Methods of Gene Transfer used in Plants

Transformation:

The steps involved are:

(a) Selection of plant tissue or explant

(b) Co-cultivation with Agrobacterium

(c) Inhibition of Agrobacterium growth

(d) Selection of transformed tissue

(e) Regeneration from selected tissue (transgenic plant)

(f) Confirmation of transgenic plant

(a) Selection of plant tissue or explant:

Suitable plant tissue, to be used as a source of explants (which has good regeneration ability), is removed from the donor plant and sterilized (if the plant is not grown in sterile condition). The explants may be decapitated seedlings, cells, protoplasts or leaf tissue, callus, etc.

(b) Co-cultivation with Agrobacterium:

The tissue or explant is cut into small pieces and placed into a culture of Agrobacterium (which contains the suitable vector containing foreign gene) for about 30 min., a process known as co- cultivation. During this period, the bacteria attach to the plant tissue, and the excess culture is blot­ted off and placed on medium for co-cultivation.

(c) Inhibition of Agrobacterium growth:

The incubation of the explants with Agrobacterium is allowed to continue for 2-3 days to permit the transfer of T-DNA to the plant cells. Then the explants are removed from the medium and washed in an antibiotic solution and further trans­ferred onto antibiotic (bacteriostatic) containing medium to inhibit the growth of Agrobacterium.

(d) Selection of transformed plant cells:

The explants are then transferred onto the selective media containing proper selective agent to encourage the growth of transformed tissue.

(e) Regeneration from the transformed tissue:

The selected tissue part (putatively trans­formed), grown or selective media, are then transferred onto the regeneration media for shoot regeneration either by organogenesis or by embryogenesis in presence of proper selective agent. The shoot apices come out, those are then transferred in rooting media to get the whole plant.

(f) Confirmation of the putatively trans­formed plant:

The transgene expression is examined either through foreign protein expres­sion or any phenotypic character expression. The presence of foreign DNA can be examined either through PGR or Dot Blot or Southern Blot experi­ment. The confirmed transgenic plants then are transferred to soil to get the next generation plant. The whole process has been depicted in Fig. 18.24.