Direct Somatic Embryogenesis and Plant Regeneration in Peucedanum Oreoselinum (L.) Moench

Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench.

Recepived for publication, January 15, 2011 Accepted, August 14, 2011

A. COSTE, B. OLTEAN, A. HALMAGYI, C. DELIU Institute of Biological Research, 400015 Cluj-Napoca, 48 Republicii St., Romania. E-mail: [email protected]

Abstract Experiments were performed to determine the influence of medium composition and explant characteristics on somatic embryogenesis in Peucedanum oreoselinum (L.) Moench. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from leaf, petiole and root explants cultured on Murashige and Skoog (1962) (MS) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. Somatic embryos were directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and root explants. Embryo formation was significantly affected by explant position. Adaxial-side-up orientation of leaf explants significantly promoted embryogenesis in comparison with abaxial-side-up orientation. The development of proembryos to globular, heart-shaped, torpedo and cotyledonary stages was achieved on the MS medium without plant growth regulators (PGRs) under light conditions. Embryo formation and maturation was further enhanced by addition of proline (5 mM) in MS medium. The frequency of embryo formation was significantly higher for root explants (85%) in comparison with leaf explants (57%) or petiole explants (11%). This protocol facilitated the recovery of 38 plantlets per root explant within 80 days.

Key words: direct somatic embryogenesis, leaf, petiole, root explants, Peucedanum oreoselinum

Introduction

Somatic embryogenesis is a developing process of somatic cells, which are undergoing some biochemical and morphological changes leading to the emergence of well- defined structures similar to those of the zygotic embryo. According to Emons (1994) [1], this process is defined as the development of somatic cells which follows a characteristic histodifferentiation pattern reaching the final stage, that of mature embryo. The somatic embryos may form directly, from the epidermal cells of leaves and hipocotyls, as well as from root and shoot explants, inflorescences, zygotic embryos, protoplasts or even from microspores under the influence of various factors, like the plant growth regulators, elicitors or the pH [2, 3]. Another way for induction of somatic embryos is by an indirect process, via an intermediary callus stage, induced from the same type of explants mentioned above [3]. Peucedanum is one of the numerous species (approximately 3000) that belongs to the Apiaceae family, from which in Romania are grown as spontaneous herbs approximately 120 species. Some of this species are used either as vegetables (Daucus, Petroselinum, Pastinaca etc.), or as medicinal and flavouring (or spices) plants (Anethum graveolens, Angelica archangelica, Carum carvi, Levisticum officinale, Foeniculum sp., Peucedanum sp. etc). Nowadays, some of this species also present a special pharmacological interest, due to their high content in volatile oils, oleoresins, fatty oils, flavons, coumarins and furanocoumarins, rarely even some alkaloids (Conium maculatum) [4]. Among the Peucedanum species, fourteen species could be found in Romania [5], from which only two species are quoted as medicinal plants (P. officinale and P. oreoselinum). Studies regarding Peucedanum species have been done on various aspects. Among the most interesting for practical applications, the 6450 A. COSTE, B. OLTEAN, A. HALMAGYI, C. DELIU following studies can be mentioned: botanical aspects, the content in active pharmacological compounds (furanocoumarins and volatile oils) [6, 7] and the production of liquid extracts with diuretical and antiinflamatory effects from Peucedanii herba [8]. It was shown that both the simple coumarins and furanocoumarins have various biological effects which comprise anticlotting, vessel-distending, oestrogenical, dermal photo-sensitive, antibacterial, antioxidizing, anti-HIV, antihelminthic, hipnotical and sedative, analgesic, hipothermical, cytostatical and antitumoural effects [9]. Peucedanum oreoselinum (L.) Moench is a well-spread species in Romania, rich in a series of valuable compounds such as flavonoids (rutine and quercitrozide), as well as simple coumarins (scopoletin) and furanocoumarins (angelicin, bergapten, psoralen, xanthotoxin, etc). By this reason P. oreoselinum is being frequently used in the treatment of various diseases in the form of liquid extracts with diuretical and antiinflamatory effects [6, 7, 8, 10]. There have been several reports of embryogenesis on Daucus carota, and the carrot has often been used as a model plant for studies on this phenomenon. Studies have also been reported on some other plants belonging to the Apiaceae family, e.g. Angelica sp., Petroselinum crispum, Apium graveolens [11, 12, 13, 14, 15] Levisticum officinale [16], and Carum carvi [13, 17]. If in case of some Apiaceae species such as Daucus carota, Apium graveolens Petroselinum crispum, etc, both the conventional and biotechnological propagation (somatic embryogenesis) raise no problems, in case of the genus Peucedanum some negative aspects occur. Thus, this species is difficult to multiply by vegetative propagation and their seeds lose their viability after a few months from their harvest, therefore being considered recalcitrant seeds. Peucedanum, could be considered recalcitrant even to in vitro culture, this bejng confirmed by the reduced number of published literature regarding aspects like the somatic embryogenesis or in vitro multiplication. Concerning this problems, some experiments have been performed with species that were grown in China and Japan, P. japonicum [18], P. praeruptorum [19] and P. terebinthaceum [20]. In Europe some studies were performed on the species P. palustre aiming to determine the influence of various factors on the frequency of somatic embryogenesis [21, 22]. In all of the above mentioned studies on Peucedanum species, the somatic embryogenesis was induced via callus by indirect embryogenesis. In this study we have described for the first time the induction of direct somatic embryogenesis in P. oreoselinum from leaf, shoot and root explants. Another objective of this study was to investigate the effects of different factors on the induction of somatic embryogenesis. These factors were PGRs, proline concentration, the explant orientation on the surface of the culture media. We also investigated the plant regeneration process from somatic embryos.

