Molecular and Biochemical Characterization in

Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage Mohammad Faisal, Abdularhaman A. Alatar & Ahmad K. Hegazy

Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-012-9977-0

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Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-012-9977-0

Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage Mohammad Faisal & Abdularhaman A. Alatar & Ahmad K. Hegazy

Received: 3 December 2011 / Accepted: 6 November 2012 # Springer Science+Business Media New York 2012

Abstract Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitroproliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl2. The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant. Keywords Cold storage . Genetic stability . Germplasm storage . Plant regeneration . Synthetic seed M. Faisal (*) : A. A. Alatar : A. K. Hegazy Department of Botany & Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia e-mail: [email protected] A. K. Hegazy Department of Botany, Faculty of Science, Cairo University, Giza, Egypt

Author's personal copy Appl Biochem Biotechnol

Introduction Rauvolfia tetraphylla (Apocynaceae) commonly known as “devil pepper” or “be still tree” is an endangered woody shrub native in tropical Americas. It has been cultivated widely as both an ornamental and as a source of pharmaceuticals and is now naturalized throughout the tropics including Australasia, Indochina, and India. It holds an important position in the Indian traditional system of medicine and has other immense applications. This particular plant is regarded as a rich source of a wide variety of important alkaloid constituents such as reserpine, reserpiline, raujemidine, isoreserpiline, deserpidine, aricine, ajmaline, ajmalicine, yohimbines, serpentine, sarpagine, vellosimine, and tetrphylline [1]. Reserpine is a potent alkaloid that depresses the central nervous system and lowers blood pressure. The root is also used to stimulate uterine contraction and is recommended for use in difficult childbirth cases. Indiscriminate collection from wilds and lack of organized cultivation of this plant have led to their rapid depletion from natural habitat [2]. Production of artificial seed has unraveled new vistas in plant biotechnology by providing an alternative dependable way for in vitro mass scale production, efficient delivery of cloned plantlets, and also to meet the international quarantine requirements. Recently, there has been increased interest in alginate encapsulation technology for the production of synthetic seed which has been widely utilized for in vitro propagation and conservation of medicinal and aromatic plant species [3–9]. Molecular and biochemical characterization of synseed-derived plantlets remains relatively unknown with the exception of some recent report on Dioscorea bulbifera [10], Cineraria maritima [11], Eclipta alba [12], Cannabis sativa [13], and Picrorhiza kurrooa [14]. In contrast, assessment of genet variability of cryopreserved materials via encapsulation/dehydration or verification methods has attracted more attention [15–18]. In the present investigation, we describe an efficient protocol for in vitro clonal multiplication and method of germplasm exchange and distribution through synthetic seeds in R. tetraphylla. Molecular and biochemical characterization of conserved propagules following their conversion into plantlets from encapsulated nodal segments stored at 4 °C for weeks was also assessed and compared with ex vitro-grown plants.

Materials and Methods Plant Material and Explant Source Nodal segments from a field grown plant of R. tetraphylla were used to establish in vitro cultures. Explants were washed thoroughly under running tap water for 30 min to remove adhering particles, surface sterilized with 0.1 % HgCl2 for 4 min, and rinsed four to five times with sterile distilled water. For in vitro shoot regeneration, the sterilized explants were cultured on Llyod and McCown woody plant medium (WPM) [19] supplemented with 7.5 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA). The pH of the medium was adjusted to 5.8 by using 1 N NaOH or HCL prior to addition of 0.8 % (w/v) agar. The medium was autoclaved at 121 °C at 1.1 kgcm2 for 20 min. Cultures were maintained at 24±2 °C with a 16-h photoperiod at a photon flux density (PFD) of 50 μmolm−2 s−1 from cool white fluorescent lamps. Encapsulation and Low Temperature Storage The gelling mixture, consisting of half-strength woody plant medium, 3 % (w/v) sucrose, 3 % (w/v) sodium alginate, and 100 mM CaCl2, was autoclaved at 121 °C for 20 min under


