A protocol was developed for excising and culturing cotyledon explants from mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed callus with multiple shoot buds on agar-solidified Murashige and Skoog media containing several combinations of hormones (1 μm 2,4-D; 10 μm 2,4-D; 1 μm BA + 1 μm 2,4-D; 1 μm BA + 10 μm 2,4-D; 5 μm BA; 5 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μ m 2,4-D; 5 μ m BA + 5 μm NAA; 5 μ m BA + 15 μ m NAA). After three subcultures, only tissues maintained on the medium containing 5 μm BA + 5 μm NAA continued to form shoots. Tissues transferred to other media eventually died (1 μm 2,4-D; 1 μ m BA + 10 μ m 2,4-D; 5 μ m BA; 5 μ m BA + 1 μ m 2,4-D), became unorganized (1 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μm 2,4-D; 5 μm BA + 15 μm NAA), or formed roots (10 μm 2,4-D). Whole plantlets were produced by transferring callus with buds to medium lacking hormones. The rapid regeneration of clonal plantlets from cotyledon explants may be useful for reducing variability in future developmental studies. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA).
A. Raymond Miller and Craig K. Chandler
Henry R. Owen and A. Raymond Miller
A factorial combination of pretreatments, fixatives, and stains was examined to identify the best available method for staining the chromosomes of strawberry (Fragaria × ananassa Duch. `Chandler') root-tip cells. Three pretreatments (a -bromonaphthalene, p -dichlorobenzene, and 8-hydroxyquinoline), three fixatives (Farmer's fluid, Carnoy's fluid, and Newcomer's fluid), and five stains (acetocarmine, alcoholic hydrochloric-acid carmine, altered carbol fuchsin, lacto-propionic orcein, and leucobasic fuchsin) were tested. Pretreatment with either a -bromonaphthalene (saturated aqueous) or 8-hydroxyquinoline (2 mm) for 5 hours at 14C, overnight fixation in Farmer's fluid, hydrolysis in 1 n HCl (15 minutes at 60C), and staining with altered carbol fuchsin produced chromosome preparations superior to other treatment combinations. Treatment with a-bromonaphthalene, Farmer's fluid, and alcoholic hydrochloric-acid carmine (2 days at 25C) also produced acceptable chromosome preparations.
Anusuya Rangarajan, A. Raymond Miller, and Richard Veilleux
Leptine (LP) glycoalkaloids have been demonstrated to confer natural resistance to the Colorado potato beetle (CPB) in Solanum chacoense (chc). Development of cultivated potatoes with natural resistance to CPB has the potential to reduce both costs and environmental impacts of production by reducing pesticide use. To introgress the genes conferring leptine production from chc into S. tuberosum (tbr), clones of chc have been crossed with clones of S. phureja. Leaf disks from eight hybrids were subjected to a CPB second instar feeding bioassay to determine if extent of feeding was related to LP levels. Most hybrids contained leptinidine (LD, the aglycone of LP) levels intermediate to chc and tbr, and insect feeding was suppressed 30% to 50% in hybrids containing >10 mg·g–1 DW LD. One hybrid displaying feeding suppression contained a very low level of LD, whereas another hybrid that contained higher levels of LD had higher feeding rates. The presence of LD at “threshold” levels in these hybrids will suppress feeding of CPB, but other factors affecting resistance are also present and need to be explored.
Henry R. Owen and A. Raymond Miller
A comparison of pretreatment, fixing, and staining methods for root tips of Fragaria × ananassa (2n=8x=56), a polyploid species with small chromosomes, was made to facilitate chromosome counting. Three pretreatments (8-hydroxyquinoline, α-bromonaphthalene, and p-dichlorobenzene), three fixatives (Farmer's, Carnoy's, and Newcomer's), and five stains (acetocarmine, lacto-propionic orcein, leucobasic fuchsin, altered carbol fuchsin, and alcoholic hydrochloric-acid carmine) were examined in a factorial design to determine which treatment combination produced the best chromosome preparation. Field propagated runners were grown in sand under greenhouse conditions with supplemental lighting to produce root tips for late morning collection. The treatment combinations of α-bromonaphthalene or 8-hydroxyquinoline, Farmer's fixative, and altered carbol fuchsin, or the combination of α-bromonaphthalene, Farmer's fixative, and alcoholic hydrochloric-acid carmine produced the most intensely-stained and well-defined preparations.
