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Commercial citrus trees are composed of a scion grafted onto a rootstock. Because grafting is one of the most expensive methods of plant propagation, grafting efficiency is of large practical importance. The purpose of this study was to improve citrus bud-grafting efficiency. The effects of six factors that included BA, Tween-20, DMSO, type of solvent (water or EtOH), cardinal orientation of grafted bud, and type of supplemental light (LED, metal halide, none) on forty-four bud-grafting measures were determined using a multifactor design of experiment approach. Four measures useful for identifying treatments of practical value included the number of rootstock axial buds that formed shoots, the percentage of grafted buds that formed shoots, the length of the longest shoot formed from the grafted buds, and the total leaf area of the grafted bud shoots. The factors that most affected these responses were no supplemental light to minimize the number of shoots from rootstock axial buds, a south orientation and 5 mM BA to maximize the percentage of grafted buds that formed shoots, a north orientation and 5 mM BA to maximize the length of the longest grafted bud shoot, and 5 mM BA to maximize the leaf area of the grafted bud shoots., (© 2023. Springer Nature Limited.)

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants., Competing Interests: LZ, NR, and CV were employed by the company Integrated DNA Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Cheng, Fang, Roberts, Zhang, Vakulskas, Niedz, Culver and Qi.)

Anther and microspore culture for producing haploid plants are very complex systems and include general effects where the specific effects must be identified and optimized to develop culture systems capable of producing the large numbers of haploids required by breeding programs. These general effects include genotype, physiological state of the source plant, age of the anthers and microspores, preculture treatments, culture conditions, and culture media. Design of experiments (DoE) is an experimental approach specifically designed to identify and optimize the multiple factors that make up complex systems, and is ideally suited for developing in vitro systems to produce haploids. The basic DoE strategy starts by screening multiple factors thought to affect the responses being measured. Screening identifies factors with large and small effects. Factors with large effects are used to manipulate the system, and are moved to the DoE optimization phase such as response surface methodology. Factors with small or trivial effects are eliminated from further consideration, and this simplifies the system. The basic concepts of fractional factorial designs and how to use them are explained. Fractional factorials are the most important DoE screening tool and are the first experiments run before DoE optimization experiments. To illustrate the unique properties of fractional factorials, a detailed example is provided that includes all of the calculations so that no statistical software is required., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Multivariate geometric designs for mixture experiments and response surface methodology (RSM) were tested as a means of optimizing plant mixtures to support generalist predatory arthropods. The mixture design included 14 treatment groups, each comprised of six planters and having a proportion of 0.00, 0.17, 0.33, 0.66, or 1.00 of each plant species. The response variable was the frequency of predators trapped on sticky card traps placed in each group and replaced 2 times per week. The following plant species were used: Spring 2017: Euphorbia milii, E. heterophylla, and Phaseolus lunatus; Summer 2017: E. milii, Fagopyrum esculentum, and Chamaecrista fasciculata; and, Summer 2018: E. milii, F. esculentum, and Portulaca umbraticola. Predator occurrence was influenced by: 1) Linear mixture effects, which indicated that predator occurrence was driven by the amount of a single plant species in the mixture; or, 2) Nonlinear blending effects, which indicated that the plant mixture itself had emergent properties that contributed to predator occurrence. Predator abundance was highest in the Spring 2017 experiment and both linear mixture effects and nonlinear blending effects were observed. Predator occurrence decreased in subsequent experiments, which were conducted in the warmer summer months. In both Summer experiments, only linear mixture effects were observed, indicating that predator occurrence was driven by the amount of a single plant species in the test mixtures: Euphorbia milii in 2017 and Portulaca umbraticola in 2018. The results showed that not only did the species composition of a plant mixture drive predator occurrence but that proportionality of species contributed to the outcome as well. This suggests that, when formulating a plant mixture to aid in conservation biological control consideration should be given to the proportion of each plant species included in the mixture. RSM can be an important tool for achieving the goal of optimizing mixtures of plants for conservation biological control., Competing Interests: The authors have declared that no competing interests exist.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important pest of maize (Zea mays L.). Published WCR diets contain corn root powder, which is not available for purchase, thereby limiting the practical use of diets containing this ingredient. We applied response surface modeling combined with mixture designs to formulate a WCR diet that does not require corn root powder. We developed the new formulation by systematically exploring eight protein ingredients from animal, plant, and yeast sources based on simultaneous evaluation of three life history parameters (weight, molting, and survival). This formulation (WCRMO-2) without corn root powder supported approximately 97% of larval survival and successful molting. Larval weight gain after 10 days of feeding on WCRMO-2 was 4-fold greater than that of larvae feeding on the current best published WCR diet. Additionally, there was no significant difference in these larval performance traits when larvae were reared on WCRMO-2 and the best proprietary WCR diet. A commercial version of WCRMO-2 was tested and found to perform comparably for these traits. These improvements met our goal of a diet comprised of available ingredients that supports performance of WCR larvae equal to or better than publicly available formulations and proprietary formulations.

The northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, is a major pest of maize (Zea mays L.). This pest has developed resistance to insecticides and adapted to crop rotation and may already be in the early stages of adaptation to toxins produced by Bacillus thuringiensis (Bt). Toxicity bioassays using artificial diet have proven to be valuable for monitoring resistance in many species, but no artificial diet has been developed specifically for NCR larvae. Toward this end, we first evaluated known Diabrotica diets to identify a starting media. We then developed a specialized diet for NCR using an iterative approach. Screening designs including 8 diet components were performed to identify the principal nutritional components contributing to multiple developmental parameters (survival, weight, and molting). We then applied mixture designs coupled with response surface modeling to optimize a blend of those components. Finally, we validated an improved NCR diet formulation that supports approximately 97% survival and molting, and a 150% increase in larval weight after 10 days of feeding compared with the best previously published artificial diet. This formulation appears suitable for use in diet bioassays as a tool for evaluating the resistance of NCR populations to insecticides.

There is interest in using ligands of chemosensory binding proteins (CBP) to augment an insect's responsiveness to chemosensory cues. We showed previously that combining a synthetic ligand of a CBP with limonene, a common citrus volatile, enhanced the probing response of Asian citrus psyllid ( Diaphorina citri ). Here, we determined whether synthetic compounds, which were ligands of D . citri olfactory binding protein (OBP) DCSAP4, influenced the settling and aggregation levels of psyllids on young citrus shoots. The test ligands and Cmac scent were dispensed from a droplet of an emulsified wax product (SPLAT) placed on the bottom of each vial. The shoots were presented: (1) alone (shoot + blank SPLAT), (2) with a mixture of citrus volatiles ("Cmac scent") (shoot + SPLAT with Cmac scent), or (3) with different concentrations of test ligands (shoot + SPLAT with test ligand at concentration 1, shoot + SPLAT with test ligand at concentration 2, etc.). Depending on the availability of test ligands, sprigs, and psyllids, each test included from two to four replicates of each treatment (i.e., shoot only, shoot + Cmac scent, shoot + test ligand at concentration 1, shoot + test ligand at concentration 2, etc.); only a single test ligand was presented in each test. For each test, 200 D. citri were released in the test area and the numbers of psyllids on each sprig were counted 24 h later. Sprigs with ≥7 psyllids were considered to be an aggregation. A total of seven ligands were tested individually. Four of the ligands (654, 717, 784, and 861) modulated psyllid settling and aggregation response, causing greater settling and aggregation to sprigs presented with the Cmac scent than to those sprigs with blank SPLAT. Presentation of one of the ligands (019) resulted in an opposite effect in which psyllid settling and aggregation levels were lower on sprigs with Cmac scent than on those with blank SPLAT. There were no differences in settling levels in the different treatment vials in the Ligand 905 experiment. In the Ligand 937 experiment, settling levels did not vary significantly between treatment vials although settling levels were relatively high in all treatment vials and there was a significant treatment effect. Increased settling and aggregation levels were largely not observed with in the vials with only the test ligands, and there was little effect of ligand concentration on psyllid response levels. This suggests that the test ligands themselves did not attract the psyllids but rather modulated the psyllid's response to the Cmac scent. The results suggest that synthetic ligands of D . citri CBPs can be used to increase the effectiveness of citrus scent lures used to attract psyllids to monitoring traps and attract and kill devices.

