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Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant
FITOTE-02709; No of Pages 6 Fitoterapia xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Fitoterapia
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Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant
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Kuldeep Yadav a, Ashok Aggarwal b, Narender Singh a,⁎
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Plant Tissue Culture Laboratory, Department of Botany, Kurukshetra University, Haryana 136119, India Mycology and Plant Pathology Laboratory, Department of Botany, Kurukshetra University, Haryana 136119, India
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journal homepage: www.elsevier.com/locate/fitote
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Article history: Received 19 March 2013 Accepted in revised form 11 June 2013 Available online xxxx
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Keywords: Gloriosa superba Micropropagation Genetic fidelity Molecular markers RAPD ISSR
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Malabar glory lily (Gloriosa superba L.) is a medicinally potent plant species used for the production of alkaloid colchicine. With ever increasing demand, there is a pressing need to conserve it through biotechnological approaches. A large number of complete plantlets were obtained by direct regeneration from the non-dormant tuber explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) + 0.5 mg/l α-naphthalene acetic acid (NAA). Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, the genetic stability of micropropagated clones was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. During the study a total of 80 (50 RAPD and 30 ISSR) primers were screened, out of which 10 RAPD and 7 ISSR primers produced a total of 98 (49 RAPD and 49 ISSR) clear, distinct and reproducible amplicons. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. This is the first report that evaluates the use of genetic markers to establish genetic fidelity of micropropagated G. superba using RAPD and ISSR, which can be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays for colchicine production to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses. © 2013 Published by Elsevier B.V.
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1. Introduction
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Malabar glory lily (Gloriosa superba L.), the sole species in the family Colchicaceae is a perennial tuberous climbing herb widely scattered in tropical and sub-tropical parts of Africa and Southeast Asia. In India, it is usually found in the Himalayan foot-hills, Tamil Nadu, Andhra Pradesh and Bengal
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Abbreviations: dNTPs, deoxyribonucleotide triphosphates; PCR, polymerase chain reaction; BAP, 6-benzylaminopurine; NAA, α-naphthalene acetic acid; ISSR, inter simple sequence repeats; RAPD, random amplified polymorphic DNA. ⁎ Corresponding author at: Department of Botany, Kurukshetra University, Haryana 136119, India. Tel.: +91 1744 238410; fax: +91 1744 238277. E-mail address: [email protected] (N. Singh).
[27]. Its attractive wavy edged orange–red flower is the national flower of Zimbabwe and also the state flower of Tamil Nadu in India. The tubers and seeds of this plant contain more colchicine content than the genera Colchicum [3]. Colchicine alkaloid (Fig. 1) is highly demanded due to its uses for treating arthritis, Mediterranean fever, gout, rheumatism, inflammation, ulcers, bleeding piles, skin diseases, leprosy, impotency and snakebites [7]. Colchicine also has antimitotic activity, preventing growth of cancer cells by interacting with microtubules, which could lead to the design of better cancer therapeutics [29]. Due to the action of colchicine on spindle-fiber formation during celldivision, it is also capable of inducing polyploidy and used in cytological and plant breeding research [10].
0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.06.009
Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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2.1. Plant material
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Colchicine (C22H25NO6) Fig. 1. Colchicine (C22H25NO6).
