Central European Journal of Biology

Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production

* E-mail: mlucchesini@agr.unipi.it

Received 01 August 2009; Accepted 23 November 2009

Abstract: Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L-1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L-1 2,4-dichlorophenoxy acetic acid and 5 mg L-1 kinetin (control medium). Basal medium was also added with 2000 mg L-1 casein hydrolysate (CH) or 900 mg L-1 myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Keywords: Axillary propagation • Callus culture • Asein hydrolysate • Genistin • Isoflavones • Myo-inositol

1Department of Biology of Agricultural Plants, University of Pisa, 56124 Pisa, Italy

2Department of Bio-organic Chemistry and Biopharmacy, University of Pisa, 56100 Pisa, Italy

3Sant’Anna School of Advanced Studies, 56100 Pisa, Italy

Mariella Lucchesini1*, Alessandra Bertoli2, Anna Mensuali-Sodi3, Elisa Cappelli1, Cecilia Noccioli2, Laura Luciardi2, Luisa Pistelli2

Research Article

1. IntroductionCytisus is an important genus belonging to the Fabaceae family (subfamily Faboideae, tribe Genisteae). Cytisus aeolicus Guss. ex Lindl., is an endemic species of the Aeolian Islands (Sicily), where it grows especially in Vulcano, Alicudi and Stromboli isles. It is a shrub or a little tree with dark and hard leaves, yellow flowers and hard seed coats and is a pioneer species with a good ability to colonize landscape compromised by fire, adapted to live in a hard environment. C. aeolicus is considered a rare and endangered plant [1] due to low

genetic variability, limited presence of adult plants in a narrow ecological niche and high human pressure. It is cited in the Convention on the Conservation of European Wildlife and Natural Habitats (http://conventions.coe.int/Treaty/FR/Treaties/Html/104-1.htm) and listed in the first category of protected plants. Papers about the in vitro propagation of this genus are only referred to Cytisus purpureus and C. austriacus [2] or to some Genista spp. a genus strictly related to Cytisus [3-6]. C. aeolicus plants are rich in flavonoids and other secondary metabolites, for example the alkaloid lupine [7]. Isoflavones are well known for their therapeutic properties such as radical

Cent. Eur. J. Biol. DOI: 10.2478/s11535-009-0067-4

Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production

scavenging, anti-inflammatory and antibiotic activities and exhibit estrogenic, anti-estrogenic and anticancer properties [8]. Taking into account the pharmacological value of this class of compounds, callus culture of several species belonging to Leguminosae family [6,9-12] were established to obtain isoflavone mixtures. Till now, no reports are available on bioactive compounds production from cell cultures of C. aeolicus Guss. ex Lindl. The present paper outlines the establishment of C. aeolicus tissue cultures and experiments focused on the establishment of callus cultures and on the evaluation of cell ability to provide useful secondary metabolites. Moreover, the aptitude of this species to be in vitro cultured could be the first basis to develop a preservation program of C. aeolicus.

2. Experimental Procedures2.1 Plant material Legumes of C. aeolicus Guss. ex Lindl. were collected at Vulcano (Aeolian Islands, Sicily) in April 1999 by Dr A. Bader with the licence of Ispettorato Dipartimentale delle Foreste of Messina and Catania (Italy). A specimen is stored in the “Erbario Siciliano Storico- Erbario TAL” n° 286 at the Department of Botany, University of Palermo (Italy).

2.2 Growth conditionsAll the cultures were incubated in a growth chamber at 22±1°C under a 16 h photoperiod at 70 µM m-2 s-1.

