Indian Journal of Experimental Biology Vol. 54, April 2016, pp. 245-253 Somatic embryogenesis, pigment accumulation, and synthetic seed production in Digitalis davisiana Heywood Sandeep Kumar Verma*#, Gunce Sahin & Ekrem Gurel Department of Biology, Abant Izzet Baysal University, 14280 Bolu, Turkey Received 02 August 2014; revised 08 January 2015 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. Keywords: Alanya foxglove, Anthraquinones, Auxin, Callus initiation, Cardenolides, Herbal, Micropropagation Digitalis davisiana, a biennial or perennial herb, rarely a systematic cultivation of wild material make this small shrub, is an endemic and endangered species from species vulnerable and it is protected in Turkey. Plantaginaceae family. Commonly known as Alanya Natural propagation of D. davisiana through seeds foxglove, D. davisiana is the most distributed species though possible, is not effective in producing among the nine Digitalis species grown in Turkey1. sufficient stock of plantlets as their germination Digitalis spp. are the main source of cardiac glycosides frequency is poor. Hence, in vitro culture protocols and have therapeutical applications as well as for propagation of Digitalis are quite common8-12. ornamental value2. In Ayurvedic medicine, it is used to Though in vitro regeneration of various economically treat wounds and burns. Digitalis glycosides are also and medicinally important plants is not uncommon13-19, used for myocardial infarction, oedema, angina, cardiac there is hardly any such work on D. davisiana5.Same disfunction, hypertrophy and arterial hypertension3. is the case with somatic embryogenesis20-25. In this Cardiac glycosides are known to have anticancer report, we have proposed a protocol for somatic properties4. D. davisiana is a source of many embryogenesis, pigment accumulation, organogenesis, pharmacologically interesting compounds, such as synthetic seed production and plant regeneration from cardenolides5 and anthraquinones6,7. different explants of D. davisiana. Large scale indiscriminate collection, low propagation response, slow growth rate and meagre Materials and Methods —————— Plant material and culture conditions *Correspondence: Seeds of D. davisiana were obtained from a wild E-mail: [email protected] population growing in the vicinity of Alanya, #Present address: Turkey (altitude at 1100 m and N 36°31 and Department of Horticulture, Institute of Agricultural & Life Science, E 032°14). Seeds were surface disinfected with Gyeongsang National University, Jinju -660701, South Korea. freshly prepared 20% commercial bleach for 10 min, 246 INDIAN J EXP BIOL, APRIL 2016 and finally washed 3-4 times in sterile distilled water. Effect of plant growth regulators (PGRs) Seeds were aseptically placed in Petri dishes The effect of PGRs in the alginate matrix was containing 30 mL of the MS26 hormone free medium studied on plant development from encapsulated (MSO) consisting of vitamins with 3% w/v sucrose shoot tips. NAA (0.25 mg/L) and BAP (1.0 mg/L) and solidified with 0.8% w/v agar-agar. After were added to the MS-sodium alginate, and MS-CaCl2 adjusting the pH to 5.8, it was autoclaved at 121°C solution, before encapsulation and polymerization. and 1.06 kg/cm2 pressure for 15 min. The cultures Under in vitro conditions, synthetic seeds were cultured were incubated under 16 h light:8 h dark photoperiod on MS medium containing NAA (0.25-0.5 mg/L) (irradiance at 50 mol–2 s–1). Hypocotyl, cotyledonary and BAP (0.5-1.0 mg/L), MSO and half MSO, leaf and root segments (length 5-8 mm) as explants for conversion into plantlets. After germination, the were used for culture initiation. synthetic seeds were transferred to the same medium. Four weeks old shoots with fully expanded leaves Callus initiation, somatic embryo development, pigment were placed on MSO or MS medium containing accumulation and indirect shoot regeneration 0.25 mg/L NAA with 1.0 mg/L BAP to induce root Callus