Spinach hybrid SVVC5883 and parents thereof

Abstract

The invention provides seeds and plants of spinach hybrid SVVC5883 and spinach line SSB-S019-1303F. The invention thus relates to the plants, seeds, plant parts, and tissue cultures of spinach hybrid SVVC5883 and spinach line SSB-S019-1303F and to methods for producing a spinach plant produced by crossing such plants with themselves or with another plant, such as a spinach plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to plants, seeds, plant parts, and tissue cultures of spinach hybrid SVVC5883 and spinach line SSB-S019-1303F comprising introduced beneficial or desirable traits.

Claims

1. A spinach plant comprising at least a first set of chromosomes of spinach line SSB-S019-1303F, a sample of seed of said line having been deposited under NCMA Accession No. 202104017.

2. A spinach seed that produces the plant of claim 1.

3. The plant of claim 1, wherein the plant is a plant of said spinach line SSB-S019-F.

4. The plant of claim 1, wherein the plant is a plant of spinach hybrid SVVC5883, a sample of seed of said hybrid having been deposited under NCMA Accession No. 202104018.

5. The seed of claim 2, wherein said seed is a seed of said spinach line SSB-S019-1303F.

6. The seed of claim 2, wherein the seed is a seed of spinach hybrid SVVC5883, a sample of seed of said hybrid having been deposited under NCMA Accession No. 202104018.

7. A plant part of the plant of claim 1, wherein said plant part comprises a cell of said plant, and wherein said cell comprises at least said first set of chromosomes of spinach line SSB-S019-1303F.

8. A spinach plant having all of the physiological and morphological characteristics of the plant of claim 1.

9. A tissue culture of regenerable cells of the plant of claim 1, wherein said cells comprise at least said first set of chromosomes of spinach line SSB-S019-1303F.

10. A method of vegetatively propagating the plant of claim 1, the method comprising the steps of: (a) collecting tissue capable of being propagated from the plant of claim 1; and (b) propagating a spinach plant from said tissue.

11. A method of introducing a trait into a spinach line, the method comprising: (a) utilizing as a recurrent parent the plant of claim 3 by crossing said plant with a donor plant that comprises a trait to produce F.sub.1 progeny; (b) selecting an F.sub.1 progeny that comprises the trait; (c) backcrossing the selected F.sub.1 progeny with a plant of the same line used as the recurrent parent in step (a) to produce backcross progeny; (d) selecting a backcross progeny that comprises the trait and otherwise comprises all of the morphological and physiological characteristics of the recurrent parent line used in step (a); and (e) repeating steps (c) and (d) three or more times to produce a selected fourth or higher backcross progeny.

12. A spinach plant produced by the method of claim 11, wherein said plant comprises the trait and otherwise comprises all of the physiological and morphological characteristics of spinach line SSB-S019-1303F.

13. A method of producing a spinach plant comprising an added trait, the method comprising introducing a transgene conferring the trait into the plant of claim 1.

14. A spinach plant produced by the method of claim 13.

15. A spinach plant comprising at least a first set of chromosomes of spinach line SSB-S019-1303F, a sample of seed of said line having been deposited under NCMA Accession No. 202104017, further comprising a transgene.

16. The plant of claim 15, wherein said transgene confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect resistance, pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism, and modified protein metabolism.

17. A spinach plant comprising at least a first set of chromosomes of spinach line SSB-S019-1303F, a sample of seed of said line having been deposited under NCMA Accession No. 202104017, further comprising a single locus conversion.

18. The plant of claim 17, wherein said single locus conversion confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect resistance, pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism, and modified protein metabolism.

19. A method for producing a seed of a spinach plant derived from spinach hybrid SVVC5883 or spinach line SSB-S019-1303F, the method comprising the steps of: (a) crossing the plant of claim 1 with itself or a different spinach plant; and (b) allowing a seed of a spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant to form.

