ENHANCED PLANT REGENERATION AND TRANSFORMATION BY USING GRF1 BOOSTER GENE

Abstract

The present invention relates to the field of plant breeding and biotechnology and in particular to the generation of plants from cells and other tissues. More particularly, the invention provides methods and means for improving plant regeneration, especially from transformed or genetically modified plant cells using GRF1 booster gene.

Claims

1. A method for transforming a plant cell, comprising the steps (a1) introducing into a plant cell in parallel or sequentially i. at least one nucleotide sequence of interest; and ii. an expression cassette comprising a polynucleotide encoding a GRF1 polypeptide, mRNA encoding a GRF1 polypeptide, or GRF1 polypeptide(s); or (a2) introducing into a plant cell at least one nucleotide sequence of interest; and inducing in said plant cell in parallel or sequentially an enhanced expression level of an endogenous gene encoding a GRF1 polypeptide; and (b) optionally, cultivating the plant cell of (a1) or (a2) or a plant cell derived from the plant cell of (a1) or (a2) under conditions where in the plant cell the GRF1 polypeptide is expressed from the expression cassette, GRF1 polypeptide is translated from introduced mRNA, GRF1 polypeptide is enhanced expressed from the endogenous gene, or GRF1 polypeptide(s) are present.

2. A method for modifying the genome of a plant cell, comprising the steps (a1) introducing into a plant cell an expression cassette comprising a polynucleotide encoding a GRF1 polypeptide, mRNA encoding a GRF1 polypeptide, or GRF1 polypeptide(s); or (a2) inducing in a plant cell an enhanced expression level of an endogenous gene encoding a GRF1 polypeptide; and (b) cultivating the plant cell of (a1) or (a2) or a plant cell derived from the plant cell of (a1) or (a2) under conditions where in the plant cell the GRF1 polypeptide is expressed from the expression cassette, GRF1 polypeptide is translated from introduced mRNA, GRF1 polypeptide is enhanced expressed from the endogenous gene, or GRF1 polypeptide(s) are present; (c) modifying the genome of the plant cell of (b) by means of a single stranded DNA break (SSB) or double stranded DNA break (DSB) inducing enzyme or a base editor enzyme which preferably recognizes a predetermined site in the genome of said cell, and optionally by means of a repair nucleic acid molecule, wherein the modification of said genome at said predetermined site is selected from i. a replacement of at least one nucleotide; ii. a deletion of at least one nucleotide; iii. an insertion of at least one nucleotide; or iv. any combination of i.-iii.; and wherein step (c) is conducted simultaneously with step (a1)/(a2) and/or (b), before step (a1)/(a2), between step (a1)/(a2) and (b) or after step (b).

3. A method of producing a transgenic plant, comprising the steps (a) transforming a plant cell according to the method of claim 1, and (b) regenerating from the plant cell of (a) or from a plant cell derived from the plant cell of (a) a plant comprising at least one cell which comprises the at least one nucleotide sequence of interest as transgene.

4. A method of producing a genetically modified plant, comprising the steps (a) modifying the genome of a plant cell according to the method of claim 2, and (b) regenerating from the plant cell of (a) or from a plant cell derived from the plant cell of (a) a plant comprising in at least one cell the modification of the genome.

5. A method of producing a haploid plant embryo, comprising the steps (a1) introducing into an immature male gametophyte or a microspore an expression cassette comprising a polynucleotide encoding a GRF1 polypeptide, mRNA encoding a GRF1 polypeptide, or GRF1 polypeptide(s); or (a2) inducing in an immature male gametophyte or a microspore an enhanced expression level of an endogenous gene encoding a GRF1 polypeptide; and (c) cultivating the immature male gametophyte or the microspore of (a) under conditions where in the immature male gametophyte or the microspore the GRF1 polypeptide is expressed from the expression cassette, GRF1 polypeptide is translated from introduced mRNA, GRF1 polypeptide is enhanced expressed from the endogenous gene, or GRF1 polypeptide(s) are present; and (d) selecting haploid plant embryo derived from the immature male gametophyte or the microspore of step (b).

