Plant Regeneration from Leaf-derived Callus Cultures of Primrose (Primula vulgaris)

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  • 1 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK; and John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
  • 2 John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
  • 3 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK; and John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
  • 4 John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
  • 5 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK; and John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK

Efficient micropropagation of Primula species is important both for fundamental scientific studies and commercial applications. Primula vulgaris (Huds), along with other Primulaceae species, exhibits floral heteromorphy with two distinct forms of hermaphroditic flower. Studies to identify genes that control heteromorphic flower development require propagation of floral mutants, and efficient regeneration is a key requirement for plant transformation. Several species, including P. vulgaris cultivars and P. ×polyantha hybrids, are important horticultural crops in Europe, United States, and Japan and semidouble/double Primula varieties offer a high-end product. Vegetative propagation of sterile double forms, and as a means to increase numbers of inbred parent plants for F1 seed production is, however, slow. Micropropagation offers the most efficient way of increasing these varieties quickly and efficiently. To date, most Primula micropropagation protocols require explant material derived from in vitro grown seedlings or use floral parts as donor material with seasonal limitations. Therefore, an effective and efficient protocol was developed for in vitro regeneration of P. vulgaris via indirect organogenesis from adult leaf–derived explants. Exposure of leaf explants of P. vulgaris to media containing synthetic cytokinin, thidiazuron (TDZ), and auxin [1-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)] resulted in undifferentiated cell proliferation and followed by differentiated growth as shoot organogenesis. Silver nitrate improved in vitro callus growth and increased shoot regeneration further, with up to 72% of explants producing shoots. Regenerated plants developed normally and produced normal fertile flowers within 7 months. The system was also successfully applied for the micropropagation of sterile double-flowered P. vulgaris ‘Sue Jervis’. The protocol reported here enables propagation of P. vulgaris without seasonal limitation or destruction of valuable parent donor material. The protocol, with further development, has the potential to underpin development of a transformation system for Primula, which would be of value in studies on flower development and disease resistance in laboratory grown plants.

Abstract

Efficient micropropagation of Primula species is important both for fundamental scientific studies and commercial applications. Primula vulgaris (Huds), along with other Primulaceae species, exhibits floral heteromorphy with two distinct forms of hermaphroditic flower. Studies to identify genes that control heteromorphic flower development require propagation of floral mutants, and efficient regeneration is a key requirement for plant transformation. Several species, including P. vulgaris cultivars and P. ×polyantha hybrids, are important horticultural crops in Europe, United States, and Japan and semidouble/double Primula varieties offer a high-end product. Vegetative propagation of sterile double forms, and as a means to increase numbers of inbred parent plants for F1 seed production is, however, slow. Micropropagation offers the most efficient way of increasing these varieties quickly and efficiently. To date, most Primula micropropagation protocols require explant material derived from in vitro grown seedlings or use floral parts as donor material with seasonal limitations. Therefore, an effective and efficient protocol was developed for in vitro regeneration of P. vulgaris via indirect organogenesis from adult leaf–derived explants. Exposure of leaf explants of P. vulgaris to media containing synthetic cytokinin, thidiazuron (TDZ), and auxin [1-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)] resulted in undifferentiated cell proliferation and followed by differentiated growth as shoot organogenesis. Silver nitrate improved in vitro callus growth and increased shoot regeneration further, with up to 72% of explants producing shoots. Regenerated plants developed normally and produced normal fertile flowers within 7 months. The system was also successfully applied for the micropropagation of sterile double-flowered P. vulgaris ‘Sue Jervis’. The protocol reported here enables propagation of P. vulgaris without seasonal limitation or destruction of valuable parent donor material. The protocol, with further development, has the potential to underpin development of a transformation system for Primula, which would be of value in studies on flower development and disease resistance in laboratory grown plants.

