Environmental and Experimental Biology (2015) 13: 1–11 Review

The in vitro culture of species, important medicinal and ornamental : a review

Jaime A. Teixeira da Silva*

P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan

*Corresponding author, E-mail: [email protected]

Abstract

The genus Ceropegia of the Asclepiadaceae family encompasses a range of vines with medicinal and ornamental value. One way to clonally propagate such material is through in vitro culture. This review highlights the achievements made inin vitro culture and related biotechnologies of members of this genus. Details of protocols are provided and difficulties and hurdles that are yet to be overcome are pointed out. This short review provides a succinct synthesis of the literature on this topic and also a practical manual for researchers wishing to engage in new research involved with the in vitro culture of members of this genus. The range of studies conducted thus far is limited, and this review will serve as an impetus for new studies focusing on research themes that have not yet been explored.

Key words: in vitro regeneration, lantern flowers, growth regulator, vines. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2iP, N6-isopentenyladenine or 6-γ,γ-dimethylallylaminopurine; AC, activated charcoal; BA, N6-benzyladenine; B5, Gamborg et al. (1968) medium; CIM, callus induction medium; DDW, double-distilled water;

DSO, direct shoot organogenesis; FIM, flower induction medium; HgCl2, mercury chloride; IAA, indole-3-acetic acid; IBA, indole-3- butyric acid; ISO, indirect shoot organogenesis; ISSR, inter-simple sequence repeat; Kin, kinetin (N6-furfyladenine); MIM, microtuber induction medium; MS, Murashige and Skoog (1962) medium; NAA, α-naphthaleneacetic acid; NaOCl, sodium hypochlorite; NR, not reported; PGR, plant growth regulator; picloram, 4-amino-3,5,6-trichloropicolinic acid; PP, photoperiod; PPFD, photosynthetic photon flux density; RAPD, random amplified polymorphic DNA; RH, relative humidity; RIM, root induction medium; rpm, revolutions per minute; RTP, running tap water; SDW, sterile distilled water; SIM, shoot induction medium; TCL, thin cell layer; TDZ, thidiazuron (N-phenyl-N’- 1,2,3-thiadiazol-5-ylurea); tTCL, transverse thin cell layer

The importance of Ceropegia albuminoids, fats and crude fiber of this species are used in Indian Ayurvedic medicine, as “soma”, to treat diarrhea APG III (2009) lists 220 species within the genus Ceropegia and dysyntery (Beena et al. 2003, and references therein). (Asclepiadaceae family), commonly known as lantern C. pusilla also has a wide range of medicinal properties flowers, which are mostly distributed in tropical and sub- (Kalimuthu, Prabakaran 2013a, and references therein). In tropical regions (Bruyns 2003). Despite the size of the genus, addition to their health, medicinal and ornamental value, only a single study has used nuclear ribosomal internal C. spiralis, C. panchganiensis and C. evansii were shown to transcribed spacer and three non-coding chloroplast DNA possess 1,1-diphenyl-2-picryl hydrazyl radical scavenging sequences, including intergenic spacers trnT-L and trnL-F, activity, ferric reducing antioxidant power and metal- and trnL introns to study the phylogeny and evolutionary chelating ability and to contain phenolic compounds such relationships among Indian Ceropegia and their allies as gallic acid, vanillin, cathechol and ferulic acid (Chavan (Surveswaran et al. 2009). Many species have medicinal et al. 2013a). C. santapaui also possessed similar activities value while several others have been domesticated as (Chavan et al. 2014). Other economically important house plants and are used as ornamentals (Reynolds 2006; characteristics have been described in more detail by Chavan et al. 2011b). The highest species diversity occurs Kalimuthu and Prabakaran (2013c). Muthukrishnan et in South Africa, while ranks fourth (Murthy et al. al. (2013a) describe a factor-by-factor assessment of the 2012b), with a heavy concentration (approx. 50 species) in micropropagation of Ceropegia spp. as well as an overview Western Ghats (Karthikeyan et al. 2009), 28 of which are of ceropegin retrosynthesis. endemic to peninsular India. Unsurprisingly, there are a large number of conservation- and tissue culture-related Ceropegia in vitro studies emerging from India (Murthy et al. 2012b). For example, C. candelabrum (glabrous goglet flower) root There are two reasons for the surge in studies on Ceropegia tubers contain the alkaloid ceropegine, common to many spp.: (a) the listing of all species in India as being endangered Ceropegia spp. (Sukumar et al. 1995), starch, sugar, gum, (Murthy et al. 2012b); (b) the limited propagative capacity,

