Optimization of regeneration protocol and Agrobacterium mediated transformation in carnation (Dianthus caryophyllus L.) H.M. Kallesh Prasad1, J.B. Mythili, Tejaswini1, Lalitha Anand, H.J. Rashmi and C. Suneetha Division of Biotechnology Indian Institute of Horticultural Research Hessaraghatta Lake Post, Bangalore-560089, India E-mail: jbm@iihr.ernet.in ABSTRACT An efficient and reproducible regeneration protocol for carnation genotypes Arka Flame and IIHRS-1 has been developed from leaf and stem explants. Although IIHRS-1 showed slightly higher regeneration (55%) compared to Arka Flame (49.2%), there was no significant difference in their regeneration response. However, significant difference in regeneration potential was observed with leaf explant exhibiting higher regeneration potential (5.5 shoots/explant) as compared to (4.9) stem explant. Among various plant growth regulator combinations tested for regeneration, the best regeneration response and maximum regeneration potential was obtained in MS medium supplemented with NAA (0.1 mg/l) and TDZ (1.0mg /l) for both the explants and genotypes used. The medium also proved suitable for inducing elongation of regenerated shoots. Rooting of in vitro formed shootlets could be induced at greater frequency in MS medium supplemented with IAA (1.0 mg/l). Based on this protocol, transformation was carried out in genotype IIHRS-1 using leaf explants with Agrobacterium tumefaciens LBA 4404 with binary vector pROK2 containing baculovirus chitinase gene under the control of 35S promoter with npt II serving as selectable marker. There was regeneration of putative transformants at a frequency of 28.9%. However, great difficulty was encountered in rooting of shoots. Hence a few shoots regenerated on selection medium at random were tested for transgene integration. Out of the three shoots tested for npt II amplification, two shoots tested positive. The presence of transgene was confirmed through PCR amplification of npt II gene and dot blot analysis of chitinase gene. Key words: Carnation, genotype, morphogenesis, Agrobacterium mediated transformation J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 INTRODUCTION Carnation (Dianthus caryophyllus L.) is one of the most important commercial flowers in the world. To date, new carnation varieties have been produced mainly through traditional breeding, and are propagated vegetatively. However, high heterozygosity, a limited gene pool, and almost no knowledge of carnation’s genetic makeup, severely restrict breeding programs (Woodson, 1991). Moreover, there are no varieties available in India to match International standards. Carnation breeders at, Indian Institute of Horticultural Research, Bangalore, have recently released a variety Arka Flame, the flower quality of which is on par with International Standards. They have also identified another promising genotype IIHRS-1 that can serve as basic material for future varietal improvement (Fig 1). 1Department of Biotechnology, University of Agricultural Sciences, GKVK, Bangalore-560065, India 2Division of Ornamental Crops, Indian Institute of Horticultural Research, Hessaraghatta, Bangalore-560089, India Recent developments in plant molecular biology open the way for unprecedented opportunities to use the technique of genetic engineering for improvement and value addition of flower crops. The availability of methods to introduce a useful defined gene(s) would enable the specific alteration of a single trait and broaden the gene pool available for this crop. The most commonly used method for introduction of genes is Agrobacterium mediated transformation. An essential step towards development of transgenic plants through Agrobacterium mediated transformation is the development of an efficient regeneration protocol. Attempts have been made to regenerate carnation through tissue culture via organogenesis from petals, stem, leaf (Nugent et al, 1991;Van Altvorst et al, 1994) and somatic embryogenesis (Frey et al, 1992; Yantcheva et al, 1998). Similarly there are reports on 121 Agrobacterium mediated transformation in carnation using different