REGENERATION OF GENETICALLY MODIFIED PLANTS

Abstract

The present invention relates to the field of plant breeding 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.

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 GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or GRF5 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 GRF5 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 GRF5 polypeptide is expressed from the expression cassette, GRF5 polypeptide is translated from introduced mRNA, GRF5 polypeptide is enhanced expressed from the endogenous gene, or GRF5 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 GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or GRF5 polypeptide(s); or (a2) inducing in a plant cell an enhanced expression level of an endogenous gene encoding a GRF5 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 GRF5 polypeptide is expressed from the expression cassette, GRF5 polypeptide is translated from introduced mRNA, GRF5 polypeptide is enhanced expressed from the endogenous gene, or GRF5 polypeptide(s) are present; (c) modifying the genome of the plant cell of (b) by means of a double stranded DNA break (DSB) inducing enzyme which preferably recognize 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 GRF5 polypeptide, mRNA encoding a GRF5 polypeptide, or GRF5 polypeptide(s); or (a2) inducing in an immature male gametophyte or a microspore an enhanced expression level of an endogenous gene encoding a GRF5 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 GRF5 polypeptide is expressed from the expression cassette, GRF5 polypeptide is translated from introduced mRNA, GRF5 polypeptide is enhanced expressed from the endogenous gene, or GRF5 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 GRF5 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 GRF5 polypeptide and the PFAM domain PF08879 finds a match of at least 90% C-terminally located to the PFAM domain PF08880 in the GRF5 polypeptide.

7. The method of claim 6, wherein both matching amino acid stretches are located in the N-terminal half of the GRF5 polypeptide, preferably the amino acid stretch matching PFAM domain PF08880 is located in the N-terminal quarter of the GRF5 polypeptide.

8. The method of claim 1, wherein the GRF5 polypeptide comprises the motif [D]-[PL]-[E]-[P]-[G]-[R]-[C]-[R]-[R]-[T]-[D]-[G]-[K]-[K]-[W]-[R]-[C]-[SA]-[RK]-[ED]-[A]-[YH]-[P]-[D]-[S]-[K]-[Y]-[C]-[E]-[KR]-[H]-[M]-[H]-[R]-[G]-[RK]-[N]-[R] (SEQ ID NO: 177) with a maximum number of three mismatches, wherein preferably the motif consists of any of the amino acid sequences SEQ ID NO: 41 to SEQ ID NO: 104 or SEQ ID NO: 113 to SEQ ID NO: 176, 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 GRF5 polypeptide comprises (i) an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 106, 108, 110, 112 or 209; or (ii) an amino acid sequence comprising a sequence being at least 70% identical to the amino acid of (i).

10. The method of claim 1, wherein the polynucleotide encoding the GRF5 polypeptide comprises (i) a nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 105, 107, 109, 111, 207, 208 or 210; (ii) a nucleotide sequence comprising a sequence being at least 70% identical to the nucleotide sequence of (i); (iii) a nucleotide sequence encoding a polypeptide encoded by (i) or (ii) within the scope of the degeneracy of the genetic code; (iv) a nucleotide sequence complementary to a 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 GRF5 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 GRF5 polypeptide, mRNA encoding GRF5 polypeptide, or GRF5 polypeptide(s) or inducing in a plant cell the enhanced expression level of an endogenous gene encoding a GRF5 polypeptide results in a transient occurrence of GRF5 polypeptide(s) in the plant cell or in a progeny cell thereof.

12. The method of claim 1, wherein the polynucleotide encoding the GRF5 polypeptide is in operative linkage to at least one regulatory sequence suitable for expression of the GRF5 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, or a protoplast.

14. A plant 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

[0098] FIG. 1: Total number of developing shoots in each selection step (S1-S4). Data obtained from 5 independent transformation experiments performed either with the control construct pZFN-tdT-nptII or with pZFN-nptII-GRF5 (compare table 3).

[0099] FIG. 2: Shoot regeneration at the beginning of the selection step 3 (S3) of 14 independently transformed calli on a control experiment (A) and an experiment done with pZFN-nptII-GRF5 (B). Arrows indicate developing shoots. Experiments were performed following the method of 2.1.

[0100] FIG. 3: Shoot regeneration in sugar beet calli 10 days after bombarded with pZFN-nptII-GRF5 (A) and the control plasmid pABM70S-TurboYFP (B). The calli were co-bombarded with pUbi4-tDT plasmid to control the transient expression levels by red fluorescence. Experiments were performed following the method 2.2. Three independently bombarded calli are shown.

[0101] FIG. 4: Expression level of GRF5 in eleven independent sugar beet transgenic events.

[0102] FIG. 5: A: Sequence comparison of the sequences of GRF5 polypeptides derived from 16 different plant species and deduction of a GRF5 specific indicator motif (SEQ ID NO: 177); partial sequences of the sequences of GRF5 polypeptides as shown are set forth in SEQ ID NOs: 178-206; B: motif analysis by sequence alignment/comparison on the complete protein sequences derived from 16 different plant species with allowing up to 10 mismatches. The columns start and stop indicate the starting and ending position of sequence fragment of the GRF5 polypeptides corresponding to the indicator motif; column mismatches shows the number of mismatches between the respective GRF5 polypeptide and the indicator motif; partial sequences of the sequences of GRF5 polypeptides as shown are set forth in SEQ ID NOs: 178-184 and SEQ ID NOs: 186-206. AtGRF1_AT2G22840.1 (SEQ ID NO: 33); AtGRF2_AT4G37740.1 (SEQ ID NO: 34); AtGRF3_AT2G36400.1 (SEQ ID NO: 35); AtGRF4_AT3G52910.1 (SEQ ID NO: 36); AtGRF6_AT2G06200.1 (SEQ ID NO: 37); AtGRF7_AT5G53660.1 (SEQ ID NO: 38); AtGRF8_AT4G24150.1 (SEQ ID NO: 39); AtGRF9_AT2G45480.1 (SEQ ID NO: 40).