20. A method of producing a seed of a spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant, the method comprising the steps of: (a) producing a spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant from a seed produced by crossing the plant of claim 1 with itself or a different spinach plant; and (b) crossing the spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant with itself or a different spinach plant to obtain a seed of a further spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant.

21. The method of claim 20, the method further comprising repeating said producing and crossing steps of (a) and (b) using the seed from said step (b) for producing the plant according to step (a) for at least one generation to produce a seed of an additional spinach hybrid SVVC5883- or spinach line SSB-S019-1303F-derived spinach plant.

22. A method of producing food, the method comprising: (a) obtaining the plant of claim 1, wherein the plant has been cultivated to maturity; and (b) collecting a plant part capable of being used as food from said plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention provides methods and compositions relating to plants, seeds, and derivatives of spinach hybrid SVVC5883, spinach line SSB-S019-1303F, and spinach line SSB-S015-1249M.

(2) Spinach hybrid SVVC5883 is a very slow maturing, smooth spinach variety for the open field market. The hybrid produces thick, erect, smooth dark green leaves with high yield. Spinach hybrid SVVC5883 comprises high resistance to Downy Mildew races Pe1-7,9-18 and intermediate resistance to Stemphylium beticola.

(3) Spinach line SSB-S019-1303F is a very late bolting monecious female line for the open field and processing markets. The line comprises high resistance to Downy Mildew races Pel-7, 9, 11, 13, 15-16, 18.

A. Origin and Breeding History of Spinach Hybrid SVVC5883

(4) The parents of spinach hybrid SVVC5883 are spinach line SSB-S019-1303F and spinach line SSB-S015-1249M. The parent lines are uniform and stable, as is a hybrid produced therefrom. A small percentage of variants can occur within commercially acceptable limits for almost any characteristic during the course of repeated multiplication. However no variants are expected.

B. Physiological and Morphological Characteristics of Spinach Hybrid SVVC5883 and Spinach Line SSB-5019-1303F

(5) In accordance with one aspect of the present invention, there are provided plants having the physiological and morphological characteristics of spinach hybrid SVVC5883 or spinach line SSB-S019-1303F. Descriptions of the physiological and morphological characteristics of such plants are presented in the tables that follow.

(6) TABLE-US-00001 TABLE 1 Physiological and Morphological Characteristics of Spinach Hybrid SVVC5883 CHARACTERISTIC SVVC5883 Caladonia Ploidy diploid diploid Seedling Cotyledon length (mm) 53.4 53.4 length short short width (mm) 6.6 5.8 tip pointed pointed color medium green medium green color (RHS Color Chart Value) 146b 147b Leaf (First Foliage Leaves) shape circular ovate base straight straight tip round round margin slightly curled slightly curled upper surface color medium green medium green color (RHS Color Chart Value) 146a 146a lower surface color (compared lighter lighter with upper surface) color (RHS Color Chart Value) 146b 146b Maturity growth rate medium medium days from planting to prime 46 46 market stage Plant (Prime Market Stage) habit flat flat size medium medium spread (cm) 47.3 47.1 height (cm) 13.0 13.1 Leaf (Prime Market Stage) surface semi-savoy semi-savoy anthocyanin coloration of absent absent petioles and veins blade, intensity of green color medium/dark medium/dark blade blistering medium weak/medium blade lobing absent or very weak weak petiole attitude horizontal horizontal petiole length short short blade attitude semi-erect horizontal leaf shape elliptic ovate blade shape (excluding basal lobes) broad elliptic broad ovate blade, curving of margin incurved incurved blade, shape of apex rounded rounded blade shape in longitudinal section concave flat base lobed lobed tip round round margin slightly curved slightly curved upper surface color medium green medium green color (RHS Color Chart Value) 146a 146a lower surface color (compared lighter lighter with upper surface) color (RHS Color Chart Value) 146b 146b luster dull dull blade size large large petiole color light green light green color (RHS Color Chart Value) 144a 144a petiole red pigmentation absent absent petiole length to the blade (cm) 6.6 5.9 petiole diameter (mm) 9.3 8.3 petiole diameter large large Seed Stalk Development start of bolting (10% of plants) late late time of start of bolting (spring very late very late sown crop, 15% of plants) height of stalk (cm) 75.3 65.5 leaves on stalk of female plant many many leaves on stalk of male plant n/a many proportion of female plants very high low plants that are female (percentage) 91-100% 11-35% plants that are male (percentage) 0-10% 11-35% proportion of male plants absent or very low low plants that are monoecious 0-10% 11-35% (percentage) proportion of monoecious plants absent or very low low Seed surface smooth smooth spines (harvested seed) absent absent These are typical values. Values may vary due to environment. Values that are substantially equivalent are within the scope of the invention.

