Polyzygus tuberosus Dalz An assessment of genetic stability in micropropagated plants of Ochreinauclea missionis by RAPD markers

Chandrika M. and Ravishankar rai V*

Department of Studies in Biotechnology,University ofMysore, Manasagangotri, Mysore-570006, Karnataka,India

*For correspondence - rrai33@hotmail.com

Current Trends in Biotechnology and Pharmacy , Volume 3 (3) July - 2009

Abstract

Present study reports the use of randomly amplified polymorphic DNA (RAPD) markers, to verify the clonal fidelity between the micropropagated plants and the mother plant of Ochreinauclea missionis, an ethnomedicinal and endemic tree in Western Ghats of India. Thirty eight RAPD primers were screened, out of which thirty two primers generated a total of 245 clear, distinct and reproducible bands. Out of 245 total bands, 227 bands were monomorphic with 92.67 % and 18 bands showed polymorphic banding pattern with 7.34 %. Thus, a total of 5390 bands were generated showing uniform banding patterns for each primer that are comparable to the mother plant from which the cultures were established. Cluster analysis based on unweighted pair group method with arithmetic averaging (UPGMA) showed 97% similarity between the mother plant and micropropagated plants. The developed RAPD profiles confirmed the clonal fidelity of tissue culture raised plantlets which were reintroduced to its original habitat for conservation.

Keywords: Ochreinauclea missionis, Medicinal tree, Genetic fidelity, Polymerase chain reaction, RAPD analysis.

Introduction

Ochreinauclea missionis (Wall. ex G. Don) Ridsd. locally known as ‘Jalamdasa’, belongs to the family Rubiaceae. It is a medium sized evergreen threatened medicinal tree and is endemic to Central and Southern Western Ghats of peninsularIndia (1). The powdered bark and its decoction are used for curing cutaneous diseases like leprosy, ulcers and as an effective purgative (2). Root and root bark are employed in treating rheumatism, paralysis, skin diseases, dropsy, eye diseases, constipation, piles, jaundice, fever, edema, hepatic and haemophilic disorders (1).  O. missionis is reported as rare due to dwindling of natural forest as a result of construction of dams, hydroelectric projects and roads for agricultural purposes. To our knowledge, there have been no reports on the comparative genomic stability or variation in regenerated plants and mother plant of O. missionis. However, the vegetative propagation and in vitro regeneration through nodal explants were previously described (3, 4). In vitro clonal propagation of trees is an attractive method for obtaining high number of elite genotypes. The somaclonal variation of the micropropagated elite genotypes can be a potential draw back. However, true-to-type clonal fidelity is one of the most important prerequisites in the micropropagation of forest tree species (5).

In recent years, several DNA markers have been successfully employed to assess the genomic stability in regenerated plants including those with no obvious phenotypic alternations (6). Among the markers, RAPD technique being simple and cost effective has been used in numerous studies. RAPD analysis is particularly well suited to high-output systems required for plant breeding because it is easy to perform, fast, reliable and of relatively low cost (7). Nevertheless, RAPD technique has some limitations concerning reproducibility and an uncertain homology of co-migrating fragments in gel electrophoresis (8). But, most of these limitations can be minimized by carefully adjusting the reaction and detection conditions (9). RAPD analysis can be applied to assess the genetic fidelity of plants derived in vitro on an industrial scale as part of crop improvement programs (10). This method might be useful for monitoring the stability of in vitro germplasm collections and cryopreserved material. RAPD analysis in plants has also been widely used to detect genetic and somaclonal variations (11-14).  RAPD technique does not require DNA sequence information and species specificity and hence it is being conveniently used for assessing genetic stability and clonal fidelity of micropropagated plants in a number of genera. The RAPD markers are referred to as an appropriate tool to get rapid information about genetic similarities or dissimilarities in micropropagules. Thus, in the present study we report the assessment of genetic integrity in tissue cultured O. missionis plants with their mother plant using RAPD markers.

