The present invention relates to a novel concept in genetic engineering, according to which a eukaryotic organism is transformed and crossed so as to provide an offspring including a first exogene in a first chromosome of a chromosome pair and a second exogene in a second chromosome of the chromosome pair, the exogenes exhibiting allelic relationship, such that the exogenes obligatorily segregate to different gametes. The present invention further relates to expression cassettes for implementing the novel concept, to methods of implementing same for the obtainment of reversible male sterility in plants, and further to plants and plant products obtained thereby.
The gene is the basic functional unit of heredity. The gene concept was first set forth by Mendel in 1865 as a result of experiments conducted on pea plants. Mendel proposed that a genetic determinant of a specific character is passed on from one generation to the next as a unit without any blending of the units, a theory now known as the “one gene one trait” theory.
Two of mendel's proposed laws serve as the basis for modern genetics. Mendel's first law states that two members (alleles) of a gene pair obligatorily segregate to different gametes, such that one half of the gametes carry one member (allele) of the pair and the other half of the gametes carry the other member (allele) of the gene pair. Mendel's second law states that during gamete formation the segregation of alleles of a gene pair is independent of alleles other gene pairs.
Following Mendel's research, the discovery of chromosomes and the existence of linkage between genes closely positioned on a single chromosome have further contributed to our knowledge of inherited traits. Nowadays it is known that the second law of Mendel is valid for genes residing on different chromosomes and for genes residing on the same chromosome, provided that they are at least 50 centiMorgan apart, whereas for closer genes residing on the same chromosome, linkage dis-equilibrium is experienced.
Thus, in an individual, a genetic trait in it's simplest form is determined by a pair of closely related sequences known as alleles. Each allele, which is optionally an alternative form of a gene, resides on one of the chromosomes of a chromosome pair. The pair of alleles of a given individual can be identical in sequence or they can differ in sequence to various degree. In meiosis, which is the process of cell division which leads to the formation of sex cells (gametes), the chromosomes of any given chromosome pair segregate into different gametes. This chromosomal segregation is superseded by rearrangement of the DNA in chromosome pairs which takes place via inter chromosomal recombination events.
A specific trait of an individual is determined by a specific combination of two alleles carried by the individual. Thus, traits of an individual are determined by the genetic material inherited by that individual from both parents as determined by chromosomal segregation and recombination events in each of the individual's parents.
Alleles carried on a chromosome pair encode the same genetic trait. Allelic variance ensures a high degree of distinction between individuals.
Transformation of an exogene to a genome is typically random. Assuming one site of exogene integration, a resultant genome is said to be heterozygous of the type 1/0. By careful breeding and selection, a homozygous state for the exogene, i.e., 1/1 is obtainable. Although one can transform a genome with a second exogene (2), it is practically impossible, using conventional transformation techniques, to direct the second exogene to a given integration site, in a different genome. As such, using such conventional molecular biology and breeding techniques, it is practically impossible to achieve exogene allelism of the type 1/2.
Although it is theoretically possible to direct an exogene to a specific chromosomal site by gene knock-in techniques, which therefore could have been used to generate exogene allelism of the type 1/2, such techniques, are very poor in transformation yields. Indeed, no attempts of generating exogene allelism were made so far.
The production of hybrid plants has been practiced ever since the beginning of this century. Plant breeders discovered that crossing two distinct parental lines often resulted in a hybrid plant which displays what is termed as hybrid vigor or heterosis, which is characterized by an increased crop yield and/or adaptation to both biotic and abiotic stresses. Early on, hybrid plant production concentrated mainly on corn and sorghum which were found to benefit from crossing of well compatible parental lines. Later it was discovered that hybrid plant production is also applicable to other plant species.
Several explanations have been proposed for the existence of heterosis. It is clear that a wider allelic variety exist in hybrids plant lines as is compared to inbred plant lines, since the alleles present in a hybrid plant are inherited from two distinct parent plant lines. This allelic variety is favored as a likely explanation for hybrid vigor, however, at present no scientific evidence has been brought forth to support this explanation.
Since hybrid plants have been demonstrated to be superior to inbred lines with respect to yield and vigor, the development of hybrid seeds is one of the prime objectives of the seed industry. In addition, since hybrid plant varieties result from a unique combination, the possibility of duplicating or reusing the hybrid seeds is minimized, thus, providing breeders with an inherent commercial protection.
The production of hybrid seed on a large scale is challenging because many crops have both male and female reproductive organs (stamen and pistil) on the same plant, either within a single flower or in separate flowers. This arrangement results in a high level of self pollination and makes large scale directed crosses between parental lines to generate hybrid plants difficult to accomplish.
To guarantee that out-crossing will occur during the production of hybrid seed, breeders have either manually or mechanically removed stamens from one parental line. Although such manual emasculation is effective for some plants, such as wheat, it is labor intensive and impractical for plants with small bi-sexual flowers. To cross-pollinate such plants breeders have often resorted to using naturally occurring male sterile mutants in efforts to produce hybrid seeds. Although the use of naturally occurring male sterile mutants enables to cross pollinate plants with small bi-sexual flowers, the availability of such mutants and oftentimes the poor genetic makeup thereof severely limited wide spread use of this approach. In addition, using sterile mutants for out-crossing typically results in retention of sterility in a large fraction of the produced seeds. Such seeds when sown would then produce sterile plants which in the case of self pollinator crops such as wheat would lead to no crop yield for a fraction of the plants. To overcome the resultant low crop yield, a breeder is forced to select out the sterile seeds which can be a nearly impossible task.