Several Primula species, including P. vulgaris cultivars and P. ×polyantha hybrids are important horticultural crops in Europe, United States, and Japan. Primula plants are produced mainly for autumn and early spring bedding and pot plant markets with different cultivars and F1 hybrids developed for improved germination rate, uniform and seasonal flower timing, and flower size and color (Karlsson, 2001). Primula is one of the most popular flowering pot plants in Europe; in 2012, Primula ranked fifth for market volume of ornamental bedding plants in Germany, accounting for 5% of the €1.95 bn market, at just under €100 m (Federal Ministry of Food and Agriculture, 2014). For the commercial grower, Primula offers a good return for relatively low inputs; low growing temperatures and high plant densities at a time in the season when few other ornamental crops are available (Erwin, 1999; Karlsson, 2002).

Academically, and the reason for this study, P. vulgaris, along with many other species of the Primulaceae, provide important examples of heteromorphic flower development (Barrett and Shore, 2008). This breeding system, in which different individuals develop one of two forms of flower, known as pin and thrum, has attracted long-term attention, including that of Charles Darwin (Darwin, 1862). The underlying genes responsible for these pin and thrum flower forms are clustered within the S locus. Ongoing studies endeavor to identify the genes within the S locus that orchestrate floral heteromorphy (Li et al., 2011, 2015).

Many commercial breeders and amateur enthusiasts are involved in breeding, hybridizing, propagating, and cultivation of the numerous Primula species and their hybrids. Many new innovative named varieties are being produced in Europe, United States, and Japan (Karlsson, 2001). The attractive semidouble/double Primula varieties and named novel varieties, some of which date back to the 16th and 17th centuries (Gerard, 1597; Parkinson, 1629), offer a high-end product for the commercial grower, and an insight into the genes involved in floral development for the academic. However, these sterile double forms can only be propagated through vegetative means. Vegetative propagation may also be used to increase the population size of plants used as parents in F1 seed production. Micropropagation techniques offer the fastest way of propagating these varieties.

The first report of Primula micropropagation (Coumans et al., 1979) described propagation of Primula obconica from inflorescence tips as explants. Subsequent micropropagation studies of Primula required explant material derived from in vitro grown seedlings of Primula cuneifolia var. hakusanensis, Primula scotica, Primula veris, Primula ×pubescens Jacq, and Primula heterochroma (Benson et al., 2000; Hamidoghli et al., 2011; Morozowska and Wesolowska, 2004; Shimada et al., 1997; Takihira et al., 2007) or involved destruction of the parent plant in studies with P. vulgaris (Merkle and Götz, 1990). To date, there is only one reported micropropagation method published for P. vulgaris, which does not destroy the donor plants (Schween and Schwenkel, 2002). This method and its refinements (Schween and Schwenkel, 2003) regenerated plantlets from pedicle explants. Although reasonably efficient, the obligate requirement for floral material to initiate culture greatly limits this system. The limited amount of floral material produced per plant and the seasonal requirements for flower initiation further limit this system. In the present study, we report an efficient, robust, micropropagation system of P. vulgaris, which uses leaf material to initiate explant culture. We have successfully applied the system to micropropagate the commercially available double-flowered cultivar P. vulgaris Sue Jervis. Abundant donor material is readily available all year-round without seasonal limitation and the system is nondestructive of valuable plants. This technique also offers the potential for development of a plant transformation system for Primula species which would be of value in studies on flower development, color formation, and disease resistance in laboratory grown plants.

Material and Methods

Media.

All media were prepared at double (2×) the final required concentration, adjusted to pH 5.8 and filter sterilized. Phytagel (catalog no. P8169; Sigma), used as the gelling agent, was also prepared at double final required concentration, autoclaved, and stored at room temperature until required. To prepare the final medium, both double-concentration Phytagel and media components were heated to 60 °C in a water bath, then mixed together, and poured into 9-cm-diameter petri dishes or Sterilin jars.

The basal medium consisted of Gamborg’s B5 (Gamborg et al., 1968) macro salts with 966 mg·L−1 KNO3, half-strength Murashige and Skoog medium (MS; Murashige and Skoog, 1962) micro salts and full-strength MS vitamins, 30 g·L−1 maltose and was solidified with 1.75 g·L−1 Phytagel.

