Effect of antibiotics and gelling agents in transformation of brinjal (Solanum melongena L.) cv. Manjarigota D. P. Prakash, B. S. Deepali, R. Asokan, Y. L. Ramachandra1, Lalitha Anand and Vageeshbabu S. Hanur Division of Biotechnology Indian Institute of Horticultural Research Hessaraghatta Lake Post, Bangalore – 560 089, India E-mail: vageesh@iihr.ernet.in ABSTRACT A study was conducted to find out the effect of antibiotics and gelling agents on Agrobacterium-mediated transformation using hypocotyl explants of brinjal cv. Manjarigota. Hypocotyl explants of brinjal were found to be sensitive even to the lowest level of kanamycin (25 mg/l) tested. Explants that showed increased callus initiation and regeneration response upon cocultivation with Agrobacterium and on kanamycin at 100 mg/l were selected as this indicated a highly effective selection pressure. Cefotaxime did not affect regeneration response and at 500 mg/l, it effectively inhibited Agrobacterium overgrowth completely on Agrobacterium cocultivated hypocotyl explants. There were marked differences in regeneration response in hypocotyl explants cultured on medium solidified with various gelling agents indicating the influence of gelling agent on the activity of kanamycin in culture medium, which indirectly affects selection and recovery of transformants. Antibiotics and gelling agents could therefore affect, directly or indirectly, transformation of brinjal cv. Manjarigota. Key words: Solanum melongena, kanamycin, cefotaxime, gelling agents, transformation 1Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga, India INTRODUCTION In transformation studies, once the target cells have been transformed, the transgenic cells or plants produced from them are identified on selection medium. A marker gene is necessary because only a low proportion of the cells exposed to the transformation processes subsequently become stably transformed (Klee et al, 1987). The use of selection medium confers an advantage to those cells that stably incorporate the transgene construct and are, therefore, resistant to the specific antibiotic in the selective medium. The use of a marker gene in a transformation process thus aims to give a selective advantage to transformed cells by allowing them to grow. The selectable marker gene confers the transformed cells an ability to metabolize compounds that are not usually metabolized by untransformed cells. One such widely used marker gene for transformation of plants is nptII (neomycin phosphotransferase II) gene, which confers resistance to the aminoglycoside antibiotic kanamycin by phosphorylation of the specific hydroxyl group (Nap et al, 1992). Antibiotics are added to the culture media to control Agrobacterium that may affect the plant regeneration process (since Agrobacterium itself is basically a plant pathogen (Hanur, 2004)) and to select transformants with an antibiotic resistance that gets cotransferred with the gene of interest. Sensitivity to kanamycin varies with crop and explant (Shaw et al, 1983). Cefotaxime is a b-lactam antibiotic that inhibits bacterial cell wall synthesis. It inhibits the cross linking of peptidoglycan by binding and inactivating of transpeptidase leading to nicks in cell wall by which the cell membrane protudes into the hypotonic environment and, finally, ruptures as a consequence of osmotic shock. Although many antibiotics have been described for effective control of Agrobacterium cells, cefotaxime is known to exert minimal effect on most plant tissues (Mathias and Boyd, 1986) and has become most widely used antibiotic in Agrobacterium- mediated transformation (Yu et al, 2001; Magioli et al, 2000). Cefotaxime has been shown to have both negative and positive effect on callus formation and regeneration in crop plants. Various gelling agents differ in their affinity to bind kanamycin and inhibit the latter’s activity in the culture medium, which may indirectly affect transformation and recovery of transformants (Laine et al, 2000). Though the J. Hort. Sci. Vol. 2 (1): 19-25, 2007 mechanism is not clear, efficiency of kanamycin that inhibiting regeneration differed with various gelling agents (Chauvin et al, 1999). In brinjal transformation, the effect of antibiotics, viz., cefotaxime and kanamycin, has been studied to a lesser extent (Billings et al, 1997) and there are no reports on the effect of gelling agents in brinjal transformation. In developing an efficient