JP2003525589A - Genetic breeding method - Google Patents

Abstract

(57) [Summary] Provided is a method for screening for a trait associated with altered expression of a gene of interest in a plant. The method is a combinatorial approach using traditional plant breeding techniques to alter the expression pattern of the gene of interest. Also provided are kits for use in the method and transgenic plants generated by the method.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention is in the field of plant molecular biology. In particular, the invention relates to methods for breeding plants for improved agronomic traits.

[0002] The background of the invention up to now, almost all of the improvement of agricultural crops (crop) is, using traditional plant breeding techniques, have been achieved. These techniques involve crossing parental plants with different genetic backgrounds to produce progeny with genetic diversity. The progeny are then selected to obtain plants that express the desired trait. Then, through a number of backcrosses or selfing, the desired trait is fixed, while the deleterious trait is removed to finally obtain progeny with the desired traits. Hybrid corn (co
rn), low erucic acid oilseed rape, high oil corn, and hard white winter wheat.
eat) is an example of an important agricultural advance achieved by traditional breeding.

However, the amount of germplasm genetic diversity in a particular crop limits what can be achieved by breeding. Although traditional breeding has proven to be very powerful, as evidenced by advances in crop yield over the last century, recent data show that yield improvement rates for major food crops are high. , Suggesting a gradual decrease (
Lee (1998) Proc. Natl. Acad. Sci. USA
95: 2001-2004). The introduction of molecular mapping markers into breeding programs may accelerate the crop improvement process in the near future, but ultimately will be limited by the lack of new sources of genetic diversity. In particular, traditional breeding has proved rather ineffective in improving many polygenic traits, such as increased disease resistance.

Recently, biotechnology approaches involving the expression of a single transgene in crops have led to herbicide resistance (glyphosate or roundup resistance), insect resistance (Bacillus thuringiensis).
sis) expression of toxins) and virus resistance (overexpression of viral coat proteins), resulting in successful commercial introduction of new plant traits. But,
The list of monogenic traits of significant value is relatively small. The greatest potential of biotechnology lies in designing complex polygenic traits with respect to environmental stress, disease, plant development and structure, yield and quality traits. Currently, the design of such polygenic traits has been shown to be very difficult.

The present invention provides a novel method for rapidly identifying genes that are useful in modifying complex plant traits.

[0006] In summary one aspect of the invention, the present invention provides a plant relates to traits associated with altered expression of a gene of interest, provides a method for systematically screening.
The method comprises a first pool of donor vectors each containing a transactivator and a second pool of receptor vectors each containing a transactivator binding site operably linked to a different gene of interest. Including providing a pool of. A first plant is then transformed with a member of the donor vector pool, and a second plant is transformed with a member of the receptor vector pool, a second transformed plant containing the donor vector and a second transformed plant containing the receptor vector. To produce a transformed plant. The first and second transformed plants are crossed to produce a hybrid plant. Both the first and second transformed plants have a wild-type phenotype because the expression level of the gene of interest has not been altered from that of non-transformed plants. However, the phenotype of hybrid plants containing both the transactivator and the gene of interest may be different from the wild type because the expression level of the gene of interest is modified compared to that of non-transformed plants. There is a nature. Investigate the phenotype of these plants,
Identify hybrid plants with improved traits.

The transactivator appears to be functional in (1) a constitutive promoter, (2) an inducible promoter, (3) a tissue-active or specific promoter, or (4) a developmental stage-active or specific promoter. May be connected to. If the transactivator is linked to a constitutive promoter, changes in gene expression will be observed in all tissues and at all times, and a broad overview of gene expression effects on plants will be observed. When the transactivator is linked to a tissue-specific or inducible or developmental promoter, gene expression is expressed in specific tissues, such as seeds, roots, flowers, leaves, shoots, fruits or stems. Can be switched on or off during a specific time of development, for example during the early, middle or late stages of development, or under specific conditions, such as under specific environmental or disease stresses. The plant may be transformed with more than one receptor vector or more than one donor vector.

The gene of interest may be any gene, but is preferably a regulatory gene such as a transcription factor, a phosphatase or a protein kinase. In one preferred embodiment, the gene of interest is any transcription factor identified in plants,
For example, those identified in Arabidopsis thaliana. These genes collectively regulate the expression of all genes in plants, and thus all plant traits. The gene is
It may be in the sense direction for overexpression analysis, or may be in the antisense direction for underexpression analysis. Furthermore, the gene may be the full length coding sequence of the gene or a fragment of the gene, in particular a biologically active fragment.

The donor and receptor vectors may also include first and second selectable markers, respectively, to aid in selecting transformed hybrid plants.

In a second aspect, the present invention provides methods for breeding plants for desirable or improved traits. The method involves transactivating a member of a first pool of plants, each transformed with a donor vector containing a transactivator, operably linked to a different gene of interest. It involves hybridizing with a member of a second pool of plants that have been transformed with a receptor vector containing a binding site to produce a hybrid plant population. Using traditional plant breeding techniques, transgenic plants are obtained that have both donor and receptor vectors and that exhibit desirable or improved traits. Furthermore, the present invention provides a transgenic plant produced by the above method.

In yet another aspect, the present invention is a plant breeding kit. The plant breeding kit comprises (a) a pool of activator vectors, wherein each donor vector comprises a transactivator, and (b) a pool of receptor vectors, wherein each receptor vector comprises a different gene of interest. Comprising a transactivator binding site operably linked to. The vector may also include first and second selectable markers to assist in selecting transformed hybrid plants. The transactivator is operably linked to (1) a constitutive promoter, (2) an inducible promoter, (3) a tissue activity or specific promoter, or (4) a developmental stage activity or specific promoter. May be.

The individual donor and receptor vectors are transformed into first and second plants, respectively, and using traditional plant breeding techniques, the first and second plants have agronomically valuable traits. Generate a hybrid plant containing the two vectors.

In a further aspect, the invention is a method for modifying gene expression patterns in plants. The method begins with a first pool of donor vectors, each activating vector member comprising a transactivator, and a transactivator operably linked to each of the receptor vector members. Providing a second pool of receptor vectors containing a binding site.
The activator and receptor vector members are transformed into first and second plants. The transformed first and second plants are crossed to produce hybrid plants with modified gene expression patterns.

DETAILED DESCRIPTION OF THE INVENTION Definitions "transgenic plant or transgenic plant" refers to a plant comprising the introduced recombinant polynucleotide by transformation. Transformation means introducing a nucleotide sequence into a plant in any manner that causes stable or transient expression of the sequence. This can be accomplished by transfection with a viral vector, transformation with a plasmid such as an Agrobacterium-based vector, or introduction of naked DNA by electroporation, lipofection, or particle gun acceleration. Is possible. Transformed plant can refer to whole plants, as well as seeds, plant tissues, plant cells, or any other plant component, and the progeny of the plant.

A “vector” is a nucleic acid construct that contains a nucleic acid region that is recombinantly or synthetically produced and is capable of directing the expression of a gene. A "donor vector" is a construct for expressing a polynucleotide sequence of a transactivating gene. The transactivating gene is operably linked to a promoter.
The promoter region may include a tissue activity or specific promoter, a developmental stage activity or specific promoter, an inducible promoter or a constitutive promoter.