Primula callus induction (PCI) medium consisted of basal medium supplemented with 1.7 mg·L−1 silver nitrate (AgNO3), 4.0 mg·L−1 2,4-D, and 2.0 mg·L−1 TDZ or combinations of 2.0, 3.0, and 4.0 mg·L−1 TDZ and 0.3, 0.5, 1.0, and 2.0 mg·L−1 NAA. The medium was poured into 9-cm petri dishes.

Primula shoot regeneration (PSR) medium consisted of basal medium supplemented with 4.0 mg·L−1 2,4-D and 2.0 mg·L−1 TDZ or combinations 2.0, 3.0, and 4.0 mg·L−1 TDZ and 0.1, 0.2, 0.3, and 0.5 mg·L−1 NAA with either 0.85, 1.7, or 3.4 mg·L−1 AgNO3. The medium was poured into 9-cm petri dishes.

Primula rooting medium (PRM) consisted of woody plant medium (WPM) (Lloyd and McCown, 1981) basal salts with 2% sucrose, 0.5 mg·L−1 indole-3-butyric acid (IBA), solidified with 1.75 g·L−1Phytagel. About 25 mL medium was poured into 100-mL sterile culture jars (Sterilin 185AM).

Plant material and sterilization.

P. vulgaris plants were used as the initial explant source for this study. Parent plants were grown in 1-L pots of peat-based multipurpose compost (MPC60; Erin Horticulture, Ireland) in a glasshouse at 16 ± 1 °C day and night, relative humidity (RH) 60% under natural light. Tender leaves ≈5 cm long, were thoroughly washed with cool running water for 1 h. Under aseptic conditions, the leaves were surface sterilized with 70% ethanol (v/v) for 1 min, followed by 6 min in 10% (v/v) sodium hypochlorite (Fluka 71696) with a drop of Tween 20 (P-9416; Sigma), and subsequently, rinsed three times with distilled-sterile water.

In vitro culture.

The outer edges of the leaves were removed with a scalpel and discarded. The leaf material was cut transversely into ≈0.5-cm2 sections containing major veins. Leaf sections were placed with abaxial surface in contact with the PCI medium. Three petri dishes per treatment with five explants per petri dish were cultured. The cultures were kept in a controlled culture cabinet at 18 ± 1 °C in the dark for 4 weeks for callus initiation.

After two subcultures (4 weeks), the calli which had developed on PCI medium were transferred to PSR media. These were cultured under cool fluorescent lights (100 μmol·m−2·s−1) at 21 ± 1 °C with a 16-h photoperiod. The cultures were subcultured four times onto fresh PSR media every 2 weeks for shoot regeneration.

Rooting.

The adventitious shoots that developed on PSR media were isolated from the callus material when they had formed small rosettes of ≈1 to 1.5 cm across consisting of three to five leaves. These rosettes were transferred individually to 100-mL Sterilin jars containing PRM for further development and rooting.

Acclimatization.

Well-developed regenerated plantlets were gently removed from the jars using long forceps and all tissue culture medium washed from the roots with cool running water. They were planted in the multipurpose compost in 5-cm2 cells within propagation trays. Plants were covered with clear plastic propagator lids for ≈2 weeks to maintain high humidity around them while they established. Plants were grown in a controlled environment room at 18 ± 1 °C day (16 h) and 15 ± 1 °C night temperatures, 75% RH with 250 μmol·m−2·s−1 light intensity provided by metal halide lamps (HQI) supplemented with tungsten bulbs at 16-h photoperiod.

After a further 3–4 weeks, they were potted on into 15-cm-diameter pots. The plants were grown further until they established a good root system, visible through the drainage holes in the bottom of the pot; they were then transferred to 1-L pots. After the initial 6 weeks establishment, were fertilized with a weak 25 g·L−1 proprietary liquid feed (Phostrogen; Bayer Cropscience Ltd, UK) fortnightly to promote healthy growth.

Statistical analyses and experimental design.

Experiments were arranged in a completely randomized design. Each treatment consisted of three replicates, 15 explants per replicate. Statistical analyses were carried out using Genstat software (version 16.1). Analysis of variance was performed for each experiment. The least significant difference test at P = 0.05 was used for multiple mean comparisons.

Results and Discussion

Basal medium.

