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H ' *}J\'.«’ .1 MICHIGAN STATE umvensnv tBRARIES "7 C ‘) ’ " ’ ‘ WW d w ‘1 j" 3 ll l: mu Ill lllf’llllil, 3 1293 00627 9578 w , l 0 fl l! nor This is to certify mat the thesis entitled ORANGE FLOWER COLOR INHERITANCE IN PELARGONIUM X HORTORIJM presented by SHlFEW FAN has been accepted towards fulfillment of the requirements for . ORTICULTURE MASTER OF SCI. degreem H v .' I. r. ’1 (. L’) K_ , I /‘ lav . /.-l I 4 3 VI I ‘ ’.4 f, I' ’I ., I / _ \ ' \";h 7 ,V {/i a? I” \ (AR "(L/f (l :'I 1 I, I 1 u' ‘ V J A1. 51' ‘ ‘u. Major professor Date ///6/ 87 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution 4"e . PLACE IN RETURN BOX to remove thle checkout from your record. ' TO AVOID FINES return on or before one due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity institution ORANGE FLOWER COLOR INHERITANCE IN PELARGONIUM X HORTORUN By Shifeng Pan A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of Horticulture 1988 ABSTRACT ORANGE FLOWER COLOR INHERITANCE IN PELARGONIUM x HORTORUM By Shifeng Pan Five genes are hypothesized to control the orange flower color Inheritance in Pelargonium x hortorum . They are E, 9;, fig, 3 and l. The B, §§, and E are found to have the same function as reported in previous research. The dominant B or fig gives red, pp gives pink, and gag; gives deep salmon. The 3 gives colored plant, and gg gives white. The homozygous recessiveji is epistatic to all other genes. The 9;,1 and gg are new in this research. 9; gives crimson color; while the recessive oror with the dominant P, §g, fl and recessive modifier ii gives orange flower color. A recessive allele to 5;, ggfl,gives light salmon in the homozygous recessive condition. The l dilutes and i; intensifies the flower color in the presence of dominant In”? and E. The anthocyanin analysis results showed that the genes controlling the flower color were also controlling the specific anthocyanin pigment synthesis. The orange flowered phenotype contained only pelargonidin. ACKNOWLEDGMENTS The author expresses his appreciation to Dr. Lowell C. Ewart, the thesis adviser, for his patient guidance and encouragement during this research. Appreciation is also extended to Dr. Amy Iezzoni and Dr. Everett Everson for their counsel and as committee members. ii TABLE OF CONTENTS EAQE List of Tables ......................................... v List of Figures ....................................... x Introduction ............................... 4 ........... 1 Literature Review ..................................... 2 Material and Methods ............................... ... 8 Pollination and Seed handling ......................... 11 Plant Culture and Flower Color Classification ......... 12 Anthocyanin Analysis -- Paper Chromatography .......... 13 Anthocyanin Analysis -- Thin - Layer Chromatography....l4 Statistical Analysis .................................. 15 Results and Discussion ................................ 16 Orange Flower Color Inheritance ....................... 16 Cross Involving Crimson and Orange.......... ..... ..... 19 Cross Involving Deep Salmon and Orange ............... ..22 Cross Involving Light Salmon x Orange .................. 29 Cross Involving Orange and White... ................... 33 Cross Involving Orange and Rose/white ................. 37 Cross Involving Picotee x Orange ...................... 40 Summary of the Flower color Inheritance .............. ..42 Pigment Analysis. ..... .... ............................. 47 Pigment Analysis For the Cross of Crimson and Orange ...... . ....... . ...... 47 Pigment Analysis For the Cross of Deep Salmon and Orange......... ..... ........49 Pigment Analysis For the Cross of Light Salmon x Orange ..................... 54 iii Pigment Analysis For the Cross of White and Orange ...................... 56 Pigment Analysis For the Cross of Orange x Rose/white ..................... 59 Summary of Pigment Analysis ........................... 61 Literature Cited ..................................... ..65 Appendix ............................................... 68 iv LIST OF TABLES TABLE PAGE 10. 11. 12. Flower Color Inheritance From Craig, 1963............ 4 Population Ratios of the Orange Flower Colored Parents After Selfing........ ................ 10 Other Flower colored Parents Used In the Orange Flower Color Inheritance .................. 10 Phenotypes and the Related RHS numbers For the Parents and Their F1 Hybrids For the Orange Flower Color Inheritance In 2 x h ............. 17 F1 Results of Reciprocal Crosses For the Orange Flower Color Inheritance In B x h ............. 18 Pedigree Information Of the Cross Crimson and Orange For the Orange Flower Color Inheritance In E xh. ....... IOODODODODOODIOOD ..... 0.. 20 Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Crimson x Orange For the Orange Flower Color Inheritance In B x h ( Test For 3 : 1 ratio ).................. ....... .... 20 Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x Crimson For the Orange Flower Color Inheritance In 2 x h ( Test For 3:1 ).......... ........ ........... ........ 21 Proposed Genotypes For the Cross of Crimson and Orange For the Orange Flower Color Inheritance In E x h ............ . ........ . ............. . ........ 21 Pedigree Information Of the Cross Deep Salmon and Orange For the Orange Flower Color Inheritance In P x h ................................. 23 Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Deep Salmon x Orange For the Orange Flower Color Inheritance In B x h ( Test For 9:3:4 )... ...... ............... ........... 23 Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x Deep Salmon For the Orange Flower Color Inheritance In E x h ( Test For 9:3:4 ) ................................... 25 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Proposed Genotypes For the Cross of Deep Salmon and Orange For the Orange Flower Color Inheritance In 2 x h ............................................ Chi - Square Test On F2 Progenies Of the Hybrids Resulting From Cross of Orange x Deep Salmon For the Orange Flower Color Inheritance In 2 x h ( Test For 9:3:4 ) ................. . ................ Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x Deep Salmon For the Orange Flower Color Inheritance In 2 x h ( Test For 0:0:1 )........ ...... .................... Proposed Genotypes For the Cross of Deep Salmon and Orange For the Orange Flower Color Inheritance In B x h ................................. .... ....... Pedigree and F2 Family Numbers of the Cross Light Salmon and Orange For the Orange Flower Color Inheritance In 2 x h ......................... Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Light Salmon x Orange For the Orange Flower Color Inheritance In B x h ( Test For 9:3:4 ) ................ ......... ......... Chi - Square Test On Pooled F2 Progenies Of the Hybrids From Cross of Orange x Light Salmon For the Orange Flower Color Inheritance In 2 x h ( Test For 9:3:4 )...... ..... .......... ..... . ....... Proposed Genotypes For the Cross of Light Salmon x Orange For the Orange Flower Color Inheritance In 2 x h.............. .............................. Pedigree Information of the Cross Orange and White For the Orange Flower Color Inheritance In 2 x h..... ....................................... Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x White For the Orange Flower Color Inheritance In 2 x h ( Test For 1:2:1 ) ........ ................ .......... Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x White For the Orange Flower Color Inheritance In 2 x h (Tent For 1:2:1)eeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeee Proposed Genotypes For the Cross of White and Orange For the Orange Flower Color Inheritance In E x h ............ . ........................ . ...... vi 25 27 ..28 30 ..32 34 35 36 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Pedigree Information of the Cross Orange and Rose/White For the Orange Flower Color Inheritance In B x h ......... .. ............. . ......... . ......... 38 Chi - Square Test On F2 Progenies Of the Hybrids From Cross of Orange x Rose/White For the Orange Flower Color Inheritance In 2 x h ( Test For 1:2:1 ) ................. ........ ......... 38 Hypothesized Genotypes For the Cross of Orange x Rose/White For the Orange Flower Color Inheritance In E x h ..... . .............. . ............ ........... 39 Pedigree Information of the Cross Picotee and Orange For the Orange Flower Color Inheritance In E x h.............. ............... ......... ...... 41 Proposed Genotypes of the Parents Used In This Research For the Orange Flower Color Inheritance In 2 x h ....... . ......... .. ....... 44 Pigment Analysis Of the Cross Crimson x Orange For the Orange Flower Color Inheritance Pigment Analysis Of the Cross Orange x Crimson For the Orange Flower Color Inheritance InExb...IOIO....D0.000....IIOIDIDOIDODOOIODOOOOO.I. 50 Pigment Analysis Of the Cross Deep Salmon x Orange For the Orange Flower Color Inheritance InBXheeeeeeeeeeeeeee eeeee eeeeeeeeeeeeeeeeee eeeeee 51 Pigment Analysis Of the Cross Orange x Deep Salmon For the Orange Flower Color Inheritance In 2 x h....................... ......... . ...... ..... 