transformation protocol, finding out effect of various in vitro factors is an important step. Also, it is necessary to document the effect of such factors affecting transformation in various cultivars of a crop plant, so that importance of any particular factor at the species/varietal level can be studied. This is useful in determining factors for developing an efficient transformation protocol for a given cultivar. In this paper, an effort has been made to study the effect of antibiotics and gelling agents, which are critical factors in making a transformation protocol efficient, using hypocotyl explants in brinjal cv. Manjarigota. MATERIAL AND METHODS Plant material The genuine breeder seed material of brinjal cv. Manjarigota was obtained from the Division of Vegetable crops, IIHR, Bangalore. Seeds were soaked in gibberillic acid (100 ppm) for three hours, surface sterilized for 1 minute in 70% ethanol, washed in sterile distilled water, treated for 8-10 min. in sodium hypochlorite (approximately 4% available chlorine) solution and washed five times in sterile distilled water. They were germinated on half- strength MS (Murashige and Skoog, 1962) basal medium containing 3% sucrose (w/v), 0.8% agar and pH adjusted to 5.8 using dilute NaOH/HCl prior to autoclaving. Hypocotyls from aseptically germinated seedlings were used as the explant donor source (Hanur et al, 2006). Sterilization and culture incubation conditions Sterilization of culture medium and instruments was done by autoclaving at 121o C at 15-psi pressure for 15 min. Cultures were incubated in culture racks tilted with white, fluorescent tubes with a light intensity of 30-40µE m-2 s-1 under a 16 h photoperiod in a culture room maintained at 25o ± 2° C. Plant transformation Plasmid pBinBt-1 (the pBinAR binary vector containing CaMV35S promoter, the coding region of synthetic Cry1Ab gene, ocs terminator and nptII selectable marker cassette) (Kumar et al, 1998) was used for standardizing the transformation protocol. The nptII gene conferring kanamycin resistance served as a selectable marker. Fifteen to twenty day old hypocotyl explants were precultured for two days on shoot regeneration medium for hypocotyl explants (SRMH) containing MS medium with 2 µ M Benzyl Aminopurine (BAP) and 0.05 µ M Naphthalene Acetic Acid (NAA) (Hanur et al, 2007). Explants were transferred to a sterile petri plate, infected with overnight-grown Agrobacterium culture for 20-25 min. and placed back onto the parent medium, cocultivated for two days, transferred onto culture media containing cefotaxime (500 mg/l) for two days and then transferred to SRMH containing cefotaxime (500 mg/l) and kanamycin (100 mg/l). Hypocotyl explants cultured without Agrobacterium cocultivation on SRMH served as the control. All the explants in all the treatments were subsequently subcultured on shoot elongation medium (SEM) and rooting initiation medium (RIM) for complete plant regeneration. Kanamycin To examine kanamycin sensitivity, hypocotyl explants (without cocultivation) were cultured on SRMH containing kanamycin at 0, 25, 50, 75, 100, 125, 150, 175 and 200 mg/l. Observations were recorded on callus initiation and regeneration response at four weeks from culture initiation. Observations on explant survival were recorded weekly upto four weeks from culture initiation. Similarly, hypocotyl explants were cultured after Agrobacterium cocultivation on SRMH containing kanamycin at different levels and observations were recorded and stringent selection pressure for selecting putative transformants was worked out. Cefotaxime After cocultivation, hypocotyl explants were cultured at different concentrations of cefotaxime (100, 250, 500, 750 and 1000 mg/l) and transformation procedure thereafter remained the same as above. Observations were recorded on callus induction and regeneration response at four weeks from culture initiation. Observations on explant survival and bacterial overgrowth were recorded weekly upto four weeks from culture initiation. Callus induction and regeneration (transformation) frequency was worked out. Gelling agents Three types of gelling agent [Agar, Gelrite (Phytagel) and Agargel (Agar + Gelrite)] were used in the culture medium as solidifying agents at all stages of the 20J. Hort. Sci. Vol. 2 (1): 19-25, 2007 Prakash et al transformation protocol. Observations were recorded on callus induction and regeneration response at four weeks