CN108949773B - Method for producing transgenic plants - Google Patents

Method for producing transgenic plants Download PDF

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Publication number
CN108949773B
CN108949773B CN201710352683.0A CN201710352683A CN108949773B CN 108949773 B CN108949773 B CN 108949773B CN 201710352683 A CN201710352683 A CN 201710352683A CN 108949773 B CN108949773 B CN 108949773B
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gene
nucleic acid
development
acid molecule
plant cell
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CN108949773A (en
Inventor
蔡文杰
沈青玉
萧郁芸
刘仲健
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Lin Shizhe
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Lin Shizhe
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8233—Female-specific, e.g. pistil, ovule

Abstract

A method of producing a transgenic plant comprising: providing a nucleic acid molecule, wherein the sequence of the nucleic acid molecule is shown as SEQ ID No. 1; directing the nucleic acid molecule to a plant cell to obtain a transfected plant cell; and controlling the development of an ovule using the nucleic acid molecule of the transfected plant cell.

Description

Method for producing transgenic plants
Technical Field
The invention relates to an orchid ovule development regulating gene (gene regulation) and a transfer method thereof; in particular to an orchid ovule development regulating gene and a transfer method thereof, wherein MADS-box B-master group gene PeMADS28 and a combination thereof are selected.
Background
The related regulation technology of orchid gene is disclosed in the "gene for controlling flower development in orchid" patent publication No. I228511, which discloses a gene for controlling flower development of orchid. A nucleic acid molecule capable of controlling flower development in orchid, wherein the sequence of the nucleic acid molecule is selected from the group consisting of PeMADS2, peMADS3, peMADS4, peMADS5, reverse transcribed strands thereof and degenerate sequences thereof. Wherein PeMADS2 is used for controlling the development of the calyx, peMADS3 is used for controlling the development of the labial flap, peMADS4 is used for controlling the development of the labial flap and the coleus, and PeMADS5 is used for controlling the development of the petals and the stamens.
Another prior art technique for regulating and controlling orchid gene, such as the "gene for controlling flower type and/or flower longevity" of Taiwan patent publication No. I289143, is the invention patent which discloses a gene for controlling flower type and/or flower longevity of orchid plants. An isolated nucleic acid molecule capable of controlling flower type and/or flower longevity, wherein the nucleic acid molecule is selected from the group consisting of a molecule encoding a PeMADS6 protein and a reverse transcribed strand molecule encoding a PeMADS6 protein.
Another prior art technique for controlling the development of the stamen is disclosed in the "gene, protein and method for controlling the development of the stamen" of Taiwan patent publication No. I545197, which discloses a gene, protein and method for controlling the development of the stamen in orchid plants, which provides a nucleic acid molecule capable of controlling the development of the stamen. The nucleic acid molecule may be contained in a vector and a cell. The proteins and methods for controlling the development of the coleus are suitable for use in methods for making transgenic plants.
In fact, although the aforementioned taiwan patent publication nos. I228511, I289143 and I545197 by the same inventor have disclosed various orchid gene related regulatory technologies, there is still a potential need to further provide other related technologies of orchid genes in order to provide regulatory functions and mechanisms of other orchid genes having utility value. The foregoing patents are merely references to and descriptions of the state of the art in the background of the invention, and are not meant to limit the invention.
In view of the above, the present invention provides an ovule development regulating gene and a method thereof, wherein the ovule development regulating gene comprises a nucleic acid molecule for controlling the development of an ovule or having a function of controlling the development of an ovule, and the sequence of the nucleic acid molecule is shown in SEQ ID No.1, so as to provide the ovule development regulating gene and improve the economic value thereof.
Disclosure of Invention
The main objective of the present invention is to provide a method for producing transgenic plants, which provides an orchid ovule development regulating gene comprising a nucleic acid molecule for controlling the development of an ovule or having a function of controlling the development of an ovule, wherein the sequence of the nucleic acid molecule is shown in SEQ ID No.1, so as to achieve the purposes of providing the orchid ovule development regulating gene and improving the economic value thereof.
