International Journal of Biotechnology. Photon 114 (2015) 431-437 https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-biotechnology Original Research Article. ISJN: 3352-7304 Impact Index: 4.23

International Journal of Biotechnology

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In vitro Culture and Agrobacterium-Mediated Transformation Studies in Gherkin (Cucumis anguria L.) J. Jerome Jeyakumar*, M. Kamaraj PG and Research Department of Botany, Jamal Mohamed College (Autonomous) Thiruchirappalli – 620020, Tamilnadu, India J. Jerome Jeyakumar and M. Kamaraj are conferred with Barbara McClintock Research Award-2015 in Biotechnology Article history: Received: 09 May, 2015 Accepted: 11 May, 2015 Available online: 20 July, 2015 Key words: In vitro culture, Agrobacterium tumefaciens Abbreviations: %: Percentage, µM/l: Micro Molar/litter, 2,4-D, 2,4Dichlorophenoxy acetic acid, PGRs : Plant Growth Regulators Acetosyringone: HgCl2: Mercuric chloride, KI: Potassium iodide, Kin/KIN: Kinetin, l: Litter, m: metre, h: Hour, IAA: Indole-3-acetic acid, IBA: Indole-3-butric acidMg/l: Milligram/litter, MS mediu: Murashige & Skoog medium, NAA: 2-Napthalene acetic acid, npt ll: Neomycin phosphotransferase, °C: Degree centigrade, PCR: Polymerase chain reaction, PCV: Packed cell volume, PGR: Plant Growth Regulators, pH: Negative logarithm of hydrogen ion concentration, SE: Somatic embryogenesis, TDZ: Thidiazuron Corresponding Author: Jeyakumar J.J.* Research Scholar Email: [email protected] Kamaraj M. Assistant Professor

Abstract In the present study, Cucumis anguria L is a medicinally and economically important plant and also used as vegetable. We established an Agrobacterium tumefaciensmediated

transformation procedure for Cucumis anguria. Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPT-II). Following co-cultivation, leaf explants were cultured on MS medium supplemented with 6.6 µM 2, 4-D combined with 3.3µM BAP containing 100 mg/l kanamycin and 300 mg/l carbenicillin. Kanamycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh selection medium 8.8 µM BAP and 2.2 µM 2,4-D. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.0 µM GA3,100 mg/l kanamycin and 300 mg/1 carbenicillin. Finally, the shoots were rooted on MS basal medium supplemented with 3.0 µM IBA and 100 mg/l kanamycin. High transformation frequency was achieved by using 3-day-old precultured leaf explants. Further, the presence of acetosyringone (200 µM), infection of explants for 30 min and 3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Citation: Jeyakumar J.J., Kamaraj M., 2015. In vitro Culture and Agrobacterium-Mediated Transformation Studies in Gherkin (Cucumis anguria L.). International Journal of Biotechnology. Photon 114, 431-437 All Rights Reserved with Photon. Photon Ignitor: ISJN33527304D791320072015

1. Introduction The gherkin (Cucumis anguria L.) is an important horticultural crop, mainly cultivated and consumed in Africa, Brazil, Cuba, India, United States and Zimbabwe. The vegetable is similar in form and nutritional value as that of cucumber. The crop is popularly known as 'pickling cucumber' or 'small cucumber' among the farmers (Nabard, 2005). The fruits of gherkin are consumed as boiled, fried, Ph ton

stewed, pickled, fresh in salads and also in hamburgers. The fruit of the vegetable contain high amounts of protein, calcium, phosphorous, iron and vitamin C (Whitaker and Davis, 1962). The gherkin is also known for traditional importance in medicinal to treat stomach ache, jaundice, hemorrhoids and preventing stone formation in kidney (Baird and Thieret, 1988; Schultes, 1990).

