# Patent

## Title
Marigold male inbred line denominated KI4662

## Abstract

A new and distinct inbred plant of Tagentes erecta named KI4662 and characterized by elevated levels of zeaxanthin and little or no lutein.

## Background
RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Ser. No. 62/117,164 filed Feb. 17, 2015.


FIELD OF THE INVENTION
The present invention relates generally to marigolds (Tagetes erecta L.) and, more specifically, to a novel marigold male parent inbred line denominated KI4662 which has high levels of zeaxanthin and little or no lutein.
BACKGROUND OF THE INVENTION
Xanthophylls are yellow pigments that form one of two major divisions of the carotenoid group. Their molecular structure is similar to carotenes, which form the other major carotenoid group division, but xanthophylls contain oxygen atoms, while carotenes are purely hydrocarbons with no oxygen. Xanthophylls contain their oxygen either as hydroxyl groups and/or as pairs of hydrogen atoms that are substituted by oxygen atoms acting as a bridge (epoxide).
Like other carotenoids, xanthophylls are found in highest quantity in the leaves and/or flowers of most green plants, where they act to modulate light energy. The xanthophylls found in the bodies of animals, and in dietary animal products, are ultimately derived from plant sources in the diet. For example, the yellow color of chicken egg yolks, fat, and skin comes from ingested xanthophylls (primarily lutein, which is often added to chicken feed for this purpose).
The yellow color of the human macula lutea in the retina of the eye comes from the lutein and zeaxanthin it contains, both xanthophylls again requiring an exogenous source in the human diet to be present in the eye. These function in eye protection from ionizing blue light, which they absorb.
The group of xanthophylls includes (among many other compounds) lutein, zeaxanthin, neoxanthin, violaxanthin, and α- and β-cryptoxanthin.
There is an interest in developing products that have high levels of xanthophylls. For example, xanthophylls are known to have antioxidant properties and have been shown to prevent age-related macular degeneration (AMD).
Marigold flowers are the most commercially significant source of xanthophylls. A prominent supplier of xanthophyll pigments extracts them from a commercial marigold hybrid (Hybrid 50011, U.S. Pat. No. 6,894,208) which typically has a total xanthophyll composition of 94% lutein (L) and 6% zeaxanthin (Z); with an average L:Z ratio range of 15:1. Marigold hybrids that produce zeaxanthin as the primary xanthophyll, with minimal lutein, would provide an agronomically feasible source of zeaxanthin for the human dietary supplement industry. Chrysantis, Inc. (West Chicago, Ill.) has developed a high zeaxanthin marigold hybrid via mutagenesis.
SUMMARY OF THE INVENTION
The invention consists of a marigold male parent inbred line with altered carotenoid compositions through a chemically-induced mutation breeding process. The mutagenesis program identified three commercially useful mutant plants including a high zeaxanthin mutant plant KI-TeM2-4662 among several altered carotenoid mutants during 2010-2012. Mutant plant KI-TeM204662 was selfed to develop a stable mutant inbred line KI4662. Development of a proprietary, high flower yielding high zeaxanthin marigold hybrid from KI4662 with the potential to accumulate >3 mg/g of zeaxanthin and little to no lutein is highly valuable in developing an improved higher yielding marigold source of zeaxanthin.
The mutant KI-TeM204662 was selfed to fix the phenotype and simultaneously crossed with a female line to produce seed of a heterozygous mutant hybrid. Stabilization of the phenotype in KI-TeM204662 was achieved via selfing selected individuals (with >3 mg/g of zeaxanthin) for six generations. Inbreeding via selfing also considerably reduced the frequency of the undesirable weak phenotypes in the later generations. All the individuals within the selfed generations showed the mutant chemotype with an average zeaxanthin content of 3.4±4 mg/g. KI4662 represents a unique assemblage of homozygous alleles throughout the genome as a consequence of single seed descent selfing to inbred status. Genetic analysis revealed that the mutation for high zeaxanthin in KI4662 was recessive and did not express fully in heterozygous hybrid combinations. The wild type originating germplasm, Scarletade (hereinafter referred to as “SCR”) also showed a partial dominance for carotenoid expression in a hybrid combination. The development of a high zeaxanthin marigold hybrid will require the mutant allele to be present in both the male and female parent lines. A molecular marker-assisted introgression program results in the transfer of the mutant allele from the male parent into the female parent.
Plants of the inbred line KI4662 have not been observed under all possible environmental conditions. The phenotype may vary somewhat with variations in environment and culture such as temperature, light intensity, day length, water status, and/or fertilizer rate or type without, however, any variance in genotype.
An object of the present invention is a marigold plant with a high level of zeaxanthin and little or no lutein for use as an antioxidant in human and animal food, beverages and personal care products.
Another object of the invention is an inbred line of Tagetes erecta that is novel, stable, and uniform and has good agronomic characteristics that confers the trait of hyper-accumulation of zeaxanthin and little or no lutein to its progeny.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a chromatogram of SCR (left) and mutant KI4662 (right).
FIG. 2 is a scheme of the development of advanced generations of mutant plant KI4662 leading to inbred line KI4662.



