•There are 20 species of herbaceous perennials, shrubs and biennials that are commonly called foxglove.•Many of these have medicinal uses in improving healthcare.•And are exploited for the production ...of cardenolides by the pharmaceutical industries.•A compilation of up-to-date published research reports on in vitro culture of Digitalis L. is provided.•Different methods for the in vitro production of cardenolides have been reviewed and compared.
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 large-scale 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.
Sugar beet is recalcitrant to in vitro tissue culture. Usually, proliferation of in vitro cultured rosette explants is a prerequisite for micropropagation. Although hormonal treatments can induce ...proliferation in sugar beet rosette explants, they may also result in some side effects. In vitro culture of sugar beet explants and some hormonal treatments make them more prone to hyperhydricity. Effects of media with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin) on the proliferation and hyperhydricity of haploid sugar beet explants were investigated. It was observed that 0.2 mg L
Kin, with a reasonable amount of proliferation and minimum rate of hyperhydricity, performed better than BAP in different concentrations and combinations. The effect sizes of the treatments on the dependent variables were large. The correlation between proliferation and hyperhydricity of the treated explants was statistically negative and the association was large. However, the hormonal treatments without BAP or with the lowest amount of it produced the highest proliferation rate with the least hyperhydricity. The coefficient of determination was R
quadratic = 0.885. The results suggest that, in comparison with BAP, Kin is a potent plant growth regulator for the proliferation of sugar beet haploid explants that causes the least hyperhydricity. Although explants proliferated better in the presence of 0.01 mg L
BAP in combination with Kin than under Kin alone, the hyperhydricity of the proliferated explants decreased their suitability for in vitro propagation.
Digitalis davisiana, commonly called Alanya foxglove, from Turkey, is an important medicinal herb as the main source of cardiac glycosides, cardenolides, anthraquinones, etc. It is also known in the ...Indian Medicine for treatment of wounds and burns. It has ornamental value as well. Overexploitation of D. davisiana has led this species to be declared protected, and thereby encouraged various methods for its propagation. In this study, an optimized and efficient plant tissue culture protocol was established using cotyledonary leaf, hypocotyl and root explants of D. davisiana. Callus tissues were obtained from the cotyledonary leaf, hypocotyl and root segments cultured on Murashige and Skoog's (MS) medium containing different plant growth regulators. The maximum number of somatic embryos were achieved by the MS medium containing 6-benzyladenine (1.0 mg/L BAP) or 2,4-dichlorophenoxy acetic acids (0.1 mg/L 2,4-D), which produced an average of 8.3 ± 1.5 or 5.3 ± 1.5 embryos per cotyledonary leaf, respectively. After 3 wk of culture in MS medium supplemented with 1.0 mg/L 2,4-D, callus showed a clear accumulation of orange pigmentation. Shoot regeneration was remarkably higher (14.3 indirect shoots) in a combination of α-naphthalene acetic acid (0.25 mg/L NAA) plus 3.0 mg/L BAP than 2.0 mg/L zeatin (10.3 ± 0.5 direct shoots) alone. The shoots were successfully rooted on MS medium supplemented with NAA (0.1-1.0 mg/L). In addition, synthetic seeds were produced by encapsulating shoot tips in 4% sodium alginate solution. Maximum conversion frequency of 76.6% was noted from encapsulated shoot tips cultured on 0.25 mg/L NAA with 1.0 mg/L BAP. The encapsulated shoot tips could be stored up to 60 days at 4 °C. Regenerated plantlets of D. davisiana were successfully acclimatized and transferred to soil. This study has demonstrated successful preservation of elite genotypes of D. davisiana.
Hydrogen peroxide (H
2
O
2
) functions as an abiotic stress elicitor for plants. In this study, the effect of oxidative stress caused by H
2
O
2
on steviol glycosides (SGs) production and antioxidant ...activities of an in vitro grown antidiabetic plant,
Stevia rebaudiana
, has been investigated. Direct shoot organogenesis was performed, and after 4 weeks of culture, the shoots of
S. rebaudiana
were transferred to MS medium containing H
2
O
2
(10 or 20 mM) and incubated in growth room for 6 h. It resulted in increased SGs production, such as rebaudioside A (Reb-A) and stevioside (ST), and enhancement of total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and DPPH free radical scavenging activity when compared with control. The results clearly showed highest amount of SGs produced in MS medium containing 10 mM H
2
O
2
producing 2.97% Reb-A and 2.32% ST (w/w), followed by the medium employing 20 mM H
2
O
2
and control group. It is also evident that H
2
O
2
pre-treatment caused significant increase in non-enzymatic antioxidants that play defensive role against an oxidative stress induced by H
2
O
2
. The described protocol has a great potential to be utilized for Reb-A production on commercial scale.
