# Patent

## Title
Lettuce that is resistant to the lettuce aphid Nasonovia ribisnigri biotype 1

## Abstract

The present invention relates to lettuce plants and heads that are resistant to Nasonovia ribisnigri biotype 1 (also called herein Nr:1) as well as to progeny of the plants and propagation material for producing the plants. The invention further relates to a source of the resistance for use in breeding.

## Background
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
Reference is made to the application entitled RESISTANCE TO NASONOVIA RIBISNIGRI BIOTYPE 1 (NR:1) FROM LACTUCA SERRIOLA filed Nov. 16, 2010.
The foregoing application, and all documents cited therein or during their prosecution (“appin cited documents”) and all documents cited or referenced in the appin cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
FIELD OF THE INVENTION
The present invention relates to lettuce plants and heads that are resistant to Nasonovia ribisnigri biotype 1 (also called herein Nr:1) as well as to progeny of the plants and propagation material for producing the plants. The invention further relates to a source of the resistance for use in breeding.
BACKGROUND OF THE INVENTION
The lettuce aphid (Nasonovia ribisnigri (Mosley)) is a major pest occurring in lettuce worldwide. The problem started to be severe for lettuce production in the 1970's in North Western Europe and spread rapidly all across Europe. Then, in the 1980's, the aphid was detected in Canada. Later on, the problem was reported in the USA (California and Arizona). More recently, the lettuce aphid was found in New Zealand and Australia.
Lettuce aphids can colonize lettuce plants at any plant stage and feed preferably from younger leaves. Large amount of aphids on the plant are able to reduce plant growth and deform the shape of the head so that the lettuce heads are then not marketable. The presence of high amounts of aphids in lettuce heads is a reason for retailers to refuse to buy lettuce from growers. At young plant stage, it is possible to control the lettuce aphid using insecticide. Several products were reported efficient to control aphid population. However, resistances to chemicals were reported in some aphid population. Moreover, at maturity, it is not possible to control aphids using insecticides as chemical products cannot enter into the lettuce head.
One of the most valuable strategies to control lettuce is genetic resistance. Extensive gene bank screening was performed and some Lactuca virosa accessions were found completely resistant to Nasonovia ribisnigri (Eenink and Dieleman, Euphytica 32(3), 691-695 (1982)). However, Lactuca virosa is in the third gene pool of the Lactuca germplasm according to the definition of Harlan. Therefore, these interspecific crosses are sterile, and the use of bridge species (as L. serriola) was necessary to transfer the resistance into L. sativa. Genetic analyses showed that the resistance to Nasonovia ribisnigri was controlled by a single dominant gene (Nr gene) in a L. sativa background.
However, breeders experienced that the release of varieties resistant to lettuce aphid was not straightforward. The Nr-resistance gene was found tightly linked to recessive genes conferring strong negative side-effects. Such plants showed a reduced growth, a pale green colour and a lack of fertility in seed set. Using large-sized progeny and molecular markers enabled lettuce breeders to find resistant recombinant plants without the negative side-effect phenotype (see EP-0 921 720). These resistant plants served as the source of the resistance gene that was not linked to the negative side-effect phenotype.
After this finding, the release of varieties resistant to Nasonovia became more and more important. The resistance became a major requirement for outdoor lettuce production for processing and also for fresh market.
In 2007, populations of lettuce aphids able to infect varieties resistant to Nasonovia ribisnigri were found in four distinct areas in Europe (France and Germany but also in Belgium and Austria). Four isolates (two from France and two from Germany) were analysed further by the Netherlands Inspection Service for Horticulture (also known as Naktuinbouw). They concluded the existence of a new Nasonovia ribisnigri biotype. This biotype is officially named Nr:1 and is able to overcome the Nr resistance gene. The Nr:0 biotype of the aphid can, however, still be efficiently controlled by the Nr gene.
The presence of Nr:1 Nasonovia ribisnigri organisms on lettuce has the same disadvantages as described above for Nr:0.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a new source of Nasonovia ribisnigri resistance that is active against Nr:1.
According to the present invention a source of Nr:1 resistance is provided in the form of Lactuca serriola plants of which representative seed was deposited with the NCIMB and given accession number NCIMB 41776. The plants are further identified herein with the reference number 10G.913571. Plants carrying this number are grown from seed lot 10G.913571 of which a part was deposited under the above accession number.