15 lb pressure. Explants mixed with sodium alginate were suspended dropwise in CaCl2 solution so that each drop contained single explant. They were kept for 30 min for complete polymerization of alginate. The resulting beads were subsequently washed thoroughly with sterile distilled water to remove the excess CaCl2 and then stored in dark at different temperatures (4, 8, 12, and 16 °C) for 4 weeks. Planting Medium and Culture Conditions For conversion into plantlets, the encapsulated beads were cultured in vitro on Petri dishes containing different culture media, i.e., 1/2 WPM, 1/3 WPM, and 1/4 WPM in addition to standard WPM supplemented with 7.5 μM BA and 2.5 μM NAA. Sprouted buds were transferred to 250 ml culture flask containing the respective media as that used for shoot initiation. The media were solidified with 0.8 % (w/v) agar and the pH was adjusted to 5.8 with 0.1 M NaOH before being autoclaved at 121 °C for 20 min under 15 lb pressure. All cultures were maintained at 24°±2 °C under a 16-h photoperiod with a PFD of 50 μmolm−2 s–1, provided by cool white fluorescent lamps. Rooting and Acclimatization The regenerated shoots were excised and transferred individually to half-strength Murashige and Skoog (MS) medium containing 0.5 μM indole-3-butyric acid for root development. Rooted plantlets were removed from the culture medium, washed gently under running tap water, and transferred to plastic pots containing sterile garden soil under diffuse light (16:8 h photoperiod) conditions. Potted plantlets were covered with a transparent polythene membrane to ensure high humidity and watered every 3 days with half-strength MS salt solution. After 4 weeks, acclimatized plants were transferred to pots containing normal soil and maintained in a greenhouse under normal day length conditions. Photosynthetic Pigments and Net Photosynthetic Rate Leaf chlorophyll and carotenoids were extracted with 80 % acetone and estimated spectrophotometrically [20, 21]. Photosynthesis was measured on the fully expanded top leaf of each main axis with a portable photosynthetic system (LI-COR 6400; LI-COR Biosciences, Lincoln, NE, USA) at 900 μmolm−2 s−1 photosynthetically active radiations between 11:00 a.m. and12:00 noon. Extraction and Estimation of Alkaloids For alkaloid estimation, HPLC (Shimadzu HPLC System SCL-10) was performed using a Lichrosorb C18 column (25×0.5 cm) with methanol (CH3OH):acetonitrile (CH3CN) (60:40 v/ v) as mobile phase at 1 ml and wavelength was fixed at 268 nm. Root samples of Rauvolfia tetraphylla were dried at 60 °C for 24 h. One hundred milligrams of the powdered tissues was pulverized with petroleum ether and extracted with methanol ammonia (98:2 w/v). The alkaloids from the extracts were separated by the procedure described earlier by Roja et al. [22]. Genomic DNA Extraction and PCR Amplification Genomic DNA was extracted from synseed-derived plantlets and mother plant following the method described by Doyle and Doyle [23], modified by Weising et al. [24]. Purified total


DNA was quantified and its quality verified by spectrophotometry (UV 1700-PharmaSpec, Shimadzu, Tokyo, Japan). Regenerated plantlets were tested for clonal fidelity using 20 random amplified polymorphic DNA (RAPD) primers (Operon Kit A and Kit C) and 8 intersimple sequence repeats (ISSR) primers (UBC-825, UBC-827, UBC-834, UBC-841, UBC855, UBC-866, UBC-868, and UBC-880) for their unambiguous and reproducible band patterns. PCR amplifications were carried out as described by Williams et al. [25]. PCR reactions were performed in a volume of 25 μl containing 25 ng total DNA, 1 μl PCR buffer (Fermentas GmbH, Germany), 2.0 mM MgCl2, 200 mM dNTPs, 1 mM primer, and 1 unit Taq DNA polymerase (Fermentas). The PCR amplification was performed using a thermal cycler (T-Gradient Biometra, Göttingen, Germany). For RAPD analysis, PCR temperature profiles were used as initial DNA denaturation at 94 °C for 5 min followed by 40 cycles at 94 °C for 1 min, 35 °C for 1 min, and 72 °C for 2 min. Final cycle at 72 °C for 7 min was also performed. For ISSR markers, PCR reactions were performed with initial DNA denaturation at 94 °C for 5 min followed by 35 cycles at 94 °C (1 min) for DNA denaturation, 45.7 or 55.4 °C (1 min) for primer annealing, 72 °C (2 min) for primer extension, and final extension at 72 °C for 7 min was performed. All the amplified PCR products obtained from RAPD and ISSR markers were resolved by electrophoresis on 1.4 and 3.5 % agarose gel, respectively, for 3 h in 1× TBE buffer and stained with ethidium bromide, and the photographs were taken using Gel Documentation System (Gel-Doc EZ System, Bio-Rad, USA).

