Somatic Embryos in Catharanthus roseus : A Scanning Electron Microscopic Study

Catharanthus roseus (L.) G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis) has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM) study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L) Murashige and Skoog (MS) was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.


Introduction Introduction Introduction Introduction
Catharanthus roseus (L.) G. Don (Madagascar periwinkle) is an important species of the Catharanthus genus, native and endemic in Madagascar. The species has long been cultivated as herbal medicine and ornamental as well (Gupta et al., 2007). In traditional medicine, the plant extracts have been used to treat numerous diseases including diabetes, malaria and Hodgkin's disorder (Mukherjee et al., 2001). The primary compounds like vinblastine and vincristine, extracted from the plant are used in the treatment of leukemia, however, in vivo grown tissues contain a very low level of drugs (0.0005% dry weight basis) ( Van der Heijden et al., 2004). Cell and molecular biological studies have currently been employed to improve alkaloid yield and, in some cases, the in vitro grown tissues showed enhanced yield of alkaloids (Junaid et al., 2009;. Several key factors controlling the biosynthesis of alkaloids have also been optimized (Moreno et al., 1995;Mujib et al., 2002;Mujib et al., 2012). In plant biotechnology, various plant parts have been used in establishing culture for plant regeneration by various methods, the information on micropropagation via somatic embryogenesis in C. roseus is however, relatively new (Dhandahpani et al., 2008;Junaid et al., 2006;. The advantages of somatic embryogenesis over other methods is its unlimited potentiality in producing clones, to be used as medicinal raw material for secondary product synthesis (Mujib and Samaj, 2006). Although there is a risk of somaclonal variation in indirect embryogenesis, particularly developed from long term culture (Hao and Deng, 2002;Mujib et al., 2013), the technique has often been used in several biotechnological researches including transgenic programs (Davletova et al., 2001;Rommens et al., 2004, Walter, 2004. Even though somatic embryogenesis plays an important role in producing somaclones in numbers (Azad et al., 2009;Wang et al., 2006;Yan et al., 2010) the ontogeny, structure, development and conversion of plantlets have not always been studied in detail, as the whole process of in vitro embryogeny is quite complex (Vooková and Kormutak, 2006). In C. roseus, somatic embryos were obtained and it closely mimics zygotic embryogenesis, i.e. by producing all forms of embryo stages (globular, heart, torpedo and cotyledonary), which finally germinated into plantlets (Junaid et al., 2006). Ultra-structural studies, involving transmission electron microscopy (TEM) and scanning electron microscopy (SEM), have often been used to investigate the ontogeny, structure and development of embryos in various studied plant materials (Bradley et al., 2001;Ovečka and Bobák, 1999), in this plant no such investigation has been carried out yet. In this communication, SEM investigation was conducted and the origin of embryo generation from callus was presented, describing the morphology/structure, the growth and development of in vitro embryos in C. roseus.

Materials Materials Materials
Materials and methods and methods and methods and methods

Plant Material
Catharanthus roseus (L.) G. Don fruits were collected from herbal garden, Jamia Hamdard (Hamdard University), New Delhi and were used as experimental material; the plant was earlier authenticated for in vitro studies (Junaid et al., 2006).

Surface sterilization and in vitro seed germination
Surface sterilization was carried out following Junaid et al. (2006) method. In brief, the seeds were isolated from fruits inside a laminar hood, treated with 0.5% mercuric chloride for 2 minutes, followed by 1.0% (w/v) H2O2 for 3 minutes, and finally rinsed with sterilized double distilled water. These surface-disinfested seeds (20-25) were placed in GA-7 Magenta vessels (Sigma, St. Louis, MI, USA) containing 50 ml MS (Murashige and Skoog, 1962) medium without organic compounds and plant growth regulators. The medium was adjusted to pH 5.7 and sterilized at 121 °C for 15 minutes.

Embryogenic callus induction
Germinating seedlings were grown until they attained a height of 2 cm, later these seedlings were removed from the culture vessels. Hypocotyls (6-8 mm) of 5 to 7 days old seedlings were excised as explants and placed on MS medium supplemented with optimized concentrations of 2, 4-D (1.0 mg/L) for the induction of callus.
The induced calluses were sub-cultured at a regular interval of 4 weeks. The callus later transformed into embryogenic callus on medium supplemented with same concentration of 2, 4-D (1.0 mg/L) or in medium amended with NAA (1.0 mg/L) and BAP (1.5 mg/L). The entire embryo induction process was described earlier and was followed (Junaid et al., 2006).