The invention thus relates to a cultivated lettuce (Lactuca sativa) plant, which has the resistance against Nasonovia ribisnigri biotype 1 (Nr:1) as found in plants grown from seeds of Lactuca serriola 10G.913571, representative seeds of which were deposited on 16 Nov. 2010 with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK under deposit accession number NCIMB 41776.
A lettuce plant of the invention is obtainable by introgressing the resistance to Nasonovia risbisnigri Nr:1 as found in Lactuca serriola 10G.913571, representative seeds of which were deposited with the NCIMB under NCIMB accession number 41776, into a plant that is not resistant. “Introgression” as used herein is intended to mean introduction of a Nr:1 resistance into a plant not carrying the Nr:1 resistance by means of crossing and selection. Introgression is not limited to one cross only but encompasses the minimum number of generations needed for the plant to become resistant, preferably stably resistant.
A lettuce plant of the invention, which is resistant to Nasonovia ribisnigri biotype 1 (Nr:1), is obtainable by crossing a lettuce plant with a plant grown from seeds of Lactuca serriola 10G.913571, representative seeds of which were deposited with the NCIMB under NCIMB accession number 41776 and selecting in the progeny of the cross for plants showing the resistance. In one embodiment, selection is made in the F2 progeny that is obtained after selfing the F1 of the cross between the plant grown from deposited seed and the susceptible plant. Since the resistance is dominant, plants resistant against Nr:1 can also be found in the F1 progeny.
The invention further relates to seed of a lettuce plant as claimed and to progeny of the lettuce plant or the lettuce seeds.
According to a further aspect thereof, the invention relates to propagation material suitable for producing a plant as claimed. In one embodiment, such propagation material is formed by seeds.
In one embodiment, the propagation material is formed by parts of the plant that are suitable for sexual reproduction, in particular microspores, pollen, ovaries, ovules, embryo sacs and egg cells.
In one embodiment, the propagation material is formed by parts of the plant that are suitable for vegetative reproduction, in particular cuttings, roots, stems, cells and protoplasts, tissue cultures of regenerable cells.
In one embodiment, the propagation material is formed by parts of the plant that are suitable for preparing tissue cultures, in particular leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
Suitably, the plant produced from the propagation material shows the resistance to Nasonovia ribisnigri biotype 1 (Nr:1) according to the invention, in particular the resistance as found in Lactuca serriola 10G.913571, representative seeds of which were deposited with the NCIMB under NCIMB accession number 41776.
The invention further relates to the head of a lettuce plant as claimed. The invention further relates to a food product, comprising the lettuce head or parts thereof. In particular, the food product comprises leaves of the lettuce plant or parts thereof. The food product is for example a salad or a salad mixture comprising leaves of the lettuce plant of the invention.
According to a further aspect thereof the invention relates to the use of Lactuca serriola 10G.913571, representative seeds of which were deposited under NCIMB accession number 41776, in breeding to confer resistance against Nasonovia ribisnigri biotype 1 (Nr:1) to plants that are susceptible to Nasonovia ribisnigri biotype 1 (Nr:1).
The invention also relates to Lactuca serriola 10G.913571, representative seeds of which were deposited under NCIMB accession number 41776.
In one embodiment, the invention relates to lettuce plants of the species Lactuca sativa obtainable by crossing a first lettuce parent plant with a second lettuce parent plant that has the Nr:1 resistance as found in plants of which representative seed was deposited under deposit accession number NCIMB 41776 to obtain an F1 and selecting plants from the F2 that have the Nr:1 resistance as plants of the invention. In one embodiment, plants of the F1 are subsequently selfed to obtain an F2 and selecting plants from the F2 that have the Nr:1 resistance as plants of the invention. The Nr:1 resistance is transferred in a pattern consistent with dominant inheritance.
In one embodiment, the second lettuce parent plant is a plant grown from the deposited seeds. In one embodiment the second lettuce parent plant is a progeny plant of a plant grown from the deposited seeds. In one embodiment the second lettuce parent plant is a plant having the genetic information that encodes the Nr:1 resistance. In one embodiment this genetic information is substantially identical to the genetic information that encodes the Nr:1 resistance as found in plants of which representative seeds were deposited under accession number NCIMB 41776, in particular plants grown from seeds as deposited under deposit accession number NCIMB 41776.
In one embodiment, the invention thus relates to a lettuce plant, showing the Nr:1 resistance of the invention, which plant is obtainable by:

    
    
        a) growing plants representative seed of which was deposited under accession number NCIMB 41776;
        b) crossing a plant from step a) with a lettuce plant that does not have the Nr:1 resistance to obtain an F1 population;
        c) selfing plants from the F1 to obtain a F2 population; and
        d) identifying plants showing the Nr:1 resistance in the F2 population as Nr:1 resistant lettuce plants.
    
    


In the method described above steps c) and d) can be repeated one or more times by selfing an Fn population to obtain an Fn+1 population and identifying plants showing the Nr:1 resistance in the Fn+1 population as Nr:1 resistant lettuce plants.
Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
DEPOSITS
The Deposits with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK, under deposit accession number NCIMB 41776 was made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet the requirements of 37 CFR §1.801-1.809. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.


DETAILED DESCRIPTION OF THE INVENTION
The Nr:1 resistance is phenotypically visible because plants will not be attacked by aphids of the Nr:1 biotype. The Nr:1 resistance is however caused by the plant's genotype. The genotype as far as it concerns the Nr:1 resistance is the same or a similar genotype as found in the deposited seeds. The part of the genotype of a lettuce plant that causes the Nr:1 resistance will be called herein the “genetic information that encodes the Nr:1 resistance”. Presence of this genetic information is phenotypically visible in a resistance test and plants having this genetic information can thus be selected on the basis of this phenotypic expression of the underlying gene or genes. As used herein “genetic information” is intended to mean the gene or genes that are responsible for the resistance.
The presence of the genetic information that is responsible for the Nr:1 resistance of the invention in the genome of a resistant plant can be determined with the following test. The plant to be tested should be or should be made homozygous for the genetic information responsible for the Nr:1 resistance. The skilled person knows how to obtain a plant that is homozygous for the Nr:1 resistance to be tested.
This plant is then crossed with a tester plant that carries the genetic information that is responsible for the Nr:1 resistance of the invention in homozygous condition. If the plant to be tested has the resistance as a result of the same genetic information that is responsible for the resistance of the invention, all progeny plants of the first cross and successive generations will be resistant. If the resistance of the plant to be tested is the result of a different part of the genome, e.g. another gene or locus, segregation will occur from the F2 onward. The tester plant can be any plant that carries the genetic information of the invention in homozygous condition, such as plants of which representative seed was deposited under accession number NCIMB 41776 or plants directly grown from the deposited seeds or progeny thereof that has retained the resistance.
The resistance to Nasonovia ribisnigri biotype 1 (Nr:1) of the invention is at a locus corresponding to the locus comprised in plants grown from seed of seed lot 10G.913571, a representative sample of which was deposited with NCIMB under accession number NCIMB 41776, and preferably co-segregates with the resistance present in plants grown from seed lot 10G.913571.
The locus conferring the resistance to Nasonovia ribisnigri biotype 1 (Nr:1) is obtainable from Lactuca serriola 10G.913571 plants, representative seeds of which have the accession number NCIMB 41776, or from a progeny or ancestor of plants of 10G.913571 plants comprising said resistance. In another preferred embodiment, the locus for the resistance is derived or obtained from Lactuca serriola 10G.913571, or from a progeny or ancestor of Lactuca serriola 10G.913571 comprising said resistance.
The present invention further relates to a lettuce plant comprising a resistance to Nasonovia ribisnigri biotype 1 (Nr:1), wherein when said plant is homozygous for said resistance and said plant homozygous for said resistance is crossed with a tester plant homozygous for a monogenic and dominant resistance to Nasonovia ribisnigri biotype 1 (Nr:1), the plants of the first generation progeny resulting from said cross show a 1:0 segregation for the resistance.
When said plants of said first generation progeny are self-pollinated, plants of the resulting second generation show a 3:1 segregation for the resistance.
In one embodiment, the tester plant is a plant derived from Lactuca serriola 10G.913571 plants, representative seed of which has accession number NCIMB 41776 and comprises the monogenic and dominant resistance to Nasonovia ribisnigri biotype 1 (Nr:1) of the invention, or a progeny or ancestor plant thereof that has the resistance locus. In one embodiment, the tester plant is derived from Lactuca serriola 10G.913571 or a progeny plant thereof by cell fusion.
The present invention further relates to a lettuce plant comprising a resistance to Nasonovia ribisnigri biotype 1 (Nr:1), wherein when said plant is heterozygous for said resistance and said plant heterozygous for said resistance is crossed with a tester plant heterozygous for a monogenic and dominant resistance to Nr:1, plants of the first generation progeny resulting from said cross show a 3:1 segregation for resistance to Nr:1.
In one embodiment, when said resistant plants of said first generation progeny are further crossed with said plant heterozygous for said resistance, plants of the resulting second generation progeny show a 5:1 segregation for resistance to Nr: 1. In a further preferred embodiment, the tester plant is a Lactuca serriola 10G.913571 plant, representative seed of which has the accession number NCIMB 41776, or a progeny or ancestor plant thereof comprising the monogenic and dominant resistance to Nr:1 comprised in said Lactuca serriola 10G.913571 plants.
In one embodiment, when progenies of individual plants are scored, the resulting second generation progeny shows a 3:1, 1:0 or 1:1 segregation ratio for the resistance, depending on the genetic status of the individual plants in the first generation offspring (heterozygous, homozygous resistant or homozygous susceptible respectively).
The resistance to Nr:1 of the invention is monogenic, preferably monogenic and dominant. In one embodiment, the Lactuca sativa plant is homozygous for the resistance. In another embodiment, the Lactuca sativa plant is heterozygous for the resistance.
The Nr:1 resistance is independent of other Nr:1 resistances of a lettuce plant. The resistance of the invention can thus occur in lettuce plants that are completely different from the deposited plant in all their other characteristics, for example in different lettuce varieties.
It is clear that a parent that provides the Nr:1 resistance of the invention is not necessarily a plant grown directly from the deposited seeds. The parent can also be a progeny plant from the seed or a progeny plant from seeds that are identified to have or to have acquired the Nr:1 resistance of the invention by other means.
In one embodiment, the invention relates to lettuce plants that carry the resistance of the invention and have acquired said resistance by introduction of the genetic information that is responsible for the resistance from a suitable source, in particular Lactuca serriola 10G.913571, either by conventional breeding, or genetic modification, in particular by cisgenesis or transgenesis. Cisgenesis is genetic modification of plants with a natural gene, coding for Nr:1 resistance, from the crop plant itself or from a sexually compatible donor plant. Transgenesis is genetic modification of a plant with a gene from a non-crossable species or a synthetic gene.
