UNIVERSITY LIBRARIES Illllll lllll‘llll llfllllll'lll Iilll 31293 00900 975 This is to certify that the dissertation entitled . Interspecific Hybridization and Bacterial Blight (Xanthomonas campestris EX- pelargonii) Resistance In Pelargoniums. presented by I Shifeng Pan has been accepted towards fulfillment of the requirements for __Dh. D. degreein Plant Breeding & Genetics In Horticulture .___ figmfj Major professor Dme December 18, 1991 ____*__________ [MS U is an Affirmative Action/ Equal Opportunity Institution 0-12771 ".n 1 . L ——— UBMHY Michigan State University J — PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. E *- DATE DUE DATE DUE DATE DUE MSU Is An Affirmative ActIorVEqueI Opportunlty Institution omens-9.1 INTERSPECIFIC HYBRIDIZATION AND BACTERIAL BLIGHT (XANTHOMONAS CAMPESTRIS PV. PELARGONII) RESISTANCE STUDIES IN PELARGONIUMS BY Shifeng Pan A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Breeding and Genetics Department of Horticulture 1991 ABSTRACT INTERSPBCIIIC HYBRIDIZATION AND BACTERIAL SLIGHT (KANTHOMONAS CAHRESTRLS PV. RELARGONII) RESISTANCE STUDIES IN PELARGONIUHS BY Shifeng Pan Chromosome numbers were determined for thirty- eight Pelargonium taxa. Nineteen of them have not been previously reported, and seven show different chromosome numbers from the ones reported previously. The chromosome number of 2n=8 was verified for Pelargonium (P.) species P. elongatum. For the crossability studies, two hundred and ninety-three interspecific cross combinations were performed among different species, hybrid cultivars and inbred lines. Twenty-eight of the crosses produced viable seeds, and 19 of them showed partial seed development. One interspecific hybrid plant was produced from an ‘Inbred Ibite' (P..x hortorul) plant crossed with.P. grandiflorum, one of the squested ancestral species of the Pa x danesticul. fhpeltatum was found to be a bridge Species crossing with both P. cordifblium, another proposed Pu X'domesticun ancestor, and P; x hortorum cultivars. Twenty one Pelargoniun species, cultivars and interspecific hybrid plants were screened for bacterial blight and leaf spot (Kanthomonas campestris pv. pelargonii) resistance. There were significant differences for bacterial blight resistance among different genotypes studied. .P. odoratissi-un, P. cordifbliun,.P. cucullatun, P. grandiflorun, P. peltatun x P. cucullatun, P. grandiflorum x ‘Tiny'Tbt’, ‘Tiny Tbt' x ‘Barliana', P. betulinun x Pu cordifbliun,.P. grandiflorun x P. cucullatum, In grandiflorum x P. betulinun, P. cucullatun xiP. cordifoliun and P. scabrun x P. seritrilotun all showed high, resistance to the disease. A hybrid resulting from the cross of ‘Inbred White' (P. X'hortorum) and P. grandiflorum, a proposed ancestral species of P..x domesticun, showed a high level of tolerance. However, its seed parent ‘Ihbred White' was highly susceptible. This result indicates that the resistant gene(s) was transferred to the P. x hartarum from P. grandiflorum. Characteristics of morphology and cytogenetics of the successfully produced interspecific hybrids were studied. Plant height for the cross ‘Inbred’White' anle. grandiflarun, and the flower size in diameter for the cross It peltatun and P. cordifolium exhibited hybrid vigor. All other studied characteristics were intermediate between the 2 parents. Variable chromosome numbers and abnormal meiotic divisions were also observed in the cytogenetical studies of the interspecific hybrids. ACKNOILSDGBHBNTS I would like to express my gratitude to my major professor Dr. Lowell C. Ewart for the guidance, encouragement, and support during my graduate studies and research. ‘ I would like to thank the other members of my committee, Drs. Jim F. Hancock, Bernard H. Zandstra, David Roberts and George Hosfield for their council and very valuable suggestions during this research. I want to thank many peoples who helped me in any way during any part of this research. Finally I sincerely thank my wife Shuangling Guo and my daughter Dee Pan for their patiently understanding and love. iv TABLE or CONTENTS Page LIST OF TABLES . C O O O C . C O O O O O O C O O O O O O O O O O O O O C O O O C O O O O O I C O O . Vii LIST OF FIGURES O O I O O O O O O O O O O O O O O O O O I O O O O I O O O O O O O O O O O O 0 O O O 0 ix mm 1 O INTRODUflION O O O O O O O C O O O O O O O O O O O O O O I O O O O O O O O O O O O O 1 I. origin.CO...0.0...O...00.00.:0000OOOOOOOOOOIOOOOOOODI II. CYtOIOgYOO0.0.0.0...O.I.OOOOOOOOOOOOOOOOOOOOOOOOO0.04 III. Interspecific Hybridization.........................7 00.00.000.00000000009 IV. Bacterial Blight Resistance..... Ve Literature Cited...00....OOOOIOOCOCOOOOOOOOOO0.0.0.14 CHAPTER 2. CHROMOSOME NUMBERS AND CROSSABILITY STUDIES IN PELARGONIUMS .................... ....... 20- OOOOOOOOOOOOOOOOOOZO I. Abstract......................... II. Introduction.......................................21 III. Materials and Methods..............................22 IV. Results and Discussion.............................25 A: Chromosome number determination ................25 B: Crossability ...................................32 v. smarYeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee41 VI. ReferenceSeeeeoeeeeeeeoeeeeeeeeeeeeeeeeeoeeeeeeeeee43 CHAPTER 3. BACTERIAL BLIGHT (Kanthomonas campestris pv. pelargonii) RESISTANCE IN PELARGONIUMS. . . . ....... . . 46 I. Abstract....0..0.0....0.0...OOOOOOOOOOOOOOOOOOOOOO.46 V II. IntrOductionOOOO00.0.0000...0.00.00...0.0.0.000000047 III. Materials and Methods..............................48 IV. Results and Discussion.............................50 V. Summary............................................63 VI. References.........................................64 CHAPTER 4. MORPHOLOGY, CYTOLOGY AND IN VOTRO CULTURE STUDIES FOR THE INTERSPECIFIC HYBRIDS IN RELARGONIUMS....................................66 I. Abstract....................;......................66 II. Introduction.......................................67 III. Materials and Methods..............................67 IV. Results and Discussion.............................70 A. Morphology of the interspecific hybrids and their parents..............................70 B. Cytogenetical studies of the interspecific hybrids ...... ....................... ........ ...71 C. In vitro culture studies for ‘Inbred White', P. grandiflorum and their hybrid...............90 VI. References.........................................94 SUMMARY........................................ ..... ......95 SUGGESTIONS................................... ...... ......98 APPENDIX.eeeeeeeeeeoeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeloo. vi LIST OF TABLES CHAPTER 2 1. 2. 3. 4. S. 6. Page Chromosome number determination for the collected 38 species, cultivars and inbred lines...............26 Interspecific crosses with normal seed set in PelargoniumSOOOOOOOOOCOOOOOOOOOOIOOOOOOOOOOOOO0.0.28 Selfing results for some of the interspecific hybrids in Pelargoniums .............................30 Backcross results for some of the interspecific hYbrids in Pelargoniums I.I.00......00.00.000.000000031 Interspecific crosses with only fruit elongation in Pelargoniums...........................37 Result of crosses by chromosome number combinations.OOOOOOOOOOOOOOOOOO00.0.000000000038 CHAPTER 3 1. Xanthomonas campestris pv. pelargonii resistance ratings for Pelargonium species, cultivars and interspeCific hYbridSOCOOOOOOOOOOOOOOOOOOOOOOOOO ..... 52 2. xanthomonas campestris pv. pelargonii resistance ratings for ‘Inbred White', P. grandiflorum and their hybrid plant, and for P. peltatum, P. cordifolium and their hybrid plant............ ..... ..53 CHAPTER 4 1. 2. Morphological, cytological and other characters of ‘Inbred White', P. grandiflorum and their hYbridOOIOO0.0......00.000.000.00...OOOOOOOOOOO0.00.072 Morphological and cytological characters of P. peltatum, P. cordifolium and their hYbrid.0..O.I..00.0.0000000COOOOOOOOOOOOOOO00.000.00.73 Shoots and shoot primordia regenerated from ‘Inbred White', P. grandiflorum and their hybrid on 2 different culture mediums............................92 vii 4. Total number of shoots and shoot primordia regenerated from ‘Inbred White', P. grandiflorum and their hybrid.......... ........... 93 APPENDIX A1. Xanthomonas campestris pv. pelargonii disease resistance ratings for Pelargonium species, cultivars and some interspecific hybrids (2nd screening in 1991).......... .......... 100 A2. xanthomonas campestris pv. pelargonii disease resistance ratings for ‘Inbred White P. grandiflorum and their hybrid plants, and for P. peltatum, P. cordifOIium and their hybrid (2nd screening in 1991)................ ..... 102 A3. Pelargonium interspecific crosses performed with no indication of seed development... .......... 