DESCRIPTION OF THE INVENTION
The term “M” generation as used herein is the seed (and resulting plants) exposed to a mutagenic agent, “M2” is the progeny of self-pollinated M1 plants, “M3” is the progeny of a selected single self-pollinated M2 plant, and so on. The term “progeny” refers to the plants and seeds of all subsequent generations resulting from a selfed plant from the previous generation.
African marigold (Tagetes erecta L.) flowers are commercially extracted and utilized as a natural source of xanthophyll pigments for the poultry and human dietary supplement industries. A prominent supplier of xanthophyll pigments extracts them from a commercial marigold hybrid (Hybrid 50011, U.S. Pat. No. 6,894,208) which typically has a total xanthophyll composition of 94% lutein (L) and 6% zeaxanthin (Z); with an average L:Z ratio range of 15:1. Marigold hybrids that produce zeaxanthin as the primary xanthophyll, with minimal lutein, would provide an agronomically feasible source of zeaxanthin for the human dietary supplement industry. Chrysantis, Inc. (West Chicago, Ill.) has developed a high zeaxanthin marigold hybrid via mutagenesis. The hybrid requires the mutant allele to be present in both parents. Careful introgression of the mutant allele of the present invention into both parental lines using marker-assisted introgession and backcrossing will facilitate the development of a higher flower yielding high zeaxanthin marigold hybrid.
In pursuit of the present invention, a large-scale chemical mutagenesis program was conducted using ethyl methane sulfonate (EMS) as a mutagenic agent in a publicly available elite marigold open pollinated variety background, Scarletade. More than 4000 first generation (M1) mutant plants were generated during the first two years of the program. Subsequently about 9000 second generation mutants (M2) derived from 1500 M1 families were screened for their L:Z ratios and their carotenoid profile during the program. An optimized, rapid protocol called the ‘No Yield Incomplete Saponification’ (NYIS) method was used for rapid screening of these 9000 M2 plants for their L:Z ratios. Each M2 seed was grown to produce M2 flowering plants. Flowers from each M2 plant were collected, freeze dried and quantitated for carotenoid levels using HPLC based, industry standard AOAC method. Briefly, 0.5-1 g ground petal samples were extracted and saponified with 30 mL HEAT extraction solvent (hexane, ethanol, acetone, and toluene in volume ratios of 10:6:7:7) and 2 mL 40% methanolic potassium hydroxide (w/v) in a 56° C. water bath set to shake at 120 rpm for 20 minutes. The extract was analyzed in an HPLC with a Waters Sphersorb CN column (50×4.6 mm, 3 μm). An isocratic elution with mobile phase of hexanes (74.6%), methylene chloride (24.9%), methanol (0.4%), n,n-diisopropylethylamine (0.1%) at 1.000 mL/min flow rate was used. The column temperature was maintained at 25° C. and 10 μL of sample was injected to run for 13 minutes per assay. The diode array detector (DAD) was set at 446 nm. A standard method was used for the quantitation of lutein and zeaxanthin.
The large scale screening of M2 plants identified 25 mutants with altered carotenoid profile of which five mutants (KI132, KI935, KI4656, KI4662 and KI4871) had potentially valuable alterations to their L:Z ratio. KI132 is a mutant with a 6:1 L:Z ratio and is close to a potentially valuable AREDS2 target of 5:1. KI935, KI4656 and KI4662 are zeaxanthin enriched (high zeaxanthin) mutants, with almost no lutein to very low amounts of lutein present in the carotenoid profile. Mutants KI935 and KI4656 were not advanced further due to flower sterility issues. KI4662 was advanced from M2 to M7 stage using a single-seed descent procedure as shown in FIG. 2. Briefly KI4662 identified as a single M2 plant, was naturally self-pollinated in isolation to produce M3 seed. Eighty M3 plants were generated from M3 seed and flowers were quantitated for lutein and zeaxanthin content using the AOAC method. Based on the carotenoid profile, 2-3 M3 plants with the highest zeaxanthin content were selected and naturally self-pollinated in isolation to produce M4 seed. Using the same procedure, KI4662 was advanced from the M2 to the M6 generation producing M7 seed.
The benefits of lutein, especially for eye health have been well studied for decades. Research shows that zeaxanthin along with lutein also plays a unique, distinctive role in maintaining optimal eye health. In addition, human studies have demonstrated that both lutein and zeaxanthin are present in the skin. Animal studies have provided evidence of lutein and zeaxanthin protecting skin from light-induced skin damage, especially the ultraviolet wavelengths. The unique role of zeaxanthin in addition to lutein in human dietary requirements has been extensively studied. In this context, the high zeaxanthin mutant lines such as KI4662 with a potential to accumulate up to 3.4±0.2 mg/g of zeaxanthin with little to no lutein would be highly valuable.
