Phenolic compounds and carbohydrates in relation to bulb formation in Lachenalia ‘Ronina’ and ‘Rupert’ in vitro cultures under different lighting environments

Scientia Horticulturae 188 (2015) 23–29

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Phenolic compounds and carbohydrates in relation to bulb formation in Lachenalia ‘Ronina’ and ‘Rupert’ in vitro cultures under different lighting environments a ´ A. Bach a,∗ , A. Kapczynska , K. Dziurka b , M. Dziurka b a b

Department of Ornamental Plants, University of Agriculture in Krakow, al. 29 Listopada 54, 31-425 Krakow, Poland The F. Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland

a r t i c l e

i n f o

Article history: Received 18 July 2014 Received in revised form 25 February 2015 Accepted 26 February 2015 Available online 3 April 2015 Keywords: Lachenalia Light Bulb Phenolics Carbohydrates In vitro

a b s t r a c t The total soluble phenolic content, free and conjugated phenolic acids (cinnamic, p-coumaric, caffeic, ferulic, sinapic, chlorogenic), soluble carbohydrates and starch content were studied for the first time during the bulb formation in lachenalia (Lachenalia) in vitro adventitious shoot cultures of two cultivars which varied in their bulbing ability. Shoots of ‘Rupert’ and ‘Ronina’ were cultivated on the Murashige and Skoog (1962) medium with the addition of 3% sucrose and growth substances BA (2.5 ␮M) and NAA (0.5 ␮M) under different lighting environments (white, blue, red light and darkness). Lachenalia ‘Ronina’ formed most adventitious bulbs in darkness, whereas ‘Rupert’ produced them in all lighting environments. The results show a diversity of phenolic compounds in the storage organs of lachenalia cultivated in different lighting environments in terms of quality and quantity. In white and blue light or darkness, the total production of soluble phenolic compounds in cultures of investigated cultivars, varied from 1.1 to 2.0 mg/g d.w. and it was higher than in lachenalia exposed to red light (0.5 mg/g d.w.). Most of the examined conjugated phenolic acids occurred in bulbs at a higher concentration in the white and blue light, in comparison to the red light or in the dark. No, or only low concentration of chlorogenic acid was detected in scales of newly formed lachenalia bulbs. Bulbs of lachenalia ‘Ronina’ contained more soluble sugars, whereas bulbs of ‘Rupert’ contained more starch. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The genus Lachenalia J. Jacg. ex Murray (Hyacinthaceae) comprises 115 southern African species which differ from one another (Duncan et al., 2005). Lachenalia is considered to be a bulbous plant with excellent potential on the international floriculture market (Reinten et al., 2011). The scientific interest in this genus has led to the creation of stunning hybrids. The most striking aspects of the currently available cultivars are constituted by their different inflorescence colours (yellow, red, lilac, apricot or yellow-green) and often spotted succulent stems and leaves (Kleynhans, 2006). Traditional methods of propagation are not efficient enough for the commercial production of lachenalia (Kleynhans, 2006). Moreover, this flower bulb is susceptible to infection by Ornithogalum mosaic virus (OMV) (Burger and Von Wechmar, 1988) or by Freesia

∗ Corresponding author. Tel.: +48 126625246. E-mail address: [email protected] (A. Bach). http://dx.doi.org/10.1016/j.scienta.2015.02.038 0304-4238/© 2015 Elsevier B.V. All rights reserved.

sneak virus (FreSV) (Vaira et al., 2007). In vitro multiplication may increase production efficiency of virus-free plants. The induction and formation of storage organs in ornamental geophytes are correlated with initiation of dormancy in plants and depend on several environmental factors i.e. temperature or a level of sucrose (Niederwieser and Ndou, 2002; Ascough and Van Staden, 2010; Duncan, 2013; Okubo, 2013), the plant species and its stage of development, type of medium and quality of light (Hvoslef-Eide and Munster, 1999; Pelkonen and Kauppi, 1999; Bach and Pawłowska, 2006). Phenolics are the most common secondary metabolites of plants, possessing at least one aromatic ring with one or more hydroxyl groups. These compounds are accumulated in plants under biotic and abiotic stresses (Dixon and Paiva, 1995) and can be biochemical markers of morphogenesis in in vitro culture. The total soluble phenolic content was significantly lower in regenerable tissues than in non-regenerable ones at the proliferation stage of the callus culture of wheat, field bean and rape (Skrzypek et al., 2007). Similarly, Leucojum aestivum embryogenic callus produced fewer phenolics in comparison to the non-embryogenic callus (Ptak

