Indian Journal of Experimental Biology Vol. 54, April 2016, pp. 245-253

Somatic embryogenesis, pigment accumulation, and synthetic seed production in 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 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 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, , 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. 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 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, (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.

(Table 1). In 2 wk, these calli produced globular Pigment accumulation somatic embryos (Fig. 1 b and c). The developmental As shown in Table 2, calli derived from stages of somatic embryos in D. davisiana (Fig. 2 a-d) cotyledonary leaf, hypocotyl and root explants grown are similar to those reported earlier for other Digitalis on MS medium enriched with 2,4-D (0.5 to 2 mg/L) species10,12,27. Moreover, increasing the concentration showed pigment accumulation after 3 wk of incubation. of 2,4-D promoted callus formation and inhibits No pigmentation was observed in fresh callus (Fig. 1a), embryo development. However, callus formation and but with the passage of time, pigmentation occurred embryos development was promoted by BAP. Callus (Fig. 1b). During the period of active growth, the callus

Fig. 1 — (a and b) Pigmented and non-pigmented callus tissues; (c) somatic embryogenesis (arrowheads indicate globular-shaped) from cotyledonary leaf explants cultured on MS medium supplemented with 0.5 mg/L 2,4-D; (d) cotyledonary leaf derived cell line showing high pigment accumulation on MS medium contacting 0.5 mg/L 2,4-D; and (e and f) pigmentation increased during period in culture (III rd passage) on MS medium supplemented with 1.0 mg/L 2,4-D in hypocotyls derived callus cultures of D. davisiana.

Table 1—Development of somatic embryos from cotyledonary leaf and hypocotyl explants excised from 21-day-old in vitro grown seedlings of D. davisiana and cultured on MS medium containing different concentrations of 2, 4-D and BAP alone Callus forming somatic embryos (%) Number of somatic embryos per callus Growth Regulator (mg/L) Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 2,4-D (0.1) 33.0±2.0a 29.4±9.7 a 0.0±0.0 5.3±1.5b 4.3±1.5a 0.0±0.0 2,4-D (0.25) 23.8±1.0b 20.5±2.5ab 0.0±0.0 4.3±1.5bc 3.3±0.5a 0.0±0.0 2,4-D (0.5) 21.7±2.9b 19.9±5.9b 0.0±0.0 3.3±0.6c 2.7±1.2ab 0.0±0.0 2,4-D (1.0) 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 BAP (0.1) 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 BAP (0.25) 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 BAP (0.5) 30.6±2.5a 25.5±8.4ab 0.0±0.0 3.7±0.6c 3.0±2.6a 0.0±0.0 BAP (1.0) 24.4±2.6b 19.4±3.5b 0.0±0.0 8.3±1.5a 5.0±3.0a 0.0±0.0 Values are the mean±SD (Standard deviation) with the same letter within columns are not significantly different according to Duncan’s multiple range test at P <0.05. 248 INDIAN J EXP BIOL, APRIL 2016

Fig. 2 — Plant regeneration from cotyledonary leaf and hypocotyls explants of D. davisiana. (a) Well developed globular somatic embryos from hypocotyls leaf explants after 5 wk of culture on MS medium supplemented with 0.5 mg/L BAP.; (b) Somatic embryogenesis (arrowhead indicate globular and heart stage somatic embryos) from cotyledonary leaf explants culture; on MS medium supplemented with 1.0 mg/L BAP. (c and d) Torpedo and cotyledon stages somatic embryos from hypocotyle explants culture on MS medium supplemented 0.25 mg/L NAA with 1.0 mg/L BAP; (e and f) Indirect shoot regeneration from callus culture on MS medium supplemented 0.25 mg/L NAA with 3.0 mg/L BAP; (g) direct shoot development from hypocotyl explants after 2 months of culture on MS medium containing 2.0 mg/L Zeatin; (h) Conversion of encapsulated shoot tip (inset image) on MS medium containing 0.5 mg/L NAA with 1.0 mg/L BAP after 4 weeks of culture; (i and j) Plant regeneration from encapsulated shoot tips on MS medium containing 0.5 mg/L NAA with 1.0 mg/L BAP after 5 wk of culture; and (k) A healthy plant established in pot under field conditions.

