阅读说明：本技术 通过细胞型转移提高植物可转化性 (Enhancement of plant convertibility by cell type transfer ) 是由 D·S·斯基比 S·埃卢马莱 于 2020-03-24 设计创作，主要内容包括：一种改变或转移植物株系的细胞型的方法。特别地,将转化-顽固植物株系,例如,转化-顽固玉米系的细胞型从转化-顽固细胞型转化到可转化细胞型,使得所述株系在保留其核基因组的同时变得可转化。可以使用包括回交和/或单倍体诱导的方法来产生新的可转化株系。(A method of altering or transferring the cell type of a plant line. In particular, the cell type of a transformed-recalcitrant plant line, e.g., a transformed-recalcitrant maize line, is transformed from a transformed-recalcitrant cell type to a transformable cell type such that the line becomes transformable while retaining its nuclear genome. Methods including backcrossing and/or haploid induction can be used to generate new transformable strains.)

1. A method of increasing the transformation efficiency of a recalcitrant corn line, the method comprising:

a. obtaining recalcitrant corn plants and collecting pollen therefrom;

b. pollinating a recipient maize plant comprising a normal A ("NA") cytoplasm with pollen from said recalcitrant maize plant; and

c. obtaining progeny embryos therefrom;

wherein the progeny embryo comprises a NA cytoplasm and at least a nuclear genome of the recalcitrant maize plant, and wherein the progeny embryo has a higher transformation efficiency than the recalcitrant maize plant.

2. A method of imparting transformability to a recalcitrant corn line, the method comprising:

a. obtaining recalcitrant corn plants and collecting pollen therefrom;

b. pollinating a recipient maize plant comprising a normal A ("NA") cytoplasm with pollen from said recalcitrant maize plant; and

c. growing progeny plants therefrom;

wherein the progeny plant comprises the NA cytoplasm and the nuclear genome of the recalcitrant maize plant, and wherein the progeny plant is transformable.

3. The method of claim 1 or 2, wherein the recalcitrant maize plant comprises non-NA cytoplasm.

4. The method of claim 3, wherein the non-NA cytoplasm is selected from the group consisting of: normal B ("NB") cytoplasm, cytoplasmic male sterile C ("C" or "CMS-C") cytoplasm, cytoplasmic male sterile S ("S" or "CMS-S") cytoplasm, or cytoplasmic male sterile T ("T" or "CMS-T") cytoplasm.

5. The method of claim 1 or 2, wherein the recipient maize plant is a haploid inducer plant.

6. The method of claim 5, wherein the haploid inducer plant is a female haploid inducer plant.

7. The method of claim 6, wherein the female haploid inducer plant comprises a mutated ig1 gene or maternal gene.

8. The method of claim 6, wherein the haploid inducer plant is a male parent haploid inducer plant.

9. The method of claim 6, wherein the male parent haploid inducer plant comprises a CENH3 mutation.

10. The method of claim 1, wherein the progeny embryo is grown into a progeny plant.

11. The method of claim 2 or 10, wherein said progeny plant is backcrossed to the recalcitrant corn plant for at least one generation.

12. The method of claim 11, wherein said progeny plant is the female parent in said backcross.

13. The method of claim 11, wherein said progeny plant retains said NA cytoplasm.

14. The method of claim 11, wherein the progeny plant retains the nuclear genome of the recalcitrant maize plant.

15. A method of increasing transformation efficiency of a recalcitrant plant line, the method comprising:

a. obtaining recalcitrant plants and collecting pollen therefrom;

b. pollinating a recipient plant comprising a transformable cytoplasm with pollen from said recalcitrant plant;

c. obtaining progeny embryos therefrom; and

d. optionally, the step of (a) is carried out,

i. growing the progeny tissue into a progeny plant;

at least one cross using pollen from the recalcitrant plant to backcross with the progeny plant; and

e. transforming a tissue derived from the progeny embryo;

wherein said progeny tissue comprises the NA cytoplasm and at least the nuclear genome of said recalcitrant plant, and wherein said progeny tissue has a higher transformation efficiency than the recalcitrant plant.

16. A plant obtained by the method of claim 1, 2 or 15.

17. A method of transforming a plant, the method comprising:

a. obtaining a plurality of plant lines;

b. testing for markers indicative of NA cytoplasm;

c. selecting at least one line from the plurality of plant lines, wherein the selected line has a marker for the NA cytoplasm; and

d. transforming cells derived from at least one selected strain of step (c).

