Regeneration of plants in the presence of histone deacetylase inhibitors

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 formation and regeneration of plants from callus tissue using a histone deacetylase inhibitor.

Claims

1. A method for inducing callus formation from at least one somatic or embryonic Beta vulgaris (B. vulgaris) plant cell, comprising the step of cultivating the at least one B. vulgaris plant cell in the presence of a histone deacetylase inhibitor (HDACi), wherein the step of cultivating the at least one B. vulgaris cell comprises: (i) introducing the HDACi into the at least one B. vulgaris plant cell; (ii) inducing callus formation from the at least one B. vulgaris plant cell of step (i) using callus-inducing medium; and (iii) obtaining a callus having enhanced capability of shoot regeneration, wherein the enhanced capability of shoot regeneration is determined by comparing to the same method for producing callus and the same genotype but without the use of the HDACi, wherein the embryonic B. vulgaris plant cell is a non-haploid cell, and wherein the HDACi is trichostatin A (TSA).

2. The method of claim 1, wherein the at least one B. vulgaris plant cell is a somatic or embryonic cell or is isolated from an explant or a part thereof isolated from a B. vulgaris plant.

3. The method of claim 1, wherein the step of cultivating the at least one B. vulgaris cell comprises: (i) growing the at least one B. vulgaris cell in a medium optionally comprising the HDACi, in a concentration of 0.01 to 5.0 μM, or (ii) introducing the HDACi into the at least one B. vulgaris cell, via bombardment, electroporation or microinjection.

4. A method for regenerating shoots from a callus tissue of B. vulgaris, comprising the following steps: (a) inducing callus formation from the at least one B. vulgaris plant cell according to the method of claim 1, and (b) cultivating the callus tissue obtained in step (a) under conditions promoting the growing of shoots out of the callus tissue.

5. A method for transforming a B. vulgaris plant cell, comprising the following steps: (a) producing callus having enhanced capability of shoot regeneration from the at least one B. vulgaris plant cell according to the method of claim 1, and (b) introducing into a B. vulgaris plant cell to be used in step (a) and/or into a cell of the callus obtained in step (a) at least one nucleotide sequence of interest.

6. A method for producing a transgenic B. vulgaris plant comprising the following steps: (a) transforming a B. vulgaris plant cell according to the method of claim 5, and (b) regenerating a transgenic B. vulgaris plant from the transgenic B. vulgaris cell resulting from step (a) or from a transgenic B. vulgaris cell derived therefrom.

7. A method for modifying the genome of a B. vulgaris plant cell, comprising the following steps: (a) inducing callus formation from the at least one B. vulgaris plant cell according to the method of claim 1, and (b) modifying the genome of a B. vulgaris plant cell to be used in step (a) and/or of a cell of the B. vulgaris tissue obtained in step (a) by introducing into said B. vulgaris cell a site specific effector enzyme which recognizes a predetermined site in the genome of said B. vulgaris cell, and optionally a repair nucleic acid molecule, 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.

8. A method of producing a B. vulgaris genetically modified plant, comprising the following steps: (a) modifying the genome of a B. vulgaris plant cell according to the method of claim 7, and (b) regenerating a B. vulgaris plant from the B. vulgaris cell resulting from step (a) or from a B. vulgaris cell derived therefrom.

9. The method of claim 3, wherein the medium is a callus induction medium.

10. The method of claim 9, wherein the at least one B. vulgaris cell is incubated in a solution comprising the HDACi before being transferred to the medium.

11. The method of claim 9, wherein the medium comprises the HDACi in a concentration of 0.01 to 5.0 μM.

Description

FIGURES

(1) FIG. 1 shows the results of a qualitative analysis of callus induction in media supplemented with 0.5, 1.0 or 5.0 μM TSA. Control induction in medium without TSA is also shown. Ten explants per condition were randomly photographed.

(2) FIG. 2 shows the results of a qualitative analysis of callus induction in media supplemented with 0.01 or 0.1 μM TSA. Control induction in medium without TSA is also shown. Ten explants per condition were randomly photographed.

(3) FIG. 3 shows bar diagrams demonstrating callus induction and plant regeneration using different amounts of TSA.

(4) A: callus induction frequency of leaf explants incubated in medium supplemented with 0.5, 1.0 and 5.0 μM TSA.

(5) B: amount of callus produced under each condition. The amount was estimated based on the number of dishes with harvested calli obtained in each variant.

(6) C: shoot regeneration capacity based on the number of developed shoots per leaf explant used for each experimental condition.

(7) FIG. 4 shows bar diagrams demonstrating callus induction and plant regeneration using different amounts of TSA.

(8) A: callus induction frequency of leaf explants incubated in medium supplemented with 0.01 and 0.1 μM TSA.