Environmental and Experimental Biology ISSN 2255-9582 1 J.A. Teixeira da Silva low seed set and viability (Yadav, Kamble 2006). Several such as for Bauhinia spp., another climbing ornamental studies reported on somatic embryogenesis, for example in (Teixeira da Silva 2013), provide an important platform for C. candelabrum (Beena et al. 2003) in the presence of 4.52 the advancement of in vitro (including micropropagation) µM 2,4-dichlorophenoxyacetic acid (2,4-D) in Murashige studies in this genus. and Skoog (MS; 1962) medium, although no histological proof was provided or any photographic evidence of all Acknowledgements four stages of somatic embryogenesis. Flowers induction in vitro was possible for several species: C. bulbosa (Britto et The author thanks Prof. K. Sri Rama Murthy (Montessori Mahila al. 2003), C. attenuata (Chavan et al. 2011b) and C. pusilla Kalasala, India), Dr. Jaykumar J. Chavan (Yashavantrao Chavan Institute of Science, India), Prof. Gyan S. Shekhawat (Banasthali (Kalimuthu, Prabakaran 2013b). In vitro flowering of plants University, India), Mafatlal M. Kher (Sardar Patel University, India) allows for the production of sterile floral tissues, useful and Dr. Judit Dobránszki (University of Debrecen, Hungary) for the study of reproductive developmental processes, for providing literature and/or for advice and comments on the regeneration from floral tissues and the use of floral organs Ceropegia literature. The author declares no conflicts of interest, for genetic transformation (Teixeira da Silva et al. 2014). financial, or other. Several studies used thin cell layers (Teixeira da Silva, Dobránszki 2013) to enhance shoot production (Table 1). References The few studies on the molecular verification of the stability of in vitro clonal material exist. Chavan et APG III. 2009. An update of the angiosperm phylogeny group al. (2013b) used random amplified polymorphic DNA classification for the orders and families of flowering plants. Bot. J. Linn. Soc. 161: 105–121. (RAPD) and inter-simple sequence repeats (ISSR) markers Beena M.R., Martin K.P. 2003. In vitro propagation of the rare to detect for somaclonal variation among tissue-cultured medicinal plant Ceropegia candelabrum L. through somatic C. panchganiensis plants in comparison to mother plants, embryogenesis. In Vitro Cell. Dev. Biol. Plant 39: 510–513. finding 1.31% variation (RAPD and ISSR combined), Beena M.R., Martin K.P., Kirti P.B., Hariharan M. 2003. Rapid confirming similar values (0.81%) for similar previous in vitro propagation of medicinally important Ceropegia studies on C. spiralis using RAPD and ISSR (Chavan et candelabrum. Plant Cell Tiss. Org. Cult. 72: 285–289. al. 2013c). The same two techniques were used by Chavan Britto S.J., Natarajan E., Arockiasamy D.I. 2003. In vitro flowering et al. (2014) on C. santapaui in which RAPD detected and shoot multiplication from nodal explants of Ceropegia no variation while ISSR detected 1.2% polymorphism bulbosa Roxb var bulbosa. Taiwania 48: 106–111. Bruyns P.V. 2003. Three new succulent species of between plantlets derived from direct and indirect () from Southern Africa. Kew Bull. 58: 427– organogenic routes and mother plants. Similarly, Chavan et 435. al. (2015) used RAPD and ISSR to test for polymorphism Chandore A.N., Nimbalkar M.S., Gurav R.V., Bapat V.A., Yadav in tissue-cultured C. evansii and found no variability (0% S.R. 2010. An efficient micropropagation protocol for polymorphism) among micropropagated plants from 759 multiplication and restoration of Ceropegia fantastica Sedgw: bands generated. Dhir and Shekhawat (2013) found a higher a critically endangered plant species. Curr. Sci. 99: 1593–1596. level of polymorphism (7.8%) in RAPD banding between Chavan J.J., Gaikwad N.B., Kshirsagar P.R., Yadav S.R. 2013a. Total micropropagated and mother C. bulbosa var. bulbosa plants. phenolics, flavonoids and antioxidant properties of three The latter study was the only study to date to encapsulate Ceropegia species from Western Ghats of India. South African J. Bot. 88: 273–277. nodal segments in calcium alginate (3.0% sodium alginate + Chavan J.J., Gaikwad N.B., Kshirsagar P.R., Umdale S.D., Bhat 100 mM CaCl2 2H2O) beads, which could be stored at a low K.V., Dixit G.B., Yadav S.R. 2013c. Application of molecular temperature (4 °C) for up to 60 days with 50.7% survival. markers to appraise the genetic fidelity of Ceropegia spiralis, Murthy et al. (2013) also devised encapsulation protocols a threatened medicinal plant of South India. Curr. Sci. 105: for C. pusilla and C. spirallis. Encapsulation studies and 1348–1350. the use of synthetic seeds and low-temperature storage are Chavan J.J., Gaikwad N.B., Umdale S.D., Kshirsagar P.R., Bhat an important first step for establishing cryopreservation K.V., Yadav S.R. 2014. Efficiency of direct and indirect shoot protocols for the long-term storage of material (Sharma organogenesis, molecular profiling, secondary metabolite et al. 2013). Other than these studies, no other molecular- production and antioxidant activity of micropropagated Ceropegia santapaui. Plant Growth Regul. 72: 1–15. based analyses exist for Ceropegia spp. Chavan J.J., Gaikwad N.B., Kshirsagar P.R., Umdale S.D., Bhat Most studies thus far on Ceropegia spp. have focused on K.V., Dixit G.B., Yadav S.R. 2015. Highly efficient in vitro the in vitro propagation, primarily with a concern for the proliferation and genetic stability analysis of micropropagated conservation of endangered species. To achieve this goal, Ceropegia evansii by RAPD and ISSR markers: A critically in vitro propagation has been the only biotechnological endangered plant of Western Ghats. Plant Biosyst. 149: 442– tool used thus far. Considering that only approximately 450. one dozen species have been propagated thus far in vitro, Chavan J.J., Gaikwad N.B., Yadav S.R. 2013b. High multiplication it is expected that protocols for the remaining 2-3 dozen frequency and genetic stability analysis of Ceropegia in India will emerge soon. Syntheses of the literature, panchganiensis, a threatened ornamental plant of Western Ghats: Conservation implications. Sci. Hortic. 161: 134–142.

