Acta Physiologiae Plantarum

, Volume 34, Issue 5, pp 1987–1996 | Cite as

In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Willd: a valuable medicinal herb

Original Paper

Abstract

Talinum triangulare is a medicinally important herb and various parts of the plant are used pharmaceutically for the treatment of different diseases. In our study, a rapid and efficient protocol for micropropagation has been developed from shoot tip and nodal explants of T. triangulare. High shooting frequency (93.33 %) was achieved with shoot tip explants when cultured on Murashige and Skoog’s (MS) medium supplemented with 1.0 mg/L 6-benzyl amino purine (BAP) producing an average of 12.50 ± 0.23 shoots and 5.07 ± 0.02 cm shoot length per explant. A combination of 0.5 mg/L BAP and 0.5 mg/L kinetin was found to be more effective by producing 15.67 ± 0.25 shoots and 6.22 ± 0.02 cm shoot length per explant. The microshoots were excised and cultured on half-strength MS and full-strength MS medium containing different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA) for root induction. More number of roots (45.10 ± 0.96) with an average length of 5.46 ± 0.08 cm was obtained on half-strength MS medium supplemented with 0.5 mg/L IBA. The rooted shoots were successfully transplanted from different planting substrates to the field with a 100 % survival rate. Random amplified polymorphic DNA analysis was carried out using four random decamer primers. The amplification products were monomorphic in the micropropagated plants and were similar to the mother plant. Absence of polymorphism revealed that no variation was induced, thus maintaining the genetic integrity of the micropropagated plants of T. triangulare.

Keywords

Waterleaf Portulacaceae Micropropagation MS medium RAPD fingerprinting 

Introduction

Talinumtriangulare (Jacq.) Willd is a herbaceous perennial herb belonging to the family Portulacaceae. It is popularly known as Waterleaf because of its high moisture content of almost 90.8 % per 100 gm of edible leaf (Fontem and Schippers 2004). T. triangulare is a caulescent and glabrous plant growing to a height of 30–60 cm and is characterized by its terminal cyme on a triangular stalk. The plant is widely cultivated in the humid tropical regions as a leaf vegetable (Ezekwe et al. 2002), especially in West Africa, Asia and South America. T. triangulare, an herb with fleshy green leaves, succulent stem and pink flowers (Keay 1981) was introduced into South India from Sri Lanka and is cultivated in Tamil Nadu as Ceylon Spinach for its edible leaves (Anonymous 1976).

The family Portulacaceae is characterized by the occurrence of betalain and its production in in vitro cultures is utilized as a natural colorant in food industries, pharmaceuticals and cosmetics (Leathers et al. 1992). Betalain has gained interest because of its antifungal (Kimler 1975) and antioxidant activity (Escribano et al. 1998). Preliminary phytochemical studies on T. triangulare revealed that the plant is rich in crude protein, total lipids, essential oils, omega-3-fatty acids, cardiac glycosides, polyphenols (Sridhar and Lakshminarayana 1993) and flavonoids (Andarwulan et al. 2010). The leaves of the plant have been implicated medically in the management of cardiovascular diseases like stroke and obesity (Adewunmi and Sofowora 1980). They are used in folkloric medicine to treat polyuria (Khare 2007), internal heat, measles (Fontem and Schippers 2004) and cancer (Liang et al. 2011). In India, diabetics and invalids use the leaves as a substitute for Amaranthusgangeticus Linn. (Khare 2007). The leaf extracts of T. triangulare have been proved to possess remarkable antioxidant (Liang et al. 2011) and hepato-protective activities (Adefolaju et al. 2009; Liang et al. 2011).