The carotenoid composition of a typical marigold flower contains >85% lutein and 5% zeaxanthin. The current commercially dominant lutein marigold hybrid on average yields more lutein (18 mg/g) than zeaxanthin (1.5 mg/g) on a dry matter basis. Some high zeaxanthin hybrids have been developed but they contain measurable amounts of lutein. In this context, developing high zeaxanthin hybrids utilizing mutant inbred line KI4662 as a male parental line would add great economic value in developing zeaxanthin based products as the resulting hybrid would have higher overall flower yield, equivalent zeaxanthin on a dry matter basis and almost no lutein. Growing mutant variety KI4662 in a hybrid form will significantly increase flower yield relative to the per se inbred while also having the altered carotenoid profile change relative to the wild type parent.
The average lutein and zeaxanthin content in flowers of SCR is approximately 22.2±2.3 mg/g and 1.66±0.3 mg/g respectively under greenhouse conditions. Zeaxanthin content in mutant KI4662 is almost double, compared to wild type SCR, under the greenhouse conditions. In contrast, the lutein content in mutant line KI4662 is negligible (<0.05 mg/g) compared to the 22.2 mg/g present in the wild type SCR (FIG. 1). Ball Horticultural Company has developed high zeaxanthin mutants and has demonstrated that the high zeaxanthin mutation is on the lut2 gene which codes for lycopene ε-cyclase (personal discussion) causing constitutive, whole plant lutein deficiencies. The mutation of mutant KI4662 is likely to be a lut2 mutation (lutein-deficient mutant) which is characterized by very low lutein levels in the leaves as well as in flowers throughout the entire plant. Studies have shown that the lut2 mutation eliminates lutein production and is biochemically and genetically consistent with a disruption in the gene encoding the enzyme lycopene ε-cyclase (Pogson B J, McDonal K A, Truong M, Britton G and DellaPenna D. 1996. Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants. The Plant Cell, Vol. 8, 1627-1639; Pogson B J, Niyogi K K, Bjorkman O, DellaPenna D. 1998. Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants. PNAS. 95; 13324-13329).
In accordance with one aspect of the present invention, provided is an inbred marigold seed and plants thereof designated KI4662. The present invention further relates to a method for producing inbred marigold seeds that includes, but is not limited to, the steps of planting seed of inbred marigold KI4662 in proximity to itself or to different seed from a same family or line, growing the resulting marigold plants, cross-pollinating the resulting marigold plants, and harvesting resultant seed obtained from such inbred plants using techniques standard in the agricultural arts such as would be necessary to bulk-up seed such as for hybrid production. The present invention also relates to inbred seed produced by such a method.
In any cross between inbred marigold plant KI4662 and another inbred marigold plant, KI4662 may be designated as the male (pollen parent) or the female (seed parent). The present invention also relates to a marigold plant that expresses substantially all of the physiological and morphological characteristics of inbred marigold plant KI4662 and to a substantially homogenous population of marigold plants having all the physiological and morphological characteristics of inbred marigold plant KI4662. Any marigold plants produced from inbred marigold plant KI4662 are contemplated by the present invention and are, therefore, within the scope of this invention. A description of physiological and morphological characteristics of marigold plant KI4662 is presented in Table 2.
It should be appreciated by one having ordinary skill in the art that, for the quantitative characteristics identified in Table 2, the values presented are typical values. These values may vary due to the environment and accordingly, other values that are substantially equivalent are also within the scope of the invention.
Inbred marigold line KI4662 shows uniformity and stability within the limits of environmental influence for the traits described in Table 2. Inbred KI4662 has been self-pollinated a sufficient number of generations with careful attention paid to uniformity of plant type to ensure the homozygosity and phenotypic stability necessary to use in large scale, commercial production. The line has been increased both by hand in isolated fields with continued observations for uniformity. No variant traits have been observed or are expected in KI4662
The plants of the present invention have the taxonomic description of being genus Tagetes, species erecta, family Asteraceae and the common name marigold. Table 2 sets out the characteristic traits of KI4662 and Table 3 sets out the definitions of the traits of Table 2.