Biodegradable pH-responsive polysuccinimide nanoparticles (PSI-NPs) are synthesized for directly delivering agrochemicals to plant phloem to improve their efficacy. The PSI-NPs have an average size of 20.6 nm with negative charge on the surface. The desired responsiveness to changes in pH is demonstrated by release efficiency of the model molecule (Coumarin 6), which increases with increasing pH over 24 h. The internalization of PSI-NPs into grapefruit cells occurs in 10 min, and into nucleus in 2 h, with most of the PSI-NPs being distributed in cytoplasm and nucleus. The proportion of PSI-NPs in plant cells significantly increases with time, from 19.1% at 10 min to 55.5% at 2 h of administering. The PSI-NPs do not show significant inhibitory effects on soil microbial growth and activity. These results indicate that this smart nanodelivery system has potential of application in agriculture for mitigating phloem-limited diseases, such as citrus huanglongbing., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Field experiments were carried out to study responses of male moths of the carpenterworm, Chilecomadia valdiviana (Lepidoptera: Cossidae), a pest of tree and fruit crops in Chile, to five compounds previously identified from the pheromone glands of females. Previously, attraction of males to the major component, (7Z,10Z)-7,10-hexadecadienal, was clearly demonstrated while the role of the minor components was uncertain due to the use of an experimental design that left large portions of the design space unexplored. We used mixture designs to study the potential contributions to trap catch of the four minor pheromone components produced by C. valdiviana. After systematically exploring the design space described by the five pheromone components, we concluded that the major pheromone component alone is responsible for attraction of male moths in this species. The need for appropriate experimental designs to address the problem of assessing responses to mixtures of semiochemicals in chemical ecology is described. We present an analysis of mixture designs and response surface modeling and an explanation of why this approach is superior to commonly used, but statistically inappropriate, designs.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious insect pest of corn (Zea mays L.) in the United States and parts of Europe, and arguably one of the world's most expensive pests to control. Several diet formulations are currently used by industry and public researchers to evaluate WCR larvae in diet-toxicity bioassays. However, a publicly available diet that produces normative insects that are physiologically similar to WCR larvae reared on corn roots will accelerate development of management technologies. We report a new diet formulation that supports improved weight gain, larval development and survival compared with the only public diet for WCR that is currently available in the refereed literature. The formulation was created by using response surface methods combined with n-dimensional mixture designs to identify and improve the formulation of key ingredients. Weight gain increased two-fold, and survival and molting rates increased from 93% and 90%, respectively when reared on the public diet, to approximately 99% for both survival and molting at 11 days when reared on our new formulation. This new formulation provides a standardized growth medium for WCR larvae that will facilitate comparison of research results from various working groups and compliance with regulatory requirements.

Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (10 9 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×10 6 cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus., (Published by Elsevier Inc.)

ARS-Media for Excel is an ion solution calculator that uses "Microsoft Excel" to generate recipes of salts for complex ion mixtures specified by the user. Generating salt combinations (recipes) that result in pre-specified target ion values is a linear programming problem. Excel's Solver add-on solves the linear programming equation to generate a recipe. Calculating a mixture of salts to generate exact solutions of complex ionic mixtures is required for at least 2 types of problems- 1) formulating relevant ecological/biological ionic solutions such as those from a specific lake, soil, cell, tissue, or organ and, 2) designing ion confounding-free experiments to determine ion-specific effects where ions are treated as statistical factors. Using ARS-Media for Excel to solve these two problems is illustrated by 1) exactly reconstructing a soil solution representative of a loamy agricultural soil and, 2) constructing an ion-based experiment to determine the effects of substituting Na+ for K+ on the growth of a Valencia sweet orange nonembryogenic cell line., Competing Interests: The authors have declared that no competing interests exist.

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.

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'.

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.

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.

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.

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.

Seven putative protease inhibitor (PPI) cDNAs, representing four protein families, were isolated from a grapefruit (Citrus paradisi Macf. Cv. Marsh) immature fruit flavedo cDNA library. Cloned open reading frames encoded proteins with similarity to, and protein signatures for: legume Kuntiz inhibitors (lkiL-1, lkiL-2, lkiL-3), potato trypsin inhibitor I (ptiIL-1), serpins (serpL-1), cystatins (cystL-1), and gamma thionins (gthL-1). Response of transcript abundance to fruit development and leaf wounding was determined for all but lkiL-1 using real-time RT-PCR. Immature leaves had the highest transcript levels for all PPIs. The gthL-1 transcript in immature leaves was the most abundant transcript but was absent from healthy mature leaves. In fruit flavedo, transcripts for all PPIs were most abundant in youngest fruit (<15 mm dia. fruit), and declined during development, but displayed different patterns of developmental change. Mechanical or Diaprepes root weevil (DRW) feeding damage to leaves caused a <10-fold reduction or had no effect on transcript level with the exception of gthL-1 which, as a result of damage, increased >50-fold in mature leaves and decreased >1400-fold in immature leaves. This developmental control of transcript response to wounding in a woody perennial is opposite of what has been observed for defensive proteinase inhibitors (PIs) in other plants (typically herbaceous and/or annual plants), where younger leaves typically invoke a higher defensive proteinase inhibitor transcript accumulation than older tissues. Except for gthL-1, the PPI transcripts were minimally responsive or unresponsive to wounding. Changes in PPI transcript levels suggest diverse roles for the products of these genes in citrus, with only gthL-1 responding in a defense-like manner.