(7) TABLE-US-00002 TABLE 2 Physiological and Morphological Characteristics of Spinach Line SSB-S019-1303F CHARACTERISTIC SSB-S019-1303F SSB-66-1066F Ploidy diploid diploid Seedling Cotyledon length (mm) 7.0 6.3 length short short width (mm) 53.7 51.9 tip pointed pointed color medium green medium green color (RHS Color Chart Value) 146b 147b Leaf (First Foliage Leaves) shape ovate ovate base straight v-shape tip round round margin curled under slightly curled upper surface color dark green dark green color (RHS Color Chart Value) 147a 147a lower surface color (compared lighter lighter with upper surface) color (RHS Color Chart Value) 147b 147b Maturity growth rate slow slow days from planting to 50 52 prime market stage Plant (Prime Market Stage) habit semi-erect flat size medium medium spread (cm) 42.7 38.4 height (cm) 13.5 10.1 Leaf (Prime Market Stage) surface smooth smooth anthocyanin coloration of petioles absent absent and veins blade, intensity of green color dark dark blade blistering medium strong blade lobing weak weak petiole attitude semi-erect horizontal petiole length short short blade attitude horizontal horizontal leaf shape circular ovate blade shape (excluding basal lobes) circular broad ovate blade, curving of margin incurved recurved blade, shape of apex rounded rounded blade shape in longitudinal section flat flat base lobed lobed tip round round margin slightly curved curled under upper surface color medium green dark green color (RHS Color Chart Value) 146a 147a lower surface color (compared lighter lighter with upper surface) color (RHS Color Chart Value) 146b 147b blade size medium medium petiole color light green light green color (RHS Color Chart Value) 144a 144a petiole red pigmentation absent absent petiole length to the blade (cm) 5.7 5.4 petiole diameter (mm) 8.0 8.0 petiole diameter medium medium Seed Stalk Development start of bolting (10% of plants) late late time of start of bolting (spring very late very late sown crop, 15% of plants) height of stalk (cm) 58.0 53.0 leaves on stalk of female plant many many leaves on stalk of male plant n/a n/a proportion of female plants very high very high plants that are female (percentage) 91-100% 91-100% plants that are male (percentage) 0-10% 0-10% proportion of male plants absent or very low absent or very low plants that are monoecious 0-10% 0-10% (percentage) proportion of monoecious plants absent or very low absent or very low Seed surface smooth smooth spines (harvested seed) absent absent These are typical values. Values may vary due to environment. Values that are substantially equivalent are within the scope of the invention.

C. Breeding Spinach Plants

(8) One aspect of the current invention concerns methods for producing seed of spinach hybrid SVVC5883 involving crossing spinach line SSB-S019-1303F and spinach line SSB-S015-1249M. Alternatively, in other embodiments of the invention, spinach hybrid SVVC5883 or spinach line SSB-S019-1303F may be crossed with itself or with any second plant. Such methods can be used for propagation of spinach hybrid SVVC5883 or spinach line SSB-S019-1303F or can be used to produce plants that are derived from spinach hybrid SVVC5883 or spinach line SSB-S019-1303F. Plants derived from spinach hybrid SVVC5883 or spinach line SSB-S019-1303F may be used, in certain embodiments, for the development of new spinach varieties.