Materials and methods

Plant material For the induction of embryogenic cultures in Peucedanum oreoselinum (L.) Moench., seeds collected from a population from Ciucea region (Romania) were used. After the achene removal, the seeds were surface sterilized with 10% H2O2 (v/v) for 20 min, washed three times with sterile distilled water and than transfered for germination on ½ MS [23] medium supplemented with 1.5% (w/v) sucrose and 0.75% (w/v) agar. The pH was adjusted to 5.7 before autoclaving. The seeds were maintained at 25 ± 2oC during a 16h/8h photoperiod with a light intensity of 36 µmol m-2 s1 photosynthetic active radiation (PAR) provided by cool white fluorescent tubes.

Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 6451 Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench.

Induction of somatic embryogenesis Explants (leaf blade, petiole and root) were excised from the plantlets (18 days old) obtained after in vitro germination. Leaf explants (approx. 40 mm2) were cut from the leaf blade (excluding the basal portions), whereas explants derived from the petiole and from the root had 25 mm in length. The leaf explants were placed in abaxial (lower surface facing down) orientation and in adaxial (upper surface facing down) orientation whereas the petiole and root explants were placed horizontally on the medium surface. The medium used was MS with 3% (w/v) sucrose and 0.75% (w/v) agar supplemented with PGRs such as 2,4-D, α- naphthaleneacetic acid (NAA), picloram, 6-benzylaminopurine (BAP), kinetin (K) and thidiazuron (TDZ), and with or without the addition of proline in various concentrations (1.5, 3, 5 and 7 mM). At this stage of the induction of somatic embryogenesis, the cultures were maintained at 25 ± 2°C in total darkness. Somatic embryo proliferation For the proliferation of the globular proembryo structures towards the following developmental stages of the somatic embryos (globular→heart shaped→torpedo shaped→cotyledonary stages), the explants cultivated on MS medium with 2,4-D were transferred on the MS medium without PGRs, and with or without the addition of proline. The cultures were maintained at the temperature and ligth conditions like used for seed germination. For somatic embryos germination and conversion into plantlets, the explants with the mature embryos were cultivated on the same MS medium without PGRs and proline for a 25 days at the same temperature and ligth conditions as mentioned above. Analysis of results The treatments contained at least 10 samples representing three independent replications. The qualitative observations were made periodically (two weeks) using the stereomicroscope. The results regarding the quantitative determinations were expressed as the mean number± standard error (SE).