During development of this protocol, leaf explants were initially cultured on a range of media, macro and micro salt and vitamin combinations including MS, B5, WPM, and Anderson’s Rhododendron medium (Anderson, 1980) containing different carbon sources: sucrose, glucose, mannitol, and maltose (data not shown). None of these initial media gave callus proliferation, despite a range of plant growth regulators being used, with one exception. All but one of the media tested led to leaf browning and the release of phenolic compounds into the media was common. In medium containing B5 macro salts with 966 mg·L−1 KNO3, half-strength MS micro salts, and MS vitamins with 30 g·L−1 maltose, browning was limited and callus induction observed, therefore, this was chosen as the Primula basal medium (PBM).

P. vulgaris is a woodland species that has evolved in humus-rich, nutrient-poor soils (Jacquemyn et al., 2009). The majority of Primula species have relatively low nutrient requirements (Erwin, 1999; Karlsson, 2001). Primula is susceptible to ammonium toxicity and should be fertilized with a nitrate-based fertilizer (Erwin, 1999). The half-strength MS micro salts in our basal medium were favored by leaf-derived explants. MS medium contains two nitrogen salts, NH4NO3 (≈20 mm) and KNO3 (≈20 mm), which results in a nitrogen form ratio of NH4 to NO3 of 20:40 and gives a combined total of ≈60 mm for both nitrogen forms. This is considered to be a reasonably high nitrogen concentration, particularly for many woody species (Vinterhalter et al., 2007). For example, WPM contains about four times less nitrogen than MS medium. Gamborg et al. (1968) first demonstrated the significance of ammonium as a media component. Gamborg’s B5 medium contains only 1 mm ammonium, 20 times lower than MS medium (Vinterhalter et al., 2007), making B5 macro salts suitable for the potentially ammonium toxicity susceptible Primula. The amount of KNO3 (966 mg) in PBM is ≈40% of standard Gamborg’s B5 macro salts. The correct combination of salts with maltose was found to be essential for callus induction in leaf-derived Primula explants. After optimization of the correct media composition, the callus induction rate varied between 29% and 100% (Table 1). Callus induction started after ≈3 weeks of culture.

Table 1.

Callus formation on Primula callus induction medium containing various concentration of thidiazuron (TDZ), 1-naphthylacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D).

Table 1.

Effects of AgNO3 supplementation.

The inclusion of AgNO3 in the basal medium reduced the occurrence of tissue browning further. AgNO3 is widely used in plant tissue culture as a potent inhibitor of ethylene action (Beyer, 1976; Kumar et al., 2009); ethylene influences many aspects of growth and development throughout the plant’s life cycle (Johnson and Ecker, 1998). In vitro studies have shown that ethylene can affect callus growth, organogenesis, shoot regeneration, and somatic embryogenesis often detrimentally (Kumar et al., 2009). By limiting ethylene production or its action within tissue cultures, greater control over growth and development can be achieved (Beyer, 1976; Wang et al., 2002). In Primula, the addition of AgNO3 to media reduced tissue browning and promoted healthy-looking callus with vigorous growth. Callus formation was seen to develop from the leaf edges which had been cut previously while preparing the explants for in vitro culture.

The inclusion of AgNO3 at 0.85 mg·L−1 in PSR medium enhanced shoot regeneration from 27% to 44% (Table 2). Increasing the AgNO3 concentration from 0.85 to 3.4 mg·L−1 further enhanced the rate of shoot regeneration from 44% to 72% (Table 2). As a potent inhibitor of ethylene, AgNO3 is also known to regulate the polyamine pool in plants. Polyamines are associated with several important cellular processes including cell division, morphogenesis, protein synthesis, DNA replication, and abiotic stress response (Kumar et al., 2009). The positive effects of AgNO3 on regeneration during clonal propagation of several economically important plant species have been reported (Fuentes et al., 2000; Naik and Chand, 2003; Uliaie et al., 2008). The inhibition of ethylene by AgNO3 and subsequent regulation of the polyamine pool could, in part, be the underlying process that stimulated the increase in shoot regeneration from P. vulgaris callus.

Table 2.

