Micropropagation of Caralluma stalagmifera var. longipetala: A rare succulent medicinal plant from Karnataka, India

An efficient in vitro protocol has been developed for the multiplication of shoots and conservation of a rare succulent medicinal plant Caralluma stalagmifera var. longipetala growing wildly in Karnataka State. Proliferation of multiple shoots was achieved on Murashige and Skoog’s (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), Kinetin (Kn), indole acetic acid (IAA), αnaphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) alone or with various combinations from the nodal explants. The nodal explants cultured on medium containing BAP (2.0 mg/L) along with 0.5 mg/L Kn and 0.3 mg/L IAA produced the highest number of shoot sprouting (2.60±0.16) and maximum shoot length (3.96±0.20). The considerable frequency of callus induction and embryogenesis was noticed both in 1.0 mg/L NAA and 0.5-2 mg/L, 2, 4-D. The calli transferred to shoot induction medium containing the combination of hormones BAP (1.0 mg/L) plus IAA (0.2 mg/L) and NAA (0.1 mg/L) successfully regenerated in vitro shootlets. The in vitro rooting was achieved from both direct shoots regenerated from nodal explants and callus derived shootlets with NAA (0.2 mg/L). The in vitro rooted plantlets were successfully acclimatized (75%) in the greenhouse and gradually transferred to open field conditions.


INTRODUCTION
Caralluma stalagmifera var. longipetala Karupp. & Pullaiah was originally described from Tamilnadu state (Karuppusamy and Pullaiah, 2007) and later it was recognized that its distribution was extended in Karnataka State of Southern India. The plant is a xerophytic succulent leafless medicinal herb belonging to Apocynaceae. The succulent stem of the plant is used to cure many ailments and have noted antiobesity properties (Karuppusamy et al., 2013). The herb is a rich source of flavonoidal glycosides and alkaloids (Kunert *Corresponding author. E-mail: ksamytaxonomy@gmail.com. Author(s) (Madhuri et al., 2011). It is also reported to have significant anti-arthritic activity in kaolin induced rats (Reddy et al., 1996). The chemical principles isolated from C. stalagmifera are steroidal glycosides, stalagmosides, carumbellosides and lasiathosides (Kunert et al., 2009).
Natural population of this plant species are declining day by day because of increase demand in the pharmaceutical market coupled with over-exploitation and habitat destruction. There are no formulated agronomic or cultivation techniques for these endemic succulent medicinal species until now. For the conservation of these important medicinal plants, several other wild Caralluma species have already been developed via in vitro multiplication protocol by various authors (Aruna et al., 2012;Ugraiah et al., 2011;Sreelatha et al., 2009). So far there is only one report available on the micropropagation of C. stalagmifera from in vitro grown seedling explants (Sreelatha and Pullaiah, 2010). Micropropagation of other related Caralluma species include on Caralluma sarkariae (Sreelatha et al., 2009), Caralluma bhupenderiana (Ugraiah et al., 2011) and Caralluma adscendens var. attenuata (Aruna et al., 2012). The purpose of the study was to develop a rapid in vitro shoot multiplication and callus regeneration protocol from plant materials collected from natural populations growing in wild.

Plant material, surface sterilization and inoculation
Succulent plants of C. stalagmifera var. longipetala were collected from Muddapura of Chitradurga District in Karnataka and plants were maintained in pots containing mother soil under polyhouse condition in the Kuvempu University campus, Shivamogga ( Figure  1A and B). The tender shoot segments with six to eight inter nodes were collected from the potted plants and washed with running tap water for 15 min to remove the soil particles and other dust particles. The internodes were cut into small pieces and rinsed with 1% (v/v) Tween 20 (Merck, Bangalore, India) for 5 min. They were further rinsed in distilled water three times and taken into the laminar air flow chamber where they were rinsed with sterile double distilled water. The explants were immersed in 30% ethanol for 3 min and again washed with sterilized double distilled water. It was followed by Mercuric chloride (HgCl 2 0.1% (v/v)) treatment for 2 min. After sterilization, the explants were thoroughly rinsed with several changes of sterile double distilled water. The explants were trimmed into pieces of about (0.6 mm to 10 mm) and then inoculated into culture media. Murashige and Skoog's media (HiMedia, Mumbai, India) media was fortified with 3% (w/v) sucrose and 0.8% of agar for solidification. The pH of media was adjusted to 5.8 prior to the addition of 0.8% agar and autoclaved.