To achieve the above object, a method for producing a transgenic plant according to a preferred embodiment of the present invention comprises:
providing at least one nucleic acid molecule, wherein the sequence of the nucleic acid molecule is shown as SEQ ID No. 1;
directing the nucleic acid molecule to a plant cell to obtain a transfected plant cell; a kind of electronic device with high-pressure air-conditioning system
The nucleic acid molecules of the transfected plant cells are used to control the development of an ovule.
The nucleic acid molecules of the preferred embodiment of the invention are obtained by means of isolation.
The transformed plant cells of the preferred embodiment of the present invention are used to alter the expression of a protein in a plant that controls ovule development.
The preferred embodiment of the present invention utilizes the transformed plant cells to regenerate a transformed plant.
The plant cell of the preferred embodiment of the present invention is selected from the group consisting of an orchid plant cell.
The nucleic acid molecules of the preferred embodiments of the present invention are used to control the development of an ovule, which is an ovule of butterfly orchid, an ovule of butterfly orchid or other ovules of orchid.
Drawings
FIG. 1 is a schematic diagram showing the relationship between the MADS-box B-sister group gene and the ABCDE model of orchid and the flower wheel according to the preferred embodiment of the present invention.
FIG. 2 is a diagram showing the PeMADS28 gene at different embryo development stages after pollination of Ji Hudie orchid according to the preferred embodiment of the present invention.
FIG. 3 is a graph showing the expression level of the PeMADS28 gene in different embryo development periods after pollination of Ji Hudie orchid according to the preferred embodiment of the present invention.
FIGS. 4 (A) to 4 (J) are microscopic image views showing in situ hybridization of Ji Hudie blue PeMADS28 gene transcript in sections of ovules of Phalaenopsis taiwan during its development according to a preferred embodiment of the present invention.
FIGS. 5 (A) to 5 (D) are images of leaf phenotypes of wild-type Arabidopsis thaliana and of the Ji Hudie blue PeMADS28 gene transgenic Arabidopsis thaliana according to the preferred embodiment of the present invention.
FIGS. 6 (A) to 6 (H) are image graphs of the inflorescence phenotyping of wild-type Arabidopsis and of the Ji Hudie blue PeMADS28 gene transgenic Arabidopsis according to the preferred embodiment of the present invention.
FIGS. 7 (A) to 7 (C) are images of seed and pod phenotypes of wild-type Arabidopsis thaliana and Ji Hudie blue PeMADS28 transgenic Arabidopsis thaliana according to the preferred embodiment of the present invention.
Reference numerals
1: b-sister group genes
10: peMADS28 Gene expression period
Detailed Description
For a thorough understanding of the present invention, reference will now be made in detail to the preferred embodiments, examples of which are illustrated in the accompanying drawings and are not intended to limit the invention.
The orchid ovule development regulating gene of the preferred embodiment of the present invention is suitable for providing its gene product or its encoded protein, but it is not intended to limit the scope of the present invention. Furthermore, the orchid ovule development controlling gene and the controlling method thereof according to the preferred embodiments of the present invention are suitable for gene transfer (transgenics) into various plant cells or plant bodies by various genetic engineering, but are not intended to limit the scope of the present invention.
For example, orchids according to the preferred embodiment of the present invention include butterfly orchids (Phalaenopsis), taiwan butterfly orchids (Phalaenopsis amabilis), white butterfly orchids, ji Hudie orchids (Phalaenopsis equestris) or other orchids (e.g., wenyujin orchid or Dendrobium nobile), but are not intended to limit the scope of the present invention.
In general, a typical flowering plant, whose ovules and egg cells develop completely upon flowering, and wait for subsequent insemination (fertilization). However, since ovule development of butterfly orchid is precisely regulated by pollination (polinium), flowering is fashionable without ovules that have been fully developed.
