POJ 6(3):231-239 (2013) ISSN:1836-3644 Optimization of Agrobacterium-mediated genetic transformation in gherkin (Cucumis anguria L.) Muthu Thiruvengadam1, Jerome Jeyakumar2, Malaiyandi Kamaraj2, Ill-Min Chung1* 1Department of Applied Bioscience, College of Life and Environmental Sciences, Konkuk University, Seoul – 143 701, South Korea 2Post Graduate Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli - 620 020, Tamil Nadu, India *Corresponding author: [email protected] Abstract The present work was aimed to study various factors influencing Agrobacterium tumefaciens mediated genetic transformation of gherkin (Cucumis anguria L). Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene β- glucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII) 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 -1 (MSB5) containing 1.5 μM benzylaminopurine (BAP) with 0.5 μM naphthalene acetic acid (NAA), 100 mg L kanamycin and 300 -1 mg L carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB5 medium containing -1 -1 3.0 μM BAP, 100 mg L kanamycin and 300 mg L carbenicillin. Transgenic shoots were elongated in MSB5 medium fortified with -1 -1 2.0 μM gibberellic acid (GA3), 100 mg L kanamycin and 300 mg L carbenicillin. The transgenic elongated shoots were rooted in -1 MSB5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L 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. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and direct regeneration of C. anguria. Keywords: Acetosyringone; Agrobacterium tumefaciens; Cucumis anguria L; Genetic transformation; Growth regulators; GUS. Abbreviations: AS - Acetosyringone; BAP - 6-benzylaminopurine; GA3 - Gibberellic acid; GUS - β-glucuronidase; IBA - Indole 3- butyric acid; NAA - Naphthalene acetic acid; npt II - neomycin phosphotransferase II; PCR - Polymerase chain reaction; RT-PCR - Reverse transcription-PCR. Introduction The gherkin (Cucumis anguria L.) is an important and Mupa, 2012). Anthraquinones and saponins which are horticultural crop, mainly cultivated and consumed in Africa, present in C. anguria are used for antibacterial and antifungal Brazil, Cuba, India, United States and Zimbabwe. The activity against clinical pathogens (Senthil Kumar and vegetable is similar in form and nutritional value as that of Kamaraj, 2010). The pickled gherkins ensure worldwide cucumber (Mangan et al., 2010). The crop is popularly demand so many food companies have started to develop known as 'pickling cucumber' or 'small cucumber' among the opportunities for large scale production of gherkin. The farmers (Nabard, 2005). The fruits of gherkin are consumed favorable growing conditions for cultivating gherkin is as boiled, fried, stewed, pickled, fresh in salads and also in suitable for India which is mainly focused on exclusive hamburgers (Dzomba and Mupa, 2012; Matsumoto and exports in worldwide (Mohapatra and Deepa, 2012). Gherkin Miyagi, 2012). The fruit of the vegetable contain high has been introduced in India in the year 1989 for commercial amounts of protein, calcium, phosphorous, iron and vitamin production mainly for exports and its cultivation is driven C (Whitaker and Davis, 1962). The gherkin is also known for largely through contract farming (Venu Prasad et al., 2013). traditional importance in medicinal to treat stomach ache, Agri export zones (AEZ) have been created for cultivation of jaundice, hemorrhoids and preventing stone formation in gherkin in South India particularly in Karnataka, nearly one kidney (Baird and Thieret, 1988; Schultes, 1990). lakh small and marginal farmers are involved in gherkin Phytochemists have isolated a number of potential medical farming and the state produced 2.65 lakh tones of gherkin in components from C. anguria, such as cucurbitacin B, 50,000 hectares of land in 2010-11 (Venu Prasad et al., cucurbitacin D and cucurbitacins G (Sibanda and Chitate, 2013). The gherkin is susceptible to bacterial, fungal and 1990). Cucurbitacins B have potential to be used as a viral diseases (Alvarez et al. 2005; Srinivasulu et al., 2010; favorable phytochemical for cancer prevention (Promkan et Matsumoto and Miyagi, 2012). Although new cultivars have al., 2013). C. anguria consist of many useful compounds been developed by cross breeding (Modolo and Costa, 2004), such as flavonoids, tannins, alkaloids, saponins and steroids yet no cultivar has developed with resistance to