103 viii LIST OF FIGURES CHAPTER 1 Page 1. Geraniaceae family tree..............................3 CHAPTER 2 1. Somatic chromosome number of 2n=8 for P. elongatumOO0.0.00.0.0...COOOOOOOOOOOOOOOOOOOOO0.0027 Plant types for the cross of ‘Tiny Tot' x P. cucullatum and their hybrid plant...............34 Plant types for the cross of P. cucullatum x ‘Tiny Tot' and their hybrid p1ant.................35 CHAPTER 3 1. Xanthomonas campestris pv. pelargonii disease resistance screening for ‘Inbred White' showing enlarging spots and yellowish leaves post two weeks of inoculation........................54 Xanthomonas campestris pv. pelargonii disease resistance screening for ‘Inbred White' showing 45% of leaf tissues was blighted at 21 days after inoculation...............................55 Xanthomonas campestris pv. pelargonii disease resistance screening for P. cordifolium showing spots with typical dark brown halo during the first 3 weeks after inoculation......................56 xanthomonas campestris pv. pelargonii disease resistance screening for ‘Inbred White', P. grandiflorum and their hybrid plant (71 days post inoculation).....................57 Xanthomonas campestris pv. pelargonii disease resistance screening for P. peltatum, P. cordifolium and their hybrid plant (71 days post inoculation)...........................58 ix 6. xanthomonas campestris pv. pelargonii disease resistance screening for ‘Inbred White', P. grandiflorum and their hybrid plant (data from 21, 45 and 71 days post inoculation).........................................59 7. xanthomonas campestris pv. pelargonii disease resistance screening for P. peltatum, P. cordifblium and their hybrid plant (data from 21, 45 and 71 days post inoculation).........................................60 CHAPTER 4 1. Meiotic chromosome number of 2n=18 for ‘Inbred White’ooooooooooooooooooooooooooooo00000000074 2. Meiotic chromosome number of 2n=22 for P. grandiflomm ......OOOOOOO......OOOOOOOOO00......75 3. Mitotic chromosome number of 2n=18 for hybrid plant of ‘Inbred White' x P. grandiflorum ..........76 4. Mitotic chromosome number of 2n=19 for hybrid plant of ‘Inbred White' x P. grandiflorum ..........77 5. Mitotic chromosome number of 2n=20 for hybrid plant of ‘Inbred White' x P. grandiflorum ..........78 6. Meiotic chromosome doubling for hybrid plant Of ‘Inbred White' x P. grandiflorum ................79 7. Meiotic chromosome doubling for hybrid plant of ‘Inbred White' x P. grandiflorum ...................80 8. Meiotic chromosome laying out of equatorial at early metaphase I for hybrid plant of 81 ‘Inbred White’ x P. grandiflorum .................... 9. Meiotic chromosomes showing special end attachments and one chromosome segment at drakenesis of the 82 hybrid plant of ‘Inbred White' x P. grandiflorum ... 10. Meiotic chromosome unequal distribution to the tetrad for hybrid plant of ‘Inbred White' x ........83 P. grandiflomm eeeeeeeeeeeeeeeeeeeeeeeeeeee 11. Meiotic chromosome number of 2n=18 for .......85 P. ”Itatum eeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeee 12. Meiotic chromosome number of 2n=22 f°r ........ 86 P. COIdifOlium eeoeeoeooooéeeeoeeooe eeeeeee e 13. Mitotic chromosome number of 2n=20 for plant of P. peltatum x P. cordifolium ..............87 14. Mitotic chromosome number of 2n=21 for plant of P. peltatum x P. cardifblium ..............88 15. Mitotic chromosome number of 2n=22 for plant of P. peltatum x P. cordifolium ..............89 APPENDIX AFl. AFZ. AFB. AF4. AFS. AFG. AF7. Mitotic chromosome number of 2n=16 for P. alcnem11101des.........OOIOOOOOOCOOOOO......00.111 Mitotic chromosome number of 2n=18 for P. acetosum.’O....0.........IIOOOOOOOOOOOOOO ...... 112 Mitotic chromosome number of 2n=22 for P. tetragarum.......OOOOOOOOOOOOOOOOO0.0.0.0000000113 Plant leaf types for ‘Inbred White', P. garndiflorum and their hybrid ............... ..... 114 Flower types for ‘Inbred White', P. garndiflorum and their hybrid ........... ....... ..115 Plant leaf types for P. peltatum, P. cordifolium and their hybrid .....................116 Flower types for P. peltatum, P. cordifolium and their hybrid ............... ..... .117 xi CHAPTER 1 INTRODUCTION I. origin: The genus Pelargonium belongs to the family Geraniaceae, which is subdivided into 5 tribes: Geranieae, Biebersteiniceae, Wendtieae, Vivianeae and Dirachmeae. The Pelargonium containing tribe Geranieae, is subdivided into 5 genera: Geranium, Pelargonium, Erodium, Monsonia and Sarcocaulon. There are 16 sub-genera in the genus Pelargonium (Figure 1) (Clifford, 1958; Van Der Walt, 1977; Van Der Walt and Vorster, 1981; Taylor, 1988). Moore (1971) divided cultivated Pelargonium into 11 groups. Pelargonium, containing about 280 species and a large number of hybrids and cultivars, is native to South Africa (Dewolf, 1983; Harney, 1966). It is believed that Pelargonium, probably Pelargonium zonale was introduced into Europe by the Dutch governor of the Cape Colony in 1609 (Clifford, 1958). Between 1800 and 1830, two groups of hybrids began to be distinguishable: 1) Pelargonium x hortorum (P. X hortorum), the zonale geranium, and 2) Pelargonium x domesticum (P. x domesticum), the Martha Washington or Regal geranium. A third group, the ivy-leaved 2 Pelargonium was not introduced into Europe until after 1850 (Dewolf, 1983). P. X hortorum is considered to have evolved from the species P. inquinans, P. zonale, P. hybridium, P. trutetorum, and P. scandens with some possible contribution from P. acetosum and P. stenopetalum (Clifford, 1958). Harney (1976) from his chromatographic studies of the secondary biochemical constituents using crude alcoholic leaf extracts postulated that P. zonale, P. inquinans, P. scandens and P. frutetorum were the major contributors. Considering both morphological characters and biochemical markers, Harney (1976) suggested that P. hybridum might have contributed to P X hortorum, but P. stenopetalum was doubtful to have made any contribution. P. X domesticum is considered by many to be the most beautiful Pelargonium and has evolved from numerous interspecific crosses within the genus. Martha Washington Pelargonium, Lady Washington Pelargonium, Regal Pelargonium, and Show Pelargonium are all common names used for this group, which has been in existence for many years. In the 18805 serious breeding began in Germany, France, and England (Taylor, 1988). Because the original crosses were not recorded or details lost (Clark, 1988), the ancestry of these plants is really not known. The most important contributing species are thought to be P. cucullatum, P. em>uoa when»: I>>H ounces moods: < I 0 Andoouwv un>fiuaco ecu no nuoumoocn “Ono: cancelhem lsueuedoeu Inquoehaomoo nouoom eueueauem .lsaahndooaao lsasouuouempoo cannonau ewowuo ehnoeunfio Inaudnuuhz enanauuoo euueasu«A> nuancedo oucwncwxoeb eunkmweo ouocmmlnsm ea oucw oeofi>fio mowoodn omN nuacomueuem oeueooouo«ooum encoum Influenced oo>oma Homom monsoon o > it eeueHAeAlo condensoou I encasam 4 coasoooouem eeue>o p leuwuqunsz ownonoz I saunasowusoc noauoununwceo asuecouo canonuan _ Hooao chocomuosm e unannouncn a no: ma oucw oeofi>wo oucw oooa>fio ouocewnocm nsacnueo nuecomiocn n ouca coofi>wo neaoodm ooe lsficeuoo mofiommm ma nmwomdn on mofioodn om coasoooounm owconcox lsqvouu _ _ Ammma .h .uoflmoa soumv eons hawaom oomomwcmuoo .H oucmflh 4 grandiflorum, P. angulosum, P. fulgidUm, P. betulinum, P. capitatum, P. cordifolium and P. ignescens (Clifford, 1958; Taylor, 1988; Hanniford and Holcomb, 1982). The initial flowering season is rather short (flower only once each spring) for most of the older Regal Pelargoniums, but continual improvements have extended the length of flowering period as well as outdoor Summer performance for some new cultivars (Taylor, 1988). Special environmental factors are required for P. X domesticum for flower initiation and flower development. Temperature is the most important environmental factor affecting flowering (Crossley, 1968; Nilsen, 1975; Hackett and Kister, 1974). A four week temperature treatment at :UWC is necessary to insure bud initiation in all cultivars (Hanniford and Holcomb, 1982). This group also responds to high irradiance of long days for flower development (Van De Veen and Meijer, 1959). Nilsen (1975) and Hackett and Kister (1974) also indicated that daylength is not important in flower initiation, but it can hasten flower development. II. Cytology Basic chromosome numbers of X=8, 9, 10, and 11 have been reported for the natural Pelargonium species (Dakar, 1969; Darlington, 1955; Clifford, 1958). Species that P. X hortorum evolved from have a basic number of X=9, and are diploids (Clifford, 1958). Those species all belong to the section of Ciconium. Very recently, a new basic chromosome number of X=4 for Pelargonium species P. elongatum has been reported by Gibby and Westfold (1983; 1986). Reported diploid chromosome numbers for the genus Pelargonium are 2n=16, 17, 18, 20, 22, 35, 36, 40, 44, 45, 60, 81, 88 and 90. since the chromosome number of 2n=8 was observed for P. elongatum (Gibby and Westfold) and verified in the present research, a new number of 2n=8 can be added. This lowest chromosome count raises the question that possibly all the currently cultivated commercial Pelargonium cultivars are really polyploids. The first polyploid Pelargonium cultivars were reported by Badr and Horn (1886) as described by Harney (1975). Breeding probably started first at the diploid level with horticulturists later unknowingly selecting tetraploid cultivars (Chow and Barney, 1971). Investigations by Clifford (1958) and Philippi (1961) indicated that tetraploid cultivars cannot be crossed with diploids. Badr and Horn (1971), however, determined that parthenogenetically formed seeds developed after crosses between parents with different ploidy levels. Daker (1967; 1969) did a detailed cytological study of haploid cultivar ‘Kleine liebling' (n=9). He found the shoot tip was haploid but the roots were either haploid, mixed haploid-diploid or diploid, and each of the 3 types of roots could be found on a single cutting. The analysis of 6 microsporogenesis of the haploid plant showed the formation of a small number of bivalents. Trivalents and bivalents were found in the colchicine