Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass.
Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.
Chai
Ming-Liang
ML
Department of Horticulture, Zhejiang University, Hangzhou 310029, China. mlchai@zju.edu.cn
Wang
Bing-Liang
BL
Kim
Jae-Yeoul
JY
Lee
Jong-Min
JM
Kim
Doo-Hwan
DH
D031703:Agrostis / Q000145:classification / Q000166:cytology / Q000187:drug effects* / Q000235:genetics*; D000613:Aminobutyrates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D006540:Herbicides / Q000494:pharmacology; D018515:Plant Leaves / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D030821:Plants, Genetically Modified / Q000166:cytology / Q000187:drug effects* / Q000235:genetics*; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic
D000613:Aminobutyrates; D006540:Herbicides; C003121:phosphinothricin