Propagation through alginate encapsulation of axillary buds of Cannabis sativaL. — an important medicinal plant

Abbreviations : CaCl2.2H2O – Calcium chloride, MS – Murashige and Skoog, PGR – Plant Growth Regulator, PPM – Plant Preservative Mixture, TDZ – Thidiazuron

INTRODUCTION
Cannabis has acquired considerable importance as a medicinal crop all over the world. Indeed its medical use predates recorded history. The earliest written reference is to be found in the fifteenth century B.C., Chinese Pharmacopeia. Between 1840 and 1900, more than 100 articles on the therapeutic use of Cannabis were published in medical journals (Grinspoon and Bakalar, 1995). As a plant it is valued for its hallucinogenic and medicinal properties and has also been used to treat a variety of ailments including pain, glaucoma, nausea, asthma, depression, insomnia and neuralgia (Mechoulam et al., 1976; Duke and Wain, 1981). The therapeutic values of Cannabis derivatives have also been highlighted against HIV/AIDS (Abrams et al., 2007) and multiple sclerosis (Pryce and Baker, 2005). The pharmacologic and therapeutic potency of preparations of Cannabis sativa L. and it’s main active constituent Δ9-tetrahydrocannabinol (THC) has been extensively reviewed (Sirikantaramas et al., 2007; Long et al., 2005; Brenneisen et al., 1996; Mattes et al., 1994).
On other hand, Cannabis is one of the oldest sources of textile fiber. Hemp grown for fiber was introduced in western Asia and Egypt, and subsequently to Europe between 1000 and 2000 BCE. Cultivation of hemp in Europe became widespread after 500 CE. The crop was first brought to South America in 1545, in Chile, and to North America in Port Royal, Acadia in 1606 (Small and Marcus, 2002).
This extraordinary versatile important plant material naturally calls for development of suitable protocols for production of sufficient number of uniform planting materials from ‘elite’ Cannabis varieties through biotechnological intervention. Among the biotechnological approaches, micropropagation is one of the most viable techniques, allows efficient and rapid clonal propagation of many economically important crops. Since C. sativa is a cross pollinated crop, the allogamous (cross fertilization) nature of species makes it impossible to maintain elite cultivar/clones by seed. Synthetic seed technology can be considered an important application of micropropagation, that could be a valuable aid to economical large-scale clonal propagation and germplasm conservation of the screened and selected elite germplasm of C. sativa.
According to Bornman (1993), synthetic seeds may provide the only technology realistically amenable to the extensive scale up required for the commercial production of some clones. In addition, Mathur et al. (1989) reported that the use of this technology economized upon the medium, space and time requirements. Successful cases of synthetic seeds production and plantlet regeneration have been reported for cereals, vegetables, fruits, ornamentals, aromatic grass and conifers (Fowke et al., 1994; Piccioni and Standardi, 1995; Castillo et al., 1998; Ganapathi et al., 2001; Brischia et al., 2002; Hao and Deng, 2003). However, in most cases somatic embryos were used in the encapsulation process. Some authors (Mathur et al., 1989; Ganapathi et al., 1992; Sharma et al., 1994; Piccioni and Standardi, 1995; Pattnaik and Chand, 2000) described the encapsulation of vegetative propagules such as axillary buds or shoot tips, which could be used for mass clonal propagation as well as in long- term conservation of germplasm.
Although there have been few reports on tissue culture of C. sativa (Richez- Dumanois et al., 1986; Mandolino and Ranalli, 1999; Slusarkiewicz-Jarzina et al., 2005; Bing et al., 2007), no reports are available on in vitro propagation using synthetic seed technology. In our study, the possibility of using the non- embryogenic, in vitro-derived vegetative propagules for encapsulation has been explored. The strategy used is of particular significance for a plant like Cannabis, in which the process of somatic embryogenesis has not been documented, and therefore somatic embryos are not available for the production of synthetic seeds. This study reports a high frequency propagation of encapsulated axillary buds of a high yielding elite Mexican variety (MX-1) of C. sativa in calcium alginate gel, and their morphogenic response to various planting media.