2 Ceropegia species in vitro Reference 1998 Patil Beena, Martin 2003 Beena et al. Beena al. et 2003 , mercury , chloride; 2 shoots dipped in 49.2 µM in dipped shoots -furfyladenine); MIM, -furfyladenine); microtuber induction MIM, 6 Remarks, experimental outcome, outcome, experimental Remarks, variation and acclimatization vitro In embedded then in 15 s, for IBA of 82% and Maximum sand. sterile shoots formed 88% of explants respectively. microtubers, and embryogenesis ofA claim somatic proof. histological without but of > 500 100 mg induced callus PGR-free On embryos. somatic (maturation medium ¼ MS solid embryos 50% of somatic medium), 90% of plantlets, to converted survive could which the field. in 10 shoots/node or 7.8 shoots/node 6.9 roots/ (SIM). sub-cultures in Rooted plantlets (RIM). shoot (1:1), sand : soilrite in acclimatized the field. to then

–2 –2 -isopentenyladenine -isopentenyladenine or 6-γ,γ-dimethylallylaminopurine; AC, 6 , 25 ± , 25 ± , –1 –1 Culture Culture conditions 14-h PP, = 45 PPFD m µmol s 2 °C 16-h PP, PPFD 25 ± NR, Sub- 2 °C. cultured every 40 days. 25 16-h PP, m µmol s 2 °C ). ). C. jainii C. Culture medium, PGRs and PGRs and medium, Culture additives ½ MS MS + 9.05 µM 2,4-D (CIM). 2.22 µM BA or (SIM + 8.88 µM BA + latter The shoots). multiple for 0.25 µM spermine = FIM ( + 23.2 µM Kin ½ MS + 22.2 µM BA (RIM). ¼ MS + 2.5 µM IBA (MIM). pH 5.8. 0.2% Phytagel; 3% sucrose; or MS + 2.26–9.05 µM 2,4-D alone 2.22 µM BA with combination in Carbon (SEIM). 2.32 µM Kin or For pH 5.8. 0.8% agar; NR; source of 100 mg culture, suspension to transferred was friable callus 25 mL with flasks 100-mL conical cultures Suspension of SEIM. liquid a on the dark in incubated were 120 rpm. at rotary shaker + 2.46 µM IBA MS + 8.87 µM BA axillary and sub- (SIM shoot ½ MS + 0.49 µM IBA culture). pH 0.8% agar; 3% sucrose; (RIM). 5.8.

2 species. 2,4-D, 2,4-dichlorophenoxyacetic acid; N 2iP, for 12–14 min and and 12–14 min for Ceropegia 2 for 5 min, and 3–4 rinses in 3–4 rinses in and 5 min, for 2 explants, internode 1 cm) internode explants, 2 Sterilization procedure Sterilization + 10% water in washed Explants 0.1% Rinse RTW. in 20. Tween HgCl SDW. RTW under washed were Segments for (a detergent) 5% extran in then DDW, in washes several After min. 5 sterilized surface were segments 0.5% HgCl with each) (5 min rinsed 3 washes with 1 into cut Leaves of DDW. sterile cm washed were segments Nodal (a 5% extran in then RTW, under severalAfter 5 min. for detergent) were segments DDW, in washes 0.5% HgCl sterilizedsurface with rinsed with and 10–14 min for ofseveral washes DDW. sterile -benzyladenine [BA is used throughout even though BAP (6-benzylamino purine) may have been used in the original, according to Teixeira da Silva 2012b]; B5, B5, 2012b]; da Silva Teixeira to according been used have in the original, may (6-benzylamino purine) even BAP though is used -benzyladeninethroughout [BA 6 Explant used (2–3 Nodes from long) mm 10–12 week-old plants. Leaf and internode from segments shoot young oftips mature plants. Axillary at buds the nodes from of stems tender plants. mature

propagation and micropropagation of , , var. var. Roxb. Roxb. In In vitro

. bulbosa Genus, species Genus, cultivar and/or C. jainii, C. bulbosa var. bulbosa C. lushii candelabrum C. L. candelabrum C. ontinued Table Table 3 N BA, charcoal; activated HgCl medium; induction flower FIM, direct shoot organogenesis; double-distilled water; DSO, callus DDW, (1968) induction medium; et CIM, Gamborg medium; al. sequence kinetin indirect Kin, repeat; inter-simple (N ISSR, shoot indole-3-butyric indole-3-acetic acid; ISO, organogenesis; acid; IBA, IAA, medium; induction root RIM, humidity; relative RH, DNA; medium; MS, Murashige and polymorphic Skoog amplified (1962) random α-naphthaleneacetic medium; acid; NAA, NaOCl, sodium hypochlorite; NR, RAPD, not density; reported; flux PGR, plant growth photon 4-amino- regulator; picloram, photosynthetic PPFD, photoperiod; PP, acid; 3,5,6-trichloropicolinic sterile distilled running water; shoot tap SIM, induction water; medium; thin SDW, TCL, percell revolutions rpm, RTP, layer; minute; thidiazuron TDZ, (N-phenyl-N’- 1,2,3-thiadiazol- thin cell layer transverse tTCL, 5-ylurea); c

3 J.A. Teixeira da Silva Reference Britto et al. al. et Britto 2003 Goyal, Bhadauria 2006 al. et Nair 2007 Nikam, 2007 Savant In In flowers were smaller than in smaller than were flowers Remarks, experimental outcome, outcome, experimental Remarks, variation and acclimatization 70% 12 shoots/node. 76% of possible. acclimatization 65% and FIM flowered, in plants of thoseviable. seeds were 2.8 roots/shoot. shoots/node. 2.2. and buds apical Sub-cultured similar axillary showed buds (maximum frequencies flowering the 6 species; of 1.7–3.6 across 1.0–6.6 levels; BA to response to response the 6 species; across 30 days. within levels), sucrose vitro counterparts. planta 6.1 shoots/explant