Table 2 — Effect of different concentrations of 2, 4-D on pigmented callus from cotyledonary leaf and hypocotyl explants of D. davisiana. Pigmented callus (%) Callus colour and morphology PGR (mg/L) Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 2,4-D (0.1) 0.0±0.0 0.0±0.0 0.0±0.0 White White White 2,4-D (0.25) 0.0±0.0 0.0±0.0 0.0±0.0 White White White 2,4-D (0.5) 39.9±1.6b 36.1±2.5b 25±5.0ab White with White with White with orange patches orange patches orange patches 2,4-D (1.0) 59.4±5.8 a 58.0±3.3a 36±4.2a Orange Orange Orange 2,4-D (2.0) 31.6±2.8 b 24.9±4.5c 20±5.0b Yellowish with orange Yellowish with orange Yellowish with patches patches orange patches 2,4-D (3.0) 0.0±0.0 0.0±0.0 0.0±0.0 White White White Values are the mean±SD (Standard deviation) with the same letter within columns are not significantly different according to Duncan’s multiple range test at P <0.05. VERMA et al.: PROPAGATION OF ALANYA FOXGLOVE WITH COTYLEDON, HYPOCOTYL & ROOT EXPLANTS 249

showed patches of orange pigment surrounded by concentrations. After 4 wk of culture, shoot clumps yellowish pigment area (Fig. 1 c-d). After 5-6 wk of were transferred on the fresh media. The developed culture, almost all callus showed orange pigmentation shoots enlarged further while more shoots grew in 4 wk (Fig. 1 e-f). It was noticed that pigmented cells incubation (Fig. 2g). The frequency of organogenesis gradually increased with every cycle of subculture. On and a mean number of shoots per explant are presented the other hand, cotyledonary leaf and hypocotyl in Table 3. The explants in MSO medium as well as in derived callus showed pigmentation higher than root the higher concentration of BAP (0.5 to 2.0 mg/L) did derived callus. The maximum, percentage of pigment not produce any direct shoot but in lower concentration accumulation (36-60%) in callus was observed in the (0.1 mg/L BAP) produced direct shoots. Moreover, the presence of 2,4-D (1.0 mg/L). Callus with orange use of zeatin (01, 0.5, 1.0 or 2.0 mg/L) alone was patches from mixed white compact calli were selected observed highly effective in multiple shoot regeneration and subcultured after an interval of 21 days, in fresh MS in D. davisiana. The highest percentage (70%) of shoot medium containing 1.0 mg/L 2,4-D for maintaining induction and a mean number of shoots (10.3) per explant was developed on MS supplemented with cultures. This of pigment accumulation has already been reported before somatic embryo development in 2.0 mg/L zeatin. Overall, zeatin was considerably more cotton. Vinodhana and Ganesan30 observed a significant effective in promoting direct shoot regeneration in the amount of red pigmentation in embryogenic callus of present study (Table 3, Fig. 2g). Zeatin is generally used to induce shoot organogenesis in many plant cotton and reported that anthocyanin accumulation in 35,36 embryogenic callus is a good indicator of somatic species . However, when leaf explant of D. lamarckii was cultured on zeatin containing a medium, no shoot embryogenesis. Pigment accumulation in cultured 11 callus promoted by auxins has also been reported in regeneration occurred . In addition, it has been other plant species such as Aralia cordata, Hyoscyamus suggested that cytokinin efficiency may be affected by muticus, Ipomoea batatus, Panax sikkimensis31-34, but different affinities of cytokinin receptors involved in shoot induction process37. not in Digitalis sp. Indirect shoot regeneration Direct shoot regeneration The organogenic callus was subcultured for multiple Shoots were clearly visible after 7-12 days of shoot regeneration on MS medium containing different culture initiation and were found growing directly concentrations of BAP alone (Table 3) and in from the cut end of the cotyledon and hypocotyl combinations of NAA as shown Table 4. Callus could explants. These explants were inoculated on MS not be differentiated in different explants cultured on medium containing various concentrations of BAP, MSO medium. After 4 wk of culture, callus formation kinetin and zeatin individually. The root explants did reached 100% on MS medium containing 0.5 mg/L not show any shoot formation in any of the PGRs Table 3—Direct and indirect shoot regeneration from leaf explants of in vitro grown seedlings of D. davisiana cultured on MS medium containing different concentrations of BAP, kinetin, TDZ or zeatin alone Explants forming callus (%) No. of indirect shoots per callus No. of direct shoots per explant PGRs (mg/L) Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 BAP(0.1) 31.1±4.1g 32.7±4.2g 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 3.3±0.5bc 2.6±1.5b 0.0±0.0 BAP (0.5) 55.5±4.2d 56±0.9de 44.9±7.3b 5.0±1.7a 4 ±1.0a 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 BAP(1.0) 68.8±4.1b 66.6±6.7bc 51.6±3.3ab 8.3±2.0a 5 ±3.0a 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 BAP (2.0) 80.5±11.0a 75.8±4.5a 56.6±4.4a 5.6±2.0a 4±2.0a 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 Kinetin(0.1) 33.8±1.9g 37.2±2.5g 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 2.3±0.5c 3.3±2.5b 0.0±0.0 Kinetin(0.5) 40.5±5.8fg 46.6±2.8f 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 3.6±0.5bc 2.3±0.5b 0.0±0.0 Kinetin(1.0) 43.8±1.0ef 47.7±2.5f 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 5.3±1.5b 6.6±1.2a 0.0±0.0 Kinetin(2.0) 33.8±2.5g 37.7±2.5g 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 7.6 ±2.0b 7.6±1.2a 0.0±0.0 Zeatin (0.1) 45.5±1.0ef 50±8.6ef 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 2.6±0.5c 2.3±0.6b 0.0±0.0 Zeatin(0.5) 50.5±1.9de 53.8±1.9def 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 5.0±1.7b 2.0±1.0b 0.0±0.0 Zeatin(1.0) 58.3±1.7cd 60±0.9cd 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 8.6±1.2ab 6.6±1.2a 0.0±0.0 Zeatin (2.0) 65±4.4bc 70.5±2.5ab 0.0±0.0 0.0±0.0 0.0 ±0.0 0.0±0.0 10.3±0.5a 8.3±0.6a 0.0±0.0 Values are the mean ± SD (Standard deviation) with the same letter within columns are not significantly different according to Duncan’s multiple range test at P <0.05. 250 INDIAN J EXP BIOL, APRIL 2016