TABLE 2





Characteristics of KI4662 at vegetative and flowering stage
















Ploidy
Diploid


Chromosome number
24





Greenhouse Observations
Field Observations












Average
Range
Average
Range


















Growth Form
Upright





















Plant height at maturity
34.9
cm
31-38
cm
56.5
cm
50-67
cm


(Vegetative) (56 days)













Plant height class (flowering)
Tall


Tall




Flowering Season
Mid-Season


Mid-Season


Flower Type
Chrysanthemum


Chrysanthemum


Flower Fullness
Semi-double


Semi-double


Silhouette
Flattened


Flattened












Number of Flower heads per
8.5


13.6
8-25


plant (132 days)















Flower Head Diameter
6.5
cm


5.6
cm
4.1-6.2
cm












Flower Odor
Mild


Mild
Mild



“Marigold”


“Marigold”
“Marigold”


Flower Head Color
Orange


Orange
Orange















Days from planting to first
95
days
90-100
days
85
days
80-90
days


flower


Length of flowering season
140
days
135-145
days
120
days
115-132
days


Plant height at maturity
110.1
cm
102-118
cm
98.5
cm
92-108
cm


(Flowering) (98 days)


Plant Width at maturity
235.3
cm2
161.2-272.6
cm2


(Vegetative) (56 days)













Plant width class (flowering)
Semi-compact


Semi-compact




Leaf Type
Compound


Compound


Leaf Shape
lanceolate


lanceolate


Leaf Margin
Dentate


Dentate















Leaf Width
4.9
cm
3.0-6.0
cm






Leaf Length
15.6
cm
13.0-18.0
cm













Leaf Color
Medium green


Medium green




Petiole Anthocyanin
Mild


Mild


Stem Profile
Straight


Straight


Stem Structure
Intermediate


Intermediate















Node Length (middle of
4.2
cm
3-5
cm
5.3
cm
4.5-7
cm


plant) (vegetative) (56 days)


Node Length (middle of
4.8
cm
3-6
cm
5.26
cm
4.58-5.55
cm


plant) (flowering)(98 days)













Number of internodes below
1


1




first branch











Number of first order
8.9
 6-10
8
6-10


branches (vegetative)(56 Days)


Number of first order
36.3
32-50
8
6-10


branches (flowering)(98 days)
















TABLE 3





Description of Vegetative and Flowering Characteristics.


















Ploidy
Number of sets of chromosomes



Chromosome Number
Number of chromosomes



Plant Height
Measure length of the plant from



(Vegetative)
top of soil media to highest plant




growth



Plant Width
Plant Width was defined as how



(Vegetative)
wide the plant was from an aerial




view. The area was measured by




taking 2 squares and creating a




frame at the widest area of the




plant. The measurements were




recorded from where the frames




overlapped.