A “receptor vector” is a construct for expression of a gene of interest such as a regulatory gene. Typically, the receptor vector contains a sequence of transactivator binding sites. Construct sequences may also include promoters, operators, enhancer regions, silencer regions, polyadenylation sites, translation initiation sites and the like.

A “gene of interest” is a polynucleotide sequence of a regulatory gene, such as a transcription factor, protein kinase or phosphatase. These sequences may be in sense or antisense orientation, or may be partial or complete gene sequences.

A nucleotide sequence is “operably linked” when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or enhancer is operably linked to a gene coding sequence if the presence of the promoter or enhancer increases the expression level of the gene coding sequence.

A “pool” comprises a group of at least 2 members, preferably at least 10 members, more preferably at least 100 members. For example, the pool may include all identified genes of a particular type of plant, for example, all identified transcription factors from a plant, as exemplified by up to 1,700 transcription factors identified in Arabidopsis. Good.

The phrase “altered expression or modified expression” with respect to polynucleotide or polypeptide expression refers to an expression pattern in a transgenic plant that differs from the expression pattern in a wild-type plant or a reference plant; Is expressed in a cell type different from the cell type expressed in the wild-type plant, or at a different time than the time the sequence is expressed in the wild-type plant, or a different inducer, such as a hormone or environmental signal. Or a different expression level (higher or lower) as compared to that found in wild type plants. The term also reduces the expression level below the level of detection,
It also refers to completely abolishing expression. The resulting expression pattern may be transient or stable, constitutive or inducible.

“Trait” refers to a physiological, morphological or physical characteristic of a plant or a particular plant component. These properties may be visible to the human eye, such as germination rate or seed size, or the protein, starch or oil content of the seed, by biochemical assays, or Northern, RT PCR or microarray gene expression. It may be measurable by laboratory techniques, such as changes in the expression level of a gene by using the assay.

For the purpose of trait modification or improvement for a specific purpose, seeds, fruits, roots, flowers, leaves, stems, shoots,
Includes seedlings, etc .: increased resistance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; increased resistance to microbial, fungal or viral diseases; against nematodes Resistance, decreased herbicide sensitivity, increased tolerance of heavy metals (or increased ability to take up heavy metals)
, Enhanced growth under poor light conditions (eg, low light and / or short photoperiod),
Alternatively, it includes changes in the expression level of the gene of interest. Other traits that can be modified are
Production of plant metabolites such as taxol, tocopherols, tocotrienols, sterols, phytosterols, vitamins, wax monomers, antioxidants, amino acids, lignins, cellulose, tannins, prenyl lipids (eg chlorophylls and carotenoids) ), Glucosinolates, and terpenoids, or compositionally modified proteins or oil production (especially in seeds), or modified sugars (insoluble or soluble) and / or starch composition. Physical plant characteristics that may be modified include cell development, fruit and seed size and number, yield of plant parts such as stems, leaves and roots, seed stability during storage, seed pod characteristics (eg susceptibility to grinding). , Root hair length and amount, internodal distance, or seed coat quality. Plant growth characteristics that can be modified include growth rate, seed germination rate, plant and seedling vitality, leaf and flower senescence, male sterility, apomixis, flowering time, flower withdrawal, nitrogen uptake rate, biomass or transpiration characteristics, Includes plant structural characteristics such as apical dominance, branching pattern, organ number, organ identity, organ shape or size.

“Hybrid plant” refers to a plant produced by crossing two plants of interest, growing them with seeds or tissues, and then growing the plants. If the plants are sexually crossed, the pollination stage may include cross-pollination or self-pollination, or backcrossing with a non-transformed plant or another transformed plant. Hybrid plants include first generation and later generation plants.

The present invention provides methods for manipulating and improving plant traits. The method combines the power of genomics with plant breeding techniques. In the method, the expression level of a known gene of interest in a plant is constitutively modified or selectively modified, and tissue-specific expression, inducible expression, developmental stage-specific expression, etc. are treated with a high treatment method. It is possible to monitor. Then, phenotypic changes and the like resulting from the expression of a specific plant gene at different levels at different times under different kinds of stress in different plant tissues and the like are screened. Finally, select plants with improved traits.

The method comprises transforming a first plant with a member of a first pool of donor vectors. Each donor vector contains a transactivator placed under the control of a different promoter so that the expression of the transactivator can be regulated under different conditions. In addition, the method comprises transforming a second plant with a member of the second pool of receptor vectors. The receptor construct contains a transactivator binding site for binding of the transactivator.
The transactivator binding site is operably linked to the gene of interest so that when transactivator expression is switched on, overexpression of the gene (eg, by using a sense construct) ) Or underexpression (eg by using an antisense construct). Of particular interest are genes that affect polygenic traits, such as regulatory genes.

Then, two pools of plants: a first pool designed to contain specific regulatory sequences (eg promoters) and a second pool designed to contain genes of interest (eg regulatory genes). Specific crosses are made in a combinatorial manner between individual members of origin. The gene of interest is expressed only in the progeny plants under the control of each different promoter, providing the same effect as if each plant had been originally transformed with a particular gene-promoter combination. In this manner, it is thus possible to create a large number of specific gene-promoter combinations and to study their effects on transcriptional expression and trait improvement with minimal time and expense.

This method describes a method of transforming both donor and receptor vectors into the same plant, controlling gene expression and observing trait improvement in plants.
iu et al., US Pat. No. 5,968,793 and Guyer et al. (1998) Ge.
149: 633-639. Our method provides a higher degree of flexibility and speed in observing the effects of selective gene expression on plant traits.

By this method, it is possible to investigate any plant trait improvement. The plant
Crop plants, such as soybean, wheat, corn, potato, cotton, rice, oilseed rape (canola)
, Sunflower, alfalfa, sugarcane (sug)
arcane) and turf; or fruit or vegetable plants such as apple, banana, blackberry, blueberry, strawberry and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grape. , Honeydew, lettuce, mango, melon, onion, papaya, pea, pepper, pineapple, spinach (spinach)
), Squash, sweet corn, tobacco, tomato, watermelon, roseceous fruits (eg peaches, cherries and plums)
And brassicas vegetables such as broccoli, cabbage, cauliflower, brussels sprouts and kohlrabi. Other crops, fruits and vegetables that may have improved traits include barley, sorghum.
), Currants, avocados, citrus fruits such as oranges, lemons, grapefruits and tangerines, artichokes, currants, cherries, nuts,
For example, walnut and peanuts, pear,
Endive, leak, root vegetables, such as arrowroot (
arrowroot, beet, cassava,
Includes turnips, radish, yams, sweet potatoes and beans.

One embodiment of the present invention is illustrated in FIG. The activator plant was constructed with a construct containing one of 10 different promoters linked to the transactivating gene (in this figure, L
A fusion of the acI binding domain and the Gal4 transcriptional activation domain) is grown from seeds derived from plants. The target plant is La, which is linked to one of the more than 1,700 transcription factor genes that have been identified from Arabidopsis thaliana.
Transform with the construct containing the cI binding site. The target plant does not express plant transcription factors unless the transactivator is present. Only after the activator and the target plant are crossed, the transactivator binds to the binding site on the plant transcription factor gene construct, whereby the plant transcription factor is overexpressed or underexpressed. Thus, traits associated with selective or constitutive, over- or under-expression of plant transcription factors in plant tissue can be easily regulated and screened depending on the promoter.