(9) The development of new varieties using one or more starting varieties is well-known in the art. In accordance with the invention, novel varieties may be created by crossing spinach hybrid SVVC5883 or spinach line SSB-S019-1303F followed by multiple generations of breeding according to such well-known methods. New varieties may be created by crossing with any second plant. In selecting such a second plant to cross for the purpose of developing novel lines, it may be desired to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Once initial crosses have been made, inbreeding and selection take place to produce new varieties. For development of a uniform line, often five or more generations of selfing and selection are involved.

(10) Uniform lines of new varieties may also be developed by way of double-haploids. This technique allows the creation of true breeding lines without the need for multiple generations of selfing and selection. In this manner true breeding lines can be produced in as little as one generation. Haploid embryos may be produced from microspores, pollen, anther cultures, or ovary cultures. The haploid embryos may then be doubled autonomously, or by chemical treatments (e.g., colchicine treatment). Alternatively, haploid embryos may be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. In accordance with the invention, any of such techniques may be used in connection with a plant of the invention and progeny thereof to achieve a homozygous line.

(11) Backcrossing can also be used to improve an inbred plant. Backcrossing transfers a specific desirable trait from one inbred or non-inbred source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate locus or loci for the trait in question. The progeny of this cross are then mated back to the superior recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny have the characteristic being transferred, but are like the superior parent for most or almost all other loci. The last backcross generation would be selfed to give pure breeding progeny for the trait being transferred.

(12) The plants of the present invention are particularly well suited for the development of new lines based on the elite nature of the genetic background of the plants. In selecting a second plant to cross with spinach hybrid SVVC5883 or spinach line SSB-S019-1303F for the purpose of developing novel spinach lines, it will typically be preferred to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Examples of desirable traits may include, in specific embodiments, high seed yield, high seed germination, seedling vigor, high fruit yield, disease tolerance or resistance, and adaptability for soil and climate conditions. Consumer-driven traits, such as a fruit shape, color, texture, and taste are other examples of traits that may be incorporated into new lines of spinach plants developed by this invention.

D. Further Embodiments of the Invention

(13) In certain aspects of the invention, plants described herein are provided modified to include at least a first desired heritable trait. Such plants may, in one embodiment, be developed by a plant breeding technique called backcrossing, wherein essentially all of the morphological and physiological characteristics of a variety are recovered in addition to a genetic locus transferred into the plant via the backcrossing technique. The term single locus converted plant as used herein refers to those spinach plants which are developed by a plant breeding technique called backcrossing or by genetic engineering, wherein essentially all of the morphological and physiological characteristics of a variety are recovered or conserved in addition to the single locus introduced into the variety via the backcrossing or genetic engineering technique, respectively. By essentially all of the morphological and physiological characteristics, it is meant that the characteristics of a plant are recovered or conserved that are otherwise present when compared in the same environment, other than an occasional variant trait that might arise during backcrossing, introduction of a transgene, or application of a genetic engineering technique.

(14) Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the present variety. The parental spinach plant which contributes the locus for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental spinach plant to which the locus or loci from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.

(15) In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the single locus of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a spinach plant is obtained wherein essentially all of the morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred locus from the nonrecurrent parent.

(16) The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single locus of the recurrent variety is modified or substituted with the desired locus from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross; one of the major purposes is to add some commercially desirable trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered and the genetic distance between the recurrent and nonrecurrent parents. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele, or an additive allele (between recessive and dominant), may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

(17) In one embodiment, progeny spinach plants of a backcross in which a plant described herein is the recurrent parent comprise (i) the desired trait from the non-recurrent parent and (ii) all of the physiological and morphological characteristics of spinach the recurrent parent as determined at the 5% significance level when grown in the same environmental conditions.