induced diploid. The bivalents. found in the diploid closely resembled the ones found in the haploid. Daker (1967) suggests that these chromosomal associations in meiotic cells of the haploid and diploid plants indicate that there is interchromosomal homology within the genome. The chromosomal~pairing at meiosis in the colchicine - induced diploid could be expected normal, but Daker (1967) found that it was very similar to that in the haploid, with a large prOportion of univalents and pseudobivalents or non — chiasmata bivalents. Daker (1967) also found that in the haploid plants only a small prOportion of the pollen mother cells progressed through a second division resulting in normal quartet formation, and he suggests that this abnormal meiosis is due to the accumulation of deleterious recessive genes which adversely affect meiosis. Therefore, he concluded that nonsynapsis of the chromosomes in the cultivar, whether haploid or diploid, is due to genes rather than lack of chromosome homology, and such genes could accumulate through lack of selective pressures over a long period of vegetative propagation. Warburg (1938) prOposed that the genus Pelargonium is more advanced morphologically than the genus Geranium, and that hybridization has played an important role in the formation of polyploids. Pelargonium shows the greatest 7 level of interspecific hybridization, and many cultivars within the genus Pelargonium are polyploids. Both autotetraploids and allotetraploids have been found within the genus Pelargonium (Warburg, 1938). Philippi (1961) reported irregular segregation of chromosomes in tetraploid cultivars during the first meiotic division. The irregular segregation resulted in pollen inviability and decreased fertility. In addition, he also observed 1, 2 or 3 chromosomes which were not arranged on the metaphase plate. Those chromosomes divided and were later found isolated in the plasma of the tetrad. Chromosomal configurations typical of autotetraploids were noted at meiosis in tetraploid cultivars studied. Philippi (1961) also found a negative correlation between the number of univalents which occurred per PMC and the fertility of the tetraploid cultivars. This association was not found in diploid cultivars. III. Interspecific Hybridization Both P..X hortorum and P. X domesticum groups are the results of interspecific crosses of species within the subgenera Ciconium and Pelargonium respectively. P. X domesticum and P. X hortorum appear to be genetically incompatible. No successful interspecific hybridization has been reported between P. X hortarum and P..X demesticum. Through the medium of the section Dibrachya a transfer of 8 genetic material might be possible (Clifford, 1958). Sweet, as cited by Clifford (1958), noted that P. X succulentum could have resulted from the cross of P. X domesticum and P. peltatum (ivy-leaved Pelargonium). P. peltatum will cross with P..X hortorum (Clifford, 1958; Craig, 1971). Therefore, it might be possible that P. peltatum can be used as a bridge to cross with both P. X hortorum and P. X domesticum. Sweet also suggested that P. X rigescens might have resulted from a cross between P. cordifolium and a zoned species of Ciconum. The earliest interspecific hybrids were mentioned by Clifford (1958) but there was no data available on the cultivars that were used in the crosses (Craig, 1982). Knicely (1964) reported the results of controlled interspecific hybridization between and within P. x domesticum and P. x hortorum groups. He conducted 215 crosses and only 50 crosses within the 2 groups produced viable seeds. Thirty five crosses (between groups and within group) showed partial seed development, and the remaining 130 between group and within group crosses showed no indication of seed set. As described by Craig (1971), the crosses in Knicely's study, which only showed partial seed development, might indicate that embryos were formed but did not develop due to breakdown of the endosperm. Protocols for rescue of zygotic embryos in Pelargonium have been developed (Adams, 1967; Kate and Tokumasu, 1983; Scemama & Raguin, 1990). Adams (1967) reported an embryo 9 rescue method for diploid P. X hortorum. Kato and Tokumasu (1983) conducted interspecific hybridization between P. X domesticum and some other scented-leaved Pelargonium species using ovule culture technique, with the intention of introducing some desirable characters into P. X domesticum from the scented-leaved Pelargonium. They obtained potential ever-flowering and multiflorous type hybrid plants from crosses of P. quercifolium and ‘Grand Slam', ‘Priace Ruper’ and ‘Strawberry Sundae’ respectively. Further improvement by using anther culture and backcrossing procedures with these hybrid plants were attempted, but no result was reported. They also found that tetraploid X tetraploid (2n=44) crosses generated a higher callus formation rate (80%) than did diploid (2n=22) X tetraploid crosses (3.1-6.5%). IV. Bacterial Blight Resistance The most serious disease of Pelargonium is a vascular wilt caused by the bacterium, Xanthomonas campestris pv pelargonii (Brown) Dye (X. c. pelargonii). The disease at first was called bacterial stem rot and leaf rot (Munnecke, 1954). Later Rnauss and Tammen (1967) described it as bacterial blight, which is now the most often used name. X. c. pelargonii is a gram-negative, aerobic and rod- shaped bacterium. It is catalase positive, hydrogen sulfide positive, oxidase negative, and has one polar flagellum. It' k 10 produces a yellow extracellular polysaccharide slime called ‘Xanthan’ on nutrient agar (Smith et al., 1952). The bacterium can reduce nitrate, liquify gelatin, but does not produce indole. X. c. pelargonii is found only in association with plants or plant material, and it can be easily identified when isolated from infected plant tissue (Dye, 1980). Pelargonium leaves infected with X. c. pelargoniii show 2 types of symptoms (Stephens et a1.). One of the leaf symptoms is a wilting at the leaf margins. The infected areas of the leaf rapidly die and form "V" shaped, yellow lesions bounded by dark veins. The leaves then wilt, although the leaf petioles remain firm. The affected leaves may drop off immediately or may hang on the plant for a week or more (Nelson & Nichols, 1982). Another leaf symptom is the development of lesions which first appear as small water-soaked spots on the underside of leaves. These lesions enlarge, become well defined and slightly sunkin with a yellow halo, followed by a wilting and death of the leaf. Plants showing leaf lesions may develop the stem rot phase of bacterial blight. The stems turn gray to dull black and finally develop a dry rot (Horst & Nelson). There are two main factors affecting symptom development. First, symptoms of the disease develop more rapidly under higher temperature conditions. Symptom expression has been found to increase as temperature was 11 increased from 10 to 27°C during the first 5 weeks after inoculation (Kivilaan & Scheffer, 1958). This research also indicated that plants grown in a high-nitrogen program developed symptoms more rapidly than plants grown under a low-nitrogen level. The bacteria enter the host plant generally through wounds or stomates. Histochemical observations of plants of both susceptible and resistant Pelargonium species inoculated with the.bacteria through wounds in the stem showed that in the susceptible plants the bacteria initially spread through the plants through the xylem (Wainwright and I Nelson, 1972). Then it entered the vascular cambium and rapidly moved to the phloem cortex and epidermis. In the resistant plants, the pathogen had limited proliferation in the xylem and did not spread out of the xylem. Wainwright and Nelson (1972) also observed differences in the levels of tannin-like materials between susceptible and resistant Pelargonium species, and suggested that the tannin-like materials may be responsible, in part, to differences in resistance to bacterial blight. In addition, they found tylosis formation and deposition of suberin-like material in plants which were infected with the bacterium which may play. a role in restricting the spread of the bacterium. Heavy losses of Pelargonium X'hortorum to this disease has been reported yearly (Nelson & Nichols, 1982; Stephens & Tuinier, 1989). Due to the lack of bacterial blight .12 resistance cultivars, the most effective control is to prevent the introduction of the bacteria into production areas. The strict sanitation procedures which have been found to reduce the introduction and spread of the disease are as follows: only use disease free (culture-indexed) plants; do not grow cutting Pelargoniums near P. peltatum, P..X.hortorum or P. X domesticum; discard infected plants; sterilize planting containers, cutting knives and greenhouse tools; avoid overhead watering or misting as the bacterium spreads through splashing water droplets; and control white flies (Stephens et. a1.). There is no chemical available for controlling this disease. The culture-indexing procedure is very expensive and the plants can be lost later to bacterial infection. To really help control the bacterial blight disease in P. X hortorum, resistant cultivars are definitely needed. Useful resistance genotypes to bacteria blight have not been found in cutting-propagated P. X hortorum (Knauss & Tammen, 1967; Theiler, 1977), seed propagated P. X hortorum (Tuinier, 1985; Mojdehi & Singleton, 1990), or ivy-leaved Pelargonium (Tuinier, 1985). However, bacterial blight resistance has been observed in species from the P. X domesticum group such as P. acerifolium, P. tomentosum, P. scarboroviae, P. scabrum, P. betulinum, P. grandiflorum, P. mullicaule, and p. hispidum (Mojdeli & singleton, 1990; Knauss & Tammen, 1967), P. cordifolium, P. grandiflorum, P. betulinum, and 13 ‘Idny Tot' (Dunbar and Stephens 1989). If the transfer of this resistance from the P. x dbmesticum group into P. X'hortorum through interspecific or bridge hybridization could be accomplished, it would be a mile stone in the development of bacterial blight resistant cultivars of P.,x hortorum. This research was conducted to see if such a transfer was possible and to provide further useful information for Pelargonium bacterial blight resistance breeding. LITERATURE CITED Abo, El-nil M. M. 1980. Geranium (Pelargonium) in Handbook of plant cell culture v.5. ornamental Species. Ammirato, P. V., D. R. Evams, W. R. Sharp, and Y. P. S. Bajaj eds. McGram Hill, New York. Abo, El-nil M. M., and A. C. Hildebrandt. 