Richard E. Veilleux and A. Raymond Miller
F1 hybrids between high leptine-producing clones (8380-1, PI 458310 and 55-1) of Solanum chacoense Bitt. and anther culture competent or anther-derived clones of S. phureja Juz. & Buk. that did not produce leptines were generally weak plants that grew slowly and died before flowering. Exceptional hybrids could be found that were capable of completing a life cycle, especially during the hot summer months in the greenhouse. All F1 hybrids produced leptines in the leaves but not the tubers, albeit at lower levels than in the S. chacoense parent. Anther-derived monoploids from the F1 hybrids exhibited a range of leptine production from none to levels approaching the S. chacoense parent. Backcross populations of an F1 hybrid to the S. chacoense and S. phureja parents were examined for leptine production. Backcross hybrids were generally much more vigorous than the F1 hybrids. All of the S. chacoense backcrosses produced leptines ranging from intermediate to high levels; four of the twelve S. phureja backcrosses exhibited low leptine levels. A general dominance of leptine synthesis was therefore exhibited, although the nonleptine-producing parent affected the expression of leptines in the hybrids.
Ellen A. McComb, A. Raymond Miller, and Joseph C. Scheerens
Peroxidase activity in extracts from freeze-dried tissue of Fragaria × ananassa Duch. cv. Chandler was highest in tissue-cultured (TC) plants, followed by field-grown (FG) and lowest in greenhouse (GH) plants. Among tissue types, activity was highest in petioles, with leaves second highest. Fruit, root, and crown tissue all exhibited low or no activity. When subjected to isoelectric focusing (IEF), petiole tissue extracts exhibited more isozymes than extracts from other organs regardless of staining substrate. Using 4-chloro-1-naphthol and H2O2 as substrates, anionic and cationic isozymes were observed in TC petiole extract with nine isozyme bands ranging in pI from 3.9 to 9.5. In TC leaf extract an isozyme at pI 7.4 was observed that was not present in other organ extracts when H2O2 and benzidine, p-phenylenediamine or 3-amino-9-ethylcarbazole were used as substrates. Specific isozymes and number of isozymes varied according to plant organ and developmental stage. Mature leaves and over-ripe fruit appeared to exhibit more activity and a larger number of isozymes than developing tissues of those plant organs.
Judith A. Abbott, A. Raymond Miller, and T. Austin Campbell
Mechanical stress received by pickling cucumbers (Cucumis sativus L.) during harvest can cause physiological degeneration of the placental tissues, rendering the cucumbers unsuitable for use in some pickled products. Cucumbers were subjected to controlled stresses by dropping and rolling under weights to induce such degeneration. Following storage at various temperatures for O, 24, and 48 hours, refreshed delayed light emission from chlorophyll (RDLE) was measured and transmission electron micrographs of chloroplasts were made. Mechanical stress rapidly suppressed RDLE and induced accumulation of starch granules within the chloroplasts. Rolling usually had a greater effect on RDLE than did dropping. After 48 hours, RDLE suppression persisted; starch granules were no longer evident in chloroplasts from mechanically stressed fruit, but very electron-dense inclusions had developed in the chloroplasts. Storage temperatures affected RDLE levels but had minimal interaction with stress responses. Cucumber lots subjected to excessive mechanical stress likely could be detected using RDLE measurement.
