DNA Polymorphism among Justicia adhatoda L. Somaclones Differing in Vasicine Production

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1 Chapter 6 DNA Polymorphism among Justicia adhatoda L. Somaclones Differing in Vasicine Production 6.1. ABSTRACT The present study was to assess clonal fidelity of somaclones derived from calli of different explants of Justicia adhatoda. To assess somaclonal variation, genomic DNA was extracted from the in vitro cultured plants of fifth subculture. Fourteen Random Amplified Polymorphic DNA primers were used to analyze genetic variation between micropropagated J. adhatoda plants with their mother plant. The results showed different amplification patterns among the tissue cultured plants. They also showed some variations in morphology and secondary metabolite production. Keywords: Somaclones, Random Amplified Polymorphic DNA, clonal fidelity, Justicia adhatoda L., vasicine production

2 106 Chapter INTRODUCTION The callus derived plants exhibit huge genetic variation that could be exploited for developing superior clones/varieties particularly in vegetatively propagated plant species (Sharma et al., 2010). In vitro propagation is often associated with the incidences of somaclonal variation. Somaclonal variation, a common phenomenon in plant cell and tissue cultures, includes all types of variations that can be present among plants or cells derived from in vitro cultures (Shoja et al., 2010). Many methods (morphological, biochemical and molecular) are available to assess somaclonal variation. Morphological diagnosis is relatively simple, but it requires laborious field experiments and it is timeconsuming. Additionally, morphological characteristics are frequently affected by the developmental stage and environment. Molecular markers, such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) (Chuang et al., 2009) or inter simple sequence repeat (ISSR) provide an efficient way for identifying genetic uniformity of the micropropagated plantlets since these markers are not affected by environmental factors and present reliable and reproducible results (Chuang et al., 2010). Randomly Amplified Polymorphic DNA (RAPD) analysis using polymerase chain reaction (PCR) has been demonstrated to be sensitive and useful method in detecting variation among individuals (Shoja et al., 2010; Linu et al., 2000). The present experiment was carried out to establish a protocol for calli production from different explants of J. adhatoda as well as to assay somaclonal variation between in vitro derived plants.

3 DNA Polymorphism among Justicia adhatoda L. Somaclones 107 In the present work, both morphological characteristics and RAPD markers were used to assess the genetic fidelity of J. adhatoda plants indirectly regenerated from calli of leaf, axillary bud and root tip explants REVIEW OF LITERATURE Genetic variation is the resource for plant breeding and biotechnology and it provides an easy access to innovative genetic variation to support breeding. In vitro cultures revealed a rich array of culture induced genetic variants, termed somaclonal variations. Many unstable or nontransmissible variants may have their origin in epigenetic rather than genetic changes. Somaclonal variations may include chromosomal rearrangements, single-gene mutants (mostly recessive), gene modifications and deletions, and DNA methylation (Kim et al., 2009). Somaclonal variation can be assessed by the analysis of phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants (Jain, 2001; Smy kal et al., 2007). Although somaclonal variation may be used as a source for variation to get superior clones, it could be also a very serious problem in the plant tissue culture industry resulting in the production of undesirable plant off-types (Gaafar and Saker, 2006). Mutation breeding and biotechnological methods can offer useful tools for plant improvement. Plant tissue culture leading to somaclonal variation has been considered as one of the possible sources of inducing genetic variability in crop plants to be used in breeding programs (Sheidai et al., 2010). Somaclonal variation (SV) can be defined as genetically stable variation generated through plant tissue culture (Larkin and Scowcroft, 1981), and is a breeding approach to create greater genetic diversity and expand germplasm pool for plant improvement and cultivar development. It describes

4 108 Chapter 6 epigenetic and genetic changes in plants that become apparent either during or after in vitro culture of plant cells, callus or organs (Kaeppler et al., 2000). Somaclonal variation has been observed in many plant species and is an alternative way to create variants and expand the germplasm pool (Li et al., 2010). Nonetheless, somaclonal variation frequently occurs as a consequence of the propagation process in different plant species. Thus it is very important to detect somaclonal variation at earlier stage of plant growth to avoid economic loss (Chuang et al., 2009). Genetic variation analyses can be observed from morphological characters and other markers as protein or deoxyribonucleic acid (DNA)-based markers. Plant tissue culture leading to somaclonal variation has been considered as one of the possible sources of inducing genetic variability in crop plants to be used in breeding programs (Farahani et al., 2011). Chromosomal rearrangements are an important source of this variation. It has relevance in the clonal propagation of valuable or endangered plant germplasm, and in the production of transgenic plants. It may also be an effective means of generating useful mutants. Because of these reasons, somaclonal variation has been intensively studied by using various molecular markers in several plant species, including Arabidopsis (Polanco and Ruiz, 2002) and rice (Kim et al., 2003). Ngezahayo et al. (2007) studied Somaclonal variation at the nucleotide sequence level in rice. Somaclonal variation is not restricted to, but is particularly common in plants regenerated from callus. The variations can be genotypic or phenotypic, which in the latter case can be either genetic or epigenetic in origin. Typical genetic alterations are: changes in chromosome numbers (polyploidy and

