PHOSPHODIESTERASES INHIBITORS TO PROMOTE IN VITRO PLANT CELL REPROGRAMMING TOWARDS PLANT EMBRYOGENESIS OR MICROCALLUS FORMATION

Abstract

The present invention relates to the use of PDE inhibitors, preferably mammalian PDE inhibitors, to enhance the induction of in vitro plant cell reprogramming towards plant embryogenesis or microcallus formation for further plant regeneration. The present invention also relates to a method to promote this process comprising culturing an isolated plant material with at least one PDE inhibitor, preferably mammalian PDE inhibitor.

Claims

1. Use of at least one phosphodiesterase (PDE) inhibitor to promote in vitro plant cell reprogramming towards plant embryogenesis or microcallus formation.

2. The use according to claim 1, wherein the plant embryogenesis is microspore embryogenesis or somatic embryogenesis.

3. The use according to any of claim 1 or 2, wherein the PDE is a mammalian PDE, preferably wherein the mammalian PDE is selected from the list consisting of: PDE4, PDE7, PDE8, PDE10 and any combination thereof.

4. The use according to any one of claims 1 to 3, wherein the PDE inhibitor is selected from the list consisting of: (i) 3-(2,6-Difluorophenyl)-2-methylthio-4-oxo-3,4-dihydroquinazoline, (ii) (R)N-(3-chlorobenzyl)-1-((2-(2-fluorophenyl)-5-methyloxazol-4-yl)methyl)piperidin-3-carboxamide, (iii) 2-(2-Bromophenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazole, (iv) 3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide, (v) 4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-one and any combination thereof.

5. The use according to any one of claims 1 to 4, wherein the PDE inhibitor concentration is from 0.10 to 100 M.

6. The use according to any one of claims 1 to 5, wherein the plant is a crop plant, preferably Brassica spp., more preferably Brassica napus sp.; wherein the plant is a forest plant, preferably Quercus spp., more preferably Quercus suber sp; or wherein the plant is an herbaceous plant, preferably Arabidopsis spp., more preferably to Arabidopsis thaliana sp.

7. A method of promoting in vitro plant cell reprogramming towards plant embryogenesis or microcallus formation, comprising: a) Culturing an isolated plant material with at least one PDE inhibitor.

8. The method according to claim 7, wherein the plant embryogenesis is microspore embryogenesis or somatic embryogenesis.

9. The method according to any of claim 7 or 8, wherein the PDE is a mammalian PDE, preferably wherein the mammalian PDE is selected from the list consisting of: PDE4, PDE7, PDE8, PDE10 and any combination thereof.

10. The method according to any one of claims 7 to 9, wherein the PDE inhibitor is selected from the list consisting of: (i) 3-(2,6-Difluorophenyl)-2-methylthio-4-oxo-3,4-dihydroquinazoline, (ii) (R)N-(3-chlorobenzyl)-1-((2-(2-fluorophenyl)-5-methyloxazol-4-yl)methyl)piperidin-3-carboxamide, (iii) 2-(2-Bromophenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazole, (iv) 3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide, (v) 4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-one and any combination thereof.

11. The method according to any one of claims 7 to 10 wherein the isolated plant material is cultured in a solid medium or in a liquid medium.

12. The method according to any one of claims 7 to 11, wherein the PDE inhibitor concentration is from 0.10 to 100 M.

13. The method according to claim 12, wherein the PDE inhibitor concentration is from 0.10 to 19 M when the isolated plant material is cultured in a liquid medium.

14. The method according to claim 12, wherein the PDE inhibitor concentration is from 20 to 80 M, preferably 50 M, when the isolated plant material is cultured in a solid medium.

15. The method according to any one of claims 7 to 14, wherein the plant material belongs to a crop plant, preferably Brassica spp., more preferably Brassica napus sp.; wherein the plant material belongs to a forest plant, preferably Quercus spp., more preferably Quercus suber sp; or wherein the plant material belongs to an herbaceous plant, preferably Arabidopsis spp., more preferably to Arabidopsis thaliana sp.

Description

DESCRIPTION OF THE DRAWINGS

[0157] FIG. 1. In vitro microspore embryogenesis of B. napus. (A, B) representative micrographs of toluidine stained sections of isolated vacuolated microspore (culture initiation) (A) and proembryo (first morphological sign of embryogenesis initiation) (B). (C, D) Cotyledonary embryos developed from isolated microspore culture in liquid medium, panoramic view of a region of the culture plate (C) and detail at higher magnification (D).