6. The method of claim 1, wherein the GRF1 polypeptide comprises a PFAM domain PF08880 and a PFAM domain PF08879, preferably wherein the PFAM domain PF08880 finds a match of at least 90% coverage at or near the N-terminus of the GRF1 polypeptide and the PFAM domain PF08879 finds a match of at least 90% C-terminally located to the PFAM domain PF08880 in the GRF1 polypeptide.

7. The method of claim 6, wherein both matching amino acid stretches are located in the N-terminal half of the GRF1 polypeptide.

8. The method of claim 1, wherein the GRF1 polypeptide comprises the motif [D]-[P]-[E]-[P]-[G]-[R]-[C]-[R]-[R]-[T]-[D]-[G]-[K]-[K]-[W]-[R]-[C]-[AS]-[RK]-[ED]-[A]-[AH]-[PQS]-[D]-[S]-[K]-[Y]-[C]-[E]-[KR]-[H]-[M]-[H]-[R]-[G]-[R]-[N]-[R] (SEQ ID NO: 25) with a maximum number of two mismatches, wherein preferably the motif consists of any of the amino acid sequences SEQ ID NO: 26 to SEQ ID NO: 35, and/or wherein preferably the motif contains a sub-region of amino acid stretch matching PFAM domain PF08879.

9. The method of claim 1, wherein the GRF1 polypeptide comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 37, 39, 41, 43, 45, 47, 49, 51 and 53; or (ii) an amino acid sequence comprising a sequence being at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 37, 39, 41, 43, 45, 47, 49, 51 and 53.

10. The method of claim 1, wherein the polynucleotide encoding the GRF1 polypeptide comprises (i) a coding nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 36, 38, 40, 42, 44, 46, 48, 50 and 52; (ii) a coding nucleotide sequence comprising a sequence being at least 70% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 36, 38, 40, 42, 44, 46, 48, 50 and 52; (iii) a nucleotide sequence encoding the polypeptide encoded by (i) or (ii) within the scope of the degeneracy of the genetic code; (iv) a nucleotide sequence complementary to the nucleotide sequence of (i), (ii) or (iii); or (v) a nucleotide sequence hybridizing with a nucleotide sequence of (iv) under stringent condition.

11. The method of claim 1, wherein introducing into a plant cell the expression cassette comprising a polynucleotide encoding a GRF1 polypeptide results in a stable integration thereof into the genome of the plant cell, or wherein introducing into a plant cell the expression cassette comprising a polynucleotide encoding a GRF1 polypeptide, mRNA encoding GRF1 polypeptide, or GRF1 polypeptide(s) or inducing in a plant cell the enhanced expression level of an endogenous gene encoding a GRF1 polypeptide results in a transient occurrence of GRF1 polypeptide(s) in the plant cell or in a progeny cell thereof.

12. The method of claim 1, wherein the polynucleotide encoding the GRF1 polypeptide is in operative linkage to at least one regulatory sequence suitable for expression of the GRF1 polypeptide in a plant cell.

13. The method of claim 1, wherein the plant cell of step (a1) or (a2) is a cell of a somatic tissue, callus tissue, a meristematic tissue or an embryonic tissue, a protoplast, gametophyte, pollen, ovule or microspore.

14. A plant or part thereof obtained or obtainable by the method of claim 3, or a progeny plant thereof.

15. A plant cell or a seed of the plant of claim 14, wherein the plant cell or the seed comprises the at least one nucleotide sequence of interest as transgene or comprises the modification in the genome.

Description

FIGURES

[0115] FIG. 1. Assessment of biolistic delivery by tDT expression (24 hours after bombardment). A and C: immature embryo bombarded with tDT construct; A, RFP channel and C, bright field; B and D: immature embryo bombarded with ZmWUS2, ZmBBM and tDT three constructs; B, RFP channel and D, bright field.