18. The method of claim 17, wherein the test detects a G nucleotide at a position corresponding to position 11 of the mitochondrial DNA sequence SEQ ID NO 7.

19. The method of claim 18, wherein said testing for markers indicative of NA cytoplasm detects the presence of a sequence corresponding to SEQ ID No. 7.

20. The method of claims 17-19, wherein said test for markers indicative of NA cytoplasm comprises forward primer SEQ ID NO 5 and reverse primer SEQ ID NO 6.

21. The method of claim 20, wherein said test for markers indicative of NA cytoplasm comprises probe SEQ ID No. 7 and/or probe SEQ ID No. 8.

22. The method of claim 21, wherein the probes are differentially labeled with fluorophores.

Technical Field

The invention relates to the technical field of plant biology. In particular, the invention relates to plant transformation, including maize transformation, and methods of increasing the ability of recalcitrant lines to receive exogenous transgenes.

Background

Plant transformation, i.e., the stable integration of exogenous DNA ("transgene") into the plant genome, has been used to add new and useful traits to crop plants for decades. See, e.g., U.S. patent No. 6,051,409 (disclosing corn transformation) filed on 1996, 23/9, which is incorporated herein by reference in its entirety; U.S. patent application publication 2008/0229447 (disclosing soybean transformations) filed 3, 12, 2007, incorporated herein by reference. One recent example includes corn and even MIR604, which contains a pesticidal protein derived from bacteria (U.S. patent No. 7,361,813, incorporated by reference in its entirety). However, while some maize lines are relatively easy to transform (i.e., receive transgenic DNA), most lines are not. For example, most elite inbred lines (lines that have been inbred for several generations to obtain pure or near-pure homozygous genomes and used as parental lines to produce commercially valuable hybrids) cannot be transformed with exogenous DNA. Thus, in order to "move" a transgenic trait into an inbred line, the transgenic trait must first be transformed into a transformable maize line. Such transformed maize lines are rarely suitable for use as parental lines in breeding platforms. Thus, the transformed maize line is crossed into an inbred line to produce a progeny plant that will comprise the genomes of both the inbred parent and the transformed parent in heterozygous form. The progeny plants containing the transgene must then be backcrossed into the inbred line for approximately 6 or 7 generations in order to eliminate as much as possible the genome contributed by the transformed parent while retaining the transgenic trait. This process typically takes 2 to 3 years. At the end of this process, inbred lines containing the transgenic trait are finally obtained.

Corn is not a uniform species. It shows an extraordinary diversity, whether dent corn (also known as "field corn"), which has high starch and is used for animal feed, ethanol production or corn flour production, flint corn (also known as "Indian corn"), which is known in its various colours, sweet corn (sweet corn) (mainly for human consumption, usually as a corn cob) or popcorn. Although most of this diversity is due to differences between the genome of different maize lines, some of this diversity is also due to the genome of the mitochondria (subcellular organelles that provide all cells with chemical energy) that accompany the lines. Mitochondria have their own DNA, and the mitochondrial genome is indicative of a maize cell type. For example, maize is known to have at least five different cell types: normal A ("NA"), Normal B ("NB"), cytoplasmic Male sterile C ("CMS-C" or "C"), cytoplasmic Male sterile S ("CMS-S" or "S"), and cytoplasmic Male sterile T ("CMS-T" or "T"). Still other cell types can be found. Thus, relatively speaking, mitochondria, by virtue of their genome, may have a tremendous impact on the phenotype of the host cell. However, we are still just learning what these effects might be.

Here, we found for the first time that there is a relationship between transformability and cell type. Transformable maize lines are known to have NA cell types, whereas recalcitrant maize lines are known to have NB cell types. Therefore, we want to know: can we alter and make transformable the cell types of recalcitrant inbreeding? If so, we can revolutionize maize transformation and greatly accelerate farmers to gain new traits in commercially relevant germplasm.

Disclosure of Invention

A method for altering the cell type of a recalcitrant plant such that the recalcitrant plant is transformable is provided. This is achieved by collecting pollen from recalcitrant plants and pollinating recipient plants with transformable cell types. From this pollination event, progeny embryos are obtained that have a transformable cell type (inherited from the maternal parent) while also having in their nuclear genome a set of chromosomes inherited from the recalcitrant male parent. Such progeny embryos, whether directly transformed or induced to form callus or grown into plants, have higher transformation efficiency than their recalcitrant parent. In some cases, the recalcitrant parent has the NB cell type or the cytoplasmic male sterile cell type CMS-C, CMS-S or CMS-T.