explants viz., stem, petals, leaves (Lu et al, 1991; Zuker et al, 2001a). However, there are differences in the efficiency depending on the explant or genotype used. Keeping this in view, the present investigation was carried out to compare the two genotypes viz., Arka Flame and IIHRS-1 and explants viz., stem and leave for their morphogenetic response and to identify the suitable genotype and explant for Agrobacterium-mediated transformation. MATERIAL AND METHODS Plant Material In vitro grown carnation Arka Flame and IIHRS-1 were multiplied through nodal cuttings on MS medium containing BAP 0.25 mg/l, GA 3 0.25 mg/l and NAA 0.1mg/l. These multiplied plants served as source of explants (Fig 2). In vitro regeneration Leaf and stem explants obtained from 15-20 day old in vitro grown cultures were inoculated on MS (Murashige and Skoog,1962) medium containing 3% sucrose with factorial combinations of cytokinins and auxins, viz., benzylaminopurine (BAP) or thidiazuran (TDZ) and naphthalene acetic acid (NAA), respectively. The medium was gelled with phytagel (0.25%) Sigma Chemical Co.(USA) and pH was adjusted to 5.8 prior to autoclaving at 121oC for 15 min. Cultures were incubated in culture racks provided with cool white fluorescent tubes with a light intensity of 30-40 mmoles m-2s-1 under a 16 h photoperiod in a culture room maintained at 25oC ± 2oC. Elongation of shoot buds could be achieved on sub culturing shoot buds to the same regeneration medium. Shootlets obtained were transferred to MS medium (full or half strength) with various auxins or their combinations viz., Indole - 3 - butyric acid (IBA) or Indole-3-acetic acid (IAA) for root induction. Rooted plantlets were transferred to polybags containing autoclaved mixture of sand, soilrite and soil in the ratio of 1:2:1 watered to field capacity and were hardened adopting the closed sachet technique (Ravindra and Thomas, 1995). Transformation and regeneration Agrobacterium strain LBA4404 containing pR0K2 vector with baculovirus chitinase gene cloned at BamHI site under the control of 35S promoter and selectable marker gene npt II under the control of nos promoter was used for transformation (Fig 3). Bacterial strain was grown overnight (O/N) in Yeast Extract Mannitol (YEM) medium containing kanamycin at 50 mg/l and collected at log phase, when the absorbance at 600nm was 1.0. The O/N grown culture was centrifuged at 10,000 rpm for 5 min at 4oC and the supernatant was discarded and bacterial pellet resuspended in half strength MS medium. For transformation studies, only the leaf explant from genotype IIHRS-1, which showed maximum regeneration potential and the medium in which it was achieved, was used for transformation work. Fig 2. Source of explants Fig 3. T-DNA of plasmid pROK2 containing 1.65 kb fragment of baculovirus chitinase gene inserted at the multiple cloning site following digestion with BamH1 J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 Fig.1. Flowers of Arka Flame and genotype IIHRS-1 Optimization of regeneration protocol and Agrobacterium mediated transformation in carnation 122 The leaf explants were infected with Agrobacterium culture for 5-30 min. blotted dry with filter paper and placed in the regeneration media for co-cultivation for 1-5 days under 16h photoperiod or under complete darkness for varied periods of time. Thereafter, the explants were transferred to selection medium containing 75mg/l kanamycin and 500 mg/l of cefotaxime. The explants with putative transformed shootlets were transferred to rooting media containing 50mg/l kanamycin and 500 mg/l cefotaxime. The rooted transformed plants were hardened and transferred to pots. Confirmation of the presence of transgene PCR analysis: DNA was isolated from leaves of control, transformed plants and Agrobacterium plasmid following CTAB method (Sambrook et al, 1989). The primers of the npt II gene used were as follows: Forward primer (5’GATGGATTGCACGCAGG3’) Reverse Primer (3’GAAGGCGATAGAAGGCG5’) PCR reaction was carried out in 25 ml containing 2.5ml of 100ng of sample DNA, 0.2ml of 10 mM dNTPs mix, 2.5 ml of 10x assay buffer for