–2 , 25 ± , –1 s , 25 ± , –2 –1 Culture Culture conditions 16-h PP, 25 3000 lux, ± 2 °C 16-h PP, 25 2000 lux, ± 2 °C 40 16-h PP, m µmol s 2 °C 16-h PP, 50–80 µmol m 2 °C

3 (FIM). 0.05 mg/L 0.05 mg/L (FIM). 3 Culture medium, PGRs and PGRs and medium, Culture additives NAA + 0.05 mg/L BA B5 + 3 mg/L 0.5 mg/L (RIM). IBA 2 mg/L (SIM). GA 1 mg/L BA+ (MIM). IBA + 2 mg/L Kin + 0.58 µM GA MS + 4.44 µM BA MS + 11.42 (SIM). + 0.27 µM NAA 0.8% 3% sucrose; (RIM). µM IAA pH 5.8. agar-agar; (seedMS + 87 mM sucrose MS + 26.67 µM BA germination). 0.8% (FIM). 175 mM sucrose or pH 5.8. agar-agar; MS + 1 mM 2,4-D 5 BA MS (SIM). MS + 1 mM BA (CIM). + 5% sucrose spermidine + 6 mg/L pH 0.8% agar. 3% sucrose. (RIM). 5.8. for 4 min, and 3 and 4 min, for 2 for 2–3 min, and 3 rinses in 3 rinses in and 2–3 min, for 3X with Washed 5 min. for 2 2 Sterilization procedure Sterilization in washed (2 cm long) shoots Young in wash one then 30 min, for RTW 0.1% HgCl SDW, SDW. rinses in 4–5 with water placedtap in Stems nodal 2–3 cm long 20. Tween drops 0.05–0.10% with treated segments 10 min, for (fungicide) Bavistin 0.05–0.10% streptomycin then Each time 10 min. for (antibiotic) 0.1–0.5% SDW. 3X rinses with HgCl SDW. 10 for RTW under held Follicles 100 mL in 20 min soaked for min, Tween-20; + 2–4 drops of water tap 70% ethanol SDW. 2X with washed 0.1% SDW. 1X rinse with 1 min, for HgCl longitudinally cut Follicles SDW. the seeds obtain to sutures along conditions. aseptic under available Not Explant used from Nodes plants. garden from Nodes field-grown plants. and buds Apical axillary buds 2-week- from seedlings.old from Nodes mature indehiscent follicle

C. C. , ,

C. C. C. C. Roxb. Roxb. Hook., Hook., Hook., Hook., Wt et et Wt continued

bulbosa Genus, species Genus, cultivar and/or bulbosa C. var. Roxb. bulbosa bulbosa C. lawii C. maccannii C. Ansari., oculata sahyadrica C. et Ansari Kulkarni, bulbosa var. hirsuta Arn. sahyadrica C. & Kulk. Ans. ontinued Table 1. Table c

4 Ceropegia species in vitro - Reference Nikam et al. al. et Nikam 2008a al. et Nikam 2008b al. et Pandit 2008 Karup et pusamy 2009 al. Nikam, 2009 Savant Rathore, Shekhawat 2009; et Rathore 2010 al.

in in , , ex vitro ex in vitro in . Secondary . in vitro in shoots rooted rooted shoots growth, no evidence or data data evidence no or growth, C. maccannii, C. odorata C. maccannii, C. Remarks, experimental outcome, outcome, experimental Remarks, variation and acclimatization 6.1 shoots/explant 5.2 or ( in Acclimatization respectively). for covered soil completely garden conditions, natural then 2 days, 80% survival. about in resulting Acclimatization 5.7 shoots/node. quantified. not but reported, could buds Axillary apical and instead but tubers develop not flowered also possible. tuberization 75% 5.51 shoots/node. survival garden in acclimatization (1:1). humus forest soil : 8.3 shoots/ best: performed Nodes cerpegin Highest content explant. callus (470 μg/g dry from weight) 10.0 µM CIM with on induced of instead 2,4-D. IAA vitro In mg/L IBA. in 100 dipping after for evidence provided No vitro of somatic claims for provided and embryogenesis flowering.

–2 , 25 ± , 25 ± , 25 ± , –1 –1 –1 s s s , temp. temp. , –2 –2 –2 –1 Culture Culture conditions 16-h PP, 50–80 µmol m 2 °C 9-h PP, 18–24 µmol m 2 °C 16-h or 40 24-h PP, m µmol s NR 16-h PP, and PPFD NR. temp. 16-h PP, 20–30 µmol m 2 °C NR. Culture medium, PGRs and PGRs and medium, Culture additives + 3% sucrose MS + 7.5 µM BA ½ MS + 5% (SIM) + 0.8% agar (liquid; + 0.5 µM IBA sucrose RIM) pH 5.5-6.0. ½ MS (SIM). MS + 7.5 µM BA 3%, (RIM). + 2 µM IAA (liquid) 0.8% agar; RIM); (SIM, 5% sucrose pH 5.8. 4% (MIM). BA MS + 6 mg/L pH 5.8. 0.8% agar-agar; sucrose; MS + 5.37 (SIM). MS + 6.66 µM BA 0.8% 2% sucrose; (RIM). µM NAA pH 5.8. agar-agar; MS + 1.0 µM 2,4-D 5.0 BA (SIM). MS + 7.5 µM Kin (CIM). (RIM). NAA or 0.5-2.0 µM IAA pH 5.8. 0.8% agar; MS 2,4-D (CIM). MS + 1.0 mg/L NAA + 0.1 mg/L BA + 1.0 mg/L + 0.1 BA ½ MS + 1.0 mg/L (SIM). (RIM). AC + 0.1% NAA mg/L

