Method for producing male sterile plants
一种雄性不育、植物的技术,应用在植物学设备和方法、生物化学设备和方法、植物产品等方向,能够解决破坏程序性细胞死亡、延迟等问题
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Embodiment 1
[0090] Isolation of encoding from Arabidopsis BECLIN 1 / ATG cDNA of 6 genes
[0091] Plant material: The Columbia ecotype (Col-O) of Arabidopsis thaliana was used in the experiments described here. Plants were grown on a complex soil mixture of vermiculite / sphagnum moss / perlite (1:1:1) in a growth chamber with a 16 h light / 8 h dark light cycle and a 23 °C day / 18 °C night temperature cycle .
[0092] Artificial induction of autophagy: The third and fourth rosette leaves of 3-week-old plants were isolated and floated on deionized water in a Petri dish, adaxial side up. The leaves were incubated for 48 hours at 22±1°C in the dark.
[0093] plant BECLIN 1 / ATG6 Cloning of genes: Total RNA was extracted from autophagy-induced Arabidopsis leaves using TRI reagent (Sigma). Using NanoDrop ? ND-1000 UV-Vis spectrophotometer measures the amount of total RNA. RNA quality was checked by visualizing rRNA in ethidium bromide-stained agarose gels under UV light. 10 µg of total R...
Embodiment 2
[0095] Tapetum-specific promoters and plants BECLIN 1 / ATG Construction of chimeric gene fusions between 6
[0096] In early stages, the tapetum-specific 1 kb BamH1 / Xba1 promoter associates with the 1.5 kb Xba1 / Sac1 plant BECLIN1Gene and 250 bp Sac1 / EcoR1 Nos terminator fused in BamH1 / EcoR1 Sk+ cloning vector.
[0097] The entire expression cassette containing the fragment BamH1 / EcoR1 was further subcloned into the binary vector pBI101. The resulting pBI101 carrying the expression cassette was introduced into Agrobacterium tumefaciens strain LBA4404 according to a modified protocol (Cangelosi et al., 1991).
Embodiment 3
[0099] Transformation of Tobacco Plants
[0100] As described in Example 2, Petit Havana tobacco ( Nicotina tabacum cv. Petit Havana ), using the protocol described by Horsch et al. in 1985.
[0101] Briefly, a single isolated colony of Agrobacterium tumefaciens LBA 4404 containing the binary vector with the expression cassette described above was inoculated into a drug containing the antibiotics streptomycin (250 mg / ml), rifampicin (50 mg / ml) and kana Mycin (100mg / ml) in YEP medium and allowed to grow (200 rpm, overnight, 28°C). 50 μl of overnight culture was diluted to 100 ml in YEP medium and allowed to grow to OD 600 reached 0.8. Cells were recovered by centrifugation in an SS34 rotator (5,000 rpm, 10 min, 4°C). Suspend the pellet to OD in co-cultivation medium (MS salts, 2% glucose, 10 mM MES and 100 mM acetosyrengone, pH 5.6) 600 0.6. Tobacco leaf discs were co-cultured with Agrobacterium tumefaciens for 2 days in the dark. After co-cultivation, the leaf disc...
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