Taq polymerase containing 15mM MgCl 2 , 0.5 units of Taq DNA polymerase, 1 ml of 10mM each of forward and reverse primers. DNA was subjected to initial denaturation of 94oC for 2 min and 35 cycles of 94oC for 1 min, 60oC for 45sec and 72oC for 1.5min with a final extension of 72oC for 10min. Amplified DNA fragments were electrophoresed on 1.5% agarose gel and observed under UV light. Dot blot assay: Genomic DNA (5 µg) from PCR positive transformed plants and DNA of plasmid pROK2 were blotted on to nylon membrane (Hybond N+ Amersham pharmacia) and hybridized with a labeled baculovirus chitinase probe, washed and detected as per the manufacturer’s instructions of AlkPhos direct labeling and detection kit (Amersham Pharmacia Biotech UK Ltd). The baculovirus chitinase probe was prepared by amplifying it from the plasmid using the gene specific primers as given in Shi et al (2000). The PCR product was then labelled and used as a probe. Statistical analysis The experiment on in vitro regeneration (Tables1 & 2) was carried out in two genotypes using two explants viz., leaf and stem with 5 treatments. For each treatment, 5 tubes were used for each explant and the experiment repeated 6 times in a completely randomized design. The response from the 30 tubes was recorded with 10 tubes representing each replicate. The data indicated in the tables are means of replicated values. The data in Table 1 and 2 were transformed using Arc sine and square root transformation, respectively. The data were analyzed for three way interaction and subjected to analysis of variance (ANOVA). Comparison among treatment means were carried out using LSD values and are reported under “CD” at the end of each table RESULTS AND DISCUSSION In vitro regeneration Several factors are known to influence in vitro regeneration from cultured plant tissue. Genotypic differences in shoot regeneration ability among cultivated Table 1. Effect of different plant growth regulator (PGR) combinations on per cent regeneration of shoots from leaf and stem explants of carnation genotypes Genotype (A) Plant growth regulators (mg/l) (C) BAP NAA TDZ Explant (B) Mean Leaf Stem IIHRS-155.0(48.7) 1.0 0.1 - 23.32(28.65) 26.66(30.98) 23.33(28.77) 24.99(29.88) 1.0 0.3 - 49.12(44.55) 56.60(49.20) 50.00(44.98) 53.33(47.09) - 0.1 1.0 75.78(62.20) 86.60(72.76) 73.33(59.19) 79.96(65.97) 1.0 0.1 0.3 59.97(50.98) 63.30(53.05) 60.00(50.83) 61.65(51.94) Arka Flame 49.1 (44.5) 1.0 0.1 - 20.00(26.06) 23.33(28.77) 21.65(27.41) 1.0 0.3 - 46.66(42.98) 43.33(41.05) 44.95(42.01) - 0.1 1.0 76.66(61.90) 66.66(54.97) 71.60(58.43) 1.0 0.1 0.3 60.00(51.12) 56.66(48.91) 58.30(50.02) Mean 54.5 (48.5) 49.6 (44.7) CD (P ≤ 0.05); A= n.s ; B = n.s ; C = 12.0, (7.89); AxB = n.s ; AxC = n.s ; BxC = n.s ; AxBxC = n.s n.s.- Not significant Values in parentheses indicate arc-sine transformed values Kallesh Prasad et al J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 123 Table 2. Effect of plant growth regulator (PGR) on average number of shootlets per explant of carnation genotype Genotype (A) Plant growth regulators (mg/l) (C ) BAP NAA TDZ Explant (B) Mean Leaf Stem IIHRS-15.23 (2.25) 1.0 0.1 - 2.89(1.70) 3.13(1.77) 2.70(1.64) 2.91(1.70) 1.0 0.3 - 3.67(1.91) 3.97(1.99) 3.67(1.91) 3.82(1.95) - 0.1 1.0 7.39(2.71) 7.76(2.78) 6.80(2.62) 7.28(2.70) 1.0 0.1 0.3 6.80(2.61) 7.57(2.75) 6.23(2.54) 6.90(2.64) Arka Flame5.14 (2.22) 1.0 0.1 - 3.10(1.75) 2.63(1.62) 2.87(1.69) 1.0 0.3 - 3.50(1.87) 3.53(1.88) 3.51(1.87) - 0.1 1.0 7.60(2.76) 7.40(2.72) 7.50(2.74) 1.0 0.1 0.3 7.20(2.68) 6.20(2.49) 6.70(2.59) Mean 5.48(2.29) 4.89 (2.18) CD (P ≤ 0.05); A= n.s; B = n.s ; C = 12.0, (7.89); AxB = n.s ; AxC = n.s ; BxC = n.s; AxBxC = n.s n.s.