24

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29

et al., 2013). Phenolic acids constitute the simplest example of phenolics synthesis from phenylalanine via a phenylpropanoid pathway. Cinnamic acid is the first representative of main substrate for other phenolic acids, such as p-coumaric, caffeic, ferulic and sinapic (Dixon and Paiva, 1995). Free acids tend to occur at low levels in plant tissues, but they can significantly accumulate as conjugates for many molecules. These include glycosides, esters of saccharides, esters of other hydroxyl acids, amides of amino compounds and esters of lipids (Clifford, 1999). It was observed that blue and white light can enhance the production of phenolic acids (Engelsma, 1974; Ebisawa et al., 2008; Szopa et al., 2012; Ouzounis et al., 2014). The knowledge of physiological functions of phenolic acids in plants is limited. Chlorogenic acid is also considered to be a negative cofactor of IAA oxidase, which, when added to the media containing auxins, promotes the adventitious root formation of Streptocarpus (Floh and Handro, 2001). In contrast, caffeic acid is a potent root growth inhibitor (Batish et al., 2008; Bach et al., 2010). So far, phenolic acids have been analysed mainly in food plants, i.e. in cereals, fruits, vegetables and in some medicinal plants (Naczk and Shahidi, 2006), but not in ornamental species. Carbohydrates are the most abundant storage components found in crops, including flower bulbs (Du Toit et al., 2004). Ranwala and Miller (2008) reported that in a study of 30 species of ornamental geophytes, starch, fructan, glucomannan and soluble sugars made up between 50 and 80% of the dry weight of storage organs. The reports in the literature on carbohydrate metabolism in lachenalia species are limited. Orthen (2001) reports that although Lachenalia minima bulbs contain similar amounts of fructans and starch, it is starch which is mainly consumed as a source of carbon and energy during sprouting. On the other hand, Du Toit et al. (2004) studied the location of carbohydrate concentrations in various organs of Lachenalia ‘Ronina’ during bulb production. The roots and especially the bulbs contained mostly starch whilst the leaves and inflorescences constituted the main source of soluble sugars. It was observed that Cucumis sativus plants grown under blue light had higher total soluble sugars, sucrose and starch content as compared with white light grown plants. Moreover, red light declined the carbohydrate contents (Wang et al., 2009). The aim of this study was to evaluate the abilities to form adventitious bulbs in two cultivars of lachenalia grown in different lighting conditions in in vitro cultures. These investigations can be considered as a completely innovative attempt of describing the bulb formation of lachenalia in relation to the content of endogenous phenolic compounds and sugars in the bulbs.

2. Materials and methods 2.1. Plant material and bulb formation The experimental material consisted of secondary explants – the adventitious shoots of two lachenalia cultivars: ‘Rupert’ (lilacpurple flowers) and ‘Ronina’ (yellow flowers). The shoots originated from one-year in vitro cultures of both cultivars. They were obtained from primary explants (from bulb scales), which had been cultivated on a medium according to Murashige and Skoog (1962), in ´ the way described previously (Bach and Kapczynska, 2013). In the presented experiment the adventitious shoot explants (consisting of 10–15 mm length pieces) were placed on a medium with the composition of macro- and microelements, according to Murashige and Skoog (1962), containing sucrose in the amount of 3% and BA and NAA growth substances (2.5 ␮M and 0.5 ␮M, respectively). The pH of the medium was set at 5.8 before the addition of 0.7% w/v Difco Bacto agar. For each cultivar five shoot explants (one replicate) were placed on the 25 ml medium in a 100 ml

Erlenmeyer flask. Five flasks were used under each light treatment. The cultures were incubated at 23/21 ◦ C in a growth chamber and subcultured ones after 8 weeks. Then, the following parameters were recorded: the percentage of explants on which adventitious bulbs were formed, the number of bulbs formed per one explant and, additionally, the diameter of the adventitious bulbs. 2.2. Lighting environment The shoot explants were cultured under a 16 h photoperiod under the light of different spectra provided by a fluorescent lamp: white (390–760 nm, Tungsram 40 W F33), red (647–770 nm, peak wave length 660 nm, Philips TLD 36 W), blue (400–492 nm, peak wave length 450 nm, Philips TLD 36 W) and in darkness. The photosynthetically active radiation (PAR) was 30 ␮mol m−2 s−1 and it was measured in the horizontal plane 35 cm above the culture with a portable LI-1800 spectroradiometer (LI-COR, USA). 2.3. Extraction of phenolic compounds The whole bulbs of cultured plants were collected, lyophilised and then pulverised in a mixing mill (MM 400, Retach, Kroll, Germany) for 3 min at 30 Hz. That material was used for all chemical analyses. The samples of about 20 mg were extracted to 0.5 ml of 2% acetic acid in methanol for 1 h at 250 rpm on a rotary shaker. Then the samples were centrifuged (15 min, 22,000 × g, 10 ◦ C) and the supernatant was collected. This extract was later used for the total soluble phenolic content, as well as for free and conjugated phenolic acids estimation. 2.4. Total soluble phenolic content estimation The total soluble phenolic content was measured according to the modified method by Singelton (Singleton et al., 1999). An aliquot of the extract (50 ␮l) was diluted in 0.5 ml of deionised water and 0.2 ml of Folina–Ciocalteu reagent and after 10 min, 0.7 ml saturated Na2 CO3 was added. The samples were then mixed after 2 h incubation and transferred to 96-well plates. Then absorbance at 765 nm was read on a microplate reader (Synergy II, Biotek). Chlorogenic acid was used as a standard. The analysis of the total soluble phenolic content was carried out in five replicates. 2.5. Free and conjugated phenolic acid estimation The extract was diluted to 2.5 ml with deionised water. Then 100 ␮l concentrated H3 PO4 was added in order to reverse dissociation and allow the partition of free acidic phenolics to an organic phase to occur. The samples were shaken with 1 ml of ethyl acetate, the upper layer was collected, then the same ethyl acetate volume was added and extraction was repeated. The pooled organic layer was evaporated under an N2 stream. The dry residue was resuspended in 0.4 ml of methanol and used for free phenolic acids estimation. The remaining aqueous layer, containing bound phenolic acids, was hydrolysed with 250 ␮l of concentrated HCl at 85 ◦ C for 30 min. Then it was neutralised with saturated NaOH and acidified to pH 2 with concentrated H3 PO4 . The released phenolic acids (mostly sugar and amines conjugates) were extracted in the same way as their free fraction. After evaporation the dry residue was redissolved in 0.4 ml of methanol and used for conjugated phenolic acids estimation. All samples were filtered (0.22 ␮m nylon membrane) and analysed by HPLC. The analyses were performed with an Agilent 1200 system equipped with a degasser, a binary pump, an autosampler and a fluorescence detector (FLD). The samples (5 ␮l) were injected into Zorbax Eclipse XDB-C18 4.6 mm × 75 mm × 3.5 ␮m analytical column, and separated under a linear gradient of A: methanol and B: water with 2% acetic acid,

