1. Introduction
Rauvolfia serpentina Benth. (Apocynaceae), commonly known as Indian snakeroot or Sarpagandha, is indigenous to India and other tropical countries of Asia. The
Serpentina plant has drawn special attention all over the World in the pharmaceutical field and holds an important position because of the presence of number of bioactive chemicals it containes, including ajmaline, deserpidine, rescinnamine, serpentinine, and yohimbine. Reserpine is a potent alkaloid first isolated from this plant which is being widely used as an antihypertensive. This herbal plant is used as medicine for high blood pressure, insomnia, anxiety and other disorders of the central epilepsy [
1]. A major part of the commercial supply of the drug used in U.S.A. and European countries originates from India, Pakistan, Sri Lanka, Burma and Thailand, with India being a major supplier. Poor seed viability, low seed germination rate, and low vegetative propagation rate through rooted cuttings has hampered large scale commercial cultivation of
R. serpentina through conventional modes and over exploitation of the natural resources has led to listing of this species as “endangered” by the International Union for Conservation of Nature and Natural Resources (IUCN) [
2]. Under these circumstances, propagation through biotechnological approaches of this potential drug producing plant has assumed importance.
Production of synthetic seed has provided new opportunities in plant biotechnology. The alginate encapsulation technique is designed to combine the advantages of clonal propagation with those of seeds propagation and storage [
3,
4]. Although many reports are available on the utilization of synthetic seeds for micropropagation and conservation of various medicinal plant species [
5,
6,
7,
8,
9,
10,
11]. The genetic stability of synthetic seed-derived plantlets remains relatively unknown with the exception of some recent reports on
Ananus comosus [
12],
Cineraria maritima [
13] and
Picrorhiza kurrooa [
14]. True-to-type clonal fidelity is one of the most important pre-requisites in the
in vitro propagation of crop species. The occurrence of cryptic genetic defects arising via somaclonal variation in the regenerates can seriously limit the broader utility of the micropropagation system [
15]. It is, therefore, imperative to establish genetic uniformity of synthetic seed derived plantlets to suggest the quality of the plantlets for its commercial utility. Polymerase chain reaction (PCR)-based techniques such as random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are immensely useful in establishing the genetic stability of
in vitro-regenerated plantlets in many crop species [
16,
17]. RAPD and ISSR markers are very simple, fast, cost-effective, highly discriminative and reliable. They require only a small quantity of DNA sample and they do not need any prior sequence information to design the primer. They do not use radioactive probes as in restriction fragment length polymorphism (RFLP) [
16]; thus, they are suitable for the assessment of the genetic fidelity of in vitro-raised clones. However, no studies on genetic fidelity of synseed-derived plantlets of
R. serpentina have been reported.
We encapsulated individual microshoots of R. serpentina in sodium alginate beads and standardized a protocol for in vitro regeneration and short-term storage to ensure steady supply and exchange of quality plant materials. To guarantee that synthetic seed technology will indeed conserve the micropropagated propagules of R. serpentina, following their conversion from encapsulated nodal segments, the genetic fidelity of the synthetic seed-derived plantlets is assessed using RAPD and ISSR markers.
2. Results and Discussion
Nodal segments encapsulated in 3% (w/v) sodium alginate and 100 mM calcium chloride stored at 4 °C for 1, 2, 4, 6 or 8, placed on woody plant medium supplemented with 5.0 µM BA and 1.0 µM NAA showed emergence of shoots after 2 weeks of incubation (
Figure 1A,B). After four weeks of storage at 4 °C, the percentage conversion of encapsulated nodal segments into complete plantlets was 80%, whereas about 21% of non-encapsulated nodal segments produced plantlets. The alginate matrix, supplemented with the necessary ingredients, served as an artificial endosperm, thereby providing nutrients to the encapsulated explants for re-growth [
18,
19]. Antonietta
et al. [
19] reported that a synthetic endosperm should contain nutrients and a carbon source for germination and conversion. The conversion into plantlets from encapsulated nodal segments decreased as the period of storage increased beyond four weeks (
Table 1). The decline in the conversion response could be attributed to oxygen deficiency in the encapsulated beads, or to a loss of moisture due to partial desiccation during storage [
9,
20].
