Transformation of the endospermous legume guar (Cyamopsis tetragonoloba L.) and analysis of transgene transmission

A procedure for transformation of the large-seeded endospermous legume guar (Cyamopsis tetragonoloba L.) and a study on transmission of the transgenes to offspring generations are presented. Using Agrobacterium tumefaciens wi th a T-DNA construct harbouring a beta-glucuronidase gene (uidA) and a neom ycin phosphotransferase gene (nptII), maximum transformation frequencies of cotyledonary explants were obtained using 145 mg/l kanamycin sulfate as se lective agent. Carbenicillin and cefotaxime, used for the elimination of Ag robacterium after co-culture, displayed considerable toxicity to guar tissu es but replacing most of these beta-lactams by the non-phytotoxic beta-lact amase inhibitor sulbactam as well as addition of thidiazuron and silver thi osulfate increased transformation frequencies up to 10-fold in total. The p resence of the transgenes in the primary transformants was demonstrated by genomic DNA analysis of GUS-positive shoots. Chimaeric plants (5-10%) were identified by GUS analysis at the flowering stage and were discarded. Analy sis of the R-1 offspring from 17 independent transformants showed that in 4 1% of those, the uidA gene(s) was expressed and stably inherited consistent with Mendelian genetics. This was also found for the R-2 and R-3 generatio ns of single copy transformants. On the other hand, a large proportion (47% ) of the primary transformants gave R-1 offspring in which 100% of the plan ts were GUS-negative. Analysis of these plants by PCR revealed that, at lea st, most of the transgene sequences were absent, suggesting that they had n ot been transmitted from the parent transformants. This occurred at similar high frequencies (40-50%) irrespective of the estimated copy number of the transgenes. Thus, major parts of the transgenes, even when present in mult iple copies, displayed aberrant transmission, at a high frequency, in the p rocess of going from the primary transformants to the first offspring gener ation.