[0158] FIG. 2. Somatic embryogenesis in Q. suber. (A) Acorn. (B) Immature zygotic embryo, before induction. (C) Embryogenic masses emerging from the explant after induction. (D) Embryogenic mass in proliferation. (E) Embryos at different developmental stages, emerging from embryogenic masses and other embryos, some of them have differentiated fully mature cotyledonary embryos.

[0159] FIG. 3. Proliferating protoplasts from mesophyll tissue of Arabidopsis seedlings. Overlay images from bright field and epiflourescence recordings of protoplasts from an H2B-YFP overexpressing Arabidopsis line, recorded after immobilization for the days indicated. The nuclear localized YFP fluorescence (white signal, arrows) indicates the position and number of nuclei.

[0160] FIG. 4. Effects of small molecules FDA4.21 and FDA4.22 (PDE4 inhibitors) over embryogenesis induction efficiency in B. napus microspore cultures. Columns indicate percent change of proembryos at 4 days and refer to the mean percentage of proembryos in control culturesSEM (standard error of the mean) which has been normalized to 100%. Different letters indicate significant differences between control and treated cultures according to ANOVA and Tukey tests at P<0.05.

[0161] FIG. 5. Effects of small molecule TC2.43 (PDE7 inhibitor) over embryogenesis induction efficiency in B. napus microspore cultures. Columns indicate percent change of proembryos at 4 days and refer to the mean percentage of proembryos in control culturesSEM (standard error of the mean) which has been normalized to 100%. Different letters indicate significant differences between control and treated cultures according to ANOVA and Tukey tests at P<0.05.

[0162] FIG. 6. Effects of small molecule JHD1.48 (PDE8 inhibitor) over embryogenesis induction efficiency in B. napus microspore cultures. Columns indicate percent change of proembryos at 4 days and refer to the mean percentage of proembryos in control culturesSEM (standard error of the mean) which has been normalized to 100%. Different letters indicate significant differences between control and treated cultures according to ANOVA and Tukey tests at P<0.05.

[0163] FIG. 7. Effects of small molecule AGF4.17 (PDE10 inhibitor) over embryogenesis induction efficiency in B. napus microspore cultures. Columns indicate percent change of proembryos at 4 days and refer to the mean percentage of proembryos in control culturesSEM (standard error of the mean) which has been normalized to 100%. Different letters indicate significant differences between control and treated cultures according to ANOVA and Tukey tests at P<0.05.

[0164] FIG. 8. Proembryos in treated and untreated cultures of microspore embryogenesis of B. napus. (A) Culture after 4 days, where proembryos (arrows) coexist with non-responding and dead microspores (smaller structures). (B, C) DAPI staining reveals that proembryos from untreated (B) and treated (C) cultures contain several nuclei (white signals) into the proembryos, indicating embryogenesis initiation.

[0165] FIG. 9. Germination capacity of embryos produced in control and treated microspore cultures of B. napus. Germinating embryos from untreated (A) and treated cultures with the inhibitors TC2.43 (B), JHD1.48, (C), AGF4.17 (0), FDA4.21 (E) and FDA4.22 (F), all of them showing well-developed roots and hypocotyls in most embryos.

[0166] FIG. 10. Effects of PDE8 inhibitor on somatic embryogenesis of Quercus suber. (A) Untreated culture. (B) Culture treated with PDE8 inhibitor JHD1.48.

[0167] FIG. 11. Quantification of the effects of PDE inhibitors over somatic embryo production in Q. suber. (A) PDE7 inhibitor, (B) PDE8 inhibitor, (C) PDE4 inhibitors. Columns indicate mean number of embryos produced after 30 days per gram of embryogenic mass at culture initiationSEM (standard error of the mean), referred to the mean number of embryos in control cultures which has been normalized to 100. Grey columns: control cultures, black columns: cultures treated with the inhibitors.

[0168] FIG. 12. Microwell overview of PDE7 inhibitor-treated mesophyll protoplasts. Exemplary whole microwells of a 96-well plate with microcalli formed from immobilized Arabidopsis mesophyll protoplasts, pulse-treated with 10 M 3-(2,6-Difluorophenyl)-2-methylthio-4-oxo-3,4-dihydroquinazoline (TC2.43) (PDE7 inhibitor, right) or an equivalent volume of DMSO (left) for 48 h. Cells were further incubated in proliferation medium and images were recorded 10 days after immobilization. Microcalli are identified by their YFP labelled nuclei and larger size (arrows).