Different basal media MS, WPM, B5 and SH with 5.0 µM BA and 1.0 µM NAA were examined for inducing maximum conversion into shoots from encapsulated buds following storage for four weeks at 4 °C. Results revealed significant (P = 0.05) performance on woody plant medium to give maximum response of conversion of encapsulated buds into multiple shoot (
Figure 2). After eight weeks of culture well-developed shoot were observed on this medium (
Figure 1C). However, the lowest frequency of shoot formation from encapsulated bead was observed in SH medium. The data from our experiment with cold-stored encapsulated nodal segments were in agreement with the study of Faisal
et al. [
21] concerning the rates of encapsulated segments with axillary buds in
Rauvolfia tetraphylla stored at 4 °C. Similarly,
Eclipta alba encapsulated buds cold-stored for 60 days were found to display better performance and conversion indices than non-encapsulated one [
22]. The regenerated shoots were rooted in half-strength MS liquid medium containing 0.5 μM IAA on filter paper bridges (
Figure 1D). Plantlets with 4–5 fully expanded leaves and well-developed roots were successfully hardened off inside the growth room in planting substrates for 4 weeks and were eventually established in natural soil (
Figure 1E). Of the three different types of planting substrate examined, percentage survival of the plantlets was highest (90%) in soil-rite (
Table 2) and lowest (53.3%) in garden soil. About 90% of the micropropagated plants survived following transfer from soil-rite to natural soil and did not show any detectable variation in respect to morphology or growth characteristics. This observation is in agreement with several earlier findings [
6,
9,
13].
Figure 1.
Plant regeneration from synthetic seeds of R. serpentina formed by the encapsulation of nodal segments in 3% (w/v) sodium alginate and 100 mM calcium chloride; A. Shoot formation from encapsulated nodal segments on WPM + 5.0 μM BA + 1.0 μM NAA after 2 weeks of culture; B. 3 weeks old culture showing shoot formation from synthetic stored at 4 °C for 4 weeks. Shoot multiplication from encapsulated nodal segments on WPM + 5.0 μM BA + 1.0 μM NAA after 8 weeks of culture; D. Rooted synseed derived plantlets. E. Acclimatized plantlets derived from synthetic seeds.
Figure 1.
Plant regeneration from synthetic seeds of R. serpentina formed by the encapsulation of nodal segments in 3% (w/v) sodium alginate and 100 mM calcium chloride; A. Shoot formation from encapsulated nodal segments on WPM + 5.0 μM BA + 1.0 μM NAA after 2 weeks of culture; B. 3 weeks old culture showing shoot formation from synthetic stored at 4 °C for 4 weeks. Shoot multiplication from encapsulated nodal segments on WPM + 5.0 μM BA + 1.0 μM NAA after 8 weeks of culture; D. Rooted synseed derived plantlets. E. Acclimatized plantlets derived from synthetic seeds.
Table 1.
Effect of different duration of storage at 4 °C on the conversion of encapsulated and non-encapsulated nodal segments R. serpentina after 8 weeks of culture on woody plant medium supplemented with 5.0µM BA and 1.0 µM NAA a.
Table 1.
Effect of different duration of storage at 4 °C on the conversion of encapsulated and non-encapsulated nodal segments R. serpentina after 8 weeks of culture on woody plant medium supplemented with 5.0µM BA and 1.0 µM NAA a.
Storage duration (Weeks) | Encapsulated buds | Non-encapsulated buds |
---|
0 | 91.6 ± 2.7 a | 93.0 ± 3.0 a |
1 | 85.0 ± 2.3 ab | 57.0 ± 2.6 b |
2 | 81.4 ± 2.6 b | 40.2 ± 2.3 c |
4 | 80.0 ± 2.3 b | 21.0 ± 1.8 d |
6 | 57.3 ± 2.0 c | 17.1 ± 1.6 d |
8 | 50.0 ± 1.8 d | 7.0 ± 1.1 e |
Figure 2.
Effect of different medium supplemented with 5.0 µM BA and 1.0 µM NAA on the conversion of encapsulated nodal segments of R. serpentina after 4 weeks of storage at 4 °C a.
Figure 2.
Effect of different medium supplemented with 5.0 µM BA and 1.0 µM NAA on the conversion of encapsulated nodal segments of R. serpentina after 4 weeks of storage at 4 °C a.
Table 2.
Effect of different planting substrates for hardening off synseed-raised plantlets of Rauvolfia serpentina a.
Table 2.
Effect of different planting substrates for hardening off synseed-raised plantlets of Rauvolfia serpentina a.