Conventional methods for the propagation of T. triangulare through waterleaf cuttings and seeds are unreliable due to seasonal constraints, dormancy and poor germination under natural conditions (Agble 1970; Fawusi 1979; Nwoke 1982). Hence, an alternative tool for the large-scale production of this valuable herb is necessary. An efficient micropropagation system is required for clonal propagation, germplasm conservation and genetic improvement (Pattnaik and Chand 1996) to satisfy the pharmaceutical demand of this plant. In vitro multiplication to produce complete plantlets from axillary buds has been reported in Talinum portulacifolium L. (Thangavel et al. 2008). However, to date, there are no reports on in vitro studies of T. triangulare. Thus, the current study aims to develop a highly efficient protocol for micropropagation and establishment of complete plantlet from shoot tip and nodal explants. Subsequently, the most effective hormonal concentration for root induction and ex vitro hardening was also determined. The genetic integrity of the in vitro regenerated plants was checked using random amplified polymorphic DNA (RAPD) analysis.

Materials and methods

Plant material and explant preparation

Talinum triangulare plants were collected from the medicinal plant garden at Irula Tribal Women’s Welfare Society, Chengalpattu, Chennai, India. Botanical identification was performed by Dr. D. Narasimhan, Department of Botany, Madras Christian College, where the voucher specimen has been deposited. The plants grown in the garden were regularly cut so that it gave rise to the emergence of new shoots. Ten days after the cuttings, these young and fresh shoots 3–4 cm in length, which were light green in color were used as explants. Such shoots gave better response and showed higher percentage of sterility. Surface sterilization of shoot tip and nodal explants was performed by washing the explants in running tap water for 15 min. It was then washed with 1 % (v/v) Triton X-100 detergent solution and rinsed thoroughly with sterile water. The explants were disinfected by soaking in 0.2 % (w/v) bavistin (Bengard fungicide) for 5 min and washed three times with sterile distilled water. Surface decontamination was performed by immersing shoot tips and nodal explants in 0.2 % (w/v) mercuric chloride (Hi-Media, Mumbai, India) for 3 min and washed four times with sterile distilled water. Shoot tips and single node explants of 10–12 mm were used as the explants for micropropagation.

Medium and culture conditions

Murashige and Skoog’s (1962) basal medium was used throughout this study. This was prepared by adding 3 % (w/v) sucrose (Hi-Media, Mumbai, India) and different concentrations of plant growth regulators (PGRs) to MS basal salts and vitamins. MS medium devoid of growth regulators served as control. The pH of the medium was adjusted to 5.7 before adding 0.8 % (w/v) agar (Hi-Media, Mumbai, India). Molten medium (10 mL) was dispensed into each test tube (Borosil), plugged with non absorbent cotton plugs and autoclaved at 121 °C for 15 min. The cultures were incubated at 24 ± 2 °C in light with 16-h photoperiod supplied by white fluorescent tube lights (Phillips, India), with a light intensity of 50 μmol m−2 s−1 photosynthetic photon flux density (PPFD).

Multiple shoot induction and proliferation

Shoot tips and nodal explants of T. triangulare were inoculated on MS medium supplemented with PGRs such as 6-benzyl amino purine (BAP) and kinetin (KIN) (0.25, 0.5, 1.0, 2.0 and 4.0 mg/L) individually, in combination with α-naphthalene acetic acid (NAA) (0.05 mg/L) and equal concentration of both the cytokinins were used in this study. The shoots were subcultured three times at 2-week intervals onto their respective medium. A two-week subculture procedure was carried out to avoid extensive pink coloration in the medium. Total number of shoots per explant and the length of the shoots were measured after 6 weeks of culture.

Root induction

The proliferated shoots (2–3 cm) from the culture bottles were separated, excised and inoculated for root initiation. The root induction medium consisted of half-strength MS and full-strength MS medium containing 3 % sucrose (w/v) to determine the effect of MS salt concentration on root induction. The medium was then supplemented with various concentrations (0.1, 0.5, 1.0, 2.0 and 3.0 mg/L) of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) individually. After 4 weeks of culture, the total number of roots produced per shoot and length of the roots were measured.