from culture initiation. Callus induction and regeneration (transformation) frequency was worked out. Data analysis Sufficient numbers of replications were maintained in an experiment as required. Wherever necessary Analysis of Variance (ANOVA) was used to test significance of the results (details given below) observed. RESULTS AND DISCUSSION Effect of kanamycin on transformation and in vitro morphogenetic response of hypocotyl explants In the experiment conducted to work out the minimum concentration of kanamycin required for complete killing of untransformed plant cells, hypocotyl explants showed varied sensitivity to various levels of kanamycin without Agrobacterium co-cultivation. After 4 weeks, 100 % hypocotyl explants remained viable (green) in the control i.e., zero kanamycin. Approximately, 92, 16 and 8 % callus induciton response was observed on hypocotyl explants cultured on SRMH containing 25, 50 and 75 mg/l kanamycin, respectively. However, these failed to regenerate shoots. Hypocotyl explants cultured on SRMH containing 100 mg/l kanamycin and above showed neither callusing nor regeneration response. However, Billings et al (1997) reported no growth of any kind with control, non- inoculated leaf discs (bulging/thickening) cultured on 10- 100 mg/l kanamycin in brinjal. In the present study, initial callus induction response (bulging near the cut end) on hypocotyl explants on kanamycin containing culture medium, may be because of the nature of hypocotyl explants to respond as fast as compared to other explants like cotyledonary leaf or leaf (Curuk et al, 2002; Gaba et al, 1999). In the present study, explants gradually turned light yellow and ultimately died on SRMH containing kanamycin. Time taken for chlorosis (yellowing/white) of explants reduced with increasing kanamycin concentration and differed markedly with kanamycin concentration. Shoots (untransformed) cultured on SEM containing 50 mg/ l kanamycin did not elongate. Shoots cultured on RIM containing 25-mg/l kanamycin did not show root induction. Instead these turned yellow and died. Sensitivity to an antibiotic has been shown to differ with crop plant, cultivar and explant type (Sunilkumar and Rathore, 2001; Barcelo et al, 1998; Sriskandrajah and Goodwin, 1998; Sarmento et al, 1992) and the nature and size of explants may contribute to differed sensitivity to kanamycin (Chauvin et al, 1999). In the present study, hypocotyl explants cultured on kanamycin at 100 mg/l did not show any response. In the present study, after the hypocotyl explants were co-cultivated with Agrobacterium, their ability to show callus induction and regeneration in the presence of kanamycin increased showing that transformation of plant cells had taken place. Hypocotyl explants showed varied morphogenetic responses on various levels of kanamycin after Agrobacterium co-cultivation (Fig. 1; Table 1). Callus induction response occurred at all the levels of kanamycin tested (up to 200 mg/l). However, gradual reduction in callus initiation was observed with increase in kanamycin (>100 mg/l) concentration in the culture medium. Shoot regeneration response was observed upto 125 mg/l, which, sharply decreased at 150 mg/l or higher concentrations kanamycin. Billings et al (1997) found that 50 mg/l kanamycin was more effective for selecting transformed shoots and even transformed cells failed to regenerate at higher levels of kanamycin in cotyledonary leaves of brinjal. Table 1. Effect of kanamycin on transformation and morphogenetic response of hypocotyl explants of brinjal cv. Manjarigota Kanamycin Callus initiation response Regeneration response (mg/l) (% ± SE) (% ± SE) 25 100.0±0.00 41.86 ±2.51 50 97.6±2.22 19.04 ±1.11 75 97.7±2.22 11.11 ±2.22 100 97.6±2.56 6.90 ±0.34 125 79.5±3.57 2.27 ±2.22 150 74.4±4.03 0.00 ±0.00 175 58.3±5.87 0.00 ±0.00 200 58.3±4.44 0.00 ±0.00 Control 100.0±0.00 80.0 ±4.84 Fractions were converted into percentages; percentage data are with SE. Fig 1. Effect of kanamycin on Agrobacterium mediated transformation and morphogenetic response in hypocotyl explants of brinjal cv. Manjarigota M o rp h o g e n e ti c r e s p o n s e ( % ) Kanamycin (mg/l) J. Hort. Sci. Vol. 2 (1): 19-25, 2007 Effect of antibiotics and gelling agents on brinjal transformation 21 Higher level of kanamycin has been found to inhibit chlorophyll synthesis even in transgenic tissues (Norouzi et al, 2005). However, in almost all previously reported