II. Sample collection

A. Plant strain
Zhonghua 15 (Oryza stativa L. indica)

B. Starting material
Morphological stages
Material collected

C. Growth conditions
The seedlings from rice strain Zhonghua 15 were grown in a tray filled with soil. The one-month-old seedlings were transplanted into plastic pots and cultivated until ready for sample collecting.

D. Separation technique
Samples for morphological observation were collected from young panicles 2 mm to 6 cm in length. Semi-thin cross sections and scanning electronic microscopy were carried out according to the procedures described in Liang et al. 2003 and Bai et al. 2004. Stamens at stage 2, 3, 5 and 7 were identified using the established morphological traits (see results) and collected with pretreated dissecting needles under a dissecting microscope. Primordia of the 3rd leaf were collected under the dissecting microscope from seeds soaked for 3 days at 25°C. Differentiated leaf samples were collected from the 3rd leaf when it had just fully expended. All tools used in the sample collection were treated with 0.1% DEPC and the dissected samples were immediately removed to the Trizol extraction buffer (Promega, USA) contained in microtubes. To maximally synchronize the samples at individual stages; we generally collected stamen samples from 3-6 florets that shared the greatest identical morphology.

E. Extraction methods

• Quantity extracted:20 stamens were used as sample to extract total RNA
• Extraction source: fresh sample
• Extraction method
  
  
  • Kit:Trizol Reagent
  • Manufacturer:Invitrogen, USA
  • Procedure

Stamen and leaf primordia at various stages in the Trizol extraction buffer were homogenized with Micro Tissue Grinders (Kimble Knontes, USA) in the microtubes. Total RNA was isolated according the manufacturer's protocol except that before adding chloroform, 5 μg LPA was added into 60 μl extract solution as a RNA carrier.

• Amplification method
  
  

RNA isolated using the above procedure was amplified according to methods of Baugh et al (2001) with the following modifications: after the second cDNA strand was synthesized, the double strand cDNA was purified with Microcon YM-100 (Millipore, USA); and after in vitro transcription with a T7 ampliscribe kit (Epicentre, USA) for 4 h at 42°C, antisense RNA (aRNA) was purified by Genelute mammalian total RNA miniprep kit (Sigma, USA) according to the manufacturer's protocol. The quality of amplified aRNA was examined by electrophoresis with a 1% agarose gel (Figure S3).

Procedures in details see the file RNA amplification for small materials