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2.2. DNA extraction and PCR amplification
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Total genomic DNA was extracted using the method of Doyle and Doyle (1990) from leaf samples. The quality of extracted DNA after RNase treatment was assessed on 0.8% agarose gel and finally the DNA was quantified using spectrophotometer (Optigen 2020 plus). The DNA samples were diluted to 25 ng μl−1 with TE (Tris–EDTA) buffer before use. Samples were stored for further study at 4 °C. For RAPD analysis 50 primers (40 primers from set # 1 and 10 primers from set # 2) obtained from the University of British Columbia (UBC, Vancouver, Canada) were tested, and 10 were selected for carrying out the clonal fidelity assay depending on band reproducibility and clarity. The PCR reactions were performed in a 25 μl reaction mixture containing 1× assay buffer, 0.5 units of Taq DNA polymerase, 200 μM of each dNTPs (Bangalore Genei), 0.2 μM primers and 50 ng of template DNA. The PCR reactions were carried out in DNA thermal cycler (Model-CGI-96, Corbett Research, Australia) using a single primer in each reaction. The PCR reactions were repeated thrice for each primer to ensure the reproducibility of RAPD results. Only highly reproducible and polymorphic primers were included in the study. The PCR amplification conditions for RAPD consisted of initial extended step of denaturation at 94 °C for 4 min followed by 44 cycles of denaturation at 94 °C for 1 min, primer annealing at 37 °C for 1 min and elongation at 72 °C for 2 min followed by a final step of extension at 72 °C for 4 min. The PCR reaction products were mixed with 4 μl of 6 × DNA loading buffer and fractionated on 1.2% agarose for RAPD containing 0.5 μg μl− 1 ethidium bromide. Gels were electrophoresed until the
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The non-dormant tubers (1–1.5 cm) of G. superba plants collected from Ch. Devi Lal herbal garden, Chuharpur, Yamuna Nagar, Haryana (India) served as a source of explants for micropropagation. Highest shoot regeneration frequency was obtained on multiplication medium consisting of MS salts and vitamins [18] supplemented with BAP (2.0 mg/l) + NAA (0.5 mg/l), 3% sucrose and 0.4% agar [32] (Fig. 2a). The multiplied shoots were rooted, acclimatized and hardened following the standardized protocol described by Yadav et al. [33] (Fig. 2b). The micropropagated plants were phenotypically similar to the mother plant and no morphological variations were observed among the regenerated clones. After 100 days of hardening all the micropropagated plants were randomly selected and their RAPD and ISSR profiles were compared with the mother plant.
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2. Materials and methods
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albus [6]; Saussurea involucrata [36]; Bacopa monnieri [1]; Pogostemon cablin [21]; Sapindus trifoliatus [2]; Citrus jambhiri [24]; and Malus domestica [20]. To our knowledge, there has been no report on genetic stability analysis across the micropropagated plants, along with the donor mother plant in G. superba plantlets. So, the present study was conducted to screen tissue culture induced genetic variations (if any) in G. superba employing RAPD and ISSR–PCR assay.
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The conventional method of propagation through tubers is slow with poor multiplication ratio [9]. Its production is seasonal having susceptibility toward many pests [13]. The poor propagation coupled with over exploitation by the local population as well as pharmaceutical companies is the main factor responsible for its diminishing population size [26]. Micropropagation provides an alternate and effective means for rapid multiplication of species by the continuous production to meet the demand [34,35]. Several efforts have been made to propagate G. superba in vitro on various culture media as well as utilizing different regeneration pathways [4,5,26,28,32]. Genetic fidelity is the maintenance of the genetic constitution of a particular clone through its life span [12]. However, micropropagation protocol is severely hindered due to incidences of somaclonal variations [11]. Somaclonal variation mostly occurs as response to the stress imposed on the plant in culture conditions and is manifested in the form of DNA methylations, chromosome rearrangements, and point mutations (Phillips et al., 1994, [14]). The application of sub- and supra-optimal levels of growth regulators and the recurrent subculture for indefinite period hinder maintenance of genetic fidelity in the tissue cultured clones [15]. It is, therefore, imperative to establish genetic uniformity of micropropagated plants to confirm the quality of the plantlets for its commercial utility. Molecular techniques are at present powerful and valuable tools used in the analysis of genetic fidelity of in vitro propagated plants. In comparison to various morphological, cytological, and protein markers used for the detection of variations in tissue-cultured raised plantlets, polymerase chain reaction (PCR) techniques randomly amplified polymorphic DNA (RAPD) [31] and inter simple sequence repeat (ISSR) [37] markers have been favored because of their simplicity, costeffectiveness, stability, sensitivity, highly reproducible and reliability [23]. The use of two types of markers has been successfully applied to amplify different regions of the genome allowing better analysis of genetic stability in several micropropagated crops, such as Musa sp. [30]; Amorphophallus
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Fig. 2. (a) Micropropagated Gloriosa superba in plant tissue culture lab.; (b) established plants growing in pot experiment under glass house conditions.