When the recipient plant receives pollen from an recalcitrant plant (acting as a male parent), the recipient plant acts as a female parent. The recipient plant may be a haploid inducer plant, that is, any progeny of the recipient plant may have only half the normal number of chromosomes. For example, the recipient plant may be a male haploid inducer plant that causes some progeny to lose the maternal chromosomes (and thus retain only the paternal chromosomes). The parental haploid inducer plant may comprise a mutation in the ig1 gene or the CENH3 gene. See U.S. patent No. 7,439,416 (disclosing IG1 gene) filed on 7.1.2005 and U.S. patent No. 8,618,354 (disclosing CENH3 mutation) filed on 5.10.2010, both of which are incorporated herein by reference in their entireties. When the recipient plant is a male parent haploid inducer, the progeny produced by the claimed method will comprise the nuclear genome of the recalcitrant plant (i.e., as a haploid), but comprise the cell type of the recipient plant.

When the recipient plant is not a haploid inducer plant, the progeny produced by the claimed method will comprise the nuclear genome of the recalcitrant plant and the nuclear genome of the recipient plant (i.e., as a normal diploid) and have the cell type of the recipient plant. In such cases, backcrossing with recalcitrant plants may be required in order to increase the percentage and epigenetic state of the nuclear genome of the recalcitrant plants relative to the presence of the nuclear genome of the recipient plant in the progeny, while still retaining the desired transformable cell type. The first progeny (generation F1) produced by the claimed method will comprise the NA cytoplasm and a nuclear genome comprising the semi-acceptor parent genome and the semi-recalcitrant parent genome. After one backcross ("BC") with the recalcitrant parent pollen, the second generation (i.e., BC2 progeny) will contain the NA cytoplasm and the nuclear genome, 75% from the recalcitrant parent and 25% from the recipient parent. Progeny generations after the second generation (e.g., BC3, BC4, etc.) will have smaller and smaller ratios of acceptor nuclear genome to recalcitrant nuclear genome. By using molecular markers to select the desired parent, the selection of the desired nuclear genotype can be accelerated. The selection procedure without the marker is shown in the following table:

table 1. using recalcitrant plants as pollen providers without selection, the presence of the maternal genome was gradually reduced in backcross progeny generations.

By implementing the claimed method, transformation efficiency in an recalcitrant plant line can be increased such that when transforming tissue derived from progeny plants or embryos, it will have a higher success rate than its recalcitrant line parent.

Thus, one embodiment of the present invention is a method of increasing the success rate of inserting desired DNA into the chromosome of maize plant cells (i.e., increasing transformation efficiency in recalcitrant maize lines) by cell type transfer. This is done by: (a) collecting pollen from recalcitrant corn plants; (b) pollinating silks of another corn plant having normal a ("NA") cytoplasm with pollen from a recalcitrant corn plant; and (c) allowing the progeny embryo to form. In a successful cell type transfer, the progeny embryo has the NA cytoplasm and at least the nuclear genome of the recalcitrant maize plant (if haploid, the progeny will only have the chromosomes of the recalcitrant maize plant; if diploid, the progeny will have chromosomes from both parental plants). The progeny embryos have higher transformation efficiency than recalcitrant maize plants.

Another example is a method of imparting transformability to recalcitrant corn lines. This is done by: (a) collecting pollen from recalcitrant corn plants; (b) pollinating silks of another corn plant having normal a ("NA") cytoplasm with pollen from a recalcitrant corn plant; and (c) allowing the progeny embryo to form. In a successful cross, the progeny plant is transformable. Recalcitrant maize plants may have non-NA cytoplasm, e.g., normal B ("NB") cytoplasm, cytoplasmic male sterile C ("C" or "CMS-C") cytoplasm, cytoplasmic male sterile S ("S" or "CMS-S") cytoplasm, or cytoplasmic male sterile T ("T" or "CMS-T") cytoplasm. The recipient maize plant can be a haploid inducer plant (or more specifically, a male parent haploid inducer plant), or a male parent haploid inducer plant comprises a mutated ig1 gene or CENH3 mutation.

Once produced, the progeny embryo grows into a progeny plant. The progeny plant can be backcrossed with the recalcitrant corn plant for at least one generation. Alternatively, the progeny plant is the female parent in backcrossing. Progeny plants may retain the NA cytoplasm and/or retain the nuclear genome of recalcitrant maize plants.