Scanning Electron Microscopy (SEM)
For SEM, the embryo bearing embryogenic callus was fixed in 2% glutaraldehyde, adjusted to pH 6.8 in 0.1M phosphate buffer for 24 h at 4 °C. The tissue was washed in buffer, postfixed for 2 h in similarly buffered 1% osmium tetroxide, dehydrated in a graded ethanol series and finally coated with gold palladium.
The prepared samples were examined and photographed in a LEO 435 VP (Zeiss, Oberkochen, Germany) scanning electron microscope, operating at 15-25 kV.

Results Results
Results and discussion and discussion and discussion and discussion

Embryogenic callus induction
Hypocotyls of in vitro germinated seeds were used as explant on 2,4-D added MS medium, which induced white to yellowish callus within 8-10 days of incubation. The callus growth was prolific, which later turned into embryogenic callus within 4 weeks of culture.
The embryogenic callus was white, granular, friable, fast growing, and started to differentiate somatic embryos (Figs. 1A and 1B) within 4 -6 weeks of culture.
The importance of auxins especially 2,4-D in inducing callus and in triggering embryogenesis has been reported in a number of studied plants (Gaj, 2004;. In the case of this plant as well, 2,4-D was very efficient in producing callus and embryos; and this synthetic auxin was earlier observed to be very important in several investigated plants during callus induction and early embryogenesis stages (Pasternak et al., 2002;Su et al., 2009).
SEM study of embryo bearing embryogenic callus revealed different forms of somatic embryos appeared on callus surfaces that are presented in Fig. 2 (A-E). The embryos were developed as a heterogeneous mixture, where globular embryos were low in number. The somatic embryos were smooth surfaced, often clustered together in a common mass and could not easily be detached from the parental tissue. SEM of callus also revealed that several somatic embryos were long cylindrical structures devoid of unique notches with constricted base firmly attached to callus ( Figs. 2A and 2C).
Heart shaped embryos were maximum in number with typical notch at the tip (Fig. 3 A-D).
In several embryogenic cultures, secondary embryos were also formed at basal end of heart-and torpedo embryos. Torpedoes were less in number, compared to the cotyledonary embryos. The cotyledonary embryos showed distinct shoot end with well-defined cotyledonary leaf primordia (flanked at the two sides) and root axis (Fig. 3 E). Beside embryos' normal forms and structure, SEM study also revealed embryos of various aberrant morphological types (not shown).
Somatic embryos obtained from in vitro grown tissues had been reported in a number of plant genera (Bajaj, 1995;Mujib and Samaj, 2006;Pasternak et al., 2002;Raghavan, 1986;Thorpe, 1995) but the incident has been relatively new in C. roseus.
The formation of somatic embryos is one of the important methods of in vitro regeneration in C. roseus and having the potential of producing unlimited clones with functional root and shoot system in contrast to organogenesis where shoots and roots were induced separately on two different media. The culture of plant tissues via somatic embryos on a large scale presented the possibility of production of synthetically coated seeds/synthetic seed (Gray et al., 1995;Mehpara et al., 2012;Rai et al., 2009).
In the present study, embryogenic callus induced from hypocotyl of in vitro germinated seeds were used for SEM study, which helped describe embryo morphology and structure more efficiently; it also indicates about embryos' origin i.e. of being single cell or from pro-embryogenic mass of cells.
The present study agreed with previous similar observation, reported by other workers (Brown et al., 1995;Corredoira et al., 2003). Schlogl et al. (2012) reported that the embryogenic program expression could subsequently be monitored on transformation of pro-embryogenic clusters into first visible globular embryos. Rodríguez et al. (1995) studied different somatic embryonic stages (globular, heart, torpedo and cotyledonary) in sugarcane by SEM, in which globular embryos, with lateral notch and scutellum, were observed. In this study, advanced somatic embryos appeared to have well organized shoot meristem with root primordial ends, which later turned into leaves and roots respectively. The SEM of safflower (Carthamus tinctorius L.) also described the morphology of globular to cotyledonary embryos more efficiently and the embryo development was noted to be asynchronous in nature (Mandal et al., 2006). In Drosera spathulata (Bobak et al., 2006) and Actinidia deliciosa, SEM was used to study different developing stages of somatic embryos (Popielarska, 2006). Recently, SEM was conducted in Heliconia chartacea for studying embryogenic callus, developed in vitro (Ulisses et al., 2010). Therefore, the application of SEM along with TEM, histology and morphological investigation are very important and efficient in understanding embryo forms and in achieving successes following post embryo germination processes.

Conclusions Conclusions Conclusions Conclusions
The present work describes the ontogeny and structures of somatic embryo in C. roseus through SEM. It helps in revealing origin, development of advanced embryo from its early stage. The SEM-observed embryo types, normal progression towards maturity or even morphologically aberrant (often-observed) somatic embryos will determine the success of in vitro plant regeneration, to be used as an alternative and sustainable method of propagation.