Lily transgenic method for deleting selectable marker genes
A technology of selection markers and transgenes, applied in the field of plant genetic engineering, can solve the problems of lack of strict control of the deletion system, lower transformation efficiency, failure to find the best conditions, etc., and achieve the effect of eliminating the biosafety problems of transgenic plants
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Embodiment 1
[0048] The determination of the most suitable differentiation medium of embodiment 1
[0049] (1) Prepare medium: in MS basic medium + 3% sucrose + 0.6% agar, and add NAA (0.1, 0.2 and 0.3mg L -1 )+TDZ (0.002, 0.005 and 0.01mg·L -1 ) total of 9 culture media in different combinations, pH=5.8, packed in 100ml Erlenmeyer flasks, the volume of culture medium in each Erlenmeyer flask is about 30ml;
[0050] (2) Peel off the healthy, lesions-free, and bright complete lily 'Sorbonne' scales, wash them with running water for more than 30 minutes, then soak them in 75% alcohol for 30 seconds under sterile conditions, wash them with sterile water for 3 times, and then use Soak in 0.1% mercuric chloride solution for 10-15 minutes, rinse with sterile water 3 times, 5 minutes each time;
[0051] (3) Under sterile conditions, use a scalpel to cut scales about 1.0cm×1.5cm in size on a plate covered with filter paper, and inoculate the cut scales with the adaxial side up into MS basic medi...
Embodiment 2
[0057] Embodiment 2 Biolistic co-transformation
[0058] 1) Prepare culture medium. Add NAA 0.2mg·L to MS basic medium -1 +TDZ0.005mg·L -1 + 3% sucrose + 0.6% agar, pH=5.8, put it in a sterile petri dish with a diameter of 9cm, and the volume of the culture medium in each petri dish is about 20ml;
[0059] 2) strip off the outer scales of the aseptic lily bulblets obtained in Example 1, cut out the base of the small scales about 2 to 5 mm wide with a scalpel on a plate covered with filter paper under aseptic conditions, and place them on the cultured In the center of the dish, about 100 pieces per dish were arranged in a circle with a diameter of about 2-3 cm, and cultured in the dark for 2 days, 4 days and 6 days.
[0060] 3) The plasmid Pksb-rd29A-Cre / lox plant expression vector containing the Cre / loxp system vector (Shi Jun et al., 2009) and the drought-resistant and salt-tolerant gene vector plasmid pBPC-P5CS-F129A (Li Zhiliang et al., 2006) were mixed in a ratio of 1:1...
Embodiment 3
[0067] Example 3 Molecular detection of transgenic plants
[0068] Transformation was carried out according to the preferred transformation system established in Example 1 and Example 2. A total of 1200 explants were tested, and 25 resistant seedlings were obtained. Molecular detection was carried out on the obtained resistant seedlings. The specific steps are:
[0069] (1) Genomic DNA is extracted from the obtained resistant plants using the TIANGEN Plant Genomic DNA Extraction Kit;
[0070] (2) According to the sequence of Cre gene and bar gene, primers were designed for PCR detection. The primer sequences, reaction system and reaction conditions are as follows:
[0071] Cre gene upstream primer (5'-CATTACCGGTCGATGCAACG-3') and Cre gene downstream primer (5'-CTAATCGCCATCTTCCAG-3'). 50 μl reaction system: genomic DNA (5 μl), 10×PCR buffer (5 μl), 2.5 μM dNTP (4 μl), 10 μM upstream primer (2 μl), 10 μM downstream primer (2 μl), Ex Taq enzyme (0.5 μl) and double distilled I...
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