Micropropagation of a giant ornamental bromeliad Puya berteroniana

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Micropropagation of a giant ornamental bromeliad Puya berteroniana through adventitious shoots and assessment of their...

Article in Plant Cell Tissue and Organ Culture · January 2016 DOI: 10.1007/s11240-016-0949-x

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Plant Cell Tiss Organ Cult (2016) 125:293–302 DOI 10.1007/s11240-016-0949-x

ORIGINAL ARTICLE

Micropropagation of a giant ornamental bromeliad Puya berteroniana through adventitious shoots and assessment of their genetic stability through ISSR primers and flow cytometry Iva Viehmannova1 • Petra Hlasna Cepkova1,3 • Jan Vitamvas2 • Petra Streblova1 Jana Kisilova1

•

Received: 23 August 2015 / Accepted: 15 January 2016 / Published online: 23 January 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract An efficient micropropagation protocol has been developed for Puya berteroniana, a giant Chilean bromeliad with attractive turquoise flowers and great horticultural potential. Plant material for the experiment was multiplied via repeated sub-cultures of adventitious shoots, originally derived from a single in vitro germinated seedling, on a halfstrength Murashige and Skoog (MS) medium (Murashige and Skoog in Physiol Plant 15:473–497, 1962) containing 0.44 lM 6-benzyladenine (BA). For the in vitro propagation experiment, the shoots were cultured on a half-strength MS medium supplemented with BA (0.04–2.22 lM) or zeatin (0.05–2.28 lM) alone or in combination with a-naphthaleneacetic acid (NAA) (0.54 lM). The maximum shoots per explant (5.5), was obtained on a medium containing 0.44 lM BA. Rooting of the shoots was tested on a medium supplemented with NAA (0.54–2.69 lM) or indole-3-acetic acid (0.57–2.85 lM). The best rooting was achieved on a medium containing 2.69 or 1.61 lM NAA. The rooted plantlets were transferred ex vitro, with 98.3 % survival rate. Inter simple sequence repeat (ISSR), flow cytometry, and karyological analysis were used to evaluate true-to-type of the in vitro regenerants. Ten randomly chosen plants and control plant were used. Twenty ISSR primers produced 95 clear, distinct,

& Petra Hlasna Cepkova petrahlasna@gmail.com; hlasna@vurv.cz 1

Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Prague, Czech Republic

2

Department of Forest Ecology, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Prague, Czech Republic

3

Department of Gene Bank, Crop Research Institute, Drnovska 507/73, 161 06 Prague, Czech Republic

and reproducible bands per analysed sample. All amplified products were monomorfic, and no polymorphism was detected. Similarly, flow cytometric analysis confirmed that the ploidy level in all plantlets was stable. Karyological analysis revealed number of somatic chromosomes 2n = 50.

As no somaclonal variation was detected in culture, this micropropagation protocol can be used for the mass production of P. berteroniana plants.

Keywords Bromeliaceae Chromosome In vitro propagation Molecular marker Ploidy level Puya berteroniana Abbreviations AFLP Amplified fragment length polymorphism BA 6-Benzyladenine BSA Bovine serum albumin CTAB Cetyltrimethylammonium bromide DAPI 40 ,6-Diamidino-2-phenylindole IAA Indole-3-acetic acid IBA Indole-3-butyric acid ISSR Inter simple sequence repeat MS Murashige and Skoog (1962) NAA a-Naphthaleneacetic acid PGR Plant growth regulator RAPD Randomly amplified polymorphic DNA SSR Simple sequence repeat

Introduction The genus Puya represents one of the largest bromeliad genera, comprising more than 200 exclusively terrestrial species, diversified along the Andean cordillera (Schulte

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et al. 2010; Jabaily and Sytsma 2013). Puya berteroniana Mez is a decorative giant rosette plant, generally restricted to central Chile (Jabaily and Sytsma 2010). It has attracted attention from horticulturists as an ornamental plant potentially suitable for outdoor planting in gardens and parks, serving the purpose of forming impenetrable hedges, and possessing considerable beauty as an individual plant in the landscape (Hertrich 1953). The plant is remarkable for its typically metallic greenish-blue flower petals, having violet stamen filaments, and orange anthers (Hertrich 1953; Rauh 1985).