[0103] FIG. 6: Transformation frequency in sugar beet obtained with the control construct 70S::tdT, a constructs to overexpress AtGRF5 (cDNASEQ ID NO: 1; amino acid sequenceSEQ ID NO: 2), and the sugar beet GRF5 ortholog, BvGRF5 (synthetic DNASEQ ID NO: 207; amino acid sequenceSEQ ID NO: 4).

[0104] FIG. 7: Number of transgenic shoots per inoculated sugar beet callus obtained in transformation experiments by using constructs to overexpress either tdTomato (tDT) (control) and AtGRF5 (cDNASEQ ID NO: 1; amino acid sequenceSEQ ID NO: 2).

[0105] FIG. 8: Average number of transgenic shoots per sugar beet callus obtained from transformation experiments either with the control construct 70S-tDT (control) and the construct to overexpress AtGRF5 (70S-GRF5). Two recalcitrant genotypes A and B were used. The recalcitrance level to regenerate shoots of the genotype A is very high, whereas the genotype B displays a milder recalcitrance that A.

[0106] FIG. 9: Callus regeneration experiments of transgenic lines overexpressing AtGRF5 in sugar beet. The callus formation frequency (A) and the shoot regeneration frequency (B) were scored by using the callus induction medium and the shoot regeneration medium described in Kischenko et al., 2005 (see also example 1). A total of 5 independent events overexpressing AtGRF5 were assayed (AtGRF5-36, AtGRF5-41, AtGRF5-14, AtGRF5-94, AtGRF5-50). As control, non-transgenic sugar beet shoots (WT1 and WT2) and transgenic shoots overexpressing the tDT reporter protein (tDT-event) were used. Expression level of AtGRF5 was determined by qRT-PCR on samples isolated from the GRF5 transgenic events used in the assay (C).

[0107] FIG. 10: Transformation frequency in Corn, either with the control construct 70S::tDT, or with constructs to overexpress AtGRF5 (cDNASEQ ID NO: 1; amino acid sequenceSEQ ID NO: 2), two GRF5 Corn orthologs [ZmGRF5 version A (synthetic DNASEQ ID NO: 208; amino acid sequenceSEQ ID NO: 209) and ZmGRF5 version B (synthetic DNASEQ ID NO: 210; amino acid sequenceSEQ ID NO: 112). Transformation efficiency was calculated as the number of transgenic events divided by the number of inoculated immature embryos.

[0108] FIG. 11: Shoot development and DsRed fluorescence of shoots on soybean explants 21 days after transformation. Representative pictures of explants from DsRed control, AtGRF5, and GmGRF5 showing (A) shoot development at the primary-node and (B) DsRed fluorescence of shoots. DsRed pictures are a composite of multiple pictures and positioned in approximate position for orientation purposes.

[0109] FIG. 12: Formation of good shoot growth continued to rapidly increase from 16 to 22 days on selection. The percent of explants were scored at two timepoints to highlight the rapid production of shoots on selection for explants transformed with the three constructs.

[0110] FIG. 13: Shoot formation on soybean explants transformed with and without GRF5 variants. Pictures taken from top and side view from one repetition is shown for the three treatments (constructs). Shoot growth is more pronounced in the explants transformed with AtGRF5 (middle panel) and GmGRF5 (below panel) vs. the DsRed control (upper panel).

[0111] FIG. 14: A box-and-whisker plot visualizing the significant increase in regeneration of transgenic shoots in explants transformed with either AtGRF5 or GmGRF5 at 16 and 22 days on selection.

[0112] FIG. 15: Shoot development and DsRed fluorescence of shoots on canola explants 21 days after transformation. Representative pictures of explants from DsRed control, AtGRF5, and BnGRF5 showing (A) callus development at the hypocotyl segments and (B) DsRed fluorescence on explants.

[0113] FIG. 16: The percent of DsRed expressing explants across five Brassica napus experiments transformed with AtGRF5, BnGRF5, and DsRed control are depicted in a box-and-whisker plot. The percent of explants expressing DsRed are significantly greater in AtGRF5 and BnGRF5 compared to control constructs, with nearly a 3-fold increase in the mean.

[0114] FIG. 17: Co-transformation of sugar beet callus with the control construct 70S-tDT and the 70S-AtGRF5. The inoculation of callus in the co-transformation experiments was done with a mixture 1:1 of the Agrobacterium strains harboring each construct individually.

EXAMPLES

[0115] 1. Beta vulgaris Experiments

[0116] Production of the Binary Plasmid:

[0117] The binary vector pZFN-nptII-GRF5 was produced by following standard cloning procedures. Within the T-DNA of this vector, the cDNA encoding GRF5 (At3g13960) was cloned between the double CaMV 35S promoter and the nopaline synthase (NOS) terminator to ensure high ectopic expression levels of the GRF5 protein. The T-DNA also contains the neomycin phosphotransferase II (nptII) gene that confers resistance to a range of aminoglycoside antibiotics such as kanamycin or paromomycin and was used for the selection of transgenic plant cells and tissues. The NOS promoter and the pAG7 terminator flank the nptII gene. The backbone of the binary vector contains the colE1 and the pVS1 origins for plasmid replication in Escherichia coli and Agrobacterium tumefaciens, respectively; and the aadA gene that confers streptomycin/spectinomycin resistance for bacteria selection. The pZFN-nptII-GRF5 plasmid was transformed into AGL-1 Agrobacterium strain by a standard procedure.