Donor Constructs Donor constructs include a recombinant polypeptide sequence encoding a DNA binding domain fused to a transcription activation domain. The recombinant polynucleotide is a transactivator. A DNA binding domain is a sequence that binds to DNA with some specificity. A common feature of several activation domains is that they are designed to form amphipathic alpha helices with excess positive or negative charges (Giniger and Ptashne (1987) Nature 3).
30: 670-672, Gill and Ptashne (1987) Cell.
51: 121-126, Estruch et al. (1994) Nucl. Acids
Res. 22: 3983-3989). Examples include the transcriptional activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl. Acad.
． Sci. USA 95: 376-381; and Aoyama et al. (199).
5) Plant Cell 7: 1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51; 113-119).
And synthetic peptides (Giniger and Ptashne, supra). Typical transactivators are described in Brent and P, incorporated herein by reference.
Tashne, US Pat. No. 4,833,080 or Hasselhoff and Hodge, WO 97/30164.

A variety of promoter sequences are available that may be used to regulate the expression of transactivators. Such promoters may be utilized to initiate transcription of a nucleic acid sequence of interest operably linked to the promoter region.

Viral promoters may be utilized in plant expression vectors for constitutive expression in plants. These include 35MV RNA and 19SR of CaMV.
NA promoter (Brisson et al., Nature, 310: 511,
1984; Odell et al., Nature, 313: 810, 1985).
Species squirrel mosaic virus (Figwort Mosaic Virus)
) (FMV) (Gowda et al., J. Cell Biochem., 13D.
: 301, 1989) and the coat protein promoter of TMV (Tak).
amatsu et al., EMBO J .; 6: 307, 1987). Additional promoters include the nopaline synthase promoter (An et al. (1988
) Plant Physiol. 88: 547); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 97).
7) is included.

The donor construct may include one or more inducible promoters.
Examples of inducible promoters useful in plants are those that are induced by chemical means, such as the yeast metallothionein promoter that is activated by copper ions (Mett et al. (1993) Proc. Natl. Acad. Sci.,
U. S. A. , 90: 4567); substituted benzene sulfonamides, such as the In2-1 and In2-2 regulator sequences activated by the herbicide safeners (Hershey et al. (1991) Plant Mol.
Biol. , 17: 679); and GRE regulator sequences induced by glucocorticoids (Schena et al. (1991) Proc. Natl. A.
cad. Sci. , U. S. A. , 88: 10421). Plant promoters also include light-inducible promoters derived from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al. (1984) EMBO J., 3: 1671; Brogie et al. (1984).
) Science, 224: 838), heat (Callis et al. (1988) Pl.
ant Physiol. 88: 965; Ainley et al. (1993) Pl.
ant Mol. Biol. 22: 13-23), hormones such as abscisic acid (Marcotte et al. (1989) Plant Cell 1: 96).
9); Wounds (eg wunI, Siebertz et al. (1989) Plant C)
ell 1: 961) and chemicals such as methyl jasmine (meth
yl jasminate) or salicylic acid (Gatz et al. (1997) Ann.
． Rev. Plant Physiol. Plant Mol. Bio
l. 48: 89-108).

Tissue-specific promoters may also be used for gene expression in plants.
Tissue-specific promoters useful for transgenic plants include the cdc2a promoter and the cyc07 promoter (Ito et al. (1994) Pl.
ant Mol. Biol. 24: 863; Martinez et al. (19
92) Proc. Natl. Acad. Sci. USA, 89:73.
60; Medford et al. (1991) Plant Cell, 3: 359;
Terada et al. (1993) Plant Journal, 3: 241;
Wissenbach et al. (1993) Plant Journal, 4:41.
1). Additional tissue-specific promoters utilized in plants include the histone promoter (Atanassova et al. (1992) Plant Journal,
2: 291); cinnamyl alcohol dehydrogenase (CAD) promoter (Feuillet et al. (1995) Plant Mol. Biol., 2).
7: 651); mustard CHS1 promoter (Kaise)
r et al. (1995) Plant Mol. Biol. , 28: 231); pea grp 1.8 promoter (Keller et al. (1994) Plant M).
ol. Biol. , 26: 747); PAL1 promoter (Ohl et al. (1
990) Plant Cell, 2: 837); and chalcone synthase A.
Promoter (Plant Mol. Biol., (1990) 15: 9
5-109) are included. Furthermore, the timing of expression is related to aging (Gan and A
masino (1995) Science 270: 1986-1988);
Or late seed development (Odell et al. (1994) Plant Physiol.
106: 447-458) and can be regulated by using promoters such as those acting at

Other promoters include root-specific promoters such as US Pat. No. 5,618.
, 988, No. 5,837,848 and No. 5,905,186, or Wanapu and Shinmyo (199).
6) Ann. N. Y. Acad. Sci. 782: 107-113 or Miao et al. (1991) Plant Cell 3: 11-22 or Hir.
el et al. (1992) Plant Mol. Biol. 20: 207-218
PrxEa promoter, seed-specific promoters such as napine, phaseolin or the DC3 promoter described in US Pat. No. 5,773,697, oleic acid 12-hydroxylase from Desquelerella: desaturase promoter (Brown). Et al. (1998) Pl
ant J. 13: 201-210), the Arabidopsis oleosin promoter (Plant et al. (1994) Plant Mol. Biol. 25: 1.
93-205), the maize zein promoter (Russel et al. (199
7) Transgenic Res. 6: 157-168), rice (Wash
ida et al., Plant Mol Biol. (1999) 40: 1-12) and the maize glutelin promoter (Thomas et al. (1990) Pl.
ant Cell 2: 1171-1180), fruit-specific promoters, such as those that are active during fruit ripening (eg, the dru 1 promoter (US Pat.
, 783,393) or the 2A11 promoter (US Pat. No. 4,943,6).
74) and the tomato polygalacturonase promoter (Bird et al. (19
88) Plant Mol. Biol. 11: 651), pollen activity promoters such as PTA29, PTA26 and PTA13 (US Pat. No. 5,792).
, 929), vascular tissue activity (Ringli and Keller (1998).
Plant Mol. Biol. 37: 977-988), flower-specific (Ka
iser et al. (1995) Plant Mol. Biol. 28: 231-2
43), pollen (Baerson et al. (1994) Plant Mol. Biol.
． 26: 1947-1959), carpel (Ohl et al. (1990) Plant C).
ell 2: 837-848), pollen and ovules (Baerson et al. (1993).
) Plant Mol. Biol. 22: 255-267) specific promoters.

A preferred inducible or tissue-specific promoter is Rd 29a (Yama
guchi-Shinozaki and Shinozaki (1993) Pla
nt Physiol. Mar; 101: 1119-20), LTP1 (Th
oma et al. (1994) Plant Physiol. 105 (1): 35-4
5), STM (Long et al. (1996) Nature. 1996 379:
66-9), rbcS (Krebbers (1988) Plant Mol B.
iol. 11: 745-759), sucrose synthase (Martin et al. (1993) Plant J. 4: 367-77), EIR1 (Luschn.
ig et al. (1998) Genes Dev. 12: 2175-87), IL (B
ernhard and Matile, GenBank Deposit No. M83534),
PR1 (Lebel et al. (1998) Plant J. 16: 223-33),
AGL1 (Ma et al. (1991) Genes 5: 484-95), AP1 (M
andel et al. (1992) 360: 273-7), E4 (Cordes et al. (1
989) Plant Cell. 1 (10): 1025-34) or GL2
(Rerie et al. (1994) Genes Dev. 8 (12): 1388-.
99) is included.

