Acta Physiol Plant (2010) 32:443–452 DOI 10.1007/s11738-009-0419-8
ORIGINAL PAPER
Highly efficient shoot regeneration of Bacopa monnieri (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD S. Antony Ceasar • S. Lenin Maxwell K. Bhargav Prasad • M. Karthigan • Savarimuthu Ignacimuthu
•
Received: 24 February 2009 / Revised: 20 September 2009 / Accepted: 6 November 2009 / Published online: 26 November 2009 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2009
Abstract A two-stage culture procedure has been developed for highly efficient shoot regeneration from leaf and internode explants of Bacopa monnieri. Adventitious shoot buds were obtained on the shoot induction medium containing Murashige and Skoog’s (MS) basal salt supplemented with 1.5 mg/l thidiazuron and 0.5 mg/l naphthalene acetic acid; these shoot buds were subcultured on the multiplication (second) medium amended with BAP (benzyl amino purine). Multiplication medium containing 0.5 mg/l BAP produced more shoots (135) and longer shoots (7.8 cm) with more nodes (6). Best response of root induction with more number of roots (16.5) and longer roots (8.7 cm) was observed in half strength MS basal medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid) and 0.5 mg/l phloroglucinol. In vitro obtained plants were transferred to the field after hardening with a 100% survival rate. Random amplified polymorphic DNA analysis was carried out using five random primers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants.
Communicated by E. Lojkowska. S. Antony Ceasar S. Ignacimuthu (&) Entomology Research Institute, Loyola College, Chennai 600034, Tamilnadu, India e-mail:
[email protected] S. Antony Ceasar S. Lenin Maxwell K. Bhargav Prasad M. Karthigan Department of Biotechnology, St. Peter’s Engineering College, Avadi, Chennai 600054, India
Keywords Auxin Cytokinin Induction medium Multiplication medium Phenolic compounds Genetic integrity Abbreviations BAP N6-Benzyl amino purine CA Chlorogenic acid IBA Indole-3-butyric acid IAA Indole-3-acetic acid Kin Kinetin MS Murashige and Skoog’s (1962) NAA Naphthalene acetic acid PG Phloroglucinol PGR Plant growth regulator RAPD Random amplified polymorphic DNA SA Salicylic acid TDZ Thidiazuron
Introduction Bacopa monnieri (L.), commonly known as ‘‘Brahmi’’ is a member of the Family Scrophulariaceae. It is commonly found on the banks of rivers and lakes. B. monnieri has been placed second in a priority list of most important medicinal plants evaluated on the basis of medicinal importance, commercial value and potential for further research and development (Anonoymous 1997). It has been used for centuries in folklore and traditional system of medicine as a memory enhancer, antiinflammatory, analgesic, antipyretic, sedative and anti-epileptic agent (Stough et al. 2001). The memory enhancing effects of B. monnieri have been attributed to the active constituents bacosides A
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and B (Singh et al. 1988). Memory enhancing property of these bacosides has increased the international demand for this plant for its extensive use in several commercial preparations (Rahman et al. 2002). In addition to its unique medicinal use, B. monnieri has also been linked to phytoremediation programmes for the removal of heavy metals such as cadmium and chromium (Shukla et al. 2007; Ali et al. 2001). With increasing demand for herbal drugs, the natural populations of B. monnieri are threatened with overexploitation, leading to depletion of valuable genetic resources which has resulted in listing of this plant as a threatened species (Tiwari et al. 1998). Many reports are available for the in vitro culture of this important medicinal plant (Tiwari et al. 1998, 2000, 2001; Shrivastav and Ranjani 1999). Tiwari et al. (2006) have also reported efficient shoot bud regeneration from different explants of B. monnieri using trimethoprim and bavistin in MS (Murashige and Skoog 1962) basal medium devoid of plant growth regulators (PGRs). Although bavistin containing medium produced more shoots they failed to elongate. There is a demand for further improvement in the tissue culture protocol for the mass multiplication of B. monnieri, both for commercial farming system and later, if required for replanting in the natural habitat when the plant population declines. We have developed an innovative micropropagation system based on a two-stage culture procedure that has not been attempted so far in B. monnieri. The two-stage culture procedure was developed for the mass production of B. monnieri utilizing the least number and lowest concentrations of PGRs in a cost-effective manner. The key feature of this system relies on the treatment of explants in two different media (induction and multiplication) for shoot regeneration. This system also offers the mass multiplication of B. monnieri within a short period (7 weeks) of time. The effect of three phenolic compounds, phloroglucinol (PG), chlorogenic acid (CA) and salicylic acid (SA) on in vitro root induction of B. monneri was also tested; because these compounds have not been examined so far on in vitro root induction of B. monnieri. The genetic integrity of the in vitro regenerated plants was checked using random amplified polymorphic DNA (RAPD) analysis.