2 for 5-7 for 4–5 for 2 2 for 5 min. 4–5 washes with with 4–5 washes 5 min. for 2 Sterilization procedure Sterilization 0.1% HgCl SDW. in Wash SDW. 5X with Washed min. 0.1% HgCl SDW. in Wash SDW. 5–6X with Washed min. unclear. Protocol 6 internodes with shoots Young with washed plants garden from 4 cm nodes 15 min. for RTW 20 (5% v/v) for Tween with washed SDW, 3–4 rinses with 10–15 min, 0.1% 30 s then for 80% ethanol HgCl SDW. 0.1% HgCl SDW; in washed Stems SDW. 4 rinses with 5 min; for NR. Explant used Node, internode, to (up leaves node 6) Node, internode, leaves Axillary and of buds apical seed-derived shoots Axillary shoots 1–1.5 cm from nodes. long and Nodes from internodes garden-grown plants. from Epicotyls seedlings.

Wt & Wt C. C. continued

Genus, species Genus, cultivar and/or odorata C. Hook, maccannii Ansari hirsuta C. Arn. hirsuta, C. C. lawii, C. maccannii, C. oculata, C. sahyadrica intermedia C. sahyadrica C. bulbosa C. ontinued Table 1. Table c

5 6 J.A. Teixeira da Silva

Table 1. continued

Genus, species Explant used Sterilization procedure Culture medium, PGRs and Culture Remarks, experimental outcome, Reference and/or cultivar additives conditions acclimatization and variation

C. fantastica Nodes (1 cm Shoots from tubers were washed in MS + 1.5 mg/L BA (SIM). CaCl2- 16-h PP, 25 10.2 shoots/node. 65% Chandore et Sedgw. long). RTW for 30 min., a wash in liquid free MS + 1 mg/L IBA (RIM). 3% µmol m–2 acclimatization after potted in soil al. 2010 soap (5%, v/v) for 10 min, DDW (w/v) sucrose; 0.2% gelrite; pH 5.8. s–1, 25 ± : coco-peat (1:1) and watered with

several times, 0.1% HgCl2 for 7 min, 2 °C sucrose- and PGR-free ½ MS. 4–5 rinses in SDW. C. pusilla Wight Node, Nodes washed in RTW. 0.3% MS + 13.32 µM BA + 0.45 µM 16-h PP, Best callus induction from TCLs. Kondamudi and Arn. internode, TCLs fungicide + 0.3% bacteriocide, each 2,4-D (CIM). MS + 22.7 µM TDZ 18-24 µmol 37.54 shoots per callus clump. et al. 2010; from 1-month- for 10 min. Tween 20 (5% v/v) for 4 (SIM). MS + 13.32 µM BA + 0.49- m–2 s–1, 24 ± 60% of explants flowered with a Kondamudi,

old plant. min. 0.1% HgCl2 for 2 min. Several 1.23 µM IBA (RIM). ½ MS + 0.02 2 °C maximum of 20 flowers/plantlet. Murthy washes with SDW. mg/L IAA + 3% sucrose (FIM). 2011 Carbohydrate NR (CIM, SIM, RIM). 0.9% agar-agar; pH 5.7. C. spiralis TCLs of nodes Same protocol as Kondamudi et al. MS + 13.32 µM BA + 4.52 µM 16-h PP, 17.34 shoots/TCL. Plantlets Murthy et and internodes. 2010. 2,4-D (CIM). MS + 13.32 µM BA + 18-24 µmol obtained within 8 weeks. 90% al. 2010a, 0.54 µM NAA (SIM). ½ MS + 10.74 m–2 s–1, 24 ± survival after acclimatization. 2010b, 2012; µM NAA (RIM). Carbohydrate NR. 2 °C Murthy, 0.9% agar-agar; pH 5.7. Kondamudi 2011

C. spiralis Single nodes Stems with 5–6 nodes washed MS + 2.0 mg/L BA + 0.5 mg/L 16-h PP, 30 10.2 shoots/node, increasing to Chavan et (1 cm) of under RTW for 20 min. 0.5% (w/v) TDZ (SIM). 3% (w/v) sucrose; 0.2% µmol m–2 12.1/node on the 3rd subculture. al. 2011a greenhouse Bavistin (fungicide) for 5 min. 3 gelrite; pH 5.8. s–1, 25 ± Acclimatization on sterile soil, plants. washes with SDW. 5% Tween 20 for 2 °C sand and coco peat (1:2:1), but not 10 min. Several washes with SDW. quantified. 70% ethyl alcohol for 1 min and a

wash with SDW. 0.1% HgCl2 for 4 min; 3 rinses with SDW.

continued Table 1. continued

Genus, species Explant used Sterilization procedure Culture medium, PGRs and Culture Remarks, experimental outcome, Reference and/or cultivar additives conditions acclimatization and variation C. attenuata Single nodes Shoot segments defoliated and MS + 13.31 µM BA (SIM); 4.14 16-h PP, 33 On SIM, 12.9 shoots/explant. Chavan et Hook. of mature washed under RTW for 20 min, µM picloram (FIM); ½ MS + 2.46 µmol m–2 100% flowering achieved on FIM al. 2011b greenhouse washed with detergent, Tween 20 µM IBA (RIM). All media with 3% s–1, 25 ± within 4 weeks. 85% of in vitro plant. (5% v/v) for 10 min, Bavistin (0.5% (w/v) sucrose and 0.2% gelrite. 2 °C rooted plantlets survived in garden w/v) for 3 min then SDW. Under soil watered with ½ MS. aseptic conditions, explants dipped in 70% (v/v) alcohol for 30 s and

