IMPROVING PLANT REGENERATION

Abstract

The present invention relates to the field of plant breeding and in particular to the regeneration of plants from cells and other tissues. More particularly, the invention provides methods and means for improving callus and shoot formation and regeneration of plants using hyperphyllin or derivatives thereof.

Claims

1. A method for inducing callus formation from plant cells, comprising incubating the plant cells in the presence of hyperphyllin or a derivative thereof.

2. The method of to claim 1, wherein the plant cells are incubated in a culture medium comprising hyperphyllin or a derivative thereof and/or wherein hyperphyllin or a derivative thereof is introduced into the plant cells.

3. The method of claim 1, wherein the derivative of hyperphyllin is benzanilide substituted at one or more positions of one or both phenyl rings, in particular at ortho and/or para position(s) with respect to the amide functionality, preferably with halogen, e.g. Cl or F, or amino, e.g —NH.sub.2, and most preferably wherein the derivative is selected from ##STR00003##

4. A method for producing plant shoots, comprising (i) inducing callus formation according to the method of claim 1 to yield callus tissue, and (ii) cultivating the callus tissue under conditions suitable to induce shoot formation.

5. A method for producing a transgenic plant, comprising the following steps: (a) Inducing callus formation according to the method of claim 1 to yield callus tissue, (b) Introducing into a plant cell to be used in step (a) and/or into a cell of the callus tissue obtained in step (a) at least one nucleotide sequence of interest or at least one polypeptide of interest, (c) Cultivating the callus tissue obtained from steps (a) and (b) under conditions suitable to induce shoot formation to yield plant shoots, and (d) Regenerating a transgenic plant.

6. A method of producing a genetically modified plant, comprising the following steps: (a) Inducing callus formation according to the method of claim 1 to yield callus tissue, and (b) Modifying the genome of a plant cell to be used in step (a) and/or of a cell of the callus tissue obtained in step (a) by introducing into said cell a single stranded DNA break (SSB) inducing enzyme or a double stranded DNA break (DSB) inducing enzyme which preferably recognizes a predetermined site in the genome of said cell, and optionally a repair nucleic acid molecule, and/or a base editor fused to a catalytically impaired SSB or DSB inducing enzyme or fused to a SSB inducing enzyme which preferably recognizes a predetermined site in the genome of said cell, wherein the modification of said genome 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., (c) Cultivating the callus tissue obtained from steps (a) and (b) under conditions suitable to induce shoot formation to yield plant shoots, and (d) Regenerating a genetically modified plant.

7. A method of producing a haploid or double haploid plant, comprising the steps (a) Inducing callus formation from an immature male gametophyte or a microspore according to the method of claim 1 to yield callus tissue, (b) Cultivating the callus tissue obtained in step (a) under conditions suitable to induce shoot formation to yield plant shoots, (c) Optionally conducting chromosome doubling, and (d) Regenerating a haploid or double haploid plant.

8. A transgenic plant obtained or obtainable by the method of claim 5 or a progeny plant thereof.

9. A genetically modified plant obtained or obtainable by the method of claim 6, or a progeny plant thereof.

10. A haploid or double haploid plant obtained or obtainable by the method of claim 7 or a progeny plant thereof.

11. A plant cell or a seed of the plant of claim 8, wherein the plant cell or the seed comprises the at least one nucleotide sequence of interest as transgene.

12. A plant cell or a seed of the plant of claim 9, wherein the plant cell or the seed comprises the modification in the genome.

13. A plant cell or a seed of the plant of claim 10, wherein the plant cell or the seed comprises a haploid or double haploid set of chromosomes.

14. A method of using hyperphyllin or a derivative thereof, preferably a derivative of formula A1, A2 or A3, in a method for inducing callus formation from plant cells or in a method for indirect regeneration of a plant.

15. A method of using hyperphyllin or a derivative thereof, preferably a derivative of formula A1, A2 or A3, in a method for production of a transgenic plant cell, plant or seed, in a method for production of a genetically modified plant cell, plant or seed or in a method for production of a haploid or double haploid plant cell, plant or seed.

Description

FIGURES

[0101] FIG. 1: shows chemical formula of Hyperphyllin (HP; N-[4-Amino-2-chlorophenyl]-2,4-dichlorobenzamide; CAS #: 42480-64-8) and three different derivatives thereof A1, A2, and A3.

[0102] FIG. 2: shows maps of binary vectors pZFN-nptII-70s::AtGRF5 (A) and pZFN-nptII-70s::tDT (B).

