The transformation process involved the insertion
of the keratin gene product into the cactus' genome.
The cactus seeds were sterilized with 50% bleach
and 0.1% tween (detergent) solution, preventing
growths of microorganisms whilst in culture. The
surfaced sterilized seeds were placed in a petri
dish with nutrient medium (murashige and skoogs)
for germination.
A week later, the explants were cut and are pre-conditioned
in feeder plate, which is composed of an MS/agar
plate with a layer of the keratin gene product
suspension callus on the agar. The filter disk
used separated the explants from the feeder layer
of cells.
The feeder layer produced compounds that enthused
the Agrobacterium to transmit the DNA and help
condition the explants.
The explants were cut from seedlings (in liquid
MS) and the wounded cells created by cutting,
were the site of the DNA transfer from the Agrobacterium.
After overnight preconditioning on the feeder
plates, the explants were capable for transformation
and were exposed to the agrobacterium.
Additional Agrobacterium was blotted from the
explants on filter paper avoiding the excess development
of bacteria during co-cultivation.
The explants were co-cultivated with the agrobacterium
for 48hrs; the Agrobacterium transferred the engineered
DNA into the wounded cells. The explants were
placed in a medium containing antibiotics for
selection control of the Agrobacterium. (medium
also contains the growth regulator, zeatin riboside.)
The antibiotics and zeatin riboside are heat sensitive
and were filter sterilized then added to the media
after the media has been sterilized by autoclaving.
The MSZ (MS plates containing zeatin riboside)
were poured and allowed to dry overnight. Kanamycin
resistance was selectable marker used enabling
the selection against the wild type cells.
The zeatin riboside stimulated the transformed
cells to regenerate. The explants were placed
on MSZ plates containing kanamycin and returned
to the incubator.
The formations of the initial callus were seen
on the explants. The callus occurred where the
wounding were made and were the results of the
stimulated plant cell growth caused by the growth
regulator (zeatin riboside). Six weeks later,
the explants were cut from the calli and discarded.
Shoots had began to form and were transferred
to fresh MSZ media.
The cactus shoots were regenerated from the calli
and were then ready to be transferred to the rooting
media. This media lacked zeatin but contained
kanamycin for the selection of transformants.
Fully differentiated transgenic cactus plant rooted
in MS media with kanamycin. The rooted plantlets
were then transferred to container comprising
soil. The transgenic plantlets were then acclimated
to the air (this process took around four to five
days).
The transgenic plants have taken part in exhibitions
last summer.
