Method of establishing early-maturing ripe hereditary transform system and application
A systematic and early-maturing technology, applied in the field of agricultural biology and plant genetic engineering, can solve problems that affect the development of bluegrass transgenic work, low differentiation frequency, difficult subculture of embryogenic callus, etc.
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Embodiment 1
[0064] Embodiment 1: Induction and subculture of clustered buds: get the seeds of the bluegrass variety prize and sterilize with 70% alcohol for 1-2 minutes, 0.2% mercuric chloride (mercuric chloride) for 10 minutes, wash with sterile water for 4 The second time, the seeds were germinated on wet sterile filter paper, and the shoot tips were cut out and placed in the induction medium (adding 2,4-D 0.2mgL -1 , 6-BA 2mgL -1 ) to induce base expansion, and the expansion rate was 100%. After culturing on the induction medium for 20 days, excise the enlarged tissue (diameter 1-3mm) at the base of the shoot tip and transfer it to the subculture and clustering medium (take 2,4-D 0.1mgL -1 , 6-BA 2mgL -1 ) to induce clustered buds to occur, and after 15-20 days, most of the expanded bases differentiated clustered buds, the clustering rate was about 75%, and the number of clustered buds was mostly 15-20. After dividing the clumping bud block, transfer it to the subculture and clumpin...
Embodiment 2
[0068] Cluster bud induction and subculture: the procedure is the same as in Example 1, but the bluegrass variety is Xingeriad. The hormone combination in the selected subculture and clustering medium is 2,4-D 0.07mgL -1 , 6-BA 3mgL -1 .
[0069] Determination of the concentration of the screening agent: the screening agent is hygromycin. After hygromycin aqueous solution was filtered and sterilized, it was added to the induction medium at concentrations of 0, 2, 4, 6, 8, 10, 12, and 14 mgL -1 , that is, 8 gradient concentrations. Screening procedures and methods are the same as in Example 1. According to the survival rate after screening, determine the hygromycin selection concentration of Xingelaide seedlings as 10mgL -1 . At this concentration, the seedling survival rate is about 3%.
[0070] Gene gun bombardment transforms clustered buds:
[0071] The plasmid is pCAMBIA1300-hpt-antiport. Standard procedures and methods were used for extraction and purification (se...
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