Materials and methods 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) to MS basal salts; pH of the medium was adjusted to 5.8 using 0.1 M NaOH
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before adding 0.6% (w/v) agar (Bacteriological grade) (HiMedia, Mumbai, India) and the medium was autoclaved at 121°C for 15 min. Based on the experiment, different concentrations of PGRs were added to the MS basal medium before adding the agar. A preliminary study and shoot induction experiments were carried out in test tubes (140 mm high and 35 mm in diameter). Shoot multiplication and root induction tests were performed in 150 ml conical flasks. All the cultures were subcultured in the same medium unless otherwise mentioned at 15 days intervals. All the cultures were incubated at 25 ± 2°C in light with 16 h of photoperiod supplied by white fluorescent tube lights (Phillips, India), with a light intensity of 50 lmol m-2 s-1 photosynthetic photon flux density (PPFD). Plant material Bacopa monnieri plants were collected from the medicinal plant garden at Tampcol Farm, Aringar Anna Siddha Medical College, Chennai, India. The protocol described by Tiwari et al. (1998) has been used for the preparation of leaf and internode explants with minor modifications in surface decontamination procedure. Shoot buds were washed in sterile water for 15 min, soaked in 0.1% (w/v) bavistin containing carbendazime (BASF, Mumbai, India) for 20 min and washed three times with sterile distilled water. Surface decontamination was performed by immersing shoot tips in 70% (v/v) alcohol for 30 s, treated for 3 min with 0.1% (w/v) HgCl2 (Hi-Media, Mumbai, India) and washed six times with sterile distilled water. Shoots with a single node (2 cm) were placed in the hormone-free MS (Murashige and Skoog 1962) basal medium. Plants produced from this culture (after 3 weeks) were used as the source of leaf (1.0 9 0.5 cm) and internode (1 cm) explants for the subsequent experiments. Preliminary study and shoot induction A preliminary experiment was performed to choose the optimum concentration of individual cytokinins for efficient shoot induction. Different concentrations (0.5, 1.0, 1.5 and 2.0 mg/l) of BAP, TDZ and Kin were added separately to MS basal medium. Leaf (1.0 9 0.5 cm) and internode (1 cm) explants were grown on these media. A single explant was inoculated in each test tube and three replicates were maintained for each tube. Optimum concentration of individual cytokinins was selected based on the number of shoots produced after 3 weeks of incubation in the light. Based on the preliminary experiment, the optimum concentration (1.5 mg/l) of TDZ, BAP and Kin was chosen and used for the subsequent shoot induction experiment. Three cytokinins (TDZ, BAP and Kin), each at
Acta Physiol Plant (2010) 32:443–452 Table 1 Effect of different concentrations and combination of plant growth regulators on multiple shoot induction from leaf and internode explants of B. monnieri after 3 weeks
445
Concentration of plant growth regulators (mg/l)
Leaf Percentage of explants responding*
A. 1.5 Kin B. 1.5 Kin ? 0.5 NAA Values are average of 20 explants per treatment of 3 replications; Means followed by the same letter are not significantly different at 0.05% level based on Fisher’s LSD test * Mean
No. of shoots per explant**
Percentage of explants responding*
No. of shoots per explant**
99.2
22.2 ± 1.5a
98.6
16.6 ± 1.9a
99.8
a
97.8
24.3 ± 1.8b
a
30.4 ± 1.6
C. 1.5 Kin ? 0.5 IAA D. 1.5 TDZ
98.8 100
29.2 ± 1.2 38.2 ± 1.7b
96.4 100
18.8 ± 2.4a 35.4 ± 1.5c
E. 1.5 TDZ ? 0.5 NAA
100
56.4 ± 2.0c
100
49.3 ± 2.0c
100
c
100
31.7 ± 1.5c
c
100
25.4 ± 2.2c
c
100
40.3 ± 2.4d
F. 1.5 TDZ ? 0.5 IAA G. 1.5 BAP
42.7 ± 2.2
100
H. 1.5 BAP ? 0.5 NAA
30.2 ± 1.8
100
I. 1.5 BAP ? 0.5 IAA
** Mean ± SD
Internode
50.5 ± 1.6
b
99.4
35.0 ± 1.7
24.0 ± 2.3b
98.8
Table 2 Effect of two-stage culture procedure on shoot multiplication and elongation of Bacopa monnieri after 4 weeks Shoot induction medium used
Medium and Leaf? concentration of No. of plant growth regulators (mg/l) shoots**
B
B
B
MS
B
MS ? 0.2 BAP
Internode?? No. of fresh Shoot shoots* length**
42.7 ± 3.2a 12.3 40.6 ± 4.3
a
60.2 ± 3.6
b b
10.2 29.8
B
MS ? 0.5 BAP
72.3 ± 2.8
E
E
75.8 ± 4.4b 19.4
E
MS
E
MS ? 0.2 BAP
E
MS ? 0.5 BAP
67.4 ± 3.9
b
127.1 ± 3.2
c
135.3 ± 4.1
c b
41.9 11.0 70.4 78.9
H
H
70.2 ± 4.7
H
MS
67.1 ± 3.5b 26.8
H
MS ? 0.2 BAP
H
MS ? 0.5 BAP
78.9 ± 4.6
b
84.2 ± 4.1
d
29.9 38.6 43.9
No. of nodes**
2.9 ± 0.13a 2.1 ± 0.12 a
3.0 ± 0.11
b
4.4 ± 0.22
b
5.0 ± 0.20
2.8 ± 0.14 3.2 ± 0.15 3.1 ± 0.17
5.4 ± 0.17b 4.0 ± 0.16 b
4.6 ± 0.15
c
6.0 ± 0.19
d
7.8 ± 0.12
b
6.8 ± 0.17 6.0 ± 0.17
c
6.3 ± 0.12
No. of fresh Shoot shoots* length**
41.7 ± 3.9a 18.4
No. of nodes**
2.6 ± 0.17a 1.8 ± 0.14
35.2 ± 3.5
d
10.9
2.1 ± 0.15a 2.3 ± 0.14
48.7 ± 3.1
a
24.4
3.9 ± 0.12b 3.0 ± 0.15
54.1 ± 4.1
a
29.8
4.1 ± 0.16b 2.7 ± 0.11
61.2 ± 3.9b 11.9
4.3 ± 0.16b 3.1 ± 0.12
57.3 ± 4.0
b
8.0
4.0 ± 0.11b 2.7 ± 0.11
5.2 ± 0.19 100.7 ± 2.9
c
