Production method of light-sensitive male sterility rice
A production method and male sterility technology, applied in the field of genetic engineering and rice cultivation, can solve the problems of inability to be randomly matched, hybrid sterility between rice subspecies, etc., and achieve the effect of expanding the scope
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Embodiment 1
[0053] Example 1 Construction of expression vector pCAMBIA1304 / anti-osPDCD
[0054] The full-length cDNA of the rice programmed death gene OsPDC was cloned with primers with restriction sites, and the cDNA and the plant expression vector pCAMBIA1304 were digested with BsmFI and BstEII and ligated, so that OsPDCD was reversely inserted downstream of the 35s promoter. The antisense sequence expression vector pCAMBIA1304 / anti-osPDCD5 of OsPDCD was constructed. refer to figure 1 .
Embodiment 2
[0055] Example 2 Callus Culture and Transformation
[0056] 2.1 Culture medium for inducing rice callus
[0057] 2.1.1 Induction and subculture medium: MS+2mg / L 2,4-D.
[0058] 2.1.2 Hyperosmotic medium: MS+2mg / L 2,4-D+46.67g / L sorbitol+46.67g / L mannitol.
[0059] 2.1.3 The first round of screening medium: MS+2mg / L 2,4-D+30mg / L hygromycin.
[0060] 2.1.4 The second round of screening medium: MS+2mg / L 2,4-D+50mg / L hygromycin.
[0061] 2.1.5 Differentiation medium: MS+3mg / L 6-BA+0.5mg / L NAA+50mg / L hygromycin.
[0062] 2.1.6 Rooting and strong seedling medium: 1 / 2 MS+0.1mg / L NAA
[0063] Note: 1. The above media all contain 30g / L sucrose + 2.5g / L agar, pH5.8
[0064] 2. The culture conditions for callus induction, subculture, and screening are dark culture at 26-28°C, and 26-28°C and 16-hour photoperiod for differentiation and rooting of seedlings.
[0065] 2.2 Callus induction and treatment
[0066] 2.2.1 Take immature seeds 12-15 days after pollination, soak them with 70...
Embodiment 3
[0088] Example 3 Screening and detection of rice anti-osPDCD gene transformed plants
[0089] The surviving calli were cultured on the differentiation medium for 30 days under light. After the small plants are differentiated, the small plants are transferred to the medium for rooting and strong seedlings, and then moved to the greenhouse after growing up. The candidate resistant plants were detected by PCR amplification and genomic Southern hybridization respectively, and a total of 7 positive plants containing anti-osPDCD were obtained.
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