5 DNA Polymorphism among Justicia adhatoda L. Somaclones 109 aneuploidy), chromosome structure (translocations, deletions, insertions and duplications) and DNA sequence (base mutations). Typical epigenetic related events are: gene amplification and gene methylation. If no visual, morphogenic changes are apparent, other plant screening procedures must be applied. There are both benefits and disadvantages to somaclonal variation. The major likely benefit of somaclonal variation in plant is improvement. Somaclonal variation leads to the creation of additional genetic variability. Characteristics for which somaclonal mutants can be enriched during in vitro culture includes resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites. Micropropagation can be carried out throughout the year independent of the seasons. A serious disadvantage of somaclonal variation occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes. The increasing numbers of subculture increases the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. Regular reinitiation of clones from new explants might reduce variability over time. Another way of reducing somaclonal variation is to avoid 2, 4-D in the culture medium, as this hormone is known to introduce variation. Hu et al. (2008) stated that plant tissue culture has been successfully used in many valuable medicinal plants for efficient propagation or for selection of useful mutants. Different strategies exist for evaluation of the somaclones, including cytogenetic and C-value analyses, isoenzyme markers and various DNA molecular markers etc. (Soniya et al., 2001). Recently,

6 110 Chapter 6 several DNA markers have been successfully employed to assess the genomic stability/instability in regenerated plants. Among the markers, the randomly amplified polymorphic DNA (RAPD) and the inter-simple sequence repeat (ISSR) have been favored because of their sensitivity, simplicity, and costeffectiveness. RAPD markers had been used widely in studying the genetic diversity of somaclonal variations in various plant species (Maria and Garcia, 2000; Soniya et al., 2001) including banana (Hernandez et al., 2007). RAPD is the simplest, sensitive and useful technique for the analysis of genetic fidelity of in vitro propagated plants (Chuang et al., 2009). Because RAPD polymorphism results from either a nucleotide base change that alter the primer binding site, or from an insertion or deletion within amplified region. Polymorphism usually results in the presence or absence of amplification products from a single locus. RAPD was employed to evaluate the genetic reliability of Silybum marianum plantlets regenerated by leaf organogenesis (Mahmood et al., 2010). The molecular marker technologies have become a powerful tool in crop improvement through their use in germplasm characterization and fingerprinting, genetic analysis, linkage mapping, and molecular breeding. Identification of possible somaclonal variants at an early stage of development was considered to be very useful for quality control in plant tissue culture, transgenic plant production and in the introduction of variants (Soniya et al., 2001). RAPD analysis using polymerase chain reaction (PCR) in association with short primers of arbitrary sequence has been demonstrated to be sensitive in detecting variation among individuals. The advantages of this technique are: a) a large number of samples can be quickly and economically analyzed using

7 DNA Polymorphism among Justicia adhatoda L. Somaclones 111 only micro-quantities of material; b) the DNA amplicons are independent from the ontogenetic expression; and c) many genomic regions can be sampled with a potentially unlimited number of markers (Maria and Garcia, 2000). The necessity for sequence information for PCR was circumvented using short primers of arbitrary sequences to amplify DNA segments, namely RAPD. The speed and efficiency of RAPD analysis encouraged scientists to perform high-density genetic mapping in many plant species such as alfalfa, faba bean and apple in a relatively short time (Fevzi, 2001). One disadvantage of RAPD markers is that they are dominant; hence the statistical information generated is less per marker in F2 populations. Fevzi (2001) found a wide range of applications of RAPD markers in gene mapping, population genetics, molecular evolutionary genetics and plant and animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period compared with previous methods. In recent years, several DNA markers had been successfully employed to assess the genomic stability in regenerated plants including those with no obvious phenotypic alternations (Rahman and Rajora, 2001). 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 (Chandrika and Rai, 2009). RAPD analysis could be applied to assess the genetic fidelity of plants derived in vitro on an industrial scale as part of crop improvement programs (Bindiya and Kanwar, 2003). RAPD analysis in plants had also been widely used to detect genetic and somaclonal variations (Modgil et al., 2005; Lattoo et al., 2006). RAPD technique did not require DNA sequence information and species specificity and hence it is being