The donor construct may also include a further sequence, for example, a selectable marker linked to a constitutive promoter to select for plants containing the donor construct in a trial or tissue culture. These include chlorosulfuron
A acetoacetate synthase gene for sulfuron) resistance or a gene conferring resistance to cyanamide may also be included.

The plant transformation construct may also contain an RNA processing signal, such as an intron, which may be located upstream or downstream of the open reading frame sequence (ORF). In addition, the expression construct may also be derived from the 3'-untranslated region of plant genes, such as the 3'terminator region that increases mRNA stability of mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3'terminator region. May also include additional control sequences for.

Receptor Constructs Receptor constructs are transactivators described above, eg, US Pat. No. 4,833,080, operably linked to a gene of interest, eg, a transcription factor, phosphatase or kinase. It comprises one or more DNA binding sites, one of those disclosed. The transcription factors included in the construct may be from one or more of the transcription factor families described below.

Plant transcription factors may belong to one of the following transcription factor families: AP2 (
APETALA2) domain transcription factor family (Riechmann and M
eyerowitz (1998) J. Biol. Chem. 379: 63
3-646); MYB transcription factor family (ADDIN ENBib Martin and Paz-Ares (1997) Trends Genet. 13: 67-73.
); MADS domain transcription factor family (Riechmann and Mey
erowitz (1997) J. Biol. Chem. 378: 1079
-1101); WRKY protein family (Ishiguro and Nak
amura (1994) Mol. Gen. Genet. 244: 563-
571); ankyrin-repeat protein family (Zhang et al. (199
2) Plant Cell 4: 1575-1588); zinc finger protein (Z) family (Klug and Schwave (1995) FASE.
BJ. 9: 597-604); Homeobox (HB) protein family (Duboule (1994) Guidebook to the Hom.
ebox Genes, Oxford University Press
); CAAT-sequence binding protein (Forsburg and Guarente)
(1989) Genes Dev. 3: 1166-1178); Squamosa (
squamosa) promoter binding protein (SPB) (Klein et al. (1
996) Mol. Gen. Genet. 1996 250: 7-16);
NAM protein family (Souer et al. (1996) Cell 85:15.
9-170); IAA / AUX protein (Rouse et al. (1998) Scie.
279: 1371-1373); HLH / MYC protein family (Littlewood et al. (1994) Prot. Profile 1:63.
9-709); DNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J. 13: 2994-3002); bZI of transcription factors.
P family (Foster et al. (1994) FASEB J. 8: 192-2.
00); box P- binding protein (B ox P -binding prot
ein) (BPF-1) family (da Costa e Silva et al. (1
993) Plant J. 4: 125-135); High mobility group (HMG) family (Bustin and Reeves (1996) Prog. Nucl.
Acids Res. Mol. Biol. 54: 35-100); Scarecrow (SCR) family (Di Laurenzio et al. (1996) Ce.
86: 423-433); GF14 family (Wu et al. (1997) Pl
ant Physiol. 114: 1421-1431); Polycomb (PC
OMB) family (Kenison (1995) Annu. Rev. G
enet. 29: 289-303); the teosinte branch (
TEO) family (Luo et al. (1996) Nature 383: 794-7.
99); ABI3 family (Giraudat et al. (1992) Plant C.
ell 4: 1251-1261); triple helix (TH) family (Dehe
sh et al. (1990) Science 250: 1397-1399); EIL family (Chao et al. (1997) Cell 89: 1133-44); AT-
HOOK Family (Reeves and Nissen (1990) Journal
al of Biological Chemistry 265: 8573-
8582); S1FA family (Zhou et al. (1995) Nucleic A
cids Res. 23: 1165-1169); bZIPT2 family (
Lu and Ferr (1995) Plant Physiol. 109: 72
3); YABBY family (Bowman et al. (1999) Developm
ent 126: 2387-96); PAZ family (Bohmert et al. (1
998) EMBO J.M. 17: 170-80); including the DPBF family,
Various (MISC) transcription factors (Kim et al. (1997) Plant J. 11:
1237-1251) and the SPF1 family (Ishiguro and Na
kamura (1994) Mol. Gen. Genet. 244: 563
-571); Golden (GLD) family (Hall et al. (1998) Pla
nt Cell 10: 925-936).

Other transcription factors can be found in polynucleotide or polypeptide sequence databases, such as GenBank, Altschul et al. (1994) Nature Gen.
It can be identified by screening using sequence parallelism and homology calculations such as those described in etics 6: 119-129.
For example, NCBI basic local parallel search tool (Basic Local Ali)
gnment Search Tool) (BLAST) (Altschul et al. (1990) Basic local alignment search t.
pool. J. Mol. Biol. 215: 403-410) is a sequence analysis program, blastp, blastn, blastx, tblastp,
For use in combination with tblastn and tblastx, the National Center for Biot
It is available from several sources, including the technology information (NCBI, Bethesda, MD). Alternatively, a program that identifies specific sequence motifs, such as FIND PATTERN (GCG, Madison, WI) may be used with specific characteristic consensus sequences.

A typical transcription factor that may be used in the present invention is Z, filed September 13, 1999, entitled “Plant Gene Sequences I”.
Hang et al., U.S. patent application Ser. No. 09 / 394,519, filed on February 17, 2000, entitled "Plant Gene Sequences II", Keddie et al., U.S. patent application Ser. No. _________, March 2000. Two
"Polynucleotides for Seed T" filed on the 2nd
Keddie et al., US Patent Application No. _________, filed March 22, 2000, entitled "Polyester Alteration".
uclutides for Root Trait Alteration
Cai-Zhong et al., U.S. Patent Application No. ___________, filed March 22, 2000, entitled "Disease-Induced P."
, et al., US Patent Application No.
No. _________, filed March 22, 2000, “Stress-
Samaha, entitled "Induced Polynucleotides"
Et al., U.S. Patent Application No. _________, and "Polynucleotides for Flower Tra," filed March 22, 2000.
Included are those disclosed in Riechmann et al., U.S. Patent Application No. ___________, entitled "it Alteration".

Transcription factors include naturally occurring sequences as well as non-naturally occurring sequences that are derivatives of the transcription factors described above. For example, the amino acid sequence encoding the binding protein is not necessarily contiguous with a naturally occurring sequence, such as those shown above, or a functional domain from another sequence or source, but fused in frame as described above. It may be a non-naturally occurring sequence using the domain of the transcription factor. Further, the present invention provides
Minshull and Stemmer, “Methods and Comp”
positions for Cellular and Metabolic
US Patent No. 5,837,458 entitled "Engineering", and Stemmer and Crameri, "Methods for Generator".
ating Polynucleotides having Desired
Characteristics by Iterative Select
US Pat. No. 5,8, entitled "ion and Recombination".
The regions of the above transcription factors are reconstructed (sh
polypeptide).