Statistical Analysis All of the experiments were conducted with a minimum of seven replicates for each treatment and the experiments were repeated thrice. The cultures were observed periodically and morphological changes were recorded at regular intervals. The results were analyzed statistically using SPSS version 20 (SPSS Inc., Chicago, IL, USA). Significant differences among means were analyzed using Tukey’s test at P00.05.

Fig. 1 a Synthetic seeds formed by encapsulation of nodal segments using 3 % sodium alginate and 100 mM calcium chloride. b Shoot formation from encapsulated microshoots on woody plant medium after 8 weeks of culture

Author's personal copy Appl Biochem Biotechnol 100 a 80

% Regeneration

Fig. 2 Evaluation of medium strength supplemented with 7.5 μM BA and 2.5 μM NAA on the conversion of encapsulated nodal segments of R. tetraphylla after 8 weeks of culture

b c

60

d 40

20

0 WPM

1/2WPM

1/3WPM

1/4WPM

Medium strength

Results and Discussion A highly desirable feature of encapsulated nodal segment is their ability to retain viability in terms of sprouting and conversion potential even after a considerable period of storage, which is essential for their use in germplasm exchange or field application. In the present investigation, synseeds of R. tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl2 (Fig. 1a) stored at different temperatures (4, 8, 12, and 16 °C) for 4 weeks. The encapsulated beads stored at 4 °C, cultured on woody plant medium, and supplemented with 7.5 μM BA and 2.5 μM NAA showed highest frequency of conversion (cf. 72.0 %). After 8 weeks of culture, well-developed shoots were observed on this medium (Fig. 1b). This temperature was chosen as optimum for further experiment to standardize the best suitable media for conversion into plantlets. Low temperature above freezing (around 4 °C) was described suitable for storage and subsequent high conversion of synthetic seeds in R. serpentina [9], E. alba [12], Ananas comosus, [26], and Morus indica [27].

Fig. 3 Plants derived from synthetic seeds growing in greenhouse

Author's personal copy Appl Biochem Biotechnol Table 1 Comparison of photosynthetic pigments and net photosynthetic rate (PN) of in vitro- and in vivogrown plants Attributes

In vitro-regenerated plants

In vivo-grown plants

Total chlorophyll (mgg−1 fresh weight)

1.90±0.48

1.92±0.35

Chlorophyll a/b ratio (mgg−1 fresh weight)

2.61±0.63

2.55±0.41

Carotenoids (mgg−1 fresh weight)

0.45±0.12

0.41±0.15

PN (μmol CO2 m−2 s−1)

9.61±0.35

9.57±0.43

However, the synthetic seeds stored at 8, 12, and 16 °C could not be stored beyond 4 weeks due to shoot formation even while in storage. Effect of medium strength on shoot proliferation from encapsulated nodal segments stored at 4 °C for 4 weeks was also examined, with 1/2 WPM, 1/3 WPM, and 1/4 WPM in addition to standard WPM (Fig. 2). Proliferation of shoot was found to be best on 1/2 WPM medium and poorest on 1/4 WPM. These results indicate that the salt concentration in media influences shoot regeneration from encapsulated nodal segments. Similarly, Siddique and Anis [28] indicated that 1/2 MS medium induced shoots from encapsulated buds effectively as compared to full-strength MS medium in Ocimum sanctum. The percentage conversion of encapsulated nodal segments into complete plantlets was 80.6 % after 8 weeks of culture on half-strength woody plant medium supplemented with 7.5 μM BA and 2.5 μM NAA (Fig. 2). Regenerated plantlets with well-developed shoot and root system transferred to plastic pots containing sterile soilrite were successfully hardened off inside the growth room for 4 weeks and eventually established in natural soil (Fig. 3). About 90 % of the synseed-grown plantlets survived following transfer from soilrite to natural soil and did not show any detectable variation in respect to morphology or growth characteristics. Data on chlorophyll, carotenoids, chlorophyll a/b ratio, and net photosynthetic rate are given in Table 1. Almost the same data for total chlorophyll content were recorded in in vitro- and in vivo-grown plants. Chlorophyll a/b ratio and carotenoids content were found to be higher in regenerated plants. Net photosynthetic (PN) rate was almost the same in synseed seed-derived regenerants and ex vitro-grown plants. This observation is in agreement with several earlier findings [29, 30]. Furthermore, our results using HPLC analysis also showed homogeneity in the alkaloid (ajamline, reserpine, and serpentine) contents of the mother plant and the randomly selected Fig. 4 Comparison of alkaloid contents (percent dry weight) in mother plant and randomly selected synseed-raised plantlets of R. tetraphylla

