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Methods and compositions for rapid plant transformation

A plant, transgenic plant technology, applied in the field of plant molecular biology, can solve negative problems, inefficiency, and impact on the development timetable of genetically modified products, etc.

Active Publication Date: 2018-08-03
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, using standard transformation and regeneration protocols is time-consuming and inefficient, and negatively affects transgenic product development timelines, given that there is often a seasonally limited "priority development window" for The results determine which genetic constructs are prioritized for larger field work
Available standard methods of transformation and regeneration have a number of disadvantages that limit the speed and efficiency with which transgenic plants can be produced and screened

Method used

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  • Methods and compositions for rapid plant transformation
  • Methods and compositions for rapid plant transformation
  • Methods and compositions for rapid plant transformation

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0256] Example 1: Plasmids.

[0257] Plasmids containing the T-DNA described in Table 1 were used in the experiments described below. The plasmids listed in Table 1 have T-DNAs containing the indicated components.

[0258] Table 1. Plasmid components.

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

example 2

[0265] Example 2: Culture medium.

[0266] Various media are cited in the examples for transformation and cell culture. Media compositions are as described in Tables 2-9 below.

[0267] Table 2. Media used for sorghum transformation.

[0268]

[0269]

[0270] a PHI-I, PHI-T, PHI-U, PHI-V, PHI-X and PHI-Z media from Zhao et al. 2000

[0271] b MS vitamin stock solution: 0.1g / l niacin, 0.1g / l pyridoxine hydrochloride, 0.02g / l thiamine HCl, 0.4g / l glycine.

[0272] Table 3. Composition of Wheat Liquid Infection Medium WI 4.

[0273]

[0274] Table 4. Composition of wheat co-culture medium WC#10.

[0275]

[0276]

[0277] Table 5. Composition of wheat green tissue culture medium DBC4.

[0278]

[0279] Table 6. Composition of wheat green tissue induction medium DBC6.

[0280]

[0281]

[0282] Table 7. Composition of wheat regeneration medium MSA.

[0283]

[0284] Table 8. Composition of wheat regeneration medium MSB.

[0285]

[0286] ...

example 3

[0293] Example 3: Particle bombardment.

[0294] Before bombardment, 10-12 DAP immature embryos were isolated from ears of the pioneer inbred line PH184C and placed on medium containing 16% sucrose for 3 hours to separate the scutellum cytoplasm.

[0295] Four plasmids are typically used per particle bombardment; 1) a donor plasmid (100 ng / μl) containing the FRT flanking donor cassette for recombinase-mediated cassette exchange, 2) containing the expression cassette UBI PRO::FLPm:: A plasmid for PinII (2.5 ng / μl), 3) a plasmid for expression cassette UBI PRO::ODP2::PinII (10 ng / μl), and 4) a plasmid for expression cassette UBI::WUS2::PinII (5 ng / μl ). For DNA attachment to 0.6 μm gold particles, the four plasmids were mixed by adding together 10 μl of each plasmid (40 μl total) in a low-binding microcentrifuge tube (Sorenson Biosciences 39640T). To this suspension, 50 μl of 0.6 μm gold particles (30 μg / μl) and 1.0 μl of Transit 20 / 20 (Cat. No. MIR5404, Mirus Bio LLC) were ...

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Abstract

The disclosure pertains to methods and compositions for the rapid and efficient transformation of plants. The disclosure further provides methods for producing a transgenic plant, comprising (a) transforming a cell of an explant with an expression construct comprising (i) a nucleotide sequence encoding a WUS / WOX homeobox polypeptide; (ii) a nucleotide sequence encoding a polypeptide comprising twoAP2-DNA binding domains; or (iii) a combination of (i) and (ii); and (b) allowing expression of the polypeptide of (a) in each transformed cell to form a regenerable plant structure in the absence ofexogenous cytokinin, wherein no callus is formed; and (c) germinating the regenerable plant structure to form the transgenic plant. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

Description

technical field [0001] The present disclosure relates generally to the field of plant molecular biology, including the genetic manipulation of plants. More specifically, the present disclosure relates to rapid, efficient methods and compositions for producing transformed plants in the absence of cytokinins and without callus formation. [0002] Cross References to Related Applications [0003] This application claims priority to US Provisional Application No. 62 / 248578, filed October 30, 2015, which is hereby incorporated by reference in its entirety. [0004] References to Sequence Listings submitted as text files via EFS-Web [0005] An official copy of this Sequence Listing is submitted electronically via EFS-Web as a Sequence Listing in ASCII format, the Sequence Listing filename is "20160826_6752WOPCT_SeqList.txt", created on August 17, 2016, and has a size of 1000KB and is compatible with this The instruction manual is submitted at the same time. The Sequence Listing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C07K14/415
CPCC12N15/8201C12N15/821C07K14/415Y02A40/146A01H4/008C12N15/8213C12N15/8216
Inventor A.阿南德M.L.阿林格A.达斯瓦康塞曹W.J.戈当-卡姆C.哈斯廷格斯G.J.霍尔斯特T.M.克莱恩C.M.拉罗塔K.S.洛维S.B.蒂瓦里王宁X.E.吴
Owner PIONEER HI BRED INT INC
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