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Only clear and reproducible bands were scored for the data analysis, but a major band corresponding to a faint band in repetition was also included in the study. The presence of a particular band was scored as 1 and absence as 0 and each band was regarded as a locus. λ DNA EcoRI/HindIII double digest marker was used as a standard for the estimation of molecular weight of the RAPD products. Bands with same molecular weight and mobility were considered as a single locus. The total number of alleles, polymorphic alleles, and average number of alleles per primer were calculated.
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3. Results and discussion
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3.1. RAPD analysis
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Genetic fidelity testing of the micropropagated plantlets needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plantlets [25]. Assessment of genetic stability of the micropropagated plants was done through RAPD and ISSR analysis after 100 days of acclimatization. Of the 50 RAPD 10-mer primers used for the initial screening, only 10 primers produced reproducible and scorable bands. These 10 selected RAPD primers gave rise to
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3.2. ISSR analysis
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Out of the 30 ISSR markers screened, only 7 primers resulted in 4–12 scorable bands, which were selected for further study. These 7 ISSR primers generated 49 scorable bands ranging from 104 to 5075 bp in size. The number of bands for each primer varied from 4 (GCC816, GCC834 and GCC847) to 12 (GCC810), with an average of 7.0 bands per ISSR primer (Table 2; Fig. 3c, d). A total of 10 RAPD and 7 ISSR primers generated 98 distinct amplicons. In comparison to RAPD, ISSR marker assay resulted in the production of more number of polymorphic fragments per primer because of the occurrence of abundant SSR regions. ISSRs have been used for detecting somaclonal variations in many economical micropropagated plants (Bhatia et al., 2009; Huang et al., 2009; [11,20]). Monomorphic banding pattern was observed for all the amplified band classes across all the micropropagated plants, along with the donor mother plant with all the tested primers. No polymorphism was observed revealing the genetic integrity of in vitro regenerated plants. Similarly, Kumar et al. [11] screened a total of 48 (32 RAPD and 16 ISSR) primers, out of which 24 RAPD and 13 ISSR primers produced a total of 191 clear, distinct and reproducible amplicons in Simmondsia chinensis. Singh et al. [25] also noted monomorphic bands among the regenerants and mother plant of Dendrocalamus asper against various DNA-based markers. The results of this molecular study revealed that the micropropagated plants were genetically identical to that of the mother plant and no variation was induced during clonal propagation. Many investigators have reported genetic stability
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a total of 49 scorable bands ranging from 220 to 4268 bp (Table 1). The number of bands for each primer varied from 2 to 8 with an average of 4.9 bands per primer (Fig. 3b). The highest number of bands obtained was 8 in the case of primer GCC104 (5′ GGGCAATGAT 3′) (Fig. 3a) and the lowest number of bands, i.e. 2, was obtained in the case of primer GCC103 (5′ GTGACGCCGC 3′). RAPD mediated DNA fingerprinting has been extensively used for the detection of polymorphism among the micropropagated medicinal plants [12,22]. In contrast, Modgil et al. [16] reported the presence of somaclonal variations among the micropropagated plantlets of apple rootstock, MM 106 using RAPD markers.
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indicator dye reached 10 cm from the well at 55 mA for 4 h. After separation gels were documented using Avigene Gel Doc system (Korea). For ISSR analysis, 30 UBC primers (set # 9) were screened and finally, 7 anchored and non-anchored ISSR primers were used for the genetic diversity analysis of 8 plant accessions. PCR reactions were followed as in RAPD analysis except 1.0 unit of Taq polymerase instead of half units. In case of ISSR primers, optimal annealing temperature was found to vary according to the base composition of the primers. The amplification reaction consisted of an initial denaturation step at 94 °C for 4 min, followed by 40 cycles of three steps: denaturation at 94 °C for 30 s, annealing at specified temperature for each primer for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min.
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Table 1 Randomly amplified polymorphic DNA (RAPD) primers used to screen the genetic stability in micropropagated plants of G. superba and the amplicons generated. Primer code
Primer sequence (5′–3′)
Annealing temp.