The particular arrangement of transcription factor sequences in the transformation vector will be chosen according to the type of expression of the desired sequence. When it is desired to enhance transcription factor activity in plants, the transcription factor coding sequence is replaced by a constitutive high level promoter such as CaMV.
It may be operably linked to the 35S promoter. Generally, this will require a full length sequence encoding the transcription factor. In contrast, reduced transcription factor activity in transgenic plants can be obtained by introducing into plants antisense constructs based on the transcription factor cDNA. For antisense suppression, the transcription factor cDNA is placed in the transformation vector in the opposite orientation to the promoter sequence. The introduced sequence need not be the full-length transcription factor cDNA or gene, and the transcription factor c found in the plant species to be transformed.
It need not be exactly homologous to the DNA or gene. However, in general, if the introduced sequence is of shorter length, a higher degree of homology to the native transcription factor sequence will be required for effective antisense suppression.

A construct in which the RNA encoded by the transcription factor cDNA (or a variant thereof) is overexpressed is also used in the manner described in US Pat. No. 5,231,020 to Jorgensen, in which the endogenous transcription factor gene It may be used to obtain co-suppression. Such co-repression (also called sense repression) is a transcription factor cDNA in plant cells.
It is not required that the entire sequence be introduced, nor that the introduced sequence be exactly identical to the endogenous transcription factor gene. However, as in the case of antisense suppression, the suppression efficiency is enhanced as (1) the longer the introduced sequence and (2) the increased sequence similarity between the introduced sequence and the endogenous transcription factor gene. Will. Alternatively, suppression of transcription factor activity may be obtained by double-stranded RNA-mediated interference (Voinnet et al. (1998) Cell 95, 177-187,
Waterhouse et al. (1998) Proc. Natl. Acad. S
ci. USA 95, 13959-13964).

The receptor vector may also include additional sequences such as a selectable marker linked to a constitutive promoter for selecting plants which contain the activator construct in a working test or in tissue culture. These may include the acetoacetate synthase gene for chlorosulfuron resistance or the gene conferring resistance to cyanamide.

Any of the identified sequences may be incorporated into a cassette or vector for expression in plants. Several expression vectors suitable for the stable transformation of plant cells or the establishment of transgenic plants have been described, Weissbac
h and Weissbach (1989) Methods for Plant
Molecular Biology, Academic Press, and Gelvin et al. (1990) Plant Molecular Biolo.
gy Manual, Kluwer Academic Publisher
s are included. Specific examples include those derived from the Ti plasmid of Agrobacterium tumefaciens, along with Herrera-Estrel for dicots.
la, L.L. Et al. (1983) Nature 303: 209, Bevan,
M. , Nucl. Acids Res. (1984) 12: 8711-8.
721, Klee, H .; J. (1985) Bio / Technology
3: include those disclosed in 637-642.

Alternatively, non-Ti vectors may be used to introduce DNA into plants and cells by using free DNA delivery technology. Such methods may involve, for example, the use of liposomes, electroporation, biolistics, silicon carbide whiskers, and viruses. By using these methods, wheat, rice (Christou, P. et al. (1991) Bio / Technology 9
: 957-962) and corn (Gordon-Kamm, W. (1.
990) It is possible to produce transgenic plants such as Plant Cell 2: 603-618). Immature embryos have also been shown to use direct particle delivery technology (Weeks, T. et al. (1993) Plant P) by using a particle gun.
hysiol. 102: 1077-1084; Vasil, V .; (199
3) Bio / Technology 10: 667-674; Wan, Y .;
And Lemeaux, P .; (1994) Plant Physiol. 1
04: 37-48), and of Agrobacterium-mediated DNA transfer (Ishi.
da et al. (1996) Nature Biotech. 14: 745-75
0), an excellent target tissue.

[0049] Typically, the plant transformation vector is one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5'and 3'regulatory sequences.
NA) and a dominant selectable marker. These plant transformation vectors are
Typically also promoters (eg regulatory region regulatory inducible or constitutive, environmental or developmental control, or cell or tissue specific expression), transcription start sites, RNA processing signals (eg intron splicing sites), transcription termination sites , And / or also polyadenylation signals.

Transformation In order to understand the effect of the gene on the phenotype of the plant using standard techniques, the plant may be transformed with the above-mentioned vector and the gene of interest may be overexpressed or underexpressed in the plant. . Furthermore, it is possible to transform plants using a combination of transactivators or donor vectors and to understand the contribution of many genes of interest to the trait.

A typical plant that can be transformed can be any higher plant,
Includes monocots and dicots. A suitable protocol is the leguminous family (Legumin).
osae) (alfalfa, soybean, clover, etc.), Seriaceae (Umbel)
liferae) (carrot, celery, American parrot (parsnip))
, Cruciferae (Cabbage, Japanese radish, Brassica (ra)
pedseed), broccoli, etc.), Cucurbitaceae (Curcurbitaceae)
) (Melon and cucumber), Gramineae (Wheat, corn, rice, barley, millet, etc.), Solanaceae (Solana)
ceae) (potato, tomato, tobacco, pepper, etc.) and a variety of other crops. Ammirato et al. (1984) Handboo
k of Plant Cell Culture-Crop Species
． Macmillan Publ. Co. , Shimamoto et al. (198
9) Nature 338: 274-276; Fromm et al. (1990) Bi.
o / Technology 8: 833-839; and Vasil et al. (199).
0) See the protocol described in Bio / Technology 8: 429-434.

Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now a routine matter, and the choice of the most suitable transformation technique will be determined by the practitioner. The choice of method will vary depending on the type of plant to be transformed;
Those skilled in the art will recognize the suitability of a particular method for a given plant species. Suitable methods include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; viral transformation; plant cell micro-injection; It may include biolistics of plant cells; vacuum infiltration; and Agrobacterium tumefaciens (AT) -mediated transformation.

Successful examples of modification of plant traits by transformation with cloned cDNA sequences, which serve to illustrate current knowledge in the art and are incorporated herein, include: US Pat. No. 5,571,706; No. 5,677,175; No. 5
No. 5,510,471; No. 5,750,386; No. 5,597,945; No. 5,
, 589,615; 5,750,871; 5,268,526; 5
, 780,708; 5,538,880; 5,773,269; 5
, 736,369 and 5,610,042. Successful transformation of woody species with Agrobacterium tumefaciens, eg poplar (
poplar) and aspen transformations were performed using De Block (
1990) Plant Physiol. 93: 1110-1116. Other woody species that can be transformed include pine (
pine) is included. Of particular interest is the transformation of tomatoes, such as
Filatti et al., US Pat. No. 5,565,347.

After transformation and regeneration of plants with the transformation vector, transformed plants may be selected using the dominant selectable marker incorporated into the transformation vector.
Typically, such a marker would confer antibiotic or herbicide resistance to the transformed plant seedlings, and selection of transformants would expose the seedlings to an appropriate concentration of the antibiotic or herbicide. By doing so, it is possible to achieve.

