Rice Genetics IV - IRRI books - International Rice Research Institute

ice. Transgenic plants were also generated and tested with the sGFP(S65T) undercontrol of the d35S, ACT, UBI, and GOS2 promoters in comparison with theMON30063-GFP construct and confirmed the bombardment experiments.A construct termed d35S-sGFP:Ac (Table 2), harboring an autonomous Ac elementbetween the d35S promoter and the sGFP(S65T) gene, was introduced into ricevia Agrobacterium. The obtained transgenic calli displayed easily detectable fluorescentGFP sectors (Greco et al, submitted), indicative of Ac excision. The GFP excisionassay allowed the identification of excision at various stages during the transformationprocess and revealed a high excision rate. As GFP expression is easily visiblein the seed, this phenotypic excision assay is potentially suitable for selecting independentgerminal excision events before planting.Ac transposition behavior in riceThe transformants with the autonomous Ac transposon were analyzed by PCR andDNA blot hybridization for molecular evidence of excision and reinsertion. Table 2summarizes the results of the transformation experiments and molecular analyses ofindividual regenerants.The most remarkable discovery was that, in transformants with the d35S-sGFP:Acconstruct with an enhancer adjacent to the Ac promoter (Fig. 1), Ac transposed directlyafter transformation in the plant cell in every transformant. The evidence wasobtained by DNA sequence analysis of excision and reinsertion alleles from about 40regenerated plants (Greco et al, submitted). This has never been shown before andprobably occurs because the strong CaMV 35S enhancer adjacent to the Ac promoterTable 2. Molecular analysis of transposon construct transformants in rice.Independent Lines Indepen- %Transposon Construct Variety lines w/ entire dent active activeanalyzed a T-DNA a lines a lines bAutonomous Ac 35S-smGFP:Ac Taipei 309 1 (9) 1 (9) 1 (9) 100d35S-sGFP: Ac Taipei 309 11 (45) 11 (45) 11 (33) 100Nipponbare 8 (52) 7 (45) 6 (32) 86Total 20 (106) 19 (99) 18 (74) ––Direct DNA transfer UBI-smGFP:Ac Indica cvs. 348 – – – 278 – 80Ac total 368 (454) – – 296 (352) ––2-componentAc-Ds 2-comp. Ac-Ds Taipei 309 3 (19) 1 (13) 1 (13) 100Enhancer trap Ac-Ds ET Taipei 309 11 (45) 10 (37) 5 (11) 50Ac-Ds ET Nipponbare 10 (31) 8 (27) 4 (14) 50Ac-Ds ET with Nipponbare 219 (278) 104 (119) 63 (72) 60GFPET total 240 (354) 122 (183) 72 (97) ––Activation tag Ac-Ds AT Nipponbare 9 (15) 5 (7) 3 (3) 60Ac-Ds AT with Nipponbare 42 (75) 14 (17) 4 (4) 29GFPAT total 51 (90) 19 (24) 7 (7) –Ac transposase35s-AcTPase+ Taipei 309 15 (15) 12 (12) – ––SU1 Nipponbare 12 (15) 11 (13) – ––aTotal number of plants in parentheses. b The percentage is calculated on the number of independentlines that were not rearranged.268 Greco et al