Materials and Methods

Plant material and in vitro regeneration

Tender branches were excised from 8-10 year old mature trees of O. missionis growing along the river bank side of Seethanadhi in Udupi district, Karnataka,India. In vitro regeneration was achieved using the earlier standardized protocol (4). Multiple shoots were initiated from nodal explants on Murashige and Skoog’s (MS) medium (15) incorporated with 2 mg/l 6-benzylaminopurine (BA) and 0.3% (w/v) activated charcoal (AC). After 5 weeks, in vitro developed shoots were subcultured onto MS medium containing BA (0.5 mg/l) and naphthaleneacetic acid (NAA 1 mg/l) and AC (0.3%) for shoot elongation. For ex vitro rooting, the base of in vitro shoots was dipped in different concentrations of IBA solution for different time durations and immediately transferred into bottles and plastic pots containing sterile soilrite (equal proportions of decomposed coir and peat moss, Karnataka Explosives, Bangalore, India) and the plantlets were hardened in greenhouse for a period of six months before transferring to field conditions.

DNA extraction

Twenty one tagged regenerated plants from hardening stage were randomly selected along with single mother plant for screening their genetic integrity. Total DNA was extracted from fresh young leaves of micropropagated plants and field grown mother plant using cetyl trimethyl ammonium bromide (CTAB) method as described by Doyle and Doyle (16) with minor modifications. Quality and quantity of DNA were inspected both by gel electrophoresis and spectrometric assays using UV-Visible Double Beam PC Scanning spectrophotometer (LABOMED, Culver city,USA).

RAPD analysis

PCR amplification was performed with 38 arbitrary decamer RAPD primers (Sigma Aldrich chemicals Pvt. Ltd., Bangalore,India). A total of thirty eight RAPD primers were screened initially and 32 primers were selected in the present study.  Amplifications were performed as described in earlier report (7) in a total volume of 20 µl reaction mixture containing 2 µl of 1´ assay buffer (10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl and 0.01 % gelatin, pH 9.0), 250 µM dNTPs, 200 µM primer, 1.5 unit (U) Taq DNA polymerase (Bangalore Genei, Bangalore, India) and 50 ng genomic DNA. PCR amplification was performed using thermocycler UNO II (Biometra, Goettingen, Germany) with hot lid according to the following programme with initial denaturation of 94 C for 3 min, followed by 40 cycles for 60s at 94 C for denaturation, 1 min at annealing temperature, 2 min extension step at 72 C and a final extension step at 72 C for 10 min. The annealing temperature was adjusted according to the primers used in the PCR. The amplification products were resolved by electrophoresis on 1.5 % (w/v) agarose gel (Amersham, Uppsala,Sweden) in 1x TBE buffer (Tris–Borate–EDTA buffer) at 75-100 volts. The gels were stained with ethidium bromide solution. The amplified products were visualized and photographed under UV transilluminator and documented using Bioprofile Image Analysis System (Vilber Lourmat,France). Molecular marker λ DNA / EcoR I -Hind III double digest (Bangalore Genei, Bangalore,India) was used to estimate the size of amplification products. In order to have reproducible and clear banding patterns, PCR amplifications were repeated for atleast twice.

Data analysis

The presence and absence of bands between samples was scored and data were transcribed into binary format (1, 0 respectively) in each plant at a particular position, which was treated as an independent character regardless of its intensity. PCR amplified bands in the size range of 200 to 21,226 bp were scored with all the selected RAPD primers. Based on the matrix of genetic similarity, cluster analysis was performed. The similarity coefficients thus generated were used for constructing dendrogram using the UPGMA (unweighted pair-group method with arithmetic average) and the SHAN (sequential hierarchical agglomerative nested clustering) option in NTSYS-pc software package (17). The three dimensional principle coordinate analysis (PCA) was conducted with the same program using EIGEN module. This multivariate approach was chosen to complement the cluster analysis information. Genetic similarities between micropropagated and mother plants were used to calculate the Jaccard’s similarity coefficient (18).

