The characterization of the process from floral meristem formation to floral organ primordia for-
mation is an important part of the analysis of flower development. However, very little is known
about the mechanisms controlling how cell fates are determined in the floral meristem. Cell fate
selections include whether to form an organ pri- mordium, which type of organ to differentiate
into, and when to terminate cell proliferative and organogenic activities. Because cell fate selection
in the inflorescence and floral meristems is likely to be a very complex process, it is also likely that
many genes are involved in this process. Analysis of novel floral regulatory genes will provide new
insights into flower development. Much of the studies have been focused on dicot species. In
order to understand flower development in cereal plant species, we have cloned and characterized a
receptor kinase gene, OsLRK1, from rice
2. Materials and methods
2
.
1
. Bacterial strains Escherichia coli JM 83 was used as the recipient
for routine cloning experiments. Agrobacterium tumefaciens LBA4404 [13] containing the Ach5
chromosomal background and a disarmed helper- Ti plasmid pAL4404 was used for transformation
of rice. The f1 helper phage R408 and E. coli strain XL-1Blue were used for in vivo excision of
the pBluescript plasmid vector from the lZAPII phage Stratagene, CA.
2
.
2
. Screening of cDNA library and sequence analysis
A partial clone of the EST expressed sequence tag C22553 GenBank accession number was
isolated by PCR polymerase chain reaction us- ing
the primers,
5-GAGCTTCTCCGGT- GTTCAG-3
and 5-GATGTGGCATTGAAGTAGCT-3. A cDNA
library was constructed from mRNA prepared from young panicles length under 2 cm. Hy-
bridization was performed with 320 000 plaques using the [a-
32
P] dCTP labeled probe of the par- tial cDNA clone of OsLRK1 by the random
priming method [14]. The nucleotide sequence was determined using an automated sequencer
ABI 373A. DNA sequence comparison was per- formed using the DDBJ DNA data bank of
Japan database.
2
.
3
. RNA isolation and RNA gel blot analysis Total RNA was extracted from various tissues
of rice plants using the RNA isolation kit Tri reagent, Molecular Research Center, INC. Elec-
trophoresis of total RNA 10 mg was carried out in a 1.2 vv formaldehyde – agarose gel as de-
scribed previously [14]. The RNA was blotted onto a nylon membrane Hybond-N + , Amer-
sham and hybridized in a solution containing 0.5 M sodium phosphate pH 7.2, 1 mM EDTA, 1
BSA, and 7 SDS for 20 h at 55°C [15]. After hybridization, the blot was washed twice with a
solution containing 0.1 × SSPE and 0.1 SDS for 5 min at room temperature, followed by two
washes of the same solution at 55°C for 20 min. Hybridization was performed with the [a-
32
P] dCTP labeled probe of the partial cDNA clone of
OsLRK1 by the random priming method [14].
2
.
4
. Construction of binary 6ectors A binary vector, pGA1611, that can be used
for transformation of rice plants was constructed. This vector, a derivative of pGA482 [16], contains
the hygromycin phosphotransferase hph gene as a selectable marker under the control of the
cauliflower mosaic virus 35S promoter followed by the termination region of the 7 gene of pTiA6.
The vector also contains several unique sites HindIII, SacI, HpaI, and KpnI between the
maize ubiquitin promoter, including the first in- tron of the ubiquitin gene [17], and the nopaline
synthase nos terminator. Therefore, this vector can be used for expression of a foreign gene into
monocot plants when transferred by the Agrobac- terium co-cultivation method. The partial cDNA
clone of OsLRK1 was inserted into the multiple cloning sites in an antisense orientation, con-
structing pGA2152.
2
.
5
. Rice transformation A japonica rice variety, Dongjin, was used for
transformation by the Agrobacterium co-cultiva- tion method as described previously with the fol-
lowing modification [18,19]. Calli were induced from the scutellum of mature seeds on an N6
medium containing 2 mgl 2,4-D. A. tumefaciens strain LBA4404 carrying the pGA2152 plasmid was
grown for 3 days in an AB liquid medium supple- mented with 15 mgl hygromycin and 3 mgl tetra-
cycline. Three-week-old calli were co-cultivated with the Agrobacterium on a 2N6-As medium
supplemented with 100 mM betaine for 2 – 3 days in darkness at 25°C. The co-cultivated calli were
washed with sterile water containing 100 mgl cefotaxime and incubated on an N6 medium con-
taining 40 mgl hygromycin and 250 mgl cefo- taxime for 3 weeks. Actively growing calli were
transferred onto a regeneration medium, MS medium supplemented with 0.1 mgl NAA, 2 mgl
kinetin, 2 sorbitol, 1.6 phytagar Sigma, 50 mgl hygromcyin, and 250 mgl cefotaxime. After
2 – 3 weeks under continuous light 40 mmolm per s, the plantlets were transferred to soil and grown
in a growth chamber with 10 h light per day.
3. Results
