EFFECTS OF BENZYLAMINOPURINE, 6-(δ,δ-DIMETHYLALLYAMINO)-PURINE AND THIDIAZURON ON SHOOT ORGANOGENESIS OF NICOTIANA SPECIES

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1 EFFECTS OF BENZYLAMINOPURINE, 6-(δ,δ-DIMETHYLALLYAMINO)-PURINE AND THIDIAZURON ON SHOOT ORGANOGENESIS OF NICOTIANA SPECIES Claire (Huan) Xie, Willa Huang, and Baochun Li 1 Regenerability is an essential trait for plant varieties developed for the production of plant-made pharmaceuticals (PMPs). As part of the effort to develop optimal plant varieties for PMP production, two experiments were conducted in this study to determine and/or improve the shoot organogenic potential of Nicotiana species with important traits for PMP-oriented breeding. Effects of benzylaminopurine (BA) and 6-(δ,δ-Dimethylallyamino)- purine (2iP) on shoot organogenic potential of 46 Nicotiana accessions, representing 15 species, were examined in the first experiment. BA was found to be more effective than 2iP in enhancing shoot organogenesis for 23 Nicotiana accessions from 10 species, while 2iP was more effective than BA for only four accessions, including two from N. paniculata, one from N. obtusifolia and one from N. rustica. Effects of varying concentrations of thidiazuron () in combination with indole-3-acetic acid (IAA) on shoot organogenic potential of 12 poorly responsive Nicotiana accessions from 10 species were examined in a second experiment. was found to enhance shoot organogenic potential of two N. rustica accessions in an IAA-dependent fashion. These results suggested that BA is an effective cytokinin for shoot organogenesis of a broad range of Nicotiana species, and 2iP and may be effective for only certain species. Additional keywords: shoot organogenesis, leaf explants INTRODUCTION Tobacco (Nicotiana tabacum L.) exhibits several attributes that make it attractive for the production of plantmade pharmaceuticals (PMPs), including high-level expression systems for foreign proteins, high biomass production and non-food use (4, 7, 15). However, traditional tobacco varieties are not ideal for these applications since many lack resistance to major tobacco diseases and are not suitable for identity preservation and multiple harvests. A major project at this center is development of a fully optimized vehicle plant for PMP applications that incorporates these additional traits. As part of this effort, we have examined Nicotiana accessions for their regenerability, an essential trait for PMP production. In a previous study, we examined the shoot organogenic potential of leaf explants from 115 Nicotiana accessions, representing 53 species (13). Thirty species were found to be highly regenerable, five species moderately regenerable, 13 species slightly regenerable and five species not regenerable. Also, an exponential relationship between the shoot organogenic potential and shoot production per responsive leaf explant was established. We have since gained access to more Nicotiana accessions in our collection. In order to examine/improve the shoot organogenic potential of those Nicotiana accessions that were of greatest interest for PMP-oriented breeding, but were poor or unknown in their shoot organogenesis, we examined the effects of benzylaminopurine (BA), 6-(δ,δ- Dimethylallyamino)-purine (2iP), and thidiazuron (, N- phenyl-n'-1,2,3 thidiazol-5-yl urea) on shoot organogenesis of Nicotiana species in this study. Both BA and 2iP have been used for shoot organogenesis in Nicotiana species (1, 10, 22, 23), though BA is commonly used for tobacco shoot organogenesis (5). Thidiazuron (, N-phenyl-N'-1,2,3 Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY , USA. 1 Communicating author: Baochun Li; bli2@uky.edu thidiazol-5-yl urea), a substituted phenylurea, has effectively induced shoot organogenesis in a number of plant species (18), and was even more effective than BA for some plant species (17). In this study, we first examined the effects of BA and 2iP at their commonly used concentrations on shoot organogenic potential of leaf explants derived from the seedlings of 46 Nicotiana accessions, representing 15 species. In a second experiment, we examined the effects of varying concentrations of thidiazuron () in combination with indole-3-acetic acid (IAA) on shoot organogenic potential of 12 poorly responsive Nicotiana accessions from 10 species. MATERIALS AND METHODS Mature seeds from accessions of Nicotiana species were surface-sterilized by exposure to chlorine gas (13) for 4 h, and placed in a Petri dish (100 x 20 mm) containing a germination medium (GM) that