2.3 Seed germination C. aeolicus seeds collected from mature fruits, were stored in the Department of Biology of Agricultural Plants, University of Pisa, and used for this work after a pre-treatment with an aqueous solution of 3% (v/v) PPMTM

(Plant Preservative Mixture, Plant Cell Technology Inc., U.S.A.) with 50 mg L-1 MgSO4 for three hours. To break dormancy, two different heat treatments were performed before the sterilization. The first entailed seeds being maintained in the PPM solution and heated at 90°C or 100°C for 10 minutes. In the second treatment the seeds, after the wash with PPM, were dried in an oven at 90° for 10 min. Control seed did not have any heat treatments to break dormancy. Successively all the seeds were sterilized with 15% of sodium hypochlorite (8% chlorine active) for 15 min, followed by three rinses in sterile distilled water. A basal medium composed of mineral LS (Linsmaier and Skoog) salts [13] added with B5 vitamins [14] 300 mg L-1 reduced Glutathione (GSH), 500 mg L-1 2-(N-morpholino)ethanesulfonic acid (MES), sucrose

30 g L-1 and Difco Bacto agar (8 g L-1) was employed in order to germinate seeds. The pH was adjusted to 5.8 before adding agar and autoclaving. Seeds were germinated in 60 mm of diameter Petri dishes containing 5 ml of agarized medium.

2.4 Axillary shoot inductionSeedlings deprived of roots and with the first two leaves developed, were placed (one explant/vial) in polystyrene vials (30 ml) containing the basal medium with 0.1 mg L-1

6-benzylaminopurine (BA) and gibberellic acid (GA3). To evaluate shoot production and growth of the cultures, one node shoots (0.5 cm) was inoculated for twenty days on the basal medium added with 0.1 mg L-1 BA plus 0.1 mg L-1 GA3 and the basal medium with 0.1 mg L-1 BA.

2.5 Callus induction and callus growth curvesTissue portions of approximately 0.5 cm length were excised from cotyledons and hypocotyls and placed in Petri dishes with three different media: the basal medium added with 5 mg L-1 2,4 D and 5 mg L-1 kinetin (control medium), control medium added with 900 mg L-1

myo-inositol (MI medium) and control medium added with 2000 mg L-1 casein hydrolysed (CH medium). Calli obtained from both type of explants were subcultured (every three weeks for more than two months) in CH and MI media before evaluating their growth rate over time. Callus samples were weighted at regular intervals till the 21st day of culture.

2.6 Growth measurementsThe effect of treatments on the seed germination (5 seeds/dish, 10 dishes for each treatment) was monitored over time (until the 41st day) and described through the analysis of two parameters: the percentage germination and the germination rate expressed through T50 parameter, which can be defined as the time required for germination of half final germinated seeds [15-16]. As regards shoot development, the number of explants which differentiated new shoots and their length (mean values ± SE n=21) were recorded at the 10th and 20th day of culture on both proliferation media with and without GA3. The callus induction was evaluated as percentage of explants which produced undifferentiated tissue (5 explant/Petri dish, 10 dishes per treatment) during three weeks of culture (one record a week). The callus growth curves were calculated from the normalized fresh and dry weights of the proliferating callus cultures, both from cotyledon and hypocotyls explants, cultured either on MI or CH media. For each sample time, 6 explants from two different Petri dishes were weighted. Curves associated to the callus growth over time were

M. Lucchesini et al.

fitted by non-linear regression curves and assessed by F-test. Non-linear regressions and the data fitting were performed using GraphPad Prism version 4.00 for Windows [17]. The effect of the two different origins of the explants, or the influence of the two medium components (CH and MI), were globally compared and the two data sets fitted to the same model. Data on germination percentage, callus formation percentages and secondary metabolite contents were subjected to analysis of variance, and differences among means were evaluated using the Tukey test (P≤0.05). Percentage values were subjected to arcsine transformation before analysis. Data on shoot proliferation were analysed by Student’s test procedure (P≤0.05). All the experiments were twice repeated to confirm results observed in the first experiment.