8 112 Chapter 6 conveniently used for assessing genetic stability and clonal fidelity of micropropagated plants in a number of genera. The RAPD markers were referred to as an appropriate tool to get rapid information about genetic similarities or dissimilarities in micropropagules (Chandrika and Rai, 2009). In the present study, RAPD was employed to evaluate the genetic reliability of J. adhatoda plantlets regenerated by indirect organogenesis. On the basis of the results, it may be concluded that RAPD is a useful technology to detect variation in micopropagated plants of J. adhatoda SPECIFIC OBJECTIVES: The present investigation aimed at the large scale indirect regeneration of plantlets from explants of mature plants with a view to clone high alkaloid containing genotypes. The main objectives of the study are, 1. To assess the genetic integrity of tissue cultured plants by using Random Amplified Polymorphic DNA (RAPD). 1a) To assess the ability of somaclones for vasicine production MATERIALS ANDMETHODS DEVELOPMENT OF SOMACLONES Somaclones were developed through indirect organogenesis using calli from leaf, axillary bud and root tip of J. adhatoda QUANTITATIVE ANALYSIS OF VASICINE FROM SOMACLONES Quantitative analysis of total alkaloids was done by spectrophotometric method with tropaeolin OO (Sigma Aldrich, India). Coloured complex developed was measured at 545 nm against blank. The amount of total alkaloids in the samples was calculated using standard curve

9 DNA Polymorphism among Justicia adhatoda L. Somaclones 113 of vasicine (SPIC India Ltd, Chennai). The content of the total alkaloids was expressed as vasicine (Soni et al., 2008) ASSESSMENT OF CLONAL FIDELITY BY RAPD ANALYSIS Nine healthy rooted tissue cultured clonal plants were selected after six months of growth DNA Isolation Genomic DNA of mother plant and somaclones were isolated by modified - Doyle and Doyle, (1990) method. DNA was quantified and purity established by UV spectrophotometry by (A260/ A280) RAPD Analysis Reactions for RAPD analysis were set up using the following fourteen primers (Table 6.1). Table 6.1: Fourteen primers used for RAPD analysis Sl.No. Primer Sequence 1 RY01 5 GTGGCATCTC3 2 S01 5 CCACCACGAC 3 3 S04 5 GTTTCGCTCC 3 4 S07 5 GTAAACCGCC3 5 S129 5 CCAAGCTTCC3 6 S344 5 CCGAACACGG3 7 S346 5 TCGTTCCGCA3 8 S350 5 AAGCCCGAGG3 9 S353 5 CCACACTACC3 10 S358 5 TGGTCGCAGA3 11 S359 5 GGACACCACT3 12 S73 5 AAGCCTCGTC3 13 S79 5 GTTGCCAGCC3 14 S80 5 CTACGGAGGA3

10 114 Chapter 6 PCR were performed in a MJ Mini Biorad Personal Thermal Cycler, using the following PCR profile. i). Initial denaturation: 94 0 C for 5 min., ii) 34 Cycles of: Denaturation: 94 0 C for 1 min, Annealing Temperature: C for 1 min., Extension: 72 0 C for 2 min., and was followed by iii) Final Extension: 72 0 C for 10 min. to ensure that the primer extension reaction proceeded to completion. The PCR reaction mixture consists of 17.7µL PCR water, 2.5µL Buffer without MgCl 2 (10X), 1µLMgCl 2 (25mM), 1.5µLdNTPs Mix (2.5mM), 0.3µLTaq DNA polymerase (5U/µl) and 2µLTemplate DNA. The total volume was 25µL. A total of 10µL of PCR products were analyzed by agarose gel electrophoresis. 2% agarose gel was run using 0.5x TBE buffer with ethidium bromide. DNA was visualised using a 320 nm UV transilluminator. The experiment was repeated for 3 times and reproducible RAPD bands were used for further analysis. The bands were scored as either present (1) or absent (0), and genetic similarity was estimated on the basis of Population Genetic Analysis (POPGENE) : Dendrogram Based Nei s (1972) Genetic Distance were determined among the cultivars and used for grouping of the genotype by UPGMA method (unweighted pair-group method with arithmetic averages) modified from NEIGHBOR procedure of PHYLIP Version RESULT QUANTIFICATION OF VASICINE Vasicine content was found to be 6.92 mg ml -1, 5.05 mg ml -1, 4.87 mg ml -1, 5.08 mg ml -1, 6.80 mg ml -1, 4.55 mg ml -1, 7.32 mg ml -1, 5.20 mg ml -1 and 5.09 mg ml -1, in leaves of 1 to 9 somaclones respectively. Among