Crosses As described in FIG. 1, plants transformed with any donor vector may be crossed with plants transformed with any receptor vector. Plants transformed with a receptor vector for a particular transcription factor (sense or antisense configuration) are transformed with a variety of donor vectors containing a variety of different promoters, including constitutive, inducible or tissue-specific vectors. It may hybridize with plants, and under specific conditions such as environmental stress and disease, the effect of transcription factor expression may be investigated in the whole plant and in specific tissues such as seeds, roots, flowers, stems, leaves and fruits. . Collect seeds and grow hybrid plants to maturity. These plants may then be screened to identify plants with valuable traits.

These plants may be grown in the presence of first and second herbicide resistance selectable markers. Seeds obtained from herbicide-resistant regenerated plants may be further crossed to produce later generation hybrid plants. Further details of crop breeding techniques, particularly those relating to tomatoes, are incorporated herein by reference, US Pat. No. 5,817,91.
No. 3 is described.

Furthermore, the present invention is a plant breeding kit. The method provides a seed or plant, provides a first pool of seeds or plants transformed with a donor vector, and provides a second pool of seeds or plants transformed with a receptor vector. To be done. The plant breeder may then cross plants from the first and second pools to breed plants with improved traits, as described above.

The following examples are provided to better illustrate the practice of the invention and should not be construed as limiting the scope of the invention in any way. One of ordinary skill in the art will recognize that various modifications can be made to the methods and genes described herein without departing from the spirit and scope of the invention.

[0059]

【Example】

The present invention is illustrated in detail by the examples herein, in which six different donor vectors having constitutive, inducible or tissue-specific promoters operably linked to either of two different transactivators. To manufacture. The invention is further described in detail by the production of eight different receptor vectors containing transactivator binding sites that functionally bind transcription factors. The donor and receptor vectors are then transformed into separate tomato plants. Next, the transformed tomato plants with different donor vectors are crossed with the transformed tomato plants with different receptor vectors. In its descendants,
Improved traits can be screened.

Example 1. Cloning of the promoter in the donor vector The plasmid vector pMEN020 was the vector used in the construction of the donor and receptor vectors. The pMEN020 plasmid construct was transformed into E. coli (
E.coli) and Agrobacterium tumefaciens
ciens) A binary cloning vector containing DNA replications of both origins but without the vir gene encoding a protein essential for transfer and integration of the target gene inserted into the T-DNA region. pMEN020 requires the trfA gene product which replicates in Agrobacterium. This trfA
Agrobacterium strains containing the gene are referred to as ABI strains and are described in US Pat. Nos. 5,773,701 and 5,773,696. This cloning vector serves as an E. coli-Agrobacterium tumefaciens shuttle vector. After performing all cloning steps in E. coli,
The vector is introduced into the ABI strain of Agrobacterium tumefaciens.

Two sets of different constructs were produced, each based on the bacterial DNA binding proteins LexA and LacI. For the LexA system, the construct was manufactured in two steps. First, an intermediate construct containing the LexA protein and the gal4 activation domain was generated. In the intermediate construct, four fragments occurred apart but were fused by overlap extension PCR. The first fragment is 35
S minimal promoter and omega translational enhancer; its fragment from construct SLJ4D4 (Jones et al 1992 Transgenic Research 1: 285)
Primer O11731 (SEQ ID NO: 1):

[0062]

[Chemical 1]

[0063] And O11733 (SEQ ID NO: 2):

[0064]

[Chemical 2]

Was amplified using. The second fragment is the E. coli lexA gene;
From LexA (Clontech, Palo Alto, Calif.), Primer O11732 (SEQ ID NO: 3):

[0066]

[Chemical 3]

[0067] And O117717 (SEQ ID NO: 4):

[0068]

[Chemical 4]

Was amplified with. The third fragment is the gal4 transcriptional activation domain;
424 (aa 768-881, Clontech), primer O11715 (SEQ ID NO: 5):

[0070]

[Chemical 5]

[0071] And O11718 (SEQ ID NO: 6):

[0072]

[Chemical 6]

Was amplified by. The fourth fragment is the E9 transcription terminator (Fluhr et al 1986 EMBO J. 5: 2063); it was derived from pMON10098 using primer O11716 (SEQ ID NO: 7):

[0074]

[Chemical 7]

[0075] And O11719 (SEQ ID NO: 8):

[0076]

[Chemical 8]

Was amplified using. The four fragments were fused to each other by PCR with primers O11731 and O11719. Note that the restriction enzyme digestion sites HindIII and XbaI were added to the ends of O11731 and O11719, respectively.

The insert from the intermediate construct was cloned into the HindIII and BamHI sites of pMEN020. The lacI system was constructed in two steps similar to the lexA system. lacI
The translation start of the gene was changed from GTG to ATG, and the mutation at position 17 (Y = H
Lehming et al 1987 EMBO J 6: 3145) was introduced. The lacI gene was transformed into E.co
li primer O16400 (SEQ ID NO: 9) by PCR amplification from genomic DNA:

[0079]

[Chemical 9]

[0080] And O16401 (SEQ ID NO: 10):

[0081]

[Chemical 10]

Was cloned using. Note that the NcoI and EcoRI sites were introduced with O16400 and O16401 respectively. The lacI coding region defined by the NcoI and EcoRI sites was used to replace the lexA coding region in the previous intermediate construct. The inserts from the two intermediate constructs were cloned into the HindIII and BamHI sites of pMEN020 in a three-way ligation.

A multiple cloning site was added by O11731 upstream of the LacI (LexA) / gal4 fusion protein to facilitate cloning of the promoter fragment. To investigate the functionality of the system, the complete 35S promoter was added upstream of the LacI (LexA) / gal4 fusion protein to the restriction enzyme Hi.
It may be cloned by using ndIII and NotI. Inducible promoter from plant genomic DNA is a PC with primers flanking the promoter region and containing suitable restriction sites for cloning into an activation vector.
It can be isolated by R amplification. For example, the rd29a gene has been characterized in Japan by the group of Shinozaki (Yamaguchi-Shinozaki and
Shinozaki (1993) Plant Physiol. 101: 1119-1120). Its rd29a gene expression is induced by drought, salt stress and exogenous ABA treatment (Yamaguch
i-Shinozaki and Shinozaki (1993) Plant Physiol. 101: 1119-1120 (1993); Is
hitani et al. (1998) Plant Cell 10: 1151-1161).

A genomic clone with the rd29a promoter was confirmed by using rd29a as a search term at the NCBI www site. The sequence of the rd29a promoter is located in the region from nucleotide positions 3892 to 5390 (accession number D13044). The following two primers were designed to amplify this promoter region from Arabidopsis genomic DNA.

[0085]   rd29a-Primer 1 (SEQ ID NO: 11):

[0086]

[Chemical 11]

[0087] And rd29a-primer 2 (SEQ ID NO: 12):

[0088]

[Chemical 12]

Rd29a-primer 1 was prepared by using HindIII (AA) near the 5'end of the primer.
GCTT) restriction site and rd29a-primer 2 has a NotI (GCGGCCGC) restriction site near the 5'end of the primer.