brinjal transformation studies, high concentrations (100- 200 mg/l) of kanamycin were used to select transformants (Rotino and Gleddie, 1990; Kumar et al, 1998). Reducing kanamycin concentration to sub-lethal levels during selection led to higher number of escapes. Application of higher kanamycin concentration in the selection medium reduced regeneration response, while, transformed plants remained mostly chimeric in nature in carnation (Zuker et al, 1999). In the present study, almost all the explants showed callus initiation (>97%) with regeneration response (6.9%) after Agrobacterium co-cultivation on 100mg/l kanamycin. This shows that there is scope for further improvement in regeneration response by optimizing other factors involved in the transformation procedure. Moreover, both callus initiation and regeneration response were completely inhibited in control explants at 100 mg/l of kanamycin. Kanamycin at 100mg/l was, therefore, identified as a stringent selection pressure for selection of transformants in hypocotyl explants of brinjal cv. Manjarigota. Effect of cefotaxime on transformation and in vitro morphogenetic response of hypocotyl explants Hypocotyl explants cultured on SRMH containing cefotaxime showed a little callus production all over the surface (Plate 1). Cefotaxime did not affect callus initiation response in hypocotyl explants and callusing was seen at 100% at all levels of cefotaxime. Cefotaxime did not significantly affect regeneration response. Complete exclusion of bacteria was possible by employing a culture medium with cefotaxime at 500 mg/l. Explants cultured on cefotaxime below 500 mg/l showed Agrobacterium overgrowth. It is known that once bacteria start surviving in the plant tissue, it is difficult to control their overgrowth. The only option was to exclud such explants from the culture to prevent spread of overgrowth to other explants. Similarly, it is reported that lower levels of cefotaxime do not completely eliminate Agrobacterium in brinjal cotyledons (Billings et al, 1997). Five hundred mg/l of cefotaxime was extremely effective in eliminating Agrobacterium from explants of brinjal up to 3 months (Billings et al, 1997; Kumar et al, 1998). Effective concentrations of antibiotic for elimination of Agrobacterium overgrowth was dependent on Agrobacterium strain, explant type and crop (Sriskandarajah and Goodwin, 1998; Hoque et al, 2005). Various concentrations of cefotaxime, 200-500mg/l, were effectively used to eliminate Agrobacterium overgrowth in transformation studies in rice (Hoque et al, 2005), strawberry (Barcelo et al, 1998) and apple (Sriskandaranjah and Goodwin, 1998). In the present study hypocotyl explants cultured on higher levels of cefotaxime (>500mg/ l) turned brown at third week from culture initiation. This may be due to delayed sensitivity of explants to higher levels of cefotaxime. Similarly, it was seen that papaya callus turned brown in color on medium containing cefotaxime at 250 mg/l (Yu et al, 2001). In the present study, slightly higher amount of callus production was visually observed on explants cultured on all levels of cefotaxime compared to the control. However, no significant differences in callus initiation response and regeneration response were observed. Picoli et al (2002) found that cefotaxime enhanced callus fresh weight, it also caused a decrease in the rate of embryo regeneration in brinjal. Magioli et al (2001) however, reported that presence of cefotaxime did not affect embryogenic callus formation and development from leaf and cotyledonary explants. Billings et al (1997) reported no effect on either callus production or regeneration. Stimulation of callus growth and regeneration due to cefotaxime have been reported in barley (Mathias and Mukasa, 1987), bread wheat (Mathias and Boyd, 1986) and many horticultural crops. However, in the present study, the maximum regeneration response observed in explants cultured on 500mg/l (21.81%) cefotaxime might be due to an effective control of Agrobacterium without harming the explants (Fig 2). Negative effects of cefotaxime on callus formation and plant regeneration have been described in carrot (Okkels and Pederson, 1988). J. Hort. Sci. Vol. 2 (1): 19-25, 2007 Prakash et al 22 Plate 1. Regeneration response of hypocotyl explants after Agrobacterium cocultivation cultured on SRMH containing defferent levels of cefotaxime (mg/l):1, 0; 2, 100; 3, 250; 4, 500; 5,750 