No. of total bands
Approximate size range (bp)
Approximate size of each band (bp)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13
GCC135 GCC139 GCC119 GCC103 GCC104 GCC114 GCC117 GCC123 GCC111 GCC113
AAGCTGCGAG CCCAATCTTC ATTGGGCGAT GTGACGCCGC GGGCAATGAT TGACCGAGAC TTAGCGGTCT GTCTTTCAGG AGTAGACGGG ATCCCAAGAG
37 37 37 37 37 37 37 37 37 37
3 6 5 2 8 5 6 5 6 3
420 bp–725 bp 510 bp–2027 bp 305 bp–1584 bp 1607 bp–1825 bp 305 bp–3405 bp 407 bp–2027 bp 275 bp–2027 bp 220 bp–1904 bp 504 bp–4268 bp 795 bp–2027 bp
420 bp, 564 bp, 725 bp 510 bp, 1130 bp, 1290 bp,1584 bp, 1904 bp, 2027 bp 305 bp, 515 bp, 564 bp, 850 bp, 1584 bp 1607 bp, 1825 bp 305 bp, 350 bp, 630 bp, 755 bp, 1510 bp, 1725 bp, 2027 bp, 3405 bp 407 bp, 564 bp, 1103 bp, 1904 bp, 2027 bp 275 bp, 415 bp, 831 bp, 1375 bp, 1584 bp, 2027 bp 220 bp, 692 bp, 831 bp, 1430 bp, 1904 bp 504 bp, 752 bp, 947 bp, 1932 bp, 2027 bp, 4268 bp 795 bp, 1904 bp, 2027 bp
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To the best of our knowledge this is the first report on the genetic stability of micropropagated G. superba using RAPD and ISSR analysis. The information gained can be applied for the improvement in establishing a unique micropropagation
RAPD- GCC104
b RAPD- GCC111
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4. Conclusions
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of several micropropagated crops viz., Bambusa balcooa [19]; Anethum graveolens [8]; Zingiber rubens [17]; and B. monnieri [22] using RAPD and ISSR analysis. As observed, our result clearly indicates the genetic integrity and true-to-type nature of the in vitro regenerated plants in germplasm conservation program.
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Fig. 3. RAPD and ISSR profiles of mother plant and in vitro regenerated plants of G. superba with primers (a) RAPD–GCC104; (b) RAPD–GCC111; (c) ISSR–GCC846; (d) ISSR–GCC816. Lane L represents the 1-kb ladder, lane M: mother plant; lanes 1–7: micropropagated plantlets.
Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Table 2 Inter simple sequence repeat DNA (ISSR) primers used to screen the genetic stability in micropropagated plants of G. superba and the amplicons generated (Y = T or C, R = G or A).
t2:4
Primer code
Sequence
Annealing temp.
t2:5 t2:6
GCC807 GCC809
(AG)8T (AG)8G
54 °C 54 °C
t2:7
GCC810
(GA)8T
t2:8 t2:9
GCC816 GCC846
t2:10 t2:11
GCC834 GCC847
Approximate size of each band (bp)
6 9
340 bp–1715 bp 564 bp–5075 bp
54 °C
12
104 bp–3530 bp
(CA)8T (CA)8RT
54 °C 58 °C
4 10
815 bp–2410 bp 472 bp–4772 bp
(AG)8YT (CA)8RC
58 °C 58 °C
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230 bp–1375 bp 392 bp–1170 bp
340 bp, 675 bp, 720 bp, 803 bp, 905 bp, 1715 bp 564 bp, 630 bp, 745 bp, 1815 bp, 1885 bp, 2375 bp, 3410 bp, 4973 bp, 5075 bp 104 bp, 215 bp, 375 bp, 490 bp, 525 bp, 735 bp, 1703 bp, 1805 bp, 1945 bp, 2027 bp, 3410 bp, 3530 bp 815 bp, 1410 bp, 1805 bp, 2410 bp 472 bp, 564 bp, 650 bp, 775 bp, 831 bp, 1272 bp, 1904 bp, 2027 bp, 4564 bp, 4772 bp 230 bp, 372 bp, 1220 bp, 1375 bp 392 bp, 670 bp, 742 bp, 1170 bp
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Conflict of interest No conflicts of interest.
Acknowledgment
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The authors are grateful to Kurukshetra University, Kurukshetra for providing laboratory facilities and other institutional support. Funding for this work came from the University Grant Commission (UGC), New Delhi, India in the form of Major Research Project. Thanks are owed to Dr. Anita Grewal and Rakesh (U.I.E.T., K.U. Kurukshetra) and Govind K Vyas for assistance with PCR analyses.