2.7 Metabolites extraction and (LC-ESI-MS, HPLC-DAD) analysis

Fresh callus samples of C. aeolicus, were collected during the culture phase (10th and 20th day), freeze-dried, weighted and then the metabolites were extracted by maceration with chloroform and methanol (100 ml x 8 h, 3 times) in turn. The extracts were evaporated by rotavapor at 40°C and stocked in the freezer at -20°C before the HPLC analysis. The reference compounds

(1-10), used in the quantitative analysis as external standard (Table 1), were isolated in our laboratory after chromatographic purifications of the chloroformic and methanolic residues, obtained by extraction in a Soxhlet apparatus of dried and powdered aerial parts of C. aeolicus adult plant. The isolates were identified by NMR, UV and MS spectra [7] and used as reference material with an HPLC purity of 98-99%. Genistein 7-O-glucoside (11) was isolated from Genista morisii Colla according the separative procedure previously described by [18]. The phytochemical screening on the different extracts obtained from both adult plants and in vitro calli were carried out by LC-DAD-ESI-MS. HPLC system consisted of a Surveyor Thermofinnigan liquid chromatography pump equipped with an analytical Lichrosorb RP-18 column (250 x 4.6 mm i.d., 5 µm, Merck), a Thermofinnigan Photodiode Array Detector and a LCQ Advantage mass detector. The callus extracts were dissolved in methanol (2 mg/ml), filtered by PTFE (0.45 µm, 25 mm) luer-lock filter and injected onto the HPLC column (injection volume 20 µl, triplicate) in the same condition as each standard sample previously detected (Table 1). The HPLC analysis was performed by Waters 600E pump and a detector PDA (Photodiode Array Detector Waters 996) using the software Millennium 32® (Waters). Each sample (20 µl) was injected onto a LiChrospher RP-18 Merk column (250 x 4.6 mm i.d., 5 µm, Merck). The analysis was carried out by a linear gradient using CH3CN with 0.5% HCOOH (solvent A) and H2O with 0.5% HCOOH (solvent B) at flow rate of 1.0 ml/min. The following separate program was used: 15:85 v/v (A:B) at 0 min, 30:70 (A:B) at 25 min, 30:70 (A:B) at 40 min and 15:85 v/v (A:B) at 65 min. The analyses were detected in the 210-500 nm range and all chromatograms were registered at 260 nm.

Compound Rt UV (nm)

1 scopoletin 13.0 225, 299, 3412 genistein 35.3 228, 260 sh

3 pratensein 36.6 228, 259

4 wighteone 18.5 226, 264, 299 sh

5 lupiwighteone 35.7 226, 258

6 omopherreirine 28.2 228, 321 sh

7 eriodictiole 26.1 228, 288 sh

8 eriodictiolo 7-O-glucoside 13.4 226, 268

9 quercetin 29.0 228, 370

10 taxifolin 14.3 226, 285 sh

11 genistein 7-O-glucoside 15.9 228, 258, 327 sh

Table 1. Retention times (Rt) and λ maximum of the spectrum recorded in the UV region of the analyzed constituents from C. aeolicus plants.

Seed treatment 14th day 19th day 25th day 32nd day 41st day T50

control 0.00 b 16.17 ab 22.79 bc 24.10 b 26.56 b 19.4 a90°C (water) 29.67 a 34.10 ab 47.42 a 49.35 a 60.25 a 18.6 a

100°C (water) 18.54 a 39.35 a 41.45 ab 47.22 a 68.62 a 20.0 a

90°C dry heat 0.00 b 13.52 b 15.34 c 25.84 b 31.31 b 21.7 a

Table 2. Germination percentages and germination rate (from the 14th day to the 41st day) of C. aeolicus seeds exposed to different pre-germinative treatments. T50 defines the time required for germination of half final germinated seeds. ANOVA was performed within each sample date and differences were tested by Tukey (P≤0.05). Different letters indicate significant differences. Percentage values were subjected to arcsine transformation before statistical analysis.

Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production

3. Results3.1 Seed germination Table 2 reported germination progress from the 14th day of culture. Control seeds and seeds treated at 90°C in an oven showed a low percentage of germination (Table 2) while the treatments conducted in hot water demonstrated a marked efficacy in breaking seed dormancy making possible to double the number of germinated seeds in respect to the other treatments. Nevertheless, the speed in which germination occurred showed similar values for all the treatments (T50).

