In vitro culture of Digitalis L. (Foxglove) and the production of cardenolides: An up-to-date review

Industrial Crops and Products 94 (2016) 20–51

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In vitro culture of Digitalis L. (Foxglove) and the production of cardenolides: An up-to-date review Sandeep Kumar Verma a,b,∗ , Ashok Kumar Das c , Gunce Sahin Cingoz a , Ekrem Gurel a a b c

Abant Izzet Baysal University, Department of Biology, Bolu 14280, Turkey Department of Horticulture, Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 660701, South Korea Center for Superfunctional Materials, School of Natural Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, South Korea

a r t i c l e

i n f o

Article history: Received 30 March 2016 Received in revised form 13 August 2016 Accepted 13 August 2016 Keywords: Cardenolides accumulation Foxglove Micropropagation Somatic embryogenesis Shoot regeneration Synthetic seed production

a b s t r a c t Digitalis L. (Foxglove, Plantaginaceae) genus is a representative of several medicinal ornamental plants that are widely used in the production of herbal medicines. Since the eighteenth century, human civilizations have been using the extracts from several Digitalis species for treating heart-related ailments. The active ingredient in the medicine is cardiac glycosides. Cardenolides, which are constituents of cardiac glycosides, have an important role in tumor therapy. Certain pharmacologically active compounds including cardenolides are isolated from plants, as the structural complexity of cardenolides impede an easy chemical synthesis. In modern plant biotechnology research, production of cardenolides in largescale using in vitro techniques has become the need of the hour. The reasons are twofold: first, to reduce the excessive use of natural Digitalis population and second, to improve the plant quality vis-à-vis genetic preservation of the superior seeds for future use. The production of useful secondary metabolites depends on the overall wellness of the plant from which extraction is to be made. The subject matter of this review includes concurrent development and propagation of several Digitalis species based on direct and indirect regeneration methods. Herein, a compilation of up-to-date published research reports on in vitro culture of Digitalis L. has been presented, including the authors’ latest and yet-to-be-published work on Digitalis davisiana Heywood. The important steps to be followed for the implementation of any plant improvement/preservation program must include topic-wise requirements at various stages of micropropagation (viz., culture establishment, shoot multiplication, root induction and acclimatization) and also the requirements for plant regeneration (viz., somatic embryogenesis and organogenesis). These have been reviewed thoroughly and different methods for the in vitro production of cardenolides have been discussed. Critical comments on the prospects of highly scalable cultures and their importance to meet the ever-growing demand for Digitalis-derived products in pharmaceutical industries, have also been included. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Importance of Digitalis and the applications of Digitalis extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 In vitro propagation and cardenolides accumulation in various Digitalis species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.1. Digitalis purpurea L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.2. Digitalis lanata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3. Digitalis obscura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.4. Digitalis thapsi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.5. Digitalis minor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.6. Digitalis ferruginea (i.e. D. ferruginea subsp. ferruginea L., and D. ferruginea subsp. schischkini L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.7. Digitalis davisiana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.8. Digitalis trojana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

∗ Corresponding author at: Department of Horticulture, Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 660701, South Korea. E-mail addresses: [email protected], [email protected], sandeep [email protected] (S.K. Verma). http://dx.doi.org/10.1016/j.indcrop.2016.08.031 0926-6690/© 2016 Elsevier B.V. All rights reserved.

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5.

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3.9. Digitalis cariensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.10. Digitalis lamarckii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.11. Studies on common Turkish Digitalis species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

1. Introduction Digitalis L. constitutes flowering plants of the Plantaginaceae family; some of the species are endemic to the Mediterranean region, Turkey (D. davisiana Heywood, D. cariensis, D. lamarckii Ivan, and D. trojana Ivan), Ikaria island in Greece (D. leocophaea Sm. Subsp. leucophaea and D. leocophaea Sm. Subsp. ikarica) and Samtskhe-Javakheti in Armenia-Georgia region (D. ferruginea L.) (i.e., D. ferruginea subsp. ferruginea L., and D. ferruginea subsp. schischkini). Digitalis species are native to the Balkans, Hungary, Italy, Germany, Spain, Lebanon, Romania, Transcaucasia, Turkey, Japan and India. This genus was traditionally placed in Scrophulariaceae family, but recent phylogenetic research has placed it in the muchenlarged family of Plantaginaceae (Olmstead et al., 2001). There are 20 species of herbaceous perennials, shrubs and biennials that are commonly called foxglove. Digitalis species include D. ciliata; D. dubia; D. × fulva; D. grandiflora; D. isabelliana; D. laevigata; D. lanata; D. mariana; D. lutea; D. micrantha; D. obscura; D. parviflora; D. purpurea; D. thapsi; D. sceptrum; D. viridiflora; and D. canariensis. Many of these have medicinal uses that are highly valued in the pharmaceutical industries (Fig. 1A–J). Digitalis is also popular in many households as ornamental and decorative plants. The first known use of the D. purpurea extract was in 1785 (Goldthorp, 2009; Withering, 2014) for treating heart ailments in humans. The extract contains cardiac glycosides which helps to increase cardiac contractility in an affected patient making it easy to breath. Cardiac glycosides are also known for their antiarrhythmic action to control the heart rate. Extract of several Digitalis species is often prescribed to the patients in atrial fibrillation, especially if they have been diagnosed with congestive heart failure. Due to their usefulness in producing life-saving medicines, Digitalis species have high economic value. Owing to large-scale and uncontrolled exploitation to meet the ever-increasing demand of Digitalis by pharmaceutical industries, coupled with limited cultivation and insufficient attempts to replenishment it in the wild, the population of such an important plant species has been depleted markedly. Natural propagation of Digitalis through seeds is feasible; but this method is not as effective in producing a sufficient number of planting stocks as the germination frequency of the Digitalis seeds is rather poor. Besides these, uncontrolled collection of various Digitalis species from the natural population makes the vegetation disappear quickly, when the plants are harvested even before the seed setting. The limitations listed above can be partially overcome using in vitro propagation methods under controlled environmental conditions, facilitating the rapid multiplication of superior clones and the extraction of cardenolides throughout the year without any seasonal constraints. This necessitates establishment of superior tissue culture protocols. As the Digitalis species is biennial, plants in the open fields often fail to germinate after the second vegetation period. Therefore, an effective protocol for the propagation of Digitalis species is needed to produce large quantities of ‘quality plants’ for the extraction of valuable metabolites as well as for the ornamental plants market, while avoiding the exploitation of the wild populations. Even in the twenty-first cen-

tury, the cardenolides are still extracted from the plants, since the structural complexity of the cardenolides makes it difficult to effect a facile synthesis in the laboratory. Furthermore, genetic engineering practices are most effective in conjunction with the in vitro culture techniques (Kothari et al., 2010), which could provide cardenolides producers with opportunities to improve the quality and quantity of cardenolides derivatives raising the economic potential of the Digitalis species. In 1988, Rücker published an account on the in vitro culture, regeneration and production of cardenolides and other secondary products of the Digitalis species (Rücker, 1988). Since then many advanced techniques in biotechnology research have been developed. Similarly, the search for economically viable cardenolides production by more and more members of the Digitalis family (other than those described by Rücker) has been expanding. This has motivated the authors to make a thorough search in the current literature dealing solely with the in vitro cultures of Digitalis species and cardenolides production. The exercise resulted with the preparation of this up-to-date review. The present article includes a balanced compilation of up-todate published research reports on in vitro culture of Digitalis L. The latest, yet-to-be-published work on Digitalis davisiana Heywood, by the present authors, is also included here. Topic-wise requirements at various stages of micropropagation (viz., culture establishment, shoot multiplication, root induction and acclimatization) have been summarized. The requirements for plant regeneration (viz., somatic embryogenesis and organogenesis) have been discussed in detail. This review article is expected to serve as a guide for the use of in vitro tissue culture of Digitalis L. for the production of cardenolides and aims to provide for a hands-on knowledge of the Digitalis family, focussing on the generation, propagation and preservation of the species. 2. Importance of Digitalis and the applications of Digitalis extracts Amongst the Digitalis species, Digitalis purpurea is the bestknown and most widely used as an ornamental plant in households. The plant blossoms into vivid flowers with color range from purple tints, several shades of light gray and pure white. Besides multiple colors, the Digitalis purpurea flowers also possess eye-catching spottings and marks on them which make them more attractive. Digitalis lanata, also called as wooly foxglove or Grecian foxglove, has its distinctive appearance due to the wooly texture of the leaves. Like other Digitalis species, D. lanata is toxic and its extract is rich with powerful cardiac glycosides. The Digitalis species, namely, D. purpurea and D. lanata have been extensively studied by horticulturists as well as plantation agronomists for their medicinal, ornamental and economical values. The use of Digitalis extracts in the treatment of heart ailments was first reported by William Withering in 1785 (Withering, 2014), who made use of the active ingredient in the extract, later identified to be a cardiac glycoside (also known as a cardiotonic steroid). The molecule has a steroid-like structure including an unsaturated lactone ring. The cardiac glycosides contain sugar

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Fig. 1. Digitalis purpurea (A), Digitalis lanata (B), Digitalis obscura (C), Digitalis thapsi (D), Digitalis minor (E), Digitalis ferruginea (F), Digitalis davisiana (G), Digitalis trojana (H), Digitalis cariensis (I) and Digitalis lamarckii (J). [Source: https://www.anniesannuals.com/plants/view/?id=361; https://gardencoachpictures.wordpress.com/tag/digitalis-obscura/; http://www.elclubdigital.com/foro/ showthread.php?t=83586; http://luirig.altervista.org/flora/taxa/index2.php?scientific-name=digitalis+ferruginea; http://www.agaclar.net/forum/karadeniz-bolgesi/4982. htm]

Table 1 Effects of cardiac glycosides on cancer cells. Cancer type

Compounds tested

Cancer cell lines

References

Breast

Digitoxin, digoxin, digoxigenin, gitoxin, gitoxigenin

MCF-7,MDA-MD-435, MDA-MB-231, MDA-MB-453

Colon

Digoxin (modification of the lactone ring) Digoxin (modification of the lactone ring) Digitoxin

RKO-AS45-1, HT29

Bielawski et al. (2006); Einbond et al. (2010); López-Lázaro et al. (2005);Winnicka et al. (2007) Riganti et al. (2009); Rocha et al. (2014) Rocha et al. (2014)

Cervical Leukaemia

HeLa CEM/VBL100 , SUP-B15, K-562

Lung

Digitoxin, digoxin, digitoxigenin

A549, H1975, NCl-H460, H1355, SEM, K562, H3255, H1975, PC9, PC9/gef

Melanoma Myeloma

Digitoxin, digoxin Digitoxin, digoxin, digitoxigenin, digitonin, lanatocide C Digoxin Digoxin, digitoxin, digitoxigenin

UACC-62 8226-S, 8226-LRS, 8226-DOX-40

Pancreatic Renal

Digoxin Digitoxin, digoxin, digitoxigenin

BxPC-3 TK-10, ACHN, A498, 786-O, Caki-1, Caki-2

Skin

Digoxin, digitoxin

UACC62, VH10

Neuroblastoma Prostate

moieties in their structure and possess the cardiotonic activity. Cardiac glycosides containing one five-membered lactone ring are known as cardenolides (Fig. 2). The term derives from the ¯ and enolide refers to the card or ‘heart’ (from Greek kardia) lactone ring at C17 . Cardenolides are C23 steroids with methyl

SH-SY5Y, Neuro-2a PC3, DU-145, LNCaP, HRPC

Daniel et al. (2003); Hallböök et al. (2011); López-Lázaro et al. (2005) Elbaz et al. (2012); Hiyoshi et al. (2012); Iyer et al. (2010); Lin et al. (2015); Nolte et al. (2015); Trenti et al. (2014); Wang et al. (2011, 2012, 2009) López-Lázaro et al. (2005) Johansson et al. (2001)

Svensson et al. (2005) Leu et al. (2014); Lin et al. (2004); Nolte et al. (2015); Yeh et al. (2001) Prassas et al. (2011) Johansson et al. (2001); López-Lázaro et al. (2005); Nolte et al. (2015) Hiyoshi et al. (2012)

groups at C10 and C13 and a five-membered lactone ring positioned at C17 . These are aglycone constituents of cardiac glycosides and must have at least one C C double bond in the molecule. The class includes carda-di-enolides and carda-tri-enolides. Members include acetyl digitoxins, acetyldigoxins, cymarine, digitoxin,

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Fig. 2. Chemical structure of cardenolide, C23 H34 O2 .

Fig. 3. Chemical structure of digitoxin, C41 H64 O13 (the molecule contains five groups).

OH

Fig. 4. Chemical structure of digoxin, C41 H64 O14 (the molecule contains six groups).