[0116] FIG. 2. Assessment of biolistic delivery by target gene expression: A: tDT gene is expressed in the samples that were bombarded with tDT construct but not expressed in the wild type A188 embryos; B: ZmWUS2 gene is expressed in the samples that were bombarded with ZmWUS2 construct but not expressed in the wild type A188 embryos or the samples bombarded with only tDT construct; C: ZmBBM gene is expressed in the samples that were bombarded with ZmBBM construct but not expressed in the wild type A188 embryos or the samples bombarded with only tDT construct

[0117] FIG. 3. Expression of ZmGRF1 is up-regulated in ZmWUS2 and ZmBBM expressing samples. A: ZmGRF1 expression is higher in the samples bombarded with WUS2/BBM/tDT compared to the samples bombarded with tDT alone; B and C: ZmGRF1 expression is dramatically increased in the transgenic WUS/BBM callus samples (B) and small shoots (C) compared with tDT the control obtained from agrobacterial transformation.

[0118] FIG. 4. Transient delivery of ZmGRF1 enhances regeneration. Fluorescent embryonic structures appearing 1 month after bombardment in tDT only samples (A), ZmWUS2/ZmGRF1/tDT samples (B) and ZmWUS2/ZmPLT7/tDT (C).

[0119] FIG. 5. Quantification of fluorescent embryonic structure from FIG. 4.

[0120] FIG. 6. Increase in transformation frequency in stable transformation of A188 corn line. Transformation frequency fold changes comparing ZmGRF1 to the standard transformation construct. In experiment 1 (left column) the transgenic plants were obtained 3 weeks earlier than in a standard transformation due to the growth of the events.

[0121] FIG. 7. Increase in transformation frequency in stable transformation of A188 corn line. Event number and size after callus selection. Standard construct (left) ZmGRF1 (right)

[0122] FIG. 8. Increase in transformation frequency in stable transformation of A188 corn line. ZmGRF1 overexpressing single copy lines in the GH have no phenotype (left) and they are fertile (right)

[0123] FIG. 9. Effect of co-transformation of GRF1 on transformation efficiency. Standard Agrobacterium mediated transformation of 2 constructs (A and B) compared with the co-transformation with an Agrobacteria containing the GRF1 expressing binary vectors.

[0124] FIG. 10. Vector Map of pAMK-BdUbi10-ZmBbm

[0125] FIG. 11. Vector Map of pAMK-BdEF1-ZmWus2 FIG. 12. Vector Map of pAMK-BdEF1-ZmGRF1

[0126] FIG. 13. Vector Map of pLH-Pat5077399-70Subi-tDT

EXAMPLES

1. Biolistic Delivery:

Gene Cloning and Construct Preparation

[0127] Maize PLT7 (ZmPLT7; cDNA=SEQ ID NO: 4; protein=SEQ ID NO: 5), BBM (ZmBBM; cDNA=SEQ ID NO: 6; protein=SEQ ID NO: 7), WUS2 (ZmWUS2; cDNA=SEQ ID NO: 8; protein=SEQ ID NO: 9) and GRF1 (ZmGRF1; genomic DNA=SEQ ID NO: 1; cDNA=SEQ ID NO: 2; protein=SEQ ID NO: 3) genes were cloned by RT-PCR using total RNA isolated from maize A188 immature embryos. The boost gene fragments are cloned into expression vector pAMK-BdUbi10 or pAMK-BdEF1 at the cloning site, and expressing under the control of BdUbi10 promoter or BdEF1 promoter. Both promoters are strong constitutive promoters from Brachypodium. The sequencing-confirmed construct maps are shown in FIG. 10 to FIG. 12.

Preparing Maize Immature Embryo for Bombardment

[0128] 9-12 days post pollination, maize ears with immature embryos size 0.8 to 1.8 mm, preferred 1.0-1.5 mm were harvested. The ears were sterilized with 70% ethanol for 10-15 minutes. After a brief air-dry in a laminar hood, remove top ˜⅓ of the kernels from the ears with a shark scalpel, and pull the immature embryos out of the kernels carefully with a spatula. The fresh isolated embryos were placed onto the bombardment target area in an osmotic medium plate (see below) with scutellum-side up. Wrap the plates with parafilm and incubated them at 25° C. in dark for 4 hours before bombardment.

