Introduction

The genus Chirita (Gesneriaceae) is made up of about 140 species, mainly distributed throughout Southeast Asia (Wood 1974; Wang et al. 1990; Wang et al. 2011). Many Chirita species have ornamental value and are also used as medicinal plants (Harborne 1966; Damtoft and Jensen 1994; Zhou et al. 2001; Chen et al. 2011). Chirita swinglei (Merr.) W. T. Wang, mainly found growing in Guangxi and Guangdong provinces in China, is an evergreen perennial herb with glossy green foliage and baby blue or purple corollas. It is used as a potted ornamental plant and is also an important medicinal herb in South China, and the whole plant is used to treat ulcers and poisoning (Wang et al. 1990). C. swinglei generally reproduces by seed or is propagated through stem cuttings. However, these two methods are slow and rely on collecting or cutting mother plants. To strengthen the conservation, development, and utilization of this plant resource, it is important to establish an efficient in vitro micropropagation and plant regeneration protocol. Only a single tissue culture and plant regeneration protocol via adventitious shoot regeneration exists for C. swinglei (Yu et al. 2012). Callus-mediated induction of shoots has never been reported for this plant. In the Gesneriaceae, a callus-mediated regeneration protocol could be useful in genetic improvement programs through transgenic engineering, for example for the genus Saintpaulia (Kushikawa et al. 2001).

Studies on the in vitro propagation of members of the Gesneriaceae have mainly focused on the induction of adventitious shoots or somatic embryos (Dolendro et al. 2003; Ma et al. 2010; Yang et al. 2012, 2014). In this study, we established efficient protocols for high-frequency regeneration via two pathways, direct and indirect (i.e., callus-mediated) shoot organogenesis, from C. swinglei leaf explants. To be able to conserve plants with great value and low reproductive ability, it is useful to have alternative pathways that can lead to high-frequency and rapid regeneration. In addition, callus-mediated shoot formation is not only a useful method for plant propagation but also a powerful tool for plant genetic improvement, germplasm conservation, and production of useful compounds. We also investigated the effects of different types and concentrations of plant growth regulators (PGRs) on the induction of each of these pathways and studied the histology underlying shoot organogenesis.

Materials and Methods

Culture establishment

C. swinglei explant material was obtained from South China Botanical Garden in Guangzhou, China. About 20 immature leaves from one-yr-old mother plants were collected as explants and were washed under running tap water for 15 min. Excised leaves were surface sterilized with 70% (v/v) ethanol for 15 s, rinsed in sterile distilled water, and dipped in 0.1% (w/v) mercuric chloride solution for 10 min followed by five or six rinses with sterile distilled water. After that, they were placed on sterile filter paper and allowed to dry briefly in air. The leaves were cut into 0.5 cm2 explants and inoculated adaxial-side down on MS basal medium (Murashige and Skoog 1962). The pH of MS basal medium, which contained 30 g L−1 sucrose, was adjusted to 5.9 before being solidified with 0.7% (w/v) agar (Sigma-Aldrich, St. Louis, MO) and autoclaved at 121°C for 15 min. Culture jars (10 cm high and 6 cm in diameter) were placed in an air-conditioned culture room at 26 ± 2°C with a 12-h photoperiod under 100 μM m−2 s−1 fluorescent light (Philips, Tianjing, China). These culture conditions were identical for all experiments.

Effect of single PGRs on induction of shoot buds, adventitious shoots and callus from leaf explants

Leaf segments were inoculated adaxial-side down on MS medium supplemented with 0.5 or 2.0 μM of a single PGR: 6-benzyladenine (BA), α-naphthaleneacetic acid (NAA), thidiazuron (TDZ), or 2,4-dichlorophenoxyacetic acid (2,4-D) (Sigma-Aldrich). Induction of shoot buds, shoots, and callus was investigated after culture for a total of 45 d (Table 1). Among the PGRs tested, TDZ was filter-sterilized through a 0.24-μm filter and added after autoclaved medium had cooled while 2,4-D, BA, indole-3-butyric acid (IBA), and NAA were added prior to autoclaving at 121°C for 15 min.