Total genomic DNA was isolated from Arabidopsis thaliana (Colombian ecotype) by using the CTAB method (Ausubel et al. (1992).
Current Protocols in Molecular Biology (Greene & Wiley, New York)
). Ten nanograms of genomic DNA was used as template in a PCR reaction under the conditions suggested by the manufacturer (Boehringer Mannheim). The reaction conditions used in this PCR experiment were as follows. Segment 1: 94 ° C, 2 minutes; segment 2: 94 ° C, 30 seconds; 60 ° C, 1 minute; 72 ° C, 3 minutes for a total of 35 cycles; segment 3: 72 ° C, 10 minutes. A PCR product of 1525 base pairs is expected.
The PCR products were subjected to electrophoresis in 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragment containing the inducible promoter was excised and purified using the Qiaquick Gel Extraction Kit (Qiagen, CA).
The purified PCR product is then digested with HindIII and NotI,
LexA and LacI based donor vectors HindIII and No
It can be cloned into the tI site.

Similarly, tissue-specific promoters from plant genomic DNA can be isolated by PCR amplification. For example, non-specific lipid transfer protein (or LT
The P1) promoter is specific to the epidermal layer of plants (Thoma et al, Plant Phy
siol. 105 (1) 34-45 (1994)). The sequence of the LTP1 promoter is located in the region from nucleotide positions 1 to 1130 (accession number M80567). The following two primers were designed to amplify this promoter region from Arabidopsis genomic DNA.

[0092]   LTP1-Primer 1 (SEQ ID NO: 13):

[0093]

[Chemical 13]

[0094] And LTP1-primer 2 (SEQ ID NO: 14):

[0095]

[Chemical 14]

LTP1-Primer 1 was prepared by using HindIII (AAG
CTP) restriction site and LTP1-primer 2 has a NotI (GCGGCCGC) restriction site near the 5'end of the primer. The promoter fragment can then be cloned into an activation vector as described above.

Example 2. Construction of Receptor Vector The receptor vector contains the binding region corresponding to the transactivator factor produced above, in this case the LexA or LacI binding site, and the gene of interest such as a transcription factor.

Two variants of the cloning vector will be assembled, one for each Lexa or LacI. Lexa type has 8 copies of Lex
The a binding site and the 35S minimal promoter will be cloned upstream of the E9 terminator. For this two fragments will be generated and then fused to each other by overlapping PCR. The first fragment is primer O16417 (SEQ ID NO: 15):

[0099]

[Chemical 15]

And O11723 by lexA It will occur using the OP construct (described in the previous section) as a template. Second, the primers O11721 and O1641.
6 (SEQ ID NO: 16):

[0101]

[Chemical 16]

By using pMEN20 as a template. The two PCR products can be assembled together by PCR with primers O16417 and O16416. Note that the HindIII recognition site was designed 5 ′ of O16417 and the pMEN20 multiple cloning site would be included in the final product. This product is the modified pMEN20 HindIII and B
It will be linked to the amHI site, which should have a different selectable marker (eg a glyphosate resistance marker). lac
Type I would be the same as above, except that two copies of the lacI binding site were replaced by the lexa binding site. The lacI binding site is O16415 (SEQ ID NO: 1
7):

[0103]

[Chemical 17]

Will be included in. Primer O1641 using pMEN20 as template
The PCR product from 5 and 016416 will contain two copies of the lacI binding site and the 35S minimal promoter, and the multiple cloning site of pMEN20.

The CBF1 coding region was sequenced by PCR with primer O11700 (SEQ ID NO: 18):

[0106]

[Chemical 18]

[0107] And O11702 (SEQ ID NO: 19):

[0108]

[Chemical 19]

Can be used to amplify. The resulting PCR product of about 780 bp can be digested with SalI and NotI and cloned into the SalI and NotI sites of the receptor vector. Furthermore, the seed-specific transcription factor ATML1 (Lu et al. (1996) during embry
onic pattern formation and defines a new class of homeobox genes Plant C
ell 8 (12): 2155-68, GenBank Accession No. U37589) was PCR-stimulated to obtain primer O5184 (SEQ ID NO: 20):

[0110]

[Chemical 20]

[0111] And O5185 (SEQ ID NO: 21):

[0112]

[Chemical 21]

Can be used to amplify. The obtained PCR product of about 2,400 bp was
It can be digested with KpnI and NotI and cloned into the KpnI and NotI sites of the receptor vector. Furthermore, the root-specific transcription factor PRL2
(Newman, et al. (1994) Plant Physiol. 106,1241-1255) was obtained from the full length expressed sequence tag (GenBank Accession No. R86816). The cDNA can be isolated from the library vector by using the SalI and NotI enzymes, which can then be cloned into the SalI and NotI sites of the receptor site. In the fourth example, ATHB-12 (Lee et
(1988) Plant Mol Biol. 37: 377-84) was subcloned into the receptor vector by using SalI and NotI as previously described.

Example 3: Tomato Transformation Tomato transformation is performed using the following procedure. Tomato seeds are sterilized in 50% bleach for 20-30 minutes and washed at least 3 times with sterile water. The seeds,
Magenta jar containing the following medium, 1X MS salts, 1X Gamborg's Vitamins, 2% sucrose and 0.8% phytagar.
Put in. The seeds are illuminated for 16 hours at 25 ° C. and germinated for 7-10 days. 5 ml LB or YEP of Agrobacterium from glycerol stock solution
Inoculation with the appropriate antibiotic and the culture at 250 ° C. at 28 ° C.
Grow overnight with shaking at m. Once the optimal OD600 (1.0-1.5) was reached, the bacteria were centrifuged and resuspended in liquid germination medium to give an OD6 of 0.2.
00 concentration. Cotyledons of 7-10 day old seedlings were cut into 0.5 cm pieces, and the following 1X MS salts, 1X Gamborg's Vitamins, 3% glucose, 1 mg / L
Zeatin, 0.8% (w / v) Phythagar, pH up to 5.8, and 375u
Transfer to co-culture plate with (D1-) medium containing M acetosyringone. Pour 10-15 ml of dilute Argo solution onto the explants and incubate for 40 minutes. The explants are illuminated on the (D1-) co-culture medium for 16 hours at 25 ° C. and inverted for 48 hours. Approximately 20-25 explants in the following medium, 1X
Transfer to D1 plates containing MS salts, 1X Gamborg's Vitamins, 3% glucose, 1 mg / L zeatin, 0.8% (w / v) phytagar, pH 5.8, 300 u / ml Timentin and select for constructs. After 10-15 days, all explants were treated with the following 1X MS salts, 1X Gamborg's Vitamins, 3% glucose, 0.1 mg / L zeatin, 0.8% (w / v) Phytagar, pH 5.8, 30.
Transfer to D2 plates containing 0 u / ml Timentin and appropriate choices for constructs. Then, every 2-3 weeks from that point, the explants are transferred to fresh D2 plates. Shoots form within a few weeks. When the shoot was 1 to 1.5 cm high, cut the shoot from the callus and remove the next 1X MS salt, 1X Gambo.
rg's Vitamins, 3% glucose, 0.8% (w / v) Phythagar, pH 5.8
, 300 u / ml Timentin and appropriate choice of constructs. Transfer rooted shoots to soil in 2-inch pot ID magenta jars.
Once the plants settle in the soil, transfer them to larger pots and check for the presence of the transgene.

