Multicellular living organisms and unmodified parts thereof and – Method of using a plant or plant part in a breeding process...
Reexamination Certificate
1998-02-19
2001-12-11
Mosher, Mary E. (Department: 1648)
Multicellular living organisms and unmodified parts thereof and
Method of using a plant or plant part in a breeding process...
C800S263000, C800S264000, C800S270000, C800S278000, C800S281000, C800S284000, C800S285000, C800S286000, C800S287000, C800S298000, C800S306000, C800S312000, C536S023600, C536S024500, C435S468000, C435S415000, C435S419000
Reexamination Certificate
active
06329567
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to new methods for modulating mass and other properties of plant seeds.
BACKGROUND OF THE INVENTION
The pattern of flower development is controlled by the floral meristem, a complex tissue whose cells give rise to the different organ systems of the flower. Genetic it and molecular studies have defined an evolutionarily conserved network of genes that control floral meristem identity and floral organ development in Arabidopsis, snapdragon, and other plant species (see, e.g., Coen and Carpenter,
Plant Cell
5:1175-1181 (1993) and Okamuro et al.,
Plant Cell
5:1183-1193 (1993)). In Arabidopsis, a floral homeotic gene APETALA2 (AP2) controls three critical aspects of flower ontogeny—the establishment of the floral meristem (Irish and Sussex,
Plant Cell
2:741-753 (1990); Huala and Sussex,
Plant Cell
4;901-913 (1992); Bowman et al.,
Development
119:721-743 (1993); Schultz and Haughn,
Development
119:745-765 (1993); Shannon and Meeks-Wagner,
Plant Cell
5:639-655 (1993)), the specification of floral organ identity (Komaki et al.,
Development
104:195-203 (1988)); Bowman et al.,
Plant Cell
1:37-52 (1989); Kunst et al.,
Plant Cell
1:1195-1208 (1989)), and the temporal and spatial regulation of floral homeotic gene expression (Bowman et al.,
Plant Cell
3:749-758 (1991); Drews et al.,
Cell
65:91-1002 (1991)).
One early function of AP2 during flower development is to promote the establishment of the floral meristem. AP2 performs this function in cooperation with at least three other floral meristem genes, APETALA1 (AP1), LEAFY (LFY); and CAULIFLOWER (CAL) (Irish and Sussex (1990); Bowman,
Flowering Newsleter
14:7-19 (1992); Huala and Sussex (1992); Bowman et al., (1993); Schultz and Haughn, (1993); Shannon and Meeks-Wagner, (1993)). A second function of AP2 is to regulate floral organ development. In Arabidopsis, the floral meristem produces four concentric rings or whorls of floral organs—sepals, petals, stamens, and carpels. In weak, partial loss-of-function ap2 mutants, sepals are homeotically transformed into leaves, and petals are transformed into pollen-producing stamenoid organs (Bowman et al.,
Development
112:1-20 (1991)). By contrast, in strong ap2 mutants, sepals are transformed into ovule-bearing carpels, petal development is suppressed, the number of stamens is reduced, and carpel fusion is often defective (Bowman et al., (1991)). Finally, the effects of ap2 on floral organ development are in part a result of a third function of AP2, which is to directly or indirectly regulate the expression of several flower-specific homeotic regulatory genes (Bowman et al.,
Plant Cell
3;749-758 (1991); Drews et al.,
Cell
65:91-1002 (1991); Jack et al.
Cell
68:683-697 (1992); Mandel et al.
Cell
71: 133-143 (1992)).
Clearly, Ap2 plays a critical role in the regulation of Arabidopsis flower development. Yet, little is known about how it carries out its functions at the cellular and molecular levels. A spatial and combinatorial model has been proposed to explain the role of AP2 and other floral homeotic genes in the specification of floral organ identity(see, e.g., Coen and Carpenter, supra). One central premise of this model is that AP2 and a second floral homeotic gene AGAMOUS (AG) are mutually antagonistic genes. That is, AP2 negatively regulates AG gene expression in sepals and petals, and conversely, AG negatively regulates AP2 gene expression in stamens and carpels. In situ hybridization analysis of AG gene expression in wild-type and ap2 mutant flowers has demonstrated that AP2 is indeed a negative regulator of AG expression. However, it is not yet known how AP2 controls AG. Nor is it known how AG influences AP2 gene activity.
The AP2 gene in Arabidopsis has been isolated by T-DNA insertional mutagenesis as described in Jofuku et al.
The Plant Cell
6:1211-1225 (1994). AP2 encodes a putative nuclear factor that bears no significant similarity to any known fungal, or animal regulatory protein. Evidence provided there indicates that AP2 gene activity and function are not restricted to developing flowers, suggesting that it may play a broader role in the regulation of Arabidopsis development than originally proposed.
In spite of the recent progress in defining the genetic control of plant development, little progress has been reported in the identification and analysis of genes effecting agronomically important traits such as seed size, protein content, oil content and the like. Characterization of such genes would allow for the genetic engineering of plants with a variety of desirable traits. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of modulating seed mass and other traits in plants. The methods involve providing a plant comprising a recombinant expression cassette containing an ADC nucleic acid linked to a plant promoter. The plant is either selfed or crossed with a second plant to produce a plurality of seeds. Seeds with the desired trait (e.g., altered mass) are then selected.
In some embodiments, transcription of the ADC nucleic acid inhibits expression of an endogenous ADC gene or activity the encoded protein. In these embodiments, the step of selecting includes the step of selecting seed with increased mass or another trait. The seed may have, for instance, increased protein content, carbohydrate content, or oil content. In the case of increased oil content, the types of fatty acids may or may not be altered as compared to the parental lines. In these embodiments, the ADC nucleic acid may be linked to the plant promoter in the sense or the antisense orientation. Alternatively, expression of the ADC nucleic acid may enhance expression of an endogenous ADC gene or ADC activity and the step of selecting includes the step of selecting seed with decreased mass. This embodiment is particularly useful for producing seedless varieties of crop plants.
If the first plant is crossed with a second plant the two plants may be the same or different species. The plants may be any higher plants, for example, members of the families Brassicaceae or Solanaceae. In making seed of the invention, either the female or the male parent plant can comprise the expression cassette containing the ADC nucleic acid. In preferred embodiments, both parents contain the expression cassette.
In the expression cassettes, the plant promoter may be a constitutive promoter, for example, the CaMV 35S promoter. Alternatively, the promoter may be a tissue-specific promoter. Examples of tissue specific expression useful in the invention include fruit-specific, seed-specific (e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, or seed coat-specific) expression.
The invention also provides seed produced by the methods described above. The seed of the invention comprise a recombinant expression cassette containing an ADC nucleic acid. If the expression cassette is used to inhibit expression of endogenous ADC expression, the seed will have a mass at least about 20% greater than the average mass of seeds of the same plant variety which lack the recombinant expression cassette. If the expression cassette is used to enhance expression of ADC, the seed will have a mass at least about 20% less than the average mass of seeds of the same plant variety which lack the recombinant expression cassette. Other traits such as protein content, carbohydrate content, and oil content can be altered in the same manner.
DEFINITIONS
The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term “promoter” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recogn
Jofuku K. Diane
Okamuro Jack K.
Mosher Mary E.
The Regents of the University of California
Townsend & Townsend & Crew LLP
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