52 Pigment Analysis Of the Cross Orange x Deep Salmon For the Orange Flower Color Inheritance In E x h........... ........... . Pigment Analysis Of the Cross Light Salmon x Orange For the Orange Flower Color Inheritance In 2 x h.............. ..... ............. ..... . ...... 55 Pigment Analysis Of the Cross White x Orange For the Orange Flower Color Inheritance InEx.h-OODOOOD...0.0.0.0....00.000.00.000.IO.......57 Pigment Analysis Of the Cross Orange x White For the Orange Flower Color Inheritance InExQOOIIOOOIOO.....IIOODOOIDOOIIOODD 000000000000 58 vii 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. Pigment Analysis Of the Cross Orange and Rose/white For the Orange Flower Color Inheritance In E x h ............................................ 60 Pigments Identified For All Parents Used For the Orange Flower Color Inheritance In 2 x h .............................. . ............. 63 Backcross Result of ( Crimson x Orange ) x Orange For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ) ........... ... ............. 68 Backcross Result of ( Orange x Deep Salmon ) x Orange For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ) ........................... 68 Backcross Result of ( Orange x Deep Salmon ) x Deep Salmon For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ) ........................... 69 Backcross Result of ( Light Salmon x Orange ) x Light Salmon For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ) ...................... ..... 7O Backcross Result of ( Light Salmon x Orange ) x Orange For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ). .............. ...... ...... 7O Backcross Result of ( Light Salmon x Orange ) x Light Salmon For The Orange Flower Color Inheritance In E x h ( Test For 1:1 ) ........................... 71 Backcross Result of ( Light Salmon x Orange ) x Orange For The Orange Flower Color Inheritance In E x h ( Test For 1:1 ) .................. . ........ 71 Backcross Result of ( Orange x White ) x Orange For The Orange Flower Color Inheritance In E x h ( Test For 1:1 ) ........................... 72 Backcross Result of ( Orange x White ) x White For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 )... ...... ... ............... 72 Backcross Result of ( White x Orange ) x White For The Orange Flower Color Inheritance In 2 x h ( Test For 1:1 ) ........... ...... .......... 73 Backcross Result of ( Orange x Rose/White ) x Rose/White For The Orange Flower Color Inheritance In E x h ( Test For 1:1 ) ........................... 73 viii 51. F2 Segregation Progenies Of the Peach Hybrids From Cross of Picotee x Orange For the Orange Flower Color Inheritance In 2 x h ........ 74 ix LIST OF FIGURES Derived Single Flowered Orange Background Lines From Maxim Kovalevski Crossed With Robin Hood ....... 9 Line Chart Presentation For the Interactions of 9;, §§, £,W and l, and the Related Phenotypes..... ........ ....... ........... . ......... 45 Line Chart Presentation Of the Genotypes and the Synthesized Pigments ........................... 64 INTRODUCTION Pelargogium x hggtggum L. H. Bailey ( 2 x h ), commonly called geraniums, has been in cultivation for about 300 years, and has become one of the most important plants for the pot and bedding plant industries. Floriculture statistics showed that seed produced pelargonium sales in 1986 were $45.6 million, and $58.9 million in 1987 (1). In recent years progress has been made concerning genetic control of geranium flower color. Vegetative produced 2 x h plants with attractive orange flowers are in the trade, but the genetic inheritance of the color is not clear. The research on the inheritance of orange color was initiated as a first step to the possible development of a seed produced orange flowered P x h cultivar for the bedding plant industry. By determining the inheritance relationship between orange flower color and the other flower anthocyanin pigments in E x h, it could also lead to the development of other colors. The objectives of this research were 1) to study the inheritance of orange flower color in diploid B x h lines, and 2) to determine the major anthocyanin pigments related to the orange flower color. LITERATURE REVIEW Pelargonium, containing about 280 species and a large number of hybrids and cultivars, is native to South Africa (11,15). It is believed that Pelargonium, probably Pelargonium zonale, was introduced into Europe by the Dutch governor of the Cape Colony in 1609 (7). Between 1800 and 1830, two groups of hybrids began to be distinguishable: 1) Pelargonium x hgrtgrum, the zonale geranium, and 2) Pelargonium x domesticum, Martha Washington geranium. The third group, the ivy leaved Pelargonium was not introduced into Europe until after 1850 (11). Pelargonium x hortorum (B x h) cultivars continue to be rapidly developed especially after many new flower and foliage colors and forms appeared in the late 1800's. There are many flower color available in P x h including orange, purple, magenta, varied shades of red, salmon, pink, white and many bicolors. All of these colors in P x h are the result of water soluble pigments - anthocyanins which absorb light (10). There are three known factors that change the color of anthocyanins; namely, pH, metal chelation, and co-pigment mentation, which can operate in plant tissues (9, 13). Furthermore, the colored pigments are synthesized by a variety of chemical reactions controlled by enzymes, which are proteins determined by genes(13). Usually, several different anthocyanins exist within the same flower. The different anthocyanins may be synthesized from each other by reactions controlled by enzymes. Altering the enzyme, which is controlled by genes, might change the chemical reaction and produce a different pigment. This is the foundation of developing new flower colors and of studying flower color inheritance. Early B; x h. research reported that any colored flower was dominant to white, and that darker colors were dominant to light colors (6). Craig (9) studied seven distinct flower color phenotypes in inbred lines of 2 x h and determined that three independent genes 2, Se, and 2 controlled these colors. Dominant 2 gives solid flower color, while homozygous recessive gives variegation in the red phenotype. When all three genes were dominant, the phenotype was red. The reaction of these loci varied in different genetic backgrounds. Pink flowered phenotypes are always variegated, regardless of the conditions of the 1 locus. Recessive S; and E interact to give picotee, a flower having one basic color with a margin of another color. When these three loci interact, they produce the genotypes and phenotypes listed in table 1. Table 1. Flower color inheritance In E x h.( Craig, 1963 ) GENOTYPE PHENOTYPE P_Sa_V_ red solid color P_Sa_vv red variegated color P_sasaV_ salmon solid color P_sasavv picotee ppSa_V_ medium pink variegated ppSa_vv bright pink variegated ppsasaV_ light pink variegated ppsasavv Picotee Nugent and Snyder (17) studied a character in geranium flowers called ‘Center Color’, which seems to be the same as the variegated character identified by Craig (9). They reported that this phenotype was conditioned by one gene Q with the dominant condition producing a solid colored flower, whereas, the homozygous recessive resulted in a white area in the center of the flower. Badr and Horn (3) confirmed Craig's loci of E and S3. They also observed a locus 9 which acted as a diluter within flower groups, and a gene E which most likely was a major gene that acted with only incomplete dominance upon anthocyanin hydrolysis. Williams (26) found a locus A, and when dominant the gene conditions purple flower color in the presence of ppSaSaVV. Wernett (25) studied the inheritance of an orange flower color mutant derived by x - ray treatment. She proposed 5 loci to condition Orange flower color in diploid 2 x h, in which homozygous dominant 9 gives the greatest amount of orange color. When heterozygous, an intermediate level of orange color is present, and when homozygous recessive, a small amount of orange color is present. Two loci E and fl interact to modify the effect of locus of 9. However, either diluter or intensifier effect of E and fl is dependent on the dosage of orange color present. Homozygous recessive 3 gave a red-orange color. When M is dominant, orange color is observed. She found the 1 locus had the same function as described by Craig (9). Six basic anthocyanins are found in plants and all six have been reported in 2 x h (10). They are pelargonidin, peonidin, malvidin, cyanidin, petunidin and delphinidin. Robinson and Robinson (20) investigated 2 x h for anthocyanin content in flower petals. They found the anthocyanin in blue-red colored cultivars to be malvidin, and salmon-pink to be pure pelargonidin. They also found the red flowered cultivar ‘Henry Jacoby' to contain mostly pelargonidin with some malvidin 3,5-dimonoside. Scott-Moncriff (23) studied salmon flowered progenies derived from the self - pollinated rose - pink flowered flowered ‘Constane’. She found malvidin 3,5-dimonoside, small amount of pelargonidin, and some flavone and