3.1. Sterilization strategies for in vitro studies of S. Marianum

Sterilization is the process of making explants contamination free before establishment of cultures. Various sterilization agents are used to decontaminate the tissues. These sterilants are also toxic to the plant tissues, hence proper concentration of sterilants, duration of exposing the explant to the various sterilants, the sequences of using these sterilants has to be standardized to minimize explants injury and achieve better survival [22].

Microbial contamination is a constant problem, which often compromises development of in vitro cultures [23]. These microbes compete adversely with plant tissue cultures for nutrients, and their presence often results in increased culture mortality or can also result in variable growth, tissue necrosis, reduced shoot proliferation and reduced rooting [24].

The most important step for aseptic culture establishment is sterilization of explants. The most effective way of preventing bacterial contamination in vitrois elimination of bacteria from the initial plant explants that are introduced into the culture. Successful tissue culture of all plant species depends on the removal of exogenous and endogenous contaminating microorganisms [25].

The potential explant used from S. marinum plant (the starting tissue originated from the donor plant) consist mostly of shoot tip, nodal segments, leaf, cotyledons, hypocotyls fragments, stem and root segments from in vitrogerminated seeds. Generally, younger, more rapidly growing tissue or tissue in early developmental stage are the most effective. Therefore, the initial quality of the explants will determine the success of establishment of in vitro culture of S. marianum. The criteria for a good quality explants are: normal, true to type donor plant, vigorous and disease free. Plant fragments are initiated into axenic culture from various sterilization procedures depending of the tissue used. A successful sterilization is achieved when the explant is fully decontaminated and remains viable. The surfaces of living plant materials are naturally contaminated with microorganisms from the environment, so surface sterilization of explants in chemical solutions is a critical preparation step.

Its well known that procedure of sterilization is various, depending on plant species and part taken from the plant (explant) for sterilization. Each plant material has variable surface contaminant levels, depending on the growth environment, age and part of the plant used for micropropagation [26].

The procedure of sterilization of S. marianum seeds initially involves surface disinfection of explants with ethanol followed by treatment with Clorox or mercuric chloride (HgCl2) as disinfectant and tween 20 as surfactant. In most sterilization procedures of S. marianum seeds, ethanol at concentration of 70% was used. Time of seed exposure to ethanol was varied from 30 s. to 5 min. Commercial Clorox at concentration ranged from 2.0 to 50% was used depending on concentration of sodium hypochlorite in the solution. Time of seed exposure to sodium hypochloride varied from 5 to 20 min. However, in some cases, mercuric chloride (HgCl2) was used instead of sodium hypochlorite [19], [27], [28], [29], [30]. The most concentration of mercuric chloride used was 0.1%, while time of exposure to mercuric chloride ranged from 2 to 10 min.

Some workers imbibed seeds (before treatment with ethanol and Clorrox) in distilled water for 24 h [31] or for 90 min at 37 °C [32]. However, soaked seeds for 24 h in GA3 (3 mg/l) after treatment with ethanol and Clorox [27].

Many authors collected the field-grown plants and subjected it to sterilization process. When explants material is sourced directly from field grown plants the problem of contamination is further exacerbated which requires special procedure for successful removing of plant pathogens. In this respect, sterilized the whole wild plants were achieved by cetrimide (2%) for 15 min followed by streptomycin sulphate (0.5%) followed by solution of bavistin (1%) for 30 min [27]. The whole wild plants was sterilized using ethanol (70%) for 1 min followed by HgCI2 (0.2%) for 2 min [19]. Concerning explants excised from filed grown plants, young leaves from filed grown plants was separated and treated with sodium hypochlorite (0.5%) with few drops of tween 20 (0.05%) for 5–10 min. [33]. Explants excised from field plants were sterilized using HgCI2(0.1%) for 5 min followed by Alcohol (70%) for 1 min[27].