[0169] FIG. 13. Effects of small molecule TC2.43 (PDE7 inhibitor) on proliferation efficiency in isolated Arabidopsis mesophyll protoplasts. Immobilized protoplasts were pulse-treated with compounds indicated in proliferation medium for 48 h. Columns indicate percent change of microcallus formation after 7 days cultivation and refer to the mean percentage of microcalli in control culturesSEM (standard error of the mean) which has been normalized to 100%. Different numbers of asterisks indicate significant differences between control and samples treated with small molecules according to Student's t-tests with ***, P<0.001, *, P<0.05.

[0170] FIG. 14. Effects of small molecule JHD1.48 (PDE8 inhibitor) on proliferation efficiency in isolated Arabidopsis mesophyll protoplasts. Immobilized protoplasts were pulse-treated with compounds indicated in proliferation medium for 48 h. Columns indicate percent change of microcallus formation after 7 days cultivation and refer to the mean percentage of microcalli in control culturesSEM (standard error of the mean) which has been normalized to 100%. Asterisks indicate significant differences between control and samples treated with small molecules according to Student's t-tests with *, P<0.05.

[0171] FIG. 15. Effects of small molecule AGF4.17 (PDE10 inhibitor) on proliferation efficiency in isolated Arabidopsis mesophyll protoplasts. Immobilized protoplasts were pulse-treated with compounds indicated in proliferation medium for 48 h. Columns indicate percent change of microcallus formation after 7 days cultivation and refer to the mean percentage of microcalli in control culturesSEM (standard error of the mean) which has been normalized to 100%. Asterisks indicate significant differences between control and samples treated with small molecules according to Student's t-tests with ***, P<0.001.

EXAMPLES

1. Methods

1.1. PDE Inhibitors on In Vitro Plant Cultures to Promote Plant Cell Reprogramming Towards Plant Embryogenesis or Microcallus Formation.

[0172] Five small molecules that belong to an in-house chemical library and have shown inhibitory effects over different types of mammalian PDEs were used.

PDE Inhibitors:

[0173] 3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide (FDA4.21) [0174] 4-(3-cyclopentyloxy-4-methoxyphenyl)pyrrolidin-2-one (FDA4.22).

[0175] FDA4.21 (Roflumilast) and FDA4.22 (Rolipram) are commercial inhibitors of mammalian PDE4. [0176] 3-(2,6-Difluorophenyl)-2-methylthio-4-oxo-3,4-dihydroquinazoline (TC2.43). Inhibitor of mammalian PDE7 (Castano et al., ChemMedChem., 4(5):866-76 (2009)). [0177] (R)N-(3-chlorobenzyl)-1-((2-(2-fluorophenyl)-5-methyloxazol-4-yl)methyl)piperidin-3-carboxamide (JHD1.48). Inhibitor of mammalian PDE8 (ES2696516A1). [0178] 2-(2-Bromophenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazole (AGF4.17): Inhibitor of mammalian PDE10 (Garcia et al., Future Med Chem., 9(8):731-748 (2017)).

[0179] The molecular structure of the PDE inhibitors is shown here:

##STR00006##

[0180] First assays were performed with all the inhibitors in Brassica napus microspore cultures in liquid media, where 3 different concentrations (0.5 M, 1.0 M and 2.5 M) were tested for each of inhibitor.

[0181] After evaluation of the effects of the inhibitors on embryogenesis initiation efficiency in B. napus microspore embryogenesis, the compounds were tested in the in vitro embryogenesis system of Q. suber (cork oak) at higher concentration (50 M), because it was developed in solid-gelified media.

[0182] Moreover, some of the inhibitors, particularly TC2.43, JHD1.48 and AGF4.17, were performed at a concentration of 10 M in Arabidopsis thaliana isolated protoplasts to test their capability to increase the microcallus formation.

[0183] The inhibitors were added from culture initiation and their effect on embryogenesis or microcallus formation efficiency was assessed. Several plates of the same cultures were kept without the inhibitors, as controls (untreated cultures, i.e. DMSO solution without any PDE inhibitor).