A preferred embodiment of the invention comprises at least one nucleic acid molecule having a function of controlling the development of an ovule, and the sequence of the nucleic acid molecule is shown in SEQ ID No.1, which is the PeMADS28 gene designated as Ji Hudie blue MADS-box B-master group gene (Bs).
For example, FIG. 1 shows the relationship between the MADS-box B-master group gene and the ABCDE model (model) and the flower wheel (flower whorl) of orchid according to the preferred embodiment of the present invention. Referring to FIG. 1, the B-master group gene 1 (shown in the upper right of FIG. 1) of the orchid MADS-box according to the preferred embodiment of the present invention provides a flower type control mechanism, and controls the development of flower type organs by expression of the flower type control gene A, B, C, D, E, bs alone or in combination.
In general, in the ABCDE model, development of sepals (sepal) of orchid is determined by the activities of genes a and E, and development of petals (petal) of orchid is determined by the activities of genes A, B and E. In addition, after pollination (polium), development of stamen (stamen) of orchid is determined by the activities of genes B, C and E, and development of carpel (carpel) and coleus (gynostemium) of orchid is determined by the activities of genes C and E.
Referring again to FIG. 1, the preferred embodiment of the present invention can utilize the activity of genes Bs, D and E to determine or regulate the development of orchid ovules in the ABCDE model (ovule development), as shown on the right side of FIG. 1.
Referring again to FIG. 1, the nucleic acid molecule is provided in a vector or a shuttle (shuttle) vector, and the vector is used to store or produce the nucleic acid molecule. For example, the vector is provided in a plant cell, and the nucleic acid molecule may alternatively be provided in a protocon-like body (protocon-like body) or otherwise.
The nucleic acid molecule of another preferred embodiment of the present invention is used to encode a protein (e.g., a protein having the sequence of SEQ ID No. 2) or other gene product (gene product), i.e., it is encoded by the nucleic acid molecule. The nucleic acid molecule may be a genome DNA, cDNA, mRNA, an artificial mRNA, or any combination thereof.
FIG. 2 shows the expression pattern of the PeMADS28 gene at different embryo development stages of the Ji Hudie blue according to the preferred embodiment of the present invention from day 0 to 100 after pollination. Referring to FIG. 2, the preferred embodiment of the present invention is shown as a date (day after pollinium, DAP) after pollination during ovule development (ovule development), relative to the period of DAP 32 to 48, labeled as PeMADS28 gene expression period 10 in FIG. 2, during which the PeMADS28 gene is expressed in large amounts.
FIG. 3 shows the expression level of the PeMADS28 gene in different embryo development periods as a function of time from day 0 to day 100 after pollination of the Ji Hudie orchid according to the preferred embodiment of the invention. Referring to FIGS. 2 and 3, the preferred embodiment of the present invention is shown as time after pollination (DAP) during ovule development, and the PeMADS28 gene expression level is relatively maximized relative to the DAP period of 32 to 48 days, indicated as PeMADS28 gene expression period 10 in FIG. 3, and then gradually decreased, i.e., after fertilization (before 64 days relative to DAP) and relatively lower PeMADS28 gene expression level still occurs during seed development (seed development) relative to DAP after 64 days.
The method for transferring the orchid ovule development regulating gene in the preferred embodiment of the invention comprises the following steps: at least one nucleic acid molecule is provided, and the sequence of the nucleic acid molecule is shown as SEQ ID No. 1. The nucleic acid molecules of the preferred embodiments of the invention are obtained by isolation or other suitable means.
The method for transferring the orchid ovule development regulating gene in the preferred embodiment of the invention comprises the following steps: the nucleic acid molecule is directed to a plant cell to obtain a transfected plant cell. For example, preferred embodiments of the invention may optionally employ gene gun, vacuum infiltration (vacuum infiltration) or other suitable means for directing the nucleic acid molecule to the plant cell.
The method for transferring the orchid ovule development regulating gene in the preferred embodiment of the invention comprises the following steps: the nucleic acid molecules of the transfected plant cells are used to control the development of an ovule. The present invention then provides for the selective regeneration of a transformed plant using the transformed plant cells. For example, a predetermined plant may be selectively produced from the transformed plant cells to obtain a predetermined transformed plant.