Puyas are allogamous plants, producing copious seeds (Jabaily and Sytsma 2013). However, sexual propagation for mass production might be complicated because seedlings in most bromeliads require several years to reach maturity and to induce flowers (Mercier and Kerbauy 1997). Mature individuals of P. berteroniana are composed of several interconnected rosettes (Jabaily and Sytsma 2013; Jabaily pers. comm.). Thus, asexual propagation via offshoots is an alternative method of propagation. Asexual propagation has the advantage of avoiding the genetic variability inherent in sexual processes, as well as a shorter time for flowering. However, this method is incapable of producing large numbers of plants for commercial nurseries (Mercier and Kerbauy 1997).

In vitro propagation provides a rapid and reliable system for production of a large number of genetically uniform plantlets (Jha and Ghosh 2005). Previously, efficient regeneration protocols using direct or indirect morphogenesis have been reported for many ornamental, medicinal, fibre, fruit, or endangered bromeliad species, e.g.

Alcantarea imperialis (Aoyama et al. 2012), Nidularium innocentii and N. procerum (da Silva et al. 2012), Ananas comosus (Al-Saif et al. 2011; Scherer et al. 2013), Neoregelia cruenta (Carneiro et al. 1999), Vriesea reitzii (Alves et al. 2006; dal Vesco and Guerra 2010; dal Vesco et al. 2014), Dyckia distachya (Pompelli et al. 2005; Pompelli and Guerra 2005), and Tillandsia eizii (Pickens et al. 2006). However, an efficient in vitro propagation system for P. berteroniana has not been established.

For micropropagation, one crucial aspect is to retain genetic fidelity of the plants, and to ensure early detection of potential somaclonal variation in order to reduce losses to growers. Somaclonal variants can be detected using various techniques, which were broadly categorized as morphological, physiological, biochemical, molecular, and cytological detection techniques (Jin et al. 2008; Bairu et al.

2011). Recently, genetic fidelity or somaclonal variation in regenerated plants after in vitro propagation was routinely analysed using polymerase chain reaction (PCR) techniques such as randomly amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR), and amplified fragment length polymorphism

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(AFLP) (Aversano et al. 2011; Bairu et al. 2011). Inter simple sequence repeat is a method that overcomes most of the limitations of the other techniques, i.e., low reproducibility of RAPD, high cost of AFLP, and the need to know the flanking sequences to develop species-specific primers for SSR polymorphism. It also represents a simple and quick method. Additionally, ISSR markers are highly polymorphic (Reddy et al. 2002).

Flow cytometry for detection of ploidy level variation among in vitro regenerants is now also being widely employed (Bairu et al. 2011). Using flow cytometry, high numbers of nuclei can be analysed in a short time, making the result highly representative and statistically robust (Loureiro et al. 2010). In addition, flow cytometry allows the examination of different types of tissues and cell layers (Dhooghe et al. 2011).

The objective of the present study was to develop a micropropagation protocol through adventitious bud formation. In vitro regenerants of P. berteroniana were subjected to ISSR, flow cytometric and karyological analyses in order to exclude molecular and genomic changes. The reported propagation technique can be used for large-scale multiplication of this species, which has great ornamental potential. It can also serve as an initial study for the optimization of in vitro propagation of other Puya species with horticultural uses.

Materials and methods Plant material As initial plant material, thirty seeds of P. berteroniana were used. They were provided by the Botanical Garden of the Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague. The seeds (Fig. 1a) were surface disinfected in 70 % (v/v) ethanol for 2 min, followed by 1 % (v/ v) sodium hypochlorite (NaClO) with 0.02 % (v/v) Tween 20 for 20 min. After rinsing three times with sterile distilled water, the seeds were placed in Erlenmeyer flasks (100 ml) containing 35 ml of half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962), supplemented with 1 mg l-1 thiamine, 100 mg l-1 myo-inositol, 30 g l-1 sucrose and 8 g l-1 agar (Agar–Agar, da¨nisch, Carl Roth GmbH & CoKG, Germany), and the pH adjusted to 5.7. The cultures were incubated at 25/23 °C under a 16/8 h light/dark regime, and at a photosynthetic photon flux density of 35 lmol m-2 s-1, provided by cool-white fluorescent tubes.