Plant Height
Measure length of the plant from



(Flowering)
top of soil media to highest plant




growth



Plant Width Class
Structure of the overall plant



Plant Height Class
Height comparable with other




marigold varieties



Growth Form
Plant phenotype



Leaf Type
Type of leaf



Leaf Shape
Structure of leaf



Leaf Margin
Structure of leaf edge



Leaf width (cm)
Width of leaf structure from fully




developed leaf from mid-section




of plant



Leaf Length (cm)
Length of leaf structure from fully




developed leaf from mid-section




of plant



Leaf Color
Visual color of leaf



Petiole Anthocyanin
Red pigmentation of leaf stalk



Stem profile
Shape of Stem



Stem Structure
Pliability of stem



Node length
An internode measured at the mid-




section of the main stalk of




the plant



Number of nodes below
Visual number of internodes



first branch
below first branch



Number of first order
Number of branches generating from



branches
the main stalk



Days from planting to
Observed days between sowing to



first flower
first open flower



Length of flowering
Observed days between sowing to



season
end of flowering



Flowering Season
Designated by number of days to




flower compared to other crops



Silhouette
Petal Structure



Number of Flower heads
Number of flowers or buds per plant



per plant



Flower head diameter
Diameter of stage 3 flower



Flower Odor
Strength of flower fragrance



Flower Head Color
Petal color










In an embodiment, the plant KI4662 produces zeaxanthin comprising greater than 2 mg/g dry weight zeaxanthin and preferably greater than 3 mg/g dry weight zeaxanthin and little or no (<0.05 mg/g dry weight) lutein.
The present invention is related to the development of a novel, stable, inbred line of Tagetes erecta. This line is unique and clearly distinct from all other existing varieties of Tagetes erecta. Line KI4662 has fertile male flowers and is particularly suited for use as a male inbred line which is crossed with female plants to produce hybrid seed that will result in plants having plant tissues, particularly flower petals, that are high in zeaxanthin and little or no lutein. The mutation present in KI4662 is recessive. In order for the recessive mutation to fully express in hybrid combination, both the male and the female parent should carry the mutation. Under such circumstances, the hybrid will have one allele from each parent and the recessive mutation would appear on both the alleles in the hybrid. For the female parent to carry the mutation, mutant KI4662 should be backcrossed into the female to create a female plant that carries the mutation. Molecular markers will aid in the “surgical” transfer and introgression of the high zeaxanthin mutation from KI4662 into the female line in order to develop a female line with the mutation.
Various breeding schemes may be used to produce new inbred marigold lines from KI4662. In one method, generally referred to as the pedigree method, KI4662 may be crossed with another different marigold plant such as a second inbred parent marigold plant, which either itself exhibits one or more selected desirable characteristic(s) or imparts selected desirable characteristic(s) to a hybrid combination. Examples of potentially desired characteristics include greater flower yield, higher xanthophyll content, reduced time to crop maturity, better agronomic quality, resistance and/or tolerance to insecticides, herbicides, pests, heat and drought, and disease, and uniformity in germination times, stand establishment, growth rate, maturity and flower size. If the two original parent marigold plants do not provide all the desired characteristics, then other sources can be included in the breeding population. Elite inbred lines, that is, pure breeding, homozygous inbred lines, can also be used as starting materials for breeding or source populations from which to develop inbred lines.
Thereafter, resulting seed is harvested and resulting superior progeny plants are selected and selfed or sib-mated in succeeding generations, such as for about 5 to about 7 or more generations, until a generation is produced that no longer segregates for substantially all factors for which the inbred parents differ, thereby providing a large number of distinct, pure-breeding inbred lines.
In another embodiment for generating new inbred marigold plants, generally referred to as backcrossing, one or more desired traits may be introduced into the female inbred parent marigold plant (the recurrent parent) by crossing the KI4662 plants as a male non-recurrent donor parent with another marigold plant (referred to as the recipient or -recurrent parent) which carries the gene(s) encoding the particular trait(s) of interest to produce F1 progeny plants. Both dominant and recessive alleles may be transferred by backcrossing. The donor plant may also be an inbred, but in the broadest sense can be a member of any plant variety or population cross-fertile with the recurrent parent. Next, F1 progeny plants that have the desired trait are selected. Then, the selected progeny plants are crossed with KI4662 to produce backcross progeny plants. Thereafter, backcross progeny plants comprising the desired trait and the physiological and morphological characteristics of marigold inbred line KI4662 are selected. This cycle is repeated for about one to about eight cycles, preferably for about 3 or more times in succession to produce selected higher backcross progeny plants that comprise the desired trait and all of the desired physiological and morphological characteristics of marigold inbred line KI4662 listed in Table 2 as determined at the 5% significance level when grown in the same environmental conditions. Exemplary desired trait(s) include insect resistance, herbicide resistance, yield stability, yield enhancement and resistance to bacterial, fungal and viral disease. One of ordinary skill in the art of plant breeding would appreciate that a breeder uses various methods to help determine which plants should be selected from the segregating populations and ultimately which inbred lines will be used to develop hybrids for commercialization. In addition to the knowledge of the germplasm and other skills the breeder uses, a part of the selection process is dependent on experimental design coupled with the use of statistical analysis. Experimental