OH

digitoxigenin, digoxigenin, digoxin, medigoxin, neoconvalloside, ouabain, strophanthins and strophanthidin (Agrawal et al., 2012). Out of these, digitoxin and digoxin are the two main cardenolides that have extensive therapeutic use. These two differ with respect to molecular structures: digitoxin, C41 H64 O13 has five OH groups (Fig. 3) while digoxin, C41 H64 O14 contains six OH groups (Fig. 4). The extra OH group considerably alters the pharmacokinetics in digoxin (Rocha et al., 2014). The positive inotropic action of the digitoxin and digoxin cardenolides is believed to be due to inhibition of the cardiac sodium pump (Na/K-ATPase, a plasma membrane enzyme). The enzyme is responsible for counter-transport of 3 Na+ ions out and 2 K+ ions into a living cell, for each ATP hydrolyzed (Belz et al., 2001; Hallberg et al., 2007). Owing to this inhibiting action by Na/K-ATPase, cardiac glycosides are able to reduce the sodium gradient in the cell. As a consequence, the activity of the Na/Ca exchanger (responsible for an increase in intracellular

23

Ca2+ concentration) gets enhanced and the cardiac contractile force eases the patient (Epstein and Smith, 1998; Trenti et al., 2014). The only safe inotropic drugs available for the improvement of haemodynamics in patients with compromised cardiac function are digitoxin and digoxin (Schwinger et al., 2003). In patients with heart failure who have fluid retention, digoxin induces diuresis (Rahimtoola and Tak, 1996; Rahimtoola, 2004). One of the suggested mechanism is the inhibition of tubular reabsorption of renal Na/K-ATPase. Experiments using digitalis as a diuretic proved that it facilitates in diminishing fluid retention among patients suffering from dropsy or edema (Francis, 2008; May and Diaz, 2008). Modern pharmaceutical research embodies the replacement of digoxin with newer drugs including beta-blockers, angiotensin-converting enzyme inhibitors and the calcium-channel-blocking agents. Since the commencement of development of new synthetic pharmacotherapeutic agents, the use of digitalis extracts in medicines is expected to decrease in the near future. Important other commercial uses of D. lanata are also known. These include the use of powdered leaves in relieving asthma through inhalation, as sedatives and as diuretics in several South American countries. In India, ointments containing digitalis glycosides find their use in the treatment of minor wounds and burns (Belcastro, 2002; Feussner and Feussner, 2010). As an allopathic medicine, the permitted dosage of Digitalis is about 0.125–0.25 mg/day (Roden, 2001). In the 1980s, Stenkvist and colleagues reported their studies on the treatment of breast cancer cells. It was found that the affected cells (collected from the women who were treated with the Digitalis therapy) could be characterized by a series of more benign features compared with the cancer cells collected from control patients (Stenkvist et al., 1979a,b, 1982). Numerous subsequent in vitro and in vivo studies had verified these initial findings (Winnicka et al., 2006; López-Lázaro, 2007; Mijatovic et al., 2007). This led the series of cardiac-glycoside-based drugs to enter clinical trials for treating cancer (Mekhail et al., 2006; Mijatovic et al., 2006b; Newman et al., 2008). Recent studies have identified digoxin as a possible drug for prostate cancer treatment (Platz et al., 2011). In addition, the first in vitro evidence for the inhibition of malign cell proliferation by cardiac glycosides dates back to Shiratori’s work in 1967 (Shiratori, 1967). Since then, the antiproliferative and apoptotic effects of the cardiac glycosides had been confirmed by numerous studies. These include breast cancer (Kometiani et al., 2005; López-Lázaro et al., 2005; Bielawski et al., 2006), prostate cancer (McConkey et al., 2000; Platz et al., 2011), melanoma (Newman et al., 2006), pancreatic cancer (Newman et al., 2007), lung cancer (Frese et al., 2006; Mijatovic et al., 2006a), leukaemia (Daniel et al., 2003), neuroblastoma (Kulikov et al., 2007) and renal adenocarcinoma (López-Lázaro et al., 2005), as shown in Table 1. New findings suggest that plant-derived cardenolides, such as digoxinlike immunoreactive factors (Ihenetu et al., 2007; Elbaz et al., 2012) are responsible for the regulation of cell proliferation in human tumor and normal tissues. These observations led to the clinical trials on digoxin in patients afflicted with cancer (Mekhail et al., 2006; Mijatovic and Kiss, 2013; Slingerland et al., 2013).

3. In vitro propagation and cardenolides accumulation in various Digitalis species Having recognized that the cardenolides need to be extracted from the Digitalis species, one now looks for the ways to increase the accumulation of cardenolides in the plants (wild, cultivated or in vitro). Like the other countries of the globe, Turkey also has been experiencing a gradual depletion of the number of wild populations of the Digitalis species. Limited cultivation in the fields and insufficient attempts to replenish it in the wild have resulted into a marked depletion of this important plant species. The nat-

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ural propagation of Digitalis through seeds is fraught with a low germination frequency, making the restroration capabiliity to be very limited. The Digitalis plants require a period of about two years for optimal development and maximizing accumulation of the biologically active substances (especially, cardenolides and their derivatives) in the plantlets. These factors make the production of phytochemically consistent plantlets extremely difficult, paving the way for establishing in vitro cultures for the propagation/preservation/quality improvement in the various Digitalis species. With regards to the practical implementation of the in vitro techniques, multiple shoot culture was proved to be a viable alternative for the production of the much sought after biologically active plant constituents (Heble, 1985). The account below presents the description of the important members of the various Digitalis species with respect to their growth, in vitro production and cardenolides accumulation in the plants (Table 2 ). 3.1. Digitalis purpurea L. Digitalis purpurea L. is a member of the family Scrophulariaceae (Fig. 1A). The plant is a biennial or perennial herb which grow in the western Europe, Asia, northwest Africa, South America, New Zealand and Canada (Eric, 1968). The poisonous character of the plant is all pervading (leaves, roots, tender stem and seeds) and it contains several deadly physiological and chemically related cardiac and steroidal glycosides (Budavari, 1989). Scanning electron microscope image of D. purpurea seed showed clear picture of seed coat (Fig. 5A, B). The seed dimensions (length, width and height) are shown in Fig. 5C. Attempts to determine whether tissue culture of the plants (D. purpurea L. and D. lanata) can lead to biosynthesis of the cardiac glycosides or not, have suggested that the agar medium is most suitable for the establishment of in vitro generation of these two Digitalis species. For the purpose, seeds and roots were used for the callus development using different concentrations of auxins, 2,4-dichoro-phenoxyacetic acid (2,4-D), 2-benzothiazoleoxyacetic acid (BTOA) and 1-napthaleneacetic acid (NAA) (Staba, 1962). A scheme of biotransformation of progesterone in D. purpurea L. callus culture was suggested by Furuya et al. (1973) indicating the role of 20␣-hydroxysteroid dehydrogenase in the transformation. It had been proved that progesterone is an intermediate in cardenolide pathways in all plants (including Digitalis purpurea L.) producing cardenolides (Janeczko and Skoczowski, 2011). Nitsch and Nitsch (1969) were able to obtain highest frequency of regenerated plants of D. purpurea L. when the authors incubated the anthers in the basal medium of Nitsch medium, supplemented with 5 mg/L of 2,4-D. Explorations using several basal media (viz., Linsmaier and Skoog (1965) and Murashige and Skoog (1962)), supplemented with kinetin or auxin, resulted in a decrease of the number of regenerates (Corduan and Spix, 1975). It is worthwhile to note that sterol uridine diphosphoglucose (UDPG) glucosyltransferase enzyme, isolated from the cultured cells (calli) of D. purpurea showed 2–5 times increased activity than that of the original plant leaf extract (Yoshikawa and Furuya, 1979). Chemical properties of the purified enzyme obtained from the cultured cells of D. purpurea were similar to those of the enzyme extracted from the plant leaves, but the UDPG glucosyltransferase enzyme possesses much higher substrate specificity (Yoshikawa and Furuya, 1979). This caused an increased activity. The formation of cardenolides takes place by the differentiation of callus tissues of D. purpurea (Hirotani and Furuya, 1977). However, with progress of time, the calli lost the ability to produce certain cardenolides. In the case of roots and root-forming calli of D. purpurea, no cardenolides were detected in the cultured cells. But from leaves of the regenerated shoots, digitoxin and purpurea-glycoside A were found to

be present. The authors (Hirotani and Furuya, 1977) then inferred that the cardenolide-synthesizing ability in D. purpurea is closely related to the leaf development process. Extensive investigations of the biotransformation of steroids by plant cell culture of D. purpurea was undertaken by Hirotani and Furuya (1980a,b). The results revealed that, out of the eight biotransformation products from digitoxigenin, it was possible to isolate only three (digitoxigenone, epidigitoxigenine and epidigitoxigenin-␤-d-glucoside) for further use. The same group (Hirotani and Furuya, 1977) had reported conversions of digitoxin to digoxin, gitoxin and other compounds by D. purpurea along with the conversion of pregnenolone to progesterone and other steroids. It is noteworthy that the same authors had identified the products including 3-␤-hydroxy-5-␤-pregna20-one from 5-␤-pregnane-3, 20-dione by D. purpurea cultures (Hirotani and Furuya, 1975). High concentrations of cardiac glycosides, including digitoxin, were detected by Hagimori et al. (1980) who cultivated the shoot-forming tissue culture of D. purpurea in a fermenter. It was previously believed that for the production of secondary metabolites, differentiated tissue culture might be necessary. But there are reports showing total absence of digitoxin or digoxin in root-forming differentiated calli of D. purpurea. In contrast, considerable amounts of cardenolides were found in the shoot-forming calli. Similarly, D. lanata and D. purpurea cell tissue cultures show abundant formation of both digitoxin and digoxin. Variuos cultures of D. purpurea L. were considered by Hagimori et al. (1982) in order to compare the amount of digitoxin produced. These include undifferentiated, highly chlorophyllous cell cultures, undifferentiated white cell cultures, green shoot-forming cultures and white shoot-forming cultures. It was found that the green shoot-forming cultures accumulated the largest amount of digitoxin. These authors opined that, for the synthesis and accumulation of digitoxin, the chloroplasts are not essential requirements. Strategies based on in vitro culture methods, have been extensively used to improve the production of digitoxin by altering the nutrient media (Hagimori et al., 1983). The reduced photosynthesis observed in D. purpurea plantlets cultivated in vitro is believed to be due to the synthesis of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisco) by the plants, as suggested by Hagimori et al. (1984b). A possible scale-up technique for shoot culture of D. purpurea in bioreactors was described by Hagimori et al. (1984a) and was successfully tested in large fermenters. A limitation of this approach is low digitoxin accumulation rates in the in vitro grown plants. A no. of phenyl-propanoid glycosides have been detected or isolated from D. purpurea from the leaves (i.e., desrhamnosylacteoside, forsythiaside, purpureaside A, purpureaside B and 3, 4-dihydroxyphenethylalcohol-6-O-caffeoyl␤-d-glucoside) and from the callus tissue (i.e., purpureaside A, purpureaside B, acteoside and purpureaside C), as reported by ˇ Matsumoto et al. (1987). Cellárová and Honˇcariv (1991) had studied the morphogenetic capability of D. purpurea using leaf explants and callus culture. Gärtner et al. (1990) and Gärtner and Seitz (1993) described a regeneration protocol for D. purpurea which provides for 7–10 plantlets per leaf explant. These authors had traced the early stages in the biosynthesis of 5␤-cardenolides from sterols and found that the synthesis was mediated by the enzyme progesterone 5␤-reductase (P5␤R). Chaturvedi and Jain (1994) have reported that the shoots of in vitro-cultured D. purpurea grew normally in a modified MS medium supplemented with 0.98 ␮M indole-3-butyric acid (IBA) and 27.1 ␮M adenine sulfate. These authors observed an apparent loss of regeneration potential of the shoot cultures of D. purpurea. However, on closer examination the authors clarified that the regeneration capability was not permanently lost, but was temporarily masked due to a shift in the growth regulator requirement, from cytokinin (BAP) to gibberellic acid (GA3 ). In a similar note, an

Table 2 In vitro culture and cardenolides accumulation in Digitalis species. Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Digitalis purpurea

Seed and root

Medium A containing coconut milk, sucrose, meso-inositol, vitamin-free, enzyme-hydrolyzed casein, MS inorganic salts, iron-ethylenediamine tetraacetic acid, vitamin solution. Medium B containing coconut milk, sucrose, modified white’s inorganic salts, iron-ethylenediamine tetraacetic acid, vitamin-solution (same as a medium A), simple supplement (meso-inositol, glycine-anhydrous, and niacin), complex supplement (glucose-1-phosphate, L-glutamine, L-glutamine acid, L-aspartic acid, urea, L-arabinose, L-asparagine, chlorogenic acid, methyl-␤-l-arabinopyranoside, adenosine triphosphate disodium salt, adenine (hem) sulfate, and deoxyribonucleic acid with 2,4-dichlorophenoxyacetic acid (2,4-D), 2-benzothiazoleoxyacetic acid (BTOA), and 1-napthaleneacetic acid (NAA) Modified MS medium containing 2,4-D, Kinetin (Kn) and progesterone Modified MS, LS, Nitsch medium and White medium (White, 1943) with 2,4-D; 4-chloro-2-methyl-phenoxyacetic acid (4Mp); 2(2,4-dichlorophenoxy)-propionic acid (2,4-Dp); and 4-(2,4,5-trichlorophenoxy)-butyric acid (4-Tb) MS medium supplemented with 2,4-D and Kn

Callus

–

–

Staba (1962)

Callus suspension cultures Callus, regenerated plants

Biotransformation of progesterone –

–

Furuya et al. (1973)

–

Corduan and Spix (1975)

Callus and plant

UDPG glucosyltransferase activity Cardenolide

–

Yoshikawa and Furuya (1979)

No cardenolide was detected

Hirotani and Furuya (1977) Hirotani and Furuya (1980a) Hirotani and Furuya (1975); Hirotani and Furuya (1980b)

Seedling Anther (Haploid)

Leaves

Seedling and leaf Seedling Seedling and leaf

MS medium supplemented with 2,4-D, Indole-3-acetic acid (IAA), and NAA Modified MS medium with 2,4-D, Kn and digitoxigenin Modified MS medium supplemented with 2,4-D and Kn

Callus, regenerated shoot Cell suspension Normal callu, habituated callus, suspension culture

Biotransformation of digitoxigenin Metabolism of 5␤-pregnane-3, 20-dione, and 3␤-Hydroxy-5␤pregnan-20-one.

