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Method for the generation and cultivation of a plant cell pack

A plant cell and growth medium technology, applied in the fields of botany equipment and methods, plant cells, biochemical equipment and methods, etc., can solve the problems of bacterial overgrowth, low transformation efficiency and high production cost

Active Publication Date: 2014-10-08
FRAUNHOFER GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG EV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, the co-fermentation method disclosed in US 6740526B1 suffers from low transformation efficiency and concomitant bacterial overgrowth, although leaf-based systems achieve high transformation efficiencies, however, encounter problems in scaling up, with initial biomass production low space-time yields and rely on the production of plant biomass under controlled but not sterile conditions
The high cost of production compared to microbial systems is the main reason why these systems have not received widespread attention and use as production systems for biological products

Method used

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  • Method for the generation and cultivation of a plant cell pack
  • Method for the generation and cultivation of a plant cell pack
  • Method for the generation and cultivation of a plant cell pack

Examples

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Embodiment 1

[0132] Transient expression in suspensions compared to transient expression in cell packs

[0133] To evaluate the production of transient recombinant proteins in the cell packs of the present invention, the transient expression of DsRed and antibody 2G12 in Agrobacterium-infiltrated cell packs was compared with the prior art method of co-cultivating cell suspensions with Agrobacterium in liquid culture Compare.

[0134] Using recombinant Agrobacterium (strain GV3101::pMP90RK), which carries the expression cassettes for the heavy and light chains of a human antibody (2G12) and for plastid-targeting red fluorescent protein (DsRed) on the same T-DNA ) binary vector pTRAp-2G12FER-Ds of the expression cassette. Both 2G12 genes contain the KDEL sequence for ER-retaining antibodies ( figure 2 B). This KDEL sequence was deliberately used to avoid antibody secretion in order to directly compare the productivity of cell packs and suspension cells.

[0135] Agrobacterium strains fo...

Embodiment 2

[0144] Transient Expression of Plasmodium falciparum Antigens in Cell Packs

[0145] To test whether cell stacks can be used to produce malaria antigens, different proteins of P. falciparum were transiently produced.

[0146] Recombinant Agrobacterium strains containing a binary vector with an expression cassette for an ER-retained carboxy-terminal fragment of Msp1 (p38, p33 and p19) from Plasmodium falciparum and a plastid-targeted DsRed Two expression boxes ( Figure 4 ). Bacteria were grown and prepared as described in Example 1. Agrobacterium infiltration suspensions were serially diluted from OD1 to OD0.0625 before infecting plant cells.

[0147] Three day old BY-2 suspension cultures were used to grow cell packs as described in Example 1. The cell stack was cut into approximately 5 mm × 5 mm × 10 mm slices ( Figure 7 A). Six slices were transferred to Petri dishes and infiltrated dropwise with Agrobacterium suspensions of different optical densities to saturation ...

Embodiment 3

[0151] Using cell stacks for screening applications

[0152] Since cell packs are highly homogenized, they are also ideal for sieving purposes. Therefore, it was demonstrated in this example by evaluating the effect of the employed Agrobacterium strains and different expression vectors on transient product accumulation. Two different expression vectors were used: the binary vector pUTA-TPrfp ( figure 1 G); the binary vector pTRBO-G ( figure 1 H) (J.A.Lindbo, "TRBO: a high-efficiency tobaco mosaic virus RNA-based overexpression vector", Plant Phys145:1232-1240, 2007). Each vector was introduced into two different Agrobacterium strains. The standard vector pUTA-TPrfp was introduced into GV3101::pMP90RK and EHA105; the viral vector pTRBO-G was introduced into GV2260 and EHA105 (R.Helens et al., "A guide to Agrobacterium binary Ti vectors", TIBS5:446- 451, 2000).

[0153] pTRA-rTPgfp in GV3101::pMP90RK was used as a positive control ( figure 1 F). Liquid cultures of GV-p...

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Abstract

The present invention relates to the generation and cultivation of plant cell material in the form of a non-tissue multilayer cell pack and its use for the accumulation or harvesting of a desired product. In particular, the invention provides a method for the generation of plant cell material in the form of a medium- deprived, porous structured and non-tissue multilayer cell pack and for the subsequent maintenance of said cell pack, comprising the steps of (i) providing a cell pack by separating cells from a plant cell suspension culture, wherein the content of the liquid comprised by the cell pack is reduced and adjusted to correspond to a cell pack density between 0.1 and 0.9 g wet cell weight per cm3, thereby establishing the medium-deprived and porous structured nature of said cell pack, and (ii); incubating said medium-deprived and porous structured cell pack in a non-liquid environment under a relative humidity of 50 to 100 %.

Description

technical field [0001] The invention relates to the field of plant biotechnology. In particular, the invention relates to the production and cultivation of plant cell material in the form of non-organized multilayered cell packs, and their use for accumulation or harvesting of desired products. Background technique [0002] Over the past decade, efforts have been devoted to establishing and cultivating plant-based systems for the accumulation and harvesting of native or heterologous proteins and secondary metabolites. The literature provides a wealth of evidence material demonstrating the use of plant-based systems to produce a variety of desired substances either secreted into the culture medium or from the producing cells, tissues, organelles, or even A whole plant or a part of a plant is isolated. There are also numerous transformation protocols to ensure the establishment of stably transformed or transiently transformed plant material. However, there is still a need f...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N11/12C12P21/02A01H4/00
CPCC12N15/8205C12N5/04A01H4/00C12P21/02C12N11/12C12N15/8257
Inventor 托马斯·拉德马赫尔
Owner FRAUNHOFER GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG EV
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