1971. Differentiation of virus symptomless geranium plants from anther callus. Plant Disease Reporter 55:1017- Abo, El-nil M. M. and A. C. Hildebrandt. 1973. Origin of androgenetic callus and haploid geranium plants. Can J. Bot.51:2107-2109. Abo, El-nil M. M., A. C. Hildebrandt., and R. F.Evert. 1976. Effect of auxin-cytokinin interaction on organgenesis in haploid callus of Pelargonium hortorum. In Vitro 12:602-604. Adams, F. S. 1967. Histochemical and morphological analysis of in vitro cultured embryos of Pelargonium X hortorum Bailey, in comparison to normal in vivo embryology and seedlingsifferentiation. ph. D. Dissertation. The university of New Hemshire. Badr, M. and W. Horn 1971. Ein Beitrag zur zuchtung von Pelargonium zonale-hybriden. z. Pflanzenzuchtg. 66:278-292. Badr, M. and W. Horn 1971. Cytological Studies on Pelargonium zonale hybrids. Zeit. Pflaazenzhuchtung. VOl 66:158-194. Bennici, A. 1974. Cytological analysis of roots and plants regenerated from suspension and solid In Vitro cultures of hyploid Pelargonium. z. Pflanzenzuchtg 72:199-205. Bennici, A., M. Buiatti, and F. D’Amato. 1986. Nuclear conditions in haploid Pelargonium in vivo and in vitro. Chromosoma 24:194-201. Brown, J. T. and B. V. Charlwood. 1986. The control of callus formation and differentiation in scented Pelargoniums. J. Plant Physiol. 123:409-417. 14 15 Cassells,.A. C. and B. F. Carney. 1987. Adventitious shoot regeneration in Pelargonium X domesticum Bailey. Acta Horticulturae 212:419-423. Cassells, A. C. and G. Minas. 1983. Plant and in vitro factors influencing the micropropagation of Pelargonium cultivars by bud-tip culture. Scienta Hortic 21:53- 65. Cassells, A. C., G. Mines, and R. Long. 1980. Culture of Pelargonium hybrids from meristem and explants: chimeral and beneficially-infected varieties. in: Tissue Culture Methods for Plant Pathologists. D. 8. Ingram and J. P. Helgeson eds. Blackwell Scientific Publications, Boston. Chow, T. W. and P. M. Harney. 1970. Crossability between a diploid Pelargonium x hortorum Bailey cultivar and some of its putative ancestral species. Euphytica 19:338-348. Clifford, D. 1958. Pelargonium including the popular geranium. Blandford Press, London. Crossley, J. H. 1968. Warm Vs cool short days as preconditions for flowering of Pelargonium domesticum cultivars. Can. J. Plant Sci. 48:211-212. Daker, M. G. 1967. Cytological studies of a haploid cultivar of Pelargonium and its colchicine induced diploids. Chromosoma 21:250-271. Daker, M. G. 1969. Chromosome numbers of Pelargonium species and cultivars. J. Roy. Hort. Soc. 94:346- 353. Daker, M. G. 1969. Pelargonium ‘Kleine Liebling’ - a most unusual cultivar. J. Roy. Hort. Soc. 94:353-354. Darlington, C. D. and A. P. wylie. 1955. Chromosome atlas of flowering plants. George Allen and Unwin, London. de Wet, J. M. J. 1971. Polyploidy and evolution in plants. Taxon 20(1):29-35. Debergh, P. and L. Maene. 1977. Rapid clonal propagation of pathogen-free Pelargonium plants starting from shoot tips and apical meristems. Acta Horticulturae 78:449-454. Dewolf, G. 1983. Pelargonium. Horticulture. November P8- 9. 16 Dunbar, K. B. and C. T. Stephens. 1989. Shoot regeneration of hybrid seed geranium (Pelargonium X hortorum) and regal geranium (Pelargonium X domesticum) from primary callus cultures. Plant Cell, Tissue and Organ Culture 19:13-21. Dye, D. 1980. xanthomonas. Page 46 in: Laboratory Guid for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, Minnesota. ‘ Gibby, M and J. Westfold. 1983. A new basic chromosome number in Pelargonium (geraniaceae). Caryologia 36(1):?9-82. Gibby, M. and J. Westfold. 1986 A cytological study of Pelargonium sect. Eumorpha (Geraniaceae) Pl. Syst. Evol.153:205-222. Gonzale, L. G. G. B. Collins and N. L. Taylor. 1982. Facilitation of wide-crossing through embryo rescue and pollen storage in interspecific hybridization of cultivated allium species. Plant Breeding 98:318- 322. Hadley, H. H. and S. J. Openshow 1980. Hybridization of crop plants. American Society of Agronomy-CrOp Science Society of America, 677 S. Segoe Road, Madison. Hakkaart, F. A. and G. Hartel. 1979. Virus eradication from some Pelargonium zonale cultivars by meristem culture. Neth. J. Plant Path. 85:39—46. Hamdorf, G. 1976. Propagation of Pelargonium varieties by stem-tip culture. Acta Horticulturae 59:143-151. Hammerschlag, F. 1981. Effect of plant age on callus growth, plant regeneration, and anther culture of geranium. J. Amer. Soc. Hort. Sci. 106:114-116. Hanckett, W. P. and J. Kistor. 1974. Environmental factors affecting flowering in Pelargonium domesticum cultivars. J.Amer. Hort. Sci. 9(1):15-17. Hanniford, G. C. and E. J. Holcomb. 1982. Regal geraniums. in: J. W. Mastalerz and E. J. Holcomb eds. Penn State Geranium Mannual. 3rd edition. Harlan, J. R. and J. M. J. de Wet. 1975. The origin of polyploidy. Bot. Rev. 41:361-390. 17 Harney, P. M. 1966. A chromatographic studies of species presumed ancestral to P. x hortorum Bailey. Can. J. Genet. 8:780-787. Harney, P. M; 1976. The origin, cytogenetics, and reproductive morphology of the zonale geranium. A review. HortScience Vol 11 (3):189-194. Horst, R and P. Nelson. Disease of geraniums. Cornell Cooperative Extension Publication Bulletin #201. Horst, R. K., S. H. Smith, H. T. Horst, and W. A. Oglevee. 1976. In vitro regeneration of shoot and root growth from meristem tips.of Pelargonium X hortorum, Bailey. Acta Horticulturae 59:131-141. Jelaska, S. and B. Jelecic. 1980. Plantlet regeneration from shoot tip culture of Pelargonium zonale hybrida. Acta Bot. Croat. 39:59-63. Kameya, T. 1975. Culture of protoplasts from chimeral plant tissue of nature. Japan J. Genetics 50:417- 420. Kato, M. and S. Tokumasu. 1983. Characteristics of F1 hybrids produced by ovule-culture in ornamental Pelargonium. Acta Horticulturae 131:247-253. Kivilaan, A. and R. P. Scheffer. 1958. Factors Affecting development of bacterial stem rot of Pelargonium. Phytopathology 48:185-191. Knauss, J. F. and J. Tammen 1967. Resistance of Pelargonium to Xanthomonas pelargonii. Phytopathology 57:1178-1181. Knicely, W. W. 1964. Chromosome numbers and crossability studies in the genus Pelargonium. M. S. Thesis. The Penn State University. Mojdehi, H. and L. L. Singleton. 1990. Histopathology of wheat seedling roots infected with Pythium arrhenimanes. PhytOpathology 80:437. Moore, H. E. Jr. 1971. Pelargonium in cultivation. in: Penn State Geranium Mannual. Second Ed.(Ed. J. W. Mastalerz). Munnecke, D. E. 1954. Bacterial stem rot and leaf spot of Pelargonium. Phytopathology 44:627-632. Narayana Swami, C. and Knut Nordtog. 1964. Plant embryo culture. The Bot. Rev. 30:587-629. 18 Nelson, P. E. and L. P. Nichols. 1982. vascular wilts- bacterial blight. Page 221-224 in: Penn State Geranium. Mannual. 3rd Ed. (Ed by J. W. Mastalerz and E. J. Holcomb). Nilsen, J. H. 1975. Factors affecting flowering in Regal Pelargonium (Pelargonium X domesticum Bailey). Acta Horticulturae 51:299-309. Oglevee-O'Donovan, W. A. 1982. Culture-indexing for vascular wilts. in: Penn State Geranium Mannual III. 3rd Ed. (Ed by J. W. Mastalerz and E. J. Holcomb). Pan, S and L. Ewart. 1991. Cytology~and crossability studies in Pelargoniums. HortSci. 26(6)717 (abstract). Philippi, G. 1961. Untersuchungen uber die die Fertilitatsver-haltnisse einiger kulturformen Von Pelargonium zonale. z. Pflanzenzuchlg 44:380-402. Phillips, G. C., G. B. Collins and N. L. Taylor. 1982. Interspecific hybridization of red clover (T. pratense L.) with T. sarosience Hazsl using in vitro embryo culture. Theor. Appl. Genet. 62:17-24. Reuther, G. 1983. Propagation of disease-free Pelargonium cultivars by tissue culture. Acta Horticulturae 131:311-319. Scemama, C. and C. Raquin. 1990. An improved method for rescuing zygotic embryos from Pelargonium X hortorum Bailey. J. Plant Physiol. 135:763-765. Schulz - Schaeffer, J. 1980. Cytogenetics. Plants, animals, humans. Springer-Verlag, New York, Heidelberg, Berlin. P446. Smith, N. R., Gordon, R. E., Clark, F. E. 1952. Aerobic spore-forming bacteria. U. S. Department of Agriculture Monograph 16, 148 pp. Stebbins, G. L. 1974. Types of polypoids: their classification and significance. Adv. Genet. 1:403- 427. Stephens, C. T., S. Perry, and J. Vincent. Bacterial wilt of geraniums. North Cent reg. Ext. Publ. 171 CES, Michigan state University, East Lansing, Michigan. Stephens, C. T. and J. Tuinier. 