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29

at 0 min 5% A, to 35 min 70% A. The optimal parameters of fluorescence detection were chosen according to the absorption and emission spectra, taken for the standards online. The phenolic acid analysis was carried out in five replicates. 2.6. Total soluble sugar estimation Sugars were analysed spectrophotometrically according to Dubois et al. (1951) with modifications. About 2 mg of lyophilised and homogenised samples were extracted in 0.5 ml of ultra pure water (Elga Option R) for 5 min in a screw cap 2 ml polypropylene test tube. Then, the samples were centrifuged at 22,000 × g for 5 min. The supernatant aliquat (5 ␮l) was diluted with 35 ␮l of water in 7 ml test tubes (the pellet was used for starch estimation); afterwards, 400 ␮l of 5% water phenol solution and 2 ml of concentrated sulphuric acid were added. The samples were incubated for 20 min, then transferred to 96-well plates and absorbance at 490 nm was measured. The sugar content was estimated with the use of the standard curve, prepared for glucose. The analysis to determine the soluble sugar content was performed in triplicate. 2.7. Starch estimation Starch was estimated in the pellets remaining after the sugar analysis with the use of enzymatic hydrolysis. Alpha-amylase (from Bacillus licheniformis, Sigma–Aldrich) in 50 mM potassium phosphate buffer pH 6.9 with 6.7 mM of NaCl and amyloglucosidase (from Aspergillus niger, Sigma–Aldrich) in 200 mM sodium acetate buffer pH 4.5 were used. The pellets were rinsed twice with 1 ml of ultrapure water and centrifuged every time. Water was decanted and 350 ␮l of alpha-amylase solution (0.2 mg in 25 ml of phosphate buffer) was added; the samples were vortexed and placed in a boiling water bath for 10 min. Then the samples were cooled down and 450 ␮l of amyloglucosidase was added (1 mg in 30 ml of acetate buffer), and the samples were placed in a 50 ◦ C water bath for 1 h. After that, the samples were centrifuged and 50 ␮l of supernatant was used for the spectrophotometric estimation of released glucose as described above. The analysis of the starch assay was performed in triplicate.

25

Table 1 Effect of genotype on quality of forming adventitious bulbs, irrespective of the light. Cultivar

Number of explants (%) forming adventitious bulbs

Number of bulbs per explant

Diameter of adventitious bulbs (mm)

‘Ronina’ ‘Rupert’

36.6b 89.8a

1.5b 3.8a

3.5b 4.8a

Different letters in a column indicate differences between cultivars according to the t-test (p ≤ 0.05).

was 1.3 mm in favour of ‘Rupert’). Furthermore, the process of bulb formation was more efficient in the case of lachenalia ‘Rupert’ – the number of bulbs obtained from one explant was nearly four, which represented the value obtained with the explants of ‘Ronina’ more than twice. Analysing together the two factors examined, genotype and lighting conditions, we observed that the explants of both cultivars formed a similar number of adventitious bulbs when cultivated in the dark (Table 2). Red, blue and white light influenced the number of explants forming bulbs. These light conditions limited the number of lachenalia ‘Ronina’ explants, forming bulbs to a similar level: from 13% (red light) to 17% (blue light). Such an effect was not observed in the case of ‘Rupert’, where the number of explants forming bulbs was about six times higher as compared to ‘Ronina’ in white, blue and red light. The explants of both cultivars grown in the dark formed a similar number of bulbs (Table 3). In all conditions lachenalia ‘Rupert’, tested under lights, formed more adventitious bulbs as compared to lachenalia ‘Ronina’. The observed differences were significant, and the number of bulbs obtained from explants of ‘Ronina’ accounted for only 10%, 5.5% and 4.8% of the number of bulbs of the ‘Rupert’ cultivar for white, blue and red light respectively. Analysing the size of the bulbs obtained, we found that both in darkness and in the case of white light, the explants of the tested cultivars formed bulbs of similar diameters (Table 4). The differences in this parameter were observed when the bulbs were derived from explants treated with red and blue light – the size of the obtained bulbs of the ‘Rupert’ cultivar was higher as compared to the ‘Ronina’ cultivar.

