Method for propagating sterile male plant line

Abstract

A method for maintaining a male sterile plant in a homozygous recessive state includes providing a first plant that includes homozygous recessive male sterility alleles, providing a second plant that includes homozygous recessive male sterility alleles the same as that in the first plant and a nucleotide construct in which the construct exists in a heterozygous state. The first nucleotide sequence of the nucleotide construct encodes a first protein that restores male fertility of the first plant after expression in the first plant. The second nucleotide sequence of the nucleotide construct allows for distinguishing the grains with or without the construct by observation through naked eyes or devices. The first nucleotide sequence and the second nucleotide sequence are tightly connected with each other and coexist in a plant. The method further includes fertilizing female gametes of the first plant with male gametes of the second plant.

Claims

1. A method for maintaining a maize male sterile plant in a homozygous recessive state, the method comprising: providing a first maize plant that comprises homozygous recessive ms45 male sterility alleles; providing a second maize plant comprising homozygous recessive ms45 male sterility alleles that are the same as in the first maize plant, and a nucleotide construct, wherein the construct exists in a heterozygous state, and wherein the construct comprises: i. a first nucleotide sequence comprising a wild type allele of a male fertility regulatory gene Ms45 which results in expression of protein Ms45 having SEQ ID No: 4, in the progenies of the first plant; and ii. a second nucleotide sequence comprising a nucleotide sequence that inhibits the expression of the protein represented by SEQ ID No: 5, wherein the nucleotide sequence that inhibits the expression of the protein represented by SEQ ID No: 5 comprises SEQfor-X-SEC).sub.rev,wherein the SEQ.sub.for comprises a nucleotide sequence of positions 14-276 of SEQ ID No:2; the SEQ.sub.rev comprises the nucleotide sequence of positions 401-663 of SEQ ID No:2; the X is an intron forming a hairpin structure and comprises the nucleotide sequence of positions 277-400 of SEQ ID No:2; and fertilizing female gametes of the first plant with male gametes of the second plant to produce progenies maintaining the homozygous recessive state of the first plant.

2. The method according to claim 1, wherein the method is used to propagate a sterile male plant line; wherein the second nucleotide sequence in the second plant in a heterozygous state affects grain shape of the second plant, wherein the grain shape comprises size, length, width and/or thickness.

3. The method according to claim 1, wherein the first nucleotide sequence comprises an Ms45 encoding sequence having SEQ ID No: 8.

4. The method according to claim 1, wherein the first nucleotide sequence comprises the nucleotide sequence having SEQ ID No: 1.

5. The method according to claim 1, wherein the second nucleotide sequence in the second plant in a heterozygous state affects the grain size of the second plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows male flower phenotypes of a male fertile mutant ms45 and a wild-type Ms45.

(2) FIG. 2 shows grain phenotypes of a grain size regulating gene Mn1 mutant and a wild-type.

(3) FIG. 3 shows a schematic structure diagram of a plant expression vector pMs45-Mn1RNAi comprising a male fertility gene Ms45 expression element and an expression element of an interference fragment of a grain size regulating gene Mn1.

(4) FIG. 4 shows male flower and grain phenotypes of a plant transformed with a plant expression vector having a male fertility gene Ms45 expression element and an expression element of an interference fragment of a grain size regulating gene Mn1.

(5) FIG. 5 shows a route chart of a seed production using a nuclear male sterility gene, a grain size regulating gene and a transgenic technique.

(6) FIG. 6 shows a flow chart of a genetic transformation experiment of corn.

(7) FIG. 7 shows a grain phenotype of a 16-KDγ-prolamin dominant mutant of a grain composition regulating gene.

(8) FIG. 8 shows a schematic structure diagram of a plant expression vector pMs45-Mc 16-KDγ-zein comprising a male fertility gene Ms45 expression element and an expression element of a grain composition regulating gene, 16-KDγ-prolamin dominant allele.

(9) FIG. 9 shows male flower and grain phenotypes of a plant transformed with a vector having a male fertility gene Ms45 expression element and a grain composition regulating gene, 16-KDγ-prolamin dominant allele.

(10) FIG. 10 shows a route chart of a seed production using a nuclear male sterility gene, an endosperm composition regulating gene, and a transgenic technique.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(11) All the technical and scientific terms used herein have meanings as commonly understood by those ordinary skilled in the art to which the present invention belongs, unless otherwise specifically indicated. The techniques used and mentioned herein are standard techniques recognized by those ordinary skilled in the art, and the materials, methods and examples are provided only by way of illustration, rather than limiting.

(12) Nuclear male sterility is a result of a key gene that is mutated, inhibited or otherwise affected during the formation of microspores, and such a gene is generally referred to as a male sterility gene. The pollen developing pathway is regulated by various genes, and thus there are many genes whose mutation will finally result in male sterility. Currently, a large number of male sterile mutants (as shown in Table 1) have been identified in corn plants, and each of the male sterility genes has its specific restorer gene, that is, each of the male sterile mutants can be restored only with its wild-type allele.

(13) In the present invention, taking a corn male sterile mutant, such as ms45, in Table 1 as an example, the mutant has a male flower unable of normal pollination (as shown in FIG. 1, the left plant is a male sterile mutant, and the right plant is a wild-type), and the fertility of the mutant may be restored with a wild-type plant. In the present invention, a wild-type restorer gene is derived from a self-bred line B73, with a sequence presented by SEQ ID No: 1. The sequence comprises promoter (positions 8-542 of SEQ ID No: 1) and ORF (positions 1422-2972 of SEQ ID No: 1) sequences of a Ms45 gene. After transformation of DNA as presented by SEQ ID No: 1 into an ms45 male sterile mutant, the mutant plant exhibits fertility.