Acclimatization

Rooted plants (4–5 cm) were carefully removed from the test tubes, washed with sterile water to remove agar media and placed in the 1/4 strength MS basal liquid medium with 3 % sucrose (w/v). This helped in further elongation of the shoots and broadening of the leaves. After 2 weeks, they were transferred to paper cups (60 mm in diameter and 110 mm high) filled with sterilized garden soil and sand (1:1), coco pith and soil (3:1) and saw dust and soil (3:1) (w/w). It was supplied regularly with 1/4 strength MS basal salt solution devoid of sucrose to ensure that the plant obtained its required nutrients and prevented fungal contamination. The plants were covered with transparent polythene bags (10 × 8 cm) to prevent desiccation and maintain high humidity. These plants were maintained in the culture room at 24 ± 2 °C and 16 h photoperiod with a light intensity of 50 μmol m−2 s−1 PPFD. The relative humidity was gradually reduced and after 4 weeks the plants were removed from the cups and transferred to pots filled with 1:1 mixture of garden soil and farmyard manure (w/w). The acclimatized plants were successfully transferred and established in the field. The survival percentage of ex vitro plants was evaluated after 8 weeks and any variation in morphological characters was noted.

Data collection and statistical analysis

All experiments were performed with a minimum of ten explants per treatment and each experiment was repeated three times. The data were collected after 6 weeks (subcultured thrice at 2-week intervals) for shoot multiplication and 4 weeks for rooting experiments. For shoot proliferation, the mean (%) of explants responding, mean number of shoots per explant and shoot length were measured. In root induction, mean number of roots and root length were recorded. The data were analyzed statistically using IBM SPSS statistics 19 (SPSS Inc., Chicago, USA) and the mean values were expressed as mean ± SE of three experiments. The significance of differences among means was carried out at 5 % probability level using Duncan’s Multiple Range Test (DMRT).

DNA isolation and RAPD fingerprinting

Random amplified polymorphic DNA analysis was performed to check the genetic fidelity of the in vitro regenerated plants in the field (ex vitro: 3 months old). Five randomly selected plants obtained from different PGRs treatments were analyzed by four different primers. Total genomic DNA was isolated from young leaves of mother plant (in vivo grown: 3 months old) and ex vitro plants of T. triangulare using SDS method (Dellaporta et al. 1983) for RAPD analysis. Purity of DNA was checked by electrophoresis in 0.8 % (w/v) agarose gel. The concentration of DNA was determined by ultraviolet–visible spectrophotometer (Systronics, India). Four random decamer oligonucleotide primers (OPE 2, OPE 8, OPK 14, OPK 15) were used for the PCR reactions. The PCR was carried out in a volume of 10 μL reaction mixture consisting of 5 μL Ampliqon Taq DNA Polymerase Master Mix RED (2.0X—Ampliqon, Denmark), 0.8 μL template DNA (10 ng), 0.6 μL random primer (10 ng) and 3.6 μL sterile DNase-free water. The amplification was carried out in a DNA thermal cycler (Mastercycler gradient, Eppendorf, Germany). The PCR was performed at an initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 36 °C for 1 min and extension at 72 °C for 2 min with a final extension at 72 °C for 7 min. After amplification, the PCR product was resolved by electrophoresis in 1.8 % (w/v) agarose gel with 1× Tris–Acetic acid–EDTA (TAE) buffer. Lambda DNA/Hind III Plus marker (0.56–23 kb DNA ladder) was used as DNA marker. The amplified fragments were visualized under UV light and documented using the Gel Documentation equipment (UVP, Ultra-Violet Products Ltd, UK). DNA fingerprinting profiles were compared to evaluate clonal fidelity and genetic stability of ex vitro plants. PCR reactions were repeated at least twice to confirm the reproducibility of the results.

