ABSTRACT- - A good amount of DNA can be extracted from the species of Plumbago Linn. by following the standardized and modified protocol. The DNA isolated was quantified using a spectrophotometer at the absorbance of λ_260nm and λ_280nm. Both the species given the best result with a DNA yield of 23.8µg/µl and 0.895µg/µl in Plumbago zeylanica and Plumbago indica respectively and produced a clear band on the agarose gel. The estimation of purity of the obtained DNA was concluded by analyzing the ratio of λ_260nm/λ_280nm of the Genomic DNA which was 1.94 and 2.0 for P. zeylanica L. and P. indica L. respectively. These results are of high quality as it is in the range of 1.8-2.0 by the present optimized protocol.
Key-words- Genomic DNA standardized protocol plumbagin

INTRODUCTION
Plumbago Linn. is a genus under family Plumbaginaceae consisting 10-20 species of flowering plants. They are widely distributed in the tropics [1]. In India the plant has a wide distribution ranging from Central India to North-east India, and various parts of Southern India. Plumbago zeylanica L. and Plumbago indica L. are the two medicinally important, morphologically different species of this genus selected for the present study. These two species has been reported from North-eastern regions of India. [2]
Plumbago zeylanica L. is a multipurpose medicinal herb, commonly known as Ceylon leadwort or wild leadwort [3], however its trade name in India is Chitrak. In north-east India it is widely distributed in the wilds of Assam, Meghalaya. Roots are widely used for its medicinal properties. Roots contain napthaquinone derivatives, plumbagin, chloroplumbagin, biplumbagin, elliptinone, chitranone [4]. Plumbagin shows strong antibacterial activity against Escherichia coli, Salmonella typhi and Staphylococcus aureus, and antifungal and inhibitory outcome in contradiction of Klebsiella pneumoniae, Serratia marcescens and moderate result in contradiction of Bacillus subtilis, and less low effect contrary to Proteus vulgaris and Pseudomonas aeruginosa, and aqueous extract shows less antibacterial action as associated to organic extract. Plumbagin was shown anticancer, antimicrobial activity and antibiotic effects. [5]
Flowers are white in colour [4,7]. They are bisexual, regular, pentamerous, pedicellate and sweet-scented. The flowers are also characterized by having a tubular calyx (7–11 mm long and 5- ribbed) with glandular trichomes (hair) secreting a sticky mucilage. Flowers are insect pollinated. The mucilaginous glands aid in trapping insects and fruit dispersal by animals. The fruit of the plant is an oblong (7.5–8 mm long) five-furrowed capsule containing single seed. Roots are light- yellow when fresh and become reddish-brown on drying. The roots have a strong and characteristic odor [6]. Root extracts are effective against dysentery, diarrhea, diuretic, and peptic ulcers and intestinal parasite [7], malaria, rheumatism, intestinal parasites, anemia due to ‘stagnant blood’, internal and external trauma, toxic swelling and furunculous scabies can be treated with this plant [8]. Roots of the plant have abortifacient and vesicant effects. Paste prepared from roots of the plant is applied to the skin to treat abscesses, other skin diseases including ulcers and scabies also [9]. Plant extracts have shown potent mosquito larvicidal activity against the larvae of Aedes aegypti while showing no toxicity to fish [10]. Hexane extracts of Plumbago zeylanica have shown activity against canine distemper virus [11].
Plumbago indica L. is a medicinal herb and is also planted in gardens as an ornamental plant. They are commonly known as Indian leadwort, scarlet leadwort, however, the trade name in India is Lal Chitrak. It is known for its medicinal properties in roots which contains Plumbagin (5-hydroxy-2-methylnaphthalene-1,4 -dione), sitosterol glycoside. Perennial herb or small shrub up to 2m tall. Flowers bisexual, regular, pentamerous. In Plumbago indica L. fruits have never been found. Especially the roots have many uses: it is acrid, vesicant, alternative, digestive, stimulant and a powerful abortifacient and oral contraceptive. High doses are dangerous and may cause death. An infusion of the roots is taken to treat dyspepsia, colic, cough and bronchitis. A liniment made from bruised root mixed with vegetable oil is used as a rubefacient to treat rheumatism and headache. The milky juice of the leaves is applied on the skin in treatment of scabies, ring worm and haemorrhoids. Plumbago wissii is a species in this genus which is endemic to Namibia and tagged as ‘Least Concern (LC)’ by IUCN Red list [12].
Although the micro-propagation is the best way to preserve these species, the Genomic DNA isolation and its sequencing followed by comparing and finding out the various functions of its gene would reveal the most of the reasons behind the susceptibility of Plumbago indica L. then Plumbago zeylanica L. under environmental stress. Since the plant is ethno-medicinally important, a lot of human exploitation particularly in Northeastern parts of India is occurring. Therefore the present study aims at providing a better survival of these species of Plumbago Linn. by standardizing the protocol for the genomic DNA isolation in more quantity and in more purified form by doing a comparative analyzing between protocols for DNA isolation using CTAB by Doyle & Doyle [13] (modified) and Khanuja et al. [14] (modified).

