Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1997-11-14
2001-11-13
Hutzell, Paula K. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S290000, C800S294000, C800S295000, C800S298000, C800S306000, C435S469000, C435S471000, C435S410000
Reexamination Certificate
active
06316694
ABSTRACT:
1. INTRODUCTION
Transgenic crop plants can be produced using different transformation techniques. The method of choice for species and varieties, where plant regeneration from tissue culture is efficient, is Agrobacterium (Potrykus 1993). The
Agrobacterium tumefaciens
transformation system is simple and inexpensive (DeBlock 1993). It is the most effective method of transferring foreign DNA into a host plant (Thierfelder et al. 1993). Plants are obtained with a limited number of gene insertions, and usually insertion of DNA occurs between two defined border sequences completely and exclusively (DeBlock 1993). It is therefore not surprising that in terms of use and apparent progression to field trials, the majority of gene transfer experiments have involved the Agrobacterium system (White 1993).
Microspores of higher plants have the potential to develop, under appropriate conditions directly into haploid or doubled haploid plants. Microspores of higher plants develop in vivo into pollen grains (gametophytic pathway). Microspore culture can induce an alternative (sporophytic) pathway which leads to the formation of haploid and doubled haploid embryos. These embryos develop directly into normal fertile plants. Misunderstandingly these embryos are sometimes referred to as pollen embryos.
The time saving for the breeder working with microspore transformants compared to transformants from heterozygous plant material is estimated to be three years for the development of each transgenic variety. This is due to the immediate fixation of the genes in the homozygous doubled haploid lines. This allows a more efficient selection of agronomic and quality traits and the immediate testing of combining ability for the development of hybrid cultivars.
Therefore, since embryogenic microspores develop into homozygous fertile plants and are single haploid cells, they are theoretically the most suitable recipients for gene transfer (Huang and Keller 1989).
Several laboratories (Potrykus 1991, Heberle-Bors 1995) have unsuccessfully attempted transforming microspores with Agrobacterium and Sangwan et al. (1993) have concluded that “it is difficult if not impossible to obtain transgenic plants by infection of pollen or proembryos with Agrobacterium.” Interestingly Heberle-Bors (1995) summarizes his attempts to transform microspores stating that A. tumefaciens was not suitable to transfer DNA into microspores, contrary to his 1989 patent. The thick cell wall of microspores has been perceived to be inaccessible to Agrobacterium (Stöger et al. 1995, Jones-Villeneuve et al. 1995).
More frequently, multicellular haploid tissues such as microspore-derived embryos; (Neuhaus et al. 1987, Swanson and Erickson 1989, Huang 1992) or haploid stem segments (Arnoldo 1992) have been used.
A common misconception is the difference between microspores and microspore-derived embryos where doubled-haploid plants can be obtained from both. Microspores are single cells and can be stimulated to produce embryos. The transformation of microspores leads to non-chimeric embryos and then plants. On the other hand microspore-derived embryos are multi-celled tissues. The transformation of microspore-derived embryo segments may lead to chimeric sectors from which chimeric shoots or secondary embryos can be selected. In addition somaclonal variation is not uncommon as there is usually an intermediate callus phase associated with regeneration from microspore-derived embryo segments.
The closest report of the prior art is the publication of Pechan (1989) on microspore co-cultivation with Agrobacterium. He claimed the production of kanamycin and hygromycin resistant plants but did not prove DNA integration or the sexually transmissibility of the transgene and furthermore, the described method has not been reproducible by others (Huang 1992).
Several reports describe the use of transformation techniques on microspores. Huang (1992) reported the use of microinjection, particle bombardment and electroporation on microspores and observed some transient gene expression, but no stable transformants. The review also described the use of Agrobacterium on microspores and in over 50 experiments conducted, only one plant was regenerated where Agrobacteria were present at the proembryo stage and the event could not be reproduced. Jardinaud et al. (1993) reported only transient gene expression in experiments using electroporation on B. napus microspores and using biolistics on maize microspores (Jardinaud 1995). Microinjection has also been attempted in B. napus microspores but this was not successful (Jones-Villeneuve et al. 1995).
Particle bombardment is the only method used on embryogenic microspores that has led to the production of stable transformed plants (Jähne et al. 1994, Stöger et al. 1995). However in comparing different transformation methods, Christou (1995) concluded that with biolistics, the underlying mechanism of the gene transfer process is not known and the impossibility to control DNA content and integration patterns is a major drawback. Transformed plants normally have multi-copy (Jähne et al. 1994) and random fragmented DNA insertions, that may or may not contain the complete gene of interest.
For breeding purposes, single copy gene insertions are desirable as they can be selected easily and followed through generations with ease and gene expression is good. In addition the plant does not need to transcribe more than the necessary enzymes and phenomena such as co-suppression, i.e. the down regulation of a gene by additional copies of this gene having substantial homology (WO 90/12084), as wastes of valuable plant energy is not a consideration.
The optimal transformation vector to allow single copy gene insertions with defined border sequences is to make use of the naturally occuring transformation process of
Agrobacterium tumefaciens.
We surprisingly found that microspore transformation via A. tumefaciens is practicable and leads to fertile homozygous plants with predominantly single copy inserts which have been confirmed via Southern blots in B. rapa and B. napus genotypes. Thus fertile homozygous plants with single copy inserts can be produced in only one gene ration using embryogenic microspores and A. tumefaciens.
2. DESCRIPTION OF THE INVENTION
The present invention is directed to transformed embryogenic microspores. The invention additionally provides an improved process for the Agrobacterium mediated transformation.
The invention is directed to transformed, embryogenic microspores and progeny thereof characterized by
a) being transformed by
Agrobacterium tumefaciens
,
b) capable of leading to non-chimeric transformed haploid or doubled haploid embryos that develop into fertile homozygous plants within one generation and
c) containing stably integrated into their genome a foreign DNA, said DNA being characterized in that it comprises at least one gene of interest and at least base pairs within the right border sequence of Agrobacterium T-DNA.
The invention is furthermore directed to microspores containing integrated foreign DNA wherein the gene of interest is bordered by at least base pairs within the right border and parts or all of the left border sequence leading to the specified insertion of the gene(s) of interest. Especially preferred are microspores containing at least one insert of said foreign DNA.
The invention also encompasses transformed, embryogenic microspores capable of being produced according to a process comprising the following steps:
a) infection of embryogenic microspores with Agrobacteria
b) washing out and killing the Agrobacteria after co-cultivation during transformation.
Preferrably, the Agrobacteria are removed by washing with mucolytic enzymes after co-cultivation and the process comprises the further step of adding cellolytic enzymes during transformation.
The invention is also directed to a method for the incorporation of foreign DNA into chromosomes of microspores comprising the following steps:
a) infecting of embryogenic microspores with Agrobacteria, which contain plasmids carrying
Dormann Mathias
Oelck Michael
Wang Hung-Mei
AgrEvo Canada, Inc.
Frommer & Lawrence & Haug LLP
Hutzell Paula K.
Zaghmout O. M. F.
LandOfFree
Transformed embryogenic microspores for the generation of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Transformed embryogenic microspores for the generation of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Transformed embryogenic microspores for the generation of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2570503