– –

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Digitalis species

25

26

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Shoot-forming calli: D. purpurea; D. lanata; D. lutea; D. mertonensis; D. ambigua; and D. ferruginea Gigantea Leaf disc: D. purpurea D. lanata

Modified MS medium supplemented with BA, 2,4-D, IAA, and NAA

Callus and shoot forming callus

Digitoxin and digoxin

Hagimori et al. (1980)

Seedling

Basal medium supplemented with BA, IAA, NAA, and 2,4-D

Callus, shoot forming cultures

Digitoxin

Seedling

MS medium with mineral salts (CaCl2 , MgSO4 , FeSO4 ), minor elements was increased threefold (H3 BO3 , MnSO4 , ZnSO4 , KI, Na2 MoO4 , CuSO4 , and CoCl2 ), pH (4, 5, 6, 7, or 8) and precursors (methanol (MeOH), ethanol (EtOH), propylene glycol (PG), dimethyl sulfoxide (DMSO) and acetone Photoautotrophic culture MS salts with 3 ␮M, thiamine HCl, 17 ␮M IAA, 5.7 ␮M IAA and 1% CO2 Basal medium supplemented with BA, IAA, and thiamine-HCl. The composition of the revised medium is the same as that of the basal medium except for the three components i.e., CaCl2 ·2H2 O (one-third), KH2 PO4 (three-fold) and MgCl2 ·6H2 O MS medium contained 2,4-D, under continuous light

Liquid media, callus, shoot forming cultures

Digitoxin

Shoot-forming calli: D. purpurea (digitoxin 18.2 ␮g/g FW and digoxin 1.7 ␮g/g FW); D. lanata (digitoxin 1.2 ␮g/g FW and digoxin 4 ␮g/ g FW); D. lutea (digitoxin 9.7 ␮g/g FW and digoxin 1.4 ␮g/g FW); D. mertonensis (digitoxin 6.8 ␮g/g FW and digoxin 0.7 ␮g/g FW); D. ambigua (digitoxin 3.9 ␮g/g FW and digoxin 0.6 ␮g/g FW); and D. ferruginea Gigantea (digitoxin 0.7 ␮g/g FW and digoxin 0.3 ␮g/g FW) Leaf disc: a) D. purpurea Digitoxin ranged from 0.43 to 2.16 ␮g/g FW for first passage calli and 0.01–0.07 ␮g/g FW for second passage calli b) D. lanata Digoxin ranged from 1.8 to 20.5 ␮g/g FW for first passage calli and 0.14–1.66 ␮g/g FW for second passage calli Digitoxin ranged from 0.5 to 5.4 ␮g/g FW for first passage calli and 0.04–0.52 ␮g/g FW for second passage calli Light grown, green shoot forming culture contained digitoxin (about 20–40 ␮g/g DW). The chlorophyll content undifferentiated green cells and white cell without chloroplasts accumulated extremely low digitoxin (about 0.05–0.2 ␮g/g DW) A threefold increase in the concentration of either KI, Na2 MoO4 or CuSO4 (with pH = 6) improved digitoxin formation

Callus, shoot forming cultures

Digitoxin

The digitoxin contents were not improved by photoautotrophic culture

Hagimori et al. (1984b)

Callus, shoot forming culture

Digitoxin

Total digitoxin ranged from 300 to 1610 ␮g/jar

Hagimori et al. (1984a)

Callus culture

Isolated and identified

Matsumoto et al. (1987)

Leaf lamina and petiole

LS medium, B5 vitamins with 2,4-D, NAA, Kn and n-[2-isopentenyl] adenine (2iP)

–

ˇ and Cellárová Honˇcariv (1991)

Seedling

Modified MS medium (containing glucose, IAA and Kn)

Callus cultures,bud and shoot differentiation Shoot culture

Purpureaside A, purpureaside B, acteoside and purpureaside C –

Cardenolid

30 mg/kg DW

Gärtner et al. (1994)

Seedling

Seedling

Leaf and stem segments

Hagimori et al. (1982)

Hagimori et al. (1983)

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Explants

Table 2 (Continued) Digitalis species

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Shoot culture

MS media (lower concentration of CaCl2 ·2H2 O and a higher concentration of KH2 PO4 ) containing Indole-3-butyric acid (IBA), gibberellic acid GA3 ), and adenine sulfate Modified MS medium (containing glucose, IAA, and Kn).

Callus, shoot and plant regeneration

–

–

Chaturvedi and Jain (1994)

Mixotrophic shoot culture

All cardenolides are 5␤-configurated

Seitz and Gärtner (1994)

Callus and submerged cultures

Cardenolides and progesterone 5␤-reductase. Progesterone 5␤-reductase Cholesterol side-chain cleaving enzyme

Gärtner et al. (1994) Pilgrim (1972)

Callus

Digitoxin

Callus

Digitoxin

Plantlets Shoot regeneration, plant regeneration

Digoxin and digitoxin Digoxin and digitoxin

Purification, characterization, and partial peptide micro sequencing Tissue culture failed to show cholesterol side-chain cleaving activity, which may be the reason why tissue cultures are unable to biosynthesize cardenolide glycosides Quantifiable amounts of digitoxin were detected in all callus tissues A medium but supplemented with 40 mg/L phenobarbital contained 0.475 ␮g/g DW digitoxin The best net production of digoxin (167.6 ␮g/TIS) and digitoxin (119.9 ␮g/TIS) The best response for the content digitoxin (492.6 ␮g/g DW) and digoxin (214.2 ␮g/g DW) was on medium fortified with 200 and 300 mg/L of progesterone, respectively. On this medium, the increase in digitoxin was about nine-folds and digoxin was around 12-folds over the control

Shoot regeneration

–

–

Li et al. (2014)

Callus and plant regeneration

–

–

Pérez-Alonso et al. (2014)

Callus

–

–

Staba (1962)

Callus

Anthraquinone derivatives and 4hydroxydigitolutein Anthraquinone derivatives, 3-methylpurpurin, and Digoxin

Extraction and isolation

Furuya and Kojima (1971)

Extraction and isolation

Furuya et al. (1972)

Cotledon seedling were grown on RT (MS revised tobacco medium) agar medium withouth growth regulators. These plantlets were grown under a 16 h day-light cycle (500 fluorescent plant-grow bulbs, sears, 40 W, cool-light). The concentration of digoxin varied from 18 to 44 mg% DW over a 12 week perid

Lui and Staba (1979)

Seedling

Seedling Seedling

Seedling Hypocotyls

Seedling Node, internode and leaf

Leaf

Leaf

Digitalis lanata

Seed and root Seedling

Modified MS medium (containing glucose, IAA, and Kn) ??

MS medium supplemented with BAP, IAA, NAA, with or without phenobarbital MS medium supplemented with BAP, GA, IAA, and NAA with or without phenobarbital Temporary immersion systems (TIS), MS medium supplemented with BAP, and IAA MS medium supplemented with BA, Kn, Thidiazuron (TDZ) alone or in combination with IAA, NAA or 2,4-D. Several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). MS medium supplemented with TDZ, NAA. Agrobacterium tumefaciens-mediated genetic transformation Basal medium contained with MS salt with thiamine, myo-inositol, sucrose, and Gelrite supplemented with 2, 4-D and BAP. Agrobacterium tumefaciens-mediated genetic transformation Modified MS medium supplemented with 2,4-D, BTOA, and NAA White’s medium contained 2,4-D, yeast extract and sucrose

Shoot culture

Seedling

MS medium supplemented with IAA and Kn

Callus

Seedling

MS medium supplemented with BA, IAA, or 2,4-D and added precursor sodium glycocholate, deoxycholic acid, cholesteryl acetate, lanosterol, progesterone, 5␤-androstan-3,17-dione, and smilagenin

Leaf and root culture

Palazón et al. (1995) Bonfill et al. (1996)

Pérez-Alonso et al. (2009) Patil et al. (2013)

27

Medium + Plant growth regulators

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Explants

28

Table 2 (Continued) Digitalis species

Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Plant

MS revised tobacco liquid medium supplemented with BA or IAA. Later on cultured were transferred on new medium contained BA, IAA, and GA or mefluidide

Leaf/root culture

Digoxigenin, gitoxigenin, digitoxigenin, and digoxin

Lui and Staba (1981)

Strain I (Stem tissue), Strain V (Anthers)

Modified MS medium (Nm 0, Nm I, Nm II, Nm III, Nm IV and Nm VI) contained the usual macro- and microelements, except that the content of FeSO4 was doubled. The nutrient media were supplemented with sucrose, thiamine, pyridoxine, nicotinamide, inositol and the amino acid mixture. For this, 2, 4-D, BA, NAA, and Kn were used as plant growth regulators LS medium salts plus thiamine and sucrose BAP, IAA, KNO3 or NH4 NO3 was reduced to one-fifth Modified regeneration medium with IAA and BA.

Suspension culture, organogenesis

Digitoxigenin, digitoxin, lanatoside A, gitoxin, lanatoside B, digoxigenin, digoxin, lanatoside C, digipurpurin, digitonin.

The triol genin (digoxigenin and gitoxigenin) increased from 540 ␮g/g DW at 12 weeks while the diol genin (digitoxigenin) decreased from 1200 ␮g/g DW to 480 ␮g/g DW at 12 weeks Mefludide (50 mg/L) increased digoxin production 30% in the leaf cultures (42 mg% DW) and 40% in root culture (4.1 mg% DW) Digoxin production in leaf cultures increased 60% (52 mg% DW) upon removing the BA from the medium Cardenolides contents in white globular structures 0.010-0.5 ␮g digitoxin equivlent mg DW, green globular structures 0.03-0.1 ␮g digitoxin equivlent mg DW, embryoids 0.03-0.1 ␮g digitoxin equivlent mg DW and shoot, plantlets 0.08-0.1 ␮g digitoxin equivlent mg DW

Shoot multiplication and plant regeneration Protoplasts, calli

–

–

Erdei et al. (1981)

Callus, shoot, root, and plant regeneration Cell clusters

–

Li (1981)

–

Embryogenesis, plant regeneration

Cardenolides

Suspension cells

Digitoxin 12␤-hydroxylase activity Digitoxin and digoxin Plant regeneration and digitoxin content –

The cardenolide content of the irradiated embryos increased within three weeks from about zero to about 1 ␮g/mg DW (about 1 mg/L culture). It reached about 0.15 ␮g/mg DW (4 mg/L culture) during plantlet regenration Isolation and enzyme acivity

Diettrich et al. (1982) Kuberski et al. (1984)

Mesophyll

Cell culture Embryogenic strain VII

Cell culture

Cell culture Embryogenic strain VII

Leaf

Leaves, stem, or filaments (D. lanata, D. purpurea, and D. heywoodi) Cell lines

Modified MS medium supplemented with kinetin, 2,4-D, Kn, sorbitol, and mannitol Modified nutrient media supplemented with 2,4-D, Kn, NAA, and BAP

Cell cultures of D. lanata were cultivated as described previously (Petersen and Seitz, 1985) Cell cultures of D. lanata were the generous gift of Professor E. Reinhard, Tubingen Modified MS medium (Nm I) with mannitol, sucrose, glycerol, 2,4-D, Kn, NAA and BA Modified MS medium contained KH2 PO4 , no casein hydrolysate, IAA, Kn, and glucose

Modified MS medium contained KH2 PO4 , glycine, 2, 4-D, IAA, and Kn Modified MS medium supplemented mannitol, desalted cellulose RS Onozuka, rhozyme

Protoplasts

Suspension culture Solidified, suspension Suspension culture, callus, protoplast and vacuoles isolation Suspension culture

Callus, suspension culture

Cardenolide glucosyltransferases and glucohydrolases Digitoxin, digoxin, 16 -Oglucosltransferase, ␣- acetyldigitoxin, and ␣-acetyldigoxin

Isolation and enzyme acivity –

Petersen and Seitz (1988) Petersen and Seitz (1985) Diettrich et al. (1985)

–

Kreis and Reinhard (1985)

Enzyme acivity

Kreis and May (1990)

Characterize and localize the formation of purpurea glycoside A from digitoxin

Kreis et al. (1986)

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Shoot tip

Garve et al. (1980)

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Filament, leave and roots

Modified MS medium (Nm I, Nm II-M and Nm V-M) suplemneted with 2,4-D and NAA

Callus, somatic embryogenesis, and shoot culture

Cardenolides content in somatic embryos (1.0 mg digitoxin equivalents/g TM)

Diettrich et al. (1986)

Meristem, axillary buds

A half-strength liquid MS medium contained glucose and various combinations of phytohormones (IAA, NAA, IBA, BAP, and Kn)

Shoot regeneration, rooting of shoots, and plant regeneration

Glycoside contents of regenerated plants: Dgn-glycoside (541 mg 100/g DW), lanatoside C (422 mg 100/g DW), and Dtn-glycosides (74 mg 100/g DW). Colnes contents: Dgn-glycoside (642 mg 100/g DW; 20th passages), lanatoside C (577 mg 100/g DW), and Dtn-glycosides (238 mg 100/g DW)

Schöner and Reinhard (1986)

Strain VII

Nutrient medium NM VII-M, Nm VII-M supplemented with 2, 4-D and NAA

Somatic embryos

Regenerating roots, non-embryoid globules, adventitious embryos, and mixed morphogenesis. Deacetyllanatoside C, lanatoside A, B, C, digoxin, ␣-acetyldigoxin, ␤-acetyldigoxin, digitoxin, ␤-methyldigitoxin, digoxigeninglycosides, and digitoxigeninglycosides Cardenolides

Scheibner et al. (1987)

Strain S-1 and S-2

Modified MS medium supplemented with BA, gibberellic acid and SAN9789

Meristematic or differentiated as vascular cells

Cardenolides

Shoot tip

MS medium with increased thiamine content supplemented with BAP, IAA, and NAA MS medium contained claforan and Agrobacterium rhizogenes strain A4

Plant regeneration

Lanatoside C

Hairy root cultures

Digoxin, lanatoside C, and deslanoside

Modified MS medium (Nm 0, Nm I, Nm II, Nm III, Nm IV, and Nm VI) contained the usual macro- and microelements, except that the content of FeSO4 was doubled. The nutrient media were supplemented with sucrose, thiamine, pyridoxine, nicotinamide, inositol and the amino acid mixture. For this, 2, 4-D, BA, NAA, Kn, abscisic acid (ABA) and GA3 were used as plant growth regulators

Strain S-2 was isolated from strain S-1, when grown on a medium contained NAA and BA

Cardenolides and digitoxin

Optimum cardenolide amounts ( > 1 ␮mol digitoxin equivalents/g DW) were found in stage-II globules and bipolar embryo structures Authors suggested that the cardenolides are synthesised in the plastids and temporarily stored in plastoglobuli, before exprot to the vacuoles. Plastoglibuli contained the amount of cardenolides was about 140 ␮g/g DW Lanatoside C content ranged between 0.415% and 0.722% (on dry matter basis), the drug level being 0.558% The content of cardenolides (ranged from 0.12 to 16.47 digoxin equivalents ␮g/g DW) in green hairy roots cultured under light (16 h) condition Strain-1 was grown on medium without regulators (pH = 5.77) accumulated highest cardenolides (84.5 ␮g digitoxin/g DW)

Leaf

Strain S-1 (Anther filament)

Berggren and Ohlsson (1991)

Kubalákoyá et al. (1987) Yoshimatsu et al. (1990)

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Explants

Ohlsson (1990)

29

30

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Filament-derived strain

Modified MS medium supplemented with IAA, Kn, casein hydrolysate, and sucrose

Undifferentiated cell with yellowish color

Cardenolides in habituated strain K4 OHD contained highest amount 50.2 digitoxin equivalents/g DW

Seidel et al. (1990)

Proembryogenic masses of Strain VIII

Nutrient medium Nm1 supplemented with BA, and NAA

Propagation

Strain VIII, PEMs has specific activity of 5 -3␤-HSD [(16.0 ␮kat (kg protein)−1 )]

Finsterbusch et al. (1999)

Embryogenic strain VII

Nutrient media (NmI, Nm VIII-M, NmIX-M: NmViii-M) with supplemented with 2,4-D and ABA Strain VII was grown in nutrient medium Nm V-M supplemented with BA and NAA. The fermenter experiments were carried out using nutrient medium Nm VII-M Modified MS media supplemented with IAA, Kn, and DMSO

Somatic embryogenesis, Plant regeneration Somatic embryos development

5 -3␤hydroxysteroid dehydrogenase/5 4 ketosteroid isomerase 5 -3␤hydroxysteroid dehydrogenase (5 -3␤-HSD) Cardenolides

