Somatic embryogenesis and regeneration from shoot primordia ...

Plant Cell Tiss Organ Cult (2010) 100:349–353 DOI 10.1007/s11240-009-9645-4

RESEARCH NOTE

Somatic embryogenesis and regeneration from shoot primordia of Crocus heuffelianus Zita Demeter • Gyula Surańyi • V. Attila Molna´r Ga´bor Sramko´ • Dańiel Beyer • Zoltań Końya • Ga´bor Vasas • Ma´rta M-Hamvas • Csaba Ma´the´

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Received: 24 March 2009 / Accepted: 15 November 2009 / Published online: 11 December 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Crocus heuffelianus belongs to the C. vernus (Iridaceae) species aggregate. In the Carpathian Basin and particularly in Hungary it is considered an endangered species. Therefore our aim was to establish a tissue culture system with potential of germplasm preservation of this taxon. For in vitro culture experiments, shoot primordia from corms were the most suitable. We induced an embryogenic callus line from those explants on basal Murashige-Skoog (MS) medium supplemented with Gamborg’s vitamins, 2% (w/v) sucrose, 10 mg l-1 (53.7 lM) a-naphthaleneacetic acid (NAA) and 1 mg l-1 (4.44 lM) 6-benzyladenine (BA). Globular stage embryos developed on this medium and several culture conditions were used in an attempt to obtain mature embryos and plant regeneration. Firstly a decrease of auxin/cytokinin concentration and ratio, then secondly a decrease in the strength of culture medium and the concentration of carbon source was used, which was effective in embryogenesis and the production of plants. Regeneration medium used in the second step was fourfold diluted MS medium and Gamborg’s vitamins supplemented with 1% (w/v) sucrose, 0.05 mg l-1 (0.26 lM) NAA and 0.5 mg l-1 (2.22 lM) BA, with a 14/10 h photoperiod. Under these conditions we could detect all the stages of somatic embryo development characteristic for Iridaceae. This is the first report demonstrating the production of stable tissue culture of C. heuffelianus with potential use in germplasm

Z. Demeter G. Surańyi (&) V. A. Molna´r G. Sramko´ D. Beyer Z. Końya G. Vasas M. M-Hamvas C. Ma´the´ (&) Department of Botany, Faculty of Science and Technology, University of Debrecen, PO Box 14, 4010 Debrecen, Hungary e-mail: [email protected] C. Ma´the´ e-mail: [email protected]

preservation via plant regeneration. This study could also contribute to a better understanding of somatic embryogenesis in the Crocus genus. Keywords Crocus heuffelianus Mature somatic embryo Plant regeneration Germplasm preservation

Crocus heuffelianus Herbert belongs to the C. vernus species aggregate of spring-flowering Crocus species. It is distributed in the Carpathian Basin including North-Eastern Hungary, as well as several geographic locations of western Ukraine (Rafin´ski and Passakas 1976). It is considered as one of the endangered species in Hungary (Hungary: Country report to the FAO International Technical Conference on Plant Genetic Resource 1996). Economically, the most important member of the Crocus genus is C. sativus which is known for its culinary and medicinally important compounds and it is also the most widely studied species with respect to tissue culture (see the reviews of Kafi et al. 2006; Ascough et al. 2009). As member of the Crocus genus, C. heuffelianus is a potential resource for biologically active compounds such as crocin, crocetin, picrocrocin of safranal. Due to its ecological and potential economical importance, there is a need for germplasm preservation of this species. This could in turn contribute to the maintenance of genetic material of C. sativus allies, which is one of the major topics of saffron research (Kafi et al. 2006). One of the best approaches in this respect is plant in vitro culture (Dodds and Roberts 1986). Although tissue culture in the C. vernus aggregate has been previously reported (Chub et al. 1994), to date there is no report on somatic embryogenesis and plant regeneration of C. heuffelianus. Therefore our aim was to

