A highly efficient method for genetic transformation of Dendrobium officinale mediated by Agrobacterium
A genetic transformation method and Agrobacterium-mediated technology, applied in the field of Agrobacterium-mediated genetic transformation of Dendrobium candidum, can solve the problems of low transformation efficiency and imperfect genetic transformation system of Dendrobium candidum, and achieve improved genetic transformation efficiency and enhanced The effect of sensitivity
Active Publication Date: 2020-08-25
SICHUAN AGRI UNIV
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Problems solved by technology
[0004] Aiming at the above-mentioned problems existing in the prior art, the present invention provides an efficient Agrobacterium-mediated genetic transformation method of Dendrobium officinale, which can effectively solve the problem of the incomplete genetic transformation system of Dendrobium officinale mediated by Agrobacterium in the prior art and the low transformation efficiency. high problem
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Embodiment 1
[0035] A highly efficient Agrobacterium-mediated genetic transformation method for Dendrobium officinale comprises the following steps:
[0036] (1) Activation culture of Agrobacterium
[0037] Pick -80°C Agrobacterium transformants in 5ml liquid medium, place them at 28°C, 200r / min, and activate them. bottle), placed in 28°C and 200r / min to expand the culture until OD600=0.8, pour it into a 50ml centrifuge tube, centrifuge at 4000r / min for 10min, discard the supernatant, and collect the bacteria; among them, the liquid Medium includes: 1% yeast extract, 1% tryptone, 0.5% sodium chloride, 20mg / L rifampicin, 50mg / L kanamycin and 50mg / L gentamicin;
[0038] (2) Preparation of liquid dye
[0039] Add the bacterial cells obtained in step (1) into MS liquid medium, place them on ice to completely dissolve the bacterial cells in the medium, then adjust the bacterial liquid to OD600=0.4, 0.6, 0.8 and 1.0 with MS liquid medium, and then Add 100 μmol / L acetosyringone (AS) and surfac...
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The invention provides an efficient genetic transformation method of agrobacterium-mediated dendrobium candidum. The efficient genetic transformation method comprises the following steps: (1) performing activating culture on agrobacteria; (2) performing a dip dyeing liquid: adding the agrobacteria obtained in the step (1) into an MS liquid culture medium, putting on ice so as to enable the agrobacteria to be completely dissolved in the culture medium, adjusting bacterial liquid by using the MS liquid culture medium till OD600 is 0.4-0.8, then adding 100[mu]mol / L of acetosyringone(AS) and 0.001-0.01% of Triton-100, and uniformly shaking; (3) performing ultrasonic treatment and dip dyeing: under the conditions of 280-320W and 36-45KHz, performing ultrasonic treatment for 1-3min; (4) performing co-culture; (5) screening; (6) performing plant regeneration. A transformation system is complete; the survival rate of protocorms of dendrobium candidum is high; through optimization of the steps and parameters, plants with excellent resistance are obtained through transformation; the transformation rate is high, and the positive transformation rate is as high as 69.7%.
Description
technical field [0001] The invention belongs to the technical field of plant transgenesis, and in particular relates to an efficient Agrobacterium-mediated genetic transformation method of dendrobium officinale. Background technique [0002] Dendrobium candidum, as an important traditional Chinese medicinal material with both ornamental and medicinal value, is mainly distributed in Yunnan, Guizhou, Zhejiang, Sichuan and other places in my country. It contains multiple biologically active components such as dendrobium polysaccharides, dendrobine, amino acids, etc. It is sweet in taste and slightly cold in nature. However, due to its special requirements for the growth environment, it mostly grows in high-temperature and humid tropical and subtropical high mountains and cliffs, with slow growth and extremely low self-reproductive ability. Under the destruction of people's long-term predatory mining and living conditions, wild resources are on the verge of extinction, and the ...
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IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/62
CPCA01H4/00C12N15/8205
Inventor 田孟良陈稷王丽刘晨黄进刘帆梁欢
Owner SICHUAN AGRI UNIV