Cardenolides ranged from 3.4 to 15.7 ␮mol DE/g DW

Reinbothe et al. (1990)

Cardenolide and digitoxin

Cardenolide ranged from 0.68 to 1.08 ␮mol/g DW in somatic embryos in strain VII grown in gaslift fermenters

Greidziak et al. (1990)

Plant cell culture

Lanatoside A, ␤-methyldigitoxin, and cardenolide

Kreis et al. (1990)

Anther

Nutrient media (Nm AI, Nm I and NmV-M) supplemented with BA, 2,4-D, NAA, and Kn

Callus, somatic embryos, plant regeneration

Glucodigifucoside, odorobioside G, lanatoside A, and lanatoside C

Flowering shoot (Stain 285 and 287), leaves (Strain W 1.4), filaments (Strain VII), and Strain VIII

Strain 285, 287 and W 1.4 were subcultured in hormone-free nutrient medium (NmI). Strain VIII was propagated in nutrient medium NM VII-M, Nm VII-M supplemented with 2, 4-D and NAA

Lanatoside 15 -Oacetylesterase

Leaves

Modified MS medium contained KH2 PO4 , no casein hydrolysate, IAA, Kn, and glucose

Proembryognic masses and developmental stages of somatic embryos (globules, heart and germinating embryos) Callus, suspension culture

Maximum accumulation of cardiacglycoside in selected cell lines: (a) K 1 OHD content purpurea glycoside A (300 mg/kg); (b) K 3 OHD content deacetyl lanatoside C (119 mg/kg); (c) K 1 HL lanatoside A (323 mg/kg); K 1 OHD content ␤-methyldigoxin (29 mg/kg) Cardenolides ranged from 3.5 to 117 ␮mol digitoxin equivlent/g DW(4 weeks after transfer to greenhouse) and 1.7–50.3 ␮mol digitoxin equivlent/g DW (10 weeks after transfer to greenhouse) Cardenolide profile of regenerated plants: Glucodigifucoside ranged from 1.2 to 3.1 ␮mol/g DW; odorobioside G ranged from 1.0 to 2.3 ␮mol/g DW; lanatoside A ranged from 5.3 to 12.0 ␮mol/g DW; lanatoside C ranged from 4.2 to 6.2 ␮mol/g DW Purification and characterization

Modified MS media supplemented with BAP alone Strain 285, 287 and W 1.4 were subcultured in hormone-free nutrient medium (NmI). Strain VIII was propagated in nutrient medium NM VII-M, Nm VII-M supplemented with 2, 4-D and NAA

Lanatoside A (40 mg/L was fed on day 3 of the culture cycle and the cell analysed after 24 h of incubation. Lanatoside (A + C) ranged from 128 to 580 mg/kg FW and deacetyllanatoside (A + C) ranged from 5 to 225 mg/ kg FW 40 ␮g digitoxin/g DW

Sutor et al. (1990)

Strain S-1

Lanatoside A, C, purpureaglycoside A, decetyllanatoside C, and 15 -Oacetylesterase Digitoxin Cell wall associated lanatoside 15 -Oacetylesterase (molecular mass 120 kDa)

Partial purification and characterization

Strain VII

Cell lines

Leaf

Shoot forming culture Shoot forming cell line H1

Ernst et al. (1990)

Kandzia et al. (1998)

Berglund and Ohlsson (1993) Sutor and Kreis (1996)

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Explants

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Shoot tip

Nutrient media (Nm1, Nm 2, Nm 3, and Nm 4) supplemented with BA and NAA

–

–

Diettrich et al. (1990)

Strain VII from filaments

Nutrient media (NmI, Nm VIII-M, NmIX-M: NmViii-M) supplemented with 2,4-D and ABA A half- strength MS medium (except for NH4 NO3 and KH2 PO4 , which were full-strength) supplemented with NAA, BAP, and glucose

Shoot multiplication, rooting and clone plants Somatic embryos (globular and torpedo) Shoot cultures

Protein synthesis

–

Reinbothe et al. (1992)

Glucoverodoxin, glucodigifucoside, odorobioside G, lanatoside C, odoroside H, and lanatoside A

Glucoverodxin (187 nmol/g DW in strain D); glucodigifucoside (333 nmol/g DW in strain E); odorobioside G (283 nmol/g DW in strain E); lanatoside C (129 nmol/g DW in strain E); odoroside H (55 nmol/g DW in strain E); lanatoside A (62 nmol/g DW in strain D). Total cardenolide content was highest in 940 nmol/g DW in strain E) Cardenolides content about 280 mg/kg FW

Stuhlemmer et al. (1993)

Maxmimum activity ␤-glucuronidase in calli 20.5 ␮ kat/g protein

Lehmann et al. (1995)

??

Stuhlemmer and Kreis (1996)

Effect of 100 mg/L of pregnenolone accumulated maxmium lanatoside A (about 100 nmol/g DW); cotexone and 21-hydroxypregnenolone accumulated glucodigifucoside about 170 nmol/g DW and about 115 nmol/g DW Effect of 100 mg/L of 5 ␤-pregnane-3,20 dione accumualted maimum 175 nmol/g DW and 5 ␤-pregnane 3 ␤, 21-diol-20-one content about 120 nmol/g DW Effect of 5␤-pregnane-3␤, 14␤,21-triol-20-one 3-acetate (100 mg/L) maximum content glucodigifucoside (about 600 nmol/g DW) Effect of 23-nor05,20(22) E-choladienic acid-3␤-ol (20 mg/L) accumulated glucodigifucoside (about 325 nmol/g DW)

Haussmann et al. (1997)

Axillary buds (11 strains) or seeds (1 strain)

Axillary buds

Modified MS media

Cell suspension culture and shoot culture

Leaf discs

Nutrient medium I (NmI), NmII, NmIII supplemented with 2,4-D, Kn, NAA, and BA

Protoplasts of the embryogenic strain VII

Shoot

??

Cell suspension culture, shoot culture

Axillary buds

A half- strength MS medium (except for NH4 NO3 and KH2 PO4 , which were full-strength) supplemented with NAA, BAP, glucoseand various combinations of phytohormones (IAA, NAA, IBA, BAP, and Kn)

Shoot culture and cell suspension cultures

Digitoxigenin, glucodigitoxigenin, digitoxin, and purpurea glycoside A Agrobacterium tumefaciens, glucuronidase gene, neomycin phosphotransferase II gene Cardenolide formation & activity of pregnanemodifying enzymes Glucodigifucoside, odorobioside G, glucoevatromonoside, odoroside H, lanatoside A, and lanatoside C

Theurer et al. (1994)

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Explants

31

32

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Leaf

Agrobacterium rhizogenes, 2,4-D, NAA

Hairy roots and shoot regeneration

Cardenolides, digitoxigenin derivatives, anthraquinones, and flavonoids

Pradel et al. (1997)

Leaf

Modified MS medium contained KH2 PO4, no casein hydrolysate, IAA, Kn, and glucose

Suspension culture

Axillary buds

Modified 1/2-strength MS media (NH4 NO3 and KH2 PO4 , which were full-strength), supplemented with IAA, BA, and glucose

Shoot regeneration

A half-strength MS liquid medium contained sucrose, BA, and IAA

Shoot cultures

Feeding deacetyllanatoside C to senescent shoot cultures resulted in the formation of a new product as 21 -di-dehydro-deacetyllanatoside C The endogenous cardenolide content of light-grown shoots was about 0.3 ␮mol/g DW, amounting to less than 0.05 ␮mol/g. Glucodigifucoside, odorobioside G, and odoroside H were the major glycoside detected under grown in light

Rhenius et al. (1997)

Axillary buds or sterilized seeds

Embryogenic cell strain VIII

Nutrient media (NmI, NmII, and NmIII) supplemented with 2,4-D, Kn, and NAA

Two cyclophilin nucleotide sequences (DLCYCLO cDNA and DLCYP18 genomic DNA) were reported

Scholze et al. (1999)

Anthers, pollen

NmI and solidified NmI were prepared as described by Lehman et al. (1995); NmV:NmI without sucrose, 2,4-D and kinetin, but with maltose and KH2 PO4 ; Nm2 was prepared according to Dietttrich et al. (1990)

Somatic embryos (globules, heart, torpedo-shaped, and germinated embryos) Callus, haploid plants regenerated

Extracellular polysaccharides (ECP), xyloglucan, and arabinogalactan Cardiac glycosides, 21 -di-dehydrodeacetyllanatoside C Digitoxigen-3-one, 3-epidigitoxigenin, digoxigenin glucoside, digitoxigenin bisglucoside, odorobioside G, glucodigifucoside, digixigenin fucoside digitoxigenin fucoside and digoxigenin –

Transformed shoots clone A4/2 content highest ␣-acetyldigoxin (1.09 ␮mol/ g DW); untrasformed shoots clone P15 content neoglucodigifucoside (0.71 ␮mol/g DW); root of seed born plants content digitalinun verum and odorobioside G (0.2409 ␮mol/g DW); leaves of transformed plants clone A4/2 content lanatoside C (2.31 ␮mol/g DW). Leaves of untrasformed plants clone P15 content 2.72 ␮mol/g DW lanatoside C and leaves of seed–born plants content 1.39 ␮mol/g DW of lanatoside A ECP were isolated in yields of up to 4 mg/mL

Highest cardenolides content in diploid clone/haploid clone was 46.4/39.6 ␮mol/g DW, respectively

Diettrich et al. (2000)

Stem, leaves, roots and hypocotyls

MS supplemented with 2,4-D, NAA, IAA, IBA, and chlorophenoxy acetic acid (CPA) alone or combination with BAP or Kn

–

Fatima et al. (2009)

Callus, biomass growth, plant regeneration

Series A, B, C, and E,digitalinumverum, glucoverodoxin, glucodigifucoside, odorobioside G, deacetyllanatoside C, lanatoside C, purpurea glycoside A, and lanatoside A –

Hensel et al. (1997)

Theurer et al. (1998)

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Explants

Table 2 (Continued) Digitalis species

Digitalis obscura

Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Shoot tips

MS, NAA, and Kn, Elicitors (salicylic acid, yeast extract, and calcium chloride)

Plant regeneration

Digitoxin and digoxin

Ghanem et al. (2010)

Seedling

MS media supplemented with thiamine HCl, BAP, IAA, myo-inositol, and sucrose. Temporary immersion culture and elicitors (chitoplant, silioplant, and methyl jasmonate)

Shoot culture, plantlets

Lanatoside C and digoxin

The highest amounts of digitoxin was found 44.45 ␮g/g FW in in vitro plantlets (2 subculture) and digoxin was 1.73 ␮g/g FW in vivo plants (2 months) The highest accumulation of lanatoside C was achieved with Chitoplant (0.1 g/L), which resulted in 316 ␮g/g DWand with Silioplant (0.01 g/L, 310 ␮g/g DW), which accounted for a 2.2-fold incerase in lanatoside C content compared to non-elicited shoot culture

Hypocotyl, root and cotyledon

Basal medium (BM) contained: MS mineral salt with Nitsch vitamins supplemented with 2,4-D, NAA, IAA, BA, Kn or different combinations of auxins and cytokinins. MS medium nutrients, sucrose and different concentrations of auxins (IAA, NAA, or 2,4-D) alone or in combination with the cytokinin BA Basal medium (BM) contained MS nutrients, sucrose and agar. Ethylene, IAA, and Kn. Two controls were tested: open controls with caps allowing gas interchange, and closed controls sealed with provided with ethylene traps (Hg (ClO4 )2 in HClO4 ) Basal medium (BM) contained MS constituents with macronutrients at half-strength, sucrose and agar supplemented with 2,4-D, NAA, IAA, or IBA. BS medium, sucrose, and agar supplemented with IAA Modified MS medium supplemented with BA, casein hydrolysate, coconut Milk, 2,4-D, 2-isopentenyladenine (2ip), Kinetin, NAA, IAA and high concentration of NH4 NO3 BS medium contained MS nutrients and sucrose supplemented with IAA, BA, 2,4-D, Kn, and NAA

Callus, shoot, root, and plant regeneration

–

–

Perez-Bermudez et al. (1983)

Callus, rhizogenesis, bud, and shoot formation Shoot and root regeneration

–

–

Perez-Bermudez et al. (1984)

–

–

Perez-Bermudez et al. (1985a)

Callus, embryogenesis, bud formation, and plant regeneration Somatic embryogenesis Callus culture, Somatic embryogenesis,and plant regeneration

–

–

Perez-Bermudez et al. (1985b)

–

–

–

–

Arrillaga et al. (1986) Arrillaga et al. (1987)

Caulogenesis, somatic embryogenesis, and shoot regeneration Protoplasts, Shoot, root, regeneration, and plant regeneration Morphogenesis, bud formation

–

–

Pérez-Bermúdez et al. (1987)

–

–

Brisa and Segura (1987)

–

–

˜ et al. (1988) Lapena

Hypocotyl

Anther

Hypocotyl Hypocotyl

Root tip meristem cultured

Mesophyll tissue, Protoplasts

Modified MS medium supplemented with casein hydrolysate, and several IAA and BA combinations

Hypocotyl

Basal medium (BM) contained MS medium nutrients supplemented with IAA and BA; BM was supplemented with sucrose, maltose, glucose, galactose or monnitol on growth and bud formation.