contained MS salts (16) plus B5 vitamins (6), 30 g -1 sucrose and 0.7% agar. Seeds were germinated in a growth chamber at 24 C with a 16-h photoperiod and a light intensity of approximately 79 µmol m -2 s -1. Leaf explant preparation, shoot induction and the determination of shoot organogenic potential were performed as described previously (13) except that the germinating seeds were allowed to grow for four weeks, not eight weeks, before the leaf explants were prepared. For the first experiment, where the effects of BA and 2iP on shoot organogenesis of 46 Nicotiana accessions were examined, leaf explants were placed onto shoot induction medium (SIM) containing either 11.1 µm BA or 11.1 µm 2iP, and 5.1 µm indole-3-acetic acid (IAA) and basic components. The basic components were MS salts, B5 vitamins, 30 g -1 sucrose and 8 g -l agar. For the second experiment, the effects of varying concentrations of thidiazuron () in combination with IAA on shoot organogenic potential of 12 poorly responsive Tobacco Science (2003/2004) 46:

2 Nicotiana accessions from 10 species were examined. Leaf explants were placed on SIM with concentrations of 5, Table 1. Nicotiana species Genus Nicotiana Subgenus Rustica Section Paniculatae Effect of Benzylaminopurine (BA) or 6-(δ,δ- Dimethylallyamino-purine (2iP) on shoot organogenesis of Nicotiana species. Percentage of leaf explants producing shoots 10, or 20 µm with or without IAA at 5.1 µm. SIM with 11.1 µm BA or 5.1 µm IAA, as described previously, was used as a control. All seven treatments also Average number of shoots produced per responsive leaf explant Accession Number BA 2iP BA 2iP N. glauca S-3-1 a N. glauca S N. paniculata PI b N. paniculata Aus TRC c Section Rusticae N. rustica S N. rustica S N. rustica S N. rustica S N. rustica S Subgenus Tabacum Section Tomentosae N. glutinosa S Section Genuinae N. tabacum PI N. tabacum S Subgenus Petunioides Section Trigonophyllae N. obtusifolia PI N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S N. obtusifolia S LSD 0.05 d a S number, accession number in the collection of Kentucky Tobacco Research and Development Center (KTRDC). b PI number, A USDA plant introduction number. c AusTRC number, accession number in the collection of Australian Tobacco Research Center. d Least significant difference. Table continues on next page included the basic medium components. For both experiments, the shoot organogenic potential of Nicotiana accessions were classified into highly regenerable, moderately regenerable, slightly regenerable and not regenerable (13). Both experiments were conducted and analyzed in a completely randomized design (CRD) with a total of 92 treatments (2 X 46) for the first experiment and 84 treatments (7 X 12) for the second experiment. Each treatment was replicated five times. The experiments were repeated once, and those accessions that yielded dramatically different results compared with the first evaluation were further evaluated an additional one or two times to ensure reproducibility. Analysis of variance, mean separation with LSD (least significant difference), and regression analysis were performed (21) to separate treatment effects. RESULTS Effects of BA and 2iP on shoot organogenic potential of Nicotiana accessions. Effects of BA and 2iP on shoot organogenic potential of 46 Nicotiana accessions, representing 15 species, were examined in the first experiment. These 46 Nicotiana accessions included 21 from N. obtusifolia, five from N. rustica, four from N. debneyi, and two each from N. glauca, N. paniculata, N. suaveolens and N. tabacum. The remaining eight Nicotiana accessions were from the following eight species: N. alata, N. attenuata, N. benthamiana, N. glutinosa, N. goodspeedii, N. maritima, N. sylvestris, and N. velutina. Among the 46 Nicotiana accessions, four had a USDA plant introduction number (PI), three had an accession number in the collection of the Australian Tobacco Research Center (ATRC), and the remaining accessions each had an S number [where S is the accession number in the collection of the Kentucky Tobacco Research and Development Center (KTRDC)]. Also, the 46 Nicotiana accessions included six entries, one from each of six species, that were either highly regenerable (N. debneyi PI and N. tabacum PI ), slightly regenerable (N. goodspeedii PI and N. velutina PI ) or not regenerable (N. paniculata PI and N. obtusifolia PI ) in the previous study (13) (Table 1). Statistical analyses detected significant differences among the 92 treatments with regard to the percentage of leaf explants producing shoots (P<0.0001) and the 18 Tobacco Science (2003/2004) 46:17-23