MATERIALS AND METHODS 
Plant material 
Explants of nodal segements containing axillary buds, were excised from multiple shoot cultures of high yielding C. sativa variety (MX-1) maintained on Murashige and Skoog’s medium (Murashige and Skoog, 1962) containing 3 % (w/v) sucrose, 0.8 % (w/v) Type E Agar suppplemented with TDZ (0.5 μM) adjusted to pH 5.7 (Lata et al., 2008, accepted) The subculturing was done every four weeks on sterile medium dispensed (25 ml) in glass culture vessels (4 cm diameter x 9.5 cm high, baby food jars with magenta B caps).
Encapsulation Procedure 
Encapsulation Matrix : Sodium alginate was added in the range of 2-6 % (w/v) to full strength Murashige and Skoog’s medium (MS) with or without 3 % sucrose. The solutions were supplemented with 0.5 μM Thidiazuron (TDZ) and 2.5 μM indole-3-butyric acid (IBA). A broad spectrum fungicide, Plant preservative mixture (PPM) in a range of 0.3-0.5 % was added to the gel matrix for in vivo experiments. For complexation, different concentrations (25-100 mM) of complexing agent (CaCl2.2H2O) were prepared in liquid MS medium containing the same adjuvents as the sodium alginate matrix but excluding the PPM. Both the solutions were autoclaved separately for 15 min at a pressure of 1.1 kg cm-2 and temperature of 121 oC after adjusting the pH to 5.8.
Formation of beads : The beads were formed by dropping explants mixed with sodium alginate solution into CaCl2.2H2O in a flask, placed on an orbital shaker at 80 rpm. The resulting beads (0.5-0.8 cm in diameter) containing the entrapped nodal segments were left in the calcium chloride solution for 30 min for complexation. These were retrieved using a nylon mesh and the traces of calcium chloride was removed by washing with sterilized distilled water. The encapsulated nodal explants now called as synthetic seed / beads were inoculated onto the different planting substrata for growth and development studies as detailed in results (Fig. 1A).
Planting substrata and culture conditions for in vitro germination 
For in vitro experiments, in order to assess the germination potential, the synthetic seeds prepared were separately placed on these media viz. (a) cotton moistened with Murashige and Skoog’s medium, (b) sterile filter paper moistened with distilled water at room temperature, (c) Murashige and Skoog’s medium (MS) (Murashige and Skoog, 1962), (d) MS supplemented with cytokinin, Thidiazuron (TDZ) (0.5 μM), (e) MS supplemented with TDZ (0.5 μM) and Plant preservative mixture (PPM) (Caisson laboratories, USA) (0.075 %). Each treatment consisted of thirty beads and was repeated two times. Beads (10±1) were inoculated in glass culture vessels (4 cm diameter x 9.5 cm high, baby food jars with magenta B caps) jars and cultures were maintained at 25 ± 2 °C and 60-70 % humidity incubated with 16 h photoperiod under fluorescent light with a photon flux of 52 μmol m-2 s-1. After sprouting the encapsulated shoot buds were transferred to baby food jars with magenta B caps (two sprouted buds per flask) containing the same medium as that on which the encapsulated buds were sprouted. Three week-old shoots with fully expanded leaves emerging through the ruptured capsule were placed in half-strength MS medium fortified with 2.5 μM IBA concentrations to trigger root development. A set of non-encapsulated axillary buds were plated on the media described above and maintained under similar in order to serve as a control.
Planting substrata and culture conditions for in vivo germination 
For in vivo experiments, the synthetic seeds were directly placed in a pot containing substrates (marketed by Canna Continental, Los angeles, CA, www. canna-hydroponics. com) – non sterile potting mix-fertilome, coco natural growth medium and 1:1 potting mix- fertilome with coco natural growth medium. The substrates were divided into three groups,
1) Each moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose, 2) Each moistened with full strength MS medium + TDZ without 3 % sucrose, 3) Each moistened with full strength MS medium without TDZ supplemented with 3 % sucrose and 0.5 % PPM (Table 2). Each treatment had 30 beads and was repeated two times. The substrates were autoclaved prior to use. Encapsulated buds entrapped in calcium alginate capsules were plated onto the media, which were contained in pots that were placed in an indoor cultivation facility housed at Coy-Waller laboratory, University of Mississippi, at a temperature ranging from 25-30 °C. In order to maintain high humidity around the beads, they were covered with polyethylene sheets until the emergence of shoots and roots (7-14 days). A set of non-encapsulated axillary buds were plated onto the same substrates and maintained under similar conditions in order to serve as a control.
