Vol. 51—1997 - NorthEastern Weed Science Society

174
He bicide was added to the tissue culture medium to select transformed cells from
bombarde materials. Selection commenced 3-4 days after bombardment and continued for 8
weeks. Th bombarded tissues were then transferred to regeneration medium. Regenerants
appeared ithin 2-8 weeks. Shoots were transferred to phytatrayslM with regeneration medium
and roots : peared within 2-4 weeks. Plants were transplanted to soil and tested for herbicide
resistance the greenhouse. It takes about 6 months from tissue bombardment to obtain plants in
soil.
Pr 0 s were isolated from four-day old suspension culture cells by enzyme digestion.
Protoplas were transformed with foreign DNA using polyethylene glycol (pEG) treatment to
enhance e uptake of DNA We have developed a system for culturing the protoplasts back to cells
and sub uently to embryogenic callus cultures which then form plants by regeneration (Lee et
al., 1996). Transformed protoplasts were treated with herbicide 16 days after protoplast isolation
and resist t cell colonies were detected 3-4 weeks after selection began. Resistant colonies were
transferr to regeneration medium and plants were regenerated. Once rooted, these were
transferre to soil. It takes about 5-6 months from protoplast isolation to the production of
transgeni plants in soil.
nhouse herbicide tests: All regenerants were treated with Herbiace'M,Application
rates we determined by applying different concentrations of Herbiace to control plants by
painting t em with a brush. Herbicide at 2 mg/ml was used as this caused death of control plants in
all cases. e herbicide was applied at the rate of 120 mlltlat (24 plants/nat).
C eping bentgrass clones resistant to Herbiace were obtained from three cultivars:
Emerald, outhshore, and Cobra. In five experiments, involving 12 independent bombardment
events, s me 900 plants were regenerated for testing. Of these, 55 plants survived. The
transfo tion frequency for herbicide-resistant plants ranged from 0 - 13.7%. Thirty Cobra plants
were obu ined in 3 later bombardment experiments. Cobra transgenic plants were also obtained
from pro plast transformation. A total of 153 plants were regenerated from 2 resistant colonies
obtained rough protoplast transformation. All these plants survived the 2 mg/ml application rate.
More tha 200 transgenic plants of Emerald, Southshore, and Cobra survived 2 mg/ml herbicide
applicati ns in greenhouse tests and are resistant to 5x the field rate. Transgenic plants were
confmne by southern blot hybridization to show incorporation of the bar gene into the plant
genome d by northern blot hybridization to show expression of the bar gene.
F eld test of herbicide-resistant creeping bentgrass were conducted in the summer of 1994
and 1995 at Rutgers' Research and Development Center at Upper Deerfield, NJ. Field test permits
were obt 'ned from USDA-APHIS. Transgenic plants from bombardment experiments and
protopl t transformation of Cobra, Emerald, Putter, and Southshore were tested for resistance to
the herbi ide Ignite at Ix (O.75Ib active ingredient/acre) and 3x (2.251b AI/A) the label rate.
11plants that survived the 2 mg/ml greenhouse tests were completely resistant to both Ix
and 3x fi ld rates in the field tests. They remained green and unaffected like untreated plants in the
control lot. No control plants (Cobra, Emerald, and Southshore plants grown from seeds)
survived More than 30 transgenic clones, of creeping bentgrass from two tissue clones of
Emerald and one tissue clone of Southshore, were resistant to the 3x field rate. Cobra transgenic
plants 0 tained from protoplast transformation were all resistant to the 3x field rate.
vernali
e resistant transgenic plants from the 1994 field test were left in place over winter to
, Before flowering in the spring of 1995, a number were returned to a containment
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