Table 1. Effect of different PGRs on induction of shoot buds, adventitious shoots, and callus from leaf explants of Chirita swinglei
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Effect of NAA, BA and TDZ concentrations on induction of shoot buds, adventitious shoots and callus from leaf explants

We studied the effects of different PGRs (NAA, BA, and TDZ) concentrations and their combinations (selected by an orthogonal test; Fang and Ma 2001) on the induction of shoot buds, adventitious shoots, and callus from leaf explants. Leaf segments were inoculated adaxial-side down on MS medium supplemented with different concentrations of PGRs. Induction of shoot buds, adventitious shoots, and callus was investigated after 45 d of culture (Table 2).

Table 2. Effect of different combinations of NAA, BA, and TDZ on induction of shoot buds, adventitious shoots, and callus from leaf explants of Chirita swinglei
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Shoot proliferation

Adventitious shoots induced during the single PGR experiment were transferred to MS medium containing different concentrations and combinations of BA, NAA, and TDZ for shoot proliferation, which was investigated after culture for 45 d (Table 3). The shoot proliferation coefficient (SPC) was calculated as the ratio of the number of adventitious shoots after inoculation/number of adventitious shoots before inoculation.

Table 3. Effect of different of PGRs on shoot proliferation of Chirita swinglei
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Morphogenesis from different callus types

Type I (pink and friable), II (white and compact), and III (green-yellow and compact) callus were separated from cultured leaf blades and cut into 8–9 mm diameter callus clumps that were transferred to MS medium supplemented with 1.0 μM TDZ to observe callus differentiation (i.e., their ability to differentiate into shoots). All culture jars were placed in the dark for 10 d then transferred to a culture room with a 12-h photoperiod under 100 μM m−2 s−1 fluorescent light. After culture for 60 days, callus differentiation was investigated.

Root formation and acclimatization

Shoots with 2–3 leaves that had reached 2–3 cm in height on MS medium supplemented with 2.0 μM BA and 0.5 μM NAA were isolated from multiple shoot clusters, cut off at the base, and transferred to half-strength MS medium supplemented with 0.5 μM IBA, 0.5 μM NAA, 0.5 μM IBA, and 0.5 μM NAA or no PGRs. After culture for a total of 30 days on these media, root formation was investigated (Table 4). A total of 270 rooted plantlets with 4–5 leaves and 3–4 cm tall were removed from jars representing the above four treatments. The agar was gently washed off roots in tap water. Plantlets were transplanted to square plastic trays (50 cm × 40 cm × 10 cm) containing three substrates: sand, loessal clay, and sand: loessal clay: vermiculite (1:1:1, v/v). A total of 90 plantlets were planted per substrate type. The trays were placed in a makeshift greenhouse covered with a 90% black net and watered every 2 d with half-strength MS. The temperature in the greenhouse was 27 ± 2°C. Plant survival (%) was assessed after 30 d (Table 5).

Table 4. Effect of PGRs in rooting media (half-strength MS) on the rooting of Chirita swinglei
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Table 5. Effect of different culture media on plantlet survival of Chirita swinglei.
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Histology of shoot organogenesis from in vitro leaves

To study the ontogeny and development of C. swinglei shoots, leaf explants were incubated on shoot induction medium (SIM) supplemented with 2.0 μM TDZ, which was the optimal concentration shown to induce shoot buds in the single PGR experiment. After culture for 15, 25, and 35 days, the leaves were fixed in FAA (37% (w/v) formaldehyde:glacial acetic acid:glycerol 1:1:18 (v/v/v) in 50% (v/v) alcohol), solution at room temperature for more than 48 h, then transferred to 70% (v/v) alcohol for storage until analysis. The samples were dehydrated through an alcohol series and embedded in paraffin. Longitudinal sections 10 μm thick were made with a paraffin-compatible microtome (Fourth Shanghai Medicine Manufacturing Co., Shanghai, China). The sections were observed under a microscope (Leica DM1000 LED, Wetzlar, Germany).

Statistical analyses

All experiments were repeated three times within a 2-wk interval. Each treatment contained six explants per culture jar and five jars per treatment. The data were reported as mean ± SD (standard deviation). Percentage values were arcsine transformed prior to analysis. Means were statistically analyzed by one-way analysis of variance (ANOVA), and treatment means were considered to be significantly different from controls by Duncan’s multiple range test at P ≤ 0.05 using SPSS v. 19.0 (IBM, New York, NY).

