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Efficient protocol for in vitro propagation from bulb scale explants of Fritillaria ruthenica Wikstr. (Liliaceae), a rare ornamental species

  • Dinara S. Muraseva
  • Tatyana I. Novikova
Article

Abstract

An efficient protocol for in vitro propagation from bulb scale explants has been developed for Fritillaria ruthenica Wikstr., an endangered and rare species. The use of B5 medium supplemented with 5.0-µM 6-benzylaminopurine (BAP) and 2.0-µM α-naphthalene acetic acid (NAA) was found to be the most effective for adventitious bud induction and shoot multiplication (2.9 shoots per explant with 100% regeneration frequency). Adventitious regeneration occurred exclusively via direct organogenesis with the development of microshoots consisting of the rosette leaves, while bulb formation was not observed. The rooting microplants was carried out on a half-strength BDS hormone-free medium supplemented with activated charcoal at a concentration of 0.5 g/l resulted in 100% rhizogenesis with 5.2 roots per plantlet. Elevated sucrose concentrations (40.0 and 50.0 g/l) had no stimulating effect on bulblet differentiation at the in vitro rooting stage of F. ruthenica shoots. The regenerated plantlets were successfully transferred in a mixture of coconut fiber and sand (3:1) for acclimatization in greenhouse conditions with 72% of survival rate. The large bulbs developed only in ex vitro conditions at the end of the vegetation period.

Keywords

In vitro propagation Fritillaria ruthenica Rare species Scale explants Bulblet development 

1 Introduction

The genus Fritillaria L. (Liliaceae) comprises more than 150 species distributed within the temperate zone of the Northern Hemisphere (Rix 2001; Rønsted et al. 2005; Day et al. 2014). Primarily, fritillaries are widely known as ornamental plants. The first studies of their introduction have been mentioned since XVI century, and nowadays, the breeders have created a large number of varieties and garden forms. In addition, many of Asian fritillaries species are employed successfully in the traditional medicine as a source of valuable alkaloids (Lin et al. 2001; Xin et al. 2014).

Fritillaria ruthenica Wikstr. is a perennial, herbaceous, ephemeral bulbous plant growing in the forest-steppe and steppe zones, in the places of moderate moisture (along the steppe and meadow slopes, in forests). The species is distributed in the European Russia including the Central Black Earth Region, Caucasus, Southern Urals, Volga Region, some regions of Western Siberia, as well as in Ukraine and Kazakhstan’s North-West. F. ruthenica is listed in the Red Data Book of the Russian Federation as a rare species (Bardunov and Novikov 2008). This status is determined by the disturbance of the plant’s habitats due to agricultural practices and deforestation. An early flowering of F. ruthenica and unusual brownish-red, dark tessellated coloration of flowers can be considered as promising peculiarities for further breeding and propagation. In recent decades, biotechnological approach has become a priority in decision, the problems of mass propagation both valuable ornamental and medicinal geophytes, as well as their in vitro conservation (Teixeira da Silva and Dobránszki 2016).

Efficient propagation systems via direct/indirect organogenesis and somatic embryogenesis have been developed for many fritillaries species, including F. tubaeformis Gren. & Godr. (Carasso and Mucciarelli 2014), F. meleagris L. (Jevremović et al. 2010), F. imperialis L. (Mohammadi-Dehcheshmeh et al. 2008), F. thunbergii Miq. (Paek and Murthy 2002), F. unibracteata Hisao & K.C. Hsia (Gao et al. 1999), and others. However, the available micropropagation protocols are concerned with fritillaries mainly distributed in Europe and Western Asia (Petrić et al. 2012), whereas, for the species occurred in the Russian Federation, these studies are limited by a few reports (Erst and Erst 2011; Kulkhanova et al. 2015; Muraseva et al. 2015).

Thus, the present study was undertaken to develop an efficient protocol for rapid multiplication of F. ruthenica for mass propagation and ex situ conservation.

2 Materials and methods

2.1 In vitro initiation and shoot multiplication

Source material for in vitro culture establishment was bulbs gathered in Orenburg Oblast and planted in the collection of the Central Siberian Botanical Garden (No. USU_440534 “Collection of living plants indoors and outdoors”). The initial bulbs were maintained at 5 ± 2 °C for 3–4 weeks and used for in vitro culture induction in October–November. Surface sterilization of bulb scales was carried out by the technique developed earlier: bulb scales were immersed in 70% ethanol for 30 s, then in 0.1% HgCl2 containing 1 drop of Tween 80 per 100 ml for 30 min, and finally scales were rinsed three times in sterile distilled water (Erst et al. 2014; Kulkhanova et al. 2015). The aseptic scales were cut into the segments of 5 × 5 mm size and used as primary explants. Scale segments (for 4–5 pcs.) were placed by cut-surface down onto B5 induction medium (Gamborg and Eveleigh 1968) supplemented with 5.0-µM 6-benzylaminopurine (BAP) and 2.0-µM α-naphthalene acetic acid (NAA). By the end of the in vitro initiation stage (60 days), the regenerated microplants were transferred to media for multiplication.