Results and discussion

Multiple shoot induction and proliferation

Multiple shoots were successfully induced from 10-day old in vivo shoot tip and nodal explants of T. triangulare. Best response toward multiple shoot regeneration was observed from shoot tips (ranging from 66.67–96.67 %) than nodal segments (ranging from 40–70 %). In all hormonal combinations, the young leaves turned dark pink in color (Fig. 1). This was due to the production of the betalain pigment, which is the characteristic of the Portulacaceae family and other members belonging to the flowering plant order Caryophyllales (Cronquist 1981). Betalain biosynthesis is subject to complex regulation, and the pigments accumulate only in certain tissues and at specific stages of development. Their synthesis has been shown to be regulated by various factors such as light and growth regulators (Biddington and Thomas 1973; Kochhar et al. 1981).
Fig. 1

Micropropagation and regeneration from shoot tip explants of Talinumtriangulare. a Shoot tip explant (Bar 0.5 cm). b Shoot multiplication on MS medium supplemented with 1.0 mg/L BAP (Bar 0.5 cm), c 2.0 mg/L KIN (Bar 0.5 cm) and d 0.5 mg/L BAP + 0.5 mg/L KIN (Bar 0.5 cm), 6 weeks after initiation. e Rooting of shoots on half-strength MS medium with IBA (0.5 mg/L) after 4 weeks (Bar 1.0 cm). f Plantlet placed in 1/4 strength MS liquid basal medium (Bar 1.0 cm). g Acclimatization of plantlet in culture room (Bar 3.0 cm). h Hardening of plant in coco pith: soil (3:1) with 1/4 strength MS basal salt solution (w/o sucrose) (Bar 1.5 cm). i Ex vitro plant in pot bearing the first flower, 7 weeks after successful establishment under natural field conditions (Bar 2.5 cm)

The type and concentration of cytokinin influenced the average number of shoots per explant as well as the mean length of shoots. When shoot tip explants were used, good response (93.33 %) was observed in MS medium supplemented with 1.0 mg/L BAP alone producing an average of 12.50 ± 0.23 shoots and 5.07 ± 0.02 cm shoot length per culture (Fig. 1b; Table 1). When KIN alone (2.0 mg/L) was added to the medium, the response (86.67 %) was lower when compared to BAP (Table 1). There was also formation of white and thin roots when KIN was used (Fig. 1c). Similar results of KIN inducing root formation have been reported in Eclipta alba by Franca et al. (1995) and Baskaran and Jayabalan (2005). Cytokinins, especially BAP, have been reported to overcome apical dominance and promote shoot formation (George 1993). It has also been reported that, of the individual cytokinins, BAP was more efficient than KIN in inducing multiple shoot formation (Chishti et al. 2006). Among the Portulacaceae family, efficient multiple shoot induction using BAP has been reported in Portulaca oleracea (Safdari and Kazemitabar 2009) and Portulaca grandiflora (Srivastava and Joshi 2009). Superiority of BAP in inducing multiple shoot formation has been reported in several plants such as Medicago truncatula (Neves et al. 2001), Cypripedium flavum (Yan et al. 2006) and Justicia gendarussa (Thomas and Yoichiro 2010).
Table 1

Effect of different concentrations and combinations of PGRs on multiple shoot induction from shoot tip and nodal explants of Talinum triangulare on MS medium after three subcultures at 2-week intervals

Combination and concentration of PGRs (mg/L)

Shoot tip

Node

Cytokinins

Auxin

% Response

Average number of shoots per explant

Average length of shoots (cm)

% Response

Average number of shoots per explant

Average length of shoots (cm)

Control

40.00

1.90 ± 0.18a

1.25 ± 0.0.5a

30.00

1.70 ± 0.15a

1.02 ± 0.05c

BAP

KIN

NAA

      

