Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-08-04
2004-12-21
Kubelik, Anne (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S306000, C435S320100
Reexamination Certificate
active
06833494
ABSTRACT:
FIELD OF INVENTION
BACKGROUND OF INVENTION
Promoters viz. Cauliflower Mosaic Virus 35S (CaMV35S) and its variant, CaMV35S double enhancer, Figwort Mosaic Virus (FMV) and its variant, FMV double enhancer and others have been used for high level constitutive expression of heterologous genes in transgenic plant systems. The choice of a promoter is largely based on the required expression level(s) of the gene(s) under consideration. In case of marker genes, a threshold level of expression is necessary to enable selection of transgenic plants in vitro and/or in vivo. Among all homologous and heterologous promoters studied for constitutive expression of transgenes in plants, the CaMV35S and its variant, CaMV35S double enhancer promoters are known to induce significantly high levels of expression and are therefore widely used in transgenic plant research. Both these promoters are also characterized by strong enhancing functions and have been shown to induce a 40 to 50-fold increase in transcription of neighboring genes (Kay et al 1987, Science 236:1299-1302). The use of CaMV35S promoter for expression of a particular gene would therefore also influence expression of other genes present within the same transformation vector. This is of particular concern when one requires regulated expression of a gene by its transcriptional control under a tissue-specific promoter along with constitutive expression of a marker gene under transcriptional control of a strong constitutive promoter, both being located in the same DNA construct used for development of transgenic plants. Furthermore, presence of a strong promoter with enhancing effects could also lead to deceptive results in studies on temporal and spatial expression patterns of tissue-specific promoters in transgenic plant systems.
Production of male-sterile lines is important for development of hybrids in crop plants to enhance crop productivity. Use of hybrids for increasing crop yield is primarily based on utilization of the phenomenon of hybrid vigor or heterosis (Shull 1952, In Heterosis, Gowen Ed. 14-48 Ames: Iwo State College Press). Hybrid vigor has been exploited in plant breeding for several years. When two genetically diverse parents with compensatory agronomic characters are combined by conventional breeding methodologies, the F1 hybrid plants show higher yield than either of the parents [William 1959, Nature (Lond.) 184:527-530; Sinha and Khanna 1975, Adv. Agron. 27:123-174; Pradhan et al. 1993, Euphytica 69:219-229]. To produce hybrid progeny, cross-pollination must occur. However, there are several crop plants, which are naturally self-pollinating, for instance Brassica sp., rice and wheat. In the event of parental lines being self-pollinating, either the pollen-producing organ (anthers) must be removed or the male reproductive units (microspores) must be destroyed in one parent to facilitate cross-pollination. It is in this context, that the development of stable, normal male sterile lines assumes importance.
A simple method of generating a male sterile line is by physical removal of anthers (emasculation). Hybrids in maize, cotton and tomato are produced by this method.
Another approach for generation of male-sterile lines for hybrid seed production is the use of cytoplasmic male sterility (CMS) systems. CMS is a maternally inherited phenomenon manifesting itself as the inability to produce functional pollen grains. The genetic determinants of male sterility in CMS systems are located in genomes of the cytoplasmic organelles, the mitochondria. Restorer genes for CMS systems are dominant nuclear genes that suppress male-sterile effects of the cytoplasm (mitochondria). When incorporated into the male parent, they can function as restorers of male fertility in the F1 hybrids. CMS systems have found widespread use in the production of hybrids in sorghum, sunflower, pearl millet and sugarbeet. However, their use has been limited in corn, wheat and oilseed Brassicas due to linkage of undesirable traits such as increased disease susceptibility, chlorosis, distortion of petals, poor nectary function, etc. with CMS in these systems (McVetty et al. 1989, Can. J. Plant Sci. 69:915-918; Burns et al 1991, Can. J Plant Sci. 71:655-661; Williams 1995, Trends Biotech. 13:344-349; Buzza 1995, In Brassica oilseeds: Production and Utilisation, Kimber and McGregor, Eds. CAB International).
With the advent of recombinant DNA and plant transformation technologies, pollination control based on genetic engineering of nuclear male sterility has emerged as a tangible option for production of male-sterile plants (reviewed by Williams 1995, Trends Biotech. 13:344-349). In these methods, the plant is provided with a male-sterility gene comprising a DNA sequence coding, for example, a cytotoxic product. The cytotoxic product, in many cases, may be a lethal gene under transcriptional control of a promoter, which is predominantly active in selective tissue(s) of the male reproductive organs in plants. As an example, male sterility could be successfully induced in transgenic tobacco and oilseed rape (
Brassica napus
) by targeted expression of a ribonuclease [barnase from
Bacillus amyloliquefaciens
(Hartley 1989, Trends Biochem. Sciences 14:450-454) or Rnase T1 from
Aspergillus oryzae
] in the tapetal tissues of anthers using a tapetum-specific promoter, TA29, from tobacco (Mariani et al 1990, Nature 347:737-741). Tapetum, which forms the innermost layers of the anther wall, is one of the most important tissues associated with pollen development. Disruption of tapetal cells by the expression of toxic proteins consequently impairs pollen development leading to male sterile plants. Several other strategies for disruption of normal pollen development have subsequently been developed (reviewed by Williams 1995, Trends Biotech. 13:344-349).
The DNA constructs used for development of male sterile lines also require the presence of a selectable marker gene(s) for in vitro selection of transformed tissues and field selection of segregants that contain the male sterility-inducing gene. Use of a strong constitutive promoter to express the marker gene is therefore important for enabling selection of transgenic plants. However, use of a strong constitutive promoter with a lethal gene in the vicinity can be detrimental to the process of generating transgenic plants using the latter because enhancing functions of the strong constitutive promoter could induce deregulated expression of the (lethal) gene that is otherwise under transcriptional control of a known tissue-specific promoter. Some DNA sequences [Scaffold or Matrix Attachment Regions (SARs/MARs)] are known to buffer, to some extent, influences of surrounding regions on transgene expression in plants (Breyne et al 1992, Plant Cell 4:463-471, Mlynarova et al 1994, Plant Cell 6:417-426, Mlynarova et al 1996, Plant Cell 8:1589-1599). However, SARs are also known to possess enhancing functions (Steif et al 1989, Nature 341:343-345, Allen et al 1993, Plant Cell 5:603-613). Use of such sequences to achieve conditional expression of lethal genes is therefore not advisable.
Plant Genetic Systems have described, in EP 0344029A1, use of the barnase gene for generation of male-sterile lines for hybrid seed production. According to this patent, the barnase construct used for plant transformation contained three components:
1. a male sterility-conferring transcription unit (TA29 promoter-barnase gene),
2. a nos promoter-nptII marker gene cassette: the nptII gene encodes the enzyme neomycin phosphotransferase which confers resistance to the antibiotic, kanamycin and can therefore be used for in vitro selection of transformed tissues, and
3. a rbcs promoter-bar marker gene cassette: the bar gene (from
Streptomyces hygroscopicus
) encodes the enzyme phosphinothricin acetyl transferase which confers resistance to the herbicide, Basta. It can therefore be used for field selection of lines with the male sterility-conferring gene among segregants. The rbcs promoter used in the above construct was
Arumugam Neelakantan
Bandyopadhyay Panchali
Burma Pradeep Kumar
Gupta Vibha
Jagannath Arun
Birch & Stewart Kolasch & Birch, LLP
Kubelik Anne
University of Delhi
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