11 DNA Polymorphism among Justicia adhatoda L. Somaclones 115 the somaclones first, fifth and seventh ones produce more vasicine compared to the others and mother plant (5.07 mg ml -1 ) DNA ISOLATION Plate 6.1. Genomic DNA isolation Pattern The concentrations of isolated DNA were on an average 50 µg ml -1. This DNA was further diluted and used for analysis RAPD ANALYSIS RY01 S01 S04 S07 S73

12 116 Chapter 6 S79 S80 S129 S344 S346 S350 S353 S358 S 359 Plate 6.2. DNA Pattern of J. adhatoda L. based on PCR-RAPD analysis using 14 different decamer primers. Lane 1: mother plant and Lanes 2-10: somaclones respectively. Figure.6.1. Dendrogram of PCR-RAPD for somaclones of J. adhatoda Out of forty five random decamers only fourteen were able to give polymorphism. They produced 124 bands, out of which 33 (26.61%) were monomorphic while, 91 (73.39%) bands were polymorphic. Among the primers S01 produced the highest number of bands (12) while S358 primer produced the lowest number (4). The highest number of polymorphic bands

13 DNA Polymorphism among Justicia adhatoda L. Somaclones 117 was observed in S01 (11 bands) while the lowest number was observed in RY01 (2 bands) (Plate 6.2). Clone pop1 and pop10 formed a single cluster showing that they were genetically similar. Pop3, 4, 5, and 9 formed another cluster. Pop2 and 6 formed a third cluster. Pop7 and pop8 formed two independent branches. Pop8 showed least genetic similarity to other clusters (Figure 6.1) DISCUSSION The results indicated that there was variation among the mother plant and somaclones. Indirect micropropation did not support clonal fidelity and led to the production of variants with ability to produce more secondary metabolites. Out of the nine somaclones tested for increased production of vasicine three showed significantly higher amount of vasicine production (pop8, pop6 and pop2). These three were on par with respect to the production of vasicine. Rest of the somaclones was poor performers, and showed vasicine production on par with the mother plant. So this result may be an indication of its elite genetic status. On analysing the genetic dissimilarity of somaclones by UPGMA based dendraogram it was found that pop1 and pop10 formed a cluster, pop3 and 4 another, and 5 and 9 formed another cluster and pop2 and pop6 formed another cluster. pop7 and pop8 remained independent without clustering. It evident that pop 10 showed maximum similarity to mother plant (pop1) and pop8 and pop7 showed maximum dissimilarity. The genetic dissimilarity of pop8 to mother plant was also demonstrated in the amount of vasicine produced. So this dissimilarity may be indicative of the elite status of pop8 (Figure 6.1).

14 118 Chapter 6 The presence of specific bands/ loci in the parental plants and loss of them in the regenerated plants indicates the loss of certain loci during tissue culture due to somaclonal variation. Such specific loci are of high importance in the genetic identification of the genotype or somaclones from each other. According to Fevzi (2001) RAPD markers are more suitable for clonal organisms than sexually reproducing organisms. As they breed asexually, a polymorphic fragment among individuals can be used to determine clonal identity. Mahmood et al. (2010) stated that RAPD technology has also been utilized to analyze genetic stability of micropropagated plants. The absence of loci in one of the genotypes indicates genetic changes of the plants brought about during sub-culture. Since, even single base change at the primer annealing site is manifested as appearance or disappearance of RAPD bands, it could be suggested that tissue culture conditions might have induced genetic changes in different regenerated plants. Some of these changes appeared identical in different plants as represented by appearance of non-parental bands. The reason for such commonness of genetic variation in these plants could be because they were all derived from the same callus (Soniya et al., 2001). It was found that somaclones of different calli were not reliable in producing genetically similar pattern to the mother plant, but Rout et al. (2006) and Shoja et al. (2010) reported that somaclonal variation in the callus cultures can be a promising source for creating new cultivars in plants and for isolating cell lines with high capacity of producing desired secondary compounds. Explant source is also considered as one of the critical variable for somaclonal variation (Sheidai et al., 2010).

15 DNA Polymorphism among Justicia adhatoda L. Somaclones 119 In conclusion, we have confirmed that somaclonal variation might be useful for selection of plants for desirable traits, such as secondary metabolite production. We demonstrated that RAPD analysis can detect sufficient polymorphism to differentiate among J. adhatoda somaclones and that is suitable for the assessment of genetic fidelity. The current experiment was an initial step towards the mass production of desired somaclones from different calli and production of high valued secondary metabolite, vasicine.