design and statistical analysis are used to help determine which plants, which family of plants, and finally which inbred lines and hybrid combinations are significantly better or different for one or more traits of interest. Experimental design methods are used to assess error so that differences between two inbred lines or two hybrid lines can be more accurately determined. Statistical analysis includes the calculation of mean values, determination of the statistical significance of the sources of variation, and the calculation of the appropriate variance components. Either a five or a one percent significance level is customarily used to determine whether a difference that occurs for a given trait is real or due to the environment or experimental error. One of ordinary skill in the art of plant breeding would know how to evaluate the traits of two plant varieties to determine if there is no significant difference between the two traits expressed by those varieties. Mean trait values may be used to determine whether trait differences are significant, and preferably the traits are measured on plants grown under the same environmental conditions.
Of course, the other marigold plant may be the recurrent parent whereby the mutation associated with high zeaxanthin values and little or no lutein in KI4662 is transferred to the recurrent parent following the foregoing process. It is expected that use of such an inbred line in a cross with KI4662 would result in hybrid marigold plants with greater than 3 mg/g zeaxanthin and little or no lutein on a dry matter basis.
This method results in the generation of inbred marigold plants with substantially all of the desired morphological and physiological characteristics of the recurrent parent and the particular transferred trait(s) of interest. Because such inbred marigold plants are heterozygous for loci controlling the transferred trait(s) of interest, the last backcross generation would subsequently be selfed to provide pure breeding progeny for the transferred trait(s).
It should be appreciated by those having ordinary skill in the art that backcrossing can be combined with pedigree breeding as where inbred KI4662 is crossed with another marigold plant, the resultant progeny are crossed back to inbred KI4662 and thereafter, the resulting progeny of this single backcross are subsequently inbred to develop new inbred lines. This combination of backcrossing and pedigree breeding is useful as when recovery of fewer than all of the KI4662 characteristics than would be obtained by a conventional backcross are desired.
Once inbred lines are created, the next step is to determine if the inbreds have any value. This is accomplished by techniques of measuring the combining ability of the new inbred plant, as well as the performance of the line itself. Combining ability refers to a line's contribution as a parent when crossed with other lines to form hybrids. Specific combining ability (SCA) refers to the ability of a line to cross to another specific inbred to form a hybrid. General combining ability (GCA) refers to the ability of a line to cross to a wide range of lines to form hybrids. The methodology of forming hybrids to evaluate an inbred line's contribution as a parent for the purpose of selecting superior lines is interchangeably known as experimental, top or test crossing.
In accordance with processes of the present invention, a hybrid plant having inbred KI4662 as a parent is crossed with itself or any different marigold plant such as an inbred marigold plant or a hybrid marigold plant to develop a novel inbred line. For example, a hybrid marigold plant having inbred marigold plant KI4662 as a parent may be inbred, i.e., crossed to itself or sib-pollinated, and the resulting progeny each selfed for about 5 to about 7 or more generations, thereby providing a set of distinct, pure-breeding inbred lines wherein each of the lines received all of its alleles from the hybrid marigold plant having inbred marigold plant KI4662 as a parent. In other embodiments, a hybrid marigold plant having inbred marigold plant KI4662 as a parent is crossed with a different marigold plant that may include any inbred marigold plant that is not inbred marigold plant KI4662, any hybrid marigold plant that does not have KI4662 as a parent, another germplasm source, a mutation inducing stock, or a trait donor plant, thereby providing a set of distinct, pure-breeding inbred lines. The resulting inbred lines could then be crossed with other inbred or non-inbred lines and the resulting inbred progeny analyzed for beneficial characteristics. In this way, novel inbred lines conferring desirable characteristics could be identified.
In yet another aspect of the invention, processes are provided for producing marigold seeds or plants, which processes generally comprise crossing a first parent marigold plant with a second parent marigold plant wherein at least one of the first parent marigold plant or the second parent marigold plant is inbred parent marigold plant KI4662. In some embodiments of the present invention, the first inbred marigold plant is KI4662 and is a female and in other embodiments the first inbred marigold plant is KI4662 and is a male. These processes may be further exemplified as processes for preparing hybrid marigold seed or plants, wherein a first inbred marigold plant is crossed with a second marigold plant of a different, distinct variety to provide a hybrid that has, as one of its parents, the inbred marigold plant variety KI4662. In this case, a second inbred variety is selected which confers desirable characteristics when in hybrid combination with the first inbred line. In these processes, crossing will result in the production of seed. The seed production occurs regardless whether the seed is collected.
Any time the inbred marigold plant KI4662 is crossed with another, different marigold inbred, a first generation (F1) marigold hybrid plant is produced. As such, an F1 hybrid marigold plant may be produced by crossing KI4662 with any second inbred marigold plant. Therefore, any F1 hybrid marigold plant or marigold seed which is produced with KI4662 as a parent is part of the present invention.
