Introduction

Basella rubra L. (family Basellaceae) commonly known as Indian Spinach, is mostly cultivated as leafy vegetable. It is extremely heat tolerant plant, native of tropical Asia and originated from India (Bamidele et al. 2010). It is a succulent-branched climber and the tender shoots are used as a cool season vegetable.

The fruits are the potential source of natural colourant. Anthocyanins are found responsible for different colours present in the body parts of B.rubra (Maisuthisakul and Ritthiruangdej 2008). The natural maroon colour obtained from the ripened fruit’s saps of B. rubra used in dyeing industry as natural dye in India (Ozela et al. 2007).

The dye isolated from the fruit pulp of this plant is used as an alternative to crystal violet or safranin for the staining of microbes (Mundo et al. 1995). The purple colored extract from the fruit of Basella is also used in coloring of food items.

Basella contains some unique compounds like, basella saponins, flavonoid kaempherol, ß-carotene and betacyanin betalain. (Yang et al. 2008). The leaf extract is reported to possess vital vitamins A, C, E, K, folic acid, riboflavin, niacin, thiamine and minerals like calcium, magnesium and iron. The mucilage from B.rubra has strong suspending ability along with high viscosity, and used as a good thickening agent in pharmaceutical and cosmetic industries. The gel obtained from the mucilage used as medicines for skin problems due to its antioxidant activity (Toshiyuki et al. 2001). Histochemical staining method was performed to locate phytochemicals such as alkaloid, ascorbic acid, polyphenols, tannins and terpenoids synthesized by B.rubra plant.

Basella rubra is now cultivated for its industrial purposes and naturally propagated by the stem cuttings (Cyunel 1989), this method causes the carryover of disease causing pathogens from one generation to the next (Sankar et al. 2011). The propagation through seeds has some limitations due some special requirements in germination of seeds and proper flowering in this plant (Larkcom 1991). Plant tissue culture methods can be used for rapid multiplication of improved varieties and to produce disease free and quality planting materials (Kartha and Gamborg 1975). Some reports are available on the tissue culture and anthocyanin production using B.rubra cells (Cyunel 1989; Guo and Bin 2001). However, efficient in vitro propagation protocol for this plant species is largely deficit in the literature. The histochemical and shoot induction studies by using plant growth regulators were tested and conditions were optimized for efficient shoots, roots and plant regeneration in B.rubra during present investigation of in vitro and in vivo plants and SEM-EDX elemental analysis of roots of in vitro and in vivo plants.

Material and Methods

Collection of Plant Material and Preparation of Explants

Basella rubra plants were collected from the green house, National college, Tiruchirapalli, Tamilnadu, India. Fresh shoot sprouts were used as explants. The stems were cut into 3–4 cm long segments with 1–2 nodes. The explants were first treated with 0.1 % (w/v) Bavistin (a systemic fungicide) for 5 min, and surface sterilized with 0.1 % HgCl₂ (w/v) for 3 min with alternate shaking followed by washing with autoclaved distilled water for 3 times under aseptic conditions in a laminar air flow cabinet.

Establishment of culture

The sterilized nodal segment were inoculated on agar gelled (0.8 % agar) MS medium (Murashige and Skoog 1962) supplemented with IAA (Indole 3 acetic acid) ranging from 0.5 to 3.0 mg/l,  NAA (α-Naphthalene acetic acid) ranging from 0.5 to 2.0 mg/l and GA3 (Gibberellic acid) ranging from 0.5 to 2.0 mg/l for shoot regeneration. MS + IBA (Indole -3- butyric acid) ranging from 0.5 to 3.0 mg/l for root induction, MS + IBA ranging from 0.5 to 3.0 mg/l and GA3 ranging from 0.5 to 3.0 + IAA 0.5 to 3.0 mg/l for plant regeneration. Explants with single node were inoculated vertically in each culture tubes for bud breaking from the nodal meristem. Cultures were incubated at 1500 lux light with 12:12 hr light: dark photo period at 25 ± 2°C.

Multiplication of shoots in cultures

The shoots were multiplied by two methods (i) the original explants were repetitively transferred to fresh medium for 2–3 passages after harvesting in vitro raised shoots, and (ii) the in vitro produced shoots were cut into 2cm long segments (each with one node) and subcultured on fresh MS medium. The culture medium used for shoot multiplication was incorporated with various concentrations and combinations of IAA + IBA + NAA + GA3. The multiple shoots were regularly subcultured on fresh medium after an interval of 4–5 weeks.