Results and Discussion

Proembryos formation In order to investigate the effects of culture media composition and the explants position on the induction of somatic embryogenesis, the first experiments were performed using leaf explants. For this reason leaf explants were placed horizontally with both the abaxial and adaxial surface on the MS medium supplemented with different types and combinations of PGRs, in total darkness (Table 1). Stereomicroscopic observations revealed that in most cases after 15 to 20 days of culture (especially in the case of the explants cultivated on the MS medium supplemented with 2,4-D and proline), small white and translucent globular protuberances emerged (become visible) on the surface and at the edge of the root and leaf explants (Fig. 1). Further experiments showed that these protuberances will develop into somatic embryos, after the transfer of embryogenic root and leaf tissues on MS medium without PGRs and under light conditions. This observations lead us to the conclusion that the above mentioned protuberances are the proembryonic structures named in literature as somatic proembryos or globular proembryos [24, 25, 26, 27, 28]. Regarding this aspect it was established that there are a series of morphological, physiological and genetic factors influencing the transformation of somatic cells into somatic embryos. Hormone regimes, especially auxins or in some cases even cytokinins, have been found to be the most important factors affecting somatic embryogenesis. These inductive conditions allow dedifferentiated cells to develop into competent embryogenic cells [27]. During the first so called induction stage, the somatic tissues acquire, directly (without a dedifferentiation step) or indirectly (by dedifferentiating 6452 Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 A. COSTE, B. OLTEAN, A. HALMAGYI, C. DELIU tissues already differentiated, usually involving a callus phase), embryogenic competence. This stage is followed by the expression of somatic embryogenesis, in which the competent cells or proembryos start developing, after receiving the proper stimulus, passing through the phases characteristic of zygotic embryo development, i.e., globular, heart-shaped and torpedo- shaped stages in dicots, globular, scutellar (transition) and coleoptilar stages in monocots, and globular, early cotyledonary and late cotyledonary embryos in conifers. Finally, during maturation, somatic embryos prepare themselves for germination, by desiccating and accumulating reserves [28].

Table 1. Effects of PGRs, proline and leaf explants orientation on direct somatic embryogenesis in P. oreoselinum Abaxial orientation of explants Adaxial orientation of explants % of Plant growth regulators and % of No. of % of No. of % of explants explants other supplements* explants proembryos explants proembryos producing producing responded per explant responded per explant callus callus BA (4.44 μM) 6.3±1.49 0.3±0.07 10 11.4±1.1 2.9±0.3 20 K (4.65 μM) 3.8±1.16 0.1±0.03 6 9.1±0.8 1.2± 0.1 18 TDZ (4.5 μM) 7.3±0.64 0.2±0.06 12 12.3±1.3 5.1±0.6 25 Picloram (4.14 μM) 8.8±0.82 0.4±0.08 9 16.7±0.9 7.1± 0.7 30 2,4-D (4.5 μM) 11.4±2.97 0.5±0.09 51 29.3±2.1 23.4 ± 2.2 46 2,4-D (4.5 μM) + P* (1.5 mM) 15.5±0.19 0.8±0.11 3 43.8±1.9 33.1 ± 2.2 - 2,4-D (4.5 μM) + P* (3 mM) 18.1±0.99 0.9±0,26 4 50.4±2.9 45.4 ±3.5 - 2,4-D (4.5 μM) + P* (5 mM) 27.3±0.71 1.8±0.63 6 68.3±3.9 67.3 ±3.4 - 2,4-D (4.5 μM) + P* (7 mM) 19.2±0.46 0.8±0.33 7 55.2±2.0 55.1 ± 1.6 - *Proline The determinations were achieved after one month of culture maintainance in total darkness Values are means ± SE. Basal medium: MS + sucrose 3% + agar 0.75

A B

100 μm 100 μm

Figure. 1. Explants of P. oreoselinum with somatic proembryos (arrow pointed). A) root explant; B) leaf explant. Explants were grown on MS medium supplemented with 2,4-D (4.5 μM) and proline (5 mM) in darkness.