0.25

83.33

7.83 ± 0.16hi

3.40 ± 0.03g

53.33

3.33 ± 0.21ef

3.19 ± 0.04k

0.5

86.67

9.67 ± 0.21kl

4.60 ± 0.02m

60.00

4.50 ± 0.28hi

4.25 ± 0.03n

1.0

93.33

12.50 ± 0.23n

5.07 ± 0.02o

63.33

6.00 ± 0.24lm

4.68 ± 0.04p

2.0

80.00

8.33 ± 0.14ij

4.09 ± 0.03jk

46.67

3.67 ± 0.28fg

3.78 ± 0.03m

4.0

66.67

5.40 ± 0.22ef

2.55 ± 0.03d

40.00

2.17 ± 0.13abc

2.70 ± 0.03i

0.25

73.33

5.57 ± 0.19f

2.77 ± 0.03e

50.00

3.17 ± 0.20ef

2.59 ± 0.02h

0.5

80.00

7.37 ± 0.18h

3.18 ± 0.03f

53.33

4.17 ± 0.25gh

3.50 ± 0.03l

1.0

83.33

8.67 ± 0.11j

4.08 ± 0.03jk

56.67

5.67 ± 0.18kl

4.36 ± 0.03o

2.0

86.67

10.17 ± 0.18l

4.66 ± 0.02m

50.00

2.50 ± 0.09cd

1.69 ± 0.03e

4.0

76.67

6.67 ± 0.19g

3.51 ± 0.02h

46.67

1.83 ± 0.07ab

0.63 ± 0.03a

0.25

0.05

73.33

4.83 ± 0.16de

2.81 ± 0.02e

56.67

3.67 ± 0.18fg

1.85 ± 0.03f

0.5

0.05

76.67

5.23 ± 0.24def

3.47 ± 0.02gh

66.67

5.83 ± 0.20lm

3.17 ± 0.02k

1.0

0.05

86.67

8.50 ± 0.23j

4.23 ± 0.03l

60.00

5.17 ± 0/20jk

2.68 ± 0.03i

2.0

0.05

83.33

6.33 ± 0.17g

3.72 ± 0.02i

53.33

3.17 ± 0.23ef

1.30 ± 0.02d

4.0

0.05

66.67

4.67 ± 0.15d

2.49 ± 0.02d

43.33

1.83 ± 0.13ab

0.77 ± 0.03b

0.25

0.05

76.67

3.17 ± 0.19b

2.27 ± 0.03c

56.67

4.17 ± 0.17gh

1.75 ± 0.03e

0.5

0.05

83.33

7.53 ± 0.26h

4.15 ± 0.02k

60.00

5.67 ± 0.23kl

2.43 ± 0.02g

1.0

0.05

76.67

6.33 ± 0.22g

3.67 ± 0.02i

56.67

4.33 ± 0.14hi

3.07 ± 0.03j

2.0

0.05

70.00

4.70 ± 0.15d

2.82 ± 0.03e

50.00

2.33 ± 0.18bcd

2.73 ± 0.03i

4.0

0.05

66.67

3.83 ± 0.21c

1.79 ± 0.04b

43.33

1.67 ± 0.08a

0.63 ± 0.04a

0.10

0.10

83.33

9.57 ± 0.34k

4.84 ± 0.02n

66.67

6.33 ± 0.23m

3.71 ± 0.02m

0.25

0.25

83.33

11.33 ± 0.17m

5.15 ± 0.02p

70.00

7.67 ± 0.17n

5.30 ± 0.03r

0.5

0.5

96.67

15.67 ± 0.25o

6.22 ± 0.02r

63.33

5.50 ± 0.21kl

4.95 ± 0.04q

0.75

0.75

90.00

13.17 ± 0.20n

5.72 ± 0.02q

56.67

4.83 ± 0.17ij

4.28 ± 0.03no

1.0

1.0

73.33

7.83 ± 0.15hi

4.03 ± 0.03j

46.67

2.83 ± 0.13de

1.92 ± 0.04f

Values represent mean values ± standard error of 10 explants per treatment of three repeated experiments

% Response values represent mean percentage response

Means followed by the same letter within columns are not significantly different at 5 % probability level using Duncan’s Multiple Range Test (DMRT)

