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Use of RAPD Assay for the detection of mutation changes in aonla (Emblica officinalis Gaertn.).


Amla, scientifically known as Emblica officinalis Gaertn. (Syn. Phyllanthus emblica) belongs to the family Euphorbiaceae, ranges in status from insignificant in the Western world to highly prized plant species in tropical Asia. Its cultivation is spreading rapidly in the semi grid regions of different states viz., Maharashtra, Gujarat, Rajasthan andhra Pradesh, Karnataka, Tamil Nadu and Aravali ranges of Haryana, Kandi area in Punjab and Himachal Pradesh (Pathak and Pathak, 1993). As per the recent estimates, amla has spreaded over an area of 50,000 ha in India with an approximate production of 1,75,000 tonnes (Pathak et al., 2003). In this scenario, it is important to aim for developing dwarf tree stature of amla for medium density orchards, to develop precocious and prolific bearing cultivars and to develop excellent fruit quality (high phenolic content and tannins) for processing.

Different methods are available to investigate the effect of mutagens on plants. Molecular markers allow a direct comparison of the effects on genotypes at the DNA level. Different molecular techniques have been developed and are widely used in many fields such as agriculture, biology medicine, etc for various purposes. The exploration of random amplified polymorphic DNA (RAPD) as genetic markers has improved the effectiveness of recombinant DNA techniques. This method is widely used for the genetic mapping, taxonomic and phylogenetic studies of many organisms. This method can also be applicable for the detection of DNA alterations. The RAPD method does not require DNA probes or prior sequence information. Thus, the method is simple and requires only small amounts of DNA. The method utilizes a single, arbitrarily--chosen primer to amplify a number of fragments from a given template of DNA to generate a discrete "fingerprint" when resolved by gel electrophoresis (Danylchenko and Sorochinsky, 2005. The development of molecular markers which are based on polymorphisms found in proteins or DNA has greatly facilitated research in a variety of disciplines such as taxonomy, phylogeny, ecology, genetics and plant breeding. Radiation by gamma rays leads to the increasing level of DNA break formation. These different types of DNA damages can be detected by changes in RAPD profiles.

The aim of the present study was to investigate the possibility of mutation changes in plant (amla) DNA after the influence of gamma rays. The use of gamma ray radiation as a mutagen for inducing variation in the existing varieties of various crops is well established. However, the desired variation viz., dwarf tree stature, fruits with high vitamin C and tannin content is lacking in amla varieties. So, radiation can be used to induce mutations and thereby generate genetic variations in the existing varieties of amla from which desired mutants may be selected. Biochemical composition of leaves had a direct influence on the biochemical composition of the amla fruits. Hence, induction of variation through gamma rays was confirmed in leaves of the amla grafts of [V.sub.1][M.sub.1] and [V.sub.1][M.sub.1] generation. In the present study, amla variety B SR-1 was treated with gamma rays with different doses and the effect of these doses of gamma rays was analysed with RAPD technique.

Materials and methods


The scions of the aural variety BSR-1 were chosen for the present investigation. The scions were of pencil thickness consisting of 10 nodes (dormant buds) from seven year old mother trees. These scions were treated under a temperature range of 25 [+ or -] 2 [degrees]C. A physical mutagen (gamma rays) was employed for the treatment of scions. Gamma rays treatment was given from the gamma chamber -900 at Tamil Nadu Agricultural University, Coimbatore. Gamma ray source was 60 Co in 1000 (mCi), emitting 5000 rads per minute at the time of irradiation. The treated scions were stored in a wet gunny cloth at room temperature till treatment (12 hrs) and thereafter till grafting on the rootstock. The treated scions were cleft grafted on the same day on one year old amla seedling rootstock collected from Salem. Both the treated and untreated grafts were planted in pots and the biological effectiveness of gamma rays (Sensitivity studies) was studied.