[0118] Transformation of Micropropagated Shoots:

[0119] Shoots of sugar beets were transformed by different methods:

[0120] a) Agrobacterium mediated transformation based on Kischenko et al., 2005 Cell Biology International: [0121] 1. Micropropagated shoots of the genotype S706 were used as starting material. Shoots were multiplied in MS salts supplemented with 30 g/l sucrose and 0.25 mg/l benzyladenine (BAP). [0122] 2. To induce friable callus, leaf explants were incubated in MS salts including 15 g/l sucrose and 2 mg/l BAP at around 30 C. for several weeks. [0123] 3. Friable calli were harvested. [0124] 4. Agrobacterium AGL-1 harbouring the vector pZFN-nptII-GRF5 was grown in suitable medium supplemented with the appropriate antibiotics. [0125] 5. Calli were inoculated with Agrobacterium suspension. The co-culture of the callus tissue and the Agrobacterium was done in medium containing 440 mg/l CaCl.sub.2x2H.sub.2O, 170 mg/l KH.sub.2PO.sub.4, 1900 mg/l KNO.sub.3, 370 mg/l MgSO.sub.4, 1650 mg/l NH.sub.4NO.sub.3, 2 mg/l BAP, 40 g/l Acetosyringone, 20 g/l sucrose and 2 g/l glucose for at least 2 days. [0126] 6. Calli were subcultured to MS salts supplemented with 30 g/l sucrose, 1 mg/l GA3, 1 mg/l TDZ and 500 mg/l Timentin and incubated in the dark, for c. 1 week. [0127] 7. For the selection of transgenic cells, calli were transferred to the medium of step 6 supplemented with 100 mg/l paromomycin and incubated in the light for several weeks. [0128] 8. Transgenic calli were selected and subcultured for several times in the same medium and conditions. [0129] 9. Regenerating shoots were isolated and propagated in MS salts including 30 g/l sucrose, 0.25 mg/l BAP and 100 mg/l kanamycin. [0130] 10. Green shoots were transferred to MS salts supplemented with 30 g/l sucrose, 0.1 mg/l BAP, 250 mg/l Timentin and 200 mg/l paromomycin in order to finish the selection phase and were multiplied regularly for several times in this media. [0131] 11. Leaf explants were isolated from the green growing shoots for DNA extraction and PCR analysis, in order to confirm the putative transgenic lines. [0132] 12. Selected shoots were rooted in MS salts supplemented with 0.5 mg/l IBA, 100 mg/l cefotaxime and 10 mg/l PPT and transferred to the green house for seed production.

[0133] b) Particle bombardment of sugar beet callus [0134] 1. Friable calli were produced as previously described in the method of 2.1. [0135] 2. An osmotic treatment was carried out for several hours. [0136] 3. Preparation and DNA coating of the gold particles was done by standard procedures, as describe in the PDS-1000/He instruction manual. The plasmids pZFN-nptII-GRF5 and pUbi4-tDT (containing a red fluorescent reporter) were coprecipitated with gold particles. As control, gold particles were coated with pUbi4tDT and pABM70STurboYFP (containing a yellow fluorescent reporter). [0137] 4. Calli were bombarded with a PDS-1000/He unit (Bio-Rad), using 30 ng gold particles coated with 500 ng DNA per shot. [0138] 5. To evaluate the effect of the transient expression of GRF5 in the shoot regeneration frequency, bombarded calli were incubated in MS salts supplemented with 30 g/l sucrose, 1 mg/l GA3 and 1 mg/l Thidiazuron in light conditions for c. 2 weeks. Shoot number was scored by using a standard binocular.

[0139] Results:

[0140] The Agrobacterium tumefaciens-mediated transformation of calli derived from micropropagated shoots of sugar beet with the construct pZFN-nptII-GRF5 increases the number of regenerated shoots at the end of the selection step significantly (Table 2). As control, the construct pZFN-tdT-nptII (Control) containing a red fluorescent reporter has been used. The five independent experiments show an increase of the on average transformation frequency from 5.0% to 33.9%. Further the number of transgenic events confirmed by PCR has been increased on average from 4.8 events to 25.4 events per transformation experiment.

TABLE-US-00002 TABLE 2 Transformation frequency of 5 independent experiments performed by the transformation method of a), either with a control construct or with the pZFN-nptII-GRF5. The total number of shoots at the end of the selection phase and total number of transgenic events confirmed by PCR are also shown. On average number of developing shoots, transgenic events and transformation frequency are indicated in bold. # shoots at the # confirmed Transformation Experiment Name end of selection transgenic lines frequency (%) Control_Rep01 9 3 5.5 Control_Rep02 8 5 8.5 Control_Rep03 11 5 3.3 Control_Rep04 9 4 3.1 Control_Rep05 13 7 4.7 Control average 10 4.8 5.0 GRF5_Rep01 108 23 11.5 GRF5_Rep02 61 28 35.0 GRF5_Rep03 27 13 21.7 GRF5_Rep04 167 35 38.9 GRF5_Rep05 86 28 62.2 GRF5 average 89.8 25.4 33.9

[0141] In the callus transformation experiments the regeneration of shoots has been determined also for each individual selection step. The Quantification of regenerating shoots in each selection step shows that the overexpression of the GRF5 in calli of sugar beet accelerates the shoot organogenesis (Table 3, FIGS. 1 and 2). In another callus transformation experiment the regeneration of shoots has been determined for two recalcitrant sugar beet genotypes (FIG. 8): The transformation with construct pZFN-tdT-nptII (control) results in no transgenic shoot for genotype A and in only on average 0.3 shoots per callus for genotype B. Using AtGRF5 overexpression under the 70S promoter the average number of transgenic shoots is increased from 0 to 1.67 transgenic shoots for genotype A and from 0.3 to 4.82 shoots per callus for genotype B. This demonstrate impressively the potential of AtGRF5 and opens up the possibility to work genotype-independently with standard transformation methods.

[0142] Further experiments demonstrated that the number of transgenic events per inoculated callus could be increased by factor 9 by use of a construct for overexpression of AtGRF5 in Beta vulgaris calli (FIG. 9). As control, the construct pZFN-tdT-nptII (Control) containing a red fluorescent reporter has been used. The GRF gene as well as the reporter gene were under the control of the constitutive 70S promoter (double enhanced constitutive 35S promoter from Cauliflower mosaic virus).

[0143] The expression level of GRF5 has been determined in eleven, randomly chosen independent transgenic events of sugar beet. The expression analysis was performed with primers binding to the 3-UTR of the NOS terminator. In all analyzed transgenic events a high level of GRF5 expression have been detected (see FIG. 4).