Prior to transformation of tissue derived from progeny embryos, practitioners of the invention may use pollen from recalcitrant plants for at least one cross to backcross with progeny plants. This will increase the proportion of recalcitrant plant nuclear DNA (see table 1), but retain the new cell type, such that any plant tissue derived from BC1 generation or more has higher transformation efficiency than the original recalcitrant plant.

Another embodiment of the invention is a method of transforming plants by testing maize plant lines for markers indicative of NA cytoplasm and selecting at least one of these lines for transformation. In one aspect, the test detects a G nucleotide at a position corresponding to position 11 of the mitochondrial DNA sequence SEQ ID NO 7; or the test detects the presence of a sequence corresponding to SEQ ID NO 7. Optionally, the assay for markers indicative of NA cytoplasm further comprises forward primer SEQ ID NO 5 and reverse primer SEQ ID NO 6. Alternatively, the test for labeling indicative of the NA cytoplasm comprises probe SEQ ID NO. 7 and/or probe SEQ ID NO. 8, wherein the probes are differentially labeled with fluorophores.

Brief description of the sequences in the sequence listing

SEQ ID NOS: 1-4 are marker sets for distinguishing CMS from normal cytoplasm. The primary target was amplified by the forward and reverse primers (SEQ ID NO:1 and 2, respectively) and the CMS and normal cytoplasmic probes (SEQ ID NO:3 and 4, respectively) were labeled with different fluorophores indicating which allele was detected.

SEQ ID NOS 5-8 are marker sets for distinguishing normal A from normal B cell types. The primary target was amplified by the forward and reverse primers (SEQ ID NO:5 and 6, respectively) and the normal A and normal B probes (SEQ ID NO:7 and 8, respectively) were labeled with different fluorophores indicating which allele was detected.

Definition of

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and/or substitutions of equivalent techniques that would be apparent to one of ordinary skill in the art. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

The term "cell type" refers to the classification of the cytoplasm associated with a plant line. Cell types currently known include normal a ("NA") and normal B ("NB") cytoplasm, but also cytoplasmic male sterile cell types: cytoplasmic male sterile C ("C" or "CMS-C") cytoplasm, cytoplasmic male sterile S ("S" or "CMS-S") cytoplasm, and cytoplasmic male sterile T ("T" or "CMS-T") cytoplasm. The terms cell type and cytoplasm are used interchangeably.

As used herein, "recalcitrant" refers to a plant line that is not transformable or substantially untransformable. In other words, the conversion efficiency is 0% or substantially 0%. The term recalcitrant is synonymous with "non-convertible" and these terms are used interchangeably.

"transformable", and the like, refer to a plant, plant line or plant cell (such as callus or protoplast) that is more receptive to exogenous DNA and can stably integrate the exogenous DNA into its genome.

"transformation efficiency" or "conversion rate" means a measure of the number of successfully transformed plants compared to the total number of attempts. The measure may be expressed quantitatively, e.g. as a percentage or a raw number, or qualitatively, e.g. "low" or "high".

The term "one or more alleles" refers to any of one or more alternative forms of a gene, all of which are involved in at least one trait or characteristic. In diploid cells, both alleles of a given gene occupy corresponding loci on homologous chromosome pairs. In some cases (e.g. for QTL) it is more accurate to replace the "allele" with a "haplotype" (i.e. an allele of a chromosomal segment), but in these cases the term "allele" is to be understood as encompassing the term "haplotype". If two individuals possess the same allele at a particular locus, the alleles are said to be "identical by inheritance" if they are inherited from a common ancestor (i.e., the alleles are copies of the same parental allele). Alternatively, the alleles are "identical by state" (i.e., the alleles appear to be identical, but are derived from two different copies of the allele). The generation information identification is useful for linkage research; both generational and status information identification may be used for association studies, although pedigree information identification may be particularly useful.

The term "backcross" is understood within the scope of the present invention to mean a process by which the progeny of a hybrid are repeatedly crossed back to one of the parents.

The term "conditional male sterility" refers to a phenotype of male sterility (i.e., inability to produce viable pollen) that can be induced and/or inhibited by certain conditions. Thus, a plant can be "switched" from a male sterile phenotype to a male fertile phenotype by applying the certain conditions. Male sterility can be caused by a variety of factors and can be manifested, for example, by a complete lack of male organs (anthers), pollen degeneration, sterile pollen, and the like. The "switch" from male sterility to male fertility can be complete or incomplete based on the intensity of the conditions. Most preferably, in the context of the present invention, the term "conditional male sterility" means temperature dependent male sterility and thus means a nuclear male sterility phenotype, wherein the sterility is temperature dependent and can be restored to fertility at temperatures exceeding 35 ℃ (preferably between 35 ℃ and 43 ℃, more preferably between 37 ℃ and 40 ℃, most preferably at about 39 ℃), preferably by exposure to a preferred heat treatment time, followed by growth at room temperature).