Data analysis 
A total of ninety explants were inoculated for each replicate per treatment. Each replicate consisted of thirty explants removed at 30 day interval during the growing period of 90 days. After 30, 60 and 90 days, regrowth and conversion of plantlets were evaluated. Regrowth of encapsulated shoots is based on number of shoots obtained per explant induced in 30, 60 and 90 days. Conversion was considered as percentage of sown synthetic seeds that had performed both sprouting and rooting i.e., that had produced a complete plantlet. The data were submitted to statistical analyses by analysis of variance followed by the Tukey test with the level of significance set at 5 % using SAS version 9.1 (SAS Institute, Cary, NC, USA).
Transfer of plants to soil 
Rooted shoots were carefully taken out of the medium and washed thoroughly in running tap water to remove all traces of medium attached to the roots without damaging the roots. Plantlets were preincubated in fertilome in thermocol cups (marketed by Wal-mart Stores, Inc.) for 10 days and irrigated with tap water supplemented with 0.5 % PPM. The cups were covered with polythene bags to maintain humidity and kept in growroom and later acclimatized in non sterile 1:1 potting mix- fertilome with coco natural growth medium irrigated with regular tap water. All these plantlets were kept under similar environmental conditions grown in an indoor cultivation facility housed at Coy-Waller laboratory, University of Mississippi. Light was provided with full spectrum 1000 watts HID (high intensity discharge) lamps (Sun Systems, CA) hung on the top of plants. A hot air suction fan was attached and about 3 to 4 feet distance between plants and bulb was maintained to avoid heating due to HID bulbs. Using an automatic electric timer artificial day/night cycle was regulated with a 16 h photoperiod. Growroom temperature and relative humidity was kept at nearly 25- 30 °C and 60 % respectively.
RESULTS AND DISCUSSION 
Characteristics of encapsulated buds 
About 220-250 characteristic beads (hydrogels) were formed using 100 ml of sodium alginate and 200 ml CaC12.2H20 solution. The beads differed morphologically with respect to texture, shape and transparency, with different combinations of sodium alginate and calcium chloride, and with diameters ranging from 4.5 to 6.2 mm. An encapsulation matrix of 5 % sodium alginate with 50 mm CaC12.2H20 with an ion exchange duration of 30 min, was most suitable for the formation of ideal beads (Fig. 1). Lower concentration of Na alginate (2-4 %) resulted in the formation of too soft beads that were difficult to handle, while at higher concentrations the beads were iso-diametric but were hard enough to cause considerable delay in sprouting. The reduction in the gelling ability of lower concentrations of Na alginate after exposure to high temperatures during autoclaving has been reported by Larkin et al. (1988). The use of agar as a gel matrix was deliberately avoided because it has been described as inferior to alginate with respect to long-term storage (Bapat et al., 1987). Lower levels of CaC12.2H20 prolonged the complexation time whereas higher concentrations adversely affected the bead quality. Therefore, synthetic seeds formed with 5 % sodium alginate with 50 mm CaC12.2H20 for 30 min were used in all the subsequent experiments.
The encapsulated nodal segments containing axillary buds were cultured on different substrates and the effect of these substrates was evaluated both under in vitro and in vivo conditions (Table 1 & 2). The most desirable feature of the encapsulated explants is their capability to retain viability in terms of regrowth and conversion abilities after encapsulation (Micheli et al., 2007; Adriani et al., 2000). In the present investigation, regrowth (defined as the number of shoots obtained per explant induced in 30, 60 and 90 days) and conversion (defined as the percentage of sown synthetic seeds that had performed both sprouting and rooting i.e., that had produced a complete plantlet) were the parameters used to measure the plantlet development. Differences in regrowth and conversion were observed after different treatments of the synthetic seeds.