Table 4—Effect of different NAA/BAP combinations on morphogenesis in three explants (cotyledon, hypocotyl, and root) of D. davisiana Explants forming callus (%) Callus forming shoots (%) No. of shoots per callus PGRs (mg/L) Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Cotyledon Hypocotyl Root Control 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.1NAA+ 50.5±3.5g 47.2±3.5e 36.1±6.3e 23.8±5.4f 20.5±2.5g 0.0±0.0 4.0±1.0e 3.3±1.2c 0.0±0.0 1.0 BAP 0.25 NAA+ 59.4±2.6f 50±5.0e 39.9±1.7de 26.6±2.9f 22.7±3.8fg 0.0±0.0 5.3±2.5bcde 3.6±1.1c 0.0±0.0 1.0 BAP 0.5 NAA+ 74.4±5.9e 76.6±1.7d 46.8±9.9cd 31±2.6ef 27.2±3.4ef 0.0±0.0 7.6±2.5bcd 5.3±0.6bc 0.0±0.0 1.0 BAP 0.1 NAA+ 81.0±2.5d 80.5±3.5cd 51.1±1.0c 36±0.9 de 27.7±2.5ef 0.0±0.0 4.6±3.0de 5.6±3.2bc 0.0±0.0 2.0 BAP 0.25 NAA+ 84.9±1.7cd 81.1±1.9cd 49.9±1.7c 38±1.9de 32.2±2.5de 0.0±0.0 8.3±0.6bc 7.3±2.3ab 0.0±0.0 2.0 BAP 0.5 NAA+ 86.6±4.4cd 85.5±4.2c 55.5±3.5bc 42.7±0.9cd 35.5±2.5cd 0.0±0.0 7.9±2.0bcd 6.3±2.5abc 0.0±0.0 2.0 BAP 0.1 NAA+ 90.5±3.3bc 91.6±1.7b 52.7±6.3c 46.6±3.3bc 40.5±5.0c 0.0±0.0 9.3±0.6ab 8.3±1.1ab 0.0±0.0 3.0 BAP 0.25 NAA+ 94.9±3.3ab 93.3±2.9b 62.7±6.9b 50.5±3.9b 47.7±2.5b 0.0±0.0 14.3±2.0a 9.3±1.5a 0.0±0.0 3.0 BAP 0.5 NAA+ 99.4±1.0a 100±0.0a 75.3±3.8a 60±8.7a 57.7±2.5a 0.0±0.0 10.3±0.6ab 7.3±1.5ab 0.0±0.0 3.0 BAP Values are the mean ± SD (Standard deviation) with the same letter within columns are not significantly different according to Duncan’s multiple range test at P <0.05