TABLE-US-00003 TABLE 3 Quantification of regenerating shoots in each selection step of 5 independent callus transformation experiments done either with pZFN-tdT-nptII (Ctrl) or pZFN-nptII-GRF5 (RB). Total number of developing shoots in each step are indicated in bold. SELECTION STEPS Experiment ID Overexpressed gene S1 S2 S3 S4 Ctrl-1 tdTomato 0 0 3 6 Ctrl-2 tdTomato 0 1 3 4 Ctrl-3 tdTomato 0 0 0 11 Ctrl-4 tdTomato 0 0 4 5 Ctrl-5 tdTomato 0 0 7 6 Ctrl-Total tdTomato 0 1 17 32 RB-1 GRF5 0 40 41 27 RB-2 GRF5 0 11 20 30 RB-3 GRF5 0 9 11 7 RB-4 GRF5 0 65 77 25 RB-5 GRF5 2 27 23 34 RB-Total GRF5 2 152 172 123

[0144] In a further experiment of callus regeneration five different transgenic events overexpressing AtGRF5 in sugar beet have been analyzed. As control, on the one hand two non-transgenic sugar beet lines (WT1 and WT2) and on the other hand a transgenic line overexpressing the tdT reporter protein were used. FIG. 9 A shows that the average callus formation frequency in the control lines and the transgenic AtGRF5 events were almost comparable, perhaps slightly enhanced in the transgenic events. In FIG. 9B the average number of transgenic shoots is presented. Four of the five AtGRF5 events show a significant increase in shoot formation compared the two controls. From FIG. 9 C is becomes apparently that the positive effect on shoot formation is related to the expression level of AtGRF5 in the transgenic events.

[0145] In co-transformation experiments with the control construct 70S-tDT and the 70S-AtGRF5 has been stably integrated in the genome of sugar beet callus cells. The inoculation of callus in the co-transformation experiments was done with a mixture 1:1 of the Agrobacterium strains harboring each construct individually. The average co-transformation frequency was 31.5%. As shown in FIG. 17, the number of red fluorescent (tDT positive) events is around 8 times higher in the co-transformation than in the single transformation experiments.

[0146] The transformation via particle bombardment according to method of 2.2 showed that even the transient (over)expression of GRF5 results in a significant increase of transformation frequency. Bombarded calli show an improved regeneration capability. FIG. 3 shows that calli biolistically transformed with construct pZFN-nptII-GRF5 exhibit an increased number of regenerating shoots compared to calli with the control construct.

[0147] In additional callus transformation experiments the transformation frequency in sugar beet has been determined also using the construct pZFN-nptII-AtGRF5 compared with the same construct but expressing the GRF homolog from Beta vulgaris (BvGRF5). As control, the construct pZFN-tdT-nptII (Control) containing a red fluorescent reporter has been used. The GRF genes as well as the reporter gene were under the control of a constitutive 70S promoter. The experiments shown in FIG. 6 were stopped at an early selection phase. That explains why transformation efficiency of the tDT construct is 0%. For the construct AtGRF5 on average transformation frequency of 70% and for construct BvGRF5 on average transformation frequency of 32.5% could be achieved.

[0148] 2. Oryza sativa Experiments

[0149] Constructs Used in the Binary Plasmid:

[0150] The binary vectors were produced by standard cloning procedures. Within the T-DNA of this vector, the cDNA encoding GRF5 (At3g13960) was cloned between a suitable promoter and terminator ensuring sufficient ectopic expression levels of the GRF5 protein in rice (=single constructs). The T-DNA also contains the GFP gene that was used for the selection of transgenic plant cells and tissues. Additionally, binary vectors were produced which carrying beside the cDNA encoding GRF5 (At3g13960) including the suitable promoter and terminator ensuring sufficient ectopic expression levels of the GRF5 protein in rice and a further gene of interest under the control of a promoter and terminator (=double (stack) constructs).

[0151] Seed Sterilization and Sowing:

[0152] Wild type green seeds have been incubated in 70% ethanol and shaked for approximately 1 minute. After removal of ethanol the seeds have been washed once with sterile mQ water. Then 30 ml of 6% sodium hypochlorite solution has been added and the seeds have been shaked for 40-60 minutes. After removal of the sodium hypochlorite solution seeds have been washed 3 to 5 times with sterile mQ.

[0153] After finishing sterilization (0-3 hours) the seeds have been dried on sterile filter paper and placed onto the surface of the induction medium R001. The incubation took place under continuous light (3000 lux) at 32 C. for 6 days.

[0154] Transformation & Co-Cultivation:

[0155] For explant preparation swollen embryo's (scutellum derived calli) from the wild type seeds suitable for transformation has been selected and transferred to liquid infection medium (R002) containing Agrobacterium tumefaciens transformed with the plasmid being incorporated into the plant cells for 1.5 minutes and then to the cocultivation plates (R003). The plates have been incubated for 3 days at 25 C. in darkness. Selection of resistant tissue

[0156] Selection:

[0157] After 3 days on cocultivation the calli have been removed from the seeds, washed several times with sterile mQ water and once with sterile mQ water containing 250 mg/l cefotaxime and transferred to R004 selection medium. Incubation has been performed under continuous light (3000 lux) at 32 C. for 2 weeks.

[0158] Microcalli Isolation & Regeneration

[0159] By means of sterile forceps the microcalli have been transferred to R005 and incubated under continuous light (3000 lux) at 32 C. for 1 week. Thereafter GFP used as selection marker has been checked in the dark room. Healthy calli positive for GFP have been transfer to R006 and further incubated under continuous light (3000 lux) at 32 C. for 1 week. For continuous regeneration, the calli have been transferred to R007 for 3 weeks under continuous light (3000 lux) at 32 C. With sterile forceps, healthy plantlets have been pulled out and transferred to R008 for 2 weeks under continuous light (3000 lux) at 32 C. before the plantlets have been brought to the greenhouse.