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establish a tissue culture system with regeneration potential for this species. The collection of C. heuffelianus plants was part of a germplasm preservation programme directed by the EU CROCUSBANK Project (AGRI-2006-0265). The site of collection was Rodnei Mountains, Romania, near the locality of Bors¸ a. Whole plants of Crocus heuffelianus (sprouting corms and leaf primordia enclosed in a sheath) were collected before flowering in April 2007. Corms, roots, foliage leaves, and shoot primordia within corms were the source explants tested for callus induction. Explants were rinsed in running tap water for 2 min and surface sterilized by rinsing the explants in 10% (v/v) Domestos bleach for 30 min, followed by three rinses with sterile distilled water under aseptic conditions (5 min each wash). In case of shoot primordia, the central column of corms containing the primordia was dissected out. The prepared explants were cultured on Murashige and Skoog basal medium (MS medium, Murashige and Skoog 1962) supplemented with 2% (w/v) sucrose (Reanal, Budapest, Hungary), B5 vitamins (Gamborg et al. 1968). The medium was solidified with 0.8% (w/v) Difco Bacto agar, Voigt Global Distributions Inc., Lawrence, KS, USA. All growth regulators (hormones) were from Sigma– Aldrich, Budapest, Hungary. The auxins used were a-naphthaleneacetic acid (NAA) and indoleacetic acid (IAA), and the cytokinins were 6-benzyladenine (BA) and kinetin (KIN). For embryogenic callus induction, explants were cultured under a 14/10 (light/dark) photoperiod (white fluorescent illumination, 10 lmol m-2 s-1) with temperatures of 22/18 ± 2°C, respectively. For the maturation of somatic embryos, the same photoperiodic and temperature conditions were used, except that light intensity was increased to 60 lmol m-2 s-1. For histology, segments of embryogenic calli were sectioned by hand or fixed with 4% (v/v) formaldehyde and cryosectioned with a Leica Jung Histoslide 2000 microtome, Leica, Nussloch, Germany. Sections were stained for nuclear DNA with 3 lg ml-1 of 40 ,60 -diamidino-2-phenylindole (DAPI, Fluka, Buchs, Switzerland) for 40 min. Histological preparations were examined by bright field and fluorescence microscopy, with an Olympus Provis AX-70 fluorescence microscope (Olympus, Tokyo, Japan). The excitation wavelength used for the examination of chromatin and cell wall autofluorescence was 320–360 nm. We have investigated the genetic variability of the original explants and tissue cultures of C. heuffelianus by Amplified Fragment Length Polymorphism (AFLP). In brief, DNA from all Crocus samples was purified using TRI REAGENT RNA/DNA/Protein Isolation Reagent (Molecular Res. Center, USA). Enzymatic digestion of 200 ng total genomic DNA samples was carried out by EcoRI and TrulI restriction endonucleases (Fermentas, Lithuania). The digested DNA was analyzed with the

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Plant Cell Tiss Organ Cult (2010) 100:349–353

AFLP method adapted from Vos et al. (1995) using separated restriction cleavage and ligation of DNA samples. For the induction of embryogenic calli, we have investigated the response of different C. heuffelianus explants to different auxin/cytokinin combinations. We could not obtain callus induction from leaves and roots. Stable embryogenic callus cultures could be induced from shoot primordia of corm explants by separating them from the initial explants and subculturing at 30–60 days intervals. The best results were obtained on a full-strength culture medium supplemented with 10 mg l-1 (53.7 lM) NAA and 1 mg l-1 (4.44 lM) BA. On this medium, stable callus cultures with globular stage embryos were obtained (Fig. 1a, b, h, h’). This hormone combination was originally used for the induction of shoot regeneration from ovary explants of C. sativus (Bhagyalakshmi 1999). Another synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) is known to induce somaclonal variability (Dodds and Roberts 1986). Since the primary goal was to preserve the original genome of C. heuffelianus, 2,4-D was not suitable for this study. We applied several culture conditions in order to obtain embryo maturation and ultimately, achieving plant regeneration from the stable embryogenic calli. Those conditions are summarized in Table 1 and were partially described for other members of the Crocus genus. The conditions were as follows: (I.) The maintenance of embryogenic callus culture on a medium containing 10 mg l-1 NAA and 1 mg l-1 BA; (II.) Decrease of hormone concentrations and subsequent use of hormonefree medium (Plessner and Ziv 1999; Karamian and Ebrahimzadeh 2001; Karamian 2007), or changes of the auxin/cytokinin ratio; (III.) This culture condition involved more pronounced changes in the composition of culture medium. It included the direct transfer of embryogenic calli to hormone-free medium or a decrease of hormone concentration, the changing of auxin/cytokinin ratio (Bhagyalakshmi 1999) with the decrease of medium strength. In the case of culture condition II, we obtained mature somatic embryos that started to develop organs, but stopped their development at an early stage. In the case of culture condition III, somatic embryos underwent organogenesis, but the development of root primordia dominated over that of shoot primordia (Fig. 1c and Table 1). By using culture condition III, the effects of the decrease of hormone content, the change of auxin/cytokinin ratio and reducing the organic carbon (sucrose) content of the culture medium, directly or in two steps were investigated. The efficient production of organ (root) primordia was obtained when we changed hormone ratio and subsequently reduced the strength of basal medium with keeping sucrose concentration and the previously changed hormone content. We obtained similar results by the one-step reduction of hormone concentration concomitantly with changing of