pyrogallol tannin. A paper chromatographic method was first used for anthocyanin pigment analysis by Bate-Smith in 1948 ( l4 ). Since then the method has become the most valuable application in the identification of anthocyanins and in the study of biochemical genetics. Anthocyanins were the first group of plant substances in which the relationship between the single gene and simple biochemical differences was demonstrated ( 14). Admedullah et al (2) found pelargonidin in the B x h cultivars ‘Red Irene’, ‘Madame Salleron’ and Pelargogium cucullatum. Williams (26) in her anthocyanin studies of orange flower color in 2 x 5 suggested the presence of pelargonidin as the only anthocyanin. The source of the orange color in her study was not mentioned. She also found that purple flower petals lacked pelargonidin, but appeared to contain peonidin, malvidin and either delphinidin or petunidin. Buswell (4) studied flower color inheritance on tetraploid 2 x b. He proposed that flower color seemed to be related to a gene dosage effect with pink - flowered plants containing no dominant alleles, red-flowered plants containing a single dominant allele and orange - red flowered plants containing two or more dominant alleles. After TLC studies, he found that pink flowered phenotypes contained only peonidin and cyanidin, while both red and red-orange flowered phenotypes contained pelargonidin, peonidin and cyanidin. Increasing amounts of pelargonidin was present with increasing numbers of dominant alleles. With spectrophotometry and TLC, Wernett (25) found all orange and orange related flower color phenotypes to contain only pelargonidin 3,5-diglucoside, while red-orange and red- orange related phenotypes to contain pelargonidin 3- monoglucoside. MATERIALS AND METHODS The orange flower color used in this research originally was obtained by Dr. Lowell C. Ewart from a private grower. The cultivar, Maxim Kovalevski, was tall growing with very big orange flowers. Later the cultivar was crossed with the very short growing, cherry red colored cultivar ‘Robin Hood’ to reduce the plant height for possible pack production. After selfing the F1 hybrids, orange flowered plants were selected, and selfed for three or four generations ( Figure 1 ). The selected orange flowered plants were checked for the homozygosity of the orange color in the beginning of this research. The results are presented in Table 2. Other homozygous plants for flower color which were crossed with the selected orange flowered plants in this study are listed in Table 3. To study the orange flower color inheritance of the diploid E x h, the following crosses were made: Orange x Crimson Crimson x Orange Deep salmon x Orange Rose/white x Orange Light salmon x Orange Light pink x Orange White x Orange Orange x White Picotee x Orange Figure 1. Derived Single Flowered Orange Background Lines From Maxim Kovalevski Crossed With Robin Hood 81: 82: F1: F2: F3: F4: 78-20 Lt.org.scar 1o 80-14A 83-25C lo 85-15A X l 81-50D lo 83-25D 84-33A lo 78-14 X 78-19 78-25 orange orge.scar Lt.orge scar 1 o 80—9A 80-9C X 80-268 82-33D lo 83-103B 83-103C lo lo 84-33D 85-79A-B-C-D-E-G 85-80A-B la 85-16A-B-C 85-17A 10 Table 2. Population Ratios of the Orange Flower Colored Parents Following Selfing SEGREGATION R.H.S.COLOR DEEP PEDIGREE PHENOTYPE CHART NUMB. GENERATION ORANGE SALM. 84-33D ORANGE; 33B F3 14 4 85-15A ORANGE 33B F3 44 0 85-16A ORANGE 338 F4 27 0 85-79B ORANGE 33B F3 14 0 85-79D ORANGE 33B F3 13 0 85-79E ORANGE 338 F3 11 0 Table 3. Other Flower Colored Parents Used In the Orange Flower Color Inheritance R.H.S.COLOR HOMOZYGOSITY PEDIGREE PHENOTYPE CHART NUMB. GENERATION OF FLOWER COLOR 81-10C LIGHT SALM. 48C F5 HOMOZYGOUS 82-27A DEEP SALM. 39B F5 HOMOZYGOUS 82-60C ROSE/WHITE 57B F5 HOMOZYGOUS 83-54B CRIMSON 47B F5 HOMOZYGOUS 84-10A WHITE -a F5 HOMOZYGOUS 80-38A PICOTEE -a F5 HOMOZYGOUS -a. No R.H.S. Color Chart number available. POLLINATION AND SEED HANDLING Standard pollination techniques were used to produce the necessary seeds. For cross pollination, emasculation was done when the anthers were immature. Both small soft camel hair brushes and forceps were used in removing the anthers or applying pollen on the stigma. Self and cross pollinations were performed using fresh pollen released from the anthers and applied to the stigma of the parent. The stigma was most acceptable when the five branches of the stigma were starting to open. Usually about one month after pollination, the resulting seed was harvested, and put in a 4"C seed storage room and dried for two to three weeks before any seed treatment. 2 x h seed was treated using concentrated Sulfuric acid (H2804) to affect proper germination as described by Ewart (12). Seed was put in a 100 ml glass beaker, and concentrated H2504 added until the seed was covered. The treatment was timed after the acid was added and was carried out at room temperature (about 21°C ). The average time for treatment was seven to eight minutes. A glass rod was used to stir the seed several times during the treatment. The acid and seed were poured into a wire hand strainer at the end of the treatment, and washed with water to stop the acid action. The treated seed was dried and put into a seed bag. 11 PLANT CULTURE AND FLOWER COLOR CLASSIFICATION A11 H2804 treated seeds were sown in a peat-lite planting mix and covered with about 1/8 inch layer of fine perlite. The sowing mix was drenched with Banrot to prevent damping off problems. After sowing and watering, the seed trays were placed in clear plastic bags ( for uniform moisture and temperature ) and placed in a production room at 21 to 23 C. Cool white fluorescent light was provided for quick germination. The plastic bags were removed when the seeds germinated. The seedlings were transplanted to 24 or 32 cell flats 2 to 3 weeks after sowing. After transplanting the progeny seedlings to the flats, Banrot and Subdue were used to drench the growing medium for disease control. To keep the plants compact and to hasten flowering, Cycocel (1500ppm) was sprayed on the plants two and three weeks after transplanting. Standard greenhouse practices were followed for disease and insect control. The parental plants and all selected F1 progenies were grown in 6 inch pots containing a peat-1ite growing medium under normal greenhouse conditions. All plants were grown in a screened greenhouse to avoid uncontrolled pollinations. B x h is very responsive to cumulative light energy for early flowering (5). Therefore 24 hours supplemental light was supplied using high - pressure sodium lights. 12 13 Temperature was maintained at 24°C days and 16"C nights. Flower color of the plants was classified using the Royal Horticultural Society Color Chart ( RHS ) (21) at a constant light condition and using the first open floret. ANTHOCYANIN ANALYSIS --- PAPER CHROMATOGRAPHY One to two day old flower petals were taken from the parental and F1 plants for drying in a forced draft oven at 40°C for one week and stored in glass bottles in desiccators until needed for analysis. Half gram samples of dried flower petals were extracted with 50 m1 of 1% HCL in methanol. If dry flower petal samples were not available, ten grams of fresh flower petals were extracted with 50ml of 1% HCL in methanol. The extract was filtered and concentrated in a rotary evaporator at 40"C or in a boiling water bath for about 30 minutes. The pigment concentrate was rinsed from the flask by using a 0.05 M citrate phosphate buffer of pH 5.5. To adjust the pH of the concentrate to 2.8, a 2N NaOH solution was used. The concentrate was centrifuged at 7500 rpm for 10 minutes, and the resulting supernatant was stored in the dark at 2"C. Whatman number 1 chromatography paper was used for analysis. Three solvent systems as described by Harborne (14) were used. These solvents were BAW (n-butanol-acetic acid-water 4:1:5), 1% HCL (water-12 N hydrochloric acid 97:3), and BuHCL ( n - butanol - 2N hydrochloric acid 1:1). When using the BuHCL solvent system, the paper and the 14 chromatographic tank were equilibrated with the bottom layer of separated n-butanol - 2N HCL (1:1) for at least 24 hours before the tests started. Special attention was also paid to the BAW solvent system. The solvent was prepared 3 days before use since the length of time this solvent storage affected the Rf values (14). The pigment concentrate was applied to sheets of papers in 2.5 centimeter streaks, using drumond 10 ul pipettes. Each sample was run two times on a different sheet of chromatographic paper. The Rf values were taken by averaging the two replications. Pigments were identified by observation their color in visible light, under ultraviolet light, and by observing their Rf ( relative positions ) on the chromatographic paper as described by Harborn (14). The Rf values were calculated as, distance of the spot from the origin (cm) Rf = distance of the solvent from the origin (cm) ANTHOCYANIN ANALYSIS -- THIN-LAYER CHROMATOGRAPHY Thin - layer chromatography was also done for the parents and some of the F1 hybrids. Precoated silica gel thin—layer plates were marked 2 cm from the bottom, 15 cm from the origin ( bottom mark ). The