Another factor highly influencing the induction of direct somatic embryogenesis was the orientation of leaf explants on the culture medium surface, since only explants having an adaxial orientation, formed proembryos without a callus stage. Therefore the percentage of the adaxial oriented explants in which direct somatic embryogenesis occured is significantly higher in comparison with the abaxial oriented explants. The number of the proembryos found on the adaxial oriented explants was also higher compared with the abaxial oriented explants (Table 1). In this context, our results are similar to others using leaf explants of Oncidium [29], Phalaenopsis [26] and Dianthus [30]. An important role in the first stages of somatic embryogenesis was played by the composition of the culture media. Thus, the MS medium supplemented with 2,4-D and proline showed to be the most adequate for the induction of direct somatic embryogenesis. On

Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 6453 Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench. this culture medium leaf explants of P.oreoselinum showed the highest number of proembryos in comparison with other hormonal variants (Table 1). In the case of 2,4-D, the literature data showed that it may be considered an important factor in the induction of the embryogenic competence of plant somatic cells [27, 28]. Regarding the influence of proline in triggering somatic embryogenesis this is covered by incertitude and it was little taken into account. However, some researches confirmed that proline combined only with 2,4-D could represent a conclusive factor in the somatic embryogenesis induction [31]. The supplementation of MS or N6 media with proline, not only increased callus growth but also showed an increase in embryogenic callus formation in Oryza sativa [32]. To determine the effect of explant type on the induction of somatic embryogenesis, in the following experiments we used adaxial oriented leaf explants and horizontally placed petiole and root explants. These explants have been cultivated on the media that proved to be the most efficient for the somatic embryogenesis induction - the MS basal medium with 3% sucrose supplemented with 2,4-D (4.5 μM) and proline (3, 5 and 7 mM) respectively. The cultures were placed in total darkness. After one month, on the surface of the explants, globular proembryos, in different stages of development, were observed. Their number varied depending upon the type of explant and proline concentration. Thus, the highest frequency of somatic embryogenesis was registered for root explants (93 proembryos/explant) cutured on MS medium with 5 mM proline, while the lowest results were registered for petiole explants (18 proembryos/explant) (Table 2).

Table 2. Effects of explant type and culture medium composition on the P. oreoselinum proembryos formation.

Proline % of explant responded No. of proembryos per Medium composition (mM) explant

Leaf* Petiole Root Leaf* Petiole Root 23.4 ± 0 29 9 44 7.1±0.6 44.3±3.1 2.2 MS medium with 3% 45.4 sucrose and 0.75% 3 52 12 78 13.4±0.9 64.7±4.4 ±3.5 agar With 4.5 μM 67.3 2,4-D 5 73 14 89 17.7±1.3 93.4±6.4 ±3.4 (in darkness) 55.1 ± 7 58 10 81 16.2±0.5 74.7±5.9 1.6 *Leaf explant were placed on culture medium in adaxial orientation Values are means ± SE

Developmental stages during somatic embryogenesis The proliferation of somatic proembryos to the following developmental stages (globular, heart-shaped, torpedo shaped and cotiledonary stage), followed the embryogenic explants transfer on the MS medium lacking PGRs, and with, or without, proline addition. This cultures were maintained under a photoperiod of 16/8 hours (light/darkness). For these two treatments, somatic embryogenesis occurred directly on the surface of explants. A noticeable number of somatic embryos were formed (Table 3). Even if some authors have the opinion that the level of the emerged proembryos generally do not represent an adequate indicator of the embryogenic competence [24], our results showed that in the case of P. oreoselinum the total number of somatic embryos is proportional with the number of proembryos formed on media supplemented with 2,4-D and proline. Thus, after explants transfer from the MS medium with 2,4-D on MS medium lacking PGRs, approximately 70 of proembryos (74%) proliferated towards the following developmental stages of the somatic