(14) In an embodiment of the present invention, the present invention constructs a plant expression interfering vector of a gene of corn endosperm-specific cell wall invertase, CWI-2 (Cheng, W H et al. 1996), to silence the gene. The mutant of the gene is designated as miniature1 (mn1), and a mutation or silence of the gene will result in smaller grains. Since the gene is specifically expressed in endosperm, silence of the gene will not affect other traits of the same plant. During fertilization of a corn plant, a fertilized gamete comprising the vector will affect endosperm development, thereby resulting in smaller grains. Mn1 gene encodes a cell wall invertase, and if inactivated, will have an influence on the development of grain endosperm to result in smaller grains (as shown in FIG. 2, A is a mn1 mutant grain, and B is a wild-type grain), but have no impact on the development of embryo and the plant. Most of Mn1 mutants in nature or from artificial mutagenesis are recessive mutants. The inventors interferes a wild-type Mn1 gene at a mRNA level with a transgenic technique, so that grains will become smaller as long as having a transformed interference nucleotide fragment, as indicated by A in FIG. 4. Therefore, when the transformed fragment exists in a grain in a heterozygous state, the grain having the transformed fragment may be quickly distinguished in terms of grain size. Likewise, a similar operation may also be performed on other gene(s) that affects the development of grain shape (e.g., size, length, width, thickness, etc.) by the same method, to obtain an easily distinguishable transgenic grain. The RNAi interference sequence of Mn1 gene in the present invention is derived from B73, but not limited to self-bred line B73, and may be likewise derived from any other wild-type corn self-bred line, or a homologous gene from another species, or a synthetic nucleotide sequence. In the present invention, interference fragments of a male restorer gene Ms45 and of a grain size labeling gene Mn1 are constructed into a single vector, which may restore the fertility of a male sterile mutant ms45, and meanwhile allow grains containing the transgenic sequence to be smaller, that is, labeling the grains containing the restorer gene, so as to distinguish fertile grains (a maintainer line) and sterile grains (a sterile line). In following Examples of the present invention, interference fragments of a male fertility regulating gene Ms45 and a grain size regulating gene Mn1 of a corn are constructed into a single vector, which is transformed into HiIIAxHiIIB corn hybrid, to obtain a transgenic plant, which is then backcrossed with an ms45 male sterile plant, to thereby introduce both of the interference fragments of the Ms45 restorer gene and of the Mn1 gene into a male sterile mutant ms45. Due to the presence of a wild-type Ms45 gene, the transgenic plant exhibits fertility. When the transgenic heterozygous plant (Ms45ms45ms45) is crossed with a male sterile plant (ms45ms45), two progenies will be produced, one being male sterile, normal grains free of a transgenic sequence (a sterile line, ms45ms45), which may be restored to have fertility with any of wild-type plants (Ms45Ms45), and may act as a sterile line during seed production; the other being fertile grains with smaller grains (a maintainer line, Ms45ms45ms45), which may have homozygous, recessive, male fertility regulating sites. As a result of comprising a complement transgenic sequence, the plant exhibits fertility, and in virtue of also comprising a nucleotide sequence that affects grain size, the grains become small.

(15) In another embodiment of the present invention, a grain composition regulating gene, a 16-KDγ-prolamin dominant allele is used in the present invention. The 16-KDγ-prolamin gene encodes a prolamin. A mutant Mucronate (Mc), due to a deletion of 38 bases in 438-476 bp of 16-KDγ-prolamin gene which results in a variation in the encoding frame of the gene, has a translated protein significant different than a wild-type. This in turns affects the development of grain endosperm, leading to an opaque endosperm (as shown in FIG. 7, A is a Mc16-KDγ-prolamin mutant, and B is a wild-type), but have no impact on the development of embryo and plant. Since Mc is a dominant mutant, grains will exhibit opaque endosperm as long as they have the dominant allele of Mc. In the present invention, the Mc dominant allele is transformed into a wild-type through a transgenic technique, and is ensured to achieve a normal expression in the plant, and then the plant exhibits an opaque phenotype of endosperm, as indicated by A in FIG. 9. In such conditions, an individual grain having a transformed fragment existing therein a heterozygous state may be quickly distinguished through naked eyes or devices. Likewise, a similar operation may also be performed on other dominant gene(s) that affects endosperm composition development by the same method, to obtain an easily distinguishable transgenic grain. The nucleotide sequence of the 16-KDγ-prolamin gene in the present invention is derived from a Mc mutant, with a specific sequence as shown by SEQ ID No: 6, comprising a promoter sequence and a gene encoding frame sequence. The nucleotide sequence as described above is not limited to Mc mutant, and may also be derived from any other corn endosperm mutant, or a homologous gene of another species, or a synthetic nucleotide sequence. In the present invention, a vector of a 16-KDγ-prolamin gene (CheolSoo Kim et al. 2006) regulating corn endosperm composition is constructed. The vector will affect the development of endosperm, allowing for a corn having farinaceous endosperm that appears opaque. A mutant of the gene is Mucronate (Mc). The gene, if silenced, will not have an influence on other traits of the plant. During fertilization of a corn plant, a fertilized gamete comprising the vector will affect endosperm development, thereby resulting in opaque endosperm. In the present invention, a male restorer gene Ms45 and a Mc16-KDγ-prolamin endosperm labeling gene are constructed in a single vector, which may restore the fertility of a male sterile mutant ms45, while allowing for opaque endosperm of grains comprising a transgenic sequence, i.e., labeling the grains comprising a restorer gene, to distinguish fertile grains (a maintainer line) and sterile grains (a sterile line). In the present invention, a corn male fertility regulating gene Ms45 and an endosperm composition regulating Mc 16-KDγ-prolamin gene are constructed in a single vector, which is then transformed into a HiIIAxHiIIB corn hybrid plant, to obtain a transgenic plant. The transgenic plant is then backcrossed with an ms45 male sterile plant, to thereby introduce both of the Ms45 restorer gene and the Mc 16-KDγ-prolamin gene into a male sterile mutant ms45. Due to the presence of wild-type Ms45 gene, the transgenic plant exhibits fertility. When the transgenic heterozygous plant (Ms45ms45ms45) is crossed with male sterile plant (ms45ms45), two progenies will be produced, one being male sterile normal grains free of a transgenic sequence (a sterile line, ms45ms45), which may have fertility restored with any of wild-type plant (Ms45Ms45), and may act as a sterile line in seed production; the other being fertile grains with opaque endosperm (a maintainer line, Ms45ms45ms45), which may have homozygous recessive sites that regulate male fertility. As a result of inclusion of a complementary transgenic sequence, the plant exhibits fertility; and in virtue of also comprising a nucleotide sequence that affects endosperm composition, the grain endosperm is opaque.