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Leaf

Pérez-Alonso et al. (2012)

33

34

Table 2 (Continued) Digitalis species

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Hypocotyl and root explants

Basal medium (BM) contained MS salts and vitamins, sucrose and agar supplemented with IAA, BA, NAA, 2,4-D, Kinetin, casein hydrolysate, coconut milk and lower concentration of NH4 NO3 . MS nutrients medium supplemented with BA, 2,4-D, IAA, and NAA

Calli, adventitious shoots, roots, and embryos

–

–

Brisa and Segura (1989)

Callus, caulogenesis, embryogenesis, rhizogenesis or shoot regeneration

Embryogenic and rhizogenic accumulated maximum amount of cardenolide 3.04 digoxin equivalents ␮g/g DW

Brisa et al. (1991)

Basal medium (BM) contained MS medium nutrients and sucrose agar concentration, supplemented with NH4 NO3 , IAA and BA Basal medium (BM) contained with MS nutrients supplemented with abscisic acid (ABA), 2,4-D, GA, IAA, polyamines, putrescine, spermidine, and spermine

Caulogenesis and vitrification

Series C: Digoxigenin, digoxin, deacetyllanatoside C, and lanatoside C Series A: Digitoxigenin digitoxin, and deacetyllanatoside A Digoxigenin

Normal plants and vitrified plants content 7.92 and 4.84 digoxigenin equivalents ␮g/g DW, respectively –

˜ et al. (1992) Lapena

Digitoxigenin, purpureaglycoside A, Evatromonoside, lanatoside A, digitoxigenin bis-digitoxoside, digitoxin, gitoxigenin, gitoxin, and digoxin Digitoxigenin, purpureaglycoside A, Evatromonoside, lanatoside A, digitoxigenin bis-digitoxoside, digitoxin, and gitoxigenin

Effect of phosphate (Pi) and Mn2+ concentrations on cardenolide content in shoot-tip culture were lanatoside A (850 ␮g/g DW) and total content (1144 ␮g/g DW) in BM × 2Pi

Gavidia and Pérez-Bermúdez (1997a)

Nine months culture content lanatoside A (537 ␮g/g DW) and total cardenolides (863 ␮g/g DW), while 2 years culture content lanatoside A (1554 ␮g/g DW) and total cardenolides (2301 ␮g/g DW)

Gavidia and Pérez-Bermúdez (1997b)

Cardenolides

GA3 (1.5 ␮M) accumulated highest cardenolides (183.5 ␮g/g DW) in shoot tip culture

Gavidia et al. (1993)

Hypocotyl

Hypocotyl

Hypocotyl

Axillary buds

Basal medium (BM) contained MS nutrients with sucrose and agar with BA Modified BM media where phosphate (Pi) concentration was raised to twice or three times that of the control, or the original manganese (Mn2+ ) concentration increased by the factor of 10 or 100

Axillary buds

Modified basal medium (BM) and macronutrient concentration and N source (KNO3 , NaNO3 , NH4 NO3 , NH4 Cl, NO3 − /NH4 + , Cl− , and Na+ ). BM with either MS macronutrients reduced to one-half or one fourth strength (1/2 BM, 1/4 BM), or with different NO3 − /NH4 + ratios maintaining the original nitrogen concentration Basal medium (BM) contained MS nutrients with BA, NAA, IAA, and GA3

Hypocotyls, leaves, and shoot tips.

Suspension culture, proembryogenic masses, and somatic embryogenesis (cotyledonary embryos) Shoots and plantlets

Shoots, rooting, and plantlets

Caulogenesis, somatic embryogenesis, and shoot length, and plant regeneration

–

˜ and Brisa Lapena (1995)

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Explants

Table 2 (Continued) Digitalis species

Digitalis thapsi

Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Shoot tips

Basal Medium (BM) contained MS nutrients, BA, amitrole, streptomycin and gamma radiation

Cardenolide

MS medium containing 0.5 M sucrose

–

Plantlets developed from irradiated shoot tip presented a high variability in their cardenolides production ranged from 878 to 3291 ␮g/g DW –

Gavidia and Pérez-Bermúdez (1999)

Shoot tips

Green shoots formed and plantlets developed Shoot tips were cryopreserved

Shoot tips

MS medium with various auxins (2,4-D, NAA and IAA) alone or combination with cytokinins (BA and Kn) Basal medium containing MS medium nutrients with auxins (2,4-D, NAA and IAA) either alone or in combinations with Kn, or BA MS medium supplemented with 2, 4-D, BA, calcium, manganese, and lithium

Callus formation, shoot multiplicatio, and direct rooting Calluses, root formation, shoot formation, and plant regeneration Suspension culture and callus

–

–

Herrera et al. (1990)

–

–

Cacho et al. (1991)

Digoxin

Corchete et al. (1991)

Leaves and hypocotyl

MS medium supplemented 2,4-D, BA and removal of calcium ions from MS medium or the effect of calcium being reversed by the addition of LaCl3 or ethyleneglycol-bis(␤-aminoethylether)-N,N’-tetraacetic acid

Callus and suspension culture

Digitoxin and digoxin

Hypocotyl

MS medium supplemented with 2,4-D, BA and elimination of calcium ions from MS medium

Callus and suspension culture

Cardenolides

Leaf and hypocotyl

MS medium with 2,4-D, BA and concentration of CaCl2 ·H2 O in MS medium 3 mM

Callus and suspension culture

Digitoxin

MnSO4 (1.0 mM) and lithium chloride (100 ␮M) accumulated 1188 and 851 digoxin relative content (%) Cardenolide relative content (digitoxin and digoxin) in complete and calcium-depleted medium): Cell line H2, absolute values for 100% (day 3 of control) = 39.8 ng/g FW digitoxin and 30.8 ng/g FW digoxin. Cell line F2, absolute values for 100% (day 3 of control) = 64.4 ng/g FW digitoxin and 56.28 ng/g FW digoxin Cardenolide relative content in calcium depleted medium and grown in calcium-depleted (control) and calcium-supplemented medium: Leaf cell line (H2a), absolute hypocotyl cell line (F2a), absolute values for 100% (day 3 of control) = 180 ng/g FW digoxin Maximum cardenolides accumulated are in below treatments: 1.0 nM H2 O2 [(297.2 digitoxin equivolents (%)] afer 24 h; CAT and SOD (3 U/mL culture) 47.9 and 161.2 digitoxin equivolents (%) presence of Ca2+ in medium, respectively. BSO (0.2 mM) and LaCl3 (0.28 mM) content 60.3 and 134 digitoxin equivalents ng/g FW Percentage of increase of the cardenolides when the F2 line (257.5% increase in digitoxin equivalents) and H2 line (240.1% increase in digitoxin equivalents) grown under continuous light

Axillary bud and adventitious bud

Basal medium (BM) contained MS nutrients with IAA, BA, and NAA

Cardenolide

Shoot cultures accumulated upto 226 ␮g cardenolides/g DW

Sales et al. (2002)

Leaf explants

Bud regeneration medium (BRM) with IAA and BA

Shoot regeneration and micropropagation Agrobacterium tumefaciens Leaf disc transformation procedure

Agrobacterium tumefaciens

–

Sales et al. (2003)

Leaf, root, and hypocotyl

Leaves and roots

Cacho et al. (1995)

Paranhos et al. (1999)

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Digitalis minor

Sales et al. (2001)

Cacho et al. (1999)

35

36

Table 2 (Continued) Digitalis species

Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Digitalis ferruginea L.

Hypocotyl

MS medium supplemented with BA, IAA, NAA, TDZ, and Zeatin

Cardenolides

MS medium supplemented with GA3 for seed germination; MS with 2,4-D, BAP, NAA, IAA, and IBA

Lanatoside C and digoxin ranged from 8.0 to 131.5 mg/kg DW and 2.3–29.3 mg/kg DW, respectively Higher amounts of lanatoside C (13.2 mg 100/g DW) and digoxin (2.93 mg 100/g) were obtained from indirect shoot regenration

Verma et al. (2010)

Hypocotyl

Direct and indirect somatic embryogenesis Shoot regeneration, somatic embryogenesis, and plant regeneration

Hypocotyl, leaf, root, and flamingo-bill

MS, Gamborg’s B5(Gamborg et al., 1968); LS medium, MM (Huang and Murashige, 1977); CP (Chee and Pool, 1987); and SH (Schenk and Hildebrandt, 1972) MS medium supplemented with 2,4-D, BAP, NAA, Kn, and Zeatin, 75 mM CaCl2 , and sodium alginate

Cotyledonary leaf, hypocotyl,and root

Digitalis trojana Ivan.

Leaf

MS medium supplemented with NAA and BAP

Hypocotyl

MS medium supplemented withBAP, TDZ, 2,4-D, Kn, and Zeatin alone. TDZ combined with IAA, IBA, and NAA MS medium supplemented with TDZ and IAA. Temperature and salicylic (SA) acid treatments.

Hypocotyl

Verma et al. (2014)

Shoot regeneration, root induction, and plant regeneration

Lanatoside C and digoxin

Higher amounts of lanatoside C (3.60 mg/kg) and digoxin (12.59 mg/kg) content of in vitro regenrated plantlets

Gurel et al. (2011)

Callus, somatic embryogenesis, direct and indirect shoots regeneration, plant regeneration, and synthetic seeds formation

–

–

Verma et al. (2016)

Shoot regeneration,multiplication and plant regeneration Direct somatic embryogenesis

–

–

C¸ördük and Akı (2010)

–

–

Verma et al. (2012)

Callus

Digoxigenin, gitoxigenin, lanatoside, digoxin and digitoxin.

Highest cardenolides accumulated in callus are digoxigenin [(control: 16.93 ␮g/g DW and SA + high temperature (2 h): 52.38 ␮g/g DW); gitoxigenin (control: 8.37 ␮g/g DW and SA + high temperature (2 h): 19.33 ␮g/g DW); lanatoside C: (control: 155.93 ␮g/g DW and SA + high temperature (2 h): 357.48 ␮g/g DW); digoxin
Cingoz and Gurel (2016)

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Digitalis davisiana Heywood

Lanatoside C and digoxin,

Table 2 (Continued) Explants

Medium + Plant growth regulators

Development stage

Cardiac glycosides and derivatives

Best response for the yield of cardiac glycosides and derivatives

References

Digitalis cariensis

Cotyledonary leaf, hypocotyl, root or flamingo-bill

LS, MS, and Gamborg’s (B5) supplemented with TDZ and IAA

Seed germination, adventitious shoot regeneration, root induction, and plant regeneration

Lanatoside A, B, C, digoxin, and digitoxin

Cardenolide contents of basal leaves collected from in vitro plants including lanatoside A (59.2 mg 100/g DW), lanatoside B (30.7 mg 100/g DW), and lanatoside C (100 mg 100/g DW)

Mohammed et al. (2015)

Digitalis lamarckii

Leaf

MS medium supplemented with BAP, TDZ, Zeatin, IBA, and Kn alone and combined with IBA MS medium supplemented with BAP, NAA, IAA, and IBA LS medium supplemented with BA, NAA, IAA, and GA3

Direct shoot regeneration, and multiplication Indirect shoot organogenesis Callus, indirect shoot regeneration, root induction

–

–

Verma et al. (2011b)

–

–

Glucogitoroside, strospeside, neo-digitalinum verum, neoglucodigifucoside, neo-odorobioside G, purpurea glycoside B, glucoevatromonoside, digitoxin, lanatoside C and digoxin

Neo-odorobioside G and glucogitoroside content highest in petiole (56.4 and 53.4 mg/kg DW) and lamina (170 and 143.9 mg/kg DW), respectively

Verma et al. (2011a) Yücesan et al. (2014)

Hypocotyl

MS medium elimination of calcium (Ca) and magnesium (Mg) or both and supplemented with IAA and TDZ

Callus

Digoxigenin, gitoxigenin, lanatoside C, digoxin, and digitoxin

Hypocotyl

MS medium with IAA, TDZ and hydrogen peroxide (H2 O2 )

Callus

Lanatoside C, digitoxin, digoxigenin, gitoxigenin, and digoxin

The mean contents of total cardenolides are in D. lamarckii (2017.97 ␮g/g); D. trojana (1385.75 ␮g/g); D. cariensis (1038.65 ␮g/g); and D. davisiana (899.86 ␮g/g) when Ca and Mg were eliminated from the medium The total cardenolides in D. lamarckii (586.65 ␮g/g); D. trojana (282.39 ␮g/g); D. cariensis (376.60 ␮g/g); and D. davisiana (506.79 ␮g/g)

Cotyledonary leaf Leaf lamina

Turkish Digitalis species Digitalis davisiana, Digitalis trojana, Digitalis cariensis, and Digitalis lamarckii

Sahin et al. (2013)

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Digitalis species

Cingoz et al. (2014)

37

38

S.K. Verma et al. / Industrial Crops and Products 94 (2016) 20–51

Fig. 5. Scanning electron microscopy (SEM) images of the seed (A) and seed coat (B) of Digitalis purpurea L. The seed dimensions (length, width and height) are shown in (C).

apparent rooting deficiency of the retrieved shoots was proved to be due to shifting in the auxin requirement from IAA to IBA. Other researchers had attempted to investigate the role of enzymes which influence the cardenolide production in D. purpurea. Seitz and Gärtner (1994) established that the reduction of progesterone to 5␤-pregnane-3, 20-dione, catalyzed by progesterone 5␤-reductase is the starting point of the cardenolide pathway, which leads to the formation of digitoxigenin. In addition, progesterone-5␤-reductase is the enzyme responsible for cardenolide forming plant tissues in D. purpurea (Gärtner et al., 1994). The elucidation of the molecular structure of the gene and of the protein forming this key enzyme of cardenolide biosynthesis would be most interesting in order to shed lights into the question how the flux of steroid skeleton into the cardenolide pathways is controlled (Dräger, 2006). The exact nature of the side chain-cleaving enzyme, effective in Digitalis plants is yet to be clearly understood. Pilgrim observed side-chain cleaving activity in enriched mitochondrial fractions of D. purpurea seedlings (Pilgrim, 1972). Palazón et al. (1995) reported enhancement of the cleavage in D. purpurea callus cultures by additional application of indole-3-acetic acid (IAA), a plant growth hormone, and the P450 (the enzyme that provides the precursor, pregnenolone), for the biosynthesis of cardenolides in the foxglove plants. Digitoxin content was enhanced in callus tissues of D. purpurea, especially in those grown in the presence of IAA containing the medium, supplemented with phenobarbital, whereas, the presence of phenobarbital caused a reduction of the vacuole/cytoplasm ratio as well as chloroplastic volume (Bonfill et al., 1996). A novel temporary immersion system (TIS) based shoot culture system was described by Perez-Alonso et al. (2009). The TIS was projected to act as a reliable alternative for the steady production of biomass in D. purpurea and the reported results were digitoxin (167.6 ␮g/TIS) and digoxin (119.9 ␮g/TIS) in the plant. Sivanandhan et al. (2014) experimented on different precursor feeding strategies in plant cell/organ cultures in order to improve secondary metabolites production. It was found that the growth

characteristics were considerably reduced upon precursor treatment and wide variations exist amongst the tested plant species. Specifically, the inclusion of cholesterol and squalene in the culture medium accelerated the synthesis of digitoxin and digoxin in D. purpurea cultures, but the inclusion also brought down the biomass production (Patil et al., 2013). Recently, Li et al. (2014) have developed a regeneration protocol through direct organogenesis and subsequently established as agrobacterium-mediated genetic transformation protocol starting from D. purpurea mature leaf explants. These authors reported the regeneration of 0.62 kanamycin-resistant shoots per initial explant. In order to efficiently obtain D. purpurea transgenic plants, Pérez-Alonso et al. (2014) studied some factors that significantly affect regeneration and genetic transformation processes. The reproducible protocol described in (Pérez-Alonso et al., 2014) yielded up to 6.91 transgenic lines per initial leaf segment infected with C58C1RifR agrobacterium tumefaciens strain; providing a fast and reliable tool for metabolic engineering of cardenolide production and genetic improvement of D. purpurea.

