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: Special issue, Vol. IV: 213-217: 2013

www.theecoscan.inAN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

Proceedings of International Conference onHarmony with Nature in Context of

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Aruna Singh et al.,

Callus

DNA

PCR

INDUCTION OF CALLUS AND DNA EXTRACTION FROM SEEDS ANDFRESH LEAF TISSUES OF MARIGOLD (TAGETES ERECTA)

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ARUNA SINGH, SUPRIYA SHRIVASTAVA AND SOMA ROY*Department of Biotechnology, Ranchi Women’s College, Ranchi - 834 001

E-mail: drsomaroy9@gmail.com

*Corresponding author

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Tagetes erecta, a flowering plant of familyAsteraceae has ornamental value and widelyused in religious and decoration purposes. Itis used for its nematocide, cosmetic andmedicinal properties.Its use as a cover cropand has immense potential in ecology. In thepresent investigation we used different explantsof Marigold to study the role of growthregulators on callus initiation and regenerationpotential. 2,4-D ranging from 2.5mg/L- 5mg/Lwere suitable for callus induction in stemexplant. Cut ends of root explants did notdevelop much callus even with higherconcentration of 2, 4-D. Increase of callusformation was seen with supplemented NAAconcentration upto 5mg/L along with lowconcentration (.5mg/L - 1.5mg/L) of kinetin orBAP. We have also used standard protocol toextract DNA from explant (seeds and freshleaves) and successfully amplified the DNAby PCR using arbitrary RAPD primers. Callusinduction will be helpful for commercialproduction for plants of the family and DNAextraction and PCR amplification will helpunderstanding them at molecular level, suchas Southern blotting and restriction fragmentlength polymorphism.

ABSTRACT

INTRODUCTION

Marigolds’repressive impact on nematodes has been documented for over 50years Steiner (1941). Tyler (1938) reported 29 marigold varieties were resistant toroot-knot nematodes (Meloidogyne spp.). Early literature also indicated thatmarigolds could prevent the population increase of 14 genera of PPNs (Steiner,1941; Oostenbrink et al., 1957; Suatmadji, 1969), encompassing endoparasitic,semi-endoparasitic, and ectoparasitic nematodes (Siddiqui and Alam, 1987). Severalstudies found that T. erecta acted as a trap crop by arresting the development ofMeloidogyne spp. Juveniles (Daulton and Curtis, 1963; Rangaswamy et al., 1993;Ploeg and Maris, 1999). Outside the scientiûc community, marigold is less likely tobe recognized for its nematicidal properties and is mostly grown as an ornamentalplants in home gardens. Four species of Tagetes are commonly grown asornamentals: T. erecta, T. patula, T. tenuifolia, and T. lunulata (Soule, 1996).However, in addition to its ornamental or nematicidal uses, marigolds posses’ richnectar and can help support local populations of bees and other pollinating insects(Comba et al., 1999). Further, Tagetes oil, mainly from T. minuta, is used inperfumery and as a ûavoring constituent. Thus it has considerable impact onecology . In vitro culture of plant cells and tissue has attracted considerable interestover recent years because it provides the means to study plant’s physiological andgenetic processes in addition to offering the potential to assist in the breeding ofimproved cultivars by increasing genetic variability. The focus of this study is to testthe role of different growth regulators on callus initiation, its growth andorganogenesis from Tagetes erecta which is locally known as “Genda”. The studyfurther uses a standard protocol for DNA isolation from seeds and fresh leaves ofthe plant and to obtain DNA that is suitable for amplification by PCR.

MATERIALS AND METHODS

Tissue CultureIn the present investigation, MS (Murashige and Skoog, 1962) basal medium wasused. Components of media for growth of plant cell cultures include inorganicnutrients (macronutrients and micronutrients), amino acids and vitamins and ironsource. Growth regulators such as Auxins-2, 4D, NAA, IBA, IAA and Cytokinins-(BAP and Kinetine) were used. They were prepared as stock solutions separatelyand stored under refrigeration. Required volume of stock solution was pipetted outduring media preparation and 0.3% of sucrose, as Carbon source, was added.Medium was homogenized by boiling and continuous stirring with pH of mediumadjusted up to 5.8 before adding 0.8% agar by using 0.1 N NaOH or 0.1N HCL.Desired concentration of growth regulators were added and mixed properly. 15-20mL. medium was poured into culture tubes (15x2.5cm) which were washedthoroughly and rinsed in distilled water and oven dried. Sterilization of mediumwas done for 15-20 minutes. Tubes containing sterilized medium were left tilted toprepare slants in air conditioned room.