Results

Effect of single PGRs on induction of shoot buds, adventitious shoots and callus from leaf explants

Significant differences were observed in adventitious shoot formation among the different PGRs or PGR concentrations (Table 1). On SIM supplemented with 0.5–2.0 μM BA, many adventitious shoot buds developed directly on the leaf surface and leaf blade (Fig. 1A ). On SIM containing 2.0 μM TDZ, leaf explants became swollen after culture for 15 d. Globular-shaped shoot buds were observed after 20 (Fig. 1B ) and 25 d of culture (Fig. 1C ). These enlarged by 35 d (Fig. 1D ) and formed distinct leaf initials after culture for 45 d (Fig. 1E ). The shoot buds developed into complete shoots that could be harvested when culture period was prolonged to 60 d (Fig. 1F ). The response of shoot buds reached 93.3% and an average of 17.1 shoot buds per explant on medium containing 2.0 μM TDZ. A lower concentration of TDZ and BA (0.5 μM) could only induce fewer shoot buds but many adventitious shoots whereas a higher concentration of TDZ or BA (2.0 μM) induced both shoot buds and adventitious shoots.

Figure 1.
figure 1

Plant regeneration via shoot organogenesis from leaf explants of Chirita swinglei (bars, 2 mm). (A) Adventitious shoots were induced directly from the leaf surface on MS medium containing 0.5 μM BA after culture for 30 d. (B and C) Globular shoot buds (white arrows) were induced on MS medium with 2.0 μM TDZ for 20 and 25 d, respectively. (D) Shoot buds with different shapes were induced on MS medium containing 2.0 μM TDZ after 35 d of culture. (E) Larger shoot buds (white arrows) were induced on MS medium supplemented with 2.0 μM TDZ for 45 d. (F) A shoot developed from a shoot bud on MS containing 2.0 μM TDZ after culture for 60 d. (G) Adventitious shoots (white arrow) were induced on the leaf surface and callus (black arrow) was induced on the lamina’s abaxial side on MS medium with 2.0 μM NAA when cultured for 30 d. (H) Shoot clusters proliferated on MS medium with 2.5 μM BA after 45 d. (I) Plantlets in a plastic tray containing sand, 30 d after transplanting from in vitro conditions.

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On medium supplemented with 0.5 or 2.0 μM NAA, callus was observed on the abaxial surface of leaf blades and at the same time, many adventitious shoot buds developed directly on the adaxial leaf surface (Fig. 1G ). This callus was defined as type I callus, which was pink, friable, and slow growing. As the culture period was prolonged, adventitious roots developed from callus. Leaf explants could not induce shoot buds, adventitious shoots, or callus on SIM supplemented with 0.5–2.0 μM 2,4-D, and generally became brown and necrosed.

Effect of NAA, BA and TDZ concentrations on the induction of shoot buds, adventitious shoots and callus from leaf explants

Culture on MS medium, supplemented with 2.5 μM BA and 2.0 μM TDZ, produced globular shoot buds, which were initially white, turned light green, and emerged in large numbers. Some shoot buds were observed after 40 d of culture. Thereafter, many shoot buds gradually developed into shoots: 100% of explants formed an average of 23.1 shoot buds per explant on this SIM (Table 2).

MS medium supplemented with 1.5 μM BA combined with a low concentration of NAA (0.5 μM) and TDZ (1.0 μM) produced 92.2% of responding leaf explants, and 14.9 adventitious shoots formed per explant. Callus was only induced on MS medium when NAA was included, forming two kinds of callus, which were defined as type II and type III callus.

Type II callus was compact, white, and slow growing (Fig. 2B ). The highest callus response was 58.9% on medium with 1.0 μM NAA and 2.0 μM BA. Only 34.4% callusing response was observed on medium supplemented with 0.5 μM NAA, 1.5 μM BA, and 1.0 μM TDZ (Table 2).

Figure 2.
figure 2

Callus induction and morphogenesis from different types of callus induced from leaf explants of Chirita swinglei. (A) Type I callus (pink and friable) was induced on medium supplemented with 2.0 μM NAA after 40 d. (B) Type II callus (white and compact) was induced on medium with 1.0 μM NAA and 2.0 μM BA after culture for 40 d. (C) Type III callus (green-yellow and compact) was induced on medium containing 1.0 μM NAA, 2.0 μM BA, and 2.0 μM TDZ after culture for 40 d. (D) Type III callus differentiated into adventitious shoots (white arrow) or shoot buds (black arrow) on medium supplemented with 1.0 μM TDZ after 55 d. Bars, 2 mm.