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Phytochemists have isolated a number of potential medical components from C. anguria, such as cucurbitacin B, cucurbitacin D and cucurbitacins G (Sibanda and Chitate, 1990). Cucurbitacins B have potential to be used as a favorable phytochemical for cancer preventio. C. anguria consist of many useful compounds such as flavonoids, tannins, alkaloids, saponins and steroids which contained high level of antioxidant activity (Dzomba and Mupa, 2012). Anthraquinones and saponins which are present in C. anguria are used for antibacterial and antifungal activity against clinical pathogens (Senthil Kumar and Kamaraj, 2010). The pickled gherkins ensure worldwide demand so many food companies have started to develop opportunities for large scale production of gherkin. The favorable growing condition for cultivating gherkin is suitable for India which is mainly focused on exclusive exports in worldwide. Gherkin has been introduced in India in the year 1989 for commercial production mainly for exports and its cultivation is driven largely through contract farming (Venu Prasad et al., 2013). Agri export zones (AEZ) have been created for cultivation of gherkin in South India particularly in Karnataka, nearly one lakh small and marginal farmers are involved in gherkin farming and the state produced 2.65 lakh tones of gherkin in 50,000 hectares of land in 2010-11. The gherkin is susceptible to bacterial, fungal and viral diseases (Matsumoto and Miyagi, 2012). Although new cultivars have been developed by cross breeding (Modolo and Costa, 2004), yet no cultivar has developed with resistance to all these diseases (Matsumoto et al., 2012). Recently, genetic engineering has been presented as an alternative for obtaining plants that are resistant to viral diseases; this approach has been widely applied in cucurbit breeding(Tricoli et al. 1995; Compton et al. 2000; Klas et al.2006). However, the use of this biotechnological tool requiresan efficient in vitro culture system that allows plant regeneration. An efficient plant regeneration system is therefore necessary for genetic transformation and propagation of gherkin.Genetic improvement of C. anguria has been achieved mainly by traditional breeding methods, but recent advances in gene transformation techniques have opened new avenues for crop improvement. Objectives of Research This work has two main objectives. One is to find out the present study of in vitro culture and Agrobacterium -Mediated transformation studies in Gherkin (Cucumis anguria L.)

2. Materials and Methods 2.1 Source of explants The collected seeds were grown in the College Botanical garden of Jamal Mohamed College, Tiruchirappalli, Tamil Nadu, India, in vitro germinated plants as source of explants (Plate-1). Systematic Position (Bentham and Hooker’s 1862 - 1883) Class - Dicotyledons Subclass - Polypetalae Series - Calyciflorae Order - Passiflorales Family - Cucurbitaceae Genus - Cucumis Species - anguria L. English name - Gherkin Tamil name - Vesha vellari Description of the Plan Cucumis anguria is an herbaceous annuals, weak stemmed, tendril climbers, leaves are alternate exstipulate, simple, palmately, lobed, axillary inflorescence and solitary cyme. In flower from July to September and the seeds ripen from August to October. The flowers are monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by Insects. The plant is self-fertile. The surface of the fruits has long hairs covering a surface having warts or spines.The inner flesh is palid to green. 2.2 Plant material and explants preparation Seeds of gherkin (Cucumis anguria L.) were obtained from Nunhems Seeds Pvt., Ltd., Bangalore, India. The seeds were surface sterilized first with 70% (v/v) ethanol for 1 min and then with 25% (v/v) commercial bleach (with sodium hypochlorite as the active agent) containing 0.05% (w/v) of Tween-20 (polyethylene sorbitanmonooleate; Nutritional Biochemical, Cleveland, OH) for 20 min and then rinsed thoroughly (three times) with sterile distilled water. Disinfected seeds were germinated in the dark for 48h in a jar containing half-strength MS (Murashige and Skoog, 1962) basal salt mixture + B5 vitamins (Gamborg et al., 1968) (MSB5) supplemented with 0.8% (w/v) agar and 3% (w/v) sucrose without any growth regulators. The seedlings were grown under white fluorescent light (35 µmol m-2s-1) at a photoperiod of 16/8 h of light/dark and temperature 25 ± 2˚C. The node, shoot tip, leaf and petiole explants were trimmed into appropriate sizes (0.5 cm2) to obtain from 10day-old in vitro seedlings. 2.3 Micropropagation Shoot tip and nodal explants were inoculated on MSB5 medium supplemented with BAP 2.0 µM plus Kin 0.5 µM for multiple shoot induction.