Sensitivity studies

A preliminary study was conducted to fix up the optimal dose of gamma ray irradiation on survival of grafts (scions treated with gamma rays). The range of gamma ray (kR) doses were 1.00, 2.00, 3.00, 4.00 and 5.00.The different criteria adopted for assessing sensitivity were

Graft survival

The survival of the gamma ray treated grafts was recorded on 30, 60, 90 and 120 days after grafting and expressed as percentage.

Degree of growth inhibition

The degree of growth inhibition was expressed in terms of the following parameters measured 90 days after grafting.

* Length of the primary shoot (cm)

* Number of leaves (counted 90 days after grafting)

* Fresh weight of the primary shoot (g)

The LD 50 value for survival ranged from 1.0 to 2.0 kR (Fig.l). Thus after assessing the LD 50 value, the gamma ray dose employed for BSR-1 variety were 0.0,0.50.,0.75,1.0,1.50,1.75,2.0,2.5 and 5.0 kR. Those treated amla grafts ([V.sub.1][M.sub.1]) were planted in pots to study the vegetative and biochemical characters of amla. The [V.sub.1][M.sub.1] (Fig.2) grafts were back pruned 180 days after planting at 2nd, 4nd and 6nd node. Those shoots arising from the cut ends were the second vegetative generation ([V.sub.2][M.sub.1]) (Fig.3).



RAPD Analysis

DNA Preparation

Fresh leaf samples collected from the field were used for isolation of genomic DNA. Isolation was done following the method recommended by Gawal and Jarret (1991).



Quantification of DNA

The quantity of DNA present in each sample was determined by reading the absorbance at 260 nm in a fluorometer (Model DyNA Quant 200, Hoefer, California, USA). The quantity of DNA in different samples varied from 500-700 ng/g of leaf tissue. After quantification, all the samples were diluted to 25ng/10 ml and used for PCR reactions.

Polymerase Chain Reaction (PCR) (Williams et al., 1990)

DNA from one parent (B SR-1), 8 mutants (dwarf plants) from [V.sub.1][M.sub.1] generation and 11 mutants (leaf mutants, early flowering mutants, mutant with shoot bifurcation, mutant with multiple shoots and mutant with blind shoot) from [V.sub.2][M.sub.2] were used for RAPD analysis following the method recommended by Bhat and Jarret (1995) with required modification. The cocktail for the amplification was prepared as follows in 0.2ml PCR tubes:
DNA 25 ng/ml 3.00[micro]l
dNTPs (2.5 mM) (Banglore Genei Ltd., India 1.00[micro]l
Primer (Operon Technologies Inc. USA) 1.00[micro]l
10% assay buffer 1.50[micro]l
Taq polymerase (3 units/mL) 0.20[micro]l
(Bangalore Genei Ltd., India)
Magnesium chloride 0.18[micro]l
Sterile distilled [H.sub.2]O 8.12[micro]l

Total 15.00[micro]l

A set of seven primers were used in the study (Table 4.) The reaction mixture was given a short spin (Biofuge Pico, Germany) for thoroughly mixing of the cocktail components. Then the 0.2ml PCR tubes were loaded on to a thermal cycler (PTC-100T[TM] MJ Research Inc., USA).

Gel electrophoresis

Along with the PCR amplified products, 100 by DNA ladders as standard marker were subjected to electrophoresis in 1.5 per cent agarose gel in 0.5X TBE buffer at 8V/cm for 4-5 hours. The electronic image of the ethidium bromide stained gel was visualized and documented in gel documentation system (Model Alpha imager 1200, Alpha Innotech Corp., USA).

List of random primers used for RAPD analysis

Results and discussion

DNA polymorphism due to different doses of gamma rays could be distinguished based on the RAPD profiles. Different and distinctive finger pattern could be obtained from the mutants studied. The seven operon primers used in the RAPD analysis yielded 2-70 bands in control (BSR - 1: Lane.2) and in respect of its mutants. The number of bands for each primer varied ranging from 35 for the primer OPZ 3 to 99 for the primer OPZ 7. The RAPD patterns of the [V.sub.1][M.sub.1] and [V.sub.2][M.sub.1] generation plants exposed to gamma rays were different from the control of the amla variety BSR-1. Thus, indicating that radiation created polymorphic regions in the amla genome.

