Background
Okra (Abelmoschus esculentus Moench) is an edible hibiscus of the Malvaceae family. It is often cross-pollinated and is a popular vegetable crop in the tropics. Okra is very useful against genito-urinary disorders, spermatorrhoea and chronic dysentery (Nandkarni, 1927). It has been reported to use as medicine in curing ulcers and relief from hemorrhoids (Adams, 1975). Previous studies have reported that 100 gm of okra endow with calcium (20%), iron (15%) and vitamin C (50%) of human dietary requirements (Grubben, 1977).

Leaves of okra have high mucilaginous acidic polysaccharides content forming pectin and its main component is polygalacturonic acid associated with minerals, which presents a major problem during the purification of okra DNA. When cells are lysed nucleic acids come into contact with these polysaccrides (Loomis, 1974). In the oxidised form these polyphenols bind covalently and irreversibly to proteins and nucleic acids (Guilletmaut and Marechal-Drouard, 1992) resulting in a brown gelatinous material that reduces the yield and purity of the extracted DNA (Porebski et al., 1997; Aljanabi et al., 1999) and prevents it from redissolving completely. Furthermore, DNA which dissolves even in the presence of these polysaccharides inhibits the activity of different restriction enzymes (Sahu et al., 2012), PCR, or in vitro labeling.

The availability of high quality intact genomic DNA is a precondition for almost every molecular genetic analysis of crops. But when a plant tissue is rich in polysaccharide contaminants, Isolation of good quality DNA for PCR, gene mapping, diversity assessments and other molecular analyses is challenging, secondary metabolites and polyphenoles. Protocols for the isolation of DNA from tissues with high levels of polysaccharides and polyphenols have been reported (Dellaporta et al., 1983; Bernatzky and Taksley, 1986; Lodhi et al., 1994; Wang et al., 1996; 2011; Kim et al., 1997; Li et al., 2002; Geuna et al., 2004) but these did not give satisfactory results with okra.

Due to problems with DNA isolation, there have been few studies at the molecular level in okra. Appropriate protocol for DNA Isolation from okra is necessary for such studies as PCR amplification (RAPD analysis) and restriction endonuclease. After following published protocols and failing to obtain high quality DNA that was not contaminated with polyphenolic compounds, we have developed an extraction system for okra genomic DNA. This protocol yields 8~10 µg high-quality intact genomic DNA from 100 mg okra mature and young leaves applicable to enzymatic digestion and PCR amplification in 1.5 mL tube without using liquid nitrogen.

1 Results and Discussion
We present a simple, efficient and reliable method to extract high-quality genomic DNA from okra without liquid nitrogen. We attempted to isolate DNA using a few traditional methods (Doyle and Doyle, 1990; Cheng et al., 2003; Couch and Fritz, 1990.) but failed to obtain high quality pure intact DNA from okra. The tissues of okra have high mucilaginous acidic polysaccharides content forming pectin and its main component is polygalacturonic acid associated with minerals. This presents a major contamination problem in the purification of okra DNA. Okra is undoubtedly the most difficult plant we have worked on but this method has enabled us to prepare the DNA with good spectral qualities.

The DNA prepared with this procedure was tested for yield, purity, susceptibility to restriction endonuclease digestion and ability to be amplified by PCR. We obtained approx. µg high-quality DNA 100 mg^-1 fresh leaves, enough for 500 RAPD reactions. The A₂₆₀/A₂₈₀ of the extracted DNA ranged from 1.6~2.1 consistent with the ratio recommended for pure DNA preparations. The use of high concentration NaCl with isopropanol helps the release of DNA from polysaccharides. The polysaccharides settle in the bottom with NaCl and precipitated DNA remains suspended in the isopropanol in the upper layer. Undigested okra DNA showed a discrete band of unbreakable DNA (Figure 1).

Figure 1 Agarose gel electrophoresis of unrestricted DNA prepared from 10 cultivars of okra

DNA isolated by this protocol was accessible to a wide range of restriction enzymes. The restriction pattern of the isolated DNA from okra gave characteristics length distribution of digested chromosomal DNA. Repetitive bands were not visible (Figure 2).

Figure 2 Agarose gel electrophoresis of total DNA (300 ng) digested with Ava II prepared from 10 cultivars of okra

To check the suitability of extracted DNA for further analysis, RAPD PCR was conducted. Reproducible amplification was observed in PCR reactions in several independent extractions and replications (Figure 3).

Figure 3  RAPD PCR amplification of okra leaf DNA

To prevent residual ribonucleosides from acting as primers during PCR amplification (Porebski et al., 1997), the DNA was made RNA free by using RNaseA.