High frequency shoot multiplication was observed in MS medium supplemented with a combination of BAP and KIN in equal concentrations (Table 1). Significant shooting response (96.67 %) was observed in 0.5 mg/L BAP and 0.5 mg/L KIN combination from shoot tip explants (Fig. 1d), with a maximum of 15.67 ± 0.25 shoots and shoot length of 6.22 ± 0.02 cm per explant. Frabetti et al. (2009) and Rajeswari and Paliwal (2006) reported that an equal concentration of two cytokinins enhanced the formation of multiple shoots. Results of the present study clearly indicate that the increased shooting response was influenced by the interaction of BAP with KIN, as also reported by Singh et al. (2012) in Eclipta alba. A combination of BAP and KIN on efficient shoot induction has been well documented and proved in plant species such as Portulaca grandiflora (Srivastava and Joshi 2009), Artemisia vulgaris (Sujatha and Kumari 2007) and Vitex agnus-castus (Balaraju et al. 2008). In the current study, higher concentration of cytokinin combinations reduced the number of shoots and percentage response, as previously reported by Indhra and Dhar (2000).

The combined effect of cytokinins with auxin (NAA) in inducing multiple shoots from shoot tip explants was also studied. When NAA was used at higher concentrations (0.1–1.0 mg/L), there was formation of cream colored friable callus at the base. Lower concentration of NAA (0.05 mg/L) was suitable for shoot induction when combined with various concentrations of BAP and KIN. Among them, 1.0 mg/L BAP along with 0.05 mg/L NAA were able to produce 8.50 ± 0.23 shoots and a shoot length of 4.23 ± 0.03 cm (Table 1). The current results corroborate with the earlier findings where the addition of low-level of auxin with cytokinin promoted shoot proliferation in Coleus blumei (Rani et al. 2006) and Saussurea involucrata (Guo et al. 2007).

Among the different hormonal supplements used for multiple shoot induction of T.triangulare using nodal segments as explants, a combination of BAP (0.25 mg/L) and KIN (0.25 mg/L) showed 70 % response with an average of 7.67 ± 0.17 shoots and 5.30 ± 0.03 cm shoot length per explant. BAP (1.0 mg/L) or KIN (1.0 mg/L) when used individually resulted in 6.00 ± 0.24 and 5.67 ± 0.18 shoots per explant, respectively. Combined effects of BAP with NAA and KIN with NAA were recorded to be minimal (Table 1). However, in T. portulacifolium, Thangavel et al. (2008) reported that the combined effect of BAP and IAA induced an average of 8.0 shoots using axillary buds, which indicates a species-specific response.

Root induction

Effect of different auxins and MS salt strength (half and full-strength) on root induction of shoots has been presented in Table 2. When individual shoots were planted in half or full-strength MS basal medium free from growth regulators, few roots were elicited (1.00 ± 0.30 and 0.70 ± 0.21, respectively) with low frequency (Table 2). This may be due to the presence of endogenous auxins which were sufficient to initiate rooting. The effect of auxins on in vitro rooting was noticeably influenced by the concentration of the hormone as well the strength of the MS medium. PGRs added to half-strength MS medium produced better rooting response when compared with full-strength MS medium. Of the two auxins (IBA and IAA) tested, IBA was found to be more effective for root induction than IAA (Table 2). Likewise, efficient rooting by IBA was reported in Portulaca grandiflora (Srivastava and Joshi 2009) and Bacopa monnieri (Ceasar et al. 2010). Half-strength MS medium supplemented with 0.5 mg/L IBA induced an average of 45.10 ± 0.96 roots per shoot with a root length of 5.46 ± 0.08 cm (Fig. 1e). However, longer roots (6.19 ± 0.05 cm) were measured on full-strength MS medium supplemented with 1.0 mg/L IBA. Similar to our current study, rooting in half-strength MS medium supplemented with various concentrations of IBA has been reported to be efficient in Isodon wightii (Thirugnanasampandan et al. 2010) and Eclipta alba (Husain and Anis 2006).
Table 2

Effect of different concentrations of auxins (IBA and IAA) and MS salt concentration on root induction from in vitro raised microshoots of T. triangulare after 4 weeks of culture