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Contents lists available at SciVerse ScienceDirect
Fitoterapia
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Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant
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Kuldeep Yadav a, Ashok Aggarwal b, Narender Singh a,⁎
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Plant Tissue Culture Laboratory, Department of Botany, Kurukshetra University, Haryana 136119, India Mycology and Plant Pathology Laboratory, Department of Botany, Kurukshetra University, Haryana 136119, India
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journal homepage: www.elsevier.com/locate/fitote
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Article history: Received 19 March 2013 Accepted in revised form 11 June 2013 Available online xxxx
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Keywords: Gloriosa superba Micropropagation Genetic fidelity Molecular markers RAPD ISSR
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Malabar glory lily (Gloriosa superba L.) is a medicinally potent plant species used for the production of alkaloid colchicine. With ever increasing demand, there is a pressing need to conserve it through biotechnological approaches. A large number of complete plantlets were obtained by direct regeneration from the non-dormant tuber explants on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) + 0.5 mg/l α-naphthalene acetic acid (NAA). Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, the genetic stability of micropropagated clones was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. During the study a total of 80 (50 RAPD and 30 ISSR) primers were screened, out of which 10 RAPD and 7 ISSR primers produced a total of 98 (49 RAPD and 49 ISSR) clear, distinct and reproducible amplicons. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. This is the first report that evaluates the use of genetic markers to establish genetic fidelity of micropropagated G. superba using RAPD and ISSR, which can be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays for colchicine production to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses. © 2013 Published by Elsevier B.V.
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1. Introduction
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Malabar glory lily (Gloriosa superba L.), the sole species in the family Colchicaceae is a perennial tuberous climbing herb widely scattered in tropical and sub-tropical parts of Africa and Southeast Asia. In India, it is usually found in the Himalayan foot-hills, Tamil Nadu, Andhra Pradesh and Bengal
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Abbreviations: dNTPs, deoxyribonucleotide triphosphates; PCR, polymerase chain reaction; BAP, 6-benzylaminopurine; NAA, α-naphthalene acetic acid; ISSR, inter simple sequence repeats; RAPD, random amplified polymorphic DNA. ⁎ Corresponding author at: Department of Botany, Kurukshetra University, Haryana 136119, India. Tel.: +91 1744 238410; fax: +91 1744 238277. E-mail address: [email protected] (N. Singh).
[27]. Its attractive wavy edged orange–red flower is the national flower of Zimbabwe and also the state flower of Tamil Nadu in India. The tubers and seeds of this plant contain more colchicine content than the genera Colchicum [3]. Colchicine alkaloid (Fig. 1) is highly demanded due to its uses for treating arthritis, Mediterranean fever, gout, rheumatism, inflammation, ulcers, bleeding piles, skin diseases, leprosy, impotency and snakebites [7]. Colchicine also has antimitotic activity, preventing growth of cancer cells by interacting with microtubules, which could lead to the design of better cancer therapeutics [29]. Due to the action of colchicine on spindle-fiber formation during celldivision, it is also capable of inducing polyploidy and used in cytological and plant breeding research [10].
0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.06.009
Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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2.1. Plant material
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Colchicine (C22H25NO6) Fig. 1. Colchicine (C22H25NO6).