Seeds of ornamental plant Tagetes erecta were collected from the seed centre,

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INDUCTION OF CALLUS AND DNA EXTRACTION

Ranchi. Seeds were soaked in water for 12 hours to facilitatein vitro germination. Vegetative parts such as stem, leaf androot were taken as explant from in vitro grown seedlings.Explants were also obtained from plants growing in pots. Theseexplants were thoroughly washed with tap water and weretreated with suthol solution for about 5 minutes and againwashed with running tap water. The explants were then surfacesterilized in 0.2% of HgCl2 by immersing for 2-3 minutes andthen were rinsed in double-distilled water. These sterilizedexplants were then cut into pieces of about 1.0 to 1.5 cm. andmade ready for inoculation in order to observe callus formationand regeneration potentiality. Various concentrations rangingfrom 0.5 mg/L to 5 mg/L of Auxins and Cytikinins were used.Fully developed seedlings formation took about 15 days.Inoculated explants were used for callus induction andorganogenesis.

DNA IsolationPlant genomic mini kit (Xcelris) was used for total cellularDNA Isolation. Fresh specimens of leaves (approx. 100mg)were crushed in liquid nitrogen with mortar and pestle andtransferred it to 2 mL centrifuge tube. Three buffers (P1, P2and P3), provided in the kit were used . At first 500µl buffer P1was added and vortexed vigorously to mix and to disperse allthe clumps and incubated at 65ºC for 4 minutes. The samplewas mixed twice during incubation by inverting tubes. Afterthat 400µL buffer P2 was added and mixed well by vortexing,then incubated at 65ºC for 6 minutes, lastly 140µL buffer P3was mixed well by vortexing and again centrifuged at 10,000rpm for 10 minutes. Entire sample was applied to a DNAcolumn placed in a 2.0 mL collection tube. The column wascentrifuged at 13,000 rpm for 1 minute, then 2.0mL collectiontube and the flow-through liquid was discarded. Wash stepwas repeated with another 650µL DNA wash buffer, thencentrifuged at 13,000 rpm for 1 minute. Flow-through andcollection tube was discarded. In next step another 2.0collection tube was used. The column was placed into a newcollection tube and centrifuged at 13,000 rpm for 2 minutes.The column was transferred to a clean 1.5mL tube. 100µLElution Buffer (pre warmed to 65ºC and incubated at roomtemperature for 3-5 minutes) was added, and centrifuged at13,000 rpm for 1 minute to elute DNA. In next step another100µL Elution Buffer was used. This step was performed usinganother 1.5mL tube to maintain a higher DNA concentrationin the first elute.

PCR amplification and Gel ElectrophoresisPCR-based amplification of purified DNA was carried out in a20-µL reaction mixture. PCR-based amplification of the purifiedDNA was carried out in a 20-µL reaction mixture. The reactionmixture contained 25ng templates DNA, 0.125-U Taq DNApolymerase, 1.6 mM dNTPs, 3.75 mM MgCl2, 1X Taq DNApolymerase buffer (Shah MM, Yen Y, Gill KS, 2000) and 2mMPrimer (Himedia). Amplification of the DNA was done using aPeltier Analytical thermocycler with the following parameters:initial denaturation at 94ºC for 5 min, followed by 35 cycles ofdenaturation at 94ºC for 30 s, primer annealing at 50ºC for 1min, and extension at 72ºC for 1 min, with a final extension at72ºC for 7 min. The reaction was stored at 4ºC, until it wasloaded onto the gel. The PCR products were fractionated on0.8% agarose gel using 1X TBE buffer containing 10 mg/mL

ethidium bromide and were visualized under UV light and thegels were photographed using the UV- gel documentationsystem.

RESULTS AND DISCUSSION

Effect of Auxin and Cytokinin on ExplantsThe sterilized explants started developing seedlings in about6 days. (Fig. 1)

1. 2, 4-DWith 2, 4-D ranging from 2.5 mg/L- 5mg/L were found to besuitable for the induction and growth of the callus on stemexplant . Callus developed at cut ends of the explant but thegrowth was not much in 4 weeks time . After the transfer ofthese calli on plain MS media growth increased. No rootswere observed . Lower concentration of 2, 4-D (0.5mg/L –1.5mg/L) did not produce much callus and amount wasnegligible. Cut ends of leaves developed callus. Little calluswas observed with all concentrations used but a little higherconcentration (1.5 mg/L- 5mg/L) were found to be most suitable(Fig. 2). With the increasing concentration of 2, 4-D the growthwas also found to increase but the difference was not muchNo roots were observed in any of the cultures. Root explantsalso developed brown coloured callus but amount was notmuch and they always produced roots with hairs. Up to 5 mg/L the amount of callus did not show any more increase in size.Calli in all cases increased after subculturing on plain MSmedia and with addition of low concentration of BAP orKinetine (0.1mg/L – 0.5mg/L)

2. NAACut ends of stem explants developed callus with allconcentration of NAA used but concentration ranging from(1.5 mg/L-5mg/L) were found to be most suitable. 2-4 smallroots were also observed growing from these explants. Cutends of leaves developed callus only. No roots were observed.Young leaves developed callus all over which grew in size in4-6 weeks. 2.5 mg/L-5 mg/L concentration were found to bemore suitable. With increasing concentration (0.5mg/L- 5mg/L) callus amount gradually increased a little but the amountwas never very much. Many roots appeared on explant whichwere upto 2-3 inches long and cream coloured when young(Fig. 4). Later they turned brown. Callus formation increasedwhen the media was supplemented with NAA (upto 5mg/L)and low concentration (0.5mg/L -1.5mg/L) of kinetin or BAP.