(16) The present invention provides an effective method for seed labeling, which is applicable not only to corn plants (Zea mays), but also to crops such as rice (Oryza sativa), Sorghum (Sorghum bicolor), wheat (Triticumaestivum), soybean (Glycine max), cotton (Gossypiumhirsutum), sunflower (Helianthus annuus), and the like.

(17) Hereinafter, more detailed description is provided by way of explanation and illustration, which is not intended to limit the scope of the present invention.

Example 1. Construction of a Plant Transforming Vector pMs45-Mn1RNAi Comprising a DNA Fragment (a DNA Construct) that Regulates Corn Male Fertility and Corn Grain Size

(18) As shown in FIG. 3, a plant transforming vector pMs45-Mn1RNAi comprises a DNA fragment (a DNA construct) that regulates corn male fertility and corn grain size, and a selectable marker gene. Therein, the DNA fragment that regulates corn male fertility and corn grain size is designated as Ms45-Mn1RNAi that is a DNA fragment between LB and RB of pMs45-Mn1RNAi. Ms45-Mn1RNAi comprises an Ms45 expression element (a first nucleotide sequence) of SEQ ID No: 1 and an expression element (a second nucleotide sequence) for silencing Mn1 gene. The expression element for silencing Mn1 gene is formed by linking a promoter of Mn1 of SEQ ID No: 3 (a Mn1 promoter), a Mn1 interference fragment Mn1RNAi and a terminator. The Ms45 expression element is closely linked with the Mn1 gene silencing expression element, and both exist in a plant when pMs45-Mn1RNAi is transformed into the plant. A method for constructing pMs45-Mn1RNAi is as follows:

(19) 1. Amplification of Ms45 Wild-Type Allele (Ms45 Expression Element) for Restoring Male Fertility of a Corn Male Sterile Mutant Ms45

(20) In the present invention, the ms45 male sterile mutant in Table 1 is taken as an example for particularly illustrating an embodiment. First, a wild-type allele Ms45 of ms45 was amplified, which was derived from a self-bred line B73, with a sequence as presented by SEQ ID No: 1. Taking corn self-bred line B73 genome DNA as a template, with reference to B73 genome sequence, primers were designed to amplify the whole expression element (a promoter and an encoding frame sequence of the Ms45 gene) of the gene. The amplification primers were: Ms45F: 5′ tgaattcTGCTGAGTTCTCCTTGGGTTATCC 3′ (SEQ ID NO:9), Ms45R: 5′ tcccgggGGTTGCGCATGAAATAGGGGT 3′ (SEQ ID NO:10). The upstream amplification primer had an EcoRI recognition site added at 5′-terminus, and the downstream amplification primer had a SmaI recognition site added at 5′-terminus. The amplification reaction system was of: 2 μL template DNA, 0.5 μL primer Ms45F, 0.5 μL primer Ms45R, 1.6 μL dNTP, 2 μL 10× Buffer, 0.3 μL high-fidelity taq polymerase, and 13.1 μL ddH.sub.2O. The reaction conditions were: pre-denaturation at 95° C. for 5 min, denaturation at 95° C. for 45 s, anneal at 59° C. for 45 s, and extension at 72° C. for 3 min, 32 cycles, and post-extension at 72° C. for 10 min. The amplified target band had a full length of 3518 bp. After the amplification, the sequence was linked to a T-easy sequencing vector, and a positive clone was sequenced. The result of the sequencing indicated that the 3518 bp DNA comprised an Ms45 gene expression element as presented by SEQ ID No: 1, an EcoRI recognition site and a SmaI recognition site. In SEQ ID No:1, positions 8-542 represented a promoter, positions 1422-2972 represented an Ms45 gene encoding sequence, for encoding a Ms45 protein of SEQ ID No:4.

(21) 2. Preparation of Mn1 Interference Fragment Mn1RNAi for Silencing Mn1 Gene

(22) Mn1 gene encodes a cell wall invertase protein (with an amino acid sequence of SEQ ID No: 5), and if inactivated, will affects the development of endosperm, resulting in smaller grains (as shown in FIG. 2), but having no impact on the development of embryo and plant. In the present invention, the Mn1 gene was silenced by RNAi technique, and an interference fragment used was designated as Mn1RNAi, with a structure of Mn1SEQ.sub.for-X-Mn1SEQ.sub.rev, wherein the Mn1SEQ.sub.rev has a sequence that is reversely complementary to that of Mn1SEQ.sub.for, and X is an intron forming a hairpin structure. Mn1RNAi had a nucleotide sequence as presented by SEQ ID No:2, wherein positions 7-13 represented a BstEII recognition sequence, positions 14-276 represented the nucleotide sequence of Mn1SEQ.sub.for, positions 277-400 represented X as an intron forming a hairpin structure, positions 401-663 represented the nucleotide sequence of Mn1SEQ.sub.rev, and positions 664-669 represented a HindIII recognition sequence.

(23) 3. Cloning of Mn1 Gene Promoter (Mn1 Promoter)

(24) In the present invention, a promoter (Mn1 promoter) of a grain size regulating gene Mn1 was used to promote Mn1 interference fragment Mn1RNAi, wherein Mn1 gene was specifically expressed in endosperm, such that the mRNA transcribed with the promoter existed in only endosperm cells. The promoter was derived from corn self-bred line B73 genome DNA, with a sequence particularly presented by SEQ ID No: 3. Taking corn self-bred line B73 genome DNA as a template, with reference to B73 genome sequence, primers were designed to amplify the promoter of the gene. The amplification primers were as follows: Mn1pro bF:5′ atcccggGCTCGCATGAGAGAACAACCA 3′ (SEQ ID NO:11), Mn1pro bR:5′ gcaagcttGGGGGTGCTATTTGTACTGTGC 3′ (SEQ ID NO:12), wherein the upstream amplification primer had a SmaI recognition site added at 5′-terminus, and the downstream amplification primer had a HindIII recognition site added at 5′-terminus. The amplification reaction system was of: 2 μL template DNA, 0.5 μL primer Mn1pro bF, 0.5 μL primer Mn1pro bR, 1.6 μL dNTP, 2 μL 10× Buffer, 0.3 μL high-fidelity taq polymerase, and 13.1 μL ddH.sub.2O. The reaction conditions were: pre-denaturation at 95° C. for 5 min, denaturation at 95° C. for 45 s, anneal at 59° C. for 45 s, and extension at 72° C. for 2 min, 32 cycles, and post-extension at 72° C. for 10 min. The amplified target band had a full length of 2422 bp. After the amplification, the sequence was linked to a T-easy sequencing vector, and a positive clone was sequenced. The result of the sequencing indicated that the 2422 bp DNA was a Mn1 gene promoter fragment, and the Mn1 gene promoter fragment comprised a Mn1 gene promoter of SEQ ID No: 3, a SmaI recognition site and a HindIII recognition site.

