Most of the transformation procedures discussed so far have one major disadvantage—they rely on tissue culture for the regeneration of whole plants. As discussed, the tissue culture requirement adds significantly to the time and cost of producing transgenic plants and is perhaps the most serious limitation to the technology because it restricts the range of species amenable to genetic manipulation. Methods that circumvent tissue culture all together —in planta transformation systems—are therefore highly desirable, although until recently they were available only for the model dicot Arabi-dopsis thaliana. These techniques include seed transformation, in planta DNA inoculation, and flower dipping or infiltration, and all involve Agro-bacterium-mediated transformation.
For seed transformation, Arabidopsis seeds are imbibed and coculti-vated with Agrobacterium tumefaciens and then germinated (66). This is convenient in Arabidopsis because a large number of seeds are produced (10,000 per plant) and the seeds are very small. The procedure is relatively inefficient and is not very reproducible, but because the plant is small it is relatively easy to screen for transformants. A more efficient procedure is to inoculate plants with bacteria after severing the apical shoots (67). The bacteria infect the wounded meristematic tissue and about 5% of shoots emerging from the wound site are transgenic. Another method is to dip Arabidopsis flowers into bacterial suspension, or vacuum infiltrate the bacteria into the flowers, at around the time of fertilization (68,69). This generates plants with transformed and wild-type sectors of vegetative tissue, and these give rise to transgenic progeny at a low frequency (5-10 offspring per plant). Again, the low transformation frequency is acceptable because of the large number of seeds and the ease of screening.
In planta transformation techniques involving direct DNA transfer have also been developed. These techniques have been applied to a number of economically important crop plants, although they have yet to catch on because they have low reproducibility. Generally, these techniques fall into four classes:
1. Introduction of DNA into meristematic tissue, e.g., by electro-poration, injection of DNA/lipofectin complexes, or particle bombardment, followed by the recovery of transgenic shoots—this has been attempted in rice (70).
2. Introduction of DNA into flowers around the time of fertilization, followed by the recovery of transgenic seeds—this has been attempted in cotton (71) and rice (72).
3. Imbibing seeds with a DNA solution—this has been used for soybean (29).
4. Introducing DNA into pollen or mixing pollen with DNA before applying to stigmata—this has been attempted with rice (72) and tobacco (73).
These techniques have met with variable levels of success.
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