1.2. Microspore Embryogenesis of Brassica napus, Through Isolated Microspore Culture, without Inhibitors.

[0184] Brassica napus L. (rapeseed) cv. Topas line DH407 plants were used as donor plants. Rapeseed seeds were germinated and grew under controlled conditions (relative humidity 60%, 15 C. under long-day photoperiod 16 h light and 8 h dark at 10 C.) in a growth chamber (Sanyo MLR-351-H) in pots containing a mixture of organic substrate and vermiculite (2/1, v/v). Flower buds containing vacuolated microspores (FIG. 1A), the most responsive stage for microspore induction, were isolated for microspore culture as previously described (Prem et al., 2012 BMC Plant Biology 12, 127).

[0185] The selected buds were surface-sterilized in 5.0% (v/v) commercial bleach (5% active chlorine) for 20 min and then rinsed 6-7 times with sterile distilled water. Ten to 15 buds were crushed using a cold mortar and pestle in 5 mL of cold NLN-13 medium (Lichter, R., 1982, Zeitschrift Fur Pflanzenphysiologie 105(5), 427-434) containing 13% sucrose (w/v). The suspension was filtered through 48 m nylon mesh and the filtrate collected in 15-mL falcon centrifuge tubes. The crushed buds were rinsed with 5 mL NLN-13 to make up the volume to 10 mL and the filtrate was then centrifuged at 1100 rpm for 5 min at 4 C. The pellet was re-suspended in 10 mL of cold NLN-13 and centrifuged as mentioned above. This process was repeated three times for washing of the microspores. The final pellet was suspended in the NLN-13, and the cell density was adjusted to 10,000 cells per mL. The cell suspension was then poured into 90-mm Petri dishes (10 mL per Petri dish) and cultured in darkness.

[0186] For embryogenesis induction, microspore cultures were subjected to an in vitro stress treatment of 32 C. for several days (around 15 days). In response to the inductive treatment, responsive microspores divide and produce multicellular structures or proembryos (FIG. 1B), still confined within the microspore wall (exine). Such structures are considered to be the first sign of embryogenesis initiation; they can be found after 4-6 days in culture. When globular/heart shaped embryos were observed (around 20 days), cultures were shifted to 25 C. on a gyratory shaker at 60 rpm until complete development and maturation of the embryos was observed, normally around 30 days in culture (FIG. 1C).

1.3. Somatic Embryogenesis of Quercus suber, Through Immature Zygotic Embryos Culture, without Inhibitors.

[0187] Immature pollinated acorns were collected from Quercus suber L. (cork oak) trees in the countryside (El Pardo region, Madrid, Spain) during fruit development period (late August and September), transferred to the laboratory and kept at 4 C. for one week before in vitro culture initiation. Immature acorns (FIG. 2A) were selected at the most responsive stage to somatic embryogenesis induction; they are those with small size, around 1 cm diameter; they contain immature zygotic embryos at the early cotyledonary stage. Immature zygotic embryos (FIG. 2B) were carefully excised from the acorns by dissecting the surrounding tissues with the help of scalpel and forceps. After dissection, explants (immature zygotic embryos) were sterilized by immersion in 70% ethanol for 30 s and in 2% sodium hypochlorite for 20 min, followed by three rinses in sterile distilled water of 10 min each.

[0188] Somatic embryogenesis was induced as previously described (Testillano et al. 2018, Plant Cell Culture Protocols, eds. V. M. Loyola-Vargas & N. Ochoa-Alejo. Springer and Bussines Media. pp. 247-256). Explants were first cultured in solid induction medium (Bueno et al., 1992, Physiologia Plantarum 85, 30-34; Manzanera et al., 1993, Silvae Genetics 42, 90-93), which contains Sommer macronutrients, MS micronutrients and vitamins, 0.5 mg/L Glutamine, 30 g/L Sucrose, and 0.5 mg/L Dichlorophenoxyacetic acid (2,4-D), for one month at 25 C. and 16/8 h light/darkness. During this induction period, cell reprogramming occurred in some responsive cells which initiated the embryogenesis pathway, producing small proembryogenic masses that emerge from the surface (FIG. 2C).

[0189] Then, the explants were transferred to solid proliferation medium, with the same composition but growth regulator-free (without 2,4-D). During the next weeks of culture in the proliferation medium, proembryogenic masses (FIG. 2D) proliferated; they produce new embryogenic masses and embryos, which in turn give rise to new embryos, that developed to fully developed cotyledonary embryos, by recurrent and secondary embryogenesis (FIG. 2E).

