Genetic transformation of Labisia Pumila using Agrobacterium Tumefaciens
The present protocol was aimed to establish a routine transformation procedure via Agrobaderium tumefaciens for Labisia puml'la var. pumila. The effect of different factors on T-DNA transfer by measuring transcient expression levels of a disarmed strain lBA 4404 harbouring the binary vector...
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English |
| Published: |
2007
|
| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/19716/1/Genetic%20transformation%20of%20Labisia%20Pumila.pdf https://eprints.ums.edu.my/id/eprint/19716/ |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | The present protocol was aimed to establish a routine transformation procedure
via Agrobaderium tumefaciens for Labisia puml'la var. pumila. The effect of
different factors on T-DNA transfer by measuring transcient expression levels of
a disarmed strain lBA 4404 harbouring the binary vector pBI121 carrying
chimeric glucuronidase (GUS) and neomycin phosphotransferase (NP71l) genes
examined. Parameters optimized were light influence, types of wounding, types
of explant, co-cultivation period, bacterial concentration, shaking influence,
addition of glucose, pH, temperature, addition of acetosyringone and addition of
sucrose. Improved transformation frequencies were btained with an A.
tumefaciens strain carrying kanamycin-type vir genes and when leaves were
infected with Agrobaderium cells in the early log growth phase. Optimized cocultivation
was performed in the presence of 251-1g/1 of kanamycin. Methods
used have expressed up to 77.62% positive transformants. This result were
done using leaf explants co-cultivated for two days using a batch of
Agrobaderium grown until giving the reading of 0.9 absorbance taken at
600nm wavelength. The leaves were poked using sterile needle and co-cultivated
with Agrobacterium at room temperature with the addition of
0.02mg/1 of acetosyringone, 5g/1 of glucose and 15g/1 of sucrose in the pH 7
medium. The mixture was left with 24h lighting and 100rpm aggitation.
Expression and presence of transgene was assayed by histochemical test and
later confirmed with polymerase chain reaction. Seven percent transgenic plants
were micropropagated and successfully acclimatised. The protocol is yet t be
proven t enable it t be reproducible. |
|---|