extract was run in the chromatography tank until the solvent front moved about 15 15 cm, and then the plates were air dried in a fume hood. Pigments were identified according to their color in both visible and ultraviolet light, and by observing their Rf values ( 16,18, 19 and 24 ). STATISTICAL ANALYSIS Chi - Square analysis was used to test the fit of segregation ratios to Mendelian ratios. The non - significant null hypothesis was accepted when the probability was equal or greater than 0.05. RESULTS AND DISCUSSION ORANGE FLOWER COLOR INHERITANCE The phenotypes of the parents and their F1 hybrids are listed in table 4. Genetic studies were not possible for the crosses of red with orange and light pink with orange due to a seed setting problem. Craig(9) showed that reciprocal crosses in his flower color inheritance study did not produce different Fl hybrids. This study suggested the same conclusion from the results obtained from reciprocal crosses of crimson with orange, white with orange and white with rose/white. All these crosses and their F1 phenotypes are listed in table 5. By checking the Royal Horticulture Society Color Chart Cross Inference Table (22), it was found that the crimson, deep salmon and rose/white flower colors used in this research were very close or the same respectively to the colors of red, salmon and medium pink which were studied by Craig (9). Therefore, Craig’s genetic models for those colors were considered when hypothesizing new genetic models in this research. The gene 2, which is designed to control flower variegation by Craig (9), was not included in this research. 16 17 Table 4. Phenotypes and the Related R.H.S. numbers For The Parents and Their Cross For Orange Flower Color Inheritance In 2 x h CROSSES PARENT l RHS # PARENT 2 Orange 338 Crimson Red Deep Salm. Lt. Salm. Rose/wh. Lt. Pink Picotee White 46B 43A 39B 48C 57B 49B CROSS(F1) RHS # Crimson 47B Red 42A Red Org. 40A Red Org. 403 Red Org. 40A Red Org. 408 Peach 52C Peach 52C 2 no R.H.S. # available. 18 Table 5. Phenotypes of Reciprocal Crosses For Orange Flower Color Inheritance In E x h CROSS CROSS(F1) FEMALE R.H.S.# MALE R.H.S.# PHENOTYPE R.H.S.# 2 83-548 468 85-79E 338 86-1 478 Crimson Orange Crimson 85-79D 5‘ 333 83-548 4513 86-2 478 Orange Crimson Crimson 84-10A -y 35-7913 2 333 86-15 52c White Orange Peach 85-79D z 338 84-10A -y 86-16 52C Orange White Peach 84-10A -y 82-60C 578 86-29 69C White Rose/Wh Lt.Rose/Wh 82-60C 578 84-10A -y 86-28 69C Rose/Wh White Lt.Rose/Wh 2 Y sister plants. no R.H.S.# available. RESULTS OF THE CROSS INVOLVING CRIMSON AND ORANGE The pedigree information for the cross of crimson and orange is listed in Table 6. Three plants were selected from the cross of crimson x orange, and 2 plants for the reciprocal cross of orange x crimson to produce the F2 families. The colors of the F1 plants differed only very slightly from the crimson parent going to a color chart rating of 478 which is slightly lighter in color. The F2 segregating results from both cross combinations are listed in tables 7 and 8. A monogenic difference between these two parents is hypothesized. The crimson color used in this research ( RHS # 478 ) is very close to Craig’s red color ( HCC # 819 )(9). The red genotype in Craig’s study was reported as EESQSQVV. Possibly a new gene 9; is operating for the color difference in this cross. Dominant 9; gene is proposed to govern the crimson flower color, and the homozygous recessive 9; gene will give the orange flower color. The crimson color is completely dominant to the orange. The F2 segregation fits a 3 to 1 ratio, and the population was homogenous for both F2 populations. The hypothesis was also verified by the backcross results of Table 40 in the appendix. The small color difference of the F1 phenotype to the crimson parent was probably due to the function of a modifier gene designated as i in this research. The proposed genetic model for the cross crimson and orange is listed in Table 9. 19 Table 20 6. Pedigree Information of the Cross Crimson and Orange For the Orange Flower Color Inheritance In Pelargonium x hortorum. PEDIGREE NUMBER CROSSES P1 P2 F1 Phenotype F1 F2 and RHS # Plants Families 86 - 1 83-548 85-79E z Crimson (478) 27 3 86 - 2 135—791)” 83-548 Crimson(47B) 12 2 z sister plants. Table 7. CHi - Square test on F2 Progenies of the Hybrids From the Cross 86-1 (Crimson and Orange) For Orange Flower Color Inheritance Of 3. x Q4 OBSERVED x2 2 FAMILY CRIMSON ORANGE TOTAL D.F. (3:1) P 86-1-1 10 4 l4 1 .095 .75 86-1-6 12 8 20 1 2.400 .15 86-1-4 15 6 21 1 138 .70 Total Y 37 18 55 3 2.633 .45 Expect 41.25 13.75 55 1 1.752 .20 2 all the chi-square values are non-significant at the .05 level. y the population homogeneity P=.70 21 Table 8. Chi - Square test On F2 Progenies of the Hybrids Resulting From the Cross (86-2) of Orange x Crimson For the Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVATION x2 2 FAMILY CRIMSON ORANGE TOTAL. D.F. (3:1) P 86-2-8 17 6 23 1 .014 .90 86-2-9 12 s 17 1 .176 .70 Total " 29 11 40 2 .190 .90 Expect 30 10 40 1 .133 .75 2 all the chi-square values are non—significant at .05 level. y the popuflation homogeneity P=.95. Table 9. Proposed Genotypes For The Cross Involving Crimson and Orange For Orange Flower Color Inheritance In B x h. Crimson Orange Parents OrOrPPSaSaii ororPPSaSaii Fl Crimson z Ororii F2 Crimsonz 3 Or-ii Orange 2 l ororii Z PPSaSa is omitted. RESULTS OF THE CROSSES INVOLVING DEEP SALMON AND ORANGE The pedigree information for deep salmon and orange is listed in Table 10. In the cross combination of 86-8 and 86-24, twenty nine and forty six Fl plants respectively were obtained, which were all red orange( 40A ). The F2 families generated from these two crosses gave the same segregation phenotypes, red orange, orange and deep salmon ( Tables 11 and 12 ). The chi - square and family homogeneity tests for the 9:3:4 segregation ratio suggest that 2 genes are operating in the color inheritance of deep salmon with orange. The deep salmon and the orange parents were all homozygous for flower color ( table 2 and 3 ), and the genotype for the ( deep ) salmon flower color was proposed as PPsasaVV by Craig (9). The salmon flower color he used had a 618/620 rating from the Horticultural Color Chart of 1938 (9). The deep salmon flower color used in this research had a 398 rating from the Royal Horticultural Society Color Chart (21). These two salmon colors matched each other (22). Previously, in the cross of crimson x orange, it was hypothersized that orange flower phenotype has a genotype of ororPPSaSaii. The genotype for the (deep) salmon flower color as reported by Craig is PPsasaVV (9). Therefore, the genotypes for the deep salmon flower in this research can be postulated as ororPPsasaII, and the orange flower colored parents ( 85-79D and 85-15A ) are ororPPSaSaii, where the dominant I has a 22 23 Table 10. Parental information of the Cross Deep Salmon and Orange For the Orange Flower Color Inheritance In 2 x h. PEDIGREE Fl PHENOTYPE NUMBER CROSSES P1 P2 AND RHS # F1 F2 86-8 82-27A 85-79C RED ORANGE 29 3 ( 40A ) 86-23 84-33D 82-27A RED ORANGE 25 2 ( 40A ) DP. SALMON 25 5 ( 39B ) 86-24 85-15A 82-27A RED ORANGE 46 6 ( 40A ) Table 11. Chi - Square test On F2 Progenies of the Hybrids Resulting From the Cross 86-8 (Deep Salmon x Orange) For the Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVED XI 2 FAMILY RED ORG. ORG. DP.SALM TOTAL D.F. (9:3:4) P. 86-8-5 6 3 3 12 2 33 .80 86-8-23 15 4 6 25 2 .17 .93 86-8-32 19 7 10 36 2 .20 .90 Total y 40 14 19 73 6 .70 .95 Expect 41.06 13.69 18.25 73 2 .07 .97 2 all the chi-square values are non-significant at 0.05. y the population homogeneity P=.96. 24 diluting effect and the recessive ii has an intensifying effect in the presence of the dominant S; red gene. This cross showed a two gene epistatic inheritance. When the recessive ii gene appears with 932;, an orange color is present: while the I; with gggg shows a red orange color. These all require the presence of the dominant §a_ gene. A deep salmon flower is produced when the recessive S; appears with either the dominant or recessive of the I gene. The hypothesized genotypes for this cross are listed in Table 13. The backcross results as shown in Table 41 and 42 in the Appendix support this two gene interaction inheritance pattern. 25 Table 12. Chi - Square test On F2 Progenies of the Hybrids Resulting From the Cross 86-24 of Orange x Deep Salmon For the Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY OBSERVED TOTAL D . F. Y“ z PROB. RED ORANGE ORANGE DP.SALM. (9:3:4) 86-24-3 28 18 16 56 2 4.85 .10 86-24-9 12 2 8 22 2 2.24 .30 86-24-14 50 20 29 99 2 1.42 .50 86-24-21 5 4 4 13 2 1.90 .40 86-24-48 23 10 17 50 2 2.60 .25 86-24-57 24 9 8 46 2 .35 .80 Total 7' 142 63 87 292 12 13.36 .30 Expect 164.25 54.75 73 292 2 5.75 .06 2 all chi-square values are non-significant at 0.05 level. y the population homogeneity P=.75. Table 13. Proposed Genotypes of The Cross Deep Salmon and Orange For The Orange Color Inheritance In E x h. Deep Salmon Orange parents PPororsasaII PPororSaSaii F1 Red Orange 5 ororSasaIi Red Orange ‘2 F2 ororSaSaII 9 Orange 2 ororSaSaii 3 Deep Salmon 2 ororsasaI 4 ororgasaii z the homozygous 22 is omitted. 