3 Table 1, continued Nicotiana species Section Alatae Effect of Benzylaminopurine (BA) or 6-(δ,δ- Dimethylallyamino-purine (2iP) on shoot organogenesis of Nicotiana species. Percentage of leaf explants producing shoots average number of shoots produced per responsive leaf explant (P<0.0001) (Table 1). BA was found to be more effective than 2iP in either the percentage of leaf explants producing shoots or the average number of shoots produced per responsive leaf explant, or both, for 23 Nicotiana accessions from 10 species. The 23 Nicotiana accessions included seven from N. obtusifolia, four from N. debneyi, three from N. rustica, two from N. glauca and two from N. tabacum. The remaining five accessions included one each from N. alata, N. benthamiana, N. glutinosa, N. sylvestris and N. suaveolens. The shoot organogenic potential of the 23 accessions ranged from slightly regenerable to highly regenerable. Morphologically, shoots produced from the leaf explants cultured on SIM with 2iP tended to be larger, but with lower numbers than other treatments (Figure 1A & 1B). The 2iP treatment was more effective than BA in either the percentage of leaf explants producing shoots, the average number of shoots produced per responsive leaf explant, or both, in four Nicotiana accessions from three species. These four accessions included two from N. paniculata (Figure 1C & 1D), one from N. obtusifolia and one from N. rustica. The shoot organogenic potential of the four accessions ranged from slightly regenerable to moderately regenerable. The effect of BA and 2iP were similar in 14 of the 46 Nicotiana accessions from six species. These accessions included nine from N. obtusifolia, and one each from N. goodspeedii, N. maritima, N. rustica, N. suaveolens and N. velutina. The shoot organogenic potential of these 14 accessions ranged from slightly regenerable to highly regenerable. Five accessions from two species did not regenerate on SIM with either BA or 2iP. The five accessions included four from N. obtusifolia and one from N. attenuata. In general, among the 15 species evaluated, N. alata, N. debneyi, N. glauca, N. rustica, N. sylvestris, N. suaveolens, and N. tabacum were highly regenerable. Nicotiana benthamiana, N. glutinosa, and N. paniculata were moderately regenerable, and N. goodspeedii, N. maritima, N. obtusifolia, and N. velutina were slightly regenerable. Only one species, N. attenuata, was not regenerable. Regression analysis also detected exponential relationships between the number of shoots produced per responsive leaf explant and their shoot organogenic potential in this experiment. Using the data generated from all 92 treatments, the exponential equation is: number = x percentage 0.05 x exp [ x (100- percentage) -2 ] (R 2 =0.8159, P<0.0001). Using the data generated from the 46 Nicotiana accessions cultured on BA-containing medium, the exponential equation is: number = x percentage 0.05 x exp [ x (100-percentage) -2 ] (R 2 =0.8357, P<0.0001). Using the data generated from the 46 Nicotiana accessions cultured on 2iPcontaining medium, the exponential equation is: number = x percentage 0.05 x exp [ x (100-percentage) - 2 ] (R 2 =0.8322, P<0.0001). In these equations, number represents the number of shoots produced per responsive leaf explant, percentage represents the percentage of leaf explants producing shoots, and exp represents exponent. Average number of shoots produced per responsive leaf explant Accession Number BA 2iP BA 2iP N. sylvestris S N. alata S Section Acuminatae N. attenuata S Section Suaveolentes N. benthamiana S N. debneyi S N. debneyi PI N. debneyi Aus TRC N. debneyi S N. maritima S N. velutina PI N. suaveolens Aus TRC N. suaveolens Aus TRC N. goodspeedii PI a S number, accession number in the collection of Kentucky Tobacco Research and Development Center (KTRDC). b PI number, A USDA plant introduction number. c AusTRC number, accession number in the collection of Australian Tobacco Research Center. LSD 0.05 d d Least significant difference. Effect of on shoot organogenic potential of Nicotiana accessions. The effects of varying concentrations of thidiazuron () in combination with IAA on shoot organogenic potential of 12 poorly responsive Nicotiana accessions from 10 species (13) were examined in the second experiment. The 12 Nicotiana accessions included two from N. debneyi and two from N. rustica. The remaining eight accessions included one each from N. alata, N. attenuata, N. clevelandii, N. goodspeedii, N. goosei, N. obtusifolia, N. occidentalis, and