Process for generating male sterile plants

Details Process for generating male sterile plants Process for generating male sterile plants

1996-04-02

2001-07-17

Benzion, Gary (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of using a plant or plant part in a breeding process...

Method of breeding using gametophyte control

C800S271000, C800S286000, C800S287000, C800S295000

Reexamination Certificate

active

06262339

ABSTRACT:

1. INTRODUCTION
Male sterility is a property that is highly recognized in plant breeding. Male sterility enables the combination of traits from two parental lines, with the ultimate goal to supress negative traits from either parent with the genes from the other parent, and to superimpose positive traits carried by both parents. This results in a vigour that equals or exceeds either parental line. Several natural sources, e.g. cytoplasmic sterility or systems based on nuclear male sterility are already being used for many years. In crops were natural sources of male sterility are not available or not suitable, e.g. in tomato, hand emasculation and manual hybridization is still being carried out.
Chemical companies recognize the importance of hybridization and developed chemical hybridizing agents that affect the pollenviability (and as a consequence the fertility) of the plants on which the treatment is applied (see Mabbet, 1992).
In recent years, several molecular biological approaches have led to alternative ways for introduction of male sterility in crop plants. Mariani et al. (1990) introduced a RNAse that leads to tapetum ablation and results in transgenic plants that are male sterile.
Recently, Worall et al. (1992) published a system where the premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Mol et al. (1991) found that an antisense chalcone synthase gene expressed in the flower, could result in the absence of pigmentation of the anthers and the loss of fertile pollen. Ylstra and van Tunen (1992) reported that depletion of flavonols in tapetal cells by antisense Chalcone synthase leads to a reduction in pollen viability.
Natural mutants deficient of chalcone synthase have been described already in maize (Coe et al. 1981) and produce white disfuntional pollen. Restoration to fertility could be partially obtained by pollination on wild type stigmas or by addition of micromolar quantities of kaempferol to the pollen germination medium (Mo et al. 1992). Utilization of these characteristics led to a system for male sterility and conditional male fertility when kaempferol is supplied (Van Tunen et al. 1992). The exact role of flavonoids in the flower physiology is still unknown.
The International Patent Application WO 90/08830 is directed to methods for the production of male sterile plants by the expression of either a gene encoding a protein inhibitor, or a so-called killer gene. The expression of the genes in the male flowers leads to cell death of the anthers.
The International Patent Application WO 90/08831 teaches a method for the inhibition of cell-respiration by expression of a disrupter gene. Examples for disrupter proteins are the mammalian uncoupling protein (UCP), a mutated form of the gene for the &bgr;-1 subunit of F
1
-ATPase, and a mutated, synthetic form of the olil gene encoding subunit 9 of the F
0
-ATPase.
The International Patent Application WO 89/10396 discloses methods for the generation of male sterile plants. The plant cells are transformed with a male-sterility DNA. Examples of such male-sterility DNA are those encoding DNAses, RNAses, proteases, or enzymes of phytohormone synthesis, such as cytokinin or antisense DNA coding for a strand of DNA complementary to a strand of DNA that is naturally transcribed in the plant cells.
EP-A-0 329 308 discloses a method to produce male-sterile plants by using anti-sense DNA. The development of functional pollen grains is blocked because of anti-sense DNA directed to genes which are specifically expressed in the microspores, preferably in the premeiotic stage.
All systems described above suffer from a serious drawback in the sense that plants are terminally male sterile because of the cell death of the tapetal cells and eventually the microspores and thus the pollengrains, or the microspores directly. Maintaining lines that carry the male sterility gene requires back crossing with a wild type isogenic line. This results in a segregation and consequent loss of 50% of this backcross population due to the fact that 50% of the lines will not carry the male sterility gene, and will be male fertile.
In order to be able to perform further breeding with those plants which have desirable properties, it is necessary to restore fertility, which is very time and cost consuming.
Auxotrophic mutants of plants are characterized by the inability to synthesise one or more enzymes involved in metabolic pathways. Such mutants have been recovered in plant cells via a variety of techniques (for a review, see Pythoud and King, 1990).
These mutants in plants are very difficult to maintain due to the stringent requirement of the metabolite that is characteristic for the mutation. Most auxotrophic cells cultured “in vitro” are unable to divide unless the required compound is supplemented in the culture medium. When such mutants are not supplemented with the required compound, the cell obviously becomes starved and eventually will die. It is however possible to reinitiate division, dependent on the time of the starvation treatment by supplementing the required metabolite (Dirks et al. 1986, Negrutiu et al. 1983 and 1985).
2. DESCRIPTION OF THE INVENTION
The invention is directed to a process for the generation of male sterility in plants comprising the steps of
(a) transforming a plant cell with DNA sequences that selectively inhibit the production of essential metabolic compounds and
(b) regenerating plants from said plant cells.
The invention is further directed to a process for the generation of conditional reversible male sterility in plants by
(a) transforming plant cells with inhibitory DNA sequences
(b) under the control of a suitable male organ specific promotor that selectively inhibits the production of
(c) essential metabolic compounds which can by supplemented and
(b) regenerating plants from said plant cells.
Cells impaired in the biosynthesis of basic metabolic compounds undergo starvation and eventually die. Such pathways include amino acid biosynthesis, nucleic acid biosynthesis and other biosynthetic pathways such as citric acid cycle, pentose phosphate pathway, fatty acid metabolism, vitamin biosynthesis that will render the cell inactive due to nutrient depletion, if one or more enzymes involved in this pathway would become inactive by a variety of means.
The term inhibitory DNA encompasses any DNA that will cause that a certain metabolic pathway is blocked, e. g. an antisense DNA. The term antisense is to be understood as a DNA sequence which is complementary to a target or substrate DNA sequence, the expression of the antisense leads to expression of an inhibitory RNA and the inhibition of the expression of the target (see also EP 140 308).
The principles of antisense RNA are described by Inouye (1988) and Izant and Weintraub (1984). Inhibitory DNA might also code for ribozymes that selectively cut and therefore inhibit target sequences (EP 321 201). But there exist also other ways to inhibit gene expression and translation.
In particular, the inhibitory DNA is transcribed in the anther cells into a RNA which selectively inhibits the expression of genes coding for enzymes or proteins which are involved in the biosynthesis of amino acids.
In particular the invention relates to plants wherein the DNA sequence is expressed under the control of a tapetum-specific promotor. Plants regenerated from plant cells with an inhibitory DNA will be unable to produce functional tapetum cells and will produce non functional pollen or no pollen at all.
The invention is also related to transgenic plants containing gene constructs comprising a male organ specific promotor operably linked to inhibitory DNA sequences. The invention also pertains to male sterile plants, parts thereof and cells as well as its reproduction material. The invention further pertains to the seeds produced by the transgenic plants and all progeny that exhibit the desirable trait herein described.
The promotor contains the DNA sequence which is necessary for the initation of transcription. Further downstream, i

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