26 The cross 86-23 ( Table 10 ) produced 50 F1 plants which segregated 25 red orange ( RHS # 40A ) and 25 deep salmon ( RHS # 39B ). This segregation suggests that the 84-33D orange flowered parent had one heterozygous locus g; which was proposed to produce the deep salmon color when the homozygous recessive condition ( Table 2 ). When the orange flower line 84-33D was selfed, the segregation was very close to a 3 orange to 1 deep salmon ratio. The F2 segregation results in table 14 and 15 supported this hypothesis. The genotype for 84-33D orange flower colored parent was postulated as PPororSgsgii, and the deep salmon parent was PPororsasaII. The red orange F1 plant, with a possible genotype of PPororSasaIi, generated red orange, orange and deep salmon phenotypes in F2 ( Table 14 ). The deep salmon Fl plant PPororsasaIi generated only deep salmon F2 progenies ( Table 15 ). The hypothesized genotypes for the cross of orange ( 84-33D ) and deep salmon ( 82-27A ) are given in Table 16. The chi - square and family homogeneity tests ( Tables 11, 12, 14 and 15 ) support these hypotheses. The low number of red-orange plants produced in the family 86-23-17 may possibly be due to the chance alone because of the small population size. 27 Table 14. Chi - Square test On the F2 Progenies of the Orange Red Hybrid Plants Resulting From the Cross 86-23 of Orange x For Inheritance In Salmon Deep Orange Flower Color Pelargonium x hortorum. the FAMILY OBSERVED TOTAL D.F x2 z P RED ORANGE ORANGE DEEP SALM. (9:3:4) 86-23-9 108 32 44 184 2 1.04 .70 86—23-17 32 13 23 68 2 5.34 .07 Total 3' 140 45 60 252 4 6.38 .20 Expect 141.75 47.25 63 252 2 2.54 .45 2 all the chi-square values are non-significant at .05 level. y the population homogeneity P=.20. Table 15. F2 Results of the Deep Salmon Hybrid Plants Resulting From the Cross 86-23 of Orange x Deep Salmon For the Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY OBSERVED Z Total DP.SALM. 86-23 -3 7 7 86-23-4 24 24 86-23-13 12 12 86-23-16 17 17 86-23-18 23 23 Total 83 83 Expect 83 83 z non-segregating F2 families. 28 Table 16. Hypothesized Genotypes For the Cross 86-23 of Orange x Deep Salmon For the Orange Flower Color Inheritance In 2 x h. Orange Deep Salmon Parents PPororSasaii PPororsasaII Fl Red Orange Z ororSasaIi Deep Salmon 2 weasel; F2 From Red Orange(ororSasaIi) hybrid Red Orange 2 9 ororSa I Orange 2 3 ororSa ii Deep Salmon 2 4 ororsasaI W F2 from Deep Salmon(ororsasaIi) Hybrid Deep Salmon.z All ororsasaI orogsgsgii z Homozygous 22 is omitted. RESULTS OF THE CROSS INVOLVING LIGHT SALMON AND ORANGE The pedigree information is listed in Table 17. Two plants were selected from the F1 of cross 86-11 and 10 from 87-157 to check F2 segregation ratios ( Table 18 and 19 ). Due to a greenhouse freeze in January of 1987 before this group and some other backcross plants flowered, only a few plants survived within the family to record the flower color data. Because of the small population size, the data from all those 87-157 families were pooled. The F2 gave similar results as for the cross deep salmon and orange. The red orange flower color (RHS # 408) is slightly lighter than the the red orange (RHS #40A) in the cross of orange and deep salmon. The non-significant results for the chi-square test, the population homogeneity test and the backcross results ( Tables 43 and 44 in the Appendix ) suggest that a recessive allele to §§, g; g; interacts with EEQrQrII. The genotype for the light salmon parent is hypothesized as PPororsa‘safII and for orange PPororSaSaii. The red orange hybrid is produced when the heterozygous I; interacts with the homozygous recessive 9; orange color gene in the presence of Sasg‘ £2. The hypothesized inheritance patterns for this cross and their progenies are listed in Table 20. 29 30 Table 17. Pedigree and F2 Family numbers of the Cross Light Salmon and Orange For the Orange Flower Color Inheritance In Pelargonium x hortorum. ( for the cross of 86-11 and 87-157). PEDIGREE F1 PHENOTYPE NUMBER CROSS P1 P2 AND RHS 4 F1 F2 FAMILIES 86-11 81-10C z 85-79Dy RED ORANGE 6 2 (LT.SALM) (ORANGE) ( 408 ) 87-157 85-58 2 85-7987 RED ORANGE 53 10 (LT.SALM) (ORANGE) ( 405 ) z sister plants. y sister plants. Table 18. Chi - square test On F2 Progenies of the Hybrids Resulting From the Cross 86-11 of Orange x Light Salmon For the Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVED x2 2 FAMILY RED ORG. ORG LT. SALM TOTAL D.F. (9:3:4) P 86-11-3 16 8 6 30 2 1.34 .50 86-11-4 14 3 5 22 2 .56 .75 Total 5’ 30 11 11 52 4 1.90 .75 Expect 29.25 9.75 13 52 2 .49 .80 z all the chi-square values are non-significant at .05 level. y the population homogeneity P=.45. 31 Table 19. Chi - Square test On Pooled F2 Progenies of the Hybrids Resulting From the Cross of Orange x Light Salmon For the Orange Flower Color Inheritance In Pelargonium x hortorum. Families Observed Total D.F. X2 .2 P Red Orange orange Light Salm. (9:3:4) 87-157-1 87-157-2 87-157-4 87-157-6 87-157-8 87-157-9 87-157-11 87-157-12 87-157-13 87-157-14 Total 40 l4 14 68 2 0.73 .70 Expect 38.25 12.75 17 68 z the chi-square value is non-significant at .05 level. 32 Table 20. Hypothesized Genotypes For The Cross of Light Salmon x In the Orange and Their Progenies Color Inheritance In Pelargonium x hortorum of the Crosses 86-7 and 87-157. Parents F1 F2 Light Salmon PPororsa‘salII Orange PPororSaSaii Red Orange 2 ororSasaIIi Orange 2 3 ororSaSaii Red Orange z 9 ororSasa‘I Light Salmon 2 4 ororsa‘sa‘I ororsa‘sa‘ii 2 PE is omitted. RESULTS OF THE CROSS INVOLVING ORANGE AND WHITE Twenty-one and 22 hybrids were produced from the orange x white and reciprocal cross respectively ( Table 21). All of these plants had a peach flower color ( RHS# 52C ). Within the 2 groups, 10 F2 populations were generated. In the F2 ( Tables 23 and 24 ) progenies, there was a close fit to a 1:2:1 segregation ratio for orange, peach and white. Chi - square results, the population homogeneity test data ( Tables 22 and 23 ) and the backcross results ( Tables 47, 48 and 49 ) suggest that an incomplete dominant orange over white inheritance pattern is operating in this cross. The W gene is proposed to control this monohybrid inheritance in the presence of the homozygous recessive 9;. The homozygous recessive 3 gives white color, the homozygous dominant W gives orange color, and the heterozygous W! shows peach flower color ( Table 25 ), all in the presence of oror. 33 34 Table 21. Parental information of the Cross of Orange and White For the Orange Flower Color Inheritance In Pelargonium x hortorum (For the Crosses 86-15 and 86-16). PEDIGREE NUMBER CROSS P1 P2 F1 PHENOTYPE F1 F2 FAMILY AND RHS 4 86-15 85-79Bz 84-10A PEACH 21 3 ( 52C ) 86-16 84-10A 85-79Dz PEACH 22 7 ( 52C ) z sister plants. Table 22. Chi - Square Test Results On F2 Progenies of the Hybrids Resulting From the Cross 86-15 of Orange x White For the Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVED x1 z FAMILIES ORANGE PEACH WHITE TOT. D.F. (1:2:1) P 86-15-1 6 20 6 32 2 2.00 .40 86-15-2 9 6 4 19 2 5.21 .07 86-15-15 7 10 5 22 2 .59 .75 Total 3’ 22 36 15 73 6 7.80 .25 Expect 18.75 37.5 18.75 73 2 1.74 .45 all the szalues are non-significant at .05 level. y the population homogeneity P=.20. 35 Table 23. Chi - square Test Results On F2 Progenies of the Hybrids Resulting From the Cross 86-16 of White x Orange For the Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILIES OBSERVED Total D.F. x1 2 P ORANGE PEACH WHITE (1:2:1) 86-16—1 10 18 6 34 2 1.06 .60 86-16-2 2 11 6 19 2 2.16 .30 86-16-13 1 3 1 5 2 .20 .90 86—16-15 9 14 8 31 2 .35 .85 86-16-16 9 37 15 61 2 3.95 .18 86-16-7 2 2 4 8 2 3.00 .22 86-16-12 10 15 5 30 2 1.67 .50 Total y' 43 100 45 188 14 12.39 .60 Expect 47 94 47 188 2 .81 .70 2 all the X’values are non-significant at the .05 level. y the population homogeneity P=.50. Table 24. 36 Hypothesized Genotypes For The Crosses 86-15 and 86-16 of White and Orange and Their Progenies In the Orange Flower Color Inheritance In 2 x h. Parents Fl 2 the homozygous orange White ororWW oroggg Peach 2 W! Orange-2 1 WW Peach 2 2 W! White 2 l ww recessive oror is omitted. RESULTS OF THE CROSS INVOLVING ORANGE AND ROSE/WHITE Ten red-orange flowered plants ( RHS # 40A ) were produced from the cross of orange x rose/white ( Table 25 ). Seven red-orange Fl plants were selfed to generate F2 families. The segregation data for the F2 progenies are shown in Table 26. The data showed a monogenic difference between the orange and the rose/white flower parents. The rose/white ( RHS# 57B ) is the same color as the medium pink ( HCCR 627/1 ) which was studied by Craig (9). Craig reported the genotype for medium pink as ppSaSaVV. This genotype was confirmed later by Badr and Horn (3). With the genotype for the orange flower parent being PPSaSaWWii in the presence of gggg as proposed in this study, the F2 data suggests that the B gene, which has been reported to control red and pink ( rose/white )(9), interacts with the homozygous recessive 9; orange color gene to give orange, red orange and rose/white flowers. Thus BPgro; gives orange, pporor gives rose/white, and Eporor gives a red orange flower color. The data did not show any function of the modifier gene I proposed in previous crosses, which means that the gene 2 is probably epistatic to gene I. The hypothesis of the monogenic inheritance between orange and rose/white is accepted by chi - square and family homogeneity tests ( Table 26 ), and by the backcross results ( Table 50 in the Appendix ). The proposed genotypes for the cross of orange with rose/white and their progenies are presented in Table 27. 