Transgenic grasspea (Lathyrus sativus L.): factors influencing agrobacterium-mediated transformation and regeneration.
A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.
Barik
D P
DP
Plant Cell, Tissue & Organ Culture Facility, Post-Graduate Department of Botany, Utkal University, Bhubaneswar, 751 004 Orissa, India.
Mohapatra
U
U
Chand
P K
PK
D015139:Blotting, Southern; D004351:Drug Resistance; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007612:Kanamycin / Q000494:pharmacology; D027807:Lathyrus / Q000235:genetics / Q000382:microbiology / Q000502:physiology*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000382:microbiology / Q000502:physiology*; D012231:Rhizobium / Q000254:growth & development / Q000502:physiology*; D014170:Transformation, Genetic / Q000502:physiology*
D007612:Kanamycin; D005966:Glucuronidase