Effect of different concentrations of AgNO3 on shoot regeneration from Primula vulgaris calli on Primula shoot regeneration medium.

Table 2.

Optimization of plant growth regulators.

The concentration of TDZ and auxin (NAA or 2,4-D) used, and the interaction between them significantly influenced the callus induction rate (Table 1). TDZ and auxin concentrations were also crucial for promoting dedifferentiation and regeneration within the tissue. The media with 4.0 mg·L−1 2,4-D, 2.0 mg·L−1 TDZ, and 2.0 mg·L−1 TDZ, 0.5 mg·L−1 NAA and 3.0 mg·L−1 TDZ, 0.3 mg·L−1 NAA had the highest callus induction rates (100%). Large green-cream compact callus formation was obtained in the media containing 4.0 mg·L−1 2,4-D, 2.0 mg·L−1 TDZ (Fig. 1A). While in the medium containing 2.0 mg·L−1 TDZ and 0.5 mg·L−1 NAA, compact smaller callus structures with some red anthocyanin pigmentation were seen (Fig. 1B). Anthocyanin production in vitro is usually associated with stress (Chalker-Scott, 1999). The combination of 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA within the medium produced smaller green-compact callus structures (Fig. 1C). The callus induction of explants on a medium containing 1 mg·L−1 NAA or more was significantly reduced in comparison with media with either 0.5 or 0.3 mg·L−1 NAA (P < 0.001) with fewer explants responding. However, medium with TDZ alone was detrimental to the plant material and failed to initiate callus (Table 1). Chemically, TDZ is completely different from other commonly used auxins and cytokinins, but has been shown to exhibit both auxin and cytokinin like affects due to its ability to modulate endogenous growth regulators (Guo et al., 2011; Murthy et al., 1995; Thomas and Puthur, 2004). After 6 to 9 weeks in culture, numerous shoots differentiated from the calli (Fig. 1D and E).

Fig. 1.
Fig. 1.

Callus development, adventitious shoot development, and shoot development on Primula callus induction (PCI) medium and Primula shoot regeneration (PSR) medium with different plant growth regulators. (A) Large green-cream callus formation on PCI medium with 4.0 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg·L−1 thidiazuron (TDZ). (B) Small compact callus with anthocyanin pigmentation on PCI medium with 2.0 mg·L−1 TDZ and 0.5 mg·L−1 1-naphthylacetic acid (NAA). (C) Small green compact callus on PCI medium with 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA. (D and E) Callus induced from leaf segments on PCI medium supplemented with 4.0 mg·L−1 2,4-D, and 2.0 mg·L−1 TDZ for 4 weeks followed by 3 weeks on PSR medium 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA. Scale bar = 1 cm. (F) Shoot primordium as a unipolar structure. (G) Shoot differentiation from callus tissue. (H) Shoot development with vascular connections to callus tissue.

Citation: HortScience horts 51, 5; 10.21273/HORTSCI.51.5.558

Regeneration occurred through the initiation of shoot primordia (Fig. 1F) and over a 2- to 3-week period green shoots started to emerge (Fig. 1G), followed by leaf expansion (Fig. 1H). The regeneration was identified as adventitious shoot regeneration since the shoots were connected to callus cells by vascular tissue formation and were of a unipolar structure (Fig. 1F and G), sometimes referred to as vegetative primordium (Nunes et al., 2013). The shoot regeneration rate for P. vulgaris varied between 26.7% and 66.7% (Table 3). The greatest shoot regeneration rate (66.7%) was seen in the medium supplemented with 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA. TDZ played a critical role in promoting shoot differentiation from Primula callus. It has been shown to stimulate adventitious shoot regeneration in a large number of plant species such as Hydrangea, Sugarcane, Strawberry, and Dendrocalamus strictus (Gallo-Meagher et al., 2000; Haddadi et al., 2013; Lata et al., 2013; Ledbetter and Preece, 2004).

Table 3.

Effect of different concentrations of thidiazuron (TDZ), 1-naphthylacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on shoot regeneration from Primula vulgaris calli on Primula shoot regeneration (PSR) medium.

Table 3.

Rooting.