In the context of nucleic acid sequences, the term "corresponding to" means that when nucleic acid sequences of certain sequences are aligned with one another, nucleic acids "corresponding to" certain enumerated positions in the present invention are those aligned with those positions in the reference sequence, but are not necessarily located in these precise numerical positions relative to the particular nucleic acid sequence of the invention. For example, in the following alignment, the T at position 13 of SEQ ID NO:8 corresponds to the G at position 11 of SEQ ID NO: 7.

The term "differentially labeled" means that two or more probes each have a fluorophore that is different from each other so that the presence or absence of each probe can be detected separately, regardless of whether two or more probes are contained in the same reaction.

The term "germplasm" refers to the totality of genotypes of a population or another group of individuals (e.g., species). The term "germplasm" may also refer to plant material; for example, a group of plants that serve as a repository for various alleles. The phrase "adapted germplasm" refers to plant material that has been demonstrated to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "foreign germplasm" refer to plant material of unknown or unproven genetic value; for example, for a given environment or geographic area; as such, the phrase "unadapted germplasm" refers, in some embodiments, to plant material that does not belong to an established breeding population and has no known relationship to members of an established breeding population.

The term "haplotype" can refer to the set of alleles of an individual inherited from one parent. Thus, a diploid individual has two haplotypes. The term "haplotype" may be used in a more limited sense to refer to physically linked and/or unlinked genetic markers (e.g., sequence polymorphisms) associated with a phenotypic trait. The phrase "haplotype block" (also sometimes referred to in the literature simply as a haplotype) refers to a set of two or more genetic markers that are physically linked on a single chromosome (or a portion thereof). Typically, each block has several common haplotypes, and a subset of genetic markers (i.e., "haplotype tags") can be selected to uniquely identify each of these haplotypes.

The terms "heterosis population" and "heterosis pool" are used interchangeably and refer to the relationship between breeding pools of corn populations. In summary, the main names of the heterosis pool are: hard stem (Stiff talk) ("SS", also known as Iowa hard stem synthesis or "BSSS"), Non-hard stem (Non Stiff talk) ("NSS"), and Iodent ("IDT"). See J.v. Hweerwararden et al, Historical genetics of North American mail [ History genomics of North American corn ], Proc.Nat' l Acad.Sci [ Proc.Natl.Acad. ], 109(31), 12420-25 (2012). However, these are not exclusive and other names are known, for example, Lancaster sun Crop ("LSC"). See, e.g., the Genetic diversity of maize inbred lines with and with heterosis groups revealed by RFLP, C.Livini et al [ Genetic diversity of maize inbred lines between heterosis and heterosis groups, revealed by RFLP ], Theor.appl.Genet [ theory and App genetics ], 84:17-25 (1992).

In the context of plant breeding, the terms "hybrid," "hybrid plant," and "hybrid progeny" refer to a plant that is the progeny of genetically different parents produced by crossing plants of different lines or varieties or species, including but not limited to crosses between two inbred lines (e.g., individuals that are genetically heterozygous or mostly heterozygous). The phrase "single-cross F1 hybrid" refers to an F1 hybrid produced by a cross between two inbred lines.

The phrase "inbred line" refers to a population that is homozygous or nearly homozygous for a gene. For example, inbred lines can be obtained by several cycles of sibling/sister breeding or self-fertilization. In some embodiments, the inbreds are bred for one or more phenotypic traits of interest. An "inbred," "inbred individual," or "inbred progeny" is a separate sample from one inbred line. The term "inbred" refers to an individual or line that is substantially homozygous.

The terms "introgression", "introgressed" and "introgressing" refer to both natural and artificial processes in which a genomic region is moved from one species, variety or cultivar to another species, variety or cultivar by crossing the species, variety or cultivar with the species, variety or cultivar. This process can optionally be accomplished by backcrossing to the backcrossed parent.

The term "marker-based selection" is understood within the scope of the present invention to mean the use of a genetic marker to detect one or more nucleic acids from plants, wherein the nucleic acid is associated with a desired trait, to identify plants carrying a gene for the desired (or undesired) trait, such that those plants can be used (or avoided) in, for example, transformation procedures or selective breeding procedures. As used herein, a marker that indicates normal a cytoplasm will distinguish non-CMS plants that have normal B cytoplasm from those that do not (i.e., have normal a cytoplasm). A marker can be a mutation within a locus of the genome (e.g., a single nucleotide polymorphism ("SNP")) or a mutation within one allele.