1989. Disease symptomatology and variation in susceptibility of 4‘II--—__._ 19 seed propagated hybrid geranium varieties to xanthomonas campestris pv. pelargonii. Plant Disease 73:559-562. Taylor, J. 1988. Geraniums and Pelargoniums. The complete quid to cultivation, propagation, and exhibition. The Crowood Press, Ramsbury, Marlborough, Wiltshire SN8 2HE. 176pp. Theiler, R. 1977. In vitro culture of shoot tips of Pelargonium species. Acta Horticulturae 78:403-409. Tuinier, J. 1985. Aspects in the eoidemiology and control of bacterial wilt of geranium (M. s. Thesis). Michigan State University. Van De Veen, R. and G. Meijer. 1959. Light and plant growth. N. V. Philips Gloeilampenfabrieken, Eindboven, Netherlands. Van De Walt, J. J. A. 1977. Pelargoniums of southern Africa. Cape Town: Purnell. Van De Walt, J. J. A. and P. J. Vorster. 1981. Pelargoniums of southern Africa, Vol.2. Cape Town: Juta. Warburg, E. F. 1938. Taxonomy and its relationship in the Geraniales in light of their cytology. New Phyto 37:130-159. White, P. R. 1963. In vitro plant regeneration from hypocotyl and cotyledons of Chinese Kale (Brassica alboglabra Bailey) 2. pflan-zenphysiol 82:440-445. Yarrow, S. A., Cooking, E. C. and Power, J. B. 1987. Plant regeneration from cultured cell-derived protoplasts of Pelargonium aridum, Pelargonium X hortorum and Pelargonium peltatum. Plant Cell Reports 6:102-104. CHAPTER 2 CHROHOSONE NUMBERS AND CROSSABILITY STUDIES IN.RELARGONIUHS Additional key words: Species, Pelargonium, Interspecific Hybridization, Chromosome number. ABSTRACT Chromosome numbers were determined for 38 Pelargonium species, cultivars and inbred lines. Nineteen of them have not been previously reported, and 7 show different chromosome numbers from the reported ones. The chromosome number of 2n=8 was verified for Pelargonium elongatum. Two hundred and ninety-three crosses were performed among different species, hybrid cultivars and inbred lines. Twenty-eight of them produced plump, normal appearing seeds, and 19 of them showed only partial seed deveIOpment. One hybrid plant was produced from an inbred P..X.hortorum plant crossed with P. grandiflorum, one of the suggested ancestral species of the Regal pelargonium. P.peltatum was found to be a bridge species crossing with both P. cordifblium, another proposed P. .x damesticun ancestor, and Pa X'hartorum cultivars. 20 INTRODUCTION Only a few interspecific hybridization studies have been reported in Pelargonium (Harney and Chow, 1971; Kato and Tokumasu, 1983; Knicely and Walker, 1966). Those researchers were attempting to study genetic relationships between different-species , with the aim of bringing desired horticultural characteristics from one species into another, especially between the P. x hortorum and P. x domesticum. Bacterial blight Xanthomonas campestris pv. pelargonii (X. c. pelargonii) resistance has been reported in P. x domesticum (Craig, 1971; Dunbar and Stephens, 1989; Knauss and Tammen, 1967; Powell and Bunt, 1978; Stephens and Tuinier, 1989). But heavy losses of P. x hortorum commercial cultivars to bacterial blight caused by X. c. pelargonii are reported yearly (Ewart, 1982; Dunbar and Stephens, 1989). Interspecific hybridization could be valuable in transferring bacterial blight resistance from P. x domesticum into P. x hortorum, and also the ever flowering character from the latter into the former. Chromosome numbers reported for P.,X hortorum are 2n=17, 18, 35 and 36, and for P. x domesticum 2n=17, 18, 22, 35, 36, 42, 44 and 45 (Craig, 1971; Ewart, 1982; Knicely and Walker, 1966; Walker and Craig, 1961). The two groups have 21 'A e-h Q P! he {I} 22 no common progenitors (Ewart, 1982), but with the great variability and diverse ancestry within the genus, the possibility exists that species of one group could cross directly with species of the other groups or through a bridge species or plant (Knicely and Walker, 1966). However, no such success has been reported to date (Craig, 1971; Stephen and Tuinier, 1989). The lack of success may be due to chromosome number differences and the use of a narrow species germplasm base. The purpose of this research was to determine the chromosome numbers of Pelargonium species, hybrid cultivars and inbreds used in this study, and to study the crossabilities among different taxa. MATERIALS AND METHODS Pelargonium species: Twenty-eight Pelargonium species (obtained mostly from South Africa) and hybrid cultivars were assembled for this study (Table 1). They were: P. acetosume, P. acraem, P. betulinum, P. capitatum, P. citronellum, P. cordifblium, P. cucullatum, P. denticulatum, P. frutetorum, P. fulgidum, P. fruticosum, P. grandiflorum, P. hermanrifilium, P. hirtum, P. hispidum, P. inquinans, P. multicaule, P. peltatum, P. papilionaceum, P. reniforme, P. scabrum, P. seritrilobum, P. tetragarum, P. zonale, P. violavium, P. x domesticum cv ’Tiny Tot’ and P. x domesticum cv ’Earliana'. It has been possible to match most of those species with the color 23 plates in the book ”Pelargonium in South Africa" (Walt 1977) for proper name usage. Nine of the Pelargonium species were provided by PanAmerican Seed Inc. in West Chicago, IL. P. violavium and ‘Earliana' were obtained from.Merry Gardens in the United States. Two P. x hortorum inbred lines, ‘Picotee' (MSU80-38A), a male sterile picotee colored plant, and ‘Inbred White: (MSU87-8C), a white flower colored fertile plant from the MSU breeding\program were also used. Chromosome number determination: Where possible both root tips and pollen mother cells (PMCs) were used for chromosome number determination for each species, cultivar and inbred line. ' Vigorous roots were taken from the plants at 10:00 am, 1:00 pm and 3:00 pm. Cell division phases were found at all times. Root tips were put in cold water and stored in a 1%: refrigerator for 12 hrs, and then they were transferred to a fixative solution (absolute ethanol-chloroform-acetic acid 6-3-1) for 24 hrs. After washing in distilled water, the roots were put in 5N HCL for 10 minutes at room temperature, and then washed with distilled water. The root tips were smeared in a drop of two percent acetocarmine solution which was used for chromosome staining. The right stage for PMC meiosis was found to be in the flower buds that were about 1-3mm long. The optimal sized flower buds were picked and fixed in Farmer's solution 24 (ethanol-acetic acid 3:1) for 24 hrs at rTL Then.they were transferred to 70% alcohol and stored in a refrigerator at a temperature of 1°C until needed. The same two percent acetocarmine solution used to stain root cells was employed in the PMC chromosome determination. Ten to twenty cells were counted from each species, hybrid cultivar or inbred line. A phase contrast Olympus system microscope (model BHS) set up with an automatic photomicrographic system (model PM-10ADS) was used to study the prepared material and take the desired photos. Cross pollination procedures: Low temperature is required for P. x domesticum and derived species to initiate flower buds (Hachett et al., 1974; Hanniford et al., 1982; Powell and Bunt 1978). This material was grown at a 10°C day and night temperature with 10 hours ( 6:00AM to 4:00PM) of high pressure sodium lighting for about 3 months. The material all flowered about 75 days later. Under these environmental conditions it was possible to keep the plants flowering for several months. A total of 293 cross combinations were tried among 38 species, cultivars and inbred lines. Three to 63 flowers were pollinated for each cross depending upon the number of flower available at time of pollination. Pollinations were done during the summers in a screened greenhouse. Standard pollination procedures were applied. Reciprocal crosses were tried when possible. ¥ RESULTS AND DISCUSSION Chromosome number determination: Chromosome numbers determined for all the species, cultivars and inbreds used in this research are shown in Table 1. When compared with published counts (Chartejee and Sharma, 1970; Daker, 1969; Darlington and Wylie, 1955; Knicely and Walker, 1966; Moore, 1971; Gibby and Westfold, 1983), nineteen have not been reported, seven showed different chromosome numbers from the previous reports, and 4 showed the same basic chromosome number (ratio) but different ploidy levels. These differences in chromosome number may be due to different sources of the same species, chromosome lost or added during breeding, or incorrect identifications. P. elongatum, section Eumorpha (Clifford, 1958, Taylor, 1988), had a 2n=8 chromosome number (Figure 1), which is a new basic number (x=4) for Pelargonium (Gibby and Westfold, 1986). This research verified the previous reports of Gibby and westfold (1986). This species is fertile with small, yellow tinted, white flowers. But it is difficult to use as~ a seed parent because of very early anther dehiscence in the flower bud. The discovery of this low genome number (x=4) indicates that the currently cultivated diploid Pelargonium varieties and cultivars are actually polyploids; 25 “i . u‘vu v. e. e 0'.‘ 26 Table 1. Chromosome number determinations for the collected species, cultivars and inbreds used in this study. Plant Chrom 2n Reported 2n names No. No. ‘Tiny Tot" 22 - P.acetosun8 18 18 P.acraeum? 18 - P.betulinumy 22 - P.capitatum¥ 66 54, 66 P.citrorellun? 18 - P.cordiforlium’ 22 22 P.cucullatun? 22 22 P.denticulatumfl 22 44 ‘Earliana" 44 - P.frutetorum€ 18 18 P.fruticosun€ 22 - P.fulgidum’ 44 22, 44 P.grandiflorum€ 22 22 P.hermanrifiliumy 18 '- P.hirtum9 44 - P.hispidum¥ 44 * P.inquinansy 18 18. 