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Geranium Bronze Tolerance in Diploid and Tetraploid Ornamental

M. Alonso and M. Borja S. Herrero and J. Ferré P. Ellul and V. Moreno Departamento de I+D Departamento de Genética Laboratorio de Células y Fundación PROMIVA University of Valencia Tejidos Vegetales Boadilla del Monte Burjassot IBMCP-CSIC 28660 Madrid 46100 Valencia 46022 Valencia Spain Spain

Keywords: Breeding, , , ploidy, germplasm, management

Abstract The ornamental , Pelargonium × hortorum Bailey, is a traditional ornamental plant largely cultivated in and Northern America. Recently, a lepidopteran pest, the (Cacyreus marshalli Butler), is threatening the popularity of geraniums in Europe. An inventory of ornamental geranium samples present in remote rural areas and National Parks is currently being established to build a national germplasm bank [the Spanish Pelargonium Germplasm Bank (SPG Bank)] of garden geraniums. The objective of this study was to analyse twenty representative taxa from the SPG Bank and seven commercial cultivars of Pelargonium × hortorum to assess their ploidy level and test them for potential resistance to the geranium bronze. The ploidy level was determined by flow cytometry. All commercial cultivars tested for geranium bronze tolerance had an elevated degree of susceptibility compared to the most tolerant SPG taxa. This study indicates that there is enough variability among the SPG Bank taxa to be used as potential sources for geranium bronze-tolerant germplasm for the development of commercial Pelargonium cultivars.

INTRODUCTION The ornamental geranium, Pelargonium × hortorum Bailey, has a complex heritage (Laughner, 1993). It is thought to derive mainly from P. zonale and P. inquinans (Ciconium section), with P. hybridum, P frutetorum, P. scandens, and P. acetosum contributing traits to a lesser extent (Clifford, 1956; Harney, 1966; Chow and Harney, 1970). Geraniums are universally popular, with over 100 million plants produced and distributed commercially in 1998 in the United States (2000 Floriculture Crops Summary, National Agricultural Statistics Service, USDA) and an equivalent production and distribution in Europe. The Pelargonium consists of more than 250 species in 13 sections (Van der Walt et al., 1988), all but 33 originated from . Seven basic chromosome numbers (x=4, 7, 8, 9, 10 11, and 17) are found in the genus (Van der Walt, 1992), with ploidy leading to somatic numbers from 8 to 104. Crossability is mostly restricted to species having the same chromosome number and ploidy level (Horn, 1994). Today, most asexually produced, commercially successful taxa are tetraploid (Laughner, 1993). Currently two species are largely cultivated in Europe and northern America: Pelargonium × hortorum Bailey ("zonal geranium", 2n=2x=18 or 2n=4x=36) and Pelargonium × hederafolium Bailey ("ivy leaf geranium", 2n=2x=18 or 2n=4x=36), and more than a hundred commercial taxa are available. Geraniums are mostly clones, but F1- hybrids are also propagated though seeds (Renou et al., 1997). Today geranium is one of the traditional ornamental plants in Southern Europe, where it is grown as a perennial plant. Recently, a lepidopteran pest, the geranium bronze (Cacyreus marshalli Butler), is threatening the popularity of geraniums in Europe, since these plants are no longer easy to grow because of the biweekly insecticidal treatments that must be sprayed in order to maintain the plants free of the pest. Because of the biology of this pest, its control with conventional insecticides is not easy. The lays its eggs in the flower buds or in