N. paniculata (Table 2). Two of the 12 accessions, N. goodspeedii PI and N. obtusifolia PI , were also examined in the first experiment of this study. Significant differences were observed among the 84 treatments with regard to the percentage of leaf explants producing shoots (P<0.0001) and the average number of shoots produced per responsive leaf explant (P<0.0001) (Table 2). significantly improved the shoot organogenic potential of the two accessions from N. rustica compared with the control in a concentration-dependant and IAA-dependant fashion. The most effective treatments for shoot organogenesis of the two N. rustica accessions were Tobacco Science (2003/2004) 46:

4 Table 2. Shoot organogenesis of Nicotiana species as affected by thidiazuron (). Nicotiana species Accession number Percentage of leaf explants producing shoots Control a b Average number of shoots produced per responsive leaf explant N. alata PI c N. attenuata PI N. clevelandii PI N. debneyi PI N. debneyi PI N. goodspeedii PI N. gossei PI N. obtusifolia PI N. occidentalis PI N. paniculata PI N. rustica PI N. rustica PI LSD 0.05 d a Control, shoot induction medium that contains BA. b IAA, indole-3-acetic acid,. c PI number, a USDA plant introduction number. d Least significant difference BA 20 Tobacco Science (2003/2004) 46:17-23

5 Figure 1. Effect of BA and 2iP on shoot organogenesis of Nicotiana species. A. N. debneyi PI on SIM with BA; many shoots were produced from each responsive leaf explant. B. N. debneyi PI on SIM with 2iP; only a few large shoots were produced from each responsive leaf explant. C. N. paniculata PI on SIM with BA; no shoot was produced from any leaf explant. D. N. paniculata PI on SIM with 2iP; several shoots were produced from each responsive leaf explant. Figure 2. Effect of varying concentrations in combination with IAA on shoot organogenesis of Nicotiana species. A. N. rustica PI on the control medium; no shoots were produced from any leaf explant. B. N. rustica PI on SIM with 5 µm plus 5.1 µm IAA; a couple of shoots were produced from each responsive leaf explant. C. N. debneyi PI on the optimal tobacco shoot organogenesis medium (control); a few shoots were produced from each responsive leaf explant. D. N. debneyi PI on SIM with 5 µm plus 5.1 µm IAA; no shoots were produced from any leaf explant. concentrations of 5 µm or 10 µm with 5.1 µm IAA (Figure 2 A & 2B). For the remaining Nicotiana accessions, inclusion of in SIM, with or without IAA, reduced or inhibited the shoot organogenic potential of seven accessions from six species, compared with the control. The seven accessions included two from N. debneyi, and one each from N. alata, N. clevelandii, N. goodspeedii, N. goosei and N. occidentalis. Three accessions, one from N. attenuata, one from N obtusifolia, and one from N. paniculata, did not produce any shoots from any treatments, including the control (Table 2). DISCUSSION Diverse Nicotiana species (8, 9) constitute excellent germplasm for PMP-oriented breeding. Nicotiana germplasm includes species with resistance to the major tobacco diseases (3, 12, 14), tolerance to abiotic stresses like salt and drought (11), and varying levels of alkaloid content (20). Regenerability is an essential trait for any plant variety used for PMP production. In order to determine the regenerability of Nicotiana species, we examined the shoot organogenic potential of 115 Nicotiana accessions in a previous study (13). In this study, we examined the effects of BA, 2iP and on shoot organogenesis involving a total of 56 Nicotiana accessions in two experiments. From these studies, we have identified 31 species to be highly regenerable, six moderately regenerable, 13 slightly regenerable and three nonregenerable. These results indicated that a majority of the Nicotiana species (37/53=70%) were either highly regenerable or moderately regenerable, and easily manipulated by genetic transformations. This study also found that BA is an effective cytokinin for shoot organogenesis of a broad range of Nicotiana species. BA was more effective than 2iP and for shoot organogenesis for a majority of the Nicotiana species examined in both the first and the second experiment where the SIM containing BA was used as a control. We also evaluated the effect of BA and 2iP on shoot organogenic potential of 33 Nicotiana hybrids generated from our inhouse breeding program in the first experiment, and BA was found to be more effective than 2iP for all 33 hybrids evaluated (data not shown). The cytokinin 2iP was more effective than BA for shoot organogenesis of a few accessions. The two N. paniculata accessions examined in this study both performed much better when they were cultured on 2iP-containing SIM than on that with BA. 2iP has been used as an effective cytokinin for shoot organogenesis in transformation studies of N. paniculata (23), and based on our results, 2iP is the best cytokinin for shoot organogenesis of N. paniculata. Tobacco Science (2003/2004) 46:

6 The fact that was effective only for the two N. rustica accessions tested in the second experiment suggested the effect of on shoot organogenesis of Nicotiana species could be highly species-specific. This study also established that the stimulating effect of on shoot organogenesis of N. rustica was IAA-dependent. This IAAdependent nature may vary among different plant species. In Hypericum perforatum, the combination of and IAA in SIM was found to be deleterious for shoot organogenesis using hypocotyl segments as explants (17). We also detected exponential relationships between the shoot organogenic potential and shoot production per responsive leaf explant. This exponential relationship was first established in a previous study (13), where a dramatic increase in shoot production per responsive leaf explant occurred only when the percentage of leaf explants producing shoots reached a certain level; 80% seemed to be the critical threshold value. The mechanism that governs the formation of this relationship is unknown. Our results suggested that this exponential relationship might apply to a wide scope of Nicotiana accessions and even different SIMs. One question arising from this study is the rooting ability of shoots generated from SIMs containing different cytokinins. Rooting is a critical step for the regeneration of a whole plant in a shoot organogenesis process; it is also an important indicator in a transformation study for the stable integration of foreign DNA into a plant genome. Any shoot that carries a stably integrated foreign marker gene is more than likely to root in a medium that contains the selective compound to which the marker gene is resistant. It is generally expected that any healthy shoot generated from the shoot organogenesis process should be able to root regardless of what cytokinin is included in the induction medium. However, there are cases where shoots generated from SIMs containing different cytokinins root at different rates. For example, in Eucalyptus globulus, shoots derived from a SIM that contained kinetin rooted at a much higher rate than those from a BA-containing medium (2). Similarly, in Rhododendron PJM hybrids, shoots generated from a SIM containing 2iP rooted significantly better than those from a -containing medium (19). Therefore, for any studies involving the generation of a whole plant, rooting ability needs to be examined before choosing a cytokinin for shoot induction in a Nicotiana species. We also observed that some of the Nicotiana species with important traits for PMP-oriented breeding, such as N. obtusifolia and N. goodspeedii, were poorly responsive to shoot organogenesis. The 21 N. obtusifolia accessions evaluated in the two experiments of this study were mostly collected in the deserts of the Western US, and included accessions highly resistant to blue mold, one of the major diseases in tobacco. The N. goodspeedii accession examined in this study also was found to be resistant to blue mold. Therefore, an efficient shoot organogenesis system is needed for these Nicotiana accessions in order to implement mutagenesis studies or other approaches to identify the genetic elements that condition these traits. ACKNOWLEDGEMENT We thank Drs. Verne Sisson, Robert Miller, and the Australian Tobacco Research Center for kindly providing the seeds for Nicotiana species, Dr. Maelor Davies for his support and critical discussions on manuscript preparation, Dr. Helena Truszczynska for statistical analysis, and Troy Bass, Rich Mundell, Ruth Babbit, Peggy Rice, Bonnie Kinney, Chris Cooper, Dr. Orlando Chambers, and Dr. David Zaitlin for technical support, or seed collections, or discussions. LITERATURE CITED 1. An, G., B. D. Watson, and C. C. Chiang Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81: Bennett, I. J., J. A. McComb, C. M. Tonkin, and D. A. J. McDavid Alternating cytokinins in multiplication media stimulates in vitro shoot growth and rooting of Eucalyptus globulus Labill. Ann. Bot. 74: Clayton, E. E Resistance of tobacco to blue mold (Peronospora tabacina). J. Agric. Res. 70: Daniell, H., S. J. 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