MATERIALS AND METHODS
This study was conducted in Institutional Biotech Hub and Department of Botany, Handique Girls’ College, Guwahati, Assam, India in duration of six months from January – June 2016.

Plant material: Sapling of Plumbago zeylanica L. were collected from different areas of Greater Guwahati, Assam, India and that of Plumbago indica L. were collected from wild habitats of Amsoi hills, Nagaon, Assam, India and from Experimental Garden, Barapani; areas of Meghalaya and were raised under departmental botanical garden. Young and fresh leaves were harvested for DNA isolation.

Solutions

• 2% CTAB (100ml)- extraction buffer 2g of CTAB were dissolved in 70 ml water, followed by addition of 8.18g of NaCl, 10 ml of Tris (pH 8.0; 1M), 4ml of EDTA (pH 8.0; 0.5M). The volume was made up to 99 ml with water. Autoclaved, then added 1g of PVP.
  
• Chloroform: Isoamyl (24:1)
  Chloroform- 48ml
  Isoamyl alcohol- 2 ml
  
• Isopropanol
  
• 70% Ethanol
  95% ethanol- 73.6ml
  Sterile ddH2O- 26.4ml
  
• 0.1X TE buffer [100ml; pH 8] 100µl of 1M Tris (pH 8) and 20 µl 0f 0.5 M EDTA (pH 8) was added to water, followed by making up the volume to 100 ml and autoclaved.
  
• Ribonuclease A (10mg/ml).

DNA isolation protocol

1. 1 gm of young and fresh leaves were weighed and allowed to freeze in liquid Nitrogen.
2. The frozen tissues are grinded into fine powder with the aid of a chilled sterile mortar and pestle in the presence of liquid nitrogen.
3. 200 mg of the powder is weighed and transferred in centrifuge tube using chilled spatula. Equal volume (w/v) of hot (65°C) 2% CTAB buffer is added to the powdered sample.
4. Mixed well by inversion and kept in water bath at 60°C for 1 hour with mixing well by inversion at every 15 minutes.
5. Equal volume of Chloroform: Isoamyl (24:1) was added to the sample and mixed gently by inversion.
6. The mixture was centrifuged at 7280 g for 15 min. at 25°C-28°C.
7. The top aqueous layer was transferred to fresh tube and equal volume of chilled Isopropanol (-20°C) was added to the sample. On mixing it gently shown a clear clouded clump of nucleic acids.
8. The mixture is centrifuged for 15 min at 7280 g.
9. A clear pellet was visible down the tube. The liquid is decanted and leaving the pellet attached to the wall of the tube.
10. The pellet is washed with 70% ethanol at 4°C in centrifuge at 7280 g for 15 min.
11. The decanted tube is air dried and the pellet is dissolved in 70µl- 100µl of 0.1X TE buffer.
12. RNase treatment is given to the dissolved pellet by adding 8µl RNase and kept in water bath at 55°C for 30 min - 1 hour.

Estimation of DNA quantity and purity

The quantification and estimation of purity of the obtained genomic DNA per gram of the plant material was done using UV-VIS Spectrophotometer in the following steps-
1. 2 ml TE Buffer is taken in a cuvette and calibrated between λ₂₆₀ nm and λ₂₈₀ nm.
2. TE buffer is taken as blank.
3. 20µl of DNA sample is diluted into 1980µl TE buffer. Therefore the Dilution Factor is 100.
4. Noted the OD λ₂₆₀ nm and OD λ₂₈₀ nm values of the spectrometer.
5. Calculated the OD λ₂₆₀/OD λ₂₈₀ ratio to measure the purity of the obtained DNA sample.
6. The amount of DNA is quantified using the formula
   DNA concentration (µg/µl) = OD260 ×100(DF)× 50 ∕ 1000