In one embodiment, the source from which the genetic information is acquired is formed by plants grown from the deposited seeds of NCIMB 41776 or sexual or vegetative descendants thereof.
According to another aspect of the invention Lactuca sativa plants are provided that have all of the morphological and physiological characteristics of Nr:1 resistant lettuce plants of the invention, representative seed of which have the accession number NCIMB 41776, which plants are grown from seeds of a plant of the invention or regenerated from parts thereof, or from a tissue culture. Plants of the invention should have the morphological and physiological characteristics that correspond with the Nr:1 resistance but do not necessarily have all the other characteristics of plants of the deposited seeds. The Nr:1 resistance is broadly transferrable over multiple lettuce types and varieties.
The invention also relates to progeny of the lettuce plants of the invention. Such progeny can be produced by sexual or vegetative reproduction of a plant of the invention or a progeny plant thereof. The regenerated progeny plant shows the resistance in the same or a similar way as the plant, of which representative seed was deposited (NCIMB 41776). This means that such progeny has the same characteristics as claimed for the lettuce plants of the invention. In addition to this, the plant may be modified in one or more other characteristics. Such additional modifications are for example effected by mutagenesis or by transformation with a transgene or cisgene. Alternatively, modifications in characteristics other than the Nr:1 resistance can be introduced by introducing the Nr:1 resistance in a different background. In the first case the resistant plant is taken as the original plant and modified. In the latter case the resistance is transferred to another plant, in particular a susceptible plant.
As used herein the word “progeny” is intended to mean the offspring or the first and all further descendants from a cross with a plant of the invention that is Nr:1 resistant. Progeny of the invention are descendants of any cross with a plant of the invention that carries the Nr:1 resistance. Progeny plants are preferably also resistant.
“Progeny” also encompasses plants that carry the Nr:1 resistance of the invention which are obtained from other plants of the invention by vegetative propagation or multiplication.
The invention further relates to cells of Nr:1 resistant plants as described herein. The cells comprise the genetic information, i.e. gene(s) or locus/loci, that leads to the Nr:1 resistance as described herein. Suitably, this genetic information is substantially identical, preferably completely identical to the genetic information encoding the Nr:1 resistance of plants that have all of the morphological and physiological characteristics pertaining to the Nr:1 resistance of lettuce plants of the invention, of which representative seed has the accession number NCIMB 41776. Preferably, the cell of the invention is part of a plant or plant part, but the cell may also be in isolated form.
In one embodiment the plants of the invention are plants grown from seeds having the deposit accession number NCIMB 41776.
In one embodiment the plants of the invention are progeny plants of plants grown from seeds having the deposit accession number NCIMB 41776 that carry the Nr:1 resistance.
In one embodiment the plants of the invention are plants that carry in their genome the genetic information that is responsible for the Nr:1 resistance.
The invention, furthermore, relates to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed. In order for the hybrid seed to express the Nr:1 resistance of the invention, at least one of the parent plants need to be homozygous for the Nr:1 resistance but is not necessarily uniform for other characteristics.
The invention also relates to the germplasm of plants of the invention. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the Nr:1 resistance of the invention.
In this specification the term “Nr:1 resistance” is intended to mean having the genetic information (in particular a gene, genes, locus, loci, allele or alleles) that in heterozygous or homozygous state leads to the plant no longer being susceptible to Nasonovia ribisnigri biotype 1 (Nr:1) and which resistance is as found in plants of which representative seeds were deposited under accession number NCIMB 41776, in particular in plants grown from the deposited seeds.
Selection of plants for breeding therefore can also be independent of the phenotype of a plant and instead can be based on genetic investigations. For example, one can utilize a suitable genetic marker which is closely genetically linked to a trait of interest—the Nr:1 resistance trait in this instance, e.g., as identified using seed deposited under deposit accession number NCIMB 41776. These markers can be used to identify the presence of the trait in the offspring of a particular cross (e.g., two heterozygous plants that carry the genetic information for the Nr:1 resistance trait but do not have that phenotype because it is recessive and they are not homozygous therefor), and can be used in selection of progeny for continued breeding. This technique is commonly referred to as marker assisted selection. Any other type of genetic marker or other assay which is able to identify the relative presence or absence of a trait of interest in a plant can also be useful for breeding purposes. Methods for marker assisted selection are of particular utility in the case of recessive traits and variable phenotypes, or where conventional assays may be more expensive, time consuming or otherwise disadvantageous, and types of genetic markers which could be used in accordance with the invention include, but are not necessarily limited to, Simple Sequence Length Polymorphisms (SSLPs), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs). Reference is made to U.S. Pat. No. 7,705,206, for documents and a discussion relating to the foregoing methods; and, U.S. Pat. No. 7,705,206, and the documents cited therein, including as to the foregoing methods, are hereby incorporated herein by reference consistent with the above RELATED APPLICATIONS & INCORPORATION BY REFERENCE section.
In an embodiment this genetic information is substantially identical to the genetic information that encodes the Nr:1 resistance trait as found in plants of which representative seeds were deposited under deposit accession number NCIMB 41776, in particular plants grown from seeds as deposited under deposit accession number NCIMB 41776 (wherein “substantially identical” is identical to the genetic information in the seeds deposited under NCIMB 41776, or so similar to the genetic information in the seeds deposited under NCIMB 41776 at the locus or gene that gives rise to expression of the Nr:1 resistance trait phenotype in the seeds deposited under deposit number NCIMB 41776 such that the sample also has the Nr:1 resistance trait, e.g., a sample can be “substantially identical” in its genome to seeds deposited under deposit number NCIMB 41776 because it is different than the genome of seeds deposited under deposit numbers NCIMB 41776 due to the degeneracy of the genetic code).
In an embodiment, the invention thus relates to a plant, showing the Nr:1 resistance trait, which plant is obtainable by:
a) growing plants representative seed of which was deposited under NCIMB 41776;
b) crossing a plant from step a) with a plant that does not have the Nr:1 resistance trait to obtain an F1 population;
c) selfing plants from the F1 to obtain a F2 population; and