3.2. Digitalis lanata Digitalis lanata, commonly known as woolly foxglove or Grecian foxglove (Fig. 1B), belongs to the family Plantaginaceae. The plants are either biennial or short-lived perennials. These are native to Eastern European region and are found growing wild in Italy, Hungary, and the Balkans. This species is somewhat preferred to the D. purpurea as a source of cardiac glycosides. In 1824, Paqui isolated some white crystals from D. lanata, which he named as ‘Digitalin’. The crystals were found to exhibit an alkaline reaction towards water. Later on, Schneideberg isolated the same substance under the name ‘Digitoxin’. Furuya and Kojima (1971) had reported the first plant tissue culture protocol of D. lanata for callus development. These authors had isolated anthraquinone derivatives from the in vitro grown plants. The effect of 2,4-D on the production of anthraquinones (4-hydroxy-digitolutein (V) and digitolutein (VI))

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in D. lanata was also studied by Furuya et al. (1972). Organ cultures of leaves and roots of the D. lanata species produce cardenolides, as reported by Lui and Staba (1979). It was found that the digoxin content in the tissues increased with the age of cultures (Lui and Staba, 1981). The biosynthesis of cardenolides is proved to be connected to morphological differentiation in D. lanata, as confirmed from the observation that the cardenolides production showed an increase after the onset of differentiation (Garve et al., 1980). A protocol of in vitro propagation of D. lanata using shoot tip culture had been developed by Erdei et al. (1981). By adopting the protocol, maintenance of D. lanata clones in sterile culture had become possible. Establishment of large uniform population of heterozygous plants had become a reality following the said protocol. Plant regeneration from mesophyll protoplasts of D. lanata was reported by Li (1981). The protoplasts were prepared from D. lanata suspension cultures and the cell clusters exhibited centripetal growth of the cell wall, indicating a non-formation of a cell plate in D. lanata (Diettrich et al., 1982). Following in vitro propagation techniques in D. lanata, adventive embryos may accumulate nearly the same amount of cardenolides as plantlets (Kuberski et al., 1984). As an alternative, somatic embryos were also successful in achieving cardenolides production in D. lanata. Petersen and Seitz (1985, 1988) reported that, for biosynthesis of the cardiac glycoside digoxin in D. lanata, the P450-mediated 12␤-hydroxylation process is a precursor of the aglycone backbone glycosylation. If partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase are incubated together along with naturally occurring microsomal lipids and flavin nucleotides, it is possible to reconstitute the digitoxin 12␤hydroxylase enzyme activity (Petersen and Seitz, 1985, 1988). Diettrich et al. (1985) had observed no change in the maximum amount of cardenolides accumulation while cells of the cardenolide-forming embryogenic strain VII of D. lanata were stored in liquid nitrogen for about three years without any loss of viability. Kreis and Reinhard (1985) had described a rapid method of isolating vacuoles from suspension-cultured of D. lanata cells. Kreis et al. had reported the occurrence of lanatoside 15 -O-acetylesterase in cardenolide-transforming enzymes (acetyltransferases and glycosyl-transferases) using suspensioncultured D. lanata cells (Kreis et al., 1986; Kreis et al., 1990). According to Diettrich et al. (1986), although the typical embryonic cells are incapable to forming somatic embryos, considerable amounts of cardenolides were accumulated in adventitious embryo, whereas traces were found in non-embryoid globules. It was shown by Schöner and Reinhard (1986) that the D. lanata plants propagated by axillary-bud culture and the plants exhibited a homogeneous development. With respect to cardenolide content, the D. lanata plants were rich in digoxin and digitoxin. Stable cardenolide accumulation in cell culture was found in somatic embryos as described by Scheibner et al. (1987). It was believed that blue light photoreceptors and phytochromes were the main controlling agents in the cardenolide biosynthesis and accumulation in D. lanata plants. Berggren and Ohlsson (1991) advocated for the involvement of chloroplasts in the biosynthesis of cardenolides. This claim is disputable as cardenolides formation also occurred without any chlorophyll accumulation in D. lanata cultures (Scheibner et al., 1987). Investigations by Kubalákoyá et al. (1987) using the in vitro culture from the shoot tips in MS medium supplemented with 2.0–2.5 mg/L BAP + 0.17 mg/L IAA + thiamine (up to 1 mg/L) found no appreciable changes in the lanatoside C content in D. lanata. In the investigations on cardenolide uptake and glucosylation, Diettrich et al. (1987) found only a negligible uptake of primary glycosides. These authors had suggested model reactions representing the accumulation of glucosylated cardenolides. The cardenolide obtained from somatic embryos of D. lanata was iden-

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tified to be a glucodigifucoside and its chemical structure was established by 1 H NMR, 13 C NMR, and FABMS as digitoxigenin3␤-O-[␤-d-fucopyranosido-4 -␤-d-glucopyranoside] by Beale et al. (1988). These authors had shown that glucodigifucosides obtained from the somatic embryos of D. lanata were identical with the authentic specimens. It is still unclear to the plant biotechnologists why the early stages of somatic embryos do not synthesize cardenolides but the late embryo structures show the synthesis of cardenolides. Gibberellic acid enhances metabolic activity within pathways leading to accumulation of cardenolides in D. lanata tissue culture (Ohlsson and Björk, 1988). MnSO4 has been shown to induce higher yields of digitoxin in D. lanata (Ohlsson and Berglund, 1989). Experiments by Scheibner et al. (1989) reported the production of cardenolides in green globular stage II embryos. However, the yellowish stage I globules or the pro-embryogenic masses did not show any accumulation of cardenolides. Ohlsson et al. (1983) reported that dark-grown D. lanata tissue culture contains cardenolides. In heterotrophic or photoautotrophic green cell suspension cultures of D. lanata, the presence of chloroplasts alone did not yield any cardenolides production (Reinhard et al., 1975). Even inhibited chlorophyll synthesis in the presence of light-and-dark-grown D. lanata led to cardenolides production in somatic embryos (Scheibner et al., 1989). This leads to infer that the presence of chloroplasts do not seem to be an essential requirement for cardenolide biosynthesis (Eisenbeiß et al., 1999). Tuominen et al. (1989) had employed statistical experimental designs in order to optimize the nutrient media for cell cultures of D. lanata. The highlights of this exercise include the success of green hairy roots from D. lanata to grow appreciable levels of cardenolides (16.5 ␮g/g DW); the corresponding quantity for the dark-grown roots system is considerably small (0.02-0.70 ␮g/g DW) (Yoshimatsu et al., 1990). Ohlsson (1990) was successful in eliminating the negative effects of abscisic acid on plant growth and cardenolides, by including GA3 in the nutrient medium. Seidel et al. (1990) had shown that the conversion of pregnenolone into progesterone involves two steps. The first reaction is the NAD (5 3ˇ-hydroxysteroid dehydrogenase/5 –4 -ketosteroid isomerase (3␤-HSD))-dependent oxidation of the 3␤-hydroxy group. This yields a product 5 -pregen-3-one and is catalyzed by the 5 3␤-hydroxysteroid dehydrogenase. The C C double-bond shifts from position 5 to position 4 by the action of 5 - 4 -ketosteroid isomerase. This enzyme was isolated and characterized from D. lanata cell suspension cultures by Seidel et al. (1990), and purified by Finsterbusch et al. (1999). The role of the keto-steroid isomerase enzyme in the formation of cardenolides has thus been established. Reinbothe et al. (1990) considered the variation of ionic concentrations in the nutrient medium. Their studies established that, for the production of somatic embryos from suspension cultures of D. lanata, the optimal ratio of [NO3 − ] to [NH4 + ] in the medium must equal to 11:1. Using bioreactors which employed liquid medium, large-scale production of plants and cardenolides accumulation was achieved through the induction of somatic embryogenesis effectively (Greidziak et al., 1990). A 2.8 M aqueous solution of dimethyl sulfoxide (DMSO) was reported to have inhibited the enzymatic conversion of cardenolides by Digitalis lanata cell extract (Kreis et al., 1990). In callus culture of D. lanata, regenerates were diploid, especially on media supplemented with 2,4-D and Kinetin (Ernst et al., 1990). In Digitalis species, the main cardenolides are the lanatosides (Kandzia et al., 1998). Sutor et al. (1990) reported that the deacetylation of lanatosides to obtain purpurea-glycoside is catalyzed by an enzyme, lanatoside 15 -O-acetylesterase. In the morphogenic cell strain H1 of D. lanata lanatoside 15 -O-acetylesterase activity became detectable during the development of shoot-like structures (Sutor, 1992). According to Berglund and Ohlsson (1993), the compound, buthionine-sulfoximine (BSO) improved digitoxin accumulation in

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the embryogenic tissue culture of D. lanata. It was argued that this observation might have a bearing with the improved plastid development, proved by the increased levels of chlorophyll in the plant. However, it was also possible to reverse the positive effect of BSO on cardenolides accumulation by adding glutathione (Berglund and Ohlsson, 1993) (Table 2). In cardenolides transport, the deacetylation was proposed as a mechanism by Kreis et al. (1993). Using cell strains, without the lanatoside15 -O-acetylesterase enzyme, it was shown that acetylated cardenolides were taken up into the cells more rapidly that their 15 -O-decacetylated derivatives (Sutor and Kreis, 1996). Kandzia et al. (1998) had isolated, purified and partially sequenced the lanatoside 15 -O-acetylesterase from D. lanata. As reported by Diettrich et al. (1990), cardiac glycoside accumulation coincided with the shoot and green plantlet formation. Reinbothe et al. (1992) suggested that only a few proteins play important roles during embryogenesis in D. lanata. Studies by Stuhlemmer et al. (1993) confirmed that the dark-grown shoot cultures were unable to accumulate cardenolides, but the same cultures became successful to regain their biosynthesizing capability when transferred into light. An interesting study on the biosynthesis of cardenolides involved administering cholesterol to D. lanata, reported by Theurer et al. (1994), in order to evaluate the role of the 8 (14)-olefin, [8-3 H; 14-14 C] in glycosylation. The recovered digitoxigenin and digoxigenin showed the same 3 H/14 C ratio in the substrate. Lehmann et al. (1995) reported the first transgenic D. lanata from protoplasts of the embryonic strain VII. The enzyme is known as progesterone 5␤-reductase and is involved in the biosynthesis of the plant steroid cardenolide (Stuhlemmer and Kreis, 1996). C13 -labeled norcholanic acids used in feeding experiments indicated that some norcholanic acids, especially those with a pregnenolone structure, can serve as precursors of cardenolides (Haussmann et al., 1997). A system of producing transformed plants from exclusively root explants of D. lanata was developed by Pradel et al. (1997). These authors evaluated different wild strains of agrobacterium rhizogenes for the production of secondary products (cardenolides, anthraquinones and flavonoids) obtained from the hairy roots of Digitalis species and similar transgenic plants. Higher amounts of anthraquinones and flavonoids in the transformed hairy roots were found compared with those in un-transformed roots. For rapid isolation of a maximal variety of plant polysaccharides for pharmacological tests, suspension-cultured cells have the advantage that only a few polysaccharides in high-yields get isolated from the spent medium. Along with these, the sugar linkages were consistent with those of type II arabinogalactan (Hensel et al., 1997). Agricultural production of D. lanata is the only economically viable source of cardenolides, since cardenolides content in cell cultures is at best 10–100 times lower than in basal leaves of adult plants (Theurer et al., 1998). Improved cardenolide biosynthesis can be achieved by using certain stress factors (Luckner and Diettrich, 1985). Cardenolide production seems to be strongly linked to differentiation and specialization in D. lanata (Eisenbeiß et al., 1999). Scholze et al. (1999) observed that distinct plant cyclophilins could be conveniently regulated for developmental purpose. Haploid cell cultures from anthers and pollen grains have been generated by Diettrich et al. (2000) and it was inferred that flowers were morphologically abnormal and showed male sterility due to crippled anthers. The coding sequence for the D. lanata EHRH cardenolide 16 -O-glucohydrolase was inserted downstream of the 35S promoter in the binary vector pBI121 resulting in plant expression vector pBI121cgh (Shi and Lindemann, 2006). Explants excised from the seedling of Cucumissativus were transformed using agrobacterium rhizogenes harboring the pBI121cgh. Hairy roots were obtained from infected explants. The glycolytic activity of the recombinant CGH I was established by HPLC analysis using lanatosides as the substrates. Studies by Fatima et al.