After 8 weeks of culture, 60 % of the seeds germinated (Fig. 1b). In order to standardize the in vitro propagation experiment, all plants for the experiment were derived from a single randomly chosen seedling. The shoot was excised from the germinated seedling and transferred

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Fig. 1 Micropropagation of Puya berteroniana. a Botanical seeds (bar = 0.6 cm). b In vitro germinated seedlings (bar = 1 cm). c Shoot explant ca 2 cm in height used for shoot multiplication experiment (bar = 1 cm). d Adventitious shoot production after 4-week culture on a half-strength MS medium supplemented with

0.44 lM BA (bar = 1.5 cm). e Shoot rooted on a half-strength MS medium supplemented with 1.61 lM NAA (bar = 1.8 cm). f A wellrooted shoot before ex vitro transfer (bar = 1 cm). g Plants immediately after ex vitro transfer (bar = 3 cm). h Plants 20 weeks after ex vitro transfer (bar = 8 cm)

upright to a culture medium. Since a half-strength MS medium without plant growth regulators (PGRs) did not induce production of adventitious shoots according to our preliminary study (data not shown), a medium with 0.44 lM 6-benzyladenine (BA) was used for initial plant propagation. Adventitious shoots longer than 3 mm were excised from the original explant at the basal part and used for further propagation. In total, 680 individuals were obtained by periodic sub-culture of adventitious shoots on the same medium. Cultures were kept under the cultivation conditions described previously. Before establishment of the in vitro propagation experiment, each plantlet was subcultured once on a half-strength MS medium without PGRs.

in vitro propagation, a total of 20 explants were used per treatment in two replications. The experiment was arranged in a completely randomized design.

Shoot multiplication For the multiplication experiment, shoots ca. 1.8–2 cm in height were used (Fig. 1c). A half-strength MS medium supplemented with different concentrations of BA (0.04–2.22 lM) or zeatin (0.05–2.28 lM) alone or in combination with a-naphthaleneacetic acid (NAA) (0.54 lM), was tested for in vitro shoot multiplication. The cultures were kept under the same conditions as previously described. After 28 days of culture, the number of newly developed shoots per explant longer than 3 mm was recorded. Due to inconsistent sizes of the new rosettes, plant height was not evaluated. For

Rooting of shoots and ex vitro transfer The newly formed shoots (2–3 cm in length) were excised from the original explants and transferred to a root induction medium. For rooting, a half-strength MS medium supplemented with various concentrations of NAA (0.54–2.69 lM) or indole-3-acetic acid (IAA) (0.57–2.85 lM) was used.

After 28 days, the number of rooted plants as well as number and length of roots were evaluated. For the rooting experiment, a total of 20 explants were used per treatment in two replications. The experiment was arranged in a completely randomized design.

A total of 120 well-rooted shoots, each with at least two roots minimally 1 cm in length, were transferred to ex vitro conditions. The plants were removed from the flasks and rinsed with water to remove the adhering medium from the roots. The plants were transferred to 6 cm pots containing garden substrate (AGRO CS S.A., Czech Republic) and perlite (1:1). The pots were covered with transparent polyethylene foil to maintain high air humidity (80–90 %), temperature (ca. 25/20 °C day/night temperature), and placed into a glass house. After 2 weeks, the foil was gradually removed; from the 3rd week, the plants were

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exposed to normal glass house culture conditions (60–70 % air humidity, temperature ca. 20/17 °C day/night). The survival rate of the plants was recorded 8 weeks after ex vitro transfer. Because of their size, after 20 weeks the plants were transplanted to larger pots (12 cm-diameter) containing the same substrate.

DNA extraction and ISSR analysis Molecular analysis was carried out in ten randomly chosen plants taken from the most effective treatment for in vitro propagation, and a control plant. For ISSR analysis (Zietkiewicz et al. 1994), genomic DNA was extracted from fresh leaves using the cetyltrimethylammonium bromide (CTAB) protocol according to Doyle and Doyle (1989).