1.4. Treatment with PDE Inhibitors on Microspore Embryogenesis Cultures of B. napus in Liquid Media

[0190] The compounds were added to the microspore liquid culture media by using stock solutions of 10 mM in dimethyl sulfoxide (DMSO). Appropriate volumes of stock solutions of the compounds were added to the culture media to get the selected working concentrations of the inhibitors, keeping DMSO concentration below 0.2%. Concentrations of 0.5 M, 1.0 M and 2.5 M were tested for each of inhibitor.

1.5. Evaluation of the Effect of PDE Inhibitors Over In Vitro Embryogenesis Induction Efficiency in Isolated Microspore Cultures of B. napus

[0191] Embryogenesis induction efficiency was quantified in control (untreated) and treated-cultures by the number of proembryos formed (considered the first sign of embryogenesis initiation), as previously described (Berenguer et al., 2017, Front Plant Sci 8, 1161). Proembryos were easily identified under inverted microscope in 4 day-culture plates as rounded multicellular structures with higher size and density than microspores, still surrounded by the exine (special microspore wall). Randomly obtained micrographs from inverted microscope were collected from untreated and treated microspore culture plates. Mean percentage of proembryos per plate were obtained from three independent experiments per treatment. A minimum of 1000 proembryos were counted for each treatment. Results on proembryos were expressed as percentages (percent change) and referred to the mean percentage of proembryos in control cultures, which has been normalized to 100%.

[0192] In order to evaluate whether proembryo structures of treated cultures, identified under the inverted microscope for quantification, were actually dividing microspores, similar to the same structures from control cultures, a simply staining technique was performed to visualize nuclei inside proembryos. Samples from control and treated-cultures of 4 days at 32 C., containing proembryos, were stained with 10 g/mL 4,6-diamidine-2-phenyl indole dihydrochloride (DAPI) as previously described (Solis et al., 2008, Plant Science 174(6), 597-605). Squash preparations were analysed under fluorescence microscopy using UV excitation for observing nuclei.

1.6. Evaluation of Quality/Germination Capacity of Embryos Produced after Treatment with PDE Inhibitors

[0193] To evaluate the quality of embryos produced in microspore embryogenesis cultures in the presence of the inhibitors, embryo germination assays were performed. Brassica napus microspore cotyledonary embryos originated from control and treated-cultures were used for in vitro embryo germination and conversion to plantlets as previously described (Prem et al., 2012 BMC Plant Biology 12, 127). The 34-40 old dicotyledonous embryos, after air desiccation on sterile filter paper were germinated in MS (Murashige and Skoog) medium containing sucrose 2% (w/v) and gelled with 7 g/L bacteriological agar (w/v). Microspore derived-embryos were incubated for 15-20 days at 18 C. in darkness conditions till activation of radicle and plumule, and quantified in terms of percentage of embryos showing normal growth, similar to zygotic embryo germination.

1.7. Treatments with PDE Inhibitors on Somatic Embryogenesis Cultures of Q. suber in Solid Media

[0194] Since the in vitro system of Q. suber was two-step process in solid culture media, a different strategy than in liquid microspore cultures was applied for the treatments with the inhibitors. During in vitro embryogenesis of Q. suber, after incubation in induction medium, the transfer of explants to proliferating medium involves the multiplication of proembryogenic masses, embryogenesis initiation, by recurrent and secondary embryogenesis, and embryo development. Therefore, treatments with the inhibitors were performed during the first 15 days at 25 C. in proliferating media, and afterwards, explants with emerging embryos were transferred to fresh proliferating media without the inhibitor.

[0195] Since solid media involve much less diffusion and availability of compounds to cells in comparison with liquid media, as referred in other in vitro systems, the concentration of the inhibitors used in solid media was around 20 higher than in liquid media, particularly 50 M. Appropriate volumes of stock solutions of 10 mM in DMSO of the selected compounds were added to cooled media, before its gelling, keeping DMSO concentration below 0.2%. Mock parallel plates of the same cultures were kept as controls.

1.8. Evaluation of the Effect of PDE Inhibitors Over Somatic Embryogenesis Efficiency in Q. suber

[0196] Embryogenesis induction efficiency was quantified in control and treated-cultures by the number of embryos (torpedo to cotyledonary stage) produced by 15 days of treatment (culture medium containing the inhibitor) followed by 30 days of recovery (culture medium without inhibitor). Embryo production was estimated as the number of embryos originated per gram of embryogenic masses at culture initiation.