(9) B: amount of callus produced in each condition. The amount was estimated based on the number of dishes with harvested calli obtained in each variant.

(10) C: shoot regeneration capacity based on the number of developed shoots per leaf explant used for each experimental condition.

(11) FIG. 5 is a diagram showing quantification of leaf explants with developing friable callus at 3 time points during callus induction. Medium was supplemented with different concentrations of TSA.

(12) FIG. 6 Shoot regeneration of callus induced in medium supplemented with TSA is improved in recalcitrant genotypes. A: Average callus induction frequency of the control genotype (1), and two genotypes with either medium level (2) or high level (3) of shoot regeneration recalcitrance. Callus induction was performed in medium without TSA (white bar) or supplemented with 0.01 μM TSA (grey bar). B: shoot regeneration frequency of callus produced either in control medium (white bar) or in medium supplemented with 0.01 μM TSA (grey bar). Two experiments with 3 replicates per genotype were performed. Notice that the very recalcitrant genotype 3 is able to regenerate shoots only when the calli were produced in medium containing TSA.

EXAMPLES

(13) 1. Technical Description of the Sugar Beet Callus Induction Protocol

(14) This method is based on the publication by Kischenko et al., 2005 Cell Biology International.

(15) 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).

(16) 2. To induce friable callus, leaf explants were isolated from micropropagated shoots and incubated in medium containing MS salts including 15 g/l sucrose and 2 mg/l BAP as a control and in the same medium supplemented with 0.01 μM TSA (B1), 0.1 μM TSA (B2), 0.5 μM TSA (B3), 1.0 μM TSA (B4), and 5.0 μM TSA (B5), at 28° C. in the dark for 7 weeks.

(17) 3. Development of callus from leaf explants was monitored during the incubation in the callus induction medium at 4, 5, 6 and 7 weeks.

(18) 4. Leaf explants producing friable calli were scored in order to calculate the callus induction frequency (percentage of leaf explants that produced friable calli).

(19) An increased callus induction frequency has been observed when TSA is supplemented to the callus induction medium in a concentration range from 0.01 μM to 1.0 μM (FIG. 1, FIG. 2, FIG. 3A and FIG. 4A). The effect depends on the TSA concentration, since higher concentrations of TSA (e.g. 5.0 μM) seems to be cytotoxic. Furthermore, TSA increases the callus amount per leaf explant (FIGS. 3B and 4B).

(20) 2. Technical Description of the Shoot Regeneration Protocol

(21) 1. The friable calli of step 4 were harvested in medium containing MS salts, 30 g/l sucrose, 1 mg/l GA3 and 1 mg/l TDZ, and transferred to separate dishes.

(22) 2. The dishes were incubated under the light (16 h) at 24° C. for 10 days.

(23) 3. Developing shoots were counted under a stereomicroscope, in order to estimate the regeneration capacity (number of shoots per initial leaf explant).

(24) 3. Results

(25) An increased number of regenerated shoots per explant has been observed (FIGS. 3C and 4C). Additionally, TSA accelerates the formation of callus and therefore shorten the time to produce transgenic events (FIG. 5). Already after 28 days a high number of leaf explants with developing calli occurred. Without application of TSA such number has not been reached even after 49 days. Further, first initial tests showed that by adding TSA, genotype-dependent recalcitrance to callus formation could be reduced.

(26) Further experiments show that shoot regeneration of callus induced in medium (CIM) supplemented with TSA is improved in recalcitrant genotypes of Beta vulgaris. Genotypes 1 and 2 represent recalcitrant genotypes of Beta vulgaris from which only a small amount of plants can be regenerated from callus tissue by standard protocols. Genotype 3 is absolute recalcitrant, by known protocols a regeneration is not possible. Callus induction was performed in medium without TSA (white bar) or supplemented with 0.01 μM TSA (grey bar) (FIG. 6A). Shoot regeneration frequency of callus produced either in control medium (white bar) or in medium supplemented with 0.01 μM TSA (grey bar) (FIG. 6B). Two experiments with 3 replicates per genotype were performed. In genotype 1 the addition of TSA results in an increased formation of callus and an improved shoot regeneration capability of such callus: average callus induction frequency is increased from 66.3% to 82%, average number of shoots per explant from 4.7 to 7.7. For genotype 2 no significant increase of callus induction frequency has been observed, however the produced callus was obviously of improved quality, so that shoot regeneration capability was clearly enhanced: average number of shoots per explant is increased from 2.4 to 4.6. For the genotype 3 with the high level of recalcitrance the callus induction frequency was very low without and with TSA, perhaps slightly higher with TSA. Nevertheless, the regeneration of shoot from the produced calli were only possible if the callus has been induced in the presence of TSA.