For example, the nucleic acid molecule of the transformed plant cell controls the development of an ovule, and the ovule is a butterfly orchid ovule, a white butterfly orchid ovule, a agate butterfly orchid ovule or other orchid ovule.
The transformed plant cells of the preferred embodiment of the present invention are used to alter the expression of a protein in a plant that controls ovule development. For example, the plant cell is selected from an orchid plant cell or other plant cell (e.g., butterfly orchid).
FIGS. 4 (A) to 4 (J) show microscopic image images of in situ hybridization (in situ hybridization) of Ji Hudie blue PeMADS28 gene transcripts in sections of developmental stage ovules (ovule development) of Phalaenopsis taiwan (Phalaenopsis aphrodite subsp. Formosana) in accordance with a preferred embodiment of the present invention. Referring to FIGS. 4A and 4B, peMADS28 mRNA was detected in the placenta of ovary (planta, p) on day 4 relative to DAP on in situ hybridization of the PeMADS28 gene transcript.
Referring to FIGS. 4 (C) and 4 (D), peMADS28 mRNA was detected on the placenta of the ovary (designated p) and ovule primordia (designated op) on day 32 relative to DAP on in situ hybridization of the PeMADS28 gene transcript.
Referring to FIGS. 4 (E) and 4 (F), on day 40 relative to DAP, on in situ hybridization of the PeMADS28 gene transcript, peMADS28 mRNA was still detected in the placenta of the ovary (labeled p) and ovule primordia (labeled op).
Referring to FIGS. 4 (G) and 4 (H), peMADS28 mRNA was detected on day 48 of in situ hybridization of the PeMADS28 gene transcript with respect to DAP, in inner integument (labeled ii), outer integument (labeled oi) and embryo sac (labeled es).
Referring to fig. 4 (C) to 4 (H), the PeMADS28 gene was detected on days 32 to 48 relative to DAP, showing that the detected gene was expressed in the ovary placenta and the inner and outer beads.
Referring to FIGS. 4 (I) and 4 (J), on day 56 relative to DAP, on in situ hybridization of the PeMADS28 gene transcript, peMADS28 mRNA was still detected in the ovule (labeled ov).
FIGS. 5 (A) to 5 (D) reveal images of leaf phenotypes of wild-type (wild type, WT) Arabidopsis (Arabidopsis thaliana) and Ji Hudie blue PeMADS28 gene transgenic (transgenic) Arabidopsis according to a preferred embodiment of the present invention. Referring to FIG. 5 (A) and FIG. 5 (B), the 20-day Arabidopsis wild-type strain was compared with the 20-day PeMADS28 gene transgenic strain for observation. The PeMADS28 gene transgenic strain showed that its rotaleaf (rosette leaf) grows to form a relatively small phenotype and its rotaleaf forms a relatively curled (curled) phenotype during the early developmental period of the transgenic strain, as shown in fig. 5 (B), relative to the arabidopsis wild strain.
Referring to FIGS. 5 (C) and 5 (D), the PeMADS28 gene transgenic strain showed a phenotype in which the flowering time of the growth was relatively early, as shown in FIG. 5 (D), during the early development period of the transgenic strain, relative to the Arabidopsis wild type strain.
FIGS. 6 (A) to 6 (H) reveal images of the analysis of the inflorescence phenotype (phenotype of floral inflorescence) of wild-type Arabidopsis and of the Ji Hudie blue PeMADS28 gene transgenic Arabidopsis according to a preferred embodiment of the invention. Referring to FIGS. 6 (A) and 6 (B), a side view of a flower of wild-type Arabidopsis thaliana was compared with a side view of a flower of a transgenic Arabidopsis thaliana in which the Ji Hudie blue PeMADS28 gene according to the preferred embodiment of the present invention.