2.8. Statistical analysis The analysis of variance was used to determine the effect of different lighting environments on bulb formation as well as on total soluble phenolics, free and conjugated phenolic acids, total soluble sugars and starch content in two cultivars of lachenalia ‘Ronina’ and ‘Rupert’. All the data were analysed using the STATISTICA 10.0 (Stat-Soft, Inc., USA) software package. T-test and Duncan’s multiple range test was used to separate the means (level of significance p ≤ 0.05). 3. Results 3.1. Adventitious bulb formation After culture period (16 weeks) we observed that lachenalia shoot explants formed adventitious bulbs or single leaf-unifacial shoots or roots. The manner of regeneration depended on the genotype and on the kind of lighting environment. In the present study only the bulb formation process was analysed. The data presented in Table 1 indicate that irrespective of the lighting environment, lachenalia ‘Rupert’ (Fig. 1) formed more adventitious bulbs in the basal part of the leaf-unifacial shoots as compared to lachenalia ‘Ronina’ (Fig. 2). 89.8% explants of ‘Rupert’ formed bulbs; in the case of ‘Ronina’ – 36.6%. The diameter of ‘Rupert’ bulbs was larger in comparison to ‘Ronina’ (the difference

Fig. 1. Lachenalia ‘Rupert’ – forming mainly bulbs.

26

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29 Table 3 Effect of lighting environment and genotype on number of bulbs per explant. Cultivar

‘Ronina’ ‘Rupert’

Lighting environment White

Blue

Red

Darkness

0.3c 3.0b

0.2c 3.6ab

0.2c 4.2ab

5.4a 4.4ab

Different letters in columns and rows indicate differences between light and cultivar treatments according to the Duncan test (p ≤ 0.05).

Fig. 2. Lachenalia ‘Ronina’ – forming mainly shoots.

3.2. Total phenolics and phenolic acid content The total soluble phenolic content in the bulbs of lachenalia (Fig. 3) ranged from about 0.5 mg/g d.w. (for ‘Ronina’ exposed to red light) to 2 mg/g d.w. (for ‘Rupert’ grown in the dark). Red and blue light treatment did not induce any changes in the content of total phenolics between the studied cultivars. However, the bulbs of ‘Ronina’ exposed to white light contained significantly more phenolics than ‘Rupert’. It was the opposite case in dark-grown lachenalia plants – ‘Rupert’ bulbs contained more total phenolics than ‘Ronina’ bulbs. The bulbs of lachenalia ‘Ronina’, cultivated under white light, showed a similar amount of phenolic compounds as the bulbs obtained under blue light. A similar relationship between light treatment and total soluble phenolic content was observed for ‘Rupert’. Table 5 shows a comparison of free and conjugated phenolic acids in ‘Ronina’ and ‘Rupert’ bulbs exposed to different lighting Table 2 Effect of lighting environment and genotype on explant number (%) forming adventitious bulbs. Cultivar

‘Ronina’ ‘Rupert’

conditions. Cinnamic and caffeic acids proved to be among the predominant acids assayed for both cultivars in all lighting conditions. The remaining phenolic acids (p-coumaric, ferulic, sinapic and chlorogenic), detected in lachenalia bulbs, were present in concentrations of about one order of magnitude smaller. The highest content of free cinnamic acid (69.8 mg/g d.w.) was observed in ‘Rupert’ bulbs grown in the dark. The content of free and conjugated caffeic acid (24.1 and 112.5 ␮g/g (d.w.) respectively) in lachenalia bulbs was the highest for ‘Ronina’ cultivated in white light. Half of the concentration of this acid was found in the lachenalia ‘Ronina’ bulbs cultivated in blue light. Free ferulic and sinapic acid was not detected in ‘Rupert’ bulbs. However, free ferulic acid was observed in ‘Ronina’ bulbs, cultivated in light blue or in the dark. Moreover, conjugated ferulic acid was detected in both cultivars in all lighting environments of the cultures. Free sinapic acid was present only in ‘Ronina’ bulbs cultivated under light red, although conjugated sinapic acid was detected in the bulbs growing under white and blue light. Chlorogenic acid, both in its free and conjugated form, appeared only in the ‘Ronina’ cultivar in all lighting environments except darkness. The highest content of conjugated chlorogenic acid (7.8 ␮g/g d.w.) was reported in the bulbs of ‘Ronina’ plants exposed to white light. 3.3. Total soluble sugar and starch content ‘Ronina’ lachenalia was characterised by a significantly higher content of soluble sugars, as compared to the ‘Rupert’ cultivar under white and red light, and darkness (Fig. 4). A similar relationship was observed under blue light, but it was not statistically significant. The starch content of lachenalia bulbs ranged from 4.7 mg/g d.w. for the ‘Ronina’ cultivar in the dark to 105 mg/g d.w. for the ‘Rupert’ cultivar under blue light (Fig. 5). The highest concentration of starch was observed in ‘Rupert’ bulbs, cultivated in blue and white light. They were five and seven times higher as compared to the ‘Ronina’ cultivar under white and blue light, respectively.