It is clear that there is a strong relationship between salt combination and TDZ, auxin, and AgNO3 present in the culture media used for P. vulgaris. To develop an efficient in vitro regeneration system, effective growth regulators with an appropriate mineral salt medium capable of inducing a high level of organogenesis are necessary. Previous reported studies found that the effect of growth regulators can be strongly modified by the medium on which the culture is grown (McCown and Sellmer, 1987). The adventitious shoots were transferred to WPM medium containing 0.5 mg·L−1 IBA to root. Root primordia were seen to begin to develop after 10–15 d (Fig. 2). Several Primula species (P. cuneifolia, Primula malacoides, and P. obconica) have been reported to root on various phytohormone free media (Mizuhiro et al., 2001; Shimada et al., 1997). However, the inclusion of IBA appeared to stimulate root primordia development, which is in agreement with previous reports (Hamidoghli et al., 2011; Jia et al., 2014; Schween and Schwenkel, 2003). A subset of 20 P. vulgaris plantlets transferred to soil were successfully acclimatized and adapted to conditions within the controlled environment room.

Fig. 2.
Fig. 2.

Regeneration and rooting of Primula vulgaris plantlets. Plantlet after 4 weeks growth on a phytagel woody plant medium with 0.5 mg·L−1 indole-3-butyric acid. Scale bar = 1 cm.

Citation: HortScience horts 51, 5; 10.21273/HORTSCI.51.5.558

The optimized protocol was then tested on the sterile double-flowered P. vulgaris ‘Sue Jervis’. A 25% regeneration rate was recorded for P. vulgaris ‘Sue Jervis’ when cultured on PCI medium supplemented with 4.0 mg·L−1 2,4-D, 2.0 mg·L−1 TDZ for 4 weeks, followed by a further 8 weeks on PSR medium supplemented 3.0 mg·L−1 TDZ, 0.3 mg·L−1 NAA, and 3.4 mg·L−1 AgNO3. The isolation and rooting of plantlets on WPM began after 5 weeks on PSR medium. A subset of three P. vulgaris ‘Sue Jervis’ plantlets were transferred to soil and successfully acclimatized.

The established plants developed normally and produced normal flowers within 7 months from the start of the tissue culture process (Fig. 3A and B). The P. vulgaris plants set seed after cross pollination between pin and thrum flowers. No evidence of morphological variation has been seen in regenerated plants.

Fig. 3.
Fig. 3.

Flowering Primula vulgaris (Huds.) and P. vulgaris ‘Sue Jervis’ plants after tissue culture. (A) Mature fertile P. vulgaris (Huds.) plant derived from in vitro culture after 12 weeks in soil. (B) P. vulgaris ‘Sue Jervis’ after 16 weeks in soil. Scale bars 2 = cm.

Citation: HortScience horts 51, 5; 10.21273/HORTSCI.51.5.558

Conclusion

In summary, we have developed an in vitro regeneration system for P. vulgaris plants, which uses leaves as its starting material, and have also applied the system for the propagation of the sterile double-flowered P. vulgaris ‘Sue Jervis’. The system is not only nondestructive to valuable parent plants, but also free from the seasonal limitations of using floral tissue. The system offers a robust method that has the potential to enable breeding companies to propagate new elite cultivars quickly and efficiently. It also offers the foundation for a plant transformation system to aid fundamental research into important traits in Primula species.

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  • Merkle, S. & Götz, E.M. 1990 Micropropagation of inbred lines of Primula acaulis for breeding purposes, p. 116. Abstracts of 7th Congress Plant Tissue Cell Cultures. June 24–29, Amsterdam, The Netherlands

  • Mizuhiro, M., Kenichi, Y., Ito, K., Kadowaki, S., Ohashi, H. & Mii, M. 2001 Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica Plant Sci. 160 1221 1228

    • Search Google Scholar
    • Export Citation
  • Morozowska, M. & Wesolowska, M. 2004 In vitro clonal propagation of Primula veris L. and preliminary phytochemical analysis Acta Biol. Cracov. Ser. Bot. 46 169 175

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 497

  • Murthy, B.N.S., Murch, S.J. & Saxena, P.K. 1995 Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth-regulator levels and significance of cotyledons Physiol. Plant. 94 268 276