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2.2. DNA extraction and PCR amplification
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Total genomic DNA was extracted using the method of Doyle and Doyle (1990) from leaf samples. The quality of extracted DNA after RNase treatment was assessed on 0.8% agarose gel and finally the DNA was quantified using spectrophotometer (Optigen 2020 plus). The DNA samples were diluted to 25 ng μl−1 with TE (Tris–EDTA) buffer before use. Samples were stored for further study at 4 °C. For RAPD analysis 50 primers (40 primers from set # 1 and 10 primers from set # 2) obtained from the University of British Columbia (UBC, Vancouver, Canada) were tested, and 10 were selected for carrying out the clonal fidelity assay depending on band reproducibility and clarity. The PCR reactions were performed in a 25 μl reaction mixture containing 1× assay buffer, 0.5 units of Taq DNA polymerase, 200 μM of each dNTPs (Bangalore Genei), 0.2 μM primers and 50 ng of template DNA. The PCR reactions were carried out in DNA thermal cycler (Model-CGI-96, Corbett Research, Australia) using a single primer in each reaction. The PCR reactions were repeated thrice for each primer to ensure the reproducibility of RAPD results. Only highly reproducible and polymorphic primers were included in the study. The PCR amplification conditions for RAPD consisted of initial extended step of denaturation at 94 °C for 4 min followed by 44 cycles of denaturation at 94 °C for 1 min, primer annealing at 37 °C for 1 min and elongation at 72 °C for 2 min followed by a final step of extension at 72 °C for 4 min. The PCR reaction products were mixed with 4 μl of 6 × DNA loading buffer and fractionated on 1.2% agarose for RAPD containing 0.5 μg μl− 1 ethidium bromide. Gels were electrophoresed until the
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The non-dormant tubers (1–1.5 cm) of G. superba plants collected from Ch. Devi Lal herbal garden, Chuharpur, Yamuna Nagar, Haryana (India) served as a source of explants for micropropagation. Highest shoot regeneration frequency was obtained on multiplication medium consisting of MS salts and vitamins [18] supplemented with BAP (2.0 mg/l) + NAA (0.5 mg/l), 3% sucrose and 0.4% agar [32] (Fig. 2a). The multiplied shoots were rooted, acclimatized and hardened following the standardized protocol described by Yadav et al. [33] (Fig. 2b). The micropropagated plants were phenotypically similar to the mother plant and no morphological variations were observed among the regenerated clones. After 100 days of hardening all the micropropagated plants were randomly selected and their RAPD and ISSR profiles were compared with the mother plant.
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albus [6]; Saussurea involucrata [36]; Bacopa monnieri [1]; Pogostemon cablin [21]; Sapindus trifoliatus [2]; Citrus jambhiri [24]; and Malus domestica [20]. To our knowledge, there has been no report on genetic stability analysis across the micropropagated plants, along with the donor mother plant in G. superba plantlets. So, the present study was conducted to screen tissue culture induced genetic variations (if any) in G. superba employing RAPD and ISSR–PCR assay.
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The conventional method of propagation through tubers is slow with poor multiplication ratio [9]. Its production is seasonal having susceptibility toward many pests [13]. The poor propagation coupled with over exploitation by the local population as well as pharmaceutical companies is the main factor responsible for its diminishing population size [26]. Micropropagation provides an alternate and effective means for rapid multiplication of species by the continuous production to meet the demand [34,35]. Several efforts have been made to propagate G. superba in vitro on various culture media as well as utilizing different regeneration pathways [4,5,26,28,32]. Genetic fidelity is the maintenance of the genetic constitution of a particular clone through its life span [12]. However, micropropagation protocol is severely hindered due to incidences of somaclonal variations [11]. Somaclonal variation mostly occurs as response to the stress imposed on the plant in culture conditions and is manifested in the form of DNA methylations, chromosome rearrangements, and point mutations (Phillips et al., 1994, [14]). The application of sub- and supra-optimal levels of growth regulators and the recurrent subculture for indefinite period hinder maintenance of genetic fidelity in the tissue cultured clones [15]. It is, therefore, imperative to establish genetic uniformity of micropropagated plants to confirm the quality of the plantlets for its commercial utility. Molecular techniques are at present powerful and valuable tools used in the analysis of genetic fidelity of in vitro propagated plants. In comparison to various morphological, cytological, and protein markers used for the detection of variations in tissue-cultured raised plantlets, polymerase chain reaction (PCR) techniques randomly amplified polymorphic DNA (RAPD) [31] and inter simple sequence repeat (ISSR) [37] markers have been favored because of their simplicity, costeffectiveness, stability, sensitivity, highly reproducible and reliability [23]. The use of two types of markers has been successfully applied to amplify different regions of the genome allowing better analysis of genetic stability in several micropropagated crops, such as Musa sp. [30]; Amorphophallus
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Fig. 2. (a) Micropropagated Gloriosa superba in plant tissue culture lab.; (b) established plants growing in pot experiment under glass house conditions.
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Only clear and reproducible bands were scored for the data analysis, but a major band corresponding to a faint band in repetition was also included in the study. The presence of a particular band was scored as 1 and absence as 0 and each band was regarded as a locus. λ DNA EcoRI/HindIII double digest marker was used as a standard for the estimation of molecular weight of the RAPD products. Bands with same molecular weight and mobility were considered as a single locus. The total number of alleles, polymorphic alleles, and average number of alleles per primer were calculated.
