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Citrus is one of the most important global fruit crops, with oranges accounting for more than half the total production. Citrus can be genetically engineered for improved traits, but the process is severely limited by shoot regeneration rates, especially in commercial varieties. Although standardizing a transformation protocol is difficult due to genotype and season dependencies, use of an optimal mineral nutrient basal culture medium increases the success of recovering transgenic Citrus cells. The purpose of this study is to improve shoot regeneration from juvenile tissue in multiple Citrus genotypes. Explants of 11 Citrus cultivars were regenerated in 21 published media to determine the optimal media for each genotype. The number of shoots from juvenile tissue were counted 90 days after the explants were first cultured. The type of basal medium strongly affected the rates of shoot regeneration from Citrus juvenile tissue, and the effectiveness was negatively correlated with the level of ammonium. For each Citrus grouping, optimized media formulations were determined, and potential improvements were predicted. In general, medium R7100 appeared to be the most effective for regeneration of the Citrus genotypes tested.Key Message: Results from this study indicate that basal medium strongly affected the rates of shoot regeneration from citrus juvenile tissue, and the effectiveness was negatively correlated with the level of ammonium present. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In vitro methods for propagation and preservation are critical when seed banking is not an option for the ex situ conservation of an endangered species. Such methods rely on the ability to maintain normal growth for the ultimate goal of producing plants. Previous studies revealed that the threatened Cycladenia humilis var. jonesii can exhibit extreme hyperhydricity when cultured in vitro, but that this phenotype can be largely normalized by venting of the cultures. A design of experiments (DOE) approach was used to investigate factors contributing to hyperhydricity in this species. Using a 6-factor fractional factorial design, the effects of nitrogen, CaCl2, BAP, gelling agent, and the presence of venting on the phenotype were tested. Venting was the strongest driver decreasing hyperhydricity, but gelling agent and other factors had significant effects, depending on the response measured. A revised medium was then designed and compared with the original maintenance medium through 2–3 subcultures. Venting was again the strongest driver decreasing hyperhydricity, but the revised medium also significantly affected some responses, even in the absence of venting. The revised medium was not optimal, as shoots became progressively smaller with each transfer, but despite their size, hyperhydricity decreased. These results demonstrate the usefulness of DOE for simultaneous testing of multiple factors, but also the need for follow-up optimization experiments. DOE can be particularly useful in working with rare species. By making efficient use of limited resources, it can facilitate the development of in vitro propagation protocols for such species and help ensure their conservation. [ABSTRACT FROM AUTHOR]

This study determined the effects of a static liquid culture system on shoot regeneration from citrus epicotyl explants. A mixture-amount experiment was used to determine the effects of zeatin riboside (ZR), 6-benzylaminopurine (BA), and indole-3-acetic acid (IAA) on two citrus types—citrange (Citrus sinensis ‘Washington’ L. Osbeck. × Poncirus trifoliata L. Raf var. Carrizo) and sweet orange (Citrus sinensis L. Osbeck var. ‘Ridge Pineapple’). A liquid culture system comprising a Petri dish, cellulose filter paper, and liquid culture medium was used. Shoot regeneration experiments were conducted over 6 wk that included 2 wk in the dark followed by 4 wk in the light. Three responses were measured: (1) number of explants forming buds and/or shoots, (2) number of explants with shoots > 2 mm, and (3) overall explant and shoot quality. The effects of paper disc number, liquid medium volume, and explant size on shoot regeneration were determined. High-quality shoots were produced from explants cultured in 5.25 to 12 mL medium volume and explant sizes ranging from 2 to 15 mm. The effects of the plant growth regulators were similar for the two citrus types and were as follows: (1) use of ZR or BA resulted in high-quality shoot production; (2) ZR and BA were not synergistic; (3) culture in 20 μM ZR resulted in the highest shoot production; (4) BA and IAA were strongly synergistic, with the greatest production with BA when IAA was included in the mixture; and (5) ZR and IAA were antagonistic, particularly with Ridge Pineapple. [ABSTRACT FROM AUTHOR]

Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoog mineral salt factors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface response experimental design. Pyrus communis ‘Old Home × Farmingdale 87,’ ‘Horner 51,’ and ‘Winter Nelis’; Pyrus dimorphophylla; and Pyrus ussuriensis ‘Hang Pa Li’ shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combinations. Analysis of shoot response to these treatments identified the factors that both contributed to physiological disorders and remedied them. Undesirable callus formation was common for pear shoots cultured on standard medium and decreased on formulations with increased NH 4NO 3, Fe, and mesos (CaCl 2, KH 2PO 4, and MgSO 4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH 4NO 3. Hooked and upwardly curled new leaves were seen in most genotypes and resulted from use of low mesos in P. communis and low nitrogen for ‘Hang Pa Li’ and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simultaneously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and moderate nitrogen compounds produced normal shoots without physiological disorders. [ABSTRACT FROM AUTHOR]

Bud emergence and shoot growth from adult phase citrus nodal cultures were studied using Citrus mitis (calamondin), Citrus paradisi (grapefruit), and Citrus sinensis (sweet orange). The effects of 6-benzyladenine (BA), indole 3-acetic acid (IAA), and citrus type on shoot quality and growth of mature bud explants from greenhouse grown trees were determined using a 2-component mixture-amount × citrus type experiment. BA increased shoot number and IAA improved shoot growth. The best shoot quality (fewer shoots but large shoots) was obtained with 1 μM IAA for calamondin, 15.5 μM IAA for sweet orange, and 30 μM IAA for grapefruit. Grapefruit exhibited substantial leaf abscission compared to calamondin and sweet orange. Four factors (AgNO, silver thiosulphate (STS), CaNO, or gelling) were screened individually for their efficacy in reducing leaf abscission. Five factors (AgNO, gelling, MS ion concentration, plant growth regulator and venting) were investigated to identify potential combinations for reducing leaf abscission and maximizing shoot growth and bud emergence. The factor combination identified as most effective in minimizing leaf drop, promoting shoot growth, and maximizing bud emergence for grapefruit was 2 mg l AgNO Gelrite, 1 × MS ion concentration, 30 μM IAA, and vented. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article about ion confounding in experimental biology in the previous issue.

Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoog mineral salt factors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface response experimental design. Pyrus communis ‘Old Home × Farmingdale 87,’ ‘Horner 51,’ and ‘Winter Nelis’; Pyrus dimorphophylla; and Pyrus ussuriensis ‘Hang Pa Li’ shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combinations. Analysis of shoot response to these treatments identified the factors that both contributed to physiological disorders and remedied them. Undesirable callus formation was common for pear shoots cultured on standard medium and decreased on formulations with increased NH 4NO 3, Fe, and mesos (CaCl 2, KH 2PO 4, and MgSO 4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH 4NO 3. Hooked and upwardly curled new leaves were seen in most genotypes and resulted from use of low mesos in P. communis and low nitrogen for ‘Hang Pa Li’ and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simultaneously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and moderate nitrogen compounds produced normal shoots without physiological disorders. [ABSTRACT FROM AUTHOR]