Shoot initiation and multiplication
For the induction of shoots, nodal explants were cultured on MS medium amended with various plant growth regulators like 6benzyladenine (BA), Kinetin (Kn), and α-naphthalene acetic acid (NAA) (HiMedia, Mumbai, India) at different concentrations (0.5, 1.0, 2.0, 3.0 and 5.0 mg/L). Cultures were subcultured on to the fresh medium with every 30 days period of intervals. The in vitro response was measured in the frequency of shoot multiplication, the number of shoots per explants and the shoot lengths at the end of six week old cultures.

Callus induction and multiplication
Internodal segments of C. stalagmifera var. longipetala were cultured on MS medium fortified with auxins like 2, 4dichlorophenoxyacetic acid (2, 4 D) and α-naphthalene acetic acid (NAA) in different concentrations (0.5 to 2.0 mg/L) alone. Regenerative and embryogenic calli were transferred to fresh MS medium supplemented with different concentration of BAP (0.5 to 2.5 mg/L), IAA (0.2 mg/L) and NAA (0.1 and 0.3 mg/L) alone or in combinations for the regeneration of shoots.

Root initiation and multiplication
The regenerated in vitro shoots (4 to 5 cm height) were separated and callus induced shoots with 2 to 3 cm height were isolated and transferred for root induction on to half-strength MS medium containing different concentrations of NAA, IAA and IBA (Indole-3butyric acid). The cultures were maintained under 16 h photoperiod for one month until the micro shoots initiated the roots. In vitro rooting response were measured with number of roots and mean length of roots (Ugraiah et al., 2011).

Acclimatization and transplantation of plantlets
In vitro rooted plantlets were removed from culture tubes with at least two roots of 2 to 4 cm length. They were washed carefully with tap water to remove traces of agar and then transferred to the pots containing different potting mixtures namely: cocopeat (HiMedia, Mumbai, India), cocopeat + sand + soil (1:2:1) and cocopeat + sand (1:1). The planted pots were covered with transparent polythene to maintain humidity until the development of new rudimentary leaves and sprouting new roots (Aruna et al., 2012). After a month they were removed and the plants were maintained in the lab temperature conditions for 15 more days. After two months of hardening, the plants were transferred to new pots containing humus soil, kept in polyhouse for one month. During the first 15 days of acclimatization neither watering nor any fertilizers was provided to plants. The hardened plants were planted on nursery bed with frequent watering in natural condition.

Statistical analysis
The experiments were randomized and repeated three times. Each treatment consisted of 15 replicates. Data were statistically analyzed by analysis of variance (ANOVA) and mean readings were compared by Tukey's test at 0.05% probability level.

Effect of cytokinin on shoot regeneration
The success of micropropagation depends on the selection of suitable explants, media composition, types of growth regulators, their concentrations and combinations with culture conditions. The effect of cytokinins and its concentration on bud breaking from nodal explants cultured on MS basal medium is given in Table 1. The MS basal medium fortified with BAP 2.0 mg/L was found to have the best shoot sprouting, number and length of shoots without basal callus formation from nodal explants. The shoot buds sprouted on Kn containing medium showed only limited growth even if they were maintained for longer period of subculture. MS medium is the most efficient medium for shoot proliferation of Caralluma spp. such as Caralluma adscendens, Caralluma bhubenderiana (Ugraiah et al., 2011) and Caralluma lasiantha (Aruna et al., 2012). Nodal explants selected for shoot proliferation gave positive  (Sreelatha et al., 2009). The response of nodal explants treatment results are presented in Table 1. Out of these treatments, MS medium fortified with BAP 2.0 mg/L had a better shoot sprouting frequency (80). High concentration of BAP above 3.0 mg/L resulted in reduced shoot sprouting and frequency of response (Personal communication). Kn containing cultures produced less than 2 shoots/explants ( Table 1).