TABLE-US-00004 TABLE 4 Composition of media R001 to R008. final ingredients supplier/stock code concentration 1.00 l R001 (solid) Induction medium N6 salts Sigma - C1416 4.00 g N6 vitamins Duchefa - C0401 1x 1.00 ml L-Proline Sigma -P5607 2878 mg/l 2.88 g CasaminoAcids BD - 223050 300 mg/l 0.30 g Sucrose Duchefa - S0809 30 g/l 30.00 g pH 5.80 Gelrite Duchefa - G1101.5 4 g/l 4.00 g 2,4-D (1 mg/ml) Sigma - D7299 2 mg/l 2000.00 l R002 (liquid) Infection medium (filter sterilized) N6 salts Sigma - C1416 1x 4.00 g N6 vitamins Duchefa - C0401 1x 1.00 ml CasaminoAcids BD - 223050 300 mg/l 0.30 g Sucrose Duchefa - S0809 68.5 g/l 68.50 g D+-Glucose-Monohydrat VWR - 36 g/l 36.00 g MERC1.08342.1000 pH 5.20 acetosyringone (2M) Sigma Aldrich 2478-38-8 100 M 66.00 l preparation of acetosyringone: 2M = 392 mg in 1 ml DMSO dus: 0.04 g in 100 l DMSO R003 (solid) Co cultivation medium N6 salts Sigma - C1416 4.00 g N6 vitamins Duchefa - C0401 1x 1.00 ml CasaminoAcids BD - 223050 300 mg/l 0.30 g Sucrose Duchefa - S0809 30 g/l 30.00 g D+-Glucose-Monohydrat VWR - 10 g/l 10.00 g MERC1.08342.1000 pH 5.20 Gelrite Duchefa - G1101.5 4 g/l 4.00 g acetosyringone (2M) Sigma Aldrich 2478-38-8 100 M 66.00 l 2,4-D (1 mg/ml) Sigma - D7299 2 mg/l 2000.00 l preparation of acetosyringone: 2M = 392 mg in 1 ml DMSO dus: 0.04 g in 100 l DMSO R004 selection medium (LBA nptII) N6 salts Sigma - C1416 4.00 g N6 vitamins Duchefa - C0401 1x 1.00 ml L-Proline Duchefa - P0717 2878 mg/l 2.88 g CasaminoAcids BD - 223050 300 mg/l 0.30 g Sucrose Duchefa - S0809 30 g/l 30.00 g pH 5.80 agarose type 1 Sigma - A6013 7 g/l 7.00 g 2,4-D (1 mg/ml) Sigma - D7299 2 mg/l 2000.00 l Cefotaxime (200 mg/ml) Duchefa -C0111 100 mg/l 500.00 l Vancomycin (100 mg/ml) Duchefa - V0155 100 mg/l 1000.00 l G418 disulfate (100 mg/ml) Sigma - G1279 35 mg/l 350.00 ul R005 Pre-regeneration medium N6 salts Sigma - C1416 4.00 g N6 vitamins Duchefa - C0401 1x 1.00 ml L-Proline Duchefa - P0717 500 mg/l 0.50 g CasaminoAcids BD - 223050 300 mg/l 0.30 g Sucrose Duchefa - S0809 30 g/l 30.00 g pH 5.80 agarose type 1 Sigma - A6013 7 g/l 7.00 g Kinetin (1 mg/ml) Sigma - K0753 2 mg/l 2000.00 l NAA (1 mg/ml) Duchefa - N0903 1 mg/l 1000.00 l ABA Sigma - A1049 5 mg/ml 1000.00 l Cefotaxime (200 mg/ml) Duchefa - C0111 100 mg/l 500.00 l Vancomycin (100 mg/ml) Duchefa - V0155 100 mg/l 1000.00 l G418 disulfate (100 mg/ml) Sigma - G1279 35 mg/l 350.00 l R006 Regeneration medium with 10 g/l agarose MS salts Duchefa - M0221 1x 4.30 g MS vitamins Duchefa - 1000x/M0409 1x 1.00 ml CasaminoAcids BD - 223050 2000 mg/l 2.00 g Sucrose Duchefa - S0809 30 g/l 30.00 g Sorbitol Duchefa S0807 30 g/l 30.00 g pH 5.80 agarose type 1 Sigma - A6013 10 g/l 10.00 g Kinetin (1 mg/ml) Sigma - K0753 2 mg/l 2000.00 l NAA (1 mg/ml) Duchefa - N0903 0.02 mg/l 20.00 l Cefotaxime (200 mg/ml) Duchefa - C0111 100 mg/l 500.00 l Vancomycin (100 mg/ml) Duchefa - V0155 100 mg/l 1000.00 l G418 disulfate (100 mg/ml) Sigma - G1279 20 mg/l 200.00 l R007 Regeneration medium MS salts Duchefa - M0221 1x 4.30 g MS vitamins Duchefa - 1000x/M0409 1x 1.00 ml Sucrose Duchefa - S0809 30 g/l 30.00 g pH 5.80 agarose type 1 Sigma - A6013 7 g/l 7.00 g Cefotaxime (200 mg/ml) Duchefa - C0111 100 mg/l 500.00 l Vancomycin (100 mg/ml) Duchefa - V0155 100 mg/l 1000.00 l G418 disulfate (100 mg/ml) Sigma - G1279 20 mg/l 200.00 l R008 Development medium MS salts Duchefa - M0221 2.151045 g/l 2.15 g B5 vitamins (1000x stock) Duchefa - G0415 0.5x 0.50 ml Sucrose Duchefa - S0809 10 g/l 10.00 g NAA Duchefa - N0903 0.05 mg/l 50.00 l MgCl2.6H2O VWR - MERC1 0.75 g/l 0.75 g pH 5.80 Gelrite Duchefa - G1101.5 2.5 g/l 2.50 g

[0160] Results:

[0161] The Agrobacterium tumefaciens-mediated transformation of calli derived from immature embryos of rice with the different constructs containing GRF5 leads to a significant increase of the on average transformation frequency. As control or as comparison the transformation efficiency observed for 28 randomly selected other constructs without GRF5 has been used that shows a on average transformation efficiency of 63%. For single constructs the transformation efficiency could be increased on average from 63% to 78%, for double construct seven from 63% to 84% (Table 5).