- Not significant Values in parentheses indicate square root transformed values carnation are known to exist (Firoozabady et al, 1995). In the present study, however, no significant difference among the two genotypes was observed although slight differences in the response of genotypes to shoot regeneration was recorded with IIHRS-1 recording higher regeneration (55.0%) followed by Arka Flame with 49.2%. Differences therefore, in regeneration response have been linked to the explant used. Carnation regeneration has been reported through the use of various explants viz., petal, stem (Nugent et al, 1991) and leaf (Van Altvorst et al, 1994). Other explants such as anthers, ovule and axillary bud have been occasionally used for regeneration with differences in the regeneration ability. However, leaf and stem are the preferred explants and superior to other explants due to their high regeneration potential as well as better quality of plants regenerated. Currently most of the work on regeneration of carnation is restricted to these two explants, although there are specific reports on differences in terms of regeneration ability between these two explants. In the present study there was no significant difference in the regeneration response of the explants used, with both leaf (54.6 %) and stem (49.6%) explants giving almost similar regeneration response in both the genotypes tested. However, there was a significant difference between the two explants in their regeneration potential with leaves regenerating more shootlets per explant (5.5) as compared to stem explant (4.9) (Tables 1 and 2, Fig 4 and 5). Among the growth regulators used, it was found that both regeneration percentage and regeneration potential of the explant was expressed at its maximum level with the use of TDZ and NAA. Incorporation of two cytokinin TDZ and BAP along with NAA proved to be superior (59.9%) over BAP and NAA (23.3%). However, the best regeneration response (75.8%) was obtained in a medium supplemented with NAA (0.1 mg/l) and TDZ (1.0 mg/l) irrespective of the explant and genotype used (Table 1). There was significant reduction in the regeneration response with the use of BAP and NAA. Superiority of TDZ over BAP in regeneration of shoots from explants has been reported by Nugent et al (1991). Most of the reports on carnation regeneration have utilized combination of BAP and NAA (Nugent et al, 1991; Firoozabady et al, 1995; Van Altvorst et al, 1996). There are few reports on the use of TDZ along with NAA (Nugent et al, 1991 and Sankhla et al, 1995). The concentration of BAP and NAA has been found to influence only the regeneration potential of the explant (number of shoots/regenerating explant) and not the regeneration percentage. Highest numbers of shoots were obtained on medium containing 0.9 mg/l BAP and 0.3 mg/l IAA (Van Altvorst et al, 1994). However, Sankhla et al (1995) reported that prolonged growth in TDZ resulted in hyperhydricity. Hyperhydricity was encountered in the present study as well in both the cultivars of carnation, irrespective of the type of cytokinin used. The problem of hyperhydricity could be checked by incorporation of agar and phytagel in equal proportion in the medium for gelling and supplementing with mannitol (500 mg/l) in addition to plant growth regulators. Complete replacement of phytagel with agar resulted in cracking of the medium and poor response of explants.The regeneration media proved suitable for inducing elongation of the regenerated shoots (Fig 6). Optimization of regeneration protocol and Agrobacterium mediated transformation in carnation J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 124 Table 3. Effect of plant growth regulator (PGR) combinations on percent induction of rooted shoots in carnation genotypes Genotype (A) Plant growth regulators (mg/l) (C ) Explant (B) Mean Leaf Stem IIHRS-170.83 (58.7) IAA (1.0) 81.24 (65.8) 83.33 (66.7) 91.66 (73.4) 87.49 (70.0) IBA (1.0) 47.91 (43.8) 50.00 (45.0) 58.33 (49.9) 54.16 (47.4) Arka Flame 58.33 (50.8) IAA (1.0) 58.33 (49.8) 91.66 (73.4) 74.99 (61.6) IBA (1.0) 33.33 (35.2) 50.00 (45.0) 41.66 (40.1) Mean 56.24 (49.2) 72.91 (60.4) CD (P ≤ 0.05); A = 9.16, (5.77); B = 9.16, (5.77); C = 9.16, (5.77); AxB = n.s ; AxC = n.s; BxC = n.s ; AxBxC = n.s n.s.