|Commenced in January 1999 || Frequency: Monthly || Edition: International|| Paper Count: 1 |
Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering
Expression of Leucaena Leucocephala de Wit Chitinase in Transgenic Koshihikari Rice
The cDNA encoding the 326 amino acids of a Class I
basic chitinase gene from Leucaena leucocephala de Wit (KB3,
Genbank accession: AAM49597) was cloned under the control of
CaMV35S promoter in pCAMBIA 1300 and transferred to
Koshihikari. Calli of Koshihikari rice was transformed with
agrobacterium with this construct expressing the chitinase and β-
glucouronidase (GUS). The frequencies of calli 90 % has been
obtained from rice seedlings cultured on NB medium. The high
regeneration frequencies, 74% was obtained from calli cultured on
regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at
25°C. Various factors were studied in order to establish a procedure
for the transformation of Koshihikari Agrobacterium tumefaciens.
Supplementation of 50 mM acetosyringone to the medium during
coculivation was important to enhance the frequency to transient
transformation. The 4 week-old scutellum-derived calli were
excellent starting materials. Selection medium based on NB medium
supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were
an optimized medium for selection of transformed rice calli. The
percentage of transformation 70 was obtained. Recombinant calli and
regenerated rice plants were checked the expression of chitinase and
gus by PCR, northern blot gel, southern blot gel, and gus assay.
Chitinase and gus were expressed in all parts of recombinant rice.
The rice line expressing the KB3 chiitnase was more resistant to the
blast fungus Fusarium monoliforme than control line.