Small amounts of okra leaf tissue are sufficient for successful DNA extraction, and many samples can be processed at the same time. The chances of contamination are low as reactions are carried out in screw-top Eppendorf tubes. No liquid nitrogen is required. The protocol was standardized for 100 mg fresh leaves, which can be handled in a 1.5 mL disposable Eppendorf tube. Similar conditions on a large scale, using 1~10 g samples, can be scaled up and fit into a 50 mL screw capped tube. The method may also be beneficial for other plant species tissues containing high levels of polysaccharides.

2 Materials and Methods
Extraction buffer: [Tris-HCl (pH maintained at 7.5 and 200 mmol/L conc.), Sodium chloride (250 mmol/L), Ethylenediaminetetra acetic acid (25 mmol/L), Sodium Dodecyl sulfate (0.5% w/v)]; Isopropanol; 5 mol/L NaCl; Ethanol 75% (v/v); Tris EDTA:  [10 mmol/L Tris-HCL (pH 8.0), 0.1 mmol/L Ethylenediaminetetra acetic acid (pH 8.0)]; 10 mg/mL, RNase A (Sigma); Phenol-chloroform; Chloroform-isoamylalcohol; Absolute ethanol; 3 mol/L sodium acetate (pH 5.2).

2.1 DNA extraction
Vacuum-dry [Eyela Freeze Dryer FDU 506 (Tokyo Rikakikai Co Ltd, Japan)] 100 mg okra leaves in a 1.5 mL screw cap tube (Assist, Japan) then grind for 20 s at 3 000 r/min in multi-beads shocker (Yasui Kikai, Japan). Add 400 µL extraction buffer and mix by vortexing and leave the mixture for some time. Centrifuge at 10,000 xg. Take the supernatant; add 300 µL ice-cold isopropanol along with100 µL 5 mol/L NaCl and invert gently to mix the ingredients. Incubate at -20°C for approximately 1 hr. Transfer the upper layer gently, without disturbing the polysaccharides to a new vial. Centrifuge at 10,000 xg for 10 min at room temperature. Wash the pellet with 75% (v/v) analytical grade ethanol. Dry the pellet and dissolve in100 µL TE buffer. Add 1 µL (10 mg/mL) RNaseA (Promega) and incubate at 37°C. Add equal volume of phenol-chloroform (1:1, v/v) and centrifuge at 10,000 g for 5 min at room temperature. Transfer the supernatant to new tube and add one volume of chloroform-isoamylalcohol (24:1, v/v). Centrifuge at 10,000 xg for 5 min at room temperature and transfer the supernatant to a 1.5 mL eppendorf tube. Add 0.1 volume of 3 mol/L sodium acetate and 2.5 volumes absolute ethanol. Leave at -20°C for 20 min. Centrifuge at 10,000 xg for 10 min at 4°C and discard the supernatant then wash pellet with 75% (v/v) ethanol. Dry the pellet for 30 min and dissolve in 50 µL TE buffer.

2.2 DNA analysis
The quality of the extracted DNA was observed by electrophoresis, followed by staining with 0.5 mg^-1 ethidium bromide and photographed using gel documentation system. DNA yield and quality was calculated using a NanoDrop ND-1000 spectrophotometer (USA) at 260 nm.

2.3 Digestion of DNA with restriction enzymes
DNA (300 ng) was digested for 3 hr. at 37°C with 1U of Ava II in a total volume of 25 µL and electrophorased in agarose gel 0.8% (w/v) with undigested DNA as a control.

2.4 PCR amplification
RAPD PCR Amplification of okra leaf genomic DNA was carried out in a total reaction volume of 25 µL containing 20~25 ng  genomic DNA, 1X Taq polymerase reaction buffer, 2 mmol/L MgCl₂, 0.1 mmol/L dNTP, 0.2 µmol/L 12-mer primer 5’- GCCCCGTTAGAT-3’; Operon  A04 (Operon Technologies Inc., Alameda, Calif; USA) and 1.5 Units of polymerase (Taq DNA polymerase).

The amplifications were carried out using the icycler (BIO-RAD, USA) after 5 min at 94°C for 1 cycle, followed by 35 cycles of 1 min at 94°C, 1 min at 35°C, and 3 min at 72°C, with a final 7 min extension of 72°C. Amplification products were separated in 3% (w/v) agarose-LE (nacalai tesque, Japan) TAE gels run at 100 V for 80 min.

Acknowledgements
We are thankful to Dr S. Niikura and N. Hashimoto for kindly providing okra seeds.