The phrase "phenotypic trait" refers to the appearance or other detectable characteristic in an individual resulting from the interaction of the individual's genome with the environment.

The term "plurality" refers to more than one entity. Thus, "a plurality of individuals" means at least two individuals. In some embodiments, the term majority refers to more than one-half of the whole. For example, in some embodiments, "majority in a population" refers to more than half of the members of that population.

The term "progeny" refers to one or more descendants of a particular cross. Typically, progeny are produced from breeding of two individuals, but some species (particularly some plants and hermaphroditic animals) can be self-fertilized (i.e., the same plant acts as a donor for both male and female gametes). The one or more descendants may be, for example, F1, F2, or any descendant.

The phrase "quality trait" refers to a phenotypic trait controlled by one or several genes exhibiting a majority of the phenotypic effects. Thus, quality traits are often simply inherited. Examples in plants include, but are not limited to, flower color, cob color, and disease resistance, such as northern corn leaf blight resistance.

"phenotype" is understood within the scope of the present invention to mean one or more distinguishable characteristics of a trait controlled on a gene.

A "plant" is any plant, particularly a seed plant, at any stage of development.

A "plant cell" is the structural and physiological unit of a plant, comprising protoplasts and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organised unit such as, for example, a plant tissue, a plant organ or a whole plant.

By "plant cell culture" is meant a culture of plant units (such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages).

"plant material" means leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A "plant organ" is a distinct and distinct, structured and differentiated part of a plant, such as a root, stem, leaf, bud, or embryo.

"plant tissue" as used herein means a group of plant cells organized into structural and functional units. Including any plant tissue in a plant or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and/or functional units. The use of this term in combination or alone with any particular type of plant tissue as listed above or otherwise encompassed by this definition is not intended to exclude any other type of plant tissue.

The term "plant part" refers to a part of a plant, including single cells and cell tissues (e.g., intact plant cells in a plant), cell clumps, and tissue cultures from which a plant can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovule, leaf, embryo, root tip, anther, flower, fruit, stem, bud, and seed; and pollen, ovule, leaf, embryo, root tip, anther, flower, fruit, stem, bud, scion, rhizome, seed, protoplast, callus, and the like.

The term "population" means a genetically heterogeneous collection of plants that share a common genetic derivation.

The term "predominantly male sterile" means that no more than 10%, preferably no more than 5%, more preferably no more than 1% of the flowers on all of these plants have a functional male organ that produces viable pollen in at least 100 plants. It must be understood that a single plant may have both fertile and sterile flowers. In a preferred embodiment, no more than 10%, preferably no more than 5%, more preferably no more than 1% of the flowers on a single plant have functional male organs that produce fertile pollen.

The term "progeny" plant refers to any plant that is a vegetative or sexually reproducing progeny of one or more parent plants or progeny thereof. For example, progeny plants may be obtained by cloning or selfing of the parent plants or by crossing of two parent plants and include the selfing as well as F1 or F2 or even further generations. F1 is a first generation progeny produced from parents, at least one of which is used first as a donor for the trait, while progeny of a second (F2) or subsequent generation (F3, F4, etc.) are samples produced by selfing of F1, F2, etc. Thus, F1 may be a hybrid produced by a cross between two true breeding parents (true breeding is homozygous for the trait), while F2 may be a progeny produced by self-pollination of the F1 hybrid.

"recombination" is the exchange of information between two homologous chromosomes during meiosis. The frequency of double recombination is the product of the frequencies of the individual recombinants. For example, the frequency of recombinants found in the 10cM region was 10%, and the frequency of double recombinants was found to be 10% x 10% to 1% (1 centimorgan is defined as 1% of the recombinant progeny in the test cross).

The term "RHS" or "restored hybrid system" refers to a hybrid system based on nuclear male sterility.

As used herein, the phrase "sexually crossed" and "sexual reproduction" refers in the context of the subject matter of the present invention to the fusion of gametes to produce progeny (e.g., by fertilization, such as by pollination to produce seed in a plant). In some embodiments, "sexual crossing" or "allofertilization" is the fertilization of one individual by another (e.g., cross-pollination in a plant). In some embodiments, the term "selfing" refers to the production of seeds by self-fertilization or self-pollination; i.e. pollen and ovule from the same plant.