Reintroduction of micropropagated plants

After confirming the genetic stability, the tissue cultured plants were maintained in greenhouse for a period of six months.Nearly, five hundred micropropagated plants were reintroduced in rainy season (June - August) into their original habitat near Mani region (Udupi district, Karnataka,India) with the help of local people. After one year, the survival rate of reintroduced plants was recorded. The growth conditions were 22 ± 2 C with 70% relative humidity, in normal day light conditions.

Results

Micropropagation

Previously standardized micropropagation protocol was used to establish the large scale propagation of in vitro plants (4) for the assessment of genetic fidelity. The highest frequency of nodal explants responding (83.3%) was observed on MS medium supplemented with 2 mg/l BA and 0.3% AC. For subculture, incorporation of NAA (1 mg/l) in combination with BA (0.5 mg/l) gave a maximum of 9.7±1.2 shoots/explant. High percentage of rooting (91.6) with a maximum root length of 3.8cm was observed at 30 min exposure of multiple shoots in 10 mg/l indole-3-butyric acid (IBA) solution. Rooted plantlets were acclimatized in growthchamber (Sanyo, Moriguchi-city, Osaka,Japan) under temperature of 25±2 C, 80% relative humidity, irradiance of 50 μmol m^-2 s^-1 with 16 h of photoperiod for 4 weeks. Then plantlets were hardened in greenhouse conditions for a period of six months before transferring to their habitat.

Genetic stability by using RAPD primers

We screened thirty eight RAPD primers for this analysis but only thirty two primers were useful in reproducing the banding patterns. Therefore, remaining six primers were discarded as they were producing ambiguous and non reproducible amplification profiles. Other 32 primers produced a total of 245 clear and reproducible bands of which, 227 bands were monomorphic with 92.67% and remaining 18 bands were polymorphic with 7.34%. Indeed, all these 32 primers generated identical banding patterns in two independent amplifications that were performed for all the samples. The number of bands per each primer varied from 2 to 12 with an average of 7.65 bands per primer. A total of 5390 fragments (numbers of plantlets analyzed × number of bands in all the primers) were generated showing homogeneous RAPD banding patterns. The selected RAPD primers, their annealing temperature, total number of bands scored, their base pairs size, monomorphic bands and polymorphic bands for each primer are summarized in table 1. RAPD amplifications were observed for monomorphic pattern in primer OPE-20 (Fig. 1a) and polymorphic in primer OPC-05 (Fig.1b).

The dendrogram constructed on the basis of Jaccard’s similarity matrix, followed by UPGMA based clustering analysis (Fig. 2a) showed that the genotypes were grouped into single cluster with the donor mother plant, which comprises of twenty tissue cultured plants, while plant ‘Om1’ fell apart from this clustering. The coefficient of similarity in the dendrogram generated by the RAPD data among the regenerated plants ranged from 0.953 to 0.997 with a mean of 0.976 indicating the genetic similarity of 97%. The linear relationship among 21 micropropagated plants and donor plant are shown in PCA (Fig. 2b). In the three-dimensional plot of PCA where in vitro plants ‘Om2 to Om21’ fell within donor plant showing genetic similarity but plantlet ‘Om1’ lies apart from all showing significant changes in PCA. The PCA and dendrogram both results showed that the micropropagated plants exhibited distinct genetic similarity with mother plant. All groups showed genetic stability among each other and comes under the donor group showing monomorphism. Most of the micropropagated plants reintroduced into the Mani region (Karnataka,India) showed better establishment without any morphological variation, with 70% of survival rate.

Discussion

Plant tissue culture has been successfully used for large scale propagation of number of plant species including many medicinal plants (19). The genetic integrity of micropropagated plants can be determined with the use of various techniques. The choice of a molecular marker technique depends on its reproducibility and simplicity. In this study, RAPD technique was employed to assess the true to type clonal fidelity of O. missionis plantlets. In order to confirm genetic integrity, the DNA profile of 21 regenerated plants was compared with the DNA profile of the mother plant. Many workers suggested the role of in vitro propagation of rare and endangered plants as an effective means for conservation but there are limited reports on reintroduction of micropropagated plants in wild (20), or to ex situ conservations. Screening the tissue culture derived plants at an early stage or during hardening and prior to reintroduction using molecular markers will assist in reintroducing true-to-type plants (21) and protecting their genetic integrity (22).