Effect of auxins on rooting of shoots (in vitro)

2cm shoots were harvested individually and transferred to MS medium containing different concentrations of IBA + BAP. The cultures were incubated (1–2 days) under diffused light (800 lux) conditions and after that under the normal conditions (at 1500 lux).

Plantlet regeneration (in vitro)

The rooted shoots were transferred to MS + IBA + GA3 + IAA at various concentrations, incubated at 1500 lux light and plantlets were established in in vitro and were selected for hardening process after 45 days of culture.

Hardening of regenerated plantlets

The regenerated (in vitro) plantlets were rinsed with water, kept on a moist filter paper and transplanted immediately to the containers containing autoclaved soilrite. These plantlets were moistened with solution of one-fourth MS basal salt and shifted to greenhouse for the hardening of plantlets. After 30–35 days, the plantlets were transferred to nursery polybags and pots having perforation at in the bottom, containing potting mixture of sand, soilrite, garden soil and organic manure (1:1:1:1).

Comparative Histochemical studies of stem anatomy

Histochemical studies were conducted to compare the in vivo and in vitro plant stem anatomy for the phytochemicals like alkaloids, ascorbic acid, phenolic compounds, tannin and terpenoids. The thin hand sections were stained in different staining agents as per the procedure (Shanmugam et.al 2010). Photomicrographs were taken in the digital camera mounted on the Microscope Olympus, Magcam DC 10 with magnification 40X.

Results and Discussion

The Table-1 reveals the comparative histochemical studies of B.rubra stem anatomy of in vivo and in vitro plants.

The histochemical assay revealed some difference in the phytochemical constitutions between in vivo and in vitro plants. The presence of alkaloids is higher in in vivo plants, especially higher in epidermis hypodermis external cortex and vascular region. Ascorbic acid has shown no difference between in vivo and in vitro histochemistry. Polyphenols are higher in in vivo plant and located in external cortex and vascular region. The concentrations of tannin are identical in in vivo and in vitro plants. Terpenoids are present in higher concentration in in vivo plants especially in hypodermis and vascular regions (Fig-1 and 2). These results are comparable with the previous work of Toshiyuki et.al (2001), Ozela et.al (2007), Adenegan and Akinnubi (2015), Kumar et.al (2015a) and Kumar et.al (2015b).

The present work is aimed at the in vitro shoot induction studies of B.rubra. It has been proved that the surface sterilization of explants is very essential for establishment, pathogen free as well as optimum induction of callus and organogenesis in vitro. In the present study mercuric chloride was used to sterilize the explants. The appropriate concentration and time for surface sterilization is 1.0% mercuric chloride for 3 minutes for the stem.

Table 1: Phytochemical analysis of in vivo and in vitro B.rubra.L plant stem (anatomy) by histochemical studies.

Figure 1: In vivo Basella rubra stem anatomical section used for Histochemical studies.   Click here to view figure

Figure 2: In vitro Basella rubra stem anatomical section used for Histochemical studies.   Click here to view figure

Shoot Regeneration

In the present study following media concentration and combination were used for root and shoot induction. The following concentration and combination of hormones are used:

MS + IBA 1.0 mg/l + NAA 0.5 mg/l + IAA 1.0 mg/l

MS + IBA 2.0 mg/l + NAA 0.5 mg/l + GA3 0.5 mg/l

MS + IBA 3.0 mg/l + IAA 1.0 mg/l

Shoot induction are initiated in 10 days the other hormones like IAA, NAA have shown but much organogenesis result after inoculation on MS medium supplemented with different concentration and combination of above mentioned hormones and shoot regeneration in 20 days further growth in 30, 40 and 60 days. Among the various combinations, 100% shoot regeneration in concentration of hormones such as MS + BA (1.0 mg/l) produced multiple shoot, in addition to this Kin (0.5, 1.0 mg/l) in the presence of BA induced more number of multiple growths in Basella rubra. The present study reveals that MS+BAP (1.0 mg/l), NAA (0.5 mg/l), IAA (1.0 mg/l) as well as MS+BAP (1.0 mg/l), NAA (0.5mg/l) and GA₃ (0.5 mg/l) produced shoot regeneration in Basella rubra. The number of shoots produced in this study was on par with earlier reports by Guo and Bin (2001), Pumchaosuan and Wongroung (2009) with reference to B.alba and B.rubra.