(18) New varieties can also be developed from more than two parents. The technique, known as modified backcrossing, uses different recurrent parents during the backcrossing. Modified backcrossing may be used to replace the original recurrent parent with a variety having certain more desirable characteristics or multiple parents may be used to obtain different desirable characteristics from each.

(19) With the development of molecular markers associated with particular traits, it is possible to add additional traits into an established germ line, such as represented here, with the end result being substantially the same base germplasm with the addition of a new trait or traits. Molecular breeding, as described in Moose and Mumm, 2008 (Plant Physiol., 147: 969-977), for example, and elsewhere, provides a mechanism for integrating single or multiple traits or QTL into an elite line. This molecular breeding-facilitated movement of a trait or traits into an elite line may encompass incorporation of a particular genomic fragment associated with a particular trait of interest into the elite line by the mechanism of identification of the integrated genomic fragment with the use of flanking or associated marker assays. In the embodiment represented here, one, two, three or four genomic loci, for example, may be integrated into an elite line via this methodology. When this elite line containing the additional loci is further crossed with another parental elite line to produce hybrid offspring, it is possible to then incorporate at least eight separate additional loci into the hybrid. These additional loci may confer, for example, such traits as a disease resistance or a fruit quality trait. In one embodiment, each locus may confer a separate trait. In another embodiment, loci may need to be homozygous and exist in each parent line to confer a trait in the hybrid. In yet another embodiment, multiple loci may be combined to confer a single robust phenotype of a desired trait.

(20) Many single locus traits have been identified that are not regularly selected for in the development of a new inbred but that can be improved by backcrossing techniques. Single locus traits may or may not be transgenic; examples of these traits include, but are not limited to, herbicide resistance, resistance to bacterial, fungal, or viral disease, insect resistance, modified fatty acid or carbohydrate metabolism, and altered nutritional quality. These comprise genes generally inherited through the nucleus.

(21) Direct selection may be applied where the single locus acts as a dominant trait. For this selection process, the progeny of the initial cross are assayed for viral resistance or the presence of the corresponding gene prior to the backcrossing. Selection eliminates any plants that do not have the desired gene and resistance trait, and only those plants that have the trait are used in the subsequent backcross. This process is then repeated for all additional backcross generations.

(22) Selection of spinach plants for breeding is not necessarily dependent on 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. One of these markers can be used to identify the presence or absence of a trait in the offspring of a particular cross, 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. Procedures for marker assisted selection are well known in the art. Such methods will be 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. In addition, marker assisted selection may be used to identify plants comprising desirable genotypes at the seed, seedling, or plant stage, to identify or assess the purity of a cultivar, to catalog the genetic diversity of a germplasm collection, and to monitor specific alleles or haplotypes within an established cultivar.

(23) 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) (Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990), 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) (EP 534 858, specifically incorporated herein by reference in its entirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al., Science, 280:1077-1082, 1998).

(24) In particular embodiments of the invention, marker assisted selection is used to increase the efficiency of a backcrossing breeding scheme for producing a spinach line comprising a desired trait. This technique is commonly referred to as marker assisted backcrossing (MABC). This technique is well-known in the art and may involve, for example, the use of three or more levels of selection, including foreground selection to identity the presence of a desired locus, which may complement or replace phenotype screening protocols; recombinant selection to minimize linkage drag; and background selection to maximize recurrent parent genome recovery.

E. Plants Derived by Genetic Engineering

(25) Various genetic engineering technologies have been developed and may be used by those of skill in the art to introduce traits in plants. In certain aspects of the claimed invention, traits are introduced into spinach plants via altering or introducing a single genetic locus or transgene into the genome of a recited variety or progenitor thereof. Methods of genetic engineering to modify, delete, or insert genes and polynucleotides into the genomic DNA of plants are well-known in the art.