A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium ...supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l-¹ KIN combined with 0.3 mg l-¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l-¹ TDZ and 1.0 mg l-¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l-¹. Hardening off process resulted in a production of more than 80% healthy plantlets.
This article describes a fast, simple, and efficient plant regeneration protocol for Ocimum basilicum L. Two sets of experiments were performed. The first set was performed on Murashige and Skoog ...(MS) medium lacking zinc sulfate heptahydrate (ZnSO₄·7H₂O) and containing varying concentrations of indole-3-acetic acid (IAA). The second set used three different concentrations (8.6, 12.9, and 17.2 mg L⁻¹) of ZnSO₄·7H₂O along with varying concentrations of IAA. In the first set, without zinc sulfate, an IAA concentration of 1.0 mg L⁻¹ (MS4) was found to be most effective, producing a mean of 12.6 roots per hypocotyl explant, while shoots were not produced. In the second set, with zinc sulfate, a combination of 12.9 mg L⁻¹ ZnSO₄·7H₂O + 1.0 mg L⁻¹ IAA (MS11) produced significantly more shoots per explant (15 shoots) than a combination of 12.9 mg L⁻¹ ZnSO₄·7H₂O + 0.5 mg L⁻¹ IAA (MS10), which produced only six shoots. Later, the plantlets were successfully acclimatized (100%) and finally transferred to the greenhouse (ex vitro). In the O. basilicum plants grown using MS11 medium, total phenolic content and rosmarinic acid content were estimated from stem, shoot tip, and old leaf tissue of in vitro plantlets, ex vitro plantlets, and flowering plants. The highest amounts of total phenolic content (131.8 mg gallic acid equivalent g⁻¹ DW) and rosmarinic acid (13.0 mg g⁻¹ DW) were obtained in the old leaf tissue at flowering time. This rapid regeneration protocol for O. basilicum L. represents a major improvement over the conventional protocols for plant regeneration and propagation of this species.
Background
Although members of the
SDR
gene family (short chain dehydrogenase) are distributed in kingdom of life, they have diverse roles in stress tolerance mechanism or secondary metabolite ...biosynthesis. Nevertheless, their precise roles in gene expression or regulation under stress are yet to be understood.
Methods
As a case study, we isolated, sequenced and functionally characterized the
3β
-
HSD
promoter from
Digitalis ferruginea
subsp.
ferruginea
in
Arabidopsis thaliana
.
Results
The promoter fragment contained light and stress response elements such as Box-4, G-Box, TCT-motif, LAMP element, ABRE, ARE, WUN-motif, MYB, MYC, W box, STRE and Box S. The functional analysis of the
3β
-
HSD
promoter in transgenic Arabidopsis seedlings showed that the promoter was expressed in cotyledon and root elongation zone in 2 days’ seedlings. However, this expression was extended to hypocotyl and complete root in 6 days’ seedlings. In 20 days-old seedlings, promoter expression was distributed to the whole seedling including hydathodes aperture, vascular bundle, shoot apical meristem, trichomes, midrib, leaf primordia, hypocotyl and xylem tissues. Further, expression of the promoter was enhanced or remained stable under the different abiotic stress conditions like osmotic, heat, cold, cadmium or low pH. In addition, the promoter also showed response to methyl jasmonate (MeJA) application. The expression could not be induced in wounded cotyledon most likely due to lack of interacting elements in the promoter fragment.
Conclusions
Taken together, the
3β
-
HSD
promoter could be a candidate for the development of transgenic plants especially under changing environmental conditions.