1.9. Plant Cultivation and Protoplast Isolation of Arabidopsis

[0197] Seeds of an Arabidopsis thaliana line overexpressing histone 2B fused with yellow fluorescent protein (H2B-YFP) were surface-sterilized and sown on MS agar. After 24 h stratification, plates were cultured vertically for 7 days under long day conditions (16 h light-8 h dark) at 22 C. Prior to seedling harvest, plates were kept vertically in the dark at 4 C. for 24 h. Protoplasts were isolated from hypocotyl and cotyledons including primary leaves according to Dovzhenko et al., Protoplasma, 222(1-2):107-11 (2003).

1.10. Protoplast Immobilization and Image Acquisition

[0198] Protoplast density was determined by cell counting in a Fuchs-Rosenthal chamber. For immobilization, protoplasts were mixed with equal volumes of 1.2% (w/v) low melting agarose in protoplast buffer, prewarmed at 40 C. and immediately pipetted into a 96-well plate (Ibidi, Germany), prewarmed at 34 C. with 125 L per well. The plate was immediately centrifuged (2 min, 30 g) and incubated at 4 C. for 5 min, 200 L of cultivation medium was added on top of each well and the protoplasts were cultivated at 22 C. in the dark. Cultivation medium was composed of macro and micro elements based on Kao K N and Michayluk M R. Planta., 126(2):105-10 (1975), supplemented with 2 M of 2,4-D and 1 M of thidiazurone. For PDE inhibitors treatments, PDE inhibitors were dissolved in DMSO and added to the medium with the concentration given (liquid media). After 48 h and at 22 C., the cultivation medium was removed and replaced with fresh medium after two washing steps performed with cultivation medium.

[0199] An automated microscope (MORE1, Till I.D. GmbH, Munich, Germany) was used for the analysis of protoplast development and for the detection of YFP fluorescence. Transillumination was recorded with 100.45 and 200.8 objectives (Zeiss); epifluorescence was recorded after excitation with single-mode diode lasers (iBeam smart, Toptica) with excitation of 550 nm (YFP)/green emission filter. Image acquisition was performed using the SIAM software (Till I.D. GmbH, Munich, Germany).

1.11. Image Processing and Data Analysis

[0200] Images were recorded immediately after immobilization and 10 days after immobilization (DAIs). Image processing was performed with Fiji an image processing package distributed by ImageJ2 (Schindelin J, et al., Nat Methods, 9(7):676-82 (2012); Rueden, C. T et al., BMC Bioinformatics, 18, 529 (2017)). Raw tile image stacks were reconstituted to full-well images using the stitching plugin (Preibisch et al., Bioinformatics, 25, 1463-1465 (2009)). Image segmentation was performed with U-net (Falk et al., Nat. Methods, 16, 67-70 (2018)). Quantification of proliferating cells was performed by exploiting shape differences of dividing or closely adjacent nuclei after the application of EDM binary operations on epifluorescence images in comparison with circular fluorescence signals from non-dividing mononucleic cells (Brocher Int. J. Image Process, 8, 30-48 (2014)). Corresponding cellular shape parameters from proliferating microcalli were determined from selecting segmented transillumination images after filtering with processed epiflourescence images using the BioVoxxel binary feature extraction function. An exemplary image series of protoplasts from proliferating mesophyll in cultivation medium is shown in FIG. 3.

2. Results

2.1. Effect of PDE Inhibitors Over Microspore Embryogenesis Induction Efficiency in Brassica napus

[0201] The efficiency of embryogenesis induction was evaluated in microspore control cultures and cultures treated with the small molecule inhibitors at different concentrations. The presence of the inhibitors in the culture media affected the production of proembryos (first sign of embryogenesis initiation) in comparison with control cultures, being the proportion of proembryos different depending on the concentration used.

[0202] Inhibitors were used at 3 concentrations: 0.5 M, 1.0 M and 2.5 M. Quantification of proembryos produced, as first sign of embryogenesis initiation, in control and treated cultures showed that the two PDE4 inhibitors tested led to a significant increase of the production of proembryos, at 0.5 and 1.0 depending on the inhibitor, being the increase of embryogenesis initiation in the range of 32-47%, compared to control (FIG. 4).