Concentration of auxins (mg/L)

Half-strength MS medium

Full-strength MS medium

Average number of roots per shoot

Average length of roots (cm)

Average number of roots per shoot

Average length of roots (cm)

Control

1.00 ± 0.30a

0.40 ± 0.11a

0.70 ± 0.21a

0.24 ± 0.07a

IBA

IAA

    

0.1

36.40 ± 0.54i

4.74 ± 0.08f

15.60 ± 0.48e

5.21 ± 0.04i

0.5

45.10 ± 0.96j

5.46 ± 0.08g

17.30 ± 0.37f

5.74 ± 0.05j

1.0

29.10 ± 0.59h

4.09 ± 0.05d

21.30 ± 0.37g

6.19 ± 0.05k

2.0

18.70 ± 0.54e

3.40 ± 0.04c

11.80 ± 0.47d

4.64 ± 0.04h

3.0

15.30 ± 0.82d

2.66 ± 0.06b

7.60 ± 0.27c

3.85 ± 0.04f

0.1

17.50 ± 0.43e

4.02 ± 0.06e

7.00 ± 0.30c

3.30 ± 0.05d

0.5

23.50 ± 0.34f

4.63 ± 0.06f

11.70 ± 0.42d

3.65 ± 0.03e

1.0

27.10 ± 0.64g

3.67 ± 0.06d

15.60 ± 0.34e

4.18 ± 0.04g

2.0

13.60 ± 0.43c

3.37 ± 0.06c

6.60 ± 0.31c

2.90 ± 0.04c

3.0

6.50 ± 0.48b

2.72 ± 0.06b

4.50 ± 0.27b

2.52 ± 0.04b

Values represent mean values ± standard error of 10 shoots per treatment of three repeated experiments

Means followed by the same letter within columns are not significantly different at 5 % probability level using Duncan’s Multiple Range Test (DMRT)

Acclimatization

The rooted plantlets that were placed in 1/4 strength MS basal liquid medium showed prominent elongation of the shoots after 2 weeks. Broadening of the leaves and its color change from pink to green were also observed (Fig. 1f). Thus, when the concentration of MS basal salts was reduced, betalain pigment production was minimal. Optimization of nutrient medium composition plays an important role in maximizing the betalain content (Leathers et al. 1992; Georgiev et al. 2008). Factors such as nitrogen and phosphate ions and microelements in MS medium greatly influence the production and regulation of betalain biosynthesis in plants such as Amaranthustricolor (Bianco-Colomas and Hugues 1990), Chenopodiumrubrum (Berlin et al. 1986) and Phytolacca americana (Sakuta et al. 1986).

In all types of planting substrates examined (Fig. 1g, h), 100 % survival rate of the plantlets was recorded (Table 3) 8 weeks after establishment under field conditions. The regenerants grew well and all the plants transferred for hardening appeared healthy. The acclimatized plants that were placed under natural sunlight exhibited more branching and the first flower appeared after 50 days (Fig. 1i). The plants transferred from coco pith and soil showed best response when they were examined for average plant height (16.16 ± 0.08 cm) and total number of lateral branches (7.00 ± 0.21), leaves (44.40 ± 0.81) and flowers (6.60 ± 0.60) per plant (Table 3). There was no detectable variation among the potted regenerants of T. triangulare plants and mother plants with respect to morphological and growth characteristics.
Table 3

The effect of different planting substrates used during acclimatization on the subsequent establishment of the plants in the field

Planting substrates

Ratio

Percentage of survival (%)

Plant height (cm)

Number of lateral branches

Number of leaves per plant

Number of flowers per plant

Sand + Soil

1:1

100

15.28 ± 0.31b

5.00 ± 0.21b

36.20 ± 0.90b

4.10 ± 0.23a

Coco pith + Soil

3:1

100

16.16 ± 0.08c

7.00 ± 0.21c

44.40 ± 0.81c

6.60 ± 0.60c

Saw dust + Soil

3:1

100

14.48 ± 0.43a

4.20 ± 0.13a

33.60 ± 0.72a

5.30 ± 0.30b

Values represent mean values ± standard error of 10 plantlets per treatment, recorded after 8 weeks under field conditions