3.2 Axillary shoot induction C. aeolicus seedlings, placed in the basal medium containing BA and GA3 led to axillary shoot proliferation. However, the proliferation rate and the mean length of the newly formed shoots were very low (Figure 2) and the developed shoots showed early symptoms of hyperhydricity. The exclusion of GA3 from the medium, reduced this phenomenon giving a good quality shoots (Figure 1b) and significantly increased the number of newly developed shoots per explant and their mean length (Figure 2). At the end of multiplication phase, the in vitro shoots on BA medium developed adventitious roots (1-2 roots per explant) and could be easily acclimatized (data not shown).

Figure 1. A C. aeolicus germinated seed (A); C. aeolicus proliferating shoot on basal medium with BA (0.1 mg L-1) (B); C. aeolicus calli growing on CH medium during the growth curve experiment (C); C. aeolicus calli growing on MI medium during the growth curve experiment (D).

Figure 2. C. aeolicus axillary buds induction on basal medium with BA (0.1 mg L-1) plus GA3 (0.1 mg L-1) and basal medium with BA (0.1 mg L-1). In the figure are showed: mean values (± SE n=21) of the number and respective length (cm) of new formed shoots at the 10th and 20th day of culture. Data were analysed by Student’s test procedure (*** P≤0.0001) within each sample date.

M. Lucchesini et al.

3.3 Callus induction and growthThe cotyledon and hypocotyl explants, arising from in vitro germinated seeds and cultured on control medium, were enlarged due to absorbing water but did not produce a useful callus amount. The explants, showed cell proliferation capacity until the first week of culture on the MI and CH media (Figure 3). About 50% of the C. aeolicus explants showed a callus proliferation at the cutting surfaces in the second week of culture. In the third week, a significantly greater percentage of the cotyledon and hypocotyls explants produced callus on the medium containing casein hydrolysate (Figure 3). The calli formed from cotyledons were green with friable texture (Figure 1c); similar good features were maintained in calli arising from hypocotyls until the second week of subculture. At the end of the third week

(Figure 1d) these calli turned brown, so they had to be transferred to the fresh medium. Continuous callus growth was observed on both media for over two years maintaining the different features between the two type of callus. The growth of cotyledon and hypocotyl calli on CH and MI media was determined and the related curves were calculated. This was achieved by fitting the data sets of cotyledon and hypocotyls explants with a Boltzmann sigmoid equation typical of the cell growth:

except for the curves related to callus fresh weight growing on the MI medium which fitted with an exponential curve:

In the equations W(i) is the fresh or dry weight at the time, t, of the culture, the callus growth varies from the initial weight, w0, and the maximum weight, wmax., the time at which the weight is halfway between its start and end point is denoted as t50 and K is the slope of the curve. Non linear regression analysis provided the equations which gave the best description of the callus growth on CH and MI medium (Figure 4 and Table 3). The F ratio to perform the comparison between all the curve parameters (wmax, t50 and K) demonstrated that the two data sets regarding cotyledon and hypocotyl calli fitted separately (not share) with significant differences both for the dry and fresh weight curves. The F ratio was also calculated to evaluate the overall effect of the MI and CH medium on the increase in fresh and dry weight regardless of the callus origins. The comparison between MI and CH media demonstrated that the parameters which characterized the curves related to the dry weight accumulation differed significantly (F test=35.09; P≤0.0001) all along the culture period. In particular, callus on MI medium showed a slow growth rate (t50 17.44 and K 4.34) different from that on the CH medium (t50 8.45 and K 2.86). No differences were observed concerning the fresh weight increases.

3.4 Phytochemical analysis Different phytochemicals isolated from C. aeolicus adult plant, identified by spectroscopic methods in a previous work [7], were used as external standards for the quali-quantitative evaluation of the secondary metabolite content of C. aeolicus in vitro cultures. Only one constituent was present in the analysed C. aeolicus calli: genistein 7-O-β−D-glucopyranoside, also called genistin (Figure 5).

