A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

BACKGROUND: Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. METHODOLOGY/PRINCIPAL FINDINGS: Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. SIGNIFICANCE: The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

In vitro shoot growth of Brugmansia × candida Pers.

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida 'Creamsickle'. Several mineral nutrient experiments were conducted to determine the effect of NH4 (+), NO3 (-), K(+), FeSO4/EDTA, ZnSO4, MnSO4, and CuSO4 on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH4 (+), NO3 (-), and K(+), was conducted as a 2-component NH4 (+): K(+) mixture crossed by [NO3 (-)] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH4 (+)-K(+)-NO3 (-) experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH4 (+):K(+) ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO4 had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO4 had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH4 (+)-K(+)-NO3 (-), FeSO4/EDTA, and ZnSO4 was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO4 and CuSO4, these formulations were characterized by lower levels of NH4 (+), K(+), NO3 (-) and Zn, and higher levels of FeSO4/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. 'Creamsickle'.

A data mining approach to dinoflagellate clustering according to sterol composition: correlations with evolutionary history.

This study examined the sterol compositions of 102 dinoflagellates using clustering and cluster validation techniques, as a means of determining the relatedness of the organisms. In addition, dinoflagellate sterol-based relationships were compared statistically to 18S rDNA-based phylogenetic relationships using the Mantel test. Our results indicated that the examined dinoflagellates formed six clusters based on sterol composition and that several, but not all, dinoflagellate genera, which formed discrete clusters in the 18S rDNA-based phylogeny, shared similar sterol compositions. This and other correspondences suggest that the sterol compositions of dinoflagellates are explained, to a certain extent, by the evolutionary history of this lineage.

Artificial diet optimized to produce normative adults of Diaprepes abbreviatus (Coleoptera: Curculionidae).

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.

Sensory imbalance as mechanism of orientation disruption in the leafminer Phyllocnistis citrella: elucidation by multivariate geometric designs and response surface models.

Experimental designs developed to address mixtures are suited ideally to many areas of experimental biology, including pheromone blend studies, because such designs address the confounding of proportionality and concentration intrinsic to factorial and one-factor-at-a-time designs. Geometric multivariate designs coupled with response surface modeling allowed us to identify optimal blends of a two-component pheromone for attraction and trap disruption of the leafminer moth, Phyllocnistis citrella, a major pest in citrus growing areas around the world. Field trials confirmed that the natural 3:1 blend of (Z,Z,E)-7,11,13-hexadecatrienal:(Z,Z)-7,11-hexadecadienal was most effective as an attractant for male moths. However, the response surface generated in mating orientation trials revealed that the triene component alone was more effective than the natural blend in disrupting trap catch. Each individual component was effective at disrupting orientation in field trials, but (Z,Z,E)-7,11,13-hexadecatrienal was approximately 13 times more effective, at the same concentration, compared with (Z,Z)-7,11-hexadecadienal alone. In addition, the application of geometric design and response surface modeling to field studies provided insight into a possible mechanism of mating disruption and supported sensory imbalance as the operating mechanism for this species.

The effects of nitrogen and potassium nutrition on the growth of nonembryogenic and embryogenic tissue of sweet orange (Citrus sinensis (L.) Osbeck).

BACKGROUND: Mineral nutrients are one of the most basic components of plant tissue culture media. Nitrogen in the form of NH4+ and NO3(-) is the dominant mineral nutrient in most plant tissue culture formulations, with effects dependent on both the proportion and the amount of NH4+ and NO3(-). The effects of nitrogen nutrition on the growth of nonembryogenic and embryogenic cell lines of sweet orange (C. sinensis (L.) Osbeck cv. 'Valencia'), tissues routinely used in citrus horticultural and plant improvement research, was explored using an experimental approach free of ion confounding that included a 2-component mixture (NH4+:K+) and a quantitative factor [NO3(-)] crossed by the mixture, thereby providing ion-specific estimates of proportional and amount effects. RESULTS: First, the linear mixture component, though only a comparison of the design space vertices, was highly significant for both tissue types and showed that NH4+ was required by both tissues. Second, the NH4+ * K+ mixture term was highly significant for both tissue types, revealing that NH4+ and K+ exhibit strong synergistic blending and showed that growth was substantially greater at certain blends of these two ions. Third, though the interaction between the NH4+:K+ mixture and NO3(-) amount on fresh weight accumulation for both tissue types was significant, it was substantially less than the main effect of the NH4+:K+ mixture. Fourth, a region of the design space was identified where fresh weight growth was increased 198% and 67% over the MS medium controls for nonembryogenic and embryogenic tissues. CONCLUSION: By designing a mineral nutrient experiment free of ion confounding, a direct estimation of ion-specific proportional and amount effects on plant tissue growth is possible. When the ions themselves are the independent factors and/or mixture components, the resulting design space can be systematically explored to identify regions where the response(s) is substantially improved over current media formulations. In addition, because the response is over a defined experimental region, a specific medium formulation is more accurately interpreted as a coordinate in the specified design geometry.

Insect diets as mixtures: optimization for a polyphagous weevil.

Development or improvement of artificial insect diets can be tedious, convoluted and often under-appreciated. Using n-dimensional mixture designs, we identified a set of response-optimized meridic diets that contain fewer ingredients than the current commercial diet for Diaprepes abbreviatus, a polyphagous weevil pest of the Caribbean and southern U.S. A diet blend optimized to produce maximum adult weight was predicted to produce adult D. abbreviatus that weigh 28% more compared with adults reared on the standard commercial diet. Diet blends that produced greater individual adult weights resulted in lower survival compared with those blends that yielded adults of more modest proportions. In contrast, a simplified high cottonseed meal blend produced smaller adults more similar to field-collected individuals, and produced the greatest number of adults and the greatest biomass at relatively low cost compared with diets that yielded adult weevils of greater weight. We think that many insect-rearing programs would benefit from application of mixture design methods to situations where diet optimization is desired for researcher-selected criteria. This approach is broadly applicable to any problem that can be conceptualized as a mixture problem.

Ion chromatography of phytate in roots and tubers.

The ion chromatographic method for the quantification of phytate (InsP(6)) in foods was adapted for the analysis of roots and tubers. To maximize sensitivity, ultraviolet (UV) detection following postcolumn derivatization was compared with evaporative light-scattering detection (ELSD). Detection limits for phytate were 0.5 and 1 microg for UV and ELSD, respectively. Unidentified peaks eluting close to and after InsP(6) were removed by solid-phase extraction. Phytate was detected in 11 of 15 roots or tubers. The highest phytate levels were 0.169 and 0.133% of the fresh weight of taro (Colocasia esculenta) and yuca (Manihot esculenta), respectively. Potatoes (Solanum tuberosum) contained 0.035-0.073% phytate, whereas no phytate at a detection limit of 0.003% of fresh weight was observed in sweet potatoes (Ipomoea batatas).