cv. ‘Hamlin’ cell suspension cultures was shown to be a cytochrome P450 enzyme. This reaction required O2 and NADPH and was inhibited by CO, with partial reversal of CO-inhibition by light at 450 nm. Cytochrome P450 content ranged from 10–20 pmol (mg microsomal protein)−1. The F3′H reaction was shown to be linear in regard to protein concentration between 2.5 and 25 μg of microsomal protein. The optimum pH for the reaction was 7.4–7.6 and the temperature optimum was between 30 and 37°C. The apparent Km and Vmax for naringenin were 24 μM±3.2 and 81.4±7.9 pmol eriodictyol min−1 (mg protein)−1, respectively. The microsomal F3′H was also capable of forming dihydroquercetin from dihydrokaempferol (40 pmol min−1 (mg protein)−1) and of quercetin from kaempferol (3.25 pmol min−1 (mg protein−1). Cytochrome c and ketoconazole were the best inhibitors of WH activity followed by piperonyl butoxide and a-naphthoflavone. Light was shown to be an inducer of the F3′H almost doubling the specific activity and increasing the microsomal cytochrome P450 content by 30% over that of dark grown cells. F3′H activity was also confirmed in microsomal preparations of young (new flush) leaves from ‘Hamlin’ trees and flavedo of ‘Hamlin’ oranges, ‘Marsh’ grapefruit, and ‘Lisbon’ lemon. No activity was observed in older, hardened leaves and albedo of all the fruit tested. Initiation of embryogenesis in the ‘Hamlin’ cell suspension cultures by switching from a sucrose medium to a glycerol-based medium resulted in the down-regulation of F3′H.