0.1% (w/v) HgCl2 for 5 min. Each treatment followed by 3–4 rinses with SDW. C. juncea Shoot tips, Seeds washed with two drops of 1% Sterilized seeds germinated on 16-h PP, 60 20.65 shoots/node.78% Krishnared- nodes and Tween-20 for 20 min with constant PGR-free ½ MS. MS + 8.87 µM BA µmol m–2 acclimatization survival in garden dy et al. internodes shaking. Rinse in RTW for 30 min + 4.54 µM TDZ (SIM). MS + 4.90 s–1, 25 ± soil : forest humus (1:1). 2011 from in vitro and repeated rinses with Millipore µM IBA + 1.27 µM NAA (RIM). 2% 2 °C plantlets. water. Under aseptic conditions, sucrose; 0.6% agar; pH 5.8. seeds washed with SDW, 70% ethanol for 30 s, wash with SDW,

20% HgCl2 for 6 min and 5 rinses 5 with SDW. C. elegans Nodes from Young shoots with 6 internodes MS + 23.20 µM Kin + 5.71 µM IAA 16-h PP, 50 7.11 shoots/node. 72% Krishnared- greenhouse washed with RTW for 15 min. 4 cm (SIM). MS + 4.90 µM IBA (RIM). µmol m–2 acclimatization survival in garden dy, Pullaiah plants. nodes washed with 5% Tween 20 for 3% sucrose; 0.8% agar; pH 5.8. s–1, 25 ± soil : forest humus (1:1). 2012 15 min then rinsed 3–4 times with 2 °C SDW. 80% ethanol for 30 s, then

0.1% HgCl2 for 6 min, 4–5 rinses with SDW. C. thwaitesii Nodes (2 cm Stems washed in RTW for 20–30 MS + 13.94 µM Kin + 28.54 µM 16-h PP, 60 3.28 and 6.42 shoots/node upon Muth- Ceropegia Hook. long). min; 70% ethanol for 30 s; rinsed IAA (SIM). MS + 2.46 µM IBA µmol m–2 first subculture and subculture, ukrishnan –1 with SDW; 0.1% HgCl2 for 3 min; (RIM). 3% sucrose; 0.7% agar; pH s , 26 ± respectively. Removing agar et al. 2012, 3–5 washes with SDW. 5.8. 2 °C from medium improved shoot 2013b species regeneration and increased chlorophyll content. 73.33% in vitro acclimatization in red soil + sand

7 + coconut coir (1:1:1).

continued Table 1. continued 8 J.A. Teixeira da Silva Genus, species Explant used Sterilization procedure Culture medium, PGRs and Culture Remarks, experimental outcome, Reference and/or cultivar additives conditions acclimatization and variation C. mahabalei Node, RTW for 10 min. 0.1% Tween-20 MS + 5 µM BA (SIM). MS (liquid) 8-h PP, 40 Best regeneration from nodes with Nikam et al. –2 Hemadri & internode, for 5 min. 0.1% HgCl2 for 5 min. + 1 µM NAA (RIM). 3%, 4% µmol m a sub-culture every 3 weeks (~10 2012 Ansari and C. leaves Washed 8X with SDW. sucrose (SIM, RIM); 0.8% agar; pH s–1, 25 ± 2 shoots/explant). Acclimatization media (Huber) 5.8. °C, 60 ± in garden soil : sand (1:1), max 200 Ansari 10% RH. µmol m–2 s–1, 80 ± 10% RH under a shaded net house for 2 weeks, then transferred to field conditions (18- 32°C; max 400 µmol m–2 s–1, 55- 80% RH). 88% survival reported.

C. Single nodal Vegetative cuttings washed under Development of axillary shoots 16-h PP, 50 On SIM, 13.2 shoots/explant. 95% Chavan et panchganiensis explants from RTW for10 min. Shaken in freshly from nodes: MS + 3% sucrose + µmol m–2 of explants formed callus on CIM. al. 2013b Blatter and in vitro-derived prepared 0.5% Bavistin (w/v) for 0.2% ClariGel + 4.44 µM BA; pH s–1, 25 ± 80% of explants on FIM formed McCann shoots. 20 min, washed twice in SDW, then 5.8 MS + 13.31 µM BA + 2.69 µM 1 °C in vitro flowers. 85% of explants

surface sterilized with 0.2% HgCl2 NAA (SIM). 9.05 µM 2,4-D (CIM). formed microtubers on MIM. 96% for 6 min, then 3X rinses with SDW ½ MS + 4.44 µM BA + 175 mM of shoots rooted on RIM. From (5 min/rinse). Cuttings divided into sucrose (FIM). ½ MS + 17.74 µM RAPD and ISSR analyses, 1.31% single node explants BA + 175 mM sucrose (MIM). ½ polymorphism (syn. somaclonal MS + 7.36 µM IBA (RIM). variation) was observed. 85% of in vitro-derived plantlets could be acclimatized in a mixture of soil, sand and coco peat (1:2:1) at 60-65% RH. C. bulbosa var. Single nodal Shoot segments with single nodes MS + 8.88 µM BA (SIM and axillary 16-h PP, 40 9.7 shoots/node on SIM (100% Dhir, bulbosa segments (1–1.5 were rinsed under RTW for 45 min shoot sub-culture). ½ MS (auxin- µmol m–2 of cultures), although Kin, TDZ Shekhawat cm) from then washed with 2% cetrimide free) (RIM). 3% sucrose; 0.8% agar; s–1, 25 ± 2 and NAA also induced shoot 2013 mature plants. + 1–2 drops of Tween-20 + 1% pH 5.8. °C, 50 ± 5% formation. On RIM, as many as NaOCl for 15 min, then RTW for 30 RH 11 roots/shoot. Nodal segments min. Surface sterilization with 70% encapsulated in calcium alginate