Figure 3. Percentages of callus formation from cotyledon and hypocotyls on culture media with myo-inositol (MI) or casein hydrolysate (CH) during the first, second and third week of culture. Data are showed as mean values (±SE n=10). ANOVA was performed within each sample date and differences were tested by Tukey (P≤0.05). Different letters indicate significant differences. Percentage values were subjected to arcsine transformation before statistical analysis.

w(i) = w0 · ekt

W =1+ (w - w )

1+e(i)

max 0t -t

K50

Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production

Casein hydrolysate (CH)

W =1+ (w - w )

1+e(i)

max 0t -t

K50

myo-inositol (MI)

w(i) = w0 · ekt

Casein hydrolysate (CH)

W =1+ (w - w )

1+e(i)

max 0t -t

K50

myo-inositol (MI)

W =1+ (w - w )

1+e(i)

max 0t -t

K50

Cotyledon FW (mg)

Hypocotyl FW (mg)

Cotyledon FW (mg)

Hypocotyl FW (mg)

Cotyledon DW (mg)

Hypocotyl DW (mg)

Cotyledon DW (mg)

Hypocotyl DW (mg)

Wo 100 100 100 100 1 1 1 1

Wmax 360.5 700.2 - - 5.24 4.14 4.19 2.39

t50 12.62 25.67 12.85 20.34 9.01 7.26 17.52 16.39

K 2.18 6.20 0.054 0.034 2.43 2.95 5.10 2.67

R2 0.990 0.973 0.995 0.900 0.993 0.983 0.993 0.999

F ratio 29.78 (P≤0.0005) 62.58 (P≤0.0001) 11.82 (P≤0.0063) 84.71(P≤0.0001)

Table 3. Curve parameters which characterize the non linear regression fitted with the fresh weight and dry weight callus growth of C. aeolicus. Inside each type of medium, CH and MI, callus growth from cotyledon and hypocotyl origin was compared. The initial (w0) and the maximum fresh weight (wmax), the time at which the weight is halfway between its start and end point (t50) and the slope of the curve (K) were the curve parameters. The F test (P≤0.05) was performed, within each type of culture medium, to compare the growth curve parameters of calli derived from cotyledon and hypocotyl explants.

Figure 4. C. aeolicus callus (from hypocotyl and cotyledon explants) growth curves relative to fresh and dry weight (mg) on the basal medium with 5 mg L-1 2,4 D and 5 mg L-1 kinetin enriched with casein hydrolysed (CH) and myo-inositol medium (MI). Curves related to callus fresh weight on MI medium fit with exponential growth W(i) = W0 · ekt. Curves related to callus fresh weight on CH medium and those associated with the callus dry weight on CH and MI media fit with the Boltzmann sigmoid equation:

W =1+ (w - w )

1+e(i)

max 0t -t

K50

M. Lucchesini et al.

The quantitative analysis of genistin was performed by HPLC-DAD analysis using the external method with five concentration levels of this standard (10- 20- 50- 100- 200 μg/ml) diluted in methanol, according with the regression equation y=3E+0.7x+124494 (R2=0.9977). Genistin was produced in all the analysed in vitro callus samples and the genistin content of the analysed samples is showed in Table 4. Genistin was produced as early as the 10th day of culture in samples which demonstrated higher values for calli growth on MI medium regardless the type of explant. Also on the 20th day of culture, calli grown on the MI medium showed genistin content higher

than calli cultured on CH medium. The highest genistin content was seen in calli of cotyledon explants on MI medium.

4. Discussion C. aeolicus is a wild species growing in a narrow ecological area (Aeolian Islands; Italy) where it is considered at danger of extinction and thus was included in the LIFE99 NAT/IT/006217 EOLIFE99 project as one of the five main endemic plant species to be protected. In this work a method for C. aeolicus multiplication was described. In vitro cultures of C. aeolicus can be adopted as a feasible method for ex situ germplasm conservation through long term storage of in vitro developed shoots or acclimatization of rooted plantlets for the reinforcement of the native population. Moreover, since Cytisus plants are rich in several flavonoidic compounds, among which isoflavones (very important natural compounds for their biological and pharmacological properties), callus cultures were established in order to propose a biotechnological production of these substances.