    • Search Google Scholar
    • Export Citation
  • Naik, S.K. & Chand, P.K. 2003 Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.) Austral. J. Plant Physiol. 160 423 430

    • Search Google Scholar
    • Export Citation
  • Nunes, C.F., Santos, D.N.D., Pasqual, M., Valente, T.C.T., Oliveira, A.C.L.d., Alves, E. & Setotaw, T.A. 2013 Morphogenesis and regeneration of adventitious shoots in Jatropha curcas L Austral. J. Crop Sci. 7 1511 1519

    • Search Google Scholar
    • Export Citation
  • Parkinson, J. 1629 Paradisus in sole paradisus terrestris. Humfrey Lownes and Robert Young, London, UK

  • Schween, G. & Schwenkel, H.G. 2002 In vitro regeneration in Primula ssp via organogenesis Plant Cell Rpt. 20 1006 1010

  • Schween, G. & Schwenkel, H.G. 2003 Effect of genotype on callus induction, shoot regeneration, and phenotypic stability of regenerated plants in the greenhouse of Primula ssp Plant Cell Tissue Organ Cult. 72 53 61

    • Search Google Scholar
    • Export Citation
  • Shimada, T., Matsushita, T. & Otani, M. 1997 Plant regeneration from leaf explants of Primula cuneifolia var. hakusanensis, “Hakusankozakura” Plant Biotechnol. 14 47 50

    • Search Google Scholar
    • Export Citation
  • Takihira, M., Otani, M., Tsuchiya, S. & Shimada, T. 2007 Plant regeneration from leaf explants of auricula cultivars (Primula × pubescens Jacq.) Plant Biotechnol. 24 425 427

    • Search Google Scholar
    • Export Citation
  • Thomas, T.D. & Puthur, J.T. 2004 Thidiazuron induced high frequency shoot organogenesis in callus from Kigelia pinnata L Bot. Bul. Acad. Sin. 45 307 313

    • Search Google Scholar
    • Export Citation
  • Uliaie, E.D., Farsi, M., Ghreyazie, B. & Imani, J. 2008 Effects of genotype and AgNO3 on shoot regeneration in winter cultivars of rapeseed (Brassica napus) Pak. J. Biol. Sci. 11 2040 2043

    • Search Google Scholar
    • Export Citation
  • Vinterhalter, B., Ninković, S., Zdravković-Korać, S., Subotić, A. & Vinterhalter, D. 2007 Effect of nitrogen salts on the growth of Ceratonia siliqua L. shoot cultures Arch. Biol. Sci. Belgrade 59 217 222

    • Search Google Scholar
    • Export Citation
  • Wang, K.L.C., Li, H. & Ecker, J.R. 2002 Ethylene biosynthesis and signaling networks Plant Cell 14 S131 S151

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Contributor Notes

We are grateful to the BBSRC for support via grant BB/H019278/2 and Pam Wells for horticultural support. We also thank the University of East Anglia for support and the John Innes Centre for hosting PGs laboratory under the UEA-JIC Norwich Research Park collaboration. PG would like to thank Margaret Webster for discussions on Primula tissue culture before commencement of the work presented here.

Corresponding author. E-mail: p.gilmartin@uea.ac.uk.

  • View in gallery

    Callus development, adventitious shoot development, and shoot development on Primula callus induction (PCI) medium and Primula shoot regeneration (PSR) medium with different plant growth regulators. (A) Large green-cream callus formation on PCI medium with 4.0 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg·L−1 thidiazuron (TDZ). (B) Small compact callus with anthocyanin pigmentation on PCI medium with 2.0 mg·L−1 TDZ and 0.5 mg·L−1 1-naphthylacetic acid (NAA). (C) Small green compact callus on PCI medium with 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA. (D and E) Callus induced from leaf segments on PCI medium supplemented with 4.0 mg·L−1 2,4-D, and 2.0 mg·L−1 TDZ for 4 weeks followed by 3 weeks on PSR medium 3.0 mg·L−1 TDZ and 0.3 mg·L−1 NAA. Scale bar = 1 cm. (F) Shoot primordium as a unipolar structure. (G) Shoot differentiation from callus tissue. (H) Shoot development with vascular connections to callus tissue.