Means followed by the same letter within columns are not significantly different at 5 % probability level using Duncan’s Multiple Range Test (DMRT)

RAPD analysis

The plants raised through in vitro propagation were evaluated for their clonal fidelity by DNA fingerprinting using the random RAPD technique. RAPD is a widely employed method to detect any variation that is induced during in vitro regeneration of plant species (Valladares et al. 2006). It has the advantage of being technically simple, quick to perform and requires only small amounts of DNA (Williams et al. 1990). The RAPD-based DNA fingerprinting profiles were generated for the first time using DNA isolated from T. triangulare plants (Fig. 2). The DNA obtained from the regenerated plants was compared with the DNA of the mother plant to confirm genetic integrity. Four random primers were used for the RAPD analysis. All the tested primers produced resolvable bands. RAPD profile obtained through the amplification of genomic DNA of the ex vitro plants and that of the mother plant was similar in all respects. All the four primers produced monomorphic bands yielding a total of 47 amplicons (Table 4) with an average of 11.75 fragments ranging from ten to fifteen per primer, indicating genetic homogeneity among the regenerants and the mother plant. Figure 2a–d shows RAPD amplification patterns obtained with primers OPE 8, OPK 14, OPE 2 and OPK 15, respectively. From this molecular study it was clear that the micropropagated plants were genetically identical to that of control mother plant and no variation was induced during clonal propagation. Various researchers have used this technique to test the genetic fidelity of in vitro grown medicinal plants including Curcumalonga (Selvi et al. 2002), Plumbago zeylanica (Rout and Das 2002) and Musa paradisiaca (Venkatachalam et al. 2007).
Fig. 2

DNA fingerprinting patterns generated with four random decamer primers of mother plant and in vitro regenerated plants of T. triangulare. Amplification products obtained with primers a OPE 8, b OPK 14, c OPE 2, d OPK 15. Hind III-λ DNA marker—0.56 to 23 kb (LaneM), Mother plant (Lane 1), Randomly selected in vitro-raised field-grown plants (Lanes26)

Table 4

RAPD primers and the number of amplicons generated by four random decamer primers in T. triangulare plants

Primer name

Primer sequence

Number of base pairs (bp)

Number of amplicons

OPE 2

5′–GGTGCGGGAA–3′

10

11

OPE 8

5′–TCACCACGGT–3′

10

15

OPK 14

5′–CCCGCTACAC–3′

10

11

OPK 15

5′–CTCCTGCCAA–3′

10

10

Total

47

Conclusion

For the first time, a quick, reliable and large-scale micropropagation protocol was established from shoot tip explants of T. triangulare. A combination of BAP (0.5 mg/L) and KIN (0.5 mg/L) was found to be more effective in inducing multiple shoots when compared to BAP and KIN used individually. Half-strength MS medium with 0.5 mg/L IBA produced maximum rooting response and the complete plantlets were successfully established under field conditions with 100 % survival rate. The tissue culture protocol reported here could be used for the conservation of this valuable medicinal herb. RAPD fingerprinting was effective in confirming the genetic fidelity of plantlets regenerated through in vitro culture. Thus, the procedure described has great potential for improvement of this crop using biotechnological approaches such as genetic transformation and production of secondary metabolites. This can be achieved by adapting in vitro culture strategies to increase the biomass yield of active principles such as betalain which has recently gained interest in food and pharmaceutical industries.

Author contribution

The first author (Swarna J) has designed and carried out the experiments. Analysis and guidance was provided by the corresponding author (Ravindhran R). Both the authors have contributed equally to this paper.

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Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012

Authors and Affiliations

  1. 1.Department of Plant Biology and BiotechnologyLoyola CollegeChennaiIndia

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