all these which contained high level of antioxidant activity (Dzomba diseases (Matsumoto et al., 2012). Genetic improvement of 231 C. anguria has been achieved mainly by traditional breeding (Rajagopalan and Perl-Treves, 2005; Selvaraj et al., 2010). methods, but recent advances in gene transformation Carbenicillin was used to kill Agrobacterium after co- techniques have opened new avenues for crop improvement. cultivation with leaf explants. The obtained results were Agrobacterium-mediated transformation is a widely used and supported in C. sativus (Wang et al., 2013) and Citrullus powerful tool for introducing foreign DNA into many plant colocynthis (Dabauza et al., 1997). species. It has several advantages over physical transformation methods including its tendency to generate Evaluation of factors influencing transformation single or a low copy number of transgenes with defined ends and preferential integration into transcriptionally active Effect of pre-cultivation of leaf explants regions of the chromosomes (Birch, 1997). Agrobacterium- mediated transformation in Cucurbitaceae has been reported Pre-culture of explants is a critical factor to achieve high to be successful with Cucumis melo (Fang and Grumet, 1990; frequency of transformation. It makes the tissues competent Guis et al., 2000; Curuk et al., 2005; Rhimi et al., 2007) and enough to withstand the bacterial infection and increased the Cucumis sativus (Chee, 1990; Sarmento et al., 1992; production of GUS positive. Pre-culturing of explants prior to Nishibayashi et al., 1996; Vengadesan et al., 2005). The infection with Agrobacterium enhanced the transformation availability of an efficient in vitro regeneration system is the frequency in C. sativus (Vengadesan et al., 2005). In contrast, primary requirement for genetic transformation of most Cervera et al. (1998) stated that the pre-culture of explants plants. We have established an efficient reproducible protocol drastically reduced the regeneration of GUS positive. Higher for high frequency regeneration via organogenesis from leaf frequency of GUS positive (80%) were noted in 3 days pre- explants of gherkin. In this paper, we describe for the first cultured explants (Fig. 2a). However pre-culture of leaf time a protocol for Agrobacterium-mediated genetic explants for 4 days and 5 days showed reduction in recovery transformation via direct regeneration using leaf explants in of GUS positive (66%). The control experiments without pre- C. anguria. This optimized transformation system could be culture of leaf explant observed 35% of GUS positive (Fig. used to transform features with desirable characteristics such 2a). Similarly, pre-cultivation period of 3 days have showed as disease resistance, stress resistance and high yield of higher frequency of GUS expression in Momordica dioica gherkin. (Thiruvengadam et al., 2011) and Momordica charantia (Thiruvengadam et al., 2012). In contrary to our observation, Results and discussion Vengadesan et al. (2005) and Selvaraj et al. (2010) demonstrated that 5 days of pre-culture in C. sativus. Organogenesis from leaf explants Effect of bacterial density and infection time Different plant growth regulators can directly influence on callus induction, shoot bud formation, shoot elongation and Bacterial cell density as measured by the optical density rooting for leaf explants of C. anguria. Callus induction on (OD) of bacterial suspension is directly related to their cell MSB5 medium supplemented with 1.5 μM BAP and 0.5 μM mass or cell number (Dutt and Grosser, 2009). Selection of NAA resulted in a 90.51% induction frequency (Table 1). appropriate OD is an important factor of concern for When calluses were transferred to fresh medium containing Agrobacterium genetic transformation. Lower densities 3.0 μM BAP, shoot regeneration was induced (Table 1). This (OD600 0.2 and 0.6) were not effective for transformation, indicated that BAP was sufficient for the callus induction and whereas the highest density (OD600 1.0) decreased infection shoot bud regeneration. The calluses induced in the compact efficiency (Fig. 3a). GUS expression was significantly high were green and showed higher shoot regeneration frequency (80%) at 0.8 OD600 (Fig. 3a). Similarly, Miao et al. (2009) (35 shoots). The shoots were excised from callus and demonstrated that Agrobacterium densities OD600 0.8 was elongated (75.4%) in