    
    
        d) identifying plants showing the Nr:1 resistance trait in the F2 population as lettuce plant resistant against Nr:1.
    
    


In the method described above steps c) and d) can be repeated one or more times by selfing an Fn population to obtain an Fn+1 population and identifying plants showing the Nr:1 resistance trait in the Fn+1 population as lettuce plant resistant against Nr:1.
The genotype as far as it concerns the Nr:1 resistance trait is the same as or substantially similar or identical to genotype as found in the deposited seeds. On a nucleic acid molecule basis, this can mean a first nucleic acid molecule having at least about 95, 96, 97, 98 or 99 percent identity with a second nucleic molecule, wherein the second acid molecule is of a herein identified or exemplified or deposited plant, plant part, seed, cell or the like, and the expression of both the first and second nucleic acid molecules in a plant results in the phenotype of the Nr:1 resistance trait. The part of the genotype of a plant that causes the Nr:1 resistance trait will be called herein the “genetic information that encodes the Nr:1 resistance trait”. Presence of this genetic information is phenotypically visible and plants having this genetic information can thus be selected on the basis of this phenotypic expression of the underlying gene or genes.
As used herein “genetic information” is intended to mean the portion of the genome, e.g. gene or genes, that are responsible for the formation of significantly more leaves; which portion of the genome can be detected in the genome, e.g. by detecting polymorphisms in the genome of “Nr:1 resistance trait” plants of which representative seed was deposited under deposit accession number NCIMB 41776, and that portion of the genome, particularly the portion that gives rise to expression of the “Nr:1 resistance trait” can thus be isolated from the genome and can be an isolated nucleic acid molecule encoding the Nr:1 resistance trait (which when introduced into the genome of a regenerable cell of a plant that does not carry this genotype and when in the cell operably linked to and under the control of a suitable promoter, gives rise to the expression of the “Nr:1 resistance trait” in a plant regenerated from a tissue culture of such regenerable cells into which the isolated nucleic acid molecule has been introduced and is present in the cells operably linked to and under the control of a suitable promoter).
The presence of the genetic information that is responsible for the Nr:1 resistance trait of the invention in the genome of a plant that shows a Nr:1 resistant characteristic can be determined with the following test: The plant to be tested should be or should be made to be homozygous for the genetic information responsible for the Nr:1 resistance trait. The skilled person knows how to obtain a plant that is homozygous for the trait to be tested, e.g., via selfing or self mating or self crossing. This homozygous plant is then crossed with a tester plant that carries the genetic information that is responsible for the trait of the invention in homozygous condition. If the plant to be tested has a Nr:1 resistant characteristic as a result of the same genetic information that is responsible for the trait of the invention, all progeny plants of this first cross and successive generations will express the trait. If the Nr:1 resistant characteristic of the plant to be tested is the result of a different part of the genome, e.g. another gene or locus, segregation will occur. The tester plant can be any plant that carries the genetic information of the invention in homozygous condition, such as plants of which representative seed was deposited under accession number NCIMB 41776 or plants directly grown from the deposited seeds or progeny thereof that has retained the trait.
In an embodiment of the invention a plant is provided that comprises the Nr:1 resistance trait and thus when crossed with a tester plant, that comprises the Nr:1 resistance trait of the invention and representative seed of which as deposited with the NCIMB under accession numbers NCIMB 41776, or a progeny plant thereof that comprises the Nr:1 resistance trait comprised in plants representative seed of which was deposited with the NCIMB under accession number NCIMB 41776 or a plant derived therefrom and comprising the Nr:1 resistance trait, plants of the first generation progeny (F1) of said cross show a 1:0 segregation for the Nr:1 resistance trait. In both the tester plant and the plant of the invention the Nr:1 resistance trait is present in homozygous condition. plants of the second and further generations, if obtained by selfing also show a 1:0 segregation for the Nr:1 resistance trait. The tester plant can be a plant of which representative seed was deposited with the NCIMB under accession number NCIMB 41776. When the genetic information responsible for the Nr:1 resistance trait as contained in the deposit is present in a plant, the plant is a plant of the invention (and seeds therefrom are seeds of the invention, plant parts thereof are plant parts of the invention, etc.).
The Nr:1 resistant characteristic of the invention has a genetic basis in the genome of the plant. With the above described cross with a tester plant, plants can be identified as being plants of the invention.
The Nr:1 resistance trait is independent of other traits of a plant. The trait can thus occur in plants that are completely different in all their other characteristics, for example in different varieties.