6454 Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 A. COSTE, B. OLTEAN, A. HALMAGYI, C. DELIU embryos: globular→heart shaped→torpedo shaped→cotyledonary stages (Table 3). At the end of culture period 27% of proembryos developed to the cotyledonary stages (Fig. 2). Table 3. Effects of explant type and culture medium composition on the proliferation of P. oreoselinum proembryos Medium Proline % of explant No. of total embryos (globular +heart composition (mM) responded + torpedo + cotyledonar) per explant Leaf* Petiole Root Leaf* Petiole Root S medium with 3% 0 19 6 32 11.6±0.9 5.3±0.4 33.2±2.2 sucrose and 0.75% 3 39 8 63 34.1±2.9 9.1±0.6 48.5±4.1 agar Withouth 5 57 11 85 50.1±2.1 13.3±0.9 69.6±5.7 PGRs 7 42 7 73 41.2±1.8 11.8±0.5 56.1±3.4 (in 16h/8h photoperiod)

*Leaf explant were placed on culture medium in adaxial orientation Values are means ± SE

10 Somatic embryos (%) embryos Somatic

0 Globular Heart-shaped Torpedo-shaped Cotiledonary Germinated Different developmental stages of somatic embryos

Figure. 2. Ratio of somatic embryos (root explants) at different developmental stages after 22 days of culture on MS medium withouth PGRs supplemented with 5 mM proline (Bars represents SE).

As we mentioned above, an important role in the induction of somatic embryogenesis was played by proline, which strongly emphasized the effect of 2,4-D. The optimum concentration of this aminoacid at which all the P. oreoselinum explant types had the best response, regarding the number of somatic embryos formed per explant, was of 5 mM (Table 3). This kind of effects were also noticed in the Pimpinella anisum cultures were it has been observed that proline and the precursors of proline (ornithine and arginine), in combination with the proline analog, azetidine-2-carboxyiat significantly stimulated 2,4-D-induced embryogenic callus formation and subsequent somatic embryo development in hormone-free −1 medium [33]. In Rosa bourboniana also, the use of L-proline (800 mg l ) was found to be optimum for secondary embryogenesis [34], and in maize L-proline (10-15 mM) was found to be effective in enhancing the frequency of embryogenesis by about 15% over that of controls

Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 6455 Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench. without proline. Higher concentrations (20 and 25mM), however, decreased the embryogenic potentiality of the calli [35]. Regarding the influence of the explant type on the induction and development of somatic embryos it has been observed that all explants are positively reacting at the new medium conditions (MS medium lacking PGRs, and with, or without, proline addition) but only the root explants yielded the highest number of somatic embryos (70 embryos/explant) in comparison with the leaf (50 embryos/explant) and petiole explants (13 embryos/explant) (MS medium with 5 mM proline) (Table 3). Even if many authors consider that the leaf explant is the most adequate tissue for direct somatic embryogenesis induction [26, 29, 30], it has been found that direct somatic embryogenesis could be easily induced also from root explants in Rauvolfia micrantha [36]. Even if in the case of P. oreoselinum cultures the embryogenic response was significantly high for the root explants (85%), our results using leaf explants (57% explants with somatic embryos) being similar to the results obtained by the above mentioned authors. In Oncidium for example, the maximum embryogenic response was 80% [29]. The somatic embryogenesis induction depends on factors such as the genus, species, genotype, basal medium, growth regulators, explant orientation, etc. Thereby, for Arachis hypogaea, cotyledonary sections placed horizontally on the medium produced direct somatic embryos, however sections placed vertically did not [37]. At Epipremnum aureum somatic embryos were produced on MS medium containing 2.0 mg/l kinetin and 0.5 mg/l 2,4- D from leaf and petiole explants, MS medium supplemented with 2.0 mg/l N-(2-chloro-4- pyridyl)-N'-phenylurea and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants [38]. Stereomicroscopic observations showed the presence of green globular somatic embryos at cut edges or on the surface of explants of P. oreoselinum, 10 days after culturing on MS medium without hormones (Fig. 3A). Globular somatic embryos developed rapidly into heart, torpedo and cotiledonary stages on this medium in the light (Fig. 3B). The P.oreoselinum somatic embryos development evolves asynchronous, so that after another 15 days of culture on the same type of medium all the developmental stages of the somatic embryos can be distinguished on the explant surface, with a higher frequency of the torpedo- shaped and cotyledonary stage embryos (Fig. 3C). It can also be noticed the presence of germinated embryos (Fig. 3C). The proportion between different developmental stages of the somatic embryos after 25 days of culture revealed the same aspects that were observed on the microscope (Fig. 3B, C). Thus, the torpedo-shaped and cotyledonary embryos are present in a much higher percentages in comparison with the globular and heart-shaped ones (Fig. 2). Besides these stages, germinated embryos may also be found (11%).