(25) 4. Construction of pMs45-Mn1RNAi

(26) A plant transforming vector pMs45-Mn1RNAi, as shown in FIG. 3, was constructed by assembling the DNA components in above steps 1, 2 and 3.

(27) The plant transforming vector pMs45-Mn1RNAi, comprising a male fertility gene Ms45, a Mn1 interference fragment expression element, and a selectable marker gene bar expression element, was constructed with a plasmid pCAMBIA3301 (Center for the Application of Molecular Biology to International Agriculture (CAMBIA), Australia) as a skeleton DNA. First, the Mn1 interference fragment Mn1RNAi and pCAMBAI3301 in step 2 were digested with BstEII and HindIII. Large fragments of the Mn1 interference fragment Mn1RNAi and pCAMBAI3301 were linked, and detected for a positive clone. Then, the positive clone and the 3518 bp DNA (an Ms45 expression element) in step 1 were double digested with EcoRI and SmaI, and target bands were recovered. These two fragments were linked and detected for a positive clone. Finally, the positive clone and the Mn1 gene promoter in step 3 were digested with SmaI and HindIII, and target bands were recovered. Two fragments were linked and detected for a positive clone. A plant transforming vector pMs45-Mn1RNAi (FIG. 3) was obtained comprising the male fertility gene Ms45, the expression element of Mn1 interference fragment and the expression element of the selectable marker gene bar. pMs45-Mn1RNAi was a recombinant expression vector formed by substituting the fragment between EcoRI and SmaI recognition sites in pCAMBAI3301 with the Ms45 gene expression element as presented by SEQ ID No: 1, substituting the fragment between SmaI and HindIII recognition sites in pCAMBAI3301 with the Mn1 gene promoter as presented by SEQ ID No: 3, and substituting the fragment between BstEII and HindIII recognition sites in pCAMBAI3301 with the Mn1RNAi as presented by SEQ ID No: 2.

Example 2. Preparation of a Second Plant with Heterozygous Ms45-Mn1RNAi and Homozygous Ms45

(28) I. Transformation of a Corn Plant with the Plant Transforming Vector pMs45-Mn1RNAi in Example 1

(29) In the present invention, a transgenic plant was obtained by a method of infecting immature embryo of a corn with Agrobacterium. First, Agrobacterium tumefaciens EHA105 was transformed with the plant transforming vector pMs45-Mn1RNAi in Example 1, and then the Agrobacterium comprising the target gene was used to infect immature embryo of a corn, by a transgenic method particularly as below:

(30) During the transgenic process in the lab, a recipient plant used was an F1 hybrid from self-bred lines HiIIA and HiIIB. The self-bred lines HiIIA and HiIIB of a corn plant (Armstrong C L, Green C E and Phillips R L. Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation News Letter, 1991, 65:92-93) were used. First, corn plants of self-bred lines HiIIA and HiIIB were grown in the field, and covered with bags at the time of pollination of the self-bred lines; next was pollination, in two ways: HiIIA acting as the female parent, and HiIIB acting as the male parent; HiIIA as the male parent, and HiIIB as the female parent. An immature embryo of a grain on a pollinated ear was taken days 9-11 after pollination, and infected with Agrobacterium tumefaciens EHA105 indoors. Immature embryo infected by Agrobacterium tumefaciens EHA105 was placed on a selective medium for repeated screenings, to obtain a resistant callus, which was then regenerated into a seedling, to obtain a T0 transgenic plant. Thereafter, some female parents for seed production and Ms45 male sterility materials were crossed with pollen from the T0 transgenic plant, and phenotypes were observed. A specific flow chart in the experiment is shown in FIG. 6.

(31) With Agrobacterium tumefaciens EHA105 infection, pMs45-Mn1RNAi was introduced into an immature embryo of a recipient plant, and then screened with an herbicide, Bialaphos, to obtain a transgenic plant. Particularly, the method was as below:

(32) (I) Stripping of Immature Embryo

(33) 1. Removal of bracteal leaves. About 1 cm from the tip of an ear were cut off, and from the tip a tweezer was inserted into the ear, so that the tweezer may serve as a handle to facilitate operations. Then, the ear was placed into a beaker filled with a disinfectant. 4-6 ears may be placed into the same beaker, as desired in practice.

(34) 2. To the beaker, about 700 ml of a disinfectant (50% of a bleaching agent or 5.25% of sodium hypochlorite, with one drop of Tween 20 added) was added to immerse the ears. During 20 min disinfection, the ears were sometimes were turned around while the beaker was tapped to drive out bubbles on the surface of the grains, to thereby achieve the best disinfection. After the disinfection was completed, the ears were placed into a beaker filled with sterilized water, and washed 3 times, and then were ready for embryo stripping.

(35) 3. An end of a disinfected ear was placed on a large dish, and tips (1.5-1.8 mm) of the grains were cut off with a large scalpel, during which the tools used such as blade of the scalpel, the dish, embryo stripping knife, etc. were frequently disinfected.

(36) 4. Between embryo and endosperm, an embryo stripping knife was inserted with its knifepoint, and immature embryo was carefully pried out. The immature embryo was gently jacked up with a small scalpel tip, to protect the immature embryo from any injury. The immature embryo was placed on an N6E medium, with the hypocotyl side thereof in close contact with a filter paper on the medium. The embryos were arranged in a density of about 2×2 cm (30/dish).

(37) 5. The dish was sealed with a sealer membrane, and cultured at 28° C. for 2-3 days.