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Shoots are elongated on MSB5 medium augmented with GA3 3.0 µM. Roots occurred on MSB5 medium with 3.0 µM IBA.

medium, 3% sucrose, and (0.5 µM) L.Glutamine was highly effective for enhancing the frequency of somatic embryogenesis.

2.4 Organogenesis (i) Direct organogenesis Shoot tips were excised from 7th day old seedlings and cultured on MSB5 medium supplemented with various concentrations of BAP and or in combination with KIN. Regenerated shoots were transferred onto fresh medium with the same composition at two week intervals. Subsequently, shoots were elongated from the shoot clusters on the same media composition. The shoot clumps were repeatedly sub cultured on shoot multiplication medium for further shoot initiation. For root induction, elongated shoots (≥ 2cm) that possessed approximately 3 compound on half – strength MS medium with (1% w/v) sucrose supplemented with (0.5 - 2.5 mg/l) IBA and (0.5 2.5 mg/l) NAA alone and in combination with (0.5 mg/l) KIN were used. Well developed in vitro rooted plantlets were removed from the culture medium and the residual agar were removed by washing the roots under running tap water then transferred to plastic cups containing sterile sand and garden soil in the ratio 1:1. The cups were covered with polyethylene bags and were kept inside the tissue culture room to maintain humidity condition. After 10th days the plantlets were transferred to pots containing garden soil with organic manure and kept in the green house condition for another 2nd week before transferring to the field condition. After 15th days of acclimatization, the plantlets were shifted to the field conditions where they grew normally.

2.6 Biochemical analysis of in vitro callus Callus induced from leaf explants and biochemical analysis (protein, starch, amino acids and phenols) of callus at 15 – 30 days.

ii) Indirect organogenesis The callus induction media consisted of MSB5 medium with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar supplemented with different concentrations of (1.0 - 5.0 µM) of naphthalene acetic acid (NAA), 2, 4 - dichlorophenoxy acetic acid (2, 4-D) either separately or in combination with (1.0 - 3.0 µM) benzyl amino purine (BAP) or thiadiazuron (TDZ) were tested for callus induction. The medium was adjusted to pH 5.8 prior to autoclaving at 121 °C for 15 min. The cultures were maintained at 25±2°C under 16 hr light and 8 hr dark photoperiod with a light intensity of 40 µmol m-2 s-1 as two subculture were made at an interval of 11th days in the same induction medium. 2.5 Somatic embryogenesis Somatic embryogenesis via suspension culture from leaf callus was achieved in C. anguria. The auxins (2, 4-D) and cytokinin (KIN) were responsible for the induction of embryogenic calli and development of somatic embryos. MSB5 Ph ton

2.7 Agrobacterium-mediated genetic transformation Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene βglucuronidase intron (gus) and the marker gene neomycin phosphotransferase (npt-II) was used for transformation. Factors affecting transformation efficiency, such as Agrobacterium concentration, effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were studied. After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB5) containing 1.5 µM benzylaminopurine (BAP) with 0.5 µM naphthalene acetic acid (NAA), 100 mg/L-1 kanamycin and 300 mg/L-1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB5medium containing 3.0 µM BAP, 100 mg/L-1 kanamycin and 300 mg/L-1carbenicillin. Transgenic shoots were elongated in MSB5 medium fortified with 2.0 µM gibberellic acid (GA3), 100 mg/L-1 kanamycin and 300 mg/L-1carbenicillin. The transgenic elongated shoots were rooted in MSB5 medium supplemented with 3.0 µM indole 3-butyric acid (IBA) and 100 mg/L-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. 3. Results and Discussion 3.1 Micropropagation Multiple shoot induction was achieved from nodal and shoot tip explants of C. anguria. Cytokinins BAP, KIN and Ads were highly effective for maximum regeneration of adventitious buds. 3.2 Organogenesis Regeneration via callus phase was achieved in leaf explants of C. anguria. Combination of auxins and cytokinins were responsible for the induction of organogenic callus and shoot regeneration. GA3 and IAA played vital role on shoot elongation and rooting of regenerated shoots.