After sterilization, seeds were cultured in MS medium (Full or half strength) for germination. Most studies incubated the growing seeds in growth room at 25 °C under 16 h photoperiod or in darkness [31], [32], [34], [35].

3.2. Callus cultures of S. Marianum

The first S. marianum callus culture was reported using leaves explants [33]cultured in SH medium [36] supplemented with 0.05 mg/l benzyladenine (BA) and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (2,4-D). First isolation of S. marianum protoplasts were from leaf calli [37]. It was found that division frequency was 35.4% and no shoot differentiation occurred. However, mesophyll protoplasts were isolated from young leaves of six lines of S. marianum. It was reported that with a protoplast population density of 1 × 105/ml, division frequencies of about 75% were obtained. After these pioneers, many researchers have tried to induce and maintain successfully S. marianum cultures [14].

Different plant growth regulators have been studied for induction of callus cultures from S. marianum. Auxins, 1-naphtalenacetic acid (NAA) or 2,4-dichlorophenoxiacetic acid (2,4-D) or IBA or IAA and cytokinins, kinetin (Kin) or benzyladenine (BA) or Zeatin have been used, alone or in combination to induce callus formation from different plant explants. According to literature (Table 2), 2,4-D at levels ranged from 0.25 to 4.0 mg/l was the most auxins used alone [28]or in combination with Kin [13], [35] or BA [30], [32], [33]. BA at levels ranged from 0.05 to 5 mg/l was the most cytokinin used alone [19] or in combination with 2,4-D [30], [33] or NAA [27], [37]. Callus cultures from hypocotyls segments of 10 day-old S. marianum seedlings were obtained. Callus appeared after a month of culture on MS-medium supplemented with 1 mg/l 2, 4-D, 0.5 mg/l BA and solidified with 10 g/agar in darkness. Cell suspensions were established from 3 month-old undifferentiated hypocotyls callus in the same medium as above without agar [39].

The best medium for callus initiation from root explants was MS-medium supplemented with NAA (0.5 mg/l), BA (0.5 mg/l) and 2,4-D (0.5 mg/l). Green, friable and rhizogenic callus was observed under light conditions. However, leaf and root explants were tested on MS medium with different growth regulators for callus initiation. All calluses were induced from cut edges of leaf and root explants after one week of incubation. Callus development was observed after three weeks of culturing. The callus was either white and green or pale and brown or compact in appearance according to the type and concentration of growth regulators added to the culture medium [30].

Recently, for callus induction, cotyledons explants were cultured on solidified MS medium containing 5.0 mg/l Kin and 0.5 mg/l IAA [38]. Callus cultures were obtained after five weeks of incubation in darkness and the cultures were subcultured every 4 weeks on fresh medium for callus proliferation. The best medium which produced the greatest callus growth consisted of 0.25 mg/l 2,4-D, 0.05 mg/l BAP, 50 mg/l asparagines and 50 mg/l Inositol. Callus growth rates varied with the concentration of 2,4-D. Callus water content was strongly influenced by media composition in a time dependent manner. Also, callus volumes grown in high 2,4-D concentration combined with intermediate concentrations of asparagines, BAP and inositol produced the maximum volume after 21 d [32].

3.3. Root cultures of S. Marianum

Production of secondary metabolites using biotechnological approaches have been established through root cultures where, the undifferentiated cultures are not able to produce these compounds efficiently as compare to the root cultures [41]. Due to biochemical and genetic stability and high biomass production, root cultures considered an efficient means for production of valuable chemicals in many medicinal plants [42].

Hairy roots have attractive attention for production of secondary metabolite as it is stable, grow faster and have the same or greater biosynthetic capacity to produce the secondary metabolites compared to plant cell cultures and mother plants [43]. But the transformed hairy roots cultures produce opine like substances which are lethal to mammalian cells and not always accepted [44]. Therefore, all these observations attracted attention for many authors and have led scientists to use cultures of organs such as adventitious root culture for the production of secondary metabolites.