Proc 21st IS on Breeding Ornamentals, Part II Eds: G. Forkmann & S. Michaelis 165 Acta Hort 651, ISHS 2004 the leaves and, when the larva hatches, it feeds inside the flower bud and bores galleries in the stems. Galleries become infected with fungi (Botrytis and Pythium), which eventually leads to the plant death (Herrero et al., 2002). The geranium bronze is a butterfly from the family . Originally from southern Africa, it was introduced in Europe through the Balearic Islands, (Spain) early in the 90's (Eitschberger and Stamer, 1990). Absence of natural enemies out of its place of origin, together with its quick adaptation to cultivated geranium, allowed its fast spread, becoming the most important pest of the cultivated geraniums in Europe. Currently, this butterfly is considered a pest in several countries in Southern Europe such as Spain, (Trematerra et al., 1997), , (Germain, 1999), and is included in the EPPO A2 quarantine list: http://www.eppo.org/QUARANTINE/lists.html. Moreover, the presence of this insect has been reported in other central and northern European countries such as , The (De Vos, 2000), and Poland (Karnowski and Labanowski, 1998). Although the lower temperatures in Central and Northern Europe would impede its development in a free habitat, greenhouses with suitable plants would provide an adequate habitat for its establishment as a pest. An inventory of ornamental geranium samples present in remote rural areas and National Parks is currently being established in order to build a national germplasm bank [the Spanish Pelargonium Germplasm Bank (SPG Bank)] of garden geranium. The SPG Bank might contain enough variability for geranium bronze tolerance to be used in future breeding programs. However, since sexual crossability can only be achieved between taxa with the same level of ploidy, determination of the ploidy level is necessary before any breeding program is designed. Twenty taxa, which had traits of ornamental interest, were selected from the SPG Bank for further analysis. In the present study, these twenty taxa and seven commercial cultivars of Pelargonium × hortorum were analysed to determine the variation in tolerance to the geranium bronze and flow cytometry was applied to assess their ploidy level in order to use these taxa in future breeding programs.

MATERIALS AND METHODS

Plant Material P. x hortorum cultivars 'Americana Bright Red' (Goldsmith Plants Inc.), 'Maverick White', 'Maverick Star' (Goldsmith Seeds Inc.), 'Red Satisfaction', 'Bright Red Satisfaction' (Oglevee Inc.), 'Empress' and 'Belmonte' (Endisch), were used as commercial references for these studies. The plants were purchased from a local nursery and maintained under glasshouse conditions with a day temperature of 20±4ºC and night temperature of 16±4ºC. The 20 P. × hortorum taxa used in this study were selected from the SPG Bank, which currently has 517 taxa. The 2,037 original plants were collected throughout rural areas from Spain representing different bioclimatic areas, and had been growing in the area for at least 25 years. These 20 taxa were selected because they had ornamental breeding potential. Sample cuttings were collected for morphological characterization and initially evaluated for viral and Xhanthomonas disease by an ELISA tests. The healthy cuttings were rooted and preserved in a greenhouse under the conditions mentioned above. All the infected cuttings were discarded.

Preparation of Nuclei Suspensions Analyses were performed on young leaves obtained from plants grown in the greenhouse. Nuclei were isolated from a small amount of fresh material (1 cm2 of leaf tissue) and each taxon was analysed in triplicate. Leaf tissue was chopped with a sharp razor blade in 200 µL of nuclei isolation buffer (Partec «High Resolution DNA Kit», Type P, Solution A), at room temperature, according to Galbraith et al. (1983). The suspension was filtered through a 40 µm mesh and the nuclei suspension was stained with 800 µL of 4', 6-diamino-2-phenylindole (DAPI) (Partec «High Resolution DNA Kit», Type P, Solution B).

166 Flow Cytometric Analysis Nuclear suspensions were analysed by flow cytometry using a PARTEC Cell Analyser II (Partec, Münster, ), equipped with Flowmax software (Partec, Münster, Germany). For each sample, at least 8,000 nuclei were analysed through the focus of an high-pressure mercury vapour damp lamp (Osram HBO 100W), utilizing a TK420 dichroic mirror and GG435 longpass filter. The excitation light was converted to pulses of electric current by a photomultiplier. The electronic signals are then digitised and the binary data are stored as one-dimensional histograms (256 channel positions). The fluorescence intensity is correlated directly with the amount of DNA that is stained with DAPI. Flow cytometry is a relative method of analysis; all the samples were analysed alone and mixed with nuclei isolated from young leaves of the diploid standard (Pelargonium × hortorum Maverick cultivars).

Insects Because of the impossibility to establish and maintain a laboratory colony of the geranium bronze (adults do not mate in captivity or, if they do, their fecundity is very low), eggs and larvae of this pest were regularly collected from P. x hortorum plants, from flowerbeds in the Burjassot Campus of the University of Valencia. Collected eggs were first placed on flower petals in a petri dish containing solid 2 % agar (Hispanagar) and later transferred to flower buds (from the Empress taxa) at the stage of larva (4 days old). Field collected larvae were also maintained on flower buds. were maintained in an insect rearing room at 25ºC with 60 % RH and 16 h day-1 photoperiod until they were used in the bioassays.