was induced within 4 wk, when hypocotyl, formation. cotyledonary leaf and root segments were cultured on MS medium containing different concentrations of Short-term storage of encapsulated shoot tips 2,4-D or BAP alone (0.25-2.0 mg/L). Thereafter, Encapsulated and non-encapsulated shoot tips callused explants were transferred to fresh medium were transferred in medium containing agar and were for further induction of somatic embryos and pigment kept at low temperature (4ºC) for 15, 30, 45 and accumulation. For shoot regeneration, explants were 60 days in the dark. Encapsulated and non- cultured on MS medium incorporated with BAP alone encapsulated shoot tips, after each storage period (1.0-3.0 mg/L) or in combination with 0.1-3.0 mg/L were cultured on MS medium containing 0.25 mg/L NAA. Both, the frequency (%) and mean numbers of NAA and 1.0 mg/L BAP for conversion into plantlets. somatic embryos and/or indirect shoot regeneration Rooting and hardening off were recorded after five weeks of culture incubation. Regenerated healthy shoots were rooted easily Experiments were repeated 3 times, each using 20 on root induction medium supplemented with replicates. 0.1-1.0 mg/L NAA. The cultures showing induction of roots were transferred after 15 days into fresh Effects of cytokinins on direct shoot regeneration Hypocotyl, cotyledonary leaf and root explants were medium containing 0.1-1.0 mg/L NAA. Plantlets were separated from rooting media and roots were washed cultured in Petri plates (90 × 15 mm) on MS media containing different concentrations (0.1, 0.5, 1.0 or twice with sterile distilled water to remove the growth 2.0 mg/L) of BAP, kinetin or zeatin. Experiments medium. These plantlets were then transferred to pots containing an autoclaved mixture at a ratio of soil (1): were repeated 3 times, each using 20 replicates. Frequency (%) of explants developing direct shoot manure (2): moss (2): sand (1), and were kept initially regeneration and a mean number of shoots per explant high humidity then slowly it was decreased under growth-room conditions at 20-22 °C. was recorded after five weeks of culture. Statistical analysis Encapsulation of shoot tips Data were analyzed using the statistical programme Sodium alginate (2.5 to 4 % w/v) was added in SPSS, Version 18.0 (SPSS Inc., Chicago, IL, USA). distilled water (DW) and/or MS medium with or Analysis of variance (ANOVA) was used to calculate without growth regulators and pH was adjusted to 5.8. statistical significance. Data were expressed as mean For encapsulation, 75 mM CaCl2 was prepared in ± SD (standard deviation). MSO or MS medium containing growth regulators. Sodium alginate and CaCl2 solutions were autoclaved Result and Discussion as described above. Encapsulation was accomplished Effect of auxin and cytokinin on callus induction and somatic by mixing the shoot tips into the sodium alginate embryogenesis solution and dropping these into the calcium chloride Among all the explants used, cotyledon, hypocotyl solution. Each drop contained one shoot tip. and root explants of D. davisiana cultured on the Encapsulated shoot tips were cultured on MS medium media containing 2,4-D or BAP (0.1 to 3 mg/L), with or without growth regulators. produced white and compact or green calli, VERMA et al.: PROPAGATION OF ALANYA FOXGLOVE WITH COTYLEDON, HYPOCOTYL & ROOT EXPLANTS 247 respectively, within a week of culture incubation formed from root segments did not show somatic (Table 1, Fig. 1a). When explants were cultured on embryogenesis. In most of the reported works in MSO, they tend to be necrotic and no callus was Digitalis species, auxins (IAA, 2,4-D) induced efficient formed. The medium containing 0.1 mg/L 2,4-D or 1.0 somatic embryogenesis28,29 while, on the contrary, mg/L BAP was observed as the most efficient, embryo formation in D. lanata27 was triggered by inducing 33 and 30.6% embryogenic calli, respectively withdrawal of the auxin 2,4-D from the nutrient solution.