In S. marianum plant, different authors succeeded in producing root cultures for production of silymarin (Table 2). Root regeneration was obtained from in vitroculture of S. marianum. It was reported that better root was observed in leaf explants (2–4 mm) grown on MS solid medium containing combination of NAA (2 mg/l) and KIN (0.2 mg/l) [28]. Adventitious root cultures were obtained from young shoots cultured on MS medium supplemented with 0.1 mg/l IBA and 0.1 mg/l NAA. The produced roots were transferred to liquid medium and subjected to different physical elicitors for optimization of silymarin production [45].

Adventitious root cultures of S. marianum have been established from small segments of roots (2 cm), obtained from in vitro grown plantlets. MS liquid medium supplemented with 2 mg/l IBA was the best for initiation of adventitious root. However, adventitious roots cultures have more ability to detoxify the DPPH free radicals more than the callus cultures [30].

Thus, it is obvious that leaves, young shoots and roots were used as explants for initiation of root cultures, IBA in low concentration (0.1 to 2 mg/l) recommended to induction of roots. In this respect, it was reported that IBA at concentration 1 mg/l was suitable for the adventitious root induction in Hypericum perforatum [46].

3.4. Regeneration of S. Marianum

A successful plant regeneration protocol depends on (requires) suitable choice of plant genotype, explants type, age of explants, medium formulation, and definite growth regulators. However, physical factors which include temperature, humidity and light/dark regime are also effective. An array of research work has been achieved to explore protocols for in vitro regeneration of plants from S. marianum (Table 2).

For establishment of regeneration protocol, different explants were used (Leaves, hypocotyls, nodal segment, shoot tips), all these explants were excised from in vitro germinated sterilized seeds or from wild-grown plants [19], [27]. In some cases callus tissues was used as starting material for induction of organogenesis or somatic embryogenesis. According to literature, it could be observed that the auxin NAA at levels ranged from 0.1 to 2.0 mg/l was the most auxins used for shoot culture initiation, multiplication and rooting stages. BA at levels ranged from 0.25 to 5 mg/l was the most cytokinin used either alone or in combination with NAA in particular for initiation of shoot cultures (Table 2).

Hypocotyls calli were induced on MS medium with NAA (0.8 mg/l) and BA (0.5 mg/l). Two months after transferring calli to D6 medium, resulted in regeneration of shoots from the surface of calli. The frequency of shoot differentiation was 75%. On a MS-medium containing NAA (0.5 mg/l) and IBA (0.1 mg/l), whole plants with healthy root were obtained [37]. Mesophyll protoplasts were isolated from young leaves of six lines of S. marianum. Plant regeneration with the protocalluses on medium containing BAP led to shoot formation in only two lines. However, when the protocalluses from line M2 were treated with thidiazuron (TDZ) in a first culture step and with BAP in a second step, the shoot formation frequency rose to 22%. Shoots were rooted on hormone free MS agar medium and transferred into soil where plants grew to maturity. Similar results were obtained when protoplasts of the line M2, isolated from a suspension culture [14].

The conditions for the regeneration of S. marianum from leaf, shoot apex and nodal segments explants were reported. Indirect organogenesis was occurred with all explants used on MS medium with 0.1 mg/l NAA, 0.3 mg/l BAP and 0.3 mg/l zeatin. In these medium callus tissues was formed and upon transfer to MS medium containing 0.1 mg/l NAA and 0.5 mg/l zeatin, the callus differentiated multiple shoots followed by rooting. Direct organogenesis was occurred with nodal explants only on MS medium with IAA (0.1 mg/l) and Kin (0.5 mg/l). The produced shoots were rooted on MS medium with NAA (0.1 mg/l) and zeatin (0.5 mg/l) [27].