Since important crops such as rice, wheat are self-pollinating plants with small bi-sexual flowers, there was a need and several attempts were made to develop systems for pollination control to assist in the production of F1 hybrids.
However, several conditions must exist in order to obtain economically feasible F1 hybrid plants. The male sterile line must be 100% sterile and yet female fertile, a natural pollen transfer from the male fertile line to the male sterile line must be facilitated, and in cases of grain or fruit crops, male fertility restoration (MFR) should be enabled in order to obtain crop yield in the F1 progeny.
There exist several mechanisms of male sterility in plants. One such mechanism is cytoplasmic male sterility (CMS). In general CMS has been used in corn, rice, sorghum and onion on a limited basis, see, for example, U.S. Pat. No. 3,861,079 to Patterson.
However, reliance on a single cytoplasmic-male-sterile system for the production of all hybrid plants is undesirable because it leaves the entire hybrid stock vulnerable to plant pathogens. For example, extensive use of one corn cytotype, cmsT lead to an eptiphytic outbreak of Southern Corn Leaf Blight in the early 1970's. Thus, it is important to develop alternative methods to produce male sterile lines in plant species where only a single male-sterility system is available.
An emerging alternative to cytoplasmic sterility is a nuclear sterility. Nuclear male-sterile-based pollination systems rely upon the introduction of a male sterility trait to one parental plant followed by the introduction of a fertility-restorer gene, as a result of cross-pollination, to produce fertile hybrid plants.
Typically, genetically engineered nuclear male sterility is effected by expressing, in a controlled and targeted manner, a protein toxin which destroys anther tissues of the plant in which it is expressed. The expression of this toxin can be either silenced or antagonized in the F1 progeny by the introduction of a fertility-restorer gene, as a result of cross-pollination.
Male reproductive processes in flowering plants occur in the anther. This organ is composed of several tissues and cell types, and is responsible for producing pollen grains that contain the sperm cells. A specialized anther tissue, the tapetum, plays an important role in pollen formation. The tapetum surrounds the pollen sac early in anther development, degenerates during the later stages of development and is not present as an organized tissue in the mature anther. The tapetum produces a number of proteins and other substances that either aid in pollen development or become components of the pollen outer wall. It is known that many male sterility mutations interfere with tapetum cell differentiation and/or function. Thus tapetal tissue is believed to be essential for the production of functional pollen grains.
As such, many nuclear male-sterile-based pollination systems known in the art utilize anther specific promoters to target the destruction of the tapetum or other anther specific tissues during maturation such that subsequent pollen development is arrested.
For example, U.S. Pat. Nos. 5,409,823, 5,659,124 and 5,824,542 to Crossland describe a dual method for producing male-sterile plants. Two genetically transformed plants, parents 1 and 2, are crossed to obtain male-sterile offspring. Parent 1 is transformed with an expression cassette comprising a nucleotide sequence encoding an anther-specific promoter which is operably linked to a nucleotide sequence encoding a trans-activator. Parent 2 is transformed with an expression cassette comprising a target nucleotide sequence, which is capable of being activated by the trans-activator, operably linked to a nucleotide sequence which encodes a toxin which can be an RNA or a polypeptide and which disrupts the formation of viable pollen. Therefore, crossing parent 1 with parent 2 results in male-sterile offspring. The male-sterile plants produced are useful for producing hybrid seed. Subsequent crossing of the male sterile plant with a plant which does not include the trans-activator restores fertility.
Although this system produces desirable male sterility in a portion of the produced offspring, restoration of fertility which relies upon the segregation of the trans-activator gene from the toxin gene cannot be efficiently effected using this system. In the male sterile plant the segregation of these two genes during meiosis relies a great deal on their physical chromosomal location, as such when a male sterile plant is crossed with a plant which does not express the trans-activator, the trans-activator and the toxin can be provided by the male sterile parent as a result of which a portion of the produced progeny will retain undesirable male sterility.
Thus the method by Crossland is not efficiently applicable for self pollinators such as, for example, wheat, cotton and rice.
U.S. Pat. No. 5,929,307 to Hodges describes a recombinant expression vector comprising a suicide gene flanked by site-specific recombination sequences, which vector, when introduced into a plant leads to male sterility. This invention further relates to a second expression vector including a recombinase gene which when introduced into the male sterile plant via a cross-pollinating restorer plant, leads to the deletion of the toxin gene and as a result to the restoration of fertility.
It will be appreciated in this case that since restoration of fertility is effected by a gene product which is introduced via a cross-pollinating restorer plant, a high copy number of the restorer gene must be maintained in the restorer plant such that highly efficient restoration of fertility in the resultant progeny is effected.
Thus, the presently available nuclear male-sterility pollination systems, although generally efficient in the production of male sterile plants, suffer from limitations when restoration of such male sterility via out-crossing is desirable, such as the case for grain or fruit producing hybrid plants. Since these systems cannot enable 100% restoration of fertility to the male sterile plants produced and since the resultant male sterile hybrid seeds are not typically selected out, a suboptimal crop yield from grain or fruit producing hybrid plants produced by these systems results.
There is thus a widely recognized need for, and it would be highly advantageous to have, a male sterile plant, which plant when out-crossed with a compatible male fertile plant generates male fertile offspring in substantially 100% of it's progeny. As further detailed hereinunder, such a plant can be generated by a transformation method which results in formation of allelism between two exogenes. There is thus a widely recognized need for, and it would be highly advantageous to have, a method and expression cassettes for the generation of exogene allelism.