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3. Results and discussion
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3.1. RAPD analysis
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Genetic fidelity testing of the micropropagated plantlets needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plantlets [25]. Assessment of genetic stability of the micropropagated plants was done through RAPD and ISSR analysis after 100 days of acclimatization. Of the 50 RAPD 10-mer primers used for the initial screening, only 10 primers produced reproducible and scorable bands. These 10 selected RAPD primers gave rise to
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3.2. ISSR analysis
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Out of the 30 ISSR markers screened, only 7 primers resulted in 4–12 scorable bands, which were selected for further study. These 7 ISSR primers generated 49 scorable bands ranging from 104 to 5075 bp in size. The number of bands for each primer varied from 4 (GCC816, GCC834 and GCC847) to 12 (GCC810), with an average of 7.0 bands per ISSR primer (Table 2; Fig. 3c, d). A total of 10 RAPD and 7 ISSR primers generated 98 distinct amplicons. In comparison to RAPD, ISSR marker assay resulted in the production of more number of polymorphic fragments per primer because of the occurrence of abundant SSR regions. ISSRs have been used for detecting somaclonal variations in many economical micropropagated plants (Bhatia et al., 2009; Huang et al., 2009; [11,20]). Monomorphic banding pattern was observed for all the amplified band classes across all the micropropagated plants, along with the donor mother plant with all the tested primers. No polymorphism was observed revealing the genetic integrity of in vitro regenerated plants. Similarly, Kumar et al. [11] screened a total of 48 (32 RAPD and 16 ISSR) primers, out of which 24 RAPD and 13 ISSR primers produced a total of 191 clear, distinct and reproducible amplicons in Simmondsia chinensis. Singh et al. [25] also noted monomorphic bands among the regenerants and mother plant of Dendrocalamus asper against various DNA-based markers. The results of this molecular study revealed that the micropropagated plants were genetically identical to that of the mother plant and no variation was induced during clonal propagation. Many investigators have reported genetic stability
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a total of 49 scorable bands ranging from 220 to 4268 bp (Table 1). The number of bands for each primer varied from 2 to 8 with an average of 4.9 bands per primer (Fig. 3b). The highest number of bands obtained was 8 in the case of primer GCC104 (5′ GGGCAATGAT 3′) (Fig. 3a) and the lowest number of bands, i.e. 2, was obtained in the case of primer GCC103 (5′ GTGACGCCGC 3′). RAPD mediated DNA fingerprinting has been extensively used for the detection of polymorphism among the micropropagated medicinal plants [12,22]. In contrast, Modgil et al. [16] reported the presence of somaclonal variations among the micropropagated plantlets of apple rootstock, MM 106 using RAPD markers.
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indicator dye reached 10 cm from the well at 55 mA for 4 h. After separation gels were documented using Avigene Gel Doc system (Korea). For ISSR analysis, 30 UBC primers (set # 9) were screened and finally, 7 anchored and non-anchored ISSR primers were used for the genetic diversity analysis of 8 plant accessions. PCR reactions were followed as in RAPD analysis except 1.0 unit of Taq polymerase instead of half units. In case of ISSR primers, optimal annealing temperature was found to vary according to the base composition of the primers. The amplification reaction consisted of an initial denaturation step at 94 °C for 4 min, followed by 40 cycles of three steps: denaturation at 94 °C for 30 s, annealing at specified temperature for each primer for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min.
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Table 1 Randomly amplified polymorphic DNA (RAPD) primers used to screen the genetic stability in micropropagated plants of G. superba and the amplicons generated. Primer code
Primer sequence (5′–3′)
Annealing temp.