Callus development and shoot organogenesis of S. marianum were induced from leaf explants of wild-grown plants cultured on media supplemented with different plant growth regulators. Subsequent transfer of callogenic leaf explants onto MS medium supplemented with 2.0 mg/l GA3 and 1.0 mg/l NAA resulted in 25.5 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg/l BA and 1.0 mg/l NAA, and these shoots were rooted on a PGR-free MS basal medium [19].

Indirect organogenesis in S. marianum was studied. callus tissues from hypocotyl explants were obtained on MS medium containing 4.5 mg/l 2,4-D. Shoot initiation was obtained when callus was cultured on MS medium containing NAA at 2 mg/l and BAP at 1.5 mg/l. Shoot cultures were transferred to the shoot elongation medium (MS medium with 2 mg/l GA3). Finally shoots were rooted on MS medium containing 2 mg/l NAA [28]. Shoot tip explants from S. marianum seedlings were cultured on MS medium with BA (2.0 mg/l) developed maximum number of multiple shoots and leaves. Upon transfer of shoots to MS medium containing 1.0 mg/l BA in combination with 0.1 mg/l NAA, resulted in maximum number (25.6) of shoots per explants. The presence of IAA (0.2 mg/l) resulted in the maximum number of roots as well as highest root length, 11.0 and 2.4 cm, respectively [29]. Complete plantlets through direct organogenesis were obtained when leaf explants of S. marianum were cultured on MS medium with BA (1 mg/l) and NAA (2 mg/l). However, indirect organogenesis was occurred when leaf derived callus grown on MS medium with Kin (2 mg/l) and NAA (2 mg/l) [47].

Shoot cultures from shoot tips explants were established. The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg/l each of BA and NAA [13]. Also, nodal segment was used for shoot initiation when cultured on MS medium with Kin (0.5 mg/l) and NAA (0.1 mg/l). Multiple shoots were regenerated on MS medium with Kin (1.6 mg/l). The produced shoots were rooted on MS medium containing NAA (1.0 mg/l) [48]. Induction of direct regeneration and rooting was observed when leaf explants were grown on MS medium with IBA or NAA (1 mg/l) [30].

3.5. Hairy root cultures of S. Marianum

Neoplastic hairy root culture obtained by infection of explants with Agrobacterium rhizogenesis, a gram negative soil bacterium, which offers an efficient system for secondary metabolites production. Hairy root culture (transformed root cultures) is a type of plant tissue culture that is used for to study plant metabolic processes or to produce valuable secondary metabolites.

The hairy root phenotype is characterized by fast hormone-independent growth, lack of geotropism, lateral branching and genetic stability. The secondary metabolites produced by hairy roots arising from the infection of plant material by A. rhizogenes are the same as those usually synthesized in intact parent roots, with similar or higher yields [49]. This feature, together with genetic stability and generally rapid growth in simple media lacking phytohormones, makes them especially suitable for biochemical studies not easily undertaken with root cultures of an intact plant. During the infection process A. rhizogenes transfers a part of the DNA (transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant cells and the genes contained in this segment are expressed in the same way as the endogenous genes of the plant cells.

Unorganized plant tissue cultures are frequently unable to produce secondary metabolites at the same levels as the intact plant. The hairy root system based on inoculation with Agrobacterium rhizogenes has become popular in the two last decades as a method of producing secondary metabolites synthesized in plant roots [50].

Hairy roots are genetically and biochemically stable, the fast growing nature of hairy roots, low doubling times, biosynthetic stability, and ease of maintenance, high yield of secondary metabolites and no need of growth hormones offers an additional advantage. Moreover, transformed roots are able to regenerate whole viable plants and maintain their genetic stability during further subculturing and plant regeneration [51]. The main aim to produce hairy root culture is to find out efficient parameters for commercial production. The researchers which have been carried out in this field create enormous combinations of hairy roots and elicitors to produce high yield of secondary metabolites. In general, hairy root culture is, therefore one of the most feasible and promising approach from an industrial point of view.