Lighting environment White

Blue

Red

Darkness

16.0b 90.0a

17.0b 100.0a

13.0b 88.0a

100.0a 81.0a

Different letters in columns and rows indicate differences between light and cultivar treatments according to the Duncan test (p ≤ 0.05). Table 4 Effect of lighting environment and genotype on the diameter of adventitious bulbs (mm). Cultivar

‘Ronina’ ‘Rupert

Lighting environment White

Blue

Red

Darkness

3.8b 4.5ab

3.2b 5.0a

3.2b 5.0a

4.1ab 4.7ab

Different letters in columns and rows indicate differences between light and cultivar treatments according to the Duncan test (p ≤ 0.05).

Fig. 3. Total phenolic compounds of lachenalia bulbs grown in different light conditions.

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29

27

Table 5 Free and conjugated phenolic acid content (␮g/g (d.w.)) in bulbs of lachenalia cultivated under different lighting conditions. ‘Ronina’

Free phenolic acids Cinnamic acid Coumaric acid Caffeic acid Ferulic acid Sinapic acid Chlorogenic acid Conjugated phenolic acids Cinnamic acid Coumaric acid Caffeic acid Ferulic acid Sinapic acid Chlorogenic acid

‘Rupert’

White

Blue

Red

Darkness

White

Blue

Red

Darkness

14.7bcd 0.5ab 24.1a nd nd 3.0a

24.0b 0.1c 12.0b 0.7a nd 1.6ab

6.3cd nd 3.2cd nd 1.2a 1.5ab

28.6b 0.1c 2.4d 0.4b nd nd

17.0bc 0.3bc 7.7c nd nd nd

15.4bc 0.5ab 4.1cd nd nd nd

nd nd 3.2cd nd nd nd

69.8a 0.6a 2.8d nd nd nd

94.9a 1.0a 112.5a 14.2a 8.4a 7.8a

64.4b nd 52.8b 7.3b 5.4b 5.1b

nd 0.5b 1.6c 4.1c nd 1.2c

25.3d nd nd 0.3d nd nd

47.4bcd 0.9a 42.7b 4.5bc 3.1bc nd

33.0cd 0.7ab 48.4b 3.9c 1.8cd nd

37.4bcd nd nd 2.3cd nd nd

58.3bc nd nd 0.2d nd nd

Different letters in rows indicate differences between light and cultivar treatments according to the Duncan test (p ≥ 0.05); nd – not detected.

4. Discussion 4.1. Bulb formation The bulbing process in ornamental geophytes usually includes two steps: the induction of bulbing in meristems and the growth of bulbs (swelling) (Okubo, 2013). An important physiological feature of flowering bulbs is their efficiency of bulb formation both in terms of their number and size – under the photoautotrophic conditions the survival rate and growth of bulbous plants are higher since the bulbs are larger (Slabbert and Niederwieser, 1999). On the basis of our own observations, it follows that the number of explants which formed bulbs of ‘Rupert’ did not depend on the lighting environment but in the case of lachenalia ‘Ronina’ the greatest number of explants forming adventitious bulbs was obtained in the dark in comparison with the light of white, red and blue colours. Other studies on bulblet induction have shown a similar trend. Darkness was also a favourable environmental condition in the formation of bulbs for other geophytes, including Hyacinthus ´ 2000; Rice et al., 2011). Other and Brunsvigia (Bach and Swiderski, studies have contrasted with mentioned results (Ulrich et al., 1999; Paek and Murthy, 2002; Cheesman et al., 2010). The discrepancies in the assessment of the effect of light on the bulbing process may result from the authors’ imprecisely highlighting the steps of bulb formation: bulb induction or growth of bulbs. The differences between the two discussed cultivars were also noticed during the acclimatization process of micropropagated

Fig. 4. The content of soluble sugars of lachenalia bulbs grown under different light conditions.

plants to ex vitro conditions; the weight of ‘Ronina’ bulbs obtained after the first season of cultivation was lower (1.3–1.6 g) in ´ comparison to ‘Rupert’ bulbs (2.1–2.6 g) (Kapczynska and Bach, 2012). Comparing the different genotypes of lachenalia in terms of the number of bulbs obtained from one plant in open ground ´ (2013, 2014) found that ‘Ronina’ and cultivation, Kapczynska ‘Rupert’ produced fewer offsets than other lachenalia cultivars, e.g. ‘Namakwa’ or ‘Rosabeth’. This fact once again clearly indicates that the ability of bulb formation in the individual lachenalia genotypes may take a different course. Other factors that may influence the size of bulbs comprise medium sucrose concentration (Slabbert and Niederwieser, 1999) and temperature of in vitro culture (Ascough and Van Staden, 2010). 4.2. Phenolic compounds Jin et al. (2012) showed that the total soluble phenolic content may vary within the lily genus. Our results also show a variation in quantity and quality of phenolic compounds in the newly formed bulbs of lachenalia. The total soluble phenolic content depends on cultivar and lighting environments. ‘Ronina’ bulbs contained more phenolics than ‘Rupert’ under the white light condition. Interestingly, the differences in phenolics between ‘Ronina’ and ‘Rupert’ disappear under blue and red light treatment. Regardless of the tested cultivars, white and blue light resulted in higher level of total soluble phenolics content in comparison to red light in lachenalia bulbs. It was reported that UV-B and blue light irradiation stimulate the phenylpropanoid pathway in lettuce (Ebisawa et al., 2008). This could be explained by the fact that phenolic acid synthesis involves

Fig. 5. The starch content of lachenalia bulbs grown under different light conditions.