Planting substrate | Number of plants transferred | Number of plants survived | Plant survival (%) |
---|
Garden soil | 30 | 16 | 53.3 |
Soil-rite | 30 | 27 | 90.0 |
Vermiculite | 30 | 25 | 76.6 |
For RAPD analysis 20 primers were used for initial screening with the mother plant of
R. serpentina and 19 RAPD primers gave clear and reproducible bands. The number of scorable bands for each RAPD primer varied from 2 (OPA-1) to 7 (OPA-12) (
Table 3). The 19 RAPD primers produced 160 distinct and scorable bands, with an average of 8.4 bands per primer). No polymorphism was detected during the RAPD analysis of
in vitro-raised clones (
Figure 3). All seven ISSR primers used in the initial screening produced clear and reproducible bands. The optimum annealing temperature for ISSR markers varied from 45.7 to 49.0 °C (
Table 4). The number of scorable bands for each primer varied from eight (ISSR-01) to 17 (ISSR-06), with an average of 11.4 bands per primer. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant (Figure 4). Our results corroborate with the earlier reports on genetic stability of synthetic seed derived plantlets of
Ananus comosus [
12],
Cineraria maritime [
13] and
Picrorhiza kurrooa [
14].
Table 3.
List of RAPD primers used to verify the genetic fidelity of micropropagated plantlets of Rauvolfia serpentina.
Table 3.
List of RAPD primers used to verify the genetic fidelity of micropropagated plantlets of Rauvolfia serpentina.
S. No. | Name of primers | Primer sequence (5´ – 3´) | Number of bands |
---|
1 | OPA-01 | CAGGCCCTTC | 12 |
2 | OPA-02 | TGCCGAGCTG | 12 |
3 | OPA-03 | AGTCAGCCAC | 7 |
4 | OPA-04 | AATCGGGCTG | 14 |
5 | OPA-05 | AGGGGTCTTG | 8 |
6 | OPA-06 | GGTCCCTGAC | 1 |
7 | OPA-07 | GAAACGGGTG | 7 |
8 | OPA-08 | GTGACGTAGG | 5 |
9 | OPA-09 | GGGTAACGCC | 10 |
10 | OPA-10 | GTGATCGCAG | 13 |
11 | OPA-11 | CAATCGCCGT | 6 |
12 | OPA-12 | TCGGCGATAG | 7 |
13 | OPA-13 | CAGCACCCAC | 15 |
14 | OPA-14 | TCTGTGCTGG | 12 |
15 | OPA-15 | TTCCGAACCC | 5 |
16 | OPA-16 | AGCCAGCGAA | 0 |
17 | OPA-17 | GACCGCTTGT | 3 |
18 | OPA-18 | AGGTGACCGT | 11 |
19 | OPA-19 | CAAACGTCGG | 4 |
20 | OPA-20 | GTTGCGATCC | 8 |
Table 4.
List of ISSR primers used to verify the genetic fidelity of micropropagated plantlets of Rauvolfia serpentina a.
Table 4.
List of ISSR primers used to verify the genetic fidelity of micropropagated plantlets of Rauvolfia serpentina a.
S. No. | Name of primers | Primer sequence (5′ – 3′) | Annealing temperature °C | Number of bands |
---|
1 | ISSR-01 | ACA CAC ACA CAC ACA CT | 45.7 | 8 |
2 | ISSR-02 | ACA CAC ACA CAC ACA CG | 49.0 | 12 |
3 | ISSR-03 | AGA GAG AGA GAG AGA GYT | 49.0 | 12 |
4 | ISSR-04 | GAG AGA GAG AGA GAG AYC | 49.0 | 13 |
5 | ISSR-05 | ACA CAC ACA CAC ACA CYT | 49.0 | 15 |
6 | ISSR-06 | DBD ACA CAC ACA CAC AC | 45.7 | 17 |
7 | ISSR-07 | HVH TGT GTG TGT GTG TG | 45.7 | 13 |
The two PCR-based techniques, RAPD and ISSR, were used to test clonal fidelity because of their simplicity and cost-effectiveness. The use of two markers, which amplify different regions of the genome, allows better chances for the identification of genetic variation [
23]. In this study the number of bands generated per primer was greater in ISSR (11.4) than RAPD (8.4). These differences could possibly be due to the high melting temperature for the ISSR primers, which permits much more stringent annealing conditions and, consequently, more specific and reproducible amplification. Devarumath
et al. [
24] also revealed that ISSR fingerprints detected more polymorphic loci than RAPD fingerprinting.
Figure 3.
Agarose gel electrophoresis of RAPD and ISSR products of synseed-raised Rauvolfia serpentina plantlets and mother line obtained with primer OPA-11 (A) and ISSR-03 (B). Lanes 1–9, regenerants; Lane MP=mother plant; lane M = Lambda DNA/Eco RI + HindIII marker indicated in bp.
Figure 3.
Agarose gel electrophoresis of RAPD and ISSR products of synseed-raised Rauvolfia serpentina plantlets and mother line obtained with primer OPA-11 (A) and ISSR-03 (B). Lanes 1–9, regenerants; Lane MP=mother plant; lane M = Lambda DNA/Eco RI + HindIII marker indicated in bp.