(2009) had established sucrose to be the best sugar for plant tissue culture, as it can be easily and efficiently uptaken across the plasma membrane. The use of salicylic acid decreased total cardiac glycosides in plantlets of D. lanata, according to the reports by Ghanem et al. (2010). As plants are the only source of cardiac glycosides, attempts have been made to increase the production using elicitation. Using temporary immersion systems (TIS), treatment of D. lanata shoots with Chitoplant (100 mg/L) or methyl jasmonate (100 ␮M) for 28 days increased the lanatoside C content by 200% and 20%, respectively (Pérez-Alonso et al., 2012). Abiotic elicitors also stimulated cardiac glycoside production in D. lanata; treatment with Silioplant (10 mg/L) for 28 days showed an increased production of lanatoside C content by 200% (Pérez-Alonso et al., 2012). 3.3. Digitalis obscura Digitalis obscura L. is commonly known as the sunset foxglove or willow-leaved foxglove (Fig. 1C). It is a native to regions in Spain and Africa, but can be grown as an ornamental flower around the world. It is a perennial woody plant belonging to the family Plantaginaceae. Successful experiments on organ differentiation from hypocotyl, root and cotyledon explants of Digitalis obscura L. were reported by Perez-Bermudez et al. (1983). The D. obscura explants were cultured in vitro in MS medium supplemented BAP + NAA Perez-Bermudez et al. (1983) demonstrated that almost every part of D. obscura L. seedlings has a high morphogenic potential. Exogenously applied ethylene was shown to promote shoot formation in D. obscura tissues; bubbling ethylene gas into the culture vessels containing the nutrient medium supplemented with IAA, kinetin and mercuric perchlorate yielded better shoot formation (Perez-Bermudez et al., 1985a). Factors favoring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of D. obscura L. was determined (Perez-Bermudez et al., 1985b). It was found that the presence of an auxin was essential for cell proliferation and morphogenesis while bud differentiation was achieved only in the presence of a medium containing cytokine and auxin/cytokinin combination (Perez-Bermudez et al., 1985b). IAA was proved to be an effective auxin for promoting somatic embryogenesis in Digitalis obscura L. using hypocotyl segments from the seedlings (Arrillaga et al., 1986, 1987). Using root explants of the same species, PerezBermudez et al. (1984) obtained similar results. Plant regeneration has also been reported from protoplasts which were obtained from mesophyll tissue of D. obscura (Brisa and Segura, 1987). ˜ et al., 1988, only three-fourth According to estimations by Lapena of the required sucrose is adequate to promote optimal rates of adventitious shoot formation in D. obscura hypocotyls. The balance of sucrose osmotically regulated the morphogenesis in the plants. Regeneration of adventitious shoots, roots and embryos from the calli of hypocotyl and root explants of D. obscura L. had become possible, when the cultures were grown in MS medium supplemented with cytokinins alone or in combination with auxins (Brisa and Segura, 1989). Protoplasts have been successfully isolated from tetrads (Arnalte et al., 1991). Brisa et al. (1991) found appreciable amounts of series C cardenolides in D. obscura cultures. Shoot proliferation and vitrification in D. obscura cultures had been ˜ et al. (1992) in the presence of MS medium. studied by Lapena According to these authors’ studies, elimination of ammonium nitrate (NH4 NO3 ) from the nutrient medium imparted drastic inhibitory effects on both bud formation and shoot elongation. By reducing the concentration of NH4 NO3 to one-half or one-fourth of the original MS medium formula, the bud-forming capacity of D. obscura hypocotyls showed an increase. In addition, presence of ˜ et al., 1992). NH4 NO3 caused less vitrification of the plants (Lapena

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With respect to the influence of culture conditions, embryo development in D. obscura proceeded more effectively when 2,4d-induced proembryos were transferred to a 2,4-d-free medium ˜ and Brisa, 1995). The influence of mineral nutrition on (Lapena cardiac glycoside production was reported by Gavidia and PérezBermúdez (1997a). Changes in the nutrient medium formulation in terms of major salt concentration or nitrogen source significantly modified cardenolide production by D. obscura shoot-tip cultures (Gavidia and Pérez-Bermúdez, 1997b). Gavidia et al. (1993) found that GA3 slightly favored digitoxin synthesis in the Digitalis species. Shoot tips excised from genotype T4 of D. obscura were exposed to gamma rays (20–100 Gy) and it was found that the production of cardenolide was increased (Gavidia and Pérez-Bermúdez, 1999). Sales et al. (2001) described a four-step protocol for long-term cryopreservation of D. obscura shoot tips and it was concluded that the success of the protocol depends on the genotype and the number of subcultures of the starting material. Moreover, this protocol ensures the genetic stability of selected D. obscura genotypes, as demonstrated by RAPD analysis. 3.4. Digitalis thapsi Digitalis thapsi is commonly known as Spanish foxglove and fingerhut foxglove (Fig. 1D). It is a flowering plant native to Spain. Studies on D. thapsi L. by Herrera et al. (1990) brought out the effectiveness of the exogenous auxins along with cytokinin, for better response of multiple shoot developments in the plant. The differential response could be due to the varying concentrations of the growth regulators used in the medium and the explants types (Cacho et al., 1991). It has been reported that the elimination of CaCl2 from the growth medium induced a marked increase in the production of cardenolides in D. thapsi (Corchete et al., 1991; Cacho et al., 1995). In the absence of calcium, glutathione reductase activity had become drastically reduced in the cell cultures of D. thapsi. This also caused a change in the redox state of the cells (Paranhos et al., 1999). These authors predicted a connection between H2 O2 and cardenolide formation in D. thapsi. Studies by Cacho et al. (1999) had shed some light on the effect of calcium-deprivation on growth and production of cardenolides in D. thapsi. Two undifferentiated cell lines were maintained under three different light regimes (16 h photoperiod, continuous light and total darkness). The photoperiod did not affect cardenolides accumulation. In contrast, continuous light or total darkness increased the production of cardenolides in the leaf-derived explants. The elimination of calcium from the medium favored cardenolides production in D. thapsi, independent of the origin of the suspension cultures and the light regime (Cacho et al., 1999). 3.5. Digitalis minor Digitalis minor is known by the common names Mallorca foxglove and Majorca foxglove (Fig. 1E). This hardy perennial plant belongs to the Scrophulariaceae family. The plants are possibly the most-attractive of all foxgloves with their tiny and short branching spikes bearing pendulous delicate flowers. Almost white, trumpetshaped flowers arise above a basal rosette of narrow, white-haired leaves. The dwarf rosettes of thick and leathery leaves slowly increase, making in time a compact hairy shrub. It is one of the most beautiful endemic plant species native to the Balearic Islands in Spain (Mallorca, Menorca, and Cabrera) where it lives in rocky crags. Sales et al. (2002) had studied the shoot culture of D. minor and found that alterations made in the liquid medium drastically altered the cardenolide content in the plants. The effect was believed to be associated with the high level of hyperhydricity in the shoots. Transformation systems based on agrobacterium tume-

Fig. 6. Shoot regeneration through direct somatic embryogenesis from hypocotyl explants of Digitalis ferruginea subsp. schischkini L. (A) Well-developed globular, heart-shaped (arrowhead) somatic embryos in MS medium, supplemented with 0.5 mg/L IAA + 0.5 mg/L GA3 . Inset shows somatic embryos development (B) Developing stages of somatic embryos in MS medium supplemented with 0.05 mg/L NAA + 2 mg/L BAP. (C) Somatic embryos induction in MS medium with 0.1 mg/L NAA + 2 mg/L BAP. (D) Direct shoot regeneration from somatic embryos on MS medium with 0.1 mg/L NAA + 2 mg/L BAP.

faciens is well established for D. minor as reported by Sales et al. (2003). 3.6. Digitalis ferruginea (i.e. D. ferruginea subsp. ferruginea L., and D. ferruginea subsp. schischkini L.) Digitalis ferruginea subsp. ferruginea is commonly known as rusty foxglove (Fig. 1F), which belongs to the family Scrophulariaceae. D. ferruginea comprised of biennial or perennial herbs and occasionally of small shrubs. This species is native to the Balkans, Hungary, Italy, Lebanon, Romania, Trans-Caucasian regions and Turkey. D. ferruginea of the Samtskhe-Javakheti region are included in the list of rare species of Georgia. In this original study by the present authors, a simple and effective protocol has been developed for the induction of direct somatic embryogenesis in D. ferruginea. In order to achieve this, different concentration of IAA plus GA3 and NAA plus BAP were tested using hypocotyl explants excised from in vitro germinated seedlings. Highest mean frequency (80%) of explants developing into embryos was achieved. Similarly, largest mean number of embryos (20.7 embryos) per hypocotyl explant was found. The nutrient medium contained suitably chosen combinations of 0.5 mg/L IAA + 0.5 mg/L GA3 and 0.05 − 0.1 mg/L + 2 mg/L BAP. Somatic embryos further developed into globular and cotyledonary stages (Fig. 6A–C) and later on to shoot formation (Fig. 6D). However, indirect somatic

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Fig. 7. Somatic embryogenesis from embryogenic callus induced from the hypocotyl culture of Digitalis ferruginea subsp. ferruginea L. Embryogenic calli were transferred to suspension cultures in the presence of MS medium along with plant growth regulators (1 mg/ L 2,4-D + 0.5 mg/L BAP). (A) Globular somatic embryos (progressive development of globular embryo, arrowhead). (B–H) Embryo development stages. (I, J) Early cotyledonary stage embryo. (K) Embryo germination.

embryogenesis had been achieved in MS medium supplemented with the auxins (NAA or IAA) and cytokinins (BAP or TDZ). Onemonth old embryogenic callus was transferred into suspension culture for further embryoid induction (Fig. 7A). The embryoids were formed at the bottom of the suspension culture. The MS medium supplemented with 1 mg/L 2,4-D + 0.5 mg/L BAP showed the best response for embryoid induction (Verma et al., 2010). Somatic embryos were further subcultured in the same medium for germination (globular to cotyledonary stage) (Fig. 7A–K). Verma et al. (2014) obtained the maximum mean number of somatic embryos or shoots per callus, when the nutrient medium contained NAA plus BAP. Higher amounts of lanatoside C and digoxin accumulation were achieved when shoots were obtained by indirect regeneration. Sahbaz et al. (2011) reported the maximum frequency of shoot regeneration from hypocotyl explants, obtained in MS medium supplemented with TDZ and IAA. The highest amount of total cardenolides (1901.35 ␮g/g DW) in D. ferruginea L. subsp schischkini was estimated in the in vitro samples regenerated in MS medium containing kinetin plus IAA. Different combinations of sodium alginate and CaCl2 may be used to form calcium alginate beads. These beads differ morphologically with respect to texture, shape and transparency, depending on the actual contents of sodium alginate and CaCl2 . An encapsulated matrix of 3% sodium alginate with 75 mM CaCl2 ·2H2 O was found to be most suitable for the formation of ideal beads (Fig. S1).

Encapsulated somatic embryos sprouted within 10–12 days in MS medium containing growth regulators (0.25 mg/L NAA + 1.0 mg/L TDZ). Sprouting took longer period (15–18 days) in MSO (i.e., in MS medium without the growth regulators). Successful germination in the presence of full MSO, half-strength MSO and (MSO + 0.25 mg/L NAA + 1.0 mg/L TDZ) media was achieved as 64.3, 30.0 and 52.0%, respectively. Roots were developed from the sprouted buds in MS and MS + 0.5 mg/L IBA media within 4–6 weeks of culture (Fig. S1). Encapsulated somatic embryos cultured in the presence of 0.25 mg/L NAA + 1.0 mg/L TDZ exhibited multiple shoot induction (3–4 shoots per embryos), whereas only single shoot emergence was noted in the case of synthetic seeds cultured in MS medium. 3.7. Digitalis davisiana Digitalis davisiana belongs to the Plantaginaceae family and is commonly known as Alanya foxglove (Fig. 1G). This is a very longlived perennial of the digitalis family. This lovely Turkish plant is vaguely similar to D. grandiflora with an elongated flower spike bearing soft yellow flowers. Gurel et al. (2011) had experimented on a set of media compositions using the D. davisiana shoot cultures to study the impact of media on the growth parameters and cardenolides accumulation. The authors established that LS media was superior in terms of shoot and cardenolides production yielding a higher digoxin con-

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tent (12 ␮g/g DW). In this species, roots develop easily using low levels of auxin, or simply in a basic medium without any growth regulators. Recently, an optimized and efficient plant tissue culture protocol was established using cotyledonary leaf, hypocotyl, and root explants of D. davisiana (Verma et al., 2016). Maximum no. of somatic embryos was achieved when the MS medium contained BAP or 2,4-D. After three weeks of culture in MS medium supplemented with 2,4-D, callus showed a clear accumulation of orange color pigmentation (Fig. S2). Shoot regeneration was remarkably higher in a combination of NAA plus BAP than the zeatin alone. The shoots were successfully rooted in MS medium supplemented with NAA alone. In addition, synthetic seeds were produced by encapsulating the shoot tips in 4% sodium alginate solution. A maximum conversion frequency of 75% was achieved from encapsulated shoot tips cultured in NAA and BAP. Regenerated plantlets of D. davisiana were successfully acclimatized and transferred to soil (Verma et al., 2016).

Because of extensive dormancy of D. cariensis seeds, it is difficult to obtain quality seeds of the species. Mohammed et al. (2015) employed different pretreatments on D. cariensis seeds in order to increase germination ability. An improved seed germination was observed when seeds were pretreated by scarification followed by soaking in sterile distilled water overnight. Among all of the explants tested, only the flamingo-bill-type explant got regenerated successfully in a LS medium. Regenerated shoots were rooted in LS medium containing 1.0 mg/L IAA. Only lanatoside contents (A–C) were found, whereas digoxin and digitoxin were not detected (Mohammed et al., 2015). A protocol for direct somatic embryogenesis from hypocotyl explants has been developed in MS medium supplemented with 0.25 mg/L IAA + 0.5 mg/L BAP. Globular somatic embryos further developed into cotyledonary stage embryos after two months in MS medium supplemented with 0.5 mg/L IBA + 2 mg/L TDZ, but no heart-shaped or torpedo-stage embryos were found (Fig. S4).

3.8. Digitalis trojana

3.10. Digitalis lamarckii

Digitalis trojana Ivan is commonly known as Helen of Troy foxglove (Fig. 1H). The species is an endemic and medicinally important which has been marked as vulnerable in the Red Data Book of Turkish plants. It was found that leaf explants of D. trojana excised from in vitro germinated seedlings were most productive in shoot induction and the MS medium needed to be supplemented with varying concentrations of NAA and BAP (C¸ördük and Akı, 2010). Shoots were further cultured in MS medium along with activated charcoal for root formation. All of the in vitro regenerated plantlets were successfully acclimatized ex vitro and then grown as healthy plants. TDZ in combination with IAA produced significantly more somatic embryogenesis than TDZ alone in D. trojana. In a growth regulator-free and half-strength MS medium, somatic embryos of D. trojana gradually developed into healthy plantlets. The successful transplantation of the plantlets and their normal growth in a greenhouse environment was achieved (Verma et al., 2012). Direct shoot regeneration from cotyledonary leaf explants was achieved in MS medium supplemented with 2 mg/L BAP as shown in Fig. S3. In a recent paper, Cingoz and Gurel (2016) reported that salicyclic acid (SA) pretreatment on the callus cultures of D. trojana had brought out considerable increase in the cardenolides production (up to 472.3 ␮g/g DW). The SA pretreatment also improved the thermotolerance capacity in the callus cultures of D. trojana. While high temperature treatments caused a reduction in antioxidant enzyme activities (including superoxide dismutase (SOD) and catalase (CAT) activities), the SA pretreatment brought about an increase in these activities. An increase in proline content, total phenolics and flavonoid content in D. trojana was observed due to the SA and/or high temperature treatment (Cingoz and Gurel, 2016). Maximum increase was recorded in callus cultures treated with both high temperature (2 h) and SA together. However, CAT activity showed a reduction under high temperature applications (2 h and 4 h). Total phenolic and flavonoid content were also increased after the elicitor treatments. Increased phenolic accumulation is believed to be associated with increased stress protection. These results confirm that the SA treatments effectively induce the synthesis of cardenolides and antioxidants in D. trojana. This, in turn, plays a significant role in the resistance against high temperature stress on the plant.