[0129] Particle co-bombardment Particle bombardment gun and gold particles size 0.4 or 0.6 micron (μm) were used to deliver DNA into the scutellum cells of maize immature embryo. The plasmids were premixed with genes of interest (GOI). For 10 shots, 1 mg of gold particle in 50% (v/v) glycerol (100 μg of gold particles per shot) in a total volume of 100 microliter (μl) was pipetted into a clear low-retention microcentrifuge tube. Sonicate for 15 seconds to suspend the gold particles. While vortex at a low speed, add the following in order to each 100 μl of gold particles: [0130] Up to 10 μl of DNA (1.0-10.0 μg total DNA of pre-mixed, 100-1000 ng per each shot) [0131] 100 μl of 2.5 M CaCl.sub.2 (pre-cold on ice) [0132] 40 μl of 0.1 M cold spermidine

[0133] Close the lid and vortex the tube for 2-30 minutes at 0-10° C., and spin down the DNA-coated gold particles. After washing in 500 μl of 100% ethanol for two times, the pellet was resuspended in 120 μl of 100% ethanol. While vortexing at a low speed, pipet 10 μl of co-coated gold particles with a wide open 20 μl tip from the tube onto the center of the macrocarrier evenly since the particles tend to form clumps at this point, get the gold particles onto the macrocarriers as soon as possible. Air dry. Bombardment was conducted using a Bio-Rad PDS-1000/He particle gun. The bombardment conditions are: 28 mm/Hg vacuum, 450 or 650 psi rupture disc, 6 mm gap distance, the specimen platform is in the second position from the bottom in the chamber at a distance of 60 mm, three shots per sample (maize immature embryos) plate.

Post Bombardment Observation and Embryo Culture

[0134] After bombardment the embryos were remained on the osmotic medium for another 16 hours. Transient transformation was examined using a fluorescence microscope for the tDT expression at excitation maximum 554 nm and emission maximum 581 nm 16-20 hours after bombardment. The embryos with dense fluorescent signals under a fluorescence microscope were selected and transfer from N6OSM onto a N6-5Ag plate (˜15 embryos per plate) with scutellum-face-up for callus induction.

[0135] Osmotic medium: N6 salt, N6 vitamin, 1.0 mg/L of 2,4-D, 100 mg/L of Caseine, 0.7 g/L of L-proline, 0.2 M Mannitol (36.4 g/L), 0.2 M sorbitol (36.4 g/L), 20 g/L sucrose, 15 g/L of Bacto-agar, pH 5.8.

[0136] N6-5Ag: N6 salt, N6 vitamin, 1.0 mg/L of 2,4-D, 100 mg/L of Caseine, 2.9 g/L of L-proline, 20 g/L sucrose, 5 g/L of glucose, 5 mg/L of AgNO3, 8 g/L of Bacto-agar, pH 5.8.

2. Identification of ZmWUS2 and ZmBBM Targets:

[0137] The following constructs were used in the biolistic delivery of A188 immature embryos: pAMK-BdUbi10Zm-Bbm, pAMK-BdEF1ZmWus2, pLH-Pat5077399-70Subi-tDt

[0138] The experiment set-up is as follows:

TABLE-US-00001 Category 1 Category 2 Category 3 Non pLH-Pat5077399- pAMK-BdUbi10Zm-Bbm, bombardment 70Subi-tDt pAMK--BdEF1ZmWus2, (FIG. 13) pLH-Pat5077399- 70Subi-tDt

[0139] 24 hour after biolistic delivery, the success of plasmid delivery was assessed by tDT expression. FIG. 1 indicates that the bombardment was successful for Category 2 and 3. Further the expression of introduced genes tDT, ZmWUS2 and ZmBBM (Category 3) has been determined (FIG. 2).