Laboratory Geranium Bronze Evaluations Tolerance of the Pelargonium taxa to the geranium bronze was evaluated using a growth inhibition assay with larvae feeding on geranium flower buds. The growth inhibition assay was chosen instead of the mortality score assay because the former is known to be more sensitive (Gould et al., 1995). The higher sensitivity of the growth inhibition method allowed us to detect changes in tolerance among the different taxa that would probably not be detected using a mortality assay. Tests were performed at 25ºC with 60 % RH and a 16 h day-1 photoperiod. Preweighed third instar larvae were allowed to feed on these buds on single wells for 48 hours and then they were weighed again to determine the larval weight increase, which is proportional to the plant damage. Due to shortage of larvae, the sample size was small, and it ranged from 4 to 15 larvae per taxon. Larvae that died without inflicting any damage to the bud were not taken into consideration. Larvae that died after inflicting damage to the bud were considered the same as living larvae with zero increase in weight. Relative growth was calculated as the weight gain (final minus initial weight) divided by the initial weight for each larva.

Field Geranium Bronze Evaluations Comparative field trials were set up to evaluate the potential tolerance of the Pelargonium taxa to the geranium bronze. Ten plants from each of the taxa were planted in 40 cm x 1 m beds in April 2001. The presence of the geranium bronze was determined visually by looking for flower buds with bored holes, or by observing larvae feeding on the plants. Scouting was done three times during the season, in June, July, and September. If at least one plant of a given taxon was found with a bored hole or with larvae at the time of scouting, the result was considered positive for attack by the geranium bronze and this is indicated by a + sign in Table 2. So an arbitrary scale was derived which ranged between 0 (no plant was infected in the three observations) and +++ (at least one plant was infected in each of the three observations).

167 RESULTS AND DISCUSSION

Ploidy Analysis Standard flow cytometry of diploid tissue resulted in a histogram with two peak regions (Fig. 1). The first region consisted of cells in the G0/G1 phase of the cell cycle corresponding to the 2C DNA content. The second smaller peak, at twice the channel position of the 2C peak, consisted of cells in the G2 or M phases. Cells between the two peaks are at some point in the S phase. The surface area of the peaks is correlated with the number of cells that excite at the same fluorescence intensity, thus containing the same amount of DNA (De Schepper et al., 2001). The results obtained are illustrated in Fig. 1, which shows the histogram of leaves samples of a diploid 'Maverick white' taxa (A) and a tetraploid P. × hortorum ‘ZAZ 2’ taxa (B) as well as a mixture of nuclei from both taxa (C). All the vegetative commercial taxa ('Americana Bright Red', 'Belmonte', 'Empress', 'Red Satisfaction' and 'Bright Red Satisfaction') were determined to be tetraploid (2n=4x=36), while the seed commercial taxa ("Maverick Star" and "Maverick White") were determined to be diploid (2n=2x=18). In contrast, of the seventeen SPG Bank taxa included in this study, 12 were determined to be diploid (2n=2x=18), and 5 tetraploid (NAZ3, SEZ19, SEZ29, ZAZ2, and ZAZ11) (Table 1). This result is not surprising since it is more likely that old taxa are still diploid, while commercial taxa have been bred and selected to obtain tetraploid plants. Conventionally, microscopic chromosome counting and Feulgen scanning microspectrophotometry, using meristematic mitotic cells, have been conducted to determine chromosome number in Pelargonium (Cramer, 1999). However, these methods are time consuming and laborious. Flow cytometric analysis offers a valuable and rapid alternative to traditional chromosome counts. Flow cytometry involves the analysis of fluorescence and light scattering properties of single particles during their passage within a narrow, precisely defined, liquid stream (Dolezel, 1991). Heller (1973) was the first to use this technique for DNA analysis in plant cells. The technique is now widely used for many plant species, because, compared with conventional chromosome counting, it has the advantages of accuracy, convenience, simplicity, low cost and rapidity (Galbraith, 1990; Dolezel, 1997; De Schepper et al., 2001). While it was previously described that flow cytometry was not useful to distinguish Pelargonium somaclonal variants (Cassels et al., 1997), it was very useful to determine the ploidy of the SPG Bank taxa.