The best and significant result was recorded with MS- mediumsupplemented with 5.0 mg/L of NAA + 1.5mg/L of BAP ascompared with other supplemented media. The results werein harmony with those obtained by Osman et al., 2008).Further, the previous observation could be explained on thefact that these growth factors enhanced the protein synthesis,cell growth and cell division (Skoog and Schmitz, 1972 andGamborg and Shyluk, 1981). In this respect, Kothari andChandra (1986) found that the best medium for callusinduction from leaf explants of T. erecta was obtained byaugmented of MS-medium with 10.0 mg/L BAP + 2.0 mg/LNAA + 0.5 mg/L GA3 , whereas Belarmino et al. (1992) reportedthat 5 mg/L NAA and 0.2-0.5 mg/L BAP were the bestconcentrations for producing large yellow calli from leaf

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explants. These results indicate that callus formation had beenaffected by plant growth regulator type and concentration, theratio between auxin and cytokinin, and the type of explant.Similar results have been reported by Benavides and Caso(1993) for in vitro culture of T. erecta and T. mendocina.

On other hand, Ekes-Kretovics et al. (1993) mentioned thatthe optimal condition of callus growth was obtained whencallus was supplemented with 0.1 mg/L NAA. In general, thoseplant growth regulator combinations which induced a highfrequency of callus formation also had improved callusgrowth. These results indicate that callus formation had beenaffected by plant growth regulator type and concentration, theratio between auxin and cytokinin and the type of explant.Similar results have been reported by Benavides and Caso(1993) for in vitro culture of T. erecta and T. mendocina. Ourstudy concluded that callus production was limited and itsamount was not much when compared to other herbaceousdicot plants. However, stem, apical shoots and leaves weresuitable material for callus induction and its furtherdevelopment. NAA alone or in combination with BAP wasfound to be most suitable growth regulator for induction ofcallus; 2.4-D was next to NAA. Different concentrations of IBAand IAA were used to induce callus and regeneration but onlyroot formation was observed in each of the concentrations ofIBA and IAA.

DNA IsolationVarious protocols and their modifications were developedand employed to isolate quality DNA from different plantspecies in the past (Murray and Thompson, 1980; Dellaportaet al., 1983; Saghai-Maroof et al.,1984; Rogers and Bendich,1985; Doyle and Doyle, 1990; Suman et al., 1999; Warudeet al., 2003 ; Sarwat et al., 2006; Deshmukh et al., 2007). Themajor aim was to isolate DNA from T.erecta explants that maybe rapid with high quality and throughput T.erecta is one ofthose medicinal plant species that contain higher levels ofsec-ondary metabolites such as polysaccharides, polyphenols,flavonoids, and essential oils, which may get co-precipitatedwith DNA during its preparation, thus interfering with enzymaticanaly-sis. We report the successful DNA extraction from fresh

Figure 1: MS plain, 6 daysold seedling

Figure 2: MS+2,4D (5mg/L),4weekleaf explant callus

Figure 3: MS+NAA (5mg/L)4 weekstem showing callus

Figure 4: MS+NAA (5mg/L)-rootexplants showing callus

Figure 5: Gel Electrophoresis of total DNA isolated, lanes1, 2, 3-leaf

1 2 3 4 5 6

Figure 6: PCR amplified products with primer GL A-08, M: 100bpladder, lanes1, 2, 3-leaf tissues;4,5,6-seed tissue

M 1 2 3 4 5 6

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leaf tissues and powdered (paste) seeds. Total DNA isolatedfrom fresh leaves and dried-seeds of T.erecta was checked bymeans of agarose gel electrophoresis (Fig. 5). The purity of theDNA samples was confirmed by absorbance (A260/A280)ratio, which was 1.7.

The DNA obtained was suitable for enzymatic manipula-tionssuch as PCR and showed high intensity amplification witharbitrary RAPD primers (Fig. 6). PCR amplification also indicatesthat the DNA was of good quality. Most of the protocolsrecommend extraction of DNA from fresh tissues, butsometimes samples collected from remote and rare locationsconsist of plant parts in dry or semi-dry conditions. Also, thechemicals and resources that are routinely used in manyprotocols are too expensive to be used for routine DNAextraction. Therefore, it was necessary to establish aninexpensive and less time-consuming protocol for DNAextraction from various plant parts. We hope the present studywill be adequate for other species containing secondarymetabolites and essential oils.

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