"Selective breeding" is understood within the scope of the present invention to mean a breeding program which uses plants having or showing desirable traits as parents.

A "test" plant is understood within the scope of the present invention to mean a plant which is used to genetically characterize a trait in the plant to be tested. Typically, the plant to be tested is crossed with a "test" plant and the segregation rate of the trait in the progeny of the cross is scored.

The term "test subject" refers to a line or individual having a standard genotype, known characteristics, and established performance. "test subject parent" refers to an individual from a test subject line that is used as a parent in a sexual cross. Typically, the test subject parent is unrelated to the individual to which it is hybridized and is genetically distinct. When crossing with individuals or inbred lines for phenotypic evaluation, test subjects are typically used to generate F1 progeny.

The phrase "top cross combination" refers to the process of crossing a single test subject line with multiple lines. The purpose of generating such crosses is to determine the phenotypic performance of the hybrid progeny; that is, the ability of each line of the plurality of lines to produce a desired phenotype in hybrid progeny derived from that line is assessed by crossing the test subjects.

The terms "variety" and "cultivar" refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and/or performance.

Detailed Description

One embodiment of the invention is a method of increasing the transformation efficiency of a recalcitrant corn line, the method comprising: (a) obtaining recalcitrant corn plants and collecting pollen therefrom; (b) pollinating a recipient maize plant comprising a normal A ("NA") cytoplasm with pollen from said recalcitrant maize plant; and (c) obtaining progeny embryos therefrom; wherein the progeny embryo comprises a NA cytoplasm and at least a nuclear genome of the recalcitrant maize plant, and wherein the progeny embryo has a higher transformation efficiency than the recalcitrant maize plant. Another embodiment is a method of imparting transformability to a recalcitrant corn line, the method comprising the steps of: (a) obtaining recalcitrant corn plants and collecting pollen therefrom; (b) pollinating a recipient maize plant comprising a normal A ("NA") cytoplasm with pollen from said recalcitrant maize plant; and (c) growing progeny plants therefrom; wherein the progeny plant comprises the NA cytoplasm and the nuclear genome of the recalcitrant maize plant, and wherein the progeny plant is transformable. In one aspect, the recalcitrant maize plant comprises a non-NA cytoplasm. In another aspect, the non-NA cytoplasm is selected from the group consisting of: normal B ("NB") cytoplasm, cytoplasmic male sterile C ("C" or "CMS-C") cytoplasm, cytoplasmic male sterile S ("S" or "CMS-S") cytoplasm, or cytoplasmic male sterile T ("T" or "CMS-T") cytoplasm. In one aspect, the recipient maize plant is a haploid inducer plant, or more specifically a male parent haploid inducer plant. In another aspect, the male haploid inducer plant comprises a mutated ig1 gene or CENH3 mutation.

In another embodiment, the progeny embryo is grown into a progeny plant. In one aspect, the progeny plant is backcrossed with the recalcitrant corn plant for at least one generation. In another aspect, the progeny plant is the female parent in backcrossing. In yet another embodiment, the progeny plant retains the NA cytoplasm and/or retains the nuclear genome of the recalcitrant maize plant.

In yet another embodiment, the invention is a method of increasing the transformation efficiency of a recalcitrant plant line, the method comprising: (a) obtaining recalcitrant plants and collecting pollen therefrom; (b) pollinating a recipient plant comprising a transformable cytoplasm with pollen from the recalcitrant plant; (c) obtaining progeny embryos therefrom; and (d) optionally (i) growing the progeny tissue into a progeny plant; (ii) at least one cross with pollen from the recalcitrant plant to backcross with the progeny plant; and (e) transforming tissue derived from the progeny embryo; wherein said progeny tissue comprises the NA cytoplasm and at least the nuclear genome of said recalcitrant plant, and wherein said progeny tissue has a higher transformation efficiency than the recalcitrant plant.

Another embodiment of the invention is a method of transforming a plant, the method comprising: (a) obtaining a plurality of plant lines; (b) testing for markers indicative of NA cytoplasm; (c) selecting the at least one line from the plurality of plant lines, wherein the selected line has a marker for the NA cytoplasm; and (d) transforming cells derived from at least one selected strain of step (c). In one aspect, the test detects a G nucleotide at a position corresponding to position 11 of the mitochondrial DNA sequence SEQ ID NO 7. In another aspect, this test for markers indicative of NA cytoplasm detects the presence of the sequence corresponding to SEQ ID NO 7. Optionally, the assay for markers indicative of NA cytoplasm further comprises forward primer SEQ ID NO 5 and reverse primer SEQ ID NO 6. Alternatively, the test for labeling indicative of the NA cytoplasm comprises probe SEQ ID NO. 7 and/or probe SEQ ID NO. 8, wherein the probes are differentially labeled with fluorophores.