Geranium Bronze Tolerance Tests Growth inhibition assays with larvae of the geranium bronze showed that the plants analysed presented varied degrees of tolerance (Table 2). Relative larval growth was strongly influenced by the taxa tested, ranging from 0.17 to 2.30, the lower value reflecting the higher tolerance by the plants. The two most tolerant taxa were ZAZPE1 (relative growth of 0.17±0.15) and TOAZ1 (relative growth of 0.30 ± 0.08). While ZAZPE1 was collected from a severely infected area in Zamora (Castilla-León province), the TOAZ1 was collected in an isolated hunting palace in Toledo (Castilla-La Mancha province) were this pest had not yet been established. It is interesting to note that all commercial taxa tested had an elevated degree of susceptibility ('Maverick Star', 'Maverick White', 'Americana Bright Red', 'Belmonte', 'Empress', 'Red Satisfaction' and 'Bright Red Satisfaction'), which is coherent with the rapid expansion of the disease. The mean relative growth of the larvae fed in these seven commercial taxa was 1.60, a value that is almost 10-fold higher than the value for the larvae fed on the most tolerant taxon from the bank, ZAZPE1. This taxon seems particularly promising because, under the current bioassay conditions (two-day growth inhibition of third instar larvae), little larval growth was supported. Of the 8 larvae tested with ZAZPE1, one died, three had a reduction in weight, one did not have any increase, and just three gained weight (relative growth values of 0.10, 0.40, and 1.10). It would be interesting to see how the most tolerant taxa, and in particular ZAZPE1, could support the entire larval cycle.

168 The results from the field trial agree relatively well with those of the laboratory evaluation (Table 2). The 7 commercial taxa were all found attacked by the pest every time the scouting was done. Conversely, the two most tolerant taxa from the SPG Bank, according to the laboratory evaluation (ZAZPE1 and TOAZ1), were never found attacked in the field trial. Most of the taxa from the SPG Bank were found less attacked than the commercial taxa, one exception being ZAZ2, which is the taxon with the highest value of relative growth of all taxa tested. In conclusion the results reported here suggest that the SPG Bank is a good source of variation for geranium bronze tolerance. The tolerant taxa identified here could be used as sources for geranium bronze-tolerant germplasm for the development of additional Pelargonium commercial taxa that require fewer chemical inputs. Since the two most tolerant taxa, ZAZPE1 and TOAZ1, are both diploid, sexual crosses are on the way to transfer the genes, which confer the geranium bronze tolerance into diploid commercial taxa.

ACKNOWLEDGEMENTS We thank Pino Lago, Adolfo Fernandez, Andrés Henández, Yago Pérez, David del Pozo, José Antonio Segado and Cristina Sesma for technical assistance, This work was supported by a CICYT-FEDER grant (FEDER funds from the European Union, project No. 1FD1997-0917) and a grant from the INIA (project No. RF99/006).

Literature Cited Arumuganathan, K. and Earle, E.D. 1991. Estimation of nuclear DNA content of plants by flow citometry. Plant Mol.Biol. Rptr. 9: 229-233. Cassells, A.C., Croke, J.T. and Doyle, B.M. 1997. Evaluation of image analysis, flow cytometry, and RAPD analysis for the assessment of somaclonal variation and induced mutation in tissue culture-derived Pelargonium plants. Angew. Bot. 71: 125- 130. Chow, T.W. and Harney, P.M. 1970. Crossability between a diploid Pelargonium x hortorum Bailey cultivar and some of its putative ancestral species. Euphytica 19: 338-348. Clifford, D. 1956. The parents of the geranium. J. Roy. Hort. Soc. 81: 20-22. Compton, M.E. 2000. Interaction between explant size and cultivar affects shoot organogenic competence of watermelon cotyledons. Hortscience 35: 749-750. Cramer, C.S. 1999. Laboratory techniques for determining ploidy in plants. HortTechnology. 9: 594-596. De Schepper, S., Leus, L., Mertens, M., Van Bockstaele, E., De Loose, M., Debergh, P. and Heursel, J. 2001. Flow cytometric analysis of ploidy in Rhododendron (subgenus Tsutsusi). Hortscience. 36: 125-127. Dolezel, J. 1991. Flow citometric analysis of nuclear DNA content in higher plants. Phytochem. Anal. 2: 143-154. Dolezel, J. 1997. Application of flow citometry for the study of plant genomes. J. Appl. Genet. 38: 285-302. Eitschberger, U. and Stamer, P. 1990. Cacyreus marshalli Butler, 1898, eine neue Tagfalterart für Sie Europäische Fauna? , Lycaenidae. Atalanta 21: 101- 108. Galbraith, D.W. 1990. Flow cytometric analysis of plant genomes. Methods in Cell Biol. 33: 549-561. Germain, J.F. 1999. Cacyreus marshalli Butler, a new pest of Pelargonium in France, development of the situation in 1999. Proceeding of the Fifth International conference on pest in Agriculture. Part I, 127-133. Gould, F., Anderson, A., Reynolds, A., Bumgarner, L. and Moar, W. 1995. Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to toxins. J. Econ. Entomol. 88: 1554-1559.