28

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29

l-phenylalanine ammonia-lyase (PAL), which can be activated by light, especially blue light (Engelsma, 1974). The most abundant phenolic acids in bulbs of both tested lachenalia cultivars were cinnamic and caffeic, both in their free and conjugated forms, supposingly with sugar and amine (Dixon and Paiva, 1995). Significant amounts of these acids were accompanied by low level or absence of acids preceding or following caffeic acid on the metabolic pathway (i.e. p-coumaric, ferulic and sinapic). The accumulation of caffeic acid suggests its involvement in physiological processes examined in in vitro lachenalia culture. We observed that a lower concentration of caffeic acid accompanied an increasing amount of ‘Rupert’ bulbs. A high negative correlation with the ability of in vitro lachenalia cultures to form bulbs was exhibited with chlorogenic acid (a caffeic acid derivative), the presence of which was observed only in plants that did not form bulbs or formed only a limited number of bulbs, i.e. in ‘Ronina’ cultures in conditions of white, blue and red light. It is possible that endogenously occurring chlorogenic acid in the culture of ‘Ronina’ fulfils regulatory functions and is involved in the inhibition of bulb formation process. But, on the other hand, it can play a certain role in the induction phase of bulbing and then disappears. Phenols play some role in the organogenesis of bulbous plants. Bach et al. (2009, 2010) reported different levels of phenolics in the adventitious bulbs of Galanthus depending on the developmental stage of culture and on the content of growth regulators. In the presence of cytokinin (BA), the scales of bulbs had lower amounts of phenolics than on the medium with auxin (IAA). Al-Quadan et al. (2008) reported that phenolic compounds inhibit the activity of dehydrogenase glucose 6-phosphate (G6PDH). This enzyme is active in the distribution of carbohydrates and possibly in inducing dormancy, as shown in Puchalski et al.’s (1991) studies on the formation of grape hyacinth bulbs (Muscari sp.). Furthermore, it was found that the G6PDH enzyme is active in dormancy of apple buds (Wang et al., 1991). The G6PDH enzyme is active in the dark (Al-Quadan et al., 2008; Nee et al., 2009), which would clarify the formation of bulbs in explants of ‘Ronina’ almost exclusively in the absence of light, i.e. in conditions where there was no presence of an inhibitor of the enzyme–chlorogenic acid. Floh and Handro (2001) state that chlorogenic acid added to the media containing auxins promotes in vitro shoot rooting. Moreover, it can replace the IAA requirement for olive callus growth in vitro (Lavee et al., 1994). Further studies are needed to determine a possible explanation of the mechanism of chlorogenic acid activity in bulb formation. 4.3. Soluble sugars and starch In all of the tested specimens, except for ‘Rupert’ cultivated under blue light, higher soluble sugars were observed in the bulbs of ‘Ronina’ than of ‘Rupert’. Higher starch concentrations were noticed in the bulbs of ‘Rupert’ cultivated in blue and white light than in the bulbs of the same cultivar grown in red light or darkness. Talbott and Zeiger (1993) suggest that light quality may affect the translocation of carbohydrates, which may result in starch breakdown. Under red light the decrease of starch is observed, while blue light increases the concentration of this polysaccharide in Acetabularia plants (Vettermann, 1973). Du Toit et al. (2004) showed that mother bulbs of lachenalia can have different patterns of starch and soluble sugars during the growing season. We report, a several times higher concentration of starch in bulbs of ‘Rupert’ than in ‘Ronina’, both cultivated under white and blue light. This may indicate that the plants of ‘Rupert’ were in a different developmental stage, closer to the flowering phase, as compared to the plants from other treatments. Such an argument is supported by the study of lachenalia acclimatization – after ex vitro transfer only single ‘Ronina’ bulbs flowered, while in the same conditions even 50%