51.4
5.1 ± 0.10c 3.8 ± 0.10
6.3 ± 0.15 112.6 ± 3.5
c
62.7
6.2 ± 0.13c 2.7 ± 0.19
3.2 ± 0.13
b
6.3
5.1 ± 0.21b 2.7 ± 0.15
50.3 ± 3.7b
2.2
5.2 ± 0.16b 2.4 ± 0.13
61.5 ± 3.3
b
11.0
5.4 ± 0.15b 2.0 ± 0.18
73.4 ± 2.7
b
22.9
4.7 ± 0.18b 2.2 ± 0.10
3.4 ± 0.10
5.8 ± 0.24b 3.1 ± 0.20 c
No. of shoots**
6.1 ± 0.11 5.9 ± 0.21
52.2 ± 2.8
Values are average of 10 explants per treatment of 3 replications Means followed by the same letter are not significantly different at 0.05% level based on Fisher’s LSD test B 1.5 Kin ? 0.5 NAA, E 1.5 TDZ ? 0.5 NAA, H 1.5 BAP ? 0.5 NAA ?
3 weeks old shoot clumps obtained from leaf explant
??
3 weeks sold shoot clumps obtained from internode explant
* Mean ** Mean ± SD, shoot length (cm)
1.5 mg/l were added separately with 0.5 mg/l NAA and IAA (Table 1) to find out the combined effect of auxin and cytokinin on shoot induction of B. monnieri. Percentage explants responding and number of shoots produced by the responding explants were calculated after 3 weeks of incubation in the light. Shoot multiplication and elongation Shoot clumps containing multiple shoots (3 weeks old) obtained from the shoot induction medium containing
1.5 mg/l Kin and 0.5 mg/l NAA (B), 1.5 mg/l TDZ and 0.5 mg/l NAA (E) and 1.5 mg/l BAP and 0.5 mg/l NAA (H) were subcultured in the multiplication (second) medium for further shoot multiplication and elongation (Table 2). The multiplication medium consisted of MS supplemented with BAP (0.2 and 0.5 mg/l) and MS devoid of PGRs. One set of shoot clumps was also subcultured in the same shoot induction medium (B, E and H) to serve as control and compare the effects of multiplication medium on shoot multiplication and elongation. Total number of shoots, number of fresh axillary shoots
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446 Table 3 Effect of MS salt concentration, IBA and IAA on in vitro root induction of Bacopa monnieri, after 4 weeks
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Concentration of auxin (mg/l)
Half strength MS No. of roots
Full strength MS Root length (in cm)
No. of roots
Root length (in cm)
IBA 0.0
5.1 ± 0.11a
1.9 ± 0.12a
3.5 ± 0.12a
1.0 ± 0.10b
0.5
8.8 ± 0.12
a
a
a
1.7 ± 0.14a
10.5 ± 0.23
b
5.7 ± 0.17
b
7.7 ± 0.17
2.3 ± 0.17b
1.0
Values are mean ± SD of 10 explants per treatment of 3 replications Means followed by the same letter are not significantly different at 0.01% level based on Fisher’s LSD test
b
6.4 ± 0.14
1.5
7.6 ± 0.14a
3.7 ± 0.16c
5.3 ± 0.18a
3.3 ± 0.18c
2.0
c
a
4.9 ± 0.15
c
4.9 ± 0.19
3..7 ± 0.12c
6.0 ± 0.21
IAA 0.5
6.0 ± 0.12a
2.7 ± 0.19a
4.8 ± 0.12a
1.4 ± 0.13b
1.0
7.5 ± 0.24
b
c
2.9 ± 0.12
a
5.2 ± 0.14
2.1 ± 0.16c
1.5
6.3 ± 0.19a
3.4 ± 0.16a
4.5 ± 0.15a
2.8 ± 0.19c
2.0
c
a
c
3.0 ± 0.12a
5.2 ± 0.21
formed from shoot clumps, length of the shoots and number of nodes per shoot were calculated after 4 weeks of incubation in the light. Root induction Elongated shoots ([5 cm in length) were excised aseptically from shoot clumps and transferred to the rooting medium amended with 0.5, 1.0, 1.5 and 2.0 mg/l IBA or IAA. The PGRs were added separately to full strength and half strength MS media to determine the effect of MS salt concentration on root induction (Table 3). Total number of roots produced per shoot and length of the root were measured after 4 weeks of incubation in the light. Effect of phenolic compounds on root induction In a separate experiment, 0.2, 0.5, 1.0 and 2.0 mg/l PG, CA or SA were added to half strength MS basal medium containing 1.0 mg/l IAA or IBA to examine the effect of phenolic compounds on in vitro root induction of B. monnieri. Cultures were incubated in the light; total number of roots produced and length of the root were measured after 4 weeks. Data collection and statistical analysis Each experiment was repeated three times each consisting of three replicates. In shoot induction medium, 20 explants each of leaf and internode were used per treatment. The mean (%) of explants responding (explants producing at least one shoot) and mean number of shoots per explant were calculated after 3 weeks of incubation. In multiplication medium, 10 shoot clump explants obtained from leaf and internode explants were used per treatment; mean number of shoots produced, number of fresh shoots formed, shoot length and number of nodes were measured
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2.8 ± 0.14
4.0 ± 0.11
4.1 ± 0.19
