Plant materials: The mature embryos, immature embryos and inflorescences of eight Chinese spring wheat cultivars or lines were used to evaluate the competence of callus initiation. All materials were randomly planted in experimental field in 2002. Chuannong 16, Chuanyu 16, Chuanmai 32, Chuanyu 12 and 80-8 are elite wheat cultivars, and the remains are excellent breeding lines.

The immature inflorescences with 1.0 to 2.0 cm and the early-medium milk-stage young grains 12-16 days after anthesis were harvested. Mature and young grains, and immature inflorescences were surface-sterilized firstly in 70% ethanol for 1 mm, then 10 min in 0.1% mercuric chloride (HgCI₂), and followed by three 5-min in sterile distilled water. Mature and immature embryos were aseptically dissected from mature and young grains, respectively, and then placed on media with the embryo-axis facing upward. Immature inflorescences were cut into small pieces of 1-2 mm and placed on media. The explants were cultured in dark at 25°C about 10 days until the calli grow to 10 mm, and then transferred to differentiation medium at 26°C, 16-h photoperiod to induce plant regeneration.

Culture media: The medium compositions were listed in Table 1. Among these, 6 media (No.1 to No.6) were used for callus induction from immature embryo. MS₉ was used for callus maintenance. Two media with different content of auxins, MS₁₀ and MS_10-1, were used in plantlet differentiation and regeneration. MS₁₁ and Ms_11-1 with different concentration of phytohormone 1-naphthylacetic acid (NAA) were used to induce the root initiation. All media were autoclaved for 20 min at 121°C and 0.11 MPa. Plasmids construct: The transformation vector pDM803 was used for microprojectile bombardment. The schematic structure of pDM803 was shown in Fig.1. pDM8O3 contains a chimaeric bar gene from Streptomyces hygroscopicus (Thompson et al.1987) under control of the maize Ubi1 promoter (Christensen et al. 1992) with Agrobacterium tumifaciens nopaline synthase (nos) terminator (Bevan et al. 1983) and the E.coli uidA (β- glucuronidase) gene (Jefferson et al. 1987) under control of the rice Act1 promoter (McElroy et al. 1991) with 3' transcript termination region of rice rubisco gene terminater (rbcS-TER). One enhancer was, respectively, inserted at the upstream of β - glucuronidase and bar to enhance their expression. Particle bombardment: For each bombardment, 40 to 50 scutella were placed in the center of a 9 cm diameter Petri dish containing MS-based induction medium with 1 mg/I 2,4-D. Explants were cultured in dark at 25°C for 10 day prior to bombardment. Plasmid DNA was precipitated onto 1 mm diameter gold particles immediately prior to bombardment as detailed in Sanford et al. (1987). Bombardments were carried out using a PDS1000/He particle gun (Bio-Rad) at an acceleration pressure of 1,100 or 1,350 psi and a distance of 9cm from the stopping plate. After bombardment, explants were spread over the surface of the original medium and cultured at 25°C in darkness for 3 weeks to induce embryogenesis. Histochemical GUS analysis: A histochemical GUS assay was conducted according to Jefferson et al. (1987). Immature embryos and calli were immersed in a 0.1 M phosphate buffer solution, pH 7.2, containing 1 mg/ml 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) at 37°C for 24 h in dark, subsequently washed in 70% alcohol and observed under microscope.
Statistical analysis: A completely randomized design was used to estimate the culture responses of different explants. The frequencies of induced-callus were investigated when explants had been cultured on the callus induction medium for four weeks. The effects of genotypes on culture responses were determined by variance analysis and shortest significant ranges tests (Rong 1993).