Transformation Procedure

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We summarize transformation procedures oftwo prototype MAT vectors (pNPI132, 702) (Figs. 25.1 and 25.3). Plasmid pNPI132 has a "hit & run" cassette in which both the chimerical ipt (isopentenyl transferase)7-9 and R (recombinase) genes fused with 35S promoters are located between two directly oriented RS (recombination site) sequences (Fig. 25.1). pNPI132 transformation and selection were as follows (Fig. 25.3A):

1. Tobacco leaf segments were infected with A. tumefaciens containing a MAT vector (pNPI132), and cultured on a hormone-free MS medium without kanamycin (nonselective medium). When adventitious shoots were regenerated, they were separated from the leaf segments and transferred to the same medium;

2. After one month of cultivation, we visually identified and selected for further cultivation abnormal shoots (called ESP for extreme shooty phe-notype) that lost apical dominance;

3. Normal shoots exhibiting normal apical dominance appeared from ESP shoots. We visually identified, selected and transferred these normal shoots to the same medium. These shoots grew normally and rooted. In PCR analysis, the chimerical ipt gene was present in the chromosomal DNA of the ESP shoots, but was excised from that of the "normal" shoots along with the "hit and run" cassette. These results indicate that these nomally grown

Fig. 25.1. Construction of MAT vectors. Plasmid pNPI106 has a "hit and run" cassette in which the chimeric ipt gene with 35S promoter is inserted into Ac as a selectable marker. The pNPI132 and 702 vectors use the excision system of R/RS to remove the chimeric ipt gene or rol genes and has a "hit and run" cassette in which these marker genes and R (recombinase) genes fused with a 35S promoter are located between two directly oriented RS (recombination site) sequences. The gusA and nptII genes are unselected markers in these experiments.

Fig. 25.1. Construction of MAT vectors. Plasmid pNPI106 has a "hit and run" cassette in which the chimeric ipt gene with 35S promoter is inserted into Ac as a selectable marker. The pNPI132 and 702 vectors use the excision system of R/RS to remove the chimeric ipt gene or rol genes and has a "hit and run" cassette in which these marker genes and R (recombinase) genes fused with a 35S promoter are located between two directly oriented RS (recombination site) sequences. The gusA and nptII genes are unselected markers in these experiments.

shoots are marker-free transgenic plants containing only desired genes.

Using the pNPI106 vector, we could obtain marker-free transgenic tobacco plants from only 3 of 63 ESP lines (4.8%) by 8 months after infection of Agrobacterium.2 Also, we obtained marker-free transgenic tobacco plants from 10 of 48 ESP lines by 4 months after infection using the pNPI132 vector. Finally, marker-free transgenic tobacco plants appeared from 32 of 48 ESP lines (67%) by 8 months after infection.3 These results indicate that the MATVS could produce marker-free transgenic plants without sexual crossing and that the R/RS-type MATVS is the more practical of the two (Table 25.1).

Plasmid pNPI702 has a "hit and run" cassette in which the 7.6 kb DNA fragment containing rolA, B, C, D 9-11 and the R genes with a 35S promoter are located between two directly oriented RS sequences. pNPI170 transformation and selection were as follows (Fig. 25.3B):

1. Tobacco leaf segments were infected with A. tumefaciens containing a MAT vector (pNPI702) and cultured on a hormone-free MS medium without kanamycin (nonselective medium). Hairy roots were regenerated two weeks after infection. After one month, they were separated from the leaf segments and transferred to a shoot-inducing medium with hormones;

2. After two months half of cultivation, transgenic shoots were regenerated from such roots. We separated these shoots and transferred to a nonselec-tive medium for further cultivation.

3. We visually identified and selected normal plants from abnormal plants with wrinkled leaves, reduced apical dominance or shortened internodes.

Commonly, crossing of chimeric plants segregates out non-chimerical progenies. Therefore, we subjected the seedlings of chimerical plants to PCR analysis and observed

┬┐hit & run" casscttc gusA

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