Improvement of MAT Vectors

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In our evaluations of two prototype MAT vectors, we found several problems with practical use and developed new MAT vectors (pMAT21, 22, 101, pTL7, pIPT53, 54, prol103,

Table 25.1. Transformation efficiency of tobacco plants

ESP lines which formed normal shoots

ESP 4 months 8 months after infection after infection

pNPI 132 48 10 20.8 32 66.6

Transformation of tobacco plants using pNPI 106 and 132 vectors, and appearance of marker-free transgenic plants at hormone-free MS medium.

108) (Figure 25.4). Our improvement is as follows.

1. Cloning sites: We constructed two kinds of vectors. One is a binary cloning vector pTL7 in which multicloning sites except for an SseI site are available for desired genes, and an SseI site outside the multicloning sites, for a "hit & run" cassette of MAT vectors. The other type is MAT cassette vectors (pIPT53, 54, prol103, 108). After cloning valuable genes into multi -cloning sites, a MAT cassette is put into an Sse site.

2. Second markers: A selectable marker gene (nptII) is inserted into a "hit & run" cassette (pMAT22, pIPT53, prol108) and a reporter gene (GUS) into a cassette (pMAT21, pIPT54, prol103). Now we are evaluating MAT vectors (rol genes type) containing GFP genes which enable us to easily distinguish transgenic roots from non-transgenic ones.

3. Control of recombinase: We have detected dropouts of a MAT cassette by excision events during cultivation of Agrobacterium. Therefore, we inserted an intron of Eucalyptus histone genes into the R genes to inactivate in Agrobacterium. In addition to the 35S promoter (constitutive type), rbcS,13 parB 14 and GST II15 promoters (inducible type) are joined to the R genes for control of its expression. rbcS and parB promoters are under evaluation. Our evaluation work shows that the GST II promoter is preferable for controlling excision events during transformation.

4. Control of ipt genes: When we used the ipt genes fused with a 35S promoter to regenerate shoots at tobacco transformation, we detected inacti-vation of GUS genes in several plants. At transformation of hybrid aspen, we could get a lot of regenerated shoots, but most shoots were non-transgenic. Therefore, we examined native ipt and rbcS promoters to compare with a 35S promoter. A native ipt promoter has been isolated from A. tumefaciens PO22. On the other hand, a rbcS promoter came from tomato. In hybrid aspen, rbcS is absolutely greater than others in ESP formation. In tobacco, rbcS and native ipt are better than 35S. These results might depend on plant species.

5. Combination with iaaM/H genes: Endogenous hormonal levels are very different across plant species and plant tissues. The ipt genes are combined with iaaM/H genes16 to manipulate the auxin/cytokinin ratio according to the physiological state of plant tissues. Now the vector is in the process of evaluation.






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