(38) (II) Agrobacterium Infection

(39) 1. Agrobacterium tumefaciens EHA105 was cultured in a YEP (containing 33 mg/L of kanamycin and 100 mg/L of rifampicin) medium a week in advance, and stored in a refrigerator at 4° C. for about one month, and should be stored with glycerol at −80° C. for long term storage.

(40) 2. Agrobacterium tumefaciens EHA105 was cultured on a YEP medium at 19° C. for 3 days, with an addition of kanamycin to a concentration of 33 mg/L, and of rifampicin to a concentration of 50 mg/L.

(41) 3. After 3 days, Agrobacterium tumefaciens EHA105 was picked and placed into 5 mL infection medium in a 50 ml centrifuge tube, with adding AS(inf+AS) (having solutes as shown in Table 2, and a solvent of water), and incubated with shaking at room temperature (25° C.) at 75 rpm for 2-4 hours.

(42) 4. Infection of immature embryo. The stripped immature embryos were immediately placed into a centrifuge tube containing a liquid medium AS (inf+AS) (2 ml), about 20-100 immature embryos per tube, and washed with such medium 2 times, and then added with 1-1.5 ml of a certain concentration (OD550=0.3-0.4) of Agrobacterium. After carefully inversing the centrifuge tube 20 times, the tube was placed upright in a dark box for 5 min, ensuring that all of the immature embryos were immersed in the liquid of Agrobacterium. Vortexing should be avoided in the whole process.

(43) (III) Co-Culture

(44) 1. After infection, the infected immature embryos were transferred to a co-culture medium (having solutes as shown in Table 2, and a solvent of water), bringing the hypocotyls of the immature embryos in contact with the surface of the medium, while driving out excessive Agrobacterium on the surface of the medium.

(45) 2. The dish was sealed with a sealer membrane, and culture was performed in dark at 20° C. for 3 days.

(46) (IV) Resting Culturing

(47) After 3-day co-culture, the immature embryos were transferred onto a resting medium (having solutes as shown in Table 2, and a solvent of water), and the dish was sealed with a sealer membrane, for culture in dark at 28° C. for 7 days.

(48) (V) Selection

(49) After 7 days, all of the immature embryos were transferred onto a selective medium (having solutes as shown in Table 2, and a solvent of water) (35 immature embryos/dish), and cultured for two weeks. The selective medium contains 1.5 mg/L of Bialaphos. After two weeks, subculture was performed with a concentration of Bialaphos up to 3 mg/L.

(50) 2. With infection of about 5 weeks, cells containing a transformant would grow into observable type II calluses.

(51) (VI) Regeneration of Transgenic Plant

(52) 1. After growth on regeneration medium I (having solutes as shown in Table 2, and a solvent of water) for 3 weeks, followed by germination on regeneration medium II (having solutes as shown in Table 2, and a solvent of water) (in a light culture room), 100 corn plants transformed with T0 pMs45-Mn1RNAi were obtained.

(53) 2. The regenerated seedlings were grown until 3-4 leaves appeared, and transferred into a greenhouse. When grown into a fusule and pollination stage, these seedlings were pollinated.

(54) TABLE-US-00002 TABLE 2 Solutes and their contents in media Co- Resting- Selective- Regeneration Regeneration Type AS(inf + AS) culturemedium medium medium medium I medium II Component MS salt 2.16 g/L 4.33 g/L 4.33 g/L 4.33 g/L 4.33 g/L 2.16 g/L Sucrose 68.5 g/L 30 g/L 30 g/L 30 g/L 30 g/L 30 g/L Glucose 30 g/L L-proline 0.115 g/L 1.38 g/L 1.38 g/L 1.38 g/L 1.38 g/L Vitamin B.sub.1 0.5 mg/L 0.5 mg/L 0.5 mg/L 0.5 mg/L 2,4-D 5 mg/L 5 mg/L 5 mg/L 6-BA 0.01 mg/L 0.01 mg/L 0.01 mg/L 3.5 mg/L IBA 0.75 mg/L NAA 0.5 mg/L 4-amino-3,5,6- 1 mg/L trichloropyridine- carboxylic acid (picloram) Timentin 100 mg/L 100 mg/L 100 mg/L Bialaphos 3 mg/L 3 mg/L 3 mg/L Agar 6 g/L phytagel 3 g/L 3 g/L 3 g/L 3 g/L pH 5.7 5.7 5.7 5.7 5.7 5.7 AgNO3 3.4 mg/L 3.4 mg/L 3.4 mg/L Acetosyringone 200 μmol/L 200 μmol/L 200 μmol/L
In Table 2, MS salt was commercial available from phyto Technology Laboratories, LLC, Art. No. M524.

(55) II. Changing the Ms45Ms45 Wild-Type Self-Bred Line into an ms45ms45 Homozygous Recessive Self-Bred Line

(56) An ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 905I) as the female parent was crossed with a different self-bred line (e.g., Zheng 58) to obtain F1, which was further backcrossed with the corn self-bred line of Zheng 58 (Henan Qiule Seed Industry Science & Technology Co. Ltd., China), to obtain a BC1 population for genotype analysis. Plants identified as having a heterozygous Ms45 site was further backcrossed with Zheng 58. After 5-6 generations of backcross as such, individuals that had a heterozygous Ms45 site with the rest of the sites all from Zheng 58 were screened out using a molecular marker, and self-bred to thereby obtain an ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)). The self-bred line may serve as a sterile line, and was called the first plant.

(57) III. Preparation of a Second Plant Having Heterozygous Ms45-Mn1RNAi and Homozygous Ms45

(58) The ms45 homozygous recessive self-bred line (a female parent) was crossed with the pMs45-Mn1RNAi transformed T0 corn plant (a male parent) obtained in procedure I, and subjected to multiple backcrosses with the ms45 homozygous recessive self-bred line as a recurrent parent. The pMs45-Mn1RNAi transformed T0 corn plant obtained in procedure I was changed into a self-bred line comprising Ms45-Mn1RNAi and having a homozygous recessive ms45 site and heterozygous Ms45-Mn1RNAi. This self-bred line is the second plant having heterozygous Ms45-Mn1RNAi and homozygous ms45.