References
Adams C.F., 1975, Nutritive value of American foods in common units, U.S. Department of Agriculture, Agric Handbook, pp.29 
 
Aljanabi S.M., Forget L., and Dookun A., 1999, An improved and rapid protocol for the isolation of polysaccharide-and polyphenol-free sugarcane DNA, Plant Mol. Biol. Reptr., 17: 1-8 http://dx.doi.org/10.1023/A:1007692929505  http://dx.doi.org/10.1023/A:1017213630972 
 
Bernatzky R., and Taksley S.D., 1986, Methods for detection of single or low copy sequences in tomato on southern blots, Plant Mol. Biol. Reptr., 4: 37-41 http://dx.doi.org/10.1007/BF02672487 
 
Cheng Y.J., Guo W.W., Yi H.L., and Pang X.M., 2003, An efficient protocol for genomic DNA extraction from citrus species, Plant Mol. Biol. Rep., 21: 177a-177g http://dx.doi.org/10.1007/BF02774246 
 
Couch J.A., and Fritz P.J., 1990, Isolation of DNA from plants high in polyphenolics, Plant Mol. Biol. Rep., 8(1): 8-12 http://dx.doi.org/10.1007/BF02668875 
 
Dellaporta S.L., Wood J., and Hicks J.B., 1983, A plant DNA minipreparation: version II, Plant Mol. Biol. Reptr., 1: 19-21 http://dx.doi.org/10.1007/BF02712670 
 
Doyle J.J., and Doyle J.L., 1990, Isolation of plant DNA from fresh tissue, Focus, 12, pp. 13-15 
 
Geuna F., Maitti C., Digiuni S., and Riccardo B., 2004, A method for extracting genomic DNA suitable for medium-throughput applications from plant tissues rich in contaminants, Plant Mol. Biol. Reptr., 22: 87-87 http://dx.doi.org/10.1007/BF02773354 
 
Grubben G.J.H., 1977, Tropical vegetables and their genetic resources (Tindall H.D. & Willams J.T. eds.), IBPGR, Rome, pp.197 
 
Kim C.S., Lee C.H., Shin J.S., Chung Y.S., and Hyung N.I., 1997, A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP, Nucl. Acids. Res., 25: 1085-1086 http://dx.doi.org/10.1093/nar/25.5.1085
PMid: 9023124 PMCid: PMC146538
 
Li Y.X., Zhi X.S., and Fang C., 2002, Rapid extraction of genomic DNA from leaves and bracts of Dove tree (Davidia involucrate), Plant Mol. Biol. Reptr., 20: 185-190 http://dx.doi.org/10.1007/BF02799433 
 
Lodhi M.A., Ye G.N., Weeden N.F., and Reisch B.I., 1994, A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species, Plant Mol. Biol. Reptr., 12: 613-614 http://dx.doi.org/10.1007/BF02668658 
 
Loomis W.D., 1974, Overcoming problems of phenolics and quinines in the isolation of plant enzymes and organelles, Meth. Enzymol., 31: 528-544 http://dx.doi.org/10.1016/0076-6879(74)31057-9 
 
Margam V.M., Gachomo E.W., Shukle J.H., and Ariyo O.O., 2010, A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding, Mol. Biol. Rep., 37: 3631-3635
http://dx.doi.org/10.1007/s11033-010-0014-5 PMid: 20204529 
 
Maréchal-Drouard L., and Guillemaut P., 1995, A powerful but simple technique to prepare polysaccharide-free DNA quickly and without phenol extraction, Plant Mol. Biol. Reptr., 13: 26-30 http://dx.doi.org/10.1007/BF02668390 
 
Nandkarni K.M., 1927, Indian Meteria Medical, Nandkarni and Co., Bombay 

Porebskim S., Bailey L.G., and Baum B.R., 1997, Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components, Plant Mol. Biol. Reptr., 15: 8-15 http://dx.doi.org/10.1007/BF02772108 
 
Sahu S.k., Thangaraj M., and Kathiresan K., 2012, DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol, Mol. Biol., 1: 1-6
 
Singh V., and Kumar V., 2012, An optimized method of DNA isolation from highly mucilage-rich okra (Abelmoschus Esculentus L.) for PCR analysis, Adv. Appl. Sci. Res., 3 (3): 1809-1813
 
Wang T.Y., Wang L., Zhang J.H., and Dong W.H., 2011, A simplified universal genomic DNA extraction protocol suitable for PCR, Genet. Mol. Res., 10(1): 519-525 http://dx.doi.org/10.4238/vol10-1gmr1055 PMid: 21476197 
 
Wang X.D., Wang Z.P., and Zou Y.P., 1996, An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel, Plant Mol. Biol. Reptr., 14: 369-373
http://dx.doi.org/10.1007/BF02673369