35 P.multicau1ey 22 ' P-Peltatum’ 18 18, 36 1°.papilionaceumy 28 44 P.reniformey - 22 ' P.scabrum¥ 22 36: 22 P.semitrilotum? 22 ‘ P . tetragarumy 2 2 2 2 P.violavium¥ 36 2° P.zona1e! 18 13 ‘Inbred White'w 18 ' ‘Picotte" 18 ’ P.alchemilloides" ~ 16 16: 18: 34. 35 P.coriandrifolium" 22 13: 22 P.elongatumr 3 , 8 P.mollicomum? 22 ‘ P.myrrhidifolium' 22 " P.odoratissimmmf .16 16 P-QUinquelobatumfi 26 ‘ P. ranunculiphyllum" 36 " P.tongaense' 18 " ¥ obtained from Netherlands obtained from the Natioal Botanic Garden 0 Africa obtained from Merry Gardens in U. S. A. 14131119811 State University breeding Line: Chicago IL provided by Pan American seed Inc. , Wes r f South H" 27 Somatic chromosome number of 2n=8 for Pelargonium species P. elongatum (2500K). 28 Table 2. Interspecific Pelargonium crosses which produced plump, normal appearing seed. Cross No. flowers Fruit set No. seed Germination pollinated % obtained % ‘Tiny Tot’ x P.cucullatum 24 4.2 5 60.0 P.cucullatum x ‘Tiny Tot' 27 17.8 24 58.3 P.acraeum x P.inquinans 14 53.0 32 87.5 P.frutetorum x P.inquinans 13 10.8 7 42.9 P. frutetorum x P.zonale 13 4.4 2 0.0 P.citrorellum x P.cucullatum 7 11.4 4 50.0 P.citrorellum X ‘Tiny Tot’ 4 50.0 10 100.0 P.citrorellum x P.zonale 8 7.5 3 0.0 P.cucullatum 61 5 x P.cordifolium 20 26.0 26 . P.cucullatum 0 0 x P.hispidum 18 1.1 1 . P.cucullatum x 2 0.0 P.papilionaceum 12 3-4 P.cucullatum x 25.0 P. tetragarum 9 8.9 4 P.scabrum x 50.0 ‘Tiny Tot' 10 24 . o 12 ‘ 29 Table 2 (cont'd). Cross No. flowers Fruit set No. seed Germination pollinated % obtained % P.scabrum x P.seritrilobum 3 40.0 6 83.3 P.scabrum x ‘Earliana' 6 3.3 1 0.0 P.peltatum x r P.cordifolium 42 1.4 3 33.3 P.peltatum x ‘Picotte' 53 4.9 13 23.3 ‘Picotte’ x P.peltatum 63 7.9 25 20.0 ‘Inbred White' x P.grandiflorum 13 0.5 1 100.0 ‘Inbred White' x P. peltatum 48 0.8 2 0.0 P.grandiflorum x ‘Tiny Tot' 40 0.5 1 0.0 P.grandiflorum x P.cordifolium 10 6.0 3 100.0 P.grandiflorum 0 0 x P.cucullatum 26 0-3 1 ' P.grandiflorum x P.hetulinum 17 17. 6 15 0- 7 P.semitrilotum x ‘Tiny Tot' 8 12.5 5 40:0 P.seritrilotum 50.0 x P.cordifolium 6 13.3 4 P.tetragatum x 1 100.0 P. scabrum 20 1-0 h 30 Table 3. Results of selfing the interspecific Pelargonium hybrids. Hybrid No. flowers Seed set No. seed plant pollinated % obtained (P. grandiflorum X ‘Tiny Tot) 35 0.0 0.0 (P. peltatum X’ Pu cordifblium) 194 0.0 0.0 ‘Tiny Tot' x ‘Eariana' 65 2.8 9.0 (P..betu1inumlx P. cordifblium) 14 0.0 0.0 (P. cucullatum x ‘Tiny Tot’) 237 2.3 27.0 (P. cucullatum x P. tetragatum) 151 5.6 42.0 (‘Inbred White’ x P. grandiflorum) 125 1.1 2.0 (P. seritrilotum x P. cordifolium) 83 0.5 2.0 (P. citrorellum x P. cucullatum) 100 2.8 14.0 (P. scabrum X’ P. seritrilotum) 99 1.0 5.0 (P. grandiflorum x P. cordifolium) 94 0.0 0.0 (P. grandiflorum x P. betulinum) 47 0.0 0.0 (P. citrorellum x ‘Tiny Tot') 136 1.3 9.0 (P. cucullatum x P. cordifolium) 25 16.0 22.0 (‘Tiny Tot' x P. cucullatum) 40 10.0 20.0 (P. seritrilotum x ‘Tiny Tot’) 65 0.0 0.0 (P. scabrum x ‘Tiny Tot') 62 0.3 1.0 R 31 Table 4. Reunite of beckcnoee inter-pacific Pew hybrids. Croce No. flowers Seed not No. need pollinated S obtained (P. peltanm x P. com) 1: L m 84 0.5 2.0 ('Inblcd White’ at P. graldiflonnn) x P. gmndiflonon 68 0.0 0.0 62(‘1nbrcd White'): P. WM) x ‘Inbrod White’ 53 0.0 0.0 (‘Ilimd White’: P. grandiflorwn) ‘ x (P. pelican r P. cordifolium) 24 4.2 5.0 (P. mallow: ’Nuy Tot‘) x ‘nny 701’ 80 0.6 2.0 (P. scabrum r ‘I'Iny lot) 1: ‘Rny 701' 70 0.0 0.0 (P. scabrum x P. un‘uflorum) x P. scabrum 12 8.3 5 .0 (P. scabrum r P. sorta-110m) x P. 801011034»: 103 1.1 7.0 (P. Citrorelltan x 'finy Tot') x ‘Tlny To: ' 70 5.4 19.0 (P. claw-cilia: x ‘M Tot') x P. cinnamon 8 0.0 0.0 (P- graldiflonon x P. cordifolium) x (P. semi-110nm x P. cordxjfoliron) 70 4.9 17.0 (P. redo-{lam x P. cordifolhan) x P. cordifofltm 40 1.5 3.0 (P. tin-arena»: x P. cucullarwn) x P. cucullanen 60 38.3 115.0 (P- Wm x P. beadinum) x P. bendinum 40 0.0 0.0 ('Tiny Tot' x P. mullanan) 1: Tiny Tot’ 63 7.9 25 .0 (P. semi-Ileana r Tiny Tot’ 1: Tiny Tot‘ 43 0.0 0.0 (P. set-Inflow a: Tiny Tot’ x P. redo-(loam 40 0.0 0.0 (Tiny Tor x 'sndim') X 'Tiny Tot' 64 5.3 17.0 g 32 however, the most common chromosome numbers (2n=18, 22, 36 and 44) do not fit this basic number. Crossability: From the 293 cross combinations of the 38 species, cultivars and inbreds, twenty-eight produced seeds. This resulted in 17 groups of seedlings (Table 2). Nineteen of the 293 crosses showed only fruit elongation (Table 5). P. x domesticum cv ‘Tiny Tot’ was successfully crossed with P. cucullatum, a species reported as one of the ancestors of P. x domesticum (Clifford, 1958; Craig, 1971; Ewart, 1982; Hanniford et al., 1982; Knicely and Walker, 1966). The seed set was lower (4.2%) when ‘Tiny Tot' was used as seed parent as compared with that (17.8%) of the reciprocal cross (Table 2). The hybrid seedlings showed a maternal influence on plant height and compactness. Tiny Tot has a short, compact plant habit, and P. cucullatum has a relatively loose, taller plant habit. The hybrid plants are shorter and more compact when ‘Tiny Tot' was used as seed parent, and looser and taller plants were produced when P. cucullatum was used as the seed parent (Figures 2 and 3). This phenomenon suggests a cytoplasmic effect. The hybrids could be further selfed and backcrossed to ‘Tiny Tot'. In selfing, the seed set (10.0%) of (‘Tiny Tot’ X P. cucullatum) was higher than that (2.3%) of the reciprocal cross (Table 3). After backcrossing to ‘Tiny Tot', ‘Tiny Tot' X P. cucullatum produced a higher seed set than it's 33 reciprocal hybrid (Table 4). These results suggest that ‘Tiny Tot’ is utilized most effectively as a female plant in interspecific cross. Both P. acareum and P. frutetorum, which belong to the subgenus ciconum, hybridized successfully with P.inquinans - a proposed ancestral species of P. X'hortorum (Craig, 1971; Ewart, 1982; Walker and Craig, 1961). P. acareum x P. inguinans had a seed set of 53% and seed germination of 87.5% (Table 2). P. frutetorum successfully crossed with P. inquinans producing a seed set of 10.8% and seed germination of 42.9%. This species also crossed with P. zonale, another proposed ancestral parent of P. x hortorum, but with a very low seed set (4.4%) and no seed germination. This data suggests that P. acareum has a closer genetic relationship with P inquinans than P. frutetorum does, and P. frutetorum has a closer relationship with P. inquinans than with P. zonale. P. citrorellum hybridized with P. cucullatum, P.x domesticum cv ‘Tiny Tot', and P. zonale with a seed set of 11.4%, 50% and 7.5% respectively. However, the hybrid seeds of P. citrorellum x P. zonale did not germinate (Table 2). When P. cucullatum was crossed with ‘Tiny Tot', P. cordifolium, P. hispidum, P. papilionaceum and P. tetragarum, it produced what looked like normal seed (Table 2). but the hybrid seeds of P. cucullatum x P. hispidum and P. cucullatum x P. papilionaceum did not germinate after 34 Figure 2. Plant types for the cross of ‘Tiny Tot' x P. cucullatum and their hybrid plant. Left: ‘Tiny Tot'; Center: hybrid plant; Right: P. cucullatum Figure 3. 35 Plant types for the cross of P. cucullatum X ‘Tiny Tot' and their hybrid plant. Left: P. cucullatum; Center: hybrid plant; Right:‘Tiny Tot'. 36 hand scarification and sowing. The inviability of some of these interspecific seeds may be due to the incompatibility between the genomes of the parental species, and the incompatibility between the genotype of the hybrid zygote and the genotypes of the endosperm. P. scabrum, a scented-leaved Pelargonium, was successfully crossed with ‘Tiny Tot', and P. seritrilotum (Table 2), and the aromatic character did transfer to the hybrid seedlings. In this case the scented leaf character is probably dominant. P. scabrum is also reported to be very highly resistance to bacterial blight X. c. pelargonii (Dunbar and Stephens, 1989). A P. x hortorum ‘Inbred White' was found to be crossable with P. x domesticum species P. grandiflorum. One viable seed was obtained (Table 2). This plant had yellow tented leaves at the young seedling stage, but after 12 weeks became more vigorous with normal green foliage. P. grandiflorum is reported to be bacterial wilt resistant (Dunbar and Stephens, 1989), and present screenings (Chapter 3) confirm this information. P- peltatum, an ivy - leaved Pelargonium, is reported to cross with P. x hortorum and to be rust resistant (Craig, 1971)). It is, however, highly susceptible to bacterial wilt (Craig, 1971; Knauss and Tammen, 1967; Wainwright and Nelson, 1972). Forty two flowers of P. peltatum were 37 Table 5. Interspecific Pelargonium crosses with only fruit elongation but no seed set. Cross NO. flowers Pollinated ‘Tiny Tot' ‘Tiny Tot’ ‘Tiny Tot' ‘Tiny Tot' x P.denticulatum x P.grandiflorum x P.inquinans x P.zona1e P.capitatum x ‘Tiny Tot' P.cordifolium x ‘Tiny Tot’ P.cordifolium x P.frutetorum P.fruticosum x P.inquinans P.fruticosum x P.zonale P.fulgidum x P.fruticosum P.grandiflorum x P.fulgidum P.inquinans x P.multicaule P.zonale x P.zonale x P.peltatum P.peltatum P.peltatum P.peltatum P.peltatum ¥ P.grandiflorium P. peltatum x ‘Tiny Tot' x P.denticulatum x P.inquinans x P.scabrum x P.violavium 18 8 73 90 33 19 21 17 12 12 16 20 12 11 38 Table 6. Results of crosses between chromosome numbers of various Pelargonium species. Chromosome No.