Generation, molecular characterization
and evaluation of a genome wide library of insertion lines in rice cv. Nipponbare
We have generated an insertion
line library through Agrobacterium-mediated transformation
in the model cereal rice, cv. Nipponbare- the standard genotype of
IRGSP. The overall scheme of production and further molecular and
phenotypic characterizations of the insertion lines is illustrated
in Figure 1. A highly efficient
Agrobacterium-mediated transformation procedure (Figure
2) allowed the production of more than 46,000 primary transformants
(T0) with an average efficiency of four independent transformation
events per co-cultured callus in seven different experiments (Table 2) conducted from January 2001 to June 2003.
(For more information about the transformation technique
see protocols or refer to Sallaud
et al 2003 Theor Appl Genet, 106:1396-1408)
The library now encompasses lines harbouring a T-DNA equipped with a gusA enhancer trap
(pC-4978), a gusA enhancer trap and a Ds element (pC-4984)
and a gal4:gfp enhancer trap (pC-4956ET15) respectively (Figure
3 ). Each line contains an average of 2.2 copies of the T-DNA
at 1.4 loci (Figure 4) and of 3.2 new copies of the retrotransposon Tos17(Figure 5). The collection eventually
totalizes more than 200,000 inserts, ensuring a good coverage of the
rice genome and the possibility of creating additional inserts in
specific chromosomal regions using Ds launching pads.
(For recent results download pdf of a poster presented at Plant Gems 3, Lyon)
Phenotypic evaluation of the library
Large scale screenings of the progeny lines for presence of GUS/GFP,
grain filling, response to Magnaporthe inoculation and morphological/physiological
changes are conducted under parallel projects. Mutant phenotypes,
excluding pigmentation alterations and segregating in a Mendelian
manner, are detected in 1-3% of the lines per target trait in the
3 independent screens, which already included more than 7,000 lines.
Parallel seed increase and evaluation of 15,000 lines has already been conducted under
field conditions in Colombia in a collaborative effort with CIAT.