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Fig. 1 The induction of Crocus heuffelianus embryogenic calli and plant regeneration from somatic embryos. a Callus induction on shoot tip explants after 70 days of culture. b Callus with globular stage embryos from a stable culture grown on full-strength medium with 10 mg l-1 a-naphthaleneacetic acid (NAA) and 1 mg l-1 6-benzyladenine (BA). c Callus grown on hormone-free medium. The development of root primordia (rp) dominates over the development of shoot primordia (sp). d–m The morphology and histology of embryogenesis and regeneration of C. heuffelianus cultures. See Table 1 and text for the growth regulator content of culture medium (Regeneration medium II) in case of (d–g) and (i–m). d Embryogenic callus grown for 45 days on fourfold diluted MS basal medium and Gamborg’s vitamins, with 1% sucrose. All developmental stages of somatic embryos were present (arrows). e Mature somatic embryo from the callus culture presented in (d). Fifty days (f), then 65 days (g) of culture on fourfold diluted MS basal medium and Gamborg’s vitamins, with 1% sucrose supplemented with 0.05 mg l-1 NAA, 2 mg l-1 BA, 25 mg l-1 gibberellic acid (GA3) and 100 mg l-1

ascorbic acid results in the conversion of somatic embryos to plantlets (r-root). h Globular stage embryo stained with DAPI, from a callus grown on full-strength medium with 10 mg l-1 NAA and 1 mg l-1 BA. h’ Bright-field microscopy image of a globular stage embryo. i Torpedo stage embryo from a callus grown for 30 days on fourfold diluted MS basal medium and Gamborg’s vitamins, with 1% sucrose. j After 40 days, the cotyledon (c) and shoot primordium (sp) can be distinguished on the torpedo stage embryo. k The differentiation of vessel elements in the cotyledon of the embryo stage presented in (j). k’ Differentiated tracheary element (arrow) from a torpedo stage embryo showing autofluorescence of secondary wall thickening and the lack of nucleus. DAPI staining reveals the nucleus from an adjacent meristematic cell (arrowhead). l Cross-section of a radicle from a mature embryo, showing the location of stele initials (si) and cortex initials (ci). m Longitudinal section of a radicle from a mature somatic embryo. Scalebars: 4 mm for (a–g), 1 mm for (h–j), 60 lm for (k, k’) and 200 lm for (l, m)

hormone ratio and reducing of sucrose concentration to 1% (w/v) (Table 1). Based on these observations we developed a novel culture method (culture condition IV of Table 1). According to this, we reduced auxin and cytokinin concentration concomitantly with changing of hormone ratio

(increasing cytokinin/auxin ratio) in the first step (regeneration medium I), followed by a reduction in the strength of basal medium and that of sucrose (regeneration medium II, Table 1). This culture condition resulted in the maturation of somatic embryos (that is, all globular stage

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Table 1 The effects of strength and growth regulator content of culture media on the development of C. heuffelianus embryogenic calli. The basic culture medium was full-strength MS with Gamborg’s vitamins, except where stated. The starting calli for all culture strategies were embryogenic calli grown on full-strength medium containing 10 mg l-1 NAA and 1 mg l-1 BA. The distinct medium compositions used within a given culture condition are indicated with lowercase letters and Arabic numerals in brackets indicate the subsequent callus passages Culture condition

Final developmental stage

Culture media used

I

Embryogenic callus

a. 10 mg l-1 NAA ? 1 mg l-1 BA

II

Mature somatic embryos germinate, but organ development stops at an early stage.

a. 0.5 mg l-1 NAA ? 0.05 mg l-1 BA b. 0.05 mg l-1 NAA ? 0.5 mg l-1 BA c. (1) 0.5 mg l-1 NAA ? 0.05 mg l-1 BA (2) hormone-free medium d. (1) 0.5 mg l-1 NAA ? 0.05 mg l-1 BA (2) 0.05 mg l-1 NAA ? 0.5 mg l-1 BA

III

The development of root primordia on somatic embryos dominates over the development of shoot primordia.