after 4 weeks of incubation. In root induction, 10 individual shoots were used per treatment; mean number of roots and root length were measured after 4 weeks of incubation. Mean values were expressed as mean ± SD and the significance of differences among means was carried out using Fisher’s least significant difference test. Hardening Rooted plants were carefully removed from the culture flasks, washed with sterile water to remove agar media, placed in the paper cups (60 mm in diameter and 110 mm high) filled with sterilized vermicompost (Bharat organic fertilizers, Karnataka, India) and supplied with diluted (5 times) MS basal salts. The plants were covered with polythene bags (10 9 8 cm) to maintain high humidity. These plants were maintained in the culture room for 3 weeks with the following atmospheric conditions: Temperature, 25 ± 2°C; light, 16 h photoperiod with light intensity of 50 lmol m-2 s-1 PPFD; relative humidity, 85 ± 5%. The plants were then removed from the cups after 3 weeks and transferred to the field outside the greenhouse with 100% survival rate. The regenerated plants were grown in vivo for about 3 months. The regenerants grew well and did not show any variation in morphology and growth characteristics when compared with mother plant used for tissue culture study. The size and length of the stems, shoots and leaves were also identical to that of mother plant. RAPD analysis Random amplified polymorphic DNA analysis was performed to check the genetic fidelity of in vitro regenerated plants. Plants obtained from different shoot multiplication media containing various PGRs (Table 2) were used for DNA isolation for RAPD analysis. Twelve plants obtained
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from different PGRs treatments were analyzed by five different primers. Genomic DNA was isolated from mother plant and in vitro regenerated plants using Dellaporta et al. (1983) method for RAPD analysis. The concentration of DNA was determined by ultraviolet–visible spectrophotometer (Perkin Elmer LambdaEZ201, Germany); 5 decamer random oligonucleotide primers (P1, GTGAT CGCAG; P2, AGATGCAGCC; P3, GTCCCGACGA; P4, TGGACCGGTG; P5, CTCACCGTCC) were used for the PCR reactions. The PCR was carried out in a volume of 25 ll reactions consisting 1 ll template DNA (40–80 ng), 2.5 ll 10 9 PCR buffer, 2.5 ll MgCl2 (2.5 mM), 0.5 ll of dNTPs (10 mM), 2 ll random primer (7.5 pmol), 1 ll Taq polymerase (0.5 U) (Medox Biotech, India) and 15.5 ll sterile DNA-free water. The amplification was carried out in a DNA thermal cycler (Eppendorf, Germany). The PCR was performed at an initial denaturation at 94°C for 5 min followed by 45 cycles of 1 min denaturation at 94°C, 30 s annealing at 32°C and 1 min extension at 72°C with a final extension at 72°C for 10 min. Amplification with each primer was repeated twice to confirm reproducibility of the results. The amplified samples were electrophoresed in 1.5% agarose gels. The number of bands was recorded using a gel documentation system.
Results Preliminary study Best response of shoot induction was observed at 1.5 mg/l for all three cytokinins (BAP, TDZ and Kin) tested in the preliminary study (results not shown); this concentration (1.5 mg/l) was preferred to begin the investigation. Initially, callus was formed in TDZ, BAP or Kin at 1.5 mg/l after 2 weeks of incubation, but they did not remain as callus for longer time, soon they were changed into adventitious shoots within 3 weeks of incubation in light. Shoot induction In the shoot induction medium, 0.5 mg/l NAA and IAA were added separately to 1.5 mg/l BAP, TDZ and Kin to examine the combined effect of auxin and cytokinin on shoot induction (Table 1; a–i). Most explants responded to the treatments (Fig. 1a, b). The addition of auxin to either of the cytokinins has improved the frequency of shoot induction. Cytokinins added with NAA produced superior response over IAA (Table 1). Leaf explants were more responsive than internode explants in all the treatments. NAA (0.5 mg/l) added with TDZ (1.5 mg/l) gave more shoots (56.4) followed by NAA (0.5 mg/l) and BAP
447
(1.5 mg/l) (50.5); NAA added with Kin produced the lowest response (30.4). The addition of IAA produced fewer numbers of shoots when compared with NAA and also induced callus instead of shoots in a few cultures. The size of the shoot clumps also varied based on the concentration and combination of PGRs in induction medium and was directly proportional to the number of shoots. Shoot multiplication Shoot clumps cultured in the multiplication medium containing BAP produced significantly more shoots (Fig. 1c). Shoot clumps obtained from leaf explants gave more number of shoots than clumps obtained from internode explants (Table 2). Shoot clumps subcultured in the MS medium containing BAP produced more fresh axillary shoots than other media. The best response of shoot multiplication was observed in the MS containing 0.5 mg/l BAP for all shoot clumps. Shoot clumps obtained from induction medium containing TDZ (1.5 mg/l) and NAA (0.5 mg/l) (E) produced more shoots (135.3) in MS containing BAP (0.5 mg/l); this clump also produced more fresh shoots (78) (Table 2). However, shoot clumps obtained from the induction medium containing Kin ? NAA (B) and BAP ? NAA (H) did not produce same number of fresh shoots when compared with those obtained from medium containing TDZ and NAA (E) (Table 2). Shoot clumps cultured in the same induction medium (B, E and H) and MS medium devoid of PGRs produced least response of shoot multiplication. Shoot