Plant Regeneration from Alginate Encapsulated Shoot Tips of Boucerosia umbellata (Haw.) Wight & Arn. (Syn.: Caralluma umbellata Haw.)

The shoot apices of multiple shoots of Boucerosia umbellata under in vitro conditions were selected for encapsulation studies. Gel matrix Sodium Alginate at different concentrations was exposed to complexing agent Calcium Chloride for different durations. The seeds thus produced were stored at 4°C under aseptic conditions to study their viability a nd were inoculated on nutrient medium to study their morphogenetic response. The germination percentage decreased after storage of these synthetic seeds. Immediate germination gave a germination percentage of 98 and gradually reduced during storage. After one month of storage, the germination percentage decreased to 74%. After 6 months of storage at 4°C the germination was as low as 36%.


INTRODUCTION
The genus Caralluma has been included in the family Apocynaceae (sub family Asclepiadoideae) in APG III classification. The plants of species of Caralluma found in India are edible. The medicinal properties of Caralluma includes anti-inflammatory, anti-ulcer, antidiabetic, carminative, febrifugal, anti-pyretic and anti-oxidant effects, besides a number of phytochemical compounds were isolated from the genus Caralluma [1]. Caralluma extracts have also been found to be appetite suppressant, a property well known to Indian tribals and hunters [1]. Indian folklore records its use as a potent appetite suppressant and weight loss promoter [1]. Some Caralluma species are used in the treatment of obesity [1]. The extract of Caralluma species in the form of capsules has been released under trade name GENASLIM for body weight control [2]. The genus Boucerosia, which was earlier included in the genus Caralluma, is now separated from the genus Caralluma (sensu stricto) mainly by the pseudoumbellate terminal cymes (in Boucerosia) Boucerosia umbellata (Haw.) Wight & Arn. (Syn: Caralluma umbellata Haw.) is a succulent leafless herb, distributed in the dry hilly regions of Andhra Pradesh, Karnataka, Tamil Nadu and Kerala. Tender stems of Caralluma umbellata locally known as Pedda saara are consumed as vegetable in both the forest areas and plains of Andhra Pradesh [4]. Stems of Caralluma umbellata are used in stomach disorders and abdominal pains [5], for the treatment of kidney stones by the ethnic practitioners of Nalgonda district [6]. According to Neelima et al. [7], external applications of latex of Caralluma umbellata heals mouth ulcers. Ramesh et al. [8] reported antinociceptive and anti-inflammatory activity of carumbelloside-I isolated from Caralluma umbellata. The literature survey indicates pregnane glycosides were isolated from various parts of the plant [1]. Bellamkondi et al. [9] reported antihyperglycemic activity of Caralluma umbellata.
The encapsulated vegetative propagules could be used for mass clonal propagation of high economically important plants when somatic embryos are not available for encapsulation [10][11][12][13]. Synthetic seed technology studies have several advantages such as ease of handling and exchange of plant material, genetic uniformity of plantlets, delivery to the soil, shortening the breeding cycle and reduction of storage space. During the last four decades, synthetic seed technology has gained considerable importance in plant biotechnology as a potential viable and valuable system for ex situ conservation of commercially important plants [14]. Available reports indicate that there is no work on encapsulation in any species of the genus Caralluma and hence the present investigation has been undertaken.
We reported earlier enhanced axillary proliferation of Boucerosia umbellata from nodal explants; on MS medium supplemented with BAP (2 mg/l) and TDZ (1 mg/l) gave maximum response [15]. We report here encapsulation of shoot tips for development of synthetic seeds in Boucerosia umbellata.