37 38 Table 25. Pedigree information of the Cross of Orange and Rose/White (86-20) For the Orange Flower Color Inheritance In Pelargonium x hortorum. PEDIGREE Fl PHENOTYPE NUMBER CROSS P1 P2 AND RHS # F1 F2 PLANTS FAMILIES 86-20 85-16A 82-60C RED ORANGE 10 7 (ORANGE) (ROSE/WHITE) (40A) Table 26. Chi - square test On F2 Progenies of the Hybrids Resulting From the Cross 86-20 of Orange x Rose/white For the Orange Flower Color Inheritance In 2 x h; FAMILIES OBSERVED TOTAL D.F. x1 P ORANGE RED ORANGE ROSE/WH. <1:2:1>z 86-20-1 4 5 5 14 2 1.29 .60 86-20-3 33 59 19 111 2 3.97 .15 86—20-4 5 5 9 19 2 5.95 .05 86-20-6 2 5 4 11 2 .82 .70 86—20-7 7 9 5 21 2 .81 .70 86-20-8 2 3 1 6 2 .33 .85 86—20-9 30 43 19 92 2 3.02 .20 Total 7’ 83 129 62 274 14 16.19 .13 Expect 68.5 137 68.5 274 2 4.15 .13 2 all the Xtvalues are non-significant at the .05 level. y the population homogeneity P=.40. 39 Table 27. Hypothesized Genotypes For The Cross 86-20 of Orange x Rose/white and Their Progenies In the Orange Flower Color Inheritance In 2 x h. Orange Rose/White Parents PPororSaSaWWii ppororSaSaWWII F1 Red Orange 2 Eporor Orange 2 l PPoror F2 Red Orange2 2 Eporor Rose/White z 1 QQOI'OI z SaSaWWIi is omitted. RESULTS OF THE CROSS INVOLVING PICOTEE 3 ORANGE From the cross of picotee x orange, four peach hybrids were selected to produce F2 families ( Table 29 ). The F2 result is similar to the cross of white x orange in that a diluting factor in the picotee parent diluted the orange color to the peach color. There were six distinctive flower color phenotypes in the F2 segregation. They were peach, orange, rose/white, deep salmon, light pink and picotee. All the colors have been described above except the light pink. The F2 segregation data ( Table 51 in the Appendix ) suggests a trihybrid inheritance, but no suitable genetic model could be derived to explain the segregation ratio. This may be due to the possibility that the picotee parent was not really homozygous and can appear as two different genotypes (9). Further research will be necessary to determining the inheritance for the cross of picotee and orange by first determine the actual genotype of possible picotee parent plants through testing them with other known colored plants and then crossing them with the orange genotype for analysis. 40 41 Table 28. Pedigree information For The Cross of Picotee and Orange For the Orange Flower Color Inheritance In 2 x h. PEDIGREE NUMBER CROSS P1 P2 Fl PHENOTYPE F1 F2 AND RHS # PLANTS FAMILIES 86-27 80-38 85-15A PEACH 40 4 (PICOTEE) (ORANGE) ( 52C ) SUMMARY OF THE ORANGE FLOWER COLOR INHERITANCE Five genes have been hypothesized to control the orange flower color inheritance in Pelargonium x hortorum. They are 2, 9;, SQ, fl and i. The E and SQ genes have been previously reported by Craig (9): The E and SQ give red, 22 gives pink, §Q§Q gives deep salmon. The S gene in this research has the same function as that reported by Badr and Horn (3). fl_ gives colored phenotype and 3! gives white. The recessive ! is epistatic to all other genes. An allele to SQ, gafgives light salmon in the homozygous recessive condition. Q;_ gives crimson, 252; gives orange. The S gene dilutes the flower color and it’s homozygous recessive ii intensifies the flower color in the presence of the dominant SQ and 3 genes. Both complete and incomplete dominant inheritance patterns have been found for the inheritance of orange flower color in this research. The 9; gene is different from the 9 gene hypothesized to control the X - ray mutation originated orange flower color in 2 x h studied by Wernett (25). The homozygous recessive 9; gene combining with dominant 2, SQ, !, and recessive intensify gene ;; gives the orange flower color in this research. The difference in the results between Wernett’s and the present research may be due to the different sources of the orange material used. Wernett used the orange, picotee and blush sister plants as parents, which came from the same X - ray originated plant. 42 43 Besides Or, the gene I and the allele 3:1 are new in this study. Considering all 5 genes, the genotypes for the parents used in this research are shown in Table 29. The line chart form (19) is used to represent all the genotypes and the related phenotypes ( Figure 2 ) which were studied in this research. The genes are listed in a horizontal line with the dominant gene on top of the line, and the incomplete dominant and the recessive gene below the line. The epistatic genes are represented by parallel lines down from the hypostatic genes to the right a short distance below the one from the epistatic genes. To determine the genotype of any given phenotype start at the selected color and follow the line to the left and upward until all genes are accounted for. 44 Table 29. Proposed Genotypes Of The Parents Used In This Research For the Orange Flower Color Inheritance In E x g Parents Phenotype Genotype 83-548 Crimson OrOrPPSaSaWWii 82-27A Deep Salm. ororPPsasaWWII 85-798 Orange ororPPSaSaWWii 85-79D Orange ororPPSaSaWWii 84-33D Orange ororPPSasaWWii 81-10C Light Salmon ororPPsa‘sa‘WWII 82-60C Rose/white ororppSaSaWWII 84-10A White ororPPSaSawwII 45 Figure 2. Line Chart Presentation For The Interactions Of Q£,§Q(SQ ),P,W and l, and The Related Phenotype; FOP the Inheritance Of Orange flower color in Pelargonium x hortorum. W Or P Sa I ------ — ------- 1 - ? i--- Crimson L P 33 _______ _ ——------- Orange or i P . ______________ --— Orange Sasa i P I r ______________ ~ Deep Salmon sa P — — --— Deep Salmon sa Ii P a _________________ Deep Salmon sa i P I u ————————————————————— Light Salmon sa1 P ————————————————— Light Salmon sa1 Ii P 1. _____________________________ Light Salmon 1 46 Figure 2. ( cont’d ) W P P _________ _ _ or Sasa Ii P Sasa1 11 Sa Pp Ii Sa I L- _- _________ Sa fix or Ii P Sa I Red Orange Red Orange Red Orange Rose/white PIGMENT ANALYSIS Paper chromatography (P. C.) and thin - layer chromatography (T.L.C.) methods were conducted to analyze the anthocyanin pigments of the parents and their F1 hybrids. In the P.C. method the three solvents BAW, BuHCL and 1% HCL were tried. The 1% HCL solvent system gave very poor separation results and was discarded. To confirm the results of the P.C. method, the T.L.C. procedure was used with the BAW solvent system. In each cross combination, the Rf values, band numbers, visible band colors and band colors under ultra violet light for both the parents and their Fl hybrids were investigated, and the anthocyanin pigments were identified. Piggent Analysis For The Cross 9: Crimson Qng QgQQgg In the cross of Crimson x orange ( Table 30 ), two bands were found in the crimson parent (83-548 ). They were orange and purple respectively under visible light. Under U.V. light they were fluorescent yellow and dull purple colors. After comparing these band colors and the tested Rf values with published standards ( 13, 15, 17 and 23), they were identified as petunidin. The orange parent ( 85-79E ) showed only one band, which gave orange color under visible light, and fluorescent yellow color under U.V. light. It was identified as pelargonidin. The F1 hybrid showed both bands which had been appeared in the crimson parent. 47 1 rs”! |ll .eee_e=ooa - .oa xe_oeeoote_oa - .oa m .283 :3 .. .36 “26:9. 2838:: .. S.» N .282. .. .3 "mace; .. ..8 H 48 .oa mm. co. as. .a.o .aa N 1_coeo axe come—to ea. s-ee .oa .me. e”. an. .a.e .to a o e e e eQe e ea as so 2N 6 ea N eeeoeo nee eooeeeo a eom-me .oa me. Ne. em. .a.e .to s .oa mo. 61. mm. .x.e .46 e “Hummm amm ooeeeo a mox-mo 6.8 mecca 3.383 .895: 8.395.: 53828 23.5: EU ceee . max 6 _a U moo1e_oeoe_ 3am BU==e gee Mmhma e 22.51; E 9.; Rudd 5b: .3; I! d a x m =_ mecca—emzcm xo_ou euro—m macexo use Lou maceeo x comet“. 9:362: $95 93 co c.3365 2.253 .8 ~33 49 This result further confirmed the hypothesis that the crimson color is completely dominant to the orange flower color. The 9; gene, supposed to control the crimson color, is hypothesized also to control the pelargonidin and petunidin biosynthesis. When it is homozygous recessive, gggg, only pelargonidin is produced. This hypothesis is further supported by the pigment analysis result in the reciprocal cross orange x crimson ( Table 31 ). Pigment Analysis £2; [£3 Cross _§ Sggg Salmon Qgg Orange Two bands were found in the deep salmon ( 82-27A ) parent. They were identified as pelargonidin and delphinidin. The orange parent 85-79D, 84-33D and 85-15A all showed only one band, identified as pelargonidin ( Tables 32, 33 and 34 ). F1 hybrids ( 86-8 ) of deep salmon ( 82- 27A ) x orange ( 85-798 ) showed three bands ( Table 32 ). Two of them were identified as pelargonidin, and one band was delphinidin. The reason for the two pelargonidin bands appearing is not quite clear. This may possibly be caused by the modifier gene S which was postulated in the genetic studies of the cross involving deep salmon and orange. The genes 9!, SQ and l which control flower colors in this cross are proposed also to control the pigment synthesis. Thus ororSaSaii controls the pelargonidin production, and ororsasaII controls the pelargonidin and delphinidin production. The hybrid ororSasaIi produces both pelargonidin and delphinidin. 