The effect of hydrogen peroxide (H2O2) on callus cultures of four Digitalis species (Digitalis lamarckii, Digitalis trojana, Digitalis davisiana and Digitalis cariensis) increased catalase (CAT), ...superoxide dismutase (SOD), total phenolic, proline activity and cardiotonic glycoside production. Callus derived from hypocotyl explants was cultured on Murashige and Skoog medium supplemented with 0.25 mg L−1 indole-3-acetic acid (IAA) and 0.5 mg L−1 thidiazuron (TDZ). After a month of culture, callus was transferred to MS medium containing 10 mM H2O2 and then incubated for 6 h. The amount of five cardenolides (Lanatoside C, Digitoxin, Digoxigenin, Gitoxigenin and Digoxin) as well as CAT, SOD, total phenolic, proline activity from Digitalis species were compared. No digoxin was detected in all treatments and control groups. The total cardenolides estimated were in the order of D. lamarckii (586.65 μg g−1 dw), D. davisiana (506.79 μg g−1 dw), D. cariensis (376.60 μg g−1 dw) and D. trojana (282.39 μg g−1 dw). It was clear that H2O2 pre-treatment resulted in an increase in enzymatic and nonenzymatic antioxidants. However, a significant negative relationship between cardenolides production and overall activities of CAT, SOD, total phenolic and proline was evident. The described protocol here will be useful for the development of new strategies for a large-scale production of cardenolides.
•Exposure of H2O2 resulted in the highest level of cardiac glycosides.•The predominant cardiac glycoside was lanatoside C.•Oxidative stress increases activities of CAT and SOD in cultures.•Treatment of callus with H2O2 drastically increased phenolic and proline content.
The present study reports the role of auxin and its transport inhibitor during the establishment of an efficient and optimized protocol for the somatic embryogenesis in
Digitalis trojana
Ivan. ...Hypocotyl segments (5 mm long) were placed vertically in the Murashige and Skoog medium supplemented with three sets indole-3-acetic acid (IAA) alone or 2,3,5-triiodobenzoic acid (TIBA) alone or IAA–TIBA combination of formulations of plant growth regulators, to assess their differential influence on induction and proliferation of somatic embryos (SEs). IAA alone was found to be the most effective, at a concentration of 0.5 mg/l, inducing ~ 10 SEs per explant with 52% induction frequency. On the other hand, the combination of 0.5 mg/l of IAA and 1 mg/l of TIBA produced significantly fewer (~ 3.6 SEs) and abnormal (enlarged, oblong, jar and cup-shaped) SEs per explant with 24% induction frequency in comparison to that in the IAA alone. The explants treated with IAA–TIBA exhibited a delayed response along with the formation of abnormal SEs. Our study revealed that IAA induces high-frequency SE formation when used singly, but the frequency gradually declines when IAA was coupled with increasing levels of TIBA. Eventually, our findings bring new insights into the roles of auxin and its polar transport in somatic embryogenesis of
D. trojana
.
► An effective protocol for direct somatic embryogenesis was developed. ► TDZ alone at 0.5 or 1.0mg/l was more effective for somatic embryo induction. ► IAA was more effective than NAA or IBA for ...somatic embryogenesis. ► Combination of low IAA and high TDZ was more effective for somatic embryogenesis. ► Plantlet development was achieved after culturing on half-strength MSO medium.
This study describes, for the first time, an in vitro protocol for the direct development of somatic embryos and subsequent plant regeneration from hypocotyl segments excised from 21-days-old in vitro-germinated seedlings of Digitalis trojana Ivan (Helen of troy foxglove). Two sets of experiments were carried out, the first compared different concentrations of four cytokinins: N6-benzyladenine BAP, 6-furfurylaminopurine kinetin, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea TDZ, and 6-(4-hydroxy-3-methylbut-2-enylamino) purine zeatin alone, while the second set tested TDZ or BAP combinations with IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA (α-naphthalene acetic acid). In the first set of experiments, TDZ was found the most effective at 1.0mg/l concentration, producing a mean of 10.7 somatic embryos per explant. In the second set, a combination of 1.0mg/l TDZ with 0.5mg/l IAA produced significantly more somatic embryos per explant (13.8 embryos) than with BAP (8.8 embryos). During subculture on growth regulator-free half-strength MS medium, somatic embryos gradually developed into plantlets. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large-scale production of cardenolides and genetic transformation of this valuable medicinal plant for its further improvement.
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