[0203] Regarding the assays with PDE7, PDE8 and PDE10 inhibitors, the results showed significant increases in microspore embryogenesis induction efficiency in the range of 32-35%, in cultures treated with the three inhibitors. (FIGS. 5, 6 and 7).

[0204] To confirm that proembryos quantified in treated cultures were multicellular microspores that have initiated embryogenesis, squash preparations from control and treated cultures at 4 days were stained with DAPI and observed under fluorescence microscopy. Results showed that proembryos from treated cultures contained several nuclei, as in control cultures, indicating that they were actually dividing microspores that likely initiated embryogenesis (FIG. 8).

[0205] The quality of the embryos produced in microspore cultures treated with the inhibitors was evaluated by germination assays. Fully developed cotyledonary embryos from control and treated 30-40 days cultures were desiccated and cultured under germination conditions. Results showed that embryos from treated cultures germinated very well, producing roots and hypocotyl, similarly and in the same proportion than embryos from control cultures (FIG. 9).

2.2. Effect of PDE Inhibitors Over Somatic Embryogenesis Cultures of Quercus suber

[0206] In order to evaluate the possibility to extend the findings from B. napus to more distant species and processes, the inhibitors of PDE4, PDE7 and PDE8 were applied to a forest woody species Q. suber. PDE inhibitors were applied to the in vitro system of somatic embryogenesis from immature zygotic embryos, a two-step culture in solid media.

[0207] Inhibitor treatments were applied on embryogenic masses at concentration around 20 higher than in liquid media, particularly 50 M, because of the lower diffusion and availability of compounds in gelled medium. The effects of the compounds over embryogenesis efficiency were assessed by the quantification of the embryos produced after 15 days of treatment followed by 30 days in fresh medium.

[0208] Control (untreated) cultures developed a number of somatic embryos (FIG. 10A, white structures), while cultures treated with the PDE8 inhibitor showed higher number of somatic embryos (FIG. 10B).

[0209] Quantification of the number of embryos per gram of embryogenic masses at the beginning of culture showed that treatments with the three types of inhibitors increased embryogenesis induction efficiency and lead to higher embryo production, being the increment of 42-36% for inhibitors of PDE7 and PDE8 (FIGS. 11A, 11B), and around 20% for inhibitors of PDE4 (FIG. 11C).

2.3. Effect of PDE Inhibitors Over Mesophyll Protoplast Microcallus Induction Efficiency in Arabidopsis thaliana

[0210] PDE inhibitors were tested for their capability to increase the proliferation rate of Arabidopsis protoplasts isolated from leaves. For this, immobilized protoplasts were overlaid with cultivation medium supplemented with inhibitors in concentrations from 1 to 10 M for 48 h, inhibitors were removed by washing and cultivation medium was added and cultivation continued. Microscopic images were recorded 10 days after immobilization and proliferating microcalli were identified by the presence of multiple fluorescing nuclei visualized by exploiting the nuclear-encoded H2B-YFP marker (see FIG. 3). As mock control, protoplasts were treated with the equivalent volume of DMSO. This revealed for the small molecule inhibitor of PDE7 a visibly increased number of microcalli formed (see FIG. 12).

[0211] Proliferation rates were quantified and normalized to the number of protoplasts immobilized in each well. This revealed an increased rate of microcalli formation of 140% for 1 M TC2.43 while 10 M TC2.43 increased the rate by 200% (FIG. 13), each relative to the untreated control.

[0212] Similarly, if the small molecule JHD1.48, which inhibits mammalian PDE8 was applied with 10 M concentration to mesophyll protoplasts for 48 h, microcalli formation rate was increased by 50% (FIG. 14).

[0213] Moreover, application of AGF4.17 showed the strongest induction of microcallus formation among the inhibitors tested with 250% compared with the untreated control. (FIG. 15).

3. Conclusions

[0214] The PDE inhibitors described before have demonstrated capacity to increase plant cell reprogramming, embryogenesis induction and embryo production yield in two different species, a crop and a forest plant, as well as plant cell reprogramming and proliferation in protoplast cultures in Arabidopsis. Moreover, treatments with these inhibitors have been successfully applied to different in vitro protocols, in liquid or solid media. These results suggest that the use of these compounds could be extended to promote in vitro plant cell reprogramming a proliferation towards plant embryogenesis, either somatic or microspore embryogenesis, or microcallus formation in a wider range of plant species and in vitro systems, for further plant regeneration.