169 Harney, P.M. 1966. A chromatographic study of species presumed ancestral to Pelargonium x hortorum Bailey. Can. J. Genet. Cytol. 8: 780-787. Heller, F.O. 1973. DNA-Bestimmungen an Keimwurzeln von Vicia faba mit Hilfe der Impulszytophotometrie. Berl. Deutsch Bot. Ges. 86: 437-441. Herrero, S., Ferre, J., Hernandez, C.S., Escriche, B. and Borja, M. 2002. Uso de Bacillus thuringiensis en el control del taladro del geranio (Cacyreus marshalli Butler). Phytoma. 141: 36-44. Horn, W. 1994. Interspecific crossability and inheritance in Pelargonium. Plant Breeding 113: 3-17. Karnowski, W. and Labanowski, G. 1998. Cacyreus marshalli. A potential pest of Pelargonium in Poland. Ochrona Roslin. 42: 10-13 Laughner, L.J. 1993. History, p. 363-371. In: White, J. (ed.) Geraniums IV. Ball Publishing, Geneva, USA. Renou, J.P., Aubry, C., Serveau, M. and Jalouzot, P. 1997. Evaluation of genetic variability in the genus Pelargonium using RAPD markers. J. Hort. Sci. 72: 229-237. Trematerra, P., Zilli, A., Valentini, V. and Mazzei, P. 1997. Informatore Fitopatologico 47: 7-8. Van der Walt, J.J.A. and Vorster, P. 1988. of Southern Africa 3. National Botanic Gardens, Kirstenbosch, Republic of South Africa. Van der Walt, J.J.A. 1992. Discovering the world of Pelargoniums. Third International Geranium Conference, Odense Denmark, 15-27. De Vos, R. 2000. Migration lepidoptera in 1999 (sixtieth report). Entomologische Berichten Amsterdam. 60: 217-230

Tables

Table 1. Evaluation of ploidy by flow cytometric analysis of nuclei isolated from leaf tissue of different Pelargonium × hortorum taxa".

Taxa name Cutting source Ploidy 'Americana Bright Red' Commercial 2n=4x=36 'Belmonte' Commercial 2n=4x=36 'Bright Red Satisfaction' Commercial 2n=4x=36 'Empress' Commercial 2n=4x=36 'Maverick Star' Commercial 2n=2x=18 'Maverick White' Commercial 2n=2x=18 'Red Satisfaction' Commercial 2n=4x=36 AVZ1 Castilla-León 2n=2x=18 BAZ12 Extremadura 2n=2x=18 M18112 Madrid 2n=2x=18 NAZ3 Navarra 2n=4x=36 PAZ2 Castilla-León 2n=2x=18 PAZ4 Castilla-León 2n=2x=18 SEZ19 Andalucía 2n=4x=36 SEZ29 Andalucía 2n=4x=36 TOAZ1 Castilla-La Mancha 2n=2x=18 XZ13 Euskadi 2n=2x=18 ZAZ25 Castilla-León 2n=2x=18 ZAZPE1 Castilla-León 2n=2x=18 ZAZPE5 Castilla-León 2n=2x=18 ZAZPE8 Castilla-León 2n=2x=18 ZAZ2 Castilla-León 2n=4x=36 ZAZ4 Castilla-León 2n=2x=18 ZAZ11 Castilla-León 2n=4x=36

170

Table 2. Evaluation of Pelargonium x hortorum for geranium bronze tolerance. Relative growth (weight gain divided by initial weight) was determined after two-days of laboratory exposure to flower buds from the different taxa. The field infection was determined three times during the trial (in June, July and September).