When inbred marigold plant KI4662 is crossed with another inbred plant to yield a hybrid, the original inbred can serve as a paternal plant contributing to some of the characteristics in the hybrids. Both paternally and maternally inherited characteristics may express differently in the hybrid. However, often one of the parental plants is preferred as the maternal plant because of its male sterility. It is generally preferable to use KI4662 as the male parent.
For a decision to be made to advance a hybrid, it is not necessary that the hybrid be better than all other hybrids. Rather, significant improvements must be shown in at least some traits that would create value for some applications or markets. Some testcross hybrids are eliminated despite being similarly competitive relative to the current commercial hybrids because of the cost to bring a new hybrid to market requires a new product to be a significant improvement over the existing product offering.
All plants produced using inbred marigold plant KI4662 as a parent are within the scope of this invention, including plants derived from inbred marigold plant KI4662. This includes plants essentially derived from inbred KI4662 with the term “essentially derived variety” having the meaning ascribed to such term in 7 U.S.C. §2104(a)(3) of the Plant Variety Protection Act, which definition is hereby incorporated by reference. This also includes progeny plant and parts thereof with at least one ancestor that is inbred marigold plant KI4662 and more specifically where the pedigree of this progeny includes 1, 2, 3, 4, and/or 5 or cross pollinations to inbred marigold plant KI4662, or a plant that has KI4662 as a progenitor. All breeders of ordinary skill in the art maintain pedigree records of their breeding programs. These pedigree records contain a detailed description of the breeding process, including a listing of all parental lines used in the breeding process and information on how such line was used. Thus, a breeder would know if KI4662 were used in the development of a progeny line, and would also know how many breeding crosses to a line other than KI4662 were made in the development of any progeny line. A progeny line so developed may then be used in crosses with other, different, marigold inbreds to produce first generation F1 marigold hybrid seeds and plants with superior characteristics.
Accordingly, another aspect of the present invention is methods for producing an inbred marigold line KI4662-derived marigold plant. This method for producing a KI4662-derived marigold plant, comprises: (a) crossing inbred marigold plant KI4662 with a second marigold plant to yield progeny marigold seed; and, (b) growing the progeny marigold seed, (under plant growth conditions), to yield the KI4662-derived marigold plant. Such methods may further comprise the steps of: (c) crossing the KI4662-derived marigold plant with itself or another marigold plant to yield additional KI4662-derived progeny marigold seed; (b) growing the progeny marigold seed of step (d) (under plant growing conditions), to yield additional KI4662-derived marigold plants; and (e) repeating the crossing and growing steps of (c) and (d) from 0 to 7 times to generate further KI4662-derived marigold plants. Still further, this may comprise utilizing methods of haploid breeding and plant tissue culture methods to derive progeny of the KI4662-derived marigold plant.
With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and to express foreign genes, or additional, or modified versions of native or endogenous genes (perhaps driven by different promoters) to alter the traits of a plant in a specific manner. Such foreign, additional and/or modified genes are referred to herein collectively as “transgenes.” The present invention, in particular embodiments, also relates to transformed versions of the claimed inbred marigold line KI4662 containing one or more transgenes, particularly genes that encode resistance to a herbicide.
Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.
The foregoing methods for transformation would typically be used for producing transgenic inbred lines. Transgenic inbred lines could then be crossed, with another (non-transformed or transformed) inbred line, in order to produce a transgenic hybrid marigold plant. Alternatively, a genetic trait which has been engineered into a particular marigold line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite line into an elite line, or from a hybrid marigold plant containing a foreign gene in its genome into a line or lines which do not contain that gene.
In addition to phenotypic observations, a plant can also be described by its genotype. The genotype of a plant can be described through a genetic marker profile which can identify plants of the same variety, a related variety or be used to determine or to validate a pedigree. Genetic marker profiles can be obtained by techniques such as Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) which are also referred to as Microsatellites, and Single Nucleotide Polymorphisms (SNPs), Isozyme Electrophoresis and Isolelectric Focusing.
Particular markers used for these purposes are not limited to the set of markers disclosed herewithin, but are envisioned to include any type of genetically stable marker and marker profile which provides a means of distinguishing varieties. In addition to being used for identification of inbred parents, a hybrid produced through the use of KI4662 or its parents, and identification or verification of the pedigree of progeny plants produced through the use of KI4662, the genetic marker profile is also useful in breeding and developing backcross conversions.
DEPOSIT INFORMATION
Applicants have made a deposit of at least 2,500 seeds of inbred Tagetes erecta plant KI4662 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110 USA, under ATCC Accession No. PTA-121906. The seeds received by the ATCC on Jan. 14, 2015, were taken from a deposit maintained by Kemin Industries, Inc. since prior to the filing date of this application. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant will make the deposit available to the public pursuant to 37 C.F.R. §1.808. This deposit of the Marigold inbred line KI4662 will be maintained in the ATCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has or will satisfy all of the requirements of 37 C.F.R. §§1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant has no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce.
The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