The deposited seeds contain in their genome the genetic information that encodes the Nr:1 resistance trait. The deposited seeds are thus a source for the genetic information that leads to the trait. The skilled person is capable of introducing the trait into any other plant he desires. A plant resulting from the initial cross between a first parent plant with a second parent plant that contains the genetic information responsible for the Nr:1 resistance trait, cannot yet be identified as being a plant of the invention. Therefore, an F2 generation is produced by selfing plants of the F1 and assessing the number of leaves of the F2 progeny plant and comparing it with the number of leaves of the first parent plant. If this number is at least 1.25 times higher than the number of leaves in the first parent plant, the progeny plant is a plant of the invention.
In one embodiment, the source from which the genetic information is acquired is formed by plants grown from the deposited seeds or sexual or vegetative descendants therefrom.
It is clear, however, that a parent that provides the trait of the invention is not necessarily a plant grown directly from the deposited seeds. For example, the parent can also be a progeny plant from the seed or a progeny plant from seeds that are identified to have or to have acquired the trait of the invention by other means.
In an embodiment, the invention relates to plants that carry the trait of the invention and have acquired said trait by introduction of the genetic information that is responsible for the trait from a suitable source, either by conventional breeding, or genetic modification, in particular by cisgenesis or transgenesis. Cisgenesis is genetic modification of plants with a natural gene, coding for an (agricultural) trait, from the crop plant itself or from a sexually compatible donor plant. Transgenesis is genetic modification of a plant with a gene from a non-crossable species or a synthetic gene.
Just as useful traits that can be introduced by backcrossing, useful traits can be introduced directly into a Nr:1 resistance trait plant of the invention, by genetic transformation techniques; and, such Nr:1 resistant plants that have additional genetic information introduced into the genome or that express additional traits by having the DNA coding therefor introduced into the genome via transformation techniques, are within the ambit of the invention, as well as uses of such plants, and the making of such plants.
Genetic transformation may therefore be used to insert a selected transgene into the plant of the invention or may, alternatively, be used for the preparation of transgenes which can be introduced by backcrossing. Methods for the transformation of plants are well known to those of skill in the art.
Vectors used for the transformation of cells are not limited so long as the vector can express an inserted DNA in the cells. For example, vectors comprising promoters for constitutive gene expression in cells (e.g., cauliflower mosaic virus 35S promoter) and promoters inducible by exogenous stimuli can be used. Examples of suitable vectors include pBI binary vector. The cell” into which the vector is to be introduced includes various forms of cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus. A vector can be introduced into cells by known methods, such as the polyethylene glycol method, polycation method, electroporation, Agrobacterium-mediated transfer, particle bombardment and direct DNA uptake by protoplasts.
To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wound tissues in a controlled manner.
A particularly efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a surface covered with target cells. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species, including Nr:1 resistance trait of the invention.
Agrobacterium-mediated transfer is another widely applicable system for introducing gene loci into plant cells. An advantage of the technique is that DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations. Moreover, advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene locus transfer. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells, including plant cells, is well known in the art (See, e.g., U.S. Pat. Nos. 7,250,560 and 5,563,055).
Transformation of plant protoplasts also can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments.
A number of promoters have utility for plant gene expression for any gene of interest including but not limited to selectable markers, scoreable markers, genes for pest tolerance, disease resistance, nutritional enhancements and any other gene of agronomic interest. Examples of constitutive promoters useful for plant gene expression include, but are not limited to, the cauliflower mosaic virus (CaMV) P-35S promoter, a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S), the nopaline synthase promoter, the octopine synthase promoter, the figwort mosaic virus (P-FMV) promoter (see U.S. Pat. No. 5,378,619), an enhanced version of the FMV promoter (P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform virus promoter, a commelina yellow mottle virus promoter, the promoter for the thylakoid membrane proteins from (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS) (see U.S. Pat. No. 7,161,061), the CAB-1 promoter from (see U.S. Pat. No. 7,663,027), the promoter from maize prolamin seed storage protein (see U.S. Pat. No. 7,119,255), and other plant DNA virus promoters known to express in plant cells. A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals can be used for expression of an operably linked gene in plant cells, including promoters regulated by (1) heat, (2) light (e.g., pea rbcS-3A promoter, maize rbcS promoter, or chlorophyll a/b-binding protein promoter), (3) hormones, such as abscisic acid, (4) wounding (e.g., wunl, or (5) chemicals such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific promoters.
Exemplary nucleic acids which may be introduced to the Nr:1 resistance trait of this invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Many hundreds if not thousands of different genes are known and could potentially be introduced into a Nr:1 resistance trait plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a plant include one or more genes for insect tolerance, pest tolerance such as genes for fungal disease control, herbicide tolerance, and genes for quality improvements such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s).