Larva weight Weight Relative Field trial Taxa name N (mg) ± SE gain Growth infectiony Initialz Finalz 'Americana Bright Red' 5 18 ± 3 42 ± 7 24 ± 5 1.40 ± 0.2 +++ 'Belmonte' 5 16 ± 3 44 ± 5 26 ± 3 2.00 ± 0.5 +++ 'Bright Red Satisfaction' 5 17 ± 3 39 ± 7 22 ± 6 1.40 ± 0.4 +++ 'Empress' 15 12.4± 1.1 30 ± 4 18 ± 4 1.50 ± 0.3 +++ 'Maverick Star' 10 12 ± 2 29 ± 6 17 ± 4 1.30 ± 0.3 +++ 'Maverick White' 4 14 ± 3 36 ± 7 22 ± 3 1.80 ± 0.4 +++ 'Red Satisfaction' 9 15 ± 2 39 ± 6 24 ± 5 1.60 ± 0.3 +++ AVZ1 9 15 ± 3 29 ± 6 13 ± 3 0.98 ± 0.18 +++ BAZ12 5 17 ± 5 39 ± 11 22 ± 7 1.30 ± 0.3 ++ M18112 4 12 ± 2 25 ± 8 12 ± 7 0.80 ± 0.4 + NAZ3 9 15 ± 4 26 ± 7 11 ± 4 0.69 ± 0.18 + PAZ2 9 15 ± 2 32 ± 5 18 ± 4 1.30 ± 0.3 ++ PAZ4 5 19 ± 4 36 ± 7 18 ± 5 1.00 ± 0.3 ++ SEZ19 4 15 ± 3 31 ± 8 16 ± 6 1.10 ± 0.3 + SEZ29 4 17 ± 8 23 ± 9 6 ± 3 0.50 ± 0.4 + SSZ15 9 12.6± 1.4 32 ± 5 19 ± 4 1.50 ± 0.3 ++ SSZ6 4 16 ± 3 41 ± 9 25 ± 7 1.60 ± 0.4 ++ TOAZ1 11 15 ± 3 20 ± 4 5.1± 1.7 0.30 ± 0.08 - XZ13 5 17 ± 6 38 ± 10 21 ± 9 1.60 ± 0.6 ++ ZAZ25 5 13 ± 3 29 ± 5 16 ± 3 1.25 ± 0.12 ++ ZAZ49 9 13 ± 2 27 ± 5 14 ± 3 1.00 ± 0.2 + ZAZPE1 8 15 ± 2 17 ± 2 2.1± 1.6 0.17 ± 0.15 - ZAZPE5 5 18 ± 7 36 ± 9 18 ± 5 1.20 ± 0.4 + ZAZPE8 5 17 ± 4 44 ± 10 27 ± 8 1.70 ± 0.7 ++ ZAZ2 5 15 ± 4 45 ± 12 30 ± 8 2.30 ± 0.7 +++ ZAZ4 8 15 ± 2 28 ± 6 13 ± 4 0.80 ± 0.2 ++ ZAZ11 4 14 ± 3 21 ± 5 7 ± 2 0.48 ± 0.13 - y Natural infestation was determined in 10 plants from each taxa, 3 times during the season. Each + sign indicates that at least one plant from a given taxona showed conspicuous signs of attack by the geranium bronze (holes in the buds or larvae feeding on the plant).

171 Figures

2C A Number of events events of Number 4C

Relative nuclear DNA content

B 4C Number of events events of Number

8C

Relative nuclear DNA content

C 4C

2C

Number of events events of Number 8C

Relative nuclear DNA content

Fig. 1. Histograms of relative fluorescence intensity obtained after flow cytometric analysis of DAPI stained nuclei isolated from leaf tissue of the diploid P. x hortorum 'Maverick white' taxa (A), the tetraploid P. × hortorum ‘ZAZ2’ taxa (B) and by mixing nuclei from the diploid P. × hortorum 'Maverick white' taxa and ‘ZAZ2’ taxa (C). 172