(26) In specific embodiments of the invention, improved spinach lines can be created through the site-specific modification of a plant genome. Methods of genetic engineering include, for example, utilizing sequence-specific nucleases such as zinc-finger nucleases (see, for example, U.S. Pat. Appl. Pub. No. 2011-0203012); engineered or native meganucleases; TALE-endonucleases (see, for example, U.S. Pat. Nos. 8,586,363 and 9,181,535); and RNA-guided endonucleases, such as those of the CRISPR/Cas systems (see, for example, U.S. Pat. Nos. 8,697,359 and 8,771,945 and U.S. Pat. Appl. Pub. No. 2014-0068797). One embodiment of the invention thus relates to utilizing a nuclease or any associated protein to carry out genome modification. This nuclease could be provided heterologously within donor template DNA for templated-genomic editing or in a separate molecule or vector. A recombinant DNA construct may also comprise a sequence encoding one or more guide RNAs to direct the nuclease to the site within the plant genome to be modified. Further methods for altering or introducing a single genetic locus include, for example, utilizing single-stranded oligonucleotides to introduce base pair modifications in a spinach plant genome (see, for example Sauer et al., Plant Physiol, 170(4):1917-1928, 2016).

(27) Methods for site-directed alteration or introduction of a single genetic locus are well-known in the art and include those that utilize sequence-specific nucleases, such as the aforementioned, or complexes of proteins and guide-RNA that cut genomic DNA to produce a double-strand break (DSB) or nick at a genetic locus. As is well-understood in the art, during the process of repairing the DSB or nick introduced by the nuclease enzyme, a donor template, transgene, or expression cassette polynucleotide may become integrated into the genome at the site of the DSB or nick. The presence of homology arms in the DNA to be integrated may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination or non-homologous end joining (NHEJ).

(28) In another embodiment of the invention, genetic transformation may be used to insert a selected transgene into a 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 that are well-known to those of skill in the art and applicable to many crop species include, but are not limited to, electroporation, microprojectile bombardment, Agrobacterium-mediated transformation, and direct DNA uptake by protoplasts.

(29) 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.

(30) An 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.

(31) 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. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species.

(32) 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. Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations (Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Moreover, recent technological 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 described 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.

(33) 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 is well known in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S. Pat. No. 5,563,055).

(34) Transformation of plant protoplasts also can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, for example, Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature, 312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986; Marcotte et al., Nature, 335:454, 1988). Transformation of plants and expression of foreign genetic elements is exemplified in Choi et al. (Plant Cell Rep., 13:344-348, 1994), and Ellul et al. (Theor. Appl. Genet., 107:462-469, 2003).

(35) 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, which confers constitutive, high-level expression in most plant tissues (see, for example, Odel et al., Nature, 313:810, 1985), including in monocots (see, for example, Dekeyser et al., Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S); the nopaline synthase promoter (An et al., Plant Physiol., 88:547, 1988); the octopine synthase promoter (Fromm et al., Plant Cell, 1:977, 1989); the figwort mosaic virus (P-FMV) promoter as described in 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; and other plant virus promoters known to express in plant cells.

(36) A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, or developmental signals can also be used for expression of an operably linked gene in plant cells, including promoters regulated by (1) heat (Callis et al., Plant Physiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4) wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5) chemicals, such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific promoters (e.g., Roshal et al., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988; Bustos et al., Plant Cell, 1:839, 1989).

(37) Exemplary nucleic acids which may be introduced to plants 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 the same 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.

(38) Many hundreds if not thousands of different genes are known and could potentially be introduced into a spinach plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a spinach plant include one or more genes for insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pest tolerance such as genes for fungal disease control, herbicide tolerance such as genes conferring glyphosate 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). For example, structural genes would include any gene that confers insect tolerance including but not limited to a Bacillus insect control protein gene as described in WO 99/31248, herein incorporated by reference in its entirety, U.S. Pat. No. 5,689,052, herein incorporated by reference in its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference in their entirety. In another embodiment, the structural gene can confer tolerance to the herbicide glyphosate as conferred by genes including, but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety, or glyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporated by reference in its entirety.