- Not significant Values in parentheses indicate angular transformed values Rooting of in vitro formed shoots was achieved in MS medium supplemented with auxins viz., IAA and IBA. Among these two, IAA (1.0 mg/l) proved to be superior in inducing rooting (81.2%) as compared to IBA (47.9%) at the same concentration. Among the genotypes, highest induction of rooting (70.8%) was noticed in IIHRS-1 compared to Arka Flame (58.3%). Similarly shoots regenerated from stem explants recorded highest rooting (72.9%) as compared to shoots regenerated from leaf (56.2%) (Table 3, Fig 7). There was no difference among genotypes in ex vitro establishment of rooted shoots. They established with 75-80% success in ex vitro poly bags containing sand, soilrite and soil in the ratio of 1:2:1 and subsequently transferred to earthen pots (Fig 8). Transformation The genotype IIHRS-1 was used for transformation work due to its higher regeneration response. Although leaf and stem explants gave almost similar regeneration response, leaf explants were used due to their significantly higher regeneration potential. Further it was found that stem explants produced only callus on selection media and the callus turned brown subsequently without any shoot regeneration. Agrobacterium mediated transformation of carnation has been successful with the use of various explants viz., stem (Lu et al, 1991; Zuker et al, 2001a), leaf (Firoozabady et al, 1995) and petals (Van Altvorst et al, 1996; Miroshnichenko and Doglov, 2000). Among these, both stem and leaf explants have been widely used. The use of petals for transformation has been less successful, despite the extremely high regeneration potential of theseFig 6. In vitro shoot elongation Fig 7. Rooted shoots Fig 4. Regeneration from leaf explant of IIHRS-1 Fig 5. Regeneration from stem explant of IIHRS-1 J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 Kallesh Prasad et al 125 explants because of their ability to induce premature flowering in vitro and difficulty to transfer ex vitro. There was an increase in the percent regeneration response with increase in inoculation time up to 20 min. There after, there was a decline in percent explant regeneration due to Agrobacterium overgrowth and death of explants (Table 4). Hence an inoculation time of 20 minutes with an undiluted Agrobacterium culture grown overnight with an O.D of (0.9-1.0) at 600nm followed by 5 days of co-cultivation was required for sufficient infection to take place. It was observed that carnation leaf explants were resistant to infection by Agrobacterium as evidenced by the lack of Agrobacterium overgrowth even after 3-4 days of co-cultivation. The waxy nature of carnation leaves may be the reason for its resistance to Agrobacterium infection. Such a long co-cultivation time has been recommended in carnation (Ahroni, 1996). Light was another important factor, which was found to influence the growth of Agrobacterium and retention of healthy explants and their regeneration following inoculation with Agrobacterium. Incubating the explants after inoculation for 3 days under complete darkness followed by incubation under 16h photoperiod for 2 days resulted in greater number of explant regeneration (51.6%) as compared to incubation of explants under 16h photoperiod (28.3%) or total darkness (33.3%) on all 5 days for co- cultivation. (Table 4) Co-cultivation under 16h photoperiod and total darkness for all 5 days resulted in Agrobacterium overgrowth by 3rd day itself as compared to overgrowth appearance on 4th day when co-cultivation was carried out 3 days under complete dark followed by 2 days in 16hour photoperiod condition (Data not shown). Five days co-cultivated explants were transferred to selection medium containing cefotaxime (500 mg/l) and kanamycin (75 mg/l). There was no need to transfer the co-cultivated explants to cefotaxime medium prior to transfer to selection medium, as there was no Agrobacterium overgrowth. In the present study 75mg/l was the concentration at which there was no regeneration from the leaf explant without Agrobacterium co-cultivation and hence Table 4. Effect of inoculation time (min.) and photoperiod (light) during co-cultivation with Agrobacterium on percent healthy explants Time (min)(A) Photoperiod (B) Mean (A) 3 days dark and 16h photoperiod Complete dark 16 h photoperiod (2days) (5days) (5days) % healthy explants 5 59.0 (50.1) 55.8 (48.8) 55.8 (48.5) 56.9 (49.2) 10 57.4 (49.4) 55.2 (48.1) 51.2 (46.1) 54.6 (47.9) 15 57.2 (49.2) 54.6 (47.9) 51.2 (45.7) 54.3 (47.6) 20 57.4 (49.6) 55.0 (48.0) 51.8 (46.1) 54.7 (47.9) 30 49.0 (44.4) 46.2 (42.8) 40.6 (39.3) 45.3 (42.1) Mean (B) 56.0 (48.6) 53.4 (47.1) 50.1 (45.1) CD (P ≤ 0.05); A = n.s ; B = n.s ; AxB = n.s n.s.