RESULTS AND DISCUSSION
A comparative analysis between the present modified method of Khanuja et al. [14] and the standard method of Doyle & Doyle [13] draws clear and contrast difference in the both the amount and purity of DNA isolated from the two species of Plumbago Linn: P. zeylanica L. and, P. indica L. The observations and results obtained after following the two methods are as follows-



Fig. 1: Well No. 1&2 Loaded with sample extracted from P. zeylanica,
Well. No. 3 Loaded with sample extracted from P. indica By Modified method of Doyle & Doyle ^[13]

Method: I: Doyle & Doyle [13] (Modified)

The following observations were recorded under this method-
1. By following this method very less amount of DNA were able to extract. The width and dense of the band shows the less amount of Genomic DNA extracted through the modified method of Doyle & Doyle [13].
2. While performing the steps in the protocol pellets were barely visible.
3. Spectrophotometer analysis showed the DNA concentration to be 1.15µg/µl from P. zeyalnica L. and 0.62µg/µl from P. indica L. (Table 1).
4. The ratio of λ_260nm/λ_280nm of the Genomic DNA extracted by this method is 0.6, and 0.4 for P. zeylanica L. and P. indica L. respectively. (Table 1)

Fig. 2: Well No. 1 & 2 Loaded with sample extracted from P. zeylanica
Well No. 3 & 4 Loaded with sample extracted from P. indica by Modified method of Khanuja et al. ^[14]

Method: II: Khanuja et al. [14] (Modified)

The following observations were recorded under this method-
1. By following the modified method of Khanuja et al. [14], a noticeably larger amount of Genomic DNA bands were observed in agarose gel electrophoresis analysis.
2. A thick coagulated mass were observed at the step when chilled Isopropanol were added in this method, which on centrifugation settled into a pellet.
3. Spectrophotometer analysis showed the DNA concentration to be 2.38µg/µl from P. zeyalnica L. and 0.895µg/µl from P. indica L. (Table 1).
4. The ratio of λ_260nm/λ_280nm of the Genomic DNA extracted by this method is 1.94 and 2.0 for P. zeylanica L. and P. indica L. respectively (Table 1).
   
   The quality and quantity of the DNA was checked using spectrophotometer. The quantification of DNA was done by recording its absorbance at λ_260nmtoλ_280nm nm using a UV-VIS Spectrophotometer-
   
   
   • The extracted DNA of the two species of Plumbago Linn: P. zeylanica L. and P. indica L. with the modified protocol of Khanuja et al. [14], was of high quality as its ratio of λ_260nmλ_280nm is 1.94 and 2.0 which is in the range of 1.8-2.0, in comparison to the yield of DNA by the modified protocol of Doyle & Doyle [13] where the ratio of ?260/?280 is 0.6 and 0.4 in respectively.
   • The absorption in the UV range ?260 nm is due to nucleic acids and the obtained yield is 2.38 µg/µl and 0.895 µg/µl form Khanuja et al. [14] (Modified) and from Doyle & Doyle [13] (Modified) only 1.15 µg/µl and 0.62µg/µl in Plumbago zeylanica L. and Plumbago indica L. respectively (Table 1).
   • In the present study selective precipitants viz, addition of 1g PVP in Extraction buffer, fixing the centrifugal force to 7820g, fixing the incubation period to 1 hour with mixing well by inversion at every 15 minutes etc were added/modified to remove the contaminants one by one and yield pure DNA, modification in centrifugation speed and time was also found effective in the yield of DNA.
   
   
   Table 1: DNA obtained from different species of Plumbago Linn. using Doyle & Doyle [13] (modified) and Khanuja et al. [14] (modified)
   
   

   	Modified method of Doyle & Doyle [13]				Modified method of Khanuja et al. [14]
   Species	Optical density at λ260nm	Optical Density at λ280nm	Optical Density λ260nm/λ280nm	DNA conc. µg/µl	Optical Density at λ260nm µg/µl	Optical Density at λ280nm	Optical Density λ260nm/λ280nm	DNA conc. µg/µl
   Plumbago zeylanica L.	0.23	0.345	0.6	1.15	0.476	0.245	1.94	2.38
   Plumbago indica L.	0.124	0.256	0.48	0.62	0.179	0.087	2.0	0.895