TABLE-US-00005 TABLE 5 Transformation frequency of independent experiments, either with a single construct with GRF5 or with GRF5 in a stack with another gene XY in Rice. In total, 12 different genes in the stack with GRF5 from Arabidopsis thaliana (GRF5, nucleotide sequence as set forth in SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2) have been tested, whereby 3 experiments have been repeated two times (indicated by Rep01 and Rep02). Also, GRF5 from Sorghum bicolor (GRF5-Sorghum, nucleotide sequence as set forth in SEQ ID NO: 17, amino acid sequence of SEQ ID NO: 18) was tested alone and in a stack. On average transformation frequency are indicated in bold. Transformation Single/Stack Gene #1 Gene #2 frequency [%] Single GRF5 72 Single GRF5 62 Single GRF5 73 Single GRF5 88 Single GRF5 93 Average: 78 Double GRF5 Gene XY-01 77 Double GRF5 Gene XY-02 86 Double GRF5 Gene XY-03_Rep01 91 Double GRF5 Gene XY-03_Rep02 92 Double GRF5 Gene XY-04 92 Double GRF5 Gene XY-05_Rep01 75 Double GRF5 Gene XY-05_Rep02 100 Double GRF5 Gene XY-06 79 Double GRF5 Gene XY-07 89 Double GRF5 Gene XY-08 79 Double GRF5 Gene XY-09 81 Double GRF5 Gene XY-10_Rep01 80 Double GRF5 Gene XY-10_Rep02 82 Double GRF5 Gene XY-11 76 Double GRF5 Gene XY-12 77 Average: 84 Single GRF5-Sorghum 67 Average: 67 Double GRF5-Sorghum Gene XY-13 78 Average: 78 Control: Average: 63

[0162] 3. Zea mays Experiments

[0163] Constructs Used in the Binary Plasmid:

[0164] The binary vectors were produced by standard cloning procedures. Within the T-DNA of these vectors, a) the cDNA encoding AtGRF5 (SEQ ID NO: 1), b) the synthetic DNA encoding ZmGRF5 (version A) (SEQ ID NO: 208), and c) the synthetic DNA encoding ZmGRF5 (version B) SEQ ID NO: 210) were cloned between a suitable promoter (e.g. BdEF1 promoter) and terminator ensuring sufficient ectopic expression levels of the GRF5 proteins in corn. As control a construct containing the tDT reporter gene under control of the 70S promoter with the ZmUbi Intron has been used.

[0165] Transformation:

[0166] The Agrobacterium tumefaciens-mediated transformation has been conducted by standard method of transforming monocotyledon by using scutellum of immature embryo (e.g., WO 95/06722).

[0167] Results:

[0168] As shown in FIG. 10 the Agrobacterium tumefaciens-mediated transformation of immature embryos with construct containing the GRF derived from Arabidopsis thaliana leads to a significant increase of the on average transformation frequency, from 8% to 14%. In contrast thereto, the use of the both ZmGRF5 versions derived from different Zea mays genotypes boosted the transformation efficiency much stronger. For version A of ZmGRF5 the efficiency is increased from 8% to 52%, and for version B of ZmGRF5 from 8% to 48%.

[0169] 4. Glycine max (Soybean) Experiments

[0170] Soybean Transformation:

[0171] Glycine max transformation was performed using Agrobacterium rhizogenes for T-DNA delivery into the epicotyl's axillary meristem cells located at the primary-node of soybean seedlings of cultivar Jake (according to Olhoft P. M., Bernal L. M., Grist L. B., Hill D. S., Mankin S. L., Shen Y., Kalogerakis M., Wiley H., Toren E., Song H.-S., Hillebrand H., and Jones T. 2007, A novel Agrobacterium rhizogenes-mediated transformation method soybean [Glycine max (L.) Merrill] using primary-node explants from seedlings, In Vitro Cell. Dev. Biol.-Plant 43:536-549; US 2014237688, WO 2006024509, WO 2005121345).

[0172] Production of Binary Plasmids:

[0173] Three binary plasmids were produced by following standard cloning procedures. The first binary plasmid was the control plasmid used for the experiments (referred to the DsRed control) and is the base vector used for the other vectors. It contains within the T-DNA a DsRed gene that was used for phenotypic scoring of transgenic plant cells and tissues and the AtAHAS gene that was used for preferential selection of transgenic cells. Both genes were cloned with suitable promoter and terminators that ensure sufficient ectopic expression to serve abovementioned purposes. The second binary plasmid contains the base vector with the cDNA encoding AtGRF5 (SEQ ID NO. 2) cloned between a suitable promoter and terminator ensuring sufficient ectopic expression levels of the GRF5 protein in soybean. The third binary plasmid contains the base vector with the cDNA encoding GmGRF5 (SEQ ID NO. 106) cloned between a suitable promoter and terminator ensuring sufficient ectopic expression levels of the GRF5 protein in soybean.

[0174] Seed Sterilization and Germination:

[0175] Soybean seeds of Jake cultivar were sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCl into 100 ml bleach. After sterilization, approximately 65 seeds were plated on solid germination medium (1B5 salts and vitamins, 2% sucrose, 0.8% Noble agar (A5431 Sigma-Aldrich); pH 5.8) in PlantCons. The seedlings were germinated in the light (150 m.sup.2s.sup.2) at 26 C. for 7 days and used as explant material for transformation.