The visual assessment of over hundred plants derived from the axillary bud culture did not reveal any morphological variation in the micropropagated plants of Eucalyptus tereticornis and E. camaldulensis (23) and Tylophora indica (24). The identification of variability in micropropagated plants derived from the same donor mother plant as in Populus deltoides (11) and Piper longum (25) using RAPD and in few other cases however provides evidences for the existence of variants. Thus, suggesting visual phenotypic evaluation may not be sufficient for characterization of in vitro plants. Screening for DNA variations among several millions of base-pair could be more problematic and exhaustive than scoring for a few morphological variations. The RAPD technique was found useful in examining genetic fidelity of tissue culture-clones. With the use of RAPD markers, clonal fidelity of tissue cultured plants has been determined in micropropagated species of Drosera anglica and D. binata (26), Cedrus (27), Swertia chirata, (28), Macadamia tetraphylla (29), sugarcane (30) and Mucuna pruiens (31).

The 21 randomly tagged plants were found phenotypically normal and essentially identical with their mother plant at hardening stage which partly suggest the minimal or absence of somaclonal variations. A total of 32 primers resulted in 245 distinct and reproducible bands showing homogeneous RAPD patterns. Band intensity of each gel confirmed their monomorphic nature of 92.67% and with low level of genetic variation of 7.34% in RAPD primers among the plantlets analyzed. Similarly, an average of about 97% genetic fidelity was maintained among the 18 micropropagated populations raised from shoot tip explants of sugarcane (32), 97.4% homology and 7.6% polymorphism were observed in 15 micropropagated plants of Syzygium travancoricum (22) and 7.74% polymorphism and 92.25% genetic similarity were seen in in vitro plants of Gypsophila paniculata (33). Whereas, high level of polymorphic variations were observed in regenerants of Codonopsis lanceolata, a medicinal plant, with 24.9% of polymorphism, 23.2% polymorphism were seen in micropropagated apple root stock plants (12) and 32% in micropropagated plants of Robinia pseudoacacia (10). By using ISSR primers, low polymorphism of 3.92% among the twenty one in vitro grown Dictyospermum ovalifolium plantlets was reported (34).

In O. missionis plantlets, a total of 5390 bands were generated using 32 RAPD primers. Similarly, a total of 6520 reproducible bands were obtained in Quercus serrata plantlets (35), 1056 bands from micropropagated plants of Syzygium travancoricum (22), 1856 and 2320 scorable bands in Drosera binata and Drosera anglica respectively (26) and 925 bands in ten tissue culture clones of Mucuna pruriens (31). In O. missionis, the number of bands of each primer varied from 2 to 12, with an average of 7.65 bands per primer. On similar findings, bands varied from 1 to 7, with an average of 3.02 bands per primer in Quercus serrata (35), 2 to 8 bands, with an average of 4.6 per primer in micropropagated plants of Syzygium travancoricum (22) and in Hagenia abyssinica regenerated plantlets, number of bands per primer ranged from 4 to 11 with an average of 7.2 bands per primer (5). In conclusion, a simple, efficient and high fidelity protocol for comparative assessment of genetic stability in micropropagated plantlets and mother plant of O. missionis has been established. The present study indicates that the molecular analysis of plants at the hardening stage provides an early evaluation of plants for clonal fidelity. The result presented demonstrate that the RAPD technique proved to be effective in generating reproducible results useful in assessment of genetic fidelity of micropropagated plants in O. missionis. Since O. missionis is threatened medicinal plant, production of genetically true to type plants is important for the germplasm conservation.

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Table 1 Number of amplification products generated with RAPD primers in the analysis of O. missionis plants.
(A.T-Annealing Temperature, P.B-Polymorphic Bands, M.B-Monomorphic Bands)