´ of ‘Rupert’ bulbs flowered (Kapczynska and Bach, 2012). Ohyama et al. (2006) observed that according to the developmental stage of mother and new bulblets of tulip cultivated in the open field not only accumulation but also the type of carbohydrates may differ. 5. Conclusions The ability to form bulbs in lachenalia in vitro varied with cultivar and lighting conditions. Lachenalia ‘Ronina’ formed adventitious bulbs mostly in the dark whereas ‘Rupert’ formed them in all lighting conditions. The obtained results showed different contents of phenolic compounds, both qualitatively and quantitatively, depending on the light conditions and genotype. White and blue light stimulated the production of phenolics. We suggest that the bulb formation process may depend on chlorogenic acid – no, or only low concentration of chlorogenic acid was detected in scales of newly formed lachenalia bulbs. The higher amount of starch was observed in bulbs of cultivar ‘Rupert’ in comparison with ‘Ronina’ bulbs, which were characterised by higher concentration of soluble sugars. Acknowledgments This study was financed by The State Committee for Scientific Research in Warsaw (KBN) within the grant N N310 309934 and by the Polish Ministry of Science and Higher Education, within the project “The use of biotechnological methods in intensifying the production of selected ornamental plants” (DS 3500/KRO/20132014). References Al-Quadan, F., Ibrahim, A., Al-Charchafchi, F.M.R., 2008. Effect of chlorogenic and caffeic acids on activities and isoenzymes of G6PDH and 6PGDH of Artemisia herba Alba seeds germinated for one and three days in light and dark. Jordan J. Biol. Sci. 1, 85–88. Ascough, G.D., Van Staden, J., 2010. Micropropagation of Albuca bracteata and A. nelsonii – indigenous ornamentals with medicinal value. S. Afr. J. Bot. 76, 579–584. ´ Bach, A., Swiderski, A., 2000. The effect of light quality on organogenesis of Hyacinthus orientalis L. in vitro. Acta Biol. Cracov. Bot. 42, 115–120. Bach, A., Pawłowska, A., 2006. Effect of light qualities on cultured in vitro ornamental bulbous plants. In: Teixeira da Silva, J.A. (Ed.), Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. Global Science Books, United Kingdom, pp. 271–276. Bach, A., Pawłowska, B., Hura, K., 2009. The level of phenolic compounds at various development at stages in in vitro cultures of Galanthus elwesii Hook. Adv. Agric. Sci. Probl. Issues 534, 13–21. Bach, A., Pawłowska, B., Hura, K., 2010. The effect of the exogenous phenolic compound, caffeic acid on organogenesis of Galanthus elwesii Hook. cultured in vitro. Biotechnologia 2, 139–145. ´ Bach, A., Kapczynska, A., 2013. Initiation of in vitro cultures of Lachenalia. Ann. Warsaw Univ. Life Sci. – SGGW. Hortic. Landsc. Archit. 34, 3–12. Batish, D.R., Singh, H.P., Kaur, S., Kohli, R.K., Yadav, S.S., 2008. Caffeic acid affects early growth, and morphogenetic response of hypocotyl cuttings of mung bean (Phaseolus aureus). J. Plant Physiol. 165, 297–305. Burger, J.T., Von Wechmar, M.B., 1988. Rapid diagnosis of Ornithogalum and Lachenalia viruses in propagation stock. Acta Hortic. 234, 31–38. Cheesman, L., Finnie, J.F., Van Staden, J., 2010. Eucomis zambesiaca baker: factors affecting in vitro bulblet induction. S. Afr. J. Bot. 76, 543–549. Clifford, M.N., 1999. Chlorogenic acids and other cinnamates – nature, occurrence and dietary burden. J. Sci. Food Agric. 79, 362–372. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085–1097. Du Toit, E.S., Robbertse, P.J., Niederwieser, J.G., 2004. Plant carbohydrate partitioning of Lachenalia cv. Ronina during bulb production. Sci. Hortic. 102, 433–440. Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F., 1951. A colorimetric method for the determination of sugars. Nature 168, 167–168. Duncan, G.D., Edwards, T.J., Mitchell, A., 2005. Character variation and a cladistic analysis of the genus Lachenalia Jacq f. ex Murray (Hyacinthaceae). Acta Hortic. 673, 113–120. Duncan, G.D., 2013. Geophyte research and production in South Africa. In: Kamenetsky, R., Okubo, H. (Eds.), Ornamental Geophytes. From Basic Science to Sustainable Production. CRC Press Taylor & Francis Group, pp. 485–503. Ebisawa, M., Shoji, K., Kato, M., Shimomura, K., Goto, F., Yoshihara, T., 2008. Supplementary ultraviolet radiation B together with blue light at night increased