50 .cercauma . pa “catacomxo_ma 1 .ma m .mpaeso __:u . .a.c ”soppmz pcmummxoapm . .x.» N .mFaxza -.aa momentouxo H .ea . . . . . . ------ we we AH a 6 ea N estate mxo eooaeeo He N-ea .aa me. Na. am. .x.e .to 2 .ea me. me. 2N. .a.c .aa N ------ eeeoeo moo eooeeco a mom-mm .oa me. NH. em. .a.e .to H .oa mme. es. mum. .x.e .to a “flummm mam ooceto a mox-mm o a» one as: on» cam no peace 1 En: eoeeweeoea 3am oozes 3am moeo Leo—co ocean o e 6 too 6 ca 16 8 one m econ econ mzm » —a 855: E 6.: 1:13.? LEV Pas/l . m x m :H moccuexmzcn Lopou Lmzopu manage on» com comsexu x moccxo.m:e>_o>:u mmoxu we» we mama—mc< “seamed .flm opens 51 .eeoee_eaHoa - .ao scenecomonma . .mo m HmHoeaa HHsu . .a.u Honme pcmummxoaHe u .».e N .omccxo no; . .o.x HoHaxaa . .aa .umppHEo we Loco m>Hmmmumx msomxuoso; x “mmcoxo . ..S H .ae am. so. mH. .a.c .aa .ma 1 mo. cm. .1.6 .o.x wmmmmw .: 23> m x m cH wocmupemscH LoHou xmond macexo mg» Lou mmcceo x cos—om noon mcH>Ho>=H mmoxu as» to mHmaHmc< “swamps .mm mHnmh 52 . . .cHnwcwsaHoa . .ao HcHuwcommcHoa n .ma m .mHoaza HHau r .a.c_monme accommeoaHe . .».e N mHaxaa t .aa memento . .10 H .umpoweo NH xoxo m>wmmmumx unomANoeo; one a can I e . e on». 0:“ III-cell .oa - Mm. mm. .x.w .2“ m HHemeo Nam eoeHem He ammo . ea. I . e e .e .e e . III-III: a mo . NN a 6 ea N HHeoam (co oaeato Ha NN-eN toa - NH. NN. .x.e .26 H . cox .ae am. No. NH. .a.o .aa N .oa .Nm. HH. mN. xx.e .26 H HHNNNN Nam eocHem a aNN-NN . aooe om. . . o a . ext oko 11!!!! a. No 6H am e H Hemmem mmm ooeoto a emm-eN *. o co xo co m com maauocmu amass: maxuocmga copuoxocmw topaz: NooHeHHeocH 34m Hozzm 3am Mensa H once 6 xxx HceHa H5521 E o: 13.: .>.: on; m x m cH mocmHHLmscH eoHoo xmonu macexo mg» Lou 55.3 aooo x. cacao 65:25 395 25 .6 c.5322 Home: .8 £92 53 .cHuHchquo . .ao HcHeHcomxcHoa - .oa m .oHaeaa some n .q.v «so—Hos NcoummeoaHe . .x.e N .mHaeaa . .aa memento . .10 w .mHaoHHc>c Ho: m. came one i .coHNHEo NH Loco m>Hmmmome ozomaNoeo; one a .ae - so. mH. .a.o .aa N -------- - .oa - NH. NN. .x.e .26 H eHeoem ace ooemmw Ha eN 6N .ae NN. Ho. NH. .a.o .aa N -------- - .oa am. HH. NN. .x.e .to H HHeoeo mom eofiwww a «AN NN on o o o ox. o llllllll a No ac am e to H HHemaN NNN ooeeto a «NH-NN . _ a cam x 5:: on» ozmH co cameo x as: ooHLPoeoos 3am 3o:=N 3am moHoo HtoHoo mecca oa H o ea 6 .a HH e one m econ econ mzm u Ha 1 65.3: Eb: E3; 5.: gm; a x m :H muccuHLoch eoHou gaze—u macexo oz» god .853 noon x mucosa one—39:: 36.6 9: co c.3385 Hausa—a .3 ~23 54 In the cross 86-23 ( orange 84-33D x deep salmon 82-27A ), two different colored plants appeared, red orange and deep salmon. Both hybrids showed the same pigments of pelargonidin and delphinidin. As shown in Table 33, the 84-33D orange parent showed only one pelargonidin band, and the deep salmon parent ( 82- 27A ) showed 2 bands which were identified as pelargonidin and delphinidin. Similarly as that in cross 86-8, the genes of 9;, SQ and l, which are hypothesized to control the flower color inheritance in this cross, are also proposed to control the pigment production. Thus, ororsasaII controls pelargonidin and delphinidin production; while ororSasaii controls only pelargonidin production. The hybrids ororSasaIi and ororsasaIi produce both pelargonidin and delphinidin. For the cross of 86-24 ( Table 34 ), the orange parent ( 85-15A ) which is hypothesized as ororSaSaii, produces only pelargonidin, while The deep salmon parent ( 82-27A ) OrOrsasaII produces pelargonidin and delphinidin. Their F1 hybrids ororSasaIi Contains both pelargonidin and delphinidin. mmmmwfiwmal ahead-Mane The results for the cross involving light salmon and orange are shown in Table 35. Only one band was found for each parent. In the light salmon parent ( 84-10A ), the band was magenta and dull magenta color under visible and 55 .cHu_:omtona . .ma HcHuwcch . .xu .3oHme pcmummxosHm . .a.m "museums HHau . .e.v .mmccto . .xo Hepcmmce . .me .cmeHso mH Loco m>Hmmmume NaomaNoEo; use a v—INO’) exo . mm. No. 0N.. .e.o .oe N HHHomem Noe ooeeeo He HH-eN .oa Ne. HH. oN. .x.e .eo H not .3 8. OH. NN. :3 .6 H -HHNWNW- _ NNN ooeeto a SEN .ao me. No. HN. .e.o .62 H HHMNNMNN Nae eocHoe a ooH-HN . HeoHH 11 MW . s oo o co m :6 max one a as: on» o: coH exocm x as: ooHeeHcooH 3am oozes 3am moH H1 h c N H e on H oea .o o MmeHa N 6:68 e 66 max Hcoeoea em use ea.o.a .m>»=, .amr> . . m x m :H mocmpmeccH onou Loonm moccxo use tom mmcmxo use coeHem psme m=H>Ho>cH mmoxu ms» co memszc< acmEmHa .mm mHno» 56 U.V. light respectively. It was identified as cyanidin. The band in the orange parent ( 85-79D ) was identified as pelargonidin, which is proposed to be controlled by ororSaSaii. The genotype ororsa‘sa‘II, which is hypothesized to control the light salmon flower color, allowing cyanidin 1 production. Their hybrid contains both pelargonidin and ~ cyanidin produced by the genotype ororSasQ’I'. The results 5 l in this cross show that the SQ‘, an allele to 5Q, does not produce pelargonidin and delphinidin but cyanidin. Thus the resulting flower color is light salmon. B;gmggg Analysis For Eng Cross 9; White And Orange In cross 86-15, no band appeared for the white parent ( 84-10A ), thus no pigment is being produced ( Table 36 ). One band was found for the orange parent ( 85-798 ). This band was identified as pelargonidin. A second band appeared besides the pelargonidin band in their F1. This second band was identified as petunidin. Assuming homozygous recessive 3 controls white flower color and non - pigment production; W! colored flower phenotype governs the pelargonidin production. Their hybrid 23 gives not only pelargonidin but also the pigment petunidin. This new pigment is obviously related to the recessive suppresser of the 3 gene, but not the modifier(s), such as 1, since the recessive g is epistatic to any other genes in function as proposed in the flower color segregation study of white and 57 .eHoHeaeoa - .Ha meeoeeooeaHoa - .oa .mHoxaa HHan . .a.c Honme pcmummxoaHm . .x.m mecca anchH> o: u o: .mHaxza . .sa Hmmcmxo . .xo mecca mHneHmH> o: u o: .mmaHo> mHno>xmmno o: . HNM .Ha am. No. HN. .a.o .aa N satoto on eoeoa He .oa me. HH. mN. .a.e .to H .oa No. me. NN. .x.e .46 H mammmm NNN ooeeto a o: n u u o: o: o: -mmmmmm- on open: a mHnmm mmmamm Ho>cH mmoxu we» to mmeHoc< acmEmHa .mm ane» toHou mucem maXuocow amass: maxuocosa cowucchmo topaz: unmpd 58 .eHoHeaeoa - .Ha HeHcceooteHoa - .oa meoHtHeeooH Heocoea oe - 6: .chcaa HHau .c.c.monme HcmummcoaHe . .x.e .chxaa.- m HcoHou ace econ cheHmH> cc 1 oc N H .za "muccco . .co onHoo ucc econ cheHmH> oc . cc .mmzHc> chHmH> oc .Ha. mm. as. HN. .a.e .aa N ------ zzcoco umm comma He oHuom .oa Ne. HH. HN. .x.t .to H oe - - - ea 6: cc mmummm o: open: a coH-eN .oa me. oH. NN. .a.e .to H mmumum NNN ooeeeo a eoN-mN u A m :c a» c c ace c 5:: cmmcwucmnH 3»=. .Hmr> . m x m cH mucepmeccH coHou cmsoHu omcmco ecu cod 3.23 x mmcoco @5302: 396 9: .5 3922.4 ..HcmcaE .3633 59 orange. This hypothesis is further verified by the result of the pigment analysis for the reciprocal cross orange x white ( Table 37 ). Pigment Analysis For The Cross 9_ annge g RoseZWhite In Table 38, the orange parent ( 86-16 ) contained only pelargonidin. The rose/white parent ( 82-60C ) showed only one band identified as cyanidin. Their red orange hybrid gave both pelargonidin and cyanidin. The ppororSaSaII genotype which controls rose/white flower color would also control the cyanidin pigment production and the PPororSaSaii genotypes which controls orange flower color would control pelargonidin production. Their hybrid EpororSaSaIi synthesized both cyanidin and pelargonidin. This result matches the flower color segregation results showed in table 26. 60 .oHnoHHo>o poc NH ouoe . .cHeHconu 1 .HQ HcHeHcomcona . .ma m .opcomoe HHoe . .e.e HonHox ucmumocooHe . .>.e N .opcmmoe 1 .me Homcoco . .10 H .emfiHEo me “mum 65388 $8.322 one c .No - No. NN.. .c.o .oc N -- - HHNa coo ooeoeo H1 ON-oo .oa - HH. NN. .x.e .to H cot .ao mm. on. NH. .c.o .oc H HHNN . mam oeeea\oooc a ooo-NN .oa no. an. em. .H.e .to H HHmm NNN ooeoto a aoH-mo eoHeHHcoeH 3Ho>cH mmoco one co mHma—oc< ucoEch .mm oHno» SUMMARY OF PIGMENT ANALYSIS The identified pigments for each parents used in this research are shown in table 39. The pigment analysis results show that OrOrSaSaii synthesizes pelargonidin and petunidin, and ggongsaII synthesizes pelargonidin and delphinidin in the presence of BEES. The homozygous recessive 9; gene controls only pelargonidin synthesis in the presence of EB, SQSQ, fl! and recessive gene 1;. In the presence of gggg, 22 gene together with recessive sa1 sa1 or SQ together with homozygous 22 control cyanidin production; while the genotype PPSasa or EpSaSa produces both pelargonidin and cyanidin. The 3 in the homozygous recessive condition governs no pigment production, proposed as epistatic to all other genes. Figure 3 shows all the pigment analysis results. These results fit what would be expected from the flower color inheritance results. The result that the orange flower color contains only pelargonidin agrees with previous researches ( 25, 26 ). The genetic segregation results which differ from the Wernett’s study (25) may possibly be explained as: l) the lack of a wide range of crosses between orange and other flower colors in that research; 2) the different sources of the orange flower color; 3) the same problem that a flower color is controlled by both dominant and recessive gene(s) from different research has been found in some other ornamental plant species (19). 