Figure 5. HPLC profile and UV spectrum of the main peak of the methanolic extracts obtained from C. aeolicus in vitro callus (A) in comparison with genistin standard reference (B).

0.00

0.02

0.04

0.06

0.08

Minutes20.00 40.00 60.00

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

227.6

258.3

330.8

B

AU

0.00

0.05

0.10

0.15

0.20

Minutes20.00 40.00 60.00

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00

226.4

258.3

324.8

AA

U AU

AU

Culture daysCH MIcotyledon hypocotyl cotyledon hypocotyl

10th 10296.80 a 6977.78 a 21150.44 b 14683.54 b

20th 10137.93 a 12266.67 a 34158.42 c 26886.79 b

Table 4. Genistin yields (µg/100 g DW in vitro callus) during two different phases of the in vitro callus culture of C. aeolicus (10th and 20th day of culture) obtained from cotyledon and hypocotyl explants. Callus was cultured on the basal medium with 5 mg L-1 2,4 D and 5 mg L-1 kinetin enriched with casein hydrolysed (CH) or myo-inositol (MI). ANOVA was performed within each sample date and differences were tested by Tukey (P≤0.05). Different letters indicate significant differences.

Cytisus aeolicus Guss. ex Lindl. in vitro cultures and genistin production

For the establishment of an in vitro culture it was necessary to develop an efficient method for aseptic germination of C. aeolicus seeds. Sterile plant material (up to 100%) was obtained using sodium hypochlorite and PPM, without affecting the vitality of the seeds [19]. Leguminosae plants, highly represented in the Mediterranean flora, are characterized by waterproof and extremely hard teguments in their seeds; many of this recalcitrant species showed a maximum germination percentage of 25%, therefore they need to be scarified to facilitate the germination process [20-21]. High temperatures as pre-treatment to break seed dormancy has been used for seeds of several plants typical of the Mediterranean areas subjected to the environmental pressures of routine fire [21]. Some Authors in fact found a beneficial effect of boiling water treatments on seed germination percentage with a great variability of responses among the Cytisus genus varying from values between 80% and 25%. In the present work heat and water (90° and 100°C water) were utilized as synergistic factors to improve the number of germinated C. aeolicus seeds which reached the 68% as the maximum mean value. The effectiveness of this treatment mainly lay in the time required for breaking dormancy, not in the speediness of the germination process as demonstrated by the T50 values. This treatment is a type of scarification that could prime the germination process simulating the high temperature effect typical of the Aeolian Island climate [21]. The procedure to propagate axillary shoots from in vitro seedlings allowed the in vitro multiplication of C. aeolicus plantlets. The presence of the gibberellic acid (GA3) in the induction phase of the culture, often used for other Cytisus species [22], negatively affected the successive multiplication stage and the quality of the culture which resulted damaged by hyperhydricity [23]. The GA3 removal from the medium gave a good multiplication rate and quality of the propagated shoots. This has also been observed in plants belonging to the Fabaceae family [24-25] cultured on medium with BA as unique growth regulator. Cytisus plants are rich in isoflavones, which are very important natural compounds for their biological and pharmacological properties. Due to this callus cultures were established to obtain an additional source of these substances. Cell culture on solid medium is the first step to develop a liquid culture as a profitable source for scaling up secondary metabolite production [12]. The origin and nature of the explant influence callus formation and its morphogenetic ability in many species [26]. As regard to the C. aeolicus callus biomass production, the type of explant was a critical factor to obtain the greatest callus production. Even if both cotyledon and hypocotyl