No. of total bands
Approximate size range (bp)
Approximate size of each band (bp)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13
GCC135 GCC139 GCC119 GCC103 GCC104 GCC114 GCC117 GCC123 GCC111 GCC113
AAGCTGCGAG CCCAATCTTC ATTGGGCGAT GTGACGCCGC GGGCAATGAT TGACCGAGAC TTAGCGGTCT GTCTTTCAGG AGTAGACGGG ATCCCAAGAG
37 37 37 37 37 37 37 37 37 37
3 6 5 2 8 5 6 5 6 3
420 bp–725 bp 510 bp–2027 bp 305 bp–1584 bp 1607 bp–1825 bp 305 bp–3405 bp 407 bp–2027 bp 275 bp–2027 bp 220 bp–1904 bp 504 bp–4268 bp 795 bp–2027 bp
420 bp, 564 bp, 725 bp 510 bp, 1130 bp, 1290 bp,1584 bp, 1904 bp, 2027 bp 305 bp, 515 bp, 564 bp, 850 bp, 1584 bp 1607 bp, 1825 bp 305 bp, 350 bp, 630 bp, 755 bp, 1510 bp, 1725 bp, 2027 bp, 3405 bp 407 bp, 564 bp, 1103 bp, 1904 bp, 2027 bp 275 bp, 415 bp, 831 bp, 1375 bp, 1584 bp, 2027 bp 220 bp, 692 bp, 831 bp, 1430 bp, 1904 bp 504 bp, 752 bp, 947 bp, 1932 bp, 2027 bp, 4268 bp 795 bp, 1904 bp, 2027 bp
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RAPD- GCC104
b RAPD- GCC111
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4. Conclusions
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of several micropropagated crops viz., Bambusa balcooa [19]; Anethum graveolens [8]; Zingiber rubens [17]; and B. monnieri [22] using RAPD and ISSR analysis. As observed, our result clearly indicates the genetic integrity and true-to-type nature of the in vitro regenerated plants in germplasm conservation program.
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Fig. 3. RAPD and ISSR profiles of mother plant and in vitro regenerated plants of G. superba with primers (a) RAPD–GCC104; (b) RAPD–GCC111; (c) ISSR–GCC846; (d) ISSR–GCC816. Lane L represents the 1-kb ladder, lane M: mother plant; lanes 1–7: micropropagated plantlets.
Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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Table 2 Inter simple sequence repeat DNA (ISSR) primers used to screen the genetic stability in micropropagated plants of G. superba and the amplicons generated (Y = T or C, R = G or A).
t2:4
Primer code
Sequence
Annealing temp.
t2:5 t2:6
GCC807 GCC809
(AG)8T (AG)8G
54 °C 54 °C
t2:7
GCC810
(GA)8T
t2:8 t2:9
GCC816 GCC846
t2:10 t2:11
GCC834 GCC847
Approximate size of each band (bp)
6 9
340 bp–1715 bp 564 bp–5075 bp
54 °C
12
104 bp–3530 bp
(CA)8T (CA)8RT
54 °C 58 °C
4 10
815 bp–2410 bp 472 bp–4772 bp
(AG)8YT (CA)8RC
58 °C 58 °C
4 4
230 bp–1375 bp 392 bp–1170 bp
340 bp, 675 bp, 720 bp, 803 bp, 905 bp, 1715 bp 564 bp, 630 bp, 745 bp, 1815 bp, 1885 bp, 2375 bp, 3410 bp, 4973 bp, 5075 bp 104 bp, 215 bp, 375 bp, 490 bp, 525 bp, 735 bp, 1703 bp, 1805 bp, 1945 bp, 2027 bp, 3410 bp, 3530 bp 815 bp, 1410 bp, 1805 bp, 2410 bp 472 bp, 564 bp, 650 bp, 775 bp, 831 bp, 1272 bp, 1904 bp, 2027 bp, 4564 bp, 4772 bp 230 bp, 372 bp, 1220 bp, 1375 bp 392 bp, 670 bp, 742 bp, 1170 bp
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Conflict of interest No conflicts of interest.
Acknowledgment
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The authors are grateful to Kurukshetra University, Kurukshetra for providing laboratory facilities and other institutional support. Funding for this work came from the University Grant Commission (UGC), New Delhi, India in the form of Major Research Project. Thanks are owed to Dr. Anita Grewal and Rakesh (U.I.E.T., K.U. Kurukshetra) and Govind K Vyas for assistance with PCR analyses.
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Please cite this article as: Yadav K, et al, Evaluation of genetic fidelity among micropropagated plants of Gloriosa superba L. using DNA-based markers — a potential medicinal plant, Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.06.009
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