These and other embodiments of the present invention will be more fully understood from the following non-limiting examples.

Examples of the invention

Example 1. cell type transformability and cell type transfer.

Markers that distinguish between NA, NB and CMS cytoplasm were developed based on the NA and NB Mitochondrial Genomes disclosed in James O.Allen et al, incorporated herein by reference, Comparisons Among Two Fertilie and Three Man-Sterile Mitochondrial Genomes of Maize [ comparison between Two Fertile and Three Male Sterile Mitochondrial Genomes in Maize ], Genetics [ Genetics ], 177:1173-1192 (10 months 2007) ]. Cytoplasmic genotyping was performed on 100 lines with molecular markers that distinguish normal cytoplasm from CMS cytoplasm (marker set 1; SEQ ID NOS: 1-4), followed by NB marker only (marker set 2, SEQ ID NOS: 5-8). In this case, the NA genotype is inferred when marker set 1 is positive (meaning the cell type is not CMS) and marker set 2 is positive for the alternative allele (meaning the cell type is not NB). The transformation frequency of the 11 inferred NA lines was tested using standard transformation procedures (e.g., the procedure disclosed in U.S. patent application publication No. 2015/0113681 filed on 2013, 10, 23, which is incorporated herein by reference in its entirety). The strain NP2222 was highly transformable and used as a control benchmark for transformation frequency.

Table 2. selected transformation frequency of NA cytoplasmic maize lines.

Strain 17 and strain 18 were tested in a second assay performed separately.

TABLE 3 NB strains and reciprocal crosses with NP 2222.

In the cross, the recipient system (i.e., the female parent) is listed first, and the pollen donor (i.e., the male parent) is listed second. Without wishing to be bound by theory, it is believed that the NP2222 parent also confers some other nuclear genetic factor that improves the transformability of progeny. The current transformation protocol is believed to be biased towards NP2222 and its derivatives. Even so, a 6% improvement (e.g., in hybridization group 3) is a significant improvement in convertibility and is a surprisingly good improvement in this regard.

By introducing NA cytoplasm into the progeny, transformation rates were significantly increased in most lines compared to the recalcitrant parent with NB cell type. Even a modest increase in conversion rate (i.e., 0.0% to 0.8%) is a significant improvement in non-transformability relative to NB cell types.

Example 2.ig1 mediated cell type transfer.

Maize mutants do not determine that gametophyte 1(ig1) produces 1% -10% maternal and paternal haploid progeny (Kindinger, 1994). NA transformable lines were crossed with heterozygous ig1 individuals, PCR genotyped to identify heterozygous vectors, and self-pollinated to generate NA versions of ig1 stock. F2 progeny were PCR genotyped and homozygous mutant ig1 individuals were pollinated by line 13. A paternal haploid is identified and pollinated by recurrent parent of line 13. The transformation test from normal B to normal a was unsuccessful.

Example 3. no adverse effect on yield.

The effect of cell type on hybrid yield was evaluated by crossing line 7, line 9, line 10, line 11 and NP2222 positively and negatively with normal B test species and growing the progeny at approximately 10 yield test sites. No phenotypic differences were apparent in the progeny of the crosses in both positive and negative crosses, and no statistically significant differences were observed between the hybrids, the sites, or any interaction terms. This finding indicates that the cell type is an attribute that is not expected to affect any significant plant attributes, and further illustrates the importance of differential effects on transformation capacity.

Although the present invention has been described in considerable detail, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the versions contained herein.

All the features disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

Sequence listing

<110> Syngenta Crop Protection, LLC

Skibbe, David

Elumalai , Sivamani

<120> improvement of plant transformability by cell type transfer

<130> 81844-WO-REG-ORG-P-1

<150> US 62/827,450

<151> 2019-04-01

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 1

atggtgccaa ttcgtaattt aagtt 25

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 2

acccctctgg ttgcctctct 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 3

tatgaagaag aataccatcc 20

<210> 4

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 4

aataccctcc agtttc 16

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 5

gtattcgcac ctactctgcc g 21

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 6

aaagcaaaaa taccattgca acc 23

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 7

cgtaaatttt gtttgatgc 19

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 8

atcgtaaatt ttttttgatg c 21