A. Bach et al. / Scientia Horticulturae 188 (2015) 23–29 quercetin content and flavonol synthase gene expression in leaf lettuce (Lactuca sativa L.). Environ. Control Biol. 46, 1–11. Engelsma, G., 1974. On the mechanism of the changes in phenylalanine ammonialyase activity induced by ultraviolet and blue light in gherkin hypocotyls. Plant Physiol. 54, 702–705. Floh, E.I.S., Handro, W., 2001. Effect of photoperiod and chlorogenic acid on morphogenesis in leaf discs of Streptocarpus nobilis. Biol. Plant. 44, 615–618. Hvoslef-Eide, A.K., Munster, C., 1999. Light quality effect on somatic embryogenesis of Cyclamen persicum Mill. in bioreactors. In: Shcwenkel, H.G. (Ed.), Reproduction of Cyclamen persicum Mill. through Somatic Embryogenesis Using Suspensor Culture Systems. European Commision, Brussels, pp. 79–84. Jin, L., Zhang, Y., Yan, L., Guo, Y., Niu, L., 2012. Phenolic compounds and antioxidant activity of bulb extracts of six lilium species native to China. Molecules 17, 9361–9378. ´ Kapczynska, A., Bach, A., 2012. Acclimatization of lachenalia (Lachenalia sp.) regenerants propagated in vitro in different light conditions. Biotechnologia 93, 191. ´ Kapczynska, A., 2013. Effect of plant spacing on the growth, flowering and bulb production of four lachenalia cultivars. S. Afr. J. Bot. 88, 164–169. ´ Kapczynska, A., 2014. Effect of bulb size on growth, flowering and bulb formation in lachenalia cultivars. Hortic. Sci. 41, 89–94. Kleynhans, R., 2006. Lachenalia, spp. In: Anderson, N.O. (Ed.), Flower Breeding & Genetics: Issues, Challenges, and Opportunities for the 21st Century. Springer, pp. 491–516. Lavee, S., Avidan, N., Pierik, R.L.M., 1994. Chlorogenic acid – an independent morphogenesis regulator or a cofactor. Acta Hortic. 381, 405–412. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Naczk, M., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J. Pharm. Biomed. 41, 1523–1542. Nee, G., Zaffagnini, M., Issakidis-Bourguet, E., 2009. Redox regulations of chloroplastic glucose-6-phosphate dehydrogenase: a new role for f-type thioredoxin. FEBS Lett. 583, 2827–2832. Niederwieser, J.G., Ndou, A.M., 2002. Review on adventitious bud formation in Lachenalia. Acta Hortic. 570, 135–139. Ohyama, T., Komiyama, S., Ohtake, N., Sueyoshi, K., Teixeira da Silva, J.A., Ruamrungsri, S., 2006. Physiology and genetics of carbon and nitrogen metabolism in tulip. In: Teixeira da Silva, J.A. (Ed.), Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. Global Science Books, United Kingdom, pp. 12–25. Okubo, H., 2013. Dormancy. In: Kamenetsky, R., Okubo, H. (Eds.), Ornamental Geophytes. From Basic Science to Sustainable Production. CRC Press Taylor & Francis Group, pp. 233–259. Orthen, B., 2001. Sprouting of the fructan- and starch-storing geophyte Lachenalia minima: effects on carbohydrate and water content within the bulbs. Physiol. Plant. 113, 308–314. Ouzounis, T., Fretté, X., Rosenqvist, E., Ottosen, C.O., 2014. Spectral effects of supplementary lighting on the secondary metabolites in roses, chrysanthemums, and campanulas. J. Plant Physiol. 171, 1491–1499.

29

Paek, K.Y., Murthy, H.N., 2002. High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii. Plant Cell Tissue Organ 68, 247–252. Pelkonen, V.P., Kauppi, A., 1999. The effect of light and auxins on the regeneration of lily (Lilium regale Wil.) cells by somatic embryogenesis and organogenesis. Int. J. Plant Sci. 160, 483–490. Ptak, A., El Tahchy, A., Skrzypek, E., Wojtowicz, T., Laurain-Mattar, D., 2013. Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus. Cent. Eur. J. Biol. 8, 591–599. Puchalski, J., Puchalska, M., Saniewski, M., 1991. Glucose-6-phosphate dehydrogenase electrophoretic forms during differentiation of benzyladenine induced bulblets of Muscari. Acta Hortic. 91, 241–245. Ranwala, A.P., Miller, W.B., 2008. Analysis of non-structural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection. New Phytol. 180, 421–433. Reinten, E.Y., Coetzee, J.H., Van Wyk, B.E., 2011. The potential of South African indigenous plants for the international cut flower trade. S. Afr. J. Bot. 77, 934–946. Rice, L.J., Finnie, J.F., Van Staden, J., 2011. In vitro bulblet production of Brunsvigia undulata from twin-scales. S. Afr. J. Bot. 77, 305–312. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods Enzymol. 299, 152–178. ´ ˛ Skrzypek, E., Szechynska-Hebda, M., Dabrowska, G., 2007. The influence of phenolic accumulation on callus regeneration abilities of chosen plant species. Adv. Agric. Sci. Probl. Issues 523, 203–212. Slabbert, M.M., Niederwieser, J.G., 1999. In vitro bulblet production of Lachenalia. Plant Cell Rep. 18, 620–624. Szopa, A., Ekiert, H., Szewczyk, A., Fugas, E., 2012. Production of bioactive phenolic acids and furanocoumarins in in vitro cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. under different light conditions. Plant Cell Tissue Organ Cult. 110, 329–336. Talbott, L.D., Zeiger, E., 1993. Sugar and organic acid accumulation in guard cells of Vicia faba in response to red and blue light. Plant Physiol. 102, 1163–1169. Wang, S.Y., Jiao, H.J., Faust, M., 1991. Changes in metabolic enzyme activities during thidiazuron-induced lateral budbreak of apple. HortScience 26, 171–173. Wang, H., Gu, M., Cui, K., Zhou, Y., Yu, J., 2009. Effect of light quality on CO2 assimilation, chlorophyll-fluorescence quenching, expression of Calvin cycle genes and carbohydrate accumulation in Cucumis sativus. J. Phytochem. Phytobiol. B. 96, 30–37. Ulrich, M.R., Davies Jr., F.T., Koh, Y.C., Duray, S.A., Egilla, J.N., 1999. Micropropagation of Crinum ‘Ellen Bosanquet’ by tri-scales. Sci. Hortic. 82, 95–102. Vaira, A.M., Kleynhans, R., Hammond, J., 2007. First report of freesia sneak virus infecting Lachenalia cultivars in South Africa. Plant Dis. 91, 770. Vettermann, W., 1973. Mechanism of the light-dependent accumulation of starch in chloroplasts of Acetabularia, and its regulation. Protoplasma 76, 261–278.