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Type III callus was green-yellow, compact, and grew the slowest among the three types of callus. In some cases, type II and type III callus were induced on the same medium and the amount of type III callus was higher than type II callus.

Shoot proliferation

Adventitious shoots grew slowly and SPC was 5.1 after culture for 45 d on PGR-free MS medium. After 45 days of culture, SPC reached 15.5 on medium containing 2.5 μM BA and was 12.7 on medium containing 2.5 μM TDZ (Fig. 1H ). On medium supplemented with 2.5 μM BA alone or in combination with 0.5 μM NAA, there was no significant difference in SPC. Similarly, there was also no significant difference in SPC when the medium was supplemented with 2.5 μM TDZ alone or combined with 0.5 μM NAA (Table 3).

Morphogenesis from different types of callus

MS medium supplemented with 1.0 μM TDZ, produced type I callus which gradually became brown and necrotic, and never differentiated. Type II callus showed no response within the first 15 d of culture, but after culture for 25 d, it began to differentiate and green granular structures became visible. As culture period was extended, 46.7% of callus produced adventitious shoots. Moreover, type II callus could convert into type III callus as culture period progressed. Type III callus began to proliferate rapidly then differentiated into adventitious shoots (shoot response was 55.6%) and green shoot buds (globular shoot bud response was 34.4%) by the 60th day of culture (Fig. 2D ).

Root formation and acclimatization

Plantlets could induce adventitious roots on all rooting media. However, root development was slow and the rooting response only reached 91.1% on half-strength PGR-free MS medium. 100% of plantlets could develop roots when medium contained 0.5 μM IBA, 0.5 μM NAA, or 0.5 μM IBA and 0.5 μM NAA (Table 4). On rooting medium with any auxin, callus was visible at the base of shoots, but this callus had no effect on plant survival during acclimatization. When plantlets were transplanted to sand 30 days later, 94.4% of plantlets survived after 30 d. Only 57.8% of plantlets could survive in loessal clay (Fig 1I , Table 5).

Histology of shoot organogenesis from in vitro leaves

Histological analysis revealed that meristematic tissues and some darkly stained primordial cell clumps were visible after 15 d of culture (Fig. 3A, B ). As culture period approached 25 days, some globular shoot buds were visible on both the abaxial and adaxial leaf surfaces (Fig. 3C, D , respectively). After leaf explants were cultured for 35 days, some adventitious shoots formed (Fig. 3E ). As culture period was prolonged, shoot buds formed leaves (Fig. 3F ).

Figure 3.
figure 3

Histology of shoot organogenesis on MS medium supplemented with 2.0 μM TDZ from leaves of Chirita swinglei. (A and B) Meristematic tissue was visible after culture for 15 d (white arrows). (C and D) Globular shoot buds (white arrows) were visible protruding from the leaf surface after culture for 25 d, (E) Adventitious shoots were visible after 35 d (white arrow). (F) Shoot buds (white arrow) after culture for 35 d. Bars, 0.2 mm.

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Discussion

In several species of the Gesneriaceae, shoot organogenesis has been reported. For example, the use of BA and kinetin is a popular means to propagate African violet (Saintpaulia ionantha) from leaf explants (Dewir et al. 2015). In Metabriggsia ovalifolia, MS medium with 5.0 μM TDZ and 0.5 μM BA could induce adventitious shoots from in vitro leaf explants and MS medium with 5.0 μM BA and 0.5 μM NAA could induce adventitious shoots from petiole explants (Ma et al. 2011a). In Titanotrichum oldhamii, adventitious shoots formed on medium with 0.1 μM BA from in vitro leaf explants (Takagi et al. 2011). In vitro leaf explants of Dayaoshania cotinifolia also induced adventitious shoots on MS medium with 1, 3, or 5 μM TDZ, BA or NAA alone (Yang et al. 2014). In vitro leaf explants of Chirita flavimaculata, Chirita eburnea, and Chirita speciosa formed adventitious shoots on MS medium containing 0.5 μM NAA and 0.4 μM BA (Nakano et al. 2009). MS medium supplemented with 0.5–2.0 μM NAA produced adventitious shoots formed from in vitro leaf explants of Lysionotus pauciflorus (Godo et al. 2010). Yu et al. (2012), using in vitro leaves as explants, noted that optimal SIM was MS medium supplemented with 0.5 μM BA and 0.1 μM NAA. The optimal differentiation medium was MS medium supplemented with 0.3 μM BA and 0.1 μM NAA. The optimal rooting medium was half-strength MS medium supplemented with 0.1 μM NAA and 1.0 g L−1 activated charcoal. In our experiments, BA, TDZ, and NAA could induce adventitious shoots from leaf explants (Fig. 4).