[0103] FIG. 3: demonstrates promotion of callus induction in recalcitrant genotype A treated with HP in the concentration of 30 μM and 50 μM. upper panel: 30 μM HP dramatically promotes callus induction in recalcitrant genotype A; lower panel: 50 μM HP has the similar effect but no further increased efficiency. Arrows indicate callus formation. DMSO without HP serves as control.

[0104] FIG. 4: shows the callus induction efficiency without (−HP) and with HP (+HP) and demonstrates the boosting effect of HP for the recalcitrant genotype A in two repetitions and additional genotype B.

[0105] FIG. 5: shows that HP (30 μM)-induced calli (+HP) produce much more amount of callus tissue compared to control without HP (−HP).

[0106] FIG. 6: shows the comparison of plantlets regenerated from calli induced without (−HP) and with HP (+HP). HP-mediated induction results in more regenerated plants from callus.

[0107] FIG. 7: shows fluorescence imaging of HP induced calli of amenable genotype C and of recalcitrant genotype A seven days after Agrobacteria infection. Both genotypes have been transformed with 70s::tDT construct (see also FIG. 2B). Bright spots and sectors indicate successful Agrobacterium-mediated transformation.

[0108] FIG. 8: shows that a HP induced and genetically modified calli of a few recalcitrant genotypes like genotype A could be only regenerated to a whole plant by co-transformation with a regeneration booster gene like AtGRF5.

[0109] FIG. 9: shows that HP derivate A2 (50 μM) promotes callus induction in recalcitrant genotype (right column). Left column: control without HP treatment.

EXAMPLES

1. Description of the Binary Plasmids

[0110] The binary vectors (FIG. 2) pZFN-nptII-70s::AtGRF5 and pZFN-nptII-70s::tDT (both constructs are binary) were produced by following standard cloning procedures. As shown in FIG. 2, within the T-DNA of this vector, the cDNA encoding ArGRF5 (DNA: SEQ ID NO: 1; amino acid: SEQ ID NO: 2) and tDTomato (tDT) as fluorescent marker were cloned between the double CaMV 35S promoter and the terminator of nopaline synthase (NOS) gene. 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.

[0111] Both plasmids were transformed into AGL-1 Agrobacterium strain by standard procedure.

2. Sugar Beet Callus Induction

[0112] 1. Micropropagated shoots of various proprietary genotypes were used as starting material: Genotypes A and B are recalcitrant to callus induction and plant regeneration. For example, genotype A does not show sufficient callus induction and an extremely bad callus quality. Therefore, such calli were never transformable. Genotype C is less recalcitrant. Shoots were multiplied in MS salts+30 g/l sucrose and 0.25 mg/l benzyladenine (BAP). [0113] 2. To induce friable callus, leaf explants were isolated from micropropagated shoots and incubated in callus induction medium (CIM) containing MS salts including 15 g/l sucrose and 2 mg/l BAP at 28° C. for 7 to 8 weeks. To the medium either 2 ml/L DMSO as control, 30 μM HP dissolved in 2 ml/L DMSO or 50 μM HP dissolved in 2 ml/L DMSO has been added. HP is Hyperphyllin, a small molecule with the chemical formula N-[4-Amino-2-chlorophenyl]-2,4-dichlorobenzamide (CAS #: 42480-64-8).

[0114] After incubation period the induction and formation of callus has been analysed. FIG. 3 shows that in the recalcitrant genotype A HP in the concentration of 30 μM and 50 μM promotes callus formation. There is no significant difference in induction efficiency between these two concentrations. The quantification of callus induction efficiency with and without HP demonstrates the boosting effect of HP for different recalcitrant genotypes (A and B), even though the increase in efficiency was higher for genotype A (FIG. 4A) than for genotype B (FIG. 4B). After transfer of induced callus on individual plates it has been observed that much more amount of callus tissue could be induced from leaves by application of 30 μM HP compared to control without HP (FIG. 5).

3. Sugar Beet Shoot Induction and Propagation

[0115] 3. Friable calli of step 2 were harvested from the induction and transferred to the shoot induction medium containing MS salts, 30 g/l sucrose, 1 mg/l GA3 and 1 mg/l Thidiazuron (TDZ). The calli were incubated at 24° C. in the light/dark cycle (16 h/8 h) for 1 to 2 weeks. [0116] 4. Regenerated shoots were mounted and cultured in the medium of step 1. The plants are grown at 24° C. in the light/dark cycle (16 h/8 h).

[0117] FIG. 6 shows the comparison of plantlets regenerated from calli induced with and without HP. More plants could be regenerated from HP induced callus. All plants induced with HP showed a normal phenotype.