3. RNAseq Analysis:

[0140] 24 hours after biolistic delivery, immature embryos were harvested and flash-frozen in liquid nitrogen. All the samples were subsequently sent for RNA extraction, library preparation and RNAseq that was done by external sequencing provider.

[0141] Several candidates were identified by comparison between the samples only containing tDT (Category 2) and samples contains ZmWUS2, ZmBBM, and tDT (Category 3). Sixteen genes showed an up-regulated expression in response to the expression of ZmWUS2 and ZmBBM, five genes a down-regulated expression. After confirmation by qRT-PCR the candidate ZmGRF1 has been selected for further analysis. FIG. 3A shows that ZmGRF1 expression is higher in the samples bombarded with Constructs containing ZmWUS2, ZmBBM and TDt (Category 3). In addition, ZmWUS2 and ZmBBM have been stably integrated by Agrobacterium-mediated transformation. The expression of ZmGRF1 has been assessed in callus samples and small shoots of corn plants. Compared to the control obtained by agrobacterial transformation solely of tDT gene the expression of ZmGRF1 was significantly increased in WUS2/BBM overexpressing plant material (FIGS. 3B and C).

4. Transient Assay for Testing ZmGRF1

[0142] A188 immature embryos were bombarded with the following constructs:

TABLE-US-00002 Category 1 Category 2 Category 3 pLH- pAMK--BdEF1ZmWus2 pAMK--BdEF1ZmWus2, Pat5077399- pMK-BdEF_ZmGRF1-GRF pABM-BdEF1_ZmPLT7 70Subi-tDt pLH-Pat5077399- pLH-Pat5077399- 70Subi-tDt 70Subi-tDt

[0143] Fluorescent embryonic structures were counted one month after bombardment. Such structures indicate enhanced regeneration capability. Samples only transiently transformed with tDT showed less pronounced embryonic structure (Category 1; FIG. 4A). From the combination of ZmWUS2 and ZmPLT7 it is known that a boosting in regeneration could be achieved. Thus, this category 3 served as positive control (FIG. 4C). Even more impressive is that the occurrence of fluorescent embryonic structures in samples transiently expressing ZmWUS2 and ZmGRF1 together with tDT (Category 2) was more pronounced than in the positive control (FIG. 4B). The quantification confirms the visual result and showed that the percentage of fluorescent embryonic structures in samples transiently expressing ZmWUS2 and ZmGRF1 together with tDT (Category 2) was more than 5 fold higher than in category 1 and more than 2 fold higher than in category 3 (FIG. 5).

5. Maize Transformation Methods

[0144] Corn transformation (line A188) was performed using Agrobacterium following a standard protocol (Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens Nat Biotechnol 14:745-750). Briefly, immature embryos of the A188 line were inoculated with the Agrobacteria and co-cultured for 7 days. After the initial embryogenic callus production a selection media was used where a herbicide selected for the transformed events. DNA of the putative transgenic plants was extracted and analyzed through qPCR for the presence of the selectable marker gene. Transformation rate was calculated as the number of transgenic events per inoculated embryos (%). When a co-transformation was performed, equal amounts of the Agrobacterias containing the construct and the GRF1 gene were mixed together prior inoculation.

[0145] The results showed that compared to the standard transformation, i.e. without the additional delivery of ZmGRF1, up to 4 times higher transformation efficiencies have been reached. Of advantage was in addition that transgenic plants were obtained approximately 3 weeks earlier due to the rapid growth of the events. FIG. 6 shows the quantitative analysis of two independent experiments, FIG. 7 the visual comparison. Corn plant lines with ZmGRF1 single copy events grown in the greenhouse have no undesired phenotype and are fertile (FIG. 8).

[0146] Further the effect of co-transformation of GRF1 on transformation efficiency has been investigated. Standard Agrobacterium mediated transformation of 2 constructs (A and B) has been compared with the co-transformation with an Agrobacteria containing the GRF1 expressing binary vector. The transformation rates were 2.3 and 1.8 fold higher when GRF1 was used (FIG. 9). Additionally, the co-transformation experiment was 36% faster than the standard protocol.