A. Raymond Miller, Thomas J. Kelley, and Brian D. White
A nondestructive method was developed utilizing a modified Trebor 101 watercore tester to evaluate the internal quality of pickling cucumbers. The method involved measuring the relative amount of visible-infrared light passing through the longitudinal midsection of whole cucumber fruit. Light transmission was quantified on a unitless sigmoid scale from 1 to 10, with light transmission and scale values positively related. Immediately after hand harvest, size 3F (47 to 51 mm in diameter) cucumbers exhibited transmission values between 2 and 3, regardless of cultivar. Following a mechanical-stress treatment, which simulated bruising incurred during harvesting and handling of cucumbers, the internal quality of the fruit declined and was associated with an increase to a value of 6 in light transmission compared to non-stressed fruit. Light transmission increased as the severity of stress applied to the fruit increased, and high light transmission values were evident throughout a 48 h storage period at room temperature. Light transmission values increased as fruit diameter decreased, but values within a particular size class of undamaged, hand-harvested fruit were consistent. Machine-harvested fruit (size 3F), evaluated just before processing, exhibited light transmission values from 2 to 8, but the majority of fruit fell within the transmission range of 2 to 3. When fruit exhibiting different light transmission values were speared (cut longitudinally into sixths), processed, and then visually evaluated by panelists, spears prepared from fruit exhibiting high transmission values were judged to be of lower quality than those prepared from fruit exhibiting low transmission values. Visible-infrared light transmission may be a valuable tool for detecting poor quality cucumbers before processing, and could allow the mechanical selection of high quality fruit on a large scale basis.
Anusuya Rangarajan, A. Raymond Miller, and Richard E. Veilleux
Leptine glycoalkaloids in leaves of the weedy diploid potato, Solanum chacoense Bitt., have been shown to reduce feeding by Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). Development of cultivated potatoes with natural resistance to CPB has the potential to reduce costs and environmental impacts of production by reducing pesticide use. Through efforts to move the genes controlling leptine biosynthesis into cultivated potato, a series of hybrids was generated between the high leptine producing S. chacoense and a cultivated type, S. phureja Juz. and Buk. These hybrids were evaluated for solanine (+chaconine), leptinins, leptines, and total steroidal glycoalkaloid content. All hybrids contained leptines, but at different levels (ranging from 117 to 802 mg·g-1 dry weight of leptine aglycon). Some hybrids appeared to convert solanine (+chaconine) to leptinine and leptine efficiently and had no detectable solanine in sampled leaves. To verify the biological significance of these glycoalkaloids, leaf tissue was subjected to feeding assays with second instar CPB. CPB feeding rate ranged from 38 to 87 mm2·d-1 and was most closely correlated with leptine concentration. A minimum leptine level of 300 mg/100 g fresh leaves suppressed feeding by 50%, and levels below this had no effect on CPB feeding.
Mustafa Ozgen, Artemio Z. Tulio Jr., A. Raymond Miller, R. Neil Reese, and Joseph C. Scheerens
In preliminary studies, we found that relative and absolute antioxidant (AO) levels varied within and among small fruit types. AO levels were affected by assay method used, time of reaction, volume of sample, and the ratio of reactants to total AO activity. To identify the physicochemical parameters that affect accuracy and reproducibility, a series of experiments were conducted to test the roles of AO assay, different AOs, and AO concentration on measured AO content and reaction kinetics. Three assays (DPPH, FRAP, ABTS) were used to evaluate AO capacity of seven fruit types (black and red raspberry, blackberry, strawberry, grape, elderberry, and cranberry) and nine purified AOs (ascorbic, caffeic, chlorogenic, gallic, and ellagic acids, α-tocopherol, trolox, cyanidin-3-glucoside, and quercetin). Ascorbic acid, trolox, caffeic acid, chlorogenic acid, and α-tocopherol exhibited simple reaction kinetics and reached endpoints quickly, regardless of assay. Gallic and ellagic acids, quercetin, cyanidin-3-glucoside, and all fruit extracts exhibited more complex kinetics and long reaction times (>70 min) to reach an endpoint. Moreover, the latter four AOs had the highest AO capacity among the compounds tested. We observed differences in reactivity between assays, compounds and fruit extracts, but relative AO activity was comparable, although the absolute values differed. Since AO capacity of fruit extracts is a composite of the individual AOs present, it is important that reactions progress to near steady state, assay reactants are in excess of (30–50×) the AO capacity being measured, more than one assay is used to describe the total AO activity of fruit samples. Thus, there may not be a single AO assay method that completely defines the AO activity of a given fruit.