11 mmmm C A 500 μm B

1 mm

Figure. 3. Somatic embryos of P. oreoselinum in different developmental stages regenerated on MS hormone- free medium with 5 mM proline (after 22 days of culture under light conditions). A) Globular and heart-shaped stages on root explant; B) Torpedo-shaped and cotyledonary stages; C) General view. 6456 Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 A. COSTE, B. OLTEAN, A. HALMAGYI, C. DELIU

Plant regeneration from somatic embryos In order to attain the complete conversion of the somatic embryos into plantlets, the explants with somatic embryos were kept further on MS medium lacking PGRs and proline under a 16h/8h photoperiod. In this case the conversion period was of 25 days and the plantlets derived from this embryos were well-developed. The conversion of the embryos in plantlets with well developed shoot and root reached 60% (Table 4 and Fig. 4A). In the case of somatic embryos transfer on MS medium supplemented with BA (4.44 μM) and ANA (0.54 μM), even if the conversion percentage was much higher (85%), in comparison with that attained on hormone-free media, the plantlets were smaller, presenting vitrification signs and almost lacking the root system which was (if present) poorly developed (Table 4 and Fig. 4B).

Table 4. Effect of different media on somatic embryos conversion into plantlets in P. oreoselinum, after 25 days of culture. Total number of Plantlets formation Hyperhydric Media embryos cultured (%) plantlets (%) MS withouth PGRs 69.6 ± 3.4 80.5 ± 4.6 9.2 ± 0.9 MS with BA (4.44 μM) 72.7 ± 4.4 95.6 ± 5.4 71.4 ± 6.1 and ANA (0.54 μM) Values are means ± SE

A B

Figure. 4. Plantlets regenerated from somatic embryos. A) on MS growth regulators-free medium; B) on MS medium supplemented with BAP (4.44 μM) and NAA (0.54 μM).

Conclusions

Two steps were necessary for the induction of somatic embryogenesis in Peucedanum oreoselinum. In the first step the explants excised from different organs (root, petiole and leaf) were cultivated on MS medium supplemented with various combinations of PGRs and proline in different concentrations. Among these, the optimum variant showed to be the one containing 2.4-D (4.5 µM) and proline (5 mM). The explants showing a maximum embryogenic response proved to be the root explants (85%), followed by the leaf explants (57%) (when they had an adaxial orientation on the culture medium surface). In this stage the cultures were kept under darkness. In the second step all the embryogenic explants were subcultivated on the MS medium PGRs free and with or withouth proline addition, and maintained under a photoperiod of 16h light/8h darkness. In this step emerged embryos evolved from the globular to the cotiledonary stage and after 22 days they began germinating. Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 6457 Direct somatic embryogenesis and plant regeneration in Peucedanum oreoselinum (L.) Moench.

In order to obtain the conversion of the embryos into well-configured, healthy plantlets it was necessary to maintain the explants for another 25 days on MS medium withouth PGRs and proline. This variant also proved to be the best type of culture medium for the regeneration of plantlets (60%).

Acknowledgements

This study has been supported by a research project from the Romanian Ministry of Education and Research on the “BIOTECH Programme”.

References

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