Root Regeneration

In the present study following media concentration and combination were used for root induction:

MS + IBA 1.0 mg/l, MS + BAP 1.0 mg/l

MS + IBA 3.0 mg/l

Root induction are formed in 15 days after inoculation on MS medium supplemented with different concentration and combination of above mentioned hormones and further growth occurred in 30, 40, 60 days (Fig-4 a to d). Among the different combination MS + IBA 1.0 mg/l induced root induction in B.rubra. The worked carried out by Guo and Bin (2001), Pumchaosuan and Wongroung (2009), Shekhawat and Manokari 2016 support the present findings.

Plantlet Regeneration

In the present study following media concentration and combination were used for plant regeneration induction:

MS + IBA 0.5 mg/l + GA3 0.5 mg/l + IAA 0.5 mg/l,

MS + IBA 0.5 mg/l + GA3 1.0 mg/l + IAA 1.0 mg/l and

MS + IBA 0.5 mg/l + GA3 1.5 mg/l + IAA 1.0 mg/l.

Plant regeneration is maximum in the concentration MS + IBA 0.5mg/l + GA3 1.5 mg/l + IAA 1.0 mg/l (Fig-3) According to Hong et.al (2003) propagation of B.rubra by stem cutting into MS + BAP 1.0 mg/l + NAA 0.5 mg/l resulted good response the present work reveals that MS + IBA 1.0 mg/l combination has better response to plant regeneration in B.rubra These results are comparable with the work of Guo and Bin (2001), Tanikan Pumchaosuan and Sasitorn Wongroung (2009) and Shekhawat and Manokari (2016).

Protocol for Basella Rubra Stem Explants In Vitro Propagation

The selected stem explants were brought to the laboratory and kept in refrigerator for two days.

Surface sterilization with 75% alcohol for 50 seconds and 0.1% HgCl₂ for 3 minutes.

Washed thrice in double distilled water.

Shoot induction with MS + IBA 1.0 mg/l + NAA 0.5 mg/l + IAA 1.0 mg/l.

(Or)

MS + BAP 1.0 mg/l + NAA 0.5 mg/l + GA3 0.5 mg/l.

Root induction with MS IBA 1.0 mg/l + IBA 2.0 mg/l.

Plant regeneration with MS + IBA 0.5 mg/l + GA3 1.5 mg/l +IAA 1.0mg/l

Incubated in light 1500 Lux at 25°C ± 2°C.

Figure 3: Basella rubra stem explants in vitro propagation.   Click here to view figure

Nutrient Uptake Studies by In Vivo and In Vitro Same Plants by SEM- EDX Analysis

The SEM-EDX elemental analysis of in vivo from B.rubra have shown the elements carbon, oxygen, sodium, magnesium, silica, phosphorus, sulphur, chlorine and potassium. The concentration of potassium was 5.82%. The in vitro plants have shown carbon, oxygen, magnesium, silica, chlorine, potassium, calcium and iron (Table – 2). The result has shown that there is a significance difference in the absorption of nutrients. The in vivo plant absorbs sodium, phosphorus and sulphur. Whereas the in vitro plants are capable of absorb calcium and iron from the medium (Fig-4). The present study of nutrient uptake by SEM-EDX analyses is a tool to identify selective absorption of minerals by in vitro and in vivo plants as reported by Abubacker and Sathya (2017).

Table 2

Figure 4: SEM – EDX Elemental analysis of In vitro Basella rubra L.   Click here to view figure

Figure 5: SEM–EDX Elemental analysis of In vitro Basella rubra L.   Click here to view figure

Conclusion

Due to over exploitation of plant source from natural environment for medical uses, the standardization and optimization of shoot regeneration protocol for B.rubra because imminent. The present histochemical and shoot induction studies would facilitate the phytochemical constituents and conservation of this important plant species.

Acknowledgements

The authors express thanks to Padmavibhushan Dr.V. Krishnamurthy, President, Sri K.Ragunathan, Secretary, Dr. K. Anbarasu, Director and Dr. R. Sundararaman Principal, National College, Tiruchirapalli for providing the infrastructure facilities, supports and encouragement given to PG and Research Department of Biotechnology to carryover the research work.

Conflict of Interest

There is no conflict of interest.

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