(59) For above purpose, 50 of the pMs45-Mn1RNAi transformed T0 corn plants (a male parent) obtained in procedure I were crossed with the ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)) (a female parent) obtained in procedure II. From the hybrid progenies, those grains with a small size were selected and sowed in the field, and subsequently sprayed with 200 mM Bialaphos. Survived plants were further backcrossed with the ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)) obtained in procedure II. After 5-6 generations of backcross as such, individuals that had a heterozygous transgenic site (Ms45-Mn1RNAi) and a homozygous recessive Ms45 site with the rest of the sites all of Zheng 58 background were screened out using a molecular marker. Such an individual is the second plant having heterozygous Ms45-Mn1RNAi and homozygous ms45, and was called the second plant of Zheng 58 (Ms45ms45ms45).

(60) Above second plant of Zheng 58 (Ms45ms45ms45) as a male parent was crossed with the ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)) (a female parent) obtained in procedure II, to produce progenies, which included not only a male sterile line of Zheng 58 (ms45ms45) with a normal grain size (as indicated by B in FIG. 4), but also a maintainer line of the male sterile Zheng 58 (ms45ms45), Zheng 58 (Ms45ms45ms45), with a smaller grain size (as indicated by A in FIG. 4). Specific process for selection and culture is shown in FIG. 5.

(61) IV. Analysis of the pMs45-Mn1RNAi Transformed T0 Corn Plant Obtained in Procedure I

(62) The pMs45-Mn1RNAi transformed T0 corn plant and progenies thereof obtained in procedure I were assessed in terms of the overall morphology of the plant, and particularly analyzed for pollen and grain phenotypes. Except for grains, no difference in morphology was observed between a pMs45-Mn1RNAi transformed T0 corn plant and a non-transgenic control plant. When the pMs45-Mn1RNAi transformed T0 corn plant was crossed with an ms45 male sterility material, among the hybrid progenies, those plants comprising Ms45-Mn1RNAi exhibited fertility, as indicated by C in FIG. 4, while the hybrid progenies free of Ms45-Mn1RNAi exhibited complete sterility. This suggested that the performance of male sterility of the homozygous recessive ms45 was compensated by the expression of Ms45 gene, and meanwhile those grains comprising Ms45-Mn1RNAi were smaller than the normal grains free of Ms45-Mn1RNAi, and had a phenotype the same as the mn1 mutant, as indicated by A in FIG. 4. This suggested that the interference fragment of Mn1 gene could have a normal function in all transgenic plants. Moreover, the small size grains and normal grains that have been subjected to the grain phenotype identification were sowed by the inventors in the field, and all of these grains were capable of normal germination, with no significant difference in germination rate in comparison with normal grains. When the plants were grown to appear 4-5 leaves, 200 mM bialaphos was sprayed. As a result, all of the small size grains could survive as normal, with no inhibition of growth, however, all of the seedlings from the normal grains were dead. This suggested that all of the Ms45 male fertility gene and the mn1 interference fragment as well as the selectable marker gene bar could function as normal, and these three genes allowed for a linkage inheritance. In the progenies produced by the hybridization of the pMs45-Mn1RNAi transformed plant with the male sterile mutant ms45, a ratio of the normal grains: the smaller grains was 1:1.

(63) The specific experimental method of FIG. 4 is the same as that in procedure III.

Example 3. Large-Scale Propagation of an Ms45 Male Sterile Self-Bred Line Using the Male Sterile Maintainer Line in Example 2

(64) Taking a self-bred line of Zheng 58 as an example, the male sterile line of Zheng 58 (ms45ms45) in Example 2 and the male sterile maintainer line of Zheng 58 (Ms45ms45ms45) in Example 2 were sowed in the field alternately with 5 rows of the sterile line and 1 row of the maintainer line, in the condition of ensuring no additional corn planted around, allowing for a natural pollination between the sterile line and the maintainer line. The maintainer line would receive only its own pollen to produce progenies. Since the grains having a homozygous transgenic composition (Ms45-Mn1RNAi) were indistinguishable from heterozygous grains in the produced progenies, these grains were discarded. The normal size grains (having a large size) might serve as a sterile line. The male sterile line of Zheng 58 (ms45ms45) received the pollen from the male sterile maintainer line of Zheng 58 (Ms45ms45ms45) to produce progenies, wherein normal size grains belonged to a sterile line without a transgenic composition, and smaller size grains belonged to a maintainer line with a transgenic composition. The maintainer line all was used to propagate the sterile line and the maintainer line in the next year, and most of the sterile line was used to produce commercial seeds, and a small remaining part was used to propagate the sterile line and the maintainer line in the next year. A specific flow chart of the production is shown in FIG. 5.

Example 4. Large-Scale Production of Hybrid Seed Using the Male Sterile Line in Example 3

(65) The sterile line produced in Example 3 is a homozygous recessive sterile line regulated by cell nucleus, and such a sterile line may have fertility restored with any wild-type plant (Ms45Ms45). Therefore, as long as a self-bred line, such as Chang 7-2, that has high combining ability with the male sterile (ms45ms45) self-bred line, e.g., male sterile Zheng 58 (ms45ms45), is selected for hybridization, hybrid seeds having excellent agronomic traits may be produced. For this purpose, the inventors sowed the male sterile self-bred line and the wild-type self-bred line alternately in the field, with ensuring no additional corn planted around within 300 meters, so that the ears of the sterile line would receive only the pollen from the wild-type self-bred line, while the wild-type self-bred line was only capable of selfing. Thus, the seeds produced on the ears of the sterile line were hybrid seeds.

Example 5. Construction of a Plant Transforming Vector pMs45-Mc 16-KDγ-Zein Comprising a DNA Fragment (a DNA Construct) that Regulates Corn Male Fertility and Corn Grain Endosperm Composition

(66) The plant transforming vector pMs45-Mc 16-KDγ-zein, as show in FIG. 8, comprised a DNA fragment (a DNA construct) that regulated corn male fertility and corn grain endosperm transparency and a selectable marker gene. Therein, a DNA fragment that regulated corn male fertility and corn grain size was designated as Ms45-Mc 16-KDγ-zein, i.e., a DNA fragment between LB and RB in pMs45-Mc 16-KDγ-zein. Ms45-Mc 16-KDγ-zein comprised an Ms45 expression element (a first nucleotide sequence) of SEQ ID No: 1 and an Mc16-KDγ-prolamin gene expression element (a second nucleotide sequence) of SEQ ID No: 6. The Ms45 expression element was closely linked to the Mc16-KDγ-prolamin gene expression element, and when the pMs45-Mc 16-KDγ-zein was transformed into a plant, these two expression elements coexisted in the plant. The pMs45-Mc 16-KDγ-zein was constructed by a method as below:

(67) 1. Amplification of an Ms45 Wild-Type Allele (the Ms45 Expression Element) Restoring the Male Fertility of Corn Male Sterile Mutant Ms45

(68) The step is same as step 1 in Example 1.