ethanol for 30 s, then 0.05% HgCl2 beads could form plantlets within for 3 min, 5 rinses with SDW. 4 weeks, which could then be fully acclimatized within a further 2 weeks. 7.8% polymorphism shown by RAPD. continued Table 1. continued

Genus, species Explant used Sterilization procedure Culture medium, PGRs and Culture Remarks, experimental outcome, Reference and/or cultivar additives conditions acclimatization and variation C. pusilla Nodes from Shoots with 5-6 nodes washed with MS + 11 µM BA + 5.35 µM NAA 16-h PP, 50 3.5 shoots/explant. 90-95% of Kalimuthu, field-grown RTW for 15 min. Nodes (1–2 cm) (SIM). MS + 2 mg/L BA + 1.0 g/L µmol m–2 callus formed shoots. In vitro Prabaka- plants. washed with Tween 20 (5% v/v) for NAA (CIM). MS + 6.66 µM BA + s–1, 25 ± 2 flowering induced within 32 ran, 2013b; 5 min and 3–4 rinses in SDW. 70% 5.35 µM NAA (MIM). ½ MS + 4.44 °C. days and microtubers in 90% of Prabakaran

ethanol for 30 s, 0.12% (w/v) HgCl2. µM BA + 2.46 µM IBA (FIM). 3% explants within 20-25 days. 81% et al. 2013; 3–4 rinses with SDW. sucrose; pH 5.8. of acclimatized plants survived in Kalimuthu decomposed coir. et al. 2014

C. bulbosa Seedling- Seeds surface sterilized with 0.1% PGR-free MS (seed germination). 12-h PP, Callus-derived shoots. Maximum Phulwaria

derived HgCl2 for 4-5 min, 2–3 rinses with MS + 1 mg/L 2,4-D (CIM). MS + 1 30-40 µmol of 75% of explants formed white, et al. 2013 epicotyls. SDW. mg/L 2,4-D + 0.5 mg/L BA (callus m–2 s–1, 26 ± friable callus. Shoots could be proliferation). MS + 1 mg/L BA + 2 °C rooted ex vitro when dipped in 0.1 mg/L NAA (SIM). 100 mg/L IBA for 3 min and acclimatized (100%) in organic manure, clay soil and sand (1:1:1). C. santapaui Cotyledonary Seeds from mature plants were MS + 2 mg/l 2iP (SIM – DSO). MS 16-h PP, 50 8.1 shoots/cotyledonary node Chavan et Wadhwa and nodes and separated from follicles, washed + 1.5 mg/L picloram (CIM – ISO). µmol m–2 in SIM – DSO. A maximum of al. 2014 Ansari cotyledons from and shade-dried for 3 days, surface MS + 2.5 mg/L BA + 0.4 mg/L IBA s–1, 25 ± 97.5% of cultures produced callus

15-day-old sterilized with 0.1% HgCl2 for 5 (SIM – ISO). All media with 3% 1 °C in CIM – ISO and 19.7 shoots/ seedlings. min under aseptic conditions and sucrose + 0.1 g/L myo-inositol + callus piece in SIM – ISO. Plants rinsed 2–3 times with SDW. Seeds 0.2% ClariGel; pH 5.8. were acclimatized in the in vitro were germinated on PGR-free MS growth room under same light medium. and temperature conditions, watering soil, sand and coco peat (1:2:1) with ½ MS. Two-week- old plantlets were transferred to a glasshouse (30 °C; 70% RH; –2 –1

50 µm m s ). No variation was Ceropegia detected in DSO- and ISO-derived plantlets by RAPD, relative to

mother plants, but ISSR detected species 1.2% polymorphism. The choice

of PGRs affected thein vitro in vitro secondary metabolites and 9 antioxidant capacity of extracts. continued Table 1. continued 10 Genus, species Explant used Sterilization procedure Culture medium, PGRs and Culture Remarks, experimental outcome, Reference J.A. Teixeira da Silva and/or cultivar additives conditions acclimatization and variation C. bulbosa tTCLs from Nodal segments rinsed under RTW MS + 4.5 µM 2,4-D + 2.2 µM BA 16-h PP, 95% of explants formed callus. Dhir, young nodal for 30 min then washed with 2% (CIM). MS + 8.88 µM BA (direct PPFD NR, 15.6 shoots/tTCL (direct route) or Shekhawat segments (1- cetrimide + few drops of Tween-20 SIM). MS + 8.88 µM BA + 0.27 µM 25 ± 2 °C, 22.2 (via callus route). On RIM, 2014