- Not significant Values in parentheses indicate angular transformed values Table 5. Effect of inoculation time (min) and photoperiod (light) during co- cultivation with Agrobacterium on percent explant regeneration Time (min.) (A) Photoperiod (B) Mean (A) 3 days dark and Complete dark 16 h photoperiod 16 h photoperiod (2 days) (5 days) (5 days) % explant regeneration 5 15.1 (22.6) 10.0 (18.3) 3.3 (8.6) 12.7 (16.3) 10 25.0 (29.7) 13.3 (21.2) 11.7 (19.9) 15.8 (23.6) 15 38.3 (38.2) 21.6 (27.5) 18.3 (25.1) 26.4 (30.3) 20 51.6 (45.9) 33.3 (35.2) 28.3 (32.0) 35.5 (37.7) 30 33.3 (35.2) 20.0 (26.0) 16.7 (23.3) 23.7 (28.2) Mean (B) 30.0 (34.3) 21.6 (25.6) 16.9 (21.7) CD (P ≤ 0.05); A = 7.22, (5.12); B = 5.56, (3.97); AxB =12.5, (8.87). Values in parentheses indicate angular transformed values J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 Optimization of regeneration protocol and Agrobacterium mediated transformation in carnation 126 the same concentration of kanamycin was used for selecting transformants. There was regeneration of 28.9 % of putative transformants on selection medium (Fig 9) and 50% of the regenerated transformants showed elongation in the same medium (Fig 10). However, great difficulty was encountered in rooting of these shoots. Incorporation of antibiotics in selection media has been reported to reduce multiplication and rooting rates (Cassels, 1991). The rooting ability of transgenic carnation plants was enhanced dramatically with rolC gene from A. rhizogenes (Zuker et al, 2001c). It was also observed that induction of rooting was considerably reduced in shoots regenerated from leaf explants (56.2%) as compared to shoots regenerated from stem explants (72.9%) in control plants. This may be another reason for encountering difficulty in rooting of shoots as leaf explants were used for transformation. Zuker et al (1999) on the other hand have developed a highly efficient procedure for Agrobacterium mediated transformation following wounding of stem explants through particle bombardment. This procedure gave rise to 20% transformation efficiency. Hence, few shoots regenerated in selection medium at random were tested for transgene integration through PCR analysis of the npt II gene using the specific primer sequence. Out of the 3 shoots tested for npt II amplification 2 shoots tested positive with a single 750bp band amplifying in the both plasmid and transformed plants but absent in control plant (Fig 11) and these transformed shoots tested positive in dot blot assay as well (Fig 12). Hitherto, the focus of carnation breeding has been on the development of novel floral traits, although the grower would desire carnation plants with improved agronomic performance especially for resistance to diseases. Among the various diseases afflicting carnation, wilt disease caused by Fusarium oxysporum f.sp. dainthi is a major one. The methods currently used to control this soil borne fungus are very hazardous as well as ineffective and costly. Classical breeding efforts to identify and select for resistant phenotype based on extensive and costly screening in infected soil is proving difficult. Under these circumstances, development of transgenic lines by incorporating specific genes that could Fig 9. Regeneration of putative transformants Fig 10. Elongation of putative transformants Fig 11. Amplification of nptII gene in putatively transformed plants (IIHRS-1) Fig 8. Hardened plants Fig 12. Dot blot assay of transgenic (Tr1,Tr2)plants and plasmid pROK2 (P). Membrane probed with Alk Phos labelled baculovirus chitinase gene (1.65 Kb) PCR fragment J. Hortl. Sci. Vol. 4 (2): 120-127, 2009 Kallesh Prasad et al 127 impart resistance to the target organism would be a more effective approach. 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