(39) 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 (see, for example, Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product (see, for example, Gibson and Shillito, Mol. Biotech., 7:125,1997). 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.

F. Definitions

(40) In the description and tables herein, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided:

(41) Allele: Any of one or more alternative forms of a genetic locus, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

(42) Backcrossing: A process in which a breeder repeatedly crosses hybrid progeny, for example a first generation hybrid (F.sup.1), back to one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions or transgenes from one genetic background into another.

(43) Crossing: The mating of two parent plants.

(44) Cross-Pollination: Fertilization by the union of two gametes from different plants.

(45) Diploid: A cell or organism having two sets of chromosomes.

(46) Emasculate: The removal of plant male sex organs or the inactivation of the organs with a cytoplasmic or nuclear genetic factor or a chemical agent conferring male sterility.

(47) Enzymes: Molecules which can act as catalysts in biological reactions.

(48) F.sub.1 Hybrid: The first generation progeny of the cross of two nonisogenic plants.

(49) Genotype: The genetic constitution of a cell or organism.

(50) Haploid: A cell or organism having one set of the two sets of chromosomes in a diploid.

(51) Linkage: A phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent.

(52) Marker: A readily detectable phenotype, preferably inherited in codominant fashion (both alleles at a locus in a diploid heterozygote are readily detectable), with no environmental variance component, i.e., heritability of 1.

(53) Phenotype: The detectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.

(54) Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.

(55) Resistance: As used herein, the terms “resistance” and “tolerance” are used interchangeably to describe plants that show no symptoms to a specified biotic pest, pathogen, abiotic influence, or environmental condition. These terms are also used to describe plants showing some symptoms but that are still able to produce marketable product with an acceptable yield. Some plants that are referred to as resistant or tolerant are only so in the sense that they may still produce a crop, even though the plants are stunted and the yield is reduced.

(56) Regeneration: The development of a plant from tissue culture.

(57) Royal Horticultural Society (RHS) Color Chart Value: The RHS Color Chart is a standardized reference which allows accurate identification of any color. A color's designation on the chart describes its hue, brightness, and saturation. A color is precisely named by the RHS Color Chart by identifying the group name, sheet number, and letter, e.g., Yellow-Orange Group 19A or Red Group 41B.

(58) Self-Pollination: The transfer of pollen from the anther to the stigma of the same plant.

(59) Single Locus Converted (Conversion) Plant: Plants which are developed by a plant breeding technique called backcrossing or genetic engineering of a locus, wherein essentially all of the morphological and physiological characteristics of a spinach variety are recovered in addition to the characteristics of the single locus.

(60) Substantially Equivalent: A characteristic that, when compared, does not show a statistically significant difference (e.g., p=0.05) from the mean.

(61) Tissue Culture: A composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.

(62) Transgene: A genetic locus comprising a sequence which has been introduced into the genome of a spinach plant by transformation or site-specific modification.

G. Deposit Information

(63) A deposit of at least 625 seeds of spinach hybrid SVVC5883 and spinach line SSB-S 019-1303F, disclosed above and recited in the claims, has been made with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA), 60 Bigelow Drive, East Boothbay, Me., USA. The date of deposit for spinach hybrid SVVC5883 and spinach line SSB-S 019-1303F is Apr. 12, 2021. The accession numbers for those deposited seeds of spinach hybrid SVVC5883 and spinach line SSB-S019-1303F are NCMA Accession No. 202104018 and NCMA Accession No. 202104017, respectively. Upon issuance of a patent, all restrictions upon the deposits will be removed, and the deposits are intended to meet all of the requirements of 37 C.F.R. §§ 1.801-1.809. The deposits have been accepted under the Budapest Treaty and will be maintained in the depository for a period of 30 years, 5 years after the last request, or the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.

(64) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.

(65) All references cited herein are hereby expressly incorporated herein by reference.