NAA and 3.0 mg/L BAP. With increasing concentrations addition of auxins increased the frequency of leaf of NAA and BAP, the frequency of callus induction explants in D. minor. Recently, a combination of and shoots per callus from cotyledon, hypocotyl and 1.0 mg/L BAP and 0.1 mg/L IAA was also used for root explants increased slightly. Callus from root establishment of shoot cultures in D. lanata by Perez- explants did not regenerate into shoots. The Alonso et al.41. In contrast, the inclusion of auxins combination of BAP with NAA was more effective IAA, NAA, and 2,4-D together with BAP, KN, and (Table 4) in shoot proliferation than BAP alone (Table 3). TDZ inhibited the response of shoot regeneration in The highest shoot growth was observed in a D. purpurea26. combination of NAA (0.5 mg/L) and BAP (3.0 mg/L) Synthetic seed germination performing the regeneration frequency (60%) with In all the treatments, synthetic seed germination highest number of shoots per explant (14.3, Fig. 2 e-f). occurred within 10 days of incubation primarily by After 10-15 days of culture initiation, callus produced breaking open the calcium alginate matrix resulting in green nodular protrusions with indirect shoot the emergence of shoots (inset image Fig. 2h). organogenesis. Further 6 wk of incubation, the shoot This was followed by the emergence of roots after a length reached 1.5-2.0 cm (data not provided). These week. The development of shoots and roots was 10 results were similar to those obtained by Verma et al. simultaneous and resulted in rapid growth of plantlets in D. lamarckii where MS medium containing 2.0 mg/L within 5-6 wk. Some of the synthetic seeds failed to BAP in combination with 0.5 mg/L NAA was used for germinate and shoot buds turned brown in the matrix 38 organogenesis. Hagimori et al. established shoot within 10-15 days of culture. The encapsulated shoot forming cultures of D. purpurea on MS medium tips prepared in distilled water showed no germination 5 containing 1.0 mg/L BAP plus 1.0 mg/L IAA. Gurel et al. response when cultured on MSO medium, MS reported regeneration of D. davisiana on LS medium medium and also on ½ MSO medium supplemented supplemented with 0.5 mg/L TDZ in combination with with NAA (0.25-0.5 mg/L) and BAP (0.5-1.0 mg/L). 39 0.25 mg/L IAA. Similarly, Perez-Alonso et al. also The maximum conversion rate of encapsulated shoot established shoot forming cultures on MS medium tips into plantlets was 76.6% after 6 wk of culture on supplemented with 1.0 mg/L BAP in conjunction with MS medium supplemented with 0.25 mg/L NAA plus 0.1 mg/L IAA in D. purpurea. Sales et al.40 reported 1.0 mg/L BAP (Fig. 2h and Fig. 3a). The plantlets adventitious bud differentiation with BAP, but the thus obtained were maintained on 0.25 mg/L NAA VERMA et al.: PROPAGATION OF ALANYA FOXGLOVE WITH COTYLEDON, HYPOCOTYL & ROOT EXPLANTS 251