elongation The effect of multiplication medium on shoot elongation was calculated by measuring the mean length of shoots after 4 weeks of incubation. Shoot length increased in all MS media supplemented with BAP (Fig. 1d); MS medium containing 0.5 mg/l BAP produced longer shoots than 0.2 mg/l BAP (Table 2). Highest length (7.8 cm) of shoot with more nodes (6.3) was observed in the cultures obtained from induction medium containing TDZ and NAA (E). The shoot clumps subcultured in the same induction medium (B, E and H) and MS medium devoid of PGRs did not significantly increase the length of the shoots and number of nodes (Table 2). Root induction Elongated shoots were separated from the shoot induction medium and transferred to the rooting medium (Fig. 1e). Root induction was observed in all flasks supplemented with PGRs (Fig. 1f). PGRs added to half strength MS medium produced a better rooting response when
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Acta Physiol Plant (2010) 32:443–452
Fig. 1 Micropropagation of Bacopa monnieri (L.) using a two-stage culture method. a Adventitious shoot bud induction in MS medium containing 1.5 mg/l TDZ and 0.5 mg/l NAA from internode explant (Bar 0.4 cm), b from leaf explant (Bar 0.3 cm) after 3 weeks of incubation. c Multiplication of shoot clump cultured in the second medium containing MS and 0.2 mg/l BAP(Bar 1.3 cm). d
Multiplication and elongation of shoot clump cultured in the second medium containing MS and 0.5 mg/l BAP (Bar 1.5 cm). e Elongated shoots inoculated in the root induction medium (Bar 1.3 cm). f Rooted plants (Bar 1.5 cm). g Hardened plant (Bar 1.2 cm). h Fully grown plant established in the field (Bar 2.5 cm)
compared with full strength MS (Table 3). Of the two auxins (IBA and IAA) tested, the best response was observed with IBA at all four concentrations. More roots (10.5) and longer roots (5.7 cm) were obtained on half strength MS medium supplemented with 1.0 mg/l IBA (Table 3).
RAPD analysis
Effect of phenolic compounds on root induction The addition of phenolic compounds in root induction medium containing 1 mg/l IBA or IAA induced more roots. The optimum concentration of PG, SA and CA for root induction was 0.5 mg/l. The best response was observed with PG; CA gave medium response and SA gave poor response. Phenolic compounds added with IBA gave superior response over IAA at all concentrations tested. More number of roots (16.5) and longer roots (8.7 cm) were obtained at 0.5 mg/l PG and 1.0 mg/l IBA (Table 4).
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The DNA of regenerated plants was compared with the DNA of the mother plant to confirm genetic integrity. Five random primers were used for the RAPD analysis. All the tested primers produced resolvable bands. The number of bands produced by each primer ranged from 5 to 12. RAPD profile obtained through amplification of genomic DNA of the in vitro grown plants and that of the mother plant was similar in all respects. All the five primers produced monomorphic bands confirming the genetic homogeneity of the in vitro regenerated plants; 12 plants obtained from different PGRs treatments produced monomorphic bands for these five primers. Figure 2a, b shows RAPD amplification patterns obtained with primers P1 and P2, respectively. The band profile was similar in control and micropropagated plants with the rest of the primers also; no RAPD polymorphism was observed in the
Acta Physiol Plant (2010) 32:443–452 Table 4 Effect of phloroglucinol, chlorogenic acid and salicylic acid on root induction of B. monnieri, after 4 weeks
Concentration of phenolic compounds (1.0 mg/l)
449
MS ? IBA (1.0 mg/l)
MS ? IAA (1.0 mg/l)
No. of roots
Root length (in cm)
No. of roots
0.0
10.3 ± 0.21b
4.2 ± 0.17a
7.3 ± 0.21a
2.9 ± 0.12a
0.2
11.8 ± 0.11a
4.8 ± 0.13a
8.4 ± 0.12a
3.7 ± 0.12a
0.5
16.5 ± 0.26
b
8.7 ± 0.18
b
b
6.3 ± 0.10b
9.6 ± 0.18
a
6.7 ± 0.14
c
a
5.3 ± 0.13c
7.0 ± 0.22
c
5.9 ± 0.16
a
c
5.9 ± 0.17
4.7 ± 0.16c
Root length (in cm)
PG
1.0 2.0 CA 0.2 0.5 Values are mean ± SD of 10 explants per treatment of 3 replications Means followed by the same letter are not significantly different at 0.01% level based on fisher’s LSD test CA chlorogenic acid, PG phloroglucinol, SA salicylic acid
1.0 2.0
12.7 ± 0.15
7.3 ± 0.13
11.0 ± 0.17a
3.7 ± 0.11a
9.0 ± 0.11a
7.4 ± 0.19b
13.5 ± 0.25
b
6.9 ± 0.19
c
a
5.9 ± 0.17c
10.0 ± 0.17
a
5.4 ± 0.13
a
a
4.3 ± 0.13c
8.0 ± 0.20
c
4.0 ± 0.16
a
c
5.2 ± 0.16
3.0 ± 0.18a
2.7 ± 0.11a
6.3 ± 0.11a
5.4 ± 0.19b
4.9 ± 0.19
c
a
3.9 ± 0.17c
3.4 ± 0.13
a
a
3.7 ± 0.13c
3.2 ± 0.16
a
c
2.5 ± 0.18a
10.2 ± 0.16
7.5 ± 0.12
SA 0.2 0.5 1.0 2.0
micropropagated plants. The remaining three primers (P3, P4 and P5) also produced similar monomorphic bands revealing the genetic integrity of in vitro regenerated plants. From this molecular study, it was clear that the micropropagated plants were genetically identical to that of control plant and no variation was induced during clonal propagation.