[0176] Agrobacterium Preparation:

[0177] Agrobacterium rhizogenes (WO 2006024509) was transformed with one of the following vectors containing: (1) pSUPER:DsRed and pPcUBI:AHAS selectable marker as a control, or the control vector plus either (2) pPcUbi-AtGRF5 or (3) pPcUBI-GmGRF5. A. rhizogenes was grown and resuspended in 50 ml liquid inoculation medium ( 1/10.sup.th B5 salts (G768 Phytotech), 3% sucrose, 20 mM MES, 1 Gamborg's vitamins, 200 M acetosyringone, 1.44 M gibberellic acid, 5.0 M Kinetin; pH 5.4.) in a Falcon tube to an OD.sub.600 of 1.5. The Agrobacterium suspension was then placed in a deep petri dish for receiving prepared explants.

[0178] Explant Preparation and Transformation:

[0179] Seedling explants were prepared from the 8-day-old seedlings by removing the roots and majority of the hypocotyl, one cotyledon, the axillary tissue growth at cotyledonary node, and the epicotyl above primary-node including all preformed leaves. After preparing the explants, approximately 45-50 explants were incubated with the Agrobacterium suspension in the petri dish for 30 minutes. Explants were then placed in petri dishes on a wet filter paper containing co-cultivation medium ( 1/10.sup.th B5 salts (G768 Phytotech), 3% sucrose, 20 mM MES, 0.5% Noble agar (A5431 Sigma-Aldrich), 1 Gamborg's vitamins, 200 M acetosyringone, 1.44 M gibberellic acid, 5.0 M kinetin, 4.1 mM L-cysteine, 0.5 mM dithiothrietol, 0.5 mM sodium thiosulfate; pH 5.4), and sealed in a container for 5 days at room temperature.

[0180] Shoot Development and Selection:

[0181] After 5 days, explants were transferred to selection medium (1B5 salts and vitamins (G398 Phytotech), 3% sucrose, 3 mM MES, 1 M 6-benzyl-aminopurine, 5 M Kinetin, 250 mg/l Timentin STK, 3 M imazapyr, 0.8% Noble agar (A5431 Sigma-Aldrich); pH 5.6) five per plate and cultivated at 26 C. The explants had significant growth at the primary-node axillary meristem after 16 days on selection and were first scored for shoot development (regeneration). After 22 days on selection (the end of selection), the explants were removed from the solid media and placed on Oasis growing media prior to scoring for (1) quality of shoot formation and (2) DsRed fluorescence on shoots developing at the primary-node.

[0182] Experimental Design and Results:

[0183] One experiment was conducted across four researchers and three constructs (Table 6). Soybean primary-node axillary meristems were transformed with DsRed control, AtGRF5, and GmGRF5. In total, 524 seedling explants were transformed and scored after 16 days on selection (21 days after transformation) and 22 days on selection (27 days after transformation). After 16 days on selection, shoots were rapidly growing at the primary-node and were a combination of small, compact shoot pads and larger, elongating shoots (FIG. 11). Regeneration [(number of explants with shoots)/total explants*100] at the target tissue, the primary-node, was between 80-100% for all constructs, and was fixed at 16 days on selection. Between 16 and 22 days, the shoots on the explants continued to grow rapidly. For both timepoints, the explants were subjectively scored for the presence of healthy, elongated shoot growth with a morphology (good) that is predictive of successful formation of a rooted, transgenic plant (FIG. 12). There was an increase in good shoot formation on explants transformed with all three constructs from the two timepoints, especially for explants transformed with AtGRF5 and GmGRF5. Across the four replicates, the explants transformed with either form of GRF5 tended to have more advanced shoot formation (larger shoots, more elongated) when compared to the DsRed control (FIG. 13).

[0184] To get an early measurement of transformation efficiencies, the explants were scored at 16 and 22 days on selection for the presence or absence of DsRed fluorescing shoots at the primary-node, which is due to transgenic cells expressing the DsRed protein. The DsRed expression was markedly more intense in the constructs containing AtGRF5 or GmGRF5 than the DsRed control at both timepoints; however more so at 16 days on selection (FIG. 11). The explants that were characterized as having good shoot morphology generally had shoots that had DsRed expression (FIG. 11). The percent of explants with DsRed expressing shoots was significantly greater (at =0.05) in explants that were transformed with either AtGRF5 or GmGRF5 than compared to explants transformed with the DsRed control at both timepoints (Table 6; FIG. 14). Of the explants transformed with the DsRed control construct, 54.5% showed DsRed expressing shoots compared to 70.2% and 74.6% for AtGRF5 and GmGRF5, respectively, after 22 days on selection (Table 6). The data indicate that at 27 days after transformation, soybean explants transformed with either AtGRF5 or GmGRF5 have significantly more transgenic shoots regenerating at the primary-node than explants not transformed with either form of GRF5.

TABLE-US-00006 TABLE 6 Regeneration of transformed shoots was significantly increased in explants transformed with either AtGRF5 or GmGRF5 at 16 days and 22 days on selection. The mean and the range in regeneration and DsRed fluorescing shoots are shown. Regeneration (%).sup.1 Explants (%) with DsRed shoots.sup.2 16 d on selection 16 d on selection 22 d on selection Construct n Mean Range (%) Mean Range (%) Mean Range (%) DsRed 162 89.8 a 81.8-95.2 31.4 a 20.5-40.5 54.5 a 50.0-57.1 control AtGRF5 180 88.4 a 82.6-91.3 58.0 b 52.2-66.7 70.2 b 65.2-78.6 GmGRF5 182 94.6 a 85.4-100 53.3 b 45.2-61.9 74.6 b 62.5-86.0 Constructs that were significantly different (at = 0.05) are followed with different letters; .sup.1= [(number of explants with shoots at primary-node/total number {n} of explants inoculated) 100]; .sup.2= [(number of explants with DsRed shoots at primary-node/total number {n} of explants inoculated) 100]

[0185] 5. Brassica napus (Canola) Experiments

[0186] Canola Transformation:

[0187] Brassica napus transformation was performed using Agrobacterium rhizogenes for T-DNA delivery into hypocotyl segments of B. napus seedlings of genotype BNS3.