## Claims

1. A seed of marigold inbred line designated KI4662, or a part of the seed, representative seed of the line having been deposited under ATCC Accession No. PTA-121906.

2. A substantially homogenous population of seeds comprising at least one of the marigold seed of claim 1.

3. A method for producing marigold seed, said method comprising the steps of:
(a) planting the seed of claim 1 in proximity to another seed of inbred line KI4662 or to a seed of a different line;
(b) growing plants from the seed;
(c) pollinating said plants; and,
(d) harvesting the resultant seed.

4. A marigold seed produced by the method of claim 3.

5. A marigold plant produced by growing the seed of claim 1.

6. A part of the marigold plant of claim 5, selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, pollen, an ovule, a flower, a seed, a petal and a leaf.

7. Pollen of the plant of claim 5.

8. An ovule of the plant of claim 5.

9. A marigold plant, or a part of the marigold plant, having all the physiological and morphological characteristics of the marigold plant, or a part of the marigold plant, of claim 5.

10. A substantially homogenous population comprising at least one of the marigold plant of claim 5.

11. A method for producing a marigold plant, said method comprising the step of: (a) crossing inbred marigold plant KI4662, representative seed of the line having been deposited under ATCC Accession No. PTA-121906, with another different marigold plant to yield progeny marigold seed.

12. The method of claim 11, wherein the other, different marigold plant is an inbred marigold plant.

13. The method of claim 11, further comprising the steps of:
(b) growing the progeny marigold seed from step (a);
(c) under self-pollinating or sib-pollinating conditions for 5 to 7 generations; and
(d) harvesting resultant seed.

14. The method of claim 11, further comprising the step of selecting plants obtained from growing at least one generation of the progeny marigold seed for a desirable trait.

15. A method of introducing a desired trait from marigold inbred line KI4662, representative seed of the line having been deposited under ATCC Accession No. PTA-121906, said method comprising the steps of:
(a) crossing KI4662 plants with plants of another marigold line that comprise a desired trait to produce F1 progeny plants;
(b) selecting F1 progeny plants that have the desired trait;
(c) crossing selected progeny plants with said other marigold line plants to produce backcross progeny plants;
(d) selecting for backcross progeny plants that comprise the desired trait and physiological and morphological characteristics of marigold inbred line KI4662; and
(e) performing steps (c) and (d) one or more times in succession to produce the selected or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of said other marigold inbred line as determined at the 5% significance level when grown in the same environmental conditions.

16. A method for producing a hybrid marigold seed comprising crossing a first inbred parent marigold plant with a second inbred parent marigold plant and harvesting resultant hybrid marigold seed, wherein the first inbred marigold plant or the second inbred marigold plant is the marigold plant of claim 5.

17. A method for producing a hybrid marigold seed, the method comprising:
(a) planting in pollinating proximity a seed of the inbred marigold plant of claim 5 and a seed of a second, different inbred marigold plant;
(b) cultivating the seeds into plants that bear flowers;
(c) cross pollinating at least some of the cultivated plants; and
(d) harvesting seeds produced from at least one of the first or second inbred marigold plants.

18. The method according to claim 17, wherein the KI4662 marigold plant is the male parent.

19. The method according to claim 17, wherein the KI4662 marigold plant is the female parent.

20. A hybrid marigold seed produced by the method according to claim 17.

21. A hybrid marigold plant, or parts thereof, produced by growing the hybrid marigold seed of claim 20.