Fig. 3—(a) Effect of different media (MSO, 0.25 or 0.5 mg/L NAA plus 1.0 mg/L BAP, and Half MSO) on conservation of encapsulated shoot tips into plantlets of D. davisiana; (b) Effect of different storage durations (0, 15, 30, 45 and 60 days) on conversion of encapsulated shoot tips cultured on MS medium supplemented with 0.25 mg/L NAA plus 1.0 mg/L BAP. plus 1.0 mg/L BAP for 6 wk (Fig. 2i). After which the Table 5—Rooting of in vitro regenerated shoots of developing plantlets were transferred to soil. The D. davisiana cultured on MS medium containing different plantlets were gradually acclimatized to the concentration of NAA environment and successfully grown in the field Auxin (mg/L) Mean no. of roots per % of shoots that (Fig. 2k). These studies indicated that the presence of rooted shoot rooted nutrients and PGRs in the encapsulation matrix are Control 0 0 more important than in the inoculation medium. NAA (0.1) 2.6±0.5b 100a Moreover, artificial microenvironment supplemented NAA (0.25) 3.3±0.5ab 100a with adequate PGR additives encapsulated within NAA (0.5) 4.6±2.0ab 100a gelling matrix served as an artificial endosperm, NAA (1.0) 5.3±0.5a 100a providing enrichment to the encapsulated explants for Values are the mean±SD (Standard deviation) with the same letter plant regrowth. However, in recent years, the within columns are not significantly different according to encapsulation of shoot tips for the formation of Duncan’s multiple range test at P <0.05. synthetic seeds has received considerable attention42-45. 46 shoot tips into complete plantlets was 33%, whereas Ballester et al. also suggested that, shoot tip explants about 0% of non-encapsulated shoot tips showed plantlet are more responsive than other non-embryogenic formation on MS medium supplemented with 0.25 mg/L propagules because of greater mitotic activity in the NAA plus 1.0 mg/L BAP (Fig. 3b). It assumed that . Furthermore, shoot tips encapsulated decline in the conversion of encapsulated propagules stored synthetic seeds of D. davisiana are expected to be at low temperatures may be due to the inhibited respiration superior to conventional synthetic seeds as it would of plant tissues because of the alginate cover44,48. obviate the need of somatic embryos which are not Similarly, the conversion frequency of encapsulated nodal induced with ease in this species. segments of Punica granatum also showed marked 48 Short-term storage of encapsulated shoot tips decline following storage at low temperature . Plantlets The conversion rate of encapsulated and non- with well-developed shoots and roots obtained from encapsulated shoot tips decreased slowly with encapsulated shoot tip explants were successfully increasing storage duration at 4°C (Fig. 3b). Similar acclimatized and transferred to soil (Fig. 2k). results were obtained in several other plant species such as Phyllanthus amarus43, Withania somnifera47, In vitro rooting of shoots and Momordica dioica44. Encapsulated shoot tips Regenerated shoots of 4-5 cm in length were showed higher resistance to storage at 4ºC than non- transferred to MS medium having 0.5 mg/L NAA. encapsulated shoot tips. After 60 days of storage at Roots emerged from the cut ends of the shoots within 4ºC, percentage response for conversion of encapsulated 2 wk of transfer to the rooting medium. Absolute 252 INDIAN J EXP BIOL, APRIL 2016

rooting (100%) could be achieved in NAA (0.5 mg/L) 3 Chevallier A, Encyclopedia of , (Dk Publishers, was within 35 days of culture incubation (Table 5 and London), 2000. 4 Slingerland M, Cerella C, Guchelaar H, Diederich M & Fig. 2j). All the emerged roots were healthy having Gelderblom H, Cardiac glycosides in cancer therapy: from many root hairs. Similar results were obtained when preclinical investigations towards clinical trials. Investl New different explants of D. thapsi were cultured on Drug, 31 (2013) 1087. 0.5 mg/L NAA49,50. However, the hormone-free 5 Gurel E, Yucesan B, Aglic E, Gurel S, Verma SK, Sokmen M medium (MSO) did not produce any root. On the & Sokmen A, Regeneration and cardiotonic glycoside production in Digitalis davisiana Heywood (Alanya contrary, in D. davisiana, root formation was induced Foxglove). Plant Cell Tiss Org Cult, 104 (2011) 217. 5 100% on LSO medium . This may be attributed to the 6 Imre S, Sar S & Thomson RH, Anthraquinones in Digitalis higher thiamine content of LS medium, and possibly, species. Phytochemistry, 15 (1976) 317. thiamine was responsible for stimulating root 7 Büyüktimkin N, Imre S & Thomson RH, An anthraquinone from the roots of Digitalis davisiana. Phytochemistry, 20 formation and/or growth in the culture medium. (1981) 2441. 8 Verma SK, Yucesan BB, Gurel S & Gurel E, Indirect Hardening of regenerated plants somatic embryogenesis and shoot organogenesis from Ninety percent of the plantlets survived during cotyledonary leaf segments of Digitalis lamarckii Ivan., an acclimatization (Fig. 2k). The acclimatized plants endemic medicinal species. Turk J Biol, 35 (2011) 743. were transferred to open field conditions. These plants 9 Verma SK, Yücesan, BB, Şahin G, Gürel S & Gürel E, did not show variations in morphology or in growth Direct shoot regeneration from leaf explants of Digitalis lamarckii, an endemic medicinal species. Turk J Bot, 35 characteristic as compared to their parent donor plant. (2011) 689.