DNA quality was determined by 0.8 % agarose gel electrophoresis and using a Nanodrop Spectrophotometer (Thermo Scientific, USA). The final concentration of all DNA samples was adjusted to 20 ng/ll for PCR, and stored at -20 °C. Out of 30 tested primers (Integrated DNA Technologies, Belgium), 20 produced at least three clear and scorable bands, and these primers were used for further PCR analysis. DNA amplifications using the PCR were carried out in a reaction volume of 20 ll with the following composition: 10 ll 29 PPP Master Mix [150 mM Tris– HCl, pH 8.8 (25 °C), 40 mM (NH4)2SO4, 0.02 % Tween 20, 5 mM MgCl2, 400 lM dATP, 400 lM dCTP, 400 lM dGTP, 400 lM dTTP, 100 U/ml Taq-Purple DNA polymerase, monoclonal antibody anti-Taq (38 nM), stabilisers and additives (Tob-Bio, Czech Republic)], 7.3 ll PCR H2O (Top-Bio, Czech Republic), 2 ll template DNA (20 ng/ll), 0.5 ll primer (0.1 lM), and 0.2 ll bovine serum albumin (BSA) (20 mg/ml) (Thermoscientific, Lithuania).

PCR reactions were carried out in a T100TM Thermal Cycler (Bio-Rad, USA), programmed with an initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min.; annealing temperatures ranged from 47 to 53 °C (Table 3) for 1 min, and extension at 72 °C for 2 min, with the final extension at 72 °C for 8 min.

Amplified products were electrophoretically separated on agarose gel, prepared from 2 g agarose dissolved in 100 ml of 1 % TBE buffer (P-Lab, Czech Republic), and stained with ethidium bromide (Carl Roth GmbH, Germany). The final concentration of ethidium bromide in the gel and running buffer was 0.5 lg/ml. Gels were run at 55 V for about 2.5 h at 4 V cm-1. The amplified stained products were visualized on a gel with a UV transilluminator (Cleaver Scientific, UK). The size of the amplified products was estimated using a GeneRuler 100 pb Plus DNA Ladder (Thermoscientific, Lithuania). ISSR profiles were scored visually as either having the presence (1) or absence (0) of bands in the gel. Only clear, distinct bands were scored.

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Flow cytometric analysis The relative DNA content of ten regenerated plants, obtained from the most efficient treatment for in vitro propagation, was assessed by flow cytometry. Samples for analysis were prepared following a simplified two-step procedure according to Dolezel et al. (2007) using Otto buffers and 40 ,6-diamidino-2-phenylindole (DAPI) as a fluorescent dye. As an internal standard, Glycine max cv.

Polanka (2C = 2.50 pg; Dolezel et al. 1994) was used. For each sample, the relative fluorescence intensity of at least 3000 nuclei was measured using a CyFlow Space flow cytometer (Partec GmbH, Mu¨nster, Germany). The data were analysed using FloMax software, 2.4d (Partec GmbH, Mu¨nster, Germany). For each analysed sample, the DNA ratios were counted by dividing the mean of the G1 peak of the studied plant by the mean of the G1 peak of the internal standard.

Chromosome counts Chromosome numbers were determined in the somatic cells of actively growing root tips using the protocol of Dyer (1963). The root tips, excised from a randomly chosen ex vitro plant, were pre-treated with a saturated solution of p-dichlorbenzene for 3 h. Thereafter, the samples were fixed in ice-cold 3:1 ethanol:acetic acid fixative overnight, macerated in a 1:1 hydrochloric acid:ethanol for 30 s, rinsed in water, and stained with lacto-propionic orcein. The root-tip chromosome counts were determined using a Carl Zeiss NU microscope (Jena, Germany) equipped with an Olympus Camedia C-2000 Z camera (Tokyo, Japan).

Statistical analysis The statistical evaluation of the data obtained from the micropropagation was performed by analysis of variance (ANOVA), and the significantly different means were identified by using Tukey’s HSD test at the 5 % level of significance (P B 0.05) (STATISTICA 12.0, StatSoft, Inc. (2013), USA).

Results Formation of adventitious shoots Adventitious shoots started to form at the basal part of explants from the 2nd week of culture in most of the treatments. Thereafter, the shoots developed asynchronously within the experiment duration, resulting in formation of a cluster composed of rosettes of various sizes.