Discussion Two explant types were used for adventitious shoot induction, and in all treatments, leaf explants gave the best response. This confirms the earlier reports for shoot
Fig. 2 RAPD analysis of mother plant and in vitro regenerated plants of B. monnieri. L 100-bp DNA ladder, MP mother plant, IR1–IR4 in vitro regenerated plants. a Amplification products obtained with primer P1. b Amplification products obtained with primer P2
9.1 ± 0..17a 10.5 ± 0.25
b
8.4 ± 0.17
a
5.3 ± 0..20
c
7.6 ± 0.16 5.9 ± 0.12 3.9 ± 0.16
induction from different explants of B. monnieri (Tiwari et al. 1998, 2000, 2001; Shrivastav and Ranjani 1999), where the leaf explant was more responsive for shoot induction than other explants. In the preliminary experiment, best response was observed at 1.5 mg/l for all three cytokinins (BAP, TDZ and Kin) tested; this was in agreement with earlier studies by Tiwari et al. (1998, 2001) who have obtained efficient shoot induction at 1.5 mg/l of cytokinin. Among the three cytokinins tested, TDZ was superior to BAP and Kin on shoot induction of B. monnieri in this study. TDZ has been used most frequently in recent times for the efficient shoot induction of many plants. It showed high cytokinin activity in promoting growth of cytokinin-dependent cultures (Mok et al. 2005). TDZ also stimulated conversion of cytokinin nucleotides to more biologically active nucleotides (Capelle et al. 1983) and stimulated accumulation of endogenous purine cytokinins (Thomas and Katterman 1986). Therefore, addition of TDZ is more helpful for the efficient shoot induction of B. monnieri. Addition of auxin (NAA and IAA) to cytokinin had significantly increased the frequency of shoot induction. NAA produced more shoots than IAA when added with either of the cytokinins. More shoots were produced in the MS containing TDZ (1.5 mg/l) and NAA (0.5 mg/l). This may be due to the synergistic effect of auxin and cytokinin on shoot induction. Synergistic effect of PGRs on efficient shoot induction has been well documented and proved in the following plant species: Dianthus chinesis; Rhodiola rosea; Salvia nemorosa. In these plants, addition of two different PGRs in the regeneration medium has augmented
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the shoot induction and produced more shoots than addition of single hormone (Kanita and Kothari 2002; Dimitrov et al. 2003; Skala and Wysokinska 2004). Based on the present study, it is clear that addition of two different hormones is essential for increased shoot induction of B. monnieri. Shoot clumps obtained from shoot induction medium were subcultured in the multiplication medium containing a lower concentration of BAP. This produced more shoots (135); both the number and length of shoots were increased significantly following BAP (0.5 mg/l) addition. This is the highest number reported in B. monnieri. In an earlier study, Tiwari et al. (2006) reported 81 and 98 shoots from internode and leaf explants, respectively, in MS basal medium containing bavistin; shoots obtained from bavistin containing medium failed to elongate and remained stunted. In this report, both the number of shoots and length of shoots increased significantly following BAP (0.5 mg l-1) treatment. Two-stage culture procedure seems to be a versatile protocol for efficient shoot induction and elongation. This provides rapid propagation of B. monnieri in an effortless manner by treating the explants in two different media. Treatment of explants in two different media containing least number and low concentration of PGRs produced more shoots which were also very long in this study. This is a very efficient system and also offers mass multiplication of B. monnieri within 7 weeks of time. Two-stage culture method has also been proved efficient in other plants such as Cassia angustifolia (Iram and Anis 2007), Manihot esculenta (Konan et al. 1994), Loblolly pine (Tang and Guo 2001), Gladiolus corms (Sen and Sen 1995), Pterocarpus marsupium (Husain et al. 2007), Rosa hybrida (Ibrahim and Debergh 2001) and Ipomoea batatas (Ramana et al. 1995). In these reports, the treatment of explants in the induction medium containing auxin and cytokinin caused the induction of adventitious shoots and subsequent transfer of these shoots to the second medium containing auxin or lower level of BAP improved the shoot multiplication and elongation. In two-stage culture methods, second (multiplication) medium containing BAP alone found to be appropriate for the efficient shoot multiplication and elongation (Geetha et al. 1998). This study validates that the addition of 0.5 mg/l BAP in the multiplication medium improved shoot multiplication; this method can be utilized in future for the mass multiplication of B. monnieri. In root induction, half strength MS medium containing auxin (IBA or IAA) produced more roots than full strength MS. Many plant species have been reported to root best in the half strength MS medium; examples are: Tylophora indica (Faisal and Anis 2005), Solanum melongena (Magioli et al. 2004), Trapa japonica (Hoque and Arima 2002) and Rhodiola fastigiata (Liu et al. 2006). The IBA treatment was superior to IAA for the root induction of B. monnieri in this study. IBA and IAA are the most