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of 2, 4 - D and Glutamine were mandatory for embryo formation in the present study. 3.4 Biochemical studies of callus To induce the callus from in vitro grown seedling explants. The effect BAP and NAA have been investigated,the combination of BAP and NAA results in the higher callus induction. The callus was further analyzed for biochemical changes at 15 and 30 days of intervel. The steep increase in phenol in 30 days callus compared to 15 days old callus, which could be one of the reason for browning of callus.

3.3 Somatic embryogenesis Somatic embryos have been produced in the way of direct and indirect. Based on our results direct somatic embryogenesis occurred in leaf explants of C. anguria. The combination of 2, 4-D and BAP with Glutamine was suitable plant growth regulators for induction of somatic embryogenesis. It was found that the presence of low concentration

3.5 Agrobacterium - mediated transformation studies Transgenic plants were regenerated from the nodal explants of in C. anguria via Agrobacterium transformation.Three days preculture and 3-days co-cultivation increased the transformation frequency.Kanamycin 100 mg/L-1 was suitable selection agent.After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB5) containing1.5 µM benzylaminopurine (BAP) with 0.5 µM naphthalene acetic acid (NAA), 100 mg/L-1 kanamycin and 300 mg/L1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB5 medium containing 3.0 µM BAP, 100 mg/L1 kanamycin and 300 mg/L-1carbenicillin. Transgenic shoots were elongated in MSB5 medium fortified with 2.0 µM gibberellic acid (GA3), 100 mg/L-1 kanamycin and 300 mg/L-1 carbenicillin. The transgenic elongated shoots were rooted in MSB5 medium supplemented with 3.0 µM indole 3-butyric acid (IBA) and 100 mg/L-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. Transformation was confirmed by GUS, PCR and Southern Blot analysis. In our study, GUS was used as marker gene and npt-II as reporter gene.

Table 1: Effect of IBA, IAA and NAA for initiation of roots from In vitro Culture and Agrobacterium-tumefaciens of Cucumis anguria L. Plant growth Concentration Percentage of Rooting response regulators (mg/l) Shoot Nodal Leaf Internodal Somatic Agrobacterium tip embryogenesis tumefaciens 1.0 58 48 52 41 53 62 IBA 2.0 62 68 50 43 64 74 3.0 76 70 73 76 79 90 4.0 50 54 62 56 68 67 1.0+0.5 66 70 72 76 69 78 IBA+NAA 2.0+0.5 90 88 84 86 89 84 3.0+0.5 98 94 91 92 96 92 4.0+0.5 82 88 89 84 72 87 1.0+0.5 54 41 62 65 52 54 IAA+NAA 2.0+0.5 41 50 49 55 57 56 3.0+0.5 65 65 66 73 67 72 4.0+0.5 65 59 55 48 64 50

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Figure 1: Effect of different combination of BAP, KIN and IBA in MSB5 medium on shoot formation direct organogenesis shoot tip and nodal explants of Cucumis anguria L.

Figure 2: Effect of different combination of NAA, IBA in MSB5 medium on shoot formation indirect organogenesis leaf and internodal explants of Cucumis anguria L.

Figure 3: Effect of different combination of NAA, IBA in MSB5 medium leaf explants for callus induction shoot formation on somatic embryogenesis of Cucumis anguria L.