MATERIALS AND METHODS
The plants of B. umbellata were collected from Penukonda hills, Anantapur district and maintained in earthen pots. Young sprouts were collected from 3-month old garden plants for In vitro propagation to obtain multiple shoots. The possibility of using aseptic propagules like shoot apices obtained during direct shoot organogenesis for synthetic seed production was undertaken. Shoot tips measuring 0.5-1 cm were excised from proliferated In vitro multiple shoots ( Fig. 1 A). The following variables were tested to study encapsulation (firm transparent bead formation). All the experiments were conducted with a minimum of 10 replicates per treatment. The experiments were repeated three times. Standard error of means was calculated in each experiment. The data was subjected to statistical analysis using one-way analysis of variance (ANOVA) and means compared using the DMR test at 0.05% level of significance.

RESULTS AND DISCUSSION
Gel matrix Sodium Alginate at different concentrations was exposed to complexing agent Calcium Chloride for different durations. Propagules in 3% Sodium Alginate exposed to 50 mM CaCl 2 for duration of 20 minutes produced firm transparent beads (Table 1, Fig. 1 B & C). Lower concentrations of sodium alginate and calcium chloride (2% and 25 mM respectively) resulted in very soft beads irrespective of the duration of exposure. The very soft beads were very difficult to handle. Increase in the concentrations of sodium alginate and calcium chloride resulted in hard beads and these were unfavourable for the germination purpose even though they were comfortable to handle. These beads were collected in sterilized tea strainer and rinsed several times in sterile water to remove traces of calcium chloride. The seeds thus produced were stored at 4°C under aseptic conditions to study their viability and inoculated on nutrient medium to study their morphogenetic response. They were germinated on MS+BAP (2 mg/l) at different time intervals. On germination, the encapsulated shoot apex started proliferating by increasing its axis. The apex as it grew, slit open the gel matrix of the Alginate and emerged to the exterior (Fig. 1 D). Later on the shoot apex produced multiple shoots more profusely than in vivo field grown explants ( Fig. 1E & F). The germination percentage decreased on storage of these synthetic seeds ( Table  2). Immediate germination gave a germination percentage of 98 and gradually reduced during storage. After one month of storage, the germination percentage decreased to 74%. After 6 months of storage at 4°C the germination was as low as 36%.   In some species the unipolar axillary shoot buds and/ or apical shoot tips have been encapsulated to produce synthetic seeds. In vitro shoot apex was also very commonly utilized to produce synthetic seeds through encapsulation. Ganapathi and collaborates [12] produced synthetic seeds of Banana while Nyende et al. [16] encapsulated the shoot apices of four cultivars of Potato. Encapsulation of shoot apices was earlier reported in Actinidia deliciosa [17], Phyllanthus amarus [18], Rhododendron [19], Rauvolfia serpentina [20], Trichosanthes dioica [21], Solanum nigrum [22], Salvia officinalis [23], Brassica oleracea var. botrytis [24] and Oxalis triangularis [25].
In Salvia officinalis Izabela and Halina [23] reported that an encapsulation was better with 3% Sodium alginate and 50 mM CaCl 2 H 2 O was ideal for the formation of microshoot beads compared with other concentrations while Daud et al. [26] and Bukhari et al. [27] used 3% Sodium alginate with 100 mM CaCl 2 H 2 O and Siong et al. [24] used 4% Sodium alginate with 100 mM CaCl 2 H 2 O to obtain satisfactory synthetic seeds. In our present study 3% Sodium Alginate exposed to 50 mM CaCl 2 for 20 minutes produced firm transparent beads.
Decrease in conversion response during storage could be attributed to inhibition of tissue respiration by the alginate matrix, or a loss of moisture due to partial desiccation during storage as reported earlier by Danso and Llyod [28], Faisal et al. [29], and Faisal and Anis [30].

CONCLUSION
Three percent of Sodium alginate with 50 mM CaCl 2 for 20 minutes was optimum for producing firm transparent beads. These synthetic seeds can be stored for one month with 74% of germination. The present study describes a simple, reproducible and efficient protocol for synthetic seed production. This may facilitate cold storage of encapsulated shoot tips, offers possibility for germplasm conservation and exchange between laboratories.