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Table 1 Effect of plant growth regulators on adventitious shoot formation in Puya berteroniana after 28 days of culture

Root induction and ex vitro transfer

PGR (lM)

Shoots obtained from the multiplication experiment did not develop roots, not even those in vitro regenerants cultivated on media supplemented with a combination of auxin and cytokinins.

For rooting, three concentrations of NAA and IAA were tested, and roots started to develop after 1 week of cultivation. After the 4th week, the greatest number of roots per explant was produced on a medium containing 2.69 lM NAA (6.9), followed by a medium supplemented with 1.61 lM NAA (4.6). However, increasing the concentration of NAA decreased the length of the roots which developed. Thus, a concentration of 2.69 lM NAA, which provided the greatest number of roots, also produced the shortest roots (0.8 cm). The plants on the medium with 1.61 lM NAA had a well-balanced ratio between the number and length of the roots (Fig. 1e). The percentage of rooting in treatments 1.61 lM NAA and 2.69 lM NAA was comparable (92.5 % for 1.61 lM NAA, and 97.5 for 2.69 lM NAA, respectively).

The most effective IAA concentration tested was 1.71 lM providing the highest number of roots per explant (3.9). The roots in this treatment were the longest of the entire rooting experiment (2.0 cm). Nevertheless, the root percentage was rather low, reaching only 65 % (Table 2).

With the exception of the control, all tested treatments provided satisfactory rooting efficiency enabling ex vitro transfer of the plantlets. A total of 120 well rooted shoots, each with at least two roots that were minimally 1 cm in length, were transferred to ex vitro conditions (Fig. 1f, g).

The survival rate of plants after 8 weeks of culture in a glass house reached 98.3 %. No morphological abnormalities were

BA

Zeatin

NAA

Number of shoots per explant (mean ± SE)

0

0

0

1.2 ± 0.3 cde

0.04

1.1 ± 0.1 cde

0.44

5.5 ± 0.9 a

1.33

4.0 ± 0.4 ab

2.22

1.2 ± 0.1 cde

0.04

0.54

1.4 ± 0.3 cde

0.44

0.54

2.6 ± 0.5 bc

1.33

0.54

3.0 ± 0.6 bc

2.22

0.54

1.6 ± 0.2 cde

0.05

0.2 ± 0.1 e

0.46

2.4 ± 0.3 bcd

1.36

2.4 ± 0.5 bcd

2.28

2.4 ± 0.8 bc

0.05 0.46

0.54 0.54

1.0 ± 0.2 cde 0.5 ± 0.2 de

1.36

0.54

1.7 ± 0.3 cde

2.28

0.54

1.8 ± 0.4 cde

Mean values in a column, followed by different letters, were significantly different according to the Tukey’s HSD test (P B 0.05) PGR plant growth regulator, BA 6-benzyladenine, NAA a-naphthaleneacetic acid, SE standard error

After 28 days of cultivation, plants grown on a medium supplemented with 0.44 lM BA produced the highest number of new shoots per explant (5.5) (Fig. 1d). Lower and higher concentrations of this cytokinin decreased shoot multiplication (Table 1). Although statistically significant differences were not detected for 0.44 and 1.33 lM BA, those shoots which developed on a medium with the addition of 0.44 lM BA showed better growth without morphological abnormalities. Shoots grown on media with higher concentrations of BA were vitrificated.

Overall, BA proved to be more effective for in vitro propagation of P. berteroniana than did zeatin. The latter cytokinin provided the highest number of new adventitious shoots at the highest concentration tested, 2.28 lM (2.6).

However, this result was still significantly lower than that from the most effective concentration of BA. When the effect of auxin on adventitious shoot formation was investigated, no positive response was observed.

The combination of NAA with BA or zeatin led to the formation of comparable or even a lower numbers of shoots when compared to treatments without auxin. The addition of NAA to 0.44 lM BA significantly decreased the average number of shoots per explant.

Table 2 Effect of auxin on in vitro rooting in Puya berteroniana after 28 days of culture Auxin (lM) NAA

IAA

0

0

Rooting (%)

No. roots per shoot (mean ± SE)

Root length (cm) (mean ± SE)

27.5

0.6 ± 0.1 d

1.1 ± 0.3 abc

0.54

55.0

2.7 ± 0.6 bcd

1.9 ± 0.3 a

1.61 2.69

92.5 97.5

4.6 ± 0.5 ab 6.9 ± 0.7 a

1.1 ± 0.1 bc 0.8 ± 0.1 c

0.57

57.5

2.0 ± 0.3 cd

1.6 ± 0.3 ab

1.71

65.0

3.9 ± 1.0 bc

2.0 ± 0.2 a

2.85

62.5

2.6 ± 0.3 bcd

1.0 ± 0.2 bc

Mean values in a column, followed by different letters, were significantly different according to the Tukey’s HSD test (P B 0.05) NAA a-naphthaleneacetic acid, IAA indole-3-acetic acid, SE standard error

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Table 3 List of ISSR primers, annealing temperatures, numbers and sizes of the amplified fragments No.

Primer code

Primer sequence (50 –30 )a

Annealing temperature (°C)

Total no. bands amplified

1

‘UBC807’

(AG)8T

49.3

44

2

‘UBC809’

(AG)8G

50

77

3 4

‘UBC810’ ‘UBC812’

(GA)8T (GA)8A

48 47

5

‘UBC813’

(CT)8T

6

‘UBC814’

(CT)8A

7

‘UBC815’

8 9

No. scorable bands per primer

No. and frequency of polymorphic bands

Range of amplification (pb)

4

0

200–550

7

0

200–1200

66 33

6 3

0 0

350–1200 550–750

49.3

33

3

0

880–1380

53.6

44

4

0

450–700

(CT)8G

47

33

3

0

450–700

‘UBC816’

(CA)8T

49.3

44

4

0

400–750

‘UBC824’

(AG)8G

47

55

5

0

550–850 300–550

10

‘UBC826’

(AC)8C

50

33

3

0

11

‘UBC827’

(AC)8G

47

33

3

0

450–700

12

‘UBC834’

(AG)8YT

47

66

6

0

550–750

13

‘UBC840’

(GA)8YT

51.3

66

6

0

480–900

14

‘UBC843’

(CT)8RA

49.4

77

7

0

380–1500

15

‘UBC844’

(CT)8RC

51.3

66

6

0

400–1150

16

‘UBC845’

(CT)8RG

49.4

77

7

0

280–1800

17

‘UBC847’

(CA)8RC

49.4

44

4

0

450–1500

18 19

‘UBC848’ ‘UBC859’

(CA)8RG (TG)8RC

53 49.4

44 44

4 4

0 0

700–1100 300–1500

20

‘UBC866’

(CTC)6

Total

–

a

53 –

66

6

0

850–1750

1045

95

0

–

Single letter abbreviations for mixed-base positions: Y = (C,T), R = (A,G)

observed. The plants grew continuously and new leaves were produced. Plants did not develop any further adventitious shoots under glass house conditions. After 20 weeks, the plants were transplanted to larger pots (Fig. 1h).

ISSR analysis The genetic stability of regenerated shoots was assessed by ISSR primers. Twenty ISSR primers of the 30 primers initially tested, produced at least three scorable and clear bands, and these primers were used for further analysis. Ninety-five amplification fragments were generated for each plant, and fragment sizes ranged from 200 to 1800 bp (Table 3). The number of bands per primer varied from three to seven, with an average of 4.6. A total of 1045 bands were generated using ISSR primers, giving rise to monomorphic patterns across 10 in vitro regenerants and control plant analysed, indicating no genetic variation among samples. Representative monomorphic amplification patterns obtained with ISSR primer ‘UBC843’ are shown in Fig. 2.

Flow cytometric analysis and chromosome counting Genetic stability of in vitro regenerants was additionally assessed with flow cytometry. In all cases, linear histograms

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of relative nuclear content showed two peaks: the first representing nuclei in the G0/G1 phase of the cell cycle, and belonging to the Puya sample; the second corresponding to nuclei of the internal standard Glycine max cv. Polanka in the G0/G1 phase (Fig. 3a, b). No significant differences in DNA-ratios were detected among in vitro regenerants (0.344–0.351) and control plant (0.345), suggesting that the plants obtained via direct shoot multiplication had an unchanged ploidy level. Coefficients of variation for all measurements ranged between 3.8 and 5.1. Karyological analysis revealed that ploidy level detected by flow cytometry corresponds to 2n = 50 chromosomes (Fig. 3c).

Discussion For in vitro propagation of P. berteroniana, cytokinins BA and zeatin alone, or in combination with NAA, were used.

The best results were obtained using BA at a relatively low concentration (0.44 lM). This cytokinin had been reported to be optimal for bud induction in many species of Bromeliaceae, for example, Vriesea scalaris (da Silva et al. 2009), V. fosteriana (Mercier and Kerbauy 1992), Hohenbergia penduliflora (Perez et al. 2013), and Neoregelia cruenta (Carneiro et al. 1999). In contrast to our experiment, higher

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Fig. 2 ISSR profile of control plant and in vitro regenerants (1–10) of Puya berteroniana using primer ‘UBC843’ (L ladder, C control plant)

concentrations ranging from 4.44 to 22.19 lM BA were more effective in shoot proliferation of these species.

Perez et al. (2013) found that the addition of NAA further increased the proliferation rate induced initially by BA in Hohenbergia penduliflora. Most of the in vitro propagation protocols for Bromeliaceae plants are based on a combination of BA and NAA at various concentrations.

In some species, NAA is not essential to increase the shoot proliferation rate; rather, as Mercier and Kerbauy (1992) suggested, to stop adventitious proliferation induced by BA, and to re-establish apical growth of the shoots. Unlike these studies, in P. berteroniana, the addition of auxin in the medium did not lead to an improvement of the propagation rate, or to elongation of shoots (data not shown).

This indicated that a combination of PGRs in this species did not play a key role in the process of propagation. A low concentration of BA provided sufficient numbers of shoots, without any need to use auxins for elongation of shoots.

The effect of zeatin as an alternative cytokinin to BA on induction of adventitious shoot was also tested. For many plants from various botanical families, for example, for Cymbalaria muralis (Thwe et al. 2012), Drosera intermedia (Rejthar et al. 2014), or Rubus pubescens (Debnath 2004), zeatin represents a much more efficient solution than other cytokinins. Nevertheless, in P. berteroniana, zeatin was observed to be less effective in the formation of adventitious shoots when compared to BA. According to Bairu et al. (2007), where the effects of various cytokinins on shoot formation in Aloe polyphylla were compared, BA produced a greater number of shoots than zeatin, up to

concentration of 7.5 lM; in higher concentrations, zeatin exceeded the effect of BA. In our experiment, an increasing tendency toward shoot formation was observed with higher concentrations of zeatin. Since zeatin represents a less affordable cytokinin than BA (Bairu et al. 2007), further increasing its concentration would not be economically reasonable, especially when BA was effective at very low concentrations.

Adventitious shoots developed from individual explant showed different sizes depending on the time of their formation. These findings were congruent with the observations of Pickens et al. (2006), which showed, using light microscopy, that bud development of Tillandsia eizii was asynchronous. This fact in P. berteroniana complicated the statistical evaluation of the plant height. Thus, in our study we did not carry out this measurement.

Rooting efficiency varied depending on the auxin and the concentration used. The best rooting was achieved on a medium with 2.69 lM NAA or 1.61 lM NAA. The plants on the former medium had the greatest number of roots, but they were rather small. The latter medium provided a wellbalanced ratio between the number and length of the roots, enabling easier manipulation with the plants within ex vitro transfer. However, satisfactory results were obtained by both tested auxins (NAA, IAA) at all concentrations. Martins et al. (2013) compared the rooting efficiency of NAA and indole-3-butyric acid (IBA) in Neoregelia concentrica and found that NAA accelerated and increased root development; 3.22 lM was the most promising concentration for rooting. The same concentration was recommended by

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Fig. 3 Flow cytometric and karyological analysis of Puya berteroniana in vitro regenerants. a, b Representative flow cytometric histograms of DAPI stained nuclei obtained from in vitro plantlets of P. berteroniana: a control plant, b randomly chosen in vitro plantlet.

Peak indicated as ‘‘*’’ corresponds to the internal reference standard (Glycine max). c Chromosomes of a root tip cell from ex vitro plant (2n = 50) (bar = 5 lm)

Perez et al. (2013) for Hohengergia penduliflora; providing enough roots per shoot and adequate root length. However, a similar NAA concentration in P. berteroniana radically decreased root length, and proved to be less suitable than lower NAA concentrations. In Vriesea scalaris, the formation of roots was observed even without auxin (da Silva et al. 2009). Martins et al. (2013) attributed this phenomenon to the higher rhizogenic potential of some bromeliads. However, P. berteroniana did not respond to an absence of auxin with rooting, suggesting that auxin was indispensable for root induction in this species.

After in vitro culture, genetic stability of regenerated plantlets was assessed using ISSR primers. No genetic variability among regenerants of P. berteroniana was detected.

Comparable to this study, ISSR was found to be a reliable method for the genetic analysis of in vitro regenerants in many species. It was successfully used to assess genetic stability in regenerated shoots from nodal segments of Alhagi maurorum (Agarwal et al. 2015), in multiplied shoots from the capitulum explants of Gerbera jamesonii (Bhatia et al.

2011), in shoots proliferated from the apex of 17 genotypes of Zea mays (Ramakrishnan et al. 2014), and in plants of Solanum trilobatum regenerated via direct shoot organogenesis using nodal and shoot tip explants (Shilpha et al. 2014). In Rhaponticum carthamoidese, ISSR analysis identified the heterogeneity of plants regenerated from callus culture via shoot organogenesis (Skala et al. 2015). In Smallanthus sonchifolius, ISSR proved to be a powerful technique to reveal genetically distinct in vitro plantlets regenerated through indirect somatic embryogenesis. Using this method, several new somaclones were selected for further breeding purposes of this species (Viehmannova et al. 2014). In our study, an identical set of ISSR primers were used. However, they did not reveal any genetic variability, indicating the uniformity of in vitro regenerants of P. berteroniana.

The data obtained by ISSR were further completed with flow cytometric analysis in order to reveal the potential occurrences of genomic changes. The results confirmed ploidy stability of in vitro regenerants. In most cases, somaclonal variation was associated with plant regeneration via indirect morphogenesis (Bairu et al. 2011); indeed, variations of relative DNA content were detected in plants of Titanotrichum oldhamii regenerated via indirect shoot organogenesis, while direct shoot formation in the same species did not result in any ploidy alterations (Takagi et al.

2011). In vitro regenerants of Populus x beijingensis obtained through somatic embryogenesis using antherderived callus showed various ploidy levels (Huang et al.

2015). Neither direct shoot organogenesis can exclude ploidy variation, as shown in Cucurbita pepo in vitro regenerants from cotyledon explants, where numerous mixoploids were detected (Kathiravan et al. 2006). Thus, it is highly advisable to complete each micropropagation protocol with a reliable system of assessment of in vitro raised plants in order to determine the success of a protocol. In our experiment, all tested regenerants were detected to be diploids, confirming that the micropropagation system optimized here produces plants of stable ploidy level.

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Conclusion This is the first report on the micropropagation of P. berteroniana, a plant with great ornamental potential. The protocol optimized here represents a reliable method for the propagation of true-to-type plants. Given that factors such as the length of in vitro culture or the concentration of PGRs may influence the occurrence of genetic changes (Bairu et al. 2011; Pierik 1987), the risk of somaclonal variation can be minimized by application of an

Plant Cell Tiss Organ Cult (2016) 125:293–302

appropriate micropropagation system. The protocol described in this study represents a relatively safe method, consisting of short-term (28 day) cultivation of shoots on a medium with a very low concentration of BA (0.44 lM).

By combining techniques revealing both molecular and genomic changes, an effective system of the assessment of in vitro regenerants was developed.

The protocol described here could be employed for effective mass propagation of P. berteroniana for commercial and conservation purposes, and it can serve as a first step for optimization of in vitro propagation of other ornamental Puya species.

Acknowledgments The authors are grateful for assistance with the chromosome counting provided by Vlasta Jarolimova (Institute of Botany ASCR). This research was financially supported by the Internal Grant Agency of the Czech University of Life Science Prague CIGA (Project No.20144207), and the Internal Grant Agency of Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague IGA (Project No. 20145020).

Authors contribution I.V. conceived the idea, designed the experiments, statistically analysed the results and prepared the manuscript, P.H.C. conducted molecular analysis, J.V. analysed samples using flow cytometer, and P.S. and J.K. carried out in vitro experiments.

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