Randall P. Niedz

Green fluorescent protein: an in vivo reporter of plant gene expression

SummaryProtoplasts 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.

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T1 progeny contained T-DNA. Molecular characterization of one primary transformant and its T1 and T2 progeny plants established that T-DNA was integrated into the host genome.

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. ‘Hales 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.

An expressed sequence tag (EST) set from Citrus sinensis L. Osbeck whole seedlings and the implications of further perennial source investigations

Abstract Compared with the large-scale single pass cDNA sequencing entries from annual plants, the NCBI database has very little sequence information from perennial plant species. Although similar to annuals in many biochemical pathways, perennials are unique in the fact that they posses long generation times. Without short cycle reproduction as an escape mechanism, perennials have evolved alternative survival mechanisms to pathogen attack and environmental stresses. The study of these alternate strategies by way of functional genomics will greatly increase the understanding of the biochemical changes underlining stress responses in perennials. Herein we analyze a set of expressed sequence tags (ESTs) produced from a 180-day-old whole immature sweet orange citrus seedling cDNA library. From this library, 7680 cDNAs were single pass sequenced from the 5′ end, generating 6443 high quality ESTs. In the analysis, 2272 ESTs (35%) were found to significantly match ( E -value≤10 −10 ) proteins with known function in the public databases using blastx . Additionally, 1457 ESTs (23%) significantly matched proteins with unknown function and 1619 ESTs (25%) matched to proteins described as putative. The remaining 1095 ESTs (17%) failed to match with significance to any protein sequence found in the public databases. ESTs matching to the photosynthetic proteins chlorophyll A/B binding protein, plastocyanin and ribulose-1,5-bisphosphate carboxylase were abundant, 6.0% of the total. Interestingly, stress related proteins such as low molecular weight heat shock proteins, peroxidases, lipid transfer proteins and metallothionein-like proteins were also abundant, 3.6% of the total, suggesting a role for these genes in citrus seedling development .

Control of in vitro contamination of explants from greenhouse- and field-grown trees

SummaryControlling 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.

Growth of embryogenic sweet orange callus on media varying in the ratio of nitrate to ammonium nitrogen

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.

Glycerol stimulation of chlorophyll synthesis, embryogenesis, and carboxylation and sucrose metabolism enzymes in nucellar callus of Hamlin' sweet orange

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.

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Improving in vitro mineral nutrition for diverse pear germplasm

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NORMALIZING SWEET ORANGE ( C. SINENSIS (L.) OSBECK) SOMATIC EMBRYOGENESIS WITH SEMI-PERMEABLE MEMBRANES

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