61 62 It has been shown from this research that the orange flower color is controlled by multi-loci. It seems that the only way to produce a seed produced hybrid orange flower colored B x S is by crossing orange inbreds together. This is true at least for the materials used in this research. Fir—f1 " 63 Table 39. Pigments Identified For All Parents Used For The Orange Flower Color Inheritance In 2 x h. Phenotype Genotype Pigments Identified Crimson PPOrOrSaSaWWii Pelargonidin and Petunidin Orange PPororSasaWWii Pelargonidin Deep Salmon PPororsasaWWII Light Salm. PPororsa‘sa‘WWII White EPOIQI§§S§WWII Rose/white DEDEDESESEEEII Pelargonidin and Delphinidin Cyanidin None Cyanidin 64 Figure 3. Line Chart Presentation Of The Genotypes And The Synthesized Pigments For The Orange Flower Color Inheritance In 2 x h. W Or P Sa ---. ———————————————————————————————— Pelargonidin i & Petunidin P Sa L ------------------------------- Pelargonidin or i P I ————————————————————————————————— Pelargonidin or sa & Delphinidin P H HHHHHHHHHHHH — ——————————— Pelargonidin or Sasa1 Ii & Cyanidin l I ---------------- Cyanidin sa1 Sa HHHHHHHHHH — — -—-----—- Pelargonidin or Pp Ii & Cyanidin Sa I __ ———————————————— Cyanidin P Or P Sa I L —————————————————————————————————————————— No Pigment w P Sa I __ — — --—- -- ~-—- No Pigment LITERATURE CITED 1. Agricultural Statistical Board. 1988. Floriculture Crop, 1987 Summary. Washington D.C., USDA, NASS. Apr. Sp Cr 6-1 (88), PP9-10. 2. Ahmedullah, M., W. J. Carpenter and H. L. Mitchell. 1963. Identification of anthocyanins in three cultivars of geranium (Pelargonium hortorum) by chromatographic and spectrophotometric methods. Proc. Am. Soc. Hort. Sci. 83:769-771. 3. Badr, M. and Horn, W., 1971. Cytooligische untersuchungen ber pelargonium zonale - hybriden. z. pflanzenzuchtg. 66: 203-220. 4. Buswell, G. E. 1978. Flower color and anthocyanin inheritance of tetraploid pelargonium x hortorum Bailey. Ph.D. Thesis. The Pennsylvania State University. 5. Carlson, W. 1986. One to grow on. 1986 seed geranium trails. Greenhouse Grower. September. p12-l3. 6. Chittenden, R. J. 1927. Inheritance of variegation. Biblogr. Genet. 3:355-439. 7. Cliford, C.D. 1970. Pelargonium including the popular geranium. Blandford Press, London. 2nd edit. 350pp. 8. Craig, R. 1962. Geranium pollination techniques. Geraniums Around the World 10(2):29-30. 9. Craig, R. 1963. The inheritance of several characters in the geranium, Pelargonium hortorum Bailey. Ph.D. Thesis. The Pennsylvania State University. 65 66 10. Craig, R. 1982. Chromosomes, genes, and cultivar improvement. In J. W. Mastalerz. Geraniums. A manual on the culture geraniums as a greenhouse crop. 3rd. edt. 350p. 11. Dewolf, G. 1983. Pelargonium. Horticulture. Nov. p8-9. 12. Ewart, L.C. 1982. Geranium seed treatment with H2804. Personal communication. 2p. 13. Griffiths, J. F. Anthony and Ganders R. Fred. 1983. Wild Flower Genetics. A field guide for British Columbia and the Pacific Northwest. 14. Harborne, J. B. 1958. The chromatographic study of species presumed ancestral to p. x hortorum Bailey. Can. J. Genet. 8:780-787. 15. Harney, P.M. 1966. A chromatographic studies of species presumed ancestral to P. x hortorium Bailey. Can. J. Genet. 8:780-787. 16. Mullick, D.B. 1968. Thin layer chromatography of anthocyanidins. Techniques and solvents for two- dimensional chromatography. J. Chromatog., 39:291-301. 17. Nugent, P. E. and R. J. Snyder, 1966. The inheritance of flower color and plant habit in pelargonium hortorum. Proc. XVII Int. Hort. Congr. Vol.1:20. 18. Nybom, Nils. 1964. Thin - layer chromatographic analysis of anthocyanidins. Phisiol. Plant. Vol.17:157- 165. 67 19. Paris, D. Clark, W. J. Haney, and G. B. Wilson. 1960. A survey of the interactions of gene for flower color. Michigan State University. Agr. Exp. Sta. Tech. Bul. 281. 20. Robinson, G. M. and R. Robinson. 1932. A survey of anthocyanins II. Biochem. J. 26:1647-1664. 21. Royal Horticultural Society Color Chart. 1966. Royal Hort. Society, London. 22. Royal Horticultural Society Color Chart. Table of cross - references. 1966. Royal Horticultural Society, London. 23. Scott-Moncrieff, R. 1936. A survey of some Mendelian factors for flower color. J. Genet. 32:117-170. 24. Stahl, Egon. 1969a. Thin - layer chromatography. A laboratory handbook, by H. R. Bollinger and others, English translation by Cambridge Consultants. Berlin, New York, Springer - Verlag. 553p. 25. Wernett, Heidy. 1982. The inheritance of orange flower color in Pelargonium x hortorum Bailey. M.S. Thesis. The Pennsylvania State University. 26. Williams, Susan. 1978. Inheritance of flower color in pelargonium x hortorum Bailey. M.S. Thesis. The Pennsylvania State University. APPENDI X 68 Table 40. Backcross Result Of ( Crimson x Orange ) x Orange For The Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVED xz FAMILY CRIMSON ORANGE Total D.F. (1:1) P 87-163 9 7 16 1 .250 .55 EXPECT 8 8 16 Table 41. Backcross Result Of ( Orange x Deep Salmon ) x Orange For The Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY CROSS OBSERVED Total D.F. xi’ P RED ORANGE ORANGE (1:1) 87—251 86-24-3 x 85-15A 9 7 16 1 .25 .60 87-220 86-24-57 x 85-15A 4 3 7 1 .14 .70 Total 2 13 10 23 2 .39 .80 Expect 11.5 11.5 23 1 .39 .53 z the population homogeneity P=1.0. 69 Table 42. Backcross result Of ( Orange x Deep Salmon ) Deep Salmon Inheritance In Pelargonium x hortorum. X For The Orange Flower Color FAMILY CROSS OBSERVED Total D.F. x2 RED ORANGE DP. SALM. (1:1) 87-216 86-24-3 x 82-27A 3 4 7 1 .14 87-217 86-24-14 x 82-27A 14 14 28 1 .00 1. 87-218 86—24-23 x 82—27A 3 2 5 1 .20 87-219 86-24-57 x 82-27A 8 10 18 1 .22 Total 3 28 30 58 4 .56 Expect 29 29 58 1 .07 z the population homogeneity P=.94. 70 Table 43. Backcross Result Of ( Light Salmon X Orange ) x Light Salmon For The Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY CROSS OBSERVED Total. D.F. x1 P RED ORANGE LT. SALM. (1:1) 87-192 86—11—3 x 81-10C 5 3 8 1 .50 50 87-194 86—11-4 x 81-10C 7 6 13 1 .08 80 87—196 86—11-5 x 81-10C 5 8 13 1 .69 .40 Total 5 l7 17 34 3 1.27 .75 Expect l7 17 34 l 0.00 1.00 z the population homogeneity P=.55. Table 44. Backcross result Of ( Light Salmon x Orange ) x Orange For The Orange Flower COlor Inheritance In Pelargonium x hortorum. FAMILY CROSS OBSERVED Total D.F. x1~ P ORANGE RED ORANGE (1:1) 87-195 86-11-5 x 85—79D 8 9 l7 1 .06 .80 8.5 17 Expect 8.5 71 Table 45. Backcross Result 0f ( Light Salmon X Orange ) x Light Salmon For The Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILIES OBSERVED x2 RED ORANGE LIGHT SALMON Tot. D.F. (1.1) P Total 63 57 120 1 .30 .50 Expect 60 60 120 1 Table 46. Backcross Result of ( Light Salmon x Orange ) x Orange For The Orange Flower COlor Inheritance In Pelargonium x hortorum. FAMILIES OBSERVED Xz RED ORANGE ORANGE Total D.F. (1:1) P 87-246 87-249 87-256 87-264 87-269 87-276 Total 19 21 40 l .10 .75 Expect 20 20 40 1 72 Table 47. Backcross Result of ( Orange x White ) x Orange For the Orange Flower Color Inheritance In Pelargonium x hortorum. OBSERVED xi FAMILY CROSS ORANGE PEACH Total D.F. (1:1) P 87-37 85-79B x 85-15—1 7 6 13 1 .08 .75 EXPECT 6.5 6.5 13 Table 48. Backcross Result of ( Orange x White ) x White For The Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY CROSS OBSERVED Total D.F. X2 P PEACH WHITE (1:1) 87-38 86-15-2 X 84-10A 13 ll 24 l .17 .65 EXPECT 12 12 24 73 Table 49. Backcross Result of ( White x orange ) x White For The Orange Flower Color Inheritance In PelQrgoniug x hortorum. FAMILIES CROSSES OBSERVED Total D.F. x2 P PEACH WHITE (1:1) 87-46 86—16-7 x 84—10A 5 8 13 1 .69 .45 87-48 86-16-12 x 84-10A 3 2 5 1 .20 .60 87-202 86-16-16 x 84-10A 7 6 l3 1 .08 .75 Total2 15 16 31 3 .89 .80 1 .03 .85 EXPECT 15.5 15.5 31 z the population homogeneity P=.40. Table 50. Backcross Result of ( Orange x Rose/White ) x Rose/White For The Orange Flower Color Inheritance In Pelargonium x hortorum. FAMILY CROSS OBSERVED Total D.F. X2 P RED ORANGE ROSE/WH. (1:1) 86-20-7 X 82-60C 8 7 15 l .067 .97 EXPECT 7.5 7.5 15 74 Table 51. F2 Segregation Progenies of the Peach Hybrids Resulting From the Cross of Picotee x Orange For the Orange Flower Color Inheritance In Pelargonigm x hortorgm. FAMILY OBSERVED PEAC. ORANGE ROSE/ DEEP LT. PICT. TOTAL WHITE SALM. PK. 86-27-8 43 15 15 17 3 6 99 86-27-12 37 12 13 15 5 6 88 86-27-36 33 11 13 18 5 3 83 86-27-43 40 14 12 10 7 5 88 Total 153 52 53 60 20 20 358 "11111111111111“