(2n) No. cross No. cross combination attempted producing seed 8 x 18 1 0 18 x 8 4 0 18 x 18 53 10 18 x 22 74 2 18 x 28 2 1 18 x 36 3 0 18 x 44 17 0 18 x 66 5 0 22 x 18 43 0 22 x 22 54 12 22 x 28 1 0 22 x 36 2 0 22 x 44 19 2 22 x 66 1 0 36 x 8 1 0 44 x 18 6 0 44 X 22 2 1 44 x 66 1 0 66 x 18 2 66 X 22 2 Tot-:51 """""""""""""" 5;; """"""""" 3 E """" 39 pollinatedwith the pollen of P. cordifolium, and produced three hybrid seeds (seed set of 1.4%). This is the first successful report of a cross between the 2 species groups, and the first successful cross of P. peltatum and P. cordifolium. One of the seeds germinated and developed into a vigorous plant. The hybrid plant is more like P. cordifolium in leaf type, and like P. peltatum for plant type with multiple branched stem(s). The results from this research and previous studies (Craig, 1971), indicate that P. peltatum could be a usable bridge species between P..X hortorum and P..X domesticum. One second generation (G2) plant was obtained from 125 self pollinations of the hybrid plant ‘Inbred White’ X P. grandiflorum after the hybrid plant was moved from a 10%: greenhouse to another 23°C greenhouse. This G2 plant is partially fertile. Twenty four flowers of this plant were pollinated with the pollen from the hybrid plant P. peltatum X P. cordifolium, and 5 seeds were obtained (Table 4). If these seeds can be germinated, this germplasm will possibly bring the gene(s) for bacterial blight resistance from the species P. grandiflorum and P. cordifblium together with P. x hortorum and P. peltatum. But more work still needs to be done in solving the self incompatibility problems of hybrid plants derived from the cross of P. peltatum and P. cordifolium, as well as the backcross incompatibility to both parents in the cross of ‘Inbred White' X.P. 40 granditlorum. During this study, however, two seeds were obtained when this interspecific hybrid plant was selfed after being moved from a 10°C greenhouse into one of 23°C. This might indicate that a higher average growing temperature could possibly help break down the self incompatibiltity for this cross. If the bridge would prove to be successful, the bacterial blight resistance transferred from P. x domesticum to P. x hortorum would become more realistic. Other horticulturally desired characters, such as expanded flower coloration for P. x hortorum, and non-temperature controlled flower initiation for P. x domesticum might also be possible. The nineteen crosses which showed only fruit elongation (Table 5) may have been due to early endosperm abortion, as well as the incompatible interaction of related embryo, endosperm and maternal tissues. Those crosses might prove to be successful by using embryo rescue to culture the early stage embryos as has been done by previous research (Kato and Tokumasu, 1983). Crosses between chromosome numbers of various Pelargonium species showed that 18 x 18 and 22 x 22 were the I most successful but other combinations were possible (Table 6). SUMMARY Chromosome numbers were determined for a total of 38 species, cultivars and inbreds. Nineteen of the taxa have not been reported previously, and 7 others had chromosome numbers differing from previous reports. Due to the great variability and diverse ancestry that is found within P. x hortorum and P. x domesticum it has been suggested that successful crosses could be accomplished if a common progenitor could be found ( Nicely and Walker, 1966). The successful cross between ‘Inbred White’ and P. grandiflorum has supported this hypothesis. Besides the success of the direct cross of P. x hortorum ‘inbred white’ and P. grandiflorum( an ancestor to P. x domesticum), a possible bridge species (P. peltatum) has been found which can cross with both P. x hortorum and the P. x domesticum related species P. cordifblium. One interspecific hybrid seedling of P. x hortorum ‘Inbred White’ x P. grandiflorum and one Pu peltatum x P. cordifolium seedling were obtained. This is the first successful report on such crosses. Since P. grandiflorum and P. cordifblium have been found highly resistance to bacterial blight .X. c. pv. pelargonii (Chapter 3), the success of these crosses has great possible value for 41 42 transferring the bacterial blight resistance to P. x hortorum . ' REFERENCES Chow, T.W. and P. M. Harney. 1970. Crossability between a diploid Pelargonium x hortorum Bailey cultivar and some of its putative ancestral species. Euphytica 19:338-348. Chartejee, A. and A. K. Sharma. 1970. Chromosome study in Geraniales. Nucleus 13:179-200. Clifford, D. 1958. Pelargonium Including the Popular ‘Geranium’, a monograph. Blanford press, Lodon. Craig, R. 1971. Cytology, genetics and breeding of the geranium. P315-346 In: J. W. Mastalerz (ed.) Geraniums II. Penn. Flower Grower, University Park. Daker, M. G. 1969. Chromosome numbers of Pelargonium species and cultivars. J. Roy. Hort. Soc. 94:346-353. Darlington, C. D. and A. D. Wylie. 1955. Chromosome Atlas of flowering plants. Allen and Urwin Ltd., London. Ewart, L. C. 1982. Utilization of flower germplasm. HortScience Vol.16(2):135-138. Gibby, M and J. Westfold. 1983. A new basic chromosome number in Pelargonium (Geraniaceae). Caryologia 36(1):?9-82. Gibby, M and J. Westfold. 1986. A cytological study of Pelargonium sect. EUmorpha (Geraniaceae). Pl. Syst. EVOl.153:205-222. ' b. Hachett W. P. J. kister and A. T. Y. Tse. 1974 Flower induction of Pelargonium domesticum Bailey cv ‘Lavender Grand Slam’ With exposure to liw temperature and low light intensity. HortSc ence. 9(1):63-65. 1982. Hanniford Glenn G. and E. jay Holcomb. Regal Geranium. P161-169 In: J. . Mastalergaéfid.) Geraniums. Penn. Flower Grower, Univer51 y . 43 44 Harney, Patricia M. and T. W. Chow. 1971. Crossability between some Pelargonium Species. Euphytica 20:286-291. Kato, M. and S. Tokumasu. 1983. Characteristics of F1 hybrids produced by ovule - culture in ornamental Pelargonium. Acta Hort. 131:247-252. Dnubar, K, and C. Stephens. 1989. An in vitro screen for detecting resistance in Pelargonium somaclones to bacterial blight of geranium. Plant Disease Vol.73(11):910-912. Knauss, J. F. and J. Tammen. 1967. Resistance of Pelargonium to Xanthomonas Pelargonii. Phytopathology 57:1178-1181. Knicely, W. W. and D. E. Walker. 1966. Chromosome counts and crossability studies in the genus Pelargonium. Proc. XVII Intern. Hort. Congr. 1,209. Moore, M. J. Index to plant chromosome numbers 1967-1971. Utrech, Netherlands. Published by Oosthoek’s Uitgeversmaats-chappij B. V., Domstraat 5-13, Utrecht, Netherlands for the international Bureau for plant taxonomy and nomenclature. P252- 254 0 Nelson, P. E. and Nichols L. P. 1982. Bacterial blight. P221-223 In:J. W. Mastlerz (ed.) Geraniums. Penn. Flower Grower, UniverSIty Park. Pan, 8., J. Bacher and L. Ewart. 1990. Genetics of orange flower color in P. x hortorum. ACTA Horticulturae 272:53-57. Powell, M. C. and A. C. Bunt. 1978. The effect of temperature and light on flower development in Pelargonium x domesticum. Sc1entia Horticulture 8:75-79. Ste hens C. T. and Tuinier J. 1989. Disease . P symptomatology and variation in susceptibility of seed-propagated hybrid geranium vaiietéizntonis. Xanthomonas Compestris pv pelargon . 73:559-562. . ' . The complete Tylor, J. 1988. Geraniums and Pelargoniugid exhibition- The cultivation propagation, . EggggogoPress, Ramsbury, Marlborough, Wiltshire SN2 45 2HE. 176pp. wainwright, S. H. and P. E. Nelson. 1972. Histopathology of Pelargonium species infected with xanthomonas pelargonii. Phytopathology 62:1337-1347. Walker, D. E. and R. Craig. 1961. Breeding: The further of geranium. P93-94 In: J. W. Mastlerz (ed.) Geraniums I. Penn. Flower Grower, University Park, PA. Walt, J. J. A. Van Der. 1977. Pelargonium of Southern Africa. Vol. 1 Purnell and Sons. Cape Town. ' ~ CHAPTER 3 BACTERIAL BLIGHT (xanthomonas campestris‘pv.pe1argonii) RESISTANCE IN PELARGONIUHS Additional key words. Disease, Xanthomonas pelargonii, Resistance, Susceptible, Screening, Inoculation ABSTRACT Twenty one Pelargonium species, cultivars and hybrid plants were screened for bacterial blight (Xanthomonas campestris pv. pelargonii) resistance. The statistical results indicated that there were significant differences for bacterial blight resistance among different genotypes studied. P. odoratissimum, P. cordifolium, P. cucullatum, P. grandiflorum, P. peltatum x P. cucullatum, P. grandiflorum x ‘Tiny Tot’, ‘Tiny Tot’ x ‘Earliana’, P. betulinum x P. cordifolium, P. grandiflorum x P. cucullatum, P. grandiflorum x P. betulinum, P. cucullatum x P. cordifolium and P. scabrum x P. seritrilotum showed high resistance to the disease. A hybrid resulting from the cross of ‘Inbred White’ and P. grandiflorum, a proposed ancestral species of P. x domesticum, showed great tolerance. However, its seed parent ‘Inbred White’ was 46 47 highly susceptible to this disease. This indicates that resistance gene(s) was transferred from P. grandiflorum, and that resistance is an inheritable character. INTRODUCTION Bacterial blight caused by X. c. pelargonii is recognized as the most serious disease in Pelargonium X hortorum, and heavy losses of Pelargoniums to this disease has been reported yearly in greenhouses (Nelson and Nichols, 1982; Stephens and Tuinier, 1989). The disease is common in cuttings and seedlings of P X hortorum, as well as in the ivy-leaved pelargoniums (P. peltatum). All commercial cultivars of P. X hortorum are highly susceptible (Stephens et al.), and no genotypes of this species group are known to posses useful resistance genes (Craig, 1971; Knauss and A Tammen, 1967; Munnecke, 1954). There is also no chemical control