4. Agrobacteria-Mediated Transformation of Sugar Beet Callus

[0118] a. Sugar beet calli were induced as described in steps 1 and 2. [0119] b. Friable calli were mounted in medium containing MS salts, 30 g/l sucrose, 1 mg/l GA3 and 1 mg/l Thidiazuron (TDZ), and kept for 1 week in the dark, 24° C. [0120] c. Agrobacterium AGL-1 harbouring the vector pZFN-nptII-70s::AtGRF5 and pZFN-nptII-70s::tDT was grown in medium (5 g/l tryptone+2.5 g/l yeast extract+1 g/l NaCl+5 g/l mannitol+0.1 g/l MgSO.sub.4×7H.sub.2O+0.25 g/l KH.sub.2PO.sub.4+1 g/l glutamic acid, pH 7.0) supplemented with the appropriate antibiotics, at 28° C., for 24 h. [0121] d. Calli were inoculated with Agrobacterium suspension (medium: 440 mg/l CaCl.sub.2×2H.sub.2O+170 mg/l KH.sub.2PO.sub.4+1.9 g/l KNO.sub.3+180.7 mg/l MgSO.sub.4+1.65 g/l NH.sub.4NO.sub.3+2 mg/l BAP+40 μg/l Acetosyringone+20 g/l sucrose+2 g/l glucose, pH 6.0) at an OD600 of 0.8. The callus tissue and the Agrobacterium were incubated in medium comprising 440 mg/l CaCl.sub.2×2H.sub.2O, 170 mg/l KH.sub.2PO.sub.4, 1.9 g/l KNO.sub.3, 180.7 mg/l MgSO.sub.4, 1.65 g/l NH.sub.4NO.sub.3, 2 mg/l BAP, 40 μg/l Acetosyringone, 20 g/l sucrose and 2 g/l glucose, at 21° C. for 3 days in the dark. [0122] e. Calli were sub-cultured to medium comprising MS salts, 30 g/l sucrose, 1 mg/l GA3, 1 mg/l TDZ and 500 mg/l Timentin, and incubated in the dark, at 24° C. for 1 week. [0123] f. To select the transgenic calli, samples were transferred to medium containing MS salts, 30 g/l sucrose, 1 mg/l GA3, 1 mg/l TDZ, 500 mg/l Timentin and 100 mg/l Paromomycin, and incubated at 24° C. in the light/dark cycle (16 h/8 h) for 3 weeks. [0124] g. Transgenic calli were selected and sub-cultured for several times in the same medium and conditions. [0125] h. Regenerating shoots were isolated and propagated in medium containing MS salts, 30 g/l sucrose, 0.25 mg/l benzyladenine (BAP) and 100 mg/l kanamycin. [0126] i. Leaf explants were isolated from the green growing shoots for DNA extraction and PCR analysis, in order to confirm the putative transgenic lines. [0127] j. Selected shoots were rooted in medium (MS salts+30 g/l sucrose+6.25 mg/l NAA) and transferred to the green house for seed production.

[0128] FIG. 7 shows that HP induced callus from recalcitrant genotype can be infected by agrobacteria. Nevertheless, the regeneration of transgenic plants from calli of recalcitrant genotypes was not always possible. Transgenic plants could be obtained from HP induced callus often only by additional expression of a regeneration booster gene like AtGRF5 (see PCT/EP2018/086902) (FIG. 8).

[0129] Additionally, different concentrations (10, 30, 50, 100, 500 μM, and 1 mM) of HP has been tested according the above described process. Only 30 and 50 μM showed a strong callus inducing effect. However, 10 and 100 μM caused at least a modest effect, whereby higher concentration showed no effect (Table 1). Further, also HP derivates A1 and A2 have been tested in concentrations of 30 and 50 μM. Derivate A2 led to strong callus-inducing effect at 30 μM (FIG. 9) and only a weak effect at 50 μM. Derivate A1 worked only at a concentration of 30 μM (Table 1).

TABLE-US-00001 TABLE 1 Test of different HP derivates in different concentrations Concentration Callus Chemical (dissolved in induction compound 2 ml/L DMSO) effect HP 10 μM + HP 30 μM ++ HP 50 μM ++ HP 100 μM + HP 500 μM − HP 1 mM − HP derivate A1 30 μM + HP derivate A1 50 μM − HP derivate A2 30 μM ++ HP derivate A2 50 μM + − no callus induction effect; + modest callus induction effect; ++ strong callus induction effect