Analysis of Genes for Stigma Coloration in Rice - IRRI books

µg DNA/10 6 protoplasts, and the plasmid-treated protoplasts were embedded in
KPR agarose medium and cultured at 28 °C in the dark.
• Detection of ß-glucuronidase activity in rice tissues. According to Jefferson's
(1987) method the callus and plants derived from plasmid-treated protoplasts
were extracted in the extraction buffer and reacted with 4-methyl umbelliferyl
glucuronide (Sigma M-9130). Then fluorescence of the reaction product was
measured by fluorimeter under a 360-nm excitation spectrum and a 452-nm
emission spectrum. The standard curve of µmoles 4-methyl umbelliferone
(4Mu) (Sigma 1508) against fluorescence density was made under the same
conditions. The corresponding product produced in the reaction buffer (that is ß-
glucuronidase [GUS] activity of the specimen) was found from the standard
curve.
• Regeneration of the transformed rice protoplasts. The protoplast-derived
microcolonies grew to 1 mm in diameter and were transferred into differentiation
medium (Abdullah et al 1986). One month later, shoots and roots came out, and
the setups were transferred into the light. When the plantlets were 10 cm high,
they were transferred to normal soil. The plants grew well if the temperature was
kept at 28-32 °C and the relative humidity at 60-70%, and if the proper fertilizers
were supplied.
• Confirmation of transgenic rice plants. Transgenic rice plants were confirmed
by Southern (1975) blot. When the plants grew up, 1.5 g of leaves was used for
DNA extraction according to the method of Dellaporta et a1 (1983). The plant
DNA was digested with restriction enzyme Hin d III. The 3.0-kb Hin d III fragment
of the endotoxin protein gene was labeled with 32 P as a probe. DNA hybridization
was done by the Southern method.
Results and discussion
We transferred the B. thuringiensis insecticidal crystal protein gene into rice protoplasts
and successfully obtained transgenic rice plants.
Isolation and culture of rice protoplasts
The rice protoplasts isolated from cell suspension culture (Fig. 2a) numbered 5 × l0 7 /
g fresh cells. More than 90% of the protoplasts were viable, as shown by staining with
FDA. The purified protoplasts were embedded in KPR agarose medium. The first
division was observed after 3-5 d of isolation (Fig. 2b). The second division of most
cells was observed on the 7th to 10th day of isolation (Fig. 2c). Plating efficiency was
about 1%.
Transformation of d -endotoxin protein gene from B. thuringiensis into rice
protoplasts
Recombinant plasmids pGY61 and pGYCK63 were transferred into rice protoplasts
separately. pGY61 contained the complete d -endotoxin protein gene fragment (3.8 kb),
Transgenic rice plants 607