Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform

a technology of planta and manufacturing process, applied in the field of in vitro planta transformation methods for plants or plant materials with a genetic construct, can solve the problems of time-consuming, expensive, elite varieties, time-consuming and costly, and the process of regenerating plants from in vitro transformed materials is time-consuming and expensive, so as to achieve stable introduction/integration, versatile and efficient

Inactive Publication Date: 2018-05-24
KWS SAAT SE & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0015]The primary object of the present invention was thus the provision of methods for the genotype independent in planta transformation of a plant or a plant material, which is suitable for both, the transient as well as the stable introduction / integration of a genetic construct of interest into at least one meristematic cell of a meristematic tissue belonging to the immature inflorescence of a plant. Furthermore, it was an object to provide methods for the manufacturing of a transgenic or a genetically manipulated plant based on an in planta approach for transforming a plant or plant material. Finally it was an object to obtain a plant or a progeny of a plant manufactured by the methods according to the present invention or plant cells or a plant material, or derivatives thereof or to provide and use a plant transformed by the methods according to the present invention for the manufacture of a transgenic or genetically manipulated plant or plant material. Finally, it was an object to define meristem access and meristem delivery methods suitable for a variety of different target plants, and to define genetic constructs and delivery methods to provide a versatile and efficient genome editing approach to modify a variety of relevant crop plants in a targeted and efficient way, either transiently or in a stable way, where all methods can be performed completely in planta obviating the need for cumbersome in vitro cultures.

Problems solved by technology

The process of regenerating a plant from the in vitro transformed material is, however, time consuming, expensive and the efficiency and the parameters to follow are strongly genotype dependent.
In addition, only few genotypes are suitable for genetic transformation, requiring backcrosses of the generated transgenic material with elite varieties which is time consuming and costly.
Said approaches, however, intrinsically require the cumbersome regeneration of the transformed plant cell or tissue to obtain a transgenic or genetically modified plant.
However, there is no follow-up of the technology, especially no follow up in planta or for other important crop plants.
1987) at high velocity fast enough (˜1500 km / h) to penetrate the cell wall of a target tissue, but not harsh enough to cause cell death.
Despite transformation methods based on biological approaches, like Agrobacterium transformation or viral vector mediated plant transformation, and methods based on physical delivery methods, like particle bombardment or microinjection, have evolved as prominent techniques for introducing genetic material into a plant cell or tissue of interest over the last years, there are still several problems associated therewith hampering the broad approach for both transient as well as stable introduction of a construct of interest in any kind of plant to be transformed.
There are, however, no examples concerning either the achievement of stable transformation of the genetic construct or any advice of how to use the technology for major crop plants in plant organs other than leaves.
The tissues “in planta” that can develop reproductive organs are limited.

Method used

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  • Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform
  • Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform
  • Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform

Examples

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example 1

sel Development

[0148]In corn development, the apical meristem differentiates into tassel primordia in the V6 stage (“Vegetative stage 6” or when the plant has 6 leaves) or 25-30 days after germination (Cheng et al. 1983; Abendroth et al. 2011). Therefore, it was tried to target the cells of those immature tassels that will further develop in the anther tissue that will produce pollen. One set of experiments focused on maize plant, namely A188 plants. Already 1 month old plants have developing tassels instead of the classical apical meristems in these plants see (FIGS. 3 and 4). It was observed that the growth of the immature tassel is very fast, going from less than 1 cm to more than 8 in 11 days (FIG. 4). We also looked at the developmental stage of the spikelets and decided that the target age will be between 34 and 37 days after sowing, when the tassel is easy to be detected and exposed but the spikelets are not formed (FIG. 4). This is an important parameter, in case the immatur...

example 2

the Immature Inflorescence of a Growing Maize Plant

[0149]To facilitate the genetic manipulation or the introduction of a genetic construct into the maturing tassel tissue, several methods were analyzed. It was found that a window could artificially be introduced into the region, where the meristematic tissue of the immature inflorescence is located. For maize, this region is located near the axil in the shoot axis, the culm, of the plant. An exemplary plant comprising an artificially introduced window is shown in FIG. 5. The tassel is pushed upwards very fast during plant development. When the tassel is exposed through a window, the natural pathway to grow upwards is disrupted. Sometimes the tassel grows out of the plant or other is trapped between the growing leaf mass, making the plants look abnormal in comparison to a non treated plant (see e.g. FIG. 7 (D to E)), which, however, did not disturb the overall plant and tassel development. For a maize plant, it is thus recommendable ...

example 3

Transformation of Exposed Immature Tassel with Particle Bombardment In Vitro

[0151]One of the questions to be answered by this technology was if the exposed immature tassel tissue is amenable for transformation, especially in planta, but also ex vivo. For that, several experiments were conducted in which the immature tassel was separated from the plant and bombarded in the lab using the bench gene-gun PDS-1000 / He (BIO-RAD). Particle bombardment turned out to be a suitable method for all experiments conducted with immature meristematic tissues and isolated cells from several plants including maize, barley and wheat varieties for both in vitro as well as in planta bombardment. Experiments with several test constructs of dsDNA, RNA and proteins as well as virus particles were used in the different settings. During this series of experiments it turned out that the parameters for bombardment are critical to influence, whether a transient or stable expression of a dsDNA construct will be a...

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Abstract

The present invention relates to in planta transformation methods for plants or plant materials with a genetic construct, wherein at least one meristematic cell of a meristematic tissue of an immature inflorescence able to differentiate into a gamete of a pollen or of an ovule is exposed and then transformed, wherein the transformation can be performed to yield a stable integration or to yield a transient introduction of a genetic construct of interest. Further provided are methods for manufacturing a transgenic plant, or methods for manufacturing a genetically manipulated plant based on the in planta transformation methods according to the present invention. In addition, there is provided a plant or a progeny thereof manufactured according to the transformation and/or manufacturing methods according to the present invention.

Description

TECHNICAL FIELD[0001]The present invention relates to in planta transformation methods for plants or plant materials with a genetic construct, wherein at least one meristematic cell of a meristematic tissue of an immature inflorescence able to differentiate into a gamete of a pollen or of an ovule is exposed and then transformed, wherein the transformation can be performed to yield a stable integration or to yield a transient introduction of a genetic construct of interest. Further provided are methods for manufacturing a transgenic plant, or methods for manufacturing a genetically manipulated plant based on the in planta transformation methods according to the present invention. In addition, there is provided a plant or a progeny thereof manufactured according to the transformation and / or manufacturing methods according to the present invention. Finally, there is provided the use of the inventive methods and plants for the manufacture of a transgenic or genetically manipulated plan...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82C12N15/113A01H5/04A01H5/12
CPCC12N15/8207C12N15/113A01H5/04C12N15/8203C12N15/8205C12N15/8213A01H5/12C12N15/8201C12N2310/20
Inventor MARTIN-ORTIGOSA, SUSANAHARLING, HINRICHNIESSEN, MARKUS
Owner KWS SAAT SE & CO KGAA
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