Search Results
Several characteristics of amylases involved in starch degradation were studied in extracts from immature (30 days before harvest) `d'Anjou' pears (Pyrus communis L.). Enzyme activity was not detected until after at least 60 minutes of incubation in frozen or lyophilized tissues. Activity increased significantly after 90 minutes and increased linearly after 2 to 12 hours of incubation. Activity was greater, however, in frozen than in lyophilized tissues. Three buffers (acetate, tris-HCl, and imidazole-HCl) were used at a range of pH levels (4.6-8.2) to ascertain the optimum assay system. Highest specific activity was recorded with acetate buffer at pH 5.6. The Km value in this system was 1.43 × 10-3g·ml-1. Specific activity increased as Ca concentration in the reaction mixture increased from 1 to 15 mm CaCl2 but did not change as Ca concentration increased from 15 to 25 mm CaCl2. The `d'Anjou' pear amylases were purified 5.7-fold using ammonium sulfate fractionation.
Abstract
Stomatal resistance of leaves, water absorption through roots, and water content of stem internodes was evaluated during cold acclimation of red-osier dogwood (Cornus stolonifera Michx.). Four weeks of short-days at 20/15°C resulted in a significant reduction in stem water content. This reduction was related to water lost from pith cells as this tissue senesced sequentially in successively younger internodes from the stem base to its apex at autumn growth cessation. A 1.5 fold decrease in relative stomatal resistance of leaves and a 3.5 fold increase in resistance to water flow though roots at 0.15 bars tension occurred prior to hardening to -12°C.
Additional hardening to -40°C resulted in a 28 fold increase in resistance to forced water flow through roots and equalization of water content in all internodes. Thereafter, water content remained constant but stems continued to harden to temperatures below -65°C.
Comparison of resistance to forced water flow through living and dead roots from hardy and tender plants suggested there was a decrease in the permeability of root cells to water during the initial (-12°C) stage of acclimation. Root surfaces became suberized, as acclimation progressed, giving rise to an additional barrier to water uptake in roots of very hardy plants (-45°C). It seems likely that the decrease in hydration of overwintering stems during cold acclimation results from a decreased stomatal resistance and increased root resistance to water movement.
The colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.
The Colorado potato beetle [Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)] is a destructive pest of the cultivated potato (Solanum tuberosum L.) in northern latitudes. Combining resistance mechanisms of leptine glycoalkaloids and glandular trichomes with the synthetic Bacillus thuringiensis Berliner (Bt) cry3A gene in potato may be an effective strategy for controlling the Colorado potato beetle. Bt-cry3A transgenic plants were developed for three potato lines with differing levels of resistance to Colorado potato beetle ['Yukon Gold' (susceptible control), USDA8380-1 (leptine glycoalkaloids), and NYL235-4 (glandular trichomes)]. Polymerase chain reaction, and Southern and northern blot analyses confirmed integration and transcription of the cry3A gene in the transgenic lines. Detached-leaf bioassays of the cry3A engineered transgenic lines demonstrated that resistance effectively controlled feeding by first instar Colorado potato beetles. The susceptible `Yukon Gold' control suffered 32.3% defoliation, the nontransformed high foliar leptine line (USDA8380-1) had 3.0% defoliation, and the nontransformed glandular trichome line (NYL235-4) had 32.9% defoliation. Mean percentage defoliation for all transgenic lines ranged between 0.1% and 1.9%. Mean mortality ranged from 0.0% to 98.9% among the Bt-cry3A transgenic lines, compared to 20% for the susceptible `Yukon Gold' control, 32.2% for USDA8380-1, and 16.4% for NYL235-4. Results indicate that genetic engineering and the availability of natural resistance mechanisms of potato provide the ability to readily combine host plant resistance factors with different mechanisms in potato.
The Colorado potato beetle, Leptinotarsa decemlineata (Say), is the most serious insect pest of potatoes throughout the eastern and north central United States. Host plant resistance to the Colorado potato beetle has been identified in wild Solanum species and Bt-transgenic potato lines. Detached-leaf bioassays (72 h) were conducted on insecticide-resistant, first instar Colorado potato beetles to study the effectiveness of individual and combined host plant resistance traits in potato. Potato lines tested include non-transgenic cultivars (`Russet Burbank', `Lemhi Russet', and `Spunta'), a line with glandular trichomes (NYL235-4), a line with high foliar leptines (USDA8380-1), and transgenic lines expressing either codon-modified Bt-cry3A or Bt-cry5 (Bt-cry1Ia1). Bt-cry3A transgenic lines, foliar leptine line, and foliar leptine lines with Bt-cry5 had reduced feeding compared to non-transgenic cultivars. Glandular trichome lines and glandular trichome lines with Bt-cry5 did not reduce feeding in this no-choice feeding study. Some Bt-cry5 transgenic lines, using either the constitutive promoters CaMV35s or (ocs)3mas (Gelvin super promoter), were moderately effective in reducing larval feeding. Feeding on Bt-cry5 transgenic lines with the tuber-specific patatin promoter was not significantly different than or greater than feeding on the susceptible cultivars. Mortality of first instars was highest when fed on the Bt-cry3A lines (68% to 70%) and intermediate (38%) on the Bt-cry5 `Spunta' line SPG3 where the gus reporter gene was not included in the gene construct. Host plant resistance from foliar leptines is a candidate mechanism to pyramid with either Bt-cry3A or Bt-cry5 expression in potato foliage against Colorado potato beetle. Without multiple sources of host plant resistance, long-term sustainability is questionable for a highly adaptable insect like the Colorado potato beetle.