   
   
   
   It was interesting to note that except in some steps both species reacted to the reactants in same manner.
   • The first exception came to notice was their interaction with the CTAB extraction buffer (60°C). The powdered leaves of P. zeylanica L in liquid nitrogen interacted very well with the CTAB extraction buffer without changing its fresh green colour and its powdered particles nicely got spread with the buffer, on the other hand the powdered leaves samples of P. indica L. interacted with the CTAB extraction buffer in a more clumped manner, i.e. in contact with the CTAB buffer they got organized in a clumped form. With repetitive tapping and mixing by vigorous inversion only they got unclamped and allowed the extraction buffer to act on them. P. indica L. during the 1 hour incubation time with the CTAB extraction buffer changed its colour from green to lead (grayish black) color.
   • The second exception was the time taken in the RNase action on the individual species. In P. zeylanica L. the RNase took 30 min. to degrade its RNA contaminant and on the other hand P. indica L. took 1 hour to totally degrade the amount of RNA present in the sample as a contaminant in 55°C as the suitable temperature.
   
   The statistical analysis shows the DNA concentration obtained by following the protocols of Doyle & Doyle [13] (modified) and Khanuja et al. [14] (modified). In the graph a clear, noticeable difference of the yield of the DNA from each protocol is observed. Therefore the method of Khanuja et al. [14] (modified) protocol is best suited to obtain genomic DNA from Plumbago Linn (Fig. 3)
   The statistical analysis of the Extracted DNA purity by the two methods- Doyle & Doyle [13] (Modified) and Khanuja et al. [14] (modified) is observed in this Graphical representation of the ratio λ_260nm/λ_280nm. This ratio clearly shows the purity of the obtained DNA yield. From the graphical representation it is clear that Khanuja et al. [14] (modified) protocol is best suited to obtain a purified DNA yield from Plumbago Linn (Fig. 4).
   
   
   
   Fig. 3: Bar diagram showing DNA concentration
   
   
   
   Fig. 4: Bar diagram showing extracted DNA purity
   
   From the above two statistical representation it can be suggested that in Plumbago Linn, Plumbago zeylanica L. genomic DNA was obtained in higher quantity than Plumbago indica L. in both the protocols- Doyle & Doyle [13] (modified) and Khanuja et al. [14] (modified) with a p-value 0.0455. This may be due to the presence of more interfering secondary metabolites in P. indica L. than in P. zeylanica L. (Fig. 3-4). Selective percipients like extraction buffer, PVP etc were added to remove the contaminants one by one and yield pure DNA, modification in centrifugation speed and time also found effective in the yield of DNA. The extracted DNA was of high quality as it ranges from 1.94 to 2.0 in P. zeylanica L. and P. indica L. respectively.
   Successful isolation of RNA free DNA was found to be 2.38 µg/µl and 0.895µg/µl. after following the present optimized protocol of Khanuja et al. [14] with modifications. Therefore it can be concluded that this is the best suited protocol for Genomic DNA isolation from Plumbago Linn.
   
   CONCLUSIONS
   A good amount of DNA can be extracted from Plumbago sp. by following the protocol of Khanuja et al. [14] (modified). Both the species gave the best result with a DNA yield of 2.38 µg/µl and 0.895 µg/µl and the ratio of λ_260nm/λ_280nm of the Genomic DNA was 1.94 and 2.0 for P. zeylanica L. and P. indica L. respectively. The estimation of purity of the obtained DNA was concluded by analyzing This result demonstrates that genomic DNA ranging from 1.8-2.0 after following the present optimized protocol is of high quality. Fresh, tender and new leaves are mandatory as they contain secondary metabolites in less content and they are believed to be resistant to other parasites. Therefore further study and analysis of the genomic DNA of Plumbago Linn. will help to develop genetically engineered plants which will lead to more enhanced production of plumbagin and would have more resistance to environmental stress and secure survival is possible to this precious and ethno- medicinally important plant species.
   
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   How to cite this article: Ravindran A, Gayan J, Das BN: A Standardized Protocol for Genomic DNA Isolation from the Species of Plumbago Linn. Int. J. Life. Sci. Scienti. Res., 2017; 3(5):1345-1349. DOI:10.21276/ijlssr.2017.3.5.13 Source of Financial Support:Nil, Conflict of interest: Nil