10 Verma SK, Sahin G, Yucesan B, Eker I, Sahbaz N, Conclusion Gurel S & Gurel E, Direct somatic embryogenesis from The micropropagation method reported in this hypocotyl segments of Digitalis trojana Ivan and subsequent study is characterized by rapid proliferation of shoots. plant regeneration. Ind Crops Prod, 40 (2012) 76. This is highly advantageous for the conservation of 11 Patil JG, Ahire ML, Nitnaware KM, Panda S, Bhatt VP, Kishor PB K & Nikam TD, In vitro propagation and this species, production of secondary metabolites and production of cardiotonic glycosides in shoot cultures of possible genetic modification. Increasing the auxin L. by elicitation and precursor feeding. (2,4-D) concentrations in medium, the somatic Appl Microbiol Biotechnol, 97 (2013) 2379. embryos induction percentage and a number of 12 Sahin G, Verma S & Gurel E, Calcium and magnesium somatic embryos per callus considerably decreased elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey. Plant but the pigment accumulation was increased. Physiol Biochem, 73 (2013) 139. Successful plant retrieval from encapsulated shoot 13 Ghosh A, Hossain MM & Sharma M, Mass propagation of tips indicates that this method could be potentially Cymbidium giganteum Wall. ex Lindl. using in vitro used to preserve elite genotypes of D. davisiana and seedlings. Indian J Exp Biol, 52 (2014) 905. facilitate their transport to laboratories and extension 14 Shriram V, Nanekar V, Kumar V & Kishor PBK, In vitro regeneration and ploidy level analysis of Eulophia ochreata centres of distant places. Further studies will be Lindl. Indian J Exp Biol, 52 (2014) 1112. required to identify natural pigment from callus 15 Mangal M, Sharma D, Sharma M & Kumar S, In vitro culture of D. davisiana. regeneration in olive (Olea europaea L.) cv, ‘Frontio’ from nodal segments. Indian J Exp Biol, 52 (2014) 912. Acknowledgment 16 Pant NC, Agarrwal Ruchi & Agrawal S, Mannitol-induced The authors are grateful to the TUBITAK for drought stress on calli of Trigonella foenum-graecum L. Var. financial support (project no: TOVAG-106O470) and RMt-303. Indian J Exp Biol, 52 (2014) 1128. 17 Shibu BS, Wesley PS, Moin S & Devi BC, In vitro the Turkish Government for providing scholarship to regeneration of Coelogyne nervosa A. Rich. and Eria SK Verma through the Ministry of Human Resource pseudoclavicaulis Blatt., threatened orchids of Western Development, Department of Higher Education, Ghats, India. Indian J Exp Biol, 52 (2014) 658. Government of India. 18 Bhat B & Sharma VD, In vitro propagation of the Garden Heliotrope, Valeriana officinalis L.: Influence of pre-chilling and References light on seed germination. Indian J Exp Biol, 53 (2015) 184. 1 Davis PH, Cullen J & Coode MJ, Flora of Turkey: and the 19 Choudhary R, Chaudhury R, Malik SK & Sharma KC, East Aegean Islands,(Supplement) Vol.1 (Edinburgh University An efficient regeneration and rapid micropropagation Press, Edinburgh), 1988, 10. protocol for Almond using dormant axillary buds as explants. 2 Sales E, Muller-Uri F, Nebauer SG, Segura J, Kreis W & Indian J Exp Biol, 53 (2015) 462. Arrillaga I, Digitalis wild crop relatives: genomic and 20 Kumar NU, Gnanaraj M, Sindhujaa V, Viji M & Manoharan K, breeding resources. (Springer, Berlin), 2011, 73. A microdroplet cell culture based high frequency somatic VERMA et al.: PROPAGATION OF ALANYA FOXGLOVE WITH COTYLEDON, HYPOCOTYL & ROOT EXPLANTS 253

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