Referring to FIGS. 6 (C) and 6 (D), the upper view of the flower of wild-type Arabidopsis thaliana was compared with the upper view of the flower of a transgenic Arabidopsis thaliana transformed with the Ji Hudie blue PeMADS28 gene according to the preferred embodiment of the present invention. Referring to FIGS. 6 (A) to 6 (D), the flowers of the PeMADS28 gene transgenic strain, as shown in FIGS. 6 (A) and 6 (C), are shown to have significantly smaller flower sizes and sepal rupture (sepal rupture) than the flowers of the Arabidopsis wild strain, as shown in FIGS. 6 (B) and 6 (D).
Referring to FIGS. 6 (E) and 6 (F), a side view of an inflorescence (floral inflorescence) of a wild-type Arabidopsis thaliana was compared with a side view of an inflorescence of a Ji Hudie blue PeMADS28 gene-transfected Arabidopsis thaliana according to a preferred embodiment of the present invention.
Referring to FIGS. 6 (G) and 6 (H), a side view of the cauliflower (leaf) of wild-type Arabidopsis thaliana was compared with a side view of the cauliflower (leaf) of a transgenic Arabidopsis thaliana in which the Ji Hudie blue PeMADS28 gene of the preferred embodiment of the present invention was expressed.
FIGS. 7 (A) to 7 (C) show images of phenotypic analysis of seed (seed) and pod (silique) of wild type Arabidopsis thaliana and of the Ji Hudie blue PeMADS28 gene transgenic Arabidopsis thaliana according to the preferred embodiment of the present invention. Referring to FIG. 7 (A), a comparison is made between a side view of a seed of wild type Arabidopsis thaliana (shown on the left side of FIG. 7 (A)) and a side view of a seed of a Ji Hudie blue PeMADS28 gene-transfected Arabidopsis thaliana (shown on the right side of FIG. 7 (A)) according to a preferred embodiment of the present invention. The PeMADS28 transgenic seeds showed a phenotype that is relatively large in size (i.e., relatively heavy in weight) relative to the seeds of wild-type Arabidopsis thaliana, as shown in Table 1.
Referring to FIG. 7 (B), a side view of the pod of wild-type Arabidopsis thaliana (shown in the upper side of FIG. 7 (B)) was compared with a side view of the pod of a Ji Hudie blue PeMADS28 transgenic Arabidopsis thaliana (shown in the lower side of FIG. 7 (B)) according to the preferred embodiment of the present invention. The pepads 28 gene-transfected pods showed a relatively short phenotype in length relative to the pods of wild-type arabidopsis thaliana, as shown in table 1.
Referring to fig. 7 (C), the number of seeds in the pod was observed by comparing the cross-sectional view of the pod of wild-type arabidopsis thaliana (shown on the upper side of fig. 7 (C)) with the cross-sectional view of the pod of the Ji Hudie blue PeMADS28 gene-transfected arabidopsis thaliana (shown on the lower side of fig. 7 (C)) according to the preferred embodiment of the present invention. The number of seeds in the pod of the PeMADS28 gene transgenic relative to the number of seeds in the pod of the wild-type arabidopsis thaliana showed a phenotype having a relatively small number of seeds, as shown in table 1.
Table 1: pod length, number of seeds in pod, and seed weight of wild type and pepads 28 gene transgenic arabidopsis thaliana
The foregoing description of the preferred embodiments merely illustrates the invention and its technical features, and the technology of this embodiment can be implemented in various substantially equivalent modifications and/or alternative ways as appropriate; the scope of the invention should, therefore, be determined with reference to the appended claims.
SEQUENCE LISTING
<110> Xiao Yuyun
Lin Shizhe
Liu Zhongjian
<120> orchid ovule development regulating gene and method thereof
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 723
<212> DNA
<213> Phalaenopsis sp.