showed a good initial callus induction, C. aelicus calli from cotyledons maintained a good quality during the successive subcultures demonstrating that cotyledon was the best explants. This is in agreement with another literature report on Genista species, which also shows that that cotyledon was the best explants for callus production [12]. Another critical factor was the organic components of the culture medium. As observed for other species of Genista species, plants belonging to Cytisus genus were difficult to start callus cultures on conventional media without additional organic components [12]. Addition of myo-inositol and casein hydrolysate to the cultures was necessary to induce C. aeolicus callus formation. These results were verified by other literature data which reported the use of casein hydrolysate and myo-inositol in culture media as the best choice to induce and proliferate callus cultures of Leguminosae species [11-12,27]. myo-inositol is a staple component of several media used to growth plant tissue even if the positive role of myo-inositol in cell growth and division has not been determined [28-30]. As regards casein hydrolysate, its positive role in tissue culture may be ascribed to its formula which contains twenty amino acids. It is frequently used as nitrogen source, rich in amino form, in many plant culture media and several studies reported a beneficial effect when it was added to the medium. The analysis of the non-linear regression linked with a F test allowed us to discriminate between the callus growth curves of the different cultures. The kinetics of the growth curves regarding the two types of explants on CH medium exhibited a sigmoid shape typical of plant cell cultures growth. Regardless of the origin of the explants, the addition of casein hydrolysate to the medium increased the growth rate of callus whereas the supply of myo-inositol caused a significant deceleration of callus growth with a delay in reaching the stationary phase as regards to the dry weight accumulation. This behaviour might be ascribed to the chemical property of the myo-inositol, a natural cyclitol, which may contribute to increase the osmotic potential of the medium hindering the nutrient availability to the callus cultures [31]. Supporting this opinion, the curve data set relative to the callus fresh weight fitted with the exponential model demonstrated that the cultures continued to absorb water from the medium. The colour of these cultures changed during the growth cycle from light green to dark brown during the post-exponential and senescence phase. Callus browning, generally linked to the phenolic oxidation, could be considered as a response to stress due to the deficit of medium components [32]. Although leaves and legumes of C. aeolicus and other Cytisus species were rich in various flavonoid

M. Lucchesini et al.

compounds [7,33], in all the callus cultures analyzed in this work, genistin was identified as unique compound. This constituent has already been isolated in the genus Cytisus (in vivo plants) [34] and is commonly found in Genista species [35], however, this is the first time it has been identified in the C. aeolicus extracts from callus cultures. Callus cultures from Genista species showed increased genistin production (from 0.9 to 3 g/100 g of callus dry weight) in comparison to C. aeolicus (from 0.070 to 0.341 g/100 g of callus dry weight) [12]. Therefore, the peculiarity of C. aeolicus calli was not the ability to produce a great amount of genistin but the selective synthesis of only one compound. It is well known that the secondary metabolism of in vitro plants is sometimes quite different from that of the corresponding in vivo plants. For example, transferring Genista plants to in vitro conditions, the production of flavones changed in comparison with the in vivo plants [6,12]. In the present work genistin accumulation was affected by medium supplements. The major genistin production took place on MI medium from cotyledon explant, at the end of the culture. Genistin accumulation might be related to the presence of the high dose of myo-inositol in the medium which could affect the osmolarity and the callus mineral uptake as above mentioned. The addition of myo-inositol, as an abiotic elicitor, might activate an array of defense mechanisms in callus tissue, improving the biosynthesis of secondary

metabolites [36]. Callus of C. aeolicus growing on the CH medium proliferated well until the end of the culture period but showed less genistin content. This result suggested that, in this case, CH was not considered as a promoter in isoflavone biosynthesis but employed to sustain the biomass growth, whereas in other Leguminosae plants, such as Pueraria lobata, casein promote the cell growth and the isoflavone synthesis at the same time [37]. Moreover, in tissue cultures of Rudbekia hirta [38] and Taxus cuspidata [39], which do not synthesize isoflavones, there is evidence that the CH addition to the medium improved secondary metabolite production. In conclusion, C. aeolicus can be propagated by tissue culture procedures. In vitro plant material could be considered in germplasm preservation programs for restoration purposes. Moreover, it is possible to establish C. aeolicus callus cultures from which genistin can be obtained. This compound was not previously identified in C. aeolicus wild plants, but it was de novo produced in callus cultures in this work. It is likely that the in vitro conditions caused considerable changes in the isoflavone biosynthesis so that C. aeolicus cell cultures selectively produced genistin. These results could be used in further research to establish in vitro cell suspension cultures and increase genistin cell production which could be a prospective for the pharmaceutical industry.

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