Alternatively, the DNA coding sequences can affect these phenotypes by encoding a non-translatable RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense- or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product. Thus, any gene which produces a protein or mRNA which expresses a phenotype or morphology change of interest is useful for the practice of the present invention. (See also U.S. Pat. No. 7,576,262, “Modified gene-silencing RNA and uses thereof”)
U.S. Pat. Nos. 7,230,158, 7,122,720, 7,081,363, 6,734,341, 6,503,732, 6,392,121, 6,087,560, 5,981,181, 5,977,060, 5,608,146, 5,516,667, each of which, and all documents cited therein are hereby incorporated herein by reference, consistent with the above RELATED APPLICATIONS & INCORPORATION BY REFERENCE section, are additionally cited as examples of U.S. patents that may concern transformed and/or methods of transforming plants or plant cells, and techniques from these US patents, as well as promoters, vectors, etc., may be employed in the practice of this invention to introduce exogenous nucleic acid sequence(s) into the Nr:1 resistance plant (or cells thereof) of the invention, and exemplify some exogenous nucleic acid sequence(s) which can be introduced into the Nr:1 resistant plant (or cells thereof) of the invention, as well as techniques, promoters, vectors etc., to thereby obtain further Nr:1 resistance trait plants, plant parts and cells, seeds, other propagation material harvestable parts of these plants, etc. of the invention, e.g. tissue culture, including a cell or protoplast, such as an embryo, meristem, cotyledon, pollen, leaf, anther, root, root tip, pistil, flower, seed or stalk.
The invention further relates to propagation material for producing plants of the invention. Such propagation material comprises inter alia seeds of the claimed plant and parts of the plant that are suitable for sexual reproduction. Such parts are for example selected from the group consisting of seeds, microspores, pollen, ovaries, ovules, embryo sacs and egg cells. In addition, the invention relates to propagation material comprising parts of the plant that are suitable for vegetative reproduction, for example cuttings, roots, stems, cells, protoplasts.
According to a further aspect thereof the propagation material of the invention comprises a tissue culture of the claimed plant. The tissue culture comprises regenerable cells. Such tissue culture can be derived from leaves, pollen, embryos, cotyledon, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems. (See generally U.S. Pat. No. 7,041,876 on being recognized as a plant that can be regenerated from cultured cells or tissue).
According to another aspect of the invention plants are provided that have all of the morphological and physiological characteristics corresponding to the Nr:1 resistance trait of Nr:1 resistant plants of the invention, representative seed of which having been deposited under NCIMB Accession No NCIMB 41776, which plants are grown from seeds of a plant of the invention or regenerated from parts thereof, or from a tissue culture. plants of the invention should have the morphological and physiological characteristics that correspond with the Nr:1 resistance trait but do not necessarily have all the other characteristics of plants of the deposited seeds. The trait is broadly transferrable over multiple types and varieties.
The invention further relates to cells of Nr:1 resistant plants as described herein. The cells comprise the genetic information that leads to the Nr:1 resistance trait as described herein. Suitably, this genetic information is substantially identical, preferably completely identical to the genetic information encoding the Nr:1 resistance trait of plants that have all of the morphological and physiological characteristics pertaining to the Nr:1 resistance trait of Nr:1 resistant plants of the invention, representative seed of which having been deposited under accession number NCIMB 41776. Preferably, the cell of the invention is part of a plant or plant part, but the cell may also be in isolated form.
In an embodiment the plants of the invention are plants grown from seeds having the deposit accession number NCIMB 41776.
In an embodiment the plants of the invention are progeny plants of plants grown from seeds having the deposit accession number NCIMB 41776 that carry the Nr:1 resistance trait.
In an embodiment the plants of the invention are plants that carry in their genome the genetic information that is responsible for the Nr:1 resistance trait by causing the plant to form significantly more leaves than a plant not having the said genetic information in its genome.
The invention, furthermore, relates to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed. In order for the hybrid seed to express the trait of the invention, both parent plants need to be homozygous for the Nr:1 resistance trait but not necessarily uniform for other traits.
The invention also relates to the germplasm of plants of the invention. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the Nr:1 resistance trait of the invention.
The invention also relates to the Nr:1 resistant leaves that are produced by the plants of the invention and marketed as vegetables, either as fresh vegetables or processed, i.e. cooked, and optionally frozen.
The invention further relates to a container comprising one or more plants of the invention in a growth substrate for harvest of leaves from the plant in a domestic environment. This way the consumer can pick very fresh leaves for use in salads. More generally, the invention includes one or more plants of the invention wherein the plant is in a ready-to-harvest condition, including with the consumer picking his own, and further including a container comprising one or more of these plants.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.