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commonly used PGRs for in vitro root induction of many shrubs (Gonza´lez et al. 1991; David 1983). The frequency of root induction was markedly enhanced by augmenting the medium with phenolic compounds. The addition of either of the phenolic compound in the medium containing auxin (IBA or IAA) promoted the frequency of root induction; PG added with IBA gave the best response of root induction. The proficient role of PG on in vitro rooting was also tested and confirmed in Decalepis hamiltonii (Giridhar et al. 2005) and Malus pumila (Zanol et al. 1998). In these studies, addition of PG along with auxin in the rooting medium has been shown to have a significant effect on root induction. Enhancement of root induction is due to auxin–phenol synergism resulting in the suppression of the peroxidase activity in the culture, thereby protecting the endogenous auxin from peroxidase catalyzed oxidation (De Klerk et al. 1999). In this report, it has been confirmed that the addition of phenolic compounds in the root induction medium along with IBA enhanced the in vitro root induction of B. monneri; this can be used in future for the efficient root induction of same or other plant species. RAPD profile obtained through amplification of genomic DNA of the in vitro regenerated plants and that of the source plant were similar and no polymorphic bands were seen. This clearly indicates the genetic integrity and true-to-type nature of the in vitro regenerated plants. RAPD is becoming a widely employed method in the detection of genetic diversity because it has the advantage of being technically simple, quick to perform and requires only small amounts of DNA (Williams et al. 1990). RAPD has been proven to be a suitable molecular technique to detect the variation that is induced or occurs during in vitro regeneration of plant species (Shu et al. 2003). We have already applied this technique in Curcuma amada (Prakash et al. 2004). RAPD was also applied by various researchers for testing the genetic fidelity of in vitro grown medicinal plants including Drosera anglica and Drosera binata (Kawiak and Lojkowska 2004), Plumbago zeylanica (Rout and Das 2002), Zingiber officinale (Rout et al. 1998), Curcuma longa (Selvi et al. 2002), Tylophora indica (Jayanthi and Mandal 2001) and Musa paradisiacal (Venkatachalam et al. 2007). In conclusion, a two-stage culture procedure has been proved to offer efficient mass multiplication of B. monnieri. This innovative two-stage culture system might offer some interesting possibilities in terms of mass production of B. monnieri within a short time and low cost; mass produced plants can be utilized for secondary metabolite production. Addition of phenolic compounds in half strength MS basal medium increased the rate of root induction. RAPD analysis confirmed the genetic integrity of micropropagated plants. This protocol may be harnessed in future for the efficient micropropagation of B. monnieri and can be extended to other important medicinal plants.
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References Ali G, Shrivastav PS, Iabal M (2001) Responses of Bacopa monnera cultures to cadmium toxicity. Bull Environ Con Tox 66:342–349 Anonoymous (1997) Indian medicinal plants: a sector study. Occasional paper No. 54. Export-Import Bank of India, Quest publications, Bombay, India Capelle S, Mok D, Kirchner S, Mok S (1983) Effects of thidiazuran on (D2-isopentyl) (8–14C) adenosine in callus tissues of phaseolus linatus L. Plant Physiol 73:796–802 David JJ (1983) Adventitious root formation ‘in vitro’ in apple rootstocks (Malus pumila) I. Factors affecting the length of the auxin-sensitive phase in M9. Physiol Plant 5:149–153 De Klerk GJ, Vander Kreiken W, De Jong JC (1999) The formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol Plant 35:189–199 Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation. Version II. Plant Mol Biol Rep 1:19–21 Dimitrov B, Tashera K, Zagorska N, Evstatiera L (2003) In vitro cultivation of Rhodiola rosea L. Gene Breed 32:3–6 Faisal M, Anis M (2005) An efficient in vitro method for mass propagation of Tylophora indicia. Biol Plant 49:257–260 Geetha N, Venkatachalam P, Prakash V, Lakshmi Sita G (1998) High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeonpea (Cajanus cajan L.). Curr Sci 75:1036–1041 Giridhar P, Gururaj H, Ravishankar G (2005) In vitro shoot multiplication through shoot tip cultures of Decalepis hamiltonii Wight & Arn., a threatened plant endemic to Southern India. In Vitro Cell Dev Biol Plant 41:77–80 Gonza´lez A, Casares TR, Sa´nchez Rodrı´guezR (1991) Adventitious root induction in Corylusvellana L. cotyledon. In Vitro Cell Dev Bio Plant 27:125–131 Hoque A, Arima S (2002) Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov.). In Vitro Cell