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    Regeneration and rooting of Primula vulgaris plantlets. Plantlet after 4 weeks growth on a phytagel woody plant medium with 0.5 mg·L−1 indole-3-butyric acid. Scale bar = 1 cm.

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    Flowering Primula vulgaris (Huds.) and P. vulgaris ‘Sue Jervis’ plants after tissue culture. (A) Mature fertile P. vulgaris (Huds.) plant derived from in vitro culture after 12 weeks in soil. (B) P. vulgaris ‘Sue Jervis’ after 16 weeks in soil. Scale bars 2 = cm.

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  • Mizuhiro, M., Kenichi, Y., Ito, K., Kadowaki, S., Ohashi, H. & Mii, M. 2001 Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica Plant Sci. 160 1221 1228

    • Search Google Scholar
    • Export Citation
  • Morozowska, M. & Wesolowska, M. 2004 In vitro clonal propagation of Primula veris L. and preliminary phytochemical analysis Acta Biol. Cracov. Ser. Bot. 46 169 175

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 497

  • Murthy, B.N.S., Murch, S.J. & Saxena, P.K. 1995 Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth-regulator levels and significance of cotyledons Physiol. Plant. 94 268 276

    • Search Google Scholar
    • Export Citation
  • Naik, S.K. & Chand, P.K. 2003 Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.) Austral. J. Plant Physiol. 160 423 430

    • Search Google Scholar
    • Export Citation
  • Nunes, C.F., Santos, D.N.D., Pasqual, M., Valente, T.C.T., Oliveira, A.C.L.d., Alves, E. & Setotaw, T.A. 2013 Morphogenesis and regeneration of adventitious shoots in Jatropha curcas L Austral. J. Crop Sci. 7 1511 1519

    • Search Google Scholar
    • Export Citation
  • Parkinson, J. 1629 Paradisus in sole paradisus terrestris. Humfrey Lownes and Robert Young, London, UK

  • Schween, G. & Schwenkel, H.G. 2002 In vitro regeneration in Primula ssp via organogenesis Plant Cell Rpt. 20 1006 1010

  • Schween, G. & Schwenkel, H.G. 2003 Effect of genotype on callus induction, shoot regeneration, and phenotypic stability of regenerated plants in the greenhouse of Primula ssp Plant Cell Tissue Organ Cult. 72 53 61

    • Search Google Scholar
    • Export Citation
  • Shimada, T., Matsushita, T. & Otani, M. 1997 Plant regeneration from leaf explants of Primula cuneifolia var. hakusanensis, “Hakusankozakura” Plant Biotechnol. 14 47 50

    • Search Google Scholar
    • Export Citation
  • Takihira, M., Otani, M., Tsuchiya, S. & Shimada, T. 2007 Plant regeneration from leaf explants of auricula cultivars (Primula × pubescens Jacq.) Plant Biotechnol. 24 425 427

    • Search Google Scholar
    • Export Citation
  • Thomas, T.D. & Puthur, J.T. 2004 Thidiazuron induced high frequency shoot organogenesis in callus from Kigelia pinnata L Bot. Bul. Acad. Sin. 45 307 313

    • Search Google Scholar
    • Export Citation
  • Uliaie, E.D., Farsi, M., Ghreyazie, B. & Imani, J. 2008 Effects of genotype and AgNO3 on shoot regeneration in winter cultivars of rapeseed (Brassica napus) Pak. J. Biol. Sci. 11 2040 2043

    • Search Google Scholar
    • Export Citation
  • Vinterhalter, B., Ninković, S., Zdravković-Korać, S., Subotić, A. & Vinterhalter, D. 2007 Effect of nitrogen salts on the growth of Ceratonia siliqua L. shoot cultures Arch. Biol. Sci. Belgrade 59 217 222

    • Search Google Scholar
    • Export Citation
  • Wang, K.L.C., Li, H. & Ecker, J.R. 2002 Ethylene biosynthesis and signaling networks Plant Cell 14 S131 S151

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