Tomato Breeding for Trait Improvement Using the above protocol, the first pool of tomatoes was found to have a constitutive promoter (3
The 5S promoter) or an inducible promoter (rd29a or ltp1) can be transformed with a vector containing the lexA / Gal4 or lacI / Gal4 transactivator in combination. Tomatoes in the second pool
The lexA or lacI binding site can be either CBF, PRL2, ATML1 or A
The vector containing in combination with THB-1 can be used for transformation.

The first and second pool of tomato plants are components of a plant breeding kit. The following definitions are used. T0: First transgenic plant resulting from tissue culture. S1 to S4 or F1 to F4: S number is for self-pollination, but F number is 2
Crosses between lines: For example, a T0 plant can be self-pollinating (produced seed S1) or the first cross to a donor line (produced seed F1). F1S2
Are considered to be the offspring of the F1 parent.

Transformed T0 plants will grow to maturity in the greenhouse (they can be scored with the cyanamide herbicide, even if escape is a problem). Some flowers will be crossed to T0 plants containing the donor vector. (The F1 progeny of this cross would have been segregated for both genes).
. Some flowers will be crossed to obtain at least 100 seeds. Self-pollinating seeds (S1) will be stored for later crosses to different activator promoters.

F1 progeny are screened by spraying with chlorsulfuron and cyanamide to find progeny that contain both donor (chlorsulfuron resistant) and receptor (cyanamide resistant) vectors (heterozygous for both). Will Since both chlorsulfuron resistance and cyanamide resistance are predominant in their action, a phenotype resistant to both herbicides, and thus containing both genes, should be distinguishable in the F1 progeny. The presence of two distinct herbicide markers greatly facilitates genotyping.

F1 progeny can be screened by RT-PCR. F1S
Two plants are segregated for both activator and receptor genes.
F1S2 plants are grown and sprayed with chlorsulfuron and cyanamide to find plants containing both transgenes (3/4 × 3/4 = 9/16) and self-pollinate these plants. 1/9 of these (1/16 of the first planting before spraying) F1S2S3 seeds from these F1S2 plants were homozygous for both transgenes and were homozygous for both homozygous offspring (both chlorsulfuron and cyanamide). 100%
Tolerance). A single plant analysis of these F1S2S3 plants can be performed. Since they are homozygous for both donor and receptor vectors, they can be studied more fully in this generation. Alternatively, seeds from the F1S2S2 plants can be expanded for detailed study.

Plants are screened for the desired plant phenotype for each gene. First, studies can be performed with a two component system in which transcription factor expression is activated using the CaMV 35S activator gene. Next, a transcription factor that is thought to play a role in an interesting phenotype is identified by a specific receptor vector and a tissue-specific such as a fruit-specific activator line, or an environment- or disease-inducing line or a stage-specific line. Further studies can be carried out by crossing with specific or inducible activator lines.

Specific traits of interest in tomato include increased carotenoid levels (particularly lycopene), increased soluble solids levels, and enhanced disease resistance.

All references (publications and patents) are incorporated herein by reference in their entirety for all purposes. Although the present invention has been described above with respect to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Therefore, the present invention is limited only by the claims.

[Brief description of drawings]

[Figure 1]   FIG. 1 illustrates one embodiment of the present invention.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C R, CU, CZ, DE, DK, DM, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, K Z, LC, LK, LR, LS, LT, LU, LV, MA , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA (72) Inventor From, Michael             California 94707,             Kensington, Anson Way 32 (72) Inventor Chan, James             California 94303, USA             Palo Alto, Amarillo Avenue             951 F term (reference) 2B030 AA07 CA15 CA17 CA19                 4B024 AA08 BA80 CA04 DA01 DA05                       DA06 EA04 EA05 FA02 GA11

Claims (33)

[Claims] 1. A method for screening an improved trait in a plant, comprising: (a) a first pool of donor vectors each containing a transactivator and each capable of functioning to a gene of interest. Providing a second pool of receptor vectors containing transactivator binding sites ligated as follows: (b) transforming a first plant with a member of a donor vector pool; (c) a second (E) to transform the first and second transformed plants to produce a hybrid plant; and (e) to identify improved traits. Said method comprising characterizing the phenotype of said hybrid plant. 2.   The method of claim 1, wherein the gene of interest is a regulatory gene. 3.   The method of claim 2, wherein the control gene is a transcription factor. 4. The method of claim 1, wherein the transactivator is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage-specific promoter. 5.   The method of claim 1, wherein steps (b) to (e) are performed more than once. 6. The method of claim 1, wherein the first plant is transformed with more than one donor vector member. 7. The second plant is transformed with more than one target vector member,
The method of claim 1. 8. The method of claim 1, wherein the donor vector further comprises a first selectable marker. 9. The method of claim 1, wherein the receptor vector further comprises a second selectable marker. 10. (a) a first pool of donor vectors each containing a transactivator; and (b) each transactivator binding site operably linked to a different gene of interest. A plant breeding kit comprising a second pool of receptor vectors comprising. 11.   The kit according to claim 10, wherein the gene of interest is a regulatory gene. 12. The method according to claim 12,   The kit according to claim 11, wherein the control gene is a transcription factor. 13. The kit of claim 10, wherein the donor vector further comprises a first selectable marker. 14. The receptor vector further comprising a second selectable marker.
Kit. 15. The kit of claim 10, wherein the transactivator is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage-specific promoter. 16. A method for breeding plants for improved traits, comprising: (a) providing a first plant transformed with a donor vector containing a transactivator; b) providing a second plant transformed with a receptor vector comprising a transactivator binding site operably linked to a gene of interest; (c) said first and second traits. Said method comprising the step of crossing the transformed plants to produce hybrid plants; and (d) selecting hybrid plants with improved traits. 17.   17. The method of claim 16, wherein the gene of interest is a regulatory gene. 18.   18. The method of claim 17, wherein the control gene is a transcription factor. 19. The method of claim 16, wherein the transactivator is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage-specific promoter. 20.   17. The method of claim 16, wherein steps (a) to (d) are performed more than once. 21. The method of claim 16, wherein the first plant is transformed with more than one donor vector member. 22. The second plant is transformed with more than one targeting vector member.
The method of claim 16. 23. The method of claim 16, wherein the donor vector further comprises a first selectable marker. 24. The receptor vector further comprising a second selectable marker.
the method of. 25.   A transgenic plant produced according to the method of claim 16. 26. A method for modifying a pattern of gene expression in a plant, comprising: (a) a first pool of donor vectors, each containing a transactivator, and each functional to a regulatory gene. A second pool of receptor vectors comprising transactivator binding sites so linked; (b) transforming the first plant with a member of the donor vector pool; (c) a second plant. Of the receptor vector pool; and (d) crossing the first and second transformed plants to produce hybrid plants having a modified pattern of gene expression. . 27.   27. The method of claim 26, wherein the control gene is a transcription factor. 28. The method of claim 26, wherein the transactivator is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage-specific promoter. 29.   27. The method of claim 26, wherein steps (a) to (d) are performed more than once. 30. The method of claim 26, wherein the first plant is transformed with more than one donor vector member. 31. The second plant is transformed with more than one targeting vector member.
27. The method of claim 26. 32. The method of claim 26, wherein the donor vector further comprises a first selectable marker. 33. The method of claim 26, wherein the receptor vector further comprises a second selectable marker.
the method of.