Ajmaline

Reserpine

Serpenine 0.19

0.17

0.12

0.11

0.028

Regenerated plant

0.031

Mother plant


Table 2 RAPD primers used to assess the genetic fidelity of synseed grown plantlets of Rauvolfia tetraphylla

Sample no. Name of Primer sequence (5′–3′) Number of bands primers 1

OPA-01

CAGGCCCTTC

01

2

OPA-02

TGCCGAGCTG

05

3

OPA-03

AGTCAGCCAC

09

4

OPA-04

AATCGGGCTG

05

5

OPA-05

AGGGGTCTTG

07

6 7

OPA-06 OPA-07

GGTCCCTGAC GAAACGGGTG

0 05

8

OPA-08

GTGACGTAGG

0

9

OPA-09

GGGTAACGCC

06

10

OPA-10

GTGATCGCAG

02

11

OPC-01

TTCGAGCCAG

01

12

OPC-02

GTGAGGCGTC

09

13

OPC-03

GGGGGTCTTT

0

14 15

OPC-04 OPC-05

CCGCATCTAC GATGACCGCC

08 05

16

OPC-06

GAACGGACTC

0

17

OPC-07

GTCCCGACGA

03

18

OPC-08

TGGACCGGTG

13

19

OPC-09

CTCACCGTCC

04

20

OPC-10

TGTCTGGGTG

03

clones propagated through synthetic seeds following storage at 4 °C for 4 weeks (Fig. 4). These results confirm that the biochemical mechanism followed to produce the synthetic seeds maintains the same metabolic profile of the mother plant and, therefore, synthetic seeds can be used as a cost-effective mechanism for the short-term conservation and mass propagation of true-to-type plants of R. tetraphylla. RAPD and ISSR analyses were also carried out to test genetic fidelity of ten randomly selected synseed seed-derived plantlets and compared with those of the donor plant. Twenty RAPD primers were screened, and 17 primers yielded clear, reproducible bands with a total of 86 bands and on average of 4.3 bands per primer (Table 2). For ISSR, eight primers were Table 3 ISSR primers used to assess the genetic fidelity of synseed grown plantlets of R. tetraphylla Sample no.

Name of primers

Primer sequence (5′–3′)

Annealing temperature, °C

Number of bands

1

UBC-825

ACA CAC ACA CAC ACA CT

45.7

11

2

UBC-827

ACA CAC ACA CAC ACA CG

49.0

10

3

UBC-834

AGA GAG AGA GAG AGA GYT

49.0

12

4

UBC-841

GAG AGA GAG AGA GAG AYC

49.0

14

5

UBC-855

ACA CAC ACA CAC ACA CYT

49.0

15

6 7

UBC-866 UBC-868

CTC CTC CTC CTC CTC CTC GAA GAA GAA GAA GAA GAA

55.4 45.7

13 14

8

UBC-880

GGG TGG GGT GGG GTG

49.0

17


used to screen the regenerated plants. All the eight primers gave clear and scorable bands and were used for ISSR-PCR. Average number of bands for each primer is 13.2 with a total of 106 bands (Table 3). All the RAPD- and ISSR-tested primers produced monomorphic pattern across all the plants and the mother plant, confirming the genetic uniformity of the synseed-derived plantlets (Fig. 5a, b). Findings of this study corroborate earlier reports of the genetic fidelity of synthetic seed-derived plantlets [11–14] and cryopreserved tissues of various plant species [31, 32]. To conclude, the synthetic seed technology described in this paper provides an alternative method of clonal propagation of this endangered medicinal plant. The comparison of physiological, biochemical, and molecular parameters showed considerable similarities. Successful plant retrieval from encapsulated nodal segments stored at 4 °C for 4 weeks indicates that the method could be potentially used to conserve desirable elite genotype of R. tetraphylla over a short period.

M

D

1

2

3

4

5

6

7

8

9

10

A

B

Fig. 5 Representative RAPD and ISSR profiles of donor and synseed-derived plants of R. tetraphylla. a RAPD with primer OPC-02. b ISSR with primer UBC-866. Lane M DNA molecular weight marker; lane D donor plant; lanes 1–10 randomly selected regenerated plants

Author's personal copy Appl Biochem Biotechnol Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project number RGP-VPP-175.

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