According to Erdem and Oldacay (2004), radiation is one of the best known physical mutagens. It dissociates the atoms of water molecule and causes the generation of hydroxyl radicals that are the most reactive. They react with most of the biomolecules including DNA and scavenge the electrons from them. The oxidation of biomolecules by the radicals damages the DNA structure and biological activity. Hence, genetic alterations occur on the DNA molecules, which is the cause of mutations depend on radiation.

The main changes in the RAPD profiles of the present investigation were the appearance or disappearance of different bands with variation in their intensity. These effects might be due to the structural rearrangements in DNA caused by different types of DNA damages. RAPD method is applicable for the detection of changes in the DNA structure after different genotoxical treatments (radiation). Thus, the variation in band intensity and disappearance of some bands may correlate with the level of photoproducts in DNA template after radiation, which can reduce the number of binding sites for Taq polymerise. Appearance of new bands can be explained as a result of different DNA structural changes (Breaks, transpositions, deletions, etc) (Danylchenko and Sorochinsky, 2005). Thus, the estimate on the existence of mutation and structural alterations in plant DNA after impact of radiation on the bases of DNA patterns could be obtained after RAPD with the set of primers.

From the present study, polymorphism was detected in most of the variants. and hence can be suggested that radiation (gamma rays) Oas a physical mutagen could be used for mutant selection in breeding of amla for crop improvement.


Bhat, K.V. and R.L. Janet, 1995. Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genetic Resources and Crop Evaluation, 42: 07-118.

Danylchenko, O. and B. Sorochinsky, 2005. Use of RAPD assay for the detection of mutation changes in plant DNA induced by UV-B and R-rays BMC plant. Biol., 5(1): 59.

Erdem, G. and S. Oldacay, 2004. Employment of RAPD technique to assess the genetic stability of Helianthus annuus treated with different mutagenic agents. J. Applied Sciences, 4(2): 277-281.

Gawal, N.J. and R.L. Janet, 1991. A modified CTAB DNA extraction procedure for Musa and Ipomea. Plant Mol. Bio. Rep., 9: 262-266.

Pathak, R.K. and R.A. Pathak, 1993. Improvement of minor fruits. In: Advances in Horticulture. Eds. K.L. Chadha and O.P. Pareek., 1(1): 408-421.

Pathak, R.K., D. Pandey, G. Singh and D. Mishra, 2003. Approaches and strategies for precision farming in aonla. In: Proc. of Nat. Seminar--cum--workshop on Hi-Tech. Horticulture and Precision farming, CISH, Lucknow, 2002, pp: 176-190.

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Corresponding Author: B. Senthamizh Selvi, Faculty of Horticulture, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore- 641 003. Tamil Nadu, India

B. Senthamizh Selvi, V. Ponnuswami and P.S. Kavitha

Faculty of Horticulture, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore- 641 003. Tamil Nadu, India

B. Senthamizh Selvi, V. Ponnuswami and P.S. Kavitha: Use of RAPD Assay for the Detection of Mutation Changes in Aonla (Emblica officinalis Gaertn.): Adv. in Nat. Appl. Sci., 2(3): 129-134, 2008
The thermal cycler was programmed as follows

Profile 1: 94[degrees]C for 5 minute Initial denaturation
Profile 2: 94[degrees]C for 1 minute Denaturing
Profile 3: 37[degrees]C for 1 minute Annealing
Profile 4: 72[degrees]C for 2 minute Extension
Profile 5: 72[degrees]C for 10 minutes Final extension
Profile 6: 4[degrees]C for 30 minutes

Profiles 2, 3 and 4 were programmed to run for 44 cycles.

S.No. Primer Sequence (5' to 3')

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Title Annotation:Original Article; Random Amplification of Polymorphic DNA
Author:Selvi, Senthamizh B.; Ponnuswami, V.; Kavitha, P.S.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2008
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