(69) 2. Artificial Synthesis of Mc Mutant 16-KDγ-Prolamin Gene

(70) According to the report of the mutant in CheolSoo Kim et al. 2006 and the instruction of the sequence (Gene accession no. DQ826676), the gene was synthesized into an Mc16-KDγ-prolamin gene expression element as presented by SEQ ID No:6, with a HindIII restriction site added at 5′-terminus and a BstEII restriction site added at 3′-terminus. In SEQ ID No:6, positions 9-1149 represented a promoter sequence, positions 1244-1780 represented encoding sequence of Mc16-KDγ-prolamin gene, encoding Mc16-KDγ-prolamin of SEQ ID No:7.

(71) 3. Construction of a Plant Transforming Vector pMs45-Mc 16-KDγ-Zein Comprising a Male Fertility Gene Ms45 and a Mc Mutant 16-KDγ-Prolamin Gene Expression Element as Well as a Selectable Marker Gene

(72) With a plasmid pCAMBAI3301 as a skeleton DNA, a plant transforming vector pMs45-Mc 16-KDγ-zein comprising a male fertility gene Ms45 and a Mc16-KDγ-prolamin expression element as well as a selectable marker gene bar was constructed. First, the Mc16-KDγ-prolamin expression element and pCAMBIA3301 (Center for the Application of Molecular Biology to International Agriculture (CAMBIA), Australia) were digested with BstEII and HindIII, and large fragments of the Mc16-KDγ-prolamin expression element and pCAMBAI3301 were linked, and detected for a positive clone. Thereafter, the positive clone and the Ms45 wild-type allele in step 1 were double digested with EcoRI and SmaI, and target bands were recovered. These two fragments were linked, and detected for a positive clone, to obtain a plant transforming vector pMs45-Mc 16-KDγ-zein comprising the male fertility gene Ms45 and the Mc 16-KDγ-prolamin expression element as well as the selectable marker gene bar (the constructed vector is shown in FIG. 8). pMs45-Mc 16-KDγ-zein was a recombinant expression vector in which the fragment between EcoRI and SmaI recognition sites of pCAMBAI3301 was substituted with the Ms45 gene expression element as presented by SEQ ID No: 1, and the fragment between BstEII and HindIII recognition sites of pCAMBAI3301 was substituted with the Mc16-KDγ-prolamin gene expression element as presented by SEQ ID No: 6.

Example 6. Preparation of the Second Plant Having Heterozygous Ms45-Mc 16-KDγ-Zein and Homozygous Ms45

(73) I. Transformation of a Corn with the Plant Transforming Vector pMs45-Mc 16-KDγ-Zein in Example 5

(74) In the present invention, a transgenic plant was obtained by a method of infecting immature embryo of a corn with Agrobacterium. First, Agrobacterium tumefaciens EHA105 was transformed with the plant transforming vector pMs45-Mc 16-KDγ-zein in Example 5, and then the Agrobacterium comprising the target gene was used to infect the immature embryo of an F1 hybrid corn of self-bred lines HiIIA and HiIIB. The Agrobacterium infected immature embryo was placed on a selective medium for repeated screenings, to obtain a resistant callus, which was then regenerated into a seedling, to obtain a T0 transgenic plant. Thereafter, pollen from the T0 transgenic plant were used to cross to some female parents and Ms45 male sterility materials in seed production, and phenotypes were observed. Specific experimental method is the same as procedure I in Example 2.

(75) II. Changing the Ms45Ms45 Wild-Type Self-Bred Line into an ms45ms45 Homozygous Recessive Self-Bred Line

(76) An ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 905I) as the female parent was crossed with a corn self-bred line of Zheng 58 (Henan Qiule Seed Industry Science & Technology Co. Ltd.) to obtain F1, which was further backcrossed with the corn self-bred line of Zheng 58 (Henan Qiule Seed Industry Science & Technology Co. Ltd., China), to obtain a BC1 population for genotype analysis. Plants identified as having a heterozygous Ms45 site was further backcrossed with Zheng 58. After 5-6 generations of backcross as such, individuals that had a heterozygous Ms45 site with the rest of the sites all from Zheng 58 were screened out by using a molecular marker, and self-bred to thereby obtain an ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)). The self-bred line may serve as a sterile line, and was called the first plant. Specific experimental method is the same as procedure II in Example 2.

(77) III. The Second Plant Having Heterozygous Ms45-Mc 16-KDγ-Zein and Homozygous Ms45

(78) The ms45 homozygous recessive self-bred line (a female parent) was crossed with the pMs45-Mc 16-KDγ-zein transformed T0 corn plant (a male parent) obtained in procedure I, and subjected to multiple backcrosses with the ms45 homozygous recessive self-bred line as a recurrent parent. The pMs45-Mc 16-KDγ-zein transformed T0 corn plant obtained in procedure I was changed into a self-bred line comprising Ms45-Mc 16-KDγ-zein and having a homozygous recessive ms45 site and heterozygous Ms45-Mc 16-KDγ-zein. This self-bred line is the second plant having heterozygous Ms45-Mc 16-KDγ-zein and homozygous ms45.