<400> 1
atggggagag ggaagattga gataaagagg attgatagca ttaccaatag gcaagtgacg 60
ttctccaaga gaagaggagg gcttctgaag aaagctcatg agctcgctgt tcttacagat 120
gcccgtgtag gcgttatgat cttctctgct agtggcaggc tctatcaata caacagccct 180
ggtgaaagca tggaaagaat catagaagac tatcttaaag ttcaccatat ccagttggag 240
gaccttaaca gccaccagct acttcagttc gagctagcaa aaataaagca ggacacacat 300
gtgcttcagg cgagcattag gcagtttact ggggaagact tgactggcct aaccattaat 360
gatcttaacc tgttagaaga gcagcttgat tactctgttt ctaagattcg gacacgcaag 420
catgaactcc tccatcaaca actcgagaac ctccgccgaa aggagcacat attagcagac 480
cagaacacct gcttatacag atctatcatg ccagagcatg aagtggagat ggagcaggag 540
cggcagatgg aagaggaaca taagttaagg gaaatgagtc ttctagaacc cttcggtcat 600
tttctagaag attctagaag catgcttcag ctgggcccac aaacacttcc atttcagctg 660
cagcctaccc agcctaatct gcagtactct gaactccatg gccatggact gcagctctgg 720
taa 723
<210> 2
<211> 240
<212> PRT
<213> Phalaenopsis sp.
<400> 2
Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Asp Ser Ile Thr Asn
1 5 10 15
Arg Gln Val Thr Phe Ser Lys Arg Arg Gly Gly Leu Leu Lys Lys Ala
20 25 30
His Glu Leu Ala Val Leu Thr Asp Ala Arg Val Gly Val Met Ile Phe
35 40 45
Ser Ala Ser Gly Arg Leu Tyr Gln Tyr Asn Ser Pro Gly Glu Ser Met
50 55 60
Glu Arg Ile Ile Glu Asp Tyr Leu Lys Val His His Ile Gln Leu Glu
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Asp Leu Asn Ser His Gln Leu Leu Gln Phe Glu Leu Ala Lys Ile Lys
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Leu Asp Tyr Ser Val Ser Lys Ile Arg Thr Arg Lys His Glu Leu Leu
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His Gln Gln Leu Glu Asn Leu Arg Arg Lys Glu His Ile Leu Ala Asp
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Gln Asn Thr Cys Leu Tyr Arg Ser Ile Met Pro Glu His Glu Val Glu
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Met Glu Gln Glu Arg Gln Met Glu Glu Glu His Lys Leu Arg Glu Met
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Ser Leu Leu Glu Pro Phe Gly His Phe Leu Glu Asp Ser Arg Ser Met
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Leu Gln Leu Gly Pro Gln Thr Leu Pro Phe Gln Leu Gln Pro Thr Gln
210 215 220
Pro Asn Leu Gln Tyr Ser Glu Leu His Gly His Gly Leu Gln Leu Trp
225 230 235 240

Claims (6)

1. A method of producing a transgenic plant comprising:
providing a nucleic acid molecule, wherein the sequence of the nucleic acid molecule is shown as SEQ ID No. 1;
introducing the nucleic acid molecule into a plant cell to obtain a transfected plant cell, wherein the plant cell is a butterfly orchid plant cell or an arabidopsis thaliana plant cell; a kind of electronic device with high-pressure air-conditioning system
The nucleic acid molecule or a protein expressed by the nucleic acid molecule is used for controlling the development of an ovule of the transfected plant cell, and the sequence of the protein is shown as SEQ ID No. 2.
2. A method of producing a transgenic plant according to claim 1, wherein the butterfly orchid plant cell is a white flower butterfly orchid plant cell or a agate butterfly orchid plant cell.
3. The method of claim 1, wherein the transformed plant cell is used to alter the expression of a protein in a plant that controls ovule development.
4. The method of claim 1, wherein said transgenic plant is regenerated using said transgenic plant cell to produce a transgenic plant.
5. The method of claim 1, wherein the nucleic acid molecule is provided in a vector.
6. The method of producing a transgenic plant of claim 5, wherein the vector is a shuttle vector.
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WO2021076346A1 (en) * 2019-10-18 2021-04-22 Pioneer Hi-Bred International, Inc. Maize event dp-202216-6 and dp-023211-2 stack

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