[0188] Seed Sterilization and Germination:

[0189] Seeds were surface sterilized by placing 200-300 seeds for 2 minutes in 70% ethanol in a 50-mL Falcon tube. After gently shaking for 2 minutes, the ethanol was removed and 40 to 50 ml 30% Clorox bleach with 1 drop Tween was added. The seeds were incubated for 10 minutes with occasional mixing. The liquid was removed, then the seeds rinsed with sterile water three times before placing seeds on germination medium (MS salts/vitamins, 10 g l.sup.1 sucrose, Phyto Agar 7g l.sup.1; pH 5.8) in PlantCon boxes. The boxes were placed in a chamber for 4 to 5 days in the dark at 23 C.

[0190] Agrobacterium Preparation:

[0191] Agrobacterium rhizogenes was transformed with one of the following vectors: (1) pSUPER:DsRed and PcUBI:AHAS selectable marker as a control, or the control vector plus either (2) PcUbi-AtGRF5 or (3) PcUBI-BnGRF5 (SEQ ID NO. 108). Agrobacterium rhizogenes was grown to OD.sub.600 of 1.0 and subsequently diluted to 0.1 with liquid infection medium (1MS salts/vitamins, 30 g l.sup.1 sucrose; pH 5.8). The Agrobacterium suspension was then placed in a dish for receiving prepared hypocotyl explants.

[0192] Explant Preparation and Transformation:

[0193] Hypocotyl explants were prepared from the four to five-day etiolated seedlings by removing from germination box and placing on sterile filter paper wetted with infection medium without Agrobacterium to keep seedlings turgid. Hypocotyl segments 7 to 10 mm in length were prepared by after removing the root, cotyledon and epicotyl. After cutting, the explant was dipped in the Agrobacterium suspension, blotted on dry, sterile filter paper, and finally placed on filter paper on top of co-culture medium (1MS salts/vitamins, 30 g l.sup.1 sucrose, 0.6 g l.sup.1 MES, 18 g I-mannitol, 7 g l.sup.1 Phyto Agar, 1 mg l.sup.1 2,4-D, 100 mg l.sup.1 acetosyringone, 200 mg l.sup.1 L-cysteine; pH 5.6) in plates. Once 50 explants were plated, the plates were sealed with micropore tape and cultivated in the light at 23 C. at a 16 h light/8 h dark photoperiod for 3 days.

[0194] Callus Development and Shoot Initiation:

[0195] After 3 days, all explants were transferred to recovery medium (1MS salts/vitamins, 30 g l.sup.1 sucrose, 0.6 g l.sup.1 MES, 18 g l.sup.1 mannitol, 7 g l.sup.1 Phyto Agar, 1 mg l.sup.1 2,4-D, 300 mg l.sup.1 Timentin; pH 5.6), sealed with micropore tape, and cultivated at 23 C. for 7 days. Explants were transferred to selection medium #1 (1MS salts/vitamins, 30 g l.sup.1 sucrose, 0.5 g l.sup.1 MES, 7 g l.sup.1 Phyto Agar, 3 mg l.sup.1 BAP, 0.1 mg l.sup.1 NAA, 0.1 mg l.sup.1 GA.sub.3, 2.5 mg l.sup.1 AgNO.sub.3, 100 nM imazethapyr, 300 mg l.sup.1 Timentin; pH 5.8) after one week, and cultivated for 14 days at 23 C. under 16 h light/8 h dark photoperiod. After two weeks of selection, the explants were transferred to Selection Medium 2 (1MS salts/vitamins, 30 g l.sup.1 sucrose, 0.5 g l.sup.1 MES, 7 g l.sup.1 Phyto Agar, 0.5 mg l.sup.1 BAP, 0.1 mg l.sup.1 GA.sub.3, 2.5 mg l.sup.1 AgNO.sub.3, 100 nM imazethapyr, 300 mg l.sup.1 Timentin; pH 5.8).

[0196] Experimental Design and Results:

[0197] Five experiments over time were conducted across 3 researchers with a minimum of one replicate per researcher (Table Canola-1). In experiment 1, Brassica napus hypocotyls were transformed with DsRed control and BnGRF5 constructs for experiment 1 only; in the other experiments, explants were transformed with DsRed control, AtGRF5, and BnGRF5 constructs. In total, 3,156 explants were inoculated and scored for DsRed fluorescence after 16 to 22 days. At this point in time, explants were rapidly developing callus, especially on the cut ends of the hypocotyl. The explants were scored for presence or absence of DsRed fluorescence on explants, regardless of size, intensity, and frequency on a single explant. The frequency of DsRed expressing sectors is an early indicator of transformation efficiency for a treatment.

[0198] The DsRed expression was markedly more intense in the constructs containing AtGRF5 or BnGRF5 than the DsRed control (FIG. 15). The percent of explants with DsRed expressing shoots was significantly greater in explants that were transformed with either AtGRF5 or BnGRF5 than compared to explants transformed with the DsRed control (Table 7; FIG. 16). Of the explants transformed with the DsRed control construct, 20.1% showed DsRed expressing shoots compared to 56.6% and 58.5% for AtGRF5 and GmGRF5, respectively. The data indicate that at 21 days after transformation, B. napus explants transformed with either AtGRF5 or BnGRF5 have nearly 3-fold more transgenic callus sectors than explants not transformed with either form of GRF5 (statistically significant =0.05). Significant differences between researchers and experiments was not detected across the treatments (constructs).

TABLE-US-00007 TABLE 7 Transformed callus was significantly increased on explants transformed with either AtGRF5 or BnGRF5 at 21 days after inoculation. The mean and the range in regeneration and DsRed fluorescing callus are shown. Explants Explants (%) with DsRed.sup.1 Construct Exp. Researchers Replicates inoculated Mean Range Control 5 3 20 1202 20.1 a .sup.13-28.7 AtGRF5 4 3 15 822 56.6 b 40.8-70.3 BnGRF5 5 3 20 1132 58.5 b 44.0-78.4 Constructs that were significantly different (at = 0.05) are followed with different letters; .sup.1= [(number of explants with DsRed callus/total number {n} of explants inoculated) 100].