Somatic embryogenesis (SE) is the ultimate developmental pathway by which somatic cells develop into structures that resemble zygotic embryos (that is, bipolar and without vascular connection to the parental tissue) through an orderly series of characteristic embryological stages without fusion of gametes (Jimenez, 2005). SE has been traditionally divided into two main stages, namely induction and expression. In the former, somatic cells acquire embryogenic characteristics by means of gene expression (Feher et al., 2002). The ontogeny, physiological, biochemical and media properties are required for somatic embryogenesis (Victor, 2001). Typical globular, heart, torpedo and cotyledonary stages of somatic embryos are different from various kinds of explants. True-to–type nature of the somatic embryo derived plantlets has been reported (Tokuhara and Mii, 2001). As a result any plant which needs to be altered by genetic engineering and transgenesis requires a pre developed protocol for successful regeneration through somatic embryogenesis (Birch, 1997; Thangavel et al., 2014). Developing a protocol for plant regeneration through somatic embryogenesis will immensely benefit the plant conservation programs too as it is a shorter and viable method for producing a large number of plantlets.

The genus Rumex belongs to family Polygonaceae that comprises about 150 species widely distributed around the World. The main chemical constituents of Rumex are anthraquinones and flavanoids (Cunningham, 1993). Rumex vesicarius L. (English: bladder dock, Hindi: Chooka, Sanskrit: Amlavetasa, Telugu: Chukkakura) a common green leafy vegetable is also used in herbal and ayurvedic formulations. It is a branched succulent herb and is distributed throughout India (Alam, 2012). The plant extract have been used to reduce cholesterol levels, biliary disorders (Rechinger, 1984; Mona et al., 2013) and also it showed significant effect on antioxidant (Palani and Ramakrishnan, 2011; 2012; Sakkir et al., 2012) and antimicrobial activities (Al Akeel et al., 2014; Ramesh and Asha, 2013).

In vitro regeneration of R. vesicarius L. has been achieved by researchers using explants like shoot tips, nodal explants, leaves and callus (Panduraju et al., 2009; Abo El-soud et al., 2012; Nandini et al., 2013; Lavanya et al., 2013). However, there is no report on the induction of somatic embryogenesis. This is an alternative method for plant propagation over regeneration via organogenesis. The plants regenerate via somatic embryogenesis is of single cell origin with true-to-type and are produced in large numbers within a short period (Ammirato et al., 1983; Lavanya et al., 2014).

Many researchers have emphasized that somatic embryogenesis is preferred method for rapid in vitro multiplication of plants (Moon et al., 2013), production of artificially synthetic seeds (Ravi and Anand, 2012), Agro bacterium mediated genetic transformation studies and regeneration of transgenic plants (Satyanarayana et al., 2012).  In the present study we made an attempt to establish a reliable and efficient protocol for the induction of somatic embryogenesis and plant regeneration from leaf explants of R. vesicarius L.

Culture medium and conditions

The seeds of R. vesicarius were collected from the plants grown in the research field of Department of Biotechnology, K L University. They  were soaked  in sterile  distilled  water for 24 h, later  cleaned with 5% tween-20 (w/v) and thoroughly washed in running tap water 3 to 4 times. Subsequently, they were surface sterilized with 0.1% w/v HgCl₂ for 2 to 3 min followed by rinsing with sterile distilled water for 2 to 3 times and germinated aseptically on SH medium (Schenk and Hildebrandt, 1972). Finally, these seeds were flame sterilized with Whatman filter paper and supplemented on the surface of the nutrient culture medium SH without growth regulators. Effective plantlets developed from these seeds within one week. After two weeks, leaves were taken as explants for callus induction. We found that compared to ex vitro, the in vitro leaf explants was found to be appropriate as it was responding well under in vitro conditions.

Embryo germination and plantlet formation

For germination and plantlet formation cotyledonary stage somatic embryos were transferred to SH medium supplemented with 0.5 mg/l 2,4-D + 0.5 to 3.0 mg/l BA.