and 6, 1000 mg/l. In tomato, cefotaxime did not by itself inhibit callus growth in the culture medium, but it clearly decreased shoot differentiation. Together with kanamycin, cefotaxime showed a strong negative effect on callus growth, shoot regeneration and transformation frequency in tomato (Ling et al, 1998). Okkels and Pederson (1988) reported stimulation of plant regeneration at low concentrations of cefotaxime (less than 100 mg/l) in carrot and inhibition at high concentration (300 mg/l). Enhanced callus formation and shoot regeneration in culture medium containing antibiotic may be possibly caused by release of auxin-like compounds (Halford and Newbury, 1992; Robert et al, 1989; Ling et al, 1998). Hence, it appears that the effect of cefotaxime on any tissue depends on the crop plant, genotype, explant type, concentration of cefotaxime and other transformation conditions. The present study showed that cefotaxime could promote a slight increase in callus production in explants without a marked effect on regeneration response of hypocotyl explants in brinjal cv. Manjarigota. Effect of gelling agent in the culture medium on transformation and in vitro morphogenetic response of hypocotyl explants In the present study, three types of gelling agents, viz., agar, agargel (agar + Phytagel at 1:1 ratio) and Phytagel were assessed for their effect on regeneration response of Agrobacterium cocultivated hypocotyl explants in the presence of kanamycin (Table 2). Gelling agents did not affect callus initiation response from hypocotyl explants and callus induction response was found to be 100% on all the gelling agents tried. Explants cultured on medium solidified with agargel showed better regeneration (35.71%) than Phytagel (31.42%) or agar (27.14%). Though the effect of gelling agent was not significant, it is clear that kanamycin has the maximum activity in a medium solidified with agar than with agargel and Phytagel. Similarly, agar encouraged maximum effect of kanamycin, whereas Phytagel and agargel showed reduced inhibitory effect of kanamycin in flax transformation studies (Laine et al, 2000). Kanamycin appears to bind to gelrite with higher affinity than to agar; hence, inhibition of regeneration by kanamycin on a medium solidified with gelrite was less, compared to that with agar (Chauvin et al, 1999; Wilmink and Dons, 1993). In the present study, agargel, an intermediate form of agar and Phytagel, showed the highest regeneration response and less kanamycin activity compared to that with agar and Phytagel. Cassells and Collins (2000) reported that physical and chemical grading of gelling agents was not related to biological performance of the gelling agents. However, the small differences observed in regeneration response of cocultivated explants cultured on selection medium solidified with various gelling agents suggest that the gelling agent found to be optimal with respect to sensitivity of the explants to kanamycin should be continued to be used in transformation studies. It should not be changed at any stage of the transformation experiment because it may lead to altered response from explants between the experiments. Change in solidifying agent may either reduce regeneration response or increase regeneration of escapes due to change caused in the effect of kanamycin (selection). Generally, differences were observed in explants cultured on media solidified with various gelling agents in tobacco (Chauvin et al, 1999). So, there is also a possibility of the gelling agent itself affecting regeneration response of explants without kanamycin in the culture medium. However, in brinjal, reports indicate agar to be the best gelling agent for the realization of better in vitro regeneration response (Perrone et al, 1992). It can be therefore concluded that gelling agents have a role in deciding kanamycin activity, Table 2. Ef fect of gelling agents on transformation and morphogenetic response from hypocotyl explants of brinjal cv. Manjarigota Gelling agent Callus Regeneration response initiation (%) (% ± SE) Agar 100 27.14±2.8 Agar+ Phytagel (Agargel) 100 35.71±2.0 Phytagel 100 31.42±2.6 Fractions were converted into percentages; percentage data were subjected to angular transformation; values in the parentheses are transformed values, CD=34.30, SEm=3.81. Differences among treatments were non-significant at 5%. J. Hort. Sci. 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