Dev Biol Plant 38:342– 346 Husain MK, Avis M, Shahzad A (2007) In vitro propagation of Indian kino (Pterocarpus marsupium. Rokb.) using Thidiazuron. In Vitro Cell Dev Biol Plant 43:59–64 Ibrahim R, Debergh PC (2001) Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.). Sci Hortic 88:41–57 Iram S, Anis M (2007) In vitro shoot multiplication and plantlet regeneration from nodal explants of Cassia angustifolia (Vahl.), a medicinal plant. Acta Phys Plant 29:233–238 Jayanthi M, Mandal PK (2001) Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plants in Tylophora indica (Burm. F. Merrill.). In Vitro Cell Dev Biol Plant 37:576–580 Kanita A, Kothari SL (2002) High efficiency adventious shoot bud formation and plant regeneration from leaf explants of Dianthus chinesis L. Sci Hortic 90:205–212 Kawiak AA, Lojkowska E (2004) Application of RAPD in the determination of genetic fidelity in micropropagated drosera plantlets in vitro cell. Dev Biol Plant 40:592–595 Konan NK, Sangwan RS, Sangwan-Norreel BS (1994) Efficient in vitro shoot-regeneration systems in Cassava (Manihot esculenta Crantz). Plant Breed 113:227–236 Liu HJ, Xu YAN, Liu Y-J, Liu C-Z (2006) Plant regeneration from leaf explants of Rhodiola fastigiata. In Vitro Cell Dev Biol Plant 42:345–347 Magioli C, Rocha AP, Oiveira DE (2004) Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron. Plant Cell Rep 7:661–663
451 Mok MC, Mok DW, Amstrong DJ, Shudo K, Isogai Y, Okamanto T (2005) Cytokinin activity of N-phenyl-N0 -1, 2, 3-thiadiazol-5urea (thidiazuron). Phytochemistry 21:1509–1511 Murashige T, Skoog F (1962) A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiol Plant 15:497–573 Prakash S, Elangomathavan R, Seshadri S, Kathiravan K, Ignacimuthu S (2004) Efficient regeneration of Curcuma amada Roxb plantlets from rhizome and leaf sheath explants. Plant Cell Tissue Organ Cult 78:159–165 Rahman LU, Verma PC, Singh D, Gupta MM, Banerjee S (2002) Bacosides production by suspension cultures of Bacopa monnieri (L). Biotechnol Lett 24:1427–1429 Ramana M, Gosukonda, Ananta Porobodessai, Essie Blay, Channapatna S, Prakasha Curt M, Peterson (1995) Thidiazuroninduced adventitious shoot regeneration of sweet potato (Ipomoea batatas). In Vitro Cell Dev Biol Plant 31:65–71 Rout GR, Das (2002) An assessment of genetic integrity of micropropagated plants of Plumbago zeylanica by RAPD markers. Biol Plant 45:27–32 Rout GR, Das P, Goel S, Raina SN (1998) Determination of genetic stability of micropropagated plants of ginger using random amplified polymorphic DNA (RAPD) markers. Bot Bull Acad Sin 39:23–27 Selvi DN, George L, Eapen S (2002) Micropropagation and field evaluation of micropropagated plants of turmeric. Plant Cell Tissue Organ Cult 68:143–151 Sen J, Sen S (1995) Two-step bud culture technique for a high frequency regeneration of Gladiolus corms. Sci Hortic 64:133–138 Shrivastav N, Ranjani M (1999) Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennel. Plant Cell Rep 18:919–923 Shu QY, Liu GS, Qi DM, Chu CC, Liu J, Li HJ (2003) An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust. Plant Cell Rep 22:175–180 Shukla OP, Pubey S, Ravi VN (2007) Preferential accumulation of cadmium and chromium: toxicity in Bacopa monnieri. L. under mixed metal treatments. Bull Environ Contam Toxicol 78:252– 257 Singh HK, Rastogi RP, Sriman RC, Dhawan BN (1988) Effect of bacoside A and B on avoidance response in rats. Phytother Res 2:70–75 Skala E, Wysokinska H (2004) In vitro regeneration of Salvia nemorosa L. from shoot tips and leaf explants. In Vitro Cell Dev Biol Plant 40:596–602 Stough C, Lloyd J, Clarke J, Downey LA, Hutchison CW, Rodgers T, Nathan PJ (2001) The chronic effects of an extract Bacopa monniera (Brahimi) on cognitive function in healthy human subjects. Psychopharmacology 156:481–484 Tang W, Guo Z (2001) In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regul 33:25–31 Thomas JC, Katterman FR (1986) Cytokinin activity induced by thidiazuran. Plant Physiol 81:681–683 Tiwari V, Deo Singh B, Nath Tiwari K (1998) Shoot regeneration and Somatic embryogenesis from different explants of Brahmi (Bacopa monniera (L.) Wettst.). Plant Cell Rep 17:538–543 Tiwari V, Tewari KN, Singh BD (2000) Suitability of liquid cultures for in vitro multiplication of Bacopa Monniera (L.) Wettst. Phytomorphology 50:337–342 Tiwari V, Tewari KN, Singh BD (2001) Comparative studies of cytokinin on in vitro propagation of Bacopa monniera. Plant Cell Tissue Organ Cult 66:9–16 Tiwari V, Tewari KN, Singh BD (2006) Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin. Plant Cell Rep 25:629–635
123
452 Venkatachalam LE, Sreedhar RV, Bhagyalakshmi N (2007) Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers. Plant Growth Regul 51:193–205 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535
123
Acta Physiol Plant (2010) 32:443–452 Zanol GC, Fortes GR, de L, Campos AD, Centellas AQ, da Silva JB (1998) In vitro rooting and peroxidase activity of apple rootstock treated with indole butyric acid and phloroglucinol. Res Brasil Fisiol Veg 10:65–68
