Grantee Research Project Results

1999 Progress Report: Genetically Engineered Potato Plants Which Do Not Produce O3-induced Ethylene A Mechanism of O3 Tolerance?

EPA Grant Number: R823193
Title: Genetically Engineered Potato Plants Which Do Not Produce O3-induced Ethylene A Mechanism of O3 Tolerance?
Investigators: Pell, Eva J. , Sinn, Judy , Arteca, Richard N.
Current Investigators: Pell, Eva J. , Arteca, Richard N.
Institution: Pennsylvania State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 30, 1998
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $412,615
RFA: Exploratory Research - Environmental Biology (1995) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences , Human Health

Objective:

The objectives of this project are to: (1) produce transgenic potato plants that do not produce ethylene in response to ozone stress; and (2) test these transgenic plants to determine which ozone responses are ethylene dependent.

Progress Summary:

We have previously isolated two ozone-induced ACC synthase cDNAs, ST-ACS4 and ST-ACS5 from ozone-treated potato foliage. In previous progress reports, we described the antisense constructs developed for each of these two genes. We made several changes in the transformation/regeneration system for potato to reduce the number of "escapes" and achieve positive transformations. Since the last report, we have been successful in generating multiple lines of cv. FL1607 that carry the antisense ACS contructs.

Regenerated plants demonstrating kanamycin resistance were screened for presence of the antisense genes by Southern analysis. Southerns showed that some lines carried one copy of the antisense, while others carried multiple copies. Those plants carrying antisense genes were transplanted to soil-less growth medium and placed in growth chambers. When plants were approximately 2 months old and had about 12 leaves, they were screened for ozone-induced ethylene production.

Plants were given 2-hour exposures to 250 ppb ozone, and leaves were collected for ethylene measurement and Northern analysis. During ozone exposures, companion sets of transgenic plants were kept in an ambient-air chamber and subjected to the same analyses. Nontransformed plants of the cv. FL1607 also were subjected to the ozone and ambient-air treatments on each exposure day, enabling us to compare the transgenic response to that of the nontransformed plants.

In initial trials, some transgenic lines (e.g., L5-3, L5-17, G5-2, G4-18, and G4-31) produced much less ethylene than FL1607, while other lines produce only slightly less or comparable amounts of the hormone in response to ozone. Northern analysis is ongoing to determine which of the five ACC synthase genes are being expressed in ozone-challenged and nonexposed plants.
After receiving approval from USDA APHIS, we established a field study with six of the antisense potato lines. Lines G4-9, G4-18, and G4-40 carry the antisense for ST-ACS4; lines L5-3, L5-14, and L5-17 carry antisense to ST-ACS5. Also, included in the study were the nontransformed FL1607 plants as a "control" line. Prior to transplanting the FL1607 and transgenic lines, the field was planted with the cv. Chieftain, a relatively ozone tolerant, late-maturing red-skinned variety. This variety served as a border planting around our experimental plants, allowing us to harvest the white-skinned tubers of the FL1607 and transgenic plants without confusing lines during tuber harvest.

After planting the border crop, 12 open-topped gas exclusion chambers were placed in the field. Six chambers were assigned to the antisense plants of ACS4, and six to the ACS5 antisense plants. The area inside the chambers permitted the transplanting of four pairs of plants (three antisense lines and FL1607), separated by single border plants. Throughout the course of the season, pests were controlled as in a commercial planting. Irrigation was provided as needed. Flowers were removed to prevent the spread of pollen from the transgenic lines. Plants were permitted to acclimate to field conditions for 12 days before ozone exposures were begun.

All chambers received charcoal-filtered air; six of the chambers (three per antisense gene) were supplemented with 80 ppb ozone between 1000 and 1800 hours every day. Ozone exposures were begun on July 1 and ended on September 3. By September 9, vines had reached maturity and were beginning to decline, so the vines were removed and dried for biomass comparisons. After tubers were permitted to suberize for two weeks, they were harvested for biomass comparisons.

Visual assessments of plant health were made periodically over the course of the season. Ozone symptoms (reddish stipple on leaves) began to appear on the more mature border vines in the last week of July, but were not noticeable on the transplanted lines until the first week in August. Lines L5-3, G4-18, and G4-40 did not differ from the FL1607 in their visible response to ozone, having similar vine size and severity of leaf injury. Two transgenic lines, G4-9 and L5-17, did not grow as vigorously as other lines, either in CF air or ozone. Both lines had severe ozone injury and exhibited leaf rolling and earlier vine decline in all chambers.

Future Activities:

Additional ozone exposures of the transgenic plants are being conducted in the greenhouse and in growth chambers to characterize the ozone response of the various lines. Northern analysis will be conducted to determine which of the five ACS genes are being expressed. Biomass of the above- and below-ground portions of the field-grown plants will be measured to compare growth and tuber yield amongst lines and between pollutant treatments.

Supplemental Keywords:

molecular biology, air pollution., Health, Scientific Discipline, Engineering, Risk Assessments, Chemistry, Biology, Environmental Chemistry, Genetics, pyridoxal phosphate inhibitor, potato plant, agrobacterium cell supression, biosynthesis, ozone, genetic engineering, germplasm

Progress and Final Reports:

Original Abstract

1996

1997

Final Report