Culture conditions

SH media were supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar (Himedia). After adding all the growth regulators, the pH of the medium was adjusted to 5.6 with 1 N NaOH or 1 N HCL and autoclaved at 121°C with 15 p.s.i pressure for 15-20 min. All the cultures were incubated at 25±2°C under a 16 h photoperiod. Light intensity of 40 to 50 µmolm^-2s^-1 was provided by using cool white fluorescent tubes. The cultures were transferred to fresh medium after an interval of four weeks. For each hormonal treatment 20 replicates were raised and the experiments repeated at least twice. Data on somatic embryogenesis and germination were statistically analyzed using Turkey’s HSD test at p=0.05 with SPSS ver.13.0. The results are expressed as Mean ± SE of two experiments.

Acclimatization

The plants were taken out from the cultured tubes and washed with sterile distilled water under aseptic conditions to remove agar medium. They were shifted to plastic pots containing sterile vermiculate: soil (1:1), covered with polythene bags in order to maintain 80 to 85% relative humidity and kept in culture room for 3 weeks. Later, they were transferred to earthen pots containing garden soil and maintained in the research field.

The in vitro leaf explants were spliced at the terminal ends using scalpels and inoculated on SH medium containing different concentrations of 2,4-D (0.5 to 3.0 mg/l) in combination with Kn (0.5 mg/l). Highly differentiated, friable callus was induced from these explants in one week (Figure 1a). Within 10 to 15 days of culture inoculation greenish friable callus was observed (Figure 1b). Green nodular embryogenic callus was noticed after three weeks of culture inoculation from these explants (Figure 1c). When the explants of embryogenic callus was cut into fragments and cultured on the same induc-tion medium for an extended period of three to four months, secondary somatic embryos with different shapes such as globular, heart, torpedo and cotyledonary embryoids were observed after four to six weeks of culture inoculation (Figure 2).

After elongation, the in vitro regenerated shoots were transferred onto rooting media containing IBA (0.5 to 2.0 mg/L). The highest rooting (90%) was noted on SH medium containing 1.0 mg/L IBA with average number of roots (6.38 ± 0.687) (Table 3, Figure 4c). Increasing or decreasing the concentrations of IBA resulted in lower rooting. Later, these in vitro regenerated plantlets were transferred to plastic pots containing sterile vermiculite: soil (1:1) mixture. Finally, they were shifted to earthen pots after hardening in the culture room and maintained in the research field under shady conditions. The survival percentage of plants was found to be 70 to 80%. The plants were normal; morphological and floral characters were found to be similar to the donor plants (Figure 4d).

In the present investigation, the results on somatic embryogenesis have shown that auxin, such as 2,4-D along with cytokinin BA are essential for inducing the somatic embryogenesis from leaf explants of R. vesicarius. The auxin/ auxin in combination with cytokinin used in the medium can greatly influence the frequency of induction and also on maturation of somatic embryos. The requirement of cytokinin in addition to auxin was observed in Solanum surattense (Rama swamy et al., 2005) whereas, somatic embryogenesis was reported on medium containing 2,4-D alone in Capsicum annuum L. (Marla et al., 1996). Direct somatic embryogenesis was also reported by adding Kn to the medium and also the number of embryos further increased by enriching the medium with 2,4-D in leaf explants of Cicer arietinum L. (Dinesh  et al., 1994).

New gene products are needed for the progression from the globular to the heart shaped stage and these new products are synthesized only, when exogenous auxin is removed (Zimmermman 1993). But according to our observation in R. vesicarius for morphogenesis of somatic embryos, auxins and cytokinins combination is required. At higher concentration of auxin, probably the population of embryogenic cells drops due to their disruption and elongation and the embryogenic potential of the culture are lost (Aboshama, 2011). Maturation process is critical step in somatic embryogenesis. Similarly, somatic embryo maturation on MS medium containing the combination of 2,4-D and Kn was observed in Brassica oleraceae and Oryza sativa L (Siong et al., 2011; Verma et al., 2011).

For induction of in vitro somatic embryogenesis, the type of primary explants, choice of genotypes and hormonal concentration plays an important role (Josephina and Van Staden, 1990). During the present investigation it was found that the high concentration of auxin in combination with less concentration of cytokinin induced the somatic embryogenesis and maturation of somatic embryos in R. vesicarius. However, for germination of somatic embryos, low level of auxin and high concentrations of cytokinin combination is required. Secondary embryogenesis observed in R. vesicarius has great potential for its mass propagation and repetitive embryogenesis can also be used for synthetic seed production and genetic transformation.

The author(s) have not declared any conflict of interest.