Effect of cytokinins with auxin on shoot multiplication
C. stalagmifera var. longipetala nodal explants were cultured on MS medium supplemented with various concentrations of cytokinins (BAP 1.5 to 2 mg/L, Kn 0.2 to 1.5 mg/L) and auxins (NAA 0.2 to 1 mg/L, IAA 0.3 to 0.6 mg/L) ( Table 2). The effect of BAP on multiple shoots proliferation has been demonstrated in Asclepiads (Aruna et al., 2009(Aruna et al., , 2012. Combination of 1.5 mg/L BAP + 0.6 mg/L Kn produced highest number of shoot sprouting frequency of about 76%, the mean shoot/explants was 2.05±0.17 and mean shoot length was 3.07±0.17 cm. Similarly the combination of BAP 1.5 mg/L +NAA 0.3 mg/L produced sprouting frequency of about 53.3% (Table 2). Mean shoot number per explants was about 1.40±0.11 and the mean shoot length is 2.12±0.14 cm.
Basal callus formed in all the shoots were healthy ( Figure  1B). The combination of BAP (2.0 mg/L) + Kn (0.5 mg/L) + IAA (0.3 mg/L) yielded maximum shoot regeneration frequency of 90%. Number of shoots per explant was 2.60±0.16 and 3.96±0.20 cm. The mean shoot length followed by other combination of hormones such as BAP (2.0 mg/L) + Kn (1.0 Mg/L) + IAA (0.3 mg/L) showed regeneration frequency of about 86% and the number of shoots per explants was 2.10±0.18 and 3.32±0.17. The mean shoot length, of other combination of cytokinins BAP with auxins showed that the medium resulted in moderate callus formation ( Table 2). The combination of BAP (2 mg/L) + NAA (0.5 mg/L) showed lower frequency of shoot sprouts (53.3%) with basal callus ( Figure 1G).

Callus induction and somatic embryogenesis
The intermodal explants cultured on various concentration of 2,4-D (0.5, 1.0, 1.5, 2.0) and NAA (0.5, 1.0, 1.5, 2.0) become swollen and generally dedifferentiated and developed friable callus after two weeks of culture. Among the different auxins tested 2,4-D at 1.5 mg/L favored the best response of callus production (90%) ( Table 3). NAA supplemented cultures showed good callus production but there was no further shoot regeneration or somatic embryogenesis when other auxins in different combinations were added (Table 3). The calli subcultured onto media containing a combination of 2,4-D (1.5 mg/L) with BAP (1.0 mg/L), IAA (0.2 mg/L) and NAA (0.1 mg/L) produced maximum number of globular embryos on the surface within four weeks ( Figure 1E). In Asclepias, studies demonstrated the need of 2, 4-D (2 mg/L) and BA (0.1 mg/L) for callus induction (Vyapari et al., 1993). Other Asclepiad members like Tylophora indica and Hemidesmus indicus produced  callus on 2, 4-D and BAP (Thomas and Philip, 2005;Sarasan et al., 1994). The somatic embryos were induced within two weeks of subculture with media containing NAA and 2, 4-D at different concentrations (Stephan and Jayabalan, 2001;Inamdar et al., 1990).

Rooting of in vitro regenerated shoots
In C. stalagmifera var. longipetala, half strength MS medium supplemented with auxins such as IAA, IBA and NAA at different concentrations showed varied effect on rooting ( Table 4). The experiment was aimed at the induction of rooting in basal portion of C. stalagmiferavar. longipetala microshoots; NAA was found more effective than the IAA and IBA hormones. The NAA showed positive response of rooting in the present study, which is similar to the observations on other Asclepiads such as Decalepis arayalpathra (Sudha et al., 2005) and Euphorbia tirucalli