EXAMPLE
Protocol of the Young-Plant Test
In order to test whether a plant is resistant to Nr:1 the following test is performed.
Seeds are sown in potting compost blocks. Twenty plants per line were evaluated. The young plants are inoculated 2 weeks after sowing by pouring aphids of the Nr:1 biotype on the top of the plants. One week after inoculation, the test is scored. The second and final score occurs two weeks after inoculation.
Nasonovia ribisnigri Isolates
The Nr:1 isolate used in this example was sampled in 2007 from Köningen (Germany) on a Nr:0 resistant variety Estelle (Nunhems): sample 279. The aphid isolate is maintained on the variety Kitare (Nr:0 resistant variety, Rijk Zwaan).
Introduction of the Nr:1 Resistance into Cultivated Lettuce
Nr:1 resistant L. serriola 10G.913571 plants (called herein “the donors”) were used to introgress the Nr:1 resistance into cultivated lettuce (L. sativa).
These donors were crossed to a cultivated lettuce variety. Cultivated varieties belonging to different lettuce types are used. Many F1 seeds were obtained. The F1s obtained were then crossed with the different varieties used in the original F1 (also called the “recurrent parents”). The BC1F1 population is propagated into BC by one cycle of inbreeding. Then, the different BC1F2 populations are placed in a Nr:1 disease resistance test as described above. At the end of the test, Nr:1 resistant plantlet are selected.
These selected plants are crossed again to the cultivated lettuce varieties (recurrent parents). The F1s obtained are propagated into F2 generation and the F2 populations are again placed in the Nr:1 disease test.
Resistant F2 plants are selected and crossed again to the recurrent parent. By performing this recurrent breeding process (also named backcrossing) the agronomic properties of the breeding material is improved and in the end the Nr:1 resistance in a cultivated lettuce is obtained.
The invention is further described by the following numbered paragraphs:
1. Lettuce (Lactuca sativa) plant, which has the resistance against Nasonovia ribisnigri biotype 1 (Nr:1) as found in plants grown from seeds of Lactuca serriola 10G.913571 representative seeds of which were deposited under NCIMB accession number 41776.
2. Lettuce plant of paragraph 1, obtainable by introgressing the resistance to Nasonovia risbisnigri Nr:1 as found in Lactuca serriola 10G.913571 representative seeds of which were deposited under NCIMB accession number 41776 into a plant that is not resistant.
3. Lettuce plant of paragraph 1 or 2, which is resistant to Nasonovia ribisnigri biotype 1 (Nr:1), and which plant is obtainable by crossing a lettuce plant with a plant grown from seeds of L. serriola 10G.913571 representative seeds of which were deposited with the NCIMB under NCIMB accession number 41776 and selecting in the F2 progeny of the cross that is obtained after selling the F1 for plants showing the resistance.
4. Seed of a lettuce plant of paragraph any one of the paragraphs 1-3.
5. Seed of paragraph 4, wherein the plant that can be grown from the seed is resistant to Nasonovia ribisnigri biotype 1 (Nr:1).
6. Progeny of a lettuce plant or lettuce seeds of any one of paragraphs 1 to 5, wherein the progeny is resistant to Nasonovia ribisnigri biotype 1 (Nr:1).
7. Propagation material suitable for producing a plant as claimed in any one of the claims 1-6.
8. Propagation material of paragraph 7, which propagation material is formed by seeds.
9. Propagation material of paragraph 7, which propagation material is formed by parts of the plant that are suitable for sexual reproduction, in particular microspores, pollen, ovaries, ovules, embryo sacs and egg cells.
10. Propagation material of paragraph 7, which propagation material is formed by parts of the plant that are suitable for vegetative reproduction, in particular cuttings, roots, stems, cells and protoplasts, tissue cultures of regenerable cells.
11. Propagation material of paragraph 7, which propagation material is formed by parts of the plant that are suitable for preparing tissue cultures, in particular leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
12. Propagation material of any one of paragraphs 7-11, wherein the plant produced from the propagation material is resistant to Nasonovia ribisnigri biotype 1 (Nr:1)
13. Head of a lettuce of paragraph of any one of paragraphs 1-6.
14. Food product, comprising the lettuce head of paragraph 13 or parts thereof
15. Food product of paragraph 14, wherein the parts are leaves or parts thereof
16. Use of Lactuca serriola 10G.913571, representative seeds of which were deposited under NCIMB accession number 41776, in breeding to confer resistance against Nasonovia ribisnigri biotype 1 (Nr:1) to plants that are susceptible to Nasonovia ribisnigri biotype 1 (Nr:1).
17. Lactuca serriola 10G.913571 representative seeds of which were deposited under NCIMB accession number 41776.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

## Claims

1. A method of breeding to confer to a progeny Lactuca plant resistance against Nasonovia ribisnigri biotype 1 (Nr:1) comprising crossing a Lactuca serriola 10G.913571 plant, representative seeds of which were deposited under NCIMB accession number 41776, with a Lactuca plant that is susceptible to Nasonovia ribisnigri biotype 1 (Nr:1), whereby there is breeding to confer to a progeny Lactuca plant resistance against Nasonovia ribisnigri biotype 1 (Nr:1), and a progeny Lactuca plant having resistance against Nasonovia ribisnigri biotype 1 (Nr:1) is obtained.

2. Lactuca serriola 10G.913571 representative seeds of which were deposited under NCIMB accession number 41776.