Figure 4.
figure 4

Simplified flow diagram of two regeneration pathways (direct and indirect) from Chirita swinglei leaf explants. Even though shoot buds and adventitious shoots are most likely the same organs (adventitious shoots), they form as distinct pathways with distinct periods of formation. They are both represented in the figure as Route 1 (i.e., direct shoot organogenesis).

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In other species of many plant families, previous reports have shown that TDZ can induce shoot organogenesis, including in Albizzia julibrissin (Hosseini-Nasr and Rashid 2002), Curcuma alismatifolia (Mahadtanapuk et al. 2006), Primulina tamiana (Padmanabhan et al. 2014), and Primulina dryas (Padmanabhan et al. 2015). In other cases, TDZ induced somatic embryos such as in Arachis hypogaea (Murch and Saxena 1999), Capsicum annuum (Khan et al. 2006), and Ricinus communis (Kumari et al. 2008). In Cajanus cajan, a low concentration of TDZ (0.05–1.0 μM) induced multiple shoots while a higher concentration (10 or 20 μM) induced somatic embryos (Dolendro et al. 2003). In Ochna integerrima, a higher concentration of TDZ (10.0–15.0 μM) could induce both somatic embryos and adventitious shoots whereas a low concentration of TDZ (5.0 μM) could only induce adventitious shoots (Ma et al. 2011b). Therefore, TDZ not only functions as a cytokinin but also as an auxin and can substitute for auxins or combinations of auxins and cytokinins (Murthy et al. 1998; Singh et al. 2003). Our results also showed that a higher level of TDZ or BA (2.0 μM) could induce both shoot buds and adventitious shoots, whereas a low level of TDZ and BA (0.5 μM) could only induce fewer shoot buds and many adventitious shoots (Table 1).

There are many reports that used 2,4-D to induce callus and plant regeneration. When MS medium was supplemented with 2,4-D combined with TDZ and BA, callus was induced and plant regeneration of Curcuma kwangsiensis took place (Zhang et al. 2011). MS medium containing 2.0 μM 2,4-D induced callus from the leaves of Lysionotus pauciflorus and showed fourfold proliferation in liquid medium after culture for 4 wk (Lu et al. 2006). In Curcuma attenuata, 4.5 μM 2,4-D could induce callus then differentiate into shoots (Kou et al. 2013). However, in this study, all explants generally become brown and necrotic on SIM with 2,4-D, which indicates that 2,4-D is not suitable for C. swinglei plant regeneration. However, NAA combined with cytokinins (BA and TDZ) could induce callus and be used for plant regeneration.

For plants with great ornamental or medicinal value but low reproductive ability, such as C. swinglei, a single tissue culture and plant regeneration protocol via adventitious shoot regeneration is sub-optimal for conservation purposes. The mass propagation and conservation of this medicinal ornamental herb would benefit from the establishment of multiple plant regeneration pathways. In addition, callus-mediated shoot regeneration is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques and is also an important technique to understand plant growth and development and mechanisms of cell differentiation. Our study expands on previous research on plant regeneration system of this species, which was limited to the simple induction of adventitious buds. This is the first efficient in vitro shoot induction and plant regeneration system for C. swinglei from young in vitro-derived leaf explants through two distinct pathways, namely indirect callus-mediated shoot organogenesis and direct shoot organogenesis (Fig. 4). This protocol provides a foundation for future transgenic and other biotechnological applications and also serves as a very practical and useful technique to mass propagate this ornamental and medicinal plant.