(79) For achieving above purpose, 50 of the pMs45-Mc 16-KDγ-zein transformed T0 corn plants (a male parent) obtained in procedure I were crossed with the ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)) (a female parent) obtained in procedure II. From the hybrid progenies, those grains with an opaque endosperm were selected and sowed in the field, and subsequently sprayed with 200 mM Bialaphos. Survived plants were further backcrossed with the ms45 homozygous recessive self-bred line of Zheng58 (Zheng 58 (ms45ms45)) obtained in procedure II. After 5-6 generations of backcross as such, individuals that had a heterozygous transgenic site (Ms45-Mc 16-KDγ-zein) and a homozygous recessive Ms45 site with the rest of the sites all of Zheng 58 background were screened out by using a molecular marker. Such an individual is the second plant having heterozygous Ms45-Mc 16-KDγ-zein and homozygous ms45, and was called the second plant of Zheng 58 (Ms45ms45ms45).

(80) Above second plant of Zheng 58 (Ms45ms45ms45) as a male parent was crossed with the ms45 homozygous recessive self-bred line of Zheng 58 (Zheng 58 (ms45ms45)) (a female parent) obtained in procedure II, to produce progenies, which included not only a male sterile line of Zheng 58 (ms45ms45) with normal grains and transparent endosperm (as indicated by B in FIG. 9), but also a maintainer line of the male sterile Zheng 58 (ms45ms45), Zheng 58 (Ms45ms45ms45), with opaque endosperm (as indicated by A in FIG. 9). Specific process for selection and culture is shown in FIG. 10.

(81) IV. Analysis of the pMs45-Mc 16-KDγ-Zein Transformed T0 Corn Plant Obtained in Procedure I

(82) The pMs45-Mc 16-KDγ-zein transformed T0 corn plant and progenies thereof obtained in procedure I were assessed in terms of the overall morphology of the plant, and particularly analyzed for pollen and grain phenotypes. Except for grains, no difference in morphology was observed between a pMs45-Mc 16-KDγ-zein transformed T0 corn plant and a non-transgenic control plant. When the pMs45-Mc 16-KDγ-zein transformed T0 corn plant was crossed with an ms45 male sterility material, among the hybrid progenies, those plants comprising a transgenic composition exhibited fertility, as indicated by C in FIG. 9, while the non-transgenic plants exhibited complete sterility. This suggested that the performance of male sterility of the homozygous recessive ms45 was compensated by the expression of Ms45 gene, and meanwhile those grains comprising the Mc mutant 16-KDγ-prolamin gene showed opaque endosperm, with a phenotype the same as the Mc mutant, as indicated by A in FIG. 9. This suggested that the Mc dominant mutant allele could have a normal function in all transgenic plants. Moreover, the opaque-endosperm grains and normal grains that have been subjected to the grain phenotype identification were sowed by the inventors in the field, and all of these grains were capable of normal germination, with no significant difference in germination rate in comparison with normal grains. When the plants were grown to appear 4-5 leaves, 200 mM Bialaphos was sprayed. As a result, all of the opaque-endosperm grains could survive as normal, with no inhibition of growth, however, all of the seedlings from the normal grains were dead. This suggested that all of the selectable marker gene bar, Ms45 male fertility gene, and Mc 16-KDγ-prolamin gene could function as normal, and these three genes allowed for a linkage inheritance. In the progenies produced by the hybridization of the transgenic plant with the male sterile mutant ms45, a ratio of the normal grains: the opaque-endosperm grains was 1:1.

(83) The specific experimental method of FIG. 9 is the same as that in procedure III.

Example 7. Large-Scale Propagation of an Ms45 Male Sterile Self-Bred Line Using the Male Sterile Maintainer Line in Example 6

(84) Taking a self-bred line of Zheng 58 as an example, the male sterile line of Zheng 58 (ms45ms45) in Example 6 and the male sterile maintainer line of Zheng 58 (Ms45ms45ms45) in Example 6 were sowed in the field alternately with 5 rows of the sterile line and 1 row of the maintainer line, in the condition of ensuring no additional corn planted around within 300 meters, allowing for a natural pollination between the sterile line and the maintainer line. The maintainer line would receive only its own pollen to produce progenies. Since the grains having a homozygous transgenic composition were indistinguishable from heterozygous grains in the produced progenies, these grains were discarded. The normal grains might serve as a sterile line. The sterile line material received the pollen from the maintainer line to produce progenies, wherein normal grains belonged to a sterile line without a transgenic composition, and opaque-endosperm grains belonged to a maintainer line with a transgenic composition. The maintainer line all was used to propagate the sterile line and the maintainer line in the next year, and most of the sterile lines were used to produce commercial article seeds, and a small remaining part was used to propagate the sterile line and the maintainer line in the next year. Specific flow chart of the production is shown in FIG. 10.

Example 8. Large-Scale Production of Hybrids Using the Male Sterile Line in Example 7

(85) The sterile line produced in Example 7 is a homozygous recessive sterile line regulated by cell nucleus, and such a sterile line may have fertility restored with any wild-type plant (Ms45Ms45). Therefore, as long as a self-bred line, such as Chang 7-2, that has high combining ability with the male sterile (ms45ms45) self-bred line, e.g., male sterile Zheng 58 (ms45ms45), is selected for hybridization, hybrid seeds having excellent agronomic traits may be produced. For this purpose, the inventors sowed the male sterile self-bred line and the wild-type self-bred line alternately in the field, with ensuring no additional corn planted around within 300 meters, so that the ears of the sterile line would only receive the pollen from the wild-type self-bred line, while the wild-type self-bred line was only capable of selfing. Thus, the seeds produced on the ears of the sterile line were hybrid seeds.

INDUSTRIAL APPLICATION

(86) In the method for propagating a sterile male plant line of the present invention, an highly effective method for seed labeling is used so that a male sterile seed of a plant may be propagated, saving manpower, reducing costs and ensuring seed purity for hybrid seed production. In the method for propagating a sterile male plant line of the present invention, a nucleotide that enables differentiation of grain shape (e.g., size, length, width, thickness, etc.) or of main endosperm nutrient material composition (e.g., starch content, oil content, presence or absence of farinaceous endosperm, etc.), a wild-type allele of a cell nuclear male sterility gene, and a transgenic technique are used to allow distinguishing fertile grains and sterile grains among the transgenic grains via grain shape (e.g., size, length, width, thickness, etc.) or endosperm nutrient material composition (e.g., starch content, oil content, presence or absence of farinaceous endosperm, etc.). The homozygous recessive male sterile plant produced by the method of the present invention may be used for producing a hybrid.