1.5 cm) from for 5–10 min, then 0.1% HgCl2 for NAA (indirect SIM). ½ MS (auxin- 50 ± 5% 10.9 roots/shoot. 89% survival mature plants. 3–5 min, then some rinses with free) (RIM). 3% sucrose; 0.8% agar; RH after acclimatization in sterile SDW. pH 5.8. vermiculite + soil (1:2). Variation among in vitro-derived plantlets using ISSR. C. evansii Nodal explants. Nodal explants washed under MS + 4 mg/L BA + 0.3 mg/L IAA 16-h PP, 40 11.6 shoots/node and 85% Chavan et RTW then 0.1% (w/v) Bavistin (SIM). ½ MS + 1 mg/L IBA (RIM). µmol m–2 shoot multiplication frequency. al. 2015 (fungicide) for 10–15 min. 0.1% Agar, carbohydrate concentration s–1, 25 ± 92% of shoots rooted with 90%

HgCl2 for 7 min; 3–4 rinses with and pH NR. 1 °C acclimatization on sterile soil, sand SDW. and coco peat (1:2:1). No genetic differences betweenin vitro- derived plantlets using RAPD and ISSR. Kalimuthu K., Prabakaran R. 2013a. Phytochemical screening and 2006. S. Bhadauria D., Goyal Gamborg O.L., Miller R.A., Ojima K. 1968. Nutrient requirements 2014. G.S. Shekhawat R., Dhir and storability Production, 2013. G.S. Shekhawat R., Dhir Gaikwad S.S., Kamble A.A., Adsul M.S., J.J., Nimbalkar Chavan ChavanJ.J., Nimbalkar M.S., GaikwadN.B., DixitG.B., YadavS.R. uty ... Knaui . 01 Rpd ho regeneration shoot Rapid 2011. R. Kondamudi K.S.R., Murthy and cytokinins of Effect 2010. R. Kondamudi K.S.R., Murthy MurashigeT., F.growthrapidSkoog for medium 1962.revised A 2011. T. Pullaiah S., P.V., Karuppusamy Krishnareddy 2012. T. P.V., Pullaiah Krishnareddy R.,KondamudiVijayalakshmi V., Induction2010. K.S.R. Murthy of Micropropagation 2011. K.S.R. Murthy R., Kondamudi 2009. S. Moorthy M., Sanjappa S., Karthikeyan Karuppusamy S., Kiranmani C., Aruna V., Pullaiah T. 2009. 2014. S. Jeyaraman S., Paulsamy R., Prabakaran K., Kalimuthu Kalimuthu K., Prabakaran R. 2013c. Kalimuthu K., Prabakaran R. 2013b. GC-MS analysis of methanol extract of extract methanol of analysis GC-MS bulbosa 158. suspensionof cultures soybean rootof cells. in transverse thin cell layer culture and homogeneity assessment of threatened plant species. evaluation fidelity regenerated genetic axillary encapsulated and alginate meristems of response morphogenic Biotechnol. endangered plant and Micropropagation 2011b. V.A., S.R. Yadav Bapat R.V., Gurav G.B., Dixit N.B., India. peninsular of plant ornamental potential rare and endemic 2011a. rm hn el ae epat o a ednee medicinal endangered an asclepiad of explants layer cell thin from 37. andWight on auxins 473–497. cultures.tissue tobacco with assays bio and 813–818. of propagation Biotechnol. Ceropegiaelegans Ceropegia pusilla in regeneration shoot multiple and callus morphogenetic of medicinal Asclepiad. pusilla Ceropegia 160–164. India, of Dicotyledons south India. of conservation endangered medicinal plant. of Microtuberization plant medicinal threatened and Ceropegia rare of conservation for 3: 35–42. of explants vitro Ceropegia bulbosa tuber. Proc. Nat. Acad. Sci. India B In vitro In using segment.nodal species –areview. species eoei spiralis Ceropegia in vitro in 20: 276–282. 11: 12443–12449. Int. J. Inv. Pharm. Sci. C. pusillaC. Ceropegia pusilla Ceropegia Ceropegia bulbosa African J. Biotechnol. propagation of of propagation eoei juncea Ceropegia Ceropegia intermediaCeropegia Wight and Arn. , an endemic plant of South India.South , plantendemicof an t & r. a ednee ad rare and endangered an – Arn. & Wt. flowering of endangered of flowering Ceropegia attenuata Roxb. Wight & Arn.& Wight J. Trop. Med. Plants . Vol.I, Acanthaceae-Avicenniaceae),pp. eoei pusilla Ceropegia n vitro in Indian Crops Prod. n vitro In J. Plant Growth Reg. L. Eur. J. Med. Plants Int. J. Biol. Technol. Wight and Arn. and Wight Indian J. Biotechnol. : A pharmaceutically important Ceropegia spiralisCeropegia In vitro ln Tsu Cl. Biotechnol. Cult. Tissue Plant n vitro In In vitro 1: 376–383. flowering of endemic and endemic of flowering 8: 236–239. Biotechnology 81: 120–126. Roxb. propagation of of propagation n vitro In Phytomorphology Hook. – an endemic plant of of plant endemic an – and micropropagation floweringfrom nodal ih ad r. an Arn. and Wight 12: 41–48. rpgto using propagation Ceropegia pusilla Ceropegia f. . ln Sci. Plant J. Afr. 47: 139–144. conservation of of conservation Physiol.Plant. Ceropegia spiralisCeropegia Cell Res. Flowering Plants Flowering J. Plant Biochem. Int. J. Bot. Res.Bot.Int. J. 4: 64–74. 33: 820–830. 9: 141–148. 4: 23–36. Wight – an – Wight 5: 565–567. Ceropegia AfricanJ. n vitro in 50: 151– 60: 32– 60: n vitro In In vitro 15:

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Received 13 December 2014; received in revised form 26 January 2015; accepted 24 February 2015

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