Yield in common bean, Phaseolus vulgaris L., can be significantly reduced by high temperature (I-IT) during bloom. Ethylene production from plant tissue increases as a consequence of various stresses, including heat stress. The inheritance of leaf ethylene evolution rate (EER) of HT-stressed (35/30C day/night) progenies from crosses among bean genotypes previously categorized as HT sensitive or tolerant, based on cell electrolyte leakage, was investigated. Evidence from generation means analysis of Fl, F2, and backcross progenies shows EER to be genetically controlled, with additive, dominance, and epistatic effects indicated for low EER. The range (0.62 to 2.52 μg-1·hr-1) of EER from field-grown lines and cultivars suggests the existence of considerable genetic variability. EER was associated (r = –0.70) with heat tolerance, as estimated by cell electrolyte; leakage.
Research was done to determine if enhanced resistance to potato (Solanum tuberosum L.) late blight could be obtained by combining host plant resistance and engineered resistance. Late blight susceptible cultivars, Atlantic, and Spunta and the partially resistant cultivar Libertas were transformed with a fungal glucose oxidase gene, resulting in lines which ranged in transgene copy number from 1 to 8. Glucose oxidase enzyme activity ranged from 0.00 to 96.74×10-5 units/mg plant tissue. There was no correlation between copy number and level of transgene mRNA, level of transgene mRNA and enzyme activity, or between level of enzyme activity and disease resistance. Field and growth chamber evaluation of late blight response demonstrated little to no effect of the glucose oxidase transgene in either late blight susceptible or partially late blight resistant cultivars. However, enzyme activity levels were much lower than levels reported in previous research, which may account for the lack of effect of glucose oxidase against Phytophthora infestans. Twenty-one percent of the transgenic lines were phenotypically off-type compared to nontransgenic controls. Most of the off-type transgenic lines (four out of seven) were derived from `Libertas'. Because several off-type lines did not express the glucose oxidase protein, this phenomenon could not be attributed solely to the glucose oxidase transgene. Based on these results, transgenic lines produced for this study do not increase resistance to P. infestans even in combination with moderate host plant resistance. However, production of greater numbers of transgenic lines with the current construct or, production of transgenic lines in which a different constitutive promoter drives the expression of the glucose oxidase gene might result in greater disease resistance. However, the usefulness of any small increase in resistance would need to be evaluated against the time and cost required for development of transgenic potato cultivars and the potential for off-type tubers and plants.
The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L.) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic 'Spunta' clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to 'Spunta'. These Bt-cry5 transgenic potato plants with high potato tuber moth resistance have value in integrated pest management programs.
Pierce's disease (PD), caused by the xylem-limited bacterium Xylella fastidiosa, is endemic to the coastal plain of the southeastern United States. Although native southern grapevines are tolerant to X. fastidiosa, all varieties of Vitisvinifera grown in the region will succumb to PD. Genetic transformation to add disease resistance genes, while not disturbing desirable phenotypic characters, holds promise for expanding the southeastern U.S. grape industry by allowing use of established fruit and wine varieties. We utilize embryogenic cell cultures and Agrobacterium strain EHA105 to refine transformation systems for Vitis species and hybrids. V. vinifera`Thompson Seedless' is employed as a model variety to test various transgenes for disease resistance, since as many as 150 independent transgenic plant lines routinely are produced from 1 g of embryogenic culture material. Transgenic plants are stringently screened for PD resistance in greenhouses by mechanical inoculation with X. fastidiosa. Transgenic plants are compared with both susceptible and resistant control plants by assessing typical PD symptom development and by assaying bacterial populations in xylem sap over time. Using these procedures, nine putative PD resistance genes have been inserted into grapevine and over 900 unique transgenic lines have been evaluated. A range of susceptible-to-resistant responses has been catalogued. Thus far, the best construct for PD resistance contains a grape codon-optimized hybrid lytic peptide gene in a high-performance bi-directional 35S promoter complex. Certain transgenic plant lines containing this construct exhibit better resistance than that of resistant control vines.
The codon-modified cryV-Bt gene (cryV-Bt) from Bacillus thuringiensis subsp. kurstaki Berliner, which is specifically toxic to Lepidoptera and Coleoptera insects, and a potato virus Yo coat protein gene (PVYocp), in which the aphid transmission site was inactivated, were cotransformed into potato (Solanum tuberosum L.) `Spunta' via Agrobacterium tumefaciens Conn. We demonstrated the integration and expression of both genes by molecular analysis and bioassays. All cryV-Bt/PVYocp-transgenic lines were more resistant to potato tuber moth (Phthorimaea operculella Zeller) and PVYo infection than nontransgenic `Spunta'. Four cryV-Bt/PVYocp transgenic lines were equal in potato tuber moth mortality to a cryV-Bt transgenic line, but of these four only two lines were equivalent in PVYo titer levels to a PVYocp-transgenic line. We identified two transgenic lines, 6a-3 and 6a-5, which showed greater resistance to potato tuber moth and PVYo than the other cryV-Bt/PVYocp transgenic lines. This study indicated that multiple genes, conferring insect pest resistance and virus resistance, could be engineered into and expressed simultaneously in a potato cultivar.