Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2000-07-10
2003-01-28
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C435S468000, C800S278000, C800S280000, C800S288000, C800S312000, C800S320100, C800S320200, C800S320300, C536S023700, C536S023720
Reexamination Certificate
active
06512165
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology.
BACKGROUND OF THE INVENTION
Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment requires precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems such as in lateral root formation). Such control of cell division is also important in organs themselves (i.e. separate from meristems per se), for example, in leaf expansion and secondary growth.
A complex network controls cell proliferation in eukaryotes. Various regulatory pathways communicate environmental constraints, such as nutrient availability, mitogenic signals such as growth factors or hormones, or developmental cues such as the transition from vegetative to reproductive. Ultimately, these regulatory pathways control the timing, frequency (rate), plane and position of cell divisions.
Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not migrate, and thus only cell division, expansion and programmed cell death determine morphogenesis. Organs are formed throughout the entire life span of the plant from specialized regions called meristems.
In addition, many differentiated cells have the potential to both dedifferentiate and to reenter the cell cycle. The study of plant cell cycle control genes is expected to contribute to the understanding of these unique phenomena. O. Shaul et al.,
Regulation of Cell Division in Arabidopsis, Critical Reviews in Plant Sciences,
15(2): 97-112 (1996).
Current transformation technology provides an opportunity to engineer plants with desired traits. Major advances in plant transformation have occurred over the last few years. However, in many major crop plants, serious genotype limitations still exist. Transformation of some agronomically important crop plants continues to be both difficult and time consuming.
For example, it is difficult to obtain a culture response from some maize genotypes. Typically, a suitable culture response has been obtained by optimizing medium components and/or explant material and source. This has led to success in some genotypes. While, transformation of model genotypes is efficient, the process of introgressing transgenes into production inbreds is laborious, expensive and time consuming. It would save considerable time and money if genes could be more efficiently introduced into and evaluated directly into inbreds.
There is evidence to suggest that cells must be dividing for transformation to occur. It has also been observed that dividing cells represent only a fraction of cells that transiently express a transgene. Furthermore, the presence of damaged DNA in non-plant systems (similar to DNA introduced by particle gun or other physical means) has been well documented to rapidly induce cell cycle arrest (W. Siede,
Cell cycle arrest in response to DNA damage: lessons from yeast, Mutation Res.
337(2:73-84). Methods for increasing the number of dividing cells would therefore provide valuable tools for increasing transformation efficiency.
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly growing meristems, in callus, in suspension cultures, or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into literally thousands of cells, yet transformants are recovered at frequencies of 10
−5
relative to transiently expressing cells.
Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest and accumulating evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Reference Bowen et al., Tucson International Mol. Biol. Meetings). Therefore it would be desirable to provide improved methods capable of increasing transformation efficiency in a number of cell types.
While advances have been made in the transformation of elite inbreds of maize, it would be desirable to increase frequencies of transformation. Present model systems, designed around fast growing and highly embryogenic cultures, produce high frequencies of transgenic events in the hybrid GS3 and in model maize inbreds. Because of the high frequencies, these models, instead of the elite inbred genotypes, are frequently the standard target germplasm for product development.
SUMMARY OF THE INVENTION
The present invention provides a method for increasing transformation frequencies, especially in recalcitrant plants or explants. The method comprises transforming a target cell with at least one polynucleotide of interest operably linked to a promoter. The target cell has previously been stably modified to stimulate growth of the cell and has gone through at least one cell division.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
As used herein, “polypeptide” and “protein” are used interchangeably and mean proteins, protein fragments, modified proteins, amino acid sequences and synthetic amino acid sequences. The polypeptide can be glycosylated or not.
As used herein, “polynucleotide” and “nucleic acid” are used interchangeably. A polynucleotide can be full-length or a fragment and includes polynucleotides that have been modified for stability. Unless otherwise indicated, the term includes reference to a specific sequence or its complement.
As used herein, “growth stimulation polynucleotide” means a polynucleotide capable of influencing growth of a cell. The polynucleotides fall into several categories, 1) cell cycle stimulatory polynucleotides 2) developmental polynucleotides 3) anti-apoptosis polynucleotides other than
baculovirus p
35 or
baculovirus
iap 4) hormone polynucleotides or 5) silencing constructs targeted against cell cycle repressors.
The following are provided as examples of each category and are not considered a complete list of useful polynucleotides for each category: 1) cell cycle stimulatory polynucleotides including plant viral replicase genes such as RepA, Cyclins, E2F, prolifera, cdc2 and cdc25; 2) developmental polynucleotides such as Lec1, Kn1 family, WUSCHEL, Zwille, and Aintegumenta (ANT); 3) anti-apoptosis polynucleotides other than
baculovirus
p35 or
baculovirus
iap such as CED9, Bcl2, Bcl-X(L), Bcl-W, A1, McL-1, Mac1, Boo, Bax-inhibitors; 4) hormone polynucleotides such as IPT, TZS, Baby Boom (BBM) and CKI-1; 5) Silencing constructs targeted against cell cycle repressors, such as Rb, CKl, prohibitin, wee1, etc. or stimulators of apoptosis such as APAF-1, bad, bax, CED-4, caspase-3, etc. and repressors of plant developmental transitions such as Pickle and WD polycomb genes including FIE and Medea. The polynucleotides can be silenced by any known method such as antisense, cosuppression, chimerplasty, or transposon insertion.
As used herein, “growth stimulation vector” means a vector capable of altering the expression of polynucleotides resulting in growth stimulation.
As used herein, “plant” includes but is not limited to plant cells, plant tissue, plant parts, and plant seeds.
As used herein “recalcitrant plant or explant” means a plant or explant that is more difficult to transform than model systems. In maize such a model system is GS3. Elite maize inbreds are typically recalcitrant. In soybeans such model systems are Peking or Jack.
As used herein “responsive target plant cell” is a plant cell that exhibits increased transformation efficiency after transformation with a growth stimulation vector compared to a corresponding plant cell that has not been transformed with the growth stimulation vector.
As used herein “Stable Transformat
Bidney Dennis L.
Church Laura A.
Gordon-Kamm William J.
Hill Patrea M.
Hoerster George J.
Fox David T.
Kruse David H
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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