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Figure 4: Effect of BAP, 2, 4-D and IBA in MSB5 medium leaf explants for callus induction shoot formation on Agrobacterium tumefaciens of Cucumis anguria L.

Conclusion The present investigation in Cucumis anguria belonging to the family Cucurbitaceae, it’s vegetable and medicinally importance. The techniques used for micropropagation, organogenesis, somatic embryogenesis, Agrobacterium mediated genetic transformation and biochemical analysis of callus. Micropropagation using the growth hormones BAP and KIN of node and shoot tip explants. Organogenesis using the growth regulators NAA, BAP and TDZ of petiole explants. Somatic embryogenesis using 2,4-D of leaf explants. Callus induced in vitro and biochemical analysis of protein, starch, amino acids and phenols of callus at 15 – 30 days. Agrobacterium mediated genetic transformation using leaf explants were confirmed by molecular analysis of GUS, PCR and Southernbloting. Acknowledgement The authors are thankful to the management of Jamal Mohamed College (Autonomous), Thiruchirappalli 620 020, Tamil Nadu for offering facilities to carry out this study. References Baird J.R. and Thieret J.W., 1988. The gherkin (Cucumis anguria var.anguria, Cucurbitaceae). Economic Botany, 42,447–451. Bentham G. and Hooker J.D., 1862 - 1883. Genera Plantarum. London, 3. Murashige T. And Skoog F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiologia Plantarum. 5, 473–497.

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Compton M.E. Gray D.J., Gaba V.P., 2000. Use of tissue culture and biotechnology for the genetic improvement of watermelon. Plant Cell, Tissue and Organ Culture, 77, 231-243. Dzomba P. and Mupa M., 2012. Wild Cucumis anguria leaves phytochemical profile and antioxidant capacity. Asian Pacific Journal of Tropical Medicine, 1–5. Gamborg O.L., Miller R.A., Ojima K., 1968. Nutrient experiments of suspension culture of soybean root callus. Experimental Cell Research, 80, 150–158. Klas F.E. Fuchs M., Gonsalves D., 2006. Comparative spatial spread Overtime of zucchini yellow mosaic virus ZYMV and watermelon mosaic virus WMV in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and it fields of non transgenic squash. Transgenic Research, 15, 527–541. Matsumoto Y. and Miyagi M., 2012. Evaluation of the wilt and inheritance of the resistant gene. Journal of resistance in gherkin Cucumis anguria L. to Fusarium Agricultural Science, 4, 145- 149. Modolo V.A. and Costa C.P., 2004. Gherkin elite line selection. Crop Breeding and Applied Biotechnology, 4, 63–67. Nabard, 2005. National bank of agriculture and rural development gherkin AEZ-Karnataka. Evaluation study in Karnataka. 13. Schultes R.E., 1990. Biodynamic cucurbits in the new world tropics. In Bates D.M. Robinsion R.W., Biology and utilization of the cucurbitaceae. Cornell University Press, Ithaca. 39. Sibanda S. and Chitate N., 1990. Some Constituents of Cucumis anguria. Fitoterapia, 16, 381. Senthil Kumar S. And Kamaraj M., 2010. Analysis of phytochemical constituents and antimicrobial activities of Cucumis anguria L. against clinical pathogens.

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American Eurasian Journal of Agricultural Environmental Sciences, 7, 176–178.

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Tricoli D., Carney K.J., Russell P.F., Mc Master J.R., Groff D.W., Hadden K.C., Himmel P.T., Hubbard J.P., Boeshore M.L., Quemada H.D., 1995. Field Evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus. Nature Biotechnology, 13, 1458–1465. Venu Prasad H.D. Singh P. Kumar S. and Singh B.K., 2013. Performance and constraints of gherkin contract farming. Indian Journal of Education and Information Management, 13, 112–116. Whitaker T.W. and Davis G.N.,1962. Cucurbits botany, cultivation and utilization. Leonard Hill, Interscience, London, 249.

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