available for this disease. Culture-indexing is used for disease control; but the procedure is expensive, and the culture-indexed plants are not resistant and can be lost to later infection. Most species and cultivars of P. x domesticum are, however, resistant to this disease (Knauss and Tammen, 1967), although there has been no report of successful resistant gene transfer from P. X domesticum to P. X hortorum. A worthy objective would be to transfer resistance genes from P. x domesticum to P. x hortorum. This research 48 was undertaken with the purposes of screening a broad base of Pelargonium germplasm for bacterial blight resistance, and providing useful breeding information directed toward the development of resistant cultivars of P. x hortorum. MATERIALS AND METHODS Plant Materials: Pelargonium species P. cordifolium, P. cucullatum, P. betulinum, P. scabrum, P. seritrilotum and P. peltatum were obtained from the National Botanic Gardens of South Africa. P. achemelloides, P. elongatum, P. mollicomum, P. odoratissimum, P. quinquelobatum P. ranunculiphyllum and P. tongaense were obtained from the Pan-American Seed Company, West Chicago, IL. P. X hortorum ‘Inbred White’ is a Michigan State University (MSU) breeding line. All Plants were grown in a screened greenhouse, supplemented with ten hours (daily) of high pressure sodium lightings, and following recommended growing procedures. Plants were inoculated when they were three month old. After inoculation the plants were grown at a temperature of 24%: day and 22°C night to promote growth of the bacterial blight organism. Inoculation: Bacterial strain X-l (from Kansas) provided by Dr. K. Dunbar in Dr. C. Stephens’ laboratory of the Department of Botany and Plant Pathology of MSU was used. The strain was 49 used because of its aggressive property (Dunbar and Stephens, 1989). The x-1 strain was stored in a saline solution (0.85% NaCl) for about one month at 4°C was streaked onto Difco nutrient agar in petri dishes and incubated at 23°C. After 3-5 days a single colony was transferred to 25 ml of modified Lederberg’s complete broth (elimination of glucose, Dunbar and Stephens, 1989). The broth culture was ‘ incubated at 25°C on a rotary-shaker at 125 rpm for 2 days. Then the bacteria were pelleted by centrifugation at 900 g for 15 min. The pellets were suspended in water, and diluted to the desired concentration (107) of colony forming units (cfu) based on standard dilution plating and turbidimetric techniques (Dunbar and Stephens, 1989). Five plants (cuttings) of each Species, cultivar and hybrid plant were sprayed to runoff using a hand sprayer, and placed in closed clear plastic bags for 3 days. Plants were randomly arranged on the bench, and grown at 24°C in the greenhouse to promote disease development. The control plants were sprayed with water. Three observations were taken at 21, 45 and 71 days respectively. The following numerical disease ratings were made on the percent of tissue blighted (yellowed and dried). 1 = 510% of the leaf tissue blighted; 2 = 11-30% of the leaf tissue blighted; 31-50% of the leaf tissue blighted; to II 51-70% of the leaf tissue blighted; .5 II 50 5 = 270% of the leaf tissue blighted; 6 whole plant died. Statistical Hathods: Experimental data were analyzed using MSTAT microcomputer software. The significance F test and the least significant differences (LSD) test were performed at the a=0.05 level. Disease resistance means were calculated for 5 replications of 21 plant genotypes on the last 2 observations (45 days and 71 days). The disease ratings from the first observation (21 days) were not used since the disease symptoms were developing at that time. RESULTS AND DISCUSSION Disease ratings for each species, cultivar, breeding line and plant are listed in Table 1. There was a significant difference (a=0.05) for the disease resistance among species from different groups. P. x hortorum ‘Inbred White', P. alchemilloides, P. elongatum, P. mollicomum, P. quinquelobatum, P. peltatum, P. ranumculiphyllum, and P. tongaense were very susceptible to bacterial blight. The inoculation spots on the plants of this susceptible group spread quickly, and the inoculated leaves became yellowish within the first two weeks (Figure 1). Many of the plants were dead 45 days after inoculation (Tabla 1) . ‘Inbred White', a line from the MSU breeding program 51 showed enlarging spots at 7 days after inoculation. About 45 percent of the leaf tissues was blighted in 21 days (Figure 2). At 45 days all the plants were entirely blighted, and the plants were dead by day 71. P. grandiflorum, a P. x domesticum ancestor, showed very tiny spots with a dark brown halo one week after inoculation. Plants developed a few small spots by 21 days, and the spotted leaves fell off at 45 days.‘ At 71 days the plants no longer showed symptoms. The hybrid plant derived from the cross of the above two (‘Inbred White' X P. grandiflorum) developed some spots at 21 days after inoculation. About 25 per cent of the leaf tissue of plants was blighted at 21 days, and remained about the same at 45 days. At 71 days about 30 per cent of the leaf tissue of the plants was blighted, but new shoots were developing (Figure 4 a Figure 6). The hybrid plants of P. cordifolium (a P. X domesticum progenitor species) and P.peltatum (an ivy-leaved Pelargonium) and parent plants were also screened (Tabla 1). The pollen parent P. cordifolium developed very small spots with a dark brown halo around the centers at 21 days after inoculation (Figure 3). Less than 20 per cent of the leaf tissue of the plants was blighted at day 45. No new spots had developed at 71 days, and the plants started to produce new shoots. The seed parent P. peltatum had developed very large spots on the leaves by the end of the first week after 52 Table 1. xanthomonas campestris. pv. pelargonii resistance ratings (1-best, 6-worst) for Pelargonium species, cultivars and hybrid plants Items Disease resistance rating means‘ Speciesy ‘Inbred White' 5.8 A P. grandiflorum 1.2 PC P. cordifolium 1.2 PC P. cucullatum 1.5 EF P. peltatum 5.7 A P. alchemilloides 5.8 A P. elongatum 6.0 A P. mollicomum 4.3 B P. odoratissimum 2.0 CD P. quinquelobatum 4.3 B P. ranunculiphyllum 5.8 A P. tongaense 6.0 A F1(‘Inbred Wh.’ X P.grandiflorum) 2.2 C F1(P.peltatum x P.cordifolium) 1.7 DE F1(P.grandiflorum x ‘Tiny Tot’) 1.2 PC F1(‘Tiny Tot' X ‘Earliana') 1.0 G F1(P.betulinum X P.cordifolium) 1.2 FG F1(P.grandiflorum X P.cordifolium) 1.7 DE F1(P.grandiflorum X P. betulinum) 1.3 EFG F1(P.cucullatum X P.cordifolium) 1.0 G F1(P.scabrum X P.seritrilotum) 1.3 EFG 1.300,, 0.45 Days post inoculation‘ 21 days 1.6 B 45 days 3.2 A 71 days 3.3 A 1.80005 0.15 ‘ means with the same letters are not significantly different, those with different letters are significantly different at a=0.05 by Least Significant Difference (LSD) test. ’ means from 5 replications on the last 2 observation times (45 days and 71 days) ‘ means from 21 genotypes and 5 replications for each genotype Table 2. 53 xanthomonas campestris pv. pelargonii resistance ratings (1-best, 6-worst) for ‘Inbred White', P. grandiflorum and their hybrid plants, and for ‘P. peltatum, P. cordifolium and their hybrid plants. Items Disease resistance rating means‘ Genotypey ‘Inbred White' 5.8 A P. grandiflorum 1.2 C ‘Inbred White’ x P. grandiflorum 2.2 B Lsnw 0.50 £12222; """""""""""""""""" 3'3""; """"""" P. cordifolium 1.0 C P. peltatum x P. cordifolium 1.8 B LSD“, 0.41 ‘ means with the same letters are not significantly different, those with different letters are significantly different at a=0.05 by Least Significant Difference (LSD) test. Q days and means from 5 replications and last 2 observation times(45 71 days) 54 Figure 1. Bacterial blight (X. c. pelargonii) resistance screening for ‘Inbred White’ showing enlarging inoculation spots and yellowish leaves at 10 days after inoculation. 55 Figure 2. Bacterial blight (X. c. pelargonii) resistance screening for ‘Inbred White’ showing about 45% of leaf tissues was blighted at 21 days post inoculation. 56 Figure 3. Bacterial blight (X. c. pelargonii) resistance screening for P. cordiforlium showing spots with the typical dark brown halo during the first three weeks after inoculation. 57 Figure 4. Bacterial blight (X. c. pelargonii) resistance screening for the ‘Inbred White', P. grandif. and their hybrid plant (71 days post inoculation). Left:‘Inbred White, plant completely dead; Center: hybrid plant, showed less symptoms; Right: P. grandiflorum, no symptoms. 58 Figure 5. Bacterial blight (X. c. pelargonii) resistance screening for P. peltatum , P. cordifolium and their hybrid plant (71 days post inoculation). Left: P. peltatum, plant completely dead; Center: hybrid plant, showed less symptoms; Right: P. cordifolium, no symptoms. of ’/n bred White ’, P. grand/'f/orum and their hybrid to X. ‘. Res/5 [an ce Figure 6 campesz‘r/s pv. pe/argon/i. ..... ............ ............. ............ ............. v ' . .‘ z.‘ ;.:.'.'-;.‘.‘. :.....'<.‘. . v. 3.3""? ...l. ; . g. . ... ’ ' -.' _. . .;.:.;.~ ‘43.: 4.151 '. '.'.‘~‘.'.’.' 2‘ -:v;-. -. v’.‘.‘.':.‘. We . .' ‘.'-'u' .‘p' ‘3‘ - . .‘. l .5. ‘.'l.- 3' .1 (...:I ...~_.‘. . . ' ...-h v . -. 'u'.: u l .'.' ' OOOOOOO Resistance of P. peltatum, P. cord/'fo/ium and their hybrid to X. Figure 7.. campestr/s pv. pe/argonii. 60 00.90".”Oanoooo ...-000000.00... ..... .... 0...”. 6“.“0000 :Qomuuutuoc ::oo¢~~ouum 00-0....” ..... :zooooonooooooopo ......OOM“. :0...“ ....... Q—(DLIJ4WLLI Ofi