a. Hormone-free medium b. (1) 0.5 mg l-1 NAA ? 0.05 mg l-1 BA (2) fourfold diluted MS basal medium and vitamins with 1% sucrose, 0.05 mg l-1 NAA ? 0.5 mg l-1 BA c. (1) 0.05 mg l-1 NAA ? 0.5 mg l-1 BA (2) fourfold diluted MS basal medium and vitamins with 2% sucrose, 0.05 mg l-1 NAA ? 0.5 mg l-1 BA d. Fourfold diluted MS basal medium and vitamins with 1% sucrose, 0.05 mg l-1 NAA ? 0.5 mg l-1 BA

IV

Mature somatic embryos develop plantlets with well defined shoots and roots.

embryos converted into mature embryos) and plant regeneration (Fig. 1d–g). All calli produced somatic embryos. The number of somatic embryos per callus was 10.5 ± 0.5 (SE) and their length was 6.5 ± 0.3 (SE) mm. Somatic embryos differentiated into plantlets (Fig. 1f, g). Plant production could be enhanced by the increase of cytokinin content of culture medium from 0.5 to 2 mg l-1 (Table 1) together with the use of 25 mg l-1 (72 lM) gibberellic acid (GA3) and 100 mg l-1 (568 lM) L-ascorbic acid (Fig. 1f, g). Under such conditions, the number of plantlets developed on a single embryogenic callus was 4.9 ± 0.8 (SE). After 65 days of culture, the shoots of plantlets reached a size of 20.7 ± 1.8 mm. Therefore, the increase of cytokinin/auxin ratio otherwise known to induce shoot development (Dodds and Roberts 1986) is not sufficient for plant regeneration in Crocus heuffelianus tissue cultures. It must be followed by the decrease of the strength of culture medium and especially the decrease in the content of organic carbon source. There are several studies reporting that decreasing of sucrose content induces maturation of somatic embryos in plant tissue cultures (Li and Wolyn 1997; Lee et al. 2001). AFLP analysis revealed the genetic identity of the original explants with all tissue cultures including embryogenic calli and regenerants. The genetic stability was maintained after long-term (2 years) subculture. Thus no somaclonal variation occurred during in vitro culture.

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a. (1) 0.05 mg l-1 NAA ? 0.5 mg l-1 BA (regeneration medium I) (2) fourfold diluted MS basal medium and vitamins with 1% sucrose, 0.05 mg l-1 NAA ? 0.5–2 mg l-1 BA (regeneration medium II)

Therefore the in vitro approach proved to be suitable for germplasm preservation purposes. Histological investigation revealed all stages of somatic embryo development in C. heuffelianus cultures. Calli grown on full-strength medium with 10 mg l-1 NAA and 1 mg l-1 BA developed numerous embryos of globular stage (Fig. 1b, h, h’). Globular embryos consisted of undifferentiated cells (Fig. 1h, h’). Culture condition IV allowed their further development. Embryos of torpedo stage were detected at 30 days of culture (Fig. 1i). Those embryos were typically bipolar, and after 40 days, they developed a single cotyledon (Fig. 1j). Tracheary elements with reticulate cell wall thickenings differentiated within cotyledons and shoot primordia as well (Fig. 1k). Their maturation was proven by the lack of nucleus and the autofluorescence of secondary walls indicating their lignification (Fig. 1k’). Further development of torpedo stage embryos led to the formation of mature bipolar embryos with well defined shoot and root primordia as well as a single cotyledon (Fig. 1e). Organ primordia had a histological structure otherwise characteristic to embryos of the Iridaceae family (Fig 1l, m). During somatic embryogenesis of C. heuffelianus, bipolar embryos differentiated directly from globular stage embryos. Such embryo development was slightly different to that reported previously for C. sativus tissue cultures by Blazquez et al. (2009). In the latter case, monopolar embryos developed


firstly, and the typical bipolar structures appeared at a later culture stage. The somatic embryos closely resembled zygotic and somatic embryogenesis in the Crocus genus and of other Monocotyledonous geophytes like Narcissus (Chichiricco` 1989; Sage et al. 2000). This work developed for the first time an efficient tissue culture system that originated from a natural population of Crocus heuffelianus. As this species is known to be endangered, this approach is of particular importance in terms of germplasm preservation. The embryogenic cultures have genetic material identical to that of the original explants. Therefore the in vitro culture is suitable for germplasm preservation in this species. The efficient somatic embryogenesis could be a powerful tool for the biochemical and cytological studies of developmental processes in the Crocus genus. This taxon includes C. sativus, important for culinary and medicinal purposes (Kafi et al. 2006) and therefore such embryological studies are of particular importance. Acknowledgments This study was supported by the EU Project CROCUSBANK AGRI-2006-0265. The authors would like to thank K. Ja´mbrik and I. Tańdor for their help in preparing this work.

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