Digitalis lamarckii Ivan. also known as Digitalis heldreichii Jaub. & Spach and Digitalis orientalis Lam., has a common identity as dwarf foxglove (Fig. 1J). This represents the least concerned endemic species belonging to the family Plantaginaceae. The plant is a native to Anatolia (Asian Turkey). Verma et al. (2011b) had studied direct shoot regeneration from leaf explants and also indirect somatic embryogenesis and shoot organogenesis from cotyledonary leaf segments of D. lamarckii Ivan. TDZ has been known to stimulate in vitro shoot regeneration more effectively than several commonly used cytokinins. It was demonstrated that TDZ promoted direct shoot regeneration from leaf explants of D. lamarckii. For shoot multiplication, a combination of IBA with TDZ produced significantly more shoot than BAP alone. Further, a treatment with 0.5 mg/L IAA had been identified as an appropriate treatment for D. lamarckii root formation (Verma et al., 2011a). Successful development of somatic embryos were observed within 4–6 weeks of culture from embryogenic calli derived from cut leaf segments. The development passed through several distinct stages: protrusions on the surface of the cut leaf, enlargement into bipolar structures, formation of globular, heart, torpedo and cotyledonary stages. For the shoot regeneration from calli, a combination of higher auxin and higher cytokinin concentration was the most effective. Rooted regenerates were then transferred to the pots, where these grew well and attained maturity (Verma et al., 2011a). A similar observation was recorded when lamina explants, cultured in LS medium containing different concentrations of BAP and NAA, developed into calli. Indirect shoot regeneration was achieved when the callus explants were cultured in LS medium supplemented with varying concentrations of BAP and/or gibberellic acid (GA3 ). Amongst the cardenolides, neo-odorobioside G (170.3 mg/kg DW) and glucogitoroside (143.9 mg/kg DW) were the two which were present in large abundance in lamina extracts, analyzed by the reverse-phase high-performance liquid chromatography (RPHPLC) (Yücesan et al., 2014). In the present study, a simple protocol has been developed for direct and indirect shoot regeneration from cotyledonary leaf in MS medium supplemented with 0.5 mg/L IBA + 0.5 mg/L BAP (Fig. 8A, B) and 0.25 mg/L NAA + 2 mg/L BAP (Fig. 8C, D), respectively. In addition, direct microshoot regeneration (or secondary shoot) from the midrib of indirect regenerated shoots of cotyledonary leaf explants was achieved, when cultured in MS medium supplemented with 0.5 or 2 mg/L BAP (Fig. S5). For the first time, this study reports, BAP plus NAA induced cardenolides accumulation in in vitro culture of D. lamarckii. In order to achieve this, several combinations of BAP and NAA were used for direct microshoots induction (Figs. 9 and 10). The study was further extended to determine cardenolides accumulation

3.9. Digitalis cariensis Digitalis cariensis Boiss. ex Jaub. et Spach (Plantaginaceae) is commonly known as Mugla foxglove (Fig. 1I). This is an endemic and medicinally important species.

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Fig. 8. (A, B) Direct shoot organogenesis from cotyledonary leaf of Digitalis lamarckii L. in MS medium supplemented with (A) 0.5 mg/L IBA + 0.5 mg/L BAP; (B) 0.25 mg/L NAA + 2 mg/L BAP. (C, D) Indirect shoot organogenesis from organogenic callus derived from leaf segments of Digitalis lamarckii L. in MS medium supplemented with 2 mg/L BAP.

(Fig. 11) in the plants. For the experiments, hypocotyl explants were excised from 45-days old seedlings of the in vitro germinated plants. The two cut ends showed the appearance of tiny shoot clumps by the end of one week, after culture initiation. The MS medium was supplemented with BAP alone or with combinations of BAP + NAA. Small globular structures had developed on the cut surface of the hypocotyls within the next two weeks. Thereafter, small globular structures turned into well-developed microshoots and were observed within four weeks of incubation (Fig. 9A–F). This study established that increasing the auxin concentration has a decreasing effect on the microshoots regeneration in D. lamarckii. Highest mean frequency (88%) of explants developing into microshoots and largest mean number of microshoots (60 microshoots) per hypocotyl explant were achieved using this protocol, in which the nutrient MS medium was supplemented with 0.25 mg/L NAA + 0.5 mg/L BAP. It was found that combinations of auxin and cytokinin were effective for direct microshoots initiation (Fig. 9). In an earlier report by the same authors, it was demonstrated that cytokinin alone and/or combination with auxins promotes shoot regeneration from leaf explants of D. lamarckii (Verma et al., 2011a,b). As shown in Figs. 10 and 11, microshoots obtained in MS medium with either (0.25 mg/ L NAA + 0.5 mg/L BAP) or (0.5 mg/L NAA + 0.5 mg/L BAP) produced the highest amount of digoxin (92.2 mg/kg) followed by gitoxigenin (43.6 mg/kg) and lanatoside C (2.7 mg/kg). In the present study, combinations of auxin and cytokinin were found to be effective for direct microshoots generation and cardiac glycoside production. A similar observation was also reported in the case of D. lanata (Kuberski et al., 1984) where increasing the cytokinin:auxin ratio in the medium formed large numbers of adventive embryos and started to produce cardic glycosides. In an earlier study (Verma et al., 2014), it was reported

that, when shoot was regenerated in MS medium containing auxin (NAA) and cytokinin (BAP), higher amounts of lanatoside C and digoxin were produced in D. ferruginea subsp. ferruginea L (Verma et al., 2014). Working with D. lamarckii, the predominant cardenolide was digoxin, followed by gitoxigenin and lanatoside C. All of the detected glycosides belonged to the series B and C. In contrast, comparison of cardenolides content in D. lamarckii, petiole and lamina extract from 4 month-old regenerated plantlets did not produce digitoxin, digoxin and lanatoside C, as reported by Yücesan et al. (2014). However, the previous study (Verma et al., 2014) demonstrated the callus culture from hypocotyl explant of D. lamarckii produced a good amount of digitoxin, gitoxigenen, digoxigenin and lanatoside C but digoxin was not detected (Sahin et al., 2013). It should be noted that callus and plantlets did not produce digoxin, but direct microshoots could be the cause of higher digoxin production. It is well known that lanatoside C is transformed into digoxin by deglycosylation using digilanidase, present in the leaves and subsequent deacetylation (Ikeda et al., 1992). Brisa et al. (1991), identified cardenolides of the series C in both wild plants and morphogenic cultures of D. obscura, while on the contrary, odoroside H and four related cardenolides were isolated from cultured somatic embryos of D. lanata, but no cardenolides such as lanatoside C were found (Seidel et al., 1990). 3.11. Studies on common Turkish Digitalis species The callus cultures of Digitalis davisiana, Digitalis lamarckii, Digitalis trojana and Digitalis cariensis showed increased cardenolides production through the elimination of calcium, magnesium or both from the nutrient medium (Sahin et al., 2013). Callus was induced from the hypocotyl segments of one-month-old seedlings which were cultured in MS medium containing TDZ plus IAA. A hormone-

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Fig. 9. Direct microshoot induction from hypocotyl segments of Digitalis lamarckii Ivan. (A, D) Nodular masses protruding from the cut end of hypocotyl explants after 10 days of culture in MS medium supplemented with 0.25 or 0.5 mg/L NAA + 0.5 mg/L BAP. (B, C) Microshoots regeneration in MS medium supplemented with 0.25 or 0.5 mg/L NAA + 0.5 mg/L BAP. (E, F) Well-developed microshoots protruding from the cut end of hypocotyl explants after 30 days of culture in MS medium supplemented with 0.25 or 0.5 mg/L NAA + 0.5 mg/L BAP.

Fig. 10. Development of direct microshoots from hypocotyl explants excised from 45 days-old in vitro germinated seedlings of Digitalis. lamarckii and cultured in MS medium containing NAA combined with BAP. Data were collected after five weeks of culture.

free MS medium (MSO) as well as Ca-free or Mg-free or both-free medium was used for the growth of the callus cultures after 30 days from culture initiation. The amount of five cardenolides from D. davisiana, D. lamarckii, D. trojana and D. cariensis was compared.

Fig. 11. Amount of cardenolides accumulation in in vitro microshoots of Digitalis lamrckii Ivan.

When the calli of the four Digitalis species were incubated in MS medium lacking both Ca and Mg, higher amounts of five cardenolides and total cardenolides were obtained. The mean contents of total cardenolides obtained were in the order of D. lamarckii > D. trojana > D. cariensis > D. davisiana, when both Ca and Mg were eliminated from the medium. The presented protocol proved to be

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useful for the development of new strategies for the large-scale production of cardenolides in the Digitalis species (Sahin et al., 2013). The effect of hydrogen peroxide (H2 O2 ) on callus cultures of the four Digitalis species mentioned above was examined by Cingoz et al. (2014). In all of the four species, the authors detected increased catalase (CAT), superoxide dismutase (SOD), total phenolic content, proline activity and cardiotonic glycoside production. Cingoz et al. (2014) had used the calli derived from hypocotyl explants which were cultured in MS medium supplemented with IAA + TDZ. After the culture completed four weeks, the calli were transferred into MS medium containing 10 mM H2 O2 and then incubated for six hours. Interestingly, no digoxin was detected in all the treatments as well as in control groups. The total cardenolides estimated were found to follow the order, D. lamarckii > D. davisiana > D. cariensis > D. trojana. These studies confirmed that H2 O2 pretreatment caused an increase in enzymatic and nonenzymatic antioxidants in all the four Digitalis species. The cardenoides production and overall activities (of CAT, SOD, total phenolics and proline activity) bear an inverse relation in Digitalis species, due to the H2 O2 pretreatment. Specifically, highest total cardenolide production was found in D. lamarckii and lowest total cardenolides was found in D. cariensis. In comparison, overall activities of CAT, SOD, total phenolic and proline activity were lowest in D. lamarckii and highest in D. cariensis (Cingoz et al., 2014).

5. Concluding remarks The present review includes an up-to-date compilation of the published work on micropropgation, tissue culture and cardenolides accumulation of several selected Digitalis species. There are a total of twenty Digitalis species known for their cardiac glycosides production. About nine of those grow wild in the flora of Turkey. However, major research publications mostly deal with these three: D. purpurea, D. lanata and D. obscura. Total regeneration and propagation protocols have so far been established for only ten Digitalis species. A direct implication of the published work points to the fact that the use of plant biotechnology techniques on the Digitalis species is still at a nascent phase. The explorations need to be widened encompassing the usable species and their extracts, with a major focus on improving the human healthcare. The authors have achieved successful regeneration by direct and indirect somatic embryogenesis, microshoot regeneration, synthetic seed and cardenolides production of four selected Turkish Digitalis species using a series of simple and reliable steps. Further experimentation is necessary in order to improve the efficiency of somatic embryogenesis and also to produce genetically modified Digitalis species emboding all of the characteristics, required for commercial interest. It is a well-established fact that somatic embryos are important alternatives to achieve in vitro production of cardenolides. Further, this is the first extensive report on direct and indirect somatic embryogenesis in D. ferruginea (suspenion culture), D. cariensis (from hypocotyls), D. trojana (direct shoot regeneration), D. davisiana (pigment accumulation), D. lamarckii (cardenolides accumulation) and D. ferruginea (germplasm conservation using synthetic seeds). The protocols for direct microshoots induction and cardenolides accumulation in D. lamarckii species, developed in the laboratory (at Abant Izzet Baysal University) is the first well-documented published report. The results described in this review show that microshoots accumulate cardenolides belonging to the series B and C and produce higher amount of digoxin. Moreover, the microshoots might be more suitable (than callus or plantlets) for the mechanistic investigations on the triggering of digoxin accumulation. The microshoots are easier to handle biotechnologically than

other more complex structures. Thus, microshoots are expected to play more significant roles in further investigation of the digoxin formation in Digitalis in vitro cultures. The cardenolides derived from Digitalis species have a high commercial value. Due to large-scale and unrestricted exploitation of the species, combined with very limited cultivation and inadequate attempts to replenishment it in the wild, the population of such an important plant species has been reduced drastically. The reason is because cardenolides are still isolated from the plants, since their structural complexity make it difficult to work out a feasible and easy chemical synthesis in the laboratories. Natural propagation of Digitalis through seeds is possible; however, this method is not as effective in producing sufficient quantity of plant stock, as the germination frequency of the seeds is rather poor. These are generally biennial plants and seed setting takes two years after planting. Therefore, using in vitro tissue culture methods, mass production of the Digitalis species becomes very important. In vitro propagation methods under controlled environmental conditions need to be exploited, facilitating rapid multiplication of superior clones and the extraction of cardenolides throughout the year without any seasonal constraints. The use of tissue cultures for the production of cardenolides was examined quite extensively using suspension cultures, morphogenic or embryogenic cell cultures as well as shoots or root cultures. It is important to note that, cell cultures without clear tissue or organ differentiation (undifferentiated cultures), are unable to produce considerable amount of cardenolides. In contrast, biosynthesis of cardenolides is restored as soon as morphogenesis is induced. Amongst all the known Digitalis species, D. lanata is the main source of glycosides for pharmaceutical industry. Due to the commercial importance of D. lanata, many agricultural and biotechnological companies in Germany have investments on large scale production for cardenolides. The Turkish Digitalis species have not yet been exploited fully in terms of production or regeneration. Clinical studies with some wild Digitalis species in Turkey show high toxic effects on animal treatments. Of those, D. lamarckii and D. trojana showed toxicity and antimicrobial actions (Benli et al., 2009) and antiproliferative activities (Kirmizibekmez et al., 2014), based on studies using leaf extracts. It is expected that the present study will contribute to the biotechnological research in the areas of cardenlides isolation and mass production of Digitalis. An increasing number of pharmacological publications reveal significant interest in the biological activities of digoxin and digitoxin (Scott and Thomas, 2000; Doss et al., 2001; Fabricant and Farnsworth, 2001; Prassas and Diamandis, 2008). This review is expected to function as a platform for future research in the field of cardenolides derivatives and micropropagation of the Digitalis species. Besides these, molecular studies on different enzymes involved in the cardenolide biosynthesis, different cell and tissue cultures, and biotransformation reactions for the production of digoxin and digitoxin are still important research areas for the development and production of important drugs for the treatment of congestive heart failure as well as cancer therapy. Conflict of interest The authors jointly declare that there are no conflicts of interests. Acknowledgments A small part of the studies reported in this manuscript was funded by TUBITAK (Project no. TOVAG-106O470, a research grant for international researcher programme (2216), awarded to Sandeep Kumar Verma. The Turkish Government has been gener-

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ous in providing a research scholarship to Sandeep Kumar Verma through the Ministry of Human Resources Development (MHRD), Government of India. The authors are grateful to the Department of Biology, Abant Izzet Baysal University, for providing the lab facilities.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2016.08. 031.

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