At the shoot multiplication stage, B5 and BDS (Dunstan and Short 1977) media supplemented with cytokinins (BAP and thidiazuron, TDZ) at a concentration of 0.1–10.0 µM and auxin (2.0-µM NAA) were used. The pH was adjusted to 5.5 before autoclaving the medium at 121 °C for 20 min. All cultures were incubated at 23 ± 2 °C, under a light intensity of 54 μmol m−2 s−1 under a 16-h photoperiod. The regeneration frequency (%) and shoot number per explant were counted after 35–40 days at the end of the passage. The number of multiplication subcultures was limited to 9–10 cycles.

2.2 In vitro rooting and ex vitro acclimatization

At the in vitro rooting stage, half-strength hormone-free B5, BDS, or MS (Murashige and Skoog 1962) media and 1/2 BDS containing 1.5- or 2.5-µM NAA were tested. All media were supplemented with activated charcoal (0.5 g/l). Rooting experiments were carried out at a temperature of 7 °C, under a light intensity of 20 μmol m−2 s−1 in a light thermostat (Rumed, Germany) under a 16-h photoperiod. The rhizogenesis parameters (root and leaf number per plantlet, root length, and rosette width) were registered after 6–8 weeks.

To evaluate the sucrose effect as a trophic factor on differentiation and bulblet growth, regenerated shoots were transferred on hormone-free 1/2 BDS supplemented with 40.0- or 50.0-g/l sucrose. 1/2 BDS containing 30.0 g/l sucrose was applied as a control. The passage lasted 6–8 weeks at 7 °C and a light intensity of 20 μmol m−2 s−1 under a 16-h photoperiod.

Regenerated plants with a developed root system were transferred to ex vitro conditions for acclimatization. A mixture of shredded coconut fiber and sand (3:1) was used as a substrate. Potted plants were grown in the cold section of the greenhouse at 5–10 °C at the beginning this period (mid-January) under light intensity range from 41 to 75 μmol m−2 s−1 (natural light). For 2 weeks after the beginning of acclimatization, a high humidity (approximately 80–90%) was maintained to avoid wilting the leaves. The acclimatization stage lasted for the 2 months.

All experiments were repeated twice and a minimum of 15 microplants were used for each treatment. The multiple comparisons were performed using the one-way ANOVA followed by the Tukey’s HSD test to distinguish the statistical difference among the means using the R software environment (The R Foundation). The significance level accepted was P ≤ 0.05. Data are presented as mean ± standard error (M ± SE).

3 Results and discussion

3.1 In vitro initiation and shoot multiplication

The used surface sterilization method was recorded as being highly effective, the yield of aseptic explants being 81%. Development of buds on the explant surface was observed within 39–45 days after inoculation on the nutrient medium, whereas well-developed microbulbs were formed 2.5–3 weeks later. The morphogenic response of using explants on B5 induction medium supplemented with 5.0 µM BAP and 2.0 µM NAA was high (78%) which resulted in formation of 1.7 ± 0.2 buds per explant. Adventitious bud regeneration was noted in the vicinity of the cut surface. Morphological observation of de novo structures consisting of 2–3 rounded scales revealed the direct gemmogenesis processes in the primary explant tissues (Fig. 1a). Hereafter, the microplants obtained at the induction stage were used for further optimization of the shoot multiplication.
Fig. 1

Adventitious regeneration of Fritillaria ruthenica. a Direct organogenesis in the primary explant tissue, B5 supplemented with 5.0-µM BAP and 2.0-µM NAA. b Microshoots (arrows) formed at the multiplication stage, B5 supplemented with 5.0-µM BAP and 2.0-µM NAA. c Characteristic microshoot consisting of the rosette leaves (arrows) with enlarged base; BDS supplemented with 5.0-µM TDZ and 2.0-µM NAA

The one-way ANOVA which was performed did not show a statistical difference in the number of adventitious shoots depending on nutrient media (for all variant tested P > 0.05) (Table 1). Considering this, the most effective nutrient media were defined by the regeneration frequency. This parameter was higher on hormone-free B5 than on BDS—41 and 21%, respectively. The addition of 0.5-µM TDZ or 0.5-µM BAP to BDS increased regeneration frequency almost twofold in comparison with hormone-free BDS. In general, the presence of plant growth regulators on BDS stimulated regeneration; however, it was not exceeded 54%. Analysis of the influence of the growth regulators supplemented to B5 did not show a direct effect on regeneration frequency, because microshoot formation varied significantly. Maximum regeneration frequency 100% was obtained on B5 supplemented with 5.0 µM BAP and 2.0 µM NAA, that allowed to consider the medium to be the best for F. ruthenica micropropagation.
Table 1

Effect of the nutrient medium components on the regeneration of Fritillaria ruthenica adventitious microshoots

Growth regulators, µM

Medium

BDS

B5

Regeneration frequency, %

Shoot number per explant

Regeneration frequency, %

Shoot number per explant

Hormone-free (control)

21

1.3 ± 0.3

41

2.1 ± 0.2

BAP 0.1

15

1.8 ± 0.3

BAP 0.5

54

2.5 ± 0.5

BAP 5.0 + NAA 2.0

54

2.1 ± 0.2

100

2.9 ± 0.5

TDZ 0.1

37

2.0 ± 0

42

1.9 ± 0.2

TDZ 0.5

57

3.4 ± 0.8

TDZ 10.0 + NAA 2.0

41

3.4 ± 0.7

10

2.1 ± 0.3

Data present as M ± SE

– no data

In our previous study, the influence of mineral composition on shoot formation and morphogenesis type of F. meleagris culture was revealed. The cultivation of bulblets on B5 resulted in active gemmogenesis without callus formation, while on BDS indirect gemmorhizogenesis was obtained (Muraseva et al. 2015). On the contrary, an exclusive characteristic feature of F. ruthenica morphogenesis was the direct regeneration without callus formation on all nutrient media studied. At the multiplication stage, bulblet differentiation did not take place; only de novo development of shoots consisting of the rosette leaves with green linear or lanceolate lamina with enlarged base was observed (Fig. 1b, c). In vitro morphogenesis of F. ruthenica was differed considerably from those of F. sonnikovae and F. meleagris: for F. sonnikovae only bulblet development was characterized, whereas, for F. meleagris, both microbulblets and shoots were observed (Kulkhanova et al. 2015; Muraseva et al. 2015).

3.2 In vitro rooting and ex vitro acclimatization

Successful rooting of regenerated shoots of geophytes is carried out on both media supplemented with auxins and hormone-free nutrient media (Nasircilar et al. 2011; Yin et al. 2013; Teixeira da Silva and Dobránszki 2016). NAA and indole-3-butyric acid are often used for stimulating rhizogenesis of monocotyledonous geophytes (Bacchetta et al. 2003; Liu and Yang 2012).

The comparison of rooting data on hormone-free nutrient media showed that the rooting efficiency of F. ruthenica microplants was higher on 1/2 BDS and 1/2 MS, excluding the root length which did not have statistical difference between the media. Among the variant tested, maximum (100%) in vitro rooting frequency was achieved only on 1/2 BDS medium which induced high growth of root and shoot systems (Table 2). Addition of 2.5 µM NAA to 1/2 BDS was resulted in 100% rooting too. However, cultivation on this medium led to the decrease of the number of leaves per plantlet and rosette size, but did not have an effect on the number of root and their length. Decreasing the concentration of NAA to 1.5 µM inhibited both the rhizogenesis and plantlets development of F. ruthenica. In general, the high rooting frequency (more than 75%) as well as the number of roots were evidence of the successful rooting ability of F. ruthenica in all treatments tested.
Table 2

Effect of medium and NAA concentration on the growth and development of Fritillaria ruthenica plantlets at the in vitro rooting stage, 7 °C

Medium

Rooting frequency, %

Root number per plantlet

Root length, mm

Leaf number per plantlet

Rosette width, mm

½ MS

86

4.5 ± 0.4abc

20.0 ± 2.1ab

7.7 ± 0.9a

8.7 ± 0.4a

½ B5

94

2.4 ± 0.3bc

26.3 ± 1.6a

4.2 ± 0.3b

5.9 ± 0.6b

½ BDS

100

5.2 ± 0.7a

22.5 ± 2.2a

7.5 ± 0.8a

8.5 ± 0.5a

½ BDS + NAA 1.5 µM

75

1.0 ± 0bc

7.3 ± 1.6b

4.8 ± 0.6b

4.6 ± 0.2bc

½ BDS + NAA 2.5 µM

100

3.4 ± 0.5ab

31.9 ± 5.2a

4.0 ± 0.5b

5.4 ± 0.3bc

Data present as M ± SE

Means followed by different letters in the same column are significantly different (P ≤ 0.05) according to Tukey’s test

One of the main factors for in vitro propagation of bulbous plants is the differentiation and growth of a bulb, that define successful rooting and acclimatization of microplants to field conditions (Podwyszyńska 2012). Some researchers noted the positive effect of high sucrose concentration in a medium on in vitro fritillary bulb development and growth (Seon et al. 1999; Petrić et al. 2015). According to our data, increasing the sucrose concentration up to 40.0 or 50.0 g/l did not have an effect on number of leaves (4.8 ± 0.2 and 4.9 ± 0.3 leaves per plantlet correspondingly) but resulted in the enlargement of rosette width (5.7 ± 0.2 and 6.8 ± 0.6 mm correspondingly). However, as shown in Fig. 2, the morphometric bulb growth parameters in a control medium containing 30.0 g/l sucrose were significantly higher. Thus, in the present study, it was established that an elevated sucrose concentration (up to 50.0 g/l) did not stimulate the bulb formation process. Our data are not in agreement with the results that showed a positive effect of high sucrose concentrations on the bulb differentiation process reported for other bulbous geophytes (Takayama and Misawa 1979; De Klerk et al. 1992). Despite the high frequency of F. ruthenica rhizogenesis, the bulb development at the rooting stage was failed to stimulate.
Fig. 2

Influence of sucrose concentration on bulb differentiation of Fritillaria ruthenica microplants at the rooting stage (7 °C), 1/2 BDS. Data presents as M ± SE

De Klerk (2012) suggested a possible decision planting microshoots of bulbous species in a soil without the bulb differentiation stage that resulted in bulb formation during the first growing season. Considering the complexity of bulblet formation at the in vitro rooting stage, the rooted shoots of F. ruthenica were planted in the soil mixture for further bulblet development and acclimatization.

The bulblets from in vitro culture are known to be dormant, as a rule, and sprout only after maintaining at low temperature for some weeks (Langens-Gerrits et al. 2003). Activating growth processes at a low temperature reflects the normal seasonal rhythm of developing spring-flowering bulbous ephemeral plants characterized by the sprouting at the temperature transition up to 5 °C (Sedelnikova 2002). Taking into account the information mentioned above, after in vitro rooting at 7 °C, the regenerated shoots planted in the substrate for acclimatization were transferred to the cold section of the greenhouse where the average temperature was 8.5 ± 1.4 °C (mid-January). The leaves formed during in vitro cultivation gradually died off, and thus, the acclimatization degree was assessed by the number of plants with leaves formed ex vitro. The beginning of leaf emergence was observed 60 days after start of the acclimatization at 16 ± 1.4 °C and vegetation period lasted 53–66 days. Use of the mixture of shredded coconut fiber and sand allowed 72% of the plants to be obtained with well-developed leaves and root systems. After the death of the rosette leaves, a bulb differentiation with well-formed scales (2–3 pcs.) was observed. As a result, semitunicate bulbs (diameter 7.5–11.0 mm) consisted of the fleshy scales were formed (Fig. 3).
Fig. 3

In vitro rooting and ex vitro acclimatization stages of Fritillaria ruthenica microplants. a Microplants at the rooting stage, ½ BDS. b Rooted microshoots consisting of rosette leaves, ½ BDS. c Sprouting shoots in the mixture of shredded coconut fiber and sand (3:1), greenhouse. d Bulbs with well-developed scales formed after the end of vegetation period

In conclusion, we developed an efficient method for in vitro propagation of rare species F. ruthenica using bulb scales as explants. From media tested B5 medium supplemented with 5.0-µM BAP and 2.0-µM NAA was found to be optimal for stimulating a 100% shoot regeneration frequency and 2.9 ± 0.5 shoots per explant at the shoot multiplication stage. Adventitious shoot regeneration occurred via direct organogenesis, resulting in the formation of rosette leaves with an enlarged base. The highest rooting frequency and root number were achieved with 1/2 BDS hormone-free medium supplemented with activated charcoal (0.5 g/l) at 7 °C. The regenerated plants were acclimatized successfully in greenhouse conditions in a mixture of coconut fiber and sand (3:1). The development of large bulbs of F. ruthenica was observed only at the end of the vegetation period, so the propagation cycle occupied 9 months.

Since F. ruthenica is a rare species with ornamental properties, the present study can be used for commercial mass propagation as well as contribute to this plant’s conservation.

Notes

Acknowledgements

In our study, material from the collection of the Central Siberian Botanical Garden SB RAS—USU_440534 “Collection of living plants indoors and outdoors” was used.

Funding

The work was carried out with the financial support of the budgetary project of the Central Siberian Botanical Garden, SB RAS “Assessment of morphogenetic potential of North Asian plant populations by experimental methods” (no. AAAA-A17-117012610051-5) within the